The Time of Cortical Granule Breakdown and Sperm Penetration in Mouse and Hamster Eggs Inseminated in vitro

Transcription

1 BIOLOGY OF REPRODUTION 19, (1978) The Time of ortical Granule Breakdown and Sperm Penetration in Mouse and Hamster Eggs Inseminated in vitro Y. FUKUDA1 and M.. HANG2 Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts ABSTRAT The time of sperm attachment and cortical granule breakdown in intact and zona-free mouse eggs and zona-free hamster eggs at various times after insemination with capacitated sperm was examined by means of a vital staining technique. Three to 68% intact mouse eggs had sperm passing through the zona pellucida and attached to their vitellus 5-15 mm after insemination and 4-78% eggs had no or very few cortical granules mm after insemination. The attachment of sperm to the vitellus in the zona-free eggs was immediate, but the breakdown of cortical granules was observed in 2-15% of the mouse eggs and 65-85% of the hamster eggs within 5-15 mm after insemination. When zona-free hamster eggs were exposed to mouse sperm, the breakdown of cortical granules was observed in 4-47% of the eggs mm after insemination. It appears that the breakdown of cortical granules after the attachment of homologous sperm occurs sooner in the hamster eggs than in the mouse eggs, but occurs much slower in hamster eggs exposed to mouse sperm, although more mouse sperm penetrated into and attached to the hamster vitellus. INTRODUTION The presence of cortical granules in the unpenetrated, but not in the penetrated hamster eggs examined under a phase contrast microscope, was first described by Austin (1956). Examination of tubal eggs with the electron microscope has shown that the presence of cortical granules is a general feature of unfertilized mammalian eggs (Szollosi, 1962). Although vital stain of cortical granules by Janus Green in sea urchin eggs was reported (Motomura, 1941), this method was unsatisfactory for hamster eggs (Austin, 1956). It has been reported that the disappearance or breakdown of cortical granules after sperm penetration is initiated by the fusion of gamete membranes; simple contact is not sufficient and sperm penetration is not needed (Gwatkin et al., 1976). Since the zona reaction to block the further entry of spermatozoa (Braden et al., 1954) was attributed to the breakdown of cortical granules after sperm penetration (Austin and Braden, 1956) and direct evidence was presented (Barros and Yanagimachi, 1971, Accepted February 8, Received December 12, On leave from Department of Animal Science, Kitasato University Towada-shi, Aomori, 034, Japan. 2 Research areer Awardee (HD 18,334) of National Institute of hild Health and Human Development. 1972; Gwatkin et al., 1973), a study of cortical granule breakdown is of importance to under stand the block to entry of supplementary sperm. The time required for the cortica4 granule breakdown has been reported to be mm in the zona-free hamster eggs (Barros and Yanagimachi, 1972; Gwatkin, et al., 1976), 45 mm-i h in the intact and corona-devoid rabbit eggs (Fraser et al., 1972) and 1 h in the zona-free mouse eggs (Wolf et al., 1976) after insemination. The present study, utilizing a vital staining technique, was designed to determine the time sequence of sperm attachment, cortical granule breakdown, sperm penetration and the activation of eggs in the intact and zona-free mouse eggs and in zona-free hamster eggs inseminated with capacitated hamster or mouse spermatozoa. MATERIALS AND METHODS A modified Krebs Ringer bicarbonate solution (Toyoda et al., 1971) was used for in vitro fertilization and 2 parts of Tyrode s solution with 1 part of agaxnma human serum (North American Biochemicals, Inc.) was used for preincubation of hamster spermatozoa. The medium was prepared by dissolving all ingredients without serum in double distilled water and 0.01 ml 1% phenol red (Difco) was added to 100 ml medium. It was then gassed with humid 5% O2 in air for 20 mm and stored in a tightly stopped flask at 4#{176}. Bovine serum albumin or agamma human serum was added just before use and the medium was 261

2 262 FUKUDA AND HANG sterilized with a Millip ore filter (Swinnex-1 3 filter unit, Millipore o.). Epididymal sperm suspension was prepared according to Toyoda et al. (1971) for the mouse and according to Barros and Yanagimachi (1972) for the hamster. In order to capacitate sperm in vitro, sperm suspensions in concentrations of X 10 cells/mi (mouse) and 1-2 X 1O cells/mi (hamster) covered with oil were incubated at 37#{176}under 5% O2 in air for 2 h (mouse) or 6 h (hamster). For the study of mouse eggs, 0.1% of Bismark Brown (National Aniline and hemical) was added to the medium. This was first prepared and diluted to a concentration of 0.01%, then 0.2 ml of this solution was added to 0.2 ml medium covered with oil in a dish mm before the introduction of eggs. Mature female D-i Swiss albino mice were injected i.p. with 5 lu PMSG (Sigma) followed 48 h later with 5 iu hg (A.P.L. Ayerst Ltd.). Mature female golden hamsters were similarly injected with 20 IU PMS and 20 IU h (Yanagimachi, 1969) to induce superovulation. The females were killed h after injection of h and their eggs were recovered by breaking the ampulla of oviducts in a dish containing the appropriate medium without Bismark Brown covered with oil. To obtain zonafree eggs, the egg clots of hamster and mouse were first treated with hyaluronidase (150 units/mi) for 15 mm and then with 0.02% a-chymotrypsin (3 X crystallized, Type I, Sigma) for a few mm at 30#{176} in the medium without serum albumin and Bismark Brown. After the dissolution of the zona pellucida, the eggs were washed 3 times. The intact mouse eggs in cumulus clot and the zona-free mouse eggs were introduced into 0.4 ml preincubated (capacitated) sperm suspensions in a concentration of about 25 X io cells/mi prepared mm earlier from the original sperm suspension in the presence of 0.005% Bismark Brown. For the study of the zona-free hamster eggs, 5 l of the preincubated sperm suspension was added to 50 l medium containing 0.005% Bismark Brown immediately after introduction of eggs. After introduction of sperm, the preparations covered with oil were kept in the incubator at 37#{176}saturated with 5% O2 in air. At various times, eggs were selected and mounted on a slide (hang, 1952). If the eggs were still in cumulus clot, the egg clot was treated with hyaluronidase and the eggs were washed before mounting. They were then examined under a phase contrast microscope with an oil immersion objective for determination of the presence or absence of cortical granules. The eggs were then fixed and stained (Toyoda and hang, 1974) to determine whether the sperm head was only attached to the vitellus or enlarged in the vitellus and whether or not the eggs were activated. A spermatozoon attached to the vitellus without obvious change was considered as sperm attachment. An enlarged sperm head in the vitellus was considered to have penetrated into the vitellus. An egg that resumed the second meiotic division was considered an activated egg. RESULTS The motility of mouse spermatozoa was maintained very well in the presence of Bismark Brown during incubation. The motility of hamster sperm, however, was reduced in the medium either with or without Bismark Brown, showing that the reduction of motility of hamster sperm is not due to the toxic effect of Bismark Brown. The egg cytoplasm was stained light yellowish brown immediately after introduction to the medium with dye and cortical granules were stained black and showed good contrast to the color of egg cytoplasm. The cortical granules of hamster eggs were larger than those of mouse eggs and more distinctive after being stained (Figs. 1, 2). Although there were many granules of different sizes in the whole inner part of the cytoplasm, by careful focusing, the cortical granules of relatively uniform size were located on the outer layer of the cytoplasm and appeared to be darker than other granules. As shown in Table 1, the sperm penetration through the zona pellucida in the intact mouse could occur 5 mm after insemination with capacitated mouse spermatozoa, but 68-96% of the eggs had sperm passing through their zonae pellucidae mm after insemination and 30% of the eggs had enlarged sperm heads 30 mm after insemination. The breakdown of cortical granules in the intact mouse eggs started 15 mm after insemination and 78% of the eggs had no or very few cortical granules 30 mm after insemination. It seems that about 5-15 mm are required for a spermatozoon to pass through the zona pellucida and to attach to the vitellus and about mm are required for the breakdown of cortical granules after insemination in the intact mouse eggs. In the zona-free mouse eggs, the attachment of sperm to the vitellus was immediate, but breakdown of cortical granules started 5-15 mm after insemination and 84% of the eggs had no or very few cortical granules 30 mm after insemination. The resumption of second maturation division started 15 mm after insemination in the zona-free mouse eggs (11%), but 30 mm after insemination in the intact mouse eggs (56%). Although cortical granules were seen in a few eggs mm after insemination, most of the activated eggs had no cortical granules. When zona-free hamster eggs were inseminated with capacitated hamster spermatozoa, the sperm attachment to the vitellus was immediate. Sixty-five to 85% of the eggs had no or few cortical granules 5-15 mm after insemination. When zona-free hamster eggs were inseminated with capacitated mouse

3 ORTIAL GRANULE BREAKDOWN IN MOUSE AND HAMSTER EGGS 263 FIG. 1. ortical granules of a hamster egg. FIG. 2. ortical granules of a mouse egg, enlarged to twice the size of Fig. 1. spermatozoa, sperm attachment to the vitellus was immediate. Although 97% of the zonafree hamster eggs had enlarged mouse sperm heads 30 mm after insemination, only 47% of the eggs showed cortical granule breakdown and 45% of the eggs resumed their second maturation division 60 mm after insemination with capacitated mouse sperm. During this study, 2 cases of fusion of 2 eggs into 1 egg were observed mn the zonafree mouse eggs examined 60 mm after incubation and in 9 cases, fusion was observed in the zona-free hamster eggs examined mm after incubation. This was shown by the relatively large size of the eggs that contained 2 second maturation spindles. DISUSSION Due to their small size, cortical granules in mammalian eggs can be observed accurately with the electron microscope. However, a convenient method of staining cortical granules for light microscopic study is lacking. Several vital dyes such as Janus Green B, Nile BlueA and others were tested for the staining of cortical granules of mammalian eggs. We found that none of them was satisfactory. Bismark Brown in the culture medium at a concentration of 0.005% as described in the present study was neither toxic to the spermatozoa, as shown by their good motility nor to the eggs, as shown by their pronuclear formation during incubation. The contrast between the coloration of cytoplasm (yellow) and that of cortical granules (black) and other granules can be easily distinguished by focusing the microscope. It has been reported that the breakdown of cortical granules occurred within 5 mm after sperm attachment to the vitellus of hamster eggs (Barros and Yanagimachi, 1972; Gwatkin et al., 1976). Wolf et al. (1976) stated that cortical granules in the zona-free mouse eggs disappeared following 1 h exposure to sperm. In the present study, we have observed that after exposure to capacitated mouse spermatozoa, the cortical granule breakdown started earlier in the zona-free mouse eggs (5 mm) than in the intact egg (15 mm). The breakdown of cortical granules was also slower in the intact eggs (4%) than in the zona-free eggs (15%) 15 mm after insemination. Thirty mm after insemination, there was very little difference in the disappearance of cortical granules between the intact eggs (78%) and zona-free mouse eggs (84%). This is expected because of the relatively large number of sperm attached to the zona-free eggs and the small number of sperm in the perivitelline space

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5 ORTIAL GRANULE BREAKDOWN IN MOUSE AND HAMSTER EGGS 265 of the intact eggs. It seems that the cortical granule breakdown can be induced sooner and faster if a large number of sperm are attached to the vitelline membrane. In confirmation of the work by Barros and Yanagimachi (1972) and Gwatkin et al. (1976), we have observed that 65% of the zona-free hamster eggs had no or very few cortical granules 5 mm after insemination with capacitated hamster spermatozoa. In comparison with zona-free mouse eggs inseminated with capacitated mouse spermatozoa, the disappearance of cortical granules 15 mm after insemination with homologous spermatozoa is faster in the zona-free hamster eggs (85%) than in the zona-free mouse eggs (15%). When zona-free hamster eggs were inseminated with capacitated mouse sperm, although attachment of spermatozoa to the vitellus was immediate, the cortical granule breakdown started 15 mm after insemination and only 47% of the zona-free hamster eggs had no or very few cortical granules 60 mm after insemination. The proportion of hamster eggs activated by mouse sperm (45%) was also lower than that of eggs activated by spermatozoa of the same species (83-98%) 60 mm after insemination. The slow rate of cortical granule breakdown and the inefficiency of egg activation by foreign sperm are thus demonstrated. The average number of spermatozoa attached to the vitellus in the intact mouse eggs increased from sperm/egg 5-60 mm after insemination and the number of sperm attached to the zona-free mouse and hamster eggs ranged from during incubation, but the average number of mouse sperm attached to the zona-free hamster eggs ranged from The reason for a large number of mouse sperm attached to the hamster vitellus is unknown. Polyspermy was frequently observed (18-46%) in the intact mouse eggs and especially in the zona-free mouse and hamster eggs inseminated with sperm of the same species when examined mm after insemination, although vitelline block, unlike the zona reaction, may require a longer time to develop (Barros and Yanagimachi, 1972). Seventy-five to 100% of these polyspermic eggs were dispermic or trispermic and the average number of enlarged sperm heads ranged from In the case of zonafree hamster eggs inseminated with capacitated mouse sperm, a higher proportion of polyspermy (90-98%) was observed and 41 or 10% of these eggs were dispermic or trispermic, but the average number of enlarged sperm heads ranged from These facts may demonstrate that the mechanism of the block to polyspermy in the hamster is rather weak or that mouse sperm do not induce an efficient blocking mechanism in the hamster eggs. It has been stated that activation of eggs by sperm starts from the breakdown of cortical granules and is immediately followed by the resumption of the second maturation division (Austin, 1956; Barros and Yanagimachi, 1972). As shown in the present study, although the presence of cortical granules was observed in 1-9% activated eggs, the majority of activated eggs were without cortical granules (Table 1). The biochemical nature of cortical granule breakdown leading to the block to further sperm entry in the vitelline membrane and followed by the resumption of the second maturation division, is still to be investigated. AKNOWLEDGMENTS This work was supported by a grant (HD 03472) from NIHD and a grant from the Ford Foundation. Thanks are due to Dr. M. B. Maddock for reading the manuscript and to Mrs. Rose Bartke and E. Senior for assistance. REFERENES Austin,. R. (1956). ortical granules in hamster eggs.exp. ell. Res. 10, Austin,. R. and Braden, A. W. H. (1956). Early reactions of the rodent egg to spermatozoon penetration. J. Exp. Biol. 33, Barros,. and Yanagimachi, R. (1971). Induction of zona reaction in golden hamster eggs by cortical granule material. Nature, Lond. 233, Barros,. and Yanagimachi, R. (1972). Polyspermypreventing mechanisms in the golden hamster egg. J. Exp. Zool. 180, Braden, A. W. H., Austin,. R. and David, H. A. (1954). The reaction of the zona pellucida to sperm penetration. Aust. J. BioI. Sci. 7, hang, M.. (1952). Fertilizability of rabbit ova and the effects of temperatue in vitro on their subsequent fertilization and activation in vivo. J. Exp. Zool. 121, Fraser, L. R., Dandekar, P. V. and Gordon, M. K. (1972). Loss of cortical granules in rabbit eggs exposed to spermatozoa in vitro. J. Reprod. Fert. 29, Gwatkin, R. B. L., Rasmusson, G. H. and Williams, D. T. (1976). Induction of the cortical reaction in hamster eggs by membrane-active agents. J. Reprod. Fert. 47,

6 266 FUKUDA AND HANG Gwatkin, R. B. L., Williams, D. T., Hartmann, J. F. and Kniazuk, M. (1973). The zona reaction of hamster and mouse eggs: production in vitro by a trypsin-like protease from cortical granules. J. Reprod. Fert. 32, Motomura, 1. (1941). Materials of the fertilization membrane in the eggs of echinoderms. Tohoku Imp. Univ. Sci. Rep., 4th Ser. 16, Szollosi, D. G. (1962). ortical granules: a general feature of mammalian eggs? J. Reprod. Fert. 4, Toyoda, V. and hang, M.. (1974). Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. J. Reprod. Fert. 36, Toyoda, V., Yokoyama, M. and Hosi, T. (1971). Studies on the fertilization of mouse eggs in vitro. I. In vitro fertilization of eggs by fresh epididymal sperm. Jap. J. Anim. Reprod. 16, Wolf, D. P., lnoue, M. and Stark, R. A. (1976). Penetration of zona-free mouse ova. Biol. Reprod. 15, Vanagimachi, R. (1969). In vitro capacitation of hamster spermatozoa by follicular fluid. J. Reprod. Fert. 18,