Biochemical and fluorescence studies of mammalian sperm membranes

Abstract

Enzymatic characterization of the sperm plasma membrane fraction by estimating the activity of a number of standard cell surface marker enzymes clearly shows that the sperm plasma membrane fraction is enriched in cation?activated ATPases, alkaline and acid phosphatases, 5??nucleotidase, and acetylcholinesterase. The ATPases have been implicated in a number of important functions related to the mechanochemical generation of energy required for sperm motility, the transport of metabolites from seminal plasma into sperm cells, and the maintenance of ionic equilibria. Under conditions of strenuous homogenization, the plasma membrane?associated acetylcholinesterase of bull spermatozoa appears to dissociate from the lipid matrix, suggesting that it may be a loosely bound peripheral membrane protein, similar to the enzyme of the erythrocyte membrane. A particularly interesting feature is the detection and localization of a Ca²??ATPase, which could be an integral component of the plasma membrane Ca²? pump and may actively participate in a number of important processes linked to the phenomenon of fertilization. Further, the exclusive association of this enzyme with the plasma membrane suggests that the Ca²??ATPase could serve as a cell surface marker enzyme for mammalian spermatozoa.

The distribution pattern of acrosomal enzymes in the various subcellular fractions suggests that some of these hydrolytic enzymes exist both in bound and soluble forms within the sperm. While glycosidases exist largely in the soluble form, most of the hyaluronidase and acrosin appear to be present in a relatively insoluble and bound state. This differential solubility of sperm acrosomal enzymes may reflect differences in their functions during fertilization.

Membrane?bound Ca²? appears to play an active role in maintaining the functional integrity of the sperm cell, since depletion of this divalent cation appears to introduce irreversible transformations that drastically affect the activity of membrane?associated enzymes. Electrophoretic analysis (SDS?PAGE) of the plasma and acrosomal membranes reveals differences in the polypeptide profiles of the two membranes. Fluorescence experiments using lipid fluidity probes such as pyrene and 1,6?diphenyl?1,3,5?hexatriene (DPH) show that the plasma membranes are more fluid than the acrosomal membranes, consistent with observed differences in cholesterol distribution between the two membranes. These findings constitute clear evidence for marked differences in lipid phase mobility in the plasma and acrosomal membranes of mammalian spermatozoa. The relatively lower fluidity of the plasma membrane may be an important factor in determining its susceptibility to fusion phenomena during the acrosome reaction and gamete union. Studies employing the fluorescence properties of probes such as ANS (8?anilinonaphthalene?1?sulfonic acid), dansylcadaverine, and terbium ions (Tb³?) reveal striking differences in surface charge characteristics and Ca²??binding sites between the two membrane systems. The isolated plasma membrane fraction may have been derived only from a specific region of the cell and therefore may not represent the entire cell boundary; this is important in view of the diverse morphology and function of the head and tail regions of the male gamete. The next chapter deals with the regionally differentiated state of mammalian spermatozoan surface membranes.