Abstract.A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model. FoR fertilization to occur the ability of sperm to reach the egg is as critical as the ensuing events of spermegg adhesion and fusion. In mammals a collection of physiological processes including sperm motility, ciliary action in the oviduct, and muscular activity in the vaginal, cervical, and oviductal walls allow the sperm and egg to approach each other in the female reproductive tract (10). After the gametes approach each other, the sperm must have a means to penetrate the structures surrounding the egg in order to reach, and ultimately fuse with, the egg plasma membrane.A single ovulated mammalian (mouse) egg is surrounded by about 3,000 cumulus cells. Before ovulation the spacing between cumulus cells increases as the result of synthesis of increased extracellular matrix (ECM) 1 components, including hyaluronic acid (HA). This process, termed cumulus expansion, is regulated by gonadotropins and soluble factor(s) from the oocyte (31, 32). Expansion results in a cumulus cell/ECM layer that presents a potentially formidable obstacle to an approaching sperm cell.Various ideas of how sperm may penetrate the cumulus layer have been proposed by different investigators. In an Address all correspondence to Y. Lin, Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030. Abbreviations used in this paper:CNBr, cyanogen bromide; cPH-20, cynomolgus monkey; ECM, extraceUular matrix; GPI, glycosyl phosphatidylinositol; gpPH-20, guinea pig PH-20; HA, hyaluronic acid; hPH-20, human PH-20; mPH-20, mouse PH-20; roT, modified Tyrode's medium; PI-PLC, phosphatidylinositol-specific phospholipase C; R-123, rhodamine-123. early model, sperm were believed to acrosome react outside the cumulus releasing their soluble, acrosomal contents including hyaluronidase. The soluble hyaluronidase was thought to yield sufficient HA cleavage in the ECM of the cumulus cells to allow the acrosome-reacted sperm to penetrate the cumulus layer and proceed to fertilize the egg (20,34,41).This early model proved to be inconsis...
The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acrosome reaction.
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