JEZ 826 THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:185–188 (1997) Cumulus Cell Dispersal From Murine Oocytes by an Epididymal Guanidinobenzoatase STELLINE Y. PEDOTO,1 DAVID J. AARONS,2 SHONDRIA B. YOUNG,2 1 AND GARY R. POIRER * 1 Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294 2 Department of Biology, Talladega College, Talladega, Alabama 35160 ABSTRACT Guanidinobenzoatase (GB), a serine proteinase with a molecular weight of 71,000, is found both free in the epididymal fluids of the mouse and bound to the sperm surface. Microgram quantities of the enzyme, purified from epididymal fluid, will completely disperse follicle cells from freshly ovulated ooctyes after 15 min of incubation. Purified GB exhibits no hyaluronidase activity as determined by the acid albumin assay. The ability of GB to disperse follicle cells is blocked by a proteinase inhibitor endogenous to the male reproductive tract. The inhibitor has no effect on bovine testicular hyaluronidase. Although the function of GB has not been defined, the observations presented here indicate that it may play a role in cumulus matrix penetration during fertilization. J. Exp. Zool. 279:185–188, 1997. © 1997 Wiley-Liss, Inc. The ovulated mouse oocyte is covered by two distinct layers, the cumulus oophorus and the zona pellucida. Fertilization requires that the sperm penetrate both layers. It is generally believed that passage through these layers is, at least in part, an enzymatic process involving hyaluronidase for penetration of the cumulus layer and acrosin for the zona (Yanagimachi, ’88). The cumulus layer is composed of approximately 3,000 cells (Lin et al., ’94) embedded in a matrix of hyaluronic acid and protein (Talbot, ’85). Ultrastructural analysis of the matrix reveals a combination of granules and filaments. The filaments are sensitive to hyaluronidase and the granules to trypsin (Talbot and DiCarlantonio ’84). These observations suggest that a protease, in addition to the more commonly implicated hyaluronidase, could be involved in cumulus matrix digestion during sperm penetration. Data presented here indicate that a serine proteinase, (GB), isolated from epididymal fluids (though also found on sperm, Benau and Storey, 1987; Barksdale et al., 1997) is able to disperse follicle cells from recently ovulated oocytes. Seminal vesicle inhibitor (SVI) isolation and assay The inhibitor was isolated from seminal vesicle tissue as previously described (Poirier and Jackson, ’81). This includes gel filtration (G-75 Sephadex), affinity chromatography with trypsin as the ligand, and ion exchange chromatography (SPSephadex C-25). The purified material was dialyzed against PBS and stored at –20°C until used. Proteinase inhibitor activity was measured against porcine pancreatic trypsin using N-benzoyl-DL-arginine-P-nitroanilide (BAPNA) as the substrate in Ca+2-free triethanolamine hydrochloride buffer, pH 7.8, 0.2M (Fritz et al., ’74). Each 3-ml assay contained 6.0 µg trypsin, 0.77 mM BAPNA, and various amounts of inhibitor. An inhibitor unit (IU) is defined as the amount of inhibitor that reduces the hydrolysis of BAPNA by 1 µmol/min. Isolation of guanidinobenzoatase Animals The caudae epididymides and ducti deferens were removed and placed in PBS, pH 7.2. Sperm were stripped from the ducti. The caudal epididymides were punctured and the sperm were allowed to swim free. The sperm suspensions were Random bred mice 6–12 weeks of age were used throughout the study. These animals were subjected to controlled lighting (16 L:8D) and constant temperature (22 ± 2°C) and had free access to food and water. *Correspondence to: Gary R. Poirier, Dept. of Biology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294. Received 18 December 1996; Revision accepted 21 April 1997. MATERIALS AND METHODS © 1997 WILEY-LISS, INC. 186 S.Y. PEDOTO ET AL. then centrifuged and the supernatants contained GB activity. GB was purified in two steps using molecular sieving (Sephadex G-75) and affinity chromatography with agmatine as the ligand (Barksdale et al., ’97). Guanidinobenzoatase assay Guanidinobenzoatase activity was measured fluorimetrically using 4-methylumbelliferyl-pguanidinobenzoate (MUGB) as the substrate. The typical assay contained 0.1 to 0.5 ml of the enzyme (GB) and PBS to a total of 2 ml. Ten microliters of MUGB (2 mg/ml in dimethylsulfoxide) was added, final concentration of 26 µM, and the hydrolysis was immediately monitored (excitation wavelength of 360 nm and an emission wavelength of 450 nm) for 5 min at room temperature in a Sequoia-Turner Model 450 Fluorimeter equipped with a chart recorder. One enzyme unit (U) corresponds to the formation of 1,000 ng of product (4-methylumbelliferone) per min. One inhibitor unit (IU) inhibits the formation of product by 1,000 ng per min. Hyaluronidase assay The assays were performed according to procedures suggested by Dorfman (1955). One-half milliliter of GB solutions was mixed with 0.5 ml of enzyme buffer (0.02M phosphate buffer, 0.45% NaCl, 0.01% BSA, pH 7.0) followed by the addition of 1 ml of substrate buffer (0.3 M KH2PO4/ Na2HPO4, pH 5.3) containing 0.5 mg of hyaluronic acid. The mixture was incubated for 45 min at 37°C. Turbidity was then developed by adding 5 ml of an acid albumin solution (1 mg/ml albumin in Na acetate buffer, 0.05M pH 3.7). The OD (600 nm) was determined 5 min after the addition of the acid albumin. Bovine testicular hyaluronidase (Sigma Chemical Co. St. Louis, MO) containing 0.3 U/µg was used to establish standard curves. cell dispersal through an inverted microscope. Follicle cells were considered dispersed when the oocytes were clearly visible. RESULTS GB, isolated from the epididymal fluids, showed a molecular weight band of 71,000 on a 4–20% gradient SDS polyacrylamide gel (Fig. 1). The preparation had a specific activity of 1.6 U/mg. Under the conditions of the assay, GB effectively dispersed follicle cells after a 15-min incubation (Fig. 2, Table 1). Bovine testicular hyaluronidase (100 µg/ml) was used as a positive control and PBS as a negative control (Table 1). SVI is able to block the ability of GB preparations to disperse follicle cells but had no effect on hyaluronidase. GB has no detectable hyaluronidase activity and SVI does not inhibit bovine testicular hyaluronidase in the acid albumin assay (Fig. 3). Hyaluronidase, from 0.1 to 1.0 mg/ml, does not hydrolyse MUGB (data not shown). DISCUSSION GB is a serine proteinase whose presence on sperm and in seminal fluids was first described by Steven and Al-Ahmad (’83). GB has been implicated in tumor metatasis, cell migration, and tissue remodeling (Poustis-Delpont et al., ’92). Benau and Storey (’87) characterized the enzyme on murine sperm and considered it to be a trypsin inhibitor sensitive site, which participated in zona binding. Recently the soluble form, derived from Murine ovulated oocytes and cumulus dispersal Mature female mice were superovulated by IP injections of 10 IU of pregnant mare’s serum (PMS) gonadotropin followed 48 hr later by 10 IU of human chorionic gonadotropin (hCG). Ovulated eggs, in cumulus masses, were recovered from oviducts approximately 16 hr after the hCG injection. The masses were washed twice in PBS and transferred to 50 µl of test solution in wells of 96well microtiter plates. The plates were then incubated for 15 min at 37°C and observed for follicle Fig. 1. SDS-PAGE (4 to 20% gradient gel) of ~ 4 µg of GB isolated from an epididymal sperm supernatant (lane b). Molecular weight standards are in lane a. CUMULUS CELL DISPERSAL 187 TABLE 1. Cumulus dispersal summary1 Components tested PBS GB GB + SVI Hdase 0/32 28/28 0/30 35/35 Hdase + SVI 29/29 1 Number of cumulus free oocytes after a 15 min incubation/total number of oocytes. GB, guanidinobenzoatase, used at 65/µg/ml, ~1.6 U/ mg. SVI, proteinase inhibitor of seminal vesicle origin, used at 200 mIU/ml, ~50 µg/ml. Hdase, bovine testicular hyaluronidase, used at 100 µg/ml, 0.3 U/µg. epididymal fluids, has been characterized (Barksdale et al., ’97). Whole zonae, biologicaly active ZP3, and proteinase inhibitors, including SVI, block its ability to hydrolyse MUGB (Barksdale et al., ’97). These data support the contention that the trypsin inhibitor sensitive site functions in zona binding. Recently mouse PH-20, localized on the sperm head, was shown to have hyaluronidase activity (Lin et al., ’94). Purified GB was unable to hydrolyse hyaluronic acid and hyaluronidase did not hydrolyse MUGB. GB is inhibited by SVI while bovine testicular hyaluronidase is not. Even though the molecular weight estimate (71,000) for GB is only slightly higher than the 68,000 suggested for murine glycosylphosphatidylinositollinked hyaluronidase (Thaler and Cardullo, ’95), it appears that GB is distinct from hyaluronidase. The data presented here indicate that GB is able to disperse cumulus cells, presumably by matrix hydrolysis. Since the matrix is made of protein granules as well as hyaluronic acid filaments it seems appropriate that a fertilizing sperm could use both enzyme types, perhaps synergistically, Fig. 3. Effects of adding increasing amounts of purified SVI (Z) on the ability of 20 µg of bovine testicular hyaluronidase to hydrolyse hyaluronic acid. Failure of various amounts of purified GB (5) to hydrolyse hyaluronic acid. Standard curve using bovine testicular hyaluronidase (2) to hydrolyse hyaluronic acid. Fig. 2. The effects of incubating a freshly ovulated egg mass in a solution containing 65 µg/ml of purified GB. a: An egg mass at zero time. b: The same egg mass after 15 min of incubation at 37°C. × 550. 188 S.Y. PEDOTO ET AL. in matrix penetration. For an enzyme to function in murine matrix penetration during in vitro fertilization it should be bound to the surface of acrosome intact sperm (Lin et al., 1994). GB activity is present both on twice washed epididymal sperm and in epididymal fluids (Benau and Storey, 1985; Barksdale et al., 1997). If the spermbound form of GB is like the soluble form then it seems appropriate to suggest that is has a role in matrix penetration. ACKNOWLEDGMENTS The authors are grateful to Mrs. Isabell Boyd and Mrs. Freddie Oglesby for excellent secretarial help. LITERATURE CITED Barksdale, Z., S.C. Caldwell, D.J. Aarons, S.B. Young, and G.R. Poirier (1997) Characterization of the guanidinobenzoatase in epididymal fluids of the mouse. Mol. Reprod. Dev., 47:204–209. Benau, D.A., and B.T. Storey (1987) Characterization of the mouse sperm plasma membrane zona-binding site sensitive to trypsin inhibitors. Bio. Reprod., 36:282–293. Dorfman, A. (1955) Mucopolysaccharidases. Methods Enzymol., 1:166–173. Fritz, H., I. Trautschold, and E. Werle (1974) Protease inhibitors. In: Methods of Enzymatic Analysis. H.U. Bergmeyer, ed. Academic Press, New York, pp. 1064–1080. 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DiCarlantonio (1984) The oocyte-cumulus complex: Ultrastructure of the extracellular components in hamster and mice. Gamete Res., 10:127–142. Thaler, C.D. and R.A. Cardullo (1995) Biochemical characterization of a glycosylphosphatidylinositol-linked hyaluronidase on mouse sperm. Biochemistry, 30:7788–7795. Yanagimachi, R. (1988) Mammalian fertilization. In: The Physiology of Reproduction. E. Knobil and J.D. Neill, eds. Raven Press, Ltd., New York, pp. 136–185.