Ronald S. Swerdloff

THE TESTOSTERONE TRIALS: THE DESIGN OF SEVEN COORDINATED TRIALS TO DETERMINE IF TESTOSTERONE TREATMENT BENEFITS ELDERLY MEN

Background The prevalence of low testosterone levels in men increases with age, as does the prevalence of decreased mobility, sexual function, self-perceived vitality, cognitive abilities, bone mineral density, and glucose tolerance, and of increased anemia and coronary artery disease. Similar changes occur in men who have low serum testosterone concentrations due to known pituitary or testicular disease, and testosterone treatment improves the abnormalities. Prior studies of the effect of testosterone treatment in elderly men, however, have produced equivocal results. Purpose To describe a coordinated set of clinical trials designed to avoid the pitfalls of prior studies and to determine definitively whether testosterone treatment of elderly men with low testosterone is efficacious in improving symptoms and objective measures of age-associated conditions. Methods We present the scientific and clinical rationale for the decisions made in the design of this set of trials. Results We designed The Testosterone Trials as a coordinated set of seven trials to determine if testosterone treatment of elderly men with low serum testosterone concentrations and symptoms and objective evidence of impaired mobility and/or diminished libido and/or reduced vitality would be efficacious in improving mobility (Physical Function Trial), sexual function (Sexual Function Trial), fatigue (Vitality Trial), cognitive function (Cognitive Function Trial), hemoglobin (Anemia Trial), bone density (Bone Trial), and coronary artery plaque volume (Cardiovascular Trial). The scientific advantages of this coordination were common eligibility criteria, common approaches to treatment and monitoring, and the ability to pool safety data. The logistical advantages were a single steering committee, data coordinating center and data and safety monitoring board, the same clinical trial sites, and the possibility of men participating in multiple trials. The major consideration in participant selection was setting the eligibility criterion for serum testosterone low enough to ensure that the men were unequivocally testosterone deficient, but not so low as to preclude sufficient enrollment or eventual generalizability of the results. The major considerations in choosing primary outcomes for each trial were identifying those of the highest clinical importance and identifying the minimum clinically important differences between treatment arms for sample size estimation. Potential limitations Setting the serum testosterone concentration sufficiently low to ensure that most men would be unequivocally testosterone deficient, as well as many other entry criteria, resulted in screening approximately 30 men in person to randomize one participant. Conclusion Designing The Testosterone Trials as a coordinated set of seven trials afforded many important scientific and logistical advantages but required an intensive recruitment and screening effort.

Testis—Endocrinology Overview RSS

The hypothalamic-pituitary-testis axis regulates androgen production and spermatogenesis. Neurons in the hypothalamus secrete gonadotropin-releasing hormone (GnRH), which stimulates gonadotropin secretion by the pituitary. Luteinizing hormone release leads to increased testosterone production in the Leydig cells. Follicle stimulating hormone leads to increased production of regulatory proteins by the Sertoli cells and stimulates spermatogenesis. GnRH release is mediated by a hypothalamic neuronal network involving kisspeptin. Steroidogenesis involves multiple enzymes that catalyze the conversion of cholesterol to testosterone. Testosterone has effects on multiple organ systems, and hypogonadism can have various effects, depending on the patients stage of development.

POTENTIAL MALE CONTRACEPTIVE TARGET: GERM CELL-SPECIFIC VASA PROTEIN LOCALIZATION IN MONKEY AND HUMAN TESTES.: 385

We previously demonstrated that pachytene and diplotene spermatocytes and round spermatids are the most susceptible cells to undergo apoptosis in response to either a withdrawal of intratesticular testosterone, increased testicular temperature, or a combination of both in rats and monkeys. Our study focuses on vasa (DDX4 or MvH), a specific meiotic and/or postmeiotic molecule essential for germ cell survival and development. Vasa is a member of the DEAD (Asp-Glu-Ala-Asp) box family of ATP-dependent RNA helicases and participates in RNA unwinding, translation initiation, and RNA turnover. Previously, the vasa gene was found to be conserved across species in Caenorhabditis elegans, Xenopus, zebra fish, mice, and rats. In mice, vasa protein has been localized in spermatocytes and round spermatids and vasa knockout mice were infertile due to an arrest of spermatogenesis at zygotene spermatocytes. The objective of this study is to compare vasa protein expression and localization in rat, monkey, and human testes. Testicular samples were obtained from groups of four adult rats, four adult monkeys, and four fertile men. Bouin9s fixed, paraffin-embedded testicular sections were used for vasa immunohistochemistry. Vasa proteins were predominantly localized in the cytoplasm of pachytene and diplotene spermatocytes and round spermatids across all three species. The vasa proteins were condensed in the chromatoid bodies of late spermatocytes and round spermatids. Our studies also confirm vasa as an evolutionary conserved germ cell-specific molecule since vasa proteins were present in rat, monkey, and human testes. The function of vasa will be determined in the monkey and human testes after interventions to regulate spermatogenesis. Further investigation of vasa in the human testes may lead to a potential target for male contraception.

ROLE OF C-JUN NH2-TERMINAL KINASE SIGNALING IN MALE GERM CELL APOPTOSIS IN MONKEYS AFTER MILD TESTICULAR HYPERTHERMIA AND/OR INTRATESTICULAR TESTOSTERONE DEPRIVATION.: 384

Objective In earlier studies, we have shown the involvement of the mitochondria-dependent intrinsic pathway for induction of male germ cell apoptosis in monkeys after transient testicular warming or administration of exogenous testosterone. The JNK (c-Jun NH2-terminal kinase) signaling pathway has been implicated in the activation of apoptosis in various cell systems by stimulating the intrinsic pathway, but its role in testicular germ cell death is unclear. The goal of this study was to define the role of JNK in male germ cell apoptosis in monkeys after mild testicular hyperthermia or deprivation of intratesticular T or the combination of both interventions. Study Design Groups of eight adult cynomolgus monkeys received one of the following treatments: (1) two empty Silastic implants (C); (2) two 5.5 cm T-implants (T); (3) daily exposure of testes to heat (43°C for 30 minutes) for 2 consecutive days (H); and (4) two T-implants plus exposure of the testes to heat for 2 consecutive days (T + H). Testicular biopsies were performed before and at 3, 8, and 28 days during treatment. Results Activation of JNK, as evidenced by increase in phospho-c-Jun in testis lysates, was detected in all treatment groups on day 3. Compared with controls, where no staining was detected, a strong phospho-c-Jun staining was detected in the nuclei of apoptotic germ cells in all treatment groups and in the Sertoli cell nuclei at day 8 in H and H + T groups. To further define the role of JNK in apoptotic signal transduction, we examined the expression of JNK1, JNK2, and JNK3 in testes after these interventions. In the control testes, the expression of JNKs was localized in the Sertoli cell cytoplasm. Costaining for JNK2 and -3 and for TUNEL shows expression of both of these isoforms only in those germ cells undergoing apoptosis when compared with controls where these proteins were detected in cytoplasm. In contrast, JNK1 was detected in the Sertoli cell nuclei at day 8 in H and H + T groups. Conclusion Our results indicate that (1) the JNK pathway may play a role in male germ cell apoptosis in monkeys; (2) JNK isoforms could have preferential effects on testis function; and (3) Sertoli cells participate in germ cell apoptosis triggered by heat stress via JNK signaling.

ROLE OF CASPASE 2 IN APOPTOTIC SIGNALING OF PRIMATE MALE GERM CELLS.: 180

Objective Caspase 2 is an initiator caspase whose activation has been found to promote apoptosis through mitochondria-dependent intrinsic pathway signaling in various cell systems, including the oocyte. Previously, we have shown that the intrinsic pathway is the key pathway for male germ cell apoptosis in rodents, monkeys, and men. The present study investigates if germ cell apoptosis induced by mild testicular hyperthermia or deprivation of intratesticular testosterone (T) or after combined interventions involves activation of caspase 2. Study Design Groups of eight adult cynomolgus monkeys received one of the following treatments: (1) two empty silastic implants (C); (2) two 5.5 cm-T implants (T); (3) daily exposure of testes to heat (43°C for 30 minutes) for 2 consecutive days (H); and (4) two T-implants plus exposure of the testes to heat for 2 consecutive days (H + T). Testicular biopsies were performed before and at 3, 8, and 28 days during treatment. Results Mean incidence of germ cell apoptosis increased significantly by d 3 in the H-alone group and by d 8 in the T-alone group but peaked at d 3 in the H + T group. Maximum activation of caspase 2 in respective treatment groups, as evidenced by immunocytochemistry and immunoblotting using an active caspase 2 antibody, coincided with the increased incidence of apoptosis. In control testes, we detected moderate immunostaining for active caspase 2 in Sertoli cells with little or no expression of germ cells. In contrast, we found a strong staining for active caspase 2 in apoptotic germ cells and in the Sertoli cells Co-staining for TUNEL and active caspase 2 further confirmed activation of caspase 2 only in those germ cells undergoing apoptosis. Conclusion Caspase 2 is activated in male germ cell apoptosis in nonhuman primates after heat stress, hormonal deprivation, or combined interventions. Future studies aimed at determining the expression of inhibitor of apoptosis proteins in testis will be needed to determine why Sertoli cells are not dying in spite of enhanced expression of caspase 2.

TESTICULAR HYPERTHERMIA BUT NOT HORMONE DEPRIVATION RESULTS IN STAGE- AND CELL-SPECIFIC ACTIVATION OF ERK AND INACTIVATION OF BCL-2.: 179

In earlier studies, we have shown that the mitochondria-dependent (intrinsic) pathway is the key pathway for male germ cell apoptosis across species. To characterize the upstream signaling pathways that activate this death pathway, here we investigated the contributions of ERK (extracellular signal-regulated kinase) and JNK (c-Jun NH2-terminal kinase) to male germ cell apoptosis in rats after hormone deprivation or heat stress. In the hormone deprivation model, rats were given a daily injection of vehicle for 14 days or GnRH-antagonist, acyline (1.6 mg/kg BW) for 2, 5, and 14 days. In the hyperthermia model, scrota of rats were exposed once to a temperature of 22°C (control) or 43°C for 15 minutes and killed at ½, 2, and 6 hours after heat treatment. Testicular hyperthermia but not hormone withdrawal led to sustained activation of ERK. Immunocytochemistry further revealed ERK activation only in the Sertoli cells involving exclusively heat-susceptible early (I-IV) and late (XII-XIV) stages within ½ hour of heating. By 6 hours, immunostaining for active ERK was, however, evident, mostly in germ cells. Co-staining for TUNEL and phospho-ERK confirmed activation of ERK only in those germ cells undergoing apoptosis. Heat-induced germ cell apoptosis was also preceded by JNK activation, as evidenced by an increase in phospho c-Jun in testis lysates within ½ hour of heating. Unlike ERK, phospho-c-Jun immunostaining was detected only in those heat-susceptible germ cells. Because activation of both ERK and JNK is known to induce BCL-2 phosphorylation leading to its inactivation, we next examined whether the increased germ cell apoptosis after heat stress is associated with BCL-2 phosphorylation. Compared with control, where no staining was detected, we found marked increase in serine-phosphorylated form of inactive BCL-2 only in heat-susceptible germ cells. Co-staining for TUNEL and phospho-BCL-2 confirmed phosphorylation of BCL-2 only in those germ cells undergoing apoptosis. The BCL-2 phosphorylation was not apparent during hormone deprivation-induced apoptosis. These results suggest that (1) ERK and JNK promote male germ cell apoptosis via inactivation of BCL-2 through serine phosphorylation, and (2) ERK signaling is decisively different between these two paradigms and may represent an important step in the signal transduction pathway by which heat stress induces male germ cell apoptosis.