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Human Sperm Capacitation and in Vitro Fertilization*
Vol. 24, No.6, June 1973 Printed in U.S.A.
FERTILITY AND STERILITY
Copyright © 1973 by The Williams & Wilkins Co.
HUMAN SPERM CAPACITATION AND IN VITRO FERTILIZATION* PIERRE SOUPART, M.D., PH.D.,
AND
LARRY L. MORGENSTERN, M.D.t
Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Department of Obstetrics and Gynecology, United States Army Tripier General Hospital, APO San Francisco, California 96438
From observations made in some mammals, it appears that normal fertilization results only from the encounter of a mature oocyte with a capacitated spermatozoon. 1 On the basis of such observations, although limited to a few species, the need for sperm capacitation is considered to be an absolute prerequisite for mammalian fertilization. However, there is as yet no direct experimental evidence that sperm capacitation is a feature of human fertilization. The functional dependence of sperm upon the female reproductive tract prior to fertilization, which has been termed capacitation,2 was first recognized in 1951 independently by Chang 3 and by Austin.' The phenomenon was initially defined as the ability acquired by sperm in the female genital tract to penetrate through the egg investments, the matrix of the cumulus oophorus, and the zona pellucida. All sperm observed within the zona pellucida or in the perivitelline space of the ovum have lost the outer acrosomal membrane and the corresponding plasma membrane, leaving exposed the inner acrosomal membrane. This morphologic change is termed acrosome reaction, and it is assumed that the fertilizing spermatozoon has undergone this change as well. It seems important to distinguish between a "false" acrosome reaction,5 in which the outer acrosomal and sperm plasma membranes are lost sepaReceived January 4, 1973. • Supported by U.S.P.H.S. Contract NIH-70-2162 and Grant HD 03769, and by United States Army Medical Research and Development Command. t Present address: Department of Obstetrics and Gynecology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87106.
rately, and a "true" acrosome reaction,6 in which exposure of the inner acrosomal membrane involves fusion between outer acrosomal membrane and plasma membrane in an orderly vesiculation process. 6 The former is said to represent a nonspecific degenerative change, while the latter appears to be physiologic and occurs in the immediate vicinity of the ovum or within the cumulus and corona cell mass. 7 The acrosome appears to be a modified lysosome and, as such, contains hydrolytic enzymes,8 two of which are of particular interest. The first one, hyaluronidase, has long been known as being able to dissolve the matrix of the cumulus oophorus. 9 The second one, a trypsin-like enzyme,10 presently known as the acrosomal proteinase,l1 appears to act on the zona pellucida while still firmly bound to the inner acrosomal membrane. 12 The latter property would explain why localized dissolution, in the form of a sharp penetration slit, marks the passage of sperm through the zona pellucida rather than complete dissolution, as observed when the zona pellucida is treated with extracted acrosomal enzymes. 12 Since the original definition of capacitation was formulated, a trend has developed to restrict its meaning to that of some conditioning that sperm undergo in the female genital tract, which permits the occurrence of a "true" acrosome reaction in the immediate vicinity of the zona pellucida. Uncapacitated sperm, that is sperm that have not been submitted to this preliminary conditioning, cannot undergo a "true" acrosome reaction and are unable to penetrate through the zona pellucida.
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Since there is presently no demonstrated morphologic concomitant of capacitation, the fact that capacitation has occurred has to be assessed functionally. As far as human spermatozoa are concerned, such an assessment requires the use of an in vitro fertilization system. The need for capacitation of human sperm was strongly suggested when unquestionable evidence of in vitro fertilization of mature human oocytes was obtained 13, 14 under conditions provided by the modification of a culture medium which had proven effective in capacitating hamster sperm in vitro.15 In these experiments, sperm penetration through the zona pellucida of the human ovum suggested that the capacitation requirements of human sperm were similar to those of hamster sperm: namely, that such requirements could be no more than suitable environmental conditions allowing for the spontaneous occurrence of the capacitation process. 15 In experiments such as those mentioned above, the in vitro fertilization system was composed of four elements: the fertilization medium, washed ejaculated spermatozoa, oocytes, and the follicle cells that surround them. The 00cytes used in the most successful of these experiments were recovered by laparoscopic technic from follicle in situ just prior to induced ovulation. 14, 16 Ovulation was induced in the ovum donors in order to obtain oocytes having completed much of their maturation in the ovary. The ovum donors, who were volunteer women of infertile married couples, were selected on the basis of recognized fallopian tube anomalies, but their ovarian function was apparently normal. 16 It seemed reasonable to assume that not only the recovered oocytes but also their satellite follicle cells had reacted to the hormones used for inducing ovulation. Given such conditions, it was not possible to decide whether sperm penetrating ability resulted from the sole effect of the fertilization medium or whether some cellular interaction was also part of
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the process. The present study was designed to investigate the possible effect on sperm penetrating ability of hormone addition to the in vitro fertilization system. The hormones selected for this purpose were those (follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (HCG)) which are used in combination for inducing ovulation in women. The observations presented below indicate that human sperm must undergo capacitation prior to fertilization and suggest, furthermore, that follicle cells are involved in the process. MATERIALS Al'.ID METHODS
Preoperative Treatment of Oocyte Donors. Table 1 summarizes the preoperative treatment, the day of menstrual cycle, and the endometrial status of the oocyte donors at the time of oocyte recovery for the control and experimental groups. Preoperative suppression therapy is defined as the utilization of combination oral contraceptives during the cycle in which gonadotropins and/or clomiphene citrate were administered or operation was performed. Four oocyte donors received human menopausal gonadotropin (HMG, Pergonal, Cutter Laboratories, Berkeley, Calif.) in equally divided doses (75 LU. of FSH and 75 LU. of LH) on Days 1, 4, and 8 with human chorionic gonadotropin (HCG, APL. Ayerst Laboratories, New York, N.Y.) administered on Day 11. Two donors received clomiphene citrate (Clomid, Wm. S. Merrill Co., Cincinnati, Ohio) orally in doses of 50 mg./day (Patient C. J.) and 100 mg./day (Patient L. T.) on Days 1 through 5, inclusive, with HCG given on Day 13. Seven donors received only HCG the day before operation and one donor had no preoperative treatment. The dose of HCG given to all donors was 20,000 I.U., and oocyte recovery was performed 20.5-30.5 hr. after HCG administration. Oocyte Recovery and Evaluation. Oocyte recovery was performed by direct
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SOUPART AND MORGENSTERN TABLE 1. Preoperative Treatment of Oocyte Donors
Patient
Age
Suppression therapy prior to surgery'
Preoperative treatmentt
Interval HCG· oocyte recovery
Days of menstrual cycle at recovery
Condition of endometrium at recovery
No. of oocytes cultured
Control series G.G. L. S.
33 24
HMGIHCG HCG
23.5 26
19
24 34 20 34 26 24 23
None Pregnancy (5 wk.) + Ovral None None Norinyll + 80 None None None None
M.H. C.Y. B.C. M.B. J.D. E.W. K. S.
HMG/HCG HCG HCG None HMGIHCG HCG HMG/HCG
26.5 27 24.5
14 23 16 3 8 18 24
P.V.P.
34
Enovid lOmg
HCG
20.5
16
M.S. L. T.
33 32
Norinyll None
2l.5 24
A.B. C.J.
36 30
None Ovulen
B.T.
29
None
HCG Clomiphene citrate/HCG HCG Clomiphene cittrate/HCG HMGIHCG
26.5 30.5 28.5
Secretory Decidual and chorionic villi Proliferative Proliferative No data Proliferative Proliferative Proliferative Secretory
3 2 5 2 3 2 3 3
Experimental series
+ 80
16 23
Proliferative with focal glandular ectasia Proliferative Proliferative
4 2
24 26.5
24 ?
Secretory Proliferative
8 3
27.5
27
Secretory
2
* Ovral, Wyeth Laboratories, ethinyl estradiol 50 /lg. + Norgestrel 0.5 mg.; Norinyl 1 + 80, Syntex Laboratories, mestranol 80 /lg. + norethindrone 1 mg.; Enovid 10 mg., G. D. Searle Company, mestranol 150 /lg. + norethynodrel 9.85 mg.; Ovulen, G. D. Searle Co., mestranol 100 /lg. + ethynodiol diacetate 1 mg. t See text for dose schedule.
aspiration of follicle on ovaries in situ in the course of elective gynecologic surgery, or on excised ovaries, using the ovum recovery unit (ORU) previously described. 17 The evaluation of oocyte initial quality was made by direct observation using the dissecting microscope at the time of examination of follicular aspirates. Morphologic criteria used for evaluating oocyte quality are summarized in Table 2. All oocyte manipulations were carried out in a sterile plastic bench enclosure at 37° C. in an atmosphere of 5% CO 2 in air. Preparation of Culture Media. Triple glass distilled water was used in preparation of all media. All glassware had been soaked in Haemo-Sol detergent, rinsed five times in tap water, rinsed three more times in triple glass distilled water, and sterilized
by dry heat at 150° C. for 1 hr. All cultures were carried out in sterile plastic culture dishes (Falcon Plastics 3010) under 1 ml. of paraffin oil USP (Saybolt viscosity 125/135). The oil had been previously sterilized by dry heat at 150°C. for 1 hr., and equilibrated with a gas phase of 5% CO 2 in air and either the maturation medium or the fertilization medium. Bavister's medium, as modified for human oocytes, 14 was further modified by adding 1,000 units of sodium heparin, 100,000 units of penicillin G and 50 mg. of streptomycin sulfate per liter, and is thereafter referred to as HPS Bavister's medium. The addition of heparin was intended to prevent coagulation of follicular aspirates which sometimes became contaminated with blood. Prior to use in the maturation or the fertilization
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HUMAN SPERM CAPACITATION AND FERTILIZATION
TABLE 2. Classification of Oocyte Qunlity Based on Observation with Dissecting Microscope Prior to in Vitro Maturation Culture Type of oocyte
Cumulus mass
Corona radiata cells
Vitellus
Estimated potential for maturation
No. of oocytes
Nil
12 (27.3)
Good
24 (54.5)
%
Degenerate
None
None or few
Nonovulatory
None
Several compact layers
Preovulatory
Extensive, sticky, silvery, and loosely applied
Loosely applied
Nonhomogenous and/ or shrinkage within zona pellucida When details observable, germinal vesicle generally observed When details observable, germinal vesicle may be seen
Excellent
Total
system, the HPS Bavister's medium was sterilized by filtration through a 0_2 J.I. pore size Millipore filter, using a Swinney adapter and disposable plastic syringe_ Maturation Cultures_ The maturation medium was composed of one part HPS Bavister's medium and three parts follicular fluid. 18 The human follicular fluid used was obtained by aspiration of 10-15 mm. size follicles from the same or a different ovary, and consisted of a pale straw-colored fluid uncontaminated with blood_ All cellular elements were removed from the follicular fluid before use and the pH and tonicity were 7_80 and 294 mosm./kg., respectively. The final maturation medium was equilibrated with a gas phase of 5% CO 2 in air. The contents of the ORV glass vial were transferred to depression slides for examination and the vials were rinsed three times with HPS Bavister's medium. After identification under the dissecting microscope, the recovered oocytes were transferred to the maturation medium within 10-20 min. following follicle aspiration_ The oocytes were maintained in 40J.l.l. droplets of maturation medium under oil at 37° C. in the incubator in an atmosphere of 5% CO 2 in air saturated with water vapor_ Oocytes were transferred to fertilization medium after 38-48 hr. of in-
8 (18.2)
44 (100)
cubation in maturation medium_ Thirteen oocytes, belonging to experimental series, were cultured in a maturation medium containing FSH, LH, and HCG (see "Results"). Fertilization Cultures. For the control series, the fertilization medium consisted of HPS Bavister's medium only. For the experimental series, the fertilization medium consisted of HPS Bavister's medium supplemented with 1 ampule of menotropins (Pergonal, Cutter Laboratories, 75 LV. of FSH and. 75 LV. of LH) and 5000 LV. of human chorionic gonadotropin (APL, Ayerst) per 250 ml. After equilibration with paraffin oil and 5% CO 2 in air, the experimental fertilization medium had a pH of 7.60 and a tonicity of 327 mosm./kg. Semen specimens were obtained from healthy fertile donors within 1 hr. prior to initiation of the fertilization cultures. Spontaneous liquefaction and sedimentation of coarse particulate matter in the ejaculate were allowed for 15 min. in a 10-ml. graduated cylinder at room temperature. The uppermost milliliter was removed with a pipet and diluted to 10 ml. with the particular version of fertilization medium under study_ The latter suspension was centrifuged at 1000 x g for 5 min., the supernate discarded, and the cell sedi-
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SOUP ART AND MORGENSTERN
ment resuspended in 10 ml. of the fertilization medium under study. The concentration of motile spermatozoa in this suspension was determined by counting the number of immotile sperm in fertilization medium and calculating the difference from total sperm count using water as diluent. An aliquot of the sperm suspension was then diluted further with the medium under study to obtain a concentration of 2 X 10 6 motile spermatozoa per ml. The working sperm suspension was then examined for progressive motility and presence of abnormal forms. The final fertilization cultures consisted of a 50-~l. droplet of sperm suspension containing approximately 100,000 motile spermatozoa. One oocyte was transferred to each 50-~l. droplet with as little maturation medium as possible. Culture in the fertilization medium was carried out for 24-28 hr. Examination of Specimens. During the transfer of oocytes from maturation to fertilization cultures, the specimens were briefly examined for evidence of maturation using the dissecting microscope. Assessment of maturation, however, was not completed until fertilization cultures were terminated. Maturation criteria were the presence of an extruded first polar body in the perivitelline space and the identification of a second metaphase plate of chromosomes within the vitellus, in an area close to the extruded polar body. Alternatively, evidence of fertilization was taken as implying that maturation had effectively occurred. The criteria used to assess fertilization were the presence of two pronuclei in the vitellus and that of two or three polar bodies in the perivitelline space. As a third criterion, a search for the fertilizing sperm tail was conducted, the results of which are discussed below. After observation and photographing in the fresh state, the specimens were fixed (glacial acetic acid-absolute ethanol 1: 3) and stained (saturated solution of carmine in 45% acetic acid) in order to demonstrate
the presence of chromatin. No specific treatment (enzymic dissolution of zona pellucida and hypotonic treatment) was applied which could have led to the precise identification of maturation stages earlier than completion of the first meiotic division. For observation, the oocytes were mounted in saline between a slide and a coverslip supported by a grease dot at each corner. This served to free the oocytes from remaining corona cells, by rolling the specimen between slide and coverslip. The specimens were examined and photographed using Nomarski's differential interference contrast optics. The location of sperm within the zona pellucida and in the perivitelline space was precisely determined using the rolling technic. RESULTS
Oocyte Recovery. Fifteen ovum donors were involved in this study. Oocytes were recovered from ovaries in situ exposed through the vaginal route in 8 patients, and from ovaries exposed through the abdominal route in 5 patients. In another patient, oocytes were recovered from one ovary in situ, exposed through the abdominal route, and, in the laboratory, from the second ovary which was excised. In still another patient, both ovaries were excised and oocyte recovery was performed in the laboratory. A total of 176 follicles were aspirated which led to the recovery of 50 oocytes, averaging at 3.3 oocytes per patient. Six oocytes were lost or damaged during subsequent manipulations, reducing to 44 the number of oocytes used in this study. Initial Classification of Oocytes. The evaluation of oocyte quality, based on observation with the dissecting microscope at the time ofrecovery, is shown in Table 2. Twelve out of 44 oocytes (27.3%) were found to be degenerate at recovery. Since the degenerate oocytes had intact zonaepellucidae and since the purpose of this investigation was the study of sperm pene-
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HUMAN SPERM CAPACITATION AND FERTILIZATION
tration through the zona, these oocytes were treated together with the apparently healthy ones. Among the 32 remaining oocytes, 24 were classified as nonovulatory (54.5%), and 8 as preovulatory (18.2%). The estimated potential for maturation in culture ranged from good in the former category, to excellent in the latter. Oocyte Maturation in Culture. Only 00cytes having completed the first meiotic division were recorded as having matured in culture. Morphologic criteria used for assessing maturation in culture are illustrated in Figs. 1-3. Figure 1 shows the significant area of an oocyte having completed the first meiotic division in culture, photographed in the fresh state using Nomarski's optics, without any other preparation than the removal of corona cells. The first polar body, containing chromatin, is
467
in close apposition to the vitelline membrane, and the second metaphase plate is seen within the vitellus in a marginal location close to that of the extruded polar body. Figure 2 shows the edge of a similar oocyte after fixation and staining. Figure 3 shows details of the polar body seen in Fig. 2. The presence of double-stranded chromosomes, one of which is in sharp focus in Fig. 3, identifies this polar body as the first one. Thirty-one oocytes (i.e., all those belonging to control series and the first seven of experimental series in Table 3) were cultured for maturation in medium without exogenous hormones. Only 25 of these oocytes were initially classified as likely to mature in vitro, 7 of which (28.0%) reached metaphase II. The 13 remaining oocytes were cultured for maturation in presence of exogenous hormones (75 mU. of FSH and
FIG. 1. Human oocyte having completed the first meiotic division in culture. The first polar body, containing chromatin, lies on the vitelline membrane in the perivitelline space (top right). The second metaphase plate of chromosome is at lower left. The small spherical mass at lower center is a microdrop of oil stuck to the outer surface of the zona pellucida and is out of focus as is the zona. Photographed in the fresh state after denudation, using Nomarski's optics. Direct magnification. x 400.
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FIG. 2. Human oocyte matured in culture, having been inseminated (see sperm at lower center) but not fertilized. Fixed with ethanol-acetic acid (3: 1) and stained with acetocarmine. The second metaphase plate of chromosome is seen at right of center. The first polar body is at left of center outside the vitelline membrane. Photographed using Nomarski's optics. Direct magnification. x260.
LR, and 5 LD. of RCG/mi.). Only 7 of these 13 oocytes were initially classified as likely to mature in vitro, 4 of which (57.1 %) reached metaphase II. Although higher maturation rate suggested maturation enhancement in the presence of exogenous hormones, the difference was not statistically significant, owing to the small size of the sample. Among a total of 32 oocytes initially classified as likely to mature in culture, only 11 (34.4%) did actually reach metaphase II. Sperm Penetration within and through the Zona Pellucida, and Fertilization in Vitro. At the end of fertilization cultures (24-28 hr.) oocytes were freed from remaining cumulus and/or corona cells when such cells were still present. The location of sperm within the zona pellucida and/or in the perivitelline space was precisely determined by rolling the ova between slide and
coverslip. Sperm penetration of fertilization data are summarized in Table 3. In the presence of exogenous hormones (FSR, LR, and RCG), the incidence of sperm penetration within and through the zona pellucida was found to be considerably increased as compared to controls. The differences between sperm penetration incidences in control and experimental series were found to be highly significant (p < 0.01) when analyzed by the x 2 method for the effect of added hormones. All 00cytes that were already naked at the start of fertilization cultures had sperm attached to the outer surface of their zona pellucida, but no degree of penetration had been achieved. One ovum only was found to be fertilized, which belonged to the experimental series. This fertilized ovum, as first viewed through the microscope, is shown in Fig. 4. It was characterized by
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HUMAN SPERM CAPACITATION AND FERTILIZATION
469
FIG. 3. First polar body of the human ovum shown in Fig. 2. The double-stranded chromosome in sharp focus at lower right of center identifies that polar body as the first one. Fixed with ethanol-acetic acid (3: 1) and stained with acetocarmine. Photographed using Nomarski's optics. Direct magnification. x400.
•
two sharply defined pronuclei in close apposition to each other. Each pronucleus contained eight nucleoli. An additional spermatozoon was trapped in the thickness of the zona pellucida and is visible at 6 o'clock in Fig. 4. Upon rolling between slide and coverslip, two polar bodies lying in the perivitelline space were brought into view while only one of the pronuclei remained in focus, as shown in Fig. 5. These observations were made 28 hr. after the initiation of the fertilization culture. The fixed and stained specimen is illustrated in Fig. 6. With the fixation process used, the zona pellucida of cultured oocytes is lost. The oocyte, in culture medium, is slightly compressed between slide and coverslip, and the culture medium is displaced first by fixative, then by stain. The zona constricts immediately into a few tenuous filaments which are pulled away from the oocyte by
the flow of fixative. One of the polar bodies floated away during fixation but was observed to contain chromatin. The other polar body remained close to the vitellus but is obscured by ooplasm that flowed through damaged vitelline membrane (at about 4 o'clock in Fig. 6). This polar body was also observed to contain chromatin. During the fixation process, the two pronuclei pulled apart from each other and the nucleoli disappeared. Remnants of the fertilizing sperm tail could not be observed either in the fresh or in the fixed specimen with the light microscope. Effects of Preoperative Treatment. Table 4 analyzes the maturation rate, incidence of spermatozoa penetration, and fertilization of the cultured oocytes as a function of the preoperative treatment. The data for HMG-HCG and clomiphene citrate-HCG treatment have been com-
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SOUPART AND MORGENSTERN
TABLE 3. Sperm Penetration and Fertilization of Human Oocytes as Related to Presence of Gonadotropins in Fertilization Medium
No. of oocytes cultures·
No. of ovum donors
No. of oocytes not fertilized but with sperm in Perivitelline space
Zona pellucida
No. of fertilized oocytes
%
%
Bavister's medium without hormone 9
24
6 (25.0)
1 (4.2)
o (0)
Bavister's medium + FSH, LH, and HCG 6
20
13t
9t
(65.0)
(45.0)
1 (12.5):j:
• All oocytes included (see text). differences between these figures and corresponding ones in the first series (controls) are highly significant (p < 0.01). :j: Fertilization rate, as percentage of the number of oocytes (8) having completed the first reduction division.
t The
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bined since they both induce follicular growth, oocyte maturation, and ovulation, although through different mechanisms. The treatment of oocyte donors with HMG or clomiphene citrate 11 and 13 days, respectively, prior to HCG administration did not improve the incidence of sperm penetration within and through the zona pellucida over treatment with HCG alone 20.5-30.5 hr. before oocyte recovery. Also, sperm penetration within or through the zona pellucida was not related to whether oocytes had matured, since there were more oocytes showing sperm within the zona (19) or in the perivitelline space (9) than the total number of oocytes (11) having reached metaphase II (Table 4). A comparison of the maturation rate among oocytes classified as likely to mature in culture and obtained from donors treated with HMG or clomiphene citrate prior to HCG (46.7%) with the maturation rate among the same class of oocytes obtained from donors
FIG. 4. Human ovum fertilized in vitro. Two sharply defined pronuclei in close apposition and each containing eight nucleoli are visible in the vitellus. A supplementary spermatozoon is visible in the thickness of the zona pellucida at about 6 o'clock. Photographed in the fresh state, using Nomarski's optics, 28 hr. post insemination. Direct magnification. x 260.
.
,
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471
FIG. 5. Human ovum fertilized in vitro. Same ovum as in Fig. 4. The ovum has been rolled to bring into focus the two polar bodies lying in the perivitelline space. Only one of the pronuclei is still in focus. Photographed in the fresh state, using Nomarski's optics, 28 hr. postinsemination. Direct magnification. x260.
treated with HCG alone (26.7%) suggested that the former treatment might have augmented in vitro oocyte maturation by initiating the maturation process in vivo. Statistical analysis, however, indicated that the observed difference was not significant. DISCUSSION
Accurate evaluation of oocyte status at recovery is highly desirable if the yield of oocyte maturation in culture is to be defined with precision. Such an evaluation was found to be technically difficult. Analytical procedures that can be applied for this purpose without endangering the oocyte suffer from strong limitations, and the classification of oocyte shown in Table 2 is at best an approximation. This is exemplified by the fact that among 32 oocytes classified as likely to mature in culture (Table 2) only 11 (34.4%) had completed meiosis at the end of the 44- to 48-hr.
culture period and were apparently fit for fertilization. The mean over-all maturation rate observed in this study compares with that reported by Chandley (30%).19 Zamboni, Thompson, and Moore Smith 20 in a series of 159 oocytes cultured for maturation, observed 61 inactive oocytes, 21 undetermined, and 77 maturing or mature oocytes. Various culture durations were used by these authors and, in the subseries of oocytes cultured for over 40 hr., which compares to the present study, 29 out of 59 oocytes critically evaluated (49.1%) were found at metaphase II at the end of the culture period. Edwards, Bavister, and Steptoe 13 have reported 60% oocytes reaching metaphase II in culture, and Kennedy and Donahue 21 observed 40.8% and 50% oocytes at metaphase II, respectively, in 199 and Ham's FlO culture media. Thus, maturation rates of cultured human 00cytes reported in the literature vary quite widely, which might be accounted for by
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<
,
FIG. 6. Human ovum fertilized in vitro. Same ovum as in Figs. 4 and 5, after fixation and staining. The two pronuclei have pulled apart. The nucleoli have disappeared. The vitelline membrane was damaged during fixation and the flowing cytoplasm obscured one of the polar bodies (4 o'clock). The zona pellucida was dissolved by the fixative and the other polar body floated away during fixation. Remnants of the sperm tail were not visible. Photographed using Nomarski's optics. Direct magnification. x260.
differences in the initial quality of the oocytes and in culture techniques. In the present study, the fact that a lower proportion of oocytes than those initially classified as likely to mature in culture did actually complete the first meiotic division might be accounted for by the following observation. Figure 7 shows an oocyte which was classified as nonovulatory but in good condition when examined with the dissecting microscope. Immediately after this first examination, the oocyte was denuded and re-examined by differential interference contrast optics (Fig. 7). The germinal vesicle is sharply defined and exhibits a prominent nucleolus containing several inclusions. Two irregular granular masses of a size similar to that of the nucleolus were in close apposition to it, representing nucleolus-associated chromatin. The appearance of the vitellus sug-
gests, however, that discrete vacuoles are distributed throughout the cytoplasm, which could be the sign of early degeneration, as described in the electron microscope study of Zamboni et al. 20 Ordinary phase contrast microscopy did not reveal such an alteration, but it became evident with the use of differential interference contrast optics, especially when the structure of the vitellus shown in Fig. 7 was compared with that of the pronuclear ovum shown in Figs. 4 and 5. The presence or absence of exogenous hormones (FSH, LH, and HCG) in the maturation culture medium did not significantly influence the maturation rate. The endogenous hormonal content, possibly contributed to the maturation medium by the follicular fluid, was unknown. Quality evaluation of 00cytes matured in culture is further complicated by the fact that obvious nuclear
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HUMAN SPERM CAPACITATION AND FERTILIZATION
maturation is not necessarily an adequate criterion for normal developmental potential. Rabbit oocytes for instance, when "matured" outside their follicle and fertilized in vivo, failed to form blastocysts. 22 In contrast, rabbit oocytes, matured in vivo following HCG-induced ovulation, have been fertilized in vitro and produced live young when transferred into synchronized foster mothers. 23-25 Also, some of the human oocytes matured and fertilized in culture have displayed cytoplasmic fragmentation. 16 Although transfer of 00cytes from their follicles to culture media results in nuclear maturation, it appears that the factor or factors essential to complete maturation have been missing from the various culture media which have been used thus far. An explanation for the almost total inability of in vitro matured oocytes to develop into viable fetuses was provided by Thibault and Gerard. 26 Rabbit oocytes having undergone nuclear maturation in culture were penetrated and activated by sperm in vitro. A female pronucleus developed apparently normally, but the sperm nucleus failed to swell. The female pronucleus and the unswollen sperm nucleus came in close apposition to
each other in the center of the vitellus. Subsequently, about half of these abnormally fertilized ova cleaved, apparently normally. Thibault and Gerard attributed the abnormal behavior of the sperm nucleus to the lack of a "male pronucleus growth factor" in oocytes matured in culture. Hence, until complete in vitro maturation requirements are better understood, the best source of fertilizable human 00cytes remains the follicles that have been stimulated to grow by exogenous hormone administration. In the investigations of Steptoe and Edwards,16 the maturing oocytes were obtained by aspirating large follicles just prior to ovulation induced by sequential therapy with HMG and HCG. It has been estimated that an interval of 30-36 hr. is required between the abrupt increase in LH activity and oocyte maturation with subsequent ovulation. 27 In the present study, clomiphene citrate and/or exogenous gonadotropins were given preoperatively in an attempt to initiate the oocyte maturation process in vivo. Several considerations make it difficult to directly evaluate the effectiveness of the preoperative treatment: (1) The donors were essentially
TABLE 4. Maturation, Sperm Penetration, and Fertilization of Human Oocytes as Related to Preoperative
Treatment No. ovum donors
Total oocytes cultured
No.oocytes classified as likely to mature in culture
No.oocytes showing maturation
No. oocytes not fertilized but with sperm in Zona pellucida
%
Perivitelline space
No.oocytes fertilized
%
HMG or clomiphene citrate - HCG 7
19
15
7
23
15
7* (46.7)
9 (47.4)
3 (15.8)
10 (43.6)
6 (26.1)
0
0
0
HCG only 4* (26.7) No treatment 1
2
2
0
* The difference between these two values was not statistically significant.
0
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.
FIG. 7. Immature human oocyte. The corona cells were removed by rolling the specimen between slide and coverslip. The spherical dictyate nucleus, or germinal vesicle, is visible in the center of the vitellus. It contains one prominent nucleolus with several inclusions. Beside the nucleolus are two irregular granular masses of nucleolus-associated chromatin. The appearance of the vitellus suggests that discrete vacuoles are distributed throughout the ooplasm and that this oocyte is undergoing degeneration. Photographed in the fresh state using Nomarski's optics. Direct magnification. x260.
normal women with an endogenous supply of FSH and LH activity characteristic for the day of the menstrual cycle on which operation was performed; (2) some of the donors were on suppressive hormonal therapy at the time of the treatment and operation; (3) logistic considerations regarding the operation schedule made it impossible to administer LH activity in the form of HeG more than 20.5-30.5 hr. before oocyte recovery. However, several indirect observations may be made regarding the effectiveness of the preoperative treatment in spite of its heterogeneity resulting from restrictive clinical considerations. Firstly, previous studies have established that oral contraceptive preparations are unable to abolish an ovarian response to exogenous gonadotropins.28, 29 Secondly, our results (Table 4) suggest that exoge-
nous FSH activity does not augment the incidence of zona pellucida penetration by spermatozoa over that seen with exogenous LH activity (HeG) alone. The stimulating role of HeG in sperm capacitation in the rabbit has been reported, 30 and the possibility exists that a similar phenomenon is important in the human. Thirdly, pretreatment with clomiphene citrate or exogenous gonadotropin may augment in vitro maturation of oocytes by initiation of the maturation process in vivo. It was not possible to determine unequivocally by direct observation whether maturation had been initiated in vivo, thus making it difficult to control the timing of oocyte maturation before insemination. That is, the addition of 20.5-30.5 hr. of possible in vivo maturation to the 38-48 hr. of in vitro maturation results in a 58.5- to 78.5-hr. interval be-
,.
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HUMAN SPERM CAPACITATION AND FERTILIZATION
tween HCG administration to the patient and insemination in vitro. Such a total maturation time would be excessive and might have resulted in the degeneration of otherwise normal mature oocytes before insemination, and therefore in a reduction of the fertilization rate. Normally, the mature ovulated oocyte is released by a follicle.which was submitted to the sequential influence of FSH and LH, and the cellular components of the follicle were influenced by both hormones. In vitro fertilization of human oocytes was obtained by Edwards et al. 13, 14 under culture conditions provided by an adaptation of Bavister's medium, which had proven effective in capacitating hamster sperm in vitro.15 This medium was considered by these authors as conducive to human sperm capacitation as well. In these investigations, however, capacitation occurred in the presence of both cumulus and corona cells, and the possible participation of these cells in the process could not be ruled out. If they were indeed involved, it seemed likely that their activity could be stimulated by one or several of the hormones used for inducing ovulation. Such an interaction could then be reflected by an increased incidence of sperm penetration within and through the zona pellucida. In order to test our working hypothesis, the hormones used for inducing ovulation were added to the fertilization medium in the same concentration ratios as the dosages administered to ovum donors (300 LU. of FSH and LH to 20,000 1. U. of HCG per liter),16 and the effects of such stimulation on sperm penetrating ability were examined. The increase in sperm penetrating ability in the presence of exogenous hormones (Table 3) was found to be statistically highly significant (p < 0.01) when tested for the effect of added hormones. Which factor or factors among the three added hormones are responsible for the observed effect remains to be determined, but LH or the LH-like HCG seem to be
475
prime candidates. These observations call for two main conclusions. First, the fact that sperm penetrating ability can be enhanced by modifying the fertilization medium demonstrates that some conditioning is necessary for human sperm to penetrate the egg investments. That is to say, human sperm require capacitation prior to fertilization as do sperm from other mammalian species. If this were not the case, the observed fertilization rate should have been close or equal to the maturation rate in culture, and all oocytes present should have had their investments penetrated, none of which was observed. The second conclusion is derived from present knowledge of the mechanism of hormone action and indicates that the sperm penetration enhancement effect had to be mediated by the hormonally stimulated follicle cells. All oocytes that were naked at the start of fertilization cultures were found later on with sperm attached to the outer surface of their zonae pellucidae, but no degree of penetration had been achieved at the end of the individual cultures. Penetration to various degrees was observed only in 00cytes initially surrounded by follicle cells. It is now well established that the effects of a hormone such as LH, for instance, are mediated by the second messenger cyclic adenosine mono phosphate (cAMP). 31 According to current concept, cAMP acts by facilitating a cascade of cellular events which involve an active nucleus and result in the synthesis and/or release of specific factors. At the time at which sperm capacitation is occurring in the fertilization milieu, the sperm nucleus, highly condensed, is very unlikely to be functional. On the other hand, the mature oocyte has no nucleus as such. Its genetic material, being present in the form of the second metaphase plate of chromosomes, is also very unlikely to be functional. The only cellular elements, present in the milieu, possessing an active nucleus and therefore most susceptible to responding to hormonal stimu-
476
SOUPART AND MORGENSTERN
lation are the follicle cells. The nature of the specific factors, synthesized and released by hormonally stimulated follicle cells, and capable of acting on sperm membranes, is a matter for further investigation. Thus, it appears that conditions for human sperm capacitation in vitro might be quite similar to those required by hamster sperm. It also appears that the process in both cases, instead of being simply the result of environmental conditions favoring the spontaneous occurrence of capacitation as previously suggested,13, 15 is indeed mediated by follicle cells, since Gwatkin, Andersen, and Hutchinson 32 have recently demonstrated a key role for the cumulus cells in association with one or more low molecular weight components ofthe cumulus matrix in the hamster sperm capacitation process. The pronuclear ovum, shown in the fresh state in Figs. 4 and 5, and in the fixed state in Fig. 6, fulfilled only two of the three criteria of fertilization. Two clearly defined pronuclei were present in close apposition in the center of the vitellus, and two polar bodies containing chromatin were present in the perivitelline space. The ovum appeared to be in late pronuclear stage as judged by close apposition of pronuclei and the fact that they both contained eight nucleoli, the number of which is known to increase with pronuclear age. 33 , 34 The fertilizing sperm tailor its remnants were not observed, either in the fresh state or after fixation and staining. To satisfy this last criterion of fertilization, the specimen should have been examined either earlier than 28 hr. after the initiation of fertilization culture, or it should have been examined with the electron microscope in order to identify remnants of the sperm tail not visible by light microscopy. The observations of the fertilizing sperm tail within the vitellus reported by Bavister, Edwards, and Steptoe,18 were made on specimens examined from 11-14.5 hr. after the initiation of fertilization cultures. The early
Vol. 24
disappearance of the fertilizing sperm tail is common in the rabbit. By contrast, in the rat, it can still be observed in one of the blastomeres following the first cleavage. The timing of disappearance of the fertilizing sperm tail in the human ovum is not presently known. Owing to the failure to identify by light microscopy remnants of the fertilizing sperm tail in the pronuclear ovum described here, the possibility of artificial activation has to be considered. According to Austin,33 there are nine theoretically possible kinds of ootid. The nine types differ from each other in the number and ploidy of polar bodies and female pronuclei. Each theoretical type is determined by whether a block of either one or both meiotic divisions has occurred and the stage of division (either metaphase or anaphase) at which the block was effective. The particular mechanism that could have led to an ovum exhibiting the features of the pronuclear ovum shown in Figs. 4 and 5, in the absence of the fertilizing sperm, could have operated as follows. The first polar body would have failed to form after the first meiotic division had gone through to anaphase, two distinct groups of chromosomes remaining within the vitellus. Then, the mechanism of the second meiotic division would have operated normally, affecting both groups of chromosomes and resulting in the simultaneous extrusion of two second polar bodies. Thus, by artificial activation without sperm penetration, combined with incomplete first meiotic division, a pronuclear and polar body configuration could theroretically be obtained, which mimics exactly the configuration of the normally fertilized ovum. The differences between these two types of pronuclear ova would be that in the artificially activated ovum the two polar bodies would be haploid, and the two pronuclei, also haploid, would be of female origin. The probability of such an occurrence in human ova is not presently documented, but it seems likely to be very low. Thus,
,.
.
~
June 1973
HUMAN SPERM CAPACITATION AND FERTILIZATION
although the possibility of artificial activation could not be ruled out definitely, it seems much more likely that the pronuclear ovum described here had undergone normal monospermic fertilization but was first observed at a late pronuclear stage, a stage at which elements of the fertilizing sperm tail had already disintegrated beyond recognition by means of light microscopy. Recent studies 35 in our laboratory have indeed shown that in ova analyzed at late pronuclear stages, remnants of the fertilizing sperm flagellum not observable by light microscopy are nevertheless demonstrable by electron microscopy. In the same studies, all sperm observed traversing the zona pellucida had undergone the acrosome reaction, which, in the light of the current concept of capacitation, implies that they were capacitated. From the viewpoint of contraception, it is worth mentioning that the pronuclear ovum described here was obtained from a donor using an oral contraceptive. Oocyte recovery in this patient was conducted on Day 15 of her cycle. She had been using Norinyl 1 + 80 up to Day 13 of that cycle. Only HCG (20,000 LV.) was administered 21 hr. 40 min. prior to follicle aspiration in the course of a vaginal hysterectomy performed for elective sterilization. A total of six follicles were aspirated. What appeared to be a follicle of about 2 mm. size at the upper pole of the right ovary was productive of about 2 ml. of pale straw-colored fluid, uncontaminated with blood, indicating that this subsurface follicle was indeed much larger than its apparent size suggested. In the process of aspirating other identified follicles, the content of the ovum recovery unit became contaminated with blood. It is of interest to note that the initial blood contamination of the follicular aspirates did not prevent subsequent fertilization of one of the four oocytes recovered. The oocyte that became fertilized was initially classified as preovulatory, being encased in a rather loose, sticky and silvery
477
cumulus mass. At the end of fertilization culture, this ovum was found to be completely free of corona cells. SUMMARY
Forty-four human oocytes were recovered from 15 patients undergoing elective gynecologic surgery. Oocytes were recovered by direct follicle aspiration, mainly from ovaries in situ exposed in the course of surgery, and from excised ovaries. These oocytes, divided in two lots of approximately equal size, were submitted to maturation culture and to fertilization culture, the latter in the absence or presence of the hormones (FSH, LH, and HCG) used for inducing ovulation. Fertilization cultures in the presence of exogenous hormones resulted in a highly significant (p < 0.01) increase in the incidence of sperm penetration within and through the zona pellucida. The hormonally induced enhancement of sperm penetrating ability demonstrates the need for capacitation of human sperm. Present knowledge on the mechanism of hormone action strongly suggests that the observed effect had to be mediated by follicle cells. An oocyte, recovered from a patient using an oral contraceptive, became fertilized and is described in this study. Acknowledgments. The authors wish to acknowledge the skillful technical assistance of Mrs. Lois Campbell-Taylor, Mrs. Debra Jarashow, Mrs. Simone Soupart, and Mr. John E. Repp, as well as the secretarial skills of Miss Nicqui Hailey. Thanks are also due to Mr. Homer A. Sprague, Department of Biostatistics, Vanderbilt University School of Medicine, for performing statistical analysis of the data, and to the operating room staff of United States Army Tripier General Hospital. REFERENCES 1. AUSTIN, C. R. "Sperm capacitation-Biological
Significance in Various Species." In Advances in the Biosciences (Vol. 4). Schering Symposium on Conception, Pergamon, Oxford, 1969, p. 5. 2. AUSTIN, C. R. The "capacitation" of the mam-
478
SOUPART AND MORGENSTERN
malian sperm. Nature (London) 170: 326, 1952. 3. CHANG, M. C. Fertilizing capacity of spermatozoa deposited in the Fallopian tube. Nature (London) 168:687, 1951. 4. AUSTIN, C. R Observations on the penetration of sperm into the mammalian egg. Aust J Sci Res B 4:581, 1951. 5. BEDFORD, J. M. Sperm capacitation and fertilization in mammals. Bioi Reprod (Suppl. 2):128, 1970. 6. BARROS, C., BEDFORD, J. M., FRANKLIN, L. E., AND AUSTIN, C. R Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell Bioi 34:C 1, 1967. 7. BEDFORD, J. M. Ultrastructural changes in the sperm head during fertilization in the rabbit. Amer J Anat 123:329, 1968. 8. ALLISON, A. C., AND HARTREE, E. F. Lysosomal enzymes in the acrosome and their possible role in fertilization. J Reprod FertiI21:501, 1970. 9. BRADEN, A. W. H. Properties of the membranes of rat and rabbit eggs. Aust J Sci Res B 5:460, 1952. 10. STAMBAUGH, R, AND BUCKLEY, J. Zona pellucida dissolution enzymes of the rabbit sperm head. Science 161:585, 1968. 11. NEWSLETTER FROM THE EDITORS. Acrosomal enzyme. Bioi Reprod 6:176,1972. 12. STAMBAUGH, R, AND BUCKLEY, J. Identification and subcellular localization of the enzymes effecting penetration of the zona pellucida by rabbit spermatozoa. J. Reprod Fertil 19:423, 1969. '13. EDWARDS, R G., BAVISTER, B. D., AND STEPTOE, P. C. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (London) 221:632, 1969. -14. EDWARDS, R G., STEPTOE, P. C., AND PURDY, J. M. Fertilization and cleavage in vitro of preovulator human oocytes. Nature (London) 227:1307,1970. 15. BAVISTER, B. D. Environmental factors important for in vitro fertilization in the hamster. J Reprod Fertil 18:544, 1969. 16. STEPTOE, P. C., AND EDWARDS, R G. Laparoscopic recovery of preovulator human oocytes after priming of ovaries with gonadotrophins. Lancet 1 :683, 1970. 17. MORGENSTERN, L. L., AND SOUPART, P. Oocyte recovery from the human ovary. Fertil Steril 23:751, 1972. 18. BAVISTER, B. D., EDWARDS, R G., AND STEPTOE, P. C. Identification of the midpiece and tail of the spermatozoon during fertilization of human eggs in vitro. J Reprod Fertil 20:159, 1969. 19. CHANDLEY, A. C. Culture of mammalian oocytes. J Reprod Fertil (Suppl. 14):1, 1971.
Vol. 24
20. ZAMBONI, L., THOMPSON, R S., AND MOORE SMITH, D. Fine morphology of human oocyte maturation in vitro. Bioi Reprod 7:425, 1972. 21. KENNEDY, J. F., AND DONAHUE, R P. Human oocytes: Maturation in chemically defined media. Science 164:1292, 1969. 22. CHANG, M. C. The maturation of rabbit oocytes in culture and their maturation, activation, fertilization and subsequent development in the Fallopian tubes. J Exp Zool 128:379, 1955. 23. CHANG, M. C. Fertilization of rabbit ova in vitro. Nature (London) 183:466, 1959. 24. THIBAULT, C., AND DAUZIER, L. Analyse des conditions de la fecondabilite in vitro de l'oeuf de la lapine. Ann Bioi Anim Biochim Biophys 1:277, 1961. 25. BRACKETT, B. G. Effects of washing the gametes on fertilization in vitro. Fertil Steril20:127, 1969. 26. THIBAULT, C., AND GERARD, M. Facteur cytoplasmique necessaire a la formation du pronucleus male dans l'ovocyte de lapine. C R Acad Sci Paris
D 270:2025, 1970. 27. EDWARDS, R G. "Stages of the Development of the Human Egg." In Advances in the Biosciences (Vol. 4). Raspe, G., Ed. Pergamon, Oxford, 1970, p. 235. 28. PUJOL-AMAT, P., URGELL-RoCA, J. M., ESTEBANALTIRRIBA, J., AND MARQUEZ-RAMIREZ, M. Studies on ovarian biopsies from women cyclically treated with the combination ethynodiol diacetate + mestranol alone and together with menopausal gonadotropin (Pergonal) + HCG. Gynaecologia 167:237, 1969. 29. JOHANISSON, E., TILLINGER, K. G., ANDDICZFALUSY, E. Effect of oral contraceptive on the ovarian reaction to human gonadotropins in amenorrheic women. Fertil Steril 16:292, 1965. 30. SOUPART, P. Effects of human chorionic gonadotrophin on capacitation of rabbit spermatozoa. Nature (London) 212:408, 1966. 31. JOST, J. P., AND RICKENBERG, H. V. Cyclic AMP.
Ann Rev Biochem 40:741, 1971. 32. GWATKIN, R B. L., ANDERSEN, O. F., AND HUTCHINSON, C. F. Capacitation of hamster spermatozoa in vitro: the role of cumulus components. J Reprod Fertil 30:389, 1972. 33. AUSTIN, C. R The Mammalian Egg. Blackwell, Oxford, 1961, p. 27. 34. SOTELO, J. R, AND PORTER, K. R An electron microscope study of the rat ovum. J Biophys
Biochem CytoI5:327, 1959. 35. SOUPART, P., AND STRONG, P. A. Ultrastructural observations on human oocytes fertilized in vitro. In preparation.
FERTILITY AND STERILITY
Copyright © 1973 by The Williams & Wilkins Co.
HUMAN SPERM CAPACITATION AND IN VITRO FERTILIZATION* PIERRE SOUPART, M.D., PH.D.,
AND
LARRY L. MORGENSTERN, M.D.t
Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Department of Obstetrics and Gynecology, United States Army Tripier General Hospital, APO San Francisco, California 96438
From observations made in some mammals, it appears that normal fertilization results only from the encounter of a mature oocyte with a capacitated spermatozoon. 1 On the basis of such observations, although limited to a few species, the need for sperm capacitation is considered to be an absolute prerequisite for mammalian fertilization. However, there is as yet no direct experimental evidence that sperm capacitation is a feature of human fertilization. The functional dependence of sperm upon the female reproductive tract prior to fertilization, which has been termed capacitation,2 was first recognized in 1951 independently by Chang 3 and by Austin.' The phenomenon was initially defined as the ability acquired by sperm in the female genital tract to penetrate through the egg investments, the matrix of the cumulus oophorus, and the zona pellucida. All sperm observed within the zona pellucida or in the perivitelline space of the ovum have lost the outer acrosomal membrane and the corresponding plasma membrane, leaving exposed the inner acrosomal membrane. This morphologic change is termed acrosome reaction, and it is assumed that the fertilizing spermatozoon has undergone this change as well. It seems important to distinguish between a "false" acrosome reaction,5 in which the outer acrosomal and sperm plasma membranes are lost sepaReceived January 4, 1973. • Supported by U.S.P.H.S. Contract NIH-70-2162 and Grant HD 03769, and by United States Army Medical Research and Development Command. t Present address: Department of Obstetrics and Gynecology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87106.
rately, and a "true" acrosome reaction,6 in which exposure of the inner acrosomal membrane involves fusion between outer acrosomal membrane and plasma membrane in an orderly vesiculation process. 6 The former is said to represent a nonspecific degenerative change, while the latter appears to be physiologic and occurs in the immediate vicinity of the ovum or within the cumulus and corona cell mass. 7 The acrosome appears to be a modified lysosome and, as such, contains hydrolytic enzymes,8 two of which are of particular interest. The first one, hyaluronidase, has long been known as being able to dissolve the matrix of the cumulus oophorus. 9 The second one, a trypsin-like enzyme,10 presently known as the acrosomal proteinase,l1 appears to act on the zona pellucida while still firmly bound to the inner acrosomal membrane. 12 The latter property would explain why localized dissolution, in the form of a sharp penetration slit, marks the passage of sperm through the zona pellucida rather than complete dissolution, as observed when the zona pellucida is treated with extracted acrosomal enzymes. 12 Since the original definition of capacitation was formulated, a trend has developed to restrict its meaning to that of some conditioning that sperm undergo in the female genital tract, which permits the occurrence of a "true" acrosome reaction in the immediate vicinity of the zona pellucida. Uncapacitated sperm, that is sperm that have not been submitted to this preliminary conditioning, cannot undergo a "true" acrosome reaction and are unable to penetrate through the zona pellucida.
462
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HUMAN SPERM CAPACITATION AND FERTILIZATION
Since there is presently no demonstrated morphologic concomitant of capacitation, the fact that capacitation has occurred has to be assessed functionally. As far as human spermatozoa are concerned, such an assessment requires the use of an in vitro fertilization system. The need for capacitation of human sperm was strongly suggested when unquestionable evidence of in vitro fertilization of mature human oocytes was obtained 13, 14 under conditions provided by the modification of a culture medium which had proven effective in capacitating hamster sperm in vitro.15 In these experiments, sperm penetration through the zona pellucida of the human ovum suggested that the capacitation requirements of human sperm were similar to those of hamster sperm: namely, that such requirements could be no more than suitable environmental conditions allowing for the spontaneous occurrence of the capacitation process. 15 In experiments such as those mentioned above, the in vitro fertilization system was composed of four elements: the fertilization medium, washed ejaculated spermatozoa, oocytes, and the follicle cells that surround them. The 00cytes used in the most successful of these experiments were recovered by laparoscopic technic from follicle in situ just prior to induced ovulation. 14, 16 Ovulation was induced in the ovum donors in order to obtain oocytes having completed much of their maturation in the ovary. The ovum donors, who were volunteer women of infertile married couples, were selected on the basis of recognized fallopian tube anomalies, but their ovarian function was apparently normal. 16 It seemed reasonable to assume that not only the recovered oocytes but also their satellite follicle cells had reacted to the hormones used for inducing ovulation. Given such conditions, it was not possible to decide whether sperm penetrating ability resulted from the sole effect of the fertilization medium or whether some cellular interaction was also part of
463
the process. The present study was designed to investigate the possible effect on sperm penetrating ability of hormone addition to the in vitro fertilization system. The hormones selected for this purpose were those (follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (HCG)) which are used in combination for inducing ovulation in women. The observations presented below indicate that human sperm must undergo capacitation prior to fertilization and suggest, furthermore, that follicle cells are involved in the process. MATERIALS Al'.ID METHODS
Preoperative Treatment of Oocyte Donors. Table 1 summarizes the preoperative treatment, the day of menstrual cycle, and the endometrial status of the oocyte donors at the time of oocyte recovery for the control and experimental groups. Preoperative suppression therapy is defined as the utilization of combination oral contraceptives during the cycle in which gonadotropins and/or clomiphene citrate were administered or operation was performed. Four oocyte donors received human menopausal gonadotropin (HMG, Pergonal, Cutter Laboratories, Berkeley, Calif.) in equally divided doses (75 LU. of FSH and 75 LU. of LH) on Days 1, 4, and 8 with human chorionic gonadotropin (HCG, APL. Ayerst Laboratories, New York, N.Y.) administered on Day 11. Two donors received clomiphene citrate (Clomid, Wm. S. Merrill Co., Cincinnati, Ohio) orally in doses of 50 mg./day (Patient C. J.) and 100 mg./day (Patient L. T.) on Days 1 through 5, inclusive, with HCG given on Day 13. Seven donors received only HCG the day before operation and one donor had no preoperative treatment. The dose of HCG given to all donors was 20,000 I.U., and oocyte recovery was performed 20.5-30.5 hr. after HCG administration. Oocyte Recovery and Evaluation. Oocyte recovery was performed by direct
464
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SOUPART AND MORGENSTERN TABLE 1. Preoperative Treatment of Oocyte Donors
Patient
Age
Suppression therapy prior to surgery'
Preoperative treatmentt
Interval HCG· oocyte recovery
Days of menstrual cycle at recovery
Condition of endometrium at recovery
No. of oocytes cultured
Control series G.G. L. S.
33 24
HMGIHCG HCG
23.5 26
19
24 34 20 34 26 24 23
None Pregnancy (5 wk.) + Ovral None None Norinyll + 80 None None None None
M.H. C.Y. B.C. M.B. J.D. E.W. K. S.
HMG/HCG HCG HCG None HMGIHCG HCG HMG/HCG
26.5 27 24.5
14 23 16 3 8 18 24
P.V.P.
34
Enovid lOmg
HCG
20.5
16
M.S. L. T.
33 32
Norinyll None
2l.5 24
A.B. C.J.
36 30
None Ovulen
B.T.
29
None
HCG Clomiphene citrate/HCG HCG Clomiphene cittrate/HCG HMGIHCG
26.5 30.5 28.5
Secretory Decidual and chorionic villi Proliferative Proliferative No data Proliferative Proliferative Proliferative Secretory
3 2 5 2 3 2 3 3
Experimental series
+ 80
16 23
Proliferative with focal glandular ectasia Proliferative Proliferative
4 2
24 26.5
24 ?
Secretory Proliferative
8 3
27.5
27
Secretory
2
* Ovral, Wyeth Laboratories, ethinyl estradiol 50 /lg. + Norgestrel 0.5 mg.; Norinyl 1 + 80, Syntex Laboratories, mestranol 80 /lg. + norethindrone 1 mg.; Enovid 10 mg., G. D. Searle Company, mestranol 150 /lg. + norethynodrel 9.85 mg.; Ovulen, G. D. Searle Co., mestranol 100 /lg. + ethynodiol diacetate 1 mg. t See text for dose schedule.
aspiration of follicle on ovaries in situ in the course of elective gynecologic surgery, or on excised ovaries, using the ovum recovery unit (ORU) previously described. 17 The evaluation of oocyte initial quality was made by direct observation using the dissecting microscope at the time of examination of follicular aspirates. Morphologic criteria used for evaluating oocyte quality are summarized in Table 2. All oocyte manipulations were carried out in a sterile plastic bench enclosure at 37° C. in an atmosphere of 5% CO 2 in air. Preparation of Culture Media. Triple glass distilled water was used in preparation of all media. All glassware had been soaked in Haemo-Sol detergent, rinsed five times in tap water, rinsed three more times in triple glass distilled water, and sterilized
by dry heat at 150° C. for 1 hr. All cultures were carried out in sterile plastic culture dishes (Falcon Plastics 3010) under 1 ml. of paraffin oil USP (Saybolt viscosity 125/135). The oil had been previously sterilized by dry heat at 150°C. for 1 hr., and equilibrated with a gas phase of 5% CO 2 in air and either the maturation medium or the fertilization medium. Bavister's medium, as modified for human oocytes, 14 was further modified by adding 1,000 units of sodium heparin, 100,000 units of penicillin G and 50 mg. of streptomycin sulfate per liter, and is thereafter referred to as HPS Bavister's medium. The addition of heparin was intended to prevent coagulation of follicular aspirates which sometimes became contaminated with blood. Prior to use in the maturation or the fertilization
I~ June 1973
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HUMAN SPERM CAPACITATION AND FERTILIZATION
TABLE 2. Classification of Oocyte Qunlity Based on Observation with Dissecting Microscope Prior to in Vitro Maturation Culture Type of oocyte
Cumulus mass
Corona radiata cells
Vitellus
Estimated potential for maturation
No. of oocytes
Nil
12 (27.3)
Good
24 (54.5)
%
Degenerate
None
None or few
Nonovulatory
None
Several compact layers
Preovulatory
Extensive, sticky, silvery, and loosely applied
Loosely applied
Nonhomogenous and/ or shrinkage within zona pellucida When details observable, germinal vesicle generally observed When details observable, germinal vesicle may be seen
Excellent
Total
system, the HPS Bavister's medium was sterilized by filtration through a 0_2 J.I. pore size Millipore filter, using a Swinney adapter and disposable plastic syringe_ Maturation Cultures_ The maturation medium was composed of one part HPS Bavister's medium and three parts follicular fluid. 18 The human follicular fluid used was obtained by aspiration of 10-15 mm. size follicles from the same or a different ovary, and consisted of a pale straw-colored fluid uncontaminated with blood_ All cellular elements were removed from the follicular fluid before use and the pH and tonicity were 7_80 and 294 mosm./kg., respectively. The final maturation medium was equilibrated with a gas phase of 5% CO 2 in air. The contents of the ORV glass vial were transferred to depression slides for examination and the vials were rinsed three times with HPS Bavister's medium. After identification under the dissecting microscope, the recovered oocytes were transferred to the maturation medium within 10-20 min. following follicle aspiration_ The oocytes were maintained in 40J.l.l. droplets of maturation medium under oil at 37° C. in the incubator in an atmosphere of 5% CO 2 in air saturated with water vapor_ Oocytes were transferred to fertilization medium after 38-48 hr. of in-
8 (18.2)
44 (100)
cubation in maturation medium_ Thirteen oocytes, belonging to experimental series, were cultured in a maturation medium containing FSH, LH, and HCG (see "Results"). Fertilization Cultures. For the control series, the fertilization medium consisted of HPS Bavister's medium only. For the experimental series, the fertilization medium consisted of HPS Bavister's medium supplemented with 1 ampule of menotropins (Pergonal, Cutter Laboratories, 75 LV. of FSH and. 75 LV. of LH) and 5000 LV. of human chorionic gonadotropin (APL, Ayerst) per 250 ml. After equilibration with paraffin oil and 5% CO 2 in air, the experimental fertilization medium had a pH of 7.60 and a tonicity of 327 mosm./kg. Semen specimens were obtained from healthy fertile donors within 1 hr. prior to initiation of the fertilization cultures. Spontaneous liquefaction and sedimentation of coarse particulate matter in the ejaculate were allowed for 15 min. in a 10-ml. graduated cylinder at room temperature. The uppermost milliliter was removed with a pipet and diluted to 10 ml. with the particular version of fertilization medium under study_ The latter suspension was centrifuged at 1000 x g for 5 min., the supernate discarded, and the cell sedi-
466
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SOUP ART AND MORGENSTERN
ment resuspended in 10 ml. of the fertilization medium under study. The concentration of motile spermatozoa in this suspension was determined by counting the number of immotile sperm in fertilization medium and calculating the difference from total sperm count using water as diluent. An aliquot of the sperm suspension was then diluted further with the medium under study to obtain a concentration of 2 X 10 6 motile spermatozoa per ml. The working sperm suspension was then examined for progressive motility and presence of abnormal forms. The final fertilization cultures consisted of a 50-~l. droplet of sperm suspension containing approximately 100,000 motile spermatozoa. One oocyte was transferred to each 50-~l. droplet with as little maturation medium as possible. Culture in the fertilization medium was carried out for 24-28 hr. Examination of Specimens. During the transfer of oocytes from maturation to fertilization cultures, the specimens were briefly examined for evidence of maturation using the dissecting microscope. Assessment of maturation, however, was not completed until fertilization cultures were terminated. Maturation criteria were the presence of an extruded first polar body in the perivitelline space and the identification of a second metaphase plate of chromosomes within the vitellus, in an area close to the extruded polar body. Alternatively, evidence of fertilization was taken as implying that maturation had effectively occurred. The criteria used to assess fertilization were the presence of two pronuclei in the vitellus and that of two or three polar bodies in the perivitelline space. As a third criterion, a search for the fertilizing sperm tail was conducted, the results of which are discussed below. After observation and photographing in the fresh state, the specimens were fixed (glacial acetic acid-absolute ethanol 1: 3) and stained (saturated solution of carmine in 45% acetic acid) in order to demonstrate
the presence of chromatin. No specific treatment (enzymic dissolution of zona pellucida and hypotonic treatment) was applied which could have led to the precise identification of maturation stages earlier than completion of the first meiotic division. For observation, the oocytes were mounted in saline between a slide and a coverslip supported by a grease dot at each corner. This served to free the oocytes from remaining corona cells, by rolling the specimen between slide and coverslip. The specimens were examined and photographed using Nomarski's differential interference contrast optics. The location of sperm within the zona pellucida and in the perivitelline space was precisely determined using the rolling technic. RESULTS
Oocyte Recovery. Fifteen ovum donors were involved in this study. Oocytes were recovered from ovaries in situ exposed through the vaginal route in 8 patients, and from ovaries exposed through the abdominal route in 5 patients. In another patient, oocytes were recovered from one ovary in situ, exposed through the abdominal route, and, in the laboratory, from the second ovary which was excised. In still another patient, both ovaries were excised and oocyte recovery was performed in the laboratory. A total of 176 follicles were aspirated which led to the recovery of 50 oocytes, averaging at 3.3 oocytes per patient. Six oocytes were lost or damaged during subsequent manipulations, reducing to 44 the number of oocytes used in this study. Initial Classification of Oocytes. The evaluation of oocyte quality, based on observation with the dissecting microscope at the time ofrecovery, is shown in Table 2. Twelve out of 44 oocytes (27.3%) were found to be degenerate at recovery. Since the degenerate oocytes had intact zonaepellucidae and since the purpose of this investigation was the study of sperm pene-
-1
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HUMAN SPERM CAPACITATION AND FERTILIZATION
tration through the zona, these oocytes were treated together with the apparently healthy ones. Among the 32 remaining oocytes, 24 were classified as nonovulatory (54.5%), and 8 as preovulatory (18.2%). The estimated potential for maturation in culture ranged from good in the former category, to excellent in the latter. Oocyte Maturation in Culture. Only 00cytes having completed the first meiotic division were recorded as having matured in culture. Morphologic criteria used for assessing maturation in culture are illustrated in Figs. 1-3. Figure 1 shows the significant area of an oocyte having completed the first meiotic division in culture, photographed in the fresh state using Nomarski's optics, without any other preparation than the removal of corona cells. The first polar body, containing chromatin, is
467
in close apposition to the vitelline membrane, and the second metaphase plate is seen within the vitellus in a marginal location close to that of the extruded polar body. Figure 2 shows the edge of a similar oocyte after fixation and staining. Figure 3 shows details of the polar body seen in Fig. 2. The presence of double-stranded chromosomes, one of which is in sharp focus in Fig. 3, identifies this polar body as the first one. Thirty-one oocytes (i.e., all those belonging to control series and the first seven of experimental series in Table 3) were cultured for maturation in medium without exogenous hormones. Only 25 of these oocytes were initially classified as likely to mature in vitro, 7 of which (28.0%) reached metaphase II. The 13 remaining oocytes were cultured for maturation in presence of exogenous hormones (75 mU. of FSH and
FIG. 1. Human oocyte having completed the first meiotic division in culture. The first polar body, containing chromatin, lies on the vitelline membrane in the perivitelline space (top right). The second metaphase plate of chromosome is at lower left. The small spherical mass at lower center is a microdrop of oil stuck to the outer surface of the zona pellucida and is out of focus as is the zona. Photographed in the fresh state after denudation, using Nomarski's optics. Direct magnification. x 400.
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FIG. 2. Human oocyte matured in culture, having been inseminated (see sperm at lower center) but not fertilized. Fixed with ethanol-acetic acid (3: 1) and stained with acetocarmine. The second metaphase plate of chromosome is seen at right of center. The first polar body is at left of center outside the vitelline membrane. Photographed using Nomarski's optics. Direct magnification. x260.
LR, and 5 LD. of RCG/mi.). Only 7 of these 13 oocytes were initially classified as likely to mature in vitro, 4 of which (57.1 %) reached metaphase II. Although higher maturation rate suggested maturation enhancement in the presence of exogenous hormones, the difference was not statistically significant, owing to the small size of the sample. Among a total of 32 oocytes initially classified as likely to mature in culture, only 11 (34.4%) did actually reach metaphase II. Sperm Penetration within and through the Zona Pellucida, and Fertilization in Vitro. At the end of fertilization cultures (24-28 hr.) oocytes were freed from remaining cumulus and/or corona cells when such cells were still present. The location of sperm within the zona pellucida and/or in the perivitelline space was precisely determined by rolling the ova between slide and
coverslip. Sperm penetration of fertilization data are summarized in Table 3. In the presence of exogenous hormones (FSR, LR, and RCG), the incidence of sperm penetration within and through the zona pellucida was found to be considerably increased as compared to controls. The differences between sperm penetration incidences in control and experimental series were found to be highly significant (p < 0.01) when analyzed by the x 2 method for the effect of added hormones. All 00cytes that were already naked at the start of fertilization cultures had sperm attached to the outer surface of their zona pellucida, but no degree of penetration had been achieved. One ovum only was found to be fertilized, which belonged to the experimental series. This fertilized ovum, as first viewed through the microscope, is shown in Fig. 4. It was characterized by
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HUMAN SPERM CAPACITATION AND FERTILIZATION
469
FIG. 3. First polar body of the human ovum shown in Fig. 2. The double-stranded chromosome in sharp focus at lower right of center identifies that polar body as the first one. Fixed with ethanol-acetic acid (3: 1) and stained with acetocarmine. Photographed using Nomarski's optics. Direct magnification. x400.
•
two sharply defined pronuclei in close apposition to each other. Each pronucleus contained eight nucleoli. An additional spermatozoon was trapped in the thickness of the zona pellucida and is visible at 6 o'clock in Fig. 4. Upon rolling between slide and coverslip, two polar bodies lying in the perivitelline space were brought into view while only one of the pronuclei remained in focus, as shown in Fig. 5. These observations were made 28 hr. after the initiation of the fertilization culture. The fixed and stained specimen is illustrated in Fig. 6. With the fixation process used, the zona pellucida of cultured oocytes is lost. The oocyte, in culture medium, is slightly compressed between slide and coverslip, and the culture medium is displaced first by fixative, then by stain. The zona constricts immediately into a few tenuous filaments which are pulled away from the oocyte by
the flow of fixative. One of the polar bodies floated away during fixation but was observed to contain chromatin. The other polar body remained close to the vitellus but is obscured by ooplasm that flowed through damaged vitelline membrane (at about 4 o'clock in Fig. 6). This polar body was also observed to contain chromatin. During the fixation process, the two pronuclei pulled apart from each other and the nucleoli disappeared. Remnants of the fertilizing sperm tail could not be observed either in the fresh or in the fixed specimen with the light microscope. Effects of Preoperative Treatment. Table 4 analyzes the maturation rate, incidence of spermatozoa penetration, and fertilization of the cultured oocytes as a function of the preoperative treatment. The data for HMG-HCG and clomiphene citrate-HCG treatment have been com-
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TABLE 3. Sperm Penetration and Fertilization of Human Oocytes as Related to Presence of Gonadotropins in Fertilization Medium
No. of oocytes cultures·
No. of ovum donors
No. of oocytes not fertilized but with sperm in Perivitelline space
Zona pellucida
No. of fertilized oocytes
%
%
Bavister's medium without hormone 9
24
6 (25.0)
1 (4.2)
o (0)
Bavister's medium + FSH, LH, and HCG 6
20
13t
9t
(65.0)
(45.0)
1 (12.5):j:
• All oocytes included (see text). differences between these figures and corresponding ones in the first series (controls) are highly significant (p < 0.01). :j: Fertilization rate, as percentage of the number of oocytes (8) having completed the first reduction division.
t The
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bined since they both induce follicular growth, oocyte maturation, and ovulation, although through different mechanisms. The treatment of oocyte donors with HMG or clomiphene citrate 11 and 13 days, respectively, prior to HCG administration did not improve the incidence of sperm penetration within and through the zona pellucida over treatment with HCG alone 20.5-30.5 hr. before oocyte recovery. Also, sperm penetration within or through the zona pellucida was not related to whether oocytes had matured, since there were more oocytes showing sperm within the zona (19) or in the perivitelline space (9) than the total number of oocytes (11) having reached metaphase II (Table 4). A comparison of the maturation rate among oocytes classified as likely to mature in culture and obtained from donors treated with HMG or clomiphene citrate prior to HCG (46.7%) with the maturation rate among the same class of oocytes obtained from donors
FIG. 4. Human ovum fertilized in vitro. Two sharply defined pronuclei in close apposition and each containing eight nucleoli are visible in the vitellus. A supplementary spermatozoon is visible in the thickness of the zona pellucida at about 6 o'clock. Photographed in the fresh state, using Nomarski's optics, 28 hr. post insemination. Direct magnification. x 260.
.
,
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471
FIG. 5. Human ovum fertilized in vitro. Same ovum as in Fig. 4. The ovum has been rolled to bring into focus the two polar bodies lying in the perivitelline space. Only one of the pronuclei is still in focus. Photographed in the fresh state, using Nomarski's optics, 28 hr. postinsemination. Direct magnification. x260.
treated with HCG alone (26.7%) suggested that the former treatment might have augmented in vitro oocyte maturation by initiating the maturation process in vivo. Statistical analysis, however, indicated that the observed difference was not significant. DISCUSSION
Accurate evaluation of oocyte status at recovery is highly desirable if the yield of oocyte maturation in culture is to be defined with precision. Such an evaluation was found to be technically difficult. Analytical procedures that can be applied for this purpose without endangering the oocyte suffer from strong limitations, and the classification of oocyte shown in Table 2 is at best an approximation. This is exemplified by the fact that among 32 oocytes classified as likely to mature in culture (Table 2) only 11 (34.4%) had completed meiosis at the end of the 44- to 48-hr.
culture period and were apparently fit for fertilization. The mean over-all maturation rate observed in this study compares with that reported by Chandley (30%).19 Zamboni, Thompson, and Moore Smith 20 in a series of 159 oocytes cultured for maturation, observed 61 inactive oocytes, 21 undetermined, and 77 maturing or mature oocytes. Various culture durations were used by these authors and, in the subseries of oocytes cultured for over 40 hr., which compares to the present study, 29 out of 59 oocytes critically evaluated (49.1%) were found at metaphase II at the end of the culture period. Edwards, Bavister, and Steptoe 13 have reported 60% oocytes reaching metaphase II in culture, and Kennedy and Donahue 21 observed 40.8% and 50% oocytes at metaphase II, respectively, in 199 and Ham's FlO culture media. Thus, maturation rates of cultured human 00cytes reported in the literature vary quite widely, which might be accounted for by
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<
,
FIG. 6. Human ovum fertilized in vitro. Same ovum as in Figs. 4 and 5, after fixation and staining. The two pronuclei have pulled apart. The nucleoli have disappeared. The vitelline membrane was damaged during fixation and the flowing cytoplasm obscured one of the polar bodies (4 o'clock). The zona pellucida was dissolved by the fixative and the other polar body floated away during fixation. Remnants of the sperm tail were not visible. Photographed using Nomarski's optics. Direct magnification. x260.
differences in the initial quality of the oocytes and in culture techniques. In the present study, the fact that a lower proportion of oocytes than those initially classified as likely to mature in culture did actually complete the first meiotic division might be accounted for by the following observation. Figure 7 shows an oocyte which was classified as nonovulatory but in good condition when examined with the dissecting microscope. Immediately after this first examination, the oocyte was denuded and re-examined by differential interference contrast optics (Fig. 7). The germinal vesicle is sharply defined and exhibits a prominent nucleolus containing several inclusions. Two irregular granular masses of a size similar to that of the nucleolus were in close apposition to it, representing nucleolus-associated chromatin. The appearance of the vitellus sug-
gests, however, that discrete vacuoles are distributed throughout the cytoplasm, which could be the sign of early degeneration, as described in the electron microscope study of Zamboni et al. 20 Ordinary phase contrast microscopy did not reveal such an alteration, but it became evident with the use of differential interference contrast optics, especially when the structure of the vitellus shown in Fig. 7 was compared with that of the pronuclear ovum shown in Figs. 4 and 5. The presence or absence of exogenous hormones (FSH, LH, and HCG) in the maturation culture medium did not significantly influence the maturation rate. The endogenous hormonal content, possibly contributed to the maturation medium by the follicular fluid, was unknown. Quality evaluation of 00cytes matured in culture is further complicated by the fact that obvious nuclear
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HUMAN SPERM CAPACITATION AND FERTILIZATION
maturation is not necessarily an adequate criterion for normal developmental potential. Rabbit oocytes for instance, when "matured" outside their follicle and fertilized in vivo, failed to form blastocysts. 22 In contrast, rabbit oocytes, matured in vivo following HCG-induced ovulation, have been fertilized in vitro and produced live young when transferred into synchronized foster mothers. 23-25 Also, some of the human oocytes matured and fertilized in culture have displayed cytoplasmic fragmentation. 16 Although transfer of 00cytes from their follicles to culture media results in nuclear maturation, it appears that the factor or factors essential to complete maturation have been missing from the various culture media which have been used thus far. An explanation for the almost total inability of in vitro matured oocytes to develop into viable fetuses was provided by Thibault and Gerard. 26 Rabbit oocytes having undergone nuclear maturation in culture were penetrated and activated by sperm in vitro. A female pronucleus developed apparently normally, but the sperm nucleus failed to swell. The female pronucleus and the unswollen sperm nucleus came in close apposition to
each other in the center of the vitellus. Subsequently, about half of these abnormally fertilized ova cleaved, apparently normally. Thibault and Gerard attributed the abnormal behavior of the sperm nucleus to the lack of a "male pronucleus growth factor" in oocytes matured in culture. Hence, until complete in vitro maturation requirements are better understood, the best source of fertilizable human 00cytes remains the follicles that have been stimulated to grow by exogenous hormone administration. In the investigations of Steptoe and Edwards,16 the maturing oocytes were obtained by aspirating large follicles just prior to ovulation induced by sequential therapy with HMG and HCG. It has been estimated that an interval of 30-36 hr. is required between the abrupt increase in LH activity and oocyte maturation with subsequent ovulation. 27 In the present study, clomiphene citrate and/or exogenous gonadotropins were given preoperatively in an attempt to initiate the oocyte maturation process in vivo. Several considerations make it difficult to directly evaluate the effectiveness of the preoperative treatment: (1) The donors were essentially
TABLE 4. Maturation, Sperm Penetration, and Fertilization of Human Oocytes as Related to Preoperative
Treatment No. ovum donors
Total oocytes cultured
No.oocytes classified as likely to mature in culture
No.oocytes showing maturation
No. oocytes not fertilized but with sperm in Zona pellucida
%
Perivitelline space
No.oocytes fertilized
%
HMG or clomiphene citrate - HCG 7
19
15
7
23
15
7* (46.7)
9 (47.4)
3 (15.8)
10 (43.6)
6 (26.1)
0
0
0
HCG only 4* (26.7) No treatment 1
2
2
0
* The difference between these two values was not statistically significant.
0
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.
FIG. 7. Immature human oocyte. The corona cells were removed by rolling the specimen between slide and coverslip. The spherical dictyate nucleus, or germinal vesicle, is visible in the center of the vitellus. It contains one prominent nucleolus with several inclusions. Beside the nucleolus are two irregular granular masses of nucleolus-associated chromatin. The appearance of the vitellus suggests that discrete vacuoles are distributed throughout the ooplasm and that this oocyte is undergoing degeneration. Photographed in the fresh state using Nomarski's optics. Direct magnification. x260.
normal women with an endogenous supply of FSH and LH activity characteristic for the day of the menstrual cycle on which operation was performed; (2) some of the donors were on suppressive hormonal therapy at the time of the treatment and operation; (3) logistic considerations regarding the operation schedule made it impossible to administer LH activity in the form of HeG more than 20.5-30.5 hr. before oocyte recovery. However, several indirect observations may be made regarding the effectiveness of the preoperative treatment in spite of its heterogeneity resulting from restrictive clinical considerations. Firstly, previous studies have established that oral contraceptive preparations are unable to abolish an ovarian response to exogenous gonadotropins.28, 29 Secondly, our results (Table 4) suggest that exoge-
nous FSH activity does not augment the incidence of zona pellucida penetration by spermatozoa over that seen with exogenous LH activity (HeG) alone. The stimulating role of HeG in sperm capacitation in the rabbit has been reported, 30 and the possibility exists that a similar phenomenon is important in the human. Thirdly, pretreatment with clomiphene citrate or exogenous gonadotropin may augment in vitro maturation of oocytes by initiation of the maturation process in vivo. It was not possible to determine unequivocally by direct observation whether maturation had been initiated in vivo, thus making it difficult to control the timing of oocyte maturation before insemination. That is, the addition of 20.5-30.5 hr. of possible in vivo maturation to the 38-48 hr. of in vitro maturation results in a 58.5- to 78.5-hr. interval be-
,.
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HUMAN SPERM CAPACITATION AND FERTILIZATION
tween HCG administration to the patient and insemination in vitro. Such a total maturation time would be excessive and might have resulted in the degeneration of otherwise normal mature oocytes before insemination, and therefore in a reduction of the fertilization rate. Normally, the mature ovulated oocyte is released by a follicle.which was submitted to the sequential influence of FSH and LH, and the cellular components of the follicle were influenced by both hormones. In vitro fertilization of human oocytes was obtained by Edwards et al. 13, 14 under culture conditions provided by an adaptation of Bavister's medium, which had proven effective in capacitating hamster sperm in vitro.15 This medium was considered by these authors as conducive to human sperm capacitation as well. In these investigations, however, capacitation occurred in the presence of both cumulus and corona cells, and the possible participation of these cells in the process could not be ruled out. If they were indeed involved, it seemed likely that their activity could be stimulated by one or several of the hormones used for inducing ovulation. Such an interaction could then be reflected by an increased incidence of sperm penetration within and through the zona pellucida. In order to test our working hypothesis, the hormones used for inducing ovulation were added to the fertilization medium in the same concentration ratios as the dosages administered to ovum donors (300 LU. of FSH and LH to 20,000 1. U. of HCG per liter),16 and the effects of such stimulation on sperm penetrating ability were examined. The increase in sperm penetrating ability in the presence of exogenous hormones (Table 3) was found to be statistically highly significant (p < 0.01) when tested for the effect of added hormones. Which factor or factors among the three added hormones are responsible for the observed effect remains to be determined, but LH or the LH-like HCG seem to be
475
prime candidates. These observations call for two main conclusions. First, the fact that sperm penetrating ability can be enhanced by modifying the fertilization medium demonstrates that some conditioning is necessary for human sperm to penetrate the egg investments. That is to say, human sperm require capacitation prior to fertilization as do sperm from other mammalian species. If this were not the case, the observed fertilization rate should have been close or equal to the maturation rate in culture, and all oocytes present should have had their investments penetrated, none of which was observed. The second conclusion is derived from present knowledge of the mechanism of hormone action and indicates that the sperm penetration enhancement effect had to be mediated by the hormonally stimulated follicle cells. All oocytes that were naked at the start of fertilization cultures were found later on with sperm attached to the outer surface of their zonae pellucidae, but no degree of penetration had been achieved at the end of the individual cultures. Penetration to various degrees was observed only in 00cytes initially surrounded by follicle cells. It is now well established that the effects of a hormone such as LH, for instance, are mediated by the second messenger cyclic adenosine mono phosphate (cAMP). 31 According to current concept, cAMP acts by facilitating a cascade of cellular events which involve an active nucleus and result in the synthesis and/or release of specific factors. At the time at which sperm capacitation is occurring in the fertilization milieu, the sperm nucleus, highly condensed, is very unlikely to be functional. On the other hand, the mature oocyte has no nucleus as such. Its genetic material, being present in the form of the second metaphase plate of chromosomes, is also very unlikely to be functional. The only cellular elements, present in the milieu, possessing an active nucleus and therefore most susceptible to responding to hormonal stimu-
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lation are the follicle cells. The nature of the specific factors, synthesized and released by hormonally stimulated follicle cells, and capable of acting on sperm membranes, is a matter for further investigation. Thus, it appears that conditions for human sperm capacitation in vitro might be quite similar to those required by hamster sperm. It also appears that the process in both cases, instead of being simply the result of environmental conditions favoring the spontaneous occurrence of capacitation as previously suggested,13, 15 is indeed mediated by follicle cells, since Gwatkin, Andersen, and Hutchinson 32 have recently demonstrated a key role for the cumulus cells in association with one or more low molecular weight components ofthe cumulus matrix in the hamster sperm capacitation process. The pronuclear ovum, shown in the fresh state in Figs. 4 and 5, and in the fixed state in Fig. 6, fulfilled only two of the three criteria of fertilization. Two clearly defined pronuclei were present in close apposition in the center of the vitellus, and two polar bodies containing chromatin were present in the perivitelline space. The ovum appeared to be in late pronuclear stage as judged by close apposition of pronuclei and the fact that they both contained eight nucleoli, the number of which is known to increase with pronuclear age. 33 , 34 The fertilizing sperm tailor its remnants were not observed, either in the fresh state or after fixation and staining. To satisfy this last criterion of fertilization, the specimen should have been examined either earlier than 28 hr. after the initiation of fertilization culture, or it should have been examined with the electron microscope in order to identify remnants of the sperm tail not visible by light microscopy. The observations of the fertilizing sperm tail within the vitellus reported by Bavister, Edwards, and Steptoe,18 were made on specimens examined from 11-14.5 hr. after the initiation of fertilization cultures. The early
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disappearance of the fertilizing sperm tail is common in the rabbit. By contrast, in the rat, it can still be observed in one of the blastomeres following the first cleavage. The timing of disappearance of the fertilizing sperm tail in the human ovum is not presently known. Owing to the failure to identify by light microscopy remnants of the fertilizing sperm tail in the pronuclear ovum described here, the possibility of artificial activation has to be considered. According to Austin,33 there are nine theoretically possible kinds of ootid. The nine types differ from each other in the number and ploidy of polar bodies and female pronuclei. Each theoretical type is determined by whether a block of either one or both meiotic divisions has occurred and the stage of division (either metaphase or anaphase) at which the block was effective. The particular mechanism that could have led to an ovum exhibiting the features of the pronuclear ovum shown in Figs. 4 and 5, in the absence of the fertilizing sperm, could have operated as follows. The first polar body would have failed to form after the first meiotic division had gone through to anaphase, two distinct groups of chromosomes remaining within the vitellus. Then, the mechanism of the second meiotic division would have operated normally, affecting both groups of chromosomes and resulting in the simultaneous extrusion of two second polar bodies. Thus, by artificial activation without sperm penetration, combined with incomplete first meiotic division, a pronuclear and polar body configuration could theroretically be obtained, which mimics exactly the configuration of the normally fertilized ovum. The differences between these two types of pronuclear ova would be that in the artificially activated ovum the two polar bodies would be haploid, and the two pronuclei, also haploid, would be of female origin. The probability of such an occurrence in human ova is not presently documented, but it seems likely to be very low. Thus,
,.
.
~
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HUMAN SPERM CAPACITATION AND FERTILIZATION
although the possibility of artificial activation could not be ruled out definitely, it seems much more likely that the pronuclear ovum described here had undergone normal monospermic fertilization but was first observed at a late pronuclear stage, a stage at which elements of the fertilizing sperm tail had already disintegrated beyond recognition by means of light microscopy. Recent studies 35 in our laboratory have indeed shown that in ova analyzed at late pronuclear stages, remnants of the fertilizing sperm flagellum not observable by light microscopy are nevertheless demonstrable by electron microscopy. In the same studies, all sperm observed traversing the zona pellucida had undergone the acrosome reaction, which, in the light of the current concept of capacitation, implies that they were capacitated. From the viewpoint of contraception, it is worth mentioning that the pronuclear ovum described here was obtained from a donor using an oral contraceptive. Oocyte recovery in this patient was conducted on Day 15 of her cycle. She had been using Norinyl 1 + 80 up to Day 13 of that cycle. Only HCG (20,000 LV.) was administered 21 hr. 40 min. prior to follicle aspiration in the course of a vaginal hysterectomy performed for elective sterilization. A total of six follicles were aspirated. What appeared to be a follicle of about 2 mm. size at the upper pole of the right ovary was productive of about 2 ml. of pale straw-colored fluid, uncontaminated with blood, indicating that this subsurface follicle was indeed much larger than its apparent size suggested. In the process of aspirating other identified follicles, the content of the ovum recovery unit became contaminated with blood. It is of interest to note that the initial blood contamination of the follicular aspirates did not prevent subsequent fertilization of one of the four oocytes recovered. The oocyte that became fertilized was initially classified as preovulatory, being encased in a rather loose, sticky and silvery
477
cumulus mass. At the end of fertilization culture, this ovum was found to be completely free of corona cells. SUMMARY
Forty-four human oocytes were recovered from 15 patients undergoing elective gynecologic surgery. Oocytes were recovered by direct follicle aspiration, mainly from ovaries in situ exposed in the course of surgery, and from excised ovaries. These oocytes, divided in two lots of approximately equal size, were submitted to maturation culture and to fertilization culture, the latter in the absence or presence of the hormones (FSH, LH, and HCG) used for inducing ovulation. Fertilization cultures in the presence of exogenous hormones resulted in a highly significant (p < 0.01) increase in the incidence of sperm penetration within and through the zona pellucida. The hormonally induced enhancement of sperm penetrating ability demonstrates the need for capacitation of human sperm. Present knowledge on the mechanism of hormone action strongly suggests that the observed effect had to be mediated by follicle cells. An oocyte, recovered from a patient using an oral contraceptive, became fertilized and is described in this study. Acknowledgments. The authors wish to acknowledge the skillful technical assistance of Mrs. Lois Campbell-Taylor, Mrs. Debra Jarashow, Mrs. Simone Soupart, and Mr. John E. Repp, as well as the secretarial skills of Miss Nicqui Hailey. Thanks are also due to Mr. Homer A. Sprague, Department of Biostatistics, Vanderbilt University School of Medicine, for performing statistical analysis of the data, and to the operating room staff of United States Army Tripier General Hospital. REFERENCES 1. AUSTIN, C. R. "Sperm capacitation-Biological
Significance in Various Species." In Advances in the Biosciences (Vol. 4). Schering Symposium on Conception, Pergamon, Oxford, 1969, p. 5. 2. AUSTIN, C. R. The "capacitation" of the mam-
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malian sperm. Nature (London) 170: 326, 1952. 3. CHANG, M. C. Fertilizing capacity of spermatozoa deposited in the Fallopian tube. Nature (London) 168:687, 1951. 4. AUSTIN, C. R Observations on the penetration of sperm into the mammalian egg. Aust J Sci Res B 4:581, 1951. 5. BEDFORD, J. M. Sperm capacitation and fertilization in mammals. Bioi Reprod (Suppl. 2):128, 1970. 6. BARROS, C., BEDFORD, J. M., FRANKLIN, L. E., AND AUSTIN, C. R Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell Bioi 34:C 1, 1967. 7. BEDFORD, J. M. Ultrastructural changes in the sperm head during fertilization in the rabbit. Amer J Anat 123:329, 1968. 8. ALLISON, A. C., AND HARTREE, E. F. Lysosomal enzymes in the acrosome and their possible role in fertilization. J Reprod FertiI21:501, 1970. 9. BRADEN, A. W. H. Properties of the membranes of rat and rabbit eggs. Aust J Sci Res B 5:460, 1952. 10. STAMBAUGH, R, AND BUCKLEY, J. Zona pellucida dissolution enzymes of the rabbit sperm head. Science 161:585, 1968. 11. NEWSLETTER FROM THE EDITORS. Acrosomal enzyme. Bioi Reprod 6:176,1972. 12. STAMBAUGH, R, AND BUCKLEY, J. Identification and subcellular localization of the enzymes effecting penetration of the zona pellucida by rabbit spermatozoa. J. Reprod Fertil 19:423, 1969. '13. EDWARDS, R G., BAVISTER, B. D., AND STEPTOE, P. C. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (London) 221:632, 1969. -14. EDWARDS, R G., STEPTOE, P. C., AND PURDY, J. M. Fertilization and cleavage in vitro of preovulator human oocytes. Nature (London) 227:1307,1970. 15. BAVISTER, B. D. Environmental factors important for in vitro fertilization in the hamster. J Reprod Fertil 18:544, 1969. 16. STEPTOE, P. C., AND EDWARDS, R G. Laparoscopic recovery of preovulator human oocytes after priming of ovaries with gonadotrophins. Lancet 1 :683, 1970. 17. MORGENSTERN, L. L., AND SOUPART, P. Oocyte recovery from the human ovary. Fertil Steril 23:751, 1972. 18. BAVISTER, B. D., EDWARDS, R G., AND STEPTOE, P. C. Identification of the midpiece and tail of the spermatozoon during fertilization of human eggs in vitro. J Reprod Fertil 20:159, 1969. 19. CHANDLEY, A. C. Culture of mammalian oocytes. J Reprod Fertil (Suppl. 14):1, 1971.
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