BIOLOGY OF REPRODUCTION 67, 13271336 (2002)

DOI 10.1095/biolreprod.102.003830

Progesterone Receptor as an Indicator of Sperm Function

Sushama Gadkar,2 C.A. Shah,2 Geetanjali Sachdeva,2 Urmila Samant,3 and C.P. Puri1,2
Institute for Research in Reproduction,2 Indian Council of Medical Research, Parel, Mumbai 400 012, India
Tata Memorial Hospital,3 Parel, Mumbai 400 012, India
ABSTRACT
Expression of progesterone receptor (PR) localization on
spermatozoa was determined in men with normal and abnormal
spermiograms. Studies were also carried out to evaluate the potential of PR as a marker of sperm function. Progesterone receptor expression on spermatozoa from men with normozoospermia (n 5 8), oligozoospermia (n 5 7), asthenozoospermia
(n 5 8), oligoasthenozoospermia (n 5 7), and teratozoospermia
(n 5 11) was analyzed using an immunocytochemical method
with monoclonal antibodies against PR, and flow cytometry using a cell-impermeable fluorescein-tagged progesterone coupled
to BSA complex (P-FITC-BSA). Both methods revealed significantly fewer (P , 0.05) PR-positive spermatozoa in men with
oligozoospermia, asthenozoospermia, oligoasthenozoospermia,
and teratozoospermia compared with men with normozoospermia, thereby suggesting that down-regulation of PR expression
in spermatozoa may be one of the causes of male infertility.
Spermatozoa from men with normozoospermia (n 5 12), oligozoospermia (n 5 12), asthenozoospermia (n 5 12), oligoasthenozoospermia (n 5 9), and teratozoospermia (n 5 10) were exposed to low osmotic conditions in the hypoosmotic swelling
(HOS) test and then analyzed for PR expression using P-FITCBSA complex. A significantly higher percentage (P , 0.05) of
spermatozoa with physiologically active plasma membrane
(HOS1) lacked PR expression (HOS1PR2) in all categories of
men with infertility, thereby suggesting that compared to the
HOS test, PR expression is a better indicator of sperm function.
Furthermore, PR expression in spermatozoa showed a strong (P
, 0.05) positive correlation with their ability to undergo an in
vitro acrosome reaction. This was observed in all study groups
(i.e., normozoospermia, r 5 0.8545; oligozoospermia, r 5
0.8711; asthenozoospermia, r 5 0.7645; oligoasthenozoospermia, r 5 0.9003; and teratozoospermia, r 5 0.8676). This suggests a potential role for PR in the events leading to the acrosome reaction in sperm.

acrosome reaction, progesterone receptor

INTRODUCTION

A reliable tool to assess the fertilizing potential of human spermatozoa is not yet available. Therefore, it is not
surprising that the causes of infertility remain undetectable
in a large percentage of men. Routine semen analyses that
include estimation of sperm concentration, motility, and
morphology have limitations as fertility indicators [13].
These analyses lack the potential to ascertain the functional
capacity of spermatozoa, and they cannot predict the possible occurrence of both in vivo and in vitro conception [4
Correspondence. FAX: 91 22 4139412, 4964853;
e-mail: vichin@vsnl.com and dirirr@vsnl.com

Received: 29 January 2002.

First decision: 15 February 2002.
Accepted: 28 May 2002.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org

6]. In the last few years, efforts have been directed toward
the development of various functional tests such as the
zona-free hamster oocyte sperm penetration test [7, 8], the
hypoosmotic swelling (HOS) test [9], and the acrosome reaction (AR) [10] to assess the sperms functional capacity.
The zona-free hamster oocyte sperm penetration test
[1113] has been developed as an indicator of the ability
of human spermatozoa to capacitate, acrosome react, and
fuse with the vitelline membrane of the oocyte [14, 15].
However, some reports have suggested that assessment of
sperm fertilizing capacity using this test does not have significant clinical benefit in diagnosing infertility [7, 16].
Dead or immotile spermatozoa also penetrate zona-free
hamster eggs under certain conditions [15, 17]. Furthermore, this assay is time-consuming, complex, and subject
to many variables such as capacitation time [18], sperm
concentration and incubation conditions [19], medium composition [15], and the age of the ovum after ovulation [20].
The HOS test can predict the fertilizing capacity of human spermatozoa [9, 21, 22]. A positive HOS test is an
indicator of the functional integrity of the sperm membrane
as judged by a swollen head and curled tail of spermatozoa
when exposed to hypo-osmotic conditions [9]. A decrease
in the number of HOS-positive forms in the ejaculates of
men with oligozoospermia, asthenozoospermia, and oligoasthenozoospermia compared with men with normozoospermia has been reported [23]. Positive correlation was
also observed between HOS and the fertilizing ability of
sperm [21, 22, 2426], however, others have not found a
significant correlation between HOS and in vitro fertilization (IVF) outcome [2730]. Thus, the clinical utility of the
HOS test in assessment of sperm function is still debatable.
The AR, which is one of the prerequisites for successful
fertilization, seems to be a more relevant parameter for assessing sperm function [2, 7, 3133]. AR is an irreversible,
exocytotic, postcapacitational event that involves fusion
and fenestration of the outer acrosomal membrane with the
plasma membrane [10]. It has been demonstrated that progesterone facilitates the AR [3437]. Studies have also
been carried out to determine whether an assessment of a
sperms responsiveness to progesterone may predict its fertilizing ability in vitro [3844]. A significant correlation has
been found between the outcome of IVF and progesteronestimulated Ca21 influx [45, 46]. A physiological role for
progesterone in AR has also been suggested by other reports demonstrating a relationship between the ability of
sperm to respond in vitro to progesterone and male infertility [4749].
The mechanism by which progesterone elicits an AR and
subsequent events probably involves its interaction with a
cell surface receptor on spermatozoa [5052]. The presence
of membrane receptors that specifically bind to progesterone has been demonstrated on human spermatozoa [51, 53,

1327

1328

GADKAR ET AL.

54], and there is evidence to suggest that blocking these

surface receptors inhibits progesterone-induced AR [55,
56]. This prompted us to investigate the possibility of an
aberration in the expression of progesterone receptor (PR)
on spermatozoa in men with an abnormal spermiogram and
whether it has any effect on sperm functions (i.e., the AR).
Studies were also conducted to compare the predictive value of PR with that of other tests such as HOS for sperm
function.
This is the first report to assess the characteristics of
HOS and positive PR in the same sperm samples. To our
knowledge, no such attempt has been made until now to
analyze the expression of a surface protein of potential
functional relevance on spermatozoa, which is categorized
on the basis of membrane activity. This is also the first
study in which PR expression in spermatozoa from fertile
and infertile groups of men was evaluated by both immunocytochemistry and flow cytometric analysis.
MATERIALS AND METHODS

Samples
Semen samples were collected by masturbation after 3648 h of sexual
abstinence from healthy and proven fertile (n 5 20) volunteers. These
fertile men had fathered a child within the last 12 mo. Semen samples
were collected from infertile (n 5 76) men attending the infertility clinic
at Wadia Maternity Hospital, Mumbai. The wives of the volunteers had
normal hormonal levels, menstrual cycle length, ovulation, and endometrial function. Informed consent was obtained from each subject before
samples were collected.
Samples were classified in accordance with World Health Organization
guidelines [6] by a trained andrologist. They were categorized either as
normozoospermic (n 5 20; count $20 million/ml, progressive motility
$50%, morphology $30% normal forms), oligozoospermic (n 5 19;
count ,20 million/ml, progressive motility $50%, morphology $30%
normal forms), asthenozoospermic (n 5 20; count $20 million/ml, progressive motility ,50%, morphology $30%), oligoasthenozoospermic (n
5 16; count ,20 million/ml, progressive motility ,50%, morphology
$30% normal forms), or teratozoospermic (n 5 21; count $20 million/
ml, progressive motility $50%, morphology ,30% normal forms).
Spermatozoa from men with normozoospermia (n 5 8), oligozoospermia (n 5 7), asthenozoospermia (n 5 8), oligoasthenozoospermia (n 5
7), and teratozoospermia (n 5 11) were analyzed for PR expression by
flow cytometric and immunocytochemical methods. Spermatozoa from
men with normozoospermia (n 5 12), oligozoospermia (n 5 12), asthenozoospermia (n 5 12), oligoasthenozoospermia (n 5 9), and teratozoospermia (n 5 10) were used in the HOS test followed by PR localization
and AR analysis.
Each ejaculate was allowed to liquefy at room temperature for 30 min,
washed twice with 13 PBS pH 7.4, centrifuged at 980 3 g for 10 min,
and assayed by all three techniques.

Collection of Follicular Fluid

Follicular fluid was obtained by aspiration of antral follicles from
women undergoing laparoscopy for IVF and embryo transfer. Follicular
fluids were pooled and centrifuged at 2800 3 g at 48C to remove cellular
debris, and progesterone content was measured by radioimmunoassay [57].
Follicular fluid was then aliquoted and stored at 2208C until used.

Preparation of Stock Solutions

Progesterone 3-(O carboxymethyl) oxime:BSA conjugate (510 mol of
progesterone per mole of BSA) labeled with fluorescein isothiocyanate
(FITC; P-FITC-BSA; Sigma, St. Louis, MO) was stripped of free progesterone using dextran-coated charcoal [58]. FITC was conjugated with BSA
according to the method described by Goding [59]. The conjugate solution
was lyophilized and reconstituted in PBS to obtain a solution of 1 mg/ml.
FITC-conjugated Pisum sativum lectin (Sigma) was dissolved in PBS at
1 mg/ml and stored frozen (2208C) until used.

Immunocytochemistry
Approximately 1 3 106 spermatozoa in 10 ml of PBS were smeared
on clean, grease-free slides, air-dried, and fixed in chilled methanol for 30
min. The slides were washed twice with 13 PBS and once with PBS-T
(0.5% Tween 20 in 13 PBS) for 10 min at room temperature. After washing, the sperm membrane was permeabilized with 0.1% sodium deoxycholate in 13 PBS at 48C for 30 min. Smears were washed with PBS-T
for 5 min, and blocked in 1% BSA in 13 PBS at room temperature for
2025 min. Slides were incubated with mouse antisera (Affinity Bioreagents, Golden, CO) against the carboxyl terminus of PR (clone PRAT4.14) diluted 1:100 in 13 PBS at 48C for 1820 h. The slides were
washed with PBS-T for 5 min and then incubated with biotin-conjugated
goat anti-mouse secondary antibody diluted 1:500 in 13 PBS for 1 h at
room temperature. The slides were washed twice with 13 PBS and incubated with avidin-biotin complex (Vectastain kit, Vector Laboratories,
Burlingame, CA) for 30 min at room temperature. The slides were then
washed twice with 13 PBS at room temperature for 30 min each, followed
by treatment with 0.05% diaminobenzidine in 13 PBS with 0.06% H2O2
for 10 min. The slides were counterstained with 1% hematoxylin for 2
min, dehydrated in ascending grades of alcohol for 10 min each, and kept
in xylene for 2 h followed by mounting in DPX. Specificity of the staining
was evaluated by running a negative control (1% BSA without antibody).
The slides were examined using the 1003 objective of a brightfield
microscope (Olympus, Tokyo, Japan) with a double-blind technique. At
least 200 spermatozoa were counted in 20 microscopic fields selected at
random. Counterstained spermatozoa showing a brown color at the head
region were counted as PR-positive. Spermatozoa that did not react with
the antibodies against PR had no brown precipitates and appeared blue
throughout due to counterstaining and were counted as PR-negative.

Direct Fluorescence
Four million sperm were incubated with 50 ml of 0.1% digitonin in
PBS at 48C for 30 min. These samples were washed twice with 13 PBS
and centrifuged at 1000 3 g for 10 min. Washed pellets were incubated
with either 0.1 mM P-FITC-BSA or FITC-BSA in 50 ml of PBS at 48C
for 1618 h. Unbound fluorescein-labeled compounds were removed by
washing with 13 PBS and centrifuged at 1000 3 g. The pellets were
suspended in 1 ml of 13 PBS. Ten microliters of each cell suspension
was processed for direct fluorescence analysis and the remaining for flow
cytometry.
Ten microliters of cell suspension was smeared on a clean, grease-free
slide, mounted, and visualized with a 1003 objective of the fluorescence
microscope (Olympus) using a triple-band filter (Cube U-MNIBA, DM505, BP-470-550, and BA-515-550). Positive staining following incubation with P-FITC-BSA resulted in a green color at the acrosomal region,
whereas negative staining as well as incubation of spermatozoa with FITCBSA resulted in a red color.

Flow Cytometric Analysis

Approximately 4 million plain or digitonin-treated spermatozoa were
incubated with P-FITC-BSA or FITC-BSA as mentioned in the previous
section. The samples were washed and suspended in 1 ml of PBS. These
samples were analyzed using a FACScan cytometer (Becton Dickinson,
San Jose, CA) equipped with a 15 mW air-cooled 488 nm argon-ion laser.
FL1 (FITC) signals were detected through a 530/30 nm bandpass filter. To
distinguish spermatozoa from debris, a dot plot distribution of spermatozoa
according to a forward angle light scatter (correlating with cell size) and
right angle light scatter (correlating with cell density) were used to determine a sperm window as described by Haas and Cunningham [60]. The
fluorescence intensity of 10 000 spermatozoa within the sperm window
was computed twice in list mode and analyzed using Lysis II software
(Becton Dickinson).
Intensity of the fluorescence of stained cells was presented in log scale
on the X-axis and number of cells showing fluorescence on the Y-axis in
flow cytograms. Channels M1, M2, and M3 represented low, moderate,
and high intensity staining, respectively. Flow cytometry data for PR positivity were compared with the data for PR positivity and evaluated with
the immunocytochemical method.

Localization of PR on Sperm Subjected to the HOS Test

The HOS test was performed on 0.1 ml of the original ejaculate mixed
with 1 ml of hypo-osmotic solution of sodium citrate and fructose (150
mOsm/L) [9]. After incubation for 40 min at 378C, 200 spermatozoa were

SPERM PROGESTERONE RECEPTOR IN FERTILE AND INFERTILE MEN

1329

FIG. 1. Detection of PR using antibodies

against conventional PR in spermatozoa
from men with normozoospermia (b), oligozoospermia (c), asthenozoospermia (d),
oligoasthenozoospermia (e), and teratozoospermia (f). Negative control (no antibody) is shown in a. Brown color indicates
positive staining for PR. Blue color appears
because of counterstaining with hematoxylin. Spermatozoa were visualized with an
3100 oil immersion objective.

observed at a magnification of 4003 with a phase contrast microscope.

The number of spermatozoa with head and tail changes typical of swelling
(positive for hypoosmotic swelling) was counted [9]. Spermatozoa subjected to HOS were pelleted by centrifugation at 1000 3 g for 5 min and
smeared on clean, dry, grease-free slides. Smears were fixed in methanol
for 10 min at 48C. Smears were treated with 0.1% digitonin at 48C for 30
min and washed three times with 13 PBS. The spermatozoa were incubated with 0.1mM P-FITC-BSA or FITC-BSA at 48C for 16 h and washed
three times with 13 PBS to remove unbound stain. The smears were then
mounted with glycerol in PBS (1:9). Stained slides were observed using
a 1003 objective of a fluorescence microscope (Olympus). Spermatozoa
subjected to HOS showing green color at the acrosomal or equatorial region were counted as PR-positive spermatozoa. PR staining was assessed
in both HOS-positive and HOS-negative spermatozoa from fertile and infertile men, respectively. This evaluation of PR positivity was made by
two different observers in a blinded manner. For quantitative assessment
of PR at least 200 spermatozoa were examined in randomly selected 20
fields.

Acrosome Reaction
Semen samples were suspended in Biggers Whitten Whittingham
(BWW) medium [61] and separated from seminal plasma by centrifugation
at 1000 3 g at room temperature. Washed sperm (12 3 106) were overlaid with 1 ml of BWW medium supplemented with 3.5% human serum
albumin (HSA-BWW) and incubated at 378C in 5% CO2 and 95% air for
6 h to allow capacitation [62]. Capacitated spermatozoa were divided into
two equal aliquots and centrifuged at 1000 3 g. The acrosome reaction
was induced by incubating the capacitated spermatozoa in 20 ml of 20%
follicular fluid in HSA-BWW for 20 min at 378C in 5% CO2 and 95% air.
In another aliquot, 20 ml of HSA-BWW instead of follicular fluid was
used to take into account the number of spermatozoa undergoing a spontaneous AR or an unstimulated AR. The sperm cells were then separated
from the medium by mild centrifugation at 1000 3 g to visualize the AR.

Visualization of Acrosomal Status

Separated sperm cells were diluted (approximately 1 3 106/ml) in
BWW medium and smeared on clean, dry, grease-free slides. The smears
were fixed with methanol for 10 min, washed with PBS, and allowed to
react with FITC-labeled Pisum sativum lectin (1 mg/ml) for 1618 h at
48C. Excess stain was removed by washing with PBS. The smears were
then mounted with glycerol in PBS (1:9) and examined under a fluorescence microscope (Olympus) using an oil immersion objective.
Two hundred spermatozoa per sample were counted to determine
whether they were intact (stained acrosomal cap) or acrosome reacted
(patchy and equatorially stained and acrosome lost). The percentage of AR
was expressed as the difference between stimulated and unstimulated
(spontaneous) acrosome reacted sperm population.

Statistical Analysis
All statistical analyses were performed using the Statistical Program
for the Social Sciences (SPSS; Chicago, IL), version 9. Percentages of PRpositive sperm evaluated by immunocytochemistry and flow cytometry
were compared with one-way ANOVA.
Percentages of HOS1PR1, HOS1PR2, HOS2PR2 and acrosomereacted sperm populations in different study groups were also compared
via one-way ANOVA. The coefficients of correlation between %HOS,
%HOS1PR1, %HOS1PR2, and %AR were determined with SPSS version 9.

RESULTS

PR Expression on Spermatozoa

Immunocytochemical localization using antibodies

against the conventional PR demonstrated localization of
PR, as evidenced by brown staining at the acrosomal region

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GADKAR ET AL.

FIG. 2. Localization of PR using P-FITCBSA on spermatozoa from men with normozoospermia (b), oligozoospermia (c),
asthenozoospermia (d), oligoasthenozoospermia (e), and teratozoospermia (f). Negative control (FITC-BSA) is shown in a.
Green color indicates positive PR and red
color indicates negative staining. Spermatozoa were visualized with an 3100 oil
immersion objective.

TABLE 1. Percentage of PR-positive spermatozoa in fertile and infertile

groups assessed by immunocytochemical localization and flow cytometry.a
Groups
Normozoospermic
(n 5 8)
Oligozoospermic
(n 5 7)
Asthenozoospermic
(n 5 8)
Oligoasthenozoospermic
(n 5 7)
Teratozoospermic
(n 5 11)

Immunocytochemical
analysis

Flow cytometric
analysis

83.99 6 1.8

83.69 6 2.04

48.7 6 4.51*

58.11 6 4.38*

33.47 6 7.47*

47.75 6 8.44*

38.82 6 10.31*

46.27 6 7.97*

61.69 6 4.35*

61.95 6 7.07*

Values are mean 6 SEM.

* P , 0.05, significance of difference when compared with normozoospermic group.

(Fig. 1). Aliquots of the samples processed for flow cytometric analysis were also subjected to direct fluorescence
using fluorescein-tagged ligand (P-FITC-BSA). This also
demonstrated a similar pattern of PR localization (i.e., at
the acrosomal region) in spermatozoa from both fertile and
infertile groups (Fig. 2).
Immunocytochemical and flow cytometric analysis revealed a significant decrease (P , 0.05) (Table 1) in the
number of PR-positive spermatozoa in infertile groups
compared with those that were fertile (Figs. 1 and 3).
PR was also localized to spermatozoa with abnormal
morphology (Fig. 4). It was interesting that the number of
PR-positive spermatozoa was higher in men with teratozoospermia than it was in men with other types of infertility. Very weak positive correlation was observed between
PR expression and the traditional semen parameters such
as motility and morphology (data not shown).
HOS Positivity in Spermatozoa

A significant decrease in the percentage of HOS-positive

spermatozoa (P , 0.05) was observed in all infertile

SPERM PROGESTERONE RECEPTOR IN FERTILE AND INFERTILE MEN

1331

groups, except in the teratozoospermic group, compared

with those that were fertile (Table 2).
PR Expression and HOS Positivity in Spermatozoa

Spermatozoa subjected to hypoosmotic conditions revealed three patterns of PR expression (Fig. 5): 1)
HOS1PR1: HOS positive spermatozoa with a functionally
active plasma membrane demonstrating PR expression at
the equatorial or acrosomal region. 2) HOS1PR2: HOSpositive spermatozoa that lacked PR expression. 3)
HOS2PR2: Spermatozoa that lacked a functionally active
membrane as well as PR expression.
A significant decrease (P , 0.05) in HOS1PR1 and a
significant increase (P , 0.05) in HOS1PR2 spermatozoa
was observed in all infertile groups compared with normozoospermic men (Table 3; Fig. 6). However, no significant change was observed in the number of HOS2PR2
spermatozoa in infertile men except in those with oligoasthenozoospermia compared with fertile men.
PR Expression and AR in Spermatozoa

Significantly fewer (P , 0.05) spermatozoa underwent

an in vitro AR in all infertile groups compared with those
in the normozoospermia group (Table 2).
A weak positive correlation was found between the percentage of HOS-positive forms and the percentage of AR
spermatozoa in all groups (normozoospermia, r 5 0.4982;
oligozoospermia, r 5 0.4958; asthenozoospermia, r 5
0.4680; oligoasthenozoospermia, r 5 0.5800), whereas a
negative correlation (r 5 20.2204) was observed in men
with teratozoospermia (Table 4). In contrast, a very strong
positive correlation was observed between the percentage
of HOS1PR1 and the percentage of AR spermatozoa in
men with normozoospermia (r 5 0.8545), oligozoospermia
(r 5 0.8711), asthenozoospermia (r 5 0.7645), oligoasthenozoospermia (r 5 0.9003), and teratozoospermia (r 5
0.8676) (Table 4). No strong positive correlation was found
between the percentage of HOS1PR2 and the percentage
of AR spermatozoa in any group (Table 4).
DISCUSSION

Culmination of the sperm-egg interaction into an event

of successful fertilization requires sperm to undergo the
AR, which is an irreversible, exocytotic, postcapacitational
event. This consists of fusion and fenestration of the outer
acrosomal membrane with the plasma membrane. AR dysfunctions have been proposed to be one of the causes of
reduced fertility in men. The assessment of AR in vitro is
thus expected to yield additional information on the fertilization potential of spermatozoa, which has not been derived from conventional semen analysis [63].
Progesterone, which is secreted by cumulus cells, has
been indicated as a physiological stimulus or costimulus
for initiating the AR in spermatozoa. It is speculated that
progesterone-induced effects (i.e., an increase in calcium
influx, hyperactive motility, zona pellucida binding, and
zona-free oocyte penetration) are mediated via distinct,
nongenomic PR [55, 56]. It is also speculated that these
sperm functions probably involve three different types of
receptorsa plasma membrane Ca21 ion channel, a GABAA receptor, and a membrane-associated protein tyrosine
kinase [64]. Our previous studies on the biochemical and
molecular characterization of these membrane-bound progesterone binding sites on human spermatozoa have re-

FIG. 3. Flow cytometric analysis to quantitate the number of PR-positive

spermatozoa in samples from men with normozoospermia (a), oligozoospermia (b), asthenozoospermia (c), oligoasthenozoospermia (d), and teratozoospermia (e). Respective controls (samples stained with FITC-BSA)
are shown in the left panel. Comparison of PR positivity for each category
was done by comparing the fluorescence in M2 channel.

vealed similarities as well as dissimilarities of these receptors with the conventional intracellular PR [54, 65]. These
studies demonstrated that these progesterone binding sites
on spermatozoa are membrane-bound, heat-labile, and localized at the acrosomal region [54]. These sites were found
to be masked in spermatozoa of ejaculates from normal
men. It has been speculated that the reshuffling of membrane components during in vivo capacitation, the AR, or
both facilitates unmasking of these progesterone binding

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GADKAR ET AL.

FIG. 4. Immunolocalization of PR on human spermatozoa with abnormal morphology. Spermatozoa with head defects (cf), midpiece defects (g and
h), and tail defects (i and j) were stained with antibodies against the conventional PR. Brown staining indicates PR positivity and blue color appears
due to counterstaining with hematoxylin. Spermatozoa with normal morphology are shown in b. Negative control (without antibody) is shown in a.
Sperm were visualized with an 3100 oil immersion objective.

sites. The present study was undertaken to investigate

whether PR expression on spermatozoa has any significance
for evaluating infertility in men.
In contrast to other reports demonstrating that only 11%
of sperm in ejaculates from fertile men bear PR [47, 66],
its expression was observed in 70%80% of spermatozoa
from fertile men in the present study. Our results are in
agreement with a report demonstrating significant responsiveness to progesterone in 93% of spermatozoa from fertile donors [67]. It can be hypothesized that either the coating of the sperm PR with proteins of testicular or epidid-

ymal origin or masking of these sites may stearically hinder

the binding of progesterone to its receptor. It is likely that
treatment of spermatozoa with mild detergents (i.e., digitonin) as carried out in this study unmasks these sites and
thereby allows detection of a higher percentage of PR-positive cells. In the present study, the functional assessments
(acrosome reaction) of live spermatozoa were conducted in
the absence of detergent because these events led to reshuffling of membrane components in spermatozoa and
probably subsequent unmasking of PR. However, for localization of PR, fixed sperm smears were treated with mild

TABLE 2. Spermatozoa showing positivity for hypoosmotic swelling test and acrosome reaction in fertile and infertile men.a
Group
Test
Positive for the
hypoosmotic
swelling test
Acrosome-reacted
a

Normozoospermic
n 5 12

Oligozoospermic
n 5 12

Asthenozoospermic
n 5 12

83.5%
(79.4487.55)

69.08%*
(55.4882.68)

67.08%*
(58.1676.0)

24.0%
(21.5326.46)

11.08%*
(7.8314.32)

11.41%*
(8.0214.8)

Oligoasthenozoospermic
n 5 09
49.55%*
(35.8163.3)
9.22%*
(4.9613.48)

Values are mean; * P , 0.05, compared with normozoospermic group. Values in parenthesis indicate range.

Teratozoospermic
n 5 10
72.1%
(64.9179.28)
17.2%*
(12.4221.97)

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TABLE 3. Percentage of spermatozoa with different patterns of PR expression detected following HOST.a
Pattern
HOST1PR1
HOST1PR2
HOST2PR2

Normozoospermic
n58
70.75 6 2.80
8.41 6 1.23
11.16 6 2.35

Oligozoospermic
n57
43.0 6 6.92*
28.0 6 5.65*
20.25 6 5.2 (NS)

Asthenozoospermic
n58
39.5 6 3.56*
27.2 6 4.0*
15.33 6 3.85 (NS)

Oligoasthenozoospermic
n57
28.22 6 4.07*
23.44 6 4.15*
26.55 6 5.75*

Teratozoospermic
n 5 11
49.20 6 4.79*
23.0 6 5.82*
14.30 6 3.21 (NS)

a Values are mean 6 SEM. HOST1, Swollen head and coiled tail (cell with functionally active plasma membrane); HOST2, straight tail (damaged
plasma membrane); PR1, stained equatorial or acrosomal region as indicated by yellow or green color; PR2, unstained equatorial or acrosomal region
as indicated by red color. NS, Not significant.
* P , 0.05, compared with the normozoospermic group.

detergent (either 0.1% digitonin or 0.01% sodium deoxycholate).

Evidence supporting a physiological role for progesterone in the AR comes from reports suggesting a relationship
between male infertility and the inability of spermatozoa to
respond to progesterone in vitro [4749]. However, these
studies were conducted on spermatozoa from selected
groups (either patients with unexplained infertility or oligozoospermia or teratozoospermia). The present study was
undertaken to analyze PR expression in spermatozoa from
men with various seminal abnormalities.
A significant decrease in the number of PR-positive spermatozoa was detected in infertile men in the present study.
To our knowledge, this is the first study in which two methods (flow cytometry and immunocytochemistry) were used
to evaluate PR expression in spermatozoa from fertile and
infertile men. Percentages of PR-positive spermatozoa using both immunocytochemical and flow cytometric methods were found to be similar. These two techniques were
used to rule out the possibilities of measuring postacrosomal fluorescence (due to diffusion of P-FITC-BSA into
dead cells) and of manual bias in counting the positive
cells, respectively. A slightly higher percentage of PR-positive spermatozoa was recorded by flow cytometry in some
infertile groups compared with that assessed by immunocytochemistry. This may arise because of the cumulative
fluorescence (postacrosomal, acrosomal, and equatorial)
measured by the flow cytometer, whereas the data collected
using immunocytochemistry took into account only those
cells that showed PR localization at the acrosomal region.
The failure to express PR by spermatozoa from infertile
men may reflect an underlying pathological mechanism.
It was intriguing to find that only 24% of spermatozoa
from normal men undergo inducible AR. This may be attributed to the possibility of incomplete unmasking of PR
on human spermatozoa or the requirement of additional inducers for AR. The other plausible reason for the observed
difference in the numbers of PR-positive spermatozoa and
in vitro acrosome-reacted spermatozoa is that plain washed
(not digitonin-treated) spermatozoa were subjected to the
AR. It is also likely that the concentration of AR stimulator
(follicular fluid in the present study) may not precisely
mimic the effects of physiological inducers. In the present
study, the use of follicular fluid was preferred over chemical inducers (i.e., calcium ionophore), because these chemical inducers are known to be cytotoxic. Although follicular
fluid has other AR-inducing components in addition to progesterone, and it would have been more appropriate to evaluate only progesterone-induced AR and rule out the contribution of other follicular fluid components to AR, follicular fluid is an AR inducer that closely mimics physiological conditions. Further, the same batch of follicular fluid
was used to stimulate the AR in spermatozoa from both
fertile and infertile groups.

In the present study, a significant decrease in the percentage of acrosome-reacted spermatozoa was observed in
men with oligozoospermia, asthenozoospermia, oligoasthenozoospermia, and teratozoospermia compared with
those with normozoospermia. These results are in agreement with the findings of Fuse et al. [40]. A decrease in
the percentage of spermatozoa with the potential to undergo
the AR in infertile groups is suggestive of aberrations in
the cascade of events leading to the AR. This may arise
because of the lower PR expression on spermatozoa in infertile men observed in this study.
The potential role of PR in the AR is also indirectly
evident by the observation of a higher percentage of both
acrosome-reacted as well as PR-positive spermatozoa in
men with teratozoospermia compared with those with oligozoospermia, asthenozoospermia, and oligoasthenozoospermia. It is interesting that no strong correlation was

FIG. 5. PR localization on spermatozoa exposed to hypoosmotic conditions. a) Double positive, HOS1PR1, coiled tail (indicative of HOS
positivity), and green color (indicative of PR positivity); b) Single positive,
HOS1PR2, coiled tail, and red color (PR negativity); c) double negative,
HOS2PR2, uncoiled tail, and red color. Sperm were visualized with an
3100 oil immersion objective.

1334

GADKAR ET AL.

FIG. 6. Detection of PR on spermatozoa

previously exposed to hypoosmotic conditions in men with normozoospermia (b),
oligozoospermia (c), asthenozoospermia
(d), oligoasthenozoospermia (e), and teratozoospermia (f). Negative control (FITCBSA) is shown in a. Green-yellow color
indicates PR positivity and red indicates
negative staining.

found between PR expression and other seminal parameters

such as motility and morphology (data not shown). In light
of these observations, it is not surprising that spermatozoa
with abnormal morphology retain their fertilization potential as evident by in vitro fertilization studies [49, 68], and
abnormal morphology is not necessarily accompanied by a
failed AR [63].
Attempts have been made in the past to use the functional activity of the plasma membrane (i.e., the HOS test)
as an indicator of sperm function [21, 22, 25, 26, 69]. However, its diagnostic usefulness is still debatable [2730]. The
ability of the sperm tail to swell in the presence of a hypoosmotic solution is an indicator of the membrane activity.
In the present study, significantly fewer HOS1 spermatozoa were found in men with oligozoospermia, astheno-

zoospermia, and oligoasthenozoospermia than in men with

normozoospermia, thereby indicating an impaired membrane integrity of spermatozoa in men with infertility. However, HOS positivity alone failed to serve as an indicator
of sperm function in men with teratozoospermia. Further,
when an attempt was made to find a correlation between
HOS positivity and AR in spermatozoa, only a weak correlation was found. Although the HOS test and the AR
require a functionally active sperm membrane, these seem
to be independent events. In contrast, a strong positive correlation was found between the percentage of HOS1PR1
and the percentage of AR sperm cells. Significantly fewer
HOS positive spermatozoa from men with normozoospermia lacked PR expression compared with those in men with
abnormal spermiograms. This probably suggests that not

TABLE 4. Coefficient of correlation between % HOS1/% HOS1PR1/% HOS1PR2 and % AR spermatozoa in fertile and infertile men.
Group
Coefficient of
correlation (r)
% HOS1 and % AR
%HOS1PR1 and % AR
%HOS1PR2 and % AR

Normozoospermic
n 5 12

Oligozoospermic
n 5 12

Asthenozoospermic
n 5 12

Oligoasthenozoospermic
n 5 09

Teratozoospermic
n 5 10

0.4982
0.8545
20.2906

0.4958
0.8711
20.5246

0.4680
0.7645
0.1912

0.5800
0.9003
0.0902

20.224
0.8676
20.8596

SPERM PROGESTERONE RECEPTOR IN FERTILE AND INFERTILE MEN

only the integrity of the plasma membrane, but that PR

expression, are both required for the AR. This is the first
study in which the expression of a surface antigen or membrane-bound molecule was analyzed in spermatozoa exposed to hypoosmotic conditions.
Evaluation of PR expression on spermatozoa may give
insight into the functional competence of sperm because
the PR expression on sperm bears a strong correlation with
its ability to undergo in vitro AR. Further studies need to
be undertaken to understand the mechanism of progesterone-induced AR through PRs. Nevertheless, the present
study demonstrates that compared to HOS, PR expression
seems to be a better predictor of sperm function. In men
with an abnormal spermiogram, a higher percentage of
HOS-positive spermatozoa lacked PR expression. Further,
the detection of PR on human spermatozoa is fast, easy to
perform, and does not require expensive equipment. It is
possible to analyze the slides, and they can be permanently
preserved and reassessed. Hence, analysis of PR expression
on spermatozoa is a useful method for evaluating sperm
function.
ACKNOWLEDGMENTS
The authors acknowledge Dr. Vandana Walvekar, the dean of Wadia
Maternity Hospital, Mumbai, for providing the semen samples. We appreciate the suggestions by Dr. Geeta Vanage and Dr. Usha Natraj (assistant
directors, Institute for Research in Reproduction, Parel, Mumbai) for immunocytochemical studies. The authors also thank Dr. M. Hansotia of the
Fertility Clinic in Mumbai for providing human follicular fluid. The authors also thank Mr. D. Balaiah (assistant director, Institute for Research
in Reproduction, Parel, Mumbai) for assisting in statistical analysis.

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