GC-2spd Cells Research Articles

GALNTL5, which is restricted to mouse spermatids, impairs endoplasmic reticulum (ER) function through direct interaction with ER chaperone proteins

Polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) was identified as a pp-GalNAc-T family gene. Nevertheless, GALNTL5 has no glycosyltransferase activity. In mice, Galntl5 expression is restricted to differentiating spermatids, and haploinsufficiency leads to immotile spermatozoa with an aberrant protein composition. Moreover, heterozygotic deletions of human GALNTL5 have been detected in patients diagnosed with asthenozoospermia (low sperm motility). Although these findings indicate that GALNTL5 is a functional molecule essential for mature sperm formation in mammals, the exact function of GALNTL5 in spermiogenesis remains unknown. To clarify this role, we established the mouse spermatocyte cell line GC-2spd(ts), which exhibits drug-inducible GALNTL5 expression. Interestingly, continuous GALNTL5 expression in the resultant cell lines caused apoptosis with cell shrinkage, and GALNTL5 was localized in the endoplasmic reticulum (ER) and was associated with two ER-resident chaperone proteins, calnexin and BiP (GRP78). Calnexin recognized and strongly bound to the N-glycans on GALNTL5 molecules modified in the ER. In contrast, ER-resident BiP likely attached to GALNL5 regardless of its glycosylation. GALNTL5 expression abolished the binding between calnexin and misfolded substrate proteins, indicating that GALNTL5 directly blocks calnexin function. Furthermore, the interaction between GALNTL5 and calnexin decreased the level of BiP protein, and consequently also the expression levels of proteins that are resident in the ER, Golgi apparatus, and cytoplasm. These reduced protein levels were confirmed by loss of calnexin or BiP function in the GC-2spd(ts) cell line using siRNA knockdown. Further, sustained expression of GALNTL5 resulted in cell structure changes, including the position of the cis-Golgi apparatus and alterations in the ER network. These results strongly suggest that GALNTL5 contributes to alteration of the cell structure specific to differentiating spermatids by blocking ER function.

Exploring the Biological Effects of Polystyrene Nanoplastics on Spermatogenesis: Insights From Transcriptomic Analysis in Mouse Spermatocytes.

The presence of polystyrene plastics in the human testis has raised concerns, yet their biological activity remains poorly characterized. This study aimed to investigate the biological effects and potential regulatory genes of polystyrene nanoplastics on spermatocyte line, GC-2spd(ts). After a 24-h exposure to polystyrene nanoplastics, the results indicated cell membrane disruption, impairment of mitochondrial membrane potential, increased levels of reactive oxygen species (ROS), and induced DNA damage. Furthermore, a comprehensive transcriptomic analysis was conducted, revealing differential gene expression patterns in GC-2spd(ts) cells in response to polystyrene nanoplastics. A total of 134 differentially expressed genes (DEGs) were identified, with 48 genes upregulated and 86 genes downregulated. The Gene Ontology analysis highlighted the involvement of these genes in various spermatogenesis-related biological processes, including acrosome reaction, sperm mitochondrial organization, sperm annulus, and outer acrosomal membrane. Subsequently, the quantification of gene expression through qRT-PCR identified five key genes (NSUN7, SEPTIN4, TRIM36, EQTN, and SYT8) screened from the DEGs. In conclusion, this study provides valuable insights into the biological effects of polystyrene nanoplastics on mouse spermatocytes using comprehensive transcriptomic analysis, contributing to the establishment of a foundation for future investigations into these relevant pathways.

Protective effect of trehalose on sperm chromatin condensation failure and semen quality decline in BDE-209-exposed mice.

BDE-209 exposure induced male reproductive toxicity with sperm quality decline. However, the role of autophagy in this was unclear. The purpose was to evaluate the protective effect and its potential mechanism of trehalose (Tre, autophagy inducer) on reproductive damage during spermiogenesis induced by BDE-209. We used 2% w/v Tre and 75mg/kg/d BDE-209 cotreated mice for 42 days. GC-2 spd cells were cotreated with Tre, chloroquine (CQ, inhibition of autophagic flux), compound C (CC, AMPK inhibitor), and BDE-209. Tre intake significantly recovered decrease in sexual organ ratio and poor sperm quality in BDE-209-exposed mice. Supplementation with Tre rescued sperm head malformation by improving aberrant histone-protamine exchange in BDE-209-exposed mice. However, Tre intake couldn't restore the acrosome biogenesis. In addition, Tre supplementation improved testicular damage induced by BDE-209. BDE-209 blocked autophagic flux with increased P62 and LC3BⅡ/Ⅰ levels. Mechanistically, CQ treatment aggravated elevation of P62 and LC3BⅡ/Ⅰ levels induced by BDE-209, otherwise, CC and Tre treatments inhibited the rise in p-AMPK, p-ULK1, P62 and LC3BⅡ/Ⅰ levels induced by BDE-209. Tre supplementation improved reproductive injury in BDE-209-exposed mice by regulating autophagic flow via AMPK-ULK1 signaling pathways, which providing a new theoretical basis and possible therapeutic targets for male reproductive toxicity.

Integrated transcriptomic and metabolomic analysis reveals the underlying mechanisms for male reproductive toxicity of polystyrene nanoplastics in mouse spermatocyte-derived GC-2spd(ts) cells

BackgroundPolystyrene nanoplastics (PS-NPs), are ubiquitous pollution sources in human environments, posing significant biosafety and health risks. While recent studies, including our own, have illustrated that PS-NPs can breach the blood-testis barrier and impact germ cells, there remains a gap in understanding their effects on specific spermatogenic cells such as spermatocytes. Methods and resultsHerein, we employed an integrated approach encompassing phenotype, metabolomics, and transcriptomics analyses to assess the molecular impact of PS-NPs on mouse spermatocyte-derived GC-2spd(ts) cells. Optimal exposure conditions were determined as 24 h with 50 nm PS-NPs at 12.5 μg/mL and 90 nm PS-NPs at 50 μg/mL for subsequent multi-omics analysis. Our findings revealed that PS-NPs significantly influenced proliferation and viability, causing alterations in transcriptome and metabolome profiles. Transcriptomics analysis of GC-2spd(ts) cells exposed to PS-NPs indicated the pivotal involvement of cell proliferation and cycle, autophagy, ferroptosis, and redox reaction pathways in PS-NP-induced effects on the proliferation and viability of GC-2spd(ts) cells. Furthermore, metabolomics analysis identified major changes in amino acid metabolism, cyanoamino acid metabolism, and purine and pyrimidine metabolism following PS-NP exposure. ConclusionOur integrated approach, combining metabolomics and transcriptomics profiles with phenotype data, enhances our understanding of the adverse effects of PS-NPs on germ cells.

Wdr17 Regulates Cell Proliferation, Cell Cycle Progression and Apoptosis in Mouse Spermatocyte Cell Line.

We identified Wdr17 as a highly expressed gene in pachytene spermatocytes by transcriptomic analysis of mouse testis. Germ cell-deficient infertile mouse models had significantly reduced Wdr17 expression. We performed gene interference and overexpression in the mouse spermatocyte cell line GC-2spd(ts) and investigated how Wdr17 affects spermatocyte growth and development. Our results showed that Wdr17 suppression significantly decreased cell growth rate and increased cell apoptosis in GC-2spd(ts) cells. Wdr17 suppression also arrested the cell cycle at the G1 phase. On the contrary, Wdr17 overexpression significantly promoted cell proliferation and inhibited cell apoptosis in GC-2spd(ts) cells. More cells were enriched at the S stage with a concomitant reduction of cells at the G1 stage. Wdr17 promotes mouse spermatocyte proliferation by advancing cell cycle progression and inhibiting cell apoptosis, indicating its potential role in regulating spermatogenesis in the mouse.

Cadmium disrupts spermatogenic cell cycle via piRNA-DQ717867/p53 pathway

Cadmium (Cd) is a harmful environmental pollutant that disrupts public health, including respiratory, digestive, and reproductive systems. In this study, male rats were exposed to CdCl2 at a dose of 3 mg/kg by oral for 28 days to investigate the impact on spermatogenesis. Testis tissue samples were collected after sacrifice, and piRNA expression levels were measured using piRNA microarray and qPCR. PiRNAs, specialized molecules involved in spermatogenesis, were examined. CdCl2 exposure led to disrupted piRNA expression, particularly in piRNA-DQ759395 in rats. This piRNA was found to have a binding site with p53, and a similar piRNA-DQ717867 was discovered in mice. In GC-2spd cells, CdCl2 exposure increased piRNA-DQ717867 expression, which resulted in cell cycle arrest and abnormal expression of cell cycle-related proteins. The activation of p53-related pathways and disruptions in cell cycle regulation were also observed. Antagomir-717867 transfections and PFT-a pretreatment in GC-2spd cells supported the involvement of piRNA-DQ717867 in regulating cell cycle-related proteins. This study suggests that Cd exposure induces abnormal expression of piRNA-DQ759395 in rat testis and that piRNA-DQ717867 may regulate p53, causing cell cycle abnormalities in GC-2spd cells. These findings help understand the mechanisms of male reproductive toxicity caused by Cd exposure and emphasize the role of piRNAs in cell cycle regulation and male reproductive health.

Perturbation of IP3R-dependent endoplasmic reticulum calcium homeostasis by PPARδ-activated metabolic stress leads to mouse spermatocyte apoptosis: A direct mechanism for perfluorooctane sulfonic acid-induced spermatogenic disorders

Perfluorooctane sulfonic acid (PFOS) as an archetypal representative of per- and polyfluoroalkyl substances (PFAS) is ubiquitously distributed in the environment and extensively detected in human bodies. Although accumulating evidence is suggestive of the deleterious effects of PFOS on male reproduction, the direct toxicity of PFOS towards spermatogenic cells and the relevant mechanisms remain poorly understood. The aims of the present study were to explore the direct effects and underlying molecular mechanisms of PFOS on spermatogenesis. Through integrating animal study, transcriptome profiling, in silico toxicological approaches, and in vitro validation study, we identified the molecular initiating event and key events contributing to PFOS-induced spermatogenic impairments. The mouse experiments revealed that spermatocytes were involved in PFOS-induced spermatogenic disorders and the activation of peroxisome proliferator-activated receptor delta (PPARδ) was linked to spermatocyte loss in PFOS-administrated mice. GC-2spd(ts) cells were treated with an increased gradient of PFOS, which was relevant to environmental and occupational exposure levels of PFOS in populations. Following 72-h treatment, cells was harvested for RNA sequencing. The transcriptome profiling and benchmark dose (BMD) modeling identified endoplasmic reticulum (ER) stress as the key event for PFOS-mediated spermatocyte apoptosis and determined the point-of-departure (PoD) for perturbations of ER stress signaling. Based on the calculated PoD value, further bioinformatics analyses combined with in vitro and in vivo validations showed that PFOS caused metabolic stress by activating PPARδ in mouse spermatocytes, which was responsible for Beclin 1-involved inositol 1,4,5-trisphosphate receptor (IP3R) sensitization. The disruption of IP3R-mediated ER calcium homeostasis triggered ER calcium depletion, leading to ER stress and apoptosis in mouse spermatocytes exposed to PFOS. This study systematically investigated the direct impacts of PFOS on spermatogenesis and unveiled the relevant molecular mechanism of PFOS-induced spermatogenic disorders, providing novel insights and potential preventive/therapeutic targets for PFAS-associated male reproductive toxicity.

Curcumin attenuates diphenyl phosphate-induced apoptosis in GC-2spd(ts) cells through activated autophagy via the Nrf2/P53 pathway.

Diphenyl phosphate (DPhP) is one of the frequently used derivatives of aryl phosphate esters and is used as a plasticizer in industrial production. Like other plasticizers, DPhP is not chemically bound and can easily escape into the environment, thereby affecting human health. DPhP has been associated with developmental toxicity, reproductive toxicity, neurodevelopmental toxicity, and interference with thyroid homeostasis. However, understanding of the underlying mechanism of DPhP on the reproductive toxicity of GC-2spd(ts) cells remains limited. For the first time, we investigated the effect of DPhP on GC-2spd(ts) cell apoptosis. By decreasing nuclear factor erythroid-derived 2-related factor (Nrf2)/p53 signaling, DPhP inhibited autophagy and promoted apoptosis. DPhP reduced total antioxidant capacity and nuclear Nrf2 and its downstream target gene expression. In addition, we investigated the protective effects of Curcumin (Cur) against DPhP toxicity. Cur attenuated the DPhP-induced rise in p53 expression while increasing Nrf2 expression. Cur inhibited DPhP-induced apoptosis in GC-2spd(ts) cells by activating autophagy via Nrf2/p53 signaling. In conclusion, our study provides new insights into the reproductive toxicity hazards of DPhP and demonstrates that Cur is an important therapeutic agent for alleviating DPhP-induced reproductive toxicity by regulating Nrf2/p53 signaling.

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.

Reactive Oxygen Species-Mediated Mitophagy and Cell Apoptosis are Involved in the Toxicity of Aluminum Chloride Exposure in GC-2spd.

Aluminum chloride is an inorganic polymeric coagulant commonly found in daily life and various materials. Although male reproductive toxicity has been associated with AlCl3 exposure, the underlying mechanism remains unclear. This study aimed to examine the impact of AlCl3 exposure on mitophagy and mitochondrial apoptosis in testicular tissue and mouse spermatocytes. Reactive oxygen species (ROS) and ATP levels were measured in GC-2spd after AlCl3 exposure using a multifunctional enzyme labeler. The changes in mitochondrial membrane potential (MMP) and TUNEL were observed through confocal laser microscopy, and the expression of proteins associated with mitophagy and apoptosis was analyzed using Western blot. Our results demonstrated that AlCl3 exposure disrupted mitophagy and increased apoptosis-related protein expression in testicular tissues. In the in vitro experiments, AlCl3 exposure induced ROS production, suppressed cell viability and ATP production, and caused a decrease in MMP, leading to mitophagy and cell apoptosis in GC-2spd cells. Intervention with N-acetylcysteine (NAC) reduced ROS production and partially restored mitochondrial function, thereby reversing the resulting mitophagy and cell apoptosis. Our findings provide evidence that ROS-mediated mitophagy and cell apoptosis play a crucial role in the toxicity of AlCl3 exposure in GC-2spd. These results contribute to the understanding of male reproductive toxicity caused by AlCl3 exposure and offer a foundation for future research in this area.

Transcriptome-based analysis of the toxic effects of aluminum chloride exposure on spermatocytes

Aluminum chloride (AlCl3) exposure is pervasive in our daily lives. Numerous studies have demonstrated that exposure to AlCl3 can lead to male reproductive toxicity. However, the precise mechanism of action remains unclear. The objective of this study is to investigate the mechanism of aluminum-induced toxicity by analyzing the alterations in the global transcriptome gene profile of mouse spermatocytes (GC-2spd cells) exposed to AlCl3. GC-2spd cells were exposed to concentrations of 0, 1, 2, and 4 mM AlCl3, and high-throughput mRNA-seq was performed to investigate the changes in the transcriptome after exposure to 4 mM AlCl3. Our findings indicate that exposure to AlCl3 led to an increase in oxidative stress, disrupted glutathione metabolism, reduced cell viability, and altered gene expression in mouse spermatocytes. Gene enrichment analysis revealed that the differentially expressed genes (DEGs) were associated with various biological functions such as mitochondrial inner membrane, response to oxidative stress. Furthermore, these DEGs were found to be enriched in pathways including proteasome, glutathione metabolism, oxidative phosphorylation, and Hif-1 signaling pathway. Real-time PCR and western blot were employed to validate the expression alterations of pivotal genes, and the outcomes exhibited concordance with the mRNA-seq findings. This study provides a theoretical basis for revealing the potential mechanism of male reproductive toxicity caused by aluminum exposure.

Yiqi Jiedu decoction attenuates radiation injury of spermatogenic cells via suppressing IκBα/NF-κB pathway-induced excessive autophagy and apoptosis

Ethnopharmacological relevanceThe prescription of Yiqi Jiedu decoction (YQJD) originated from the classic Chinese herbal prescriptions of Danggui Buxue Decoction and Wuzi Yanzong Pill. A previous study has shown that 4 Gy irradiation induced the apoptosis of spermatocytes and revealed autophagosomes in cells exposed to radiation. YQJD decoction has the effect of preventing radiation injury. Aim of the studyWe used spermatocytes (GC-2spd cell line) to investigate the relationship between autophagy and apoptosis of spermatogenic cells after radiation, and the mechanisms of YQJD decoction. Materials and methodsEstablish an in vitro radiation injury model by irradiating GC-2spd cells with 60Co γ-rays (4 Gy or 8 Gy). Autophagy agonists, autophagy inhibitors and YQJD were used to intervene cells. Cell apoptosis and inflammatory factors were measured. NF-κB localization was observed by immunofluorescence. Autophagy and apoptosis-related proteins and IκBα/NF-κB pathway factors were detected. ResultsIonizing radiation promoted the growth of spermatogenic autophagosomes. After radiation, NF-κB was translocated to the nucleus, inflammatory factors were secreted, and IκBα/NF-κB pathway was activated, which promoted autophagy and apoptosis. YQJD decoction can inhibit the phosphorylation of IκBα/NF-κB pathway related factors, regulate the expression of Beclin-1 and Bcl-2 proteins, and inhibit the occurrence of autophagy and apoptosis of irradiated spermatocyte. ConclusionsThe research results indicate that ionizing radiation can activate the IκBα/NF-κB signaling pathway in spermatocytes, promote cell autophagy and apoptosis by regulating the expression of Beclin-1 and Bcl-2 factors. The YQJD decoction inhibits the IκBα/NF-κB signaling pathway so as to regulate Beclin-1 and Bcl-2.

Investigation on the interaction of aromatic organophosphate flame retardants with human serum albumin via computer simulations, multispectroscopic techniques and cytotoxicity assay

Organophosphate flame retardants (OPFRs) are newly emerging estrogenic environmental pollutants, which attracted widespread public interest owing to their potential threats to human. Here, the interaction between two typical aromatic OPFRs, TPHP/EHDPP and HSA was researched by different experiments. Experimental results indicated that TPHP/EHDPP can insert the site I of HSA and be encircled by several amino acid residues, Asp451, Glu292, Lys195, Trp214 and Arg218 played vital roles in this binding process. At 298 K, the Ka value of TPHP-HSA complex was 5.098 × 104 M−1, and the Ka value of EHDPP-HSA was 1.912 × 104 M−1. Except H-bonds and van der Waals forces, the π-electrons on the phenyl ring of aromatic-based OPFRs played a pivotal role in maintaining the stability of the complexes. The content alterations of HSA were observed in the present of TPHP/EHDPP. The IC50 values of TPHP and EHDPP were 157.9 μM and 31.14 μM to GC-2spd cells, respectively. And the existence of HSA has a regulatory effect on the reproductive toxicity of TPHP/EHDPP. In addition, the results of present work implied Ka values of OPFRs and HSA are possible to be a useful parameter for evaluating their relative toxicity.

Evaluation of the cytotoxic activity of triphenyl phosphate on mouse spermatocytes cells

Triphenyl phosphate (TPhP) is one of the most commonly found organophosphorus flame retardants (OPFRs) in the environment and the general population. Continuous daily exposure to TPhP may adversely impact male reproductive health. However, few researches were conducted to investigate the direct effects of TPhP on the progress of sperm growth and development. In this study, mouse spermatocyte GC-2spd (GC-2) cells were selected as an in vitro model, the impact of oxidative stress, mitochondrial impairment, DNA damage, cell apoptosis and the related molecular mechanisms were investigated using high content screening (HCS) system. Our study indicated that cell viability was decreased significantly in a dose-dependent manner after TPhP treatment with the half lethal concentration (LC50) at 105.8, 61.61 and 53.23 μM for 24, 48 and 72 h. A concentration-related apoptosis occurrence was observed in GC-2 cells after TPhP exposure for 48 h. In addition, the elevated intracellular reactive oxygen species (ROS) and the total antioxidant capacity (T-AOC) also observed after exposing to 6, 30 and 60 μM of TPhP. Furthermore, based on the enhancement of pH2AX protein and alteration of nuclear morphology or DNA content, DNA damage might be induced by higher concentration of TPhP treatment. Simultaneously, alteration of mitochondrial structure, enhancement of mitochondrial membrane potential (MMP), reduction of cellular adenosine triphosphate (ATP) content, altered expression of Bcl-2 family proteins, release of cytochrome c and increase of caspase-3 and caspase-9 activity demonstrated that caspase-3 dependent mitochondrial pathway might play a key role in the process of GC-2 cell apoptosis. Taken together, these results showed that TPhP was a mitochondrial toxicant and apoptotic inducer, which might trigger alike responses in human spermatogenic cells. Therefore, the potential reproductive toxicity of TPhP should not be ignored.

Effect of autophagy in cadmium chloride induced apoptosis of mouse spermatogenic cells

To study the effect of autophagy in cadmium chloride(CdCl_2)-induced apoptosis of mouse spermatocytes(GC-2 spd) cells and explore the underlying molecular mechanisms. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 μmol/L) for 24 h. Hoechst33342 staining and monodansylcadaverine(MDC) were performed to explore the formation of autophagosomes and apoptotic bodies. The apoptosis of cadmium-treated cells was examined by TUNEL staining. Autophagy inhibitor 3-methyladenine(3-MA)(60 μmol/L), apoptotic inhibitorCaspase inhibitor Z-VAD-FMK( zVAD-FMK)(50 nmol/L), autophagy inducer rapamycin(RAPA)(50 nmol/L) and lysosomal inhibitor chloroquine(CQ)(10 μmol/L) were added to cell culture in the presence/absence of CdCl_2(10 μmol/L) to treat GC-2 spd cells for 24 h. The expression levels of autophagy-related proteins LC3, P62, and pro-apoptotic proteins cleaved Caspase-3 and cleaved Caspase-9 were examined by Western blot. Autophagosomes aggregated and the number of apoptotic cells increased after exposure to CdCl_2 for 24 h. Western blot result showed that in the 5 and 10 μmol/L CdCl_2 exposure groups, the protein expression levels of LC3II/LC3I increased to 9.23±0.81 and 12.15±0.80 compared with the control group(5.50±0.56)(P<0.05), LC3II protein expression level increased to 3.35±0.14 and 3.47±0.32 compared with the control group(2.35±0.34)(P<0.05), P62 protein expression level increased to 1.48±0.12 and 1.80±0.22 compared with the control group(0.83±0.09)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of LC3II/LC3I, LC3II, P62, cleaved Caspase-9 and cleaved Caspase-3 after 3-MA treatment decreased to 0.90±0.07(CdCl_2 group: 1.47±0.06), 1.57±0.14(CdCl_2 group: 2.45±0.29), 0.82±0.05(CdCl_2 group: 1.44±0.18), 0.18±0.01(CdCl_2 group: 0.28±0.01) and 0.61±0.84(CdCl_2 group: 1.15±0.04)(P<0.05). Compared with the CdCl_2-treated group, the protein expression levels of cleaved Caspase-9 and cleaved Caspase-3 after zVAD-FMK treatment decreased to 0.12±0.01(CdCl_2 group: 0.28±0.01) and 0.34±0.01(CdCl_2 group: 1.15±0.04)(P<0.05), while those of LC3II/LC3I, LC3II and P62 had no significant change(P>0.05). Compared with the CdCl_2-treated group, RAPA enhanced cadmium-induced LC3II/LC3I, LC3II and P62 protein expressions to 2.22±0.21(CdCl_2 group: 1.56±0.06), 3.72±0.21(CdCl_2 group: 2.97±0.15) and 2.41±0.19(CdCl_2 group: 1.52±0.35)(P<0.05). Western blot result showed that compared with the CdCl_2 group, the protein expressions of LC3II/LC3I, LC3II, P62 and cleaved Caspase-3 in the CdCl_2 and CQ treatment groups increased to 3.21±0.31(CdCl_2 group: 2.09±0.25), 4.49±0.43(CdCl_2 group: 2.72±0.26), 2.59±0.19(CdCl_2 group: 1.84±0.19) and 2.43±0.23(CdCl_2 group: 1.50±0.27)(P<0.05). Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosome-lysosomal fusion and prompting abnormal aggregation of autophagosomes.

GRIM-19 in asthenozoospermia regulates GC-2 spd cell proliferation, apoptosis and migration

Asthenozoospermia (AZS) is a severe form of male infertility with no clear pathogenesis, despite numerous research efforts, there is no consensus on this. This study was to investigate the expression of gene-associated with retinoid-interferon-induced mortality 19 (GRIM-19) in the sperm of patients with asthenozoospermia and the regulation of GC-2 spd cell proliferation, apoptosis and migration. We analyzed the sperm samples from 82 asthenozoospermia and normal patients were collected in the First People's Hospital of Shangqiu and the First Affiliated Hospital of Zhengzhou University. Immunofluorescence, western blots and RT-qPCR analyses were used to verify the expressions of GRIM-19. MTT assays were used to assess cell proliferations, flow cytometry was performed to assess cell apoptosis, wound‑healing was performed to measure cell migration. Immunofluorescence showed that GRIM-19 is predominantly expressed in the sperm mid-piece, the mRNA expressions of GRIM-19 in sperms of the asthenozoospermia group were significantly low, relative to the normal group (OR 0.266; 95% CI = 0.081–0.868; P = 0.028). The protein expressions of GRIM-19 in sperms of the asthenozoospermia group were significantly lower than that of the normal group as well (GRIM-19/GAPDH: 0.827 ± 0.063 vs 0.458 ± 0.033; P < 0.001). GRIM-19 overexpression promotes GC-2 spd cell proliferation and migration and reduces apoptosis, while GRIM-19-silenced reduces GC-2 spd cell proliferation and migration and increased apoptosis. GRIM-19 is closely related to the occurrence of asthenozoospermia and promotes GC-2 spd cell proliferation and migration and reduces apoptosis.

Hypoxia induces alterations in tRNA modifications involved in translational control

BackgroundAdaptation to high-altitude hypobaric hypoxia has been shown to require a set of physiological traits enabled by an associated set of genetic modifications, as well as transcriptome regulation. These lead to both lifetime adaptation of individuals to hypoxia at high altitudes and generational evolution of populations as seen for instance in those of Tibet. Additionally, RNA modifications, which are sensitive to environmental exposure, have been shown to play pivotal biological roles in maintaining the physiological functions of organs. However, the dynamic RNA modification landscape and related molecular mechanisms in mouse tissues under hypobaric hypoxia exposure remain to be fully understood. Here, we explore the tissue-specific distribution pattern of multiple RNA modifications across mouse tissues.ResultsBy applying an LC-MS/MS-dependent RNA modification detection platform, we identified the distribution of multiple RNA modifications in total RNA, tRNA-enriched fragments, and 17–50-nt sncRNAs across mouse tissues; these patterns were associated with the expression levels of RNA modification modifiers in different tissues. Moreover, the tissue-specific abundance of RNA modifications was sensitively altered across different RNA groups in a simulated high-altitude (over 5500 m) hypobaric hypoxia mouse model with the activation of the hypoxia response in mouse peripheral blood and multiple tissues. RNase digestion experiments revealed that the alteration of RNA modification abundance under hypoxia exposure impacted the molecular stability of tissue total tRNA-enriched fragments and isolated individual tRNAs, such as tRNAAla, tRNAval, tRNAGlu, and tRNALeu. In vitro transfection experiments showed that the transfection of testis total tRNA-enriched fragments from the hypoxia group into GC-2spd cells attenuated the cell proliferation rate and led to a reduction in overall nascent protein synthesis in cells.ConclusionsOur results reveal that the abundance of RNA modifications for different classes of RNAs under physiological conditions is tissue-specific and responds to hypobaric hypoxia exposure in a tissue-specific manner. Mechanistically, the dysregulation of tRNA modifications under hypobaric hypoxia attenuated the cell proliferation rate, facilitated the sensitivity of tRNA to RNases, and led to a reduction in overall nascent protein synthesis, suggesting an active role of tRNA epitranscriptome alteration in the adaptive response to environmental hypoxia exposure.

N-acetylcysteine rescues meiotic arrest during spermatogenesis in mice exposed to BDE-209.

Deca-bromodiphenyl ethers (BDE-209) has been widely used in electronic devices and textiles as additives to flame retardants. Growing evidence showed that BDE-209 exposure leads to poorer sperm quality and male reproductive dysfunction. However, the underlying mechanisms of BDE-209 exposure caused a decline in sperm quality remains unclear. This study aimed to evaluate the protective effects of N-acetylcysteine (NAC) on meiotic arrest in spermatocytes and decreased sperm quality in BDE-209-exposed mice. In the study, mice were treated with NAC (150mg/kg BW) 2h before administrated with BDE-209 (80mg/kg BW) for 2weeks. For the in vitro studies, spermatocyte cell line GC-2spd cells were pretreated with NAC (5mM) 2h before treated with BDE-209 (50μM) for 24h. We found that pretreatment with NAC attenuated the oxidative stress status induced by BDE-209 in vivo and in vitro. Moreover, pretreatment with NAC rescued the testicular histology impairment and decreased the testicular organ coefficient in BDE-209-exposed mice. In addition, NAC supplement partially promoted meiotic prophase and improved sperm quality in BDE-209-exposed mice. Furthermore, NAC pretreatment effectively improved DNA damage repair by recovering DMC1, RAD51, and MLH1. In conclusion, BDE-209 caused spermatogenesis dysfunction related to the meiotic arrest medicated by oxidative stress, decreasing sperm quality.

Chemical Characteristics and Cytotoxicity to GC-2spd(ts) Cells of PM2.5 in Nanjing Jiangbei New Area from 2015 to 2019

PM2.5 is an air pollutant with complex components. After entering the body through respiration, PM2.5 can not only cause respiratory diseases, but also break through the blood-testis barrier and influence the reproductive system. PM2.5 with different components may result in different toxic effects. In the first five years of Nanjing Jiangbei New Area, industrial transformation would change the concentration and chemical fraction of PM2.5 in the local environment to a certain extent. In this study, PM2.5 collected in Nanjing Jiangbei New Area every autumn and winter from 2015 to 2019 was analyzed. PM2.5 concentration generally decreased year by year. The large proportion of secondary inorganic ions indicated the presence of secondary pollution at the sampling site. PM2.5 was mainly emitted from fossil fuel combustion and vehicle exhaust. The cytotoxicity of PM2.5 samples was evaluated by PM2.5 exposure to mouse spermatocytes (GC-2spd(ts) cells). Cell viability was relatively low in 2016 and 2018, and relatively high in 2017 and 2019. Reactive oxygen species levels and DNA damage levels followed similar trends, with an overall annual decrease. The cytotoxicity of PM2.5 on GC-2spd(ts) cells was significantly correlated with water-soluble ions, water-soluble organic carbon, heavy metals and polycyclic aromatic hydrocarbons (p < 0.01). According to principal component analysis and multiple linear regression, fossil fuel combustion, secondary transformation of pollutants and construction dust were identified as the major contributors to cytotoxic effects, contributing more than 50%.

Phosphodiesterase 10A inhibitor PF-2545920 as a prospective agent for the clinical promotion of sperm motility.

Phosphodiesterase (PDE) inhibitors can improve sperm motility in patients with asthenozoospermia. However, the most commonly reported nonselective PDE inhibitor pentoxifylline and PDE5 inhibitor sildenafil have the disadvantages of requiring a high concentration and destroying sperm integrity. We examined the PDE10A inhibitor PF-2545920 to compare its ability to promote sperm motility with that of pentoxifylline and sildenafil. After seminal plasma was discarded, several semen samples were subjected to four treatments (control, PF-2545920, pentoxifylline, and sildenafil) to evaluate their ability to affect motility, viability, and spontaneous acrosome reactions. Intracellular calcium and adenosine triphosphate (ATP), mitochondrial membrane potential, and penetration through viscous medium were assessed by flow cytometry, luciferase, and hyaluronic acid after treatment with PF-2545920. Statistical analyses were performed using the analysis of variance statistical test. PF-2545920 elevated the percentage of motile spermatozoa compared to the control, pentoxifylline, and sildenafil groups at 10 µmol l -1 ( P < 0.01). It is less toxic to GC-2spd mouse spermatocytes cells and spermatozoa and causes fewer spontaneous acrosomal reactions ( P < 0.05). PF-2545920 also increased mitochondrial membrane potential ( P < 0.001) and altered intracellular calcium ( P < 0.05) in a dose-dependent manner, including increasing sperm hyaluronic acid penetrating ability ( P < 0.05). Therefore, PF-2545920 might be an excellent choice for stimulating the sperm motility.