[
International Worm Meeting,
2007]
The mechanism(s) by which motile sperm find oocytes in the female reproductive tract is not understood. To investigate this mechanism, we have developed an in vivo assay for tracking the movement of sperm in the uterus of C. elegans hermaphrodites. Previous results support the hypothesis that oocytes generate sperm-recruiting signals derived from polyunsaturated fatty acids (PUFAs). These signals direct sperm migration to the spermatheca, the site of fertilization. In mammals, PUFAs are synthesized into eicosanoids, which direct T-cell migration to sites of inflammation. I am using RNA-mediated interference (RNAi) to identify genes that function in oocytes to control directional sperm motility. Genome-wide DNA microarray (Reinke et al. 2004) and in situ hybridization (Kohara 2001) databases were screened for genes expressed in oocytes. I have identified eight genes that are required in hermaphrodites for the directional movement of wild-type sperm. Several of these genes are related to human genes implicated in eicosanoid synthesis and transport. I am currently focusing on the genes T28F3.1 and C01F6.1, which encode proteins called copines. Copines are conserved calcium-dependent membrane binding proteins with unknown functions. They are characterized by having two Ca(2+)-binding C2-domains at the N-terminal region and an A-domain at the C-terminal region. We hypothesize that C01F6.1 and T28F3.1 function in oocytes to target PUFA-modifying enzymes to the plasma membrane where they can generate sperm-recruiting signals. I am currently conducting phenotypic analysis of loss of function mutations in C01F6.1 and T28F3.1. Results from this analysis and site of action studies will be presented.
[
Development & Evolution Meeting,
2008]
The eicosanoids, which include prostaglandins, leukotrienes and thromboxanes, are signaling molecules derived from polyunsaturated fatty acids or PUFAs. In mammals, eicosanoids are important mediators of inflammation, immunity, reproduction and central nervous system function. However, the roles of eicosanoids at the cellular level and the molecular mechanisms that control these actions are not well understood. We have shown that C. elegans oocytes generate sperm-attracting signals derived from PUFAs. I conducted an RNAi screen to identify genes that function outside of sperm to regulate their motility. These genes are candidates for mediating the synthesis or secretion of the sperm attractant. I screened approximately forty genes with oocyte-enriched transcription profiles and predicted functions in lipid metabolism and transport. The screen resulted in the identification of five genes encoding proteins implicated in prostaglandin metabolism. I am focusing on the glutathione S-transferase K08F4.7, which has significant sequence similarity to human prostaglandin D synthase. Genetic analysis using a method to track sperm motility in vivo confirms that K08F4.7 is required for sperm guidance. Wild-type sperm in K08F4.7 null mutants move with reduced velocity and severely reduced directional velocity. These results support the hypothesis that prostaglandin D2 or a structurally similar prostaglandin functions as a sperm attractant. Consistent with this hypothesis, the microinjection of human prostaglandin D2 into the uterus of PUFA-deficient hermaphrodites stimulates sperm motility. Currently, we are collaborating with the UAB mass spectrometry facility to directly test whether oocytes synthesize prostaglandins. I am also testing the site of K08F4.7 action and whether recombinant K08F4.7 can function as a prostaglandin D synthase in vitro. In addition to K08F4.7, I am characterizing two other genes identified in the RNAi screen, the copines C01F6.1 and T28F3.1. Copines are evolutionarily conserved proteins with predicted lipid binding domains, but largely unknown functions. Loss of function mutations in C01F6.1 and T28F3.1 do not cause sperm motility defects as severe as those observed with RNAi. The C. elegans genome encodes 5 copine family members and I am currently testing whether these genes exhibit functional redundancy. Triple- and quadruple-mutant strains are being constructed for further phenotypic analysis.