Testicular heat exposure enhances the suppression of spermatogenesis by testosterone in rats: The "two-hit" approach to male contraceptive development

The objectives of the study were to determine stage-specific changes in the kinetics of germ cell apoptosis induced by administration of exogenous tes tosterone (T) alone and to examine whether addition of a single testicular heat exposure would enhance the induction of germ cell apoptosis and the su ppression of spermatogenesis by T. Adult male rats were implanted with 3-cm SILASTIC brand capsules (Dow Coming 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 incide nce 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 apo ptosis 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 t he 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 subderm al empty polydimethylsilozane implant, 2) exposure to a single testicular h eating (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 ani mals were killed at the end of 6 weeks. In the heat-treated group, testis w eight and testicular sperm counts were decreased to 65.4% and 28.9% of cont rol levels, respectively. The corresponding values in the T-treated group w ere 49.7% and 24.9% of control levels, respectively. Notably, addition of h eat to T further reduced testis weight to 31.1% of control levels and testi cular sperm counts to near zero. Histomorphometric analysis showed that all treatments reduced seminiferous tubular diameter and epithelial and lumina l volume, with the greatest decrease after combined T and heat treatment. H eat exposure in animals bearing T implants markedly reduced the number of p achytene spermatocytes and round spermatids through apoptosis, resulting in tubules devoid of mature spermatids. Spermatogonia and preleptotene sperma tocytes remained unaffected. These results clearly demonstrate that II exog enous T reduces intratesticular T and induces apoptosis mainly at stages VI I-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 spermatoge nesis in response to T plus heat treatment. These findings suggest that a c ombination of hormonal treatment such as T and a physical agent (heat expos ure) is more effective in suppressing spermatogenesis than either treatment alone. We hypothesize that combination of two antispermatogenic agents ("t wo hit") working at separate stages of the spermatogenic cycle will lead to greater male contraceptive efficacy.