Abstract. Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig. Key words: Apoptosis, Autophagy, LC3, Maternal gene, 3-MA, Rapamycin (J. Reprod. Dev. 58: [576][577][578][579][580][581][582][583][584] 2012) A utophagy is an intracellular, bulk degradation process in which a portion of the cytoplasm is sequestered in an autophagosome and subsequently degraded upon fusion with a lysosome [1][2][3]. Autophagy plays a critical role during fertilization [4] and an essential role in differentiation and development, as well as in the cellular response to stress. Our previous study was the first to report that mitochondrial stress influences autophagy in porcine parthenotes developing in vitro [5]. However, the role of autophagy in early porcine parthenotes is still poorly understood.A balance between the formation and degradation of cellular proteins is required for cell survival. In mammals, protein degradation is accelerated shortly after fertilization and is apparent by the early two-cell stage in mice [6] and the four-cell stage in pigs. Early embryogenesis may rely on maternal protein stores as nutrients. After fertilization, maternal proteins in oocytes are...
Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.
Meiotic maturation in many species is initiated by the activation of maturation-promoting factor (MPF) with concomitant inactivation of counteracting phosphatases, most notably protein phosphatase 2A (PP2A). Recently, Greatwall (GWL) has been identified as a cell cycle regulator that inhibits PP2A activity. In this study, we demonstrate that GWL is required for meiotic maturation in porcine oocytes. GWL expression increases from germinal vesicle (GV) to metaphase II (MII) stages of porcine oocytes and dramatically decreases with progression of the meiotic cell cycle. GWL is initially localized in the nucleus of GV oocytes and is associated with spindle fibers following GV breakdown. Depletion of GWL inhibited or delayed meiotic maturation secondary to defects in chromosome congression and spindle formation. Conversely, overexpression of GWL overcame meiotic arrest and initiated progression to MII stage. However, these oocytes had severe spindle defects. Furthermore, MII oocytes depleted of GWL progressed to pronuclear formation. Taken together, our data demonstrate that GWL is required not only for meiotic maturation but also for maintenance of MII arrest in porcine oocytes.
BackgroundSeries of epigenetic events happen during preimplantation development. Therefore assistant reproduction techniques (ART) have the potential to disrupt epigenetic regulation during embryo development. The purpose of this study was to investigate whether defects in methylation patterns in blastocyst due to superovulation originate from abnormal expression of Dnmts.MethodsLow- (6 IU) and high- (10 IU) dosage of PMSG was used to stimulate the female mice. The metaphase II(MII) oocytes, zygotes and blastocyst stage embryos were collected. Global methylation and methylation at H3K9 in zygote, and methylation at repeated sequence Line 1 and IAP in blastocysts were assayed. In addition, expression of Dnmts was examined in oocytes and zygotes.ResultsGlobal DNA methylation and methylation at H3K9 in zygotes derived from females after low- or high-dosage hormone treatment were unaltered compared to that in controls. Moreover, DNA methylation at IAP in blastocysts was also unaffected, regardless of hormone dosage. In contrast, methylation at Line1 decreased when high-dose hormone was administered. Unexpectedly, expression of Dnmt3a, Dnmt3b, Dnmt3L as well as maintenance Dnmt1o in oocytes and zygotes was not disrupted.ConclusionsThe results suggest that defects in embryonic methylation patterns do not originate from the disruption of Dnmt expression.
Biochanin A (BCA) is a natural organic compound of the phytoestrogenic isoflavone class that has antioxidant and metal chelator properties in the presence of transition metal ions, however, its efficacy in animal models is still obscure. Therefore, the objective of this study was to investigate the protective effects of BCA against arsenic-induced hepatic injury and hematotoxicity in rats. The results suggest that arsenic intoxicated rats showed significantly higher levels of plasma hepatic markers than normal control rats. Furthermore, an increase in lipid peroxidation with depletion of reduced glutathione (GSH) and activities of superoxide dismutase (SOD) and catalase (CAT) occurred in the livers of rats exposed to arsenic. Administration of BCA (20 mg/kg¨bw/day) and selenium (3 mg/kg¨bw/day) resulted in a significant reversal of hepatic and oxidative stress markers in arsenic-intoxicated rats. A low dose of BCA (10 mg/kg¨bw/day) did not show any preventive effect, while a high dose of BCA (40 mg/kg¨bw/day) partially prevented all hepatotoxicity events. These biochemical perturbations were supported by histopathological observations of the liver. Our results suggest that administration of BCA (20 mg/kg¨bw/day) attenuated the arsenic hepatotoxicity, a property that could contribute to the therapeutic approaches for chronic liver diseases.
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