GERM-CELL LOCALIZATION OF A TESTICULAR GROWTH HORMONE-RELEASING HORMONE-LIKE FACTOR

GH-releasing hormone (GHRH)-like mRNA and immunoreactivity (t-GHRH-li) are present in the testes of rats and humans. To learn more about the physiology of t-GHRH-li mRNA, we performed a series of experiments th at disrupted various testicular functions. We employed ethylene dimeth anesulfonate, a Leydig cell toxin, to assess the effects of Leydig cel l ablation on t-GHRH-li mRNA and protein levels in prepubertal and pos tpubertal male rats. The ethylene dimethane-sulfonate treatment result ed in decreases in serum testosterone, but had no effect on t-GHRH-li mRNA or peptide levels. To assess the effect of GHRH on Leydig cell st eroidogenesis, Leydig cells were isolated by Percoll gradient centrifu gation and cultured in the presence or absence of hCG, GHRH, or hCG pl us GHRH. GHRH had no effect on steroidogenesis by Leydig cells, either alone or in combination with hCG. To localize t-GHRH-li mRNA within r at testis, in situ hybridization analysis was performed on testicular sections from normal rats, using a [S-35]GHRH riboprobe. Grains were d etected in spermatogenic cells with the antisense probe, whereas none was detected with the sense strand (control) probe. To verify these re sults, Northern blot analysis of RNA from separated testicular cells w as performed. t-GHRH-li mRNA was detected in spermatocytes and round s permatids and to a lesser extent in Sertoli cells, but not in elongati ng spermatids, Leydig cells, or peritubular myoid cells. t-GHRH-li mRN A was also not found in epididymis. Since our experiments localized t- GHRH-li mRNA to spermatogenic cells, methoxyacetic acid (MAA), a pachy tene spermatocyte toxin, was administered to postpubertal rats to dete rmine whether t-GHRH-li is expressed primarily in pachytene spermatocy tes. MAA treatment caused a decrease in testicular weight, which gradu ally returned to control levels by 42 days. Serum FSH levels in the tr eated animals fluctuated over the course of the experiment, while thos e in control animals remained steady. However, there was no difference in testicular GHRH-li mRNA levels between control and treated animals at any treatment time. Insulin-like growth factor-I and -II mRNA leve ls were also unaltered by the MAA treatment. We conclude from these re sults that t-GHRH-li is synthesized in early spermatogenic cells, but not in mature sperm, and that testicular GHRH-li does not play a major role in steroidogenesis by the Leydig cell.