Hyaluronidase dissolves a component in the hamster zona pellucida

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 229:309-316 (1984)

Hyaluronidase Dissolves a Component in the Hamster Zona Pellucida PRUDENCE TALBOT Department o f Biology, University o f California, Riverside, California 92521

ABSTRACT Mammalian sperm must pass between cumulus cells and corona radiata cells before reaching the surface of the zona pellucida which surrounds the oocyte. The cumulus and corona radiata cells are separated from each other by a n extracellular matrix (ECM) containing hyaluronic acid. The structure of this ECM and of the zona pellucida was investigated in the hamster oocyte-cumulus complex (OCC) using transmission electron microscopy (TEM) following processing in ruthenium red. When fixed in the presence of ruthenium red, the ECM of the OCC and the zona pellucida were well preserved and highly structured. The ECM between corona radiata cells was comprised of a network of granules and filaments which resembled hyaluronic acid containing matrices described in other systems. The outer one-third to one-half of the zona pellucida was porous; the ECM of the corona radiata extended into these pores. Bovine testicular hyaluronidase, Streptomyces hyaluronidase, and hamster sperm extracts containing hyaluronidase each dispersed the cumulus cells and most of the corona radiata cells. TEM examination revealed that brief (5-10 min) hyaluronidase treatment of OCCs removed the matrix filaments and caused clumping of the granules in both the corona radiata and zona pellucida. Longer hyaluronidase treatments (15-30 min) removed both filaments and granules. Our observations are consistent with the ideas that: 1)the ECM between corona radiata cells contains hyaluronic acid, and 2) hyaluronic acid is present in the outer one-third to one-half of the zona pellucida. The function of sperm hyaluronidase in penetration of the OCC has not yet been established with certainty. Our data show that the substrate for sperm hyaluronidase is present in the zona pellucida as well as in the corona radiata and cumulus layer and suggest that the function of this enzyme in fertilization should be examined further. Before fusing with the oolemma, the sperm of most mammals must penetrate the extracellular matrix (ECM)between cumulus cells and corona radiata cells and then bind to and penetrate the zona pellucida. Although it is generally agreed that the acrosome reaction occurs prior to sperm passage through the zona pellucida, the role of the reaction in penetrating the cumulusicorona is incompletely understood. According to one paradigm, hyaluronidase, which is released during the normal acrosome reaction, hydrolyzes a portion of the ECM between cumulus and corona radiata cells; this is sufficient to allow sperm to penetrate these layers but not necessarily to disperse the cells of the cumulus and corona radiata (reviewed by Yanagimachi, '81).This idea is supported by the fact

$2 1984 ALAN R. LISS, INC.

that sperm acrosomes contain hyaluronidase (reviewed by Morton, '761, the oocyte-cumulus complex (OCC) contains hyaluronic acid (Eppig, '79; Ball et al., '82) and dissolves in solutions of hyaluronidase (McClean and Rowlands, '421, and both synthetic inhibitors of hyaluronidase (e.g., myocrisin, Reddy et al., '79; Perrault et al., '80) and antibodies raised against hyaluronidase (e.g., Dunbar et al., '76) can block fertilization. Acrosome-reacting sperm have been observed among cumulus cells collected in vivo (Austin and Bishop, '58; Cummins and Yanagimachi, '821, although the site of reaction of the fertilizing sperm has not yet been determined. Guinea pig sperm release hyaluronidase during the normal acrosomal reaction (Talbot and Franklin, '74b; Rogers and Yanagimachi, '75)

310

P. TALBOT

and bind to the zona pellucida only after the reaction is complete (Huang et al., '81). However, recent evidence suggests the role and release of hyaluronidase may be more complex than originally thought. Gwatkin et al. ('76) have observed that capacitated, but not yet reacted, hamster sperm can bind to the hamster zona pellucida and then fertilize hamster oocytes. They interpreted their data to mean that the acrosome reaction occurs after binding to the zona pellucida in this species. More recently, it has been shown using scanning and transmission electron microscopy that capacitated mouse and hamster sperm bind to the zona pellucida with their acrosomes intact whereas reacted sperm fail to bind at all (Saling and Storey, '79; Phillips and Shagli, '80a,b). Moreover, the mouse zona pellucida contains a sperm receptor, ZP-3, which may have acrosome reaction inducing capability (Bleil and Wasserman, '80; Florman and Storey, '82). Florman and Storey ('82) have recently shown experimentally that the physiologically relevant acrosome reaction of the mouse sperm occurs on the zona pellucida surface, and thus sperm that react prematurely are probably infertile. The observation that physiological numbers (1-10) of capacitated hamster sperm can penetrate freshly ovulated hamster cumulus masses prior to undergoing a n acrosome reaction (Corselli and Talbot, unpublished) demonstrates that the reaction is not required for cumulus penetration in this species. When considered together, these data seem most consistent with the idea that in several species the acrosome reaction occurs after binding to the zona pellucida surface. Information on the precise localization of hyaluronidase in the sperm would be valuable in interpreting its function and time of release. Light-microscopic analyses using labeled antibodies to hyaluronidase have shown the enzyme to be present in the acrosoma1region (Mancini et al., '64; Morton, '75; Flechon and Dubois, '74). In these studies, however, it is not possible to distinguish between presence in the plasma membrane, acrosomal membrane, or acrosomal vesicle. The results of one ultrastructural study demonstrate hyaluronidase activity within the acrosomal vesicle, but are not sufficiently high in resolution to clarify questions relating to the membranes (Gould and Bernstein, '75). Biochemical studies on denuded cells establish that sperm from several species retain some hyaluronidase activity even after the acrosomal vesicle is removed (Brown, '75;

Anand et al., '77). This indicates that a portion of the total complement of hyaluronidase probably resides in the inner acrosomal membrane. Some hyaluronidase may also be associated with the plasma membrane as suggested by the following observations: 1) seminal fluids contain hyaluronidase which should be available for absorption to the sperm surface (Hechter and Hadidian, '47); 2) antibodies to hyaluronidase neutralize 50% of the total complement of this enzyme in intact rabbit sperm (Metz et al., '72); 3) intact rat sperm are able to hydrolyze hyaluronic acid and chondroitin sulfate (Lewin et al., '81); and 4) hamster sperm release a significant amount of hyaluronidase before and independently of a n acrosome reaction (Rogers and Morton, '73; Talbot and Franklin, '74a). The above observations suggest that hyaluronidase is located in more than one site within or on sperm and may thus have more than one role in fertilization. If so, we would expect to find hyaluronic acid in more than one location in the oocyte's investing layers. It is generally accepted that hyaluronic acid is located between cumulus cells and corona radiata cells, although this has been demonstrated biochemically only for the mouse (Eppig, '79) and bovine OCC (Ball et al., '82). Several light-microscopicstudies suggest that hyaluronic acid is also present in the zona pellucida of rabbits (da Silva Sasso, '55; Flechon, '74) and mice (Tadano and Yamada, '78). The purpose of this study has been to examine the hamster OCC for the presence of hyaluronic acid using cytochemical techniques. We have fixed OCCs in the presence of ruthenium red which stabilizes hyaluronic acid. We will show that a n ECM with the morphological characteristics of hyaluronic acid-containing matrices is present between corona radiata cells and in the outer portion of the zona pellucida. Moreover this matrix is removed by bovine testicular hyaluronidase, Streptomyces hyaluronidase, and hamster sperm extracts containing hyaluronidase. MATERIALS AND METHODS

Oocyte-cumulus complexes (OCCs) Fresh hamster OCCs were obtained in the following manner. Females were injected with 25 IU of PMSG on the morning of the vaginal discharge. On the evening of day 3, 25 IU of hCG was injected, and females were sacrificed 12-13 h later. The ovaries were removed, dissected free of surrounding tis-

HYALURONIC ACID AND OOCYTE INVESTMENrS

sue, and placed in culture dishes containing Earle’s balanced salt solution (EBSS). Holes were poked in the apex of mature follicles using a 0-gauge insect pin, and the OCCs were allowed to ooze out. OCCs were transferred with a Pasteur pipette to fresh droplets of EBSS under mineral oil and held a t room temperature until the start of a n experiment (not more than 30 min following collection).

Incubation i n hyaluronidase solutions Hyaluronidase solutions from the following three sources were tested 1)bovine testicular hyaluronidase (Sigma, H-3506; Worthington lot No. 52P329), 2) Streptomyces hyaluronidase (Calbiochem No. 389561), and 3) hamster sperm extracts containing hyaluronidase activity. The extracts were prepared by washing cauda epididymal sperm twice in normal saline, then freeze-thawing the sperm twice, and collecting the supernatant. Supernatants, which were free of sperm, were capable of dispersing cumulus cells. The bovine and Streptomyces hyaluronidase were dissolved in EBSS a t activity levels of 10 NFUlml. Hamster sperm extracts were adjusted to disperse the cumulus cells in a length of time equivalent to that of the other two batches of enzyme. Previous studies have shown that the time of cumulus dispersion is directly related to hyaluronidase activity (Talbot and Franklin, ’74c);thus activity levels in all three solutions were assumed to be similar. The bovine testicular hyaluronidase (Sigma and Worthington) and Streptomyces hyaluronidase (Calbiochem) were tested for proteinase contamination using Bio-Rad casein-agar plates and the Rinderknecht assay on hide powder conjugated to Azure-I1 (Rinderknecht et al., ’68). None of these batches of hyaluronidase showed any activity with the hide powder assay; however, the Sigma and Worthington bovine testicular hyaluronidase (No. 52P329) did show activity on the casein substrate. This proteinase activity could be inhibited by inclusion of benzamidine HC1 (1 mgiml), soybean trypsin inhibitor (1 mglml), p-chloromecuribenzoic acid (1 mgiml), and pepstatin A (50 pgiml) in the incubation medium. In experiments in which bovine testicular hyaluronidase was used to challenge the OCC , these proteinase inhibitors were included. They were not used with the Streptornyces hyaluronidase as it had no detectable proteinase activity. Aliquots (100200 p1) of enzyme or of EBSS were placed in Falcon tissue culture dishes under mineral

311

oil a t room temperature. Washed OCCs were transferred to enzyme solutions and incubated for 5-10 or 15-30 min. Dishes were swirled several times during incubation; at the end of the incubation interval, free oocytes or OCCs were fixed for electron microscopy.

Electron microscopy OCCs were fixed and stained with ruthenium red using the following modification of Luft’s procedure (’71).Enzyme solutions were aspirated off OCCs with a Pasteur pipette; all solution aspirations and additions were done while observing OCCs with a dissecting microscope. Cells were fixed 1.5 h in 3%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 0.5% ruthenium red. OCCs were washed 3-4 times in cacodylate buffer, then postfixed 3 h in 1%osmium tetroxide in cacodylate buffer containing 0.5% ruthenium red. Osmium fixation was done in the dark and was followed by several washes in cacodylate buffer. In some experiments, OCCs or oocytes were transferred into 2% agar before additional processing. Tissue was dehydrated in a graded series of ethanol, infiltrated in ethano1:plastic overnight, and embedded in Spurr’s plastic (‘69). Thin sections through the oocyteizona pellucidalcorona radiata were cut on a Sorval MT-2B ultramicrotome, picked up on copper grids, and examined with a Hitachi H-500 transmission electron microscope. RESULTS

Streptomyces hyaluronidase, bovine testicular hyaluronidase, and hamster sperm extracts containing hyaluronidase were each capable of dispersing cumulus cells of fresh hamster OCCs, although some corona radiata cells were often retained adjacent to the zona pellucida in dispersion tests. OCCs or free oocytes were fixed for transmission electron microscopy 5-10 or 15-30 min after incubation in enzyme solutions. At these times, cumulus and corona radiata cells were either partially (5-10 min) or completely (1530 min) dispersed. When hamster OCCs are fixed using routine procedures, the ECM between cumulus and corona radiata cells is amorphous and difficult to resolve (not shown). However, when a n OCC is fixed in the presence of ruthenium red, a highly structured ECM is observed between cells (Figs. 1, 2). The matrix is composed of small electron-dense granules measuring 37.4 2 5.1 nm in diameter.

P. TALBOT

Fig. 1. Micrograph showing the hamster zona pellucida (ZP) and a portion of a polar body (PB). Ruthenium red treatment has stabilized the ECM (arrows) between corona radiata cells (not shown). This matrix extends down into pores in the outer region of the zona pellucida. The pores in the zona pellucida a r e most numerous in the outer third of this layer; a few extend to t h e perivitelline space. Magnification, x 10,000.

Fig. 2. Micrographs showing the matrix o f the corona radiata and the outer surface of the zona pellucida a t higher magnification. Figure 2a shows the ECM of the corona radiata; part of a corona cell microvillus with attached matrix is present. The matrix between corona radiata cells is composed of electron-dense g a n u l e s and filaments. Figure 2b shows that this matrix extends down into the pores of the zona pellucida (arrows). Magnification, ~ 6 0 , 0 0 0x. 34,000.

HYALURONIC ACID AND OOCYTE INVESTMENTS

The granules are interconnected by thin (5.0 + 2.1 nm) filaments. After fixation in the presence of ruthenium red, the zona pellucida is fibrous and its outer margin is porous. The pores usually extend into the outer one-third to one-half of the zona, although they were occasionally seen extending to the perivitelline space (also see Phillips and Shagli, '80a,b). The pores are continuous with the extracellular spaces of the corona radiata. The ECM of the corona radiata extends down into the pores of the zona pellucida (Fig. 2). Each enzyme we tested removed the granules and filaments in the corona radiata and in the pores of the zona pellucida. Brief (5-10 min) hyaluronidase treatment removed the filaments in both regions; some granules were often still present, but tended to be aggregated (Fig. 3). After longer incubations in hyaluronidase solutions (15-30 min), all the granules and filaments were absent (Fig. 4). Control OCCs incubated 5-10 or 15-30 min in EBSS without enzyme prior t o fixation appeared identical to the fresh OCCs shown in Figures 1 and 2. A full complement of granules and filaments were present in the extracellular spaces of the corona radiata and in the pores of the zona pellucida. DISCUSSION

Our electron micrographs demonstrate an ECM composed of granules and filaments between corona radiata cells and in the outer portion of the zona pellucida of hamsters. This matrix can be removed by hamster sperm extracts containing hyaluronidase, bovine testicular hyaluronidase, and Strepte myces hyaluronidase. Results with each batch of enzyme require slightly different interpretations. The hamster sperm extracts are not pure and contain enzymes in addition to hyaluronidase. Thus our data with extracts show that hamster sperm contain an enzyme capable of digesting this ECM but do not establish which enzyme. Bovine testicular hyaluronidase degrades hyaluronic acid, chondrontin, and chrondroitin sulfates (Leppi and Stoward, '65; Zergibe, '62). Thus data obtained with the bovine preparation indicate the matrix contains at least one of these substrates and are reassuring in demonstrating that testicular hyaluronidase can remove the matrix. Streptomyces hyaluronidase degrades only hyaluronic acid (Yamada, '73; Ohya and Kaneko, '70). Removal of the matrix by the Streptoinyces preparation thus supports the contention that this matrix contains hyaluronic acid.

313

At the ultrastructural level, this ECM resembles hyaluronic acid-containing matrices previously described in primate arteries (Wight and Ross, '751, pulmonary connective tissue (Takusagawaet al., '82), synovium and ear cartilage (Myers, '76) and epiphyseal cartilage (Thyberg et al., '73; Smith, '70; Hascall, '80). Others have suggested that the filaments in such matrices are hyaluronic acid whereas the granules correspond to proteoglycans (Hascall, '80; Singley and Solursh, '80). Our results were obtained using unfixed OCCs; thus both granules and filaments might be expected to wash out after hyaluronidase treatment. In fact prolonged treatment did remove both components, but in short enzyme incubations, filaments appeared more sensitive to hyaluronidase than did the granules. The demonstration by cytochemical procedures and enzyme sensitivity that the zona pellucida contains hyaluronic acid is in agreement with previous light-microscopic studies (de Silva Sasso, '55; Flechon, '74) and has implications in our understanding of the role of hyaluronidase in fertilization. Hyaluronic acid, a substrate for sperm hyaluronidase, is found both in the ECM of the corona radiata and cumulus layer and in the zona pellucida. The presence of hyaluronic acid in these sites suggests that sperm hyaluronidase is involved in penetration of these three investments. When these observations are coupled with data in the literature, two mechanisms can be proposed to account for the role of hyaluronidase in penetration of the OCC: First, some hyaluronidase is probably absorbed onto the sperm plasma membrane (evidence is given in the introduction). This hyaluronidase, while attached to the membrane or after release, could assist sperm in cumuluslcorona radiata passage by degrading hyaluronic acid between cells of these layers. Acrosome-intact sperm could then bind to the zona pellucida, undergo a normal acrosome reaction, and discharge acrosomal vesicle hyaluronidase, which would digest the hyaluronic acid component of the zona pellucida. Second, sperm may undergo normal acrosome reactions during passage through the cumulus matrix as observed by others (Austin and Bishop, '57; Cummins and Yanagimachi, '82). These sperm could bind to the zona pellucida following the reaction, and hyaluronidase retained by the inner-acrosomal membrane could assist in zona pellucida penetration. Both of these mechanisms are supported by evidence in the literature; however we do not yet have sufficient data to know which is generally correct. The

3 14

P. TALBOT

HYALURONIC ACID AND OOCYTE INVESTMENTS

mechanism might be characteristic of a particular species; for example, hamster and mouse sperm might utilize the first mechanism and guinea pig sperm the latter. Differences have already been reported for these animals in the time course of hyaluronidase release which occurs before and independently of the acrosome reaction in hamsters (Talbot and Franklin, '74a), but during the reaction of guinea pig sperm (Talbot and Franklin, '74b; Rogers and Yanagimachi, '75). Moreover, sperm binding to the zona pellucida appears to occur by way of the plasma membrane in hamsters (Phillips and Shagli, 'Bob) and mice (Saling and Storey, '79; Florman and Storey, '82) but by the inner acrosomal membrane in guinea pigs (Huang et al., '81). It will be important in future work to extend our observations on the role of hyaluronidase in fertilization and sperdzona binding to other species. There are sufficient differences at present between the mouse1 hamster and guinea pig to suggest that true species differences in the mechanism of cumulus penetration and zona binding do exist. Only further comparative work will establish if one mechanism is more general in its occurrence. Should future studies establish that fertilizing sperm do indeed generally bind to the zona pellucida by their plasma membrane, then sperm that reacted before binding to the zona would be infertile and the function of the acrosome reaction would require reevaluation. ACKNOWLEDGMENTS

I thank Gayle DiCarlantonio for her excellent technical help with portions of this work, and Gary Martin for his valuable suggestions regarding the manuscript. Tami Schick and Stuart Hiroyasu very conscientiously prepared the micrographs for publication, and Wendy Shipley deciphered my handwrit-

Fig. 3. The outer surface of the zona pellucida following brief (5-10 min) treatment with hyaluronidase. The matrix between corona radiata cells and in the pores of the zona pellucida is partially dissolved. The filaments are no longer present, and remaining granules appear aggregated. Magnification, x 30,000. Fig. 4. Similar to Figure 3 except the preparation was treated with hyaluronidase for 15-30 niin before fixation. All granules and filaments have been removed from the spaces between corona radiata cells and the pores of the zona pellucida. Magnification, X 26,000.

3 15

ing. I gratefully acknowledge support of a n intramural grant, NIH grant 11684, and a Research Career Development Award from NIH. LITERATURE CITED Anand, S.R., S. Kaur, and P. Chaudhry (1977) Distribution of 6-N-acetylglucosamimidase, hyaluronoglucosaminidase, and acrosin in buffalo and goat spermatozoa. Hoppe-Seyler's 2. Physiol. Chem., 358:685-688. Austin. C.R.. and M.W.H. BishoD (1958) Role of the rodent acrosome and perforatorium in fertilization. Proc. R. SOC.Lond. Ser. B, 149:241-248. Ball, G.D., M.E. Bellin, R.L. Ax, and N.L. First (1982) Glycosaminoglycans in bovine cumulus-oocyte complexes: Morphology and chemistry. Mol. Cell. Endocrinol., 28r113-122. Bleil, J., and P. Wasserman (1980) Mammalian spermegg interaction: Identification of a gtycoprotein in the mouse zona pellucida possessing receptor activity for sperm. Cell, 20,873-882. Brown, C.R. (1975) Distribution of hyaluronidase in the ram spermatozoan. J. Reprod. Fertil., 45:537-539. Cummins, J.M., and R. Yanagimachi (1982) Sperm-egg ratios and the site of the acrosome reaction during in vivo fertilization in the hamster. Gamete Res., 5: 239-256. Dunbar, B., M. Munoz, C.T. Cordle, and C.B. Metz (1976) Inhibition of fertilization in vitro by treatment of rabhit sperm with univalent isoantibodies to rabbit sperm hyaluronidase. J. Reprod. Fertil., 47t381-384. Eppig, J.J. (1979) FSH stimulates hyaluronic acid synthesis by oocyte-cumulus cell complexes from mouse preovulatory follicles. Nature, 281:483-484. Flechon, J.E. (1974) Application of cytochemical techniques to the study of maturation of gametes and fertilization in mammals. Multipurpose use of glycolmethacrylate embedding. Histochem. J., 6t65-67. Florman, H., and B. Storey (1982) Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro. Dev. Biol., 91t121-130. Gould, S.F., and M.H. Bernstein (1975) The localization of bovine hyaluronidase. Differentiation, 3t123-132. Gwatkin, R.B.L., W.H. Carter, and H. Patterson (1976) Association of mammalian sperm with cumulus cells and the zona pellucida studied by scanning electron microscopy. In: Scanning Electron Microscopy. H. Johari and R.P. Becker, eds. I.T.T. Res. Inst., Chicago, Vol. 2, pt. 2, pp. 379-384. Hascall, G. (1980) Cartilage proteoglycans: Comparison of sectioned and soread whole molecules. J. Ultrastruc. Res., 70: 369-37 5 .. Hechter, O., and 2. Hadidian (1947)Hyaluronidase activity of spermatozoa. Endocrinology, 41204-205. Huang, T.T.F., A.D. Fleming, and R. Yanagimachi (1981) Only acrosome reacted spermatozoa can hind to and penetrate the zona pellucida: A study using the guinea pig. J. Exp. Zool., 217:287-290. Leppi, T.J., and P.J. Stoward (1965) On the use of testicular hyaluronidase for identifying acid mucins in tissue sections. J. Histochem. Cytochem., 13:406-407. Lewin, L.M., 2. Nevo, and R. Weissenberg (1981) The role of hound hyaluronidase in rat sperm-cumulus oophorous interaction. Ann. N.Y. Acad. Sci., 383t473474. Luft, J.H. (1971) Ruthenium red and violet. 11. Fine structural localization in animal tissues. Anat. Rec., 171:369-416. Mancini, R.E., A. Alonso, J. Barquet, B. Alvarez, and M. Nemirousky (1964)Histo-immunological localization of

3 16

P. TALBOT

hyaluronidase in the bull testis. J. Reprod. Fertil., 8:325-332. McClean, D., and I.W. Rowlands (1942) Role of hyaluronidase in fertilization. Nature, 150:627-628. Metz, C.B., A.C. Seiger, and A.L. Castro (1972) Inhibition of the cumulus dispersing and hyaluronidase activities of sperm by heterologous and isologous antisperm antibodies. Proc. SOC.Exp. Biol. Med., 140r766781. Morton, D.B. (1975) Acrosomal enzymes: Immunological localization of acrosin and hyaluronidase in ram spermatozoa. J. Reprod. Fertil., 45:375-378. Morton, D.B. (1976) Lysosomal enzymes in mammalian spermatozoa. In: Lysosomes in Biology and Pathology. J.D. Dingle and R.T. Dean, eds. ElsevierlNorth-Holland, Amsterdam, Vol. 5 , pp. 203-255. Myers, D.B. (1976) Electron microscopic autorddiography of 3!is0 labelled material closely associated with collagen 8b;ils in mammalian synovium and ear cartilage. Histochem. J., 8:191-198. Ohya, T., and Y. Kaueko -(1970) Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta, 198:607. L. Zaneveld, and B.J. Rogers (1980) InhiPerreault, S., bition of fertilization in the hamster by sodium aurothiomalate, a hyaluronidase inhibitor. J. Reprod. Fertil., 60:461-467. Phillips, D., and R. Shagli (1980a) Surface architecture of the mouse and hamster zona pellucida and oocyte. J. Ultrastruc. Res., 72:l-12. Phillips, D.M., and R.M. Shagli (1980b) Surface properties of the zona pellucida. J. Exp. Zool., 213:l-8. Reddy, J.K., C. Joyce, and L. Zaneveld (1979) Role of hyaluronidase in fertilization: The antifertility activity of myocrisin, a nontoxic hyaluronidase inhibitor. J. Androl., 1:28-32. Rinderknecht, I., M.C. Goekas, P. Silverman, and B.J. Haverback (1968)A new ultrasensitive method for the determination of proteolytic activity. Clinica Chimica Acta, 21:197-203. Rogers, B.J., and B.E. Morton (1973) The release of hyaluronidase from capacitating sperm. J. Reprod. Fertil., 35:477-487. Rogers, B.J., and R. Yanagimachi (1975) Release of hyaluronidase from guinea pig spermatozoa through an acrosome reaction initiated by calcium. J. Reprod. Fertil., 44:135-138. Saling, P., J. Sowinski, and B. Storey (1979) An ultrastructural study of epididymal mouse sperm binding to the zona pellucida in vitro: Sequential relationship to the acrosome reaction. J. Exp. Zool., 209t229-238.

da Silva Sasso (1955)Existence of hyaluronic acid at the zona pellucida of the rabbit ovum. Acta Anat., 36: 352-357. Singley, C.T., and M. Solursh (1980) The use of tannic acid for the ultrastructural visualization of hyaluronic acid. Histochemistry, 6593-102. Smith, J.W. i1970) The disposition of protein polysaccharide in the epidermal plate cartilage of the young rabbit. J. Cell Sci., 6:843-864. Spurr, A.R. (1969)A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruc. Res., 26:31-43. Tadano, Y., and K. Yamada (1978)The histochemistry of complex carbohydrates in the ovarian follicles of adult mice. Histochem., 57:203-215. Takusagawa, K., F. Ariji, K. Shida, T. Sato, N. Asoo, and K. Konno (1982) Electron microscopic observations on pulmonary connective tissue stained by ruthenium red. Histochem. J., 14:257-271. Talbot, P., and L.E. Franklin (1974a) Hamster sperm hyaluronidase. 11. Its release from sperm in vitro in relation to the degenerative and normal acrosome reaction. J. Exp. Zool., 189~321-332. Talbot, P., and L.E. Franklin (1974b)The release of hyaluronidase from guinea pig sperm during the course of the normal acrosome reaction in vitro. J. Reprod. Fertil., 39:429-432. Talbot, P., and L.E. Franklin (1974~)Hamster sperm hyaluronidase. I. A bioassay procedure based on cumulus dispersion rate. J. Exp. Zool., 189:311-320. Thyberg, J., S. Lohmander, and U. Freiberg (1973) Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage. J. Ultrastruc. Res., 45:407-427. Wight, T.N., and R. Ross (1975)Proteoglycans in primate arteries: I. Ultrastructural localization and distribution in the intima. J. Cell Biol., 67.660-674. Yamada, K. (1973) The effect of digestion with Streptomyces hyaluronidase upon certain histochemical reactions of hyaluronic acid containing tissues. J. Histochem. Cytochem., 21:794-803. Yanagimachi, R. (1981) Mechanisms of fertilization in mammals. In: Fertilization and Embryonic Development In Vitro, L. Mastrolanni and J.D. Biggers, eds. Plenum, New York, pp. 81-182. Zergibe, F.T. (1962) The demonstration of the individual acid mucopoly saccharides in human aortas, coronary arteries and cerebral arteries. I. The methods. J. Histochem. Cytochem., 10:441-447.