Isolation and partial characterisation of the plasma and outer acrosomal membranes of goat spermatozoa

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In order to achieve successful fertilization, the essential steps include maturation, species-specific binding and fusion of gametes followed by activation of the ovum. Implicit in this are a variety of molecular events, the most important among them being the receptor–ligand interaction. A protocol is described to isolate highly enriched fractions of goat sperm plasma and the outer acrosomal membranes and present data on the ultrastructure and enzyme assays of these membrane domains. The sperm, washed with 1.3 M sucrose sedimentation, was sonicated and subjected to discontinuous sucrose density gradient centrifugation, which allowed the separation of isolated plasma and the outer acrosomal membranes in a single step. While the isolated plasma membranes formed vesicles, the cap shaped acrosomal segments retained its characteristic morphology, as was seen in the ultrastructure. The plasma membrane fraction was relatively pure and free of any contamination of other cell organelles. The matrix associated with the isolated acrosomal membranes gave it a relatively higher density and a crescent shape. Enzyme assays showed high alkaline phosphatase activity in the plasma membrane fraction, with low activity of the acrosome bound enzymes. The acrosomal fraction had a higher specific activity of acrosin and hyaluronidase and a low alkaline phosphatase activity. Suggesting that both the membranes were pure. Membranes, thus purified could be used as initial material for elucidating the lipid as well as the antigenic composition and in understanding the mechanism involved in capacitation, gametic interaction and acrosome reaction, which eventually lead to fertilization.