Germ Cell Apoptosis In Testis Research Articles

PBA alleviates cadmium-induced mouse spermatogonia apoptosis by suppressing endoplasmic reticulum stress

ObjectiveEndoplasmic reticulum (ER) stress mediates Cd-caused germ cell apoptosis in testis. The effects of 4-phenylbutyric acid (PBA), a classical chaperone, were investigated on Cd-induced apoptosis in mouse GC-1 spermatogonia cells. MethodsThe cells were pretreated with PBA before Cd exposure. TUNEL and flow cytometry assays were applied to determine apoptosis. Some key biomarkers of ER stress were analyzed using RT-PCR and western blot. Resultsas expected, the apoptotic cells exposed to Cd apparently increased. The mRNA and protein expression levels of GRP78 and ATF6α, were elevated in the Cd groups. Additional experiments displayed that Cd notably increased IRE1α and JNK phosphorylation, and upregulated XBP-1 mRNA and protein expression. Moreover, p-eIF2α and CHOP expressions were clearly elevated in the Cd groups. Interestingly, PBA almost completely inhibited ER stress and protected spermatogonia against apoptosis induced by Cd. ConclusionPBA alleviated Cd-induced ER stress and spermatogonia apoptosis, and may have the therapeutic role in Cd-induced male reproductive toxicity.

Autophagy in Sertoli cell protects against environmental cadmium-induced germ cell apoptosis in mouse testes.

Cadmium (Cd) was an environmental pollutant, which could result in germ cell apoptosis in testes. Sertoli-germ cell communication was vital for germ cell development and maturity. However, little was known about the effect of Sertoli cell autophagy on Cd-induced germ cell apoptosis. Here, we used male Amh-Cre+/Atg5flox/flox (Atg5-/-) mice, loss of autophagy-related gene 5 (Atg5) in testicular Sertoli cells, to explore the obscure effects. Atg5-/- and Wild-type (WT) mice were given with cadmium chloride (CdCl2, 2.0mg/kg) for 0-24h. Our results showed that Cd triggered testicular germ cell apoptosis, as evidenced by the increment of TUNEL-labeled germ cells, cleaved caspase3 and cleaved poly (ADP-ribose) polymerase protein level. Additionally, Cd induced testicular autophagy, as determined by elevating the level of autophagy-related proteins, including Atg5, Atg7, LC3B-II, and the gathering of LC3 puncta. 3-methyladenine, a specific autophagy inhibitor, exacerbated Cd-caused germ cell apoptosis. Inversely, rapamycin, an autophagy inducer, relieved Cd-stimulated germ cell apoptosis. Interestingly, we found that autophagy in Sertoli cells was activated in Cd-treated WT mouse testes as evidenced by the increment of LC3 puncta surrounding SOX9, a specific Sertoli cell marker. More importantly, loss of autophagy in Sertoli cells aggravated Cd-triggered germ cell apoptosis. Taken together, these data indicate that autophagy in Sertoli cells alleviates Cd-triggered germ cell apoptosis in mouse testes.

N-acetylcysteine protects against microcystin-LR-induced endoplasmic reticulum stress and germ cell apoptosis in zebrafish testes

Previous studies have shown that microcystin-LR (MCLR) is a reproductive toxicant that induces germ cell apoptosis in the testes, but the underlying mechanisms have not been well understood. In this study, we investigated that MCLR induces germ cell apoptosis is through activation of endoplasmic reticulum (ER) stress and N-acetylcysteine (NAC), an antioxidant could protect against germ cell apoptosis by inhibiting the ER stress. Healthy male zebrafish were intraperitoneally injected with NAC (500 nM), beginning at 2 h before different doses of MCLR (0, 50, 100, 200 μg/kg). As expected, acute MCLR exposure resulted in oxidative stress and germ cell apoptosis in zebrafish testes. Further analysis showed that NAC significantly alleviated MCLR-induced testicular germ cell apoptosis and inhibited the caspase-dependent apoptotic proteins. Meanwhile H&E staining showed that NAC could rescue testicular damage induced by MCLR. Moreover, MCLR induced activation of ER stress which consequently triggered apoptosis in zebrafish testes. Interestingly, NAC was effective in improving the total antioxidant capacity (T-AOC) level and activity of antioxidant enzymes in NAC pretreated groups. NAC significantly attenuated MCLR-induced upregulation of GRP78 in testes. In addition, NAC significantly attenuated MCLR-triggered testicular eIF2s1 and MAPK8 activation, indicating that NAC counteracts MCLR-induced unfolded protein response (UPR) in testes. Taken together, the results observed in this study suggested that ER stress plays a critical role in germ cell apoptosis exposed to MCLR and NAC could protect against apoptosis via inhibiting ER stress in zebrafish testes.

Testosterone regulates granzyme K expression in rat testes.

Testosterone depletion induces increased germ cell apoptosis in testes. However, limited studies exist on genes that regulate the germ cell apoptosis. Granzymes (GZM) are serine proteases that induce apoptosis in various tissues. Multiple granzymes, including GZMA, GZMB and GZMN, are present in testes. Th us, we investigated which granzyme may be testosterone responsive and possibly may have a role in germ cell apoptosis aft er testosterone depletion. Ethylene dimethane sulfonate (EDS), a toxicant that selectively ablates the Leydig cells, was injected into rats to withdraw the testosterone. The testosterone depletion effects after 7 days post-EDS were verified by replacing the testosterone exogenously into EDS-treated rats. Serum or testicular testosterone was measured by radioimmunoassay. Using qPCR, mRNAs of granzyme variants in testes were quantified. The germ cell apoptosis was identified by TUNEL assay and the localization of GZMK was by immunohistochemistry. EDS treatment eliminated the Leydig cells and depleted serum and testicular testosterone. At 7 days post-EDS, testis weights were reduced 18% with increased germ cell apoptosis plus elevation GZMK expression. GZMK was not associated with TUNEL-positive cells, but was localized to stripped cytoplasm of spermatids. In addition, apoptotic round spermatids were observed in the caput epididymis. GZMK expression in testes is testosterone dependent. GZMK is located adjacent to germ cells in seminiferous tubules and the presence of apoptotic round spermatids in the epididymis suggest its role in the degradation of microtubules in ectoplasmic specializations. Thus, overexpression of GZMK may indirectly regulate germ cell apoptosis by premature release of round spermatids from seminiferous tubule lumen.

Molecular and Cellular Mechanisms of Apoptosis during Dissociated Spermatogenesis.

Apoptosis is a tightly controlled process by which tissues eliminate unwanted cells. Spontaneous germ cell apoptosis in testis has been broadly investigated in mammals that have an associated spermatogenesis pattern. However, the mechanism of germ cell apoptosis in seasonally breeding reptiles following a dissociated spermatogenesis has remained enigmatic. In the present study, morphological evidence has clearly confirmed the dissociated spermatogenesis pattern in Pelodiscus sinensis. TUNEL and TEM analyses presented dynamic changes and ultrastructural characteristics of apoptotic germ cells during seasonal spermatogenesis, implying that apoptosis might be one of the key mechanisms to clear degraded germ cells. Furthermore, using RNA-Seq and digital gene expression (DGE) profiling, a large number of apoptosis-related differentially expressed genes (DEGs) at different phases of spermatogenesis were identified and characterized in the testis. DGE and RT-qPCR analysis revealed that the critical anti-apoptosis genes, such as Bcl-2, BAG1, and BAG5, showed up-regulated patterns during intermediate and late spermatogenesis. Moreover, the increases in mitochondrial transmembrane potential in July and October were detected by JC-1 staining. Notably, the low protein levels of pro-apoptotic cleaved caspase-3 and CytC in cytoplasm were detected by immunohistochemistry and western blot analyses, indicating that the CytC-Caspase model might be responsible for the effects of germ cell apoptosis on seasonal spermatogenesis. These results facilitate understanding the regulatory mechanisms of apoptosis during spermatogenesis and uncovering the biological process of the dissociated spermatogenesis system in reptiles.

Opipramol for antidepressant therapy in early pregnancy

Society of Toxicologic Pathology has recommended mDF to fix testes since 2002. However, subsequent studies showed that false TUNEL-positive cells were observed in mDF-fixed testes. This study compared the effects of different fixation methods on histology and TUNEL staining in mouse testes. Results showed that fixation for 24 or 36 h in mDF provided better morphologic details in untreated testes, but markedly enhanced false TUNEL-positive staining. To optimize the fixation, testes were fixed using mDF for 6 h and then PFA for 18 h. Interestingly, fixation using mDF/PFA manifested better morphologic details, and rarely caused false TUNEL-positive cells in testes. Finally, we examined germ cell apoptosis in testes using mDF/PFA fixation in cadmium-treated mice. As expected, cadmium triggered germ cell apoptosis which was well visualized in the mDF/PFA fixed testes. Taken together, mDF plus PFA fixation not only minimizes false TUNEL-positive cells, but also provides integrated morphologic details in testes.

Different fixative methods influence histological morphology and TUNEL staining in mouse testes.

Society of Toxicologic Pathology has recommended mDF to fix testes since 2002. However, subsequent studies showed that false TUNEL-positive cells were observed in mDF-fixed testes. This study compared the effects of different fixation methods on histology and TUNEL staining in mouse testes. Results showed that fixation for 24 or 36h in mDF provided better morphologic details in untreated testes, but markedly enhanced false TUNEL-positive staining. To optimize the fixation, testes were fixed using mDF for 6h and then PFA for 18h. Interestingly, fixation using mDF/PFA manifested better morphologic details, and rarely caused false TUNEL-positive cells in testes. Finally, we examined germ cell apoptosis in testes using mDF/PFA fixation in cadmium-treated mice. As expected, cadmium triggered germ cell apoptosis which was well visualized in the mDF/PFA fixed testes. Taken together, mDF plus PFA fixation not only minimizes false TUNEL-positive cells, but also provides integrated morphologic details in testes.

Extremely low frequency magnetic fields induce spermatogenic germ cell apoptosis: possible mechanism.

The energy generated by an extremely low frequency electromagnetic field (ELF-EMF) is too weak to directly induce genotoxicity. However, it is reported that an extremely low frequency magnetic field (ELF-MF) is related to DNA strand breakage and apoptosis. The testes that conduct spermatogenesis through a dynamic cellular process involving meiosis and mitosis seem vulnerable to external stress such as heat, MF exposure, and chemical or physical agents. Nevertheless the results regarding adverse effects of ELF-EMF on human or animal reproductive functions are inconclusive. According to the guideline of the International Commission on Non-Ionizing Radiation Protection (ICNIRP; 2010) for limiting exposure to time-varying MF (1 Hz to 100 kHz), overall conclusion of epidemiologic studies has not consistently shown an association between human adverse reproductive outcomes and maternal or paternal exposure to low frequency fields. In animal studies there is no compelling evidence of causal relationship between prenatal development and ELF-MF exposure. However there is increasing evidence that EL-EMF exposure is involved with germ cell apoptosis in testes. Biophysical mechanism by which ELF-MF induces germ cell apoptosis has not been established. This review proposes the possible mechanism of germ cell apoptosis in testes induced by ELF-MF.

Involvement of soluble Fas Ligand in germ cell apoptosis in testis of rats undergoing autoimmune orchitis

Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying immune and germ cell (GC) interactions. EAO is characterized by severe damage of seminiferous tubules (STs) with GCs that undergo apoptosis and sloughing. Based on previous results showing that Fas–Fas Ligand (L) system is one of the main mediators of apoptosis in EAO, in the present work we studied the involvement of Fas and the soluble form of FasL (sFasL) in GC death induction. EAO was induced in rats by immunization with testis homogenate and adjuvants; control (C) rats were injected with adjuvants; a group of non-immunized normal (N) rats was also studied. Activation of Fas employing an anti-Fas antibody decreased viability (trypan blue exclusion test) and induced apoptosis (TUNEL) of GCs from STs of N and EAO rats, an effect more pronounced on GCs from EAO STs. By Western blot we detected an increase in sFasL content in the testicular fluid of rats with severe EAO compared to N and C rats. By intratesticular injection of FasL conjugated to Strep-Tag molecule (FasL-Strep, BioTAGnology) and its immunofluorescent localization, we demonstrated that sFasL is able to enter the adluminal compartment of the STs. Moreover, FasL-Strep induced GC apoptosis in testicular fragments of N rats. By flow cytometry, we detected an increase in the number of membrane FasL-expressing CD4+ and CD8+ T cells in testis during EAO development but no expression of FasL by macrophages. Our results demonstrate that sFasL is locally produced in the chronically inflamed testis and that this molecule is able to enter the adluminal compartment of STs and induce apoptosis of Fas-bearing GCs.

Ascorbic acid protects against cadmium-induced endoplasmic reticulum stress and germ cell apoptosis in testes

Cadmium (Cd) is a testicular toxicant which induces endoplasmic reticulum (ER) stress and germ cell apoptosis in testes. This study investigated the effects of ascorbic acid on Cd-evoked ER stress and germ cell apoptosis in testes. Male mice were intraperitoneally injected with CdCl2 (2.0mg/kg). As expected, a single dose of Cd induced testicular germ cell apoptosis. Interestingly, Cd-triggered testicular germ cell apoptosis was almost completely inhibited in mice treated with ascorbic acid. Interestingly, ascorbic acid significantly attenuated Cd-induced upregulation of GRP78 in testes. In addition, ascorbic acid significantly attenuated Cd-triggered testicular IRE1α and eIF2α phosphorylation and XBP-1 activation, indicating that this antioxidant counteracts Cd-induced unfolded protein response (UPR) in testes. Finally, ascorbic acid significantly attenuated Cd-evoked upregulation of CHOP and JNK phosphorylation, two components in ER stress-mediated apoptotic pathway. In conclusion, ascorbic acid protects mice from Cd-triggered germ cell apoptosis via inhibiting ER stress and UPR in testes.

The Effects of Di(2-Ethylhexyl)Phthalate Exposure and Selenium Nutrition on Sertoli Cell Vimentin Structure and Germ-Cell Apoptosis in Rat Testis

This study aimed to investigate the effects of di(2-ethylhexyl)phthalate (DEHP) on Sertoli-cell vimentin filaments and germ-cell apoptosis in testes of pubertal rats at different selenium (Se) status. Se deficiency was produced in 3-weeks old Sprague-Dawley rats by feeding them ≤ 0.05 Se mg/kg diet for 5 weeks, Se supplementation group was on 1 mg Se/kg diet, and DEHP was applied at 1000 mg/kg dose by gavage during the last 10 days of the feeding period. The diet with excess Se did not cause any appreciable alteration in vimentin staining and apoptosis of germ cells, but Se deficiency caused a mild decrease in the intensity of vimentin immunoreactivity and enhanced germ-cell apoptosis significantly (approximately 3-fold, p <0.0033). DEHP exposure caused disruption and collapse of vimentin filaments and significantly induced apoptotic death of germ cells (approximately 8-fold, p <0.0033). In DEHP-exposed Se-deficient animals, compared with the control, collapse of vimentin filaments was more prominent; there was serious damage to the seminiferous epithelium; and a high increment (approximately 25-fold, p <0.0033) in apoptotic germ cells was observed. Thus, Se deficiency exacerbated the toxicity of DEHP on Sertoli cells and spermatogenesis, whereas Se supplementation provided protection. These results put forward the critical role of Se in the modulation of redox status of testicular cells and emphasize the importance of Se status for reproductive health.

Crosstalk Between Endoplasmic Reticulum Stress and Mitochondrial Pathway Mediates Cadmium-Induced Germ Cell Apoptosis in Testes

Cadmium (Cd) is associated with male infertility and poor semen quality in humans. Increasing evidence demonstrates that Cd induces testicular germ cell apoptosis in rodent animals. However, the molecular mechanisms of Cd-induced testicular germ cell apoptosis remain poorly understood. In the present study, we investigated the role of endoplasmic reticulum (ER) stress on Cd-evoked germ cell apoptosis in testes. We show that spliced form of XBP-1, the target of the IRE1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an ER chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly caused eIF2α and JNK phosphorylation in testes, indicating that the unfolded protein response pathway in testes was activated by Cd. Interestingly, phenylbutyric acid (PBA), an ER chemical chaperone, attenuated Cd-induced ER stress and protected against germ cell apoptosis in testes. In addition, PBA significantly attenuated Cd-evoked release of cytochrome c from mitochondria to cytoplasm in testes. Taken together, these results suggest that crosstalk between ER stress signaling and mitochondrial pathway mediates Cd-induced testicular germ cell apoptosis.

Melatonin alleviates cadmium‐induced cellular stress and germ cell apoptosis in testes

Increasing evidence demonstrates that melatonin has an anti-apoptotic effect in somatic cells. However, whether melatonin can protect against germ cell apoptosis remains obscure. Cadmium (Cd) is a testicular toxicant and induces germ cell apoptosis. In this study, we investigated the effects of melatonin on Cd-evoked germ cell apoptosis in testes. Male ICR mice were intraperitoneally (i.p.) injected with melatonin (5 mg/kg) every 8 hr, beginning at 8 hr before CdCl(2) (2.0 mg/kg, i.p.). As expected, acute Cd exposure resulted in germ cell apoptosis in testes, as determined by terminal dUTP nick-end labeling (TUNEL) staining. Melatonin significantly alleviated Cd-induced testicular germ cell apoptosis. An additional experiment showed that spliced form of XBP-1, the target of the IRE-1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an endoplasmic reticulum (ER) chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly increased testicular eIF2α and JNK phosphorylation, indicating that the unfolded protein response (UPR) pathway was activated by CdCl(2). Interestingly, melatonin almost completely inhibited Cd-induced ER stress and the UPR in testes. In addition, melatonin obviously attenuated Cd-induced heme oxygenase (HO)-1 expression and protein nitration in testes. Taken together, these results suggest that melatonin alleviates Cd-induced cellular stress and germ cell apoptosis in testes. Melatonin may be useful as pharmacological agents to protect against Cd-induced testicular toxicity.

Fenvalerate induces germ cell apoptosis in mouse testes through the Fas/FasL signaling pathway

Fenvalerate has a potentially adverse effect on male reproduction and spermatogenesis, whereas the precise mechanism remains obscure. The present study investigated the effects of fenvalerate on germ cell apoptosis in testes. Adult male mice were administered with fenvalerate (15 or 60mg/kg) by gavage for 28days. Germ cell apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). The number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with fenvalerate in a dose-dependent manner. TUNEL+ germ cells were observed mainly in stages VII-VIII and also stages IX-XII seminiferous tubules in testes. Additional experiments showed that fenvalerate increased the level of active caspase-8 and caspase-3 in testes. In addition, fenvalerate upregulated the expression of Fas and FasL in testes. No significant difference on the expression of Bcl-2 and Bax in testes was observed between fenvalerate-treated mice and controls. Fenvalerate did not affect the leakage of cytochrome c from mitochondria into cytoplasm. In addition, fenvalerate did not cause the activation of caspase-9 in testes. Taken together, these results suggest that fenvalerate induces germ cell apoptosis in testes through the Fas/FasL signaling pathway.

Testicular oxidative damage and role of combined antioxidant supplementation in experimental diabetic rats

The present study was designated to assess oxidative damage and its effect on germ cell apoptosis in testes of streptozotocin (STZ)-induced diabetic rats. The role of antioxidant supplementation with a mixture of vitamins E and C and alpha lipoic acid for protection against such damage was also evaluated. Forty-five adult male rats were randomly divided into three groups: groupI, control, non-diabetic rats; groupII, STZ-induced, untreated diabetic rats; groupIII, STZ-induced diabetic rats supplemented with a mixture of vitamins E and C and alpha lipoic acid. Glycated hemoglobin (HbA(1C)), glucose, and insulin levels were estimated in blood samples. Malondialdehyde (MDA), the activities of the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and caspase-3 in addition to testosterone (T) level were all determined in testicular tissues. Histopathological studies using H&E stain, as well as, immunohistochemical detection of apoptosis using (TUNEL) method were also performed. Blood glucose and HbA(1c) were significantly increased while insulin was significantly decreased in STZ-induced diabetic rats as compared with controls. In rat testicular tissues, MDA, and caspase-3 activity were significantly elevated while SOD and GPx enzymatic activities as well as T level were significantly decreased in diabetic rats as compared with control group. Antioxidant supplementation to diabetic rats restored the testicular enzymatic activities of SOD and GPx to almost control levels, in addition, MDA and caspase-3 activity decrease while T increase significantly as compared with untreated diabetic group. Prominent reduction of germ cell apoptosis was found in diabetic rats supplemented with antioxidants. An important role of testicular oxidative damage and germ cell apoptosis in diabetes-induced infertility could be suggested, treatment with antioxidants has a protective effect by restoring SOD and GPx antioxidant enzymatic activity.

Mitochondrial signaling pathway is also involved in bisphenol A induced germ cell apoptosis in testes

Bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. An earlier study showed that BPA-induced germ cell apoptosis through the Fas/FasL apoptotic pathway. In the present study, we aimed to investigate whether the mitochondrial pathway is also involved in the process of BPA-mediated germ cell apoptosis in testes. Male mice were administered with BPA (160 or 480 mg/kg) by gavage daily from postnatal day 35 (PND35) to PND49. Germ cell apoptosis in testes was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). As expected, the number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with BPA during puberty. TUNEL+ germ cells were observed mainly in stages VII–VIII seminiferous tubules in testes. An increase in the level of Fas and FasL was observed in testes of mice exposed to BPA during puberty. In addition, pubertal BPA exposure evoked the activation of caspase-8 and caspase-3 in testes. Interestingly, pubertal BPA exposure also caused the translocation of cytochrome c from mitochondria into cytosol. In addition, pubertal BPA exposure upregulated the level of Bax and active caspase-9 in testes. Taken together, these results suggest that pubertal BPA exposure induces germ cell apoptosis in testes through not only the Fas/FasL signaling pathway but also the mitochondrial apoptotic pathway.

Diabetes-induced Testicular Disorders Vis-a-vis Germ Cell Apoptosis in Albino Rat: Remedial Effect of Hexane Fraction of Root of Musa paradisiaca and Leaf of Coccinia indica

The present study was designed to investigate diabetes induced germ cell apoptosis in testis and its protection by treatment with hexane fraction of hydro-methanolic extract of root of Musa paradisiaca and leaf of Coccinia indica in composite manner. Testicular oxidative stress injury was reflected by diminution in the activities of antioxidant enzymes such as catalase, peroxidase, superoxide dismutase and glutathione-s-transferase along with elevation in levels of conjugated diene and thiobarbituric acid reactive substances in diabetic condition. Serum testosterone and insulin levels were decreased in association with elevation in apoptosis of pancreatic islet cells in diabetic rats. Giant cells number along with elevation i nt he number of apoptotic cells were noted in seminiferous tubules in diabetic model animals. Significant improvements in the levels of blood glucose, serum insulin and testosterone and testicular oxidative stress parameters were noted versus control after treatment of said fraction at a dose of 2 mg (1 : 3)/0.2 ml olive oil per 100 g body weight per day for 45 days to diabetic rats. Numbers of giant cells in seminiferous tubules, apoptotic germ cells and apoptotic pancreatic islet cells were decreased significantly in fraction treated diabetic group versus control. From UV-spectroscopic and TLC studies in connection with phytochemical screening of the said fraction, phenol, flavonoid and alkaloid types of compound were found. From these results it may be concluded that the active ingredient(s) present in hexane fraction of root ofMusa paradisiaca and leaf of Coccinia indica have the potential to correct diabetes-induced testicular germ cell apoptosis.

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.

Activation of NF-κB Associated With Germ Cell Apoptosis in Testes of Experimentally Induced Cryptorchid Rat Model

To evaluate the behavior of NF-kappaB in the cryptorchid testes, we investigated the testes of an experimentally induced cryptorchid rat model. Cryptorchid testes exhibit degenerative changes in the germinal epithelium with germ cell apoptosis, which results in future infertility. However, the mechanisms of apoptosis in the germ cells are multifactorial and have not been completely elucidated. NF-kappaB is a transcription factor considered to play an important role in the apoptosis of various cells. The cryptorchid rat model was generated by the administration of an antiandrogenic agent during the fetal period. The unilateral cryptorchid testes and contralateral descended testes were removed at 7-10 weeks after birth, and histopathologic examination and Western blot analysis were performed. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, and the expression of NF-kappaB and IkappaB proteins was investigated using immunohistochemistry. In the present model, spermatogenesis was arrested at the spermatid differentiation stage, and elongated spermatids or spermatozoa were not detected. Apoptosis was noted predominantly in the primary spermatocytes, and Sertoli cells and Leydig cells did not undergo apoptosis. On immunohistochemistry, NF-kappaB signals were increased and focused on the nucleus of the germ cells in the cryptorchid testes, consistent with TUNEL-positive cells. On Western blot analysis, the NF-kappaB and IkappaB proteins were detected more intensely in the cryptorchid testes. The activation of NF-kappaB was associated with germ cell apoptosis in experimentally induced cryptorchid testes, suggesting that NF-kappaB has certain roles in germ cell apoptosis. The results of the present study may guide toward new therapeutic possibilities for the impairment of spermatogenesis in patients with cryptorchidism.

Sensitivity of testicular germ cells to toxicant-induced apoptosis in gld mice that express a nonfunctional form of Fas ligand.

Germ cell apoptosis in testis is essential for functional spermatogenesis. Recent evidence suggests that the Fas signaling system is critical for the regulation of testicular germ cell apoptosis. To further evaluate the Fas signaling system in testis, we examined the incidence of germ cell apoptosis in gld mice that lack a functional Fas-signaling pathway. gld mice have a small, but significant, increase in testis weight and numbers of spermatid heads per testis compared with wild-type mice. In addition, gld mice have a small increase in the spontaneous incidence of germ cell apoptosis, as indicated by characteristic DNA fragmentation via the terminal deoxynucleotidyl-transferase-mediated deoxy-UTP nick end labeling assay. To test the role of the Fas system in toxicant-induced germ cell apoptosis, mice were exposed to either a Sertoli cell- or germ cell-specific toxicant [mono-(2-ethylhexyl)phthalate (MEHP; 1 g/kg) or 5 Gy radiation, respectively]. These two exposure paradigms induced extensive increases in germ cell apoptosis in wild-type mice. However, exposure of gld mice to MEHP caused only a minimal increase in germ cell apoptosis, whereas they were as sensitive as wild-type mice to radiation exposure. These data indicate that the Fas signaling pathway is 1) involved in regulating the numbers of germ cells in the testis, 2) crucial for the initiation of germ cell apoptosis after MEHP-induced Sertoli cell injury, and 3) differentially active in the cell-specific regulation of germ cell apoptosis that occurs as a consequence of Sertoli cell vs. germ cell injury.