Search

Testosterone deficiency resulted in increased mortality in men. Our previous work found that hydrogen sulphide (H2 S) significantly alleviated the spermatogenesis disorder. To investigate whether H2 S could regulate testosterone synthesis and the relative signalling pathways. Disorder model of testosterone synthesis was constructed in vitro and in vivo. The cell viability was detected using CCK-8 method. The concentration of H2 S and testosterone were examined using ELISA kits. The relative mRNA and protein expression of CBS, PDE4A, PDE8A and proteins related to testosterone synthesis were detected by RT-qPCR and western blotting. PAS staining was used to detect the inflammatory status of testis. The sulfhydryl level of PDE4A and PDE8A was determined by Biotin Switch Technique. CBS overexpression inhibited while knockdown promoted LPS + H2 O2 induced injury in testosterone synthesis of MLTC-1 cells, though regulating the level of H2 S. The LPS + H2 O2 induced inhibition on cAMP and p-PKA was recovered by CBS overexpression, while addition of the specific inhibitor of PKA had opposite effects. CBS overexpression alleviated the inflammation status in testis and promoted the expression of StAR, P450scc, P450c17 and 3β-HSD. CBS could also exhibit its protective role through promoting sulfhydrylation of PDE4A and PDE8A. H2 S catalysed by CBS could recover testosterone synthesis in vitro and in vivo through inhibiting PDE expression via sulfhydryl modification and activating cAMP/PKA pathway.

Background The post-translational modifications of proteins control various physiological and pathological events in cells. Objectives In this study, we investigated the influences of the X-linked deubiquitination enzyme USP26 in mediating androgen receptor (AR) deubiquitination in the physiological events of sperm maturation and spermatogenesis through the AR signaling pathway. Material and methods The cell cycle results detected with flow cytometry (FCM) showed that both of the proteins, USP26 and AR, could facilitate the transition of G1-G2 phase in the Leydig cells (TM3). This effect also promoted the proliferation of the Leydig cells. Results The cell cycle studies performed using FCM detected that the 2 proteins, USP26 and AR, could facilitate the transition of G1-G2 phase in the Leydig cells (TM3). This effect also promoted the proliferation of the Leydig cells. Moreover, the results from co-immunoprecipitation (CO-IP), immunofluorescence and western blot assays showed that the physiological process due to USP26 interacted with AR and influenced AR deubiquitination, thus upregulating the proteins CCND1 and SPATA46 - which are associated with cell cycle progression and spermatogenesis - as well as decreasing the expression of TP73. Thus, these processes took place through the AR signaling pathway. Furthermore, the USP26 mimic plasmid transfection enhanced these activities, while, conversely, USP26 and AR inhibitor plasmid transfection suppressed the physiological events. Conclusions Taken together, the effects of AR deubiquitinated by USP26 could modulate sperm maturation and spermatogenesis through the androgen receptor signaling pathway.

During sperm cryopreservation, the most significant phenotype of cryodamage is the decrease in sperm motility. Several proteomics studies have already been performed to search for key regulators at the protein level. However, sperm functions are known to be highly regulated by phosphorylation signaling. Here, we constructed a quantitative phosphoproteome to investigate the expression change of phosphorylated sites during sperm cryopreservation. A total of 3107 phosphorylated sites are identified and 848 of them are found to be significantly differentially expressed (DE). Bioinformatics analysis showed that the corresponding genes of these regulated sites are highly associated with sperm motility, providing a connection between the molecular basis and the phenotype of cryodamage. We then performed kinase enrichment analysis and successfully identified glycogen synthase kinase-3α (GSK3A) as the key kinase that may play an important role in the regulation of sperm motility. We further constructed a GSK3A centric network that could help us better understand the molecular mechanism of cryodamage in sperm motility. Finally, we also verified that GSK3A was abnormally activated during this process. The presented phosphoproteome and functional associations provide abundant research resources for us to learn the regulation of sperm functions, as well as to optimize the cryoprotectant for sperm cryopreservation.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3’UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

Background: N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Results: We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3’UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell analysis revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Conclusions: Our results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle-derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.

This study was designed to explore the effects of histone deacetylase inhibitor romidepsin (FK228) treatment on the morphology, proliferation and karyotype of porcine foetal fibroblast cells and early developmental competence of somatic cell nuclear transfer (SCNT) embryos. We found that the treatment of foetal fibroblast cells with 0.1μM FK228 for 24 h did not alter normal morphology, proliferation rate and chromosome number of donor cells, while other treatments with different does and durations altered the above characteristics of donor cells seriously. Simultaneously, fusion rate, blastocyst rate and total cell number per SCNT blastocysts from different donor cell treatment groups were similar to the control group. We further found that the treatment of SCNT embryos with low-dose FK228 did not affect their developmental efficiency, but treatment with high dose dramatically caused blastocyst formation failure. In addition, 0.1μM FK228 treatment for 36 h significantly elevated total cell number per SCNT blastocysts. Finally, combined treatments of both donor cells and embryos significantly improved the cleavage rate of cloned embryos, but did not affect the blastocyst rate and total cell number. Taken together, histone deacetylase inhibitor FK228 with optimal dose and exposure duration can enhance early developmental efficiency of porcine SCNT embryos and blastocyst quality.

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

WD repeat-containing protein 5 (WDR5), a member of conserved WD40 protein family, is an essential component of the mixed lineage leukemia (MLL) complexes, which are crucial for numerous key biological processes including methylation of histone H3 lysine 4 (H3K4), self-renewal of embryonic stem cells, and formation of induced pluripotent stem cells. The expression pattern and functional role of WDR5 during porcine preimplantation embryonic development, however, remain unknown. Our results showed that the transcripts and protein of WDR5 exhibited stage-specific expression pattern in porcine early embryos. Moreover, blastocyst rate and total cell number per blastocyst were reduced by RNAi-mediated silencing of WDR5 or pharmacological inhibition of WDR5. Knockdown of WDR5 also disturbed the expression of several pluripotency genes. Interestingly, tri-methylation of H3K4 (H3K4me3) level was dramatically increased by WDR5 depletion. Further analysis revealed that loss of MLL3 phenocopied WDR5 knockdown, triggering increased H3K4me3 level. Simultaneously, WDR5 depletion significantly decreased the levels of histone H4 lysine 16 acetylation (H4K16ac) and its writer males absent on the first (MOF). Last but not least, WDR5 knockdown induced DNA damage and DNA repair defects during porcine preimplantation development. Taken together, results of described studies establish that WDR5 plays a significant role in porcine preimplantation embryos probably through regulating key epigenetic modifications and genome integrity.

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle-derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.