REGULATION AND FUNCTION OF SPARC IN MALE REPRODUCTION

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Spermatogenesis in the mammalian testis is regulated in part by a complex set of interactions among the nongerminal Sertoli, Leydig, and peritubular cells that create a unique environment for the maturation of spermatozoa. A calcium-binding protein, SPARC (Secreted Protein, Acidic and Rich in Cysteine) has been identified in the cytoplasm of a population of murine Sertoli cells bearing Late-stage spermatids. Both SPARC mRNA and protein have been localized to murine interstitial Leydig cells. In addition, several monoclonal antibodies that reacted with mouse sperm surface antigens and inhibited in vitro fertilization were shown to cross-react with SPARC. SPARC, initially described as a differentiation- associated product of murine extraembryonic endoderm and teratocarcinoma cells, has been molecularly cloned. The complete cDNA sequence revealed a cysteine-rich "finger" sequence partially homologous to the protease inhibitor ovomucoid, and a Ca+2 binding "EF Hand" similar to those present in calmodulin and parvalbumin. Our observations suggest SPARC plays a role in male reproduction, and we propose to study, in the murine testis, the function and regulation of this well-characterized molecule. Accordingly, the distribution of SPARC mRNA and protein in embryonic, prepubertal and adult testes, and mature sperm will be determined by immunocytochemistry and in situ nucleic acid hybridization at light and electron microscopic levels. Binding, uptake and processing of radiolabeled SPARC by testis tissue will be monitored in vivo, and putative SPARC-binding receptors will be isolated from testis tissue and fluids. We will determine if SPARC can bind to cultured Leydig and Sertoli cells to induce changes in cell shape and protein biosynthesis. We will study hormonal regulation of the Sparc gene in Leydig cell cultures exposed to hCG, and interaction of transcription factors with domains of the SPARC promoter. In cultured Leydig cells, the ability of exogeneous SPARC to alter steroid production in response to hCG will be examined. Finally, we will study the fate of sperm-associated SPARC during capacitation in vitro and effects of added SPARC and anti- SPARC antibodies on mouse in vitro fertilization. These experiments focus on a well-characterized molecule whose function and genetic regulation will be examined during testicular development and spermatogenesis.