Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress.

The present study was designed to clarify the role of radical oxygen species in testicular germ cell apoptosis induced by heat stress. Testicular cells isolated from immature rats were cultured with or without elevated temperature, and occurrence of apoptosis in these cells was defined by the appearance of DNA fragmentation following agarose gel electrophoresis and by flow cytometric quantification of apoptotic cells. At 32.5 degrees C, < 1% of cells showed signs of apoptosis throughout the culture period, whereas under heat stress, the proportion of apoptotic cells increased to 5% at 37 degrees C after 24 h of culture, or to 14% after 1-h exposure at 43 degrees C followed by 23-h culture at 32.5 degrees C. Similar to the effect of heat stress, exogenously supplied oxygen free radicals also induced apoptosis. In contrast, treatment with catalase significantly attenuated heat stress-induced apoptosis. Furthermore, heat stress of testicular cells was associated with an increased intracellular peroxide level as measured by a fluorescent probe, 2', 7'-dichlorofluorescin diacetate. In conclusion, our data indicate the involvement of radical oxygen species during testicular germ cell apoptosis induced by heat stress. This study provides a useful in vitro model for the study of testicular germ cell apoptosis.     

Calpain inhibitors prevent p38 MAPK activation and germ cell apoptosis after heat stress in pubertal rat testes

Testicular injuries like torsion or cryptorchidism can cause massive germ cell death, which could have great impact on male reproductive health. In addition, it has been proposed that modern life style, in the form of underwear or sedentary work position, could increase the testicular temperature, induce germ cell apoptosis, reduce spermatozoa quality and promote male infertility. In this work we showed that a heat stress stimulus induced massive germ cells apoptosis, which was associated with p38 mitogen-activated protein kinase (MAPK) phosphorylation along with an increase in the levels of mRNA encoding calpain 2. Synthetic calpain inhibitors prevented heat stress-induced germ cell apoptosis through inhibition of p38 MAPK phosphorylation. Thus, our results indicate that the blockage of calpains suppresses p38 MAPK phosphorylation, and identifies calpain activation (most likely calpain 2) as an early event in heat stress-induced male germ cell apoptosis. J. Cell. Physiol. 221: 296–305, 2009. © 2009 Wiley-Liss, Inc.     

HSF2 mRNA在热应激和大剂量11酸睾酮诱导恒河猴生精细胞凋亡中的表达变化

为了探讨HSF2 mRNA在热应激和超生理剂量睾酮诱导恒河猴生精细胞凋亡中的表达变化,我们建立了手术诱导单侧隐睾和注射大剂量11酸睾酮（TU）恒河猴动物模型,应用3′末端标记分析（TUNEL）和原位杂交方法,检测睾丸细胞的凋亡信号和HSF2的表达变化。TUNEL结果显示热应激和超生理剂量睾酮能够诱导生精细胞出现凋亡信号,它分别于处理后第5天和第30天达到最强,表明热应激和睾酮干扰精子发生可能是通过生精细胞凋亡的方式来实现的。HSF2 mRNA水平在生精细胞凋亡早期（凋亡信号达到最强以前）略有降低,而在凋亡高峰期之后其表达急剧下降。Hsf2基因与我们以前研究的Hsp70-2基因的表达具有时间上的相关性,表明HSF2蛋白可能调控Hsp70-2基因的表达,而且HSF2可能通过多种方式影响精子的发生以及抑制生精细胞的凋亡。     

Chemical anoxia delays germ cell apoptosis in the human testis

An understanding of testicular physiology and pathology requires knowledge of the regulation of cell death. Previous observation of suppression of apoptosis by hypoxia suggested a role for ATP in germ cell death. However, the exact effects of ATP production on germ cell death and of apoptosis on the levels of ATP and other adenine nucleotides (ANs) have remained unclear. We investigated the levels of ANs during human testicular apoptosis (analyzed by HPLC) and the role of chemical anoxia in germ cell death (detected by Southern blot analysis of DNA fragmentation, in situ end labeling of DNA, and electron microscopy). Incubation of seminiferous tubule segments under serum-free conditions induced apoptosis and concomitantly decreased the levels of ANs. Chemical anoxia, induced with potassium cyanide (KCN), an inhibitor of mitochondrial respiration, dropped ATP levels further and suppressed apoptosis at 4 h. After 24 h, many of the testicular cells underwent delayed apoptosis despite ATP depletion. Some cells showed signs of necrosis or toxicity. The addition of 2-deoxyglucose, an antimetabolite of glycolysis, did not alter the results obtained with KCN alone, whereas a toxic concentration of hydrogen peroxide switched apoptosis to necrosis. In most of the testicular cells, mitochondrial respiration appears to play a crucial role in controlling primary cell death cascades. In the human testis, there seem to be secondary apoptotic pathways that do not require functional respiration (or ATP).     

Apoptosis and cell removal in the cryptorchid rat testis

In order to determine that apoptosis is responsible for large-scale germ cell elimination, we analyzed cells from cryptorchid testes both in histological sections and among those isolated in vitro. Apoptotic testicular cells during 3 to 7 days were only 8 to 30%, reaching a maximum of 80% by the end of 15 days of cryptorchidism. A similar trend was also observed with the number of dead cells. The process of large-scale germ cell removal in the initial stages was facilitated by the formation of multinucleated giant cells, which stained negative for apoptosis. Increase in oxidative stress and decrease in intratesticular testosterone was also observed. The above findings indicate that large-scale germ cell removal, at least during initial stages of cryptorchidism is not solely as a result of apoptosis. Declined intra testicular testosterone, elevated temperature and high oxidative stress following cryptorchidism probably affect cell viability and trigger a fast pace cell removal through giant cell formation.     

Role of UCH-L1/ubiquitin in acute testicular ischemia-reperfusion injury

The most significant complication of testicular torsion is loss of the testis, which may lead to impaired fertility. Molecular mechanisms how spermatogenesis impairs owing to testicular torsion remain unknown. This investigation, by using mouse model of testicular torsion, was undertaken to gain insight into the cellular and molecular mechanism underlying torsion-induced germ cell loss. Male mice were subjected to 2 h ischemia-inducing torsion, and testes were examined at 24, 48, and 72 h after the repair of torsion (reperfusion). Ischemia-reperfusion (IR) of the testes resulted in germ cell, mostly in spermatogonia, apoptosis, which was revealed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) technique. At 24 h after torsion repair germ cell apoptosis reached peak, then decreased until 72 h repair. Western blots showed that apoptotic proteins (p53, Caspase-3 and -9) gradually were upregulated at 48 h reperfusion, however, anti-apoptotic proteins (Bcl-2 and BDNF) were downregulated in the relevant IR treatment. IR injury induced CHOP protein appearance with maximum expression at 24 h of reperfusion. Furthermore, the germ cell apoptosis triggered downregulation of ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) at both mRNA and protein levels. To test further whether ubiquitination was involved in IR stress, both mono- and poly-ubiquitin levels in IR stress condition were examined, which showed that both mono- and poly-ubiquitin expression significantly impaired. These results provide evidences of UCH-L1/ubiquitination signaling to the testis IR injury in vivo.     

HST-1/FGF-4 protects male germ cells from apoptosis under heat-stress condition

Apoptosis plays an important role in controlling the number of male germ cells and eliminating defective germ cells during testicular development and spermatogenesis. We show here that fibroblast growth factor-4 (HST-1/FGF-4) may play a critical role as a survival factor for germ cells, protecting them from apoptosis. Testes of adult male mice that received an adenovirus carrying human HST-1/FGF-4 (AxHST-1) or a control adenovirus (AxCAwt) were exposed to mild hyperthermia, which causes germ cell apoptosis. An in situ terminal-deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling (TUNEL) assay characterized germ cell apoptosis. The results indicated that HST-1/FGF-4 significantly reduced the apoptotic death of germ cells and prevented testicular weight loss and sperm count reduction. We also found that Hst-1/Fgf-4 present in testes is up-regulated in vivo when the testes are exposed to mild hyperthermia, and that endogenous Hst-1/Fgf-4 mRNA expression in Sertoli cells are also induced when the cells are exposed to mild hyperthermia in vitro. In addition, the MAPK cascade, which could increase an FGF-dependent survival signal, is activated by HST-1/FGF-4 stimuli in germ cells. On the other hand, upon HST-1/FGF-4 stimulation, lactate production from Sertoli cells were induced, which is indispensable nutrient for germ cell survival. These results suggest that HST-1/FGF-4 can act as an important physiological anti-apoptotic factor for male germ cells in stimulating lactate production of Sertoli cells upon heat stress, thereby promoting germ cell survival.     

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.     

Uncoupling protein 2 protects testicular germ cells from hyperthermia-induced apoptosis

The mRNA of the mitochondrial uncoupling protein 2 (UCP2) was up-regulated by cryptorchidism, a testicular hyperthermic condition under which germ cells undergo severe apoptosis. We investigated whether UCP2 was able to protect germ cells from hyperthermia-induced apoptosis. UCP2 was predominantly present in elongate spermatids under normal conditions, and was detected in all germ cells with its level significantly increased if the testes were exposed to 43 degrees C for 5 min. Such a short heat exposure was non-lethal and enabled the preconditioned cells to be resistant to apoptosis induced by a longer hyperthermic treatment (15 min). While hyperthermia resulted in oxidative stress in mouse testes, it did not change the total anti-oxidative capacity. Indeed, overexpression of UCP2 in the GC-2 germ cell line protected the cells from radical oxygen species (ROS)-induced apoptosis. Taken together, we propose that UCP2 may represent an effective weaponry used by germ cells to combat ROS-induced apoptosis.     

Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion

Background

Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO.

Objective

The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion.

Method and Results

EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect.

Conclusions

We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress inducing ST damage and interfering in Leydig cell function.     

P16蛋白和生精细胞凋亡对热压和11酸睾酮诱导的恒河猴无精子症和少精子症中的作用

探讨了P16蛋白和生精细胞凋亡在热压和11酸睾酮诱导恒河猴无精子症和少精子症中作用间的关系。3′末端标记分析（TUNEL）结果显示热应激和超生理剂量睾酮能够诱导生精细胞出现凋亡信号,它分别于处理后第5天和第30天达到最强,免疫组化结果显示,热压或TU主要诱导精原细胞和其它生精细胞以及Sertoli细胞P16的表达。P16蛋白的表达在生精细胞凋亡晚期,即隐睾手术第10天或注射TU第60天后迅速升高并维持在热压或11酸睾酮诱导的早期精母细胞和精子细胞的凋亡和在晚期对精原细胞有丝分裂的抑制,二者共同作用导致热压或TU诱导的恒河猴无精子症和少精子症。     

Whole‐body heat exposure causes developmental stage‐specific apoptosis of male germ cells

Humans are occasionally exposed to extreme environmental heat for a prolonged period of time. Here, we investigated testicular responses to whole‐body heat exposure by placing mice in a warm chamber. Among the examined tissues, the testis was found to be most susceptible to heat stress. Heat stress induces direct responses within germ cells, such as eukaryotic initiation factor 2α phosphorylation and stress granule (SG) formation. Prolonged heat stress (42°C for 6 hr) also disturbed tissue organization, such as through blood‐testis barrier (BTB) leakage. Germ cell apoptosis was induced by heat stress for 6 hr in a cell type‐ and developmental stage‐specific manner. We previously showed that spermatocytes in the early tubular stages (I–VI) form SGs for protection against heat stress. In the mid‐tubular stages (VII–VIII), BTB leakage synergistically enhances the adverse effects of heat stress on pachytene spermatocyte apoptosis. In the late tubular stages (IX–XII), SGs are not formed and severe leakage of the BTB does not occur, resulting in mild apoptosis of late‐pachytene spermatocytes near meiosis. Our results revealed that multiple stress responses are involved in germ cell damage resulting from prolonged heat stress (42°C for 6 hr).     

环境高温促进猪睾丸Apaf-1和Caspase-9的表达

高温热应激条件下,凋亡蛋白表达量升高,生殖细胞凋亡增加。凋亡蛋白酶活化因子1（apoptosis protease activating factor 1,Apaf-1）和凋亡蛋白酶活化起始者含半胱氨酸的天冬氨酸蛋白水解酶9,(cysteine aspartic acid specific protease 9, Caspase-9）是细胞凋亡内源途径中的重要调节蛋白,热应激条件下猪睾丸Apaf-1和Caspase 9的表达未见报道。本研究发现,夏季畜舍高温使Apaf-1和Caspase-9表达量升高。qRT-PCR和Western印迹结果显示,与对照组（正常舍温20℃）相比,短时热应激组（40～42℃,1 h/d, 7 d）和长时热应激组（40～42℃,1 h/d, 42 d）,Apaf-1和Caspase-9 mRNA和蛋白的相对表达量均显著升高。免疫组织化学研究发现,Apaf-1在猪睾丸组织中免疫反应阳性物定位于间质细胞、支持细胞和各个发育阶段生精细胞。热应激处理导致精母细胞和精子细胞Apaf-1表达量升高。在各实验猪睾丸组织中,Caspase-9定位于间质细胞、支持细胞和各个发育阶段生精细胞的胞质中。与对照组相比,热应激处理导致减数分裂以后的生精细胞和支持细胞Caspase-9表达量升高。上述结果表明,高温热应激促进Apaf-1和Caspase-9的表达,提示Apaf-1和Caspase-9表达的变化可能与猪舍高温导致的猪精液品质下降存在关联。     

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.     

Deciphering the pathways of germ cell apoptosis in the testis

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 °C for 15 min) of the testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr^cg mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.     

Effects of 60 Hz 14 microT magnetic field on the apoptosis of testicular germ cell in mice

We recently reported that continuous exposure, for 8 weeks, of extremely low frequency (ELF) magnetic field (MF) of 0.1 or 0.5 mT might induce testicular germ cell apoptosis in BALB/c mice. In that report, the ELF MF exposure did not significantly affect the body weight or testicular weight, but significantly increased the incidence of testicular germ cell death. In the present study, we aimed to further characterize the effect of a 16-week continuous exposure to ELF MF of 14 or 200 microT on testicular germ cell apoptosis in mice. There were no significant effects of MF on body weight and testosterone levels in mice. In TUNEL staining (In situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling), germ cells showed a significantly higher apoptotic rate in exposed mice than in sham controls (P < 0.001). TUNEL-positive cells were mainly spermatogonia. In an electron microscopic study, degenerating spermatogonia showed condensation of nuclear chromatin similar to apoptosis. These results indicate that apoptosis may be induced in spermatogenic cells in mice by continuous exposure to 60 Hz MF of 14 microT.     

hCG treatment raises H<Subscript>2</Subscript>O<Subscript>2</Subscript> levels and induces germ cell apoptosis in rat testis

The clinical significance of exogenous hCG treatment is to stimulate steroidogenesis and spermatogenesis in the testis. However, the pathogenesis of detrimental effects on the testis arising out of chronic hCG treatment is yet to be clearly ascertained. In the present study we have shown that hCG treatment (100 IU/day) to rats for 30 days raises testicular oxidative stress leading to germ cell apoptosis and impairment of spermatogenesis. The treatment raises testicular H₂O₂ levels along with increase in lipid peroxidation and concomitant decrease in the enzymatic antioxidant activities like superoxide dismutase, catalase and glutathione-s-transferase. The rise in the number of apoptotic germ cells was associated with up regulation of Fas protein expression and caspase-3 activity in the testis. However, serum testosterone which was elevated by 15 days of hCG treatment declined to pretreatment levels by 30 days. No significant alteration in serum gonadotropins was observed. The above findings indicate that the pathogenesis of deleterious effects following chronic hCG treatment is due to increase in testicular oxidative stress with high H₂O₂ availability leading to apoptosis among germ cells.     

Alteration in expression of estrogen receptor isoforms alpha and beta, and aromatase in the testis and its relation with changes in nitric oxide during aging in mice

The aim of present study was to investigate the changes in the testicular expression of aromatase, ER alpha, ER beta and iNOS protein and correlate these with serum testosterone and nitric oxide levels, to elucidate the role of estrogen and nitric oxide in the testis during aging. This study showed localization of aromatase and ER alpha mainly in the Leydig cell and showed close correlation of testicular aromatase level with circulating testosterone level suggesting that estrogen may be modulating testicular steroidogenesis. Localization ER alpha mainly in the mitotically active germ cell suggest possible role of estrogen in germ cell proliferation. This study showed basal level of nitric oxide during reproductively active period, whereas increased serum nitric oxide coincides with decreased testicular activity in old age. This study showed inverse correlation between aromatase and NO level. Treatment with either SNP or L-NAME on testicular steroidogenic factor (3-beta HSD/ StAR) or germ cell survival factor (Bcl2) showed that increased NO causes decreased steroidogenesis and increased germ cell apoptosis. In conclusion this study suggest that estrogen modulate steroidogenesis and germ cell survival in reproductively active period whereas in old age decreased estrogen concentration causes increased nitric oxide which in turn decreases testicular steroidogenesis and germ cell apoptosis.     

Estradiol treatment induces testicular oxidative stress and germ cell apoptosis in rats

In order to understand the pathogenesis of estradiol induced effects in the seminiferous epithelium, studies were undertaken in adult rats with estradiol-3-benzoate administered for different durations. After 30 d of treatment, a significant rise in lipid peroxidation with concomitant fall in the activities of superoxide dismutase and catalase was observed. Both, serum and intra-testicular testosterone levels were found severely depleted. Seminiferous epithelium was devoid of elongated spermatids and spermatozoa by 30 d of treatment. Number of spermatocytes and round spermatids were significantly (p < 0.001) reduced. Flowcytometric analysis confirmed a drastic reduction of the haploid cell population (1c peak). Beginning from day 10 of treatment, there was a consistent rise in the number of pyknotic/apoptotic germ cells in the seminiferous epithelium. A gradual increase in Bax protein expression was observed with the duration of treatment. The shift in Bax immunostaining from the cytoplasm and nucleus of germ cells (at 10 d of treatment) to only nuclei of cells by 30 d of treatment was also noticed. By this time testicular tissue showed three-fold increase in caspase-8 enzyme activity. Viable testicular cells isolated in vitro decreased drastically subsequent to different periods of estradiol treatment. The above findings substantiate the fact that the testicular pathogenesis of estradiol benzoate treatment may be primarily because of altered reproductive hormone levels and high oxidative stress leading to germ cell apoptosis and subsequent germ cell loss in the seminiferous epithelium.     

Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis.

Heat shock protein (HSP)105 is a testis-specific and HSP90-related protein. The aim of this study was to explore the functions of HSP105 in the rat testis. Signals of HSP105 were detected immunohistochemically in the germ cells and translocated from the cytoplasm to the nucleus at 2 days after experimental induction of cryptorchidism. In cultured testicular germ cells, a significant increase in the expression of HSP105 in response to heat stress (37 degrees C) was detected in the insoluble protein fractions. Several binding proteins were isolated from rat testis using a HSP105 antibody immunoaffinity column, and p53, the tumor suppressor gene product, was copurified with these. Furthermore, immunoprecipitation using antibodies to p53 led to coprecipitation of HSP105 together with p53 after culturing germ cells at 32.5 degrees C, but not at 37 or 42 degrees C. In conclusion, HSP105 is specifically localized in the germ cells and may translocate into the nucleus after heat shock. HSP105 is suggested to form a complex with p53 at the scrotal temperature, and dissociate from it at suprascrotal temperatures. At scrotal temperature, HSP105 may thus contribute to the stabilization of p53 proteins in the cytoplasm of the germ cells, preventing the potential induction of apoptosis by p53.