The long term goals are to elucidate the cAMP-dependent pathway that leads to motility during maturation of mammalian sperm, and to understand the mechanism by which Ca2+ induces the hyperactivated waveform that is necessary for fertilization in vivo. Previous studies have shown that ram sperm maturation involves a cAMP- dependent phosphorylation of proteins that is essential for motility, and that the phosphates on these proteins turn over, albeit slowly, in mature sperm. These phosphorylations are mediated by cAMP-dependent protein kinase (PKA). The catalytic subunit (C) of ram sperm PKA has unique properties similar to those reported for C-gamma, a testis specific isoform previously known only from molecular cloning of human cDNA and expression in transfected cells. Studies will be carried out to determine if the unusual sperm isoform of C is indeed C-gamma (or, less likely, a totally novel isoform of C). Sperm C will be characterized with regard to its primary structure, its possible occurrence in cilia, and its cAMP-dependent anchorage to the sperm tail. Differences in substrate preferences between sperm C and somatic Cs will be exploited in an attempt to develop peptide substrates or pseudosubstrates that specifically inhibit sperm C activity. Ram sperm contain several proteins that are phosphorylated in a cAMP- dependent manner that correlates with the development of motility. One of these appears to be a dynein heavy chain. This phosphoprotein will be studied to determine if it is a component of the inner or outer dynein arms, and to learn how phosphorylation effects its ability to bind to microtubules and generate force in an in vitro motility assay. Sperm tails will be fractionated to determine with which flagellar structures the other phosphoproteins are associated. Complementary DNA (cDNA) clones encoding these proteins will be obtained and sequenced. The cloned proteins will be expressed in bacteria and tested to determine if they are selective substrates for sperm C. The sequence surrounding any site that is selectively phosphorylated will be determined and the information used to develop peptide inhibitors that are specific for sperm C. Studies will be carried out to determine if Ca2+ acts directly on the axoneme to induce hyperactivation, and if the action of Ca2+ is mediated by calmodulin or calmodulin-dependent protein phosphatase. The tail structures responsible for the flagellar curvature characteristic of the hyperactivated waveform will be identified, and the mechanism by which this curvature develops will be investigated. The identification of proteins and enzymes that are unique to sperm and essential for their motility will provide targets for the development of new and highly specific pharmacological methods for inhibiting sperm function in vivo, thereby preventing fertilization, and conversely, for correcting defects that result in abnormal sperm movement and infertility.
