Spermatogenesis is a unique system of cellular differentiation that has been well studied at the physiological and morphological level. Important physiological processes such as testicular temperature regulation have significant differential effects on developing germ cells. However, our knowledge of the basis for germ cell sensitivity to elevated temperatures is exceedingly shallow. The long term objectives of this research will provide an understanding of the molecular basis by which temperature regulates the process of spermatogenesis. Such understanding could prove invaluable in correctly formulating treatment and management of fertility problems associated with cryptorchism, varicocele and other complications involving or necessitating increased testicular temperatures. It would also provide the necessary information for developing inhibitors of this very specific mechanism, vital to germ cell development. The specific goal of this proposal is to definitively examine the role of a significant mechanism of germ cell transcriptional regulation. It involves the mechanism that lowers the temperature optimum for in vitro transcription by RNA polymerase II, from the 30oC of somatic and prepuberal tissue to 20.C for adult testis. To realize this goal the proposed experiments will: 1) Test the hypothesis that temperature sensitivity is a conserved feature of RNA polymerase II transcription in the scrotal testis of large mammals exhibiting temperature sensitive spermatogenesis. 2) Test the hypothesis that the temperature sensitive component of RNA polymerase II transcription could be a common component of cellular RNA polymerases I, II and III. 3) Test the hypothesis that the temperature sensitive component of male germ cell RNA polymerase II transcription is active in earlier cell types than spermatids. 4) Chromatographically resolve and identify the factor(s) responsible for the observed 20.C temperature optimum for adult testis RNA polymerase II transcription. 5) Determine at what step in transcription complex formation the temperature sensitive factor(s) interacts with the other components of transcription to optimally initiate transcription at 20oC. 6) Isolate and clone the cDNA of the factor(s) involved in lowering the temperature optimum for in vitro transcription from the typical 30.C of somatically derived tissue extracts to the 20oC of extracts derived from adult testes. The experimental methodologies that will be used to achieve these goals include: in vitro transcription, chromatographic fractionation of protein extracts, cell separation, PCR amplification, cloning of the involved factors from cell-type specific CDNA libraries, and Sanger sequencing.
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