PENETRATION OF THE ZONA FREE HAMSTER EGG BY HUMAN SPERM*

Transcription

1 FERTILITY AND STERILITY Copyright t, 1980 The American Fertility Society Vol, 33, No, 3, March 1980 Printed in U,SA, PENETRATION OF THE ZONA FREE HAMSTER EGG BY HUMAN SPERM* ZVI BINOR, M.D. JOSEPH E. SOKOLOSKI, B.A. DON P. WOLF, PH.D.t Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Human sperm become capable of penetrating zona-free hamster eggs after preincubation in an appropriate culture medium. This observation has led to the development of an assay for characterizing the fertilizing capacity of human spermatozoa. In the present study, the incorporation of sperm by zona-free hamster eggs was quantitated, and several parameters that contribute to penetration were evaluated. The importance of the motile sperm concentration was established; no penetration was seen at concentrations lower than 6 x u;5 motile cells/ml, whereas above this level the mean number of incorporated sperm per egg was linearly related to concentration. Freeze-thawed sperm, although capable of penetrating zona-free hamster eggs, did so with lesser frequency than did fresh sperm at equal concentrations of motile sperm. Kinetic experiments indicated that eggs were maximally penetrated after 5 hours of exposure to capacitated sperm and that the cessation in sperm incorporation seen at this time resulted from egg-related changes that occurred during aging in vitro. A protocol for evaluating the "fertilizing capacity" of human sperm samples was outlined incorporating the findngs from the present study. Using these conditions, reproducible penetration levels were obtained when several ejaculates obtained from the same donor over a 3 -month interval were tested at similar motile sperm concentrations. Fertil Steril 3.3: The major barrier to interspecies fertilization in mammals appears to be the acellular zona pellucida, a glycoprotein-rich layer that surrounds the vitellus at ovulation.! Additionally, in some species the egg plasma membrane acts as a safeguard against cross-species fertilization, since only low levels of penetration of zona-free eggs by heterologous sperm have been reported. 2, 3 An exception in this regard is the hamster-zona-free eggs of this species incorporate large numbers of heterologous sperm. 3,4 Advantage has been taken of this find- Received September 4, 1979; revised October 22, 1979; accepted October 26, *Supported by National Institutes of Health Contract N01- HD , the Mellon Foundation, and National Institutes of Health Grant HD treprint requests: Don P. Wolf, Ph.D., Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, 36th and Hamilton. Walk G3, Philadelphia, Pa ing by Yanagimachi et al.,5 who described a test for evaluating human sperm capacitation; sperm became capable of penetrating zona-free hamster eggs only after preincubation in culture medium. Subsequent investigators 6-8 have used this model to subdivide clinical populations; semen specimens from infertile patients did not penetrate zona-free hamster eggs with the same frequency as did samples obtained from normal donors. More recently, the human-hamster (humster) system has been used in evaluating the influence of seminal plasma on penetration 9 and in assessing the chromosomal constitution of human spermatozoa.!o In order to adapt the humster system to the routine analysis of large numbers of specimens, a relatively simple, reproducible in vitro procedure is required. The techniques employed for this test by other authors have not produced consistent results in our laboratory. Moreover, it would seem 321

2 322 BINOR ET AL. useful to correlate sperm motility and progression with penetrability in the humster system, since these parameters continue to serve as clinical measurements offertilizing potential. In the present study, we have re-examined the humster model, quantitated penetration as the mean number of sperm per egg, and established a reproducible protocol for in vitro testing. In the process, the importance of the motile sperm population in determining penetration levels was documented. Additionally, the penetration characteristics of freezethawed sperm were examined. MATERIALS AND METHODS Mature golden hamsters (LVG, Charles River Breeding Laboratories, Wilmington, Mass.) were superovulated beginning on day 1 of the cycle (the day of postestrous vaginal discharge 11 ) via an intraperitoneal injection of pregnant mare serum (30 IV, Organon, Oss, Holland) followed 48 hours later by human chorionic gonadotropin (hcm (30 IV, Sigma Chemical Co., St. Louis, Mo.). Eggs were harvested from excised oviducts 15 to 17 hours after hcg administration and freed from surrounding cumulus by exposure to O.llk hyaluronidase (510 NF units/mg, Sigma Chemical Co.) at 37 C in Biggers, Whitten, and Whittingham (BWW) medium 12 containing 3 mg/ml human serum albumin (Sigma Chemical Co., fraction V) at ph 7.5. Following hyaluronidase treatment, eggs were washed repeatedly in BWW before their zonae were removed by exposure to 0.1% bovine pancreatic trypsin (3000 NF units/mg, NBC) for 2. to 3 minutes. This step and all subsequent steps were carried out at 37 C, unless specified otherwise. Zona-free eggs were washed repeatedly in fresh culture medium and then maintained until use in 0.2-ml drops offresh medium under silicone oil (Dow-Corning 200 dielectric fluid, Brownwell Electronics, Philadelphia, Pa.) in micro dishes (12 x 17 mm, glass; A. H. Thomas Co., Philadelphia, Pa.) under a 5% CO 2 in air environment. Human ejaculates obtained by masturbation from healthy donors were allowed to liquefy for 30 to 45 minutes at 37 C. Sperm motility and progression were assessed microscopically, and sperm concentration was quantitated with a hemocytometer. The ejaculate was then diluted to 3 to 4 times its original volume with BWW, filtered through two layers of tissue paper (Kimwipes, Kimberly Clark), and centrifuged at 600 x g for 7 minutes at ambient temperature. The final pellet was resuspended in 1 ml offresh medium (approximately 3.5 March 1980 X 10 7 sperm/mn and transferred to a sterile plastic tube (Falcon, Fisher Scientific Co., Pittsburgh, Pa.) for capacitation (4 to 5.5 hours' preincubation l. Immediately prior to insemination, the motile sperm concentration was redetermined by microscopic analysis, and the suspension was diluted to yield a concentration of 5 x 10 2 to 5 X 10 6 motile sperm/ml in the final insemination mixture. Liquefied ejaculates for freezing were dil uted 1: 1 (v/v) with a modified Krebs-Ringer bicarbonate medium containing 15% glycerol 13 and incubated for 15 minutes at 37 C. Aliquots (0.05 mn were transferred to plastic ampules, cooled to - 20 C, suspended in liquid nitrogen vapor for 2 to 3 minutes, and finally lowered into liquid nitrogen for storage until use. Samples were thawed at 37 C for 20 minutes, and sperm were prepared for insemination directly or were capacitated by preincubation for 2 hours according to the procedure described above for fresh specimens. Hamster sperm suspensions were prepared by mincing the distal end of two caudae epididymides in 3.2 ml of Tyrode's solution 11 in a sterile Petri dish, where sperm were allowed to disperse for 15 minutes. For capacitation, equal volumes of sperm suspension and heat detoxified (60 C for 30 minutes) human follicular fluid were combined, and the mixture was incubated for 2.5 hours. Immediately prior to insemination, the capacitated sperm suspension was diluted with detoxified human follicular fluid to the desired concentration of motile sperm. The insemination mixture contained 10% detoxified human follicular fluid. Kinetics of Human Sperm Penetration. Eggs were inseminated at 3.3 to 3.6 x 10 6 motile sperm/ ml with capacitated (preincubated for 5.5 hours) sperm or with freshly prepared (non-preincubated) sperm from either fresh or frozen samples. At various time intervals after insemination, eggs were removed, washed, fixed, stained with acetolacmoid, and examined microscopically for evidence of sperm incorporation. Eggs were considered penetrated when a swollen sperm head (or heads) or a male pronucleus (or pronuclei) and a corresponding sperm tail were found within the ooplasm (Fig. 1), and the results were recorded as the mean number of sperm per inseminated egg. Sperm Concentration Dependency. Zona-free eggs were inseminated with 4-hour preincubated human sperm at final concentrations of 5 x 10 2 to 5 X 10 6 motile sperm/ml. The sperm-egg mixture was then incubated under silicone oil for 8 hours in 5% CO 2 in air. Eggs were removed, washed free of adherent sperm, and mounted for scoring as de-

3 Vol. 33, No.3 PENETRATION OF THE ZONA FREE HAMSTER EGG BY HUMAN SPERM FIG. 1. Zona-free hamster egg inseminated with 5.5-hour preincubated human sperm and incubated for 8 hours. Two enlarged sperm heads with corresponding tails are visible in the ooplasm; the egg chromosomes are in telophase of the second meiotic division (phase contrast, x 3000). scribed above. Results were analyzed by the method of least squares. 14 Sperm and Egg Viability. The experimental approach used in the evaluation of sperm and egg viability is outlined in Figure 2. At the beginning of the experiment, zona-free eggs were recovered and inseminated with 4.5-hour preincubated human sperm (Fig. 2, A). Nine hours later, these eggs were divided into two groups, one of which was fixed and scored for penetration directly (AI); the other group was transferred to fresh culture medium containing capacitated hamster sperm (A 2 ). This second insemination, designed to examine the possibility of a sperm-induced block to polyspermy, was allowed to proceed for 3 hours. In a parallel experiment, uninseminated control eggs were aged in vitro (B). These eggs were then inseminated with capacitated hamster sperm (B 1 ) and scored to evaluate the effect of in vitro aging of eggs on their fertilizability. The aging effects on human sperm were evaluated (CI), and controls were run with hamster sperm (C 2 ). 323 either in the fresh state or after freeze-thawing are presented in Table 1. After washing (three or more centrifugation-resuspension cycles) and preincubation, the recovery of total sperm was consistent at approximately 25% for both fresh and freeze-thawed specimens. Recovery of motile sperm (expressed as a percentage of total sperm), however, varied dramatically in the two cases: nearly 100% for the fresh specimens and only 41 % for the frozen specimens. Thus, in order to retain acceptable concentrations of motile sperm during washing, we found it necessary to accept low recoveries of total sperm. The over-all viability or survival of sperm, as judged by motility scores of washed cells at inseminating concentrations, also differed significantly between the fresh and freezethawed suspensions. Fresh sperm maintained progressive motility over a 24-hour incubation period in BWW with only a 10% to 20% decrease in motility, whereas motility in the freeze-thawed samples was completely lost within 12 hours. Because of the lability of the freeze-thawed specimens, capacitation times for these samples were shortened to 2 hours. Kinetics of Sperm Penetration. In order to define the appropriate time for egg scoring, the kinetics of penetration were investigated. Eggs inseminated at 3.3 x 106 motile sperm/ml, with fresh or capacitated sperm, were scored at various times following insemination. Maximal levels of sperm incor- ~(B) ~ RESULTS Recovery and Viability of Sperm. We initiated the sperm evaluation studies on the assumption that the fertilizing sperm would be a member of the progressively motile population. It was therefore critical to quantitate the number of motile sperm in each preparation and to optimize sperm recoveries on the basis of this motile population. Initially, sperm motility recoveries, progressions, and concentrations in freshly ejaculated specimens were compared; results for ejaculates used FIG. 2. Experimental protocol for evaluating sperm and egg viability. See "Materials and Methods" for details.

4 324 BINORETAL. March 1980 TABLE 1. Recovery of Sperm after Washing and Preincubation Suspension Total no." % Motile of sperm sperm Fresh Initial x After washingb 46.2 x and preincubation % Recovery Frozen Initial (before 153 x freezing) After thawing" 35.2 x and washing % Recovery amean of all observations. bfive hours' preincubation. ctwo hours' preincubation. poration were similar: 2.4 sperm/egg for the fresh and 2.3 sperm/egg for the capacitated population (Fig 3, a and c). However, rates of penetration differed for the two. Initial penetration was observed after only 4 hours of sperm-egg interaction for the fresh sperm, with 100% of the eggs penetrated after 9 hours. On the other hand, penetration began sooner with capacitated sperm (the postinsemination lag time was only 2.5 hours), and the time required to attain maximal penetration levels (88%) was 5 hours. When zona-free hamster eggs were inseminated with freeze-thawed sperm suspensions (3.3 x 10 6 motile sperm/ml), penetration was seen initially at 4 hours postinsemination, with a maximum of 80% of the eggs penetrated by approximately 9 hours (Fig. 3, b). The number of sperm incorporated per egg reached a plateau at one sperm per egg. Motile Sperm Concentration and Penetration. The importance of the motile sperm concentration in determining the extent of sperm penetration Hours Post-Insemination FIG. 3. Kinetics offresh or frozen human sperm penetration of zona-free hamster eggs. Zona-free eggs were inseminated with 3.3 x 10 6 motile sperm/ml, and egg aliquots were removed for scoring at the indicated times. a, Fresh sperm without preincubation; b, freeze-thawed sperm without preincubation; c, fresh sperm with 5.5-hour preincubation. 3 x 2 1. IO~ Motile Sperm Concentration FIG. 4. The relationship between motile concentration of fresh (X) and freeze-thawed (-) sperm and penetration of zonafree hamster eggs. Eggs were inseminated with various concentrations of motile sperm (fresh and freeze-thawed sperm were preincubated for 4 hours and 2 hours, respectively) and scored after 8 hours of insemination. The log of the motile sperm concentration is plotted against the mean number of sperm incorporated per egg. Each data point represents the average value from several experiments using different donors. was evaluated. Zona-free eggs were inseminated with various concentrations of motile sperm ranging from 5 x 10 2 to 5 X 10 6 motile cells/ml and scored for penetration following an 8-hour incubation. The number of incorporated sperm is expressed as a function of the log of the concentration of motile sperm (Fig. 4). No penetration was seen at motile sperm concentrations lower than 6 x 10 5 cells/ml; above this concentration, penetration increased to a maximal value of approximately 3.6 sperm/egg. At very high sperm concentrations, recovery and scoring of zona-free eggs were impractical. To evaluate the importance of the motile sperm concentration in determining penetration levels, the correlation between sperm concentration and penetration was determined by the method ofleast squares. For the motile concentrations, individual correlation coefficients ranged from 0.75 to 0.99, and an average slope of 5.36 x 10-7 with an r value of 0.75 was calculated. Two-hour princubated freeze-thawed sperm also showed a linear relationship between concentration of motile sperm and penetration, with a correlation coefficient ofo. 76 (Fig. 4). However, maximal incorporation levels for freeze-thawed sperm were much lower than those for fresh sperm, as were the slopes for the least square plots: 2.2 x 10-7 (freeze-thawed) versus 5.4 x 10-7 (fresh). The x

5 Vol. 33, No.3 PENETRATION OF THE ZONA-FREE HAMSTER EGG BY HUMAN SPERM 325 mean number of freeze-thawed sperm incorporated per egg was lower than that obtained with fresh suspensions at equivalent concentrations of motile sperm. For example, at a concentration of 3.2 x 10 6 motile sperm/ml, freeze-thawed specimens yielded a mean of o. 7 sperm/egg as compared with 1.3 obtained for fresh suspensions. Moreover, the maximal percentage (80%) of eggs penetrated by freeze-thawed sperm was less than the 100% levels routinely achieved with fresh sperm preparations. Donor-Dependent Variability in Penetration. A single donor of proven fertility, whose semen was used in four experiments over a 3-month period, produced ejaculates with sperm concentrations ranging from 88 to 165 x 10 6 cells/ml with motile fractions between 50% and 80% ofthe total. When zona-free eggs were inseminated with capacitated sperm from this donor at approximately 3 x 10 6 motile cells/ml and scored after 8 hours of incubation,1.23 ± 0.17 (mean ± standard error) incorporated sperm were recovered per egg as compared with a least-square predicted value of 1.6 (Fig. 4). A fifth ejaculate collected from this donor had a comparatively low sperm concentration (33 x 10 6 ); however, when the sperm were washed and used to inseminate zona-free eggs at a concentration of 1.8 x 10 6 motile sperm/ml, 1.1 sperm/egg were incorporated as compared with the least square prediction of 0.9 sperm/egg. Block to Polyspermy and Gamete Aging. The cessation of sperm incorporation into the zona-free egg observed in kinetic experiments (Fig. 3) could reflect either an egg plasmalemma block to polyspermy or an artifact associated with in vitro aging of the gametes. To differentiate between these possibilities, a series of reinsemination experiments was performed, the first of which was designed to distinguish sperm changes from eggrelated changes. To test the viability of human sperm over this time course, sperm aliquots recovered from the initial insemination were used to inseminate freshly harvested zona-free eggs. After an additional 6 hours of incubation, these fresh eggs had incorporated 1.4 sperm/egg as compared with the original eggs. This finding indicated that the sperm were still viable at the time when penetration ceased in the original insemination. To examine egg viabili ty, zona-free eggs showing a mean of 1.2 incorporated sperm/egg after 9 hours of incubation with capacitated human sperm were challenged with freshly capacitated hamster sperm. No additional penetration was observed under conditions where freshly harvested zonafree eggs showed a mean of 8.5 homologous sperm incorporated/egg. Therefore, egg-related changes were responsible for the cessation of penetration. Egg-related changes may be sperm-induced or could result from aging. To differentiate between these two possibilities, zona-free eggs were aged in vitro for 10 hours and then inseminated with freshly capacitated hamster sperm. At the time of scoring 3 hours later, none of these eggs was penetrated, indicating that age-induced changes can account for the observed block to sperm penetration. Human Sperm Fertilizing Capacity. According to these findings, the highlights of a protocol for evaluating human sperm fertilizing potential can be summarized as follows: (1) Wash sperm free of seminal plasma by repeated centrifugation in BWW medium (600 x g for 7 minutes). The object of this step is to remove contaminating seminal plasma components while maximizing recoveries of motile sperm. (2) Capacitate washed sperm by preincubation in BWW at 37 C prior to insemination. We have found 5 hours to be a convenient capacitation time, although considerably longer times have been employed by others.15 (3) Inseminate zona-free eggs with ",,6 x 105 motile sperm/ ml. Comparisons of different specimens can be made only when similar motile sperm concentrations are considered. This can be accomplished by inseminating at similar concentrations or by normalizing results using a standard curve such as that shown in Figure 4. (4) Score penetration of zona-free eggs after 5 hours of insemination, at which time maximal sperm incorporation should be realized. Acetolacmoid-stained whole mounts can be used to quantitate penetration; score as mean number of sperm incorporated per inseminated egg. DISCUSSION Results ofthe present study confirm the observations of Yanagimachi et al.5 that human sperm must be exposed to capacitating conditions before they will penetrate zona-free hamster eggs. However, some uncertainty is associated with the kinetics of penetration. In our studies, maximal incorporation with capacitated sperm occurred within 5 hours of insemination, whereas fresh sperm required 9.5 hours to achieve maximal penetration levels. These results differ from those of Yanagimachi et al.,5 who observed high levels ofpenetration within 1 to 7 hours of insemination for capacitated and fresh sperm, respectively. Barros et al. 7

6 326 BINORETAL. March 1980 reported that 3 hours of preincubation and 1 to 2 hours of insemination were sufficient to achieve high levels of incorporation. The reason for these apparent discrepancies in kinetics is not presently known. However, presumably they reflect the differing abilities of the incubation conditions to effect capacitation. The term capacitation historically has been applied to homologous inseminations; however, its use seems justified here, since capacitation is known to be required for the homologous penetration of zona-free eggs15-17 and since freshly prepared sperm from several species do not penetrate zona-free hamster eggs.2, 4, 5,17,18 Penetration of. zona-free hamster eggs by capacitated human sperm is critically dependent upon the concentration of motile sperm; when this concentration is known, comparisons of ejaculates obtained from a single donor or of those obtained from different subjects are possible. This finding is extremely important in the evaluation of the fertility potential of subfertile specimens which are often low in sperm concentration, motility, and total number.19 In preliminary studies by Barros et al.,7 human sperm (5 to 27 x 106 sperm/ml) obtained from normal individuals penetrated zona-free hamster eggs more frequently (75% of the sample versus 34%) than did those obtained from patients suspected to be infertile. However, the authors also noted a difference in sperm count and percentage motility between the two groups, and the resultant penetration percentages may simply reflect these differences. Rogers15 reported the use of 5 x 106 to 1 X 107 sperm/ml during prolonged sperm preincubation (18 to 20 hours), but did not quantitate the number of incorporated sperm per egg or stress the importance of sperm concentration in determining penetration levels or in comparing different specimens. For homologous inseminations of zona-intact human and animal eggs, concentrations of 105 to 106 sperm/ml are commonly used However, in the humster system, the requirement of at least 6 x 105 motile sperm/ml, as demonstrated in the present study, differs from that of homologous zona-free inseminations, where penetration has been observed at concentrations as low as 102 sperm/ml.24,25 This high concentration requirement presumably reflects a low efficiency of spermegg interaction which may reside with the hamster egg plasma membrane; additionally, incomplete sperm capacitation may contribute. In any event, the validity ofthe humster system in assessing human sperm fertilizing capacity remains to be demonstrated. Preservation of sperm by freezing is utilized in sperm banking and in the treatment of infertility due to oligospermia. However, recent progress reports on therapeutic donor insemination indicate that freeze-thawed sperm are less fertile than fresh sperm,26,27 and it is clear that the use of freeze-thawed semen for insemination would be. facilitated greatly by prognostic tests that evaluate post-thaw survival and fertilizing potential. We have used turbidimetric estimates of sperm quality in the prediction of post-thaw motility, and in the present study we examined the penetrating capacity of these sperm in the humster system. The kinetics of penetration for pre incubated fresh and freeze-thawed sperm were similar, although the latter were less effective, since fewer sperm entered the egg. This decrease in efficiency may be related to structural damage, particularly acrosomal disruption, induced by the freezing process.28, 29 In developing the humster system for evaluating human sperm fertilizing ability, the penetration characteristics of proven fertile populations versus subfertile populations must be evaluated, and progress in this direction has been made.6-8 In future studies, we would suggest that attention be paid to the following procedures: (1) inseminations with controlled motile sperm concentrations, (2) inseminations of sufficient duration to allow maximum sperm incorporation, and (3) scoring of penetration on a quantitative basis. Acknowledgments. The authors express appreciation to Dr. Luigi Mastroianni, Jr., for samples of human follicular fluid and to Ms. Patricia Park for excellent secretarial assistance. REFERENCES 1. Yanagimachi R: Specificity of sperm-egg interaction. In Immunobiology of Gametes, Edited by M Edidin, MH Johnson. Cambridge, Cambridge University Press, 1977, p Hanada A, Chang MC: Penetration of zona-free eggs by spermatozoa of different species. BioI Reprod 6:300, Hanada A, Chang MC: Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro. J Exp Zool 203:277, Yanagimachi R: Penetration of guinea-pig spermatozoa into hamster eggs in vitro. J Reprod Fertil 28:477, 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, 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, 1979

7 Vol. 33, No.3 PENETRATION OF THE ZONA-FREE HAMSTER EGG BY HUMAN SPERM Barros C, GonzalezJ, Herrera E, Bustos-Obregon E: Fertilizing capacity of human spermatozoa evaluated by actual penetration of foreign eggs. Contraception 17:87, Barros C, Gonzalez J, Herrera E, Bustos-Obregon E: Human sperm penetration into zona-free hamster oocytes as a test to evaluate the sperm fertilizing ability. Andrologia 11:197, Kanwar KC, Yanagimachi R, Lopata A: Effects of human seminal plasma on fertilizing capacity of human spermatozoa. Fertil Steril 31:321, Rudak E, Jacobs P A, Yanagimachi R: Direct analysis ofthe chromosome constitution of human spermatozoa. Nature 274:94, Barros C, Yanagimachi R: Polyspermy-preventing mechanisms in the golden hamster egg. J Exp Zool 180:251, Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel Jr. San Francisco, Freeman and Co, 1971, p Sokoloski JE, Wolf DP, Blasco L: Unpublished data 14. Snedecor GW, Cochran WG: Statistical Methods, Sixth Edition. Ames Iowa, Iowa State University Press, 1967, p Rogers BJ: Mammalian sperm capacitation and fertilization: a critique of methodology. Gamete Res 1:165, Yanagimachi R, Noda YD: Physiological changes in the postnuclear cap region of mammalian spermatozoa: a necessary preliminary to membrane fusion between sperm and egg cells. J Ultrastruct Res 31:486c, Wolf DP: The block to sperm penetration in zona-free mouse eggs. Dev BioI 64:1, Hanada A, Chang MC: Penetr"ation of hamster and rabbit zona-free eggs by rat and mouse spermatozoa with special reference to sperm capacitation. J Reprod Fertil 46:239, Smith KD, Rodriguez-Rigau LJ, Steinberger E: Relation between indices of semen analysis and pregnancy rate in infertile couples. Fertil Steril 28:1314, Chang MC, Bedford JM: Fertilizability of rabbit ova after removal of corona radiata. Fertil Steril 13:421, Niwa K, Chang MC: Optimal sperm concentration and minimal number of spermatozoa for fertilization in vitro of rat eggs. J Reprod Fertil 40:471, McMaster R, Yanagimachi R, Lopata A: Penetration of human eggs by human spermatozoa in vitro. BioI Reprod 19:212, 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, Wolf DP, Inoue M, Stark RA: Penetration of zona-free mouse eggs. BioI Reprod 15:213, Binor Z: Unpublished results 26. Ansbacher R: Artificial insemination with frozen spermatozoa. Fertil Steril 29:375, Quinlivan WLG: Therapeutic donor insemination: results and causes of nonfertilization. Fertil Steril 32:157, Pedersen H, Lebech PE: Ultrastructural changes in human spermatozoon after freezing for artificial insemination. Fertil Steril 22:125, Alexander NJ: Surface structure of spermatozoa frozen for artificial insemination. Andrologia 9:155, 1977