Testicular Heat Exposure Enhances the Suppression of Spermatogenesis by Testosterone in Rats: The “Two-Hit” Approach to Male Contraceptive Development

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society Vol. 141, No. 4 Printed in U.S.A. Testicular Heat Exposure Enhances the Suppression of Spermatogenesis by Testosterone in Rats: The “Two-Hit” Approach to Male Contraceptive Development* YANHE LUE, AMIYA P. SINHA HIKIM, CHRISTINA WANG, MICHAEL IM, ANDREW LEUNG, AND RONALD S. SWERDLOFF Division of Endocrinology, Department of Medicine, Harbor-University of California-Los Angeles Medical Center and Research and Education Institute, Torrance, California 90509 ABSTRACT The objectives of the study were to determine stage-specific changes in the kinetics of germ cell apoptosis induced by administration of exogenous testosterone (T) alone and to examine whether addition of a single testicular heat exposure would enhance the induction of germ cell apoptosis and the suppression of spermatogenesis by T. Adult male rats were implanted with 3-cm SILASTIC brand capsules (Dow Corning Corp.) containing T for up to 6 weeks. Intratesticular T levels declined to 2.9% of control values by 1 week and remained suppressed at 2, 3, and 6 weeks after T administration. The incidence of germ cell apoptosis (expressed as numbers per 100 Sertoli cells) was low in control rats (0 –9.52). After T treatment, the mean incidence of apoptosis at stages VII–VIII increased significantly by 1 week (21.43 ⫾ 3.33) and showed further increases by 6 weeks (56.30 ⫾ 7.47); apoptotic rates remained low at early (I–VI) and later (XII–XIV) stages. To test whether the combination of T with a single testicular heat exposure resulted in more complete suppression of spermatogenesis than either treatment alone, four groups of adult rats received one of the following treatments: 1) a subdermal empty polydimethylsilozane implant, 2) exposure to a single testicular heating (43 C for 15 min) applied on day 14, 3) 3-cm T implant, or 4) 3-cm T implant and a single testicular heat exposure (applied on day 14). All animals were killed at the end of 6 weeks. In the heat-treated group, testis weight and testicular sperm counts were decreased to 65.4% and 28.9% of control W E HAVE PREVIOUSLY demonstrated in the rat that stage-specific loss of germ cells occurred exclusively by apoptosis after acute withdrawal of gonadotropins and intratesticular T by GnRH antagonist treatment. The hormone-dependent stages VII–VIII were the first to show enhanced germ cell apoptosis, occurring 5–7 days after GnRH antagonist-induced suppression of gonadotropins and the resultant decrease in intratesticular T (1, 2). Preleptotene (PL) and pachytene (P) spermatocytes as well step 7 and step 19 spermatids were most susceptible to the lack of hormonal stimulation. Although presumed to be similar to that of GnRH antagonist (suppression of serum LH or FSH), the mode of cell death during spermatogenic suppression by Received October 25, 1999. Address all correspondence and requests for reprints to: Ronald S. Swerdloff, M.D., Division of Endocrinology and Metabolism, HarborUniversity of California Los Angeles Medical Center, Box 446, 1000 West Carson Street, Torrance, California 90509. E-mail: swerdloff@gcrc. humc.edu. * Presented in part at the 81st Annual Meeting of The Endocrine Society, San Diego, California, 1999. This work was supported by grants from the Mellon Foundation Reproductive Biology Center. levels, respectively. The corresponding values in the T-treated group were 49.7% and 24.9% of control levels, respectively. Notably, addition of heat to T further reduced testis weight to 31.1% of control levels and testicular sperm counts to near zero. Histomorphometric analysis showed that all treatments reduced seminiferous tubular diameter and epithelial and luminal volume, with the greatest decrease after combined T and heat treatment. Heat exposure in animals bearing T implants markedly reduced the number of pachytene spermatocytes and round spermatids through apoptosis, resulting in tubules devoid of mature spermatids. Spermatogonia and preleptotene spermatocytes remained unaffected. These results clearly demonstrate that 1) exogenous T reduces intratesticular T and induces apoptosis mainly at stages VII–VIII within 1– 6 weeks; 2) the combined treatment of T and heat markedly inhibits spermatogenesis, resulting in near azoospermia within 6 weeks; and 3) meiosis and spermiogenesis are the most vulnerable phases of spermatogenesis in response to T plus heat treatment. These findings suggest that a combination of hormonal treatment such as T and a physical agent (heat exposure) is more effective in suppressing spermatogenesis than either treatment alone. We hypothesize that combination of two antispermatogenic agents (“two hit”) working at separate stages of the spermatogenic cycle will lead to greater male contraceptive efficacy. (Endocrinology 141: 1414 –1424, 2000) exogenous administration of testosterone (T) alone has not yet been characterized. In additional studies (3), we further confirmed and extended earlier studies (3a) by demonstrating that a single exposure (43 C for 15 min) of the rat testis to heat resulted in selective, but reversible, damage to the seminiferous epithelium through increased germ cell apoptosis. Heat-induced germ cell apoptosis predominantly occurred at early (I–IV) and late (XII–XIV) stages. Spermatocytes, including P at stages I–IV and XII, diplotene and dividing spermatocytes at stages XIII–XIV, and early (steps 1– 4) spermatids at stages I–IV, were most susceptible to heat. Stages V–VI and VII–VIII were relatively protected from heat-induced apoptosis. We have also provided evidence indicating that mild testicular hyperthermia is able to increase germ cell apoptosis at stages VII–VIII only when intratesticular T levels were decreased by the prior treatment with GnRH antagonist. We conclude from these studies that intratesticular T plays a pivotal role in protecting germ cells at stages VII–VIII against heat-induced cell death (3). In clinical studies to develop male hormonal methods of contraception, T administration resulted in reversible sup- 1414 COMBINATION OF TESTOSTERONE WITH HEAT pression of spermatogenesis. Although quite effective, the suppression was not uniform, and azoospermia was achieved in 60 –90% of men (4 – 6) only when serum T was elevated to the upper normal range. Moreover, the time required to achieve azoospermia or severe oligozoospermia usually takes more than 12 weeks. Although there are no data to support such a contention; concern has been expressed that high doses of T may have untoward effects on prostate. To develop new regimens that could rapidly induce development of azoospermia in all men with a lower dose of T, clinical studies were designed to combine T with progestins or GnRH analogs (7–9). Some of these combined regimens achieved azoospermia in over 90% of men in 8 –12 weeks. Thus, even the combined T and progestogen regimens leave room for improvement to create a faster and more complete mal (...truncated)