FERTILITY AND STERILITY
2, February 1985
Copyright © 1985 The American Fertility Society
Sperm capacitation in the human female reproductive tract*
Hovey Lambert, Ph,D, t
James W. Overstreet, M.D., Ph.D.t:!:
Patricio Morales, M.S. t
Frederick W. Hanson, M.D.t
Ryuzo Yanagimachi, Ph.D.§
School of Medicine, University of California, Davis, California, and University of Hawaii
School of Medicine, Honolulu, Hawaii
In order to fertilize an oocyte, human sperma-
tozoa must first undergo capacitation. Capacita-
tion is thought to involve a number of intracellu-
lar changes as well as alterations of sperm sur-
face, but these events are not completely under-
stood. I The process of human sperm capacitation
is accomplished relatively easily in vitro by re-
moval of spermatozoa from the seminal plasma
and incubation of the washed sperm cells in cul-
ture media. 2 Sperm capacitation in the female
reproductive tract may occur during a similar se-
quence of events, but in vivo the cellular and
extracellular mechanisms may be different. For
example, capacitation in vivo and in vitro appear
to be initiated by removal of seminal plasma con-
stituents from the sperm surface. This step, which
is accomplished in the laboratory by dilution and
centrifugation of the semen, may take place in
vivo as the sperm migrate through the cervical
mucus. 3 ,4
Since in vitro fertilization and embryo transfer
have been successful in achieving pregnancy for
Received July 27, 1984; revised and accepted October 3,
1984.
*Supported by the National Institutes of Health grants HD-
15149 and HD-03402.
tDepartment of Obstetrics and Gynecology, School of Medi-
cine, University of California, Davis.
*Reprint requests: James W. Overstreet, M.D., Ph.D., Divi-
sion of Reproductive Biology and Medicine, School of Medi-
cine, University of California, Davis, California 95616.
§Department of Anatomy and Reproductive Biology, John
A. Burns School of Medicine, University of Hawaii.
Vol. 43, No.2, February 1985
Vol. 43, No.
Prinl<!d in U.s.A.
couples with infertility due to factors other than
obstructive tubal disease,5 it seems likely that
pathophysiology of sperm transport and/or capac-
itation in the female may underlie many cases of
human infertility. To understand the etiology of
these disorders and provide rational therapy in
the future, it will be necessary to study more in-
tensively the biology of human sperm interaction
with the female reproductive tract. In this com-
munication, we report experiments which demon-
strate that human spermatozoa recovered from
the female tract have achieved and maintained
an advanced state of capacitation in vivo. We also
report the results of in vitro experiments which
suggest that this process may be accomplished
during interaction between spermatozoa and cer-
vical mucus.
MATERIALS AND METHODS
Cervical mucus was collected from women who
were patients in our program for artificial insem-
ination by donor (AID). The mucus was collected
48 to 56 hours after AID by aspiration into a
polyethylene catheter inserted 1 to 2 cm into the
cervical canal. Mucus was expelled directly into 5
ml of recovery medium, and the medium was in-
cubated for 1 hour at 37°C, which allowed time for
sperm migration into the medium. The resulting
sperm suspension was concentrated in a volume
of 30 j.Ll by centrifugation. The methods for sperm
recovery from cervical mucus have been previous-
ly reported in detail. 6 The sperm suspension was
coincubated at 37°C in 5% C0 2 /95% air with cryo-
Lambert et al. Communications-in-brief 325
Table 1. Sperm Penetration of the Human Zona Pellucida and
Zona-Free Hamster Oocyte During 1 Hour of Coincubat ion in
BWW Containing 3 mglml of Bovine Serum Albumina
Penetration of human Penetration of zona-
Source of_ _ _zo_n_a-'p:....e_ll--;:u-;-cl_·d_a_ _ _ _fr_e_e_h_am_ste---,.,r,...oo_c..:.y_te_
sperm No. pene-
No. oocytes trated b No. oocytes
Cervix 24 17 53
Semen 18 o 47
aSperm were recovered either from cervical mucus 48 to 56
hours after AID or from fresh semen.
bOne or more sperm in the perivitelline space.
cOne or more decondensing sperm heads with associated
tails.
preserved immature human oocytes (to test for
penetration of the zona pel1ucida) and/or zona-
free hamster oocytes. 6 In one set of six replicate
experiments, the Biggers, Whitten and Whitting-
ham (BWW) medium containing 3 mg/ml of bo-
vine serum albumin was used as the recovery
medium. Sperm and oocytes were coincubated for
1 hour. In a second set of six replicate experi-
ments, the recovery medium was albumin-free
phosphate-buffered saline (PBS; pH 7.4), and the
coincubation time was 4 hours.
In control experiments, spermatozoa were re-
covered from semen by layering a 0.5 ml aliquot
of the ejaculate beneath 1 ml of either BWW or
PBS. The spermatozoa were allowed 1 hour at
37°C to "swim up" into the medium, and the
sperm suspension was washed twice by centrifu-
gation and dilution with the recovery medium. 4
The final sperm suspension was adjusted to 1 x
107 motile sperm/ml and was coincubated with
human and hamster oocytes under the same con-
ditions as for the cervical sperm suspension (i.e., 1
hour for BWW and 4 hours for PBS). To deter-
mine whether incubation of cervical mucus in re-
covery medium could alter the capacitation po-
tential of the medium, six additional control ex-
periments were carried out after an initial 1 hour
of preincubation of the medium (BWW in three
experiments, PBS in three experiments) with -
120 ,...,1 of sperm-free cervical mucus at 37°C. Fol-
lowing removal of the residual mucus, washed
spermatozoa were resuspended in these "mucus-
supplemented" media to a final concentration of 1
x 107 motile sperm/ml. Sperm and human 00-
cytes (twooocytes per experiment) were coincu-
bated for 1 hour or 4 hours.in BWW and PBS,
respecti vely.
In seven other experiments, - :15 ,...,1 of sperm-
free cervical mucus was drawn into a flat capil-
326 Lambert et al. Communications-in-brief
lary tube adjacent to PBS (- 15 ,...,1) containing
immature human oocytes. Mineral oil was used to
seal each end of the tube, and - 5 ,...,1 of semen was
injected at the oil/mucus interface. 3 The tube was
No. pene-
incubated for 4 hours at 37°C, during which time
tratedC spermatozoa migrated through the 2 cm mucus
22 column and into the adjacent PBS containing the
o oocytes.
Sperm penetration of the human zona pellucida
and sperm fusion with the zona-free hamster oo-
cyte were assessed by phase-contrast microscopy.
The ultrastructural morphology of spermatozoa
in the zona pellucida was assessed by transmis-
sion electron microscopy.7
RESULTS
The cervical sperm populations tested in these
experiments ranged in concentration from 1 X
105 to 1 X 106 sperm/ml and were virtually 100%
motile. The quality of sperm movement in BWW
was consistently better than in PBS, but there
was no apparent difference in the quality of
sperm movement in the cervical sperm popula-
tions in comparison with the seminal sperm popu-
lations suspended in the same media.
The incidence of penetration by cervical sperm
and seminal sperm into the human zona pellucida
and zona-free hamster oocytes following 1 hour of
coincubation in BWW is shown in Table 1 and the
comparable results following 4 hours of coincuba-
tion in PBS are given in Table 2. In the control
experiments in which BWW and PBS were prein-
cubated with sperm-free cervical mucus, none of
12 human zonae pellucidae was penetrated. How-
ever, in experiments in which spermatozoa were
allowed to swim through a column of cervical mu-
cus into PBS, three of ten human oocytes had
sperm in the perivitelline space. Ultrastructural
Table 2. Sperm Penetration of the Human Zona Pellucida and
Zona-Free Hamster Oocyte During 4 Hours of Coincubation in
Unsupplemented PBS (pH 7.4r
Penetration of human Penetration of zona-
Source of_ _ _zo_n_a...:p_e_llu7;c,...id_a_ _ _ _fr_e_e_h_am_s_te--;:r-;-o_o-,cy:....te_
sperm No. pene- No. pene-
No. oocytes trated b No. oocytes tratedC
Cervix 20 8 55 3
Semen 16 o 69 o
aSperm were recovered either from cervical mucus 48 to 56
hours after AID or from fresh semen.
bOne or more sperm in the perivitelline space.
COne or more decondensing sperm heads with associated
tails.
Fertility and Sterility
observations of spermatozoa in the zona pellucida
following gamete interaction in BWW and PBS
confirmed that the acrosome reaction had oc-
curred. The human oocytes were always nonvi-
able at the beginning of the experiments, and
fertilization in vitro never occurred.
DISCUSSION
Human spermatozoa can be capacitated in vitro
with relative ease, and any experiment designed
to demonstrate in vivo capacitation under in vitro
conditions must be interpreted with caution.
Sperm capacitation and the acrosome reaction are
prerequisites for entry into the zona pellucida and
for fusion with the oolemma.! Therefore, sperm
penetration of the human zona pellucida and/or
incorporation into the zona-free hamster oocyte is
evidence for completion of both processes. In these
experiments, ultrastructural assessment of sper-
matozoa in the thickness of the zona pellucida
confirmed that these cells had undergone the ac-
rosome reaction.
When recovered from the native cervical mucus
into the BWW, spermatozoa were able to pene-
trate the human zona pellucida and to fuse with
zona-free hamster oocytes within 1 hour of coin-
cubation, whereas spermatozoa recovered from
semen were unable to penetrate the oocytes under
the same conditions. Sperm motility in the two
preparations was similar, and it is likely that the
difference in penetration rates is a reflection of a
previously completed (or more advanced) state of
capacitation achieved by the cervical sperm popu-
lation before addition of the oocytes. This inter-
pretation is supported by the results of experi-
ments involving sperm recovery in albumin-free
PBS, a medium which does not support capacita-
tion of human spermatozoa in vitro. Although 4
hours of sperm/oocyte coincubation were allowed
in these experiments, only sperm recovered from
the cervical mucus were able to penetrate either
oocyte. As might be expected, the rates of pene-
tration into the human zona pellucida and the
zona-free hamster oocyte were lower in PBS than
in BWW. The lower rate of zona penetration may
be related to the more sluggish motility of the
sperm cells in PBS. Nevertheless, the motility of
cervical sperm and seminal sperm was similar in
PBS; so this does not explain the inability of sem-
inal sperm to penetrate the zona pellucida in
PBS. The low levels of sperm fusion with the
hamster oocyte may be the result of an effect of
the PBS on the hamster oolemma, although this
Vol. 43, No.2, February 1985
possibility was not tested directly in these experi-
ments.
When BWW and PBS were preincubated with
cervical mucus, the zona pellucida was not pene-
trated by spermatozoa in the media, suggesting
that capacitation of cervical sperm resulted from
direct interaction with the mucus rather than
augmentation of the media by the mucus. This
hypothesis is also supported by the observation
that seminal sperm were able to penetrate the
human zona pellucida in PBS after swimming
through a column of cervical mucus in vitro.
The essential role of cervical mucus in human
sperm transport has long been recognized, 8 but
its importance in regulating the physiology of the
sperm cell may not be fully appreciated. In other
experiments, we have demonstrated that human
sperm retain the ability to penetrate the human
zona pellucida during at least 80 hours of resi-
dence in the cervical canal. 6 The present study
suggests that human spermatozoa may achieve
and maintain an advanced state of capacitation in
the cervical mucus. It seems likely that the cellu-
lar and extracellular events which mediate hu-
man sperm capacitation may differ in vivo and in
vitro.4 Further experiments of the type reported
here may elucidate these differences.
REFERENCES
1. Yanagimachi R: Mechanisms offertilization in mammals.
In Fertilization and Embryonic Development in Vitro,
Edited by L Mastroianni Jr, JD Biggers. New York,
Plenum Publishing, 1981, p 81
2. Dandekar PV, Quigley MM: Laboratory setup for human
in vitro fertilization. Fertil Steril 42:1, 1984
3. Overstreet JW, Gould JE, Katz DF, Hanson FW: In vitro
capacitation of human spermatozoa after passage through
a column of cervical mucus. Fertil Steril 34:604, 1980
4. Gould JE, Overstreet JW, Hanson FW: Interaction ofhu-
man spermatozoa with the human zona pellucida and
zona-free hamster oocyte following capacitation by expo-
sure to human cervical mucus. Gamete Res. In press
5. Mahadevan MM, Trounson AO, Leeton JF: The relation-
ship of tubal blockage, infertility of unknown cause, sus-
pected male infertility, and endometriosis to success of in
vitro fertilization and embryo transfer. Fertil Steril
40:755, 1983
6. Gould JE, Overstreet JW, Hanson FW: Assessment of
human sperm fWiction after recovery from the female
reproductive tract. BioI Reprod 31:888, 1984
7. Gould JE, Overstreet JW, Yanagimachi H, Yanagimachi
R, Katz DF, Hanson FW: What functions ofthe sperm cell
are measured by in vitro fertilization of zona-free hamster
eggs? Fertil Steril 40:344, 1983
8. Overstreet JW: Transport of gametes in the reproductive
tract of the female mammal. In Mechanism and Control of
Animal Fertilization, Edited by JF Hartmann. New York,
Academic Press, 1983, p 499
Lambert et al. Communications-in-brief 327