Pem Homeobox Gene Function During Spermatogenesis

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DESCRIPTION (provided by applicant): Knowledge of molecular mechanisms determining male fertility is lacking. As a result, development of male nonsteroidal contraceptives and treatments for infertility has been limited. Communication between Sertoli cells of the testis and germ cells is essential for spermatogenesis. Testosterone regulates spermatogenesis through binding Sertoli cell androgen receptors and resultant gene transcription. The only testosterone-regulated, Sertoli cell-specific gene known to date is the Pem homeobox gene. Appropriate regulation and expression of transcription factors such as homeobox genes are required for normal developmental events. Pem expression, therefore, may serve as a regulator of spermatogenesis. We were the first to show that Sertoli cells express Pem specifically during the testosterone-dependant seminiferous tubule stages VI-VIII. Postnatally, Pem is first expressed just prior to the first germ cell meiosis concurrent with the induction of androgen receptor in Sertoli cells. In hormone-depleted mice, our work demonstrated that testosterone alone is sufficient to induce Peru expression. Currently, the function of this tightly controlled expression of Pem is unknown. The study of Peru gene function is important because Peru may be a transcription factor that regulates genes involved in androgen responsiveness and apoptosis. The primary goal of this project is to determine the function of Peru expression. The hypothesis tested is that Pem expression regulates critical Sertoli cell to germ cell communication. The first aim to test this hypothesis will be to determine genes that are upregulated or downregulated by Pem expression in Sertoli cells. This aim will be achieved by screening a mouse microarray with cDNA from Sertoli cells transfected with a Pem expression vector or with cDNA from Sertoli cells transfected with an empty vector. Unrepresented genes or novel Pem-regulated genes will be isolated by a PCR-based subtraction hybridization method the PI has used previously. The second aim will determine the recognition element to which Pem binds. For this aim, isotope-labeled random oligonucleotides will be mixed with Pem protein alone, Pem plus Sertoli cell nuclear extract or control, immunoprecipitated, PCR amplified, confirmed by mobility shift assay, cloned and sequenced. This proposal is a focused plan that will lead to the development of molecular targets to further the understanding of spermatogenesis and male fertility.