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Titanium dioxide nanoparticles (TiO 2 NPs) can result in the reduction of sperm numbers, but the mechanisms have not been well elucidated. The purpose of this study was to investigate the effects of TiO 2 NPs on cell cycle and apoptosis in spermatogonia and to explore the role of PI3K/AKT/mTOR signaling pathway in this process. The mouse spermatogonia cell line (GC-1) was treated with TiO 2 NPs at different concentrations (0, 25, 50, 75 and 100 μg/mL) for 24 h to detect cell viability, cell cycle, apoptosis, and key proteins related to cell cycle and PI3K/AKT/mTOR signaling pathway. The agonist (IGF-1) and inhibitor (LY294002) of PI3K were used to verify the role of PI3K/AKT/mTOR signaling pathway in cell cycle and apoptosis. TiO 2 NPs significantly inhibited cell proliferation, induced cell cycle arrest at G0/G1 phase and resulted in apoptosis. TiO 2 NPs downregulated the levels of cyclin-dependent kinases (CDKs) and cyclins, including CDK4, CDK2, Cyclin D1 and Cyclin E1, while upregulated the levels of p21 and p53 proteins. Furthermore, TiO 2 NPs inhibited the PI3K/AKT/mTOR signaling pathway by decreasing the levels of p-PI3K, p-AKT and p-mTOR. IGF-1 reversed the G0/G1 phase arrest and apoptosis caused by TiO 2 NPs. However, LY294002 aggravated the G0/G1 phase arrest and apoptosis resulting from TiO 2 NPs. Collectively, TiO 2 NPs induced cell cycle arrest at G0/G1 phase and apoptosis through inhibiting the activation of PI3K/AKT/mTOR pathway, which could be the main reason for the reduction in sperm numbers caused by TiO 2 NPs., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Benzyl butyl phthalate (BBP) is a widely used plasticizer that poses various potential health hazards. Although BBP has been extensively studied, the direct mechanism underlying its toxicity in male germ cells remains unclear. Therefore, we investigated BBP-mediated male germ cell toxicity in GC-1 spermatogonia (spg), a differentiated mouse male germ cell line. This study investigated the impact of BBP on reactive oxygen species (ROS) generation, apoptosis, and autophagy regulation, as well as potential protective measures against BBP-induced toxicity. A marked dose-dependent decrease in GC-1 spg cell proliferation was observed following treatment with BBP at 12.5 μM. Exposure to 50 μM BBP, approximating the IC 50 of 53.9 μM, markedly increased cellular ROS generation and instigated apoptosis, as evidenced by augmented protein levels of both intrinsic and extrinsic apoptosis-related markers. An amount of 50 μM BBP induced marked upregulation of autophagy regulator proteins, p38 MAPK, and extracellular signal-regulated kinase and substantially downregulated the phosphorylation of key kinases involved in regulating cell proliferation, including phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase. The triple combination of N-acetylcysteine, parthenolide, and 3-methyladenine markedly restored cell proliferation, decreased BBP-induced apoptosis and autophagy, and restored mTOR phosphorylation. This study provides new insights into BBP-induced male germ cell toxicity and highlights the therapeutic potential of the triple inhibitors in mitigating BBP toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Cadmium (Cd) is a well-recognized male reproductive toxicant that can cause testicular germ cell apoptosis. However, the underlying mechanism needs investigation. CG-1 mouse spermatogonia (spg) cells were treated with 20 μM cadmium chloride (CdCl 2 ) for 24 h. Cell apoptosis was measured, and the expressions of key genes and protein biomarkers involved in endoplasmic reticulum (ER) stress were detected, respectively. Untargeted metabolomics was performed to identify different metabolites, and transcriptome analysis was conducted to screen differentially expressed genes (DEGs). Our results indicated that CdCl 2 exposure caused cell apoptosis, and DEGs were involved in several apoptosis-related pathways. Moreover, CdCl 2 exposure apparently increased the mRNA and protein expressions levels of both GRP78 and ATF6α, disrupting the expression of various metabolites, particularly amino acids. Conclusively, our study reveals the pathway of CdCl 2 toxicity on mouse spg, providing a deep understanding of CdCl 2 -induced testicular toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microcystins (MCs) and nitrites are coexisted in the environment and have reproductive toxicity. The combined toxic effect and mechanism of MCs and nitrite on spermatogenesis remain largely unclear. In the present study, co-exposure to microcystin-leucine arginine (MC-LR) and sodium nitrite (NaNO 2 ) aggravated testicular damage of Balb/c mice and mitochondrial impairment of spermatogonia, Sertoli cells, and sperm. Furthermore, MC-LR and NaNO 2 reduced sperm density with a synergistic effect. In addition, MC-LR and NaNO 2 synergistically induced oxidative stress in the reproductive system by decreasing superoxide dismutase (SOD) activity and glutathione (GSH) levels and increasing levels of mitochondrial reactive oxygen species (mtROS) and reactive oxygen species (ROS). More importantly, mitoquidone mesylate (MitoQ), an inhibitor of mtROS, blocked MC-LR and NaNO 2 -induced spermatogonia and Sertoli cell apoptosis by inhibiting high expression of Bax, Fadd, Caspase-8, and cleaved-Caspase-3. On the other hand, MitoQ suppressed pyroptosis of Sertoli cells by inhibiting the expression of NLRP3, N-GSDMD, and cleaved-Caspase-1. Additionally, MitoQ alleviated co-exposure-induced sperm density reduction and organ index disorders in F1 generation mice. Together, co-exposure of MC-LR and NaNO 2 can enhance spermatogenic disorders by mitochondrial oxidative impairment-mediated germ cell death. This study emphasizes the potential risks of MC-LR and NaNO 2 on reproduction in realistic environments and highlights new insights into the cause and treatment of spermatogenic disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microplastics have emerged as environmental hazards in recent years. This study was intended to prove the toxic effects of microplastics on the male reproductive system and further elucidate its mechanism. C57bl/6 mice were exposed to ultrapure water or different doses (0.25, 0.5 and 1 mg/d) of 5 μm polystyrene microplastics (PS-MPs) for 4 weeks, and the GC-1 mouse spermatogonium was treated with different concentrations of PS-MPs. The results showed that sperm count and motility were decreased, and sperm deformity rate was increased after exposure to PS-MPs. The morphology of testes in PS-MPs groups exhibited pathological changes, such as abnormal development of spermatogenic tubules, and inhibited spermatogonium function. Furthermore, the fluorescence intensity of TUNEL staining and the BAX/BCL2 ratio were increased. Exposure to PS-MPs resulted in impaired mitochondrial morphology of spermatogonium, decreased activity of GSH-px and SOD, and increased the MDA level. In vitro, after treatment with PS-MPs, the cell apoptosis rate of spermatogonium was significantly increased, mitochondrial membrane potential was decreased, mitochondrial morphology was damaged, and exposure to PS-MPs increased mitochondrial reactive oxygen species, inducing an oxidative stress state in spermatogonia. In summary, PS-MPs induced a decrease in sperm quality by activating spermatogonium mitochondrial oxidative stress and apoptosis, offering novel insights into mitigating the reproductive toxicity of microplastics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The estrogen-like effect of bisphenol A (BPA) disrupting the maintenance of functional male germ cells is associated with male sub-fertility. This study investigated toxicity of male germ cells induced by four bisphenol analogs: BPA, BPAF, BPF, and BPS. The investigation of bisphenol analogs' impact on male germ cells included assessing proliferation, apoptosis induction, and the capacity to generate reactive oxygen species (ROS) in GC-1 spermatogonia (spg) cells, specifically type B spermatogonia. Additionally, the therapeutic potential and protective effects of N-Acetyl Cysteine (NAC) and NF-κB inhibitor parthenolide was evaluated. In comparison to BPA, BPF and BPS, BPAF exhibited the most pronounced adverse effect in GC-1 spg cell proliferation. This effect was characterized by pronounced inhibition of phosphorylation of PI3K, AKT, and mTOR, along with increased release of cytochrome c and subsequent cleavages of caspase 3, caspase 7, and poly (ADP-ribose) polymerase. Both NAC and parthenolide were effective reducing cellular ROS induced by BPAF. However, only NAC demonstrated a substantial recovery in proliferation, accompanied by a significant reduction in cytochrome c release and cleaved PARP. These results suggest that NAC supplementation may play an effective therapeutic role in countering germ cell toxicity induced by environmental pollutants with robust oxidative stress-generating capacity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bisphenol A (BPA), one of the main components of industrial products, is clinically associated with the increased male infertility rate. However, the underlying molecular mechanism of the BPA-resulted reproductive toxicity is not fully elucidated. Voltage-dependent anion channel 1 (VDAC1) is a pore protein and located at the outer mitochondrial membrane. As a mitochondrial gatekeeper, VDAC1 controls the release of reactive oxygen species (ROS) and the metabolic and energetic functions of mitochondria, and serves as a critical player in mitochondrial-mediated apoptosis. Herein, we explored the role of VDAC1 in BPA-induced apoptosis of spermatogonia. The results showed that BPA increased spermatogonia cell line GC-1 spg cell apoptosis and intracellular ROS level, and suppressed AMPK/mTOR signaling pathway at a dose of 80 μM for 48 hr. Lentivirus-mediated short hairpin RNA targeting VDAC1 (Lv-shVDAC1) silenced VDAC1 expression and enhanced BPA-restricted cell viability. Knockdown of VDAC1 inhibited the apoptosis of BPA-treated GC-1 spg cells determined by with changes of the expressions of pro-apoptotic and anti-apoptotic proteins. Knockdown of VDAC1 also alleviated the BPA-triggered intracellular ROS generation and oxidative stress. Moreover, silence of VDAC1 increased AMPKα1/2 phosphorylation and suppressed mTOR phosphorylation under BPA exposure. Dorsomorphin, an AMPK inhibitor, partially abolished the effects of VDAC1 gene silencing on BPA-stimulated GC-1 spg cells. In conclusion, inhibition of VDAC1 attenuated the BPA-induced oxidative stress and apoptosis and promoted the cell viability in spermatogonia through modulating AMPK/mTOR signaling pathway.

Zearalenone (ZEN), a widely known mycotoxin, is mainly produced by various Fusarium species, and it is a potent estrogenic metabolite that affects reproductive health in livestock and humans. In this study, the molecular mechanisms of toxicity and cell damage induced by ZEN in GC-1 spermatogonia (spg) cells were evaluated. Our results showed that cell viability decreased and apoptosis increased in a dose-dependent manner when GC-1 spg cells were exposed to ZEN. In addition, the key proteins involved in apoptosis, cleaved caspase-3 and -8, BAD, BAX, and phosphorylation of p53 and ERK1/2, were significantly increased in ZEN-exposed GC-1 spg cells for 24 h, and cytochrome c was released from mitochondria by ZEN. Interestingly, ZEN also triggered autophagy in GC-1 spg cells. The expression levels of the autophagy-related genes Atg5, Atg3, Beclin 1, LC3, Ulk1, Bnip 3, and p62 were significantly higher in ZEN-treated GC-1 spg cells, and the protein levels of both LC3A/B and Atg12 were remarkably increased in a dose-dependent manner in ZEN-exposed GC-1 spg cells compared to the control. In addition, immunostaining results showed that ZEN-treated groups showed a remarkable increase in LC 3A/B positive puncta as compared to the control in a dose-dependent manner based on confocal microscopy analysis in GC-1 spg cells. Our findings suggest that ZEN has toxic effects on tGC-1 spg cells and induces both apoptosis and autophagy.

Imatinib, the first generation of tyrosine kinase inhibitor, is used to treat and improve the prognosis of chronic myelogenous leukemia (CML). Clinical data suggest that imatinib could cross the blood-testis barrier and reduces the fertility of patients with CML-chronic phase. However, its exact molecular mechanism has not been fully elucidated. In this study, adult male Kunming mice were treated with different doses of imatinib for 8 weeks. The fertility was evaluated, and the sex hormone levels in the blood were detected by enzyme-linked immunosorbent assay. Histological changes were detected by hematoxylin and eosin staining. The concentration of imatinib in semen and blood was detected by liquid chromatography-mass spectrometry. The ultrastructure of blood-testis barrier and apoptotic bodies were observed by transmission electron microscope. The expression of blood-testis barrier function-regulating protein, Mfsd2a, and apoptosis-associated proteins in testis tissue was detected by immunohistochemistry and Western blot. The results indicated that the fertility of male mice was significantly decreased in a dose-dependent manner after imatinib treatment. Certain hormones in the serum were increased in imatinib treatment groups. Sperm morphology and testicular tissue showed various changes after imatinib treatment. The blood-testis barrier was destroyed and the concentration of imatinib in semen was similar to that in blood after imatinib treatment. Apoptosis was significantly increased in testis tissue after imatinib treatment. Collectively, these results suggest that imatinib can alter blood-testis barrier function, induce apoptosis of spermatogonia, and adversely affect fertility by reducing the number of spermatozoa, decreasing sperm motility and increasing the deformity rate., Competing Interests: Declarations of Competing Interest The authors declare no conflicts of interest., (Copyright © 2021. Published by Elsevier B.V.)

Nonylphenol (NP) is an endocrine-disruptor chemical that negatively affects reproductive health. Testes exposure to NP results in testicular structure disruption and a reduction in testicular size and testosterone levels. However, the effects of NP on spermatogonia in testes have not been fully elucidated. In this study, the molecular mechanisms of NP in GC-1 spermatogonia (spg) cells were investigated. We found that cell viability significantly decreased and apoptosis increased in a dose-dependent manner when GC-1 spg cells were exposed to NP. Furthermore, the expression levels of the pro-apoptotic proteins increased, whereas anti-apoptosis markers decreased in NP-exposed GC-1 spg cells. We also found that NP increased reactive oxygen species (ROS) generation, suggesting that ROS-induced activation of the MAPK signaling pathway is the molecular mechanism of NP-induced apoptosis in GC-1 spg cells. Thus, NP could induce c-Jun phosphorylation; dose-dependent expression of JNK, MKK4, p53, and p38; and the subsequent inhibition of ERK1/2 and MEK1/2 phosphorylation. The genes involved in apoptosis and JNK signaling were also upregulated in GC-1 spg cells treated with NP compared to those in the controls. Our findings suggest that NP induces apoptosis through ROS/JNK signaling in GC-1 spg cells.

As a widely used plasticizer in industry, di-2-ethylhexylphthalate (DEHP) can cause testicular toxicity, yet little is known about the potential mechanism. In this study, DEHP exposure dramatically inhibited cellviability and induced apoptosis of mouse GC-1 spg cells. Furthermore, DEHP significantly increased the levels of autophagy proteins LC3-II, Beclin1 and Atg5, as well as the ratio ofLC3-II/LC3-I. Transmission electron microscopy (TEM) further confirmed that DEHP induced autophagy of mouse GC-1 spg cells. DEHP was also shown to induceoxidative stress; while inhibition of oxidative stress with NAC could increase cell viability and inhibit DEHP-induced apoptosis and autophagy. These results suggested that DEHP induced apoptosis and autophagy of mouse GC-1 spg cells via oxidative stress. 3-MA, an inhibitor of autophagy, could rescue DEHP-induced apoptosis. In summary, DEHP induced apoptosis and autophagy of mouse GC-1 spg cells via oxidative stress, and autophagy might exert a cytotoxic effect on DEHP-induced apoptosis., (© 2019 Wiley Periodicals, Inc.)

The mitochondrial proteins involved in spermatogenic cells apoptosis in zebrafish after carbon ion radiation (CIR) were screened. The relative biological effectiveness (RBE) of CIR in zebrafish testes was investigated. Apoptosis of testicular cells was measured within 24 hr following 1 and 4 Gy CIR. Immunoblotting was used to assess the levels of mitochondrial apoptotic proteins in testes, and proliferative and apoptotic spermatogenic cells were detected by immunofluorescence after CIR. Label-free quantitative (LFQ) and parallel reaction monitoring-based target proteomics (PRM) were combined to screen and validate differential mitochondrial proteins in testes between 4 Gy and control groups at 24 hr after CIR. The RBE of CIR in zebrafish testes was 1.48 ± 0.04, and induction of apoptosis by CIR was higher than that of X-rays in testicular cells. Mitochondrial apoptotic pathways play a crucial role in spermatogenic cells apoptosis after CIR, with 60 differential mitochondrial proteins identified. Among 20 target proteins, 12 were significantly upregulated, 2 were significantly downregulated in the 4 Gy CIR group. The results of PRM were consistent with label-free analysis. This is the first study to screen the differential mitochondrial proteins and provide useful information to understand the underlying mechanisms of spermatogenic cell apoptosis in zebrafish following CIR., (© 2019 Wiley Periodicals, Inc.)

Toxicological studies have demonstrated the relation between use of agrochemicals and fertility issues within males. Thus, the present study aimed to elucidate the propensity of cypermethrin (CYP) in bringing testicular germ cell apoptosis and effective attenuation by vitamins C and E in caprines. Reproductive toxicity of CYP was evaluated using histomorphological, cytological, and biochemical changes in the testicular germ cells in dose-dependent (1, 5, 10 μg/mL) and time-dependent (4, 6, 8 h) manner. Histological and ethidium bromide/acridine orange fluorescence staining exhibited that vitamins C and E (0.5 and 1.0 mM) successfully diminished the CYP-induced testicular germ cells apoptosis. CYP exposure along with vitamins C and E supplementation also resulted in significantly increased ferric reducing antioxidant power activity along with the antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, and decreased lipid peroxidation in testicular germ cells. Thus, vitamins C and E ameliorated CYP-induced testicular germ cell apoptosis, thereby preventing spermatogonial cells degeneration and male infertility., (© 2018 Wiley Periodicals, Inc.)

Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1mM, respectively) of Dox, and fixed 1h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Epidemiological reports have indicated a correlation between the increasing bisphenol A (BPA) levels in the environment and the incidence of male infertility. In this study, the protective effects of melatonin on BPA-induced oxidative stress and apoptosis were investigated in the rat testes and epididymal sperm. Melatonin (10 mg/kg body weight (bw)) was injected concurrently with BPA (50 mg/kg bw) for 3 and 6 weeks. The administration of BPA significantly increased oxidative stress in the testes and epididymal sperm. This was associated with a decrease in the serum testosterone level as well as sperm quality, chromatin condensation/de-condensation level, and the percentage of haploid germ cells in the semen. BPA administration caused a significant increase in apoptosis accompanied by a decrease in the expression of the antiapoptotic proteins Bcl-2 in the testes and epididymal sperm. The concurrent administration of melatonin decreased oxidative stress by modulating the levels of glutathione, superoxide dismutase, and catalase as well as the malondialdehyde and hydrogen peroxide concentrations in the testes and sperm. Melatonin sustained Bcl-2 expression and controlled apoptosis. Furthermore, melatonin maintained the testosterone levels, ameliorated histopathological changes, increased the percentages of seminal haploid germ cells, and protected sperm chromatin condensation process, indicating appropriate spermatogenesis with production of functional sperm. In conclusion, melatonin protected against BPA-induced apoptosis by controlling Bcl-2 expression and ameliorating oxidative stress in the testes and sperm. Thus, melatonin is a promising pharmacological agent for preventing the potential reproductive toxicity of BPA following occupational or environmental exposures., (© The Author(s) 2014.)

Melatonin exerts a range of physiological effects. However, the functional significance of melatonin in spermatogenesis and the underlying mechanisms remain unclear. MicroRNAs (miRNAs) are essential in the initiation and progression of testicular development, including spermatogenesis. Thus far, limited information is known about the role of miRNAs in melatonin-mediated spermatogenesis. In this study, the expression levels of testicular miRNA machinery genes, namely, Dgcr8 and Xpo5, were significantly increased by melatonin. The miRNA expression profile was identified in the testes of melatonin-treated mice. Ten miRNAs were significantly up-regulated, and 15 miRNAs were down-regulated. Melatonin (25 μM) enhanced cell growth and reduced apoptosis in GC-1 spg cells. Among the down-regulated miRNAs, miR-16 expression was influenced by melatonin in GC-1 spg cells. The miR-16 mimics in GC-1 spg cells significantly suppressed cell growth and promoted cell apoptosis. Conversely, transfection of the miR-16 inhibitor significantly increased cell growth and decreased cell apoptosis. The protein expression level of CCND1 (Cyclin D1) in GC-1 spg cells was decreased by the miR-16 mimics and increased by knockdown of miR-16. Moreover, bioinformatics and reporter activity analyses showed that Ccnd1 was a potential target of miR-16. These results suggested that miR-16 may function as a novel regulator of testicular functions during melatonin stimulation by targeting Ccnd1., (© 2016 by the Society for the Study of Reproduction, Inc.)

To investigate the mechanisms of endosulfan-induced reproductive toxicity, the spermatogenic cell lines (GC-1 spg) of mice were treated with 0, 6, 12, and 24 μg/ml endosulfan for 24 h in vitro The results showed that endosulfan induced apoptosis as well as oxidative stress and mitochondrial dysfunction. Reactive oxygen species and damage of mitochondrial structure were considered as major factors to GC-1 spg cells apoptosis. We further examined the expression of apoptosis-related proteins in mitochondria pathway by Western blot and immunohistochemistry analysis as well as activities. The results showed that endosulfan significantly improved the expressions of cytochrome c and B-cell lymphoma 2 (Bcl-2)-associated X protein and increased the activities of caspases 9 and 3 as well as the downregulation of the expression of Bcl-2 in GC-1 spg cells. The results suggested that exposure to endosulfan might induce the apoptosis of spermatogenic cells via mitochondria-dependent pathway mediated by oxidative stress resulting in the damage of mitochondrial structure and mitochondrial dysfunction., (© The Author(s) 2015.)

Male germ-cell apoptosis occurs naturally and can be increased by exposure to drugs and toxic chemicals. Individuals may have different rates of apoptosis and are likely to also exhibit differential sensitivity to outside influences. Previously, we reported that p-chloroamphetamine (pCA), a substance that inhibits serotonin synthesis, induced germ-cell apoptosis in prepubertal male rats. Here, we identified prepubertal rats with naturally high or low rates of germ-cell apoptosis and evaluated gene expression in both groups. Bax and Shbg mRNA levels were higher in rats with high rates of germ-cell apoptosis. Rats were then treated with pCA and the neuro-hormonal response and gene expression were evaluated. Treatment with pCA induced a reduction in serotonin concentrations but levels of sex hormones and gonadotrophins were not changed. Rats with initially high rates of germ-cell apoptosis had even higher rates of germ-cell apoptosis after treatment with pCA. In rats with high rates of germ-cell apoptosis Bax mRNA expression remained high after treatment with pCA. On the basis of category, an inverse relationship between mRNA expression of Bax and Bcl2, Bax and AR and Bax and Hsd3b2 was found. Here we provide evidence that innate levels of germ-cell apoptosis could be explained by the level of mRNA expression of genes involved with apoptosis and spermatogenesis.

Bisphenol A (BPA) is a widely used industrial plasticizer, which is ubiquitously present in the environment and organisms. As an endocrine disruptor, BPA has caused significant concerns regarding its interference with reproductive function. However, little is known about the impact of BPA exposure on early testicular development. The aim of the present study was to investigate the influence of neonatal BPA exposure on the first wave of spermatogenesis. Newborn male mice were subcutaneously injected with BPA (0.01, 0.1 and 5 mg/kg body weight) daily from postnatal day (PND) 1 to 21. Histological analysis of testes at PND 22 revealed that BPA-treated testes contained mostly spermatogonia and spermatocytes with markedly less round spermatids, indicating signs of meiotic arrest. Terminal dUTP nick-end labeling (TUNEL) assay showed that BPA treatment significantly increased the number of apoptotic germ cells per tubule, which corroborated the observation of meiotic arrest. In addition, BPA caused abnormal proliferation of germ cells as revealed by Proliferating Cell Nuclear Antigen (PCNA) immunohistochemical staining. Mechanistically, BPA-treated testes displayed a complete lack of BOULE expression, which is a conserved key regulator for spermatogenesis. Moreover, BPA significantly increased the expression of estrogen receptor (ER) α and β in the developing testis. The present study demonstrated that neonatal BPA exposure disrupted meiosis progression during the first wave of spermatogenesis, which may be, at least in part, due to inhibition of BOULE expression and/or up-regulation of ERα/β expression in BPA-exposed developing testis.

Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis, resulting in male infertility. Cr(VI) by inducing oxidative stress was cytotoxic to both male somatic cells and SSCs in a dose-dependent manner, and induced mitochondria-dependent apoptosis. Although the mechanism of Cr(VI)-induced cytotoxicity was similar in both somatic cells, the differences in sensitivity of TM3 and TM4 cells to Cr(VI) could be attributed, at least in part, to cell-specific regulation of P-AKT1, P-ERK1/2, and P-P53 proteins. Cr(VI) affected the differentiation and self-renewal mechanisms of SSCs, disrupted steroidogenesis in TM3 cells, while in TM4 cells, the expression of tight junction signaling and cell receptor molecules was affected as well as the secretory functions were impaired. In conclusion, our results show that Cr(VI) is cytotoxic and impairs the physiological functions of male somatic cells and SSCs.

Parabens are p-hydroxybenzoic acid ester compounds widely used as preservatives in foods, cosmetics, toiletries and pharmaceuticals. Some parabens, including butylparaben, exert an estrogenic activity as determined by in vitro estrogen receptor assay and in vivo uterotrophic assay, and adversely affect endocrine secretion and male reproductive function. We conducted a research study to evaluate the acute effects of butylparaben on testicular tissues of prepubertal rats. Three-week-old male rats (n=8) were given a single dose of 1000mg/kg butylparaben. The rats were sacrificed under anesthesia at 3, 6 and 24h after administration, and their testes were collected for histopathological examination. The study revealed progressive detachment and sloughing of spermatogenic cells into the lumen of the seminiferous tubules at 3h, and this effect was enhanced at 6h after administration. Thin seminiferous epithelia and wide tubular lumina were seen at 24h in the butylparaben-treated group, compared to the control. In order to clarify whether sloughed spermatogenic cells underwent apoptosis, TUNEL assay was carried out. We found a significant increase in the number of apoptotic spermatogenic cells in all the treated groups, compared to the controls and a maximal number of apoptotic cells were detected at 6h after administration. In semithin sections, apoptotic cells were easily detected by their prominent basophilia and condensed chromatin, mainly found in spermatocytes. Ultrastructurally, the condensed chromatin and shrunken cytoplasm and nucleus, hallmarks of apoptotic cell death, were observed in butylparaben-treated groups. These observations lead us to postulate that butylparaben, similar to other estrogenic compounds, also induces spermatogenic cell apoptosis., (Copyright © 2013. Published by Elsevier GmbH.)

Objectives: Acrolein (Acr) is a highly reactive α, β-unsaturated aldehyde, which can induce reactive oxygen species (ROS) generation. Several factors, including lipid peroxidation, clinical use of cyclophosphamide, fried foods, automobile exhausts, smoking and aging can increase its concentration in blood serum. Mounting evidence has suggested that Acr-induced ROS might reduce quality of sperm. Thus, the aim of this study was to examine reproductive toxicity of Acr-caused ROS in vitro and find a means to alleviate it., Materials and Methods: We investigated the effects of Acr on male germ cell (MGC)-derived GC-1 cells in vitro. Dihydroethidium and DCFH-DA fluorescent dyes were used to determine generation of intracellular ROS., Results: We found that Acr induced ROS generation, which was accompanied by reduced Bcl2/Bax ratio, substantial decline in mitochondrial membrane potential, and further promoted apoptosis of MGCs. Furthermore, Rapamycin was capable of alleviating Acr-induced ROS, reducing ROS-induced apoptosis by increasing ratio of Bcl2/Bax mRNA and proteins, and protecting MGC mitochondrial membranes., Conclusion: Rapamycin inhibited Acr-induced apoptosis by alleviating ROS-driven mitochondrial dysfunction in MGCs., (© 2014 John Wiley & Sons Ltd.)

Objective: To determine the molecular mechanism of germ cell apoptosis via investigating the effect of PFT-α on the expression of p53 and bcl-2/bax during experimental cryptorchid cell apoptosis., Methods: Male Sprague-Dawley rats were assigned into 4 groups: a sham-operated group, a cryptorchid group, a cryptorchid+p53 inhibitor (p53 inhibitor-alpha, PFT-α) group, and a cryptorchid+dissolvent of PFT-α [dimethyl sulphoxide (DMSO)] group. Unilateral cryptorchidism was surgically induced in the rats of the cryptorchid group, PFT-α group, and cryptorchid+dissolvent of PFT-α group. The rats in the PFT-α group and cryptorchid+dissolvent of PFT-α group were intra-peritoneally injected PFT-α and dissolvent of PFT-α, respectively, once a day. The rats were killed on the 7th day after the surgery. The morphology of spermatogenic epithelium at the side of surgery in the rats was observed under light microscope. The apoptosis of spermatogenic cells in the unilateral cryptorchidism was evaluated by TUNEL and flow cytometry (FCM). The protein expression levels of p53, bcl-2, and Bax were detected by Western blot and immunohistochemical assay in turn., Results: Compared with the cryptorchid groups and the cryptorchid+dissolvent of PFT-α group, the seminiferous epithelium of the cryptorchid+p53 inhibitor group appeared orderly, with thicker cell layers and lower apoptosis index, weak protein expression level of p53/Bax and strong protein expression level of bcl-2., Conclusion: PFT-α inhibits the germ cell apoptosis caused by the experimental cryptorchidism via increasing the expression of bcl-2 and decreasing the expression of p53 and bax.

There is a need for improved reproductive toxicology assays that do not require large numbers of animals but are sensitive and informative. Therefore, Staput velocity-sedimentation separation followed by culture of specific mouse testicular cells was used as such a system. The specificity of separation was assessed using immunocytochemistry to identify spermatids, spermatocytes and spermatogonia. The efficacy of the system to detect toxicity was then evaluated by analysing the effects of hydrogen peroxide (H2O2) by the terminal uridine-deoxynucleotide end-labelling (TUNEL) assay to show the rate of apoptosis induced among the different types of germ cells. We found that 2 h of treatment at both 1 and 10 μM induced increases of over ∼10-fold in the percentage of apoptotic cells (p≤0.001), confirming that testicular germ cells are prone to apoptosis at very low concentrations of H2O2. It was also demonstrated for the first time for this compound that spermatogonia are significantly more susceptible than spermatocytes, which are more affected than spermatids. This reflects the proportion of actively dividing cells in these cell types, suggesting a mechanism for the differential sensitivity. The approach should thus form the basis of a useful test system for reproductive and genetic toxicology in the future., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause testicular toxicity in animals. In the present study, the effects of ZEA on spermatogenesis and possible mechanisms involved in germ cell injury were examined in rats. Ten-week-old Sprague-Dawley rats were treated with 5 mg/kg i.p. of ZEA and euthanized 3, 6, 12, 24 or 48 h after treatment. Histopathologically, spermatogonia and spermatocytes were found to be affected selectively. They were TUNEL-positive and found to be primarily in spermatogenic stages I-VI tubules from 6 h after dosing, increasing gradually until 12 h and then gradually decreasing. Western blot analysis revealed an increase in Fas and Fas ligand (Fas-L) protein levels in the ZEAtreated rats. However, the estrogen receptor (ER)alpha expression was not changed during the study. Collectively, our data suggest that acute exposure of ZEA induces apoptosis in germ cells of male rats and that this toxicity of ZEA is partially mediated through modulation of Fas and Fas-L systems, though ERalpha may not play a significant role.

Objective: To investigate the occurrence of germ cell apoptosis and the expression of Bcl-2/Bax after aniso-doses arsenic (As2O3) administration in the testes of the adult male rats., Method: Forty healthy male Sprague-Dawley rats were divided randomly into four groups and were administered respectively with 0 (control group), 0.375, 0.75, 1.5 mg x kg(-1) of As2O3 by intragastric administration consecutively for 16 weeks. The numbers of testicular sperm head were counted and the coefficient of testicular viscera and daily sperm production (DSP) were calculated in every group. The apoptosis of germ cell were assessed by in situ terminal deoxynucleotityl transferase mediated dTUP nick end labeling (TUNEL) technique. The expression of Bel-2/Bax in spermatogenic cells of different grades were located and quantitated by the method of immunohistochemistry., Result: (1) In the testes of As2O3-treated rats, the coefficient of testicular viscera, the number of testicular sperm head and DSP in the middle and high dose groups decreased significantly, compared with those in the control group (P < 0.01); however the apoptosis index of germ cell (AI) significantly increased compared with that in the control group (P < 0.01). Bcl-2 expression of middle dose group and high dose group decreased significantly compared with that in the control group (P < 0.01). Bax expression of middle dose group and high dose group increased significantly compared with that in the control group (P < 0.01); (2)The dependability analysis between DSP and AI showed a negative correlation (r = -0. 563, P <0. 01). (3) The negative correlation was significant between AI and Bcl-2 expression of spermatogenic cells (r = -0.825, P < 0.01); The positive correlation was significant between AI and Bax expression of spermatogenic cells (r = 0.710, P < 0.01)., Conclusion: One of the mechanisms of male reproduction toxicity of As2O3 might be that Bcl-2 expression is inhibited and Bax expression is encouraged which induces the decrease of quantity of sperm cells.

Cadmium (Cd) is a ubiquitous environmental pollutant that has been associated with male reproductive toxicity in both humans and animal models. The underlying mechanism of this response, however, is still uncharacterized. To address this issue, we employed a recently developed and optimized three-dimensional primary Sertoli cell-gonocyte coculture system and examined the time- and dose-dependent effects of Cd on morphological alterations, cell viability, activation of stress signaling pathway proteins, and the disruption of the ubiquitin proteasome system (UPS). Our results demonstrated that Cd exposure lead to time- and dose-dependent morphological changes that are associated with the induction of apoptosis. In response to Cd, we also saw a disruption of the UPS as evaluated through the accumulation of high-molecular weight polyubiquitinated proteins (HMW-polyUb) as well as alterations in proteasome activity. Robust activation of cellular stress response, measured through the increased phosphorylation of stress-activated protein kinase/c-jun N-terminal kinase and p38, paralleled the accumulation of HMW-polyUb. In addition, p53, a key regulatory protein, was upregulated and underwent increased ubiquitination in response to Cd. To further characterize the role of the UPS in Cd cellular response, we compared the above changes with two classic proteasomal inhibitors, lactacystin, and MG132. The stress response and the accumulation of HWM-polyUb induced by Cd were consistent with the response seen with MG132 but not with lactacystin. In addition, Cd treatment resulted in a dose- and time-dependent effect on proteasome activity, but the overall Cd-induced proteasomal inhibition was unique as compared to MG132 and lactacystin. Taken together, our studies further characterize Cd-induced in vitro testicular toxicity and highlight the potential role of the UPS in this response.

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.

The aim of this study was to clarify effects of inflammation on spermatogenesis in LPS-administered mice. ICR mice were treated by intraperitoneal injection for 7 days with either physiological saline (control) or 0.1 mg lipopolysaccharide (LPS)/kg body weight/day. Control mice were killed at 24 h after the last injection and the LPS-treated group after 24 h or 1, 3, or 5 weeks. Sperm concentration and motility in the cauda epididymis were examined as well as immunohistochemical localization of Fas and FasL and germ cell apoptosis. Sperm concentration and motility markedly fluctuated in LPS-treated mice. Increase of apoptotic cells was common in all post-LPS treatment groups, with a peak at 24 h after LPS injection. In contrast to the lack of Fas immunoreactivity in control testes, LPS-treated groups demonstrated Fas in many germ cells, especially in spermatocytes and spermatids. Immunoreactivity for FasL, on the other hand, was positive for some Sertoli cells, Leydig cells, and germ cells in both control and LPS-treated groups at all time points. The results suggest that the Fas/FasL system mediates apoptosis of germ cells in LPS-treated mice testes. LPS-administered mice thus provide a good experimental model for the study of transient disruption of spermatogenesis.

Ethanol exposure is known to suppress male reproductive activity in laboratory animals and humans. The present study was designed to evaluate whether chronic ethanol exposure decreases proliferative activity or increases apoptosis in the testes. Ethanol (1.5 g/kg or 3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Proliferating cell nuclear antigen (PCNA) was used as a proliferative marker. Western blot analysis showed that ethanol administration significantly reduced the level of PCNA. Also, immunoreactivity of PCNA-positive cells in the spermatogonia and primary spermatocytes were decreased by ethanol exposure. However, the number of TUNEL-positive cells was significantly increased in the testicular germ cells of ethanol-treated rats. Moreover, ethanol administration significantly increased the level of activated caspase-3 in testes. In conclusion, our findings suggest that ethanol may partly contribute to the suppression of male reproductive activity through a reduction of cell proliferation and an enhancement of cell death in rat testes.

Massive apoptosis of pubertal male germ cells is important for the development of functional spermatogenesis in the adult testis. Although the trigger(s) for male germ cell loss at puberty remain undefined, we have hypothesized that transforming growth factor-betas (TGF-betas) play an active role. Here we demonstrate that the three mammalian TGF-beta isoforms, TGF-beta1, TGF-beta2 and TGF-beta3, induce distinct apoptosis of pubertal spermatogonia and spermatocytes in a dose-dependent manner. Induction of male germ cell death by activation of caspase-3 was most pronounced with TGF-beta2 compared to TGF-beta1 and TGF-beta3. Furthermore, we found colocalization of activated caspase-3 with apoptotic protease-activating factor-1 (Apaf-1) in apoptotic germ cells, thus indicating the importance of the intrinsic mitochondrial pathway in TGF-beta-induced apoptosis. The specificity of the TGF-beta effects was proven by addition of recombinant latency-associated peptide against TGF-beta1 (rLAP-TGF-beta1) which completely abolished TGF-beta1-induced and TGF-beta3-induced germ cell apoptosis. Although TGF-beta2-triggered germ cell death also was significantly reduced by rLAP-TGF-beta1, inhibition was not maximal. Our results suggest that the three TGF-beta isoforms induce apoptosis of pubertal male germ cells via the mitochondrial pathway in vitro and are thus likely candidates involved in the excessive first wave of apoptosis of male germ cells during puberty.

Apoptosis or programmed cell death is characterized morphologically by chromatin condensation, cell shrinkage, fragmentation of the nucleus and cytoplasm, and consequently formation of apoptotic bodies. It has also been best characterized by the cleavage of DNA into nucleosomal size fragments of 180-200 bp or multiples of the same. Contrary to this, under extreme conditions, the cells were found to show adaptive response to apoptosis and unable to regulate their own death; necrosis is therefore predominantly observed. In the present study, we showed induction of apoptosis in Clarias batrachus due to sublethal concentration of 2,4-D and butachlor at multiple exposure time. The first phase of the study involved light microscopy (LM) and transmission electron microscopy (TEM) for ultrastructural abnormalities of the germinal tissues. While, in the second phase of the study, DNA degradation of blood and hepatic tissue was resolved on agarose gel electrophoresis. In histopathological studies, large numbers of stage II oocytes were noted for nuclear blebbing irrespective of the test chemical. Some of the butachlor-exposed oocytes showed vacuolation and electron dense cytoplasm along with thickened nuclear envelope, having close association with the lysosomes on the cytoplasmic side. Some oocytes undergo nuclear blebbing having inner dense core and translucent cytoplasm. Leydig cells were slightly hypertrophied and few appeared pycnotic, a process involving necrotic changes in which the cell nuclei were characterized by rounding up and condensation resulting in hyperchromatic staining or pycnosis. In testicular tissue, spermatogonial nuclei had irregular large clumps of heterochromatin adjoining the nuclear membrane indicating initial stage of apoptotic cell death. Electrophoretic separation resulted in a ladder pattern of blood DNA and smear like pattern of hepatic DNA. These results indicate that the above herbicides are able to induce apoptosis both at molecular as well as cytological level. A reference dose or safety factor approach to calculate risk of human exposure to both chemicals is still awaited.

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined. As indicated by microscopy and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16- and 24-day-old rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult.

Objective: To observe the relationship between spermatogenic cell apoptosis and serum estradiol level in rats exposed to fluoride., Methods: A total of 30 male Wistar rats were allocated into six groups randomly. The six experimental groups were 28-day control group, 28-day low-dose fluoride treatment group, 28-day high-dose fluoride treatment group, 38-day control group, 38-day low-dose fluoride treatment group, and 38-day high-dose fluoride treatment group. The fluorosis model was acquired by subcutaneous injection of NaF solution. The content of NaF in testis was measured by using fluorine selective electrode. The serum estradiol level was radioimmunochemically detected. And the apoptotic spermatogenic cells were quantitatively measured by TUNEL., Results: The content of NaF in testis and the ratio of apoptotic spermatogenic cell in fluoride treatment groups significantly increased with increased experimental dosage and prolonged experimental period (P < 0.05). Meanwhile, the serum estradiol level significantly decreased (P < 0.05), which was negatively correlated with the content of NaF in testis as well as the ratio of apoptotic spermatogenic cell (P < 0.05)., Conclusion: Excessive fluoride could lead disturbance to serum estradiol level during some range of dose and time, which is an important factor to spermatogenic cell apoptosis.

Etoposide is a podophyllotoxin semiderivative that is used in a variety of chemotherapy treatments, including therapy for children tumors. This drug promotes the formation of a ternary DNA-topoisomerase II-etoposide complex that triggers apoptosis. The purpose of this work was to analyze the occurrence of apoptosis in the seminiferous epithelium of prepubertal, pubertal, and adult rats treated with 10, 20, and 40 mg/Kg of etoposide during the prepubertal phase, as well as the role of apoptosis in etoposide-induced testicular damage. The rat testes were fixed in Bouin's liquid, and the apoptotic cells were quantified by means of the hematoxylin and eosin (H&E) technique (all groups) and the terminal dUTP nick end labeling (TUNEL) method (prepubertal groups only). The results obtained from both the H&E and TUNEL methods showed an increased frequency of apoptosis in the seminiferous epithelium of treated animals, except for the subgroup that received the 10-mg/Kg dose and was sacrificed 12 hr after the treatment and for the etoposide-treated pubertal group, that did not show cells suggesting apoptosis during H&E analysis. The labeled cells were mainly primary spermatocytes and differentiated spermatogonia. The prepubertal rats showed an etoposide-dose-dependent diminution of differentiated spermatogonia. Etoposide treatment during the prepubertal phase increases the frequency of apoptosis in the seminiferous epithelium, and causes serious harm to male fertility. 2004., (Copyright 2004 Wiley-Liss, Inc.)

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause toxicity of testis in male rats. To investigate whether apoptosis is involved in ZEA-induced testicular toxicity and to identify the stage and target germ cell type, 10-week-old Sprague-Dawley male rats were treated with a single intraperitoneal (i.p.) dose of ZEA (5 mg/kg) and euthanized at 3, 6, 12, 24, or 48 h subsequently. Histopathologically, germ cell degeneration was found at stages I-VI 12 h after dosing. Degenerating germ cells were shown to undergo apoptosis as revealed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The frequency of TUNEL-labeled germ cells increased in a stage-specific manner, the peak frequency gradually progressing at stages I-VI of seminiferous tubules with time after dosing, suggesting that the damaged germ cells, especially spermatogonia and spermatocytes, gradually underwent the processes leading to apoptosis. DNA laddering on gel electrophoresis was apparent 12 h after dosing. The results demonstrated that a single dose of ZEA induces testicular germ cell apoptosis in a time-dependent and stage-specific pattern. This study has established that apoptosis is the principal mechanism contributing to germ cell depletion and testicular atrophy following ZEA exposure., (Copyright 2002 Elsevier Science Ireland Ltd.)

Purpose: The present study was performed to investigate the protective effect of leuprorelin (LH-RH analog), on spermatogonia apoptosis induced by doxorubicin (DXR) in the Sprague-Dawley rat model., Methods: Twenty-four adult male rats were divided into the following four groups: (i) control group; (ii) group given doxorubicin (intravenous injection, 8 mg/kg); (iii) group given leuprorelin (subcutaneous injection, 3 mg/kg); and (iv) group given both doxorubicin (intravenous injection, 8 mg/kg) and leuprorelin (subcutaneous injection, 3 mg/kg). Evaluation for quantification of apoptotic spermatogonia was made by the ratio of TUNEL-labeled spermatogonia versus 100 Sertoli cells in each seminiferous tubule. Two hundred seminiferous tubules of each rat were assessed., Results: The ratio of apoptotic spermatogonia versus 100 Sertoli cells at stages II-IV of the groups given DXR (groups 2 and 4) were significantly higher than those of the other groups. However, the value at stages II-IV of the group given both DXR and leuprorelin (group 4) was significantly lower than that of the group given DXR (group 2)., Conclusion: The significant prophylactic effect (P < 0.05) of LH-RH analog against doxorubicin-induced spermatogonial apoptosis was observed in a stage specific manner by microscopic evaluation with TUNEL.

We previously showed that prolactin (PRL) induces apoptosis in newt secondary spermatogonia and indicated that caspase activity is involved in the apoptosis. Since it was recently reported that Z-VAD-fmk, a pan-caspase inhibitor, blocks activity of cysteine cathepsins as well, we examined whether cathepsin is involved in the newt spermatogonial apoptosis. We found cathepsin L activity in the testis that was elevated by PRL in organ culture of testis, while E-64d, a lysosomal cysteine protease inhibitor, and Z-VAD-fmk suppressed it and chromosomal condensation. These results suggest that cathepsin L activity play a pivotal role in PRL-induced spermatogonial apoptosis.

Epidemiological surveys and animal experimental studies suggest that exposure to 2-bromopropane (2-BP) could result in reproductive and hematopoietic disorders. The objectives of this study were to investigate the role of apoptosis in 2-BP-induced testicular toxicity and whether this process involves Bcl-2 family genes and the Fas signaling system. Rats were injected percutaneously with 1350 mg/kg 2-BP for 1 to 5 days and then were euthanized at 6 or 12 h after one dose, 6 h after two, three, or five doses, and 2 or 9 days after the final treatment. Light and electron microscopic analyses, TUNEL staining of DNA fragments, agarose gel electrophoresis of low-molecular-weight DNA, and Western blotting analysis of Bcl-2 family proteins and Fas receptor and ligand were conducted. Two-day treatment resulted in selective degeneration of spermatogonia with marked nuclear chromatin condensation. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The percentage of apoptotic-positive tubules and apoptotic cell index increased time dependently. 2-BP treatment resulted in two distinct morphological changes: an immediate effect on spermatogonia and secondary apoptosis of spermatocytes 9 days after treatment. Downregulation of Bcl-2 after the first or second injection of 2-BP and upregulation of Bax after the first treatment contributed to the initiation of primary apoptosis of spermatogonia. Expression of FasL was inhibited while expression of Fas increased after the 2-BP treatment and remained at levels about two times of the control. However, it increased about sixfold of the control by day 9 after final injection, which contributed to the induction of secondary apoptosis of spermatocytes. Our results indicate that 2-BP resulted in apoptotic death of testicular germ cells and that this process involves the Bcl-2 family genes and the Fas signaling system., (Copyright 2001 Academic Press.)

Cell death is a common feature during spermatogenesis and, in some seasonal breeding animals, is often observed at the transition stage from spermatogonia to spermatocytes. In the Japanese red-bellied newt, we have previously shown that this cell death is caused by the elevated titer of plasma PRL that occurs after animals are transferred to low temperature, suggesting that cell death causes the cessation of spermatocytogenesis from late autumn to early spring. In the present report, first we show that the injection of PRL into newts causes apoptosis in spermatogonia after the sixth mitotic division, the penultimate one before spermatogonia normally enter meiosis. Second, we demonstrate in organ cultures of testes fragments that PRL acts directly on the testes. Third, we show that the action by PRL is inhibited by FSH dose dependently.

This study was undertaken to determine the extent of apoptosis and mitosis in the various testicular cell types throughout rat development from foetal day 14.5 to postnatal days 9-10. Apoptotic activity was studied by detecting DNA fragmentation (TUNEL method) in situ. A TUNEL-positive reaction was detected in gonocytes, while none of the other testicular cells were labelled. The morphology of the TUNEL-positive gonocytes was characteristic of apoptotic cells and was different from that observed in experimentally induced necrosis. The percentage of stained gonocytes peaked on day 15.5-16.5 post-conception (dpc), decreased thereafter and no TUNEL-positive gonocytes were found from foetal day 18.5 onwards. On postnatal day 2, apoptosis resumed and increased to reach a maximum on day 7. Mitosis in the gonocytes, as evaluated by the immunodetection of 5-bromo-2'-deoxyuridine (BrdU) incorporation, was present during the same developmental periods but the ratio of BrdU-positive/TUNEL-positive gonocytes was much greater in the foetal period than in the neonatal period. In an organotypic culture system, the changes in the apoptotic and mitotic activities of the gonocytes in testicular explants from foetuses on days 18.5 and 20.5 or from neonates on day 3, cultured for two days were similar to those observed in vivo. Addition of LH or FSH did not influence either apoptosis or mitosis in the germ cells. These results suggest that both apoptosis and mitosis of gonocytes are independent of gonadotrophins and are mainly controlled by intratesticular factors.

Apoptosis has been proposed as a mechanism by which testis germ cells are removed during normal and various pathological conditions. To establish a new rapid way to detect stage-specific apoptosis in male rat germ cells, their supravital morphology was examined from carefully squashed monolayers of living cells, after several established toxic treatments, using a phase contrast microscope. The results were compared with early detection of apoptosis using annexin V and propidium iodide (PI) stainings. The apoptosis of type-A spermatogonia and round spermatids proceeded in a similar way to somatic cells, while intermediate and type-B spermatogonia, and particularly the dividing spermatocytes, possessed characteristics not entirely typical for apoptosis. Death of elongated spermatids was difficult to assess owing to their compacted chromatin. As the first phases of degeneration seemed different in various germ cell classes, the final stage (karyopycnosis) was similar for most cells. Degenerating cells also showed positive reactions for annexin V and PI. The 'living cell method' provides rapid and accurate possibilities for analysis of stage-specific apoptosis during spermatogenesis. This method is not influenced by artefacts induced by fixation, embedding and sectioning. It may be developed further for routine analyses of the accurate stage-specific effects of various physical and chemical effects on mammalian and human spermatogenesis.

The present study investigated the occurrence of apoptosis in rat testis 6, 12, 24, or 48 h after administration of 1,3-dinitrobenzene (DNB)(25 mg/kg, i.p.). Apoptotic cells were visualized using TUNEL in situ detection. In untreated control animals, occasional tubules contained a few apoptotic nuclei. After DNB treatment, more tubules were apoptotic and more germ cells within individual tubules were affected. The increase in incidence and severity of apoptosis was statistically significant 24 and 48 h after exposure. Spermatocytes were the primary cell population affected. Apoptosis was primarily present in Stages VI-VIII and IX-XIII. DNA ladders, characteristic of apoptosis, were clearly present by 24 h after DNB administration, faint at 12 h, and not present in the control or at 6 h. Although initiating mechanisms may be very different for different chemicals, apoptosis is a common and late manifestation of the testicular response to numerous toxicants.

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.

The Sertoli cell toxicant 2,5-hexanedione (2,5-HD) causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few spermatogonia remain in the seminiferous epithelium. In this study, the number, type, and fate of the remaining germ cells were determined. Male Sprague-Dawley rats were exposed to 1% 2,5-HD in drinking water for 5 weeks and then sacrificed 12 or 40 weeks after the start of exposure. Cell counts determined that the stem cell population was diminished in size, but made up a significant portion of the remaining germ cells. The remaining germ cells were primarily type A spermatogonia. Modeling of spermatogonial divisions suggested that most spermatogonia undergo degeneration at the level of type A3 spermatogonia after 2,5-HD-induced atrophy. Apoptosis was demonstrated to occur in the remaining germ cells by nuclear morphology and in situ analysis of DNA fragmentation. Quantitation indicated that apoptosis occurred in a majority of stem cell progeny. We conclude that the irreversibility of 2,5-HD-induced testicular injury results from the reduced size of the stem cell population as well as a block in germ cell development at the level of type A spermatogonia.