Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with the oolemma: inhibitor studies using the golden hamster

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Abstract
Summary: The sperm plasma membrane over the equatorial segment of the acrosome gains the ability to fuse with the oolemma some time during, or after, the acrosome reaction. Since acrosin is a major component of the acrosome matrix that dissolves during the acrosome reaction, we sought to determine the effect of acrosin inhibitors on the sperm's ability to fuse with the oolemma. Five acrosin inhibitors (soybean trypsin inhibitor (SBTI), leupeptin, benzamidine,N-p-tosyl-1-lysin-chloromethyl ketone (TLCK) and phenylmethylsulphonyl fluoride (PMSF) and one non-acrosin inhibitor (N-p-tosyl-1-phenylalanine chloromethyl ketone (TPCK) were tested at non-toxic levels (below motility-disturbing concentrations). These inhibitors were added at three different times: (1) during the acrosome reaction of spermatozoa, (2) during sperm-oocyte contact and fusion, and (3) soon after sperm-oocyte fusion was completed. TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction.PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. TPCK was without any inhibitory effects, suggesting that chymotrypsin-like enzymes are not involved in gamete fusion. Although acrosin inhibitors prevented acrosome-reacted spermatozoa from becoming fusion-competent, acrosin (and trypsin) alone could not make the plasma membrane of acrosome-intact spermatozoa fusion-competent. The data suggest that (1) the plasma membrane of the acrosomal region first undergoes dramatic changes immediately before or during the acrosome reaction and (2) acrosin released from the acrosome during the acrosome reaction further alters biophysical and biochemical characteristics of the plasma membrane over the equatorial segment. Such dual changes make the plasma membrane of this specialised region of the spermatozoon competent to fuse with the oolemma. Acrosin may not be the only acrosomal enzyme to participate in these changes.