The testis contain a blood-testis barrier that restricts the movement of many dyes, steroids and peptides into and out of the seminiferous tubules. Several neuropeptides that were originally considered to be restricted to the hypothalamus or the pituitary gland have been discovered in testis and may serve as autocrine or paracrine regulators of testicular function. Growth hormone releasing hormone (GHRH), like other hypothalamic neuropeptides, has been found in extrahypothalamic tissues. We speculated that GHRH might also be produced in rat testis. In a series of preliminary experiments. we looked for the presence of GHRH in rat testis by two distinct, complementary methods: with an enzyme-linked immunosorbent assay (ELISA), we measured GHRH-like mRNA species detected in testis was of significantly larger size that mRNA that codes for a peptide with structural similarity to hypothalamic GHRH and that it is produced in testicular cells, most likely the Leydig cell, and functions as an autocrine and/or paracrine regulator of testicular activity. In the current application, we will examine this hypothesis by characterization of the rat testicular GHRH-like peptide by Western gel analysis, HPLC and immunoaffinity column chromatography. Characterization of the GHRH-like mRNA sequence(s) will ge accomplished by isolation and sequencing of rat testicular GHRH-like mRNA and in vitro translation and immunoprecipitation of GHRH-like precursor peptides. We will localize the anatomic distribution of both the peptide and its mRNA within the rat tests by immunohistochemistry and in situ hybridization techniques. Physiologic studies to define a physiologic function for this GHRH-like substance in rat testis will include in vivo and in vitro studies of the role of testicular GHRH in testicular function (spermatogenesis, testicular development and testosterone levels), the mechanism of GHRH action in rat testis, and identification of testicular GHRH receptors. In summary, the experiments proposed in the current grant application will result in detailed characterization of a novel neuropeptide which may be important in modulation of testicular function.
