Melatonin ameliorates PM2.5-induced spermatogenesis disorder by preserving H3K9 methylation and SIRT3.

There was a link between exposure to PM2.5 and male infertility. Melatonin has beneficial effects on the male reproductive processes. How PM2.5 caused spermatogenesis disturbance and whether melatonin could prevent PM2.5-induced reproductive toxicity have remained unclear. The results showed that PM2.5 could inhibit the Nrf2-mediated antioxidant pathway and distinctly increase the cell apoptosis in testes. Moreover, PM2.5 also perturbed the process of meiosis by modulating meiosis-associated proteins such as γ-H2AX and Stra8. Mechanistically, PM2.5 inhibited G9a-dependent H3K9 methylation and SIRT3-mediated p53 deacetylation, which consistent with decreased sperm count and motility rate in ApoE-/- mice. Further investigation revealed melatonin effectively alleviated PM2.5-induced meiosis inhibition by preserving H3K9 methylation. Melatonin also alleviated PM2.5-induced apoptosis by regulating SIRT3-mediated p53 deacetylation. Overall, our study revealed PM2.5 resulted in spermatogenesis disorder by perturbing meiosis via G9a-dependent H3K9 di-methylation and causing cell apoptosis via SIRT3/p53 deacetylation pathway and provided promising insights into the protective role of melatonin in air pollution associated with male infertility.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice.

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

Ferroptosis mediates decabromodiphenyl ether-induced liver damage and inflammation.

Decabromodiphenyl ether (BDE-209) is an environmental toxin. Increasing evidence showed that BDE-209 exposure induced liver injury, but the mechanism still remains unknown. The present study explored the effect and mechanism of ferroptosis on hepatotoxicity triggered by BDE-209 in vivo and in vitro. In vivo experiment, ICR mice were exposed to BDE-209 for 50 days, and then recovered for 50 days; HepG2 and L02 cells were treated with BDE-209 or/and ferrostatin-1 (Fer-1) for establishing in vitro model. In vivo, the results showed that BDE-209 accumulated in liver and induced liver damage, increased Fe2+ and MDA contents, and blocked the activation of SLC7A11/GSH/GPX4 pathway in liver; BDE-209 also activated IKK/IκB/NF-κB pathway and elevated inflammatory cytokines levels in liver after exposure for 50 days. After BDE-209 stopping exposure 50 days, the severity of liver damage, ferroptosis and inflammatory response were still higher than the corresponding control group. In vitro, ferroptosis inhibitor Fer-1 rescued ferroptotic damage and attenuated cell death in BDE-209-treated HepG2 and L02 cells. In addition, Fer-1 reversed the activation of IKK/IκB/NF-κB pathway and the increase of pro-inflammatory cytokines levels in BDE-209-treated HepG2 and L02 cells. Together, the above results suggested that BDE-209 induced tissue damage and inflammatory response by activating ferroptosis through increasing iron-dependent lipid peroxidation and blocking the activation of SLC7A11/GSH/GPX4 pathway in liver, indicating that ferroptosis is a potential mechanism for BDE-209-induced hepatotoxicity.

The role of Sertoli cells-secreted factors in different stages of germ cells development in mice exposed to BDE-209.

Decabromodiphenyl ether (BDE-209), a frequently used brominated flame retardant, readily enters the environment and is difficult to degrade with bioaccumulation. BDE-209 could cause male reproductive toxicity, but the regulatory functions of Sertoli cells-secreted factors remain uncertain. In present study, male mice were treated with 75 mg/kg BDE-209 and then stopped exposure for 50 days. Exogenous Glial cell line-derived neurotrophic factor (GDNF), a Sertoli cell-secreted factor, was injected into testes of mice treated with BDE-209 for 50 days to explore the role of GDNF in BDE-209-induced reproductive toxicity. The mouse spermatogonia cell line GC-1 spg was used in vitro to further verify regulatory effects of Sertoli cells-secreted factors on meiotic initiation. The results showed that BDE-209 inhibited expressions of the self-renewal pathway GFRα-1/RAS/ERK1/2 in spermatogonial stem cells (SSCs), and reduced expressions of spermatogonia proliferation-related pathway NRG3/ERBB4 and meiosis initiation factor Stra8. Furthermore, BDE-209 decreased the levels of both GDNF and retinoic acid (RA) secreted by Sertoli cells in testes. Importantly, the alterations of above indicators induced by BDE-209 did not recover after 50-day recovery period. After exogenous GDNF injection, the decreased expression of GFRα-1/RAS/ERK in SSCs was reversed. However, the level of RA and expressions of NRG3/ERBB4/Stra8 were not restored. The in vitro experimental results showed that exogenous RA reversed the reductions in NRG3/ERBB4/Stra8 and ameliorated inhibition of GC-1 spg cells proliferation induced by BDE-209. These results suggested that Sertoli cells-secreted factors play roles in regulating various stages of germ cell development. Specifically, BDE-209 affected the self-renewal of SSCs by decreasing GDNF secretion resulting in the inhibition of GFRα-1/RAS/ERK pathway; BDE-209 hindered the proliferation of spermatogonia and initiation of meiosis by inhibiting the secretion of RA and preventing RA from binding to RARα, resulting in the suppression of NRG3/ERBB4/Stra8 pathway. As a consequence, spermatogenesis was compromised, leading to persistent male reproductive toxicity.

Silica nanoparticles induce male reproductive toxicity via Crem hypermethylation mediated spermatocyte apoptosis and sperm flagella damage.

Exposure to silica nanoparticles (SiNPs) could causally contribute to malfunctioning of the spermatogenesis, but the underlying mechanism is rarely known. This study was designed to explore the mechanism of Crem hypermethylation in SiNP-induced reproductive toxicity. The male mice were exposure to SiNPs (0 and 20 mg/kg·bw) once every 5 days via intratracheal instillation for 35 days. After exposure stopped, half of each group was killed, and the rest were sacrificed after another 15-day feeding. GC-2 cells were treated with 0 and 20 µg/mL SiNPs. The results showed that SiNPs led to structure damage of spermatocyte and sperm, caused spermatocyte apoptosis, and decreased sperm quantity and quality. After 15 days of the withdrawal, the testicular tissue damage gradually recovered. Mechanistic study showed that SiNPs induced hypermethylation of the gene of cAMP responsive element modulator (Crem) in the promoter region. Downregulation of Crem inhibited the expression of outer dense fiber 1 (Odf1), resulting in abnormal sperm flagella structure; at the same time, Crem inhibited the expression of Bcl-xl, causing upregulation of cytochrome-C, cleaved-caspase-9/caspase-9, cleaved-caspase-3/caspase-3, resulting in mitochondrial dependent apoptotic pathway. However, 5-aza, DNA methylation inhibitor, could reverse the SiNP-induced downregulation of Crem and reverse the Crem/Bcl-xl-mediated mitochondrial dependent apoptotic pathway. These results suggested SiNPs could disrupt spermatogenesis by causing Crem hypermethylation to regulate the Odf1 and Bcl-xl in spermatocytes resulting in the sperm flagella structure and spermatocyte apoptosis. Our study provided new insights into the male reproductive toxicity mechanism of SiNPs; Crem demethylation may be a potential way to prevent reproductive dysfunction from SiNP exposure.

Dietary selenium excess affected spermatogenesis via DNA damage and telomere-related cell senescence and apoptosis in mice.

Selenium (Se) is a vital microelement for spermatogenesis and male fertility. The aim of this study was to investigate the effects of Se on the male reproductive function and possible mechanisms. Fourty male mice were randomly divided into 0, 0.1, 0.3 and 0.9 mg/kg Se supplementation groups and given with Se dietary intervention for 12 weeks. Our data showed that excessive Se intake damaged the tissue structure of testes and epididymides of the mice, resulting in decreased sperm quality and quantity. Moreover, excessive Se induced oxidative stress, causing DNA damage and activated DNA damage repair factors (Mre11/Rad50/Nbs1), and also disrupted telomere function by shortening telomere length and decreasing TERT expression. Se excess activated the senescence pathway p53/p21/p16, leading to germ cell senescence, and inhibited cell proliferation by suppressing the Sirt1/Foxo1/c-Myc pathway. All of this led to spermatogenic cell apoptosis, thereby causing a decrease of sperm quantity and quality. In conclusion, excessive Se caused reproductive toxicity via inducing telomere dysfunction due to DNA damage, leading to germ cellular senescence and apoptosis in the testes of male mice. Our research provide new proof to explain the underlying mechanism of male reproductive toxicity triggered by excessive Se intake.

MiR-5622-3p inhibits ZCWPW1 to induce apoptosis in silica-exposed mice and spermatocyte cells.

Silica nanoparticles (SiNPs) could cause damage to spermatogenesis, and microRNAs were reported to be associated with male reproduction. This research was designed to explore the toxic impacts of SiNPs induced in male reproduction through miR-5622-3p. In vivo, 60 mice were randomized into the control group and SiNPs group, in which they were exposed to SiNPs for 35 days and then recovered for 15 days. In vitro, 4 groups were set: control group, SiNPs group, SiNPs + miR-5622-3p inhibitor group, and SiNPs + miR-5622-3p inhibitor negative control (NC) group. Our research indicated SiNPs caused the apoptosis of spermatogenic cells, increased level of γ-H2AX, raised the expressions of RAD51, DMC1, 53BP1, and LC8 which were DNA damage repair relative factors, and upregulated Cleaved-Caspase-9 and Cleaved-Caspase-3 levels. Furthermore, SiNPs also elevated the expression of miR-5622-3p but downregulated the level of ZCWPW1. However, miR-5622-3p inhibitor reduced the level of miR-5622-3p, increased the level of ZCWPW1, relieved DNA damage, and depressed the activation of apoptosis pathway, thus, alleviating spermatogenic cells apoptosis caused by SiNPs. The above-mentioned results indicated that SiNPs induced DNA damage resulting in activating of DNA damage response. Meanwhile, SiNPs raised the level of miR-5622-3p targeting inhibited expression of ZCWPW1 to suppress the repair process, possibly making DNA damage so severe that leading to the failure of DNA damage repair, finally inducing the apoptosis of spermatogenic cells.