Both Testosterone and Follicle-Stimulating Hormone Independently Inhibit Spermatogonial Differentiation in Irradiated Rats

0013-7227/06/$15.00/0 Printed in U.S.A. Endocrinology 147(1):472– 482 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2005-0984 Both Testosterone and Follicle-Stimulating Hormone Independently Inhibit Spermatogonial Differentiation in Irradiated Rats Gunapala Shetty, Connie C. Y. Weng, Sarah J. Meachem, Olga U. Bolden-Tiller, Zhen Zhang, Pirjo Pakarinen, Ilpo Huhtaniemi, and Marvin L. Meistrich Department of Experimental Radiation Oncology (G.S., C.C.Y.W., O.U.B.-T., Z.Z., M.L.M.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Prince Henry’s Institute of Medical Research (S.J.M.), Clayton, Victoria 3168, Australia; Department of Physiology (P.P., I.H.), University of Turku, 20520 Turku, Finland; and Institute of Reproductive and Developmental Biology (I.H.), Imperial College London, London W12 ONN, United Kingdom itary hormones. Furthermore, in this protocol we showed that TDI in the hypophysectomized testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRHantagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 d in untreated, GnRH-ant plus flutamide-treated, or GnRH-ant plus testosterone-treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus testosterone-treated rats. We conclude that both testosterone and FSH individually inhibit spermatogonial differentiation after irradiation, but testosterone is a more highly potent inhibitor than is FSH. (Endocrinology 147: 472– 482, 2006) Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in F1 hybrids of Lewis and Brown-Norway rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for 2 wk; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold, indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 wk; testosterone also reduced the TDI, demonstrating that testosterone had a definite inhibitory effect, independent of pitu- R ADIATION AND CANCER chemotherapy deplete germ cells, causing prolonged azoospermia in rodents (1–3), monkeys (4), and humans (5). In the rat these agents cause azoospermia by disrupting the differentiation of surviving type A spermatogonia (6). In humans there is some histological evidence that radiation and cancer chemotherapeutic drugs can also block spermatogonial differentiation (5, 7), and the recovery of spermatogenesis after prolonged azoospermia also shows that surviving stem cells can be blocked from completing differentiation (8). However, in rats exposed to moderate doses of radiation or procarbazine, spermatogonial differentiation and progression of spermatogenesis can be restored by suppression of gonadotropins and intratesticular testosterone (ITT) using GnRH analogs (9, 10). Because both ITT and FSH are elevated after irradiation (9, 11), both might have inhibitory effects. We initially showed that administration of exogenous testosterone (T) strongly inhibited the GnRH-analog-induced stimulation of spermatogonial recovery in a dose-dependent manner and that the effect of T was almost completely reversed if the androgen receptor antagonist flutamide was given (11). That and subsequent studies (6, 11, 12) showed excellent inverse correlations between the ITT levels and spermatogenic recovery. However, when irradiated rats treated with GnRH antagonist (GnRH-ant) were also treated with T, FSH levels were elevated in a dose-responsive manner by the action of T on the pituitary, and when these rats were also given flutamide, there was some reduction in FSH (6, 11). Thus, there also was a good inverse correlation between FSH levels and recovery of spermatogenesis. When we examined the effects of four different androgens, we found that they all suppressed spermatogenic recovery (13), but we could not determine whether suppression was a direct effect of androgen at the testicular level or a result of the elevation of FSH levels that they produced. Overall we found a better inverse correlation of spermatogenic recovery with ITT levels than FSH levels, suggesting a more dominant role of androgens than FSH in the inhibitory process. However, when the combined effects of ITT and FSH were considered, the correlations were further improved (11, 12). Because of the parallel effects of the treatments used on ITT and FSH, none of the previous stud- First Published Online October 6, 2005 Abbreviations: FSHAb, Antiserum raised against FSH; GnRH-ant, GnRH antagonist; h, human; ITT, intratesticular testosterone; LBNF1, F1 hybrids of Lewis and Brown-Norway; rh, recombinant human; T, testosterone; TDI, tubule differentiation index. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. 472 Shetty et al. • T and FSH Inhibition of Spermatogonial Differentiation ies could prove that either ITT or FSH was individually inhibitory to spermatogonial differentiation. To show that both T and FSH have unequivocal direct effects on the inhibition of spermatogonial differentiation and to determine the role of FSH relative to T in inhibiting this process, we modulated FSH and, in some cases, T levels by three different approaches and assessed spermatogenic recovery. We first tested the effect of exogenous FSH treatment in GnRH-ant-treated, irradiated rats. Second, we compared GnRH-ant plus T treatment, which incompletely suppresses FSH, with that of hypophysectomy plus T, which produces complete FSH suppression. Third, we used an FSH antibody to reduce active serum FSH levels. Materials and Methods Materials T and dextran-coated charcoal were obtained from Sigma (St. Louis, MO). Flutamide pellets were obtained from Innovative Research of America (Sarasota, FL). SILASTIC brand tubing (catalog no. 602-305) was purchased from Dow Corning (Midland, MI). Alzet miniosmotic pumps (model 2001) were obtained from Alza Corp. (now Diuret) (Palo Alto, CA). Two GnRH-ants were used in this study: cetror (...truncated)