REGULATION OF SPERM MOTILITY BY CALCIUM(II) AND CALMODULIN

Abstract

A calcium and calmodulin regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both bovine and rat sperm flagella. The enzyme was identified by both enzymatic assay and immunoblot analysis. The calcium activated enzyme is inhibited by trifluoperazine (ID{dollar}\sb {lcub}50{rcub}{dollar} = {dollar}10\mu{dollar}M) and EGTA. The basal activity of the enzyme (as measured in the presence of EGTA), is elevated to that of the fully stimulated enzyme (as measured in the presence of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/calmodulin) by limited proteolysis. Immunoblot analysis using anti-phosphodiesterase antibodies demonstrate that bovine sperm flagella contain an immunoreactive polypeptide with subunit molecular weight of 60 kD. {dollar}\sp{lcub}125{rcub}{dollar}I-calmodulin binding to purified rat sperm flagella has been characterized and the flagellar associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical crosslinking experiments using dimethylsuperimidate.;A major 67 kD calmodulin binding protein was identifed by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca{dollar}\sp{lcub}2+{rcub}{dollar} and was totally inhibited by EGTA or excess cold calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of {dollar}\sp{lcub}125{rcub}{dollar}I-calmodulin by 50% was between 10{dollar}\sp{lcub}-10{rcub}{dollar} and 10{dollar}\sp{lcub}-11{rcub}{dollar} M. Limited proteolysis resulted in the total loss of all Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent binding of {dollar}\sp{lcub}125{rcub}{dollar}I-calmodulin to the 67 kD flagellar. Chemical crosslinking studies revealed the presence of a {dollar}\sp{lcub}125{rcub}{dollar}I-calmodulin-polypeptide complex in the 80,000 to 90,000 molecular weight range. The major 67 kD dalton calmodulin binding protein is not the Ca{dollar}\sp{lcub}2+{rcub}{dollar} and calmodulin regulated phosphodiesterase since the anti-phosphodiesterase antibody cross-reacts with a 60 kD polypeptide.;Previous studies in this laboratory have shown that a type II cAMP-dependent protein kinase is an integral component of rat and bovine sperm flagella (Horowitz et al., submitted). Since cAMP is known to be involved in the regulation of sperm motility, we have also investigated the flagellar localization of the regulatory subunit of the flagellar cAMP-dependent protein kinase (RII). Immunogold localization and biochemical analysis suggested that RII was associated with the mitochondria. The finding is important in light of the reported stimulatory effects of cAMP on both the motility and respiration of mammalian sperm. (Abstract shortened with permission of author.).