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Division of Endocrinology,3 Department of Medicine, Harbor-University of California, Los Angeles (UCLA) Medical Center, David Geffen School of Medicine at UCLA, and Los Angeles Biomedical Research Institute, Torrance, California 90509
State Key Laboratory of Reproductive Biology,4 Institute of Zoology, Chinese Academy of Science, Beijing 100080, China
Department of Endocrinology,5 First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
ABSTRACT
Male contraception has focused, to a great extent, on approaches that induce azoospermia or severe oligospermia through accelerated germ cell apoptosis. Understanding the specific steps in the germ cell apoptotic pathways that are affected by male contraceptives will allow more specific targeting in future contraceptive development. In this study, we have used a nonhuman primate model to characterize the key apoptotic pathway(s) in germ cell death after mild testicular hyperthermia, hormonal deprivation, or combined interventions. Groups of 8 adult (7- to 10-year-old) cynomolgus monkeys (Macaca fascicularis) received one of the following treatments: 1) two empty silastic implants; 2) two 5.5-cm testosterone (T) implants; 3) daily exposure of testes to heat (43°C for 30 min) for 2 consecutive days; and 4) two T implants plus testicular heat exposure for two consecutive days. Testicular biopsies were performed before and at Days 3, 8, and 28 of treatment. Treatment with T, heat, or both led to sustained activation of both mitogen-activated protein kinase (MAPK) 1/3 and MAPK14. Activation of MAPK1/3 and MAPK14 were accompanied by an increase in B-cell leukemia/lymphoma (BCL) 2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL2 and activation of the MAPK14-mediated mitochondria-dependent pathway are critical for male germ cell death in monkeys.
apoptosis, primate, spermatogenesis, testis
?>Programmed germ cell death occurs spontaneously during spermatogenesis, or can be induced in a stage- and cell-specific manner by a variety of apoptotic stimuli, including experimental male contraceptive approaches, such as mild testicular hyperthermia and deprivation of gonadotropins and reduction of intratesticular testosterone (T) concentrations by administration of a gonadotropin-releasing hormone (GnRH) antagonist, or by exogenous administration of T [1–3]. We have also shown in rats [3], and more recently in monkeys [4], that a single testicular heat exposure (hit 1), in combination with exogenous T administration (hit 2), more rapidly and markedly suppresses spermatogenesis to near azoospermia through increased germ cell apoptosis as compared to either T or heat alone. The signaling pathways by which these hormonal and nonhormonal stimuli activate germ cell apoptosis are not well understood in the primate, and are the topic of this article.
Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that function as critical mediators of a variety of extracellular signals [5–7]. Members of the MAP kinase superfamily include MAPK1/3, MAPK8, and MAPK14. Available data from various cell systems other than male germ cells suggest that MAPK1 and MAPK3 are activated in response to growth stimuli and promote cell growth, whereas both MAPK8 and MAPK14 are activated in response to a variety of environmental stresses and inflammatory signals, and promote apoptosis and growth inhibition [5–7]. The downstream signaling events that couple kinase activation with testicular germ cell apoptosis in monkey are not known. One possible mechanism by which MAPK8 and MAPK14 can induce apoptosis is through activation of mitochondria-dependent intrinsic pathway signaling [8]. The intrinsic pathway for apoptosis involves the release of cytochrome c from mitochondria into the cytoplasm, where it binds to apoptotic protease activating factor 1 (APAF1), resulting in the activation of the initiator caspase 9 and the subsequent proteolytic activation of the executioner caspases 3, 6, and 7. The active executioners are then involved in the cleavage of a set of proteins, including poly (ADP) ribose polymerase 1, lamin, actin, and gelsolin, and causes morphological changes to the cell and nucleus typical of apoptosis. Members of the B-cell leukemia/lymphoma (BCL) 2 family of proteins play major roles in governing this mitochondria-dependent apoptotic pathway, with proteins, such as BAX functioning as an inducer, and BCL2 as a suppressor of cell death [9]. Additionally, smac (second mitochondria-derived activator of caspases), also known as DIABLO, is released from mitochondria into the cytosol following apoptotic stimuli and promotes apoptosis by antagonizing inhibitor of apoptosis proteins (IAPs) [10, 11]. The other important pathway for induction of apoptosis in mammalian cells is the extrinsic pathway, which involves ligation of the death receptor (such as FAS) to its ligand, FASLG, resulting in the activation of a different set of initiator caspases—namely, caspases 8 and 10—through interaction between death domains and death effector domains of an adaptor molecule, such as FAS-associated death domain, and these caspases [8]. Caspase 8 or caspase 10 then activates the executioner caspases 3 and 7, resulting in cellular disassembly.
We have previously demonstrated that mitochondria-dependent intrinsic signaling is the key apoptotic pathway for male germ cell apoptosis in rodents [12–16]. We do not know if this pathway is a common phenomenon during germ cell apoptosis across species, or if the FAS signaling pathway plays a role in germ cell apoptosis in monkeys. The objectives of the present study were 2-fold. The first was to determine whether the mitochondria-dependent intrinsic pathway, as noted in rodents, is also the key pathway for induction of male germ cell apoptosis in nonhuman primates (monkeys) after mild testicular hyperthermia, hormonal deprivation, or after combined interventions. The second was to examine the possible role of MAPKs in activation of such death pathways. Our results provide evidence indicating that activation of the MAPK14-mediated mitochondria-dependent pathway coupled with inactivation of BCL2 through phosphorylation is critical for male germ cell death in monkeys.
A total of 32 male adult (7–10 years old) cynomolgus monkeys (Macaca fascicularis) were obtained and housed at the Guangxi Hongfeng Primate Research Center, Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS). Animal handling, experimentation, and testicular tissue harvesting protocols were in accordance with the recommendation of the American Veterinary Medical Association and were approved by both the Animal Care and Use Review Committee of the Los Angeles Biomedical Research Institute at Harbor-University of California, Los Angeles Medical Center and by the Chinese Academic Committees of IOZ, CAS. The monkeys were housed in a standard animal facility under controlled temperature (22°C) and photoperiod (12L:12D) with free access to water and monkey chow. Groups of eight adult cynomolgus male 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 min) for two consecutive days (H); and 4) two T implants plus exposure of the testes to heat for two consecutive days (T+H). T implants of 5.5-cm length were prepared from polydimethylsilozane tubing (5.5-cm length, 0.33 cm inside diameter and 0.46 cm outside diameter; Dow Corning Corp., Midland, MI), packed with T (Sigma, St. Louis, MO), and sealed with silastic medical adhesive A (Dow Corning Corp.), as described previously [3, 4]. Sterilized T-filled capsules were implanted subdermally along the dorsal surface under general anesthesia with ketamine (10 mg/kg) and atropine (0.05 mg/kg). The T dose was based on the results of a previous study, which showed that two subcutaneous 5.5-cm T implants led to suppression of biologically active gonadotropins, maintained slightly higher than physiological serum T levels, and induced azoospermia in about half of the monkeys [17]. Testicular warming in water bath was performed as described previously [4, 18]. Briefly, under light sedation with ketamine (4 mg/kg), testicular hyperthermia was conducted by immersing the monkey scrota containing the testes into a thermostatically controlled water bath at 43°C for 30 min once daily for two consecutive days. After heat treatment, animals were dried, examined for any redness or injury to the scrota, 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. Quality control of the protocol was enhanced by direct supervision of one of the investigators (Y.H.L.), who also performed all surgical procedures.
Open testicular biopsies were performed under heavy sedation with ketamine (10 mg/kg) and atropine (0.05 mg/kg) before and at Days 3, 8, and 28 after the first heat or hormone treatment [4]. Testicular biopsies were taken from one testis of three or four monkeys per group at any one time point. Each testicular biopsy was divided into two equal portions. One portion was immediately frozen in liquid N2 and stored at –70 to –80°C for subsequent analysis by Western blot assays. The remaining portion was fixed with Bouin solution (Sigma) overnight for immunocytochemical and immunofluorescence studies.
Immunohistochemical and Immunofluorescence Analyses
Bouin-fixed, paraffin-embedded testicular sections were immunostained as described previously [12–16]. Briefly, testicular sections were deparaffinized, hydrated by successive series of ethanols, rinsed in PBS, and then incubated in 2% H2O2 to quench endogenous peroxidase. Sections were then blocked with a blocking serum and subsequently incubated with the following: rabbit polyclonal BAX (1:400, no. sc-493; Santa Cruz Biotechnology, Santa Cruz, CA); BCL2 (1:200, no. sc-492; Santa Cruz Biotechnology); phospho (serine 70)-BCL2 (1:25, no. 2875s; Cell Signaling Technology, Beverly, MA); DIABLO (1:1 000, no. 567365; Calbiochem, San Diego, CA); rabbit polyclonal phospho-p44/42 (1:100; no. 9101s; Cell Signaling Technology), which detects endogenous levels of MAPK1 and MAPK3 only when dually phosphorylated at threonine 202 and tyrosine 204; and rabbit monoclonal phospho-MAPK14 (1:50, no. 4631; Cell Signaling Technology), which detects MAPK14 only when dually phosphorylated at threonine 180 and tyrosine 182 antibodies overnight at 4°C. Immunoreactivity was visualized with diaminobenzidine tetrahydrochloride (DAB) per the manufacturer's instructions (rabbit Unitect ABC Immunohistochemistry Detection System; Calbiochem). Slides were counterstained with hematoxylin. Negative and positive controls were run for every assay. The negative controls were processed in an identical manner, except the primary antibody was substituted by the rabbit immunoglobulin (Ig) G. Rat testicular sections exposed to heat [13] or hormone deprivation by GnRH-A treatment [14] were used as positive controls.
Subcellular Fractionation and Western Blotting
Cytosolic and mitochondrial fractions were prepared as described previously [13, 14, 16]. Briefly, testicular biopsies were homogenized using a dounce homogenizer in 3 ml buffer A (0.25 M sucrose, 50 mM Hepes, 10 mM NaCl, 10 mM EDTA, 2 mM dithiothreitol) supplemented with protease inhibitors (Complete Protease Inhibitors; Roche). The crude homogenates were centrifuged at 1000 x g for 10 min at 4°C, and the resultant supernatant centrifuged at 10 000 x g for 15 min at 4°C to sediment the low-speed fraction containing mainly mitochondria. The mitochondria were washed twice in buffer A and pelleted. The cytosolic and high-speed fractions were isolated following centrifugation of the 10 000 x g supernatant fraction at 100 000 x g for 60 min at 4°C. The resulting supernatant was the cytosolic fraction. The purity of the cytosolic and mitochondrial fractions was assessed by Western blotting using antibodies to actin (1:2000; Sigma) and cytochrome c oxidase subunit IV (COX 4I1; 1:500, no. A-6409; Molecular Probes, Eugene, OR), respectively, as described previously [13]. The absence of COX 4I1 in cytosolic extracts confirmed that the cytosolic preparations were free of mitochondrial contamination (data not shown).
Western blotting was performed using total testicular lysates or subcellular fractions, as described previously [13–16, 19]. In brief, proteins (50–80 µg) were separated on a 4%–12% SDS-polyacrylamide gel with 2-(4-morpholino)-ethane-sulfonic acid or 4-morpholinopropanesulfunic acid buffer purchased from Invitrogen (Carlsbad, CA) at 200 V. Gel was transferred on a Immuno-blot polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA) overnight at 4°C. Membranes were blocked in blocking solution (0.3% Tween 20 in Tris-buffered saline (TBS) and 10% nonfat dry milk) for 1 h at room temperature then probed using rabbit polyclonal MAPK1/3 (1:500), BAX (1:500), BCL2 (1:500), phospho (serine 70)-BCL2 (1:500), cytochrome c (1:2000, no.sc-7159; Santa Cruz Biotechnology), smac/DIABLO (1:5000; Calbiochem), FAS and FASLG (1:500, nos. F22120 and F37720, respectively; BD Transduction Laboratories, San Jose, CA), and caspase-8 p20 (1:500, no. sc-7890; Santa Cruz Biotechnology) antibodies for 1 h at room temperature or overnight at 4°C with constant shaking. Following three 10-min washes in TBS-T buffer, membranes were incubated in anti-rabbit (Amersham Biosciences, Piscataway, NJ) or anti-mouse IgG-horse radish peroxidase (Santa Cruz Biotechnology) secondary antibodies at a 1:2000 dilution. All antibodies were diluted in blocking buffer. For immunodetection, membranes were washed three times in TBS-T wash buffer, incubated with enhanced chemiluminescence (ECL) solutions per the manufacturer's specifications (Amersham Biosciences), and exposed to hyper film ECL. The membranes were stripped and reprobed with a mouse monoclonal actin (1:2000) or a rabbit polyclonal COX 4I1 (1:500) for normalization of the loading. Band intensities were determined using Quantity One software from Bio-Rad.
Measurements of MAPK14 Activation
Activation of MAPK14 was measured using an assay kit (Cell Signaling Technology) as described previously by us [14]. In brief, a monoclonal phosphospecific antibody to MAPK14 (Thr180/Tyr182) was used to selectively immunoprecipitate active MAPK14 from testis lysates. The resulting immunoprecipitate was then incubated with activating transcription factor (ATF) 2 fusion proteins in the presence of ATF and kinase buffer, which allows immunoprecipitated, active MAPK14 to phosphorylate ATF2. Phosphorylation of ATF2 at Thr71 was measured by Western blotting using a rabbit polyclonal phospho-ATF2 (Thr71) antibody.
Statistical analyses were performed using the SigmaStat 2.0 program (Jandel Corp., San Rafael, CA). The Student-Newman-Keuls test after one-way repeated measures ANOVA was used to determine statistical significance for Western blot analyses. Differences were considered significant at P < 0.05.
Mild Testicular Hyperthermia and Deprivation of Intratesticular T either Alone or in Combination Induce Germ Cell Apoptosis in Monkeys
To elucidate the underlying apoptotic pathway involved in testicular germ cell death in monkeys triggered by deprivation of the gonadotropic support, mild testicular hyperthermia, or both, we recently determined the temporal changes in the incidence of germ cell apoptosis after these treatments in the same group of animals [4]; the quantitative data are summarized in Table 1. In the T-treated group, the rate of germ cell apoptosis increased by 2.8-fold as compared to control values at 8 days, and remained elevated at this level thereafter. Mild testicular hyperthermia for 2 days resulted in a marked increase (
6.0- to 7.3-fold over control values) in germ cell apoptosis at 3 days and 8 days, but declined (although not fully returning to pretreatment levels) by 28 days. The number of dying cells in the T+H group was markedly higher as compared to either treatment alone at 3 days, but began to decrease at 8 days. The incidence of germ cell apoptosis still remained elevated over the base line values, but similar to that induced by T, on 28 days. The predominant germ cells undergoing apoptosis after heat and/or T treatment were pachytene spermatocytes and round spermatids [4].
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Hormone Deprivation Alone or in Combination with Testicular Hyperthermia Results in Activation of MAPK1/3 and MAPK14
To characterize the signaling events in male germ cell apoptosis in monkeys after mild testicular hyperthermia, hormonal deprivation, or after combined interventions, we examined the potential role of MAPK1/3 and MAPK14. Activation of MAPK14, as evidenced by an increase in phospho-ATF2, was detected as early as Day 3 in all treatment groups, and remained active thereafter in all but the H group, where this activation was not sustained by Day 28 (Fig. 1A). This was further corroborated by densitometric evaluation (data not shown). Activation of MAPK14 was also ascertained by immunocytochemistry (Fig. 1B, panels I–V) using a phosphospecific antibody, which detects MAPK14 only when dually phosphorylated at Thr180 and Tyr182. In control testes, phospho-MAPK14 immunoreactivity was detected mainly in Sertoli cells, but not in germ cells (Fig. 1B, panel I). Compared to controls, a marked increase in phospho-MAPK14 staining was detected in susceptible germ cells (Fig. 1B, panels III–V). No such immunostaining was noted when the primary antibody was substituted by the same volume of rabbit IgG (Fig. 1B, panel II). Given the opposing effects of MAPK1/3 and MAPK14 on apoptosis, we next examined the time course of MAPK1/3 activation by immunocytochemistry and immunoblotting. Activation of MAPK1/3, as evidenced by an increase in phospho-MAPK1/3, was detected in all treatment groups at 3 days and 8 days before starting to decline at 28 days (Fig. 2, A and B). The expression of phospho-MAPK1/3 was located in both Sertoli cells and germ cells in all treatment groups, compared with controls, where no such staining was detected (Fig. 2C).
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In the control monkey testes, little or no BCL2 was detected, either in the cytosolic (Fig. 3, A–C) or the mitochondrial fractions (Fig. 3, D–F) of the testicular lysates. Compared to controls, there was a noticeable increase in the BCL2 levels in both cytosolic (Fig. 3, A–C) and mitochondrial (Fig. 3, D–F) fractions at each time point in all treatment groups. Densitometric analysis further revealed a significant (P < 0.05) increase in BCL2 levels in both cytosolic (Fig. 4, A–C) and mitochondrial (Fig. 4, D–F) fractions in all treatment groups, except for cytosolic BCL2 levels at 3 days and 8 days in the H+T group. In untreated testes, unlike BCL2, BAX was present in both cytosolic (Fig. 5, A–C) and mitochondrial (Fig. 5, D–F) fractions. Although there appeared to be an increase in the BAX levels in the cytosolic fraction (Fig. 5, A–C) of testicular lysates at 3, 8, and 28 days posttreatment in the T and the H groups compared to controls, the increase was not statistically significant as revealed by densitometric analysis (data not shown). Notably, mitochondrial fractions showed no change in BAX expression (Fig. 5, D–F). Immunocytochemistry showed a cell type-specific increase in BAX expression involving only those germ cells susceptible to apoptosis after these interventions (Fig. 6, A–D).
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Mild Testicular Hyperthermia and Deprivation of Intratesticular T either Alone or in Combination Induce Serine Phosphorylation of BCL2 in Germ Cells
Because the phosphorylation status of BCL2 plays an important role in its prosurvival activity [20–22], and this can be induced by both MAPK1/3 and MAPK14 activation [23–25], we further examined whether increased germ cell apoptosis triggered by deprivation of intratesticular T, by heat stress, or by both interventions is associated with BCL2 phosphorylation. After apoptosis induction, using an antibody that specifically recognizes endogenous levels of BCL2 when phosphorylated at serine 70, we found increased levels of the serine-phosphorylated form of inactive BCL2, as evidenced by immunoblotting (Fig. 7) and immunocytochemistry (Fig. 8). In control testicular biopsies (Fig. 8A), a weak phospho-BCL2 expression was detected in the Sertoli cells, but not in the germ cells. Compared to controls, we clearly detected a marked increase in the inactive form of BCL2 in germ cells (Fig. 8, B–D). A modest increase in phospho-BCL2 expression was also noted in the Sertoli cells. No such immunostaining was noted when primary antibody was substituted by the same volume of rabbit IgG (Fig. 8E).
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Cytochrome c and DIABLO Are Released from Mitochondria into Cytoplasm during Heat- and/or Hormonal Deprivation-Induced Germ Cell Death
To determine whether mitochondria-dependent intrinsic pathway is also the key pathway for induction of germ cell apoptosis in monkeys, we examined the cytochrome c and DIABLO release during heat- and/or hormone deprivation-induced programmed germ cell death. As shown in Figure 9, little or no cytochrome c was detected in cytosol from control testes. In contrast, cytosolic accumulation of cytochrome c was clearly evident in all treated groups at 3 days and 8 days after treatment. Marked accumulation of cytochrome c in the cytosolic fractions of testicular lysates continued to 28 days in all but the H group, which showed a decline in its levels by 28 days posttreatment. Like cytochrome c, cytosolic accumulation of DIABLO was readily detected during the entire treatment period in the T, H, and H+T groups, and at 3 days and 8 days in the H group (Fig. 10). Densitometric analysis further revealed a significant (P < 0.05) increase in cytosolic accumulation of cytochrome c (Fig. 11A) in all treatment groups, and DIABLO (Fig. 12B) in all but the H group, where the values began to decrease at 8 days before declining to pretreatment levels by 28 days posttreatment.
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Involvement of the FAS Signaling System in Germ Cell Apoptosis Triggered by Mild Testicular Hyperthermia and Deprivation of Intratesticular T either Alone or in Combination
To evaluate the involvement of the FAS signaling system in testicular germ cell apoptosis in monkeys, we further examined the FAS and FASLG levels in testicular lysates after treatment with heat, T, or both by Western blotting. As shown in Figure 12A, no obvious alteration in FASLG levels was detected after activation of apoptosis by these stimuli. Although there appeared to be an increase in FAS levels at 28 days in the T group and at 3 days in the H and H+T groups (Fig. 12B) compared to controls, this increase was not statistically significant, as revealed by densitometric analysis (data not shown). To further explore the potential involvement of the FAS signaling system during male germ cell apoptosis, we determined the activation of caspase 8, the key executioner caspase in the FAS-FASLG pathway. Caspase 8 was activated, as evidenced by the presence of cleaved (p20) caspase 8 even in untreated testes. No additional increase in active caspase 8 was noted after H, T, or H+T treatment.
In earlier studies, we carefully defined the apoptotic pathway form male germ cell death in rats and mice [12–16], but have not shown whether the murine data will also hold true for primates. Since multiple, large-volume testicular biopsies are harder to obtain in humans, in this study we characterized the key apoptotic pathways during male germ cell death in monkeys. Our data constitute the first demonstration that the additive effects of the combination of hormonal treatment, such as T, and a physical agent, such as testicular heat exposure, on germ cell apoptosis [4] appear to be mediated by the same signal transduction pathway in monkeys. These results indicate that the activation of germ cell apoptosis under different contraceptive regimens is highly conserved across species.
In concert with our previous results in murine models [12–16], here we show that induction of male germ cell apoptosis in monkeys by testicular hyperthermia, exogenously administered T, or both is associated with cytosolic translocation of mitochondrial cytochrome c and DIABLO, the essential components of the mitochondria-dependent intrinsic pathway signaling [8–11]. It is pertinent to note here that, in certain types of apoptosis involving FAS-mediated death signaling, proper activation of effector caspases depends on caspase 8-mediated cleavage of a proapoptotic BCL2 family member, BID, and subsequent release of cytochrome c, which in turn, results in caspase 9 activation via apoptosome formation [8]. Thus, we cannot rule out the possibility that caspase 8 activation, through cleavage of BID, could also induce cytochrome c release. This possibility merits further investigation.
The release of cytochrome c from mitochondria most likely initiates caspase activation and germ cell apoptosis in monkeys by binding to APAF1 [8, 26]. Indeed, in our earlier studies in murine models of testicular hyperthermia [12, 13, 16] or hormone withdrawal [14], we found that cytochrome c release from mitochondria into the cytosol is associated with activation of the initiator caspase 9 and the executioner caspase 3. The IAPs and their counteraction by the mitochondrial protein, DIABLO, have also emerged as important regulators of caspase activation and apoptosis in various cell systems [10, 11, 27–30]. Of note, the expression of DIABLO mRNA [10, 11] as well as the protein [31] have been found to be the most abundant in the adult testis. We also found DIABLO release from mitochondria to the cytosol after induction of germ cell apoptosis in monkeys by a variety of signals. These results, together with our previous findings of DIABLO release during germ cell apoptosis in rodent testis [14, 16, 27], thus suggest that DIABLO release from mitochondria is a general feature of male germ cell apoptosis across species.
The BCL2 family of proteins governs the mitochondria-dependent pathway for apoptosis [8, 9, 32, 33]. A novel finding was that in monkeys, unlike our rat model [14], the levels of BCL2 increased, while that of BAX remained unchanged after induction of apoptosis induced by H, T, or H+T. However, the present finding is consistent with an earlier study showing increased myocyte apoptosis in the decompensated human heart, in spite of the enhanced expression of BCL2 and no change in the expression of BAX [34]. The enhanced expression of BCL2 in the absence of any changes in the levels of BAX could imply possible loss of its antiapoptotic function. A growing body of evidence indicates that serine phosphorylation of BCL2 leads to its inactivation and its ability to form dimers with BAX, and therefore results in the loss of its antiapoptotic function [20, 22, 35, 36]. Indeed, in the present study, we found increased levels of the serine-phosphorylated form of the inactive BCL2 in all treatment groups, as evidenced by immunoblotting and immunocytochemistry. Most notably, our immunocytochemical analysis further revealed an increase in phospho-BCL2 immunoreactivity in germ cells compared with controls, in which no such staining was detected. Consistent with the involvement of MAPK1/3 and MAPK14 signaling in phosphorylation of BCL2 [23–25], here we show activation of both MAPK1/3 and MAPK14 in male germ cell apoptosis in monkeys. It is conceivable that the signal for activating mitochondria-dependent pathway during apoptosis emanates from MAPK1/3- and MAPK14 K-mediated inactivation of BCL2 through phosphorylation. In contrast to its role in cell growth in nongonadal cell systems [37, 38], the present finding suggests a proapoptotic role of MAPK1/3 by provoking BCL2 phosphorylation, leading to its inactivation, thereby resulting in the perturbation of the BAX/BCL2 rheostat and the subsequent activation of the mitochondria-dependent death pathway. This may help to explain our earlier, seemingly paradoxical observation of increased heat-induced testicular germ cell apoptosis in rats in spite of enhanced expression of the antiapoptotic protein, BCL2 [19]. Together, these data further suggest an important corollary for apoptotic regulation of male germ cells by BCL2 family members after heat and/or T treatment.
In summary, the present study further underscores the importance of the mitochondria-dependent apoptotic pathway in heat- and/or hormone deprivation-induced germ cell apoptosis in monkeys. Elucidation of the mechanisms by which a physical agent alone, or in combination with a well-studied hormone-based contraceptive, induces germ cell death in a nonhuman primate model will fill a major gap in our knowledge of how the apoptotic program is controlled in the testis. We believe that defining these pathways may lead to novel targets that may be used in the future for regulation of male fertility or treatment for male infertility.
FOOTNOTES
1Supported by grants from the Mellon Reproductive Biology Center to R.S.S, C.W, Y.H.L, and A.P.S.H., National Institutes of Health grant RO1 HD39293 to A.P.S.H., R.S.S., and C.W., Major Research Plan Project grant 2006 CBOF 1001, "973" project grant 2006 CB 504001, Chinese Academy of Sciences, Chuangxi program grant KSCA2-YW-R-55, and National Nature Science Foundation of China grant 30230190. 
Correspondence: 2Christina Wang, General Clinical Research Center, Box 16, Harbor-UCLA Medical Center, 1 000 West Carson St., Torrance, CA 90509-2910. FAX: 310 533 6972; e-mail: wang{at}labiomed.org
Received: 3 November 2006.
First decision: 26 December 2006.
Accepted: 21 March 2007.
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