The long term objectives of this proposed work are investigations of the biological role of testis-specific histones and regulation of their genes during spermatogenesis. In addition, the role of estrogen in spermatogenesis Will be investigated. We will investigate presence of a domain containing testis-specific genes around the rat testis-specific TH2A and TH2B genes, presence of testis-specific dominant control region near the domain, definition of the domain by nuclear matrix binding sites, and regulation of testis-specific histone genes by the testis-specific enhancer activity of the dominant control region. TH2B gene is poorly transcribed in spermatogonia and this could be due to the spermatogonia-specific protein which binds to a site between TATAA box and the TH2B transcription initiation site. This will be investigated with partially purified protein in an in vitro transcription system. The possible role of TH1 and TH2B histones in the formation of more relaxed chromatin structure in pachytene spermatocytes will be investigated by producing the histones in yeast mutant strains lacking either Hl or H2B histone genes. The regions of the histone molecule responsible for the changes in chromatin structure will be investigated by site-directed mutagenesis. Also, the possible effect of TH1 and TH2B on the rate of meiosis in yeast will be investigated. Estrogen is produced from testosterone in Sertoli cells, and the female sex hormone may have a direct role in spermatogenesis, possibly negative regulation. Primary spermatocytes contain estrogen receptors, and this implies a direct role of estrogen in spermatogenesis. We win investigate the stages of spermatogenesis affected by estrogen in differentiating primary cultures of Sertoli-spermatogenic co-cultures and the proteins regulated by estrogen. We will clone the genes regulated by estrogen and the amino acid sequence of the gene products will be determined. The cells expressing the genes and the gene products will be investigated by in situ cytohybridization and immunocytochemistry.
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