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(178) ACROSOMAL GLYCOHYDROLASES AS MARKERS TO ASSESS MEMBRANE PRIMING THAT OCCURS DURING CAPACITATION OF MOUSE SPERMATOZOA. Abou-Haila, Aida1, Tulsiani, Daulat2, 1 UFR Biomédicale, Paris, FR2 Depts of OB/GYN and Cell Biology, Nashville, TN ABSTRACT- Successful fertilization depends on priming of sperm membranes by a multifaceted process referred to as capacitation. Only capacitated spermatozoa interact with the egg's extracellular glycocalyx, the zona pellucida, and undergo Ca2+-dependent signal transduction cascade. The net result is the fusion of the sperm plasma membrane (PM) and the outer acrosomal membrane (OAM) and the release of acrosomal contents (i.e., acrosome reaction). In this study, we have used an indirect immunofluorescence approach to examine membrane changes that occur during capacitation as well as multiple staining protocols to assess capacitation and status of the acrosome in mouse spermatozoa. For immunohistochemical studies, we used affinity purified antibodies against two glycohydrolases (beta-D-glucuronidase and beta-D-galactosidase) that cross-reacted with the acrosomal enzymes only when uncapacitated spermatozoa were permeabilized. Incubation of spermatozoa in a medium that favors capacitation induced membrane changes that enable the antibodies to interact with the acrosomal enzymes in non-permeabilized acrosome-intact cells as revealed by several distinct fluorescent patterns. Data analyses demonstrated time-dependent changes in these patterns that correlated with the chlortetracycline assay frequently used to examine capacitation of mouse spermatozoa. Inclusion of calmodulin, a 17 kD Ca2+-binding protein which signals capacitation, in the incubation medium did not alter the rate of capacitation or the acrosome reaction; however, its presence accelerated initial stages of membrane priming as evident by a significantly higher number of acrosome-intact spermatozoa showing immunopositive staining than in its absence. Combined, our data suggest that the initial step of capacitation involves diffusion of acrosomal glycohydrolases (and perhaps other acrosomal contents) through fenestrations of the OAM followed by a time-dependent formation of vesicles which establish contact between the sperm PM and OAM. The proposed diffusion of the acrosomal contents and the membrane priming events enable antibodies to recognize and bind to the acrosomal antigens on capacitating/capacitated spermatozoa without permeabilization. Supported by grants HD25869 and HD34041 from NIH. KEY WORDS: sperm acrosome, acrosomal glycohydrolases, capacitation, membrane priming |
