Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis.

Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development.

Wnt/ß-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components.

Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of ß-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.

Wt1 deficiency causes undifferentiated spermatogonia accumulation and meiotic progression disruption in neonatal mice.

Spermatogenesis is a complex process involving the regulation of multiple cell types. As the only somatic cell type in the seminiferous tubules, Sertoli cells are essential for spermatogenesis throughout the spermatogenic cycle. The Wilms tumor gene, Wt1, is specifically expressed in the Sertoli cells of the mouse testes. In this study, we demonstrated that Wt1 is required for germ cell differentiation in the developing mouse testes. At 10 days post partum, Wt1-deficient testes exhibited clear meiotic arrest and undifferentiated spermatogonia accumulation in the seminiferous tubules. In addition, the expression of claudin11, a marker and indispensable component of Sertoli cell integrity, was impaired in Wt1(-/flox); Cre-ER(TM) testes. This observation was confirmed in in vitro testis cultures. However, the basal membrane of the seminiferous tubules in Wt1-deficient testes was not affected. Based on these findings, we propose that Sertoli cells' status is affected in Wt1-deficient mice, resulting in spermatogenesis failure.

The Heat-Induced Reversible Change in the Blood-Testis Barrier (BTB) Is Regulated by the Androgen Receptor (AR) via the Partitioning-Defective Protein (Par) Polarity Complex in the Mouse.

Scrotal hypothermia is essential for normal spermatogenesis, and temporal heat stress causes a reversible disruption of the blood-testis barrier (BTB). Previous studies have shown that AR expression in primary monkey Sertoli cells (SCs) was dramatically reduced after temporary heat treatment. However, the mechanisms underlying the heat-induced reversible disruption of the BTB, including whether it is directly regulated by the AR, remain largely unknown. In this study, we demonstrated that the AR acts upstream to regulate the heat-induced reversible change in the BTB in mice. When the AR was overexpressed in SCs using an adenovirus, the heat stress-induced down-regulation of BTB-associated proteins (Zonula occludens-1 (ZO-1), N-Cadherin, E-Cadherin, α-Catenin, and ß-Catenin) was partially rescued. AR knockdown by RNAi or treatment with flutamide (an AR antagonist) in SCs inhibited the recovery of BTB-associated protein expression after 43°C heat treatment for 30 min. The results of an in vivo AR antagonist injection experiment further showed that the recovery of BTB permeability induced by temporal heat stress was regulated by the AR. Furthermore, we observed that the co-localization and interactions of partitioning-defective protein (Par) 6-Par3-aPKC-Cdc42 polarity complex components were disrupted in both AR-knockdown and heat-induced SCs. AR overexpression in SCs prevented the disruption of these protein-protein interactions after heat treatment. AR knockdown or treatment with flutamide in SCs inhibited the restoration of these protein-protein interactions after heat treatment compared with heat treatment alone. Together, these results demonstrate that the AR plays a crucial role in the heat-induced reversible change in BTB via the Par polarity complex.

Notch signaling is involved in ovarian follicle development by regulating granulosa cell proliferation.

Notch signaling is an evolutionarily conserved pathway, which regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Notch signaling are expressed in mammalian ovaries, but the exact functions of this pathway in follicle development is still unclear. In this study, primary follicles were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). We found that the cultured follicles completely stopped developing after L-658,458 and DAPT treatment, most of the granulosa cells were detached, and the oocytes were also degenerated with condensed cytoplasma. Further studies demonstrated that the proliferation of granulosa cells was dependent on the Notch signaling. L-658,458 and DAPT treatment inhibited proliferation of in vitro cultured primary granulosa cells and decreased the expression of c-Myc. Lentivirus mediated overexpression of Notch intracellular domain 2, and c-Myc could promote the proliferation of granulosa cells and rescue the growth inhibition induced by L-658,458 and DAPT. In conclusion, Notch signaling is involved in follicular development by regulating granulosa cell proliferation.

Signal pathway of GnRH-III inhibiting FSH-induced steroidogenesis in granulosa cells.

Gonadotrophin-releasing hormone type 1 and type 2 have been demonstrated to inhibit follicle-stimulating hormone (FSH)-induced granulosa cell (GC) steroidogenesis. A third type of GnRH (GnRH-III) was also purified from salmon, its action on the FSH-regulated GC function, however is not clear. In the present study we demonstrated that the FSH-induced estrogen and progesterone production in cultured DES-treated GCs was significantly inhibited by GnRH-III. Furthermore, the FSH-stimulated steroidogenic acute regulatory protein and the enzymes for steroidigenesis, such as HSD3B2,aromatase and cytochrome P450 side-chain cleavage were also significantly suppressed by this peptide. The inhibitory action of GnRH-III on the FSH-induced steroidogenenisis was demonstrated via Akt and p38 mitogen-activated protein kinase signaling pathways through suppressing its own receptor expression. Further studies indicated that FSH could stimulate NR5A2 and upstream stimulatory factor (USF) activation, and their induction was significantly suppressed by the GnRH-III. Therefore, it is suggested that GnRH-III inhibiting FSH-induced steroidogenenisis in GCs might be by suppressing FSH-induced its own receptor expression via NR5A2 and USF transcriptional factors.

A novel testis-specific Na+/H+ exchanger is involved in sperm motility and fertility.

Sodium-hydrogen exchanger as a channel for regulation of intracellular pH might be a crucial modulator of sperm capacitation and motility. Three members of this family have been identified in spermatozoa. A novel protein testis-specific sodium-hydrogen exchanger named mtsNHE was cloned in the present study. The mtsNHE localizing on principle piece of sperm flagellum contained 12 predicted transmembrane regions without cytoplasmic fragment at carboxyl terminus. Hydrophilic region was common in the sodium-hydrogen exchanger family members. Polyclonal antibodies to trans-membrane region significantly reduced sperm motility, acrosome reaction and ratio of in vitro fertilization. By in-pouring the antibodies in sperm solution, intracellular pH and calcium concentration were decreased. Muscle injection of female mice with the specific gene vaccine of mtsNHE, significantly stepped down fertility rate. Considering its specific expression and involvement in the regulation of fertility, the mtsNHE might be a potential target molecule for developing a new male contraceptive.

Testosterone induces redistribution of forkhead box-3a and down-regulation of growth and differentiation factor 9 messenger ribonucleic acid expression at early stage of mouse folliculogenesis.

Increasing evidence has shown that excess androgen may be a main cause of polycystic ovary syndrome (PCOS). However, the molecular mechanism of androgen action on the ovary is unclear. To investigate the possible impacts of androgen on early follicular development, neonatal mouse ovaries mainly containing primordial follicles were cultured with testosterone. We demonstrated that the number of primary follicles was increased after 10 d culture with testosterone treatment via phosphatidylinositol 3-kinase/Akt pathway. Androgen induced Forkhead box (Foxo)-3a activation, and translocation of Foxo3a protein from oocyte nuclei to cytoplasm, which might be a key step for primordial follicle activation. Interestingly, testosterone was also capable of down-regulating growth and differentiation factor-9 expression via its receptor. In summary, we infer that intraovarian excess androgen in PCOS might result in excess early follicles by inducing oocyte Foxo3a translocation and follicular arrest by down-regulating growth and differentiation factor-9 expression.

Immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger by oral feeding or nasal instillation reduces fertility in female mice.

OBJECTIVE: To investigate the effect of immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger (tsNHE) via oral feeding or nasal instillation on fertility in female mice and to look at its potential mechanism. DESIGN: Prospective, research study. SETTING: Institution-affiliated research laboratory. ANIMAL(S): Sexual mature BALB/c mice. INTERVENTION(S): Female mice immunized orally or nasally with the DNA vaccine at 2-week' intervals. MAIN OUTCOME MEASURE(S): Number of newborns and fertility rate of the vaccinated female mice were scored. RESULT(S): We identified a novel testis-specific sodium-hydrogen exchanger, tsNHE, which is localized to the principal piece of sperm flagellum. Immunization of female mice with the tsNHE DNA vaccine via oral feeding or nasal instillation statistically significantly decreased fertility rate and the newborn numbers compared with the controls. The antiserum or vaginal fluid from the tsNHE cDNA vaccinated female mice could specifically recognize the principal piece of sperm tail and triggered sperm agglutination. The antibodies also showed a statistically significant inhibitory effect on in vitro sperm motility and fertilization. CONCLUSION(S): The sodium-hydrogen exchanger might be an excellent target molecule for developing a new contraceptive.

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 Afaf's 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.

Male germ cell-specific protein Trs4 binds to multiple proteins.

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners.

Increased expression of heat shock protein 105 in rat uterus of early pregnancy and its significance in embryo implantation.

BACKGROUND: Heat shock proteins (Hsps) are a set of highly conserved proteins, Hsp105, has been suggested to play a role in reproduction. METHODS: Spatio-temporal expression of Hsp105 in rat uterus during peri-implantation period was examined by immunohistochemistry and Western blot, pseudopregnant uterus was used as control. Injection of antisense oligodeoxynucleotides to Hsp105 into pregnant rat uteri was carried out to look at effect of Hsp105 on embryo implantation. RESULTS: Expression of Hsp105 was mainly in the luminal epithelium on day 1 of pregnancy, and reached a peak level on day 5, whereas in stroma cells, adjacent to the implanting embryo, the strongest expression of Hsp105 was observed on day 6. The immunostaining profile in the uterus was consistent with that obtained by Western blot in the early pregnancy. In contrast, no obvious peak level of Hsp105 was observed in the uterus of pseudopregnant rat on day 5 or day 6. Furthermore, injection of antisense oligodeoxynucleotides to Hsp105 into the rat uterine horn on day 3 of pregnancy obviously suppressed the protein expression as expected and reduced number of the implanted embryos as compared with the control. CONCLUSION: Temporal and spatial changes in Hsp105 expression in pregnant rat uterus may play a physiological role in regulating embryo implantation.

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.

Mitogen-activated protein kinase regulates FSH-induced expression of tissue-type plasminogen activator through an activator protein 1 response element.

We have analyzed a possible role of mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) in the regulation of FSH-induced tissue type plasminogen activator (tPA) production in granulosa cells (GCs) prepared from DES-treated immature rats; Treatment of the cells in the presence of FSH with MAPK inhibitors, such as UO126 or SB203580, significantly decreased the FSH-induced tPA production, suggesting that multiple signaling pathways may be involved in FSH-regulated tPA expression. We further examined possible signaling action involved in FSH-activated ERK1/2 and p38 MAPK on tPA production, and observed that FSH receptor occupancy led to both ERK1/2 and p38 MAPK phosphorylation. Such action might be through a protein kinase A-dependent pathway because the observed activation was destroyed by the addition of its specific inhibitor H89 to the culture. The inhibition of ERK1/2 and p38 MAPK activation by their specific inhibitors remarkably reduced FSH-induced tPA mRNA and its protein production. We further examined whether AP-1 located in the tPA promoter is involved in FSH-regulated tPA production, and demonstrated that FSH significantly stimulated AP-1 expression, whereas inclusion of H89, UO126, or SB20358 in the culture significantly decreased FSH-induced AP-1 expression. In summary, FSH-induced ERK1/2 and p38 MAPK activation is capable of regulating tPA production in cultured primary GCs, and that the transcript factor AP-1 may be important in the regulation of FSH-induced tPA expression.

Role of caspase 2 in apoptotic signaling in primate and murine germ cells.

This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (T(e)) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (H(e)) alone group and by Day 8 in the Te alone group but peaked at Day 3 in H(e) + T(e) 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.

Effect of heat stress on expression of junction-associated molecules and upstream factors androgen receptor and Wilms' tumor 1 in monkey sertoli cells.

Sertoli cells are important in determining the fate of spermatogenic cells by providing nutrition and structural support via cell junctions. In this study, we sought to examine the effect of 43 C warming on cell junctions in seminiferous epithelium and the expression of junction-associated molecules in Sertoli cells. Electron microscopy showed the appearance of large vacuoles between Sertoli and germ cells and adjacent Sertoli cells, leading to disruption of corresponding cell junctions 24 h after terminating the heat treatment. Using primary Sertoli cells isolated from pubertal monkey testes, we demonstrated that expression of adherens junction-associated molecules, such as N-cadherin and beta-catenin, and tight junction-associated molecule zonula occludens protein 1 was significantly reduced in 24-48 h after heat treatment. In contrast, intermediate filament vimentin expression was up-regulated in 6-48 h. Androgen receptor (AR) and Wilms' tumor gene 1 expression dramatically decreased after heat treatment. Both proteins completely disappeared immediately after terminating heat treatment and began to recover after 6 h. Treatment of the monkey Sertoli cells with an AR antagonist, flutamide, could mimic the heat-induced changes in the expression of junction-associated molecules in Sertoli cells. Furthermore, overexpression of AR in the Sertoli cells up-regulated the expression of N-cadherin, beta-catenin, and zonula occludens protein 1 and down-regulated vimentin expression. Their expression after heat treatment could be rescued by the AR overexpression. These results indicate that the decreased AR expression after heat treatment is involved in heat-induced cell junction disruption.

Effect of 43 degrees treatment on expression of heat shock proteins 105, 70 and 60 in cultured monkey Sertoli cells.

AIM: To examine the possible effect of heat treatment on expression of heat shock proteins (Hsps) 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. METHODS: Western blot analysis, real-time polymerase chain reaction (PCR), and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43 degrees treatment of Sertoli cells isolated from pubertal monkey testes. RESULTS: Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hsp105 was expressed in cytoplasm of untreated Sertoli cells. Both Hsp105 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase (ERK) 1/2 inhibitor (U0126) or phosphoinositide kinase-3 (PI3K) inhibitor (LY294002) could partially block the response of Hsp105 and Hsp70 induced by heat treatment. CONCLUSION: These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.

Testosterone upregulation of tissue type plasminogen activator expression in Sertoli cells : tPA expression in Sertoli cells.

Our previous studies have demonstrated that tissue type plasminogen activator (tPA) might be involved in matrix degradation of blood-testis barrier in rat. In this study, we have further investigated the effect of testosterone (T) on tPA production in rat Sertoli cells. Our results showed that Sertoli cells isolated from rat testes at various ages in vitro secreted tPA in an age-dependent manner. The tPA activity was detected on day 20 after birth, and reached maximum on day 60. The Sertoli cells isolated from the testes on day 20 were then cultured in the presence or absence of testosterone, FSH, and forskolin, the tPA activities were upregulated by T, FSH and forskolin. Addition of H89 or U0126, both inhibited the testosterone-, FSH-, and forskolin-induced tPA expression. It is suggested that FSH- and testosterone-stimulated tPA expression in Sertoli cells may be via PKA and ERK signal transduction. Furthermore, we have observed that testosterone stimulated tPA secretion at all the stages of spermatogenesis (II-VI, VII-VIII, IX-XII and XIII-I), the highest stimulation of tPA activity was observed at stages VII-VIII. This study further suggests that testosterone-induced tPA activity in the Sertoli cells might be related to the function of blood-testis barrier opening and/or closing.

Signaling pathways for germ cell death in adult cynomolgus monkeys (Macaca fascicularis) induced by mild testicular hyperthermia and exogenous testosterone treatment.

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 degrees 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.

Heat treatment induces liver receptor homolog-1 expression in monkey and rat sertoli cells.

We demonstrated in this study that liver receptor homolog-1 (LRH-1) was expressed in the round spermatids in normal monkey testis, and no LRH-1 signal was observed in the Sertoli cells. After local warming (43 C) the monkey testis, however, LRH-1 expression was induced in the Sertoli cells in coincidence with activation of cytokeratin 18 (CK-18), a Sertoli cell dedifferentiated marker. Furthermore, we isolated rat primary Sertoli cells from testes at various stages of development and treated with 43 C water in vitro. The changes in LRH-1 as well as CK-18 expression were analyzed by confocal immunohistochemistry and Western blot. The results showed that LRH-1 was stage-dependently expressed in the Sertoli cells; no LRH-1-positive signal was detected in the cells obtained from the testes of adult rat on d 60 after birth when mature spermatozoa in the testis was completed. However, the mature Sertoli cells were warmed at the 43 C water bath for 15 min, and the LRH-1 signal was remarkably induced in a time-dependent manner, just like the changes of CK-18 expression in the Sertoli cells, suggesting that the heat-induced dedifferentiation of the mature Sertoli cells might be related to LRH-1 regulation. LRH-1 expression induced by the heat treatment was completely inhibited by the addition of ERK inhibitor U0126 in the culture, indicating that the heat-induced LRH-1 expression in the Sertoli cells may be regulated via ERK1/2 activation pathway. Testosterone was found to have no such effect on LRH-1 expression in the monkey and rat Sertoli cells.