An Ultrastructural Study of Epididymal Mouse Spermatozoa Binding to Zonae Pellucidae In Vitro: Sequential Relationship to the Acrosome Reaction PATRICIA M. SALING,' JANICE SOWINSKI AND BAYARD T. STOREY Division of Reproductive Biology, Department of Obstetrics and Gynecology, and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
ABSTRACT Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound t o the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2.Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound a t the surface of the zonae pellucidae of eggs recovered a t ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading t o fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona. Mammalian sperm, either freshly ejaculated or freshly recovered from the cauda epididymis, are incapable of fertilizing eggs of the homologous species. The sperm must reside for some defined period in the female reproductive tract, as demonstrated by Austin ('51) and Chang ('51) using delayed insemination experiments. This process is known as capacitation. It is thought to consist of reversible reactions of the sperm plasma membrane (Chang, '57; Austin, '75), the nature of which remains poorly defined (Chang and Hunter, '75), despite the development of numerous methods for the capacitation in vitro of sperm from several mammalian species (Toyoda et al., '71; Barros et al., '73; Bavister, '73; Rogers, '78). While capacitation has little discernible effect on the sperm's ultrastructure (Friend et al., '771, the acrosome reaction, J. EXP. ZOOL. (1979)209: 229-238.
which is also required for fertilization, has profound and well-defined ultrastructural correlates in mammalian sperm (Austin and Bishop, '58; Barros et al., '67; Bedford, '67; Austin, '77). As a result, the acrosome reaction has been employed as a marker for the process of capacitation. In our study of the fertilization process, we have been seeking an answer to the question: What is the sequence of membrane reactions which the mammalian sperm undergoes as an immediate prelude to the membrane fusion reaction between the gametes? The mouse is a most useful system for these studies, since mouse sperm are readily capacitated within ' Present address: Department of Obstetrics and Gynecology,Cornell University Medical Center, 515 East 71st Street, New York, New York 10021. Author to whom correspondence should be addressed.
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one hour, both in vivo (Braden and Austin, '54; McGaughey et al., '68) and in vitro (Mukherjee and Cohen, '70; Toyoda et al., '71; Wolf et al., '76). A property of mouse spermatozoa which temporally parallels the development of the capacitated state is the ability to bind to the zona pellucida of the egg (Inoue and Wolf, '75). We have shown that this ability to bind requires extracellular Ca+2 and is readily reversed by EGTA (Saling et al., '78). The binding reaction develops rapidly and is complete in 10 to 15 minutes. These characteristics imply that the early sequence of events leading to fertilization is: binding of the sperm to the zona pellucida followed by the acrosome reaction (Wolf et al., '77; Saling and Storey, '78). A few years ago, these events were commonly depicted as occurring in the reverse order (Austin, '74; McRorie and Williams, '74). More recent discussions of the topic (Bedford and Cooper, '78; Meizel, '78), however, have revealed that general agreement on the precise sequence has not yet been reached. Our previous sperm binding study (Saling et al., '78) provided an experimental preparation of eggs with sperm tightly bound to the zona surface, which is easily separated from unbound sperm and isolated by gradient centrifugation. Using this preparation, we have undertaken an ultrastructural study of the acrosome reaction in mouse sperm which defines the time course of the reaction and clarifies the sequential relation between sperm binding to the zona and the occurrence of the acrosome reaction. MATERIALS AND METHODS
Detailed descriptions of the media used and the handling of the gametes have been reported previously (Saling et al., '78). The culture medium (CM) (Inoue and Wolf, '75) used, which supports mouse fertilization in vitro, was a modified Krebs-Ringer bicarbonate medium containing sodium pyruvate (1 mM), sodium lactate (25 mM), glucose (5.56 mM), and serum albumin (20 mg BSA/ml, Fraction V, Sigma). The other two media used contained 20 mM Tris plus either 130 mM NaCl (TN) or 126 mM NaCl and 1.7 mM CaC1, (TNC). All media were sterilized by Millipore filtration (0.22 Km filter) and maintained a t pH 7.4-7.5. The divalent cation ionophore, A23187, was the generous gift of Dr. Robert Hammill of Eli Lilly and Co. (Indianapolis, Indiana).
Unfertilized tuba1 eggs were obtained from superovulated adult Swiss mice. Hyaluronidase (0.1%,10 minutes) (Sigma, Type I) in CM was used to disperse cumulus cells surrounding the eggs. Any cumulus cells still adherent were mechanically removed. Eggs were routinely manipulated and maintained in CM a t 37°C under sterile silicone oil (Dow Corning) unless otherwise indicated. Mouse sperm suspensions were prepared by mincing the excised caudae epididymides of a mature ( > 8 week) Swiss mouse in 0.4 ml TN. After allowing 10 to 15 minutes for sperm dispersion, particulate tissue was removed, and aliquots of the epididymal sperm suspension were diluted 1:5 in the preincubation medium appropriate to the experiment. Mouse eggs were inseminated and incubated according to the method of Wolf and Inoue ('761, using a final inseminating sperm concentration of 1-3 x l o 5 cells/ml. Inseminated eggs were recovered a t intervals of 10, 40 and 90 minutes. The stop-fix technique (Saling et al., '78) was employed to remove sperm not specifically bound to zonae and to fix concomitantly the eggs with bound sperm. The technique was slightly modified for optimal preservation of specimen ultrastructure by the use of 3%glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4, plus 2.25% Dextran in the lower layer of the one-step gradient. Eggs with bound sperm were further fixed for one to two hours a t room temperature with 3% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4. In experiments designed to demonstrate the morphology of the acrosome-reacted mouse spermatozoon, the divalent cation ionophore A23187 in dimethylformamide (DMF) was added a t a final concentration of 3 FM (0.1% DMF) to cumulus-free mouse eggs immediately prior t o insemination with epididymal sperm which had been preincubated for 60 minutes in CM. The silicone oil overlay was delayed for 10 minutes in these preparations, due to the high solubility of A23187 in the nonpolar phase. The eggs were recovered two hours after insemination and extensively washed with a narrow bore micropipette in CM to remove all sperm bound to the zonae. The eggs were then fixed a t room temperature for one to two hours with 3%glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4. All eggs were post-fixed with 1% osmium tetroxide in 0.1 M Na-cacodylate buffer. Following graded alcohol dehydration, eggs were
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embedded in Epon 812 (Luft, '6U, polymerized for 72 hours a t 60°C, and serially sectioned. Thin sections, 50-60 nm thick, were mounted on Fomvar-coated copper grids, stained with uranyl acetate and lead citrate (Reynolds, '63) and examined with a Hitachi HU-12A electron microscope operated at 75 kv. RESULTS
Morphology of intact us. acrosomereacted mouse sperm To determine the sequential relationship between sperm binding and the acrosome reaction, a definitive assay for the acrosome reaction in mouse sperm was required. The difference in appearance between an intact mouse sperm and one which has undergone an acrosome reaction is not readily perceptible. Unlike Iwamatsu and Chang ('691, we were unable with the light microscope to distinguish between a n intact mouse sperm and one which had undergone the acrosome reaction. Visualization with the electron microscope was therefore essential. It has been observed in several mammalian species that sperm, which have penetrated the zona pellucida and lie within the perivitelline space, have undergone a "true" acrosome reaction (Bedford, '67; Piko, '69; Austin, '75; Chang and Hunter, '75; Gwatkin, '76; Yanagimachi, '77; Bedford and Cooper, '781, as opposed to sperm in suspension which may undergo a "false" or degenerative acrosome reaction. Mouse sperm in t h e perivitelline space should, therefore, show the morphology characteristic of the non-degenerative acrosome reaction. Under the conditions used for the fertilization of mouse eggs in vitro, however, very few sperm are found within the perivitelline space. Examination of Fig. 1 Mouse epididymal spermatozoon with an intact these rare specimens in thin-section would be at the surface of the zona pellucida. The impractical. The number of sperm within t h e acrosome gametes were incubated in CM. Note the intact inner and perivitelline space can be dramatically in- outer acrosomal membranes and the homogeneity of the creased by insemination of cumulus-free eggs acrosomal contents. Key: 2, zona pellucida; N, sperm nucleus; IAM, inner i n t h e presence of t h e divalent cation acrosomal membrane; OAM, outer acrosomal membrane; ionophore, A23187 (D. P. Wolf, personal com- PM, sperm plasma membrane; the arrow points to the munication). Under these conditions, more junction of the inner and outer acrosomal membranes. than 50 sperm may penetrate the zona of a The bar represents 0.5 wm. single egg, providing a population of perivitelline sperm for ultrastructural examination. The mechanism of the ionophore effect is sponds well to t h a t previously reported currently unknown, but its presence does not (Stefanini et al., '69; Thompson et al., '74; alter significantly the kinetics of zona pene- Anderson et al., '75) for the intact state. The tration by the sperm (D.P. Wolf, personal acrosome, overlying the anterior end of the sperm head, is clearly evident, as are the incommunication). The ultrastructure of the mouse sperm (fig. tact inner and outer acrosomal membranes. In l), recently bound to the zona surface, corre- contrast, t h e sperm located within t h e
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perivitelline space (fig. 2) displays a markedly different ultrastructure. The acrosome is no longer present, and the inner acrosomal membrane is the limiting membrane of the anterior portion of the sperm head, replacing the plasma membrane. A criterion for the occurrence of a true acrosome reaction is the visualization of the fused ends of the inner acrosomal and plasma membranes a t t h e sperm's equatorial segment (Bedford, '70). Although no sections were obtained to illustrate this point clearly, we are confident that a true acrosome reaction occurred, since there were no indications that the sperm entered the perivitelline space by an abnormal route. While other micrographs (Stefanini et al., '69; Zamboni, '71; Thompson et al., '74: Anderson et al., '75; Nicosia e t al., '77) of acrosomereacted mouse sperm are available, we have found one particularly clear and explicit picture of a functionally acrosome-reacted mouse sperm within the zona for comparison (Zamboni, '71). The mouse sperm found within the perivitelline space in the presence of A23187 resemble this one and also the acrosomereacted sperm depicted in the other studies. Time course study In the following experiments, the time course of the acrosome reaction was determined by examining sperm bound to zonae a t different time intervals after insemination. For these experiments, epididymal mouse sperm were preincubated for one hour in either CM or TNC and then used to inseminate eggs in the homologous medium. When the eggs were recovered a t ten minutes after insemination by the stop-fix method, all of the sperm examined ( > 100) were bound to t h e surface of the zona pellucida and were completely intact (fig. 3). When the eggs recovered a t 40 minutes after insemination were examined, intact sperm ( > 100 examined) were observed a t the zona surface, but approximately 50%of these displayed signs of the initial stages of membrane vesiculation (fig. 4). The remaining 50%of the bound sperm had entirely intact acrosomes, as at ten minutes after insemination. A major difference was observed (fig. 5) upon examination of eggs recovered a t 90 minutes after insemination. In this group, 12% (6/50) of t h e sperm a t the zona surface had undergone an acrosome reaction. Most of these sperm were fully reacted and appeared to be initiating
zona penetration (fig. 5A). One sperm was evidently in the process of the reaction a t the zona surface (fig. 5B). The remaining bound sperm population was about equally divided among two categories: 42% (21/50) displayed membrane whorls and other signs of initial membrane breakdown (fig. 5 0 , while 46% (23/ 50) appeared entirely intact. Exposure of the gametes to the two different media, CM and TNC, was without detectable effect on the occurrence of the acrosome reaction, since sperm from both media contributed similar numbers to each of the morphologically distinct categories. In fact, the gametes did not display any morphological differences as a function of the incubation medium. During the examination of these samples, we found t h a t both the apical and lateral surfaces of the mouse sperm head were competent to bind to zonae: there was no specific orientation of the sperm head needed for binding. Unlike the events observed a t the egg plasma membrane (Wolf and Armstrong, '781, there appeared to be no "reorientation reaction" of the sperm at the zona surface preceding the acrosome reaction (compare figs. 5A and 5B). The acrosome reaction proceeded independently of binding position. Finally, evidence for s t r u c t u r a l linkages between sperm and zonae (Gwatkin et al., '76) as a function of the incubation medium, of the time of recovery after insemination or of the occurrence of the acrosome reaction was never observed. DISCUSSION
The results presented here clarify the temporal relationship between the acrosome reaction and mouse sperm binding to zonae pellucidae. All of the sperm bound to eggs recovered ten minutes after insemination were completely intact. Examination of bound sperm a t 40 minutes after insemination revealed indications of the early stages of the acrosome reaction. At 90 minutes after insemination, some completely acrosome-reacted sperm could be found at the zona surface, whose morphology was the same as t h a t of sperm which entered the perivitelline space in the presence of the ionophore A23187. But even a t 90 minutes, nearly half the sperm observed were completely intact; this gives a n operational half-time for the acrosome reaction in mouse sperm of 90 minutes. This reaction is therefore at least one order of magnitude slower than
MOUSE SPERM BINDING AND ACHOSOME REACTION
Fig. 2 Mouse epididymal spermatozoon with a reacted acrosome within the perivitelliine space, following insemination in the presence of A23167 (see text). Note the characteristic pointed perforatorium at the apex of the sperm head. A cross section through the sperm’s midpiece is seen just posterior to the sperm head, while a cross section through the principal piece of the tail, which has apparently flipped over, lies anterior to the sperm head. The inset demonstrates that the sperm lies within the perivitelline space. Key: 2,zona pellucida; 0, egg cytoplasm; PVS, perivitelline space; EM, egg plasma membrane; N, sperm nucleus; IAM, inner acrosomal membrane; P, perforatorium; M, midpiece; T, tail. The bar represents 0.5 pm.
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Fig. 3 Mouse epididymal spermatozoon bound t o the zona of an egg recovered a t ten minutes after insemination in medium TNC by t h e stop-fix technique. The sperm is completely intact. The inset shows the location of the sperm a t the zona surface. Key: Z, zona pellucida; 0, egg cytoplasm; IAM, inner acrosomal membrane; OAM, outer scrosomal membrane; PM, sperm plasma membrane. The bar represents 0.5 pm.
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Fig. 4 Mouse epididymal spermatozoon bound to the zona of an egg recovered a t 40 minutes after insemination by the stop-fix technique. The sperm nucleus appears interrupted, and the sperm head appears foreshortened because of the tangential angle of the section. The sperm’s acrosome is fundamentally intact, although signs of early membrane breakdown are visible (arrows). Gametes were exposed t o CM. The inset shows the attachment of the sperm to t h e zona by the anterior portion of the sperm head. Key: Z, zona pellucida; 0, egg cytoplasm; N, sperm nucleus; M, mitochondria in sperm’s midpiece; A, acrosome. The bar represents 0.5 &m.
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Fig. 5 Mouse epididymal spermatozoa bound t o the zonae of eggs recovered at 90 minutes after insemination by the stop-fix technique. a. Fully reacted mouse sperm a t the zona surface; note the pointed perforatorium at the cell’s apex (gametes exposed to CM). b. Mouse sperm evidently in the process of the acrosome reaction while a t the zona surface, as indicated by t h e array of vesicles surrounding the acrosomal region (gametes exposed to TNC). c. Nearly intact sperm a t the zona surface, with signs of early membrane breakdown (arrows) (gametes exposed to TNC). Key: 2,zona pellueida; N, sperm nucleus; P, perforatorium; V, vesicles; A, acrosome. The bars represent 1.0 um.
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the binding reaction, which has an operational half-time of less than nine minutes (Saling et al., ’78). Just what triggers a given mouse sperm to undergo the acrosome reaction and whether the duration of the reaction is fixed are both still unknown. However, the time required for the acrosome reaction observed in this study compares favorably with the in vitro fertilization studies of Wolf et al. (‘76), who demonstrated that the majority of capacitated mouse sperm initiate penetration of cumulusfree eggs between the first and second hour after insemination. It is also consistent with the time frame of the study of sperm attachment and cortical granule breakdown reported by Fukuda and Chang (‘78) and with the times of one to two hours reported for onset of the acrosome reaction in hamster and guinea pig reported by Talbot and Franklin (‘76, ’78). We did not pursue the events occurring after 90 minutes in this study, because mouse eggs will not tolerate the stringent conditions of incubation in TNC any longer than 120 minutes. These findings strongly support the hypothesis that the true acrosome reaction occurs after the sperm is bound to the zona and is the direct precursor to sperm penetration of the zona, a t least in the mouse system. The question of whether the sequence of reactions: sperm binding to zona, acrosome reaction, sperm penetration of zona, is the obligatory one for fertilization is not fully answered by this study. We have not ruled out the possibility that a competent sperm may react in suspension and, once fully reacted, bind to the zona surface, although this sequence could not be observed in these experiments. To test this possibility, a population of acrosome-reacted sperm in suspension is needed to challenge eggs in a binding study. Such a population cannot be assayed for the occurrence of true acrosome reactions by electron microscopy; a rapid assay applicable to unfixed, motile sperm is necessary. The requirements of such an assay and the question of obligatory sequence will be dealt with in a subsequent paper. ACKNOWLEDGMENTS
The authors are grateful to Drs. L. J. Heffner, S. V. Nicosia and D. P. Wolf for stimulating discussions and helpful counsel and to Ms. Patricia Park for secretarial and editorial aid. This work was supported by USPHS Grant HD-06274 and NSF Grant PCM-7715965.
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LITERATURE CITED Anderson, E., P. C. Hoppe, W. K. Whitten and G. S. Lee 1975 In uitro fertilization and early embryogenesis: A cytological analysis. J. Ultrastruct. Res., 50: 231-252. Austin, C. R. 1951 Observations on the penetration of the sperm into the mammalian egg. Austral. J. Biol. Sci., B4: 581-596. 1974 Recent progress in the study of eggs and spermatozoa: Insemination and ovulation to implantation. In: MTP International Review of Science. Physiology, Ser. One, Vol. 8, Reproductive Physiology. R. 0. Greep, ed. Butterworths, London, pp. 95-131. - 1975 Membrane fusion events in fertilization. J. Reprod. Fert., 44: 155-166. 1977 Spermatozoa and ova: the role of membranes in the fertilization process. In: Mammalian Cell Membranes. Vol. 111. G. A. Jamieson and D. M. Robinson, eds. Butterworths, London, pp. 206-230. Austin, C. R., and M. W. H. Bishop 1958 Role of the rodent acrosome and perforatorium in fertilization. Proc. Roy. Soc. (London), Ser. B, 149: 241-248. Barros, C., J. M. Bedford, L. E. Franklin and C. R. Austin 1967 Membrane vesiculation as a feature of the mammalian acrosome reaction. J. Cell Biol., 34: Cl-C5. Barros, C.,M. Berrios and E. Herrera 1973 Capacitation in uitro of guinea pig spermatozoa in a saline solution. J. Reprod. Fert., 34: 547-549. Bavister, B. D. 1973 Capacitation of golden hamster spermatozoa during incubation in culture medium. J. Reprod. Fert., 35: 161-163. Bedford, J. M. 1967 Experimental requirements for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J. Reprod. Fert., Suppl. 2: 35-48. 1970 Sperm capacitation and fertilization in mammals. Biol. Reprod., Suppl. 2: 128-158. Bedford, J. M., and G. W. Cooper 1978 Membrane fusion events in the fertilization of vertebrate eggs. Cell Surface Reviews, 5: 65-125. Braden, A. W. H., and C. R. Austin 1954 Fertilization of the mouse egg and the effect of delayed coitus and of heat shock treatment. Austral. J. Biol. Sci., B7: 552-565. Chang, M. C. 1951 Fertilizing capacity of spermatozoa deposited in the Fallopian tubes. Nature (London), 168: 697. 1957 A detrimental effect of seminal plasma on the fertilizing capacity of sperm. Nature (London), 179: 258-259. 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. Friend, D. S., L. Orci, A. Perrelet and R. Yanagimachi 1977 Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa. J. Cell Biol., 74: 561-577. Fukuda, Y., and M. C. Chang 1978 The time of cortical granule breakdown and sperm penetration in mouse and hamster eggs inseminated in vitro. Biol. Reprod., 19: 261-266. Gwatkin, R. B. L. 1976 Fertilization. Cell Surface Reviews, 1: 1-54. Gwatkin, R. B. L., H. W. Carter and H. Patterson 1976 Association of mammalian sperm with the cumulus cells and the zona pellucida studied by scanning electron microscopy. In: Scanning Electron Microscopy, Proceedings of t h e Workshop on SEM in Reproductive Biology, Part 11. IIT Research Institute, Chicago, pp. 379-384. Inoue, M., and D. P. Wolf 1975 Sperm binding characteristics of the murine zona pellucida. Biol. Reprod., 13: 340-346.
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Iwamatsu, T., and M. C. Chang 1969 In uitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature (London), 224: 919-920. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. McGaughey, R. W., J. H. Marston and M. C. Chang 1968 Fertilizing life of mouse spermatozoa in the female tract. J. Reprod. Fert., 16: 147-150. McRorie, R. A., and W. L. Williams 1974 Biochemistry of mammalian fertilization. Annu. Rev. Biochem., 43; 777.803. Meizel, S. 1978 The mammalian sperm acrosome reaction, a biochemical approach. In: Development in Mammals. Vol. 3. M. Johnson, ed. ElsevierlNorth Holland Biomed Press, Amsterdam, pp. 1-64. Mukherjee, A. B., and M. M. &hen 1970 Development of normal mice by in uitro fertilization. Nature (London), 228: 412-473. Nicosia, S. V., D. P. Wolf and M. Inoue 1977 Cortical granule distribution and cell surface characteristics in mouse eggs. Develop. Biol., 57: 56-74. Piko, L. 1969 Gamete structure and sperm entry in mammals. In: Fertilization. Vol. 2. C. B. Metz and A. Monroy, eds. Academic Press, New York, pp. 325-403. Reynolds, E. S. 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Rogers, B. J. 1978 Mammalian sperm capacitation and fertilization in uitro: a critique of methodology. Gamete Res., 1: 165-223. Saling, P. M., and B. T. Storey 1978 The mouse sperm needs Cat*to recognize the zona pellucida of the mouse egg. In: Biophysical and Biochemical Information Transfer in Recognition. J. G. Vassileva-Popova, ed. Plenum Publishing Corp., New York, pp. 677-685. Saling, P. M., B. T. Storey and D. P. Wolf 1978 Calcium-
dependent binding of mouse epididymal spermatozoa to the zona pellucida. Develop. Biol., 65: 515-525. Stefanini, M., C. Oura andL. Zamboni 1969 Ultrastructure of fertilization in t h e mouse. 2. Penetration of sperm into the ovum. J. Submicro. Cytol., 1: 1-23. Talbot, P., and L. E. Franklin 1976 Morphology and kinetics of the hamster sperm acrosome reaction. J. Exp. Zool., 198: 163-176. 1978 Surface modification of guinea pig sperm during in vitro capacitation: a n assessment using lectininduced agglutination of living sperm. J. Exp. Zwl., 203: 1-14. 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., M. Yokoyama and T. Hosi 1971 Studies on the fertilization of mouse eggs in uitro. 11. Effects of in uitro preincubation of sperm on the time of sperm penetration of mouse eggs in uitro. Jap. J. Anim. Reprod., 16: 152-157. Wolf, D. P., and P. B. Armstrong 1978 Penetration of the zona-free mouse egg by capacitated epididymal sperm: cinemicrographic observations. Gamete Res., I: 39-46. Wolf, D. P., M. Hamada and M. Inoue 1977 Kinetics of sperm penetration in mouse and hamster eggs inseminated in uitro. Biol. Reprod., 19: 261-266. Wolf, D. P., and M. Inoue 1976 Sperm concentration dependency in the fertilization and the zonae sperm binding properties of mouse eggs inseminated in vitro. J. Exp. Zwl., 196: 27-38. Wolf, D. P., M. Inoue and R. A. Stark 1976 Penetration of zona-free mouse ova. Biol. Reprod., 15: 213-221. Yanagimachi, R. 1977 Specificity of sperm-egg interactions. In: Immunobiology of Gametes. M. Edidin and M. H. Johnson, eds. Cambridge University Press, Cambridge, pp. 255-295. Zamboni, L. 1971 Fine Morphology of Mammalian Fertilization. Harper and Row, New York, pp. 154-155.
ABSTRACT Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound t o the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2.Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound a t the surface of the zonae pellucidae of eggs recovered a t ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading t o fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona. Mammalian sperm, either freshly ejaculated or freshly recovered from the cauda epididymis, are incapable of fertilizing eggs of the homologous species. The sperm must reside for some defined period in the female reproductive tract, as demonstrated by Austin ('51) and Chang ('51) using delayed insemination experiments. This process is known as capacitation. It is thought to consist of reversible reactions of the sperm plasma membrane (Chang, '57; Austin, '75), the nature of which remains poorly defined (Chang and Hunter, '75), despite the development of numerous methods for the capacitation in vitro of sperm from several mammalian species (Toyoda et al., '71; Barros et al., '73; Bavister, '73; Rogers, '78). While capacitation has little discernible effect on the sperm's ultrastructure (Friend et al., '771, the acrosome reaction, J. EXP. ZOOL. (1979)209: 229-238.
which is also required for fertilization, has profound and well-defined ultrastructural correlates in mammalian sperm (Austin and Bishop, '58; Barros et al., '67; Bedford, '67; Austin, '77). As a result, the acrosome reaction has been employed as a marker for the process of capacitation. In our study of the fertilization process, we have been seeking an answer to the question: What is the sequence of membrane reactions which the mammalian sperm undergoes as an immediate prelude to the membrane fusion reaction between the gametes? The mouse is a most useful system for these studies, since mouse sperm are readily capacitated within ' Present address: Department of Obstetrics and Gynecology,Cornell University Medical Center, 515 East 71st Street, New York, New York 10021. Author to whom correspondence should be addressed.
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one hour, both in vivo (Braden and Austin, '54; McGaughey et al., '68) and in vitro (Mukherjee and Cohen, '70; Toyoda et al., '71; Wolf et al., '76). A property of mouse spermatozoa which temporally parallels the development of the capacitated state is the ability to bind to the zona pellucida of the egg (Inoue and Wolf, '75). We have shown that this ability to bind requires extracellular Ca+2 and is readily reversed by EGTA (Saling et al., '78). The binding reaction develops rapidly and is complete in 10 to 15 minutes. These characteristics imply that the early sequence of events leading to fertilization is: binding of the sperm to the zona pellucida followed by the acrosome reaction (Wolf et al., '77; Saling and Storey, '78). A few years ago, these events were commonly depicted as occurring in the reverse order (Austin, '74; McRorie and Williams, '74). More recent discussions of the topic (Bedford and Cooper, '78; Meizel, '78), however, have revealed that general agreement on the precise sequence has not yet been reached. Our previous sperm binding study (Saling et al., '78) provided an experimental preparation of eggs with sperm tightly bound to the zona surface, which is easily separated from unbound sperm and isolated by gradient centrifugation. Using this preparation, we have undertaken an ultrastructural study of the acrosome reaction in mouse sperm which defines the time course of the reaction and clarifies the sequential relation between sperm binding to the zona and the occurrence of the acrosome reaction. MATERIALS AND METHODS
Detailed descriptions of the media used and the handling of the gametes have been reported previously (Saling et al., '78). The culture medium (CM) (Inoue and Wolf, '75) used, which supports mouse fertilization in vitro, was a modified Krebs-Ringer bicarbonate medium containing sodium pyruvate (1 mM), sodium lactate (25 mM), glucose (5.56 mM), and serum albumin (20 mg BSA/ml, Fraction V, Sigma). The other two media used contained 20 mM Tris plus either 130 mM NaCl (TN) or 126 mM NaCl and 1.7 mM CaC1, (TNC). All media were sterilized by Millipore filtration (0.22 Km filter) and maintained a t pH 7.4-7.5. The divalent cation ionophore, A23187, was the generous gift of Dr. Robert Hammill of Eli Lilly and Co. (Indianapolis, Indiana).
Unfertilized tuba1 eggs were obtained from superovulated adult Swiss mice. Hyaluronidase (0.1%,10 minutes) (Sigma, Type I) in CM was used to disperse cumulus cells surrounding the eggs. Any cumulus cells still adherent were mechanically removed. Eggs were routinely manipulated and maintained in CM a t 37°C under sterile silicone oil (Dow Corning) unless otherwise indicated. Mouse sperm suspensions were prepared by mincing the excised caudae epididymides of a mature ( > 8 week) Swiss mouse in 0.4 ml TN. After allowing 10 to 15 minutes for sperm dispersion, particulate tissue was removed, and aliquots of the epididymal sperm suspension were diluted 1:5 in the preincubation medium appropriate to the experiment. Mouse eggs were inseminated and incubated according to the method of Wolf and Inoue ('761, using a final inseminating sperm concentration of 1-3 x l o 5 cells/ml. Inseminated eggs were recovered a t intervals of 10, 40 and 90 minutes. The stop-fix technique (Saling et al., '78) was employed to remove sperm not specifically bound to zonae and to fix concomitantly the eggs with bound sperm. The technique was slightly modified for optimal preservation of specimen ultrastructure by the use of 3%glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4, plus 2.25% Dextran in the lower layer of the one-step gradient. Eggs with bound sperm were further fixed for one to two hours a t room temperature with 3% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4. In experiments designed to demonstrate the morphology of the acrosome-reacted mouse spermatozoon, the divalent cation ionophore A23187 in dimethylformamide (DMF) was added a t a final concentration of 3 FM (0.1% DMF) to cumulus-free mouse eggs immediately prior t o insemination with epididymal sperm which had been preincubated for 60 minutes in CM. The silicone oil overlay was delayed for 10 minutes in these preparations, due to the high solubility of A23187 in the nonpolar phase. The eggs were recovered two hours after insemination and extensively washed with a narrow bore micropipette in CM to remove all sperm bound to the zonae. The eggs were then fixed a t room temperature for one to two hours with 3%glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4. All eggs were post-fixed with 1% osmium tetroxide in 0.1 M Na-cacodylate buffer. Following graded alcohol dehydration, eggs were
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embedded in Epon 812 (Luft, '6U, polymerized for 72 hours a t 60°C, and serially sectioned. Thin sections, 50-60 nm thick, were mounted on Fomvar-coated copper grids, stained with uranyl acetate and lead citrate (Reynolds, '63) and examined with a Hitachi HU-12A electron microscope operated at 75 kv. RESULTS
Morphology of intact us. acrosomereacted mouse sperm To determine the sequential relationship between sperm binding and the acrosome reaction, a definitive assay for the acrosome reaction in mouse sperm was required. The difference in appearance between an intact mouse sperm and one which has undergone an acrosome reaction is not readily perceptible. Unlike Iwamatsu and Chang ('691, we were unable with the light microscope to distinguish between a n intact mouse sperm and one which had undergone the acrosome reaction. Visualization with the electron microscope was therefore essential. It has been observed in several mammalian species that sperm, which have penetrated the zona pellucida and lie within the perivitelline space, have undergone a "true" acrosome reaction (Bedford, '67; Piko, '69; Austin, '75; Chang and Hunter, '75; Gwatkin, '76; Yanagimachi, '77; Bedford and Cooper, '781, as opposed to sperm in suspension which may undergo a "false" or degenerative acrosome reaction. Mouse sperm in t h e perivitelline space should, therefore, show the morphology characteristic of the non-degenerative acrosome reaction. Under the conditions used for the fertilization of mouse eggs in vitro, however, very few sperm are found within the perivitelline space. Examination of Fig. 1 Mouse epididymal spermatozoon with an intact these rare specimens in thin-section would be at the surface of the zona pellucida. The impractical. The number of sperm within t h e acrosome gametes were incubated in CM. Note the intact inner and perivitelline space can be dramatically in- outer acrosomal membranes and the homogeneity of the creased by insemination of cumulus-free eggs acrosomal contents. Key: 2, zona pellucida; N, sperm nucleus; IAM, inner i n t h e presence of t h e divalent cation acrosomal membrane; OAM, outer acrosomal membrane; ionophore, A23187 (D. P. Wolf, personal com- PM, sperm plasma membrane; the arrow points to the munication). Under these conditions, more junction of the inner and outer acrosomal membranes. than 50 sperm may penetrate the zona of a The bar represents 0.5 wm. single egg, providing a population of perivitelline sperm for ultrastructural examination. The mechanism of the ionophore effect is sponds well to t h a t previously reported currently unknown, but its presence does not (Stefanini et al., '69; Thompson et al., '74; alter significantly the kinetics of zona pene- Anderson et al., '75) for the intact state. The tration by the sperm (D.P. Wolf, personal acrosome, overlying the anterior end of the sperm head, is clearly evident, as are the incommunication). The ultrastructure of the mouse sperm (fig. tact inner and outer acrosomal membranes. In l), recently bound to the zona surface, corre- contrast, t h e sperm located within t h e
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perivitelline space (fig. 2) displays a markedly different ultrastructure. The acrosome is no longer present, and the inner acrosomal membrane is the limiting membrane of the anterior portion of the sperm head, replacing the plasma membrane. A criterion for the occurrence of a true acrosome reaction is the visualization of the fused ends of the inner acrosomal and plasma membranes a t t h e sperm's equatorial segment (Bedford, '70). Although no sections were obtained to illustrate this point clearly, we are confident that a true acrosome reaction occurred, since there were no indications that the sperm entered the perivitelline space by an abnormal route. While other micrographs (Stefanini et al., '69; Zamboni, '71; Thompson et al., '74: Anderson et al., '75; Nicosia e t al., '77) of acrosomereacted mouse sperm are available, we have found one particularly clear and explicit picture of a functionally acrosome-reacted mouse sperm within the zona for comparison (Zamboni, '71). The mouse sperm found within the perivitelline space in the presence of A23187 resemble this one and also the acrosomereacted sperm depicted in the other studies. Time course study In the following experiments, the time course of the acrosome reaction was determined by examining sperm bound to zonae a t different time intervals after insemination. For these experiments, epididymal mouse sperm were preincubated for one hour in either CM or TNC and then used to inseminate eggs in the homologous medium. When the eggs were recovered a t ten minutes after insemination by the stop-fix method, all of the sperm examined ( > 100) were bound to t h e surface of the zona pellucida and were completely intact (fig. 3). When the eggs recovered a t 40 minutes after insemination were examined, intact sperm ( > 100 examined) were observed a t the zona surface, but approximately 50%of these displayed signs of the initial stages of membrane vesiculation (fig. 4). The remaining 50%of the bound sperm had entirely intact acrosomes, as at ten minutes after insemination. A major difference was observed (fig. 5) upon examination of eggs recovered a t 90 minutes after insemination. In this group, 12% (6/50) of t h e sperm a t the zona surface had undergone an acrosome reaction. Most of these sperm were fully reacted and appeared to be initiating
zona penetration (fig. 5A). One sperm was evidently in the process of the reaction a t the zona surface (fig. 5B). The remaining bound sperm population was about equally divided among two categories: 42% (21/50) displayed membrane whorls and other signs of initial membrane breakdown (fig. 5 0 , while 46% (23/ 50) appeared entirely intact. Exposure of the gametes to the two different media, CM and TNC, was without detectable effect on the occurrence of the acrosome reaction, since sperm from both media contributed similar numbers to each of the morphologically distinct categories. In fact, the gametes did not display any morphological differences as a function of the incubation medium. During the examination of these samples, we found t h a t both the apical and lateral surfaces of the mouse sperm head were competent to bind to zonae: there was no specific orientation of the sperm head needed for binding. Unlike the events observed a t the egg plasma membrane (Wolf and Armstrong, '781, there appeared to be no "reorientation reaction" of the sperm at the zona surface preceding the acrosome reaction (compare figs. 5A and 5B). The acrosome reaction proceeded independently of binding position. Finally, evidence for s t r u c t u r a l linkages between sperm and zonae (Gwatkin et al., '76) as a function of the incubation medium, of the time of recovery after insemination or of the occurrence of the acrosome reaction was never observed. DISCUSSION
The results presented here clarify the temporal relationship between the acrosome reaction and mouse sperm binding to zonae pellucidae. All of the sperm bound to eggs recovered ten minutes after insemination were completely intact. Examination of bound sperm a t 40 minutes after insemination revealed indications of the early stages of the acrosome reaction. At 90 minutes after insemination, some completely acrosome-reacted sperm could be found at the zona surface, whose morphology was the same as t h a t of sperm which entered the perivitelline space in the presence of the ionophore A23187. But even a t 90 minutes, nearly half the sperm observed were completely intact; this gives a n operational half-time for the acrosome reaction in mouse sperm of 90 minutes. This reaction is therefore at least one order of magnitude slower than
MOUSE SPERM BINDING AND ACHOSOME REACTION
Fig. 2 Mouse epididymal spermatozoon with a reacted acrosome within the perivitelliine space, following insemination in the presence of A23167 (see text). Note the characteristic pointed perforatorium at the apex of the sperm head. A cross section through the sperm’s midpiece is seen just posterior to the sperm head, while a cross section through the principal piece of the tail, which has apparently flipped over, lies anterior to the sperm head. The inset demonstrates that the sperm lies within the perivitelline space. Key: 2,zona pellucida; 0, egg cytoplasm; PVS, perivitelline space; EM, egg plasma membrane; N, sperm nucleus; IAM, inner acrosomal membrane; P, perforatorium; M, midpiece; T, tail. The bar represents 0.5 pm.
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Fig. 3 Mouse epididymal spermatozoon bound t o the zona of an egg recovered a t ten minutes after insemination in medium TNC by t h e stop-fix technique. The sperm is completely intact. The inset shows the location of the sperm a t the zona surface. Key: Z, zona pellucida; 0, egg cytoplasm; IAM, inner acrosomal membrane; OAM, outer scrosomal membrane; PM, sperm plasma membrane. The bar represents 0.5 pm.
MOUSE SPERM BINDING AND ACROSOME REACTION
Fig. 4 Mouse epididymal spermatozoon bound to the zona of an egg recovered a t 40 minutes after insemination by the stop-fix technique. The sperm nucleus appears interrupted, and the sperm head appears foreshortened because of the tangential angle of the section. The sperm’s acrosome is fundamentally intact, although signs of early membrane breakdown are visible (arrows). Gametes were exposed t o CM. The inset shows the attachment of the sperm to t h e zona by the anterior portion of the sperm head. Key: Z, zona pellucida; 0, egg cytoplasm; N, sperm nucleus; M, mitochondria in sperm’s midpiece; A, acrosome. The bar represents 0.5 &m.
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Fig. 5 Mouse epididymal spermatozoa bound t o the zonae of eggs recovered at 90 minutes after insemination by the stop-fix technique. a. Fully reacted mouse sperm a t the zona surface; note the pointed perforatorium at the cell’s apex (gametes exposed to CM). b. Mouse sperm evidently in the process of the acrosome reaction while a t the zona surface, as indicated by t h e array of vesicles surrounding the acrosomal region (gametes exposed to TNC). c. Nearly intact sperm a t the zona surface, with signs of early membrane breakdown (arrows) (gametes exposed to TNC). Key: 2,zona pellueida; N, sperm nucleus; P, perforatorium; V, vesicles; A, acrosome. The bars represent 1.0 um.
MOUSE SPERM BINDING AND ACROSOME REACTION
the binding reaction, which has an operational half-time of less than nine minutes (Saling et al., ’78). Just what triggers a given mouse sperm to undergo the acrosome reaction and whether the duration of the reaction is fixed are both still unknown. However, the time required for the acrosome reaction observed in this study compares favorably with the in vitro fertilization studies of Wolf et al. (‘76), who demonstrated that the majority of capacitated mouse sperm initiate penetration of cumulusfree eggs between the first and second hour after insemination. It is also consistent with the time frame of the study of sperm attachment and cortical granule breakdown reported by Fukuda and Chang (‘78) and with the times of one to two hours reported for onset of the acrosome reaction in hamster and guinea pig reported by Talbot and Franklin (‘76, ’78). We did not pursue the events occurring after 90 minutes in this study, because mouse eggs will not tolerate the stringent conditions of incubation in TNC any longer than 120 minutes. These findings strongly support the hypothesis that the true acrosome reaction occurs after the sperm is bound to the zona and is the direct precursor to sperm penetration of the zona, a t least in the mouse system. The question of whether the sequence of reactions: sperm binding to zona, acrosome reaction, sperm penetration of zona, is the obligatory one for fertilization is not fully answered by this study. We have not ruled out the possibility that a competent sperm may react in suspension and, once fully reacted, bind to the zona surface, although this sequence could not be observed in these experiments. To test this possibility, a population of acrosome-reacted sperm in suspension is needed to challenge eggs in a binding study. Such a population cannot be assayed for the occurrence of true acrosome reactions by electron microscopy; a rapid assay applicable to unfixed, motile sperm is necessary. The requirements of such an assay and the question of obligatory sequence will be dealt with in a subsequent paper. ACKNOWLEDGMENTS
The authors are grateful to Drs. L. J. Heffner, S. V. Nicosia and D. P. Wolf for stimulating discussions and helpful counsel and to Ms. Patricia Park for secretarial and editorial aid. This work was supported by USPHS Grant HD-06274 and NSF Grant PCM-7715965.
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