What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs?*t

Transcription

1 ,. I FERTILITY AND STERILITY Copyright ~ 1983 The American Fertility Society Vol. 40, No.3, September 1983 Printed in U.SA. What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs?*t John E. Gould, B.A.:j: James W. Overstreet, M.D., Ph.D.:j: 11 Hiroko Yanagimachi, M.S. ~ Ryuzo Yanagimachi, Ph.D.~** ~ F. Katz, Ph.D.11 Frederick W. Hanson, M.D.II University of California School of Medicine, Davis, California, and University of Hawaii School of Medicine, Honolulu, Hawaii Human spermatozoa were capacitated in media containing either high concentrations (3.5%) of human serum albumin (HSA) or low concentrations (0.3%) of bovine serum albumin (BSA). The effects of both capacitation media were assessed immediately and after overnight preincubation (18 to 24 hours) by adding a mixture of nonliving human oocytes and living zona-free hamster eggs to the sperm suspension. Overnight preincubation of sperm in media containing 3.5% HSA enhanced sperm fusion with hamster vitelli, but the capacity for zona penetration was lost. These effects could be attributed to the concentration of HSA rather than a general effect of albumin concentration, because overnight preincubation in 3.5% BSA did not interfere with zona penetration. During overnight incubation in 3.5% HSA, the percentage of acrosome reactions increased, as did alterations in the equatorial segment of the acrosome-reacted sperm. The percentage of motile sperm remained high after overnight incubation in 3.5% HSA, but both the mean swimming speed and flagellar activity on the zona surface declined. The sperm also lost their ability for strong zona binding after overnight incubation in 3.5% HSA. Fertil Steril 40:344, 1983 Received September 7, 1982; revised and accepted May 3, ' *Supported by the National Institutes of Health grant HD tpresented at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24,1982, Las Vegas, Nevada. tdepartment of Human Anatomy, School of Medicine, University of California. Recipient of the National Institutes of Health Research Career Development Award HD IIDepartment of Obstetrics and Gynecology, School of Medicine, University of California. ~Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii. **Reprint requests: Ryuzo Yanagimachi, Ph.D., Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, 1960 East West Road, Honolulu, Hawaii There has been considerable recent interest in the application of new techniques in mammalian gamete biology to the diagnosis and treatment of human infertility. Clinical studies from several laboratories have suggested that human male fertility may be predicted by the ability of human spermatozoa to fuse with zona-free hamster eggs in vitro.1-5 The biologic basis for such an association is probably sound, but false-positive and false-negative predictions must be anticipated with this assay.6, 7 We have been interested in the biologic phenomena being measured in such assays, and in their application as tests for specific abnormalities of human sperm function. In previous clinical studies, we used a mixed gamete assay system, incubating nonliving human oocytes and living 344 Gould et a!. Sperm functions assessed by zona-free hamster eggs Fertility and Sterility

2 13 -I.. in in of al.ie in gs.ald ed lein li :al m, ng zona-free hamster eggs together with the sperm suspension being tested. 8 The properties of the human zona pellucida that are related to sperm penetration appear to be similar regardless of the maturity or viability of the oocyte. 9 This has permitted the use of nonviable and/or immature human oocytes for bioassay of human sperm-zona pellucida interactions. 7, 9,10 In the mixed gamete assay, the vitellus of the mature hamster oocyte is used as a substitute for the nonfertilizable human gamete, and it is presumed to measure sperm functions required for the fusion of the sperm cell with the oolemma and its incorporation into the ooplasm. 7 The observation of sperm entry into the zona pellucida would suggest the integrity of a coordinated sequence of sperm functions that includes capacitation, zona binding, the acrosome reaction, and appropriate movements of the sperm flagellum. 11 Completion of capacitation and the acrosome reaction are also thought to be required for fusion of spermatozoa with zona-free hamster eggsp However, the sensitivity of the latter assay to other essential functions of the fertilizing sperm cell is unknown. Many of these functions can now be measured directly or can be deduced by experimental manipulation in vitro. In this paper we describe experiments with a model system that has allowed us to study in greater detail the nature of sperm functions that may be monitored by in vitro fertilization assays now being advocated for routine clinical use. MATERIALS AND METHODS PREPARATION OF THE MIXED GAMETE SUSPENSIONS Semen specimens, collected by masturbation, were obtained from five fertile donors and were allowed to liquefy at room temperature for 30 to 60 minutes. Aliquots (0.3 to 0.5 ml) of semen were injected into 12 x 75-mm plastic tubes (Falcon Plastics, Oxnard, CA) beneath 3 ml of culture medium (Biggers, Whitten and Whittingham medium [BWW]13 as modified by Yanagimachi et al.14) containing 3 mg/ml of bovine serum albumin (BSA; Fraction V, Sigma Chemical Company, St. Louis, MO). The tubes were inclined at a 45-degree angle in a 37 C incubator with an atmosphere of 5% CO2 in air. During the next 45 to 60 minutes, motile sperm cells swam upward out of the seminal plasma and into the culture medium. The layer of medium containing the spermatozoa was carefully removed and centrifuged for 5 minutes at 500 x g. The sperm pellet was washed twice by dilution with culture medium and recentrifugation. The washed sperm pellet was finally resuspended for capacitation either in the same culture medium (0.3% BSA) or in BWW medium containing 35 mg/ml of human serum albumin (HSA; Fraction V, Sigma). The latter medium (3.5% HSA) is similar to the one used in our previous study8 except that in the present studies no BSA was present in the HSA medium. Human oocytes were recovered from ovarian tissue, stored at - 80 C, and prepared for incubation as previously described. 8 These oocytes were always nonviable and were never fertilizable. They were used to assess sperm interaction with the zona pellucida. Hamster vitelli were obtained by standard methods for superovulation, ovum recovery, and removal of the zona pellucida.12 A mixture of 5 to 20 hamster vitelli and 1 to 3 human oocytes were incubated with the sperm suspension being tested. CAPACITATION OF SPERMATOZOA IN MEDIUM CONTAINING 3.5% HSA OR 0.3% BSA Five paired experiments (1 to 5) were carried out in which spermatozoa from the same semen sample were resuspended in the BSA medium and in the HSA medium. For two additional experiments (6 and 7) with BSA media and one with HSA media (8), different semen samples were used. Each sperm suspension (i.e., HSA and BSA) was divided into two aliquots. Observations were subsequently made of the behavior of the "fresh" sperm suspensions in comparison with the second sperm aliquots, which were preincubated overnight. Each fresh sperm suspension was combined with human oocytes and hamster vitelli within 30 minutes of washing, and the incubation was continued in the CO2 incubator for 4 additional hours. These incubations usually took place under paraffin oil (SV125/135, Fisher Chemical Company, Pittsburgh, PA) in 35 x 10- mm polystyrene Petri dishes (Falcon Plastics). In three experiments (3, 5, and 7) the incubations were carried out in flat capillary tubes (25 mm long, 4 mm wide, 0.4 mm deep; Microslide, Vitro Dynamics, Rockaway, NJ) and sealed with paraffin oil to facilitate direct observations of sperm motility (see below). The volume of the sperm lity Vol. 40, No.3, September 1983 Gould et ai. Sperm functions assessed by zona-free hamster eggs 345

3 suspension was 200 j.li when Petri dishes were used for incubation and 40 j.li when flat capillary tubes were used. Sperm concentrations ranged from 8 x 10 5 to 1 X 10 7 sperm/ml and were matched in HSA and BSA media within the paired experiments. The second aliquot of sperm suspension was preincubated in the CO2 incubator for 18 to 24 hours under oil in a Petri dish as described above. At the end of the preincubation period, hamster vitelli and human oocytes were added, and an additional 4 hours of incubation were allowed for gamete interaction. These final incubations were carried out either in the same Petri dishes or after transfer to a capillary tube, depending on the experiment. Human oocytes were mounted between a glass slide and cover slip and were rolled to identify spermatozoa in the perivitelline space. 9 Hamster vitelli were similarly examined, and the number of attached sperm as well as the number of decondensing sperm heads within the ooplasm was counted. 12 ASSESSMENTS OF SPERM SWIMMING SPEEDS Videomicrographic assessments of sperm swimming speeds were made in experiments 3, 5, and 7. After mixing the spermatozoa, oocytes, and vitelli in flat capillary tubes (see above), sperm motility was recorded at 37 C on videotape using a phase-contrast microscope with either a 10 x or 20 x objective lens. This equipment, together with the procedures for recording and analyzing the tapes, has been previously described. 15 Twenty-five to 50 spermatozoa were analyzed in each suspension for the percentage of motility, and a mean swimming speed was determined. After. taping, the capillary tube was incubated for 4 hours, and the oocytes and vitelli were evaluated in the standard manner. When the initial experiments showed that the human zona pellucida was not penetrated by spermatozoa preincubated in medium containing 3.5% HSA, additional experiments were carried out to determine whether the failure of zona penetration resulted from a specific effect of HSA. Three paired experiments were carried out in which spermatozoa from the same semen sample were suspended either in BWW medium containing 3 mg/ml of HSA or 35 mg/ml of BSA. One aliquot from each suspension was preincubated overnight as in the previously described experiments. REINCUBATION OF HUMAN OOCYTES WITH LABELED SPERM SUSPENSIONS We designed experiments to determine whether the failure of zona penetration after preincubation was due to a change in the sperm suspension or to an effect of the medium on the zona. In five replicate experiments, a total of nine oocytes were removed after 4 hours in a sperm suspension that had been preincubated in medium containing 3.5% HSA, and these oocytes were transferred to a fresh suspension of spermatozoa in the same type of medium. In three of the five experiments, the fresh spermatozoa were labeled with the fluorescent dye tetramethylrhodamine isothiocyanate to ensure their positive identification. 16 Labeled sperm were distinguished by their red fluorescence under ultraviolet illumination. The detailed method used for fluorescent labeling of human spermatozoa and a description of their appearance in the human zona pellucida have been previously published. 16 ULTRASTRUCTURAL OBSERVATIONS To investigate whether ultrastructural changes in the sperm cells were associated with failure of zona penetration, three experiments were carried out using the 3.5% HSA medium (one with fresh sperm and two with preincubated sperm). At the conclusion of each experiment (4 hours of in cuba- CAPACITATION OF SPERMATOZOA IN MEDIA CONTAINING 3.5% BSA OR 0.3% HSA Figure 1 A flat capillary tube (A) containing human oocytes and spermatozoa is pla~ed on a glass slide (E), to which a 5-cm length of 100 ILl capillary tube (B) has been previously glued. A flame-drawn 10 ILl capillary tube (C) is inserted through B to enter the flat tube.. Positive pressure is applied through a rubber tube W) to displace the small amount of fluid that ente~d. the tube by cal?illary action. When the positive pressure. IS mte~upted,. fluid reent:ers the tube, creating a gentle suction at Its openmg, by which the oocyte is secured. The rubber tubing is then removed, and tube C may be freely ~tated within tube B to roll the attached oocyte. A magnified view of such an oocyte is illustrated (F). 346 Gould et al. Sperm functions assessed by zona-free hamster eggs Fertility and Sterility

4 tion for fresh spermatozoa and 22 to 28 hours of incubation for preincubated spermatozoa) the spermatozoa, human oocytes, and hamster vitelli were examined with the electron microscope. The gametes were processed in the conventional manner after fixation in 2.5% glutaraldehydep Assessment was made of the spermatozoa in suspension as well as of the sperm associated with the zona pellucida and with the surface of the hamster vitellus. When the thin sections were examined, the number of spermatozoa with intact acrosomes and the number with acrosome reactions were counted. The condition of the membranes overlying the equatorial segment of the acrosome was also noted for the acrosome-reacted spermatozoa. Only spermatozoa cut sagittally or semisagittally were included in this tabulation, and spermatozoa with disrupted plasma membranes and/or disorganized intracellular components were not considered. liigh-speed VIDEOMICROGRAPHIC STUDIES OF SPERM MOVEMENT ON THE ZONA PELLUCIDA To examine what alterations in sperm flagellar activity might be involved in the failure of zona penetration, two paired experiments were carried out in which observations were made ofthe movement of fresh and preincubated spermatozoa during interaction with the zona pellucida in medium containing 3.5% HSA. This required highspeed video recordings of spermatozoa on the zona surface. The experimental conditions were the same as those in previous experiments utilizing flat capillary tubes, except that the oocyte was held by gentle suction on the tip of a fine capillary pipette (Fig. 1). Rotation of this pipette allowed the zona to be "rolled" within the capillary tube, thus bringing the flagellar envelope into the plane of focus. Video recordings were made at 60 frames/second using a high-speed video camera (PCSM6500 MF, Tritronics, Burbank, CA) and a JVC %-inch video cassette recorder (CR6600, US JVC Corporation, Elmwood Park, NJ). Phasecontrast optics with a 20 x objective lens and a 5 x ocular lens were employed. The tape was analyzed with reference to a continuous display of elapsed real time on the video screen. The tapes from each experiment were analyzed in slow motion, and the flagellar beat frequencies of 25 spermatozoa in the fresh suspension were determined. Sequential tracings of beat shapes were also made in many cases. 18 Because fewer sperm were present on the zona surface when preincubated sperm suspensions were studied, only a limited number of observa- Table 1. Penetration of Human Spermatozoa into the Human Zona Pellucida and Hamster Vitellus After Capacitation in Low-BSA Medium (0.3% BSA;a Experiment b Sperm suspension No. of hamster vitelli No. of spermatozoa C No. of human oocytes Total Penetrated Attached Penetrated Total Penetrated d 1 Fresh 12 7 (58%) Preincubated (100%) Fresh 5 2 (40%) Preincubated (100%) > 100 > Fresh 12 4 (33%) Preincubated (100%) > 50 > Fresh 8 2 (25%) NDe Preincubated (100%) > 100 >8 ND 5 Fresh 16 0(0%).1 0 ND Preincubated (100%) > ND 6 Fresh 13 2 (15%) Preincubated 11 1 (9%) Fresh (69%) Preincubated 18 6 (33%) Group total Fresh (34%) Preincubated (78%) 11 6 aan aliquot of the fresh sperm suspension was compared with an aliquot preincubated for 18 to 24 hours. The differences between percentages of hamster vitelli penetrated by preincubated and fresh sperm suspensions were at the borderline of statistical significance, as analyzed by the randomization test for matched pairs (P = 0.06). bexperiments 1 to 5 were paired comparisons with experiments 1 to 5 in Table 2. CAttached spermatozoa were calculated as the mean number per all vitelli. Penetrated spermatozoa were calculated as the mean number per penetrated vitellus. dspermatozoa within the thickness of the zona pellucida and/or within the perivitelline space. end, no data. Vol. 40, No.3, September 1983 Gould et al. Sperm functions assessed by zona-free hamster eggs 347

5 tions could be made ofthe behavior of these populations. RESULTS Following Overnight Incubation in 3.5% HSA, Spermatozoa Failed to Penetrate the Zona Pellucida, but Fusion with Zona-Free Hamster Eggs Was Enhanced Preincubation of sperm suspensions for 18 to 24 hours resulted in an increase in the percentage of penetrated hamster vitelli and a larger number of decondensing sperm heads per penetrated egg (Tables 1 and 2). This trend was apparent for spermatozoa suspended in both 3.5% HSA and in 0.3% BSA. In the latter case, the increase in the percentage of penetration failed to reach statistical significance because of the unexplained poor penetration rates observed in experiments 6 and 7. The human zona pellucida was always penetrated in medium containing 3.5% HSA when a fresh sperm suspension was tested (Table 2). However, following preincubation in 3.5% HSA, spermatozoa consistently failed to penetrate the human zona pellucida in spite of the fact that every hamster vitellus was penetrated. The percentage of motile sperm and their mean swimming speeds declined during preincubation in both HSA and BSA media, and these reductions appeared to be greater in the HSA media (Table 3). However, in view of the consistent penetration of the hamster vitelli by the same sperm suspensions, these results could not be satisfactorily attributed to a decline in sperm viability during preincubation. Failure of Zona Penetration Resulted from a Specific Effect of HSA The physiologic alterations in spermatozoa that prevented zona penetration did not occur when spermatozoa were preincubated overnight in medium containing 3.5% BSA (Table 4). Both the human zonae and the hamster vitelli were consistently penetrated in this medium. Failure of Zona Penetration After Preincubation in 3.5% HSA Resulted from Changes in the Sperm Suspension Rather than an Alteration of the Zona Pellucida All nine of the oocytes were penetrated after removal from the preincubated sperm suspension and introduction to fresh spermatozoa. Of the five oocytes with a visible perivitelline space, three contained sperm. In the three experiments in which the fresh sperm suspension carried a fluorescent label, all six oocytes had fluorescent sperm in the zona and both oocytes with perivitelline spaces contained fluorescent sperm. All spermatozoa associated with these zonae fluoresced, confirming that they were penetrated only after transfer to the second sperm suspension. After Overnight Incubation in 3.5% HSA Sperm Zona Binding Was Inhibited When the behavior of spermatozoa in medium containing 3.5% HSA was compared before and after preincubation, a striking difference was noted in the tenacity of sperm binding to the zona surface. Many sperm were associated with the zona after recovery from either fresh or preincubated sperm suspensions. However, the preincubated sperm were easily dislodged from their sites of zona attachment during gentle handling. The fresh spermatozoa, in contrast, remained firmly bound even after vigorous manipulation. The Incidence of Acrosome Reactions Increased Significantly During Overnight Incubation in 3.5%HSA The percentage of spermatozoa that had undergone the acrosome reaction increased from 8% after 4 hours of incubation to 62% after 22 to 28 hours (Table 5). Of the spermatozoa remaining on the zona surface after 4 hours' incubation, 46% had undergone the acrosome reaction, in comparison with 82% that underwent acrosome reactions on the zona of oocytes recovered from sperm suspensions incubated for a total of 22 to 28 hours (preincubated sperm). Numerous spermatozoa were seen within the thickness of the zona pellucida of oocytes recovered from 4-hour sperm suspensions; all had undergone the acrosome reaction. No spermatozoon was observed within the zona pellucida of oocytes recovered from preincubated sperm suspensions. There was little evidence of alteration of the equatorial segment (Fig. 2) in the 4-hour sperm suspensions, but the majority of acrosome-reacted sperm in the preincubated suspensions showed apparent vesiculation or loss of the equatorial segment. The spermatozoa on the surface of the hamster vitelli were almost uniformly acrosome-reacted, regardless of 348 Gould et al. Sperm functions assessed by zona-free hamster eggs Fertility and Sterility

6 Table 2. Penetration of Human Spermatozoa into the Human Zona Pellucida and Hamster Vitellus After Capacitation in High- HSA Medium (3.5% HSAya Experiment b Sperm suspension No. of hamster vitelli No. of spermatozoa C No. of human oocytes Total Penetrated Attached Penetrated Total Penetrated d 1 Fresh 10 9 (90%) Preincubated 9 9 (100%) > 100 > Fresh 14 6 (43%) Preincubated (100%) > 100 > Fresh 14 3 (21%) Preincubated (100%) > 100 > Fresh (71%) Preincubated (100%) > 100 > Fresh 17 4 (24%) Preincubated (100%) > 100 > Fresh Preincubated Group total Fresh (47%) Preincubated (100%) 10 0 aan aliquot ofthe fresh sperm suspension was compared with an aliquot preincubated for 18 to 24 hours. Percentages of hamster vitelli penetrated are significantly higher for preincubated than for fresh sperm suspensions, as verified by the randomization test for matched pairs (P < 0.05). bexperiments 1 to 5 were paired comparisons with experiments 1 to 5 in Table 1. C Attached spermatozoa were calculated as the mean number per all vitelli. Penetrated spermatozoa were calculated as the mean number per penetrated vitellus. dspermatozoa within the thickness of the zona pellucida and/or within the perivitelline space. whether or not the sperm suspensions were preincubated. Many of these sperm also showed changes in the equatorial segment (Table 5). Spermatozoa on the Zona Surface Had a Lower Beat Frequency and Reduced Flagellar Amplitude After Overnight Incubation in 3.5%HSA In the two experiments in which the movement of fresh spermatozoa on the zona surface was analyzed, the mean beat frequencies of 25 sperm per experiment were 22.0 ± 0.9 and 17.2 ± 0.7 beats/ second (mean ± standard error ofthe mean). The amplitude of the flagellar beat was 7.8 ± 0.6 and 10.1 ± 1.2,...,m in the two experiments (n = 8 sperm per experiment). The sperm appeared to be vigorously thrusting against the zona substance. In contrast, the flagella of the preincubated sperm appeared to have a lower beat frequency (12.6 ± 0.8 beats/second, n = 13) and a much lower beat amplitude (3.2 ± 0.4,...,m, n = 16). These sperm gave the appearance of quivering on the zona surface rather than thrusting against it. DISCUSSION The fertilization media used in these experiments are similar to those now being used in basic research and in clinical studies.19 In most clinical studies, low-albumin media have been used for sperm capacitation over relatively long preincubation intervals (18 to 24 hours),1-5 and highalbumin media have been used for rapid capacitation during short (4-hour) incubations. 8 Combinations of low-albumin media with short preincubations have also been employed.20, 21 In the model system studied in these experiments, rapid sperm capacitation was probably induced, and a substantial interval of postcapacitation sperm aging may have occurred before introduction of the 00- cytes. Such prolonged preincubation of spermatozoa in high-hsa media has not been frequently used in clinical assays, and our comments are not intended as a critique of specific assays now in use. Nevertheless, the results of these experiments are relevant to our understanding of the biology of these in vitro test systems. Following a 4-hour incubation in BWW medium containing 35 mg/ml of HSA, freshly washed human sperm consistently penetrated both the human zona pellucida and hamster vitellus. When the incubation time was extended to 18 to 24 hours, the spermatozoa lost their capacity to penetrate the human zona pellucida. In contrast, these identical conditions enhanced the fusion of these preincubated sperm cells with the hamster vitellus. This result was clearly attributable to an alteration in the physiology of the preincubated Vol. 40, No.3, September 1983 Gould et al. Sperm functions assessed by zona-free hamster eggs 349

7 Table 3. Percentage of Motility" and Mean Swimming Speeds b of Spermatozoa in Experiments 3,5, and 7 Percentage of Mean swimming Medium Experiment Sperm suspension No. of sperm motility speed ± SEM c 0.3% BSA medium 3 Fresh ± 3.6 Preincubated ± Fresh ± 5.0 Preincubated ± Fresh ± 4.3 Preincubated ± % HSA medium 3 Fresh ± 3.7 Preincubated ± Fresh ± 2.9 Preincubated ± 3.1 "The percentage of motility was significantly higher for fresh than for preincubated spermatozoa, as verified by the randomization test for matched pairs (P < 0.05). bswimming speed was significantly higher for fresh than for preincubated spermatozoa, as verified by analysis of variance (P < 0.001). CStandard error of the mean. IJ.m!sec sperm cells rather than a direct effect on the zona pellucida; for when unpenetrated oocytes were recovered from the preincubated sperm suspension and transferred to a second suspension of fresh spermatozoa, all were penetrated. Similarly, a change in the composition of the fertilization medium can be ruled out as an explanation for our findings; preincubated sperm, when transferred to fresh media, did not significantly increase their swimming speed and failed to penetrate the human zona. Conversely, the suspending medium from the preincubated sperm suspension did not adversely affect zona penetration by fresh spermatozoa (data not shown). Sperm capacitation and the acrosome reaction are thought to be required both for penetration of the zona pellucida and for fusion with the hamster vitellus. ll Therefore, these functions are. measured in both assays. Our studies with the electron microscope suggest that the percentage of acrosome-reacted sperm increased during preincubation, and it is likely that this was a contributing factor to the higher rate of fusion with the hamster vitelli in the preincubated sperm suspension!" _ In spite of the increase in the number of acrosome-reacted sperm in the medium after preincubation, none was able to enter the human zona pellucida. It is noteworthy that a high proportion of these acrosome-reacted spermatozoa in the sperm suspension and on the zona surface showed significant changes in the integrity of their equatorial segments. The alteration of this region may reflect a rapid rate of sperm capacitation followed by senescence of the acrosome-reacted spermatozoa in vitro. Similar alterations in equatorial segments have been observed in hamster sperm suspensions after lengthy preincubations. 22 These hamster sperm, although capable of fusing with zona-free hamster eggs, were unable to penetrate the hamster zona pellucida. 22 Thus, there appears to be a similarity between results obtained in the homologous hamster-hamster system and those obtained in the heterologous human-hamster system. A principal factor contributing to the failure of zona penetration may have involved the decline in sperm motility that occurred during preincubation. Although the percentage of motile sperm remained high, the vigor of flagellar activity declined substantially. This was reflected in a reduction in the mean swimming speed and in an apparent decrease in both the flagellar beat frequency and the amplitude of these sperm cells on the 'zona surface. Since the power output of the sperm cell would be diminished by each of these Table 4. Penetration of Human Spermatozoa into the Human Zona Pellucida and Hamster Vitellus After 18 to 24 Hours of Capacitation inbww Medium Containing Either 0.3% HSA or 3.5% BSAa Hamster vitelli O.3%HSA Human oocytes 4/6 C Hamster vitelli 3.5%BSA Human oocytes 5/5 C atbe combined data from three paired experiments are given. bnumber of vitelli penetrated per total number of vitelli. cnumber of oocytes penetrated per total number of oocytes. 350 Gould et al. Sperm functions assessed by zona-free hamster eggs Fertility and Sterility

8 Table 5. Morphologic Characteristics of Human Spermatozoa After Capacitation in Medium Containing 3.5% HSA Location of sperm Total incubation time No. of tabulated sperma Total Intact acrosome (92%) 19 (38%) 12 (54%) 5 (18%) 0(0%) 2 (7%) Acrosome reaction Altered equatorial segment b hr In suspension On surface of zona pellucida On surface of vitellus 4c 22 to 28 d 4 22 to to (8%) (62%) (46%) (82%) (100%) (93%) (25%) (77%) (40%) (82%) (58%) (64%) aonly those spermatozoa with undisrupted plasma membranes and cut sagittally or semisagittally were tabulated. bpercentage among acrosome-reacted sperm with equatorial segment partially or totally absent (Fig. 2). cfresh spermatozoa were examined after 4 hours of incubation with the oocytes. dpreincubated spermatozoa were examined after 4 hours of incubation with the oocytes. reductions,23 it is likely that these sperm cells lacked the necessary thrust for passage through the zona substance. Conversely, this decrease in sperm movement may have actually enhanced sperm-vitelline interaction, since more weakly motile sperm would tend to settle on the vitelline surface and maintain such contact for a longer interval. The precise sequence of events undergone by the human spermatozoon during penetration of the ovum has not been described. Specific receptors on the sperm surface may be involved in their attachment and binding to the zona pellucida. Our ultrastructural observations suggest that both acrosome-reacted and intact spermatozoa can attach to the zona pellucida. The fact that acrosome-reacted spermatozoa were more frequently encountered on the zona and vitelline surfaces than in the suspension suggests that loss of the acrosome may enhance sustained contact. However, other factors must also be involved, since the capacity for strong sperm-zona binding appeared to be lost by all cells during overnight incubation in high-hsa media. Some investigators believe that a specific type of binding by spermatozoa to the zona surface must occur before penetration can beginy The inability ofpreincubated sperm to tightly bind to the zona surface may therefore have contributed to their failure to penetrate. The failure of zona penetration that followed prolonged incubation in medium containing HSA was not apparent with medium containing similar concentrations of BSA. The significant diminution of sperm motility and the loss of zona binding was not observed after preincubation of spermatozoa in BWW medium containing 3.5% BSA (data not shown). The mechanism of this y Vol. 40, No.3, September 1983 unique effect of HSA on the physiology of human spermatozoa was not determined in these experiments. It could involve the properties of the albumin itself or biochemical contaminants of the albumin preparation. 24 The complexity of these in vitro systems should not be surprising in view of our current knowledge of the physiology of mammalian fertilization. It is entirely appropriate that this knowledge now be applied in a clinical context, and there is a real possibility that specific abnormalities of sperm function may soon be shown to cause human male infertility. On the other hand, we must guard against unrealistic expectations for Figure 2 Electron micrographs of the heads of human spermatozoa. (A), A sagittal section of the head of a spermatozoon with intact acrosome. The brackets denote equatorial segments of the acrosome. (B), A sagittal section of the head of an acrosomereacted spermatozoon. Notice that the equatorial segment present on the right side of the spermatozoon (bracket) is absent on the left side. Gould et a1. Sperm functions assessed by zona-free hamster eggs 351

9 this technology. The present experiments offer a clear warning that successful penetration of zonafree hamster eggs may still occur in the presence of structural and/or functional abnormalities that would preclude fertilization of human eggs in vitro. Acknowledgments. The authors gratefully acknowledge the technical assistance of Ms. Charlene Brazil, the secretarial assistance of Ms. Pat Blondheim, and the artistic assistance of Mr. Todd Bloom. REFERENCES 1. Rogers BJ, Van Campen H, Ueno M, Lambert H, Bronson R, Hale R: Analysis of human spermatozoal fertilizing ability using zona-free ova. Fertil Steril 32:664, Hall JL: Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 35:457, Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 57:620, Stenchever MA, Williamson RA, Leonard J, Karp LE, Ley B, Shy K, Smith D: Possible relationship between in utero diethylstilbestrol exposure and male fertility. Am J Obstet Gynecol 140:186, Tyler JPP, Pryor JP, Collins WP: Heterologous ovum penetration by human spermatozoa. J Reprod Fertil 63:499, Overstreet JW: Evaluation of sperm function by tests of sperm ovum interaction in vitro. In Human Fertility Factors, Edited by A Spira, P Jouannet. Paris, INSERM, 1982, p Bolanos JR, Overstreet JW, Katz DF: Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2 C to 5 C. Fertil Steril 39:536, Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW: Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 33:534, Overstreet JW, Hembree WC: Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro. Fertil Steril 27:815, Overstreet JW, Gould JE, Katz DF, Hanson FW: In vitro capacitation of human spermatozoa after passage through a column of cervical mucus. Fertil Steril 34:604, Yanagimachi R: Mechanisms of fertilization in mammals. In Fertilization and Embryonic Development in Vitro, Edited by L Mastroianni Jr, JD Biggers. New York, Plenum Publishing, 1981, p Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. BioI Reprod 15:471, Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel. San Francisco, Freeman, 1971, p Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi CA, Nicolson GL: Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril 31:562, Katz DF, Overstreet JW: Sperm motility assessment by videomicrography. Fertil Steril 35:188, Blazak WF, Overstreet JW, Katz DF, Hanson FW: A competitive in vitro assay of human sperm fertilizing ability utilizing contrasting fluorescent sperm markers. J Androl 3:165, Yanagimachi R, Noda YD: Ultrastructural changes in the hamster sperm head during fertilization. J Ultrastruct Res 31:465, Katz DF, Overstreet JW: Biophysical aspects of human sperm movement. In The Spermatozoon: Maturation, Motility and Surface Properties, Edited by DW Fawcett, JM Bedford. Baltimore, Urban and Schwarzenberg, 1979, p Rogers BJ: Mammalian sperm capacitation and fertilization in vitro: a critique of methodology. Gamete Res 1: 165, Binor Z, Sokoloski JE, WolfDP: Penetration of the zonafree hamster egg by human sperm. Fertil Steril 33:321, Zausner-Guelman B, Blasco L, Wolf DP: Zona-free hamster eggs and human sperm penetration capacity: a comparative study of proven fertile donors and infertility patients. Fertil Steril 36:771, Barros C, Fujimoto M, Yanagimachi R: Failure of zona penetration of hamster spermatozoa after prolonged preincubation in a blood serum fraction. J Reprod Fertil 35: 89, Dresdner RD, Katz DF: Relationships of mammalian sperm motility and morphology to hydrodynamic aspects of.cell function. BioI Reprod 25:920, Meizel S: Stimulation of sperm fertility in vitro by exogenous molecules. Reproduccion 5:169, Gould et al. Sperm functions assessed by zona-free hamster eggs Fertility and Sterility