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Division of Endocrinology (Y.L., C.W., A.P.S.H., C.-M.N., A.L., R.S.S.), Department of Medicine, Harbor-University of California, Los Angeles Medical Center and Los Angeles Biomedical Research Institute at Harbor-University of California, Los Angeles Medical Center, Torrance, California 90502; and State Key Laboratory of Reproductive Biology (Y.-X.L., X.-S.Z., Z.-Y.H., Y.-C.L.), Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
Address all correspondence and requests for reprints to: Ronald S. Swerdloff, M.D., Division of Endocrinology and Metabolism, Harbor-University of California, Los Angeles Medical Center, Box 446, 1000 West Carson Street, Torrance, California 90509. E-mail: swerdloff{at}labiomed.org.
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Abstract |
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Objective: The objective of the study was to determine whether T+H induces a greater suppression of spermatogenesis than either treatment alone in monkeys.
Design: The study was a randomized, placebo-controlled study.
Setting: The study was conducted at a primate center in China.
Participants: The study population was comprised of 32 adult cynomolgus monkeys.
Interventions: Groups of eight adult monkeys were treated for 12 wk with: 1) two empty implants (C); 2) two T implants (T); 3) daily testicular heat exposure (43 C for 30 min) for 2 consecutive days (H); or 4) two T implants plus testicular heat exposure (T+H). Treatment was followed by an 8-wk recovery period.
Main Outcome Measures: Measures included sperm counts and germ cell apoptosis.
Results: Serum T levels were elevated in both the T and T+H groups during treatment but not in the C or H group. Sperm counts were transiently suppressed after heat to 16.4% of baseline at 4 wk and then returned to pretreatment levels. Sperm counts were suppressed slowly after T treatment to nadir of 6.4% of pretreatment levels at 12 wk. T+H rapidly suppressed sperm output as early as 4 wk to 3.9% of pretreatment levels that was maintained throughout treatment. The decreased sperm counts were due to increased germ cell apoptosis in all treatment groups. Sperm counts recovered to the pretreatment levels in all groups by 8 wk after treatment.
Conclusion: This proof-of-concept study demonstrates that transient testicular warming enhances and hastens the effect of T implant on the suppression of spermatogenesis in monkeys.
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Introduction |
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Our earlier studies demonstrated that administration of GnRH antagonist or T implant suppressed gonadotropin and intratesticular T and induced stage-specific (stages VII–VIII) loss of germ cells through apoptosis in rats (13, 14, 15, 16). We demonstrated 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 predominantly occurring at early (I–IV) and late (XII–XIV) stages (15). We additionally demonstrated that heat exposure in rats bearing T implant markedly reduced the number of pachytene spermatocytes and round spermatids through increased germ cell apoptosis in all stages (early, mid, and late), resulting in tubules devoid of mature spermatids (16). These findings in rats allowed us to design an experiment to determine whether the combination of two treatments (two hit) has additive or synergistic actions resulting in greater efficacy in suppression of spermatogenesis in nonhuman primates.
The objectives of the present study were to determine whether the combination of mild testicular warming with administration of a low dose of T could enhance the effect of T alone by rapidly and effectively suppressing spermatogenesis to near-complete azoospermia in nonhuman primates (monkeys) and to determine whether these combined and enhanced suppression of spermatogenesis was mediated through increased germ cell apoptosis. Although the basic process of mitosis, meiosis, and spermiogenesis during spermatogenesis exhibits similarities in all mammals, there are several differences between rodents and monkeys worthy of note including differences in stem cell renewal, spermatogonia proliferation, organization and duration of spermatogenesis, and dependence of spermatogenesis on a gonadotropin drive (17, 18). Thus, the use of the nonhuman primate model will allow us to extend our proof-of-concept study in rodent and collect testicular samples to determine the underlying molecular mechanisms leading to suppression of spermatogenesis after these two interventions as a prelude to future experiments in men. Understanding these mechanisms and key regulators of suppression of spermatogenesis may result in new targets for male contraception.
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Materials and Methods |
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Thirty-two male adult (7 to 10 yr old) cynomolgus monkeys (Macaca fascicularis) were obtained and housed at the Guangxi Hongfeng Primate Research Center, Institute of Zoology, Chinese Academy of Sciences. Animal handling, experimentation, and testicular tissue harvesting protocol were in accordance with the recommendation of the American Veterinary Medical Association and were approved by both the Institutional Animal Care and Use Review Committee of Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center and the Animal Care and Use Review Committee of the Institute of Zoology, Chinese Academy of Sciences. The monkeys were housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12 h of light and 12 h of darkness) with free access to water and monkey chow. Groups of eight adult cynomolgus monkeys (Macaca fascicularis) were randomly assigned to one of the following treatments for 12 wk followed by an 8-wk recovery period: 1) group 1 received two empty SILASTIC implants (Dow Corning, Midland, MI) on d 1 (C group); 2) group 2 received two T implants (T, 5.5 cm, inner diameter 0.33 and outer diameter 0.46 cm) on d 1 (T group); 3) group 3 was given daily testicular exposure to heat (43 C for 30 min) for 2 consecutive days on d 1 and 2 (H group); and 4) group 4 was subjected to two T implants plus testicular exposure to heat for 2 consecutive days (T+H group). Three monkeys from each group were used for semen collection without any testicular surgical intervention throughout the study to ensure that the semen parameters were not affected by any surgical procedure. The remaining five monkeys from each group were subjected to testicular biopsy. Biopsies were taken alternatively on each side of testes from a given monkey at different time points. Each testis received biopsy twice throughout the study. Testicular samples were harvested before treatment and at 3, 8, 28, and 84 d during treatment phase as well as at the end of the recovery phase, respectively. Semen and blood samples were collected before and every 2 wk during and after the treatment and at the end of recovery phase.
T implant preparation and implantation
The dose of T implant was chosen based on a published study in monkeys (19), which showed that two sc 5.5-cm T implants led to suppression of biologically active gonadotropins and maintenance of slightly higher than physiological serum T levels with about half of the monkeys achieving azoospermia. We intentionally administered a submaximal dose of T to enhance the power to demonstrate the synergistic or additive effects of T plus heat treatment. T SILASTIC implants (Dow Corning) of 5.5 cm length were prepared from polydimethylsiloxane tubing (5.5 cm length, inner diameter 0.33 and outer diameter 0.46 cm), packed with T (Sigma, St. Louis, MO), and sealed with SILASTIC medical adhesive A (Dow Corning) as described previously (16, 19). Each monkey in T implant alone and T+H combined treatment groups were implanted with a pair of 5.5-cm T-filled capsules subdermally along the dorsal surface near the neck under a heavy sedation with ketamine (10 mg/kg) and atropine (0.05 mg/kg). The capsules were sterilized and rinsed in sterile saline and sealed in sterilized plastic bag before implantation. At the end of the treatment phase of 12 wk, the SILASTIC capsules (Dow Corning) were removed under light anesthesia and the monkeys were then allowed to complete an 8-wk recovery phase.
Testicular warming
Testicular warming in water bath in heat alone and in combination with T (T+H) groups was performed as described previously (20). Briefly, under light sedation with ketamine (4 mg/kg), testicular hyperthermia was conducted by immersing the scrota containing the testes into a thermostatically controlled water bath at 43 C for 30 min once daily for 2 consecutive days. The temperature of 43 C was chosen based on our earlier studies in rodent and monkey showing that testicular exposure to this temperature induces germ cell apoptosis (15, 16, 20, 23). Lower temperature may not have any effect and higher temperature may induce necrosis. After heat treatment, animals were dried, examined for any redness or injury to the scrota, and then returned to their cages and allowed to recover from the effect of the anesthesia. Inspection of the scrota after heat exposure showed no evidence of thermal injury to the scrotal skin after this short duration of modest increase in temperature.
Semen and testicular tissue collection and processing
Semen samples were collected using a SD9 square pulse stimulator (Grass Telefactor, West Warwick, RI). Monkeys from each group showed good ejaculation responses with semen parameters within the range reported in Cynomolgus monkeys (20). Briefly, monkeys were restrained in a primate chair under light sedation with ketamine (4 mg/kg) for 40 min. After recovery from light sedation, they were electroejaculated with a Grass 6 stimulator equipped with electrocardiogram pad electrodes for direct penile stimulation (20, 21). The volume of each ejaculate including both fluid and coagulum fractions was recorded, and the sperm number was determined from the fluid fraction using a hemocytometer and expressed as spermatozoa x 107/ml. Semen samples were collected from three monkeys from each treatment group on the days before (pretreatment phase) and d 14, 28, 42, 56, 70, and 84 during treatment, and d 144 (end of recovery phase) after application of the penile cuff. Five monkeys in each group were subjected to testicular biopsies either before treatment or on d 3, 8, 28, and 84 during treatment and at the end of recovery phase. Biopsies were taken alternatively on each side of testes from a given monkey at different time points. Each testis received biopsy twice throughout the study.
Open testicular biopsies were performed, under aseptic conditions and heavy sedation with ketamine (10 mg/kg) and atropine (0.05 mg/kg). After testicular biopsy, the monkeys were closely observed until recovered from sedation and returned back to cages. Testicular tissue (about 300 mg) was obtained during each biopsy from a testis, which was divided equally into three portions. One portion of testicular tissue was immersion fixed with 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4), one portion immersion fixed in Bouin’s solution, and the remaining portion snap frozen in liquid N2 or immersed into RNAlater solution (Ambion, Inc., Austin, TX) and stored at –70 to –80 C for subsequent analysis of RNA and proteins. The testicular tissues were placed into the respective fixatives overnight and processed for routine paraffin embedding for in situ detection of apoptosis and immunohistochemical studies.
Hormone assays
Blood samples were collected from arm vein of animals while briefly restrained, and serum was separated and stored at –20 C for subsequent hormone assays. Serum samples from intact monkeys (without any testicular surgery) in each group were used for hormone assays. T concentrations in serum were measured by RIA, as reported previously (22, 23). The minimal detection limit in the assay was 0.25 ng/ml. The intra- and interassay coefficients of variations were 8 and 11%, respectively.
Assessment of apoptosis
In situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections by the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) technique using an Apop Tag-peroxidase kit (Intergen Co., Purchase, NY) as described earlier (16, 24). Negative and positive controls were carried out in every assay. As negative controls, tissue sections were processed in an identical manner, except that the terminal deoxynucleotidyl transferase enzyme was substituted with the same volume of PBS. Testicular sections from rats treated with 3 cm testosterone implant for 14 d were used as positive controls (16). The method used for qualitative and quantitative assessment of germ cell was similar to that described previously (20). In brief, testicular sections were examined with an American optical microscope (Scientific Instruments, Buffalo, NY) with x40 objective and a pair of x10 eyepieces. A square grid fitted within the eyepiece provided a reference area of 62,500 µm2. Apoptotic germ cells were counted within the seminiferous tubules under the frame of grid containing 121 points and covering an area of 62,500 µm2 (20, 25). A total of 50 grid frames were counted in the testicular sections from each animal. The density of germ cell apoptosis (apoptotic index) was expressed as the number of apoptotic germ cells per unit tubular area (106 µm2).
Statistical analysis
Statistical analyses were performed using the SigmaStat 2.0 Program (Jandel Corp., San Rafael, CA). Sperm concentration, serum T levels, and the incidence of germ cell apoptosis were analyzed by two-way ANOVA, using time, group, and their interaction as factors. Post hoc differences were detected by Student-Newman-Keuls method test. Differences were considered significant if P < 0.05.
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Results |
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Mean sperm concentrations from three monkeys of each group at different time points after treatment are shown in Fig. 1
. In the C group, there was no significant change in the sperm concentration throughout the study. At 4 wk, mean sperm concentration significantly decreased (pretreatment: 83.83 ± 19.76 x 107/ml vs. 4 wk after heat: 14.60 ± 0.30 107/ml; P < 0.05) in the H-alone group; thereafter the sperm counts gradually recovered. In the T-alone group, sperm concentration decreased gradually and slowly until 12 wk when marked suppression was observed (pretreatment: 101.50 ± 13.48 x 107/ml vs. 12 wk after T implants: 6.70 ± 2.30 107/ml; P < 0.05). In contrast, by 4 wk in the combined treatment (T+H) group, mean sperm count was profoundly decreased (pretreatment: 98.80 ± 31.55 x 107/ml vs. 4 wk after T+H: 3.86 ± 1.77 107/ml; P < 0.05), and this suppression of sperm output was maintained at this very low level throughout the treatment period. Removal of the implants led to return of sperm concentrations to pretreatment values in the monkeys.
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Serum T levels before, during treatment, and at the end of the recovery phase are shown in Fig. 2. There was no significant change in serum T concentration in the C group. Serum T levels increased across the 20 wk of experiment period in both T-alone (pretreatment: 6.39 ± 1.49 ng/ml vs. 2 wk after treatment: 11.63 ± 1.71 ng/ml, P < 0.05) and T+H groups (pretreatment: 5.68 ± 0.40 ng/ml vs. 2 wk after treatment: 10.37 ± 1.04 ng/ml, P < 0.05) as early as 2 wk after T implantation. This elevated serum T levels were maintained at the upper normal range throughout the 12 wk of treatment phase in T alone (12 wk after treatment: 10.89 ± 0.89 ng/ml, P < 0.05) and T+H (12 wk after treatment: 11.32 ± 0.97 ng/ml, P < 0.05) groups. Serum T levels returned to the pretreatment levels at the end of the 8-wk recovery phase after removal of the T implants. No differences in serum T levels in the H-alone group were noted across 20 wk of experiment period as compared with the control group.
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Testicular histologic examinations were performed before and on d 3, 8, 28, and 84 of treatment as well as at the end of the recovery phase in the three groups with intervention. In T-treated group (data not shown), no morphologic changes of the seminiferous epithelium were noted except for some apparent decrease in the number of pachytene spermatocytes, round spermatids, and mature spermatozoa beginning at 28 d after treatment that showed a progressive decrease until the end of the treatment phase. These testicular morphologic changes recovered completely, and the testis morphology was similar to control animals at the end of recovery. In the H-alone group (data not shown), we observed that germ cells were detached from the epithelium in some seminiferous tubules on d 3. Degeneration of the seminiferous epithelium characterized by epithelial disorganization, vacuolization, and formation of multinucleated giant cells was observed on d 8 and 28 in the H group. At this time most of the tubules showed thinner seminiferous epithelium. Some of the seminiferous tubules contained spermatozoa, even though spermatocytes and round spermatids were absent on d 8 and 28 after H-alone treatment. The tubular morphology gradually appeared similar to the control group in majority of the seminiferous tubules on d 84 and was fully restored to normal-appearing spermatogenesis on d 144. A few seminiferous tubules exhibited a severe damage on d 8 and 28 after heat treatment, but spermatogenesis recovered later, suggesting even in these tubules, stem cells and/or early spermatogonia were retained. In the combined T+H-treated group (Fig. 3), more severe damage of the seminiferous epithelium was observed including epithelial disorganization, vacuolization, and formation of multinucleated giant cells on d 3 and 8, but the spermatogonia were not affected in most of seminiferous tubules throughout the treatment phase. Decreased number of spermatocytes, round spermatids, and mature spermatozoa was obvious on d 28 and remained very marked on d 84 after T+H treatment. There were no mature spermatozoa present in the most of the seminiferous tubules on d 84. Spermatogenesis returned to normal appearance at the end of recovery phase in the T+H group.
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Germ cell apoptosis was assessed by TUNEL assay (Fig. 4), and the quantitative data are shown in Fig. 5. Most of the apoptotic germ cells in all treated groups was pachytene spermatocytes and round spermatids. Apoptotic spermatozoa was found in areas close to the basal lamina indicating the presence of sperm retention in both the T-alone and T+H groups, but not in the H-alone group.
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Discussion |
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In the present study, we provide evidence in the nonhuman primates that submerging the testes of T-treated monkeys into warm water for 30 min on 2 consecutive days resulted in suppression of the sperm count to oligospermic levels by 4 wk. The suppression of spermatogenesis after combined treatment was much greater than that after heat exposure alone at 4–8 wk and occurred much faster than with T alone. In fact, the suppression of sperm counts with T+H at 4 wk was equal to that achieved after 12 wk of treatment with T alone. In addition to increased germ cell apoptosis, inhibition of spermatogonia proliferation may contribute to the suppression of spermatogenesis in monkeys. It has been demonstrated both in monkeys and humans that administration of exogenous T also inhibited germ cell (spermatogonial) proliferation, suggesting that more than one mechanism may lead to decreased sperm output after T treatment (19, 26, 27).
We emphasize that the present study was designed to provide a submaximal suppression of spermatogenesis by either hormone treatment or scrotal warming to enhance the power to show additive and/or synergistic effects of combined treatment. It is true that T alone has not been able to produce azoospermia in all monkeys (19, 28). The paradigm did not test whether combined treatment with higher doses of T would induce azoospermia in all animals or answer the question whether the induction of azoospermia with combined treatment would have allowed continued azoospermia in animals by continued T treatment alone.
We also acknowledge that the effects of T may be biphasic, suppressing spermatogenesis with moderate pharmacological doses in rats, monkeys, and men (through suppression of LH and FSH and secondarily intratesticular T) but sustaining spermatogenesis at very high doses (through enhanced intratesticular T or interference of GnRH analog effects on suppression of FSH) (29, 30, 31). The latter effects have been seen in a few studies in monkeys and men when spermatogenesis was suppressed by treatment with GnRH agonists or progestins, and the addition of high doses of testosterone blunted the suppression of spermatogenesis (32, 33, 34). The spermatogenesis-supporting effects of T treatment in a rare male patient with hypogonadotropic hypogonadism has been reported (35), but this must be very unusual, and even very high doses seem to lower sperm counts in men administered supraphysiological doses of T (36, 37, 38). Reversibility is one of the accepted prerequisites in contraceptive development. In this study, we demonstrated that sperm count in all groups recovered to pretreatment levels after withdrawal of perturbations, demonstrating the reversibility of this combined treatment in suppression of spermatogenesis. The reversibility was further substantiated by the histologic data showing normal spermatogenesis in all testes samples at the end of recovery phase.
In summary, we demonstrated that first, the combination of exogenous hormonal treatment such as T implants (hit 1) and physical agent (heat exposure; hit 2) is more effective in suppressing spermatogenesis than either treatment alone in nonhuman primate, and second, increased germ cell apoptosis is one of the major mechanisms to induce germ cell loss in monkeys.
Thus, this study suggests the feasibility of considering exploring new physical or nonhormonal agents acting directly on the testis in combination with well-established hormonal approaches in male contraceptive development. More importantly, through this study, we established a nonhuman primate model and an experimental paradigm to study the underlying molecular mechanisms of regulation of spermatogenesis by physical agents and exogenous hormones. We can test the potential effects of various contraceptive agents acting through different targets and their combinations on suppression of spermatogenesis in monkeys. The testes biopsies from this model will be used to further identify the pathways involved in germ cell loss in the nonhuman primate; identify potentially pharmacologically targetable critical steps in the dysregulation of spermatogenesis and induction of apoptosis; and use these insights for future studies in man in which the amount of testicular biopsy materials are more limited.
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Acknowledgments |
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Footnotes |
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This study was presented in part at the 29th Annual Meeting of the American Society of Andrology, Baltimore, Maryland, 2004.
The authors have no conflict of interest.
First Published Online November 29, 2005
Abbreviations: T, Testosterone; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling.
Received August 10, 2005.
Accepted November 22, 2005.
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