The overall goal of this proposed project is to define how the CREB transcription factor relays hormonal and environmental signals in the mammalian testis to genes within Sertoli and germ cells during spermatogenesis. The specific hypothesis to be tested is that alterations in the relative concentrations of activator and repressor CREB isoforms in the seminiferous tubules regulate the expression of cAMP-inducible genes required for the expansion and differentiation of male germ cells. One objective is to define the mechanisms responsible for the stage-specific expression of various CREB isoforms during the maturation of germs cells. The role of NF-kB and Spl activator proteins as well as CREB and CREM repressor isoforms in regulating CREB gene transcription will be investigated. Transient transfection studies will be performed using primary rat Sertoli cells to determine if the proposed regulator proteins modulate CREB promoter activity through previously identified binding motifs. The cell types and spermatogenic stages in which the regulatory proteins act will be determined using in situ histochemical and immunocytochemistry protocols. Also nuclear extracts from isolated seminiferous tubule segments representing various spermatogenic stages will be used to quantitate regulator protein levels using western immunoblot and DNA-protein binding studies. Protein-protein interactions involving CREB will be studied using coimmunoprecipitation and protein binding to column immobilized CREB-GST fusion proteins. A second objective of this study is to identify """"""""target"""""""" genes expressed in Sertoli cells that are regulated by the cAMP signaling pathway and the CREB transcription factor. A focused differential display protocol employing primary Sertoli cells infected with adenovirus vectors expressing a dominant negative CREB mutant will be used to identify and clone genes that are up-regulated through CREB after FSH stimulation. In summary, this study seeks to characterize the factors that modulate expression of the CREB gene in the testis and identify genes critical for progression of spermatogenesis that are regulated by cAMP and the CREB family of transcription factors. The results of this work may then lead to infor-mation needed to provide therapies for infertility and solutions for male contraception.
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