Kinetics of Sperm Penetration into and the Zona Reaction of Mouse Ova Inseminated In Vitro D. P. WOLF,' M. HAMADA AND M. INOUE Division of Reproductive Biology, Department of Obstetricsand Gynecology and Department of Biochemistry and Biophysics, University of Pennsylvania School ofMedicine, Philadelphia, Pennsylvania 19174
ABSTRACT The kinetics of capacitated epididymal sperm penetration into cumulus-intact and completely cumulus-free mouse ova were studied. Penetration was complete within two hours of insemination in cumulus-intact ova incubated with 2 X los spermlml, as evidenced by the attainment of constant values for the mean number of spermlpenetrated ovum. A reduction in sperm viability was not a factor in the cessation of zonae penetration, since high penetration levels were obtained when fresh ova were added to 3-hour inseminations containing "aged" sperm. With completely cumulus-free ova, capacitated sperm penetration was initiated between one and two hours post-insemination, and penetration levels increased over the entire 6-hour incubation period. Final penetration levels for cumulus-free ova were generally lower than for their intact counterparts. A constant value for the mean number of spermlpenetrated ovum was obtained after three hours of sperm exposure, indicating that the cumulus-free ovum also underwent a zona reaction. Similar results were observed when sperm were capacitated in the presence or absence of cumulus and1 or epididymal components, suggesting that inadequate capacitation was not the causative agent in the altered penetration of cumulus-free ova. These results indicate that a zona reaction occurs in both ovum types, although in many cases only after multiple sperm penetration has occurred. In addition, evidence was obtained for a cumulus-dependent enhancement in the rate of sperm penetration which could not be related to a cumulus involvement in sperm capacitation. The mouse represents a convenient model Austin and Braden, '56). In vitro, these levels system for the study of mammalian fertiliza- increase to approximately 75%(Iwamatsu and tion and early development. Methods are Chang, '71; Wolf and Inoue, "76),and the disavailable for the routine in vitro fertilization tribution of sperm recovered within zonae of superovulated ova (Whittingham, '68; under these conditions is similar to a distribuIwamatsu and Chang, '70,'71;Toyoda et al., tion based on chance interactions between '71a,b). Limited development of resultant em- gametes. Thus, the mouse ovum in vitro either bryos occurs in vitro, and development to term does not undergo a zona reaction at all or else following transfer to surrogate mothers has the reaction is non-functional. been described (Whittingham, '68; Cross and The present study was designed to characBrinster, '70; Mukherjee and Cohen, '70; terize further the zona reaction of mouse ova Mukherjee, '72). The in vitro inseminated inseminated in vitro. The kinetics of capaci-. ovum is, however, characterized by the pres- tated sperm penetration into intact and comence of abnormally high numbers of sperm pletely cumulus-free ova were examined, and within its zona and by subnormal changes in the temporal requirements of the zona block zona sperm binding properties (Inoue and were defined. Evidence was obtained for a Wolf, '75). Embryos recovered from natural cumulus involvement in the initiation of matings show a moderately functional zona ' To whom correspondenceshould be addressed. reaction with 20% containing more than one *Department of Obstetrics and Gynecology, Tokai University sperm within the zona (Braden et al., '54; School of Medicine, Bohseidai, Isehara 259-11, Japan. J. EXP. ZOOL., 201: 29-36.
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D. P. WOLF, M. HAMADA AND M. INOUE
sperm penetration. A preliminary account of this work has been presented (Wolf and Hamada, '76a).
masses in a 200 microliter drop at approximately lo6 sperm/ml. Soluble epididymal components were prepared by incubating four pairs of minced MATERIALS AND METHODS epididymides in 0.8 ml culture medium for 15 Unfertilized tuba1 ova were recovered from minutes. Particulates were removed manualsuperovulated adult virgin Swiss mice (6-12 ly, and the solution was then incubated under weeks), as described previously (Inoue and oil a t 37" and centrifuged (1,600 X gfor 30 Wolf, '74). Initial cumulus dispersal was minutes) immediately prior to use. Fifty accomplished by transferring egg masses to microliters of the supernatant solution were culture medium containing hyaluronidase added to 150 p l of culture medium containing (0.1%,Sigma, Type 1).The medium employed ova in insemination dishes. Ova were preincuin these studies was a modified Krebs-Ringer bated with epididymal components for two bicarbonate preparation containing 20 mg/ml hours a t 37' in the presence of 5%COBin air. bovine serum albumin (Inoue and Wolf, '74). In some experiments, epididymal components A Fraction V preparation (Sigma) was em- were removed immediately prior to inseminaployed in the present study, although similar tion by washing ova three times in fresh culresults were obtained with 3 times crystal- ture medium. Cumulus-intact or -free ova (30-50) in 200 lized BSA or with lipid-free BSA (Inoue and Wolf, unpublished results). Following a 10- p1 of culture medium under oil were insemiminute exposure to hyaluronidase, ova were nated with 20 pl of preincubated sperm. transferred to fresh medium, and the remain- Insemination mixtures were incubated for ing corona cells were removed completely by one to six hours in the presence of 5%COz in pipetting. Cumulus-free ova were then air (Inoue and Wolf, '74). Eggs were washed, washed through three changes of fresh me- mounted, fixed and stained for the assessment dium. In considering the role of cumulus, i t is of sperm penetration according to Toyoda and important to distinguish between the com- Chang ('74). Ova were scored as penetrated if pletely cumulus-free ova employed here and sperm were present in the perivitelline space the hyaluronidase-dispersed,cumulus-free or in the vitellus. The statistical significance ova utilized in previous studies (Fukuda et al., of the data was evaluated by the chi square '72; Hoppe, '75; Wolf and Inoue, '76) where test for equality of two independent proporlimited numbers of cumulus cells may have tions (Ipsen and Feigl, '70). been retained on the surface of some zonae. RESULTS Cumulus-intact control ova were held a t 37" The kinetics of unwashed, capacitated under silicon oil (Dow Corning 200 Fluid) until egg processing was completed and then sperm penetration into cumulus-intact and transferred to insemination dishes without free ova were examined in vitro (table 1). Inwashing. Epididymal sperm suspensions were dividual experiments employed gametes from prepared as described previously (Wolf and different animals; and, where both ova types Inoue, '76) and capacitated by a 1-to 2-hour are listed within a n experiment, a single preincubation in culture medium. Final sperm preparation was utilized. Sperm penesperm concentrations varied from 2-20 x lo5 tration of cumulus-intact ova was completed cells/ml. Sperm were washed by diluting a within two hours of insemination. Penetrafresh aliquot of the standard epididymal prep- tion of cumulus-free ova was delayed relative aration with culture medium (1:lO) and re- to intact controls, and the mean number of suspending pelleted sperm (700 X g, 5 sperm/penetrated ovum did not reach a conminutes) in fresh culture medium. This pro- stant value until three hours post inseminacedure was repeated when additional dilution tion. The low mean number value obtained a t of epididymal components was desired. five hours represents data from Experiment 1 Washed sperm were capacitated by prein- only. Maximum penetration levels at four and cubation for one to two hours in culture me- five hours for cumulus-free ova were sigdium following completion of the wash pro- nificantly (p < 0.01) lower than the correcedure. Capacitation in the presence of cumu- sponding intact ova, when data from the two lus was accomplished by preincubating (2 time points were analyzed together. In the rehours a t 37' under silicon oil) fresh epididy- sults presented in table 1, both supplemental ma1 sperm with one or two unwashed cumulus and supernumerary sperm were scored a s
ZONA REACTION IN VITRO
penetrating sperm; however, we also recorded the number of sperm within each of these categories and subsequently calculated zonal and egg plasma membrane penetration frequencies. The results are presented as monopenetrated or monofertilized, the presumed physiologically normal condition, versus time of insemination (fig. 1).Most (85%)intact ova were monospermic, despite the presence of multiple sperm in the perivitelline space in many (55-60%).A similar relationship existed with cumulus-free ova. Additionally, the percentage of this ova type, whose zonae were penetrated by a single sperm, decreased progressively from two to four hours. The kinetics of washed sperm penetration of cumulus-intact and -free ova derived from matched cumulus masses were generally similar to those observed with unwashed sperm (table 2). The percentage of penetrated cumulus-free ova was significantly (p < 0.01) reduced over intact controls following six hours of incubation. In two of three experiments with intact ova, penetration levels a t six hours were lower than those a t two hours, and the total was significantly reduced over the two hour value (0.02 < p > 0.01). Cessation of sperm penetration, as observed in these experiments, could reflect sperm exhaustion rather than the occurrence of a zona reaction. Accordingly, experiments were performed in which aged sperm present in insemination mixtures were challenged with fresh ova (table 3). Following a 3-hour incubation, sperm retained the ability to penetrate ova; however, after four hours a significant reduction in penetrating capacity was apparent. Conversely, the inclusion of additional capacitated sperm a t two hours to inseminations containing intact ova did not lead to increased penetration levels. Both direct and indirect causes were examined for the altered kinetics of penetration observed with cumulus-free ova. Epididymal components present in the sperm preparations employed here were capable of reducing the penetration of intact ova (table 41, as reported previously by Iwamatsu and Chang ('71) and Oliphant and Brackett ('73). While this inhibition may result from sperm decapacitation, the possibility remained that epididymal components could act directly on exposed zonae of cumulus-free ova. As can be seen in table 4, sperm penetration of both intact and cumulus-free ova was decreased when high concentrations of epididymal com-
a
F P w
2 Y
P
32
D. P. WOLF, M. HAMADA AND M. INOUE
IOC
50
0
I
I
I
I
2 3 4 Time (hours)
I
I
5
Fig. 1 Number of ova penetrated or fertilized by a single sperm as a function of insemination time. Penetration refers to zona passage, while fertilization includes sperm-egg fusion. Open symbols represent cumulusintact eggs. Closed symbols represent cumulus-free eggs.
TABLE 2
Kinetics of washed epididymal sperm penetration of nwuse ova Time (hour) Expt. No. Egg type
1 2 3 Totals
Intact Cumulus-free Intact Cumulus-free Intact Cumulus-free Intact Cumulus-free
2
91 29 82 38
90 48 88 40
6
(29132) 3.89 (14/48) 1.57 (27/33) 2.26 (13/34) 1.53 (69/77)2.94 (33/69) 1.39 (125/142)3.03 (60/151)1.47
91 (43147) 2.77 71 (32/45) 2.84 71 (43/61)2.63 64 (39161) 1.77 78 (101/129)2.73 55 (82/150)2.50 79 (187/237)2.71 60 (153/256)2.40
’Results reported as % penetrated, number of ova employed (in parentheses) and the mean number of sperm/penetrated ovum. Significantly reduced (p < 0.01) over intact penetration levels.
TABLE 3
The effect of sperm age on zonae penetrability in vitro Sperm penetration Sperm age
3 hours 4 hours
Egg type
Original ova
Fresh ova
Intact Cumulus-free Intact Cumulus-free
96 (24/25)2.41 81 (25/31) 2.28 91 (20/22) 2.10 71 (24/34)2.00
85 (17/20)1.65 64 (16/25)1.75 22 (8/36)1.25 23 (3/13)1.00
Sperm age represents time post insemination, as all sperm were preincubated in concentrated form for one to two hours prior to use. Penetration was scored after 3-hour incubations; % penetrated, number of ova employed (in parentheses) and the mean number of spermlpenetrated ovum. *Significantly reduced fp < 0.051 over original levels.
33
ZONA REACTION IN VITRO TABLE 4
The effect of epididymal components on sperm penetration of mouse ova in vitro 1 Sperm penetration Egg exposure to epididymal components
During preincubation and insemination During preincubation only
Egg type
Intact Cumulus-free Intact Cumulus-free
Control
Experimental
80 (227/283)3.71
16 (46/290)1.23 3 (5/182)1.27 75 (140/187)2.41 68 (188/277)4.14
58 (120/207)1.93 92 (186/203)3.96 75 (200/268)3.72
' Penetration was scored after four to five hours incubation; % penetrated,number of ova employed (in parentheses) and the mean number of spermlpenetrated ovum. *Significantly reduced (p < 0.05) over control group. TABLE 5
Sperm capacitating conditions and the penetration of m u s e ova in vitro Sperm penetration Sperm pretreatment
% penetrated, (number ova employed),
Egg type
Unwashed control: capacitated in culture medium Unwashed experimental: capacitated in the presence of cumulus masses Washed control: capacitated in culture medium Washed experimental: capacitated in culture medium containing diluted epididymal companents
mean number spermlpenetrated ova
Intact Cumulus-free
89 (97/109)2.13 60 (109/182)2.40
Intact Cumulus-free
86 (71/831 1.87 66 (122/186)1.98
Intact Cumulus-free
72 (140/196)3.11 46 a (251/54232.91
Intact Cumulus-free
76 (104/136)2.63 44 (223/50412.42
1 Penetration was scored four to five hours post insemination. Both unwashed and washed control pretreatment medium contained epididymal components carried over during the preparation of sperm suspensions. Sperm were washed three to four times and then capacitsted in fresh culture medium in the washed experimental category. 2Significantly reduced (p < 0.05) over intact eggs.
ponents were present during ovum preincubation and in the insemination mixture; however, simple preincubation of cumulus-free ova with epididymal preparations did not affect penetration. Coincidentally, pretreatment of intact ova with epididymal components led to a reduction in penetration which presumably reflected the premature dispersal of cumulus. Submaximal sperm penetration of cumulusfree ova could reflect inadequate capacitation if cumulus andlor epididymal components played a role in this process. Therefore, penetration levels for intact and cumulus-free ova were compared under different sperm capacitating conditions (table 5 ) . When sperm were preincubated in the presence or absence of cumulus or when washed sperm controls were compared with washed sperm in which epididymal components were diluted a t least 100-
fold, penetration levels for each ovum type were similar. Thus, the disparities in the penetration of intact and cumulus-free ova seen in this study were independent of the conditions employed for sperm capacitation. DISCUSSION
A significant finding from the present study was that cumulus-intact mouse ova inseminated in vitro underwent a zona reaction, even though the reaction did not occur until most zonae (55-60%)had been penetrated by more than one sperm. A distribution of penetrated sperm based on chance interactions between gametes would, therefore, be anticipated, justifying a description of the zona reaction in vitro as non-functional (Wolf and Inoue, '76).A moderately effective zonal block was reported previously for mouse ova recovered from natural matings (Austin and
34
D. P. WOLF, M. HAMADA AND M. INOUE
Braden, '561, and insufficient zona modification by cortical granule exudate was suggested as a basis for the subnormal in vitro response (Barros and Yanagimachi, '72; Mintz and Gearhart, '73). The demonstration that a zona reaction occurs in the mouse, however, argues against this possibility and suggests rather that a non-functional reaction derives from the synchrony in sperm penetration occurring a t the high sperm concentrations employed in vitro. Despite the tendency toward multiple zonal penetration of these in vitro inseminated ova, monospermy levels were high (> 80%), reflecting the relative importance of the egg plasma membrane block in limiting sperm penetration of the vitellus. The existence and temporal requirements of the plasma membrane block in zona-free mouse ova have been defined elsewhere (Wolf, '77). The temporal requirements for the zona reaction have been estimated as not less than 10 minutes nor more than two hours by Braden e t al. ('54) or within 15 minutes of sperm attachment to the vitellus (Barros and Yanagimachi, '72). Under the present culture conditions, vitelline penetration of cumulusintact ova was initially detected in whole mounts between 30 and 60 minutes following insemination and was completed by 120 minutes (Inoue and Wolf, '75 and this study). By 120 minutes, a maximum mean number of spermlpenetrated ovum was also reached, the expected result if all penetrated ova had established a zonal block. Thus, the zona reaction of intact ova in vitro requires a maximum of 90 minutes following sperm-ovum fusion. In contrast to the finding of Iwamatsu and Chang ('70), who reported a progressive increase in the mean number of spermlpenetrated ovum over a 5-hour insemination period, the zona reaction, once established in this study, was highly effective. The influence of cumulus on the fertilization of hyaluronidase-treated ova has been studied in several species (see Bedford, '73 for review). In the mouse, high levels of fertilization were seen when such ova were inseminated with epididymal sperm capacitated by exposure to bovine follicular fluid (Iwamatsu and Chang, '70), to oviduct fluid (Pavlok and McLaren, '72) or to culture medium alone (Fukuda e t al., '72; Hoppe, '75; Wolf and Inoue, '76). However, in our experience, hyaluronidase-treated ovum preparations are not completely free of cumulus cells (Inoue and
Wolf, '75), and completely cumulus-free ova do not always fertilize a t high levels (Chang and Hunter, '75; Wolf and Inoue, '76). Physical abuse resulting from the additional manipulation required to remove residual cumulus cells seemed an unlikely explanation for this reduced fertility, especially in view of the ovum's ability to withstand the physical trauma associated with zona removal by mechanical means (Wolf et al., '76). In our initial attempts with completely cumulus-free ova, fertilization levels scored a t four hours were often low. However, results from the present study indicated that, although initial penetration of cumulus-free ova was delayed, relative to intact controls, high levels of penetration were attained after six hours of incubation. Indeed, the disparity in fertilization levels seen between intact and cumulus-free ova a t six hours may simply reflect nonspecific, age-dependent changes in the gametes over these prolonged incubation times. Cumulus-free ova were capable of undergoing a zona reaction, although the reaction was not complete until three hours post insemination, as indicated by the attainment of a constant mean number of spermlpenetrated ovum. Moreover, the monospermy levels in these ova were higher than in the corresponding intact or hyaluronidase-treated ova (Iwamatsu and Chang, '69, '70; Wolf and Inoue, '76) and were similar to those reported for natural matings (Braden et al., '54; Austin and Braden, '56). While an unequivocal explanation for the delay in sperm penetration observed here with cumulus-free ova is unavailable, several possibilities can be considered. The initiation of gamete interaction is, of course, dependent upon both sperm and ova; and the first step in this process, capacitated sperm binding to zonae of cumulus-free ova, occurs rapidly in our in vitro system (20 spermlzona within 5 minutes a t 2 x lo5 cellslml) (Inoue and Wolf, '75; Wolf and Inoue, '76). Gwatkin et al. ('74) maintain that capacitation in culture medium as utilized here actually represents capacitation by contaminating epididymal or cumulus components. This observation raises the possibility that washed epididymal sperm may undergo insufficient or subnormal capacitation, thereby accounting for the observed altered kinetics of penetration. However, the conclusions of Gwatkin et al. ('74) were based principally on the absence of penetration of cumulus-free ova following a 90-minute exposure to washed (1 time) epididymal sperm.
35
ZONA REACTION IN VITRO
Since in the present study penetration of cumulus-free ova was delayed, i.e., detected initially only after 120 minutes of insemination, and occurred independently of the presence or absence of cumulus or soluble epididyma1 components in the capacitation medium, we conclude that the delay in sperm penetration of cumulus-free ova is not related to the state of capacitation. The next obvious step to consider in the delayed penetration of cumulus-free ova is the occurrence of a n acrosome reaction. The sequence of events prerequisite to sperm penetration of zonae is generally conceded to involve capacitation, the occurrence of a n acrosome reaction, sperm penetration of the cumulus matrix and binding to the zona and, finally, zonal penetration (McRorie and Williams, ‘74; Chang and Hunter, ’75). However, in this sequence, sperm adherent to the outer margin of the zonae of cumulus-intact ova should have undergone an acrosome reaction, a prediction that is not always substantiated by experimental observation (Thompson e t al., ’74; Anderson et al., ’75; Nicosia and Wolf, unpublished results). We conclude, therefore, that an acrosome reaction is not an absolute prerequisite to sperm passage through the cumulus and further suggest that the rate limiting step in penetration of intact or cumulus-free ova could be the induction of this reaction. In fact, this phenomenon may apply to zona-free ova as well, since delays in capacitated sperm incorporation into this ovum type have been observed (Wolf e t al., ’76; Wolf, ’77). Moreover, it should be noted that studies with zona-free ova discount the possibility that cumulus cells directly influence ovum integrity, since both fresh and aged zona-free ova rapidly incorporate sperm in the complete absence of cumulus cells (Wolf and Hamada, ’76). If the above situation obtains, then plausible roles for the cumulus include a direct involvement in inducing an acrosome reaction in zona-bound sperm (Gwatkin e t al., ’72; Gwatkin and Carter, ’74) and /or indirect possibilities, such as “orientation” of sperm at the zona surface or the retention or alteration of acrosome reaction-inducing substances in the zona or perivitelline space. While little experimental evidence is available concerning these possibilities, it would be interesting to assess the kinetics of acrosome vesiculation in the presence and absence of intact and cumulus-free ova.
ACKNOWLEDGMENTS
This work was supported by grants from the Ford Foundation 65-58B and USPHS HD-07635. LITERATURE CITED Anderson, E., P. C. Hoppe, W. K. Whitten and G. S. Lee 1975 In uitrofertilization and early embryogenesis: A cytological analysis. J. Ultrastruct. Res., 50: 231-252. Austin, C. R., and A. W. H. Braden 1956 Early reactions of the rodent egg to spermatozoon penetration. J. Exp. Biol., 33: 358-365. Barros, C., and R. Yanagimachi 1972 Polyspermy-preventing mechanisms in the golden hamster egg. J. Exp. Zool., 180: 251-266. Bedford, J. M. 1973 Mechanisms involved in penetration of spermatozoa through the vestments of t h e mammalian egg. In: Physiology and Genetics of Reproduction, P a r t B. Basic Life Sciences, Vol. 4. E. M. Coutinho and F. Fuchs, eds. Plenum Press, New York. Braden, A. W. H., C. R. Austin and H. A. David 1954 The reaction of the zona pellucida to sperm penetration. Aust. J. Biol. Sci., 7: 391-409. Chang, M. C., and R. H. F. Hunter 1975 Capacitation of mammalian sperm: biological and experimental aspects. In: Handbook of Physiology. Vol. V. Section 7, Endocrinology. R. 0. Greep and E. B. Astwood, eds. American Physiological Society, Washington, D.C. Cross, P. C., and R. L. Brinster 1970 In uitrodevelopment of mouse oocytes. Biol. Reprod., 3: 298-307. Fukuda, Y., 0. Okado and Y. Toyoda 1972 Studies on the fertilization of mouse eggs in uitro. 111. Fertilization of denuded eggs by capacitated spermatozoa. Jap. J. Anim. Reprod., 18: 73-77. Gwatkin, R. B. L., 0. F. Anderson and C. F. Hutchison 1972 Capacitation of hamster spermatozoa in uitro: the role of cumulus components. J. Reprod. Fert., 30: 389-394. Gwatkin, R. B. L., 0. F. Anderson and D. T. Williams 1974 Capacitation of mouse spermatozoa in uitro: involvement of epididymal secretions and cumulus oophorus. J. Reprod. Fert., 41: 253-256. Gwatkin, R. B. L., and H. E. Carter 1974 Cumulus oophorus. In: Scanning Electron Microscopic Atlas of Mammalian Reproduction. E. S. E. Hafez, ed. Igaku Shion, Tokyo. Hoppe, P. C. 1975 Fertilizing ability of mouse sperm from different epididymal regions and after washing and centrifugation. J. Exp. Zool., 192: 219-222. Inoue, M., and D. P. Wolf 1974 Comparative solubility properties of the zonae pellucidae of unfertilized and fertilized mouse ova. Biol. Reprod., 11: 558-565. 1975 Sperm binding characteristics of t h e murine zona pellucida. Biol. Reprod., 13: 340-346. Ipsen, J., and P. Feigl 1970 Bancroft’s Introduction to Biostatistics. Harper and Row, New York, p. 106. Iwamatsu, T., and M. C. Chang 1969 In uitrofertilization of mouse eggs in the presence of bovine follicular fluid. Nature (London), 224: 919-920. 1970 Further investigation of capacitation of sperm and fertilization of mouse eggs in uitro. J. Exp. Zool., 175: 271-281. 1971 Factors involved in the fertilization of mouse eggs in uitro. J. Reprod. Fert., 26: 197-208. McRorie, R. A,, and W. L. Williams 1974 Biochemistry of mammalian fertilization. Ann. Rev. Biochem., 43: 777803. Mintz, B., and J. D. Gearhart 1973 Subnormal zona pellucida change in parthenogenetic mouse embryos. Develop. Biol., 31: 178-184.
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Mukherjee, A. B. 1972 Normal progeny from fertilization in uitro of mouse oocytes matured in culture and spermatozoa capacitated in uitro. Nature (London), 237: 397-398. Mukherjee, A. B., and M. M. Cohen 1970 Development of normal mice by in uitro fertilization. Nature (London), 228: 472-473. Nicosia, S. V., D. P. Wolf and M. Inoue 1977 Cortical granule distribution and cell surface characteristics of mouse ova. Develop. Biol., 57: 208-226. Oliphant, G., and B. G. Brackett 1973 Capacitation of mouse spermatozoa in media with elevated ionic strength and reversible decapacitation with epididymal extracts. Fertil. Steril., 24: 948-955. Pavlok, A., and A. McLaren 1972 The role of cumulus cells and the zona pellucida in fertilization of mouse eggs in uitro. J. Reprod. Fert., 29: 91-97. Thompson, R. S., D. Moore-Smith and L. Zamboni 1974 Fertilization of mouse ova in uitro: An electron microscope study. Fertil. Steril., 25: 222-249. Toyoda, Y., and M. C. Chang 1974 Fertilization of rat eggs in uitro by epididymal spermatozoa and the development of eggs following transfer. J . Reprod. Fert., 36: 9-22. Toyoda, Y., M. Yokoyama and T. Hosi 1971a Studies on the fertilization of mouse eggs in uitro. I. In uitrofertilization
of eggs by fresh epididymal sperm. Jap. J. Anim. Reprod., 16: 147-151. 1971b Studies on the fertilization of mouse eggs in uitro. 11. Effects of in uitro pre-incubation of spermatozoa on time of sperm penetration of mouse eggs in uitro. Jap. J. Anim. Reprod., 16: 152-157. Whittingham, D. G. 1968 Fertilization of mouse eggs in uitro.Nature (London), 220: 592-593. Wolf, D. P. 1977 The vitelline block to polyspermy in zona-free mouse ova inseminated in uitro. Develop. Biol., in review. 1977 Involvement of a trypsin-like activity in sperm penetration of zona-free mouse ova. J . Exp. Zool., 199: 149-156. Wolf, D. P., and M. Hamada 1976a The zona reaction in mouse ova inseminated in uitro. Proc. Soc. Study of Reprod., Philadelphia, Pa., p. 21 (Abstract 12). 1976b Age-dependent losses in the penetrability of mouse ova. J. Reprod. Fert., 48: 213-214. Wolf, D. P., andM. Inoue 1976 Sperm concentration dependency in the fertilization and zonae sperm binding properties of mouse eggs inseminated in uitro. J. Exp. Zool., 196: 27-38. Wolf, D. P., M. Inoue and R. A. Stark 1976 Penetration of zona-free mouse eggs. Biol. Reprod., 15: 213-221.
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ABSTRACT The kinetics of capacitated epididymal sperm penetration into cumulus-intact and completely cumulus-free mouse ova were studied. Penetration was complete within two hours of insemination in cumulus-intact ova incubated with 2 X los spermlml, as evidenced by the attainment of constant values for the mean number of spermlpenetrated ovum. A reduction in sperm viability was not a factor in the cessation of zonae penetration, since high penetration levels were obtained when fresh ova were added to 3-hour inseminations containing "aged" sperm. With completely cumulus-free ova, capacitated sperm penetration was initiated between one and two hours post-insemination, and penetration levels increased over the entire 6-hour incubation period. Final penetration levels for cumulus-free ova were generally lower than for their intact counterparts. A constant value for the mean number of spermlpenetrated ovum was obtained after three hours of sperm exposure, indicating that the cumulus-free ovum also underwent a zona reaction. Similar results were observed when sperm were capacitated in the presence or absence of cumulus and1 or epididymal components, suggesting that inadequate capacitation was not the causative agent in the altered penetration of cumulus-free ova. These results indicate that a zona reaction occurs in both ovum types, although in many cases only after multiple sperm penetration has occurred. In addition, evidence was obtained for a cumulus-dependent enhancement in the rate of sperm penetration which could not be related to a cumulus involvement in sperm capacitation. The mouse represents a convenient model Austin and Braden, '56). In vitro, these levels system for the study of mammalian fertiliza- increase to approximately 75%(Iwamatsu and tion and early development. Methods are Chang, '71; Wolf and Inoue, "76),and the disavailable for the routine in vitro fertilization tribution of sperm recovered within zonae of superovulated ova (Whittingham, '68; under these conditions is similar to a distribuIwamatsu and Chang, '70,'71;Toyoda et al., tion based on chance interactions between '71a,b). Limited development of resultant em- gametes. Thus, the mouse ovum in vitro either bryos occurs in vitro, and development to term does not undergo a zona reaction at all or else following transfer to surrogate mothers has the reaction is non-functional. been described (Whittingham, '68; Cross and The present study was designed to characBrinster, '70; Mukherjee and Cohen, '70; terize further the zona reaction of mouse ova Mukherjee, '72). The in vitro inseminated inseminated in vitro. The kinetics of capaci-. ovum is, however, characterized by the pres- tated sperm penetration into intact and comence of abnormally high numbers of sperm pletely cumulus-free ova were examined, and within its zona and by subnormal changes in the temporal requirements of the zona block zona sperm binding properties (Inoue and were defined. Evidence was obtained for a Wolf, '75). Embryos recovered from natural cumulus involvement in the initiation of matings show a moderately functional zona ' To whom correspondenceshould be addressed. reaction with 20% containing more than one *Department of Obstetrics and Gynecology, Tokai University sperm within the zona (Braden et al., '54; School of Medicine, Bohseidai, Isehara 259-11, Japan. J. EXP. ZOOL., 201: 29-36.
29
30
D. P. WOLF, M. HAMADA AND M. INOUE
sperm penetration. A preliminary account of this work has been presented (Wolf and Hamada, '76a).
masses in a 200 microliter drop at approximately lo6 sperm/ml. Soluble epididymal components were prepared by incubating four pairs of minced MATERIALS AND METHODS epididymides in 0.8 ml culture medium for 15 Unfertilized tuba1 ova were recovered from minutes. Particulates were removed manualsuperovulated adult virgin Swiss mice (6-12 ly, and the solution was then incubated under weeks), as described previously (Inoue and oil a t 37" and centrifuged (1,600 X gfor 30 Wolf, '74). Initial cumulus dispersal was minutes) immediately prior to use. Fifty accomplished by transferring egg masses to microliters of the supernatant solution were culture medium containing hyaluronidase added to 150 p l of culture medium containing (0.1%,Sigma, Type 1).The medium employed ova in insemination dishes. Ova were preincuin these studies was a modified Krebs-Ringer bated with epididymal components for two bicarbonate preparation containing 20 mg/ml hours a t 37' in the presence of 5%COBin air. bovine serum albumin (Inoue and Wolf, '74). In some experiments, epididymal components A Fraction V preparation (Sigma) was em- were removed immediately prior to inseminaployed in the present study, although similar tion by washing ova three times in fresh culresults were obtained with 3 times crystal- ture medium. Cumulus-intact or -free ova (30-50) in 200 lized BSA or with lipid-free BSA (Inoue and Wolf, unpublished results). Following a 10- p1 of culture medium under oil were insemiminute exposure to hyaluronidase, ova were nated with 20 pl of preincubated sperm. transferred to fresh medium, and the remain- Insemination mixtures were incubated for ing corona cells were removed completely by one to six hours in the presence of 5%COz in pipetting. Cumulus-free ova were then air (Inoue and Wolf, '74). Eggs were washed, washed through three changes of fresh me- mounted, fixed and stained for the assessment dium. In considering the role of cumulus, i t is of sperm penetration according to Toyoda and important to distinguish between the com- Chang ('74). Ova were scored as penetrated if pletely cumulus-free ova employed here and sperm were present in the perivitelline space the hyaluronidase-dispersed,cumulus-free or in the vitellus. The statistical significance ova utilized in previous studies (Fukuda et al., of the data was evaluated by the chi square '72; Hoppe, '75; Wolf and Inoue, '76) where test for equality of two independent proporlimited numbers of cumulus cells may have tions (Ipsen and Feigl, '70). been retained on the surface of some zonae. RESULTS Cumulus-intact control ova were held a t 37" The kinetics of unwashed, capacitated under silicon oil (Dow Corning 200 Fluid) until egg processing was completed and then sperm penetration into cumulus-intact and transferred to insemination dishes without free ova were examined in vitro (table 1). Inwashing. Epididymal sperm suspensions were dividual experiments employed gametes from prepared as described previously (Wolf and different animals; and, where both ova types Inoue, '76) and capacitated by a 1-to 2-hour are listed within a n experiment, a single preincubation in culture medium. Final sperm preparation was utilized. Sperm penesperm concentrations varied from 2-20 x lo5 tration of cumulus-intact ova was completed cells/ml. Sperm were washed by diluting a within two hours of insemination. Penetrafresh aliquot of the standard epididymal prep- tion of cumulus-free ova was delayed relative aration with culture medium (1:lO) and re- to intact controls, and the mean number of suspending pelleted sperm (700 X g, 5 sperm/penetrated ovum did not reach a conminutes) in fresh culture medium. This pro- stant value until three hours post inseminacedure was repeated when additional dilution tion. The low mean number value obtained a t of epididymal components was desired. five hours represents data from Experiment 1 Washed sperm were capacitated by prein- only. Maximum penetration levels at four and cubation for one to two hours in culture me- five hours for cumulus-free ova were sigdium following completion of the wash pro- nificantly (p < 0.01) lower than the correcedure. Capacitation in the presence of cumu- sponding intact ova, when data from the two lus was accomplished by preincubating (2 time points were analyzed together. In the rehours a t 37' under silicon oil) fresh epididy- sults presented in table 1, both supplemental ma1 sperm with one or two unwashed cumulus and supernumerary sperm were scored a s
ZONA REACTION IN VITRO
penetrating sperm; however, we also recorded the number of sperm within each of these categories and subsequently calculated zonal and egg plasma membrane penetration frequencies. The results are presented as monopenetrated or monofertilized, the presumed physiologically normal condition, versus time of insemination (fig. 1).Most (85%)intact ova were monospermic, despite the presence of multiple sperm in the perivitelline space in many (55-60%).A similar relationship existed with cumulus-free ova. Additionally, the percentage of this ova type, whose zonae were penetrated by a single sperm, decreased progressively from two to four hours. The kinetics of washed sperm penetration of cumulus-intact and -free ova derived from matched cumulus masses were generally similar to those observed with unwashed sperm (table 2). The percentage of penetrated cumulus-free ova was significantly (p < 0.01) reduced over intact controls following six hours of incubation. In two of three experiments with intact ova, penetration levels a t six hours were lower than those a t two hours, and the total was significantly reduced over the two hour value (0.02 < p > 0.01). Cessation of sperm penetration, as observed in these experiments, could reflect sperm exhaustion rather than the occurrence of a zona reaction. Accordingly, experiments were performed in which aged sperm present in insemination mixtures were challenged with fresh ova (table 3). Following a 3-hour incubation, sperm retained the ability to penetrate ova; however, after four hours a significant reduction in penetrating capacity was apparent. Conversely, the inclusion of additional capacitated sperm a t two hours to inseminations containing intact ova did not lead to increased penetration levels. Both direct and indirect causes were examined for the altered kinetics of penetration observed with cumulus-free ova. Epididymal components present in the sperm preparations employed here were capable of reducing the penetration of intact ova (table 41, as reported previously by Iwamatsu and Chang ('71) and Oliphant and Brackett ('73). While this inhibition may result from sperm decapacitation, the possibility remained that epididymal components could act directly on exposed zonae of cumulus-free ova. As can be seen in table 4, sperm penetration of both intact and cumulus-free ova was decreased when high concentrations of epididymal com-
a
F P w
2 Y
P
32
D. P. WOLF, M. HAMADA AND M. INOUE
IOC
50
0
I
I
I
I
2 3 4 Time (hours)
I
I
5
Fig. 1 Number of ova penetrated or fertilized by a single sperm as a function of insemination time. Penetration refers to zona passage, while fertilization includes sperm-egg fusion. Open symbols represent cumulusintact eggs. Closed symbols represent cumulus-free eggs.
TABLE 2
Kinetics of washed epididymal sperm penetration of nwuse ova Time (hour) Expt. No. Egg type
1 2 3 Totals
Intact Cumulus-free Intact Cumulus-free Intact Cumulus-free Intact Cumulus-free
2
91 29 82 38
90 48 88 40
6
(29132) 3.89 (14/48) 1.57 (27/33) 2.26 (13/34) 1.53 (69/77)2.94 (33/69) 1.39 (125/142)3.03 (60/151)1.47
91 (43147) 2.77 71 (32/45) 2.84 71 (43/61)2.63 64 (39161) 1.77 78 (101/129)2.73 55 (82/150)2.50 79 (187/237)2.71 60 (153/256)2.40
’Results reported as % penetrated, number of ova employed (in parentheses) and the mean number of sperm/penetrated ovum. Significantly reduced (p < 0.01) over intact penetration levels.
TABLE 3
The effect of sperm age on zonae penetrability in vitro Sperm penetration Sperm age
3 hours 4 hours
Egg type
Original ova
Fresh ova
Intact Cumulus-free Intact Cumulus-free
96 (24/25)2.41 81 (25/31) 2.28 91 (20/22) 2.10 71 (24/34)2.00
85 (17/20)1.65 64 (16/25)1.75 22 (8/36)1.25 23 (3/13)1.00
Sperm age represents time post insemination, as all sperm were preincubated in concentrated form for one to two hours prior to use. Penetration was scored after 3-hour incubations; % penetrated, number of ova employed (in parentheses) and the mean number of spermlpenetrated ovum. *Significantly reduced fp < 0.051 over original levels.
33
ZONA REACTION IN VITRO TABLE 4
The effect of epididymal components on sperm penetration of mouse ova in vitro 1 Sperm penetration Egg exposure to epididymal components
During preincubation and insemination During preincubation only
Egg type
Intact Cumulus-free Intact Cumulus-free
Control
Experimental
80 (227/283)3.71
16 (46/290)1.23 3 (5/182)1.27 75 (140/187)2.41 68 (188/277)4.14
58 (120/207)1.93 92 (186/203)3.96 75 (200/268)3.72
' Penetration was scored after four to five hours incubation; % penetrated,number of ova employed (in parentheses) and the mean number of spermlpenetrated ovum. *Significantly reduced (p < 0.05) over control group. TABLE 5
Sperm capacitating conditions and the penetration of m u s e ova in vitro Sperm penetration Sperm pretreatment
% penetrated, (number ova employed),
Egg type
Unwashed control: capacitated in culture medium Unwashed experimental: capacitated in the presence of cumulus masses Washed control: capacitated in culture medium Washed experimental: capacitated in culture medium containing diluted epididymal companents
mean number spermlpenetrated ova
Intact Cumulus-free
89 (97/109)2.13 60 (109/182)2.40
Intact Cumulus-free
86 (71/831 1.87 66 (122/186)1.98
Intact Cumulus-free
72 (140/196)3.11 46 a (251/54232.91
Intact Cumulus-free
76 (104/136)2.63 44 (223/50412.42
1 Penetration was scored four to five hours post insemination. Both unwashed and washed control pretreatment medium contained epididymal components carried over during the preparation of sperm suspensions. Sperm were washed three to four times and then capacitsted in fresh culture medium in the washed experimental category. 2Significantly reduced (p < 0.05) over intact eggs.
ponents were present during ovum preincubation and in the insemination mixture; however, simple preincubation of cumulus-free ova with epididymal preparations did not affect penetration. Coincidentally, pretreatment of intact ova with epididymal components led to a reduction in penetration which presumably reflected the premature dispersal of cumulus. Submaximal sperm penetration of cumulusfree ova could reflect inadequate capacitation if cumulus andlor epididymal components played a role in this process. Therefore, penetration levels for intact and cumulus-free ova were compared under different sperm capacitating conditions (table 5 ) . When sperm were preincubated in the presence or absence of cumulus or when washed sperm controls were compared with washed sperm in which epididymal components were diluted a t least 100-
fold, penetration levels for each ovum type were similar. Thus, the disparities in the penetration of intact and cumulus-free ova seen in this study were independent of the conditions employed for sperm capacitation. DISCUSSION
A significant finding from the present study was that cumulus-intact mouse ova inseminated in vitro underwent a zona reaction, even though the reaction did not occur until most zonae (55-60%)had been penetrated by more than one sperm. A distribution of penetrated sperm based on chance interactions between gametes would, therefore, be anticipated, justifying a description of the zona reaction in vitro as non-functional (Wolf and Inoue, '76).A moderately effective zonal block was reported previously for mouse ova recovered from natural matings (Austin and
34
D. P. WOLF, M. HAMADA AND M. INOUE
Braden, '561, and insufficient zona modification by cortical granule exudate was suggested as a basis for the subnormal in vitro response (Barros and Yanagimachi, '72; Mintz and Gearhart, '73). The demonstration that a zona reaction occurs in the mouse, however, argues against this possibility and suggests rather that a non-functional reaction derives from the synchrony in sperm penetration occurring a t the high sperm concentrations employed in vitro. Despite the tendency toward multiple zonal penetration of these in vitro inseminated ova, monospermy levels were high (> 80%), reflecting the relative importance of the egg plasma membrane block in limiting sperm penetration of the vitellus. The existence and temporal requirements of the plasma membrane block in zona-free mouse ova have been defined elsewhere (Wolf, '77). The temporal requirements for the zona reaction have been estimated as not less than 10 minutes nor more than two hours by Braden e t al. ('54) or within 15 minutes of sperm attachment to the vitellus (Barros and Yanagimachi, '72). Under the present culture conditions, vitelline penetration of cumulusintact ova was initially detected in whole mounts between 30 and 60 minutes following insemination and was completed by 120 minutes (Inoue and Wolf, '75 and this study). By 120 minutes, a maximum mean number of spermlpenetrated ovum was also reached, the expected result if all penetrated ova had established a zonal block. Thus, the zona reaction of intact ova in vitro requires a maximum of 90 minutes following sperm-ovum fusion. In contrast to the finding of Iwamatsu and Chang ('70), who reported a progressive increase in the mean number of spermlpenetrated ovum over a 5-hour insemination period, the zona reaction, once established in this study, was highly effective. The influence of cumulus on the fertilization of hyaluronidase-treated ova has been studied in several species (see Bedford, '73 for review). In the mouse, high levels of fertilization were seen when such ova were inseminated with epididymal sperm capacitated by exposure to bovine follicular fluid (Iwamatsu and Chang, '70), to oviduct fluid (Pavlok and McLaren, '72) or to culture medium alone (Fukuda e t al., '72; Hoppe, '75; Wolf and Inoue, '76). However, in our experience, hyaluronidase-treated ovum preparations are not completely free of cumulus cells (Inoue and
Wolf, '75), and completely cumulus-free ova do not always fertilize a t high levels (Chang and Hunter, '75; Wolf and Inoue, '76). Physical abuse resulting from the additional manipulation required to remove residual cumulus cells seemed an unlikely explanation for this reduced fertility, especially in view of the ovum's ability to withstand the physical trauma associated with zona removal by mechanical means (Wolf et al., '76). In our initial attempts with completely cumulus-free ova, fertilization levels scored a t four hours were often low. However, results from the present study indicated that, although initial penetration of cumulus-free ova was delayed, relative to intact controls, high levels of penetration were attained after six hours of incubation. Indeed, the disparity in fertilization levels seen between intact and cumulus-free ova a t six hours may simply reflect nonspecific, age-dependent changes in the gametes over these prolonged incubation times. Cumulus-free ova were capable of undergoing a zona reaction, although the reaction was not complete until three hours post insemination, as indicated by the attainment of a constant mean number of spermlpenetrated ovum. Moreover, the monospermy levels in these ova were higher than in the corresponding intact or hyaluronidase-treated ova (Iwamatsu and Chang, '69, '70; Wolf and Inoue, '76) and were similar to those reported for natural matings (Braden et al., '54; Austin and Braden, '56). While an unequivocal explanation for the delay in sperm penetration observed here with cumulus-free ova is unavailable, several possibilities can be considered. The initiation of gamete interaction is, of course, dependent upon both sperm and ova; and the first step in this process, capacitated sperm binding to zonae of cumulus-free ova, occurs rapidly in our in vitro system (20 spermlzona within 5 minutes a t 2 x lo5 cellslml) (Inoue and Wolf, '75; Wolf and Inoue, '76). Gwatkin et al. ('74) maintain that capacitation in culture medium as utilized here actually represents capacitation by contaminating epididymal or cumulus components. This observation raises the possibility that washed epididymal sperm may undergo insufficient or subnormal capacitation, thereby accounting for the observed altered kinetics of penetration. However, the conclusions of Gwatkin et al. ('74) were based principally on the absence of penetration of cumulus-free ova following a 90-minute exposure to washed (1 time) epididymal sperm.
35
ZONA REACTION IN VITRO
Since in the present study penetration of cumulus-free ova was delayed, i.e., detected initially only after 120 minutes of insemination, and occurred independently of the presence or absence of cumulus or soluble epididyma1 components in the capacitation medium, we conclude that the delay in sperm penetration of cumulus-free ova is not related to the state of capacitation. The next obvious step to consider in the delayed penetration of cumulus-free ova is the occurrence of a n acrosome reaction. The sequence of events prerequisite to sperm penetration of zonae is generally conceded to involve capacitation, the occurrence of a n acrosome reaction, sperm penetration of the cumulus matrix and binding to the zona and, finally, zonal penetration (McRorie and Williams, ‘74; Chang and Hunter, ’75). However, in this sequence, sperm adherent to the outer margin of the zonae of cumulus-intact ova should have undergone an acrosome reaction, a prediction that is not always substantiated by experimental observation (Thompson e t al., ’74; Anderson et al., ’75; Nicosia and Wolf, unpublished results). We conclude, therefore, that an acrosome reaction is not an absolute prerequisite to sperm passage through the cumulus and further suggest that the rate limiting step in penetration of intact or cumulus-free ova could be the induction of this reaction. In fact, this phenomenon may apply to zona-free ova as well, since delays in capacitated sperm incorporation into this ovum type have been observed (Wolf e t al., ’76; Wolf, ’77). Moreover, it should be noted that studies with zona-free ova discount the possibility that cumulus cells directly influence ovum integrity, since both fresh and aged zona-free ova rapidly incorporate sperm in the complete absence of cumulus cells (Wolf and Hamada, ’76). If the above situation obtains, then plausible roles for the cumulus include a direct involvement in inducing an acrosome reaction in zona-bound sperm (Gwatkin e t al., ’72; Gwatkin and Carter, ’74) and /or indirect possibilities, such as “orientation” of sperm at the zona surface or the retention or alteration of acrosome reaction-inducing substances in the zona or perivitelline space. While little experimental evidence is available concerning these possibilities, it would be interesting to assess the kinetics of acrosome vesiculation in the presence and absence of intact and cumulus-free ova.
ACKNOWLEDGMENTS
This work was supported by grants from the Ford Foundation 65-58B and USPHS HD-07635. LITERATURE CITED Anderson, E., P. C. Hoppe, W. K. Whitten and G. S. Lee 1975 In uitrofertilization and early embryogenesis: A cytological analysis. J. Ultrastruct. Res., 50: 231-252. Austin, C. R., and A. W. H. Braden 1956 Early reactions of the rodent egg to spermatozoon penetration. J. Exp. Biol., 33: 358-365. Barros, C., and R. Yanagimachi 1972 Polyspermy-preventing mechanisms in the golden hamster egg. J. Exp. Zool., 180: 251-266. Bedford, J. M. 1973 Mechanisms involved in penetration of spermatozoa through the vestments of t h e mammalian egg. In: Physiology and Genetics of Reproduction, P a r t B. Basic Life Sciences, Vol. 4. E. M. Coutinho and F. Fuchs, eds. Plenum Press, New York. Braden, A. W. H., C. R. Austin and H. A. David 1954 The reaction of the zona pellucida to sperm penetration. Aust. J. Biol. Sci., 7: 391-409. Chang, M. C., and R. H. F. Hunter 1975 Capacitation of mammalian sperm: biological and experimental aspects. In: Handbook of Physiology. Vol. V. Section 7, Endocrinology. R. 0. Greep and E. B. Astwood, eds. American Physiological Society, Washington, D.C. Cross, P. C., and R. L. Brinster 1970 In uitrodevelopment of mouse oocytes. Biol. Reprod., 3: 298-307. Fukuda, Y., 0. Okado and Y. Toyoda 1972 Studies on the fertilization of mouse eggs in uitro. 111. Fertilization of denuded eggs by capacitated spermatozoa. Jap. J. Anim. Reprod., 18: 73-77. Gwatkin, R. B. L., 0. F. Anderson and C. F. Hutchison 1972 Capacitation of hamster spermatozoa in uitro: the role of cumulus components. J. Reprod. Fert., 30: 389-394. Gwatkin, R. B. L., 0. F. Anderson and D. T. Williams 1974 Capacitation of mouse spermatozoa in uitro: involvement of epididymal secretions and cumulus oophorus. J. Reprod. Fert., 41: 253-256. Gwatkin, R. B. L., and H. E. Carter 1974 Cumulus oophorus. In: Scanning Electron Microscopic Atlas of Mammalian Reproduction. E. S. E. Hafez, ed. Igaku Shion, Tokyo. Hoppe, P. C. 1975 Fertilizing ability of mouse sperm from different epididymal regions and after washing and centrifugation. J. Exp. Zool., 192: 219-222. Inoue, M., and D. P. Wolf 1974 Comparative solubility properties of the zonae pellucidae of unfertilized and fertilized mouse ova. Biol. Reprod., 11: 558-565. 1975 Sperm binding characteristics of t h e murine zona pellucida. Biol. Reprod., 13: 340-346. Ipsen, J., and P. Feigl 1970 Bancroft’s Introduction to Biostatistics. Harper and Row, New York, p. 106. Iwamatsu, T., and M. C. Chang 1969 In uitrofertilization of mouse eggs in the presence of bovine follicular fluid. Nature (London), 224: 919-920. 1970 Further investigation of capacitation of sperm and fertilization of mouse eggs in uitro. J. Exp. Zool., 175: 271-281. 1971 Factors involved in the fertilization of mouse eggs in uitro. J. Reprod. Fert., 26: 197-208. McRorie, R. A,, and W. L. Williams 1974 Biochemistry of mammalian fertilization. Ann. Rev. Biochem., 43: 777803. Mintz, B., and J. D. Gearhart 1973 Subnormal zona pellucida change in parthenogenetic mouse embryos. Develop. Biol., 31: 178-184.
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Mukherjee, A. B. 1972 Normal progeny from fertilization in uitro of mouse oocytes matured in culture and spermatozoa capacitated in uitro. Nature (London), 237: 397-398. Mukherjee, A. B., and M. M. Cohen 1970 Development of normal mice by in uitro fertilization. Nature (London), 228: 472-473. Nicosia, S. V., D. P. Wolf and M. Inoue 1977 Cortical granule distribution and cell surface characteristics of mouse ova. Develop. Biol., 57: 208-226. Oliphant, G., and B. G. Brackett 1973 Capacitation of mouse spermatozoa in media with elevated ionic strength and reversible decapacitation with epididymal extracts. Fertil. Steril., 24: 948-955. Pavlok, A., and A. McLaren 1972 The role of cumulus cells and the zona pellucida in fertilization of mouse eggs in uitro. J. Reprod. Fert., 29: 91-97. Thompson, R. S., D. Moore-Smith and L. Zamboni 1974 Fertilization of mouse ova in uitro: An electron microscope study. Fertil. Steril., 25: 222-249. Toyoda, Y., and M. C. Chang 1974 Fertilization of rat eggs in uitro by epididymal spermatozoa and the development of eggs following transfer. J . Reprod. Fert., 36: 9-22. Toyoda, Y., M. Yokoyama and T. Hosi 1971a Studies on the fertilization of mouse eggs in uitro. I. In uitrofertilization
of eggs by fresh epididymal sperm. Jap. J. Anim. Reprod., 16: 147-151. 1971b Studies on the fertilization of mouse eggs in uitro. 11. Effects of in uitro pre-incubation of spermatozoa on time of sperm penetration of mouse eggs in uitro. Jap. J. Anim. Reprod., 16: 152-157. Whittingham, D. G. 1968 Fertilization of mouse eggs in uitro.Nature (London), 220: 592-593. Wolf, D. P. 1977 The vitelline block to polyspermy in zona-free mouse ova inseminated in uitro. Develop. Biol., in review. 1977 Involvement of a trypsin-like activity in sperm penetration of zona-free mouse ova. J . Exp. Zool., 199: 149-156. Wolf, D. P., and M. Hamada 1976a The zona reaction in mouse ova inseminated in uitro. Proc. Soc. Study of Reprod., Philadelphia, Pa., p. 21 (Abstract 12). 1976b Age-dependent losses in the penetrability of mouse ova. J. Reprod. Fert., 48: 213-214. Wolf, D. P., andM. Inoue 1976 Sperm concentration dependency in the fertilization and zonae sperm binding properties of mouse eggs inseminated in uitro. J. Exp. Zool., 196: 27-38. Wolf, D. P., M. Inoue and R. A. Stark 1976 Penetration of zona-free mouse eggs. Biol. Reprod., 15: 213-221.
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