Male Germ Cell Apoptosis Research Articles

Arecoline Hydrobromide Promotes the Apoptosis of Male Germ Cells

Arecoline Hydrobromide Promotes the Apoptosis of Male Germ Cells

A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells

Germ cells preferentially induce apoptosis in response to DNA damage to avoid genomic mutations. Apoptosis of germ cells is closely related to cancer development and chemotherapy resistance; however, its regulatory mechanism is unclear. Here, we suggest that testis-specific lncRNA LINC03074 is involved in male germ cell apoptosis by regulating the expression of the proto-oncogene MDM2. LINC03074 is highly expressed in the sperm of healthy adult testes and cancer cells of testes with testicular germ cell tumors (TGCTs). LINC03074 binds to MDM2 mRNA via an Alu element, thereby reducing MDM2 protein levels. LINC03074 stimulates STAU1-mediated nuclear export of MDM2 mRNA by increasing STAU1 binding to MDM2 mRNA in the cell nucleus, thereby promoting PKR-mediated translational repression in the cytoplasm. The induction of apoptosis in TGCT cells and their responsiveness to the anticancer drug cisplatin is enhanced by LINC03074. Notably, LINC03074 increased E2F1 expression without increasing p53, the primary target of MDM2, and upregulated the apoptotic gene p73, the target gene of E2F1. LINC03074-mediated regulation of apoptosis contributes to the responsiveness of TGCTs to anticancer drug-induced DNA damage.

Cadmium induces apoptosis of mouse spermatocytes through JNK activation and disruption of autophagic flux

Cadmium has been reported to accumulate primarily in spermatogonia and spermatocytes. Exposure to cadmium results in male reproductive toxicity via germ-cell apoptosis and impaired autophagy. Apoptosis and autophagy are two physiologically conserved events that maintain cellular homeostasis. However, the precise role of autophagy in cadmium-induced apoptosis of male germ cells has yet to be addressed. The present study aimed to investigate the impact of cadmium exposure on the cytotoxicity of GC-2 spd cells, a mouse spermatocyte cell line. The results showed that cadmium exposure caused apoptotic cell death and the accumulation of autophagosomes, along with the up-regulation of ATG proteins in GC-2 spd cells. It was demonstrated that the cadmium-induced accumulation of autophagosomes contributes to the apoptosis of GC-2 spd cells. This notion is supported by the findings that the autophagy inhibitor 3-MA reduced accumulation of autophagosomes and apoptotic cell death. Conversely, the apoptosis inhibitor Z-VAD-FMK inhibited apoptosis but had little effect on the accumulation of autophagosomes. Cadmium may impede the fusion of autophagosomes with lysosomes, leading to the autophagosome buildup. Additionally, we found that the JNK pathway mediates transcriptional induction of several autophagy-related (ATG) genes involved in autophagosome formation. The cadmium-activated JNK pathway regulates apoptosis by mediating the autophagosome formation. Treatment of cells with the JNK inhibitor SP600125 attenuated the accumulation of autophagosomes, the upregulated expression of autophagosome-associated proteins and apoptotic cell death induced by cadmium. Overall, these findings suggest that cadmium enhances apoptosis of GC-2 spd cells by activating the JNK pathway and inhibiting autophagic flux.

T-2 mycotoxin Induces male germ cell apoptosis by ROS-mediated JNK/p38 MAPK pathway

T-2 mycotoxin, a type A trichothecene toxin that, specifically, causes male and female reproductive toxicity. We evaluated T-2 toxin toxicity in testes from neonatal testes after in vitro tissue cultured. Additionally, current study focuses on the molecular mechanism of toxicity and germ cell damage in GC-1 spermatogonial cells. Mouse testicular fragments were subjected to T-2 toxin (0–20 nM) during days 5 of in vitro culture. Testicular germ cell number were reduced and downregulated the expression of corresponding markers depending on the exposure concentration of T-2 toxin; however, Sertoli cell markers and steroidogenic enzyme expression increased when treated with 20 nM T-2 toxin. The cell viability decreased, apoptosis increased, and pro-apoptotic protein expression increased in 5–20 nM T-2 toxin–exposed spermatogonia. Moreover, T-2 toxin generated reactive oxygen species (ROS) and induced mitochondrial dysfunction, indicating that activation of p38 MAPK signaling triggered by ROS is involved in the apoptotic molecular mechanism of T-2 toxin. T-2 toxin induced the phosphorylation of ERK1/2, c-Jun, JNK/SAPK, p38, and p53, and the subsequent inhibition of AKT phosphorylation. The upregulation of genes related to apoptosis and MAPK/JNK signaling was consistently observed in cells exposed to T-2 toxin. These results indicate that T-2 toxin triggers apoptotic cell death in germ cells through the triggering of ROS-mediated JNK/p38-MAPK signaling pathways.

Oxidative stress-induced apoptosis and autophagy: Balancing the contrary forces in spermatogenesis

Spermatogenesis is a complex process in the testis and is a cornerstone of male infertility. The abundance of unsaturated fatty acid and high cell division rate make male germs cells prone to DNA deterioration. ROS-mediated oxidative stress triggers DNA damage, autophagy, and apoptosis in male germ cells, which are critical causative factors that lead to male infertility. The complex connection and molecular crosstalk between apoptosis and autophagy is seen at multifaceted levels that interconnect the signaling pathways of these two processes. Multilevel interaction between apoptosis and autophagy is a seamless state of survival and death in response to various stressors. Interaction between multiple genes and proteins such as the mTor signaling pathway, Atg12 proteins, and the death adapter proteins, such as Beclin 1, p53, and Bcl-2 family proteins, validates such a link between these two phenomena. Testicular cells being epigenetically different from somatic cells, undergo numerous significant epigenetic transitions, and ROS modulates the epigenetic framework of mature sperm. Epigenetic deregulation of apoptosis and autophagy under oxidative stress conditions can cause sperm cell damage. The current review recapitulates the current role of prevailing stressors that generate oxidative stress leading to the induction of apoptosis and autophagy in the male reproductive system. Considering the pathophysiological consequences of ROS-mediated apoptosis and autophagy, a combinatorial approach, including apoptosis inhibition and autophagy activation, should be implemented as a therapeutic strategy to treat male idiopathic infertility. Understanding the crosslink between apoptosis and autophagy under stress conditions in male germ cells may play an essential role in developing therapeutic strategies to treat infertility.

Fat mass and obesity-associated factor (FTO)-mediated N6-methyladenosine regulates spermatogenesis in an age-dependent manner

N6-methyladenosine (m6A) is the most prevalent reversible RNA modification in the mammalian transcriptome. It has recently been demonstrated that m6A is crucial for male germline development. Fat mass and obesity-associated factor (FTO), a known m6A demethylase, is widely expressed in human and mouse tissues and is involved in manifold biological processes and human diseases. However, the function of FTO in spermatogenesis and male fertility remains poorly understood. Here, we generated an Fto knockout mouse model using CRISPR/Cas9-mediated genome editing techniques to address this knowledge gap. Remarkably, we found that loss of Fto in mice caused spermatogenesis defects in an age-dependent manner, resulting from the attenuated proliferation ability of undifferentiated spermatogonia and increased male germ cell apoptosis. Further research showed that FTO plays a vital role in the modulation of spermatogenesis and Leydig cell maturation by regulating the translation of the androgen receptor in an m6A-dependent manner. In addition, we identified two functional mutations of FTO in male infertility patients, resulting in truncated FTO protein and increased m6A modification in vitro. Our results highlight the crucial effects of FTO on spermatogonia and Leydig cells for the long-term maintenance of spermatogenesis and expand our understanding of the function of m6A in male fertility.

Male germ cell proliferation and apoptosis in sexually immature meagre Argyrosomus regius (Asso, 1801) treated with recombinant follicle stimulating hormone

The meagre Argyrosomus regius (Asso, 1801) is a marine fish species that has an increasing aquaculture production in Europe. Lowering the age at maturity of hatchery-produced juveniles would support meagre aquaculture by reducing time between generations in selective breeding programs and reducing industrial costs for broodstock maintenance. The aim of this work was to assess the effects of a treatment with recombinant follicle stimulating hormone (rFsh), produced in ovarian cells of Chinese hamsters, on male germ cell proliferation and apoptosis in sexually immature meagre. The rFsh-treated fish had higher gonadosomatic index, larger seminiferous tubules, more abundant luminal spermatozoa, a lower density of anti-PCNA positive single A spermatogonia, a higher density of anti-PCNA positive spermatocysts and a lower incidence of germ cell apoptosis than control groups. The present study demonstrated the effectiveness of the produced rFsh in stimulating testis development and spermatogenesis in pre-pubertal meagre. Moreover, the rFsh treatment proved to be highly efficient in removing the apoptotic block of spermatogenesis observed in juvenile meagre, allowing spermatogonial survival and progress towards meiosis. The administration of rFsh did not stimulate spermatogonial self-renewal, a process whose control still needs to be elucidated.

Coding and regulatory transcriptome comparisons between fertile and infertile spermatozoa identify RNA signatures of male infertility

The aim of the present study was to identify RNA-based signatures of male infertility by sperm transcriptome analysis. In this study, deep sequencing analyses of coding (mRNA) and regulatory (miRNA) transcriptomes were performed by pooling 15 oligo/oligoasthenozoospermic infertile sperm and 9 normozoospermic fertile sperm samples. Furthermore, interesting candidates were selected for validation by real-time PCR. The comparison of miRNAs between cases and controls identified 94 differentially expressed miRNAs, of which at least 38 have known functions in spermatogenesis. In transcriptome (mRNA) data, a total of 60,505 transcripts were obtained. The comparison of coding RNAs between cases and controls revealed 11,688 differentially expressed genes. miRNA-mRNA paired analysis revealed that 94 differentially expressed miRNAs could potentially target 13,573 genes, of which 6419 transcripts were actually differentially expressed in our data. Out of these, 3303 transcripts showed inverse correlation with their corresponding regulatory miRNAs. Moreover, we found that most of the genes of miRNA-mRNA pairs were involved in male germ cell differentiation, apoptosis, meiosis, spermiogenesis and male infertility. In conclusion, we found that a number of sperm transcripts (miRNAs and mRNAs) have a very high potential of serving as infertility/sperm quality markers.

Investigation of the genetic etiology in male infertility with apparently balanced chromosomal structural rearrangements by genome sequencing.

Apparently balanced chromosomal structural rearrangements are known to cause male infertility and account for approximately 1% of azoospermia or severe oligospermia. However, the underlying mechanisms of pathogenesis and etiologies are still largely unknown. Herein, we investigated apparently balanced interchromosomal structural rearrangements in six cases with azoospermia/severe oligospermia to comprehensively identify and delineate cryptic structural rearrangements and the related copy number variants. In addition, high read-depth genome sequencing (GS) (30-fold) was performed to investigate point mutations causative of male infertility. Mate-pair GS (4-fold) revealed additional structural rearrangements and/or copy number changes in 5 of 6 cases and detected a total of 48 rearrangements. Overall, the breakpoints caused truncations of 30 RefSeq genes, five of which were associated with spermatogenesis. Furthermore, the breakpoints disrupted 43 topological-associated domains. Direct disruptions or potential dysregulations of genes, which play potential roles in male germ cell development, apoptosis, and spermatogenesis, were found in all cases (n = 6). In addition, high read-depth GS detected dual molecular findings in case MI6, involving a complex rearrangement and two point mutations in the gene DNAH1. Overall, our study provided the molecular characteristics of apparently balanced interchromosomal structural rearrangements in patients with male infertility. We demonstrated the complexity of chromosomal structural rearrangements, potential gene disruptions/dysregulation and single-gene mutations could be the contributing mechanisms underlie male infertility.

Loss of CEP70 function affects acrosome biogenesis and flagella formation during spermiogenesis

The spermatogenesis process is complex and delicate, and any error in a step may cause spermatogenesis arrest and even male infertility. According to our previous transcriptomic data, CEP70 is highly expressed throughout various stages of human spermatogenesis, especially during the meiosis and deformation stages. CEP70 is present in sperm tails and that it exists in centrosomes as revealed by human centrosome proteomics. However, the specific mechanism of this protein in spermatogenesis is still unknown. In this study, we found a heterozygous site of the same mutation on CEP70 through mutation screening of patients with clinical azoospermia. To further verify, we deleted CEP70 in mice and found that it caused abnormal spermatogenesis, leading to male sterility. We found that the knockout of CEP70 did not affect the prophase of meiosis I, but led to male germ-cell apoptosis and abnormal spermiogenesis. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis, we found that the deletion of CEP70 resulted in the abnormal formation of flagella and acrosomes during spermiogenesis. Tandem mass tag (TMT)-labeled quantitative proteomic analysis revealed that the absence of CEP70 led to a significant decrease in the proteins associated with the formation of the flagella, head, and acrosome of sperm, and the microtubule cytoskeleton. Taken together, our results show that CEP70 is essential for acrosome biogenesis and flagella formation during spermiogenesis.

Identification of abnormal protein expressions associated with mouse spermatogenesis induced by cyclophosphamide.

Cyclophosphamide (CP) is a clinical anticancer drug that can cause male reproductive abnormalities, but the underlying mechanisms for this remain unknown. The present study aimed to explore the potential toxicity induced by CP in spermatogenesis events of germ cell proliferation, meiosis, and blood‐testis barrier integrity at the molecular level. CP‐treated mice showed significantly reduced serum testosterone levels, sperm motility and concentration. The results of immunohistochemistry and Western blot showed that CP reduced the proliferation of germ cells (PCNA, PLZF) and increased germ cell apoptosis (Bax and TUNEL‐positive cells) in CP‐treated mice testes. The expression of meiotic related proteins (SYCP3, REC8, MLH1) decreased significantly in the fourth week after administration, and the expression of blood‐testis barrier related proteins (β‐catenin, ZO‐1) and sperm quality‐associated proteins (PGK2, HSPA4) decreased significantly in the first week after administration. CP leads to the apoptosis of male germ cells, inhibits the proliferation of germ cells, and affects meiosis and the blood‐testis barrier, resulting in the decline of sperm quality. This study provides information to further the study of molecular mechanism and protective strategy of CP influence.

The IL-27 component EBI-3 and its receptor subunit IL-27Rα are essential for the cytoprotective action of humanin on male germ cells†.

Humanin (HN) is a mitochondrial-derived peptide that protects many cells/tissues from damage. We previously demonstrated that HN reduces stress-induced male germ cell apoptosis in rodents. HN action in neuronal cells is mediated through its binding to a trimeric cell membrane receptor composed of glycoprotein 130 (gp130), IL-27 receptor subunit (IL-27R, also known as WSX-1/TCCR), and ciliary neurotrophic factor receptor subunit (CNTFR). The mechanisms of HN action in testis remain unclear. We demonstrated in ex-vivo seminiferous tubules culture that HN prevented heat-induced germ cell apoptosis was blocked by specific anti-IL-27R, anti-gp130, and anti-EBI-3, but not by anti-CNTFR antibodies significantly. The cytoprotective action of HN was studied by using groups of il-27r-/- or ebi-3-/- mice administered the following treatment: (1) vehicle; (2) a single intraperitoneal (IP) injection of HN peptide; (3) testicular hyperthermia; and (4) testicular hyperthermia plus HN. We demonstrated that HN inhibited heat-induced germ cell apoptosis in wildtype but not in il-27r-/- or ebi-3-/- mice. HN restored heat-suppressed STAT3 phosphorylation in wildtype but not il-27r-/- or ebi-3-/- mice. Dot blot analyses showed the direct interaction of HN with IL-27R or EBI-3 peptide. Immunofluorescence staining showed the co-localization of IL-27R with HN and gp130 in Leydig cells and germ cells. We conclude that the anti-apoptotic effects of HN in mouse testes are mediated through interaction with EBI-3, IL-27R, and activation of gp130, whereas the role of CNTFR needs further studies. This suggests a multicomponent tissue-specific receptor for HN in the testis and links HN action with the IL-12/IL-27 family of cytokines.

Calcium-/Calmodulin-Dependent Protein Kinase IV (CAMKIV): A Multifunctional Enzyme and Its Role in Various Cancer: An Update

The calcium/calmodulin-dependent protein kinase IV (CAMKIV), a multi-functional enzyme, belongs to the serine/threonine protein kinase family. It plays critical role in inflammatory response, T cell maturation, dendritic cell survival, memory consolidation, regulation of cell cycle, apoptosis, and development of neurons and male germ cells. CAMKIV activates signaling cascade and controls the activity of numerous transcription factors by phosphorylation. Overexpression and increased activity of this enzyme are linked with various types of cancer such as hepatocellular carcinoma, breast cancer, neuroblastoma, prostate cancer, and AML. CAMKIV is also associated with several syndromes like azoospermia, cerebral hypoxia, and systemic lupus. As this enzyme is overexpressed in several cancers and other human diseases, it could be a potential therapeutic target in several diseases. In this review, we tried to explore the role of CAMKIV in various cancer and immune system development and available inhibitor of CAMKIV.

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

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

Seasonal changes in the spermatogenesis of the large Japanese field mice (Apodemus speciosus) controlled by proliferation and apoptosis of germ cells

The aim of this study was to investigate the proliferation and apoptosis of male germ cells during the seasonal reproductive cycle of the large Japanese field mice (Apodemus speciosus). Male mice residing in their natural habitat were captured in Niigata, Japan. Testis sections were stained with haematoxylin and eosin, and mitotic male germ cells were identified using immunofluorescence staining for proliferating cell nuclear antigen (PCNA). Apoptosis was analysed using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. The phases of spermatogenesis during the seasonal reproductive cycle were classified as active, transitional, and inactive based on the diameter of the seminiferous tubules. The number of PCNA-positive germ cells was less during the inactive than other phases. The percentage of TUNEL-positive germ cells per seminiferous tubule was greater during the inactive than active and transitional phases. Spermatogenesis during the seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells. This species of undomesticated mice could be used as an animal model to study spermatogenesis as a valuable indicator of the effects of ecological and anthropogenic factors on animal reproduction.

Expression patterns of male germ cell markers in cryptorchid pig testes

Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.

The humanin analogue (HNG) prevents temozolomide-induced male germ cell apoptosis and other adverse effects in severe combined immuno-deficiency (SCID) mice bearing human medulloblastoma

Subfertility is a major concern of long-term cancer survivors at the reproductive age. We have previously demonstrated that a potent humanin analogue, HNG, protected chemotherapy-induced apoptosis in germ cells but not cancer cells in a metastatic melanoma allograft model. In this study, we utilized severe combined immuno-deficiency (SCID) mice bearing human medulloblastoma to study the effect of HNG in Temozolomide (TMZ) induced male germ cell apoptosis and white blood cell (WBC) suppression. Human medulloblastoma DAOY cells were injected subcutaneously into the right flank of male SCID mice. Three weeks later, groups of tumor-bearing mice received one of the following treatments: vehicle, HNG, TMZ, or TMZ + HNG. 24 h after last injection, the tumors weights, complete blood counts, liver and spleen weights, male germ cell apoptosis was assessed. HNG did not affect TMZ's significant anti-tumor action. HNG significantly prevented TMZ-induced germ cell apoptosis and attenuated the suppressed total WBC and granulocyte counts in SCID mice with or without TMZ treatment. HNG also attenuated TMZ-induced body weight loss and decrease of spleen and liver weights. In conclusion, HNG ameliorated TMZ-induced germ cell apoptosis; WBC and granulocytes loss; and decreased body/organ weights without compromising the TMZ's anti-cancer action on medulloblastoma xenografts in SCID mice.

ADAM17 As Inducer of Male Germ Cell Apoptosis

Apoptosis during mammalian spermatogenesis is important to control sperm output and to eliminate damaged unwanted cells. A deregulation in apoptosis rate has deleterious consequences and leads to low sperm production. Apoptosis in spermatogenesis has been widely studied, but the mechanism by which it is induced under physiological or pathological conditions has not been clarified. We have recently identified the metalloprotease ADAM17 (TACE) as a putative physiological inducer of germ cell apoptosis. ADAM17 is widely distributed enzyme in many tissues, its main function is to shed (release) its substrates, which leads to the activation of different signaling pathways according to the cellular context. However, in spermatogenesis, ADAM17 is involved in the control or induction of germ cell apoptosis, this seems to be unique and may reflect the complexity of spermatogenesis. In this review I will focus on the published evidence that show how this enzyme involved in the control of germ cell apoptosis during physiological condition and in different insults such as heat stress, DNA damaging agent or endocrine disruptors.

Bisphenol A induced male germ cell apoptosis via IFNβ-XAF1-XIAP pathway in adult mice

Bisphenol A (BPA) impairs male fertility by acting as an endocrine disruptor. However, the mechanisms by which BPA cause reproductive toxicity are not fully elucidated. Here, we explored the role of XAF1, a novel pro-apoptosis molecule, in BPA-induced abnormal spermatogenesis and the transcriptional regulation mechanism of BPA-induced XAF1. BPA exposure detrimentally impacted spermatogenesis by inducing excessive germ cell apoptosis. XAF1 was upregulated in germ cells after BPA exposure, which was involved in the apoptosis pathway. In addition, the expression levels of XIAP and XAF1 were inversely correlated after BPA exposure. Knockdown of XAF1 expression partially inhibited the apoptosis of GC-2 cells, suppressed the activation of caspase 3 and improved the BPA-induced XIAP expression. Moreover, IFNβ expression levels were significantly upregulated after BPA exposure both in vitro and in vivo, and these levels were positively related to the expression of XAF1. Furthermore, IFNβ knockdown reduced the expression of XAF1 and increased the expression of XIAP in BPA-treated GC-2 cells. Together, these data indicated that BPA triggers male germ cell apoptosis in mice via the IFNβ-XAF1-XIAP pathway, which may contribute to BPA-induced testis toxicity.

A commercial Roundup® formulation induced male germ cell apoptosis by promoting the expression of XAF1 in adult mice

Roundup® is extensively used for weed control worldwide. Residues of this compound may lead to side effects of the male reproductive system. However, the toxic effects and mechanisms of Roundup® of male germ cells remain unclear. We aimed to investigate the apoptosis-inducing effects of Roundup® on mouse male germ cells and explore the role of a novel tumor suppressor XAF1 (X-linked inhibitor of apoptosis-associated factor 1) involved in this process. We demonstrated that Roundup® can impair spermatogenesis, decrease sperm motility and concentration, and increase the sperm deformity rate in mice. In addition, excessive apoptosis of germ cells accompanied by the overexpression of XAF1 occurred after Roundup® exposure both in vitro and in vivo. Furthermore, the low expression of XIAP (X-linked inhibitor of apoptosis) induced by Roundup® was inversely correlated with XAF1. Moreover, the knockdown of XAF1 attenuated germ cell apoptosis, improved XIAP expression and inhibited the activation of its downstream target proteins, caspase-3 and PARP, after Roundup® exposure. Taken together, our data indicated that XAF1 plays an important role in Roundup®-induced male germ cell apoptosis. The present study suggested that Roundup® exposure has potential negative implications on male reproductive health in mammals.