1

ACTIONS OF PENTOXIFYLLINE ON SPERMATOZOA KINEMATICS, THE ACROSOME REACTION AND SPERM-ZONA INTERACTION

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGY AND BIOCHEMISTRY, BRUNEL UNIVERSITY, UXBRIDGE, UNITED KINGDOM

BY

MOSES PAUL

FERTILITY LABORATORY, DEPARTMENT OF CHEMICAL PATHOLOGY ROYAL POSTGRADUATE MEDICAL SCHOOL, QUEEN CHARLOTTE'S AND HAMMERSMITH HOSPITALS, GOLDHAWK ROAD, LONDON

1994

Thesis title

Actions of pentoxifylline on spermatozoa kinematics : the acrosome reaction and spermzona interaction.

Author

Paul, Moses.

Awarding Institution

Brunel University

Year of Award 1994

Qualification PhD thesis name

Qualification doctoral Level

Keywords

Assisted reproduction Human physiology Biochemistry Human physiology Biochemistry

BL Ref. Nos. ThOS Persistent ID uk.bl.ethos.241598 ILS catalogue number 7125998 Shelfmark

DX184829

4 ABSTRACT In many assisted reproduction procedures, the drug Pentoxifylline (PF) is used to enhance spermatozoa (sperm) motion. However, no thorough study has examined under what circumstances PF might be useful, nor what concentration is most appropriate, nor exactly what parameters of motion (and other characteristics of sperm) are affected by PF. Therefore, the overall objective was to answer these questions. The thesis then proceeds to study the effect of PF on the acrosome reaction and sperm-zona pellucida binding. This study revealed that the optimum conditions to produce the maximum stimulation with PF were one hour incubation at 37 0C and the optimal concentration was 6 mM PF/L in semen and 2.8 mM PF/L in suspensions of sperm. Results also showed a significant enhancement in curvilinear velocity and lateral head displacement. However, PF did not affect the percentage of motile sperm. It further demonstrated that each sample of sperm responded to varying degree of enhancement, with 1 in 10 samples not responding to PF stimulation. Washing alone produced an increase in motion characteristics in the control samples. However, suspensions of sperm that had been stimulated with PF and then had the drug removed by washing showed a significant reduction in the sperm motion characteristics. PF alone did not affect the proportion of sperm that had undergone the acrosome reaction; however, in the presence of Ionophore A23187, it significantly reduced the proportion of sperm that had undergone the acrosome reaction. Sperm in the presence of PF had a significantly increased tendency to bind to the zona pellucida; however, sperm pretreated with PF, which was then subsequently removed by washing, showed a decreased tendency to bind to the zona pellucida.

5 ACKNOWLEDGEMENTS

I wish to express my sincere thanks to Dr Kevin Lindsay for his guidance, advice, wisdom and encouragement throughout this project which made this research a memorable experience. I am grateful to Kevin for allowing me to use the research facilities at the Fertility Laboratory. Special thanks goes to the members of the

laboratory, Mrs Ivy Floyd and Mr Robert Swan, for technical support in routine seminology and for putting-up with my constant harassment in asking for semen samples. A special word of thanks goes to Professor John Sumpter for his intellectual guidance, constructive criticism, unfailing enthusiasm and inspiration which enabled me to carry out this research. I take this opportunity to thank the staff of Electron Microscopy Unit, Dr Tim Ryder and Miss Margaret Mobberley, for their assistance in cutting of blocks, staining, mounting of specimens and for teaching me how to use the Electron

Microscope. I am greatly indebted to Mr Vic Robinson, Consultant Obstetrician and Gynaecologist of Hillingdon Hospital, who initially allowed me to embark on this project and without his support and encouragement this course of study may not have taken off the ground. I am sincerely grateful for this. Finally, I wish to express my gratitude and thanks to my wife Hiroko for her patience, understanding and encouragement, and advice on statistical manipulation of data. Also to my 2 year old son Shimon for his tolerance as he was ignored while I worked on this project. 5

TABLE OF CONTENTS

Title page British Library, UK filing Doctorate Award Abstract Acknowledgements Table of contents List of Tables List of Figures Dedication Abbreviations

1 2 3 4 5 6 14 16 18 19

Chapter 1:

GENERAL INTRODUCTION

20

1.1

Spermatogenesis

20

1.2

Sperm structure

22

1.3

Oogenesis

27

1.4

Gamete Transport

29 6

1.5

Fertilization

31

1.6

Infertility

34

1.7

Methods to study sperm motility

38

1.8

Inducers of sperm motility

43

1.9

Pentoxifylline

45

1.10

Aim of study

47

Chapter 2:

GENERAL MATERIALS AND METHODS

48

2.1

Equipment

48

2.2

Chemicals

49

8 2.3 Sample selection and Sperm assessment 51

2.4

Sperm suspension preparation

52

2.5

CASA methodology

54

2.6

Stability of Pentoxifylline to external factors

61

2.7

Statistical analysis of experimental data

65

Chapter 3

INVESTIGATIONS INTO FACTORS AFFECTING SPERM MOTILITY 67

3.1

Introduction

67

3.2

Effect of incubation temperature on sperm motility when Pentoxifylline is present 69 8

3.3

Effect of incubation time on sperm motility when Pentoxifylline is present 72

3.4

Effect of Heparin on sperm motility when Pentoxifylline is present 77

3.5

Effect of Percoll on sperm motility

80

3.6

Effect of centrifugation on sperm motility

85

3.7

Comparison between discontinuous Percoll gradient and 'swim-up' methods of sperm suspension preparation 87

3.8

Conclusions

89

Chapter 4

ACTION OF PENTOXIFYLLINE ON SEMEN

90 9

10

4.1

Introduction

90

4.2

Materials and Methods

91

4.3

Statistical Analysis and calculations

93

4.4

Results

95

4.5

Discussion

102

Chapter 5

ACTION OF PENTOXIFYLLINE ON SUSPENSIONS OF SPERMATOZOA AND EFFECTS AFTER ITS REMOVAL BY WASHING 106

5.1

Introduction

106

5.2

Materials and Methods

108

5.3

Statistical analysis and calculations

111 10

11

5.4

Results

113

5.5

Discussion

124

Chapter 6

THE ACROSOME REACTION RESPONSE TO PENTOXIFYLLINE CHALLENGE 130

6.1

Introduction

130

6.2

Materials and Methods

133

6.3

Statistical Analysis

139

6.4

Results

140

6.5

Discussion

152

Chapter 7

THE EFFECT OF PENTOXIFYLLINE ON THE BINDING OF SPERMATOZOA TO THE ZONA PELLUCIDA 159

7.1

Introduction

159 11

12

7.2

Materials and Methods

162

7.3

Statistical analysis

172

7.4

Results

173

7.5

Discussion

179

Chapter 8

GENERAL DISCUSSION

185

Appendix A

EHBS chemical composition

195

Appendix B

Action of PF on semen - The number of sperm analyzed per PF conc. group 196

Appendix C

Action of PF on suspensions of sperm The number of sperm analyzed per PF conc. group 196

12

13

REFERENCES

197

Papers published based on this thesis 1. Actions of pentoxifylline directly on semen 2. The paradoxical effects of pentoxifylline on the binding of Spermatozoa to the human zona pellucida 3. Factors affecting pentoxifylline stimulation of sperm kinematics in suspensions.

240 241

ccc

vvv

13

14 LIST OF TABLES

Table 2.1 Table 2.2 Table 2.3

Parameter setting of Celltrack/s CASA CASA reproducibility Comparison between live and taped CASA measurements

57 59

60 62 63 64

Table 2.4 Table 2.5 Table 2.6 Table 3.1

Stability of stock PF to freezing for 6 weeks Stability of stock PF to freezing for 10 weeks Stability of stock PF to thawing at 560 C The effects of incubation temperature on sperm motion after treatment with Pentoxifylline

71

Table 3.2

Effect of PF on VCL and ALH values of sperm incubated at two different temperatures 72

Table 3.3

The effects of incubation times on sperm motion after treatment of sperm samples with Pentoxifylline 75

Table 3.4

The effect of heparin on sperm motion after treatment of sperm samples with Pentoxifylline 79 81 86

Table 3.5 Table 3.6 Table 3.7

The effect of Percoll on sperm motion characteristics The effect of centrifugation on sperm motion characteristics Comparison between Percoll gradient and 'swim-up' separation methods

88

Table 4.1

Design of experiments and number of samples per group 91

14

15 Table 4.2 Kinematic responses of sperm to various concentrations of Pentoxifylline Table 4.3 Maximum recovery of motile sperm when treated with various concentrations of PF Table 5.1 Kinematic responses of sperm in suspension to various concentrations of Pentoxifylline Table 5.2 Table 6.1 Table 6.2 Table 6.3 Table 6.4 Table 6.5 Table 7.1 Residual effects of PF after its removal by washing The effect of PF on the acrosome reaction The effect of PF+IP on the acrosome reaction The acrosome reaction of controls evaluated by TEM Motion characteristics of sperm used in the AR experiment 114 120 141 142 144 149 98 96

Comparison of methodology condition used in evaluating AR155 Effect of FITC labelling of sperm on their ability to bind to intact-zona 173 174

Table 7.2 Table 7.3

Evaluation of cutting the intact-zona into equal halves Percentage of sperm bound to intact-zona using control sperm labelled with FITC

175

Table 7.4

Percentage of sperm bound to intact-zona using PF-pretreated sperm labelled with FITC 176 177

Table 7.5 Table 7.6

Effect of PF on sperm-hemizona binding Comparison of percentage of sperm binding to intact-zona and hemizona

178

15

16 LIST OF FIGURES Figure 1.1 Diagrammatic representation of a human sperm Figure 3.1a The effect of incubation time on VCL of PF-treated sperm Figure 3.1b The effect of incubation time on ALH of PF-treated sperm Figure 3.1c The effect of incubation time on LIN of PF-treated sperm Figure 3.2a The effect of Percoll and its removal by washing on VCL Figure 3.2b The effect of Percoll and its removal by washing on ALH Figure 3.2c The effect of Percoll and its removal by washing on MOT% Figure 3.2d The effect of Percoll and its removal by washing on LIN Figure 4.1a Effect of different concentrations of PF on VCL Figure 4.1b Effect of different concentrations of PF on VSL Figure 4.1c Effect of different concentrations of PF on ALH Figure 4.1d Effect of different concentrations of PF on manual count Figure 4.2 Effect of different concentrations of PF on sperm - SI 1 Figure 4.3a Rise in VCL in individual patient's sperm in response to 6 mM PF/L Figure 4.3b Rise in ALH in individual patient's sperm in response to 6 mM PF/L Figure 5.1 Flow diagram of experimental protocol Figure 5.2a Effect of different concentrations of PF on VCL on sperm in suspension Figure 5.2b Effect of different concentrations of PF on ALH on sperm in suspension 115 16 115 101 111 101 23 75 76 76 82 83 83 84 97 97 98 99 100

17 Figure 5.3 Effect of different concentrations of PF on sperm in suspension - SI 2 Figure 5.4a Rise in VCL in individual patient's sperm suspension in response to 3 mM PF/L Figure 5.4b Rise in ALH in individual patient's sperm suspension in response to 3 mM PF/L Figure 5.5a Persistent effects of PF on VCL - Percoll gradient method Figure 5.5b Persistent effects of PF on ALH - Percoll gradient method Figure 5.6a Persistent effects of PF on VCL - 'swim-up' method Figure 5.6b Persistent effects of PF on ALH - 'swim-up' method Figure 6.1 Flow diagram of experimental protocol Figure 6.2 CTC stain - Effect of PF, PF+IP & IP on the AR Figure 6.3 Electron micrograph of sperm head x 12 000 magnification showing various stages of the human sperm AR Figure 6.4 Effect of PF, PF+IP & IP on MOT% Figure 6.5 Effect of PF, PF+IP & IP on VCL Figure 6.6 Effect of PF, PF+IP & IP on ALH Figure 7.1 Flow diagram of sperm-zona binding Figure 8.1 Flow diagram of Conclusions 146 150 150 151 168 194 118 121 121 123 123 139 143 117 116

17

18 DEDICATION

To my wife Hiroko and our son Shimon for their love, understanding, and patience

If a man will begin with certainties, he shall end in doubts; but if he will be content to begin with doubts, he shall end in certainties. Francis Bacon 1561-1626

18

19 ABBREVIATIONS

ALH AR CASA cAMP CTC EHBS

Lateral head displacement Acrosome reaction Computer assisted sperm analysis Cyclic adenosine monophosphate Chlortetracycline Earles-Hepes balanced salt

FITC-PNA Fluorescein isothiocyanate conjugated Arachis hypogea agglutinin FITC-PSA Fluorescein isothiocyanate conjugated Pisum sativum agglutinin FSH HSA IP Follicle stimulating hormone Human serum albumin Ionophore A23187

LIN Linearity LH MOT NS PBS PF ROS RT SEM SI Sperm TEM VCL VSL ZP Luteinizing hormone Motility Not significant Phosphate buffered saline Pentoxifylline Reactive oxygen species Room temperature Standard error of mean Stimulation index Spermatozoon/Spermatozoa Transmission electron microscopy Curvilinear velocity Straight line velocity Zona Pellucida

19

20 CHAPTER 1 GENERAL INTRODUCTION

The roots of education are bitter, but the fruit is sweet. Aristotle 384-322 BC

Human spermatozoa (sperm) are highly specialised cells with a haploid number of chromosomes that must display several disparate properties such as cell recognition, movement, secretion and membrane fusion, if they are to successfully participate in the reproductive process. Consequently, the fertilizing potential of a sperm will not depend on any single aspect of its biochemistry and physiology, but rather on the consolidation of several independent components, each of which contributes to the general functional competence of the cell.

1.1 Spermatogenesis The process of gametogenesis in the male occurs within the seminiferous tubules of the testes, resulting in the production of sperm. It consists of two phases, spermatogenesis and spermiogenesis (Dadoune and Demoulin, 1993).

Spermatogenic activity requires an adequate concentration of testosterone, an androgen that is produced by the Leydig cells when they are stimulated by luteinizing hormone (LH) (Roberts et al., 1991). Sertoli cells regulated by follicle stimulating hormone (FSH) play a crucial role in regulation of spermatogenesis (Matsumoto and 20

21 Bremner, 1987). By mitotic (proliferative) and meiotic (reductive) divisions, millions of spermatids are produced, each with an haploid number of chromosomes. Meiosis ensures the biological necessity of evolution through the introduction of controlled variability by the process of cross-over. Just before the first meiotic division, primary spermatocytes replicate their DNA and contain twice the normal amount (4N). In the diplotene stage of meiosis, the sister chromatids of homologous chromosomes that are linked by chiasmata, exchange their chromosomal material by cross-over. After the first meiotic division, each secondary spermatocyte contains an haploid number of chromosomes, but the total amount of DNA in each daughter spermatocyte is equal to that of a normal somatic cell (2N), since each chromosome is in a double structure. During the second meiotic division, each double-structured chromosome divides, so that each daughter cell, a spermatid (1N), contains 23 chromosomes. The possible number of recombinations of the 23 chromosomes in man is enormous (Egozcue et al., 1983; De Braekeleer and Dao, 1991).

1.1.1 Spermiogenesis The spermatids complete their development into sperm by undergoing structural changes that involve extensive nuclear and cytoplasmic reorganisation. The nucleus condenses and becomes the sperm head; the two centrioles give rise to the flagellum or axial filament; part of the Golgi apparatus becomes the acrosome; and the mitochondria concentrates into a sheath located between two centrioles (Holstein, 1976).

21

22 1.2 SPERM STRUCTURE

1.2.1 Sperm A typical human sperm (as shown in Fig. 1.1) is a complex and highly specialized cell composed of a head and tail or flagellum (Mann, 1964; reviewed by Fawcett, 1975; Yanagimachi, 1981; reviewed by Zamboni, 1992). The head is a flattened oval measuring four to six microns in length, two to four microns in width, and 0.5 to 1.5 microns in thickness. The total length of the sperm is about 60 microns.

1.2.2 Sperm head The head of a human sperm is occupied mostly by the nucleus and the acrosome, with a small amount of cytoplasm and some cytoskeletal components. The nucleus consists of dense chromatin matrix carrying the haploid genome. The major nuclear protein associated with sperm DNA is protamine, which is relatively small highly basic protein rich in arginine and cysteine (Grimes, 1986). The acrosome is a membranous structure that sits as a cap over the nucleus, occupying three-fourths of the anterior part of the sperm head. It is an organelle that originates from the Golgi complex in spermatids, and contains hydrolytic enzymes that may facilitate sperm penetration of the zona pellucida and fusion with plasma membrane of the oocyte to achieve fertilization (Yanagimachi, 1988).

22

23

23

24

1.2.3 Capacitation Capacitation was first described, independently, by Austin and Chang in 1951. They observed that sperm must reside within the Fallopian tubes (oviduct) for some time before ovulation to acquire fertilizing ability. Capacitation can be defined as a process that provides a sperm with the ability to undergo physiological modification that requires a relatively high concentration of extracellular Ca2+ (Stocks, 1990), such that sperm-oocyte fusion can occur. In the mouse, it has been demonstrated that an increase in flagellar activity known as hyperactivation is a characteristic feature of capacitation (Fraser, 1977). Hyperactivation is characterized by episodic, wide amplitude or "whiplashing" beating of the flagellum. Therefore, sperm movement is characterized by periods of nonprogression ("dancing") interrupted by a brief linear motion ("dashing") with the sperm head exhibiting an erratic figure of eight motion in human (Burkman, 1984).

1.2.4 Acrosome The acrosome is a vesicular structure, a lysosome-like organelle (Allison and Hartree, 1970), lying beneath the plasma membrane of the head. It can be divided morphologically into anterior and posterior segments, called the acrosomal cap and the equatorial segment, respectively. The acrosome contains a variety of glycoproteins, glycolipids and a large array of hydrolytic enzymes. The following enzymes are reported to be present: - hyaluronidase, acrosin, proacrosin, esterase, neuraminidase, collagenase, phosphatase, phospholipase A, -N-

acetylglucosaminidase, arylsulfatase and arylamindase (McRorie and Williams, 24

25 1974; Stambaugh and Smith, 1976; Meizel, 1984). These enzymes may be

associated with the membranes or contained within the acrosome. The equatorial segment forms a band that approximately overlies the equator of the head of spatulate sperm.

1.2.5 Acrosome reaction The acrosome reaction (acrosomal exocytosis) is initiated by multiple fusion between the plasma membrane and the outer acrosomal membrane, resulting in vesiculation that is largely confined to the acrosomal cap region (Piko, 1969; Barros et al., 1967; Bedford et al., 1978). The acrosome reaction causes the acrosomal contents to be externalized and the inner acrosomal membranes to become the limiting membrane of the anterior sperm head. Two main functions are served by the acrosome reaction:- it gives the sperm the capability to, firstly, penetrate through the zona pellucida of the oocyte and, secondly, membrane. to fuse with the oocyte plasma

1.2.6 Sperm Flagellum The flagellum of human sperm (Eddy, 1988; Satir, 1979; Linck, 1979) consist of four segments: the connecting piece (neck), the middle piece, the principal piece and the end piece. The human sperm flagellum is about 55 microns in length. A narrow neck links the sperm head to the flagellum. The sperm flagellum, which is more than one micron in diameter in the connecting piece segment, tapers progressively towards its posterior tip. The connecting piece marks the beginning of the axial filament complex, or axoneme, which forms the core of the flagellum. The 25

26 axoneme consists of nine microtubular doublets, circularly arranged to form a cylinder around and connected to the central pair of single microtubule by dynein arms. This "nine plus two" arrangement extends the full length of the flagellum. The forward progressive motile force necessary for the sperm to reach the oocyte and achieve fertilization is provided by the flagellum (Gibbons, 1979; Ishijima, 1990). The flagellar movements result from sliding of the axonemal microtubules alongside one another. The coordination of the sliding movements between the peripheral doublets and central singlets lead to creating flagellar waves. Besides an intact plasma membrane, flagellar motility requires an adequate supply of adenosine triphosphate (reviewed by Zamboni, 1992).

1.2.7 Sperm motility Sperm motility is an expression of its viability and structural integrity, and is necessary for transportation through the female tract to the site of fertilization (Katz and Drobnis, 1990). The mechano-chemical mechanisms responsible for sperm motion are complex and incompletely understood. The processes involved are intrinsic and extrinsic to the sperm (Hoskin, 1979). Intrinsic processes include the sperm metabolic activity, the structure of its axoneme, membrane integrity, and transport phenomena. Extrinsic factors include substrate availability, ionic signals and the physical properties of the sperm microenvironment (Tash and Means, 1983). It has been reported (David et al., 1981) that sperm show four important aspects in their movement: 1) flagellar beating occurs in the transverse plane of the head and always to the same side; 2) flagellar beating and rotation are synchronised; 3) rotation occurs when the flagellar wave has reached a point about 20-25 microns 26

27 distal to the neck of the sperm; 4) the velocity of wave propagation on the flagellum is highly correlated with the velocity of cell progression. On average, freshly ejaculated sperm swim at 75 M/sec (Blasco et al., 1984)

1.3 OOGENESIS The ovaries are derived from the germinal ridge during embryogenesis and descend into the pelvis in fetal life. During the fetal period, primordial cells, or oogonia, proliferate within the cortex of the fetal ovaries and subsequently become surrounded by epithelial cells to form primary follicles.

1.3.1 Oocyte maturation In the ovaries (Szllsi, 1993), each primary oocyte undergoes two specialized nuclear divisions that result in the formation of four cells containing half the number of chromosomes. In the first stage of meiosis, the primary oocyte is actively synthesizing DNA and protein in preparation for entering prophase. The DNA content doubles during prophase as each chromosome replicates. Each chromosome pair is attracted to its homologous mate to form a tetrad; chromosomes of the same parental origin are connected to one another by their centromeres. The members of the tetrad come to lay side by side. Before separation, the homologous pairs of chromosomes exchange genetic material by a process known as crossingover, which accounts for most of the qualitative differences between the resulting gametes. The subsequent meiotic stages distribute the members of the tetrad to the daughter cells so that each cell receives the haploid number of chromosomes. At 27

28 telophase, one secondary oocyte and a polar body have been formed which are no longer genetically identical, since the members of the chromosomal pairs, and parts of chromosomes, may have been exchanged. Cytoplasmic organisation of the oocyte occurs during the final stages of oocyte maturation and is regulated by steroids (Jones, 1990; Trnell et al., 1991). Meiotic and cytoplasmic maturations are stimulated by the luteinizing hormone (LH) surge. The Golgi apparatus of the oocyte synthesizes lysosomal-like granules that migrate towards the surface and may be gathered into clusters or scattered individually in the subcortical ooplasm (Gulyas, 1980). New and distinctive proteins are synthesized (Wassarman, 1983) and this activity prepares the ooplasm for fertilization. Structural changes in the zona pellucida occur at the ultrastructural level during oocyte maturation (Tesarik et al., 1988). During follicular growth, mitotic

activity of a single layer of follicular cells surrounding the oocyte results in an increase of 3-5 layers. The outermost layers of follicular cells form the granulosa cells, which differentiate into the cumulus oophorus. The oocyte synthesizes and secretes a proteoglycan-like substance between the ooplasm and the innermost follicular cells, forming the zona pellucida. It is a relatively thick, mesh-like

interconnecting filament that surrounds the oocyte, separating it from the follicle (Greve and Wassarman, 1985). The mouse zona pellucida is composed of three sulphated glycoproteins; ZP1, ZP2, ZP3 (Bleil and Wassarman, 1980a, 1980b). ZP3 induces the sperm acrosome reaction and mediates the initial binding of sperm to the oocyte. ZP2 acts as a secondary sperm receptor. ZP2 and ZP3 exist as dimers in long filaments that appear to be cross-linked by ZP1. ZP2 along with ZP3, is 28

29 biochemically modified after fertilization to provide the postfertilization block to polyspermy (Wassarman, 1988a). In the human ovaries, there are between 100,000 and 400,000 primary oocytes present at puberty (Byrd and Wolf, 1984). These germ cells are arrested as primary oocytes at the diplotene stage of the meiotic prophase until just before they are ovulated. The matured ovum, approximately 100 to 150 M in diameter, is released from the ovary at the secondary oocyte stage; the second stage of meiotic division is triggered in the oviduct by the entry of the sperm.

1.4 GAMETE TRANSPORT The human sperm, about 60 microns in length, must travel through some 3040 centimetre of male and female tract before reaching the point where fertilization occurs, the oviduct (Harper, 1988). Although over twenty million sperm per millilitre are produced by a fertile man, only one sperm is required for fertilization. Before the sperm can acquire this fertilizing capacity, they undergo a series of changes in the male and female reproductive tracts. These changes are called maturation in the male tract (Moore, 1983), and capacitation and activation in the female tract. The entire process of maturation in the male tract is crucially dependent upon adequate stimulation of the epididymis by LH and the testosterone produced by Leydig cells (Roberts et al., 1991).

29

30 1.4.1 Sperm transport in the male The sperm produced by spermatogenesis are initially completely immotile and are transported passively from the seminiferous tubules to the rete testis which acts as a reservoir (Bedford, 1975). Ten to twenty ductuli efferentes connect the rete testis to the single, long, highly convoluted duct called the epididymis (Robaire and Hermo, 1988). Ciliary activity of the luminal epithelium, contractile activities of the smooth muscular elements of the efferent duct wall, and the flow of secretions from the testis all contribute to the movement of sperm at this stage. During sperm passage through the epididymis, they acquire the ability to swim progressively and the capacity to fertilise (Bedford et al., 1973). The sperm pass from the epididymis into the vas deferens as a very densely packed mass, the movement being due to the muscular activity of the epididymis and vas deferens. During the process of ejaculation, mature sperm with seminal fluid are transported out through the urethra.

1.4.2 Sperm transport in the female The sperm deposited in the vagina travel through the cervical mucus before reaching the oviduct (Fox and Fox, 1971). Thus, sperm motility is of prime importance. The cervical canal has very thick connective tissue walls which are lined by many thick crypts. Sperm, after gaining entry into the cervical mucus, are found lodged in these cervical crypts, from which they are subsequently released to continue their journey into the uterus (Elstein et al., 1972; Harper, 1988). Sperm motility in the uterus is controlled by uterine contractions rather than by the sperm flagellar activity. In goats and cattle, it has been shown that at the uterotubal junction, certain selective filtering of sperm occurs, presumably to permit the fittest to survive 30

31 and fertilize the ova in the ampullar region of the oviduct, while the dead sperm are denied entry (Mattner, 1968). The oviduct, through which the sperm need to travel before reaching the site of fertilization, can be pictured as a tube (ampulla) of five to eight centimetres long, internal diameter ranging from one millimetre to one centimetre, with a funnel like structure (the infundibulum) at the end. The lumen of the ampulla is wider at the infundibular end than at the ampullar-isthmic junction. Motile sperm reaching the ampullar-isthmic junction are carried by the currents of the ampullar fluids secreted by the oviductal secretory epithelium, assisted by the oviductal ciliated epithelium and by the flagellar activity of the sperm (Suarez et al., 1990). Fertilization is thought to occur in the oviduct nearer to the infundibulum (Harper, 1988).

1.5 FERTILIZATION Fertilization of the oocyte by sperm, the means by which sexual reproduction takes place in all multicellular organisms, is fundamental to propagation. In both mammals and non-mammals, the pathway that leads to fusion of an oocyte with a single sperm consists of many steps that occur in a mandatory order. This sequence of steps in the mouse oocyte includes species-specific cellular recognition, intracellular and intercellular membrane fusions, and enzyme catalysed modifications of cellular investments (Wassarman, 1987; Overstreet and Cross, 1988). Fertilization, whether occurring in vivo or in vitro, proceeds in a fixed pathway:sperm capacitation, penetration through oocyte investments, acrosome reaction,

31

32 binding and penetration of the zona pellucida, fusion of the gametes, and resulting in the development of the pronucleus (Wassarman, 1988a; Kopf, 1990; Crozet, 1993).

1.5.1 Sperm penetration through oocyte investments The capacitated sperm can move through the cumulus and corona radiata cells through the action of hyaluronidase, which hydrolyses and depolymerizes the intercellular hyaluronic acid matrix (Yanagimachi, 1988). Only capacitated sperm can penetrate through cumulus cells while sperm that have lost the acrosome cap through acrosome reaction appear to stick to the cumulus surface (Cummins et al., 1986).

1.5.2 Sperm receptors After passing through the cumulus mass, the capacitated sperm attach to the zona pellucida via receptors on the surface of the oocyte and proteins present on sperm outer membranes (Wassarman, 1988b). The primary site for sperm-zona interaction is the zona pellucida (Tesarik, 1989; Shabanowitz and O' Rand, 1988a; 1988b) which is a highly differentiated acellular structure rich in carbohydrate residues that may play the key role in sperm-zona binding (Ahuja, 1985; Henderson et al., 1988; Fraser and Ahuja, 1988; Mori et al., 1993). The receptor on the mouse oocyte zona pellucida has been identified as ZP3, a glycoprotein of molecular weight 83 kDa (Bleil and Wassarman, 1980a; 1983; Wassarman, 1988c; 1990). They further showed that ZP3 acts as the acrosome reaction inducer, causing the sperm to undergo the acrosome reaction, and it also mediates the initial binding of sperm to the zona via O-linked oligosaccharide side chains. ZP2 acts as a secondary sperm 32

33 receptor. Similarly, the human zona pellucida is shown to contain the ZP3 receptor (Shabanowitz and O' Rand, 1988a; 1988b). The human genes that encode ZP3 receptor have been cloned and sequenced using mouse ZP3 cDNA as a probe (Chamberlin and Dean, 1990). Chamberlin and Dean (1990) demonstrated that there is a high degree of conservation between the coding regions of the human ZP3 and mouse ZP3; both have unusually short 5' and 3' untranslated regions and both contain a single open reading frame that is 74% identical. Further, recent sequencing studies on the human ZP2 genes has shown that the sequences of its coding regions are 70% identical with those of the mouse ZP2 (Liang and Dean, 1993).

1.5.3 Sperm-oocyte fusion The acrosome-reacted sperm 'drills' through the zona pellucida to enter the perivitelline space. The equatorial segment of the sperm head attaches to the plasma membrane of the oocyte and this process activates the oocyte (GaddumRosse, 1985). The 'activated' oocyte completes its second meiotic division; 23 double-stranded chromosomes split at their centromeres, and chromatids separate to oocyte or second polar body. This process results in a haploid number of chromosomes and a haploid amount of DNA in the oocyte. Fusion occurs between the sperm plasma membrane and the oocyte plasma membrane, gradually incorporating the sperm into the ooplasm. When the sperm nucleus is incorporated into the oocyte cytoplasm, it undergoes a series of transformations such as nuclear envelope disintegration, reduction of the disulphide bonds of DNA-associated protamines, chromatin decondensation and replacement of the sperm specific protamines by histones leading to the formation of the male and female pronuclei 33

34 from the sperm and oocyte chromatin, respectively. The final event of fertilization process involves the reorganization and pairing of maternal and paternal chromosomes and formation of the zygote (Yanagimachi, 1988). The gamete fusion also triggers the oocyte to initiate the cortical reaction and the 'block to polyspermy'.

1.5.4 Cortical reaction and the block to polyspermy The human oocyte relies primarily on the 'zona reaction' to control polyspermy (Plachot and Mandelbaum, 1990). The cortical granules, membrane bound lysosome-like organelles 200 to 600 nm in diameter, are released into the perivitelline space between the plasma membrane and zona pellucida (Gulyas, 1980). The granules contain various hydrolytic enzymes such as proteinases and peroxidase. In the golden hamster, cortical granules induce the loss of sperm receptor activity in the zona pellucida so that binding and penetration of supplemental sperm is blocked (Barros and Yanagimachi, 1971; Wolf and Hamada, 1977).

1.6 INFERTILITY

1.6.1 Male-related infertility Infertility is often defined as the inability to produce a pregnancy within one year of regular sexual intercourse without any contraceptive measures being adopted (Menning, 1980). It affects approximately 15% of couples and it is estimated that the man is subfertile in 40 to 50% of these infertile couples (reviewed by

34

35 Oehninger et al., 1992). The World Health Organisation (WHO, 1992) has recommended that the normal parameters of semen are as follows:Volume pH Sperm density Total sperm count Motility Morphology Viability White cells MAR test 2.0 mL 7.2-7.8 20x106 sperm/mL 40x106 sperm/mL 50% with forward progression 30% with normal morphology 75% live <1x106 /mL <10% sperm with adherent particles

The male-related subfertility group contains those men whose semen values is sub-optimal according to these WHO criteria. Acosta et al. (1988) have suggested that male infertility may be indicated when the basic semen analysis reveals the following values:Sperm density Motility Morphology Total recovery of motile sperm after separation techniques <10x106/mL <20x106 /mL <40% motile <14% normal forms

Male-related infertility (Irianni and Coddington, 1992; Fisch and Lipshultz, 1992) can be categorized into five groups:35

36 1) Testicular causes - factors affecting spermatogenesis 2) Post-testicular causes - obstructive problems in ducts and sexual dysfunction 3) Pre-testicular causes - hypothalamic or pituitary disorders 4) Genitourinary infections 5) Immunologic causes

1.6.1.1 Medical treatment of male infertility To treat subfertility, men have been orally treated with clomiphene citrate and tamoxifen (Vermeulen and Comhaire, 1978), which increased pituitary

gonadotrophin secretion, leading to an increase in sperm density (in some cases), but there was no concomitant improvement in sperm motility. Subfertile men with persistently low sperm motility have been treated with human chorionic gonadotropin (hCG), which did produce some improvement in sperm motility (Misurale et al., 1969). However, the failure of gonadotropin therapy is due to the induction of antibodies against hCG (Sokol et al., 1980). A limited number of subfertile men have been treated with testosterone, but the main obstacle to this therapy is that the liver metabolises the testosterone before it reaches the target cells (Johnsen et al., 1974). Treatment of subfertile men orally with Pentoxifylline is controversial (see section 1.9.2). Thus, to date, no oral therapy has been successful in stimulating spermatogenesis in situ, in order to increase the quality and quantity of sperm produced by subfertile men. Several new pharmacological compounds are currently under clinical evaluation for their ability to improve the fertility of certain group of patients. Most of these investigations are Phase-One studies.

36

37 A different approach is to treat the sperm produced by such men, rather than treat the men themselves. The aim here is to enhance the quality of sperm possessing sub-optimal characteristics in vitro with chemical inducers (Hammitt et al., 1989; Oehninger and Alexander, 1991) such as caffeine (Moussa, 1983; Rees et al., 1990), Pentoxifylline (Aparicio, 1980b, Tesarik et al., 1992a), or 2deoxyadenosine (Aitken et al., 1986) before insemination. Although the mode of action of chemical inducers on sperm motion is not clearly understood, nevertheless their potential contribution as a stimulant is significant.

1.6.2 Female related infertility Infertility (reviewed by: Corsan and Kemmann, 1991; Breckwoldt et al., 1993) in females can be caused by:1) Hypothalamic-pituitary failure - reduced or absent pituitary gonadotropin release (follicle stimulating hormone & luteinizing hormone). 2) Hypothalamic-pituitary dysfunction - menstrual cycle disturbances, including luteal phase insufficiency, anovulatory cycles or amenorrhoea. 3) Ovarian failure - with no evidence of ovarian estrogen production and with elevated follicle stimulating hormone levels. 4) Congenital or acquired genital tract disorder - anatomical disorders of the genital tract. 5) Endometriosis 6) Obstruction of the tubules 7) Infection of the uterus 8) Immunological reaction 37

38

1.6.3 Unexplained infertility In the 15% of all couples who seek assistance in conceiving, there is a proportion in whom no definite cause of infertility can be ascertained; semen quality fulfils the criteria for normality, and no defect in the woman's reproductive system can be shown. Two possible reasons have been suggested (Templeton et al., 1990): 1) defective transport of gametes to the normal site of fertilization and, 2) failure of fertilization due to defective gamete function.

1.7 METHODS TO STUDY SPERM MOTILITY Although sperm were first observed more than 300 years ago, the concept of semen analysis is relatively new. In 1929, Macomber and Sanders were the first to do sperm counts in man and to look at the differences between fertile and infertile groups. Their fertile group, which contained men who had fathered children, had sperm count over 60 million per millilitre. Macleod (1950, 1951) was the first to look at semen quality by comparing volume, sperm density, proportion of motile sperm, quality of motility and proportion of sperm with normal morphology. These studies set the foundation for modern seminology.

1.7.1 Manual assessment of semen quality Largely, seminology involved looking at sperm with an optical microscope and deducing the various parameters. The results of such analyzes were subjective and operator dependant. Various studies (Bartoov, 1980; Badenoch et al., 1990; Davis 38

39 and Boyers, 1992) have reported significant variability in the subjective estimates of sperm progression and percent motility, within a single observer and between two observers. Therefore, it is difficult to obtain a precise and accurate results with the use of traditional semen analysis methods. The problems of subjective analysis were recognised by the World Health Organisation (WHO, 1987), and led to the introduction of standards that were published in its laboratory manuals. It recommended a simplified motility grading system, based on a standard method of sperm counting, and standardization of other measured parameters. However, these measures have not resolved the problem of subjectivity, which has consequently lead people to seek methods that can measure sperm parameters objectively.

1.7.2 Development of techniques to assess semen quality Spectrophotometry (Sokoloski et al., 1977), an indirect method of measuring sperm speeds in suspension, and laser-Doppler velocimetry (Jouannet et al., 1977), which measure changes in light reflectance by moving sperm, were developed, but unfortunately these methods do not provide information on individual sperm and therefore did not gain popularity. Direct methods, such as photographic ones involving visual assessment of swimming speeds of individual sperm in real time using microscopic grids and stopwatch, were introduced (Harvey, 1960). These included time-exposure photomicrography, multiple-exposure photography and cinemicrography, and included both manual and computer-assisted analysis of the results. Time-exposure photography (Overstreet, 1979) involves photographing for one second the moving sperm using dark-field illumination. Motile sperm produce a 39

40 dark track on a film negative, while immotile sperm appear as overexposed sperm heads, and stationary sperm are blurred or appear with multiple tail images. Using this technique, specific movement characteristics can be studied and the velocity calculated by measuring the straight line track of the motile sperm. However, this method is labour intensive and unsuitable for routine work. Multiple-exposure photography was introduced by Troll and Goldzieher (1962), and involves photographing sperm samples twice, two seconds apart. The negative of one picture was superimposed on the positive of the second, allowing an estimate of sperm velocity to be determined during those two seconds. The problem was accurate superimposition of the positive and negative, which proved to be tedious. Although this method was modified by Makler (1980a), it never gained popularity. Cinemicrography is a 'movie' of sperm motion (Zorgniotti et al., 1958) taken under constant illumination with multiple film frames exposed in rapid succession. This method was further modified to record the images on video cassette tapes (Morales, 1988). Motile sperm occupy different positions on each frame. Frame by frame analysis allowed an accurate identification and estimation of motile and nonmotile sperm velocity and trajectory. Although frame by frame analysis done manually were labour intensive and laborious, it could be computerised. Computerisation technique (Pedigo et al., 1989) promised to revolutionize the study of sperm behaviour, because it could dramatically improve our ability to easily and objectively quantify motion.

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41 1.7.3 Computer-aided sperm analysis (CASA) As the name implies, this technique requires the appropriate equipment to visualise and digitize the static and dynamic images of sperm in a sample, and a video recorder to record this on magnetic tapes. Briefly, the setup consists of :1) a temperature controlled specimen stage and imaging area 2) an optical magnification system 3) a video camera and video cassette recorder 4) a monitor to view analog and digital images 5) a computer system with software for image digitization and mathematical analysis of sperm tracks 6) a keyboard and printer for data input and output

The image in CASA video camera (CCD, charge-coupled device) is generated when individual picture elements, or pixel, is activated as light strikes the CCD array (reviewed by: Boyers et al., 1989, Davis and Katz, 1989; Davis and Boyers, 1992). Each activated pixel produces a voltage proportional to the intensity of the light striking it, and this is encoded into a complex analog signal. These different voltages can be used to activate a pixel in a monitor, thus creating an image on the screen for visual inspection, or they can be encoded as numbers (digitization), to be used by the CASA. In sperm motion analysis (Davis et al., 1992), the first step is to identify digitized sperm heads from non-sperm images; this can be achieved by setting the size range acceptable as a sperm head. Motion is determined by calculating the 41

42 centroid of each digitized head in all video frames and calculating the trajectories of all sperm heads across all video frames. The centroid for sperm heads in each video frame is calculated as a simple average of the x, y coordinates of the pixel making up the object's image. If the centroid does not move from the set value between frames, it is considered nonmotile. Employing mathematical calculations, CASA can provide the following information:- motility%, count, number of cells meeting the tracking requirements, straight line velocity, curvilinear velocity, lateral head displacement, and average path velocity. Recently, due to improvements in the software, additional information like beat-cross frequency, circularity, mean angular displacement, maximum amplitude head oscillation and basic head oscillation, can also be provided by a CASA. The problems in CASA (Boyers et al., 1989; Bendvold and Aanesen, 1990; Olds-Clarke et al., 1990) are image jitters, apparent motion, Doppler shift and bump & cross. Image jitters are produced from fluctuations in the size, shape and luminosity of a sperm image, and are more serious in slow-moving sperm than in fast-moving sperm. Apparent motion is an artifact of the video interlacing process that can be reduced by adjusting the magnification of the sperm heads compared with their real size. Doppler shift is caused by the video camera scanning technique, and can be reduced by insuring that the sperm move randomly with respect to the orientation of the video array. Bump & cross is the result of two or more sperm images occupying the same space simultaneously; it can be reduced by decreasing the concentration of the sperm present in a sperm suspension being analyzed. In a study conducted by Mathur (1986), he was able to show that a computerised analysis of sperm swimming motion is a reliable and rapid technique 42

43 for evaluating semen samples, and that it offered more discrimination than routine semen analysis done manually. Manual methods provide little information on sperm motion characteristics. Therefore, the goals of computerised sperm analysis are: 1) to identify the motion characteristics of sperm that will reliably discriminate between nonfertile and fertile males; 2) to use those motion characteristics to detect the earliest signs of altered reproductive potential; and 3) to relate those parameters to the physiology of sperm in vivo. Sperm motility is believed to be the most important characteristic for evaluating the fertility potential of ejaculated sperm. CASA generated information has shown that fertilization rate in vitro correlates with sperm motility (Chan et al., 1989; Fetterolf and Rogers, 1990; Check et al., 1990; Liu et al., 1991) and that the technique can be used to assess sperm hyperactivation (Mack et al., 1989; Burkman, 1991).

1.8 INDUCERS OF SPERM MOTILITY There are many published reports suggesting that specific chemical agents can stimulate sperm motility or increase the fertilizing ability. These agents include caffeine (Garbers et al., 1971a; Traub et al., 1982), 2-deoxyadenosine (Aitken et al., 1986), Pentoxifylline (Aparicio, 1980b), theophylline (Garbers et al., 1971b; Loughlin and Agarwal, 1992), platelet-activating factor (Ricker et al., 1989), relaxin (Essig et al., 1982; Colon et al., 1986), progesterone (Mbizvo et al., 1990), prostaglandins E (Colon et al., 1986), 3-Isobutyl-1-methylxanthine, IBMX, (Jiang et al., 1984), cyclic AMP (De Turner et al., 1978) and kallikrein (Schill, 1982a; Sato and Schill, 1987). In addition, combinations of inducing agents have been used to potentiate sperm 43

44 motility, including calcium & creatine phosphate (Fakih et al., 1986), IBMX plus 2deoxyadenosine, and caffeine plus 2-deoxyadenosine (Aitken et al., 1986). Furthermore, chemicals like lanthanum and -chlorohydrin have been shown to inhibit sperm motility (Gwatkin, 1985). The effect of Pentoxifylline, a phosphodiesterase inhibitor, has recently been tested in patients with male-factor infertility (Yovich et al., 1990). The results of this work suggested there was an improvement in in vitro fertilization when suspensions of sperm were treated with 3.6 mM PF/L. The choice of 3.6 mM PF/L is empirical. PF is also known to depress the production of superoxide anions by the human sperm (Gavella et al., 1991). The superoxide anions are detrimental to sperm. Furthermore, PF has been shown to improve sperm motion characteristics in both

normozoospermic and asthenozoospermic semen samples (Tesarik et al., 1992a). Hammitt et al. (1989) have shown that caffeine, Pentoxifylline and 2deoxyadenosine significantly increased sperm motility in cryopreserved human semen. Their study also looked at the effect of cAMP, relaxin, adenosine, kallikrein and calcium in sperm motility, but none of these chemicals was found to be a significant motility stimulant. Caffeine (Rees et al., 1990) has been shown to increase lateral head displacement of sperm when a sperm suspension was incubated with 6 mM caffeine/L. The rate of glycolysis of these sperm increased by over 40%. Between 3 and 6 mM caffeine/L, when added directly to semen, showed a good stimulatory effect on the percentage motility, an effect that was statistically significant (Moussa, 1983).

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45 1.9 PENTOXIFYLLINE

1.9.1 Chemical structure Pentoxifylline, C18 H18 N4 O3, (oxpentifylline), a phosphodiesterase inhibitor of the methylxanthine group, has a molecular weight of 278.3, a melting point of 1050 C, and a solubility of 77 mg/mL in water. It was first synthesized in the laboratory by Mohler et al. (1966). The structure of Pentoxifylline is shown in figure 1.2.

Fig.1.2 Chemical structure of Pentoxifylline 1.9.2 Effects of Pentoxifylline in humans Pentoxifylline is an orally active haemorheological agent used for the

treatment of peripheral vascular disease, cerebrovascular disease and several other conditions involving defective regional microcirculation. Pentoxifylline acts by improving the oxygen supply to ischaemic areas, at least in part by increasing red cell deformability and reducing blood viscosity, with consequent improvement in blood flow through the nutritive microcirculation (Muller et al., 1981; Ward et al., 1987). Extensive open and placebo-controlled clinical trials have shown that 45

46 Pentoxifylline, orally given as 300 to 1200 mg/day for at least 6 weeks, results in good to excellent therapeutic response in 60 to 100% of patients with peripheral vascular disorders. In vitro studies using human and bovine platelets have shown that Pentoxifylline raises cAMP levels and inhibits membrane-bound

phosphodiesterase. These changes activate protein kinase that catalyses the phosphorylation of membrane protein by ATP, resulting in inhibition of platelet aggregation tendencies (Stefanovich, 1978). Aparicio et al. (1980a) showed that asthenozoospermic men, when orally treated with 400 to 1200 mg Pentoxifylline/day for two to 12 months, produced semen with significant increases in sperm concentration and motility. In a similar study by Shen et al. (1991), it was demonstrated that when PF was orally given to asthenozoospermic men for three months, the sperm motility significantly increased but sperm concentration did not increase. However, patients with oligozoospermia showed no improvement in sperm motility or in the conception rate (Schill, 1982b). Therefore, the current treatment of male factor infertility with orally administered PF is still rather controversial. Quite a few studies have been published on the use of PF to increase sperm motion characteristics in vitro, prior to use of the sperm for assisted reproductive techniques. The results of these studies are discussed in relation to my own results in the following chapters.

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47 1.10 AIM OF STUDY Although PF is already widely used to enhance sperm motility in Fertility Units, the techniques used to prepare the sperm prior to the treatment with PF are not standardized. It is obviously possible (even likely) that the techniques used might affect the magnitude of the response to PF. Therefore, one aim of this project was to standardize the incubation temperature and the length of incubation of PF in sperm suspensions, and any other factors that may affect sperm motion. As evidenced from section 1.6, there are a proportion of patients with infertility problems for whom PF stimulation of sperm motion characteristics may assist in the treatment. Therefore this project attempts to define the beneficial (or non-beneficial as the case may be) effect of Pentoxifylline on semen and in sperm suspensions, and to determine exactly what dose and conditions are optimum. Investigations were also carried out to study the effect of drug removal by washing, prior to use of the sperm for fertilization which is the normal procedure carried out in Fertility Units. The possible effect of Pentoxifylline on the acrosome reaction, a prerequisite to fertilization, was also studied. Finally, to complete the story, the action of Pentoxifylline on sperm-zona binding was examined. [Please note: When I embarked on this project about four years ago, not much was known about the effects of PF on sperm kinematics, the acrosome reaction and sperm-zona interactions. Since then a number of studies had been carried out and published by various research groups on the subject. The results of these studies are discussed together with my own results in the following chapters.]

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CHAPTER 2

GENERAL MATERIALS AND METHODS

Imagination is more important than knowledge. Albert Einstein 1879-1955

2.1 EQUIPMENT

2.1.1 Celltrak/s Motion Analyzer Motion Analysis VP110 (Motion Analysis Corp., Santa Rosa, USA) was used to analyze all sperm motility characteristics. The analyzer consists of an Olympus BH-2 microscope fitted with a TI-23A CCD camera (NEC, Japan) and a heating stage supplied by Motion Analysis Corp. The analyzer was also connected to a Panasonic NV-W1 video recorder (Panasonic, Japan) to record sperm motility video images on tapes that could be used as permanent experimental records or stored for analysis at a future date.

2.1.2 Fluorescence Microscope The fluorescence microscope used was a Diapan Large Laboratory Microscope (Reichert, Austria) fitted with fluorescent equipment for incident light excitation. The incident light was provided by a HBO-50 mercury vapour burner. The 48

49 fluorescent equipment consisted of a 0515 dichroic mirror with a BG12 excitation filter and a barrier filter (1.5 mm OG1 + 1 mm GG9). The objective used for low magnification work was a Fluor 16, giving a total magnification of 200-times, while for higher magnification work the objective used was an Iris 40, giving a total magnification of 500-times.

2.2 CHEMICALS

All general chemicals were of Analar grade and obtained from BDH (Poole, Dorset, UK.) or Sigma (Poole, Dorset, UK.) unless otherwise stated. Fine chemicals and special chemicals of tissue culture grade were obtained from Sigma. Earles medium was obtained as 10x concentrate from Gibco Ltd (Paisley, Scotland). Gentamycin sulphate BP was supplied by Gibco. Fresenius Water for injection was obtained from FL (Manufacturing) Ltd (Basingstoke, UK). Gas mixture was supplied by British Oxygen Company, UK.

2.2.1 Earles-Hepes balanced salt solution preparation The medium used throughout this study for preparation of sperm suspensions and Pentoxifylline (PF) solution was modified Earles-Hepes balanced salt1 (EHBS) solution. The medium was made from Earles 10x concentrate reconstituted with water for injection, supplemented with 20 mM N-(2-hydroxyethyl) piperazine-Nethanesulphonic acid, 20 mg/L gentamycin, 0.125 mM sodium pyruvate and with the

EHBS chemical composition is given in Appendix A.

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50 osmolality adjusted to 280-285 mOsmol/L, gassed (5% CO2 in air) and pH maintained at 7.4. It was filtered through 0.2 micron filters (Gelman Sciences, USA) and stored at 40 C. Before use, a consistent source of human serum albumin (HSA, Zenalb 20, Bioproducts Laboratory, UK) was added to the culture medium to give a final concentration equivalent to 10% serum. This medium was more robust to potential pH changes from atmospheric effects, while providing sufficient nutrients to support the viability of sperm in the culture medium.

2.2.2 Preparation of discontinuous Percoll gradient Percoll (Pharmacia, UK) is a stable, non-toxic colloidal suspension of silica particles of diameter 15-30 nm coated with polyvinylpyrrolididone (PVP). The density of 1.13 g/mL and low osmolality of <25 mOs/kg H2O are exploited to select and separate viable cells from non-viable cells. It is also easy to make isotonic solution. To harvest motile sperm, generally, a 2-layer gradient of 80 and 40% Percoll was used. The stock solution of 80% Percoll was prepared by adding 80 mL Percoll to 10 mL Earles 10x concentrate plus 10 mL 0.25 mM sodium bicarbonate. The working solution of 80% Percoll was supplemented with 10% HSA. The 40% Percoll solution was prepared by diluting the 80% stock solution with an equal volume of EHBS (without HSA) and supplementing it with 10% HSA. The 40/80% discontinuous Percoll gradient was prepared by placing 2 mL 80% working Percoll in a tube and layering on the top with 2 mL 40% working Percoll, carefully and slowly so as not to disturb the interface.

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2.2.3 Preparation of Pentoxifylline Fresh stock of PF was prepared by dissolving 0.1392 mg in 10 mL of EHBS medium (without HSA) giving a concentration of 50 mM/L. The stock was aliquoted into 2 mL portions and frozen at -200 C until required. 10 to 50 mM PF/L was made by diluting the stock with the appropriate volume of EHBS medium. The prepared working solution was kept at room temperature and discarded at the end of the day. Fresh working solution was prepared every day, either from frozen stock or from fresh stock solution.

2.3 SAMPLE SELECTION AND SPERM ASSESSMENT Semen samples were obtained from patients attending the Fertility Clinic at Queen Charlotte's and Hammersmith Hospitals. All samples used had normal count (>20x106 per millilitre) and motility (50% forward progressive, >25% rapid progressive) in accordance with WHO guidelines (1992). Subjects were requested to abstain from sexual activity for at least three days before producing the sample. Samples were obtained by masturbation and were allowed to liquefy at room temperature before routine semen analysis was performed in accordance with WHO guidelines (1992). The volume and pH (with pH paper) of the sample were measured. The viscosity of the semen was assessed by drawing up 100 L in a pipette and expelling 10 L onto a microscopic glass slide. The viscosity was classified as normal, semi-mucoid or mucoid. Mucoid samples were not used in any experiments. The expelled drop of semen was covered with a coverslip, and the general 51

52 appearance and morphology were assessed using a light microscope. Semen samples with >50% abnormal morphology of sperm and samples with >20% white cells were not included in any experiments. The motilities of the sperm were subjectively assessed by examining several fields and classified into total and progressive motility. All samples were tested for antisperm antibodies by mixed antiglobulin reaction (MAR) test and samples showing >10% of sperm bound to red cells were excluded from the study. A sperm count was done by diluting the semen 1:20 with formal saline and pipetting 10 L onto a Neubauer chamber and counting the cells under 200x magnification with a light microscope.

2.4 SPERM SUSPENSION PREPARATION

2.4.1 Centrifugation migration method Liquefied semen was mixed with 2x its volume of EHBS medium and centrifuged at 500g for five minutes. The supernatant was removed and 1.5 mL of fresh medium was added to the pellet and thoroughly mixed. The sample was centrifuged and the supernatant discarded. The sperm pellet was layered, carefully and gently so as not to disturbed it, with 1 to 1.5 mL medium and placed in a humidified 37 0 C incubator to allow motile sperm to migrate into the overlying medium. At the end of an hour incubation, 0.5 to 1.0 mL of the supernatant was removed, taking care not to disturb the lower layer. The sperm concentration was assessed by taking 20 L and heat treating at 560 C and counting the cells with a Neubauer haemocytometer. A sperm suspension of five to 10 million per millilitre was obtained by adjusting the suspension 52

53 with medium.

2.4.2 Migration centrifugation method After liquefaction, about 1 mL of semen was layered with 1 to 1.5 mL EHBS medium, minimising disturbance at the interface. The tubes were incubated in a humidified incubator at 370C. At the end of an hour incubation, 0.5 to 1.0 mL of the supernatant was removed, taking care not to disturb the lower layer. The sperm concentration was assessed by taking 20 L and heat treating at 560 C and counting the cells with a Neubauer haemocytometer. A sperm suspension of 5 to 10 million per millilitre was obtained by adjusting the suspension with medium.

2.4.3 Percoll gradient method Upto 2 mL of liquefied semen was layered gently onto the prepared 40/80% Percoll gradients (section 2.2.2). The tubes were gently placed in a bench top centrifuge and centrifuged at 500 g for 20 minutes. The tubes were then gently removed and the three top layers were aspirated, leaving behind a small pellet at the bottom of the tube. Any remaining Percoll was washed off by adding 5 mL EHBS to the pellet and thoroughly mixing and re-centrifuging at 600 g for 10 minutes. The supernatant was discarded. The pellet was suspended in 0.8 to 1.5 mL medium. The sperm concentration was assessed by taking 20 L and heat treated at 56 0 C and the cells counted with a Neubauer haemocytometer. A sperm suspension of 5 to 10 million per millilitre was obtained by adjusting the suspension with medium.

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54 2.5 CASA METHODOLOGY Sperm motion analysis was carried out on a Celltrack/s Motion Analyzer. Five microlitres of each sample was pipetted onto a prewarmed Thoma chamber (Weber Scientific, Teddington, UK) with a depth of 20 0.2 m (manufacturer's specification). This was placed on the heated stage (370C) of the Olympus BH-2 microscope and incubated for 3 minutes before analyzing under pseudo-dark field illumination with 5x objective, numerical aperture 0.12 (Watson, Falmouth). In accordance with WHO guidelines (1992), three to six fields were examined and at least 100 cells were counted and 50 cells were tracked. The total number of motile and tracked cells, and the average values (tracked cells) of the following CASA parameters were recorded: motility %, curvilinear velocity, straight-line velocity, linearity and lateral head displacement.

2.5.1 CASA calibration and parameter setting Frame rate - the number of pictures per second that are taken to sample the motion of the moving sperm. In clinical evaluation of semen samples, a value of 60 or 30 frames/sec is commonly used. Duration of data capture - is specified in frames, commonly set at 60. Minimum path length - is the minimum number of frames for an individual cell to be considered a 'valid' path. If a cell appears for less than this number of frames (due to

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55 it swimming into or out of the field of view), it is not counted as a cell at all (either motile or non-motile). Minimum motile speed - user-definable threshold speed by which motile cells are distinguished from non-motile cells. This threshold is based on the average VSL for each path. Maximum burst speed - the value should be set slightly higher than any maximum expected speed of the sperm to be tracked. For human sperm in semen, a value of 250 to 400 microns/sec is recommended. Distance scale factor - this number relates the internal video units of pixels to the units of microns, which is calibrated with a scaling grid by the user. Camera aspect ratio - different kinds of video cameras have slightly different horizontal to vertical scaling ratios, which is corrected for in this correction value. ALH smoothing factor - is the number used in computing the Mean Path for each sperm. The larger the number, the smoother the mean path. Recommended figure is 7. Centroid X search neighborhood - is the value used in the image processing frontend to distinguish separate objects from each other. The recommended setting is 4 pixels for single edge detection. Centroid Y search neighborhood - is the value used in the image processing frontend to distinguish separate objects from each other. The recommended setting is 2 pixels for all normal use Centroid cell size minimum - is the setting that allows one to discard objects in the video field that are smaller than this specified size. It is calibrated by the machine.

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56 The recommended value for normal clinical uses, where there are between 20 and 40 cells in the microscopic view, is 2 units Centroid cell size maximum - is the setting that allows one to discard objects in the video field that are larger than this specified size. It is calibrated by the machine. The recommended value for normal clinical uses, where there are between 20 and 40 cells in the microscopic view, is 6 to 8 units Path maximum interpolation - The local gaps in the paths are filled in with linearly interpolated data to bridge the gap. Once centroids are computed, the pathfinder searches through time and space to connect centroids into valid paths. The Path Maximum Interpolation instructs the pathfinder to 'bridge gaps' in possible paths where the data disappears for one or more frames. Recommended value is 0 or 1 frame for normal clinical use.

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57 The Motion Analyzer was calibrated and set as follows: Table 2.1 Parameter setting of Celltrack/s CASA

System Parameter Frame Rate Duration of Data capture Distance scale factor Camera aspect ratio Minimum path length Minimum motile speed Maximum Burst speed ALH smoothing factor Cell range Depth of sample Cent. X search neighbourhood Cent. Y search neighbourhood Path max. interpolation 2.5.2 CASA terminology

Parameter 60 60 1.7875 1.0210 59 10 500 7 1-5 20 4 2 1

Units frames/sec frames m/pixel frames m/s m/s frames pixels m pixels pixels frame

There is a standard terminology (WHO, 1992) for parameters measured by CASA systems:-

VCL - curvilinear velocity, m/s. Time-average velocity of a sperm head along its actual curvilinear trajectory, as perceived in two dimensions under the microscope. VSL - straight-line velocity, m/s. Time-average velocity of a sperm head along the straight line between its first detected position and its last position. LIN - linearity. The linearity of a curvilinear trajectory. The ratio of VSL:VCL. 57

58 ALH - amplitude of lateral head displacement, m. Magnitude of lateral displacement of a sperm head about its spatial average trajectory. It is expressed as an average of such displacement. VAP - average path velocity, m/s. It is the time-average velocity of a sperm head along its spatial average trajectory, computed and smoothed by the computer. STR - straightness. The linearity of spatial average path. The ratio of VSL : VAP. WOB - wobble. The measure of oscillation of actual trajectory about its spatial average path, the ratio of VAP to VCL. BCF - beat/cross frequency, beat/s. It is the time -average rate at which the curvilinear sperm trajectory crosses its average path trajectory. MAD - mean angular displacement, degrees. The time-average of absolute values of the instantaneous turning angle of the sperm head along its curvilinear trajectory. MOT - motility, %. It is the ratio of motile cells to non-motile cells expressed as a percentage. Total cells analyzed - It is the total cells examined and counted that satisfied the set criteria (section 2.5.1). Cells tracked - It is the number of tracked cells meeting the set criteria. In this project the following parameters were measured by the Celltrack/s Motion Analyzer:- VSL, VCL, LIN, ALH and MOT %.

2.5.3 Evaluation of CASA reproducibility Since CASA was the main equipment used in measurement of sperm motility, it was essential to find out how reliable the data obtained using it were. To do this, a sperm suspension was prepared by the Percoll gradient method (section 2.4.3) of 58

59 concentration between 3 and 15 million per mL. Each sample was measured twice by CASA (section 2.5), and each time a new Thoma slide was prepared. The results (Table 2.2) were assessed by Paired t-test and Pearson's correlation, r. Table 2.2 CASA reproducibility - Mean values of 30 samples

CASA parameter VSL - m/s VCL - m/s LIN ALH - m

1st Analysis 62.5 2.3 119 2.97 54 1.64 4.8 0.12

2nd Analysis 61.1 2.22 118 2.92 54 1.65 4.8 0.12

Correlation (r) 0.81 0.96 0.77 0.91

p value

0.0001 0.0001 0.0001 0.0001

Mean SEM; Paired t-test showed no significant mean difference between 1st and 2nd analysis. The results showed that there was no significant mean difference between the first and second readings, as shown by t-test. The Pearson's correlation between 1st and 2nd analysis was highly significantly (p<0.0001) for all CASA parameters. It follows, therefore, that the CASA system was very reliable and the repeatability of results was consistent. Hence, only single CASA measurements were taken in the experiments reported in chapters 3 to 7.

2.5.4 Comparison of CASA measurements between live and taped samples In certain experiments, it was necessary to video tape the sperm motion and subsequently play back the tape and analyze the sperm motion characteristics in a Celltrack/s Analyzer. Therefore, it was essential to investigate if there was any 59

60 difference between images analyzed from live and taped samples. 17 sperm suspension samples were prepared by the migration centrifugation method (as described in section 2.4.2) to a final concentration between 5 and 10 million sperm per millilitre. Each live sample was measured by a Celltrack/s analyzer (Section 2.5) and simultaneously the images were recorded on a video tape using a video recorder. The tapes were analyzed at a later date. The results (Table 2.3) obtained showed a non-parametric distribution; therefore, significant median differences were tested by Wilcoxon signed rank test. The precision between the two sets of data was assessed by F-test. Table 2.3 Comparison between live and taped CASA measurements

Parameter

Live sample

Taped sample

Pearson's correlatio n

Wilcoxon signed rank p value NS F-test

VSL - m/s

52 42-61

52 46-63 102 88-124 53 42-57 5.0 4.1-5.9 93 83-96

0.95

NS

VCL - m/s

96 83-117

0.97

<0.01

NS

LIN

53 43-60

0.87

NS

NS

ALH - m

4.5 3.7-5.4

0.97

<0.05

NS

MOT -%

91 79-95

0.96

<0.001

NS

Median Values with 25th and 75th centile; NS=Not Significant

60

61 The results showed there were some significant median differences between samples analyzed in the live state compared with analysis of taped sperm motion. VCL showed a significant increase of 6% (p<0.01) for taped analysis compared with live sample analysis, and a highly significant positive correlation (r = 0.97) between the two sets of data (p<0.001). Similarly, ALH showed a significant increase of 11% (p<0.05) for taped analysis, and a highly significant positive correlation (r = 0.97) between the two sets of data (p<0.001). MOT% also showed a significant increase (p<0.001) for taped analysis, again with a highly significant positive correlation (r = 0.96) between the two sets of data (p<0.01). VSL and LIN showed no significant change. This appeared to suggest that analysis of sperm characteristics done by using taped images have raised CASA values, with different parameters being affected to varying extent. These increased values obtained from taped data may be due to the inherent problem of the electronics involved in image capture. The image of each moving sperm was captured on a moving tape, while for the live sample analysis, the sample was stationary. The results of this study showed that, in any studies, all analysis must be done either on live samples or on taped samples and that the two methods of data collection should not be mixed in any one study. F-test showed that the precision between live and taped analyzes were the same. Considering this information, all data collections from CASA studies were done on tapes in chapter 6; the results for all other studies were obtained from live recordings.

2.6 STABILITY OF PENTOXIFYLLINE TO EXTERNAL FACTORS

Most published papers appear to suggest that PF must be prepared fresh. This for a routine laboratory is inconvenient and would lead to wastage of chemicals.

61

62 I decided, therefore, to test the effect of freeze-thaw and the shelf-life of a frozen stock solution of PF.

2.6.1 Effect of freezing stock for 6 and 10 weeks A solution of PF at 50 mM/L was prepared in EHBS medium and aliquoted into small vials of 2mL and stored at -200 C. At the end of six or ten weeks, the stock was thawed at room temperature (23 2) 0 C and 20 mM PF/L working

solutions prepared. A fresh working solution of 20 mM PF/L was also prepared (section 2.2.3). 50 L of PF working solution was added to 0.5 mL sperm suspension, prepared by Percoll gradient method (section 2.4.3), at a concentration between 5 and 10 million per mL. The suspension was mixed and incubated at 370 C for 1 hour before CASA measurements were taken. The results were assessed by Paired t-test.

Table 2.4 Stability of stock PF to freezing for 6 weeks.

CASA parameters

Control

2mM PF/L Freshly prep.

2mM PF/L Froz. stock

p Value

VSL - m/s VCL - m/s LIN ALH - m MOT -%

53 2.07 99 2.24 53 1.55 4.3 0.08 89 4.43

64 2.93 122 2.25 55 2.51 5.0 0.16 89 4.92

62 2.63 121 2.47 53 2.21 5.1 0.15 88 5.91

0.64 0.91 0.54 0.66 0.75

Mean SEM; n=10 62

63 Table 2.5 Stability of stock PF to freezing for 10 weeks.

CASA parameters

Control

2mM PF/L Freshly prep.

2mM PF/L Froz. stock

p Value

VSL - m/s VCL - m/s LIN ALH - m MOT - %

51.0 2.26 94 2.84 54 1.57 4.1 0.12 85 3.86

57 2.79 115 3.58 52 1.40 5.0 0.14 87 2.48

58 2.58 116 3.14 52 1.26 5.0 0.13 89 2.31

0.91 0.70 0.93 0.90 0.63

Mean SEM; n=12

The results (Tables 2.4 and 2.5) show that, when compared, PF prepared freshly and from frozen stock stimulate sperm motion characteristics to a similar extent. The results showed that there were no significant differences between the working solution prepared from frozen stock and the fresh working solution for any of the parameters.

2.6.2 Effect of heat on freeze- thawing A solution of PF at 50 mM/L was prepared in EHBS medium and aliquoted in small vials of 2 mL and stored at -200 C At the end of two weeks, the stock was thawed at 560 C in a water bath and a 20 mM PF/L working solution prepared. A fresh working solution of 20 mM PF/L was also prepared. 50 L of PF working solution was added to 0.5 mL sperm suspension, prepared by Percoll method (section 2.4.3), of concentration between 5 and 10 million per mL. The suspension

63

64 was mixed and incubated at 370 C for 1 hour before CASA measurements were done. The results were analyzed by Paired t-test.

Table 2.6 Stability of stock PF to thawing at 560C.

CASA parameters

Control

2mM PF/L Freshly prep.

2mM PF/L Froz. stock

p Value

VSL - m/s VCL - m/s LIN ALH - m MOT - %

56 2.38 105 3.05 54 1.29 4.5 0.10 92 1.46

65 1.94 127 2.51 53 1.48 5.4 0.13 92 1.41

65 2.07 125 2.21 54 1.63 5.2 0.10 93 1.26

0.93 0.68 0.69 0.34 0.36

Mean SEM; n=16

The results (Table 2.6) show that, when compared, PF prepared freshly and from frozen stock thawed at 560 C stimulated sperm motion characteristics to a similar extent. The results showed that there were no significant differences between the working solution prepared from frozen stock thawed at 56 0 C and the fresh working solution for any of the parameters.

2.6.3 Conclusions The above results showed that PF could be prepared as a 50 mM PF/L stock solution and frozen up to 10 weeks before use. The frozen stock can be thawed at 560 C without any detrimental effect. It also showed that PF was a relatively stable compound between -200 and +560 C in solution. Hence, PF stock solutions for the 64

65 experiments described in chapters 4 to 7 were routinely prepared and frozen for a maximum of six weeks.

2.7 STATISTICAL

ANALYSIS OF EXPERIMENTAL DATA

One purpose of statistical analysis is to reduce a mass of data into a more compact form that highlights general trends and show relationships between variables. The chief objective is to provide quantitative (and therefore, hopefully objective) way of distilling the essential features from the data (Godfrey, 1985a, Godfrey, 1985b). Continuous data, assessed by histogram, which show symmetrical distribution about a central value, with increasing rarity of occurrence as distance above or below this central value increased, is considered under these circumstances to fit a Normal or Gaussian distribution (Sokal and Rohlf, 1981). The mean and median value is usually equal. Any departure from normal frequency distribution is considered skewed distribution, where one tail of the curve is drawn out more than the other and the mean is not equal to the median value. A non-normal distribution can sometimes be converted to a Normal distribution by applying various types of transformation (Gladen et al., 1991; Bland and Altman, 1986). The importance of knowing the type of distribution is valuable in deciding (a) what type of statistical test can be applied and (b) can confirm or reject certain underlying hypotheses about the nature of the factors affecting the phenomenon studied. In this project, all experimental data were initially assessed by histograms. Data that conformed to a Gaussian distribution was then analyzed by one way Analysis of Variance (one-way ANOVA). An Analysis of Variance answers the question whether there are differences among the population means of the groups 65

66 being compared, but it does not pinpoint which population, if any, differ from the others (Godfrey, 1985b). Null hypothesis testing for significance of difference was done, using Paired t-test (or Two sample t-test, depending on the design of the experiment) and the significance of difference was taken at p<0.05. The average response value quoted in Tables and Figures was mean standard error mean (SEM). Data that did not conform to a Gaussian distribution were analyzed by nonparametric tests, which were more robust in handling the skewed data than parametric tests. These sets of data were analyzed by Kruskal-Wallis (analogous to one-way ANOVA) test. Null hypothesis testing for significance of difference was done by using Wilcoxon signed-rank test (analogous to Paired t-test) or Mann-Whitney test (analogous to Two sample t-test) and the significance of difference was taken at p<0.05. The average response value quoted in Tables and Figures was the median and the distribution was from lower quartile (25th percentile) to 3rd quartile (75th percentile).

66

67

CHAPTER 3 INVESTIGATIONS INTO FACTORS AFFECTING SPERMATOZOA MOTILITY

For the scientific acquisition of knowledge is almost as tedious as a routine acquisition of wealth. Eric Linklater b1899

3.1 INTRODUCTION

Spermatozoa (sperm) motility is an important factor in determining the fertilization rate in man. Therefore, much work has been done in investigating sperm motility in sperm suspensions that are invariably prepared with culture media (Quinn et al., 1985; Liu et al., 1988; Muggleton-Harris et al., 1990; Yovich et al., 1990; Ing et al., 1991). However, there is very little information in the way of published works to support the various methods currently used to prepare sperm suspensions. This project investigates what factors, if any, may affect sperm motion characteristics and, based on the results, aims to standardise the method of sperm preparation. In examining the influence of Pentoxifylline (PF) on sperm stimulation (Yovich et al., 1990; Sikka et al., 1991), all authors have used different incubation times and temperatures to show the beneficial effect of PF. Thus, there was no systematic approach in studying the incubation time or temperature in works. 67 all these published

68 The discontinuous Percoll gradient method of separating motile sperm from non-motile sperm has been promoted by Gorus and Pipeleers (1981). It was said to be a rapid, simple procedure and easily applicable in routine seminology work. It has also been shown (Aitken et al., 1988) that Percoll prevents sperm being damaged by reactive oxygen species (ROS) during sperm preparation techniques. Yet, very little is known about its adverse effect, if any, on sperm motility. When sperm stimulants are added to a sperm suspension, or when sperm suspensions are prepared, centrifugation is generally the method used in washing and preparing the sperm (Yovich et al., 1990) and in removing residual Percoll after Percoll gradient separation techniques (Pickering et al., 1989). It is surprising to know how little is known about the effect of centrifugation on human sperm motility. In the specialised treatment of infertility, procedures such as Gamete Intrafallopian Transfer (GIFT), In vitro fertilization - Embryo Transfer (IVF-ET) and Pronuclear stage tubal transfer (PROST) all involves the removal of oocytes from the ovary. The aspirated oocytes are suspended in culture medium (usually EHBS medium) containing heparin that has been used in flushing out the oocytes from the ovary. Heparin prevents blood clotting in the extraction of the oocytes. However, there were no published studies on the effect of heparin on oocytes or sperm. In summary, most of the conditions used in the preparation of sperm were empirically chosen, and therefore it lacks standardisation. This part of my project was devised to investigate the ideal optimal of incubation with Pentoxifylline, and to determine at what temperature this optimal effect on sperm motion characteristics can be obtained. Also, I studied the effect of centrifugation and presence of heparin in culture medium on sperm motion. Percoll is becoming increasingly popular in 68

69 separating motile from non-motile sperm and it is, therefore, important to know the effects of Percoll on sperm motion characteristics. Furthermore, different research groups use different methods of sperm suspensions preparation, therefore it is essential to know if these different techniques induce different motion characteristics in these preparations.

3.1.1 Material Selection All specimens used in sections 3.2 to 3.7 had normal count (>20x106 per millilitre) and motility (50% total, >25% progressive) as defined by WHO (1992). The samples were processed as described in section 2.3.

3.1.2 Earles-Hepes balanced salt solution preparation All Earles-Hepes (EHBS) medium used in section 3.2 to 3.7 were prepared as described in section 2.2.1

3.2 EFFECT OF INCUBATION TEMPERATURE ON SPERM

MOTILITY WHEN PENTOXIFYLLINE IS PRESENT

3.2.1 Materials and Methods

A total of 23 semen samples were examined and the method used for preparation of the sperm suspensions was the discontinuous Percoll gradient 69

70 method, as described in section 2.4.3. The prepared sperm suspensions, of concentration between 5 to 10 million per mL, were divided into 4 portions of 0.5 mL each. The 1st and 3rd portion were treated as controls, and had 50 L EHBS medium added to them, whereas the 2nd and 4th portion were mixed with 50 L of 20 mM PF/L (final concentration 2 mM PF/L). Preparation of PF as described in section 2.2.3. The 1st and 2nd samples were incubated at room temperature (RT=23 to 250 C) for 30 minutes. Samples 3 and 4 were incubated at 370 C for 30 minutes. At the end of the incubation period, all samples were analyzed by CASA (section 2.5) and the results of VSL, VCL, LIN, ALH and motility % were recorded. The data produced were normally distributed, and therefore they were analyzed by Analysis of Variance followed by Paired t-test to check statistical significance between groups (section 2.7).

3.2.2 Results and Discussion

Table 3.1 shows the results obtained from control sperm samples incubated either at RT or 370 C temperature, and shows no significant change in motion characteristics as assessed by t-test. The effect of temperature on VSL, LIN, and MOT % was not significant. Although the VCL value had risen from 124 m/s at RT to 129 m/s at 370 C, this increase is not significant. Similarly, the control ALH value rose from 5.2 at RT to 5.5 m/s at 370 C (p<0.1), which was statistically insignificant. Therefore, it can be concluded that motion characteristics of control sperm samples as measured by VSL, VCL, LIN, and ALH were not affected by the incubation temperatures used. 70

71 However, when PF was added to sperm samples, the picture appears to change somewhat, as shown in Table 3.1. Statistically, incubation temperature has

no influence on VSL, LIN, and MOT % of sperm samples treated with 2 mM PF/L. However, VCL and ALH were influenced by incubation temperature. The VCL of 139 m/s at RT had increased to 149 m/s at 370 C, which was significant at the p<0.01 71

72 level, and for ALH the value had risen from 6.0 m at RT to 6.4 m at 370 C (p<0.05), overall increases of about 15% above their respective controls. These significant changes are illustrated more specifically in Table 3.2.

Table 3.2 Effect of PF on VCL and ALH values of sperm incubated at two different temperatures

% Increase in response to PF above control CASA Parameter VCL - m/sec ALH - m Incubation at RT 124 139 = 12% 5.2 6.0 = 15% Incubation at 370 C 129 149 = 16% 5.5 6.4 = 19%

It can be seen that, for both motion parameters, PF caused an increase of about 15% at both temperatures.

It was apparent from this study that incubation temperature had some influence on some of the sperm motion characteristics in the presence of PF, but not in its absence. In the interest of standardization, all experiments were carried out at 370 C.

72

73 3.3

EFFECT

OF

INCUBATION

TIME

ON

SPERM

MOTILITY

WHEN

PENTOXIFYLLINE

IS PRESENT

3.3.1 Materials and Methods A total of 19 semen samples were examined and the method used for preparation of sperm suspension was the discontinuous Percoll gradient method as described in section 2.4.3. Aliquots of prepared sperm suspension of 0.5 mL were used. CASA measurements were taken and recorded which acted as control (0 time). The prepared samples were mixed with 50L 20 mM PF/L (final

concentration 2 mM PF/L), the PF having been prepared as described in section 2.2.3 and incubated at 370 C. At intervals of 30, 60, 90, 120, 150, and 180 minutes after addition of PF, CASA measurements were taken and the results recorded. The data were normally distributed and the appropriate statistical tests were done to analyze the results.

3.3.2 Results and Discussion When PF was added to sperm samples, the length of incubation time appears to be a major factor in influencing sperm motion parameters, as shown in Table 3.3. Analysis of Variance on the values for VCL, ALH and LIN over the whole range of incubation times (from 0 to 180 minutes) showed highly significant effects (p<0.001) compared with the control. For example, the VCL (Fig. 3.1a) was 123 m/s at 0

73

74 time but rose to 152 m/s after 60 minutes incubation and after that rose further, to 159 m/s at 90 minutes, when it plateaued out, indicating that the maximum effect of temperature had been attained. The ALH rose, in parallel with the change in VCL, to 6.9 m/s at 90 minutes and thereafter remained unchanged (Fig. 3.1b). The increase in VCL and ALH induced a decrease in LIN, as evidenced by Fig. 3.1c, this change being highly significant (p<0.001). The LIN value dropped very dramatically from 49 at 0 time to 34 at 60 minutes and then plateaued from 90 minutes onwards. This appeared to suggest that the sperm head moved more vigorously from side to side, making bigger wave-like movements with increased flagellar amplitude and wavelength, simultaneously making less forward progressive movement. The VSL fell initially, but then rose a little, although not back to the control value. Kay et al. (1993) showed that washed sperm from cryopreserved semen exhibited hyperactivation when incubated with 3.6 mM PF/L. On examination of the data, they showed that the maximum stimulation occurred between 15 and 75 minutes of incubation. This is consistent with my findings reported above. They (Kay et al., 1993) also showed that PF treatment increased the VCL & decreased the LIN, which again is consistent with my findings. Hyperactivation of sperm in rodents has been described by Yanagimachi et al. (1970) (Fraser, 1977) as a whiplash figure of eight pattern of movement. It produces large amplitude of proximal waves by the flagellum, resulting in high amplitude lateral head displacements. The pattern of flagellar beating causes the sperm head to move more rapidly without any net gain in forward momentum. This hyperactivation model of rodent sperm also applies to human sperm, as was shown 74 ALH, but

75 by Burkman (1984). My results of human sperm that had been incubated with PF up to 3 hours appear to indicate that the activity of the sperm is consistent with motion in the hyperactive state.

75

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76

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3.4 EFFECT OF HEPARIN ON SPERM MOTILITY

WHEN

PENTOXIFYLLINE IS

PRESENT

3.4.1 Materials and Method A total of 23 semen samples were used, and each sample split into two portions of semen. Both portions of semen were separated by the discontinuous Percoll gradient method, as described in section 2.4.3. The pellet obtained from the 1st portion of semen was suspended in Earles-Hepes medium containing 5000 iu sodium Heparin/L (Minihep, Leo Laboratories Ltd, UK). This prepared sperm suspension was divided into 2 portions of 0.5 mL each. One portion served as a control, therefore 50 L of EHBS medium was added, while the other portion was mixed with 50 L 20 mM PF/L (final concentration 2 mM PF/L), prepared as described in section 2.2.3. The pellet from the 2nd portion of semen was suspended in EHBS medium, and this suspension divided into 2 portions of 0.5 mL each. One portion served as a control, therefore 50 L of EHBS medium was added, while the other portion was mixed with 50 L 20 mM PF/L (final concentration 2 mM/L). All samples were incubated for 30 minutes at 370 C before analysis by CASA. The results of VSL, VCL, LIN, ALH and motility % of sperm were recorded. The data produced were normally distributed and the appropriate statistical tests were done to test the significance of any differences.

77

78 3.4.2 Results and Discussion Table 3.4 summarises the effects of heparin, in the presence and absence of PF, on sperm motion characteristics. In the control samples, heparin had no

influence on sperm motility parameters like VSL, VCL, LIN, ALH, and MOT %, as evidenced by the p values. Addition of 2 mM PF/L to sperm suspensions containing heparin in the culture medium made no difference to the motion characteristics, as shown by the mean values in Table 3.4. In light of this information, when there was surplus EHBS medium (with heparin) to clinical requirement in the Fertility Laboratory, rather than disposing of the media, it was used for research work on this project reported in chapter five.

78

79

79

80

3.5 EFFECT OF PERCOLL ON SPERM MOTILITY

3.5.1 Materials and Methods A total of 11 semen samples were used and the method used for preparation of sperm suspension was the centrifugation migration method as described in section 2.4.1. The prepared sperm suspensions were divided into 2 portions of 0.5 mL each per tube. The 1st tube served as the control and to the second tube, 50 L of Percoll was added. Both tubes were incubated at 370 C for 30 minutes before CASA measurements were taken and the results recorded. The second tube containing Percoll was then mixed with 4 times its volume of EHBS medium and centrifuged at 600g for 5 minutes. The supernatant was removed and the pellet was thoroughly mixed with 0.4 mL of EHBS medium. CASA measurements were taken and the results recorded as "1st wash". This procedure was repeated until 6 sets of washed results were obtained from the Percoll treated sample.

3.5.2 Results and Discussion Assessment of data showed that they were not of Normal distribution; therefore, a non-parametric approach was taken. Table 3.5 shows the median values with lower and upper quartile value. Kruskal-Wallis analysis of data from control to wash 6 showed that there was statistical significance at p<0.05

80

81

for VSL, VCL, LIN, ALH and MOT %, as indicated in Table 3.5. The significance of difference from control values was tested by Wilcoxon signed rank test. The immediate effect of Percoll on sperm was to depress all CASA parameters except LIN and MOT %. The effect on VCL parameter was highly significant at p<0.01. The increase in VCL value from control to wash 1 was not significant but from wash 3 to wash 6, it was significant. The data appear to suggest an upward trend in value from wash 1 to wash 6, as is shown graphically in figure 3.2a. The gradual increase in ALH value from control to wash 6 (Fig.3.2b) follows a similar pattern to that shown by the VCL parameter, the most significant change in ALH value being at wash 6 (p<0.01) (Table 3.5). The Pearson's correlation between VCL and ALH was r = 0.98 (p<0.0001), indicating a very high positive correlation. The results in Table 3.5 show a progressive decrease in MOT % from control to wash 6. This trend is graphically shown in Fig. 3.2c. The effect of Percoll removal 81

82 on LIN from Wash 2 to Wash 4 was to depress the values (p<0.05) and the trend is graphically shown in Fig. 3.2d. Percoll removal by washing did not affect VSL parameter. Although the initial effect of Percoll on sperm motion parameters was to depress all values, the subsequent process of multiple washing to remove added Percoll induced a decrease in overall sperm MOT %, but increased the curvilinear velocity and lateral head displacement of sperm. The centrifugation

82

83

83

84

experiment in Section 3.6 will demonstrate that the apparent rise in VCL and ALH values was not due to centrifugation. The apparent cause for this rise must then be connected with washing. This phenomenon of washing effect is the subject of investigation in the second part of chapter 5 and its connection with frequent changes in culture medium. In this context, it is of interest to note an ultrastructural study by Barthelemy et al. (1992) demonstrated that Percoll had no deleterious effects on sperm when assessed by transmission electron microscopy. And it is reassuring to know that when Percoll was injected into rabbit's ovaries, histopathological assessment showed there was no observable cellular response in the ovaries (Arora et al., 1994).

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3.6 EFFECT OF CENTRIFUGATION ON SPERM

MOTILITY

3.6.1 Materials and Methods A total of 11 semen samples were used and the method used for preparation of sperm suspension was the centrifugation migration method as described in section 2.4.1. The prepared sperm suspension was divided into 2 portions of 0.5 mL each per tube. The 1st tube served as the control and the second tube was the test. CASA measurements were taken and the results of VSL, VCL, LIN, ALH, & motility % were recorded. The second tube was then subjected to centrifugation at 600g for 5 minutes. The tube was removed and the contents were thoroughly mixed before CASA measurements were taken and the results recorded as "1st Spin". The above procedure was repeated until 4 centrifugation results were obtained from the test sample. The data were normally distributed and Paired t-test were done to analyze the results.

3.6.2 Results and Discussion The results of centrifugation and its effect on sperm motion characteristics are summarised in Table 3.6. Analysis of Variance on VSL, VCL, LIN and ALH, over the whole range of spins from control to 4th spin, showed that statistically there were no significant effects on the total of 11 samples examined, except MOT %, where there was a significant drop in motility effect (p<0.05) compared with the control value.

85

86

This result was consistent with the findings of Alvarez et al. (1993). They showed that there was a decline in percentage motility of sperm that had undergone centrifugation. Their study also showed that, if following centrifugation, a second 'swim-up' was performed, the proportion of motile sperm recovered was significantly decreased. They postulated that the decreased recovery of sperm was due to sub-lethal membrane damage induced by centrifugation. In this study, centrifuging the same sample four times, did not statistically affect the sperm motion characteristics adversely. The MOT % remained constant from spin 1 to spin 4. As sperm motility is a function of overall cell integrity (Eddy, 1988), that there was no membrane damage due to centrifuging.

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3.7 COMPARISON BETWEEN DISCONTINUOUS PERCOLL GRADIENT AND 'SWIM-UP' METHODS OF

SPERM SUSPENSION PREPARATION

3.7.1 Materials and Methods A total of 30 samples were used for the preparation of sperm suspensions by the isotonic discontinuous Percoll gradient method, as described in section 2.4.3. Another 30 samples were used for the preparation of sperm suspensions by the migration centrifugation method, as described in section 2.4.2. The 60 sperm suspensions were analyzed by CASA (section 2.5) and the results of VSL, VCL, LIN, ALH and motility % were recorded. The data produced were normally distributed, and therefore they were analyzed by Analysis of Variance followed by Two sample t-test to assess any statistical significance between the two methods of separating motile sperm.

3.7.2 Results and Discussion The results of the two methods of preparing sperm suspensions and their effects on motion characteristics are summarised in Table 3.7. The results showed that there was significant difference between the two methods. VCL, LIN and ALH values were significantly different at p<0.0001. The motion characteristics of sperm separated by Percoll exhibited higher VCL (24%) and ALH (23%) values compared with sperm separated by the 'swim-up' technique.

87

88

The LIN value was reduced by 13% in the Percoll gradient separation. The changes in VSL and MOT% were not significant. The results showed that the sub-population of sperm separated by the two methods are different. Using the Percoll gradient method, abnormal sperm, cell debris and low density sperm (irrespective of motility) were retained on the top of the Percoll layer and between the 40/80 interface, whereas sperm of similar density (irrespective of motility) to the Percoll pass through it to collect at the bottom of the tube as a pellet. This preselection of sperm produces a sub-population rather than a representative portion of the motile sperm, and therefore it was possible that this fraction may have motion characteristics different from 'swim-up' preparations. In the 'swim-up' technique, only motile cells can migrate into the upper layer. Therefore, it 88

89 is essential that the appropriate method is chosen for the preparation of sperm suspensions; the two methods are not interchangeable. In light of this information, the method of sperm separation was decided at the outset of each study.

3.8 CONCLUSION
The above studies on factors affecting sperm motion characteristics appears to suggest that on the issue of standardization of method in studying sperm motion, sperm suspensions incubated at 370 C and for one hour in the presence of a sperm stimulant produces optimal results, and hence this procedure was employed in the studies reported in chapters 4 to 7, unless otherwise stated. The reason why 60 minutes incubation time was chosen, rather than 90 minutes incubation, which

appeared to be the peak response, was that the changes in VCL and ALH values at 60 and 90 minutes were not significantly different from one another. Secondly, the one hour incubation time fitted well within laboratory working conditions, i.e. it was advantageous for practical reason. The presence of heparin and centrifugation of sperm have statistically no significant impact on sperm motion characteristics. Separation of sperm by the Percoll gradient method produced a preparation with significantly different motion parameters from the 'swim-up' separation technique and therefore it is essential to define the method of sperm separation in any studies.

89

90 CHAPTER 4 ACTION OF PENTOXIFYLLINE ON SEMEN

A man may learn wisdom even from a foe. Aristophanes c. 444-380 BC

4.1 INTRODUCTION

Pentoxifylline (PF), a phosphodiesterase inhibitor in the methylxanthine group, is well documented to stimulate spermatozoa (sperm) motility, and is used clinically (Yovich et al., 1990; Sikka and Hellstrom, 1991). Many published works (Aparico et al., 1980b; Yovich et al., 1990; Sikka and Hellstrom, 1990; Tesarik et al., 1992a) have all reported that PF, although it does not increase the number of motile sperm does, however, improve sperm motion in preselected sub-populations of sperm. Preselection is usually performed either by the 'swim-up' technique (Ing et al., 1991) or by using the isotonic discontinuous Percoll gradient method (Iizuka et al., 1988). Hammitt et al. (1989) have reported that when PF was added to cryopreserved human semen rather than preselected sperm, there was increased stimulation of motion characteristics of sperm. The concentration of PF chosen for their study was empirically selected to be 3.6 mM PF/L, which may not be the optimal dose for maximum stimulation. Sikka and Hellstrom (1990) observed that motion of washed, cryopreserved sperm was significantly stimulated in a dose-dependent manner with PF. Their drug concentrations ranged from 0.1 to 10 mM/L, and the peak response was at 3 mM PF/L after a 3 hour incubation (250 C). 90

91 Most reported studies on the action of PF have been on a selected population of sperm enriched in highly motile cells. However, patients whose semen parameters are sub-optimal may benefit if PF, when added to semen (before selection), could be shown to increase recovery rate of sperm with enhanced motion characteristics. Increased recovery and enhancement of sperm motility would be advantageous for oligozoospermic and asthenozoospermic patients undergoing either IVF or GIFT treatment, or any other form of assisted conception. The purpose of this study, therefore, was to examine PF effects directly on semen over a range of PF concentrations. A further aim was to determine the optimal PF concentration

required to achieve maximum enhancement of sperm motion characteristics.

4.2 MATERIALS AND METHODS

Semen samples were obtained from 37 normal individuals attending the Fertility Clinic at Queen Charlotte's and Hammersmith Hospitals. All samples used had normal count (>20x106 per millilitre) and motility (>50% forward progressive, >25% rapid progressive) as defined by WHO (1992). Subjects were requested to abstain from sexual activity for at least 3 days before producing the sample. Samples were obtained by masturbation and were allowed to liquefy at room temperature before routine semen analyses were performed in accordance with WHO (1992) guidelines as described in section 2.3.

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92 Table 4.1 Design of experiments and number of samples per group

Groups

Final concentration of Pentoxifylline in semen mM/L 0 0 0 0 3 4 8 1 6 5 9 2 12 6 10 3 7 11 4 8 12

Total number of samples 12 12 5 8

1 2 3 4

The patients were grouped into four blocks as shown in Table 4.I. The number of samples used per PF concentration is given in Tables 4.2 and 4.3. The reason for this block design was to cover the whole range of PF concentration from 1 to 12 mM/L, and since a single semen specimen could not be separated into enough aliquots to cover the entire range under investigation. Semen from each patient was divided into 4, 5 or 6 aliquots of 0.5 mL each, depending on the volume produced and into which group they were randomly allocated. One aliquot from each patient was treated as a control and the rest were exposed to PF. The first aliquot of semen was treated as a control, to which 0.5 mL of EarlesHepes (EHBS) media (containing 10% albumin) was added. Preparation of EHBS as detailed in section 2.2.1. An equal volume of EHBS/10% containing 2 to 24 mM PF/L was added to the rest of the samples to give final concentrations of 1 to 12 mM PF/L. Preparation of PF was as described in section 2.2.3. The tubes containing control and test samples were mixed and incubated at 370 C in humidified air. At the end of a one hour incubation period (incubation time based on the study reported in section 3.3), tubes were gently overlaid with one mL 92

93 of Earles-Hepes/10% HSA media (as described in section 2.4.2) and incubated for a further one hour at 370 C. At the end of this incubation, 0.4 mL of supernatant was removed, taking care not to disturb the lower layer. 0.2 mL of the aspirated

supernatant was analyzed by a Celltrack/s Motion Analyzer as described in section 2.5 and the results of VSL, VCL, LIN, ALH, and MOT % were recorded 2. The remaining suspension was treated with 10 l of 50% formalin in saline to kill the sperm and a manual count of sperm performed using a Neubauer haemocytometer.

4.3 Statistical Analysis and Calculations In view of the non-Gaussian distribution of the data, a non-parametric approach was taken. Medians were used as an average response to PF. Significant median differences from control values were tested by the Mann-Whitney test. In view of varying effects of PF on different CASA parameters, the overall effect of PF on sperm was studied by calculating a 'Stimulation Index'. The Stimulation Index 1 (SI 1) was calculated as follows: each median value of each group was subtracted from the median value of the control and the percentage change at each PF concentration was calculated for each parameter of interest (in this study it was VSL, VCL, ALH and manual sperm count). The SI was an unweighted addition of all the parameters at each PF concentration and represents the total response of sperm to PF effect. A graph of SI 1 against PF concentration was plotted and the best curve fitted with a quadratic equation.
1

The total number of cells analyzed, motile cells counted and the number of cells tracked per PF concentration group were recorded, and these values are reported in Appendix B.

93

94 The individual sample responses to PF stimulation were examined by calculating the percentage change of CASA parameter for each patient as follows: the difference between each patient's CASA parameter value at 6 mM PF/L and the control value was divided by the control value and multiplied by 100%. The percentage change was plotted against the patient number.

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95 4.4 RESULTS

4.4.1 Kinematics of Sperm response to PF challenge Sperm showed a significant increase in some of their motion parameters when exposed to concentrations of PF above 3 mM/L, as is summarised in Table 4.2. Curvilinear velocity (VCL), straight line velocity (VSL) and manual sperm count showed a parabolic curve fit when plotted against PF concentration, while lateral head displacement (ALH) was concentration-dependent up to 12 mM/L in a linear fashion. The peak VCL response occurred around 8 mM/L (p<0.001; Fig.4.1a) and was 18% increase (calculated from the best fit curve; range 8 to 28%) above the control value. Median VSL showed a similar pattern with the peak response occurring at a concentration of 7 mM/L (Fig.4.1b). The VSL in the presence of 7 mM PF/L was 11% (range was from 1% to 22%) above the control value. PF produced a concentration - dependent enhancement in median ALH (Fig.4.1c) up to a concentration of 12 mM/L. Regression analysis showed a linear relationship with a positive correlation of 0.93 (p<0.0001). Sperm recovery (Table 4.3), as reflected by the median manual sperm count from the top layer of culture media, was highest in the presence of 5 mM PF/L (calculated from best fit curve, Fig.4.1d). At this PF concentration, the recovery was 36% (the range was from -36 to 204%) higher than the untreated group. The recovery of motile sperm from semen that had been treated with over 10 mM PF/L was lower than the control value by about 20%. The

percentage of motile sperm was not influenced by Pentoxifylline.

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Table 4.2 Kinematic responses of sperm to various concentrations of Pentoxifylline

PF conc. mM/L 0 1 2 3 4 5 6 7 8 9 10 11 12

VSL m/s 59 49-63 53 43-62 56 49-64 60 52-64 60 54-66 64b 57-66 63b 56-66 62 56-67 62 56-71 56 46-66 59 51-69 60 51-65 60 52-66

VCL m/s 104 93-114 101 87-113 100 94-108 113b 102-121 115b 99-118 115b 106-119 119a 110-123 118a 107-121 120a 108-126 114 105-133 116b 110-137 120b 108-134 119b 113-133

LIN

ALH m

MOT % 89 76-95 92 85-94 95 90-96 91 85-95 91 89-93 93 82-97 91 81-94 93 87-97 91 82-95 92 84-93 88 80-94 85 64-99 89 80-92

No. of samples 37 8 8 20 20 12 24 12 15 5 5 5 17

55 53-58 51 45-56 56 49-61 54 52-57 54 53-57 54 51-57 55 51-59 55 51-58 55 51-61 49 43-55 51 47-55 51 47-55 51 48-56

4.3 3.9-4.8 4.5 3.8-5.0 4.4 3.8-4.7 4.9b 4.3-5.2 4.9b 4.5-5.1 4.8 4.1-5.0 4.9a 4.5-5.3 4.8b 4.5-5.3 5.0a 4.6-5.5 5.1b 4.6-5.6 5.2b 4.8-5.8 5.3b 4.7-5.9 5.3a 4.7-5.7

Median values with first and third quartile value; significant median differences from control shown by p values at a<0.01; b <0.05.

96

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97

98

98

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4.4.2 Effect of PF on motion characteristics of sperm The overall effect of PF on sperm motion characteristics was studied by calculating the "Stimulation Index 1". The SI 1 was calculated from VCL, ALH, VSL and manual count as explained in section 4.3. A graph of SI 1 against PF was plotted and the curve fitted by employing a quadratic equation. The graph of SI 1 (Fig.4.2) showed that as the concentration of PF increased, the beneficial effect on sperm increased proportionally, reaching a maximum at 6 mM/L and thereafter any further increase in the concentration of PF produced a pronounced decline in the beneficial effect. 4.4.3 Observations on individual responses to 6 mM PF/L 99

100 A subset of 24 matched-pair samples was recruited from group 1 and 2. CASA parameters VCL and ALH were examined more closely as the effect of PF was more pronounced on these parameters. Although sperm from the majority of individuals showed a maximum response at a concentration of 6 mM PF/L, there was marked inter-individual variation to PF challenge (Fig.4.3a and 4.3b). The semen of two individuals (~10% of the number analyzed) showed little or no detectable response to PF challenge. The variation in percentage rise in VCL and ALH ranged from 0% (patient number 21) to about 50% for VCL and about 45% for ALH (patient number 19), as shown in Figures 4.3a & 4.3b. Pearson's correlation between the percentage increases in VCL and ALH was 0.81 (p<0.0001).

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101

102 4.5 DISCUSSION

Most studies on the effect of PF on sperm motion characteristics report on a fixed concentration of PF done in suspensions of sperm. However, in this study I report the effect of PF, using concentrations ranging from 1 to 12 mM/L, on sperm motion characteristics done directly on semen. The results obtained in this study indicate that the optimal PF concentration required to obtain the maximum beneficial effect on sperm enhancement was 6 mM/L when the PF was added directly to semen, this result being derived from the Stimulation Index. Thus, SI is an useful mathematical tool in discovering a total maximum effect when a chemical compound has varying degrees of effect on different parameters within a sample. The peak response on VCL was at 8 mM/L (p<0.01), while for VSL it was at 7 mM/L. The response of ALH was proportional to PF concentration, describing a linear relationship (r=0.93, p<0.0001). At any concentration of PF, there was a considerable variation in sperm response. By examining a subset of 24 matched pair samples, it was observed that the responses of sperm to 6 mM PF/L show an increase which ranged from 0% to over 40% for both VCL and ALH parameters. In addition, I found that approximately 1 in 10 patient's sperm does not respond to PF challenge, a result consistent with other results from my related projects (section 5.4.1). Moohan et al. (1993) reported a large variability in the response of human sperm in suspensions to PF challenge (the response varied from 0 to above 40%), and was thus similar to this study in which PF was added directly to semen. It is, therefore, very important to do preliminary testing with PF before selecting the 102

103 appropriate drug concentration for clinical application. PF has been shown (Fuse et al., 1993; Kay et al., 1993; Tesarik et al., 1992a) to increase the VCL and ALH of sperm in suspension. Tesarik et al. (1992a) also reported a variability in the degree of increase in VCL after treatment with PF. An increase in the speed (defined as time-average velocity of the sperm head along its average trajectory) of motile sperm may play an important role in enhancing in vitro fertilization. Holt et al. (1985)

showed that increased sperm speed correlates with increased in vitro fertilization and improved sperm penetrating capability in the zona-free hamster egg assay. This beneficial effect of PF was further supported by Yovich et al. (1990), who showed that the drug improved the fertilization rate in cases of severe male factor infertility. It has also been reported (Tesarik and Mendoza, 1993) that sperm treated with PF showed improved fertilizing ability in patients with acrosome reaction insufficiency. However, in contrast, Tournaye et al. (1993a) have reported that PF-treated sperm showed no therapeutic advantage in IVF for male factor infertility in cases with previous fertilization failure. The ALH showed a concentration-dependent linear increase (Fig.4.1c) as the PF concentration increased up to 12 mM/L, but there was no corresponding increase in either VCL or VSL at the higher concentrations (>8 mM/L). This appeared to suggest that the sperm head moved more vigorously from side to side, making bigger wavelike movements, presumably with increased flagellar amplitude and wavelength, but simultaneously making less forward progressive movement. Kay et al. (1993) showed that washed sperm from cryopreserved semen exhibited 28% hyperactivation at 75 minutes when incubated with 3.6 mM PF/L, with the maximum stimulation occurring between 15 and 75 103

104 minutes of incubation. Hyperactivation has also been shown (Mbizvo et al., 1993) in a suspension of cryopreserved sperm in the presence of 3 mM PF/L.

Hyperactivation of rodent sperm has been described (Yanagimachi et al., 1970; Fraser, 1977) as a whiplash figure-of-eight pattern of movement. It produces large amplitude proximal waves by the flagellum, resulting in high amplitude lateral head displacements. The patterns of flagellar beating causes the sperm head to move more rapidly without any net gain in forward momentum. This hyperactivation model of rodent sperm may also apply to human sperm (Burkman, 1984; Robertson et al., 1988; Mortimer and Mortimer, 1990). The results of this study showed that when semen was incubated with a high concentration of PF, the sperm exhibited characteristics consistent with hyperactive-like motion. This study showed that when PF was added directly to semen before any 'swim-up' procedure was performed, incubated for an hour and subsequently subjected to the 'swim-up' process, there was increased recovery of motile cells. The effect of an optimal concentration of PF (ie. 6 mM/L) on motile sperm results in changes to movement characteristics, with increased forward movement

accompanied, presumably, by intensification of flagellar beat and increased lateral head displacement. The net effect was increased recovery of motile sperm from semen. Clinically, this could be beneficial in cases of patients with impaired sperm motility in semen. Using conventional methodology to obtain sufficient motile sperm for artificial insemination can produce low yield. However, these results demonstrate that the addition of PF to semen may improve the recovery of motile sperm, which could be useful in some male factor patients; for example, it has been shown that the in vitro fertilization rate increased in patients with severely abnormal sperm (<4% 104

105 normal) when the number of sperm added was increased 2 to 10-fold (Oehninger et al., 1988). Furthermore, addition of PF to poor quality semen directly may also decrease the time required to prepare the sperm suspensions using the current methodology.

[Please note: A paper based on the data from this chapter has been accepted by Human Reproduction to be published in 1995; 10:2 ]

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106 CHAPTER 5 ACTION OF PENTOXIFYLLINE ON SUSPENSIONS OF SPERMATOZOA AND EFFECTS AFTER ITS REMOVAL BY WASHING

Knowledge itself is power. Francis Bacon 1561-1626

5.1 INTRODUCTION

Spermatozoa must have the ability to move to achieve fertilization in the oviduct. Active movement of the sperm is due to forces generated by the flagellar activity (Dresdner et al., 1981; Kratz et al., 1989). Sperm flagellar undulation generates active forces and torques along the sperm body, resulting in forward movement. However, the generation of force and propulsion by the sperm depends upon its environment and the availability of ATP. ATP is the intracellular constituent necessary for sperm motility (Calamera et al., 1982) and its presence reflects some of the functional capability of the gamete (Calamera et al., 1986). Conversion of ATP to cAMP is catalysed by adenylate cyclase and elevated concentrations of cAMP could be translated into increased flagellar movement (Tash and Means, 1982). However, phosphodiesterase converts cAMP to AMP, and Perreault and Rogers (1982b) showed that an inhibition of phosphodiesterase, raises the concentrations of cAMP in sperm.

106

107 There are a number of chemicals that can stimulate sperm motility, as is discussed in section 1.8. Pentoxifylline (PF), a phosphodiesterase inhibitor, is the most popular drug used clinically in enhancing sperm motility in suspension. Increased sperm velocity is closely associated with fertility (Milligan et al., 1980). Hence, it has been reported (Yovich et al., 1990) that in severe male factor infertility, when 3.6 mM PF/L was added to the sperm suspension and incubated, followed by subsequent removal of the PF by washing before insemination, PF improved the fertilization rate. In another study by Tesarik et al. (1992a), also using 3.6 mM PF/L, it was shown that when the drug was added to a sperm suspension, there were increased sperm motion characteristics and the maximum motion was attained within 10 minutes of incubation with PF. Further, the activity persisted for at least two hours after drug removal. They also showed that PF does not improve the percentage of motile sperm. Most of the studies have empirically chosen to use 3.6 mM PF/L to achieve a stimulative effect in sperm suspension. In this study, the first aim was to find the optimal concentration of PF required to produce a maximum increase in sperm motion characteristics of sperm in suspension. It is a common practice in IVF laboratories, after sperm have been exposed to PF, to remove the drug by washing prior to use of the sperm suspension for fertilization, in order to minimise the risk to embryo development (Tournaye et al., 1993b). Thus, the second aim was to examine the effect of drug removal by washing, to discover if the stimulation induced by PF was retained after removal of the drug.

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108 5.2 MATERIAL AND METHODS

5.2.1 Effects of PF on sperm suspensions Semen samples were obtained from 22 normal individuals and processed as described in section 2.3. Sperm suspensions were prepared as detailed in section 2.4.3, using the Percoll gradient method. Preparation of Earles-Hepes balanced salt (EHBS) medium was as stated in section 2.2.1. Pentoxifylline was prepared as described in section 2.2.3. Each prepared sperm suspension was divided into 6 aliquots of 0.5 mL. One aliquot was treated as the control, while the remaining aliquots were treated as test. To each test aliquot, 50 L of the appropriate PF concentration was added, to give a final concentration range of 1 to 5 mM PF/L. The control contained 50 L EHBS/10% HSA medium. All tubes were mixed thoroughly and incubated at 370 C for 1 hour. At the end of incubation period, CASA measurements were taken as described in section 2.5 and the results of VSL, VCL, LIN, ALH, and MOT% were recorded3. The decision to examine the PF concentration range from 1 to 5 mM/L was based on a pilot study done on 11 sperm suspension samples that were exposed to PF concentrations ranging from 1 to 10 mM/L at 1 mM PF/L intervals. Concentrations of 5 mM PF/L and above appeared to be detrimental to sperm and resulted in decreased MOT %, VCL and ALH (data not presented); 10 mM PF/L being the most detrimental to sperm and 5 mM PF/L being the least. Concentrations between 1 and

The total number of cells analyzed, motile cells counted and the number of cells tracked per PF concentration group were recorded, and these values are reported in Appendix C

108

109 5 mM/L appeared to stimulate sperm motion, hence the choice of this concentration range.

5.2.2 Effects of removal of PF by washing on subsequent sperm motion characteristics In this case, I used both methods (Percoll gradient method and 'swim-up' method) to prepare the sperm suspensions. The decision to examine both methods of sperm suspension preparation was based on information gained in section 3.7 which showed that these two methods produced different sub-population of sperm. The sperm suspensions were treated with PF, incubated and sperm motion characteristics determined, before the sperm were washed (to remove the PF) and motion characteristics determined a second time. The object was to determine the effects, if any, of washing the sperm. The exact procedure is outlined as a flow diagram in Fig. 5.1

5.2.2.1 Series A - Separation of sperm by Percoll gradient method Semen samples were obtained from 29 normal individuals and processed as described in section 2.3. Semen samples were separated by the isotonic Percoll gradient method as detailed in section 2.4.3. The pellet so obtained was used in the centrifugation migration method (section 2.4.1) to produce a population of highly motile sperm. The supernatant was aliquoted into 3 parts of 0.5 mL each. One aliquot was treated as the control, with nothing being added, and CASA measurements were taken. The second aliquot was mixed with 50 L of EHBS/10% HSA and the third aliquot was treated with 50 L of 30 mM PF/L, to give a final 109

110 concentration of 3 mM PF/L. The second and third samples were mixed and incubated at 370 C in humidified air. At the end of a one hour incubation period, the tubes were removed, and CASA measurements were taken. The tubes were then mixed with 4 mL EHBS/10% HSA, centrifuged and the supernatant discarded. This wash was repeated. At the end of second wash, the pellet was suspended in EHBS medium to give a concentration of between 5 and 10 million cells per millilitre. These final sperm suspensions were analyzed by a Celltrack/s Motion Analyzer as described in section 2.5 and the results of VSL, VCL, LIN, ALH and MOT% were recorded.

5.2.2.2 Series B - Separation of sperm by migration centrifugation method Semen samples were obtained from 60 normal individuals and processed as described in section 2.3. Semen samples were separated by the migration centrifugation method as described in section 2.4.2. The sperm suspensions were separated into 3 aliquots of 0.5 mL each and treated as described in section 5.2.2.1 above. At the end of second wash, the sperm suspensions were analyzed by Celltrack/s Motion Analyzer as described in section 2.5 and the results of VSL, VCL, LIN, ALH and MOT% were recorded.

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5.3 Statistical analysis and calculations

5.3.1 EFFECT OF PF ON SPERM SUSPENSIONs Presentation of data by histograms showed that the data were not of Gaussian distribution, and therefore, a non-parametric approach was taken to analyze the data, as described in section 2.7. Medians were used as an average response to PF. Significant median differences from control values were tested by Wilcoxon signed-rank test. The Stimulation Index 2 (SI 2) was calculated as follows: each median value of each group was subtracted from the median value of the control and the percentage change at each PF concentration was calculated for each parameter of 111

112 interest (in this study it was VSL, VCL, ALH and MOT%). The SI was an unweighted addition of all the parameters at each PF concentration and represents the total response of sperm to PF effect. A graph of SI 2 against PF concentration was plotted and the best curve fitted with a quadratic equation. The individual sample responses to PF stimulation were examined by calculating the percentage change of CASA parameter for each patient as follows: the difference between each patient's CASA parameter value at 3 mM PF/L and the control value was divided by the control value and multiplied by 100%. The percentage change was plotted against the patient number.

5.3.2 REMOVAL OF PF BY WASHING Histograms of data showed that the data were of normal distribution, and therefore a parametric approach was taken to analyze the data, as described in section 2.7. Significant mean differences were tested by the Paired t-test.

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113 5.4 RESULTS

5.4.1 RESPONSE OF SPERM SUSPENSIONS TO PF CHALLENGE -----------------------------------------------------------------------------------------------

5.4.1.1 Kinematics of the response of sperm to PF challenge Table 5.1 shows the summary of median values with first and third centiles. There was a significant increase in both VCL and ALH parameters from a concentration of 1 mM PF/L onwards. VCL (Fig 5.2a) and ALH (Fig 5.2b) showed significant elevations in response to PF, which were best described by a parabolic curve. The peak VCL response was at 3 mM PF/L (p<0.001) and was 12% increase (calculated from best fit curve) above the control value, with a range from 122 to 145 microns/sec (25th to 75th centile). Median ALH showed a similar pattern, with the peak response occurring at about 3 mM PF/L (p<0.001). This was 16% (calculated from best fit curve) above the control value, with a range from 5.4 to 6.1 microns (25th to 75th centile). The relationship between VCL and ALH was a positive

correlation of r=0.92 (p<0.0001). There was no significant change in VSL, LIN and MOT % parameters.

5.4.1.2 Effect of PF on motion characteristics of sperm in suspension The effect of PF on sperm motion characteristics was studied by calculating the "Stimulation Index 2". The SI 2 was calculated from VCL, ALH, VSL and MOT% as explained in section 5.3.1. A graph of SI 2 against PF was plotted and the curve

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fitted by employing a quadratic equation. The graph of SI 2 (Fig 5.3) showed that, as the concentration of PF increased, the beneficial effect on sperm increased proportionally, reaching a maximum at 2.8 0.2 mM PF/L, and after that any further increase in concentration of PF appeared to produce a pronounced decline in any beneficial effect.

5.4.1.3 Observations on responses of individual samples to 3 mM PF/L The effect of PF on twenty-two matched pair samples was examined more closely by calculating the percentage change. Although the CASA parameters VCL and ALH showed maximum responses at about 3 mM PF/L, there was marked inter116

117 individual variation to PF (Fig 5.4a and Fig 5.4b). The sperm of two individuals (~10% of the number analyzed) showed no detectable response to PF. The variation in percentage rise in VCL and ALH ranged from 0% (patients 14 and 19) to about 35% for VCL and about 40% for ALH. Pearson's correlation between VCL and ALH percentage change was r=0.83 (p<0.0001).

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5.4.2 RESIDUAL EFFECTS ON SPERM AFTER REMOVAL OF PF BY WASHING

------------------------------------------------------------------------------------------------------------5.4.2.1 Sperm obtained by the Percoll gradient method

The results are summarised in Table 5.2. The Analysis of Variance of CASA parameters VSL, VCL, LIN and ALH all indicated significant changes, whereas MOT% showed no significant change. The PF treatment caused a significant 20% increase in VCL (p<0.0001), as is shown in Fig.5.5a, and a 19% increase in ALH (p<0.0001) (Fig.5.5b), in response to 3 mM PF/L. There were significant decreases in VSL (p<0.001) and LIN (p<0.0001), 118

119 and did not affect motility %. In general these effects persisted after removal of the PF by washing; VCL (p<0.0001) and ALH (p<0.0001) remained higher than the control values, LIN (p<0.05) remained lower than the control value, but the effect of PF on VSL was lost after its removal. Motility% remained unaffected. Surprisingly, washing had quite pronounced effects on the control samples; that is, those samples which had not been treated with PF. It led to a significant increase, by 13%, in VCL (p<0.0001) (Fig. 5.5a) and by 11% in ALH (p<0.0001) (Fig. 5.5b) from the '0' time control values, but did not affect VSL, LIN or motility%. There was a significant difference between PF-treated & washed samples and the control washed samples for the VCL and ALH parameters at p<0.01, and for VSL at the p<0.05 level.

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5.4.2.2 Sperm obtained by the 'swim-up' method Table 5.2 shows the residual effects on sperm separated by 'swim-up' after removal of PF. The Analysis of Variance of CASA parameters VSL, VCL, LIN, and ALH all indicated significant changes. When sperm were incubated with 3 mM PF/L, the VCL increased by 20% (p<0.0001) from the control value, as is shown in Fig.5.6a. The ALH showed a similar pattern to the VCL, as is shown in Fig. 5.6b. The ALH increased by 23% (p<0.0001) from the control value. The VSL increased significantly (p<0.01),

whereas LIN decreased (p<0.0.001). Motility% remained unaffected. In general, these effects persisted after removal of the PF by washing; VCL (p<0.0001) and ALH (p<0.0001) remained higher, and LIN (p<0.01) remained lower, than the control values, but the effect of PF on VSL was lost after its removal. Motility% decreased by 11% (p<0.0001). Washing had quite pronounced effects on the control samples, consistent with those observed when the sperm was prepared by the Percoll gradient method. After washing, the VCL was 6% higher (p<0.0001) and ALH 7% higher (p<0.0001) compared with pre-wash values. In contrast, both the LIN and MOT% decreased (p<0.0001 in both cases). The VSL remained unaffected. In general, the effects of washing were similar to those in response to PF, albeit somewhat less pronounced. There was no significant difference between PF-treated & washed samples and the control washed samples for VCL, ALH, VSL, LIN and MOT% parameters.

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124 5.5 DISCUSSION

The results of this study showed that

when sperm suspensions were

incubated with PF, it resulted in significant increases in sperm motion characteristics. The optimal concentration of PF that produced the maximum stimulation was found to be 2.8 0.2 mM PF/L, as calculated from the SI. However, there was great variability in individual responses to PF stimulation. There is a body of evidence (Sikka & Hellstrom, 1991; Tesarik et al., 1992a; Lewis et al., 1993; Moohan et al., 1993; Fuse et al., 1993) showing that PF elevates sperm motion characteristics in sperm separated by the 'swim-up' technique. My results are consistent with these findings; however, there were essential differences. All previous studies used different concentrations of PF (probably empirically chosen) and different incubation times and temperatures, making comparisons difficult. For example, Sikka & Hellstrom (1990) reported that 3 mM PF/L significantly stimulated sperm motion characteristics in washed sperm maintained at 25 o C, with the peak response occurring at three hours incubation, whereas in the study by Lewis et al. (1993), it was shown that in the presence of 3.6 mM PF/L, VCL was significantly enhanced after 15 minutes of incubation at 370 C and remained high for up to 240 minutes. Elevated sperm motion characteristics were achieved when sperm suspensions were incubated for 2 hours at room temperature with 5 mM PF/L (Fuse et al., 1993), while Moohan et al. (1993) showed that the maximum response was commonly observed at a PF concentration of 2 mM/L when incubated at 370 C for 30 minutes.

124

125 My paired-controlled study showed that the maximum enhancement of sperm motion characteristics was attained at 2.8 0.2 mM PF/L (Fig. 5.3) when the sperm were incubated at 370 C for 1 hour. The incubation temperature and time were based on information obtained from the studies reported in section 3.2 and 3.3. Although the VCL parameter showed a maximum rise at 3 mM PF/L (Fig.5.2a), whereas the maximum ALH occurred at less than 3 mM PF/L (Fig.5.2b), the total effect

(assessed by the SI 2) showed that the actual maximum elevation was at 2.8 0.2 mM PF/L. Thus, SI is a useful mathematical tool in discovering a total maximum effect when a chemical compound has varying degrees of effect on different parameters of the same sample. Consistent with other studies (Moohan et al., 1993), this study also shows a wide range of individual patients' responses to PF. Of the 22 individuals examined, the VCL and ALH parameters (Fig. 5.4 a and b) of 2 patients did not show any detectable response to PF challenge (~10% of patients), consistent with findings in section 4.3. Four patients had a VCL increase of over 30% while 3 patients had an ALH increase of over 30%. As suggested in section 4.4, preliminary testing with PF is an important issue if the drug is to be used most efficaciously. Treatment of sperm with PF, whether prepared by 'swim-up' or Percoll gradient, caused some motion parameters to increase significantly, others to decrease significantly, and others to remain unchanged. When the PF was subsequently removed by washing, in some cases the effect was "washed away" i.e. was lost (true of VSL in both types of separated sperm) but in others it remained (VCL, LIN are good examples). Sometimes, when the effect remained, it was not so pronounced i.e. some of the effect, but not all, was washed away (VCL and ALH in 125

126 'swim-up' sperm, for example), but in other cases none of the effect was lost (VCL and ALH in the Percoll gradient sperm, for example). However, washing alone had quite pronounced effects on sperm motion characteristics, with increases in VCL and ALH. These findings may appear to contradict the results of others who have reported that the effects of PF remain after washing off' of the drug. For example, Tesarik4 et al. (1992a) showed that the PF effect persisted for at least 3 hours after drug removal. In another study, Hammitt3 et al. (1989) reported that although PF significantly increased sperm velocity, however, after drug removal by washing, the linearity and velocity of stimulated and untreated sperm were similar. Detailed examination of the data in Hammitt et al. (1989) study revealed that the velocity of control sperm was elevated with time, reaching a maximum at 1 hour post-wash, and declining after that. Hence, the apparent persistence of the stimulating effects of PF may have been more to do with the process of washing which increased the motion characteristics of the untreated, control sperm than it was with any effects of PF persisting in the treated sperm. Kay2 et al. (1993) showed that after PF removal by washing, hyperactivation remained high for up to 1 hour, but returned to control values after 3 hours. Examination of the data of Kay et al. (1993) on the effects of PF removal, also reveals that the motion characteristics of the control, untreated sperm values had risen after the wash when compared with before the wash. Thus, the results of these two studies (Hammitt et al., 1989; Kay et al., 1993) are consistent with the present study, and it is interesting to note that the authors of these two

'swim-up' separation

Percoll gradient separation

126

127 papers did not make any comment on the significance in the increase in the motion parameters of the control, untreated sperm after they had been washed. Results from the studies reported in sections 3.5 and 3.6 lead one to reject the hypothesis that either the Percoll or centrifugation processes may affect sperm. This unexpected phenomenon of the increase in sperm motion characteristics of the control, untreated sperm after washing may be due to some factors being present in the culture medium which enhance sperm activity or the presence of some factors in sperm suspensions which inhibit activity until removed by washing. Discrete subpopulations of sperm have been shown to produce sudden burst of reactive oxygen species (ROS) (Aitken and Clarkson, 1987; 1988; Aitken et al., 1989). Further, leucocytes present in semen have also been shown to generate ROS (Kessopoulou et al., 1992; Aitken et al., 1992; Weese et al., 1993). These reactive oxygen species also originate from the cellular components of the sperm (Iwasaki and Gagnon, 1992) generated by the centrifugation process (Aitken and Clarkson, 1988). ROS, as well as lipid peroxides, have been shown to have deleterious effects on sperm motility (Jones et al., 1979). Besides ROS, there are other factors, like cytotoxic end-products of lipid peroxidation, which are present in semen and which can cause irreversible loss of sperm motility (Selley et al., 1991; Aitken et al., 1993). Further, decapacitation factors (DF), which are glycoproteins present on the surface of sperm, can probably cause inhibition of sperm motility and the acrosome reaction (Oliphant et al., 1985; Fraser and Ahuja, 1988; Fraser et al., 1990). When preparing a 'swim-up' preparation of sperm, all these factors might diffuse into the overlaid layer of culture medium. It follows that washing of sperm suspensions by centrifugation, discarding the supernatant and replacing it with fresh medium, 127

128 removes the toxic effects of these factors (ROS and DF), thereby preventing sperm motility being inhibited. In the washing procedures, after centrifugation, the supernatant was removed and replaced with fresh culture media that contained 10% HSA. In a study conducted by De Lamirande & Gagnon (1991), it was shown that serum stimulated motility of sperm in suspensions in a dose-dependent manner, but that this effect varied from one sperm population to another. Additional support that serum albumin is implicated in maintaining sperm motility comes from a study done by Hammitt et al. (1990). They showed that different types of proteins (preovulatory donor serum, Cohn Fr. V human serum albumin and highly purified HSA) possess an equal ability to support sperm motion characteristics. Thus the replacement of culture medium5 in the wash procedures I used might have contributed to the increase in sperm motility. However, the reasons given above do not fully explain why Percoll separated sperm (and PF-treated) retained a higher increase in motion characteristics compared with sperm (and PF-treated) obtained by the 'swim-up' method. A

possible reason could be that in Percoll separation, sperm were separated based on density of the cell, irrespective of their motility, while in the 'swim-up' separation only the progressive motile sperm swim into the upper layer of the culture media, with immotile and poorly motile sperm remaining at the semen/culture media interface. The resulting differences between these two methods of separation leads to the conclusion that different subpopulation of sperm were selected by the two methods. It is plausible that one subpopulation may respond to stimulation differently from a

contains serum albumin

128

129 different subpopulation. As this study shows, this might indeed have been the case. The results in Table 5.2 show that sperm separated by Percoll exhibit higher motion characteristic values (control - VCL=123 3.86, ALH=5.3 0.18 and when treated with PF, VCL=147 3.53, ALH=6.3 0.15) compared with sperm separated by 'swim-up' (control - VCL=104 1.69, ALH=4.4 0.06 and when treated with PF, VCL= 125 1.99, ALH=5.4 0.09). This observation was further supported by the study reported in section 3.7, where motion characteristics of Percoll separated samples were significantly different from those of 'swim-up' separated sperm. In conclusion, sperm motion characteristics were elevated in the presence of PF and the maximum response was obtained at 2.8 0.2 mM PF/L, although different subpopulations of sperm responded differently to PF challenge. This increased motion was reduced by washing; however, the removal of various 'factors' in the culture medium probably masked the true reduction in stimulated sperm samples and increased the motion values of the control samples. In the future, it may be worth investigating further the influence of various 'factors' on sperm motion characteristics.

129

130 CHAPTER 6

The ACROSOME REACTION RESPONSE TO PENTOXIFYLLINE CHALLENGE

Science is organised knowledge. Herbert Spencer 1820-1903

6.1 INTRODUCTION

Leading up to fertilization, the spermatozoa must undergo a complex cascade of events prior to union with the oocytes. Thus, the acrosome reaction that occurs as a consequence of sperm capacitation is an indispensable prerequisite for sperm passage through the zona pellucida and for its fusion with the oolemma. The

acrosome reaction (AR) is an exocytotic event that involves the fusion and vesiculation of the outer acrosomal membrane and the surrounding plasma membrane, and culminates in the dispersal and release of the acrosomal contents. Adequate supplies of free Ca2+, Na+, K+ and energy substrates all play key roles in the acrosome reaction (Fraser, 1992). The site of the acrosome reaction during fertilization has been studied in vitro in many laboratory animals. In the mouse, the AR occurs on the zona pellucida surface (Florman and Storey, 1982; Wassarman, 1987). In the guinea pig, it appears to occur at a distance from the zona pellucida, probably during sperm passage 130

131 through the cumulus (Huang et al., 1981). The hamster represents an intermediate situation in which both acrosome-intact as well as acrosome-reacted sperm bind to the zona pellucida (Cummins and Yanagimachi, 1982, 1986). In the marsupial egg, which lacks follicular cells, the AR may occur on the surface of the zona pellucida (Rodger and Bedford, 1982), whereas in the sea urchin, the AR is initiated by a fructose sulphate polymer moiety of the jelly coat that is thought to be equivalent to the extracellular matrix of the cumulus oophorus of the mammalian oocyte (Lopo, 1983). The AR of human sperm is a calcium-dependent, exocytotic process and is thought to be induced by the zona pellucida and occurs at the zona pellucida, although sperm which have already undergone the AR can bind to the zona pellucida (Cross et al., 1988). There are several inducers that can initiate the acrosome reaction. Physiological agents including albumin, zona pellucida extracts (Cross et al., 1988; Bielfeld et al., 1994), follicular fluid (Zinaman et al., 1989; Mortimer and Camenzind, 1989) neuraminidase, glycosaminoglycans, catecholamine, prostaglandins,

oestrogen and progesterone (Uhler et al., 1992) are all known to stimulate the acrosome reaction (Meizel, 1985; Yanagimachi, 1988; Meizel et al., 1990). Chemical substances like Ionophore A23187 are also known to induce the acrosome reaction (De Jonge et al., 1989; Cummins et al., 1991). Other chemical substances known to induce the AR are Ionomycin and lysophosphatidycholine (reviewed by Wolf, 1989). There is also a suggestion that Pentoxifylline may modify the sperm head surface, making it more permeable and resulting in enhanced AR (Tesarik et al., 1992b). Physical agents that are known to inhibit the AR include freeze-thaw, temperature

131

132 shock and hyperosmotic medium (reviewed by Wolf, 1989). Cryopreservation has also been shown to damage the acrosome (McLaughlin et al., 1993). The AR in human sperm can be detected by employing electron transmission microscopy (TEM) or using conventional optical microscopy. The TEM (Bartoov et al., 1980) method is labourious, time-consuming and requires an expensive instrument, and hence is beyond routine application. Therefore, light microscope techniques based on staining the acrosome with histological stains were developed; one such method is the triple stain technique (Talbot and Chacon, 1981). Subsequently, fluorescein labelled dyes were developed to study the AR (Blasak et al., 1982; Cross and Meizel, 1989). Methods now available for the study of the acrosome reaction includes fluorescein isothiocyanate conjugated Pisum sativum (pea) agglutinin (FITC-PSA) (Cross et al., 1986; Mendoza et al., 1992), fluorescein isothiocyanate conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA) (Mortimer et al., 1987; 1990), chlortetracycline-UV (CTC) (Lee et al., 1987), and use of monoclonal antibodies (Coddington et al., 1990; Zhang et al., 1990; Parinaud et al., 1993) The present study was undertaken, firstly, to evaluate the fluorescent staining techniques currently available and to adapt one of them for routine use. Secondly, to study the effect of PF on the acrosome reaction. Finally, the effect of PF plus Ionophore A23187 on the acrosome reaction.

132

133 6.2 MATERIALS AND METHODS

6.2.1 Preparation of materials Semen samples were obtained from 15 normal individuals and processed as described in section 2.3. Sperm suspensions were prepared as described in section 2.4.2, using the migration centrifugation method. Earles-Hepes balanced salt (EHBS) medium was prepared as described in section 2.2.1. Pentoxifylline was prepared as described in section 2.2.3.

6.2.2 Methods of acrosome evaluation There are number of ways in which the acrosome reaction can be evaluated. In this study, I used the fluorescent stain. I evaluated the PSA, PNA, and CTC staining methods for their efficiency, speed, reliability and ease under laboratory conditions. Incubation with Ionophore A23187 was used as a positive control of the acrosome reaction, and the vital stain bis-benzamide (Mortimer et al., 1990) was used to assess the live:dead ratio.

6.2.2.1 Ionophore A23187 challenge The influx of calcium ions across the sperm membranes is generally considered to be one of the steps in the acrosome reaction, which occurs once the sperm are fully capacitated (Fraser, 1981; Aitken et al., 1984; Fraser and McDermott, 1985). The divalent cation transporting agent, Ionophore (IP) A23187, can be used to induce the acrosome reaction, in a manner similar to that which occurs under physiological conditions (Suarez et al., 1986). This property has been exploited and 133

134 ionophore-treated samples were used as evaluation studies. The method used, was similar to that described by De Jonge et al. (1989) and Cummins et al. (1991). In the pilot study, one millilitre of prepared sperm suspension containing about 10-15 million sperm per mL was divided into two aliquots; one aliquot was challenged with ionophore A23187 (final concentration 10 M/L) in DMSO and the other aliquot, the control, was treated with 10% DMSO. Samples were incubated at 370 C for 30 minutes before the staining procedure was carried out. positive controls in my acrosome

6.2.2.2 Staining using Vital stain Cell viability was assessed using the method described by DasGupta et al. (1993). A stock solution of Hoechst bis-benzamide 33258 (Sigma) was made by dissolving 100 mg of the dye in Fresenius water for injection. The prepared stock solution was aliquoted into smaller portions and frozen until required. Before use, the frozen stock was thawed and diluted to 1:1000 in protein-free EHBS medium and then further diluted 1:100 in the sperm suspension (final concentration of dye in sperm suspensions was 1 g/mL). The suspension was incubated for 5 minutes at 370 C and subsequently washed with 3 mL 2% polyvinylpyrrolidone (PVP 40, Sigma) in PBS buffer before centrifugation at 800g for 10 minutes. The resulting pellet was resuspended in EHBS medium to obtain a concentration of about 10 million sperm per mL. The suspension was then ready for lectin or CTC staining.

134

135 6.2.2.3 Staining using fluorescein isothiocyanate conjugated Pisum sativum (pea) agglutinin (FITC-PSA stain) FITC-PSA binds to -methyl mannoside residues localized within the acrosome (Cross et al., 1986); therefore, FITC-PSA will label the acrosome contents of suitably permeabilized sperm. The method used for staining was similar to that described by Cross et al. (1986) and Hoshi et al. (1993). A microscopic slide was smeared evenly with sperm suspension and air dried for 30 minutes in a 37 0 C incubator. The sperm were then permeabilized in 95% ethanol for 30 minutes, washed twice with distilled water and air dried. The dried, smeared slide was covered with 50 L of FITC-PSA stain (100 g/mL in distilled water) and left at room temperature for an hour in the dark. The stained slides were mounted with 10 L of 0.22 M 1,4 diazabycyclo [2,2,2] - octane/L (DABCO, Sigma) in glycerol plus

phosphate - buffered saline (9:1) and covered with 22x26 mm coverslips. DABCO suspension delays fluorescence decay. The slides were examined at 200 times magnification within 24 hours using a fluorescence microscope, set up as described in section 2.1.2. . 6.2.2.4 Staining using fluorescein isothiocyanate conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA stain) FITC-PNA lectin binds to - D-galactosyl residues localized on the outer acrosomal membrane. The method used for staining was similar to that described by Mortimer et al. (1990). A microscopic slide was smeared evenly with sperm suspension and air dried for 30 minutes in a 370 C incubator. The dried, smeared slide was covered with 50 L of FITC-PNA stain (100 g/mL in distilled water) and 135

136 left at room temperature for an hour in the dark. The stained slides were mounted with 10 L of 0.22 M 1,4 diazabycyclo [2,2,2] - octane/L (DABCO, Sigma) in

glycerol plus phosphate - buffered saline (9:1) and covered with 22x26 mm coverslips. DABCO suspension delays fluorescence decay and the slides were examined at 200-times magnification within 24 hours using a fluorescence microscope set up as described in section 2.1.2.

6.2.2.5 Staining using chlortetracycline (CTC) stain The CTC fluorescent staining method used was similar to that described by Lee et al. (1987) and DasGupta et al. (1993). CTC solution was prepared fresh each day and contained 750 M CTC/L (Sigma) in 130 mM Nacl/L, 5 mM cysteine/L and 20 mM Tris-Hcl/L, producing a final pH of 7.8. The prepared solution was stored in the dark at 40 C until required. 100 L of the prepared sperm suspension containing about 5-10 million sperm per mL was added to 100 L of CTC solution and mixed thoroughly. The cells were fixed by adding 10 L of 10% paraformaldehyde in 0.5 M/L Tris-Hcl, final pH 7.4, and mixed. Microscopic slides were prepared by placing 10 L of the suspension on the glass, mixing with a drop of DABCO, and gently pressing down the cover slip to remove excess mixture. The prepared slides were stored in the dark, allowing the sperm to settle down, and the slides were read at 200-times magnification the following day using a fluorescence microscope set up as described in section 2.1.2.

6.2.3 Transmission Electron Microscopy (TEM)

136

137 0.5 mL of sperm suspension in an Eppendoff vial was mixed with 1.0 mL of 3% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2-7.4) and left to fix for 1-2 hours at room temperature. At the end of the fixation period, the fixed sperm were precipitated by centrifugation and the supernatant removed. 1 mL of 0.1M cacodylate buffer was added to the pellet and the sample was then sent for TEM. Blocking, cutting and staining were performed by the staff of the Electron Microscopy Unit. The pellet was dehydrated through a graded series of increasing concentrations of ethyl alcohol and sections (<100 nm) then embedded in Araldite resin. Ultra-thin

were cut with a diamond knife in an ultra-microtome. The

sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and allowed to air dry. The sections were examined with a Hitachi HU12A Transmission Electron Microscope (TEM) at 12,000-times magnification. All grids were viewed blind i.e. I was unaware of the cohort of origin. 100 consecutive sperm were individually assessed with regard to head shape and the state of the acrosome. The following criteria were used to classify the sperm (Stock, 1990): Group 1 consisted of acrosome-intact sperm i.e. the acrosomal cap was in place and appeared normal. Group 2 consisted of acrosome-reacting sperm i.e. the acrosomal cap was still in place but appeared swollen, less electron dense, with vesicle formation, and it was beginning to show signs of ballooning out. Group 3 consisted of acrosome-reacted sperm i.e. the acrosomal cap was absent or only scanty remnants of the acrosomal membranes were seen.

137

138 Group 4 consisted of miscellaneous cells i.e. sperm showing abnormal morphology, or where the sectioning of the sperm cell was at inclined plane and hence it could not be classified. A total of 100 cells in Group 1 to Group 3 was counted.

6.2.4 The Acrosome Reaction evaluation experiment Prepared sperm suspensions were divided into 5 aliquots:- two aliquots served as negative and positive controls of 0.2 mL each; and three aliquots served as test samples of 0.4 mL each, as described in the flow diagram of the experimental protocol (Fig. 6.1). 20 L of EHBS buffer was added to the negative control sample. 40 L of the appropriate PF concentration (10, 30, 50 mM PF/L) was added to test samples to give a final concentration of 1, 3, 5 mM PF/L. All tubes were mixed thoroughly and incubated at 370 C for 1 hour. At the end of incubation period, 0.2 mL of each test sample was removed for video taping and for staining with vital and CTC stain. The motion characteristics6 were recorded on video tapes for 2 minutes using 5 different fields employing a Celltrack/s analyzer (the tapes were analyzed at a later date). 5 L of Ionophore A23187 (final concentration 10 M/L) was added to the remaining 0.2 mL test sample; similarly, 5 L of Ionophore A23187 (final

concentration 10 M/L) was added to the positive control. All tubes were then incubated for a further 30 minutes at 370 C before video taping and staining with vital stain (section 6.2.2.2) and CTC stain (section 6.2.2.5). The slides were read by counting the cells in 10 to 15 fields covering the whole slide until 100 sperm were
In light of the information gained from the studies reported in section 2.5.4, all CASA measurements were done on tapes.
6

138

139 counted. The results of total, partial, non-reacted and dead cells were expressed as percentages of the total cells counted. CASA parameters measured from the video tapes were VSL, VCL, LIN, ALH, and MOT %. Of the 15 samples, 2 samples that had very good recovery of motile sperm were randomly selected for Transmission Electron Microscopy study of positive and negative controls to confirm that the AR was inducible with IP A23187.

6.3 Statistical Analysis All data generated were assessed by histograms. Data were of normal distribution, therefore a parametric approach was taken to analyze the data as described in section 2.7. Significant mean differences were tested by the Paired ttest.

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140 6.4 RESULTS

6.4.1 Conclusions of the pilot AR staining study A pilot AR evaluation study was performed on 6 samples employing FITCPNA, FITC-PSA, and CTC staining with the AR induced by IP A23187. Although the staining of sperm with FITC-PNA and FITC-PSA produced good fluorescence, the decay to UV light was rapid, making it very difficult to differentiate between reacted and non-reacted sperm. These two staining techniques also produced a high variability in the number of sperm which appeared to have undergone the acrosome reaction when repeated measurements were made. CTC staining procedure produced good fluorescence, and no visual decay of the fluorescence to UV light, which lasted for at least 7 days, was observed. Differentiating between reacted and non-reacted cells were easy. Results obtained from repeated measurements of the 6 samples were consistent. Hence, CTC

staining was the method of choice for the experiments described in this chapter.

6.4.2 Evaluation of the AR by CTC fluorescent staining

6.4.2.1 Effect of vital staining The number of dead cells, as assessed by vital staining constituted less than 3% of all cells, as shown in Tables 6.1 and 6.2.

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141 6.4.2.2 Effect of Ionophore A23187 on the Acrosome Reaction The positive control samples that had been induced by ionophore A23187 to undergo the AR showed that 22% (p<0.0001) of the sperm were acrosome-reacted compared with 8% of the controls which had undergone spontaneous AR (Table 6.1). This approximated to a 3-fold increase. Further, 7% of the sperm in the positive control had undergone a partial acrosome reaction, control (p<0.01). compared with 4% in the

6.4.2.3 Effect of PF on the Acrosome Reaction Pentoxifylline, at 1, 3 or 5 mM /L, did not affect the proportion of sperm that had undergone either total or partial AR, as shown in Table 6.1.

Table 6.1 The effect of PF on the acrosome reaction

Control

+ve Control

1 mM PF/L

3 mM PF/L

5 mM PF/L

Total Reacted -% Partial Reacted - % Non-Reacted - % Dead Cells - %

8 1.10 4 0.48 86 0.97 2 0.43

22 1.67 7b 1.01 69d 1.83 2 0.50

7 1.15 4 0.78 87 1.61 2 0.39

10 1.33 5 0.70 83 1.26 2 0.56

9 1.00 4 0.63 84 1.28

2 0.46

N=15; Mean values SEM Paired t-test showed significant mean differences between the +ve control values and the control values at the following p values: a<0.05; b<0.01; c<0.001; d<0.0001 Analysis of Variance showed that there were no significant mean differences between the PF-treated and the control samples.

141

142 6.4.2.4 Effect of PF + !P A23187 on the Acrosome Reaction In the presence of IP, PF-treated7 sperm showed a significant (p<0.0001) decrease in the proportion which had undergone the AR compared with the positive control values, is as shown in Table 6.2. Also the proportion which had undergone the partial AR was significantly decreased at 1 mM PF/L (p<0.05) compared with the positive control value. Thus, the proportion of non-reacted sperm was higher in the PF-treated samples (p<0.001) compared with the positive control.

Table 6.2 The effect of PF+IP A23187 on the acrosome reaction

+ve Control (+ IP)

Total Reacted -% Partial Reacted - % Non-Reacted - % 22 1.67 7 1.01 69 1.83

1 mM PF/L + IP
12d 1.42 5a 0.66 80d 1.74

3 mM PF/L + IP
13d 1.11 6 0.55 79c 1.23

5 mM PF/L + IP
11d 1.72 6 0.70 81d 1.75

Dead Cells -%

2 0.50

3 0.37

2 0.47

2 0.64

N=15; Mean SEM Paired t-test showed significant mean differences between the PF+IP samples and the +ve control at the following p values: a<0.05; b<0.01; c<0.001; d<0.0001

1, 3 or 5 mM PF/L

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143 6.4.2.5 Comparison between PF treatment and PF+IP treatment on the AR The comparison between the PF-treated sperm and PF+IP treated sperm showed that the proportion which had undergone the AR was higher in the second group, as is shown in Fig. 6.2. There was a 71% (p<0.01) greater number of sperm that had undergone the AR at 1 mM PF/L + IP 23187 compared with 1 mM PF/L. Similarly, there was a significantly higher proportion (30%) which had undergone the AR at 3 mM PF/L + IP A23187 compared with 3 mM PF/L (p<0.01). However, there was no significant difference in the number of sperm that had undergone the AR between the samples treated with 5 mM PF/L + IP A23187 and 5 mM PF/L alone (Fig. 6.2). The proportion of sperm that had undergone a partial AR were not significantly different when these two groups (PF and PF+IP treated) were compared. Also, the proportion of non-reacted sperm in these two groups were not significantly different (i.e.differences were never greater than 12%).

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144 6.4.3 Evaluation of the AR by TEM in the negative & positive controls TEM analysis of the AR showed that 5% of the sperm in the positive control had the AR induced, compared with 2% which had spontaneously undergone the AR in the negative control group. This approximated to about 3-fold increase. There was no significant change in the proportion which had undergone the partial AR between the two groups. Electron micrographs (Fig. 6.3a-d) show the various stages of the acrosome reaction. Fig. 6.3a shows a normal sperm head with intact acrosomal cap. Fig. 6.3b&c shows acrosome-reacting sperm - they are swollen and beginning to balloon out - (b), while (c) shows the vesicle formation. Fig. 6.3d shows a sperm head after it had undergone the AR.

Table 6.3 The acrosome reaction of controls evaluated by TEM Control - n1 / n2 Total Reacted - % Partial Reacted - % Non-Reacted - % Miscellaneous Cells 1/2; x=2 3/4; x=4 96/94; x=95 49/36 +ve Control - n1 / n2 4/5; x=5 3/2; x=3 93/93; x=93 44/60

n1 / n2 represents sample 1 and 2 with mean.

+ve control contains IP A23187

Miscellaneous Cells (numbers) = composed of abnormal shaped sperm counted but not included in the percentage calculation.

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145 Fig 6.3 Electron Micrograph of sperm head x 18 000 magnification showing various stages of the human sperm acrosome reaction Bar = 2.5 m

Fig 6.3a: Intact acrosome Fig 6.3b: Swollen acrosomal matrix Fig 6.3c: Raptured acrosome cap with remnants Fig 6.3d: Acrosome cap is absent

1 = Outer acrosomal membrane 2 = Swollen outer acrosomal membrane with early stage of vesicle formation 3 = Vesicles 4 = Inner acrosomal membrane 5 = Remnants of outer acrosomal membrane

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146

146

147 6.4.4 Motion characteristics of sperm used in the AR experiment The results are summarised in Table 6.4.

6.4.4.1 Effect of Ionophore (+ve control) In the presence of Ionophore A23187 (IP), overall motility was significantly reduced (p<0.05) compared with control values (Fig.6.4). ALH showed a similar significant reduction (P<0.01), although there were no significant changes in VSL, VCL and LIN.

6.4.4.2 Effect of PF The results in Table 6.4 also indicate that in the presence of 1, 3, or 5 mM PF/L, there were significant changes in VSL, VCL, and ALH, as expected. VCL (Fig.6.5) and ALH (Fig.6.6) reached peak values (p<0.0001, compared with control values) at 3 mm PF/L, when they were 16% and 12% higher than their respective control values. The values of VCL at 1 and 5 mM/L were significantly increased (p <0.0001) from the control value. The ALH value at 1 mM PF/L (p<0.05) and at 5 mM PF/L (p<0.01) was also significantly raised from the control value. The VSL was significantly higher at 1 mM PF/L (p<0.001), 3 mM PF/L (p<0.01) and at 5 mM PF/L (p<0.0001) than control value. There were no significant changes in LIN and MOT % when compared with the control values.

147

148 6.4.4.3 Effect of PF+IP The results in Table 6.4 shows that when IP A23187 was added to PF-treated sperm (PF+IP), all sperm motion characteristics declined significantly compared with those of PF-treated sperm. The MOT% decrease (Fig.6.4) at 1 mM PF/L was 12% (p<0.01) and at 3 mM PF/L was 20% (p<0.001), while the biggest decrease of 37% (p<0.001) occurred at 5 mM PF/L. Analysis of Variance showed that MOT% of PF+IP samples was significantly reduced compared with the control MOT%. The VCL (Fig.6.5) at 1 mM PF/L showed a drop of 7% (p<0.05), at 3 mM PF/L a drop of 14% (p<0.01) and at 5 mM PF/L the drop was 13% (p<0.02). Analysis of Variance showed that the VCL of PF+IP samples was not significantly different from the control VCL value. The ALH (Fig.6.6) of PF+IP samples showed a drop of 4% at 1 mM PF/L, a significant drop of 8% (p<0.05) at 3 mM PF/L and at 5 mM PF/L the drop was 6%. Similarly, the VSL of PF+IP samples showed a 25 % (p<0.05) decline in value at 1 and 3 mM PF/L while at 5 mM PF/L the drop was 35% (p<0.01). The linearity of PF+IP samples showed a decrease of 13% (p<0.05) at 3 mM PF/L while at 5 mM PF/L the drop was 24% (p<0.01) compared with PF-treated samples.

148

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149

150

150

151

151

152 6.5 DISCUSSION

The CTC staining procedure produced good fluorescence, and no visual decay of the fluorescence to UV light was observed. Differentiating between reacted and non-reacted cells was easy. The repeated measurements were consistent. Hence, CTC staining was the method of choice for the experiments described in this chapter. The TEM analysis of the negative control samples showed that lower

proportion of sperm had undergone the AR than the fluorescent staining method because, it is probable, that in the TEM analysis greater proportion of sperm were classified as abnormal under 12 000-times magnification compared with the 200times magnification used in the fluorescent technique. At 12 000- times magnification, greater details of cell ultrastructure are seen, leading me to classify any sperm that does not appear normal as abnormal and the acrosome-reacted sperm can be visually identified. However, under 200-times magnification, sperm cannot be discriminated between normal and abnormal, and furthermore, classification of the acrosome-reacted sperm is based on external fluorescent appearance. TEM analysis of the AR showed that sperm treated with IP A23187 had a 3-fold increase in the number of acrosome-reacted cells compared with the control sample, a result consistent with results from the CTC staining technique. The study also demonstrated that PF increases sperm motion characteristics, but that this enhancement was significantly reduced in the presence of Ionophore A23187. De Jonge et al. (1989) showed that Ionophore A23187 could induce the AR and that a high concentration of IP A23187 (>40 M) had a deleterious effect on 152

153 sperm motility %. This observation is supported by the findings of White et al. (1990), who showed that a high concentration of IP A23187 significantly reduced sperm motility. My results are consistent with these studies, in which the positive control sample, which was treated with Ionophore A23187, showed a 3-fold increase (p<0.001) in the proportion of sperm that had undergone the AR together with a 7% reduction (p<0.05) in MOT %. This demonstrates that even at very low concentration of IP A23187 (10 M), adverse effects on sperm motility occur. This study also showed that sperm treated with 5 mM PF/L, and in the presence of IP A23187, has a highly significant further reduction in MOT % compared with both the control and the PF-treated sperm, but that there was no corresponding significant change in VSL and VCL, although there was a significant change in LIN and ALH compared with the control values. This would suggest that a subpopulation/subset of sperm were more labile/sensitive to chemicals. These more sensitive sperm are immobilized (thereby accounting for the decrease in motility), but still move their heads vigorously (thereby accounting for the increase in the ALH value). Similarly, at a lower concentration of PF (i.e. 1 and 3 mM/L), in the presence of Ionophore A23187, similar effects on MOT % were observed, but to a lesser extent. This would indicate a dose-response relationship; the concentration of PF (in the presence of IP A23187) was proportional to the reduction in sperm MOT %. A highly significant negative correlation between PF concentration in the presence of a constant concentration of IP A23187 and MOT % reduction was found: r = -0.98 (p<0.02). It has been demonstrated (Tesarik et al., 1992b; Tesarik and Mendoza, 1993; Carver-Ward et al., 1994; Tasdemir et al., 1993) that PF does not, by itself, induce the acrosome reaction. My results agree with this conclusion, by showing that at 1, 3 153

154 and 5 mM PF/L, there was no significant change in the number of acrosome-reacted sperm. However, when sperm were treated with PF, and then exposed to Ionophore A23187, the Ionophore was less effective at stimulating the AR than it was when the sperm were not treated previously with PF. This finding contradicts the current view of the effects of PF+IP on the AR. Several authors (Tesarik and Mendoza, 1993; Carver-Ward et al., 1994; Tasdemir et al., 1993) have shown that sperm treated with PF, and challenged subsequently with Ionophore A23187, had undergone increased induced acrosome reactions. This apparent discrepancy could be due simply to the culture medium used. Table 6.5 shows the different methodologies used by the above three groups of authors and myself. Other than some minor differences in the methodology, the major difference between the studies was the culture medium used. Tesarik and Mendoza, (1993) used B2 medium, Carver-Ward et al., (1994) and Tasdemir et al., (1993) used Human Tubal Fluid (HFT) medium, whereas I have used Earles medium containing Hepes (EHBS). I suspect that there was some chemical interaction between the Hepes, PF and Ionophore A23187, giving rise to some unknown chemical molecule (or molecules). It is possible that this chemical molecule(s) may have inhibited the induction of the AR. This hypothesis requires verification.

154

155
Table 6.5 Comparison of the experimental conditions used in various studies to evaluate the AR

Conditions

Tesarik & Mendoza, 1993

Carver-Ward etal., 1994 20 'swim-up'

Tasdemir etal., 1993 33 'swim-up'

Myself 1994 15 'swim-up'

Total number of samples Sperm separation Incubation time (min) : PF IP Conc. of PF - mM/L Conc. of IP A23187 - M Incubation buffer

18 Percoll gradient

30 30 3.6 10 B2

30 45 3.6 10 HFT/BSA

60 60 7.2 10 HFT/BSA

60 30 3.0 10 EHBS/HSA + Hepes

Method of detection

PSA

Flow cytometric

PSA

CTC

Result (%) : Control IP induced PF induced PF+IP induced 10 14 11 48 10 23 10 29 8 27 8 32 8 22 10 13

155

156

A survey of the literature (Perreault and Rogers, 1982; Rogers and Perreault, 1990, Kay et al., 1993; Pang et al., 1993) appears to suggest that phosphodiesterase inhibitors not only enhance sperm motility by inhibiting cAMP breakdown, but may also be implicated in other sperm functions like capacitation, hyperactivation and the AR, and that all of these might be modulated by cAMP. The prevailing hypothesis (Tesarik et al., 1992b; Tesarik and Mendoza, 1993) suggest that PF sensitizes the complex physiological acrosome on the sperm head. Chemical stimuli like Ionophore A23187 which induce the AR may increase membrane permeability towards ionic calcium influx by acting through the Na+/Ca2+ antiporter (Roldan and Harrison, 1990; Fraser and McDermott, 1992). Increased calcium levels increase the intra-acrosomal pH by acting via a Ca2+-dependent ATPase, and this in turn activates proacrosin to acrosin (Meizel, 1984). This acrosomal membranes, leads to fusion between the plasma and outer in exocytosis of acrosomal contents

culminating

(Yanagimachi, 1981; Fraser, 1984; Hinrichsen-Kohane et al., 1984; Langlais and Roberts, 1985). The role of channels, antiporters or ATPases in the regulation of calcium is still a controversial issue. Different lines of evidence appear to suggest that mammalian sperm do not have voltage-operated channels. The reasons attributed for this assumption are based on the slowly emerging following facts:- (a) changes in membrane potential do not trigger an AR; (b) specific antagonists of voltage-operated Ca2+-channels do not inhibit Ca2+ uptake; and (c) treatment of sperm with different Ca2+-channel antagonists do not prevent the acrosome reaction (Roldan and Harrison, 1990). The lack of evidence for ion channels in sperm has resulted in people postulating that the major mechanism regulating Ca 2+ influx at the 156

157 time of the AR might be a Na+/Ca2+ antiporter. However, studies in mouse sperm have shown that the AR can be inhibited by some Ca++-channel antagonist (Fraser and McIntyre, 1989). Recent studies (Fraser et al., 1993) demonstrate that ions other than Ca 2+ may also be involved in acrosomal exocytosis. In mouse sperm, Na+ is implicated in the AR via the Na+-H+ exchange mechanism. It is thought that, at the time when the mouse sperm comes in contact with the zona pellucida, there occurs an influx of Na + into the fertilizing sperm, causing a rise in intracellular pH that in turns opens Ca 2+channels to allow the influx of Ca2+, resulting in the acrosome reaction. An increasing body of recent evidence shows that cholesterol (lipid) may be involved in acrosomal exocytosis (reviewed by Benoff, 1993). Phospholipid constitute about 60 to 75% of sperm lipids (Scott, 1973). Lipid modifications on the sperm head undoubtedly play a fundamental role in the acrosome reaction, since extensive membrane alterations are involved. Although the exact mechanism of the role played by lipid has not been addressed, electron microscopic studies have shown changes in sterol and anionic phospholipid distribution in human sperm plasma membranes during in vitro capacitation. For example, the cholesterol concentration is preferentially reduced within the plasma membrane domain overlying the acrosomal cap during capacitating incubations (Tesarik and Flechon, 1986).

In conclusion, there are a number of hypotheses to account for the molecular mechanisms leading to the acrosome reaction, but no clear evidence to support any of these hypotheses. My results, to recollect, showed that PF had no significant effect on the acrosome reaction, and that PF treatment inhibited the subsequent 157

158 effect of Ionophore A23187 in enhancing the acrosome reaction. CASA

measurements show that PF+IP greatly affects sperm motility. In the treatment of male factor infertility, it would be advantageous to the patient if it could be demonstrated that sperm treated with stimulant were positively correlated with increased acrosomal exocytosis at the site of sperm-oocyte fusion, which could lead to an improvement in fertilization rate.

158

159 CHAPTER 7

THE EFFECT OF PENTOXIFYLLINE ON THE BINDING OF SPERMATOZOA TO THE ZONA PELLUCIDA

Sciences may be learned by rote, but wisdom not. Lawrence Sterne 1713-1768

7.1 INTRODUCTION

The beginning of a new individual involves the fusion of a spermatozoon and oocyte. Therefore, the process of fertilization is fundamental to the maintenance of life in both plants and animals. In the past two decades, the understanding of the biology and chemistry of mammalian fertilization has led to improvements in the treatment and diagnosis of infertility. Although the sperm can be brought to the vicinity of the oocyte, unless the sperm binds and proceeds with the penetration of the zona pellucida (zona), there cannot be a new creation of life. Thus, understanding the fundamental mechanisms in sperm-zona interactions, which represent a complex sequence of mutually linked events, is crucial. Sperm binding and penetration of the oocyte depend on a finely tuned balance between the actions of different types of cells and intercellular matrices (Tesarik and Testart, 1989). 159

160 Binding to, and penetration of, the oocyte by the capacitated sperm is species-specific (Wassarman, 1987). After the cumulus mass and corona radiata, the zona is the next barrier the sperm has to cross in the process of fertilization. Ultrastructural studies (Sathananthan et al., 1982) in the human have shown that sperm attached to the zona have intact acrosomes and that their plasma membranes in contact with the zona are often swollen before they undergo the acrosome reaction, which is a prerequisite for fertilization. Cross et al. (1988) showed that acrosome-reacted and acrosome-intact sperm are both equally effective in initiating binding to the zona in human. The zona pellucida in mouse oocytes is a sulphated glycoprotein composed of several families of glycoproteins: ZP1(200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa) (Bleil and Wassarman, 1980a). ZP3 has been identified as the prime sperm receptor and inducer of the sperm acrosome reaction, while ZP2 has been shown to be a secondary sperm receptor (Bleil and Wassarman, 1980b, Wassarman, 1988a). Recently, the genes encoding mouse ZP2 and ZP3 have been characterized and sequenced (Kinloch et al., 1988; Kinloch and Wassarman, 1989; Lunsford et al., 1990). Examination of zona by scanning electron microscope has shown that the outer surface of the zona has a fenestrated, lattice-like appearance, whereas the inner surface appears particulated (Greve and Wassarman, 1985). Similarly, in the human, the zona pellucida consists of ZP1 (90-110 kDa), ZP2 (6478 kDa), and ZP3 (57-73 kDa) (Shabanowitz and O' Rand, 1988a; 1988b); these are significantly smaller than the molecular weights of their mouse counterparts. ZP3 has been cloned and sequenced using mouse ZP3 cDNA as a probe (Chamberlin and Dean, 1990; van Duin et al., 1992). It has been demonstrated that there is high degree of conservation between the coding regions of human ZP3 and mouse ZP3, 160

161 and similarly with human ZP2 and mouse ZP2 (Liang and Dean, 1993). Recently, the presence of steroid receptors on the membranes of human sperm was identified, and their mechanism of action partially characterised (reviewed by Revelli et al., 1994). The importance of steroid receptors lies on the fact that steroids have been shown to induce the acrosome reaction, and preserve sperm viability and motility. However, it is yet to be shown if these steroid receptors play a part in the spermzona interactions. In some cases, infertility may be caused by the failure of the sperm to interact with the oocyte, and the reason for this problem could lie with the sperm, or the oocyte, or both. Thus, understanding sperm-zona interaction could assist and improve the treatment in some cases of infertility. Pentoxifylline, a

phosphodiesterase inhibitor,

has been shown to increase sperm motion

characteristics (chapters four and five ) and it would be interesting to find out what effect, if any, the drug may have on sperm-zona pellucida binding. Evidence presented in chapter 6 showed that PF does not induce the acrosome reaction, yet there are studies (Yovich et al., 1990; Sikka and Hellstrom, 1991) showing that PF treatment of sperm was correlated with an improvement in the fertilization rate in in vitro fertilization programmes. One possible explanation is that PF enhances sperm-zona interaction. Therefore, the present study was

undertaken to investigate the effect of PF on sperm-zona binding.

161

162 7.2 MATERIALS AND METHODS To study the effect of PF on the binding of sperm to the zona pellucida, two methods were used. The first method is called the intact-zona assay, and uses whole human oocytes while the second method is called the hemizona assay, and uses bisected human oocyte.

7.2.1 Rationale of intact-zona binding assay The large variability in the number of sperm binding to any one intact-zona, together with the lack of sufficient oocytes to overcome this variability statistically, has led to a particular experimental design being adopted for this study. In this design, a single intact zona is exposed to both control sperm and test sperm simultaneously, with the two types of sperm competing for binding sites on the zona. The two sperm populations can be differentiated by labelling either the control or the test sperm with a fluorescent stain. In the first set of test experiments, the control sperm were labelled. In the second set of test experiments, the PF-treated sperm were labelled. The reason for doing this was to examine the effect of interaction between PF and fluorescent stain on the binding of sperm to the intact-zona and also to examine the effect that the fluorescent labelling process might have on the

binding. Using this experimental approach, PF must be removed by washing the sperm (now called PF-pretreated) prior to co-incubation with the intact-zona, to ensure that the control population of sperm is not exposed to the drug. Preliminary experiments were done to examine the effect of fluorescent stain on binding.

162

163 7.2.1.1 Rationale of hemizona binding assay Owing to the large variability in the number of sperm binding to any one intactzona, the hemizona assay were developed, which uses bisected oocytes. In my experiments, one half of the zona was used as the control and the other matching half was used as the test hence fluorescent staining was not required and the PFtreated sperm were not washed. Preliminary experiments were done to assess the effect of cutting the oocytes into equal halves on sperm binding.

7.2.2 Oocytes Human oocytes (usually treated like gold dust!), from the in vitro fertilization program of the Royal Postgraduate Medical School, London, that had been exposed to sperm but failed to fertilize, were kindly donated towards this study. These oocytes were non-viable, and had no developmental potential. Any residual potential was nullified by storage for 5 days or greater at 40 C. It has been shown that low temperature causes the depolymerisation of major structural protein of microtubules in the oocytes (Osborn and Moor, 1984), making the oocytes non-viable. Individual oocytes were kept in 1 mL EHBS culture medium (preparation of media as described in section 2.2.1) in capped tubes at 40 C until required. They were kept like this for up to three weeks before being discarded. [Ethical and legal implication ensures that the use of virgin oocytes for experimental research is severely restricted, although this would be the best material for this study].

7.2.3 Preparation of intact-zona 163

164 Each oocyte (now called intact-zona for the purpose of this study) was examined with an inverted microscope (Olympus CK, Olympus, Japan) and any with more than five sperm bound to it were rejected. Selected intact-zona was then washed in EHBS medium three times. Washing was done by placing a drop of 0.2 mL EHBS medium in a culture dish (Falcon 3002, Beckon Dickson, UK) and aspirating the intact-zona in and out of the EHBS medium several times, using a finely drawn glass pipette to dislodge any loosely adherent sperm. The process was repeated three times with fresh medium. The intact-zona was then transferred to 0.5 mL fresh medium in a culture dish and kept at room temperature (230 C) until required.

7.2.4 Dissection of intact-zona for hemizona assay Prepared intact-zona was examined with a dissecting microscope (Olympus SZ60, Olympus, Japan) at 60 times magnification. Each intact-zona was cut into equal hemispheres by Dr KS Lindsay with a 25G needle attached to a syringe, using the sharp edge of the needle as the knife. Great care was taken to cut into equal halves, and any portion that did not appear to be a complete half zona was rejected. Each half zona in 0.5 mL EHBS medium was kept in a separate labelled culture dish at room temperature (230 C) until required.

7.2.5 Preparation of sperm suspension Semen samples were obtained from normal individuals and processed as 164

165 described in section 2.3. Sperm suspensions of about 8 million sperm per millilitre were prepared as described in section 2.4.2 using the migration centrifugation method. Sperm suspensions for control experiments were diluted with EHBS medium to obtain a concentration of about 4 million sperm per mL.

7.2.6 Preparation of Fluorescein isothiocyanate (FITC) stain and labelling of sperm The technique of fluorescein labelling of sperm was modified from the method of Parrish and Foote (1985) as described by Liu et al. (1988). 1 mg fluorescein isothiocyanate (FITC, Sigma, UK) was dissolved in 0.1 mL of 0.1 M potassium hydroxide and diluted to 5 mL with Dulbecco phosphate-buffered saline (BioWhittaker, USA) containing D-glucose and sodium pyruvate. The fluorochrome solution was stored at 40 C in a plastic tube wrapped in aluminum foil for up to 1 week. Sperm pellets were prepared using 0.5 mL of the 8 million sperm/mL suspensions by centrifuging them at 600g for 5 minutes and discarding the supernatant. The pellet was suspended in 150 L of the fluorochrome solution and incubated at 370 C for 15 minutes. At the end of the incubation period, the sperm were recovered by centrifuging at 600g for 5 minutes and discarding the supernatant. The pellet was resuspended in 5 mL of EHBS medium and the centrifugation process repeated. The resulting fluorescein labelled pellet was resuspended in EHBS medium to obtain a concentration of approximately 6 million sperm per mL. This labelled sperm suspension was kept at 370 C in the dark until required. 165

166

7.2.7 Preparation of PF-pretreated sperm Sperm suspensions were divided into 3 parts of 0.5 mL each. Each aliquot was exposed to 50 L of 1, 30 or 50 mM PF/L (final concentration was 0.1, 3, or 5 mM PF/L). Preparation of PF was as described in section 2.2.3. [Please note: The choice of 0.1 mM PF/L was based on results from a related project which showed that sperm exposed to 0.1 mM PF/L had greater motility after 20 hours incubation compared with sperm that were exposed to 1 mM PF/L (Moohan et al., 1993)]. After a 1 hour incubation at 370 C, 4 mL of EHBS medium was added to the tubes, which were mixed thoroughly, centrifuged at 600g for 5 minutes and the supernatant discarded. The process was repeated to remove any excess PF present in the sperm suspension. The resulting PF-pretreated pellet was resuspended in fresh EHBS medium to obtain a sperm concentration of about 4 million per mL and kept at 370 C until required.

7.2.8 Effect of FITC stain labelling on sperm motility A pilot evaluation study using 6 sperm samples was done to determine whether labelling of sperm with FITC stain would affect sperm motion characteristics. Each sperm suspensions was divided into 4 portions of 0.5 mL each and separate sperm pellets prepared. The first pellet was treated as a control, to which was added 300 L EHBS medium, while to pellets 2, 3, and 4 were added 150, 300, 500 L of FITC stain and the labelling carried out as described in section 7.2.6. The labelled sperm suspension was then analysed using a Celltrack/s Motion Analyser and the following motion characteristics were measured: - VSL, VCL, LIN, ALH, and MOT %. 166

167 The results obtained showed that all motion characteristics were significantly (p<0.05) depressed when 300 and 500 L of FITC stain was added. For example, the VCL value was depressed by 30 and 43% with 300 and 500 L of FITC stain, respectively. Similarly, the ALH value was decreased by 36 and 51% with 300 and 500 L of FITC stain, respectively. However, the addition of 150 L of FITC stain to the sperm pellet did not affect any of the CASA parameters. In light of this information, all labelling of sperm pellets was done with 150 L of FITC stain.

7.2.9 Intact-zona binding assay - methodology The principle of the method used, was similar to that described by Burkman et al. (1988; 1990) and Franken et al.(1989a,1989b) but modified as shown in Fig 7.1. 100 L of sperm suspension was added to an intact-zona in 500 L of EHBS medium in a culture dish and covered. The mixture in the culture dish was mixed gently and incubated at 370 C overnight (20 hours). At the end of the incubation period, the intact-zona was removed with a finely drawn glass pipette and washed 3 times in EHBS medium. The washing was done as described in section 7.2.2. The washed intact-zona was removed with an Eppendorf Varipette 4710 pipette (Eppendorf-N-Gmbh, Hamburg, Germany) in 0.5 L EHBS medium and placed in a microwell produced by PFTE (Teflon)

167

168

168

169 coating on a disposable-diagnostic slide (Muratech Scientific, UK). The intact-zona was solubilized on the slide by adding 0.5 L of 1 M HCL and mixed with a 21G needle. At this concentration of acid, the sperm were intact and undamaged visually. The slide was covered with a coverslip and the edges sealed with nail varnish and stored in the dark until it was read within 24 hours. All the sperm present in the microwell were systematically counted from left to right field under a light microscope set at 200-times magnification. The total number of sperm (unlabelled and labelled) present was recorded. Labelled sperm were examined under a fluorescence microscope (section 2.1.1) at 450 to 490 nm wavelength, 200-times magnification. All the labelled (fluorescent green) sperm present in the microwell were manually re-counted and recorded. The difference between the total and labelled sperm value was equal to the number of unlabelled sperm bound to the intact-zona, and enabled a ratio of labelled : unlabelled sperm to be determined.

7.2.10 Hemizona binding assay - methodology The methodology used for hemizona assay was essentially the same as described above, and is shown in Fig. 7.1. However, instead of using intact-zona, bisected zona were used, the sperm were unlabelled, and PF-treated sperm were not washed. One half of the cut zona was exposed to control sperm and the other matching half was exposed to test sperm.

169

170 7.2.11 Effect of FITC labelled sperm on intact-zona binding Sperm suspensions were prepared as described in section 7.2.4. Each sperm suspension was divided into 2 parts of 0.5 mL each. The first aliquot was treated as a control (non-labelled) and the second aliquot of sperm was labelled with FITC stain as described in section 7.2.6. The motion characteristics were measured using CASA. Two intact-zona were selected and prepared as described in section 7.2.3. To the first intact-zona, initially in 0.5 mL of EHBS medium in a culture dish, 50 L of control sperm suspension was added. The second intact-zona received 50 L of labelled sperm suspension. The intact-zona binding assay was carried out as

described in section 7.2.9. All the unlabelled and labelled sperm were counted and the percentage of sperm binding to the intact-zona was calculated.

7.2.12 Assessment of cutting the intact-zona into equal halves The intact-zona was selected and prepared as described in section 7.2.3 and was cut into 2 equal halves as described in section 7.2.4. Sperm suspensions of 4 million sperm/mL concentration were prepared as described in section 7.2.5. To each hemizona, initially in 0.5 mL EHBS medium in a culture dish, 50 L of sperm suspension was added, both hemizona receiving the same sample sperm preparation. The hemizona binding assay was carried out as described in section 7.2.10. All sperm on each slide were counted and the percentage of sperm binding to the hemizona was calculated.

170

171 7.2.13 Effect of PF on intact-zona binding

Sperm suspensions, either control or PF-treated were prepared as described in section 7.2.5. Each sperm suspension was divided into 5 parts of 0.5 mL each. The first aliquot was treated as a control and was labelled with 150 L of FITC stain as described in section 7.2.6. Aliquots 2, 3 and 4 were treated with PF (final concentration of 0.1, 3, and 5 mM PF/L) as described in section 7.2.7. Aliquot 5, which served as a positive control to show the effect of PF, was exposed to 30 mM PF/L (final concentration 3 mM PF/L). At the end of an hour incubation at 37 0 C, CASA measurements were recorded. Three intact-zona were selected and prepared as described in section 7.2.3. To the first intact-zona, initially in 0.5 mL of EHBS medium in a culture dish, 50 L of labelled-control sperm was added followed by 50 L of 0.1 mM PF/L pretreated sperm. This process was repeated to intact-zona 2 and 3, using 3 and 5 mM PF/L pretreated sperm. The intact-zona binding assay was carried out as described in section 7.2.9. All the labelled and total sperm on each slide were counted and the percentage of sperm bound to the intact-zona was calculated.

[Please Note: In the second set of experiments, the PF-pretreated sperm were labelled with FITC stain.]

171

172 7.2.14 Effect of PF on hemizona binding The hemizona were prepared as described in section 7.2.4. Sperm suspensions of 4 million sperm/mL were prepared as described in section 7.2.5, and divided into 2 aliquots. One aliquot was treated as a control and received 50 L of EHBS medium. 50 L of 30 mM PF/L (final concentration was 3 mM PF/L) was added to the second aliquot, mixed thoroughly and incubated at 370 C. At the end of an hour incubation, the CASA measurements were taken. 50 L of the control sperm suspension was added to one half of the hemizona while the other matching half zona received 50 L of the PF-treated sperm. The hemizona binding assay was carried out as described in section 7.2.10. All sperm on each slide were counted under a light microscope and the percentage of sperm binding to the hemizona was calculated.

7.3 Statistical Analysis Histograms of data showed that the percentage of sperm bound to the zona were of normal distribution, therefore a parametric approach was taken to analyze the data, as is described in section 2.7. Significant mean differences between assay were tested by Two sample t-test and Paired t-tests.

172

173 7.4 RESULTS

7.4.1 Effect of FITC labelling of sperm on their ability to bind to intact-zona

The results (Table 7.1) showed that FITC stain labelling of sperm had no significant effect on the binding to the intact-zona. 50% of the labelled and 50% of the non-labelled sperm were bound to the intact-zona. The results also showed that there were great variability in the number of sperm bound to the different intact-zona; for example, the range was from 85 to 306 sperm per intact-zona.

Table 7.1 Effect of FITC labelling of sperm on their ability to bind to intact-zona Intact-zona No.
1 2 3 4 5 6

Total No. of sperm bound

232 180 781 562 288 171

No. of labelled sperm bound

121 96 306 257 153 85

% of labelled sperm bound

52 53 48 46 53 50

Mean % of labelled sperm bound to intact-zona

N=6; Mean sem

50 1.2

The motion characteristics of sperm used in the above assay control values of previous studies.

were all consistent with

173

174

7.4.2 Assessment of cutting the intact-zona into equal halves The results (Table 7.2) showed that after cutting the intact-zona into two halves, approximately equal number of sperm were bound to each hemizona. 50% of the sperm bound to the 1st half and 50% to the 2nd half of the intact-zona. Again, the results showed a great variability in the number of sperm bound to the different hemizona; for example, the range was from 124 to 873 sperm per hemizona.

Table 7.2 Evaluation of cutting the intact-zona into equal halves

Hemizona No.

Total No. of sperm bound

No. of sperm bound to 1 half 183 124 285 461 873 323 618 277
st

% of sperm bound to 1st half 54 46 48 51 53 49 51 47 50 1.4

1 2 3 4 5 6 7 8

342 268 588 897 1638 662 1207 587

Mean % of sperm bound to 1st half - hemizona

N=8; Mean sem

The motion characteristics of sperm used in the above assay were all consistent with control values of previous studies.

174

175 7.4.3 Effect of PF on intact-zona binding using control sperm labelled with FITC The calculated percentages of sperm binding to the intact-zona are summarised in Table 7.3. The results showed that PF significantly inhibited (p=0.0001) sperm binding to intact-zona at 0.1, 3, & 5 mM/L compared with the control. However, the results also showed that there were no significant differences among the various PF concentrations in the degree of inhibition of sperm binding to the intact-zona, which averaged 23%. Therefore, there were no significant differences in the percentage of labelled control sperm binding to the intact-zona at the various PF concentrations, which averaged 77%. Consistent with previous experiments, the number of sperm bound to the intact-zona varied from 65 to 530 sperm per intact-zona (data not shown).

Table 7.3 Percentage of sperm bound to intact-zona using control sperm labelled with FITC
Concentration of Pentoxifylline 0.1 mM/L Control sperm - % PF-treated sperm - % 75 3.0 25a 3.0 3 mM/L 79 2.3 21a 2.3 5 mM/L 76 5.2

24a 5.2

N=8; Mean SEM; Significant mean difference from control values shown by p value at a<0.0001

The motion characteristics of sperm used in the above experiment were analyzed by Paired t-test and showed a significant difference (p=0.05) between the control and the positive control, as expected. For example, the VCL was raised by 28% and the ALH was raised by 36% in the PF-treated sperm.

175

176 7.4.4 Effect of PF on intact-zona binding using PF-pretreated sperm labelled with FITC The calculated percentages of sperm binding to the intact-zona are summarised in Table 7.4. The results showed that PF significantly inhibited (p=0.0001) sperm binding to intact-zona at 0.1, 3, & 5 mM/L compared with the control. However, the results also showed that there were no significant differences among the various PF concentrations in the degree of inhibition of sperm binding to the intact-zona, which averaged 15%. Therefore, there were no significant differences in the percentage of control sperm binding to the intact-zona at the various PF concentrations, which averaged 85%. Consistent with previous experiments, the number of sperm bound to the intact-zona varied from 82 to 509 sperm per intact-zona (data not shown).

Table 7.4 Percentage of sperm bound to intact-zona using PF-pretreated sperm labelled with FITC
Concentration of Pentoxifylline 0.1 mM/L Control sperm - % PF-treated sperm - % 87 0.8 13a 0.8 3 mM/L 83 1.6 17a 1.6 5 mM/L 85 1.6

15a 1.6

Mean SEM; N=8; Significant mean differences from control values shown by p value at a<0.0001

The motion characteristics of sperm used in the above experiment were analysed by Paired t-test and showed a significant difference (p=0.05) between the control and the positive control as expected. For example, the VCL was raised by 24% and the ALH was raised by 26% in the PF-treated sperm.

176

177 7.4.5 Effect of PF on sperm-hemizona binding The calculated percentages of sperm binding to the hemizona are summarised in Table 7.5. The results showed that PF significantly enhanced (p=0.001) sperm binding to hemizona. In the presence of 3 mM PF/L, 60% of the PF-treated sperm were bound to the hemizona compared with 40% of the control sperm. Consistent with previous experiments, the number of sperm bound to the hemizona varied from 56 to 1340 sperm per hemizona (data not shown)

Table 7.5 Effect of PF on sperm-hemizona binding

Hemizona number 1 2 3 4 5 6 7 8 Mean % bound

% of control sperm bound to hemizona 41 41 40 39 41 45 31 42 40 1.4

% of PF-treated sperm bound to hemizona 59 59 60 61 59 55 69 58 60a 1.4

N=8; Mean SEM; Significant mean differences from control values shown by p value at a<0.001

The motion characteristics of sperm used in the above experiment were analysed by Paired t-test and showed a significant difference (p=0.05) between the control and PF-treated sperm, as expected. For example, the VCL was raised by 21% and the ALH was raised by 23% in PF-treated sample.

177

178

7.4.6 Comparison between the intact-zona binding and hemizona binding assays

At all three concentrations of PF, PF-pretreated sperm had a very much reduced (4-fold) chance (p<0.0001) of binding to the intact-zona compared to the control sperm (Table 7.6). In the hemizona assay, sperm in the presence of 3 mM PF/L had a 50% greater chance (p<0.0001) of binding to the hemizona, compared to the control sperm.

Table 7.6 Comparison of percentage of sperm binding to intact-zona and hemizona Concentration of Pentoxifylline Methodology Control Intact-zona -% 80 2.3 N=48 Hemizona -% 40 2.3 N=8 0.1 mM/L 19a 2.2 N=16 _ 3.0 mM/L 19a 2.5 N=16 60a 2.5 N=8 5.0 mM/L 20a 2.8 N=16 _

Mean SEM; Significant mean differences from the control values shown by p value at a<0.0001

178

179

7.5 DISCUSSION

The results obtained in this study show that labelling sperm with 150 L of 0.2 mg FITC/mL stain had no detectable adverse effect on sperm binding to intact-zona, a finding consistent with that of Liu et al. (1988). However, sperm that had been stained with 300 and 500 L of FITC showed a significant reduction in motion characteristics. Therefore, it is probable that staining with FITC, even at a lower concentration, may have subtle effects on sperm that might have implications for the sperm-zona binding. Sperm that had been challenged with PF, which was then removed by washing before the binding assay, showed a decreased ability to bind with intact-zona compared with the control sperm. However, in the presence of 3 mM PF, sperm showed a significantly increased ability to bind to the hemizona compared with the control sperm. The wide variation in the number of sperm bound to different zona in the same treatment group was similar to the results reported by Liu et al. (1990). Most studies (Franken et al., 1989b; Liu et al., 1990; Liu and Baker, 1992; 1994a; 1994b) on sperm-zona interaction cite the number of sperm bound to each zona as greater than 100 (if there are more than 100 sperm present on the zona) rather than counting them precisely. Technically, it is very difficult to accurately count sperm tightly attached to the zona, as the sperm tend to bind in a 3-dimensional manner on the surface of the intact-zona, which is then examined with a microscope in 2 dimensions. Further, binding occurs in clusters and clumping occurs. In my study, the problem of accurately counting the bound sperm has been 179

180 overcome by solubilising the zona in acid, which does not appear to damage the sperm integrity. Solubilization also assist in dispersing the tightly bound sperm in clumps around the microwell, making it easier to count, although the counting is tedious. This may account for the high number of sperm counted (as many as 1340) in this study. It is probable that the high numbers of sperm attached to the zona may also be attributed at least partially to 'non-specific' binding to the zona on both sides (outer coat and inner coat) in the hemizona assay. One of the main difficulties in the human sperm-zona interaction studies is obtaining sufficient number of oocytes to overcome statistically the high variability in the number of sperm binding to any one zona. Thus, the hemizona assay appeared to be a solution. Each half zona possesses the same binding characteristics, thus overcoming the variability between control and test experiments. However, the main drawback to hemizona assay is the problem of cutting an oocyte into approximately equal halves which requires micro-manipulation. Therefore, in order to study a range of PF concentrations (0.1, 3.0, 5.0 mM PF/L) on sperm-zona interaction, I used the intact-zona method with some modification. In the first set of experiments, the control sperm were labelled with FITC stain, whereas in the second set of experiments, the PF-treated sperm were labelled. It is thought that the advantages of this

procedure would be that it takes into account any effect of staining on the ability of sperm to bind, and it would also demonstrate if there was any interaction between PF and FITC stain that would affect the sperm-zona interaction. Results from this study showed that when control sperm were labelled with FITC stain, the average binding of PF-treated (across the concentrations range) sperm was 23% compared with 15% when PF-treated sperm were labelled with FITC stain. 180

181 The comparison between intact-zona and hemizona shows that in the presence of PF, which occurs only in the hemizona assay, there is increased binding of PF-treated sperm. From previous studies reported in this thesis (chapters four and five), we know that PF enhances sperm motility, and therefore this might have influenced binding to the zona. Just by random chance, a fast-moving sperm has a greater chance of contact with the zona than a slow-moving sperm. A survey of the literature shows that there has been no direct study reported on the effect of PF on sperm binding, either to human intact-zona or to hemizona, except the single study reported by Kaskar et al. (1994). Their study involved the use of teratozoospermic (> 40% abnormal sperm) samples, prepared by swim-up, and co-incubated with

human hemizona in the presence of 4 mM PF/L for 4 hours. The conclusion from their study was that PF stimulates sperm motility in teratozoospermic samples, but that after 4 hour incubation in the presence of human hemizona, there was no significant difference in the amount of binding of sperm to hemizona between the control and PF-treated sperm, i.e. the increase in sperm motility caused by

exposure to PF was not correlated to the degree of sperm-hemizona binding. These findings are at variance with the results reported in this study, which show that sperm-hemizona binding was increased by 50% in the presence of 3 mM PF/L. The possible explanation for the difference in results could lie in the concentration of PF used and the limitations of method used in counting sperm bound to the zona. From the dose-response studies reported in chapter 5, it has been shown that 2.8 mM PF/L is the optimal concentration to stimulate sperm motion characteristics. Higher concentrations of PF may have deleterious effects on sperm motion characteristics. Therefore, the concentration of 4 mM used by Kaskar et al. (1994) 181

182 might have contributed to their failure to show that PF enhances sperm-hemizona binding. Another factor that might have contributed to the lack of PF effect on spermhemizona binding reported by Kaskar et al. (1994) could be the presence of a large number of abnormal sperm in the semen which could have migrated up into the overlaid medium during the preparation of sperm suspensions. It has been demonstrated (Liu and Baker, 1992; 1994b) that when sperm suspensions containing a very low percentage of normal sperm are used in intact-zona assay, lower numbers of sperm were bound to the zona. The use of different subpopulations of sperm may have contributed to the difference in results between the two studies. In my study, all the guidelines (1992). From the findings reported in chapter 5, washing would have removed some of the stimulation of sperm motion characteristics induced by PF. Therefore, one would expect the binding of the control and PF-pretreated samples to be nearly similar in the intact-zona assay, where the PF is removed by washing. However, the results from my study of sperm binding to intact-zona indicate that PF-pretreated sperm binds much less well to the intact-zona than the control sperm that had not been exposed to PF. To explain this phenomenon, it requires knowledge on both the mechanisms of sperm-zona binding and on what changes are imposed on the sperm by PF treatment, and which remained even after the drug was removed by washing, before we can understand how PF reduces the binding ability of sperm. The chronology of fertilization has been extensively studied in the mouse (Wassarman, 1987, 1988; reviewed by: Fraser and Ahuja, 1988; Yanagimachi, 1988; Wassarman, 1990; Dean, 1992). The following stages are thought to occur in mouse 182 samples were normal, as defined by WHO

183 sperm-zona interaction:- (1) The sperm initially associate with the zona at the surface of the zona pellucida. This relatively loose, nonspecific association is called attachment. (2) The attached sperm then form a relatively tenacious, species-specific adhesion with the zona that is referred to as binding. (3) Bound sperm then complete acrosome exocytosis in preparation for penetration of the zona pellucida and fusion with the oocyte oolemma. A number of hypotheses (Yanagimachi, 1994) have been proposed to explain the mechanism of sperm penetration into the zona pellucida, namely, (a) the mechanical hypothesis and (b) the enzymatic hypothesis. The mechanical hypothesis suggests that the sperm movement into the intact-zona investment is purely mechanical, with the acrosome enzymes playing no part in the penetration. According to this hypothesis, the sole function of the AR is to expose the perforatorium, which is the inner acrosomal membrane of the sperm head. This sharply pointed perforatorium cuts the zona as the sperm is in hyperactive state with its flagellum beating vigorously. Electron micrographs support this view

(Yanagimachi, 1988). The enzymatic hypothesis suggests that the large variety of acrosomal enzymes present in the acrosome are involved in every step of sperm penetration into the oocyte. Sperm motility is of secondary importance. The acrosomal enzymes (e.g. acrosin) modulate the sperm entry, assisted by various sperm receptors (ZP3, ZP2) on the oocyte (Wassarman, 1990) and other carbohydrate determinants present on the surface of the sperm (Ahuja, 1985; Fraser and Ahuja, 1988). As mentioned earlier in this chapter, the human sperm receptor has been cloned and sequenced by Chamberlin and Dean (1990), and a high degree of similarity shown to exist between the human and mouse sperm receptors.

183

184 I think it is likely that, in the human, sperm-zona interaction uses both mechanical (stage 1 for attachment) and enzymatic (stage 2 for binding) processes to penetrate the zona. If one imagines that the zona is like multi-layers of mesh made of glycoproteins (fibrillogranular strands), then the free swimming sperm get loosely attached, leading to mechanical binding. This transient binding is possibly assisted by the highly motile and hyperactivated sperm, with its "whiplash" movement. Up to this stage the process is presumably reversible. It can be shown in sperm-zona binding assays that sperm bound to zona can be removed if the zona is washed vigorously. The loose binding, in all probability, is followed by enzymatic penetration into the zona, a process involving the acrosome reaction, and sperm receptor, ZP3. This scenario may explain why, in the presence of PF (with increases in VCL and ALH of sperm), there was increased binding of sperm to the zona in the hemizona assay. In the intact-zona study, however, PF (where the drug effect was reduced by washing) inhibited binding, possibly due to alteration in the molecular structure of the sperm head. The FITC labelling and the centrifugation process may have sensitised the surface membrane of the sperm head, leading to changes in its chemical structure. The subsequent exposure to PF may have increased the resistance to binding with the zona pellucida. This requires further investigation.

184

185 CHAPTER 8 GENERAL DISCUSSION

Philosophy is nothing but discretion. John Seldon 1584-1654

When trying to advance our knowledge of human spermatozoa, several factors must be borne in mind. The semen, fairly viscous in nature, consists of a heterogeneous population of sperm suspended in seminal fluid that is rich in acid phosphatase, lysozymes, citric acid, fructose, prostaglandins, zinc and magnesium. A typical ejaculate may consist of mature, various stages of immature, abnormal and dead sperm cells. In addition, there may be leucocytes present. The motion

characteristics of this heterogeneous population of sperm in semen can vary from zero to about 70% overall motility. As alluded to in previous chapters, sperm motility plays a crucial role in the process of procreation. However, 15% of all couples will experience primary or secondary infertility at some during their reproductive lives (Menning, 1980; reviewed by Skakkebaek et al., 1994). In approximately 50% of all these cases, the man is subfertile. It is probable that in a proportion of the male factor cases, the fundamental cause of infertility is defective sperm motion. Therefore, over the years, attempts have been made to improve the motility or the fertilizing ability of sperm by using pharmacological agents (reviewed by Lanzafame et al., 1994 and Tournaye et al., 1994a) like Pentoxifylline (PF) and caffeine. Recently, other inducers like platelet-activating factor (PAF) have been shown to 185

186 stimulate sperm motility (Calvo et al., 1989; Ricker at al., 1989; Krausz et al., 1994) and possibly increase the fertilizing potential of sperm (Angle et al., 1993). Nearly a decade ago, computer-aided sperm analysis was introduced for semen analysis in basic and clinical applications. It was thought that CASA would overcome many of the limitations (section 1.7.1) of visual semen analysis (Davis and Boyers, 1992; Bartoov et al., 1993) but, however, in the course of time it has become apparent that the cost of the computer technology and laboratory staff's resistance has severely limited its applicability (Davis and Katz, 1993). In addition, computer technology itself introduced problems like image jitters, apparent motion and bump & cross (discussed in section 1.7.2), which became apparent when CASA systems were used in research. In spite of its pitfalls, CASA has been shown to provide useful information in the diagnosis and treatment of subfertile patients (Katz and Overstreet, 1981; Chan et al., 1989; Fetterolf and Rogers, 1990; Check et al., 1990; Liu et al., 1991). In this project, I undertook to study the effects of PF on sperm motion characteristics, employing computer-aided sperm analysis, and assessed its effects on the acrosome reaction and sperm-zona pellucida interactions. Although CASA can generate data on individual sperm tracks, I have used the average values by tracking more than 50 sperm per sample. The advantage of this method is that more samples can be analyzed and it will represent the whole sample. However, tracking many individual sperm is time consuming, and therefore only a limited number of samples can be assessed in the control and test experiments. Because of this limitation, currently most of the published studies are based on the average CASA values (Check et al., 1990; Tesarik et al., 1992a; Fuse et al., 1993). 186

187 A survey of the literature showed that basic information on PF, such as the optimal dose and optimal incubation time, was lacking. So, in chapters two and three, I set out to investigate how these basic factors would influence sperm motion characteristics in the presence and absence of PF. The results showed that the conditions under which PF could induce optimal stimulation were an incubation for one hour at the temperature of 370 C. It was also found that centrifuging the sperm suspension four times did not statistically affect the sperm motion characteristics adversely, a result consistent with the findings of Alvarez et al. (1993). The use of Percoll in discontinuous Percoll gradient for the preparation of motile sperm in suspension showed that the immediate effect of Percoll on sperm was to depress all CASA parameters except LIN and MOT%, but subsequent washing to remove any remaining Percoll in the sperm suspension showed that there was an increase in the value of VCL and ALH accompanied with a decrease in MOT%. Further

investigations, reported in chapter five, showed a similar effect and it is hypothesized that the removal of decapacitation factors and reactive oxygen species which presumably accompany frequent changes of fresh culture medium may have contributed to the increase in these motion values. In the study involving comparison between Percoll gradient and 'swim-up' sperm separation, it was found that the population of sperm isolated by the two methods were essentially different. In the Percoll gradient method, separation is based on density, irrespective of sperm motility, whereas in the 'swim-up' technique, only the progressive motile cells that could swim into the overlaid culture medium were collected. Experimental results indicated that sperm separated by the Percoll gradient method showed higher values in VCL, LIN, and ALH when compared with sperm separated by 'swim-up'. Thus, the 187

188 two methods of separation represent different sub-populations/sub-sets of sperm exhibiting different motion characteristics. It thus becomes very important when

undertaking any studies on sperm motility that the method of sperm separation is standardised at the outset. Sperm stimulant has been demonstrated to improve sperm motion characteristics (Aparico et al., 1980b; Yovich et al., 1990; Tesarik et al., 1992a). Investigation of the optimal concentration of PF that would result in maximum stimulation of sperm in semen formed the basis of the study reported in chapter 4. The results gathered from the study showed that, in semen, the optimal concentration of PF that produced the maximum response was 6 mM PF/L. The figure of 6 mM/L was obtained from the appropriate Stimulation Index (section 4.3), although individual CASA parameters peaked at different concentrations of PF. Hence, Stimulation Index is a useful mathematical tool in discovering a total maximum effect when a chemical compound has varying degrees of effect on different parameters in the same sample. PF produced a significant increase in VSL, VCL, and ALH, but no significant change in MOT% or LIN. The recovery of motile sperm after 370 C incubation for one hour with 6 mM PF/L was 36% above the control group. On examining a subset of 24 matched pairs of samples, there was considerable variation (0 to 40%) in response of the sperm to PF challenge. This might be because different semen samples contain different proportion of the various sub-population/sub-sets of sperm, only some of which respond to PF stimulation. This variation in the degree of response of different semen samples was consistent with the results of other studies (Tesarik et al., 1992a; Moohan et al., 1993).

188

189 De Turner et al. (1978) demonstrated that a four-hour exposure of sperm in suspensions to cyclic adenosine monophosphate (cAMP) increased the duration of sperm activity and significantly improved the percentage of sperm with forward progressive movement. Subsequently, Tash and Means (1982) were able to demonstrate that the addition of 10 M cAMP to sperm in suspensions, produced a 2.5-fold increase in the proportion of motile sperm. In addition, the wave amplitude of sperm movement was increased, which would facilitate an increase in forward velocity. The authors were further able to show that the addition of the cAMP

inhibitor, protein kinase inhibitor (PKI), blocked the effects of cAMP on sperm motility. It is thought that PF exerts its stimulating influence on sperm motion by inhibiting cAMP phosphosdiesterase (Garbers et al., 1971b; Stefanovich, 1973), thereby increasing intracellular cAMP concentrations. An increase in intracellular cAMP

concentration has been reported to increase sperm motility (Calamera et al., 1982; Calamera et al., 1986) with enhancement of endogenous adenosine triphosphate (ATP) utilization. ATP produced in sperm mitochondria is the source of energy for sperm motion. In contrast, a study by Makler et al. (1980b) showed that addition of 10 to 1000 g of cAMP directly to semen did not have any effect on sperm motion parameters as assessed by multiple exposure photography. The theme of chapter 5 was to find the optimal concentration of PF that would produce a maximum elevation in sperm motion characteristics of sperm in suspensions, and to assess if this increase was maintained after washing. A survey of the literature (Sikka and Hellstrom, 1991; Lewis et al., 1993; Moohan et al., 1993; Fuse et al., 1993) showed that different research groups used different concentrations of PF to demonstrate the beneficial effect of PF on sperm 189

190 suspensions, and that this ranged from 2.0 to 5.0 mM PF/L, and appeared to be chosen empirically. The results obtained in this study showed the maximum increase in sperm motion characteristics occurred at 2.8 0.2 mM PF/L, as calculated from the appropriate Simulation Index (section 5.3), although individual CASA parameters peaked at different concentrations of PF. Significant increases were seen in VCL and ALH, but there were no significant changes in VSL, LIN, or MOT%. On examining responses of individual samples of sperm suspensions to 3 mM PF/L challenge, it revealed that there was considerable inter-individual variation in the increase in VCL and ALH, ranging from 0% to approximately 40%. These results were consistent with the findings in chapter 4 where sperm in semen, rather than in media, were challenged with PF. Nevertheless, the stimulation of sperm motion characteristics by PF was reduced by washing. These findings are in contrast to the results obtained by Tesarik et al. (1992a), who have reported that the stimulation of sperm by PF remained for two hours after washing. For reasons already discussed in section 5.5, the maintenance of raised sperm motion characteristics in the control samples might be due to removal of reactive oxygen species produced by the sperm (Aitken, 1994) and the removal of decapacitation factors which presumably accompanies frequent changes of culture media involved in the wash procedures. This project has so far demonstrated that PF stimulated sperm motion characteristic; however, does the enhancement of sperm motion correlate with the acrosome reaction? This question formed the basis of the research reported in chapter 6. The results obtained from the study showed that PF stimulation of sperm motion characteristics does not correlate with the acrosome reaction. There was no significant increase in the proportion of sperm that had undergone the acrosome 190

191 reaction after treatment with PF, a result consistent with the results of other researchers (Tesarik et al., 1992b; Tasdemir et al., 1993; Carver-Ward et al., 1994). However, a very recent study by Gearon et al. (1994) showed that 3.6 mM PF/L was a potent inducer of the acrosome reaction in sperm which had been prepared by the Percoll gradient method. Studies conducted in this project have shown that sperm separated by the Percoll gradient method were a sub-population with higher sperm motion characteristics than sperm separated by 'swim-up' method, a factor that might have contributed to the difference in result reported by Gearon et al. (1994)

compared to all other studies (Tesarik et al., 1992b; Tasdemir et al., 1993; CarverWard et al., 1994), in which the sperm have been prepared by the 'swim-up' method. As alluded to in section 6.5, the mechanism of the acrosome reaction is still not fully understood (Zaneveld et al., 1993). Pentoxifylline has been shown to improve the fertilising ability of sperm in some cases of infertility (Yovich et al., 1990; Tesarik and Mendoza, 1993). This issue was the subject matter of investigation in chapter 7, where the effect of PF on spermzona pellucida binding was studied in order to answer the question: does PF treatment improve sperm competence to fertilize oocyte? The results obtained showed that sperm treated with PF and subsequently washed with culture medium to remove the drug showed a decreased ability to bind with zona pellucida. However, sperm in the presence of 3 mM PF/L showed increased ability to bind with zona pellucida. Kaskar et al. (1994) demonstrated that sperm from teratozoospermic (>40% abnormal sperm) patients, in the presence of 4 mM PF/L, had increased sperm motion characteristics, but that there was no corresponding increased sperm binding with zona pellucida. The possible explanation for their apparently different 191

192 result could lie in the different populations of sperm used; in their study, the semen came from teratozoospermic patients, whereas in my study, all samples were

normozoospermic. Fertilization is one of the most complex forms of cellular interactions, involving not merely the surface contact between gametes, but also encompassing other cellular processes (Garbers, 1989) like the acrosome reaction (Liu and Baker, 1994b). The various hypothesis to explain sperm-zona pellucida interaction are discussed in section 7.5. An interesting observation was made from the studies reported in chapters six and seven which involved labelling of sperm with fluorescent stain. Although labelling of sperm with a low concentration of the stain showed no adverse effect on the motion characteristics, with higher concentration, there was a significant decrease in motion characteristics. This may imply that the use of the stain in labelling sperm could introduce subtle changes which are not easily detectable. Therefore, caution has to be exercised in its application.

Considering the above discussion, the use of PF as a sperm stimulant to enhance sperm motility to treat patients with subfertility needs careful consideration. The studies in this project show that not all sperm samples respond to PF stimulation, and hence there is a need to do preliminary testing. It may be necessary that the use of PF be tailored to each individual patients. Indiscriminate use of PF in treatment of subfertility does not improve either sperm motion characteristics or fertilization, a view strongly supported by a recently published paper (Tournaye et al., 1994b).

192

193 In conclusion (Fig. 8.1), Pentoxifylline stimulates sperm motion

characteristics, although the degree of stimulation can vary from sample to sample, and it does not appear to promote the acrosome reaction. However, in the presence of this drug there was increased sperm-zona binding. Proposed research for the future would include:1) Studying the effect of PF directly on semen in asthenozoospermic patients. 2) Investigating the effect of PF on multiple sperm samples from the same individual but obtained over a period. 3) The effect of Hepes in culture medium on the Acrosome Reaction inducible by Ionophore A23187 in the presence of Pentoxifylline.

193

194

194

195 Appendix A

Chemical composition of Earles-Hepes balanced salt (EHBS) solution

Components -------------------------

mg/L

NaCl KCl MgSO47H2O NaH2PO42H2O CaCl22H2O NaHCO3 Glucose Phenol red

6800 400 200 158 264 2200 1000 10

195

196

196

197 REFERENCES

Acosta AA, Oehninger S, Morshedi M, Swanson RJ, Scott R, Irianni F:Assisted reproduction in the diagnosis and treatment of the male factor. Obstet. Gynecol. Surv. 1988; 44:1-18.

Ahuja KK: Carbohydrate determinants involved in mammalian fertilization. Am. J. Anat. 1985; 174:207-223

Aitken RJ, Richardson DW and Clarkson J: Assessment of the fertilizing capacity of human spermatozoa. In: In vitro Fertilization & Donor insemination; Proceeding of the 12th study group of the Royal College of Obstetricians & Gynaecologist. Ed: Thompson W, Joyce DN and Newton JR. Nov. 1984; pp. 275-283

Aitken RJ, Mattei A and Irvine S. Paradoxical stimulation of human motility by 2deoxyadenosine. J. Reprod. Fertil. 1986; 78:515-527

Aitken RJ and Clarkson JS: Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil. 1987; 81:459-469

Aitken RJ and Clarkson JS: Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl. 1988; 9:367-376

197

198 Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, and Wu FCW: Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J. Androl. 1989; 10:214-220

Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos and Richardson DW: Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. J. Reprod. Fertil. 1992; 94:451-462

Aitken RJ, Buckingham D and Harkiss D: Use of a xanthine free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J. Reprod. Fertil. 1993; 97:441-450

Aitken RJ: Pathophysiology of human spermatozoa. Curr. Opin. Obst. Gyn. 1994; 6:128-135

Allison AC and Hartree EF: Lysosomal enzymes in the acrosome and their possible role in fertilization. J. Reprod. Fertil. 1970; 21:501-515

Alvarez JG, Lasso JL, Blasco L, Nunez RC, Heyner S, Caballero PP and Storey BT: Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum. Reprod. 1993; 8:1087-1092

198

199 Angle MJ, Tom R, Jarvi K and McClure: Effect of platelet-activating factor (PAF) on human spermatozoa-oocyte interactions. J. Reprod. Fertil. 1993; 98:541-548

Aprarico NJ, Schwarstein L, de Turner EA: Pentoxifylline (BL191) by oral administration in the treatment of asthenozoospermia. Androl. 1980a; 12:228-231

Aparicio NJ, de Turner EA, Schwarstein L and

Turner D: Effect of the

phosphodiesterase inhibitor pentoxifylline on human sperm motility. Androl. 1980b; 12:49-54

Arora M, Carver-Ward JA, Jaroudi KA and Sieck UV: Is Percoll safe for in vivo use? Fertil. Steril. 1994; 61:979-981

Austin CR: Observations on the penetration of the sperm into mammalian egg. Aust. J. Sci. Res. 1951; 4:581-596

Badenoch DF, Moore HDM, Holt WV Evans PR, Sidhu BS and Evans SJW: Sperm motility, velocity and migration. Br. J. Urol. 1990; 65:204-208

Barros C, Bedford JM, Frankin LE and Austin CR:Membranes of vesiculation as a feature of the mammalian acrosome reaction. J. Cell Biol. 1967; 34:C1-5

Barros C and Yanagimachi R: Induction of zona reaction in golden hamster eggs by cortical reaction material. Nature 1971; 233:268-269 199

200

Barthelemy C, Fricot G, Hamamah S, Lebos C, Lansac J and Tharanne MJ: Ultrastructural comparison of human spermatozoa along a Percoll density gradient. Int. J. Fertil. 1992; 37:362-367

Bartoov B, Eltes F, Weissenberg R and Lunenfeld B: Morphological characterization of abnormal human spermatozoa using the transmission electron microscope. Arch. Androl. 1980; 5:305-322

Bartoov B, Eltes F, Pansky M, Lederman H, Caspi E and Soffer Y: Estimating fertility potential via semen analysis data. Hum. Reprod. 1993; 8:65-70

Bedford JM, Calvin H and Cooper GW: The maturation of spermatozoa in the human epididymis. J. Reprod. Fertil. (Suppl.) 1973; 18:199-213

Bedford JM: Maturation, transport and fate of spermatozoa in the epididymis. In: Handbook of Physiology; Endocrinology, vol. 5, Male Reproductive System. Ed: Hamilton DW and Greep RO. American Physiological Society, Washington, USA. 1975; pp. 303-317

Bedford JM and Cooper NL:Membrane fusion events in the fertilization of vertebrate eggs. In: Membrane fusion, Cell surface reviews. Vol. 5 Ed: Poste E and Nicolson GL. Elsevier/North-Holland Biomedical Press, Amsterdam. 1978; pp. 65-125,

200

201 Bendvold E, and Aanesen A: The Cellsoft computerized semen analysis system. II. on the optimalization of running conditions concerning Gray scale and cell size range. Andrologia 1990; 22:42-54

Benoff S: The role of cholesterol during capacitation of human spermatozoa. Hum. Reprod. 1993; 8:2001-2006

Bielfeld P, Faridi A, Zaneveld LJD and De Jonge C: The zona pellucida-induced acrosome reaction of human spermatozoa is mediated by protein kinases. Fertil. Steril. 1994; 61:536-541

Bland JM and Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; 8:307-310

Blasco L: Clinical tests of sperm fertilizing ability. Fertil. Steril. 1984; 41:177-192

Blazak WF, Overstreet, Katz DF and Hanson FW: A competitive in vitro assay of human sperm fertilizing ability utilizing contrasting fluorescent sperm markers. J. Androl. 1982; 3:165-171

Bleil JD and Wassarman PM: Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida. Dev. Biol. 1980a; 76:185-202

201

202 Bleil JD and Wassarman PM: Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse mouse egg zona pellucidae possessing receptor activity for sperm. Cell 1980b; 20:873-882

Bleil JD and Wassarman PM: Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev. Biol. 1983; 34:317-324

Boyers SP, Davis RO, and Katz DF: Automated semen analysis. Curr. Probl. Obstet. Gynecol. Fertil. 1989; 12:168-200

Breckwoldt M, Zahrednik HP and Neulen J: Classification and diagnosis of ovarian insufficiency. In: Infertility: Male and Female. Ed: Insler V and Lunenfeld B. Churchill and Livingstone, London. 1993; pp. 229-251

Burkman LJ: Characterization of hyperactivated motility by human spermatozoa during capacitation: Comparison of fertile and oligozoospermic sperm populations. Arch. Androl. 1984; 13:153-165

Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z and Hodgen GD: The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil. Steril. 1988; 49:688-701

202

203 Burkman LJ, Coddington CC, Franken DR, Oehninger SC and Hodgen GD: The hemizona assay (HZA): Assessment of fertilizing potential by means of human sperm binding to the human zona pellucida. In: CRC Handbook of the laboratory diagnosis and treatment of infertility. CRC Press, Boston. 1990; pp. 213-228

Burkman LJ: Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermatozoa using computerized analysis. Fertil. Steril. 1991; 55:363-371

Byrd W and Wolf DP: Oogenesis, Fertilization and early development. In: Human invitro Fertilization and Embryo Transfer. Ed: Wolf DP and Quigley MM. Plenum Press, New York. 1984; pp. 213-273

Calamera JC, Brugo S and Vilar O: Relationship between motility and adenosinetriphosphate (ATP) in human spermatozoa. Andrologia 1982; 14:239-241

Calamera JC, Quiros MC, Brugo S, Nicholson RF and Vilar O: Adenosine 5'Triphosphate (ATP) concentration and acrosin activity in human spermatozoa. Their relationship with the sperm penetration assay (SPA). Andrologia 1986; 18:574-580

Calvo L, Vantman D, Banks SM, Tezon J, Koukoulis GN, Dennison L and Sherins RJ: Follicullar fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil. Steril. 1989; 52:10481054 203

204

Carver-Ward JA, Jaroudi KA, Einspenner M, Parhar RS, Al-Sedairy and Sheth KV: Pentoxifylline potentiates ionophore (A23187) mediated acrosome reaction in human sperm: flow cytometric analysis using CD46 antibody. Hum. Reprod. 1994; 9:71-76

Chamberlin ME and Dean J: Human homolog of the mouse sperm receptor. Proc. Natl. Acad. Sci. 1990; 87:6014-6018

Chan SYW, Wang C, Chan STH, Ho PC, So WWK, Chan YF, and Ma HK: Predictive value of sperm morphology and movement characteristics in the outcome of in vitro fertilization of human oocytes. J. In Vitro Fert. Embryo Transf. 1989; 6:142-148

Chang MC: Fertilizing capacity of spermatozoa deposited in fallopian tubes. Nature 1951; 168:997-998

Check JH, Bollendore A,Lee MA, Nazari A and Nowroozi

K: Correlation of

computerized semen analysis with successful fertilization of oocytes in an in vitro fertilization program. Arch. Androl. 1990; 24:229-234

Coddington CC, Fulgham DL, Alexander NJ, Johnson D, Herr JC and Hodgen GD: Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody. Fertil. Steril. 1990; 54:504-508

204

205 Colon JM, Ginsburg F, Lessing JB, Schoenfeld C, Goldsmith LT, Amelar RD, Dubin L and Weiss G: The effect of relaxin and prostagladin E2 on the motility of human spermatozoa. Fertil. Steril. 1986; 46:1133-1139

Corsan GH and Kemmann E: The role of superovulation with menotropins in ovulatory infertility: a review. Fertil. Steril. 1991; 55:468-477

Cross NL, Morales P, Overstreet JW and Hanson FW: Two simple methods for detecting acrosome-reacted human sperm. Gam. Res. 1986; 15:213-226

Cross NL, Morales P, Overstreet JW and Hanson FW: Induction of acrosome reactions by the human zona pellucida. Biol. Reprod. 1988; 38:235-244

Cross NL and Meizel S: Methods for evaluating the acrosomal status of mammalian sperm. Biol. Reprod. 1989; 41:635-641

Crozet N: Fertilization in vivo and in vitro. In: Reproduction in Mammal and Man. Ed: Thibault C, Levasseur MC and Hunter RHF. Ellipses, Paris. 1993 pp. 327-347

Cummins JM and Yanagimachi R: Sperm-egg ratios and the site of the acrosome reaction during in vivo fertilization in the hamster. Gamete Res. 1982; 5:239-256

205

206 Cummins JM and Yanagimachi R: Development of ability to penetrate the cumulus oophorous by hamster spermatozoa capacitated in vitro, in relation to the timing of the acrosome reaction. Gamete Res. 1986; 15:187-212

Cummins JM, Pember SM, Jequier AM, Yovich JL and Hartmann PE: A test of the human sperm acrosome reaction following ionophore challenge; Relationship to fertility and other seminal parameters. J. Androl. 1991; 12:98-103

Dadoune JP and Demoulin A: Structure and function of the testis. In: Reproduction in Mammal and Man. Ed: Thibault C, Levasseur MC and Hunter RHF. Ellipses, Paris. 1993 pp. 327-347

DasGupta S, Mills CL and Fraser LR: Ca2+-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay. J. Reprod. Fertil. 1993; 99:135-143

David G, Serres C, and Jouannet P: Kinematics of human spermatozoa. Gam. Res. 1981; 4:83-95

Davis RO, and Katz DF: Computer-aided sperm analysis (CASA): Image digitization and processing. Biomat. Art. Cells. Art. Org. 1989; 17:93-116

Davis RO, Niswander PW and Katz DF: New measures of sperm motion: Adaptive smoothing and harmonic analysis. J. Androl. 1992; 13:139-152 206

207

Davis RO and Boyers SP: The role of digital image analysis in reproductive biology and medicine. Arch. Pathol. Lab. 1992; 116:351-363

Davis RO and Katz DF: Computer-aided sperm analysis: technology at a crossroads. Fertil. Steril. 1993; 59:953-955

De Braekeleer M and Dao TN: Cytogenetic studies in male infertility: a review. Hum. Reprod. 1991; 6:245-250

De Jonge CJ, Mack SR and Zaneveld LJD: Synchronous assay for human sperm capacitation and the acrosome reaction. J. Androl. 1989; 10:232-239

De Lamirande ED, and Gagnon C: Quantitative assessment of the serum-induced stimulation of human sperm motility. Int. J. Androl. 1991; 14:11-22

De Turner EA, Aparicio NJ, Turner D and Schuwarzstein L: Effect of two phosphodiesterase inhibitors, cyclic adenosine-3'-5'-monophosphate, and a blocking agent on human sperm motility. Fertil. Steril. 1978; 29:328-331

Dean J: Biology of mammalian fertilization: Role of the zona pellucida. J. Clin. Invest. 1992; 89:1055-1059

207

208 Dresdner RD and Kratz DF: Relationship of mammalian sperm motility and morphology to hydrodynamics aspects of cell function. Biol. Reprod. 1981; 25:920930

Eddy EM: The spermatozoon. In: The physiology of reproduction. Vol. 1 Ed: Knobil E and Neill JD. Raven Press, New York. 1988; pp. 27-68.

Egozcue J, Templado C, Vidal F, Navarro J, Morer-Fargas F and Marina S: Meiotic studies in a series of 1100 infertile and sterile males. Hum. Genet. 1983; 65:185-188

Elstein M, Moghissi KS and Borth R: Cervical Mucus in human reproduction. Scriptor, Copenhagen. 1972; pp. 163-170

Essig M, Schoenfeld C, Amelar RD, Dubin L and Weiss G: Stimulation of human sperm motility by relaxin. Fertil. Steril. 1982; 38:339-343

Fakih H. MacLusky N, DeCherney A, Wallimann T and Huszar G: Enhancement of human sperm motility and velocity in vitro: effects of calcium and creatine phosphate. Fertil. Steril. 1986; 46:938-944

Fawcett DW: The mammalian spermatozoon. Devel. Biol. 1975; 44:394-436

Fetterolf PM, and Rogers BJ: Prediction of human sperm penetrating ability using computerized motion parameters. Mol. Reprod. Devel. 1990; 27:326-331 208

209

Fisch H and Lipshultz LI: Diagnosing male factors of infertility. Arch. Pathol. Lab. 1992; 116:398-399

Florman HM and Storey BT: Mouse gamete interactions: the zona pellucida is the site of the acrosome reaction leading to fertilization in vitro. Dev. Biol. 1982; 91:121130

Fox CA and Fox B: A comparative study of coital physiology with special reference to the sexual climax. J. Reprod. Fertil. 1971; 24:319-336

Franken DR, Oehninger S, Burkman LJ, Coddington CC, Kruger TF, Rosenwaks Z, Acosta AA and Hodgen GD: The hemizona assay (HZA): A predicator of human sperm fertilizing potential in in vitro fertilization (IVF) treatment. J. Vitro Fert. Embryo. Transf. 1989a; 6:44-50

Franken DR, Burkman LJ, Oehninger SC, Coddington CC, Veeck LL, Kruger TF, Rosenwaks Z and Hodgen GD: Hemizona assay using salt-stored human oocytes: Evaluation of zona pellucida capacity for binding human spermatozoa. Gam. Res. 1989b; 22:15-26

Fraser, LR: Motility patterns in mouse spermatozoa before and after capacitation. J. Exp. Zool. 1977; 202:439-444 209

210

Fraser L: Ca2+ is required for mouse sperm capacitation and fertilization in vitro. J. Androl. 1981; 3:412- 419

Fraser LR: Mechanism controlling mammalian fertilization. In: Oxford Reviews of Reproductive Biology, Vol. 6 Ed:Finn CA. Clarendon Press, Oxford. 1984; pp. 174225

Fraser LR and Ahuja KK: Metabolic and surface events in fertilization. Gamete Res. 1988; 20:491-519

Fraser LR and McIntyre K: Calcium channel antagonists modulate the acrosome reaction but not capacitation in mouse spermatozoa. J. Reprod. Fertil. 1989; 86:223233

Fraser LR, Harrison RAP and Herod JE: Characterization of a decapacitation factor associated with epididymal mouse spermatozoa. J. Reprod. Fertil. 1990; 89:135-148

Fraser LR and McDermott CA: Ca2+-related changes in the mouse sperm capacitation state: a possible role for Ca2+-ATPase. J. Reprod. Fertil. 1992; 96:363377

Fraser LR: Requirements for successful mammalian sperm capacitation and fertilization. Arch. Pathol. Lab. 1992; 116:345-350 210

211

Fraser LR, Umar G and Sayed S: Na+-requiring mechanisms modulate capacitation and acrosomal exocytosis in mouse spermatozoa. J. Reprod. Fertil. 1993; 97:539549

Fuse H, Sakamoto M, Ohta S and Katayama T: Effect of pentoxifylline on sperm motion. Arch. Androl. 1993; 31:9-15

Gaddum-Rosse P: Mammalian gamete interactions: what can be gained from observations on living eggs? Am. J. Anat. 1985; 174:347-356

Garbers DL, First NL, Sullivan JJ and Lardy HA: Stimulation and maintenance of ejaculated bovine spermatozoan respiration and motility by caffeine. Biol. Reprod. 1971a; 5:336-339

Garbers DL, Lust WD, First NL and Lardy HA: Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry. 1971b; 10:1825-1831

Garbers DL: Molecular basis of fertilization. Annu. Rev. Biochem. 1989; 58:719-742

Gavella M, Lipovac V, and Marotti T: Effect of pentoxifylline on superoxide anion production by human sperm. Int. J. Androl. 1991; 14:320-327

211

212 Gearon CM, Mortimer D, Chapman MG and Forman RG: Artificial induction of the acrosome reaction in human spermatozoa. Hum. Reprod. 1994; 9:77-82

Gibbons BH: Studies on the mechanism of flagellar movement. In: The spermatozoon - maturation, motility, surface properties and comparative aspects. Ed:Fawcett DW and Bedford JM. Urban & Schwarzenberg, Baltimore. 1979; pp. 9197

Gladen BC, Williams J and Chapin RE: Issues in the statistical analysis of sperm motion data derived from computer-assisted systems. J. Androl. 1991; 12:89-97

Godfrey K: Simple regression 313:1629-1636

in medical research. New Engl. J. Med. 1985a;

Godfrey K: Comparing the means of several groups. New Engl. J. Med. 1985b: 313:1450-1456

Gorus FK and Pipeleers DG: A rapid method for the fractionation of human spermatozoa according to their progressive motility. Fertil. Steril. 1981; 35:662-665

Greve J and Wassarman P: Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat. J. Mol. Biol. 1985; 181:253392 212

213

Grimes SR Jr.: Nuclear proteins in spermatogenesis. Comp. Biochem. Physiol. 1986; 83:495-500

Gulyas BJ: Cortical granules of mammalian eggs. Inter. Rev. Cytol. 1980; 63:357392

Gwatkin RBL: Effects of chemicals on fertilization. Reproductive Toxicology Edited by Dixon RL, Raven Press, New York 1985:209-218

Hammitt DG, Bedia E, Rogers PR, Syrop CH, Donovan JF and Williamson RA: Comparison of motility stimulants for cryopreserved human semen. Fertil. Steril. 1989; 52:495-502

Hammitt DG, Walker DL and Williamson RA: Survival and in vitro fertilization potential of sperm following washing and incubation with different protein supplements. Int. J. Fertil. 1990; 35:46-50

Harper MJK: Gamete and zygote transport. In: The physiology of reproduction. Vol. 1 Ed: Knobil E and Neill JD. Raven Press, New York. 1988; pp. 103-134

Harvey C: The speed of human spermatozoa and the effect on it of various diluents with some preliminary observations on clinical material. J. Reprod. Fertil. 1960; 1:8495 213

214

Henderson CJ, Hulme MJ and Aitken RJ: Contraceptive potential of antibodies to the zona pellucida. J. Reprod. Fertil. 1988; 83:325-334

Hinrichsen-Kohane AC, Hinrichen MJ and Schill WS: Molecular events leading to fertilization. Andrologia. 1984; 16:321-341

Holstein AF: Ultrastructural observations on the differentiation of spermatids in man. Andrologia 1976; 8:157-165

Holt WV, Moore HDM and Hillier SG: Computer-assisted measurements of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays. Fertil. Steril. 1985; 44:112-119

Hoshi K, Sugano T, Endo C, Yoshimatsu N, Yanagida K and Sato A: Induction of the acrosome reaction in human spermatozoa by zona pellucida and effect of cervical mucus on zona-induced acrosome reaction. Fertil. Steril. 1993; 60:149-153

Hoskin DD, Johnson D, Brandt E and Acott TS: Evidence for a role for a forward motility protein in the development of sperm motility. In: The spermatozoon maturation, motility, surface properties and comparative aspects. Ed:Fawcett DW and Bedford JM. Urban & Schwarzenberg, Baltimore. 1979; pp. 43-53

214

215 Huang TTF, Fleming AD and Yanagimachi R: Only acrosome-reacted spermatozoa can bind to and penetrate zona pellucida: A study using the guinea pig. J. Exp. Zool. 1981; 217:287-290

Iizuka R, Kaneko S, Kobanawa K and Kobayashi T: Washing and concentration of human semen by Percoll density gradients and its application to AIH. Arch. Androl. 1988; 20:117-124

Ing RMY, Li DQ, Harding AM and Jones WR: A comparison of swim-down and swim-up methods for the extraction of high motility sperm. Fertil. Steril. 1991; 55:817819

Irianni FM and Coddington CC: Male factor infertility and assisted reproductive technologies. Curr. Opin. Obst. Gyn. 1992; 4:712-719

Ishijima S: Changes in the beating pattern of spermatozoa during maturation and capacitation. In: Fertilization in mammals. Ed: Bavister BD, Cummins J and Roladan ERS. Serono Symposia, USA. 1990; pp. 205-238

Iwasaki A and Gagnon C: Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil. Steril. 1992; 57:409-416

215

216 Jiang CS, Kilfeather SA, Pearson RM and Turner P: The stimulatory effects of caffeine, theophylline, lysine-theophylline and 3-isobutyl-1-methylxanthine on human sperm motility. Br. J. Clin. Pharmacol. 1984; 18:258-262

Johnsen SG, Bennet EP and Jensen VG: Therapeutic effectiveness of oral testosterone. Lancet 1974; 2:1473-1475

Jones R, Mann T and Sherins R: Peroxidative breakdown of phospholipids by human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil. Steril. 1979; 31:531-537

Jones GS: Corpus luteum: composition and function. Fertil. Steril. 1990; 54:21-27

Jouannet P, Volochine B and Deguent P: Light scattering determination of various characteristic parameters of spermatozoa motility in a series of human sperm. Andrologia 1977; 9:36-49

Kaskar K, Franken DR van der Horst and Kruger TF: The effect of pentoxifylline on sperm movement characteristics and zona pellucida binding potential of teratozoospermic men. Hum. Reprod. 1994; 9:477-481

Katz DF and Overstreet JW: Sperm motility assessment by videomicrography. Fertil. Steril. 1981; 35:188-193

216

217 Katz DF, Andrew JB and Overstreet JW: Biological basis of in vitro test of sperm function. In: Sperm Measures and Reproductive Success: Institute for Health Policy Analysis, Forum on Science, Health, and Environmental Risk Assessment. 1989, pp. 95-106

Katz DF and Drobnis EZ: Analysis and interpretation of the forces generated by spermatozoa. In: Fertilization in mammals. Ed: Bavister BD, Cummins J and Roldan ERS. Serono Symposia, USA. 1990; pp. 125-137

Kay VJ, Coutts JRT and Robertson L: Pentoxifylline stimulates hyperactivation in human spermatozoa. Hum. Reprod. 1993; 8:727-731

Kessopoulou E, Tomlinson MJ, Barrat CLR, Bolton AE and Cooke ID: Origin of reactive oxygen species in human semen: spermatozoa or leucocytes? J. Reprod. Fertil. 1992; 94:463-470

Kinloch RA and Wassarman PM: Nucleotide sequence of the gene encoding zona pellucida glycoprotein ZP3 - the mouse sperm receptor. Nucl. Acids Res. 1989; 17:2861-2863

Kinloch RA, Roller RJ, Fimiani CM, Wassarman DA: Primary structure of the mouse sperm receptor polypeptide determined by genomic cloning. Proc. Natl. Acad. Sci. 1988; 85:6409-6413

217

218 Kopf GS: The zona pellucida-induced acrosome reaction: A model for sperm signal transduction. In:Fertilization in mammals. Ed: Bavister BD, Cummins J and Roladan ERS. Serono Symposia, USA. 1990; pp. 253-266

Krausz Cs, Gervasi G, Forti G and Baldi E:Effect of platelet-activating factor on motility and acrosome reaction of human spermatozoa. Hum. Reprod. 1994; 9:471476

Langlais J and Roberts KD: A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res. 1985; 12:183-224

Lanzafame F, Chapman MG, Guglielmino A, Gearon CM and Forman RG: Pharmacological stimulation of sperm motility. Hum. Reprod. 1994; 9:192-199

Lee MA, Trucco GS, Bechtol KB, Wummer N, Kopf GS, Blasco L and Storey BT: Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay. Fertil. Steril. 1987; 48:649-658

Lewis SEM, Moohan JM, and Thompson W: Effects of pentoxifylline on human sperm motility in normospermic individuals using computer-assisted analysis. Fertil Steril 1993; 59:418-423.

218

219 Liang LF and Dean J: Conservation of mammalian secondary sperm receptor genes enables the promoter of the human gene to function in mouse oocytes. Dev. Biol. 1993; 156:399-408

Linck RW: Advances in the ultrastructural analysis of the sperm flagellar axoneme. In: The spermatozoon - maturation, motility, surface properties and comparative aspects. Ed: Fawcett DW and Bedford JM. Urban & Schwarzenberg, Baltimore. 1979; pp. 81-90

Liu DY, Lopata A, Johnston WI and Baker HWG: A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro. Fertil. Steril. 1988; 50:782788

Liu DY, Lopata A and Baker HWG: Use of oocytes that failed to be fertilized in vitro to study human sperm-oocyte interactions: Comparison of sperm-oolemma and sperm-zona pellucida binding, and relationship with results of IVF. Reprod. Fertil. Dev. 1990; 2:641-650

Liu DY, Clarke GN and Baker HWG: Relationship between sperm motility assessed with the Hamilton-Thorn analyser and fertilization rates in vitro. J. Androl. 1991; 12:231-239

Liu DY and Baker HWG: Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro. J. Reprod. Fertil. 1992; 94:71-84 219

220

Liu DY and Baker HWG: A new test for the assessment of sperm-zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro. Hum. Reprod. 1994a; 9:489-496

Liu DY and Baker HWG: Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro. Hum. Reprod. 1994b; 9:673-679

Lopo AC: Sperm-egg interactions in vertebrates. In: Mechanism and control of animal fertilization. Ed:Hartman JF. 1983; pp. 269-324. Academic Press, New York

Loughlin KR and Agarwal A: Use of theophylline to enhance sperm function. Arch. Androl. 1992; 28:99-103

Lunsford RD, Jenkins NA, Kozak CA, Liang LF, Silan CM, Copeland NG and Dean J: Genomic mapping of murine Zp-2 and Zp-3, two oocyte-specific loci encoding zona pellucida proteins. Genomics 1990; 6:184-187

Mack SO, Tash JS and Wolf DP: Effect of measurement conditions on quantification of hyperactivated human sperm subpopulations by digital image analysis. Biol. Reprod. 1989; 40:1162-1169

220

221 MacLeod J: The male factor in fertility and infertility: An analysis of ejaculate volume in 800 fertile men and in 600 men in infertile marriage. Fertil. Steril. 1950; 1:347-361

MacLeod J: Semen quality in one thousand men of known fertility and in eight hundred cases of infertile marriage. Fertil. Steril. 1951; 2:115-139

Macomber D and Sanders MB: The spermatozoa count: Its value in the diagnosis, prognosis and treatment of sterility. N. Engl. J. Med. 1929; 200:981-984

Makler A: Use of the elaborated multiple exposure photography method (MEP) in routine sperm motility analysis and for research purposes. Fertil. Steril. 1980a; 33:160-166

Makler A, Makler E, Itzkovitz J and Brandes J: Factors affecting sperm motility. IV. Incubation of human semen with caffeine, kallikrein, and other metabolically active compounds. Fertil. Steril. 1980b; 33:624-630

Mann T: The biochemistry of semen and of the male reproductive tract. John Wiley & Sons, New York. 1964; pp. 17-36

Mathur S: A computerized sperm motion analysis. Fertil. Steril. 1986; 46:484-488

221

222 Matsumoto AM and Bremner WJ: Endocrinology of the hypothalamic-pituitarytesticular axis with particular reference to the hormonal control of spermatogenesis. Bailliere's Clin. Endocrinol. Metab. 1987; 1:71-95

Mattner PE: The distribution of spermatozoa and leucocytes in the female genital tract in goats and cattle. J. Reprod. Fertil. 1968; 17:253-261

Mbizvo MT, Thomas S, Fulgham DL and Alexander NJ: Serum hormone levels affect sperm function. Fertil. Steril. 1990; 54:113-120

Mbizvo MT, Johnston RC and Baker GHW: The effect of the motility stimulants, caffeine, pentoxifylline, and 2-deoxyadenosine on hyperactivation of cryopreserved human sperm. Fertil.Steril. 1993; 59:1112-1117

McLaughlin EA, Ford WCL and Hull MGR: Effects of cryopreservation on the human sperm acrosome and its response to A23187. J. Reprod. Fertil. 1993; 99:71-76

McRorie RA and Williams WL: Biochemistry of mammalian fertilization. Ann. Rev. Biochem. 1974; 43:777-803

Meizel S: The importance of hydrolytic enzymes to an exocytosis event, the mammalian sperm acrosome reaction. Biol. Rev. 1984; 59:125-157

222

223 Meizel S: Molecules that initiate or help stimulate the acrosome reaction by their interaction with the mammalian sperm surface. Am. J. Anat. 1985; 174:285-302

Meizel S, Pillai MC, Diaz-Perez E and Thomas P: Initiation of the human sperm acrosome reaction by components of human follicular fluid and cumulus secretions including steroids. In:Fertilization in mammals. Ed: Bavister BD, Cummins J and Roladan ERS. Serono Symposia, USA. 1990; pp. 205-238

Mendoza C, Carreras A, Moos J and Tesarik J: Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J. Reprod. Fertil. 1992; 95:755-763

Menning BE: The emotional needs of infertile couples. Fertil. Steril. 1980; 34:313319

Milligan MP, Harris S and Dennis KJ: Comparison of sperm velocity in fertile and infertile groups as measured by time-lapse photography. Fertil. Steril. 1980; 34:509511

Misurale F, Cagnazzo G and Storace A: Asthenozoospermia and its treatment with HCG. Fert. Steril. 1969; 20:650-653

Mohler W, Bletz I and Reiser M: Die Struktur von Ausscheidungsprodukten des 1Hexyl-3, 7-dimethyxanthins. Archiv. der Pharmazie. 1966; 5:448-456 223

224

Moohan JM, Winston RML, and Lindsay KS: Variability of human sperm response to immediate and prolonged exposure to pentoxifylline. Hum. Reprod. 1993; 8:16961700

Moore HDM: Physiological and in vitro models of sperm maturation. In: Proceedings of the Serono Clinical Colloquia on reproduction; Ed: Crosignani PG and Rubin BL. 1983; pp. 9-38

Morales P, Katz DF, Overstreet JW, Samuels SJ and Chang RJ: The relationship between the motility and morphology of spermatozoa in human semen. J. Androl. 1988; 9:241-247

Mori K, Daitoh T, Kamada M, Maeda N, Maegawa M, Hirano K, Irahara M and Aono T: Blocking of human fertilization by carbohydrates. Hum. Reprod. 1993; 8:17291732

Mortimer D, Curtis EF and Miller RG: Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon. J. Reprod. Fertil. 1987; 81:127-135

Mortimer D and Camenzind AR: The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro. Hum. Reprod. 1989; 4:169-174

224

225 Mortimer ST and Mortimer D: Kinematics of human spermatozoa incubated under capacitating conditions. J. Androl. 1990; 11:195-203

Mortimer D, Curtis EF and Camenzind AR: Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa. Hum. Reprod. 1990; 5:99-103

Moussa MM: Caffeine and sperm motility. Fertil. Steril. 1983; 39:845-845

Muggleton-Harris AL, Findlay I and Whittingham DG: Improvement of the culture conditions for the development of human preimplantation embryos. Hum. Reprod. 1990; 5:217-220

Muller R, and

Lehrach F: Haemorheology and cerebrovascular disease:

multifunctional approach with pentoxifylline. Curr. Med. Res. Opin. 1981; 7:253263

Oehninger S, Acosta AA, Morshedi M, Veek L, Swanson RJ, Simmons K and Rosenwaks Z: Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities. Fertil. Steril. 1988; 50:283-287

Oehninger S, and

Alexander NJ: Male infertility: the focus shifts to sperm

manipulation. Curr. Opin. Obst. Gyn. 1991; 3:182-190

225

226 Oehninger S, Stecker JF, Acosta AA: Male infertility: the impact of assisted reproductive technologies. Curr. Opin. Obst. Gynecol 1992; 4:185-196

Olds-Clarke P, Baer HM and Gerber WL: Human sperm motion analysis by automatic (Hamilton-Thorn Motility Analyzer) and manual (Image-80) Digitization Systems. J. Androl. 1990; 11:52-58

Oliphant G, Reynolds AB and Thomas TS: Sperm surface components involved in the control of the acrosome reaction. Am. J. Anat. 1985; 174:269-283

Osborn JC and Moor RM: Oocyte maturation and developmental competence. In:In vitro Fertilization and Donor Insemination; Proceedings of the 12th study group of the Royal College of Obstetricians and Gynaecologist. Ed: Thompson W, Joyce DN and Newton JR. 1984; pp. 275-283

Overstreet JW, Katz DF and Hanson FW: A simple inexpensive method for objective assessment of human sperm movement characteristics. Fertil. Steril. 1979; 31:162172

Overstreet JW and Cross NL: The biology of human sperm-zona pellucida interaction. Annal. N.Y. Acad. Sci. 1988; 541:337-345

226

227 Pang SC, Williams DB, Huang T, Wang C: Effects of pentoxifylline on sperm motility and hyperactivated motility in vitro :a preliminary report. Fertil. Steril. 1993; 59:465467.

Parinaud J, Labal B, Vietez, Richoilley G and Grandjean H: Comparison between fluorescent peanut agglutinin lectin and GB24 antibody techniques for the assessment of acrosomal status. Hum. Reprod. 1993; 8:1685-1688

Parrish JJ and Foote RH: Fertility differences among male rabbits determined by heterospermic insemination of fluorochrome-labelled spermatozoa. Biol. Reprod. 1985; 33:940-945

Pedigo NG, Vernon MW and Curry TE: Characterization of a computerized semen analysis system. Fertil. Steril. 1989; 52:659-981

Perreault SD and Rogers BJ: Capacitation pattern of human spermatozoa. Fertil. Steril. 1982a; 38:258-260

Perreault SD, and Rogers BJ: Relationship between fertilizing ability and cAMP in human spermatozoa. J. Androl. 1982b; 3:396-401

Pickering SJ, Fleming TP, Braude PR, Bolton VN and Gresham GAG: Are human spermatozoa separated on a percoll density gradient safe for therapeutic use? Fertil. Steril. 1989; 51:1024-1029 227

228

Piko L: Gamete structure and sperm entry in mammals. In: Fertilization. Vol. 2 Ed: Metz C and Monroy A. Academic Press, New York. 1969; pp. 325-403

Plachot M and Mandelbaum J: Oocyte maturation, fertilization and embryonic growth in vitro. Brit. Med. Bull. 1990; 46:675-694

Quinn P, Warnes GM, Kerin JF and Kirby C: Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann. N.Y. Acad. Sci. 1985; 442:195203

Rees JM, Ford WCL, Hull MGR: Effect of caffeine and of pentoxifylline on the motility and metabolism of human spermatozoa. J. Reprod. Fertil. 1990; 90:147-156

Revelli A, Modotti M, Piffaretti-Yanez A, Massobrio M and Balerna M: Steroid receptors in human spermatozoa. Hum. Reprod. 1994; 9:760-766

Ricker DD, Minhas BS, Kumar R, Robertson JL and Dodson MG: The effects of platelet-activating factor on the motility of human spermatozoa. Fertil. Steril. 1989; 52:655-658

Robaire B and Hermo L: Efferent ducts, epididymis, and vas deferens: structure, functions and their regulation. In: The physiology of reproduction. Vol. 1 Ed: Knobil E and Neill JD. Raven Press, New York. 1988; pp. 999-1080 228

229

Roberts KP and Zirkin BR: Androgen regulation of spermatogenesis in rat. Ann. NY. Acad. Sci. 1991; 637:90-107

Robertson L, Wolf DP and Tash JS: Temporal changes in motility parameters related to acrosomal status: Identification and characterization of populations of hyperactivated human sperm. Biol. Reprod. 1988; 39:797-805

Rodger JC and Bedford JM: Separation of sperm pairs and sperm-egg interaction in the opossum, Didelphis virginiana. J. Reprod. Fertil. 1982; 64:171-179

Rogers BJ and Perreault SD: Importance of glycolysable substrates for in vitro capacitation of human spermatozoa. Biol. Reprod. 1990; 43:1064-1069

Roldan ERS and Harrison RAP: Molecular mechanism leading to exocytosis during the sperm acrosome reaction. In:Fertilization in mammals. Ed: Bavister BD, Cummins J and Roladan ERS. Serono Symposia, USA. 1990; pp. 179-196

Sathananthan AH, Trounson AO, Wood C and Leeton JF: Ultrastructural observation on the penetration of human sperm into the zona pellucida to the human egg in vitro. J. Androl. 1982; 3:356-364

Satir P: Basis of flagellar motility in spermatozoa: Current status. In: The spermatozoon - maturation, motility, surface properties and comparative aspects. 229

230 Ed:Fawcett DW and Bedford JM. Urban & Schwarzenberg, Baltimore. 1979; pp. 81-90

Sato H and Shill WB: Temperature dependent effects of the components of kallikrein-kinin system on sperm motility in vitro. Fertil. Steril. 1987; 47:684-688

Schill WB: Kinin releasing pancreatic proteinase kallikrein. In:Treatment of Male Infertility. Ed: Bain J, Schill WB, and Schuwarzenstein L. Springer-Verlag, Berlin. 1982a; pp. 125-130

Schill WB: Pharmacological therapy of male infertility of non-infectious etiology. Fertilitas. 1982b; 1:23-32

Scott TW: Lipid metabolism of spermatozoa. J. Reprod. Fertil. 1973; 18:65-76

Selley ML, Lacey MJ, Bartlett MR, Copeland CM and Ardlie NG: Content of significant amounts of a cytotoxic end-product of lipid peroxidation in human semen. J. Reprod. Fertil. 1991; 92:291-298

Shabanowitz RB and O'Rand MG: Molecular changes in the human zona pellucida associated with fertilization and human sperm-zona interactions. Ann. N.Y. Acad. Sci. 1988a; 541:621-632

230

231 Shabanowitz RB and O'Rand MG: Characterization of the human zona pellucida from fertilized and unfertilized eggs. J. Reprod. Fertil. 1988b; 82:151-161

Shen MR, Chiang PH, Yang RC, Hong CV and Chen SS: Pentoxifylline stimulates human sperm motility both in vitro and after oral therapy. Br. J. Clin. Pharmacol. 1991; 31:711-714

Sikka SC and Hellstrom WJG: Functional evaluation and motility parameters of Pentoxifylline-stimulated cryopreserved human sperm. ARTA. 1990; 1:309-319

Sikka SC, and Hellstrom WJG: The application of pentoxifylline in the stimulation of sperm motion in man undergoing electroejaculation. J. Androl 1991; 12:165-170

Skakkebaek NE, Giwercman A and de Kretser D: Pathogenesis and management of male infertility. Lancet 1994; 343:1473-1479

Sokal RR and Rohlf FJ: Biometry. In:The normal probability distribution. WH Freeman and Company, NY. 2nd Edition 1969; pp. 98-126

Sokol RZ, Swerdloff RS, McClure RD and Peterson M: Gonadotropin therapy failure secondary to HCG induced antibodies. Proceedings of the International Congress of Endocrinology, Melbourne, Australia. 1980; Abstr. No. 484, pp. 451

231

232 Sokoloski JE, Blasco L and Storey BT: Turbidemetric analysis of human sperm motility. Fertil. Steril. 1977; 28:1337-1341

Stambaugh R and Smith M: Sperm proteinase release during fertilization. J. Exp. Zool. 1976; 197:121-125

Stefanovich V: Effect of 3,7-dimethyl-1-(5-oxohexyl)-xanthin and 1-hexyl-3,7dimethyl-xanthine on cyclic AMP phosphodiesterase of the human umbilical cord vessels. Res. Commun. Chem. Pathol. Pharmacol. 1973; 5:655-662

Stefanovich

V:

The

biochemical

mechanism

of

action

of

pentoxifylline.

Pharmatherapeutica 2. 1978; 1:5-16

Stock CE: An ultrastructural study of the human sperm acrosome reaction. PhD thesis; University of London. 1990

Suarez SS, Wolf DP and Meizel S: Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res. 1986; 14:107-121

Suarez SS, Drost M, Redfern K and Gottlieb W: Sperm motility in the oviduct. In: Fertilization in mammals. Ed: Bavister BD, Cummins J and Roldan ERS. Serono Symposia, USA. 1990; pp. 111-124

232

233 Szollosi D: Oocyte Maturation. In: Reproduction in Mammal and Man. Ed: Thibault C, Levasseur MC and Hunter RHF. Ellipses, Paris. 1993 pp. 327-347

Talbot P and Chacon RS: A triple-stain technique for evaluating normal acrosome reactions of human sperm. J. Exp. Zool. 1981; 215:201-208

Tasdemir M,

Tasdemir I, Kodama H and Tanaka T: Pentoxifylline - enhanced

acrosome reaction correlates with fertilization in vitro. Hum. Reprod. 1993; 8:21022107

Tash JS and Means AR: Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium. Biol. Reprod. 1982; 26:745-763

Tash JS and Means AR: Cyclic adenosine 3', 5' monophosphate, calcium and protein phosphorylation in flagellar motility. Biol. Reprod. 1983; 28:75-104

Templeton A, Fraser C, and Thompson B: The epidemiology of infertility in Aberdeen. BMJ 1990; 301:148-152

Tesarik J and Flechon J-E: Distribution of sterols and anionic lipids in human sperm plasma membrane: effects of in vitro capacitation. J. Ultrastruct. Mol. Struct. 1986; 97:227-237

233

234 Tesarik J, Pilka L and Travnik P: Zona pellucida resistance to sperm penetration before the completion of human oocyte maturation. J. Reprod. Fertil. 1988; 83:487495

Tesarik J: Appropriate timing of the acrosome reaction is a major requirement for the fertilizing spermatozoon. Hum. Reprod. 1989; 4:957-961

Tesarik J and Testart J: Human sperm-egg interactions and their disorders: implications in the management of infertility. Hum. Reprod. 1989; 4:729-741

Tesarik J, Thebault A, and Testart J: Effect of pentoxifylline on sperm movement characteristics in normozoospermic and asthenozoospermic specimens. Hum. Reprod. 1992a; 7:1257-1263.

Tesarik J, Mendoza C and Carreras A: Effects of

phosphodiesterase inhibitors

caffeine and pentoxifylline on spontaneous and stimulus-induced acrosome reactions in human sperm. Fertil. Steril. 1992b; 58:1185-1190

Tesarik J and Mendoza C: Sperm treatment with pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency. Fertil. Steril. 1993; 60:141-148

Trnell J, Billig H and Hillensj: Regulation of oocyte maturation by changes in ovarian levels of cyclic nucleotides. Hum. Reprod. 1991; 6:411-422 234

235

Tournaye H, Janssens R, Camus M, Staessen C, Devroey P and Steirteghem AV: Pentoxifylline is not useful in enhancing sperm function in cases with previous in vitro fertilization failure. Fertil. Steril. 1993a; 59:210-215

Tournaye H, Linden MVD, Abbeel EVD, Devroey P and Steirteghem AV: Effects of pentoxifylline on in-vitro development of preimplantation mouse embryos. Hum. Reprod. 1993b; 8:1475-1480

Tournaye H, van Steirteghem AC and Devroey P: Pentoxifylline in idiopathic malefactor infertility: a review of its therapeutic efficacy after oral administration. Hum. Reprod. 1994a; 9:996-1000

Tournaye H, Janssens R, Verheyen G, Camus M, Devroey P and Steirteghem AV: An indiscriminate use of pentoxifylline does not improve in-vitro fertilization in poor fertilizers. Hum. Reprod. 1994b; 9:1289-1292

Traub AI, Earnshaw JC, Brannigan PD and Thompson W: A critical assessment of the response to caffeine of human sperm motility. Fertil. Steril. 1982; 37:436-437

Troll J, and Goldzieher JW: Quantitative evaluation of the motility of spermatozoa. Fertil. Steril. 1962; 13:72-83

235

236 Uhler ML, Leung A, Chan SYW and Wang C: Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil. Steril. 1992; 58:1191-1198

van Duin M, Polman JE, Verkoelen CC, Bunschoten H, Meyerink JH, Olijve W and Aitken RJ: Cloning and characterization of the human sperm receptor ligand ZP3: evidence for a second polymorphic allele with a different frequency in the Caucasian and Japanese populations. Genomics. 1992; 14:1064-1070

Vermeulen A and Comhaire: Hormonal effects of an antiestrogen, Tamoxifen, in normal and oligospermic men. Fertil. Steril. 1978; 29:320-327

Ward A, and Clissold SP: Pentoxifylline: A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic efficacy. Drugs 1987; 34:50-97

Wassarman P: Oogenesis: Synthetic events in the developing mammalian egg. In: Mechanism and control of animal fertilization. Ed: Hartmann J. Academic Press, New York. 1983; pp. 1-54

Wassarman PM: The biology and chemistry of fertilization. Science 1987; 235:553560

Wassarman PM: Zona pellucida glycoproteins. Annu. Rev. Cell Biol. 1988a; 57:415442 236

237

Wassarman PM: The mammalian ovum. In: The physiology of reproduction. Vol. 1 Ed: Knobil E and Neill JD. Raven Press, New York. 1988b; pp. 69-102

Wassarman PM: Fertilization in mammals. Scientific American 1988c; Dec.:52-58

Wassarman PM: Profile of a mammalian sperm receptor. Development 1990; 108:117

Weese DL, Peaster ML, Himsl KK, Leach GE, Lad PM and Zimmern PE: Stimulated reactive oxygen species generation in the spermatozoa of infertile men. J. Urol. 1993; 149:64-67

White DR, Phillips DM and Bedford JM: Factors affecting the acrosome reaction in human spermatozoa. J. Reprod. Fertil. 1990; 90:71-80

WHO laboratory manual for the examination of human semen and sperm -cervical mucus interaction. Cambridge University Press. Cambridge. 2nd ed. 1987

WHO laboratory manual for the examination of human semen and sperm -cervical mucus interaction. Cambridge University Press. Cambridge. 3rd ed. 1992

237

238 Wolf DP and Hamada M:Induction of zonal and egg plasma membrane blocks to sperm penetration in mouse eggs with cortical granule exudate. Biol. Reprod. 1977; 17:350-354

Wolf DP: Acrosomal status quantitation in human sperm. Am. J. Reprod. Immunol. 1989; 20:106-113

Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fertil. 1970; 23:193-196

Yanagimachi R: Mechanism of fertilization in mammals. In:Fertilization and Embryonic development in vitro. Ed: Mastroianni L and Briggers JD. Plenum Press, New York. 1981; pp. 82-182.

Yanagimachi R: Mammalian fertilization. In: The physiology of reproduction. Vol. 1 Ed: Knobil E and Neill JD. Raven Press, New York. 1988; pp. 134-186

Yanagimachi R: Mammalian fertilization. In: The physiology of reproduction. Vol. 1 Ed: Knobil E and Neill JD. Raven Press, New York. 1994; pp. 189-318

Yovich JM, Edirisinghe WR, Cummins JM, and Yovich JL: Influence of pentoxifylline in severe male factor infertility. Fertil. Steril. 1990; 53:715-722

238

239 Zamboni L: Sperm structure and its relevance to infertility. Arch. Pathol. Lab. 1992; 116:325-343

Zaneveld LJD, Anderson RA, Mack SR and De Jonge CJ: Mechanism and control of the human sperm acrosome reaction. Hum. Reprod. 1993; 8:2006-2008

Zhang J, Boyle MS, Smith CA and Moore HD: Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs. Mol. Reprod. Dev. 1990; 27:152-158

Zinaman M, Drobnis EZ, Morales P, Brazil C, Kiel M, Cross NL, Hanson FW and Overstreet JW: The physiology of sperm recovered from the human cervix: Acrosomal status and response to inducers of the acrosome reaction. Biol. Reprod. 1989; 41:790-797

Zorgniotti AW, Hotchkiss RS and Wall LC: High-speed cinephotomicrography of human spermatozoa. Med. Radiog. Photog. 1958; 34:44-49

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Data from this thesis was used to publish the following papers:-

1. Actions of pentoxifylline directly on semen

2. The paradoxical effects of pentoxifylline on the binding of Spermatozoa to the human zona pellucida

3. Factors affecting pentoxifylline stimulation of sperm kinematics in suspensions.

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