Yin-Chuan Li

Signaling Pathways for Germ Cell Death in Adult Cynomolgus Monkeys (Macaca fascicularis) Induced by Mild Testicular Hyperthermia and Exogenous Testosterone Treatment

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.

Role of Caspase 2 in Apoptotic Signaling in Primate and Murine Germ Cells

Abstract This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (Te) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (He) alone group and by Day 8 in the Te alone group but peaked at Day 3 in He + Te group. We found activation of caspase 2 in both germ cells and Sertoli cells after induction of apoptosis. Most notably, active caspase 2 immunoreactivity was detected only in those germ cells susceptible to apoptosis compared with controls, where little or no such staining is detected. To further explore the role of caspase 2 in regulating male germ cell death, we next evaluated the efficacy of caspase 2 inhibition in preventing or attenuating heat-induced germ cell apoptosis in rats. Caspase 2 inhibition significantly (P < 0.05) prevented such heat-induced germ cell apoptosis. The protection offered by the caspase 2 inhibitor occurred upstream of mitochondria, involving suppression of mitogen-activated protein kinase (MAPK) 14 activation and inducible nitric oxide synthase (NOS2) induction and, in turn, suppression of cytochrome c-mediated death pathway. Together, our results show that caspase 2 is activated in male germ cells undergoing apoptosis in nonhuman primates after heat stress, hormonal deprivation, or after combined interventions. Blockade of caspase 2 activation prevents heat-induced germ cell apoptosis in rats by suppressing the MAPK14- and NO-mediated intrinsic pathway signaling..

Afaf, a novel vesicle membrane protein, is related to acrosome formation in murine testis

As a cell‐specific organelle, acrosome (Acr) and its formation are an important event for spermiogenesis. However, the Acr formation is far more complicated than has been proposed. In this study, we have cloned a novel membrane protein Afaf (Acr formation associated factor) that was expressed abundantly in the round spermatids, localized in the inner and outer membrane of forming Acrs, and declined in the maturing Acrs. In the transfected Hela cells, Afaf protein was localized in the plasma membrane, EEA1‐positive early endosomes (EEs) and occasionally in the nuclei. Therefore, we propose that EEs and plasma membrane may be also directly involved in the Acr biogenesis.

Expression of Nitric Oxide Synthase During Germ Cell Apoptosis in Testis of Cynomolgus Monkey After Testosterone and Heat Treatment

This study investigates the possible involvement of nitric oxide synthase (NOS) in activating germ cell death in monkeys after mild testicular hyperthermia and/or hormonal deprivation. Groups of 8 adult male monkeys received 1 of the following treatments for 12 weeks: 1) 2 empty Silastic implants, 2) 2 testosterone (T) implants, 3) daily exposure of testes to heat (43 degrees C for 30 minutes) for 2 consecutive days, or 4) 2 T implants plus testicular heat exposure. Testicular biopsies were performed before and on days 3, 8, 28, and 84 of the treatment. In control monkey testes, endothelial NOS (eNOS) was observed mainly in Sertoli cells and spermatogonia. No obvious alteration in eNOS levels was detected in any of the treatment group as assessed by Western blotting. Induction of inducible NOS (iNOS) in testes of the 3 treated groups was detected by immunoblotting as early as day 3 after treatment compared with that of controls. Immunocytochemistry further revealed a small increase in iNOS expression in both germ cells and Sertoli cells after T treatment. However, treatment of heat or heat in combination with T markedly induced iNOS expression in germ cells. These data suggest that iNOS, but not eNOS, may be involved in monkey testicular germ cell death after heat and/or T treatment.

Acrosome formation-associated factor is involved in fertilization

OBJECTIVE To investigate the effects of a novel acrosome formation-associated factor (Afaf) on fertilization by its regulation of acrosomal exocytosis and endosomal trafficking. DESIGN Controlled laboratory study. SETTING Institution-affiliated state key laboratory. SUBJECTS ICR mice. INTERVENTION(S) Sperm penetration assay and in vitro fertilization experiment were performed to study the effects of the Afaf antibody on acrosome reaction and fertilization. Acrosome exocytosis (AE) with streptolysin O (SLO) permeabilization was conducted to test the Afafs action in calcium events. Colocalization and coimmunoprecipitation was done to determine the interaction between Afaf and SNAP25 (synaptosome-associated protein of 25,000 daltons). Transferrin (Tf) uptake assay was performed to demonstrate the impact of Afaf on endosomal pathway. RNAi was used to rescue the inhibition of Afaf on Tf uptake. MAIN OUTCOME MEASURE(S) Number of penetrated sperms, in vitro fertilization rate. Acrosomal exocytosis index, relative Tf fluorescence. RESULT(S) The Afaf antibodies were capable of significantly inhibiting sperm penetration of the eggs, therefore reducing the rate of in vitro fertilization. Acrosome formation-associated factor was involved in calcium-triggered AE by acting upstream of the calcium efflux from the acrosome inside. Acrosome formation-associated factor might exert an interaction with SNAP25, which is a crucial component in both exocytosis and endosomal trafficking. Acrosome formation-associated factor was also involved in the endocytic pathway by down-regulating Tf endocytosis in the HeLa cells, and the miRNA-mediated RNAi could rescue this alternation induced by Afaf. CONCLUSION(S) Acrosome formation-associated factor might play an important role in membrane trafficking during acrosome formation and participate in fertilization.

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.