The major goal of this study is to elucidate the molecular mechanisms regulating gene expression during spermatogenesis in the mouse testis. The basic hypothesis to be investigated is that the precision and regularity of this program of germ cell proliferation and differentiation is regulated primarily at the level of gene transcription. The experimental strategies proposed include mRNA differential display assays and PCR- select cDNA subtractions to identify and isolate gene transcripts differentially expressed in early germ cell populations. The stage and cell-type specific patterns of expression of these low abundant gene products will be determined using PCR-based """"""""virtual"""""""" Northern blots assays. Rapid isolation of promoter regions of selected stage-specific gene products will be facilitated by using ligation-mediated single-sided- PCR genomic strategies. In vivo DNase 1 footprinting studies will be utilized to identify the cis-acting promoter elements that direct temporal gene transcription in germ cells. Electrophoretic mobility gel shift assays (EMSA's), Southwestern analysis and protein microsequencing will be used to characterize transcription factors that interact with these cis- regulatory elements. The experimental strategies proposed will result in the identification of a number of germ-cell-specific gene products that display spatial and temporal expression patterns during mouse spermatogenesis. Furthermore, a detailed analysis of the regulatory promoter elements of the genes encoding these proteins will facilitate elucidation of the molecular mechanisms that determine their restricted cellular transcription patterns during germ cell differentiation. Elucidation of the molecular mechanisms regulation mouse spermatogenesis will make significant contributions towards the development of therapeutic interventions for cellular pathologies restricted to the male reproductive tract.
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