Penetration of Zona-Free Eggs by Spermatozoa of

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1 BIOLOGY OF REPRODUCTION 6, (1972) Penetration of Zona-Free Eggs by Spermatozoa of Different Species A. HANADA AND M. C. CHANG2 Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts Received September 25, 1971 Recently ovulated eggs of mice, rats, and hamsters were treated with hyaluronidase and subsequently with trypsin or pronase to disperse the corona radiata and zona pellucida. When these zona-free eggs were inseminated with epididymal or uterine sperm of rats, epididymal sperm of hamsters or mice in the presence of either heated bovine follicular fluid or heated blood serum and incubated for 1-12 hr, low proportions of mouse eggs were penetrated by rat or hamster sperm (1.3-5%), rat eggs by hamster sperm (9%) and hamster eggs by rat sperm (8.7-26%). Although enlargement of the sperm head was observed in these cases, the transformation of the sperm head into a male pronucleus did not occur, the activation of eggs to resume the second maturation division was rare, and the formation of the female pronucleus never occurred. Very high proportions of rat eggs (95%) or hamster eggs (73%) were penetrated by mouse sperm and polyspermy occurred as incubation time increased. In these cases, the transformation of many sperm heads into male pronuclei, the initiation of the second maturation division, and the formation of a female pronucleus occurred normally. The transformation of mouse sperm heads into male pronuclei in the rat eggs appeared to be earlier than that of mouse sperm in the hamster eggs. Although the incorporation or fusion of eggs with foreign sperm may depend on the possibility of sperm capacitation, physiological affinity between the sperm of one species and the vitellus of another is also an important controlling factor, but the zona pellucida appears to be the major block for interspecific fertilization. Mammalian spermatozoa require a certain length of time, either in the female tract or under certain other conditions to develop their fertilizing capacity. This process is termed capacitation of sperm (for review see Bedford, 1970; Chang and Hunter, 1972). The penetration of rat eggs by rat sperm in vitro after the dissolution of zona pellucida has been described by Toyoda and Chang (1968). Incorporation and fusion of capacitated hamster sperm, but not uncapacitated spermatozoa, with zona-free hamster eggs in vitro have been reported by Yanagimachi 1 Research Fellow supported by the Agency of Science and Technology from the Office of the Prime Minister, Japan. Present address: National Institute of Animal Industry, Chiba-shi, Japan. 2 Research Career Awardee of National Institute of Child Health and Human Development (K6-HD 18,334). Copyright 1972 by the Society for the Study of Reproduction 300 and Noda (1970). This paper reports a series of experiments dealing with the penetration of zona-free eggs by spermatozoa of different species, the morphological changes of sperm heads and female chromatin following the penetration of eggs by foreign sperm, and the possibility of sperm capacitation under various conditions in a few laboratory animats. MATERIALS AND METHODS All the animals used in this study were kept in airconditioned rooms (at 21-23C) under 12 hr of artificial light from 7.00 to hr. On the day when postestrous vaginal discharge was observed, mature golden hamsters (Mesocricetus auratus) and immature Sprague-Dawley rats (25-35 days old) were injected ip with 25 IU/0.2 ml PMSG (Equinex, Ayerst Lab.) to increase the number of maturing follicles. They were injected again hr later with 25 IU/O.2 ml HCG (APL, Ayerst Lab.) for the induction of ovulation. Mature Swiss-Webster albino mice were simi-

2 PENETRATION OF EGGS BY FOREIGN SPERM 301 larly injected with 10 IU/O.2 ml PMSG and HCG. They were killed hr after the injection of HCG for the recovery of recently-ovulated eggs. The oviducts were placed in 0.5-mi warm Tyrode s solution (Difco Laboratories) containing 100 unit/mi of potassium penicillin G and 50 pg/mi streptomycin sulfate in a watch glass (3.8 cm in diameter). The solution was sterilized by filtration through a millipore filter. After breaking the dilated portion of the ampulla, the released eggs in the cumulus oophorus were transferred to another watch glass containing 0.2 ml of 0.1% hyaluronidase (Sigma Chemical Co.) in Tyrode s solution. This preparation was then covered with warm light paraffin oil (Fisher Co.) that previously had been bubbled for 1 hr with 5% CO2 in air in the presence of a small volume of Ringer s solution. After the dispersion of the cumulus clot and corona radiata, the denuded hamster eggs were then picked up and placed in 0.1 ml Tyrode s solution containing 0.1% trypsin (Sigma Chemical Co.). Bicarbonate-free Hank s solution containing 0.5% pronase (Calbiochem Co.), known to dissolve the zona pellucida (Mintz, 1962), was used for the rat and mouse eggs. The zona pellucida was completely dissolved within 2-5 mm in the hamster eggs, and within 4-6 mm in the rat and mouse eggs. To avoid the effect of the enzyme on the zona-free eggs, they were transferred as soon as possible to another watch glass containing heated bovine follicular fluid or heated serum known to induce capacitation of sperm (Yanagimachi, 1969, 1970). This was repeated twice before finally transferring the eggs into 0.05 ml bovine follicular fluid or serum either in a Falcon plastic tissue culture dish or in a watch glass covered with oil. These preparations were then kept in an incubator saturated with 5% CO2 in air maintained at 37C with 100% relative humidity. Sperm suspensions were prepared by placing the cauda epididymis (hamsters and mice) or vas deferens and cauda epididymis (rats) into ml warm Tyrode s solution in a watch glass. The cauda was cut open and sperm from the vas were squeezed out by means of a fine forceps. After gentle shaking of the watch glass for a few minutes, an even suspension of sperm was taken up by means of a tuberculin syringe connected to a 25-gauge needle. Then 0.05 ml of the sperm suspension was added to the droplet containing eggs. Thus the final medium during incubation consisted of an equal volume of bovine follicular fluid or serum and Tyrode s solution in a volume of 0.1 ml. For the recovery of rat sperm from the uterus, mature female rats were kept on a reversed light schedule, i.e., from 3.00 to hr for 4 to 6 weeks. At hr in the morning following the finding of a proestrous smear, one female was placed in a cage with two active males. The female was killed 4 hr after being separated from the males and sperm were recovered by flushing each uterine horn with 0.2 ml Tyrode s solution in a tuberculin syringe connected to a 23-gauge needle. Only sperm samples showing motility were used for insemination. After insemination the preparations were kept in the incubator for 1, 4, 8, or 12 hr and examined for the assessment of sperm penetration. Follicular fluid of cow ovaries obtained from a slaughterhouse was prepared according to the method described by Yanagimachi (1969). Rat or hamster serum was prepared by centrifugation of blood obtained from the aorta of mature females under sodium pentobarbital anesthesia. Both bovine follicular fluid and blood serum were sterilized by filtration through a millipore filter (pore size of 0.22,.jm) and kept in air tight test tubes at 2-4C. The samples were heated either at 56C for 35 mm or at 60C for 15 mm just before use. All of the instruments, syringes, glass pipettes and watch glasses were sterilized and aseptic precautions were strictly observed during the manipulation of sperm and eggs. After incubation the eggs were picked up with a fine glass capillary pipette and washed twice in Hank s solution containing 1 mg/ml of polyvinylpyrolidone. They were then placed on a slide in the center of four vaseline spots and anchored between a cover slip. After examination under a phase-contrast microscope, the slide was placed in 10% neutral formalin overnight at 4C and then stained with 0.25% Lacmoid in 45% acetic acid. The eggs were then examined under a phase-contrast microscope for morphological details. Since the sperm head normally enlarges soon after penetration into the vitellus, the criterion for sperm penetration was the presence of enlarged sperm heads and their corresponding tails. RESULTS Penetration of Eggs by Foreign Sperm. From the results presented in Table 1, it can be seen that although zona-free mouse eggs can be penetrated either by epididymat rat sperm (5 %) in the presence of rat serum, or by epididymat hamster sperm (1.3%) in the presence of bovine fotlicular fluid, the proportion of penetrated eggs was no more than 2% (hamster sperm) to 9 % (rat sperm), and that the penetration occurred only 4 hr after insemination in both cases (exp. 1 and 2). When zona-free rat eggs were inseminated with epididymal hamster sperm in the presence of bovine fotlicular fluid, 4% were penetrated 1 hr after insemination, and 13% 4 hr after insemination, but no

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4 PENETRATION OF EGGS BY FOREIGN SPERM 303 further increase was observed 8-12 hr later (9-14%, exp. 4). In the case of zona-free rat eggs inseminated with epididymal mouse sperm in the presence of bovine follicular fluid, a very high proportion of eggs (89 %) were penetrated within 1 hr and all eggs were penetrated at 8 to 12 hr after insemination (exp. 5). Similarly, a very high proportion of zona-free hamster eggs were penetrated by mouse sperm in the presence of bovine follicular fluid (73%) but the proportion of penetrated eggs increased only 4-12 hr after insemination (78-100%, exp. 6). The penetration of zona-free hamster eggs did not occur after insemination with either epididymal rat sperm (exp. 7) or with rat sperm recovered from the uterus in the presence of bovine follicular fluid (exp. 10). However, in exp. 8, where zona-free hamster eggs were again inseminated with epididymal rat sperm, but in the presence of rat blood serum, 3 % of the eggs were penetrated 4 hr after insemination and 14-16% were penetrated 8-12 hr after insemination. In exp. 9, zona-free hamster eggs were again inseminated with rat sperm recovered from the uterus in the presence of bovine follicular fluid and 20 of 24 (83 %) hamster eggs were penetrated by rat sperm only 12 hr after insemination. In this case, sperm motility was extremely even 12 hr after incubation. It appears that zona-free hamster eggs can be penetrated by rat sperm after a longer time of exposure if the physiological integrity of rat sperm can be properly maintained and the capacitation of rat sperm can be achieved in vitro. Spontaneous activation of eggs, as shown by the presence of the telophase of the second maturation spindle or by the formation of the second polar body in the absence of an enlarged sperm head, occurred in a small proportion of eggs ( %) when zona-free eggs were inseminated with foreign sperm. However, a very high proportion of spontaneously activated eggs (85 %) were observed when zona-free rat eggs were inseminated with hamster sperm (exp. 4). When zona-free rat eggs were similarly incubated without sperm, the proportion of activated eggs was also high. It thus appears that the activation of rat eggs is an inherent property of the rat eggs themselves, and not due to the stimulation of hamster sperm. When rat eggs were inseminated with mouse sperm (exp. No. 5), sperm penetration occurred within 1 hr. Subsequent activation of these eggs was only 4%. The incidence of spontaneous activation was lower (0-14%) when mouse or hamster eggs were inseminated with foreign sperm than when mouse eggs were inseminated with mouse sperm (18%, exp. 3) or hamster eggs with hamster sperm (28%, exp. 11). This may reflect some stimulation of eggs by the sperm of its own species. The low proportion of zona-free mouse eggs penetrated by mouse sperm (exp. 3) may indicate some adverse effect of pronase on the unfertilized mouse eggs. The lower proportion of zona-free hamster eggs penetrated by hamster sperm in the presence of hamster serum (44%, exp. 11), as compared with zona-free hamster eggs penetrated by mouse sperm in the presence of bovine follicular fluid (73%, exp. 6), may indicate that bovine follicular fluid is a better agent for the capacitation of mouse sperm than hamster serum for that of the hamster sperm. Morphology of Penetrated Eggs. Although the enlargement of rat (Fig. 1) or hamster sperm heads (Fig. 2) in the vitellus of mouse eggs occurred in a few cases, not one of the enlarged sperm heads was transformed into the male pronucleus, and only 1 of 5 mouse eggs was at telophase in the presence of the enlarged hamster sperm head. The transformation of the enlarged sperm head did not occur when enlarged hamster sperm heads were found in the rat eggs (Fig. 3). Normal activation of rat eggs was not initiated by hamster sperm (only 2 of 24 eggs were at telophase, Fig. 4), and the egg

5 Fios With the exception of the egg shown in Fig. 6, which was photographed as a fresh specimen, all the others were photographed after being stained with Lacmoid: phase contrast microscopy, lox ocular, X40 objective. I. A mouse egg with an enlarged rat sperm head (arrow), and the second maturation spindle (arrows). (Approx. X375) 2. A mouse egg with an enlarged hamster sperm head (arrow). (Approx. X375) 3. A rat egg with an enlarged hamster sperm head (arrow). (Approx. X375) 4. A spontaneously activated rat egg inseminated with hamster sperm, showing extrusion of the second polar body (arrow). (Approx. x250) 5. A rat egg inseminated with hamster sperm showing scattered chromosomes (arrow) 12 hr after incubation: no penetration of sperm. (Approx. X250) 6. A rat egg penetrated by mouse sperm at the stage of pronuclear formation (arrows) photographed before fixation. (Approx. X250) 7. A rat egg penetrated by mouse sperm at the stage of pronuclear formation showing two male pronuclei (MPN), one female pronucleus (FPN), one enlarged sperm head (SH) with a sperm tail (ST). (Approx. X250) 8. Four rat eggs penetrated by mouse sperm at the stage of pronuclear formation showing several male pronuclei with female pronucleus and enlarged sperm head and tails. (Approx. X250) 304

6 PENETRATION OF EGGS BY FOREIGN SPERM 305 chromatin were scattered (Fig. 5) in most cases at the end of incubation. When hamster sperm were added to zonafree mouse or rat eggs, the enlarged sperm head appeared not to be inside the vitellus. Whether this is due to some peculiarity of hamster sperm in reaction to mouse or rat eggs, to some components which leaked out of these eggs, or to certain artifacts during manipulation of eggs is hard to say. When an enlarged hamster sperm head was found in the mouse or rat eggs, the corresponding sperm tail was either absent or on the surface of the vitellus. This may indicate the rejection of the hamster sperm tail by foreign eggs. By contrast, the transformation of the sperm head into a male pronucleus and the activation of eggs from the extrusion of the second polar body to the formation of a female pronucleus proceeded normally when mouse sperm penetrated into the zonafree rat or hamster eggs (Figs. 6-10). In general, polyspermy occurred in both cases and the number of sperm per egg increased as the incubation time increased (an average FIGS All eggs were photographed under a phase-contrast microscope, lox ocular, X40 objective, after being stained with Lacmoid. (Approx. X 375) 9. A hamster egg penetrated by many mouse sperm, showing nine enlarged sperm heads. 10. A hamster egg penetrated by many mouse sperm, showing one female pronucleus (FPN) and one enlarged sperm head (SH); the other male pronuclei are not in focus. II. A hamster egg with two enlarged rat sperm heads (arrows). 12. A hamster egg with enlarged mouse sperm heads (SH) and two second maturation spindles (arrows).

7 306 HANADA AND CHANG number of 4-7 when rat eggs were penetrated by mouse sperm; an average of 1-l7 when hamster eggs were penetrated by mouse sperm). Since several enlarged sperm heads and several male pronuclei could be found in the same eggs when rat or hamster eggs were penetrated by mouse sperm, it appears that the formation of one pronucleus does not inhibit the formation of other male pronuclei. However, the rate of transformation of the mouse sperm head into the male pronucleus in the rat eggs was higher as compared with that of the mouse sperm head in the hamster egg, as shown in Text Fig. 13. When zona-free hamster eggs were inseminated with either rat epididymal or uterine sperm, sperm penetration occurred, as shown by the enlargement of the sperm head (Fig. 11) and, in general, the number of sperm per egg also increased (from 1 to 5) as the incubation time increased, but none of the sperm heads transformed into a male pronucleus even 12 hr after insemination. The activation of hamster eggs by rat sperm was also rare because only 2 of 36 eggs extruded their second polar bodies. A hamster egg with two second maturation spindles was observed (Fig. 12). In general, in those cases where a low proportion of eggs were penetrated (mouse eggs by rat or hamster sperm, rat eggs by C 3. too #{176} 20 IROI. eggs / / penetrated / / / / Hamster by by 0 mouse mouse eggs sperm sperm penetrated p Hours of incubation FiG. 13. Percentages of penetrated eggs with male pronuclei. hamster sperm, or hamster eggs by rat sperm) only one or two sperm entered the egg. In those cases where a high proportion of eggs were penetrated (rat or hamster eggs by mouse sperm), polyspermy occurred. The Motility and Acrosome Reaction of Sperm. The motility of sperm at various times after incubation with eggs of different species is presented in Table 2. The acrosome reaction, i.e., the elevation or the loss of the acrosome, was obvious after incubation for 1 hr in the case of mouse or hamster sperm, but in rat sperm the acrosome reaction could not be ascertained even after incubation for 12 hr. Since elevation or loss of the acrosome, besides certain postmortem acrosomal changes, is a indication of the capacitation of sperm (Austin and Bishop, 1958; Yanagimachi, 1966; Iwamatsu and Chang, 1969), the possibility of penetration by foreign sperm may be closely related to the possible occurrence of sperm capacitation under certain circumstances. The motility of hamster sperm was poor at the end of incubation (Table 2) and the proportion of zona-free mouse or rat eggs penetrated by hamster sperm was also at a low level (Table 1, exps. 2 and 4). By contrast, in one experiment (Table 1, exp. 9) the motility of rat uterine sperm was very at the end of incubation; a high proportion of hamster eggs were penetrated by rat sperm at the end of incubation. DISCUSSION The enlargement of the sperm head soon after penetration into the vitellus of the egg is a common feature during normal fertilization (Sobotta, 1895; Chang and Hunt, 1962). In the present study, the presence of an enlarged sperm head and the corresponding sperm tail were taken as evidence of sperm penetration. Although when mouse eggs were penetrated by rat or hamster sperm, or rat eggs by hamster sperm, and no corresponding sperm tail could be found for each sperm head, we are fairly confident

8 PENETRATION OF EGGS BY FOREIGN SPERM 307 TABLE 2 MOTILITY OF SPERM AT VARIOUS TIMES AFTER INCUBATION Sperm of Sperm from Suspension medium with eggs 1 hr Motility after various times of incubation 4 hr 8 hr 12 hr Mouse epididymis Hamster epididymis Rat vas and epididymis uterus s Tyrode s and bovine follicular fluid Tyrode s and rat serum Tyrode s and bovine follicular fluid, loss of acrosome, loss of acrosome, Acrosome reaction not clear, Acrosome reacion not clear, Acrosome action clear re- not sluggish sluggish, Acrosome reactionnotclear, Acrosome reaction not clear sluggish, Acrosome reaction not clear almost motionless almost motionless fair almost motionless In one experiment, very motility was observed 12 hrs after incubation with zona-free hamster eggs and 20 of 24 eggs were penetrated. that those eggs with sperm heads were truly penetrated and did not represent artifacts because the enlarged heads were deep inside the vitellus. Rat sperm are known to need capacitation in the female tract before they can penetrate eggs in vivo (Austin, 1951, 1952; Noyes, 1953). In a study of the penetration of rat eggs by rat sperm in vitro after the dissolution of the zona pellucida by treatment with chymotrypsin, Toyoda and Chang (1968) concluded that either capacitation of sperm can be achieved in a short time in the presence of chymotrypsin or that capacitation of sperm is necessary only for penetration through the zona pellucida. Hamster sperm also need capacitation in the female tract to develop their fertilizing capacity (Chang and Sheaffer, 1957; Yanagimachi, 1966) but capacitation of hamster sperm in vitro can be achieved in the presence of hamster follicular fluid (Barros and Austin, 1967), heated bovine follicular fluid (Yanagimachi, 1969), or blood serum (Yanagimachi, 1970). Capacitation of mouse sperm in the female tract requires no more than 1 hr (Braden and Austin, 1954; Mc- Gaughey, Marston and Chang, 1968). Capacitation of mouse sperm in vitro can be achieved in the presence of bovine follicular fluid within 3-4 hr (Iwamatsu and Chang, 1970). When hamster sperm capacitated in vitro by treatment with heated bovine follicular fluid for 3 hr were added to the zona-free hamster eggs, it was found that only capacitated sperm, but not uncapacitated, could be incorporated and fused with the vitellus (Yanagimachi and Noda, 1970). In many cases in the present study when uncapacitated epididymal or vas sperm were mixed with eggs in the presence of bovine follicular fluid or blood serum, sperm penetration or the incorporation of foreign sperm with vitellus

9 308 HANADA AND CHANG was observed. If sperm penetration or incorporation and fusion of zona-free eggs with foreign sperm can be achieved only by capacitated sperm, we must assume that capacitation of sperm was achieved during the period of incubation and that the success or failure of sperm penetration may be closely correlated to the degree of capacitalion or the number of sperm capacitated under our experimental conditions. In this respect the capacitation of rat sperm and the fertilization of rat eggs in vitro have not been demonstrated previously and the penetration of zona-free mouse or hamster eggs by rat sperm was very low (5-8.7%). This may reflect the difficulty of achieving capacitation of rat sperm in vitro. In only one experiment where rat sperm may have been capacitated in the uterus, was there a relatively high proportion of hamster eggs penetrated by rat sperm (26%, Table 1, exp. 9). Mouse sperm can readily be capacitated in vitro, and are able to fertilize mouse eggs in vitro (Iwamatsu and Chang, 1969, 1970, 1971; Toyoda, Yokoyama, and Hosi, 1971). In the present study, when mouse sperm were used to inseminate zona-free eggs of rats and hamsters, a high proportion of rat or hamster eggs (73-95 %) were penetrated. By contrast, hamster sperm can easily be capacitated in vitro and they are able to fertilize hamster eggs in vitro (Yanagimachi and Chang, 1963; Yanagimachi, 1966, 1970; Barros and Austin, 1967; Gwatkin and Anderson, 1969), but when hamster sperm were used to inseminate mouse or rat eggs, only a very small proportion of eggs (1.3-9 #{182}4) were penetrated. Thus, it appears that the possibility of penetration of zona-free eggs by foreign sperm is not entirely dependent on the possibility of sperm capacitation. Although it has not been proven that rat blood serum can induce capacitation of rat sperm, we have found that the motility of rat sperm was much better in the presence of rat blood serum than in the presence of bovine follicular fluid, and a few mouse eggs (5%, Table 1, exp. 1) or hamster eggs (8.7%, Table 1, exp. 8) were penetrated by rat sperm in the presence of heated rat blood serum. Although the enlargement of a foreign sperm head in the vitellus was observed in many cases, the transformation of the enlarged sperm head into the male pronucleus was observed only when mouse sperm were incorporated with zona-free rat or hamster eggs. Similarly, the normal activation of eggs, that is, the resumption of the second maturation division and the formation of the female pronucleus, were also observed only when mouse sperm were incorporated with zonafree rat or hamster eggs. Normal activation of eggs did not occur when zona-free mouse eggs were incorporated with rat or hamster sperm, rat eggs with hamster sperm, or hamster eggs with rat sperm, although enlargement of the sperm head had occurred. It appears that the physiological reaction necessary for the enlargement of the foreign sperm head is not so specific. The transformation of a foreign sperm head into a male pronucleus and the activation of eggs are more specific and closely related to each other. It has been reported that only 1 of 673 rat eggs had a rabbit spermatozoon in the perivitelline space when rat eggs were transferred to the oviducts of mated rabbits (Dickmann, 1962). Barros (1968) observed that none of 280 mouse eggs were pene. trated by hamster sperm, but 1 of 294 rat eggs had 2 hamster sperm in the perivitelline space when mouse or rat eggs were incubated with hamster sperm. Yanagimachi (1972), however, reported the penetration of all of 71 zona-free hamster eggs by capacitated guinea pig sperm in vitro. In view of the fact that fertilization never occurred following the insemination of mice, rats or hamsters with heterologous sperm of these species in our laboratory, it must be

10 PENETRATION OF EGGS BY FOREIGN SPERM 309 concluded that the zona pellucida is the major block for the penetration of eggs by foreign sperm. ACKNOWLEDGMENT This work was supported by a grant from the National Institute of Child Health and Human Development (HD 03472) and a grant from the Ford Foundation. Thanks are due to Mrs. Rose Bartke for assistance. REFERENCES AUSTIN, C. R. (1951). Observations on the penetration of sperm into the mammalian egg. Aust. J. Sci., Ser. B., 4, AUSTIN, C. R. (1952). The capacitation of the mammalian sperm. Nature London 170, 326. AUSTIN, C. R., AND M. W. H. BISHOP (1958). Role of rodent acrosome and perforatorium in fertilization. Proc. Roy. Soc., B., 149, BARROS, C. (1968). In vitro capacitation of golden hamster spermatozoa with Fallopian tube fluid of the mouse and rat. J. Reprod. Fert. 17, BARROS, C. AND C. R. AUSTIN (1967) In vitro fertilization and the sperm acrosome reaction in the hamster. J. Exp. Zoo!. 166, BEDFORD, J. M. (1970). Sperm capacitation and fertilization in mammals. Biol. Reprod., Suppl. 2, BRADEN, A. W. H., AND C. R. AUSTIN (1954). Fertilization of the mouse egg and the effect of delayed coitus and of hot-shock treatment. Atist. J. Biol. Sci., B. 7, CHANG, M. C., AND DOROTHY M. HUNT (1962). Morphological changes of sperm head in the ooplasm of mouse, rat, hamster and rabbit. A,iat. Rec. 142, CHANG, M. C., AND R. H. F. HUNTER (1972). Capacitation of mammalian sperm: Biological and experimental aspects. Handbook of Physiology, Section of Endocrinology, The Amer. Physiol. Society. In press. CHANG, M. C., AND D. SHEAFFER (1957). Number of spermatozoa ejaculated at copulation, transported into the female tract, and present in the male tract of the golden hamster. J. Heredity 48, DICKMANN, Z. (1962). Experiments on interspecific sperm penetration through the zona pellucida. J. Reprod.Fert.4, GWATKIN, R. B. L. AND 0. F. ANDERSON (1969). Capacitation of hamster spermatozoa by bovine follicular fluid. Nature Londo,, 224, IWAMATSU, T., AND M. C. CHANG (1969). In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature London 224, IWAMATSU, T., AND M. C. CHANG (1970). Further investigation of capacitation of sperm and fertilization of mouse eggs in vitro. f. Exp. Zoo!. 175, IwAMATSu, T., AND M. C. CHANG (1971). Factors involved in the fertilization of mouse eggs ii, vitro. J. Reprod. Fer;. 26, MCGAUGHEY, R. W., J. H. MARSTON, AND M. C. CHANG (1968). Fertilizing life of mouse spermatozoa in the female tract. J. Reprod. Feri. 16, MINTZ, BEATRICE (1962). Experimental study of the developing mammalian egg: Removal of the zona pellucida. Science 138, NOYES, R. W. (1953). The fertilizing capacity of spermatozoa. West. J. Sorg. Obsiet. Gynec. 61, SOBOTTA, J. (1895). Die Befruchtung und Furchung des Eis der Maus. Arc/i. Mikr. A,,w. 45, TOVODA, Y., AND M. C. CHANG (1968). Sperm penetration of rat eggs in vitro after dissolution of zona pellucida by chymotrypsin. Nature 220, TOYODA, Y., M. YOKOYAMA, AND T. Hosi (1971). Studies on the fertilization of mouse eggs ii, vitro. 1. In vitro fertilization of eggs by fresh epididymal sperm. fop. I. Anim. Reprod. 16, YANAGIMACHI, R. (1966). Time and process of sperm penetration into hamster ova in vivo and in vitro. J. Reprod. Fert. 11, YANAGIMACI-II, R. (1969). 1, vitro acrosome reaction and capacitation of hamster spermatozoa by bovine follicular fluid and its fractions. J. E.vp. Zoo!. 170, YANAGIMACHI, R. (1970). Iii vitro capacitation of golden hamster spermatozoa by homologous and heterologous blood serum. Biol. Reprod. 3, YANAGIMACHI, R. (1972). Penelration of guinea pig spermatozoa into hamster eggs ii, vitro. J. Reprod. Fert. In press. YANAGIMACHI, R., AND M. C. CHANG (1963). Fertilization of hamster eggs in vitro. Nature London 200, YANAGIMACHI, R., AND Y. D. NODA (1970). 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,