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.

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.

Qualitative Proteome-wide Lysine Crotonylation Profiling Reveals Protein Modification Alteration in the Leukocyte Extravasation Pathway in Systemic Lupus Erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a severe systemic autoimmune disease with multiple manifestations. Lysine crotonylation (Kcr) is a newly discovered posttranslational modification epigenetic pattern that may affect gene expression and is linked to diseases causally. METHODS: We collected blood samples from 11 SLE individuals and 36 healthy subjects. Then, we used highly sensitive liquid chromatography-mass spectrometry technology to carry out proteomics and quantitative crotonylome analysis of SLE peripheral blood mononuclear cells in this investigation, which indicated the unique etiology of SLE. Finally, we verified the expression of critical protein in the leukocyte extravasation pathway by online database analysis and Western blot. RESULTS: There were 618 differentially expressed proteins (DEPs), and 612 crotonylated lysine sites for 272 differentially modified proteins (DMPs) found. These DEPs and DMPs are primarily enriched in the leukocyte extravasation signaling pathway, such as MMP8, MMP9, and ITGAM. CONCLUSIONS: This is the first study of crotonylated modification proteomics in SLE. The leukocyte extravasation signaling pathway had a considerable concentration of DEPs and DMPs, indicating that this pathway may be involved in the pathogenic development of SLE.

Direct 3D Printing of Recycled PET/PP Granules by Shear Screw Extrusion.

This article introduces a one-step extrusion-based fused deposition modeling (FDM) approach for the challenging separation of polypropylene (PP) and polyethylene terephthalate (PET) during recycling. A shear screw printer (SSP) with shear elements was designed, and it was compared to a conventional single-screw printer (CSP) to investigate the differences in print stability, degradation levels, tensile performance, molecular orientation, and crystallization when preparing recycled PP and recycled PET blends. Although the retention effect of the SSP screw slightly increases the degradation of the blended rPP/rPET, the strong shear (2.6 × 104 s-1) applied near the extrusion exit improves the blending efficiency. The SSP also enhances molecular orientation, modulus of the parts, and reduces performance fluctuations. Additionally, the SSP has the potential to simplify the recycling process, enabling the transformation of blended recycled materials into products with just one melt process.

Proteomic analysis of lysine 2-hydroxyisobutyryl in SLE reveals protein modification alteration in complement and coagulation cascades and platelet activation Pathways.

BACKGROUND: Post-translational modifications (PTMs) are considered to be an important factor in the pathogenesis of Systemic lupus erythematosus (SLE). Lysine 2-hydroxyisobutyryl (Khib), as an emerging post-translational modification of proteins, is involved in some important biological metabolic activities. However, there are poor studies on its correlation with diseases, especially SLE. OBJECTIVE: We performed quantitative, comparative, and bioinformatic analysis of Khib proteins in Peripheral blood mononuclear cells (PBMCs) of SLE patients and PBMCs of healthy controls. Searching for pathways related to SLE disease progression and exploring the role of Khib in SLE. METHODS: Khib levels in SLE patients and healthy controls were compared based on liquid chromatography tandem mass spectrometry, then proteomic analysis was conducted. RESULTS: Compared with healthy controls, Khib in SLE patients was up-regulated at 865 sites of 416 proteins and down-regulated at 630 sites of 349 proteins. The site abundance, distribution and function of Khib protein were investigated further. Bioinformatics analysis showed that Complement and coagulation cascades and Platelet activation in immune-related pathways were significantly enriched, suggesting that differentially modified proteins among them may affect SLE. CONCLUSION: Khib in PBMCs of SLE patients was significantly up- or down-regulated compared with healthy controls. Khib modification of key proteins in the Complement and coagulation cascades and Platelet activation pathways affects platelet activation and aggregation, coagulation functions in SLE patients. This result provides a new direction for the possible significance of Khib in the pathogenesis of SLE patients.

Genetic and Epigenetic Evidence for Nonestrogenic Disruption of Otolith Development by Bisphenol A in Zebrafish.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical (EDC) that has estrogenic activities. In addition to disrupting reproductive development and function via estrogenic signaling pathways, BPA can also interfere with nonreproductive functions through nonestrogenic pathways; however, the mechanisms underlying such nonestrogenic activities are not well understood. In this study, we demonstrated that BPA could disrupt otolith formation during the early development of zebrafish with long-lasting ethological effects. Using multiple mutants of estrogen receptors, we provided strong genetic evidence that the BPA-induced otolith malformation was independent of estrogen signaling. Transcriptome analysis revealed that two genes related to otolith development, otopetrin 1 (otop1) and starmaker (stm), decreased their expression significantly after BPA exposure. Knockout of both otop1 and stm genes could phenocopy the BPA-induced otolith malformation, while microinjection of their mRNAs could rescue the BPA-induced abnormalities of otolith formation. Further experiments showed that BPA inhibited the expression of otop1 and stm by activating the MEK/ERK-EZH2-H3K27me3 signaling pathway. Taken together, our study provided comprehensive genetic and molecular evidence that BPA induced the otolith malformation through nonestrogenic pathway during zebrafish early development and its activities involved epigenetic control of key genes (e.g., otop1 and stm) participating in otolith formation.

Effects of exposure to PM2.5 during pregnancy on the multigenerational reproductive outcomes of male mouse offspring and the role of Sertoli cells.

There is a paucity of studies on the multigenerational reproductive toxicity of fine particle matter (PM2.5) exposure during pregnancy on male offspring and the underlying mechanisms. This study explored the effects of PM2.5 exposure during pregnancy on the spermatogenesis of three consecutive generations of male mouse offspring. We randomized pregnant C57BL/6 mice into the control group, the Quartz Fiber Membrane control group, and two experimental groups exposed to different concentrations of PM2.5 (4.8 and 43.2 mg/kg B.Wt.). Pregnant mice from experimental groups received intratracheal instillation of PM2.5 of different doses on a three-day basis until birth. F1 mature male offspring from PM2.5-exposed pregnant mice were mated with normal female C57BL/6 mice. Likewise, their F2 mature male followed the same to produce the F3 generation. The results showed that PM2.5 exposure during pregnancy led to decreased body and tail length, body weight, and survival rates, decreased sperm concentration and sperm motility, and increased sperm abnormality rates significantly in F1 male offspring. We barely observed significant impacts of PM2.5 on the birth number, survival rates, and index of testes in the F2 and F3 offspring. Further exploration showed that PM2.5 exposure during pregnancy caused the morphological abnormality of Sertoli cells, downregulated androgen receptor (AR) and connexin43, upregulated anti-Müllerian hormone (AMH), cytokeratin-18 (CK-18), caspase-3, and cleaved caspase-3, decreased thyroid-stimulating hormone (TSH) and testosterone (T), and increased triiodothyronine (T3) in F1 male mouse offspring. Overall, we hypothesize that PM2.5 exposure during pregnancy mainly negatively impacts spermatogenesis in the F1 offspring. The possible mechanism could be that PM2.5 exposure during pregnancy disrupts endocrine hormone release in the F1 generation, thereby influencing the maturation and proliferation of their Sertoli cells and hindering spermatogenesis. This study for the first time investigates the role of Sertoli cells in the reproductive toxicity of PM2.5 on offspring.

Amorphous silica nanoparticles cause abnormal cytokinesis and multinucleation through dysfunction of the centralspindlin complex and microfilaments.

BACKGROUND: With the large-scale production and application of amorphous silica nanoparticles (aSiNPs), its adverse health effects are more worthy of our attention. Our previous research has demonstrated for the first time that aSiNPs induced cytokinesis failure, which resulted in abnormally high incidences of multinucleation in vitro, but the underlying mechanisms remain unclear. Therefore, the purpose of this study was firstly to explore whether aSiNPs induced multinucleation in vivo, and secondly to investigate the underlying mechanism of how aSiNPs caused abnormal cytokinesis and multinucleation. METHODS: Male ICR mice with intratracheal instillation of aSiNPs were used as an experimental model in vivo. Human hepatic cell line (L-02) was introduced for further mechanism study in vitro. RESULTS: In vivo, histopathological results showed that the rate of multinucleation was significantly increased in the liver and lung tissue after aSiNPs treatment. In vitro, immunofluorescence results manifested that aSiNPs directly caused microfilaments aggregation. Following mechanism studies indicated that aSiNPs increased ROS levels. The accumulation of ROS further inhibited the PI3k 110ß/Aurora B pathway, leading to a decrease in the expression of centralspindlin subunits MKLP1 and CYK4 as well as downstream cytokines regulation related proteins Ect2, Cep55, CHMP2A and RhoA. Meanwhile, the particles caused abnormal co-localization of the key mitotic regulatory kinase Aurora B and the centralspindlin complex by inhibiting the PI3k 110ß/Aurora B pathway. PI3K activator IGF increased the phosphorylation level of Aurora B and improved the relative ratio of the centralspindlin cluster. And ROS inhibitors NAC reduced the ratio of multinucleation, alleviated the PI3k 110ß/Aurora B pathway inhibition, and then increased the expression of MKLP1, CYK4 and cytokinesis-related proteins, whilst NAC restored the clustering of the centralspindlin. CONCLUSION: This study demonstrated that aSiNPs led to multinucleation formation both in vivo and in vitro. ASiNPs exposure caused microfilaments aggregation and inhibited the PI3k 110ß/Aurora B pathway through excessive ROS, which then hindered the centralspindlin cluster as well as restrained the expression of centralspindlin subunits and cytokinesis-related proteins, which ultimately resulted in cytokinesis failure and the formation of multinucleation.

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.

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.

Silica nanoparticles suppressed the spermatogenesis via downregulation of miR-450b-3p by targeting Layilin in spermatocyte of mouse.

Silica nanoparticles (SiNPs) suppressed spermatogenesis leading to male reproductive toxicity, while the precise mechanism remains uncertain. Here, this study explored the role of miR-450b-3p in male reproductive toxicity induced by SiNPs. In vivo study, we found that SiNPs caused apoptosis of spermatocytes, decreased quantity and quality of sperms, up-regulated the cytoskeleton proteins (Layilin, Talin, and Vinculin), activated the Hippo pathway (Rho A, Yap, and p73), downregulated the expression of miR-450b-3p, damaged the compactness and density of desmosomes between spermatocytes and the basal of the testis. Moreover, in vitro study, we confirmed that SiNPs increased the expressions of cytoskeleton proteins, activated the Hippo pathway, and suppressed miR-450b-3p expressions. Meanwhile, miR-450b-3p mimic inhibited the up-regulation of cytoskeleton proteins, suppressed the activation of the Hippo pathway, and relieved the adhesion and traction stress. Eventually, atomic force microscopy (AFM) was performed to validate the traction stress and adhesion between GC-2spd cells enhanced by deregulation of miR-450b-3p. Taken together, we concluded that SiNPs suppressed spermatogenesis via inhibiting miR-450b-3p, in turn up-regulating the expression of cytoskeleton proteins, then inducing apoptosis via activating the Hippo pathway and enhancing the traction force and adhesion between GC-2spd cells. This work provides novel evidence for the study of reproductive toxicity and risk assessment of SiNPs.

BDE-209 induced spermatogenesis disorder by inhibiting SETD8/H4K20me1 related histone methylation in mice.

Past studies have observed that decabromodiphenyl ether (BDE-209) induces reproductive and developmental toxicity, but the specific mechanism remains unclear. Based on our previous work, male mice were orally given BDE-209 at 75 mg/kg/d via continuous exposure for one spermatozoon development period (50 days) and then stopping exposure for another 50 days. The mouse spermatocyte line GC-2spd was used to examine the toxic effects of BDE-209 on histone methylation and spermatogenesis. The findings indicated that BDE-209 damaged testis and epididymis structure, induced spermatogenic cell apoptosis, and decreased sperm quantity and quality after the 50-day exposure. Furthermore, BDE-209 lowered the levels of SETD8/H4K20me1 and activated the upstream signaling of DNA damage response (Mre11/Rad50/NBS1), thereby causing spermatogenic cell cycle arrest and apoptosis. Downregulation of meiotic promoter Stra8 was associated with a decrease in SETD8 after BDE-209 exposure. After stopping the exposure for 50 days, reproductive system damage and meiosis and cell cycle inhibition due to histone methylation did not improve. In vitro experiments revealed that Setd8 overexpression upregulated the histone methylation and Stra8 expression but did not promote the cell cycle in GC-2 cells. Therefore, BDE-209 exposure impaired spermatogenesis by affecting SETD8/H4K20me1-linked histone methylation and inhibiting meiosis initiation and cell cycle progression, thereby resulting in long-term male reproductive toxicity.