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The regulatory network between signaling pathways and transcription factors (TFs) is crucial for the maintenance of pluripotent stem cells. However, little is known about how the key TF OCT4 coordinates signaling pathways to regulate self‐renewal and lineage differentiation of porcine pluripotent stem cells (pPSCs). Here, we explored the function of OCT4 in pPSCs by transcriptome and chromatin accessibility analysis. The TFs motif enrichment analysis revealed that, following OCT4 knockdown, the regions of increased chromatin accessibility were enriched with EOMES, GATA6, and FOXA1, indicating that pPSCs differentiated toward the mesoendoderm (ME) lineage. Besides, pPSCs rapidly differentiated into ME when the WNT/β‐catenin inhibitor XAV939 was removed. However, the ME differentiation of pPSCs caused by OCT4 knockdown did not rely on the activation of WNT/β‐catenin signaling because the target gene of WNT/β‐catenin signaling, AXIN2 was not upregulated after OCT4 knockdown, despite significant upregulation of WLS and some WNT ligands. Importantly, OCT4 is directly bound to the promoter and enhancers of EOMES and repressed its transcription. Overexpression of EOMES was sufficient to induce ME differentiation in the presence of XAV939. These results demonstrate that OCT4 can regulate WNT/β‐catenin signaling and prevent ME differentiation of pPSCs by repressing EOMES transcription. [ABSTRACT FROM AUTHOR]

Objectives: BCL2‐associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24‐skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis. Materials and Methods: CRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high‐testosterone mice model. H&E staining, qRT‐PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis. Results: Bag6 exon24 knockout mice show sub‐fertility along with partially impaired blood‐testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24‐deficient testes and sterol regulatory element‐binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up‐regulated, which causes a high serum testosterone level. Additionally, serine/arginine‐rich splicing factor 1 down‐regulates BAG6 exon24‐skipped transcripts in porcine Sertoli cells by binding to 35–51 nt on BAG6 exon24 via its N‐terminal RNA‐recognition domain. Conclusions: Our findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals. [ABSTRACT FROM AUTHOR]

The faithful execution of molecular programme underlying oocyte maturation and meiosis is vital to generate competent haploid gametes for efficient mammalian reproduction. However, the organization and principle of molecular circuits and modules for oocyte meiosis remain obscure. Here, we employed the recently developed single‐cell RNA‐seq technique to profile the transcriptomes of germinal vesicle (GV) and metaphase II (MII) oocytes, aiming to discover the dynamic changes of mRNAs and long non‐coding RNAs (lncRNAs) during oocyte in vitro meiotic maturation. During the transition from GV to MII, total number of detected RNAs (mRNAs and lncRNAs) in oocytes decreased. Moreover, 1,807 (602 up‐ and 1,205 down‐regulated) mRNAs and 313 (177 up‐ and 136 down‐regulated) lncRNAs were significantly differentially expressed (DE), i.e., more mRNAs down‐regulated, but more lncRNAs up‐regulated. During maturation of pig oocytes, mitochondrial mRNAs were actively transcribed, eight of which (ND6, ND5, CYTB, ND1, ND2, COX1, COX2 and COX3) were significantly up‐regulated. Both DE mRNAs and targets of DE lncRNAs were enriched in multiple biological and signal pathways potentially associated with oocyte meiosis. Highly abundantly expressed mRNAs (including DNMT1, UHRF2, PCNA, ARMC1, BTG4, ASNS and SEP11) and lncRNAs were also discovered. Weighted gene co‐expression network analysis (WGCNA) revealed 20 hub mRNAs in three modules to be important for oocyte meiosis and maturation. Taken together, our findings provide insights and resources for further functional investigation of mRNAs/lncRNAs in in vitro meiotic maturation of pig oocytes. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) regulate a variety of physiological and pathological processes. However, the biological function of lncRNAs in mammalian germ cells remains largely unexplored. Here we identified one novel lncRNA (lncRNA2193) from single‐cell RNA sequencing performed on porcine oocytes and investigated its function in oocyte meiosis. During in vitro maturation (IVM), from germinal vesicle (GV, 0 hr), GV breakdown (GVBD, 24 hr), to metaphase II stage (MII, 44 hr), the transcriptional abundance of lncRNA2193 remained stable and high. LncRNA2193 interference by small interfering RNA microinjection into porcine GV oocytes could significantly inhibit rates of GVBD and the first polar body extrusion, but enhance the rates of oocytes with a nuclear abnormality. Moreover, lncRNA2193 knockdown disturbed cytoskeletal organization (F‐actin and spindle), and decreased DNA 5‐methylcytosine (5mC) and histone trimethylation (H3K4me3, H3K9me3, H3K27me3, and H3K36me3) levels. The lncRNA2193 downregulation induced a decrease of 5mC level could be partially due to the reduction of DNA methyltransferase 3A and 3B, and the elevation of 5mC‐hydroxylase ten‐11 translocation 2 (TET2). After parthenogenetic activation of MII oocytes, parthenotes exhibited higher fragmentation but lower cleavage rates in the lncRNA2193 downregulated group. However, lncRNA2193 interference performed on mature MII oocytes and parthenotes at 1‐cell stage did not affect the cleavage and blasctocyst rates of pathenotes. Taken together, lncRNA2193 plays an important role in porcine oocyte maturation, providing more insights for relevant investigations on mammalian germ cells. [ABSTRACT FROM AUTHOR]

As a member of parabens (PBs), Isobutylparaben (IBP) has a broad‐spectrum antimicrobial activity and widely used in personal care products and cosmetics. Recent studies have indicated that usage of IBP poses a potential threat to reproductive health. In this study, we aimed to reveal the effects of acute exposure to IBP on the meiotic maturation of porcine cumulus oocyte complexes. Initial study showed that 200 μM of IBP significantly reduced the rate of the first polar body extrusion with no significant effect on cumulus cell expansion; however, 400 μM of IBP could significantly affect both. Further research revealed that abnormal spindles, misalignment chromosomes, and aberrant distributed actin filaments were detected in IBP‐treated oocytes, which indicates that the cytoskeleton architecture of oocyte could be the target of IBP. At the same time, ROS level and apoptosis rate of oocyte were significantly increased by IBP exposure. Moreover, the levels of H3K9me3 and H3K27me3 were significantly induced in oocytes by IBP. Collectively, these results demonstrate that acute exposure to IBP could disrupt porcine oocyte maturation through affecting cytoskeleton, oxidative stress, viability and epigenetic modification. Environ. Mol. Mutagen. 2020. © 2020 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose‐dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA‐seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL‐exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL‐exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL‐exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL‐induced deterioration of porcine oocyte quality. [ABSTRACT FROM AUTHOR]

Tris(1,3‐dichloro‐2‐propyl) phosphate (TDCPP) is a kind of additive flame retardants (FRs) and was found to affect early embryonic development in zebrafish; however, there are few studies to investigate whether TDCPP will disturb the development of early mouse embryos. In our studies, we used mouse embryos as models to study the toxicology of TDCPP on the early embryos. The results showed that TDCPP disturbed the development of early mouse embryos in a dose‐dependent manner. 10 μM TDCPP decreased the blastocyst formation and 100 μM TDCPP was a lethal concentration for the mouse embryos. We proved that TDCPP was detrimental to embryonic development potential by increasing the reactive oxygen species level and inducing early apoptosis. Furthermore, TDCPP changed the DNA methylation patterns of imprinted genes in treated blastocysts. The methylation of H19 and Snrpn promoter regions was increased from 37.67% to 46.00% and 31.56% to 44.38% in treated groups, respectively. In contrast, Peg3 promoter region methylation was declined from 86.55% to 73.27% in treated embryos. Taken together, our results demonstrated that TDCPP could adversely impair the early embryonic development in mouse. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Objectives: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self‐renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. Materials and methods: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. Results: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four‐cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. Conclusions: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine‐induced pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Fluorene‐9‐bisphenol (BHPF), a substitute of bisphenol A (BPA) used in the production of the so‐called "BPA‐free" plastics, has now been shown to be released from commercial plastic bottles into drinking water and has strong anti‐estrogenic activity in mice, which suggests that BHPF is also an environmental toxin. However, whether BHPF exposure has effects on mouse oocyte development is unknown. In this study, the influence of acute exposure to BHPF (50–150 μM, 12 hr) on mouse oocyte maturation and its possible mechanisms were investigated. Of note, 50‐μM BHPF had no effects on the maturation of mouse oocytes, whereas 100‐ and 150‐μM BHPF significantly blocked germinal vesicle breakdown and led to the failure of first polar body extrusion. Particularly, 100‐μM BHPF exposure severely decreased the cellular adenosine triphosphate in a time‐dependent manner, which finally brought out the loss of spindles. In addition, the actin cytoskeleton was also impaired. The defective mitochondrial dynamics and decreased mitochondrial DNA implied the damage of mitochondria in BHPF‐treated oocytes. Increased PINK1, Beclin1, and LC3B protein level and decreased TOMM20 and TOMM17A protein level illustrated that mitophagy was induced, which also confirmed that BHPF exposure impaired the cellular mitochondria. Moreover, BHPF induced reactive oxygen species accumulation and early apoptosis. Oocyte quality was also impaired by BHPF exposure through altering histone modifications evidenced by increased H3K9me3 and H3K27me3 levels. Collectively, our results indicated that BHPF exposure disrupted mouse oocyte maturation and reduced oocyte quality through affecting cytoskeleton architecture, mitochondrial function, oxidative stress, apoptosis, and histone modifications. Environ. Mol. Mutagen. 60:243–253, 2019. © 2018 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Sodium fluoride (NaF) is used as a medicine to prevent tooth decay; however, excessive NaF could cause a pathological damage to the health. Recent studies showed that NaF impaired mouse oocyte maturation, included of abnormal spindle configuration, actin cap formation, cortical granule‐free domain formation, and the following development after fertilization. However, few studies used large animals as models to study the toxicology of NaF on oocytes maturation. We proposed a hypothesis that NaF would affect the nuclear and cytoplasmic maturation of porcine oocytes and DNA methylation pattern of imprinted genes in oocytes. Our results showed that NaF affected cumulus expansion, polar body emission, spindle morphology, cortical granule distribution, early apoptosis, and the following development after parthenogenetic activation during porcine oocyte maturation. Moreover, NaF increased the DNA methylation of NNAT and decreased its expression, which disturbed the glucose transport in oocytes. These results suggest that NaF impairs the porcine oocytes maturation epigenetically, which provides a new toxicological mechanism of NaF on the oocyte maturation. Environ. Mol. Mutagen. 59:223–233, 2018. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Griseofulvin is an orally administered antifungal drug that affects microtubule formation in vitro and interferes with microtubule dynamics in vivo as clearly shown for mitotic cells in several cell systems. This article reports the effects of griseofulvin on in vitro maturation of porcine oocytes and subsequent effects on embryo development. Our results revealed a concentration-dependent effect on meiotic spindles with 20-40 μM griseofulvin affecting oocyte maturation, and 40 μM affecting fertilization and embryo development. These concentrations of griseofulvin did not affect mitochondrial and cortical granule distribution that also depend on microtubule and cytoskeletal functions during oocyte maturation. Specific effects on the meiotic spindle included spindle disorganization and aberrant chromosome separation displayed as prominent chromosome clusters in oocytes treated with 40 μM griseofulvin. These results strongly suggested that griseofulvin affected porcine oocyte in vitro maturation and following embryo development by disturbing microtubule dynamics. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The article focuses on a research about the dynamic changes of centrosomes and the microtubule cytoskeleton in porcine oocytes to restore aging oocytes' spindle integrity. It cites the importance of centrosomes in sustaining meiotic spindle integrity in the meiosis II (MII) stage. Findings show that caffeine can rescue aging oocytes' centrosome deterioration through the porcine system. It also reveals caffeine's potential to serve as treatment during in vitro fertilization (IVF).

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. Developmental Dynamics 237:640-648, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]