A method involving the treatment of ram spermatozoa with MgCl2 followed by Hyamine 2389 and Triton X-100 is described, which gave selective removal of the acrosomal membranes and acrosomal enzymes. The effect on the individual membranes of ram spermatozoa was evaluated by electron microscopy. Treatment with MgCl2 removed or altered the integrity of the plasma and outer acrosomal membranes, allowing the release of material from the acrosome. The inner acrosomal membrane and the electron-dense material of the equatorial segment were frequently unaffected by this treatment. Subsequent treatment of these spermatozoa with Hyamine and Triton removed the inner acrosomal membrane and the electron-dense material from the equatorial segment. The MgCl2 extract contained acrosomal proteinases and hyaluronidase. The detergent extract contained sperm neuraminidase. The specific activities and yields of the enzymes obtained in the MgCl2 step were much higher than those of the enzymes obtained by the detergent treatment. Slight alterations in the conditions of the treatment had different effects on the acrosomal membranes in that one or more of the membranes were removed or altered in varying proportions resulting in the release of one or more enzymes. Only the plasma membrane and the acrosome appeared to be affected by these treatments as selected mitochondrial enzymes were not detectable in the extracts.
The sperm membrane protein, designated as YWK-II protein/APLP2, is a member of the amyloid precursor protein (APP) superfamily and is a type I transmembrane protein involved in fertilization. Here, the structure-function of the domains of YWK-II protein was examined. Five segments with overlapping ends encompassing the entire extracellular region of mouse YWK-II gene were prepared, cloned and separately expressed in E. coli. The recombinant YWK-II segments were fused with glutathione S-transferase (GST), purified and evaluated for their antifertility activities by measuring their capacity to block in vitro mouse sperm-egg interaction. The structural domain(s) involved in the fertilization process was identified. The polypeptide segment corresponding to position 22-207 of YWK-II-763 inhibited the early stage of fertilization when the spermatozoa interacted with zona-free eggs; whereas the polypeptide segment 201-395 (lacking 309-364) of YWK-II-763 blocked sperm-egg membrane fusion. The remaining three segments, 201-395, 389-574 and 517-704 (lacking 613-624) of YWK-II-763, did not influence the in vitro fertilization process. The present results suggest that segment 22-308 of YWK-II-763 participates in the binding and fusion of sperm and egg plasma membranes thereby promoting fertilization.
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