Sperm must be motile following their deposition in the female reproductive tract to fertilize ovulated eggs. Factors that regulate the power stroke and waveform of the sperm flagellum are poorly understood. Although several components of mammalian sperm flagella have been characterized, relatively little is known about proteins comprising the central apparatus in the axoneme core, which are thought to play a major role in controlling flagellar function. The long term goal of this research project is to characterize the regulatory proteins of the mammalian sperm axoneme and to identify their roles in governing flagellar activity.
Four specific aims are proposed: 1) To determine the roles of the mammalian homologues of the Chlamydomonas proteins encoded by the PF16 and PF2O in sperm flagellar activity; 2) To generate targeted deletion mutants in mice of the PF16 gene, and later the PF2O gene, to assess their role in flagellogenesis and sperm motility; 3) To characterize the temporal and spatial patterns of expression of PF16, PF2O as well as other proteins during assembly of the sperm axoneme; and 4) To identify interactions among these axonemal proteins using genetic, biochemical and ultrastructural methods. We will examine the ability of antibodies and recombinant proteins to disrupt the function of PF16 and PF2O in reactivated demembranated or permeabilized sperm flagella. The overall hypothesis to be tested is that central apparatus proteins control the microtubule motors through an ordered sequence of protein-protein interactions. The function of PF16 will be probed by targeted deletion of the murine PF16 gene. The anticipated phenotype of the knockout is male infertility due to paralyzed or dysfunctional sperm flagellar activity. The murine model will also allow us to examine the role of PF16 in the maintenance of the structural integrity of the central apparatus. To test the hypothesis that the central apparatus is assembled prior to the formation of other flagellar components including the fibrous sheath, we will define the temporal and spatial patterns of expression of PF16 and PF20 relative to the fibrous sheath protein, AKAP82. Finally, in an effort to gain a more complete understanding-of the proteins comprising the mammalian axoneme, we will identify interacting partners of PF16 and PF2O using the yeast two-hybrid system and alternative biochemical methods. Knowledge gained from these experiments will provide a molecular framework for understanding some sperm motility defects that cause male infertility and possibly offer new avenue for contraception through the disruption of purposeful sperm motion.
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