GERM CELL LOCALIZATION OF TESTICULAR GROWTH HORMONE RELEASING HORMONE (GHRH)

Hypothalamic releasing hormones have been found outside of the CNS, however, their extrahypothalamic function remains largely unknown. We have identified a testicular GHRH-like peptide and mRNA in human and rat testis. Human testis, like hypothalamus and placenta, contains an abundant 750bp GHRH mRNA. Rat testis has been shown to produce a larger mRNA of 1750bp. This study was designed to localize GHRH production in testis. Rat testes were fractionated following collagenase and trypsin digestion. Germ cells were further separated by sedimentation, using a staput device. Total RNA was extracted from each cell population by the guanidinium thiocyanate method. Northern analysis, using a 32P-labeled riboprobe complementary to a GHRH cDNA (gift of R. Evans), revealed an abundant 1750bp transcript in spermatocytes, round spermatids and, to a lesser extent, in Sertoli cells. No transcript was detected in RNA from elongating spermatids, Leydig cells, or epididymis. Further localization by in sint hybridization using this probe, revealed prominent labeling of early spermatogenic cells. Because human germ cell cancers are the result of dysregulated growth of undifferentiated spermatogenic cells, we probed the Tera-2 and NT2D1 human germ cell cancer lines with the GHRH riboprobe. Like hypothalamus and human testis, a 750bp transcript was clearly delectable in both cell lines. To determine if GHRH might play a role as an autocrine mitogenic factor in these cancers, we evaluated thymidine incorporation into Tera-2 celis following treatment with GHRH (100nM). There was a 52% increase in thymidine incorporation suggesting that GHRH can induce DNA synthesis in these cells. We conclude that GHRH is produced in normal and cancerous germ cells and speculate that it may function as a local testicular growth factor.