MOLECULAR REPRODUCTION A N D DEVELOPMENT 25:339-344 (1990)
Identification of Extracellular Proteins in the Rat Cumulus Oophorus NASSIM VIRJI,' DAVID M. PHILLIPS,l
AND BONNIE S. DUNBAR2 ' T h e Population Council, N e w York, New York; 'Department of Cell Biology, Baylor College of Medicine, Houston, Texas
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W e have examined the proABSTRACT teins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte-cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by highresolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postovulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW -81,000 and 100,000). The other two proteins were more basic and occurred at MW -90,000and 150,000. O u r results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.
cumulus interaction. One holds that the cumulus has an important function(s) in fertilization. Evidence has been presented supporting roles in capacitation, hyperactivation, and the acrosome reaction. The other school of thought is that the cumulus has no role in fertilization. Supporting this is evidence that spermatozoa are capacitated, hyperactivated, and acrosome intact when they reach the cumulus and remain so until they reach the zona pellucida. Most interest has been in the role of the cumulus in inducing the acrosome reaction and the role of acrosoma1 enzymes that could hydrolyze the cumulus (McLean and Rowlands, 1942; Leonard and Kurzrok, 1946; Fekete and Duran-Reynals, 1943). Light and electron microscopic studies have suggested that in vivo spermatozoa undergo the acrosome reaction in the ampullary fluid or the cumulus (Austin, 1963; Yanagimachi, 1966; Bedford, 1968; Cummins and Yanagimachi, 1982; Yanagimachi and Phillips, 1984). There are also a number of studies demonstrating that components in the cumulus can induce the acrosome reaction (Tesarik, 1985; Meizel, 1985; Meizel and Turner, 1986; Siiteri and Meizel, 1988). These data suggest that the cumulus may induce the acrosome reaction and that acrosomal enzymes released by this process hydrolyze the extracellular material of the cumulus to facilitate Key Words: Fertilization, Oocyte investments, sperm penetration. Cumulus matrix Other workers have come to the opposite conclusion, that the acrosome reaction does not occur until spermatozoa reach the zona pellucida. Supporting this conINTRODUCTION clusion are observations that mouse spermatozoa reach At the time of fertilization, the mammalian egg and the zona pellucida with intact acrosomes (Florman and zona pellucida are surrounded by cumulus cells embed- Storey, 1982; Saling and Storey, 1979; Storey et al., ded in extracellular material. The cumulus matrix does 1984). Also, it has been shown that rabbit, mouse, hamnot have a tough consistency as does connective tissue ster, and human zonae can induce the acrosome reacor basement membrane but rather is easily deformed, tion and that ZP3 (one of the 3 glycoproteins that make so much so that it has been referred t o as mucous up the zona pellucida) binds to mouse spermatozoa and (Dekel and Phillips, 1978). On its way to the egg, the fertilizing spermatozoa must pass through the extracellular material of the cumulus. Although the interactions between the fertilizing spermatozoon and the cumulus during this short journey have attracted a great deal of interest among reproductive biologists for nearly 50 years, answers regarding sperm-cumulus in- Received June 8, 1989; accepted September 25, 1989. teractions remain obscure. Address reprint requests to David M. Phillips, The Population CounThere are two schools of thought regarding sperm- cil, 1230 York Avenue, New York, NY 10021.
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induces the acrosome reaction (O’Rand and Fisher, 1987; Cherr et al., 1986; Bleil and Wassarman, 1983; Wassarman, 1988; Cross et al., 1988). Motile cells that do not contain hydrolytic enzymes are also capable of penetrating the cumulus (Talbot et al., 1985). If the cumulus does induce the acrosome reaction, one might expect that the cumulus matrix, the mucous material in which the cumulus cells are embedded, would contain a component not present in the surrounding environment of the oviduct. This component should be capable of inducing the acrosome reaction. It is known that the cumulus matrix contains hyaluronic acid (Eppig, 1979; Ball et al., 1982) and other glycoproteins; however, there has been no detailed analysis of this material to date. Since the cumulus matrix is very hydrated, the total amount of protein is very small, making such analysis difficult. We present here a description of the proteins that make up the cumulus matrix with use of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We have compared these proteins to those of follicular fluid and ampullary fluid, which make up the cumulus environment, to determine which are enriched in or unique to the cumulus matrix. Our results indicate that the extracellular component of the cumulus contains many proteins, some of which are common to follicular fluid or ampullary fluid and some of which are not.
MATERIALS AND METHODS Reagents Pregnant mare serum gonadotropin (PMSG) was purchased from Diosynth (Holland) and human chorionic gonadotropin (hCG) was from Sigma Chemicals Co., (St. Louis, MO). Streptomyces hyaluronidase was obtained from Miles Scientific (Naperville, IL). Acrylamide was from Serva Chemicals (Garden City Park, NY), and wide-range ampholytes (pH 3.5-10) from Pharmacia LKB (Piscataway, NJ). Other electrophoresis reagents were from Bio-Rad Laboratories (Richmond, CAI. Animals Twenty-four-day-old female Wistar rats (Taconic Farms, Germantown, NY) were injected subcutaneously with 15 international units (IU) PMSG in 0.15 ml saline, followed 48 h r later by I P injection of 4 IU hCG in 0.1 ml saline. The animals were killed 16-20 h r after hCG injection , and their oviducts were placed in small volumes (-200 pl for each oviduct) of normal saline.
Preparation of the Cumulus Extracellular Fraction The cumulus-oocyte complex (COC) and the ampullary fluid were liberated into 200 pl of saline by piercing the dilated wall of the ampulla of the oviduct with fine steel dissecting scissors. The COC was washed
twice in saline and then transferred into 200 pl 10 IU/ml hyaluronidase (Miles Streptomyces) in saline containing 1mM of the protease inhibitor phenylmethylsufonyl fluoride (PMSF) and incubated for 10 min at 37°C. During this time, the cumulus matrix was hydrolyzed. Cumulus cells and oocytes were subsequently removed by centrifugation at 1 O O g for 10 min in 5 ml plastic tubes and were saved for further treatment as discussed below. The supernatant was then further centrifuged a t 12,OOOg for 10 min in 1.8 ml microfuge tubes to remove possible smaller debris. The supernatants were pooled and stored a t -70°C.
Collection of Ampullary Fluid After the cumulus mass was removed a s discussed above, the saline with the ampullary fluid that exudes into it was centrifuged as above and stored at -70°C. Collection of Follicular Fluid Twenty-four-day-old female rats were given PMSG as described above and killed 48 h r later. The ovaries were removed and washed in saline, dried gently on filter paper, and placed on a piece of parafilm under the dissecting microscope. Large follicles were punctured with a drawn glass capillary connected to a mouth piece. The follicular fluid was carefully drawn up and expelled into -200 pl of saline or into 10 IUiml Streptomyces hyaluronidase in saline and incubated for 10 min at 37°C. This was then centrifuged as described above to remove cells. The supernatant was stored a t -70°C. Oocyte and Cumulus Cell Extract The pellet of cumulus cells and oocyte obtained above was resuspended in a n equal volume of distilled water and lysed by repeated freezing and thawing. The extract was centrifuged a t 12,00Og, and the supernatant was stored at -70°C. Protein Assay The cumulus matrix protein extracts obtained as described above were concentrated and desalted in Amicon Centricon-10 microconcentrators with a molecular weight cut-off at 10,000. Total proteins were then assayed by the dye-binding method of Bradford (1976), using the Bio-Rad protein assay kit and standard assay procedure (Bio-Rad Bulletin 1069). High-Resolution 2D-PAGE High-resolution 2D-PAGE was performed as described by Dunbar (1987a,b). First-dimension isoelectric focusing (IEF) was carried out a t 25°C for 16 h r a t 750 V, for a total of 12,000 V-hr after solubilization of the samples in IEF solubilization buffer containing 9 M urea, 4% Nonidet P-40, 2% 2-mercaptoethanol, and 2% ampholines (pH 35-10]. Second-dimension linear gradient slab gels of 10-20% polyacrylamide (including methylenebis-cross linker) were prepared using the
PROTEINS OF THE CUMULUS OOPHORUS Pace linear gradient maker (Isolabs, Akron, OH) and the Anderson-DALT gel-casting system (Electronucleonics, Inc., Oak Ridge, TN); electrophoresis was carried out using the Anderson DALT electrophoresis tank (Electronucleonics). The gels were stained with the silver-based color stain a s described by Sammons et al. (1981, 1984) to detect the proteins.
RESULTS Serum Seven protein families are apparent in the acidic region of the serum (Fig. 1, spots 1-7). Anderson and Anderson (1977) have characterized the human serum protein patterns in detail. Some typical proteins are albumin and transferrin (Fig. 1). Follicular Fluid Many of the proteins in the rat follicular fluid are similar to those of rat serum (Fig. 2). The proteins common to these fluids are the typical albumin and transferrin. There was no difference in the protein patterns of follicular fluid when diluted with saline only or when treated with hyaluronidase. Ampullary Fluid Many proteins of the ampullary fluid are similar to those of serum and/or follicular fluid. These consist of heterogenously charged proteins a t the acidic end of the gel that are also heterogenous in size and the common proteins albumin and transferrin (Fig. 3). Protein Extract of the Cumulus Matrix Approximately 300 pg total protein was obtained from 100 rats. The pattern of the protein extract of the cumulus matrix (Fig. 4)shows the presence of albumin and transferrin. A predominant protein family, c, heterogenous in size and charge, is visible a t the acidic end of the gel. This family does not correspond to any of the heterogenous families 1-7 observed in the serum, follicular fluid, and ampullary fluid (Figs. 1-3). The apparent molecular weight range of component c of the cumulus matrix is -83,000-100,000 (Fig. 4). A heterogenous component, d, a t the extreme acidic end of the gel is faintly visible in the cumulus matrix a t a n apparent MW of 81,000. Two other components also detectable only in the cumulus matrix appear a t MW -90,000 (f) and 150,000 (g) (Fig. 4). These components occur a t the more basic end and appear to have different color staining properties as revealed by the silverbased color stain. The cumulus matrix also shows spots (h) at MW 42,000 corresponding to actin. Oocyte-Cumulus Cell Extract Two predominant spots (h) are observed in addition to albumin (Fig. 5). These spots are also identified in serum, follicular fluid, and ampullary fluid. The MW of these proteins is -42,000, which corresponds to the
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molecular weight of actin (Anderson and Anderson, 1977).
Streptomyces Hyaluronidase The Streptomyces hyaluronidase that we used to digest the cumulus matrix did not contain significant contaminants (Fig. 6).
DISCUSSION In addition to the predominant albumin, transferrin, and other proteins characteristically present in the serum, follicular fluid, and ampullary fluid, the cumulus matrix exhibits the presence of four other prominent proteins as characterized by 2D-PAGE after hyaluronidase digestion of the matrix. The molecular weight and charge heterogeneity, the apparent increase in molecular weight, the color, as well a s the acidic orientation of two of these proteins, c and d, suggest that these are probably glycoproteins (Dunbar, 1 9 8 7 ~ )typical of secretory proteins. The protein patterns of these proteins were consistent among replicate gels. Therefore, proteolysis, which is easily detected by 2D-PAGE analysis, was not observed in any of these samples. The proteins found in the fluid numbered 1-7 each represent proteins containing multiple charge species, all of which stain the same color, typical of secretory glycoproteins that have undergone extensive posttranslational modification (Dunbar, 1 9 8 7 ~ ) . Predominant cumulus matrix proteins (families c and d) a t the acidic region of the gel were proteins that were not found in any of the other fluids or extracts. The protein pattern of the oocyte-cumulus cell extract is considerably different from that of the cumulus matrix. The heterogenous protein patterns of the two cumulus matrix proteins are similar to those of proteins known to contain sulfates a s well as carbohydrates (Dunbar, 1 9 8 7 ~ )It. is possible that hyaluronic acid was not completely removed from these proteins during hyaluronidase treatment and therefore would contribute to some of this charge heterogeneity of these proteins. The protein molecular weights and charges would be expected to be random if incomplete digestion was occurring. Because identical protein patterns were obtained in multiple samples, this does not appear to be the case. There appears to be minor proteins that are separated as discrete globular spots and are most likely cellular proteins, such as actin (spot h) and the group of proteins 61, which are found in most cell types when analyzed by 2D-PAGE (Dunbar, unpublished observations). We have shown the presence of four major proteins characteristic of the cumulus matrix. These proteins are not apparent in serum, follicular fluid, ampullary fluid, or the oocyte-cumulus cells and are therefore much enriched in, or are unique to, the cumulus extracellular material. The identification of these components in the cumulus matrix is interesting; several in
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Fig. 1. High-resolution ZD-PAGE of rat serum proteins visualized with the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.6), second dimension SDS-PAGE in 10.20% acrylamide gradient gels. Albumin is spread across the gel at 68,000. a, albumin; b, transferrin; h, actin; 1-7, heterogenous acidic proteins.
Fig. 3. High-resolution 2D-PAGE of rat ampullary fluid proteins visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.61, followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. a, albumin; b, transferrin; h, actin; j , cellular proteins; 1-7, heterogenous acidic proteins.
Fig. 2. High-resolution ZD-PAGE of rat follicular fluid proteins visualized with the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.6), followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. a , albumin; b, transferrin; h, actin; j , cellular proteins; 1-7, heterogenous acidic proteins.
Fig. 4. High-resolution 2D-PAGE of rat cumulus matrix protein extract visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.6), second dimension SDS-PAGE in 10-20% acrylamide gradient gels. a, albumin; b, transferrin; c, d, heterogenous acidic proteins of cumulus; g, f, basic proteins of cumulus; h, actin; j , cellular proteins; 1-7, heterogenous acidic proteins.
PROTEINS OF THE CUMULUS OOPHORUS
Fig. 5. High-resolution 2D-PAGE of rat oocyte and cumulus cell extract visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.61, followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. a, albumin; h, actin; j , cellular proteins.
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Fig. 6. High-resolution 2D-PAGE of Streptomyces hyaluronidase (Miles Scientific) visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.61, followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. Arrowheads indicate silver-stain mercaptan artifact a t 69,000.
vivo studies with the golden hamster (Cummins and pecny, 1986) and bound to human sperm incubated Yanagimachi, 1982; Yanagimachi and Phillips, 19841, with cumulus in vitro (Tesarik e t al., 1984). the Chinese hamster (Yanagimachi, 19831, and the ACKNOWLEDGMENTS rabbit (Bedford, 1972) suggest that the acrosome reaction occurs in the cumulus matrix. Although the cumuWe thank Ms. Claire Lo for excellent technical assislus matrix is known to contain hyaluronic acid and tance and Dr. C. Yan Cheng for criticism and help durother mucopolysaccharides, there has not been a de- ing this study. We also thank J. Schweis and D. Myers tailed analysis of its molecular composition. If the cu- for their assistance in the preparation of the manumulus matrix interacts with the spermatozoa to ini- script. This work was supported in part by grants from tiate the acrosome reaction or alter motility, i t may do the Andrew W. Mellon Foundation and U.S. Departso via unique molecular species not present in the sur- ment of Agriculture grant 8701171 to D.M.P. During a rounding field. In vitro studies supporting the role of portion of the work, N.V. was supported by a fellowship the cumulus in the induction of the acrosome reaction from The Rockefeller Foundation. and enhancement of fertilization have been carried out in the hamster (Bavister, 1982; Gwatkin et al., 1972) REFERENCES and in the human (Siiteri and Meizel, 1988). Also, Anderson L, Anderson NG (1977): High resolution two-dimensional transmission electron microscopy studies of the fertilelectrophoresis of human plasma proteins. Proc Natl Acad Sci USA 745421-5425. ized human oocyte-cumulus complex obtained after CR (1963);Acrosome loss from the rabbit spermatozoa in renatural insemination revealed the presence of several Austin lation to entry into the egg. J Reprod Fertil 6:313-14. apparently normal, acrosome-reacted human sperm lo- Ball GD, Bellin ME, Ax RL, First ML (1982): Glycosaminoglycansin cated in the cumulus matrix (Pereda and Coppo, 19851, bovine cumulus-oocyte complexes: Morphology and chemistry. Mol Cell Endrocinol 28:113-122. suggesting that the acrosome reaction may occur Bavister BD (1982): Evidence for a role of post-ovulatory cumulus within the cumulus matrix in vivo in humans. components in supporting fertilizing ability of hamster spermatoOur findings are also of interest with regard to the zoa. J Androl 3:365-372. possible glycoprotein nature of the identified proteins. Bedford JM (1968):Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat 123:329-358. Some of these proteins may be related to the zona pellucida proteins, being similar in apparent MW to Bedford M (1972):An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am J Anat 133:213-254. mouse zona pellucida glycoproteins (Wassarman, Bleil JD, Wassarman PM (1983): Sperm-egg interactions in the 1988a). Carbohydrate-containing macromolecules synmouse: sequence of events and induction of the acrosome reaction thesized by human cumulus have been detected in close by a zona pellucida glycoprotein. Dev Biol 95317-324. association with the zona pellucida (Tesarik and Ko- Bradford M (1976):A rapid and sensitive method for the quantitation
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of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248-252. Cherr GN, Lambert H, Meizel S, Katz DF (1986): In vitro studies of the golden hamster sperm acrosome reaction: Completion on the zona pellucida and induction by homologous soluble zona pellucidae. Dev Biol 114:119-131. Cross NL, Morales P, Overstreet JW, Hansen FW (1988):Induction of acrosome reactions by human zona pellucida. Biol Reprod 38:23544. Cummins JM, Yanagimachi R (1982):Sperm-egg ratios and the site of acrosome reaction during in vivo fertilization in the hamster. Gamete Res 5:239-256. Dekel N, Phillips DM (1987):Maturation of rat cumulus oophorus: A scanning electron microscopic study. Biol Reprod 21:27-31. Dunbar BS (1987a): Protein analysis using high resolution two-dimensional polyacrylamide gel electrophoresis. In: “Laboratory Methods Manual for Hormone Action and Molecular Endocrinology, 11th ed.” Houston: Houston Biological Associates, Inc., pp 151-15-36. Dunbar BS (1987bi: “Two Dimensional Electrophoresis and Immunological Techniques.” New York: Plenum Press, pp 1-368. Dunbar BS (1987~): The use of two-dimensional electrophoresis combined with immunological techniques. BioTechniques 5218-226. Eppig JJ (1979):FSH stimulates hyaluronic acid synthesis by oocytecumulus cell complexes from mouse preovulatory follicles. Nature 281:483-484. Fekete E, and Duran-Reynals, F (1943): Hyaluronidase in the fertilization of mammalian ova. Proc SOCExp Biol Med 52:119-21. Florman HM, Storey BT (1982):Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro. Dev Biol 91:121-130. Gwatkin RBL, Anderson OF, Hutchinson CF (1972): Capacitation of hamster spermatozoa in vitro: The role of cumulus components. J Reprod Fertil 30:389-394. Leonard LS, Kurzrok R (1946): Inhibitors of hyaluronidase in blood sera and their effect on follicle cell dispersal. Endocrinology 39: 85-90. McLean D, Rowlands TW (1942): Role of hyaluronidase in fertilization. Nature 150:627-28. Meizel S (1985): Molecules that initiate or help stimulate the acrosome reaction by their interaction with the mammalian sperm surface. Am J Anat 174:285-302. Meizel S, Turner KO (1986): Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm. J Exp Zool 237:137-139.
ORand MG, Fisher SJ (1987): Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae. Dev Biol 119551-559. Pereda J , Coppo M (1985): An electron microscopic study of sperm penetration into human egg investments. Anat Embryo1 173:247252. Saling PM, Storey BT (1979): Mouse gamete interactions during fertilization in vitro. J Cell Biol 83:544-555. Sammons DW, Adams LD, Nishizawa EE (1981): Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis 2:135-41. Sammons DW, Adams LD, Vidmar TJ, Hatfield CA, Jones DH, Chuba PJ, Crooks SW (1984): Applicability of color silver stain (Gelcode System) to protein mapping with two-dimensional gel electrophoresis. In Celis J, Bravo R (eds): “Two-Dimensional Gel Electrophoresis of Proteins.” Orlando, FL: Academic Press, pp 112-26. Siiteri J E , Meizel S (1988): Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells. J Exp Zool 246:71-80. Storey BT, Lee MA, Muller C, Ward CR, Wirtshafter DG (1984):Binding of mouse spermatozoa to the zona pellucidae of mouse eggs in cumulus: Evidence that the acrosomes remain substantially intact. Biol Reprod 3:1119-1128. Talbot P, DiCarlantoni G , Zao P, Penkala J, Haimo LT (1985): Motile cells lacking hyaluronidase can penetrate the hamster oocyte cumulus complex. Dev Biol 108:387-398. Tesarik J (1985):Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 74:383-388. Tesarik J, Kopecny V (1986): Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte-cumulus-complex extracellular matrices. Histochemistry 85523-528. Tesarik J, Kopecny V, Dvorak M (1984):Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro. Fertil Steril 41:919-925. Wassarman PM (1988): Zona pellucida glycoproteins. Annu Rev Biochem 57:415-442. Yanagimachi R (1966): Time and process of sperm penetration into hamsters ova in vivo and in vitro. J Reprod Fertil 11:359-70. Yanagimachi R (1983): Fertilization. In Crosignani PG, Rubin BL (eds): “In Vitro Fertilization and Embryo Transfer.” London: Academic press, pp 65-100. Yanagimachi R, Phillips DM (1984): The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo. Gamete Res 9:l-19.
Identification of Extracellular Proteins in the Rat Cumulus Oophorus NASSIM VIRJI,' DAVID M. PHILLIPS,l
AND BONNIE S. DUNBAR2 ' T h e Population Council, N e w York, New York; 'Department of Cell Biology, Baylor College of Medicine, Houston, Texas
-
W e have examined the proABSTRACT teins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte-cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by highresolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postovulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW -81,000 and 100,000). The other two proteins were more basic and occurred at MW -90,000and 150,000. O u r results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.
cumulus interaction. One holds that the cumulus has an important function(s) in fertilization. Evidence has been presented supporting roles in capacitation, hyperactivation, and the acrosome reaction. The other school of thought is that the cumulus has no role in fertilization. Supporting this is evidence that spermatozoa are capacitated, hyperactivated, and acrosome intact when they reach the cumulus and remain so until they reach the zona pellucida. Most interest has been in the role of the cumulus in inducing the acrosome reaction and the role of acrosoma1 enzymes that could hydrolyze the cumulus (McLean and Rowlands, 1942; Leonard and Kurzrok, 1946; Fekete and Duran-Reynals, 1943). Light and electron microscopic studies have suggested that in vivo spermatozoa undergo the acrosome reaction in the ampullary fluid or the cumulus (Austin, 1963; Yanagimachi, 1966; Bedford, 1968; Cummins and Yanagimachi, 1982; Yanagimachi and Phillips, 1984). There are also a number of studies demonstrating that components in the cumulus can induce the acrosome reaction (Tesarik, 1985; Meizel, 1985; Meizel and Turner, 1986; Siiteri and Meizel, 1988). These data suggest that the cumulus may induce the acrosome reaction and that acrosomal enzymes released by this process hydrolyze the extracellular material of the cumulus to facilitate Key Words: Fertilization, Oocyte investments, sperm penetration. Cumulus matrix Other workers have come to the opposite conclusion, that the acrosome reaction does not occur until spermatozoa reach the zona pellucida. Supporting this conINTRODUCTION clusion are observations that mouse spermatozoa reach At the time of fertilization, the mammalian egg and the zona pellucida with intact acrosomes (Florman and zona pellucida are surrounded by cumulus cells embed- Storey, 1982; Saling and Storey, 1979; Storey et al., ded in extracellular material. The cumulus matrix does 1984). Also, it has been shown that rabbit, mouse, hamnot have a tough consistency as does connective tissue ster, and human zonae can induce the acrosome reacor basement membrane but rather is easily deformed, tion and that ZP3 (one of the 3 glycoproteins that make so much so that it has been referred t o as mucous up the zona pellucida) binds to mouse spermatozoa and (Dekel and Phillips, 1978). On its way to the egg, the fertilizing spermatozoa must pass through the extracellular material of the cumulus. Although the interactions between the fertilizing spermatozoon and the cumulus during this short journey have attracted a great deal of interest among reproductive biologists for nearly 50 years, answers regarding sperm-cumulus in- Received June 8, 1989; accepted September 25, 1989. teractions remain obscure. Address reprint requests to David M. Phillips, The Population CounThere are two schools of thought regarding sperm- cil, 1230 York Avenue, New York, NY 10021.
0 1990 WILEY-LISS, INC.
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induces the acrosome reaction (O’Rand and Fisher, 1987; Cherr et al., 1986; Bleil and Wassarman, 1983; Wassarman, 1988; Cross et al., 1988). Motile cells that do not contain hydrolytic enzymes are also capable of penetrating the cumulus (Talbot et al., 1985). If the cumulus does induce the acrosome reaction, one might expect that the cumulus matrix, the mucous material in which the cumulus cells are embedded, would contain a component not present in the surrounding environment of the oviduct. This component should be capable of inducing the acrosome reaction. It is known that the cumulus matrix contains hyaluronic acid (Eppig, 1979; Ball et al., 1982) and other glycoproteins; however, there has been no detailed analysis of this material to date. Since the cumulus matrix is very hydrated, the total amount of protein is very small, making such analysis difficult. We present here a description of the proteins that make up the cumulus matrix with use of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We have compared these proteins to those of follicular fluid and ampullary fluid, which make up the cumulus environment, to determine which are enriched in or unique to the cumulus matrix. Our results indicate that the extracellular component of the cumulus contains many proteins, some of which are common to follicular fluid or ampullary fluid and some of which are not.
MATERIALS AND METHODS Reagents Pregnant mare serum gonadotropin (PMSG) was purchased from Diosynth (Holland) and human chorionic gonadotropin (hCG) was from Sigma Chemicals Co., (St. Louis, MO). Streptomyces hyaluronidase was obtained from Miles Scientific (Naperville, IL). Acrylamide was from Serva Chemicals (Garden City Park, NY), and wide-range ampholytes (pH 3.5-10) from Pharmacia LKB (Piscataway, NJ). Other electrophoresis reagents were from Bio-Rad Laboratories (Richmond, CAI. Animals Twenty-four-day-old female Wistar rats (Taconic Farms, Germantown, NY) were injected subcutaneously with 15 international units (IU) PMSG in 0.15 ml saline, followed 48 h r later by I P injection of 4 IU hCG in 0.1 ml saline. The animals were killed 16-20 h r after hCG injection , and their oviducts were placed in small volumes (-200 pl for each oviduct) of normal saline.
Preparation of the Cumulus Extracellular Fraction The cumulus-oocyte complex (COC) and the ampullary fluid were liberated into 200 pl of saline by piercing the dilated wall of the ampulla of the oviduct with fine steel dissecting scissors. The COC was washed
twice in saline and then transferred into 200 pl 10 IU/ml hyaluronidase (Miles Streptomyces) in saline containing 1mM of the protease inhibitor phenylmethylsufonyl fluoride (PMSF) and incubated for 10 min at 37°C. During this time, the cumulus matrix was hydrolyzed. Cumulus cells and oocytes were subsequently removed by centrifugation at 1 O O g for 10 min in 5 ml plastic tubes and were saved for further treatment as discussed below. The supernatant was then further centrifuged a t 12,OOOg for 10 min in 1.8 ml microfuge tubes to remove possible smaller debris. The supernatants were pooled and stored a t -70°C.
Collection of Ampullary Fluid After the cumulus mass was removed a s discussed above, the saline with the ampullary fluid that exudes into it was centrifuged as above and stored at -70°C. Collection of Follicular Fluid Twenty-four-day-old female rats were given PMSG as described above and killed 48 h r later. The ovaries were removed and washed in saline, dried gently on filter paper, and placed on a piece of parafilm under the dissecting microscope. Large follicles were punctured with a drawn glass capillary connected to a mouth piece. The follicular fluid was carefully drawn up and expelled into -200 pl of saline or into 10 IUiml Streptomyces hyaluronidase in saline and incubated for 10 min at 37°C. This was then centrifuged as described above to remove cells. The supernatant was stored a t -70°C. Oocyte and Cumulus Cell Extract The pellet of cumulus cells and oocyte obtained above was resuspended in a n equal volume of distilled water and lysed by repeated freezing and thawing. The extract was centrifuged a t 12,00Og, and the supernatant was stored at -70°C. Protein Assay The cumulus matrix protein extracts obtained as described above were concentrated and desalted in Amicon Centricon-10 microconcentrators with a molecular weight cut-off at 10,000. Total proteins were then assayed by the dye-binding method of Bradford (1976), using the Bio-Rad protein assay kit and standard assay procedure (Bio-Rad Bulletin 1069). High-Resolution 2D-PAGE High-resolution 2D-PAGE was performed as described by Dunbar (1987a,b). First-dimension isoelectric focusing (IEF) was carried out a t 25°C for 16 h r a t 750 V, for a total of 12,000 V-hr after solubilization of the samples in IEF solubilization buffer containing 9 M urea, 4% Nonidet P-40, 2% 2-mercaptoethanol, and 2% ampholines (pH 35-10]. Second-dimension linear gradient slab gels of 10-20% polyacrylamide (including methylenebis-cross linker) were prepared using the
PROTEINS OF THE CUMULUS OOPHORUS Pace linear gradient maker (Isolabs, Akron, OH) and the Anderson-DALT gel-casting system (Electronucleonics, Inc., Oak Ridge, TN); electrophoresis was carried out using the Anderson DALT electrophoresis tank (Electronucleonics). The gels were stained with the silver-based color stain a s described by Sammons et al. (1981, 1984) to detect the proteins.
RESULTS Serum Seven protein families are apparent in the acidic region of the serum (Fig. 1, spots 1-7). Anderson and Anderson (1977) have characterized the human serum protein patterns in detail. Some typical proteins are albumin and transferrin (Fig. 1). Follicular Fluid Many of the proteins in the rat follicular fluid are similar to those of rat serum (Fig. 2). The proteins common to these fluids are the typical albumin and transferrin. There was no difference in the protein patterns of follicular fluid when diluted with saline only or when treated with hyaluronidase. Ampullary Fluid Many proteins of the ampullary fluid are similar to those of serum and/or follicular fluid. These consist of heterogenously charged proteins a t the acidic end of the gel that are also heterogenous in size and the common proteins albumin and transferrin (Fig. 3). Protein Extract of the Cumulus Matrix Approximately 300 pg total protein was obtained from 100 rats. The pattern of the protein extract of the cumulus matrix (Fig. 4)shows the presence of albumin and transferrin. A predominant protein family, c, heterogenous in size and charge, is visible a t the acidic end of the gel. This family does not correspond to any of the heterogenous families 1-7 observed in the serum, follicular fluid, and ampullary fluid (Figs. 1-3). The apparent molecular weight range of component c of the cumulus matrix is -83,000-100,000 (Fig. 4). A heterogenous component, d, a t the extreme acidic end of the gel is faintly visible in the cumulus matrix a t a n apparent MW of 81,000. Two other components also detectable only in the cumulus matrix appear a t MW -90,000 (f) and 150,000 (g) (Fig. 4). These components occur a t the more basic end and appear to have different color staining properties as revealed by the silverbased color stain. The cumulus matrix also shows spots (h) at MW 42,000 corresponding to actin. Oocyte-Cumulus Cell Extract Two predominant spots (h) are observed in addition to albumin (Fig. 5). These spots are also identified in serum, follicular fluid, and ampullary fluid. The MW of these proteins is -42,000, which corresponds to the
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molecular weight of actin (Anderson and Anderson, 1977).
Streptomyces Hyaluronidase The Streptomyces hyaluronidase that we used to digest the cumulus matrix did not contain significant contaminants (Fig. 6).
DISCUSSION In addition to the predominant albumin, transferrin, and other proteins characteristically present in the serum, follicular fluid, and ampullary fluid, the cumulus matrix exhibits the presence of four other prominent proteins as characterized by 2D-PAGE after hyaluronidase digestion of the matrix. The molecular weight and charge heterogeneity, the apparent increase in molecular weight, the color, as well a s the acidic orientation of two of these proteins, c and d, suggest that these are probably glycoproteins (Dunbar, 1 9 8 7 ~ )typical of secretory proteins. The protein patterns of these proteins were consistent among replicate gels. Therefore, proteolysis, which is easily detected by 2D-PAGE analysis, was not observed in any of these samples. The proteins found in the fluid numbered 1-7 each represent proteins containing multiple charge species, all of which stain the same color, typical of secretory glycoproteins that have undergone extensive posttranslational modification (Dunbar, 1 9 8 7 ~ ) . Predominant cumulus matrix proteins (families c and d) a t the acidic region of the gel were proteins that were not found in any of the other fluids or extracts. The protein pattern of the oocyte-cumulus cell extract is considerably different from that of the cumulus matrix. The heterogenous protein patterns of the two cumulus matrix proteins are similar to those of proteins known to contain sulfates a s well as carbohydrates (Dunbar, 1 9 8 7 ~ )It. is possible that hyaluronic acid was not completely removed from these proteins during hyaluronidase treatment and therefore would contribute to some of this charge heterogeneity of these proteins. The protein molecular weights and charges would be expected to be random if incomplete digestion was occurring. Because identical protein patterns were obtained in multiple samples, this does not appear to be the case. There appears to be minor proteins that are separated as discrete globular spots and are most likely cellular proteins, such as actin (spot h) and the group of proteins 61, which are found in most cell types when analyzed by 2D-PAGE (Dunbar, unpublished observations). We have shown the presence of four major proteins characteristic of the cumulus matrix. These proteins are not apparent in serum, follicular fluid, ampullary fluid, or the oocyte-cumulus cells and are therefore much enriched in, or are unique to, the cumulus extracellular material. The identification of these components in the cumulus matrix is interesting; several in
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Fig. 1. High-resolution ZD-PAGE of rat serum proteins visualized with the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.6), second dimension SDS-PAGE in 10.20% acrylamide gradient gels. Albumin is spread across the gel at 68,000. a, albumin; b, transferrin; h, actin; 1-7, heterogenous acidic proteins.
Fig. 3. High-resolution 2D-PAGE of rat ampullary fluid proteins visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.61, followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. a, albumin; b, transferrin; h, actin; j , cellular proteins; 1-7, heterogenous acidic proteins.
Fig. 2. High-resolution ZD-PAGE of rat follicular fluid proteins visualized with the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.6), followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. a , albumin; b, transferrin; h, actin; j , cellular proteins; 1-7, heterogenous acidic proteins.
Fig. 4. High-resolution 2D-PAGE of rat cumulus matrix protein extract visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.6), second dimension SDS-PAGE in 10-20% acrylamide gradient gels. a, albumin; b, transferrin; c, d, heterogenous acidic proteins of cumulus; g, f, basic proteins of cumulus; h, actin; j , cellular proteins; 1-7, heterogenous acidic proteins.
PROTEINS OF THE CUMULUS OOPHORUS
Fig. 5. High-resolution 2D-PAGE of rat oocyte and cumulus cell extract visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.61, followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. a, albumin; h, actin; j , cellular proteins.
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Fig. 6. High-resolution 2D-PAGE of Streptomyces hyaluronidase (Miles Scientific) visualized by the silver-based color stain. Isoelectric focusing in the first dimension using ampholines (relative pH range 4-9.61, followed by SDS-PAGE in 10-20% acrylamide gradient gels in the second dimension. Arrowheads indicate silver-stain mercaptan artifact a t 69,000.
vivo studies with the golden hamster (Cummins and pecny, 1986) and bound to human sperm incubated Yanagimachi, 1982; Yanagimachi and Phillips, 19841, with cumulus in vitro (Tesarik e t al., 1984). the Chinese hamster (Yanagimachi, 19831, and the ACKNOWLEDGMENTS rabbit (Bedford, 1972) suggest that the acrosome reaction occurs in the cumulus matrix. Although the cumuWe thank Ms. Claire Lo for excellent technical assislus matrix is known to contain hyaluronic acid and tance and Dr. C. Yan Cheng for criticism and help durother mucopolysaccharides, there has not been a de- ing this study. We also thank J. Schweis and D. Myers tailed analysis of its molecular composition. If the cu- for their assistance in the preparation of the manumulus matrix interacts with the spermatozoa to ini- script. This work was supported in part by grants from tiate the acrosome reaction or alter motility, i t may do the Andrew W. Mellon Foundation and U.S. Departso via unique molecular species not present in the sur- ment of Agriculture grant 8701171 to D.M.P. During a rounding field. In vitro studies supporting the role of portion of the work, N.V. was supported by a fellowship the cumulus in the induction of the acrosome reaction from The Rockefeller Foundation. and enhancement of fertilization have been carried out in the hamster (Bavister, 1982; Gwatkin et al., 1972) REFERENCES and in the human (Siiteri and Meizel, 1988). Also, Anderson L, Anderson NG (1977): High resolution two-dimensional transmission electron microscopy studies of the fertilelectrophoresis of human plasma proteins. Proc Natl Acad Sci USA 745421-5425. ized human oocyte-cumulus complex obtained after CR (1963);Acrosome loss from the rabbit spermatozoa in renatural insemination revealed the presence of several Austin lation to entry into the egg. J Reprod Fertil 6:313-14. apparently normal, acrosome-reacted human sperm lo- Ball GD, Bellin ME, Ax RL, First ML (1982): Glycosaminoglycansin cated in the cumulus matrix (Pereda and Coppo, 19851, bovine cumulus-oocyte complexes: Morphology and chemistry. Mol Cell Endrocinol 28:113-122. suggesting that the acrosome reaction may occur Bavister BD (1982): Evidence for a role of post-ovulatory cumulus within the cumulus matrix in vivo in humans. components in supporting fertilizing ability of hamster spermatoOur findings are also of interest with regard to the zoa. J Androl 3:365-372. possible glycoprotein nature of the identified proteins. Bedford JM (1968):Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat 123:329-358. Some of these proteins may be related to the zona pellucida proteins, being similar in apparent MW to Bedford M (1972):An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am J Anat 133:213-254. mouse zona pellucida glycoproteins (Wassarman, Bleil JD, Wassarman PM (1983): Sperm-egg interactions in the 1988a). Carbohydrate-containing macromolecules synmouse: sequence of events and induction of the acrosome reaction thesized by human cumulus have been detected in close by a zona pellucida glycoprotein. Dev Biol 95317-324. association with the zona pellucida (Tesarik and Ko- Bradford M (1976):A rapid and sensitive method for the quantitation
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