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Shen, Xing-Hui,Han, Young-Joon,Cui, Xiang-Shun,Kim, Nam-Hyung CSIRO Publishing 2010 Reproduction, fertility, and development Vol.22 No.4
<P> MicroRNA-mediated RNA interference appears to play a role in early development and differentiation processes in preimplantation embryos. However, the expression of its key effectors, including Ago2, a key component of the RNA-induced silencing complex, and GW182, a critical component of GW bodies (GWBs), has not been assessed in preimplantation embryos. To characterise the roles of Ago2 and GW182 in early embryo development, we determined their transcription and protein synthesis in mouse embryos. Transcript levels of Ago2 and GW182 increased steadily from the one-cell stage through to the blastocyst stage when data were not normalised against an internal reference. However, when normalised against the internal standard, transcript levels for both genes were highest in four-cell stage embryos and decreased steadily through to the blastocyst stage. Indirect immunocytochemistry showed that both AGO2 and GW182 proteins were expressed in each stage in the early embryo and were observed to colocalise in the morula and blastocyst stages. Specific silencing of mRNA expression by short interference (si) RNA against Ago2 or Dicer1 decreased the expression of selected apoptosis- and development-related microRNAs, but did not inhibit development up to the blastocyst stage. However, transcription levels of Oct3/4, Nanog and Sox2 were decreased in both Ago2- and Dicer1-knockdown embryos at the blastocyst stage. Furthermore, although knockdown of these genes did not change transcript levels of GW182, GW182 protein synthesis was decreased in blastocyst stage embryos. These results suggest that Ago2 and Dicer1 regulate GW182 protein expression in mouse embryos, which is linked to microRNA biogenesis and likely to be important for differentiation in the blastocyst stage. </P>
Shen, Xing-Hui,Cui, Xiang-Shun,Lee, Sung-Hyun,Kim, Nam-Hyung Japanese Society of Animal Reproduction 2012 Journal of Reproduction and Development Vol.58 No.4
<P>Signal transducer and activator of transcription-3 (Stat3) plays a central role in interleukin-6 (IL-6)-mediated cell proliferation by inhibiting apoptosis in a variety of cell types. The Stat3 pathway is essential for embryonic development. The aim of this study was to determine the effects of recombinant IL-6 on the viability and development of porcine diploid parthenotes cultured in vitro. Four-cell parthenotes, derived in vitro, were cultured to the blastocyst stage, with or without recombinant IL-6. The addition of 10 or 100 ng/ml of recombinant swine IL-6 into PZM3 medium increased the development rate of parthenotes to the blastocyst stage (P<0.05). When supplemented with 10 ng/ml of recombinant swine IL-6, the number of parthenotes at the blastocyst stage increased (P<0.05) and apoptosis decreased (P<0.05). Real-time RT-PCR experiments revealed that the addition of recombinant swine IL-6 decreased the mRNA expression of the pro-apoptotic gene Caspase3 (P<0.01) but increased the expression levels of the anti-apoptotic genes Bcl2l1 and Survivin. IL-6 receptors and Stat3 mRNA expression were upregulated after treatment with 10 ng/ml recombinant swine IL-6. Immunoblots and fluorescence labeling experiments showed that the levels of phosphorylated Stat3 were upregulated. These results suggest that recombinant swine IL-6 prevents apoptosis of porcine parthenotes and enhances porcine embryo viability through the IL-6/Stat3 signaling pathway in vitro.</P>
Shen, Xing-Hui,Han, Young-Joon,Zhang, Ding-Xiao,Cui, Xiang-Shun,Kim, Nam-Hyung Wiley Subscription Services, Inc., A Wiley Company 2009 Molecular reproduction and development Vol.76 No.9
<P>Signal transducers and activators of transcription-3 (Stat3) plays a central role in interleukin-6 (IL-6)-mediated cell proliferation by inhibiting apoptosis in a variety of cell types. MicroRNA-21 (miRNA-21), a ubiquitous miRNA, acts as an anti-apoptotic factor that seems to be indirectly but strictly linked to Stat3. In order to determine whether the IL-6 induced Stat3 anti-apoptosis pathway is linked with miRNA-21, we first determined the effects of recombinant mouse IL-6 on Stat3 expression, mouse embryo viability, and the mRNA levels of apoptosis related genes and miRNA-21 during mouse embryo development in vitro. Addition of 10 or 100 ng/ml of recombinant IL-6 to the culture medium did not affect the developmental ability of 2-cell stage embryos into blastocysts. However, total cell number was significantly increased and apoptosis was reduced in blastocyst stage embryos cultured in the presence of 100 ng/ml of recombinant IL-6. Furthermore, addition of recombinant IL-6 to the culture medium significantly increased the copy numbers of anti-apoptotic miRNA-21, up-regulated Bcl2l1, and down-regulated casp3. Similarly, the injection of mature miRNA-21 into cells up-regulated Bcl2l1 and down-regulated casp3. These results suggest that the induction of the Stat3 anti-apoptotic pathway by IL-6 is linked to miRNA-21 expression, which possibly results in the regulation of cell apoptosis in early mouse embryo development. Mol. Reprod. Dev. 76: 854–862, 2009. © 2009 Wiley-Liss, Inc.</P>
Zhang Yu-Xing,Tan Qiao,Shen Chen-Hui,Wu Jian-Jian,Wu Yi-Kuan,Li Wen-Ze,Jin Lin,Li Guo-Qing 한국응용곤충학회 2022 Journal of Asia-Pacific Entomology Vol.25 No.2
Henosepilachna vigintioctomaculata is one of the most serious defoliates attacking potatoes. However, studies on functional genes have greatly been limited due to the insufficiency of effective and stable endogenous references to normalize RT-qPCR data. In this report, nine housekeeping genes (RPL4, RPL6, RPL13, RPL32, RPS18, ACT, EF1α, GAPDH and α-TUB) involved in different biological processes were selected. Their expression levels under diverse experimental conditions including developmental stages, tissues, temperatures and host plants were determined using RT-qPCR technology. The tested candidate genes were comprehensively ranked based on five alternative stability analysis methods (Ct value, geNorm, NormFinder, BestKeeper and ReFinder). The results revealed that the optimal internal reference genes varied under different experimental conditions. Any gene pair among the five candidates (RPL4, RPL13, RPL32, RPS18 and EF1α) was a suitable reference gene set under different temperatures and on different host plants. A combination of RPL6 and RPL13 was recommended as the best reference gene set across different developmental stages. A pair of RPS18 and EF1α was ranked as the optimal reference gene combination within different tissues. The most suitable reference genes were RPS18 and RPL13 under four different experimental conditions. Our findings not only establish an accurate and reliable normalization of RT-qPCR data, but also lay a solid foundation for further functional gene researches in H. vigintioctomaculata
Cui, Xiang-Shun,Li, Xing-Yu,Shen, Xing-Hui,Bae, Yong-Ju,Kang, Jason-Jongho,Kim, Nam-Hyung JOHN WILEY & SONS LTD 2007 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.74 No.2
<P>To gain insight into early embryo development, we utilized microarray technology to compare gene expression profiles in four-cell (4C), morula (MO), and blastocyst (BL) stage embryos. Differences in spot intensities were normalized, and grouped by using Avadis Prophetic software platform (version 3.3, Strand Genomics Ltd.) and categories were based on the PANTHER and gene ontology (GO) classification system. This technique identified 622 of 7,927 genes as being more highly expressed in MO when compared to 4C (P < 0.05); similarly, we identified 654 of 9,299 genes as being more highly expressed in BL than in MO (P < 0.05). Upregulation of genes for cytoskeletal, cell adhesion, and cell junction proteins were identified in the MO as compared to the 4C stage embryos, this means they could be involved in the cell compaction necessary for the development to the MO. Genes thought to be involved in ion channels, membrane traffic, transfer/carrier proteins, and lipid metabolism were also identified as being expressed at a higher level in the BL stage embryos than in the MO. Real-time RT-PCR was performed to confirm differential expression of selected genes. The identification of the genes being expressed in here will provide insight into the complex gene regulatory networks effecting compaction and blastocoel formation. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc.</P>
Xu, Yong-Nan,Shen, Xing-Hui,Lee, Seung-Eun,Kwon, Jung-Suk,Kim, Deuk-Joong,Heo, Young-Tae,Cui, Xiang-Shun,Kim, Nam-Hyung Japanese Society of Animal Reproduction 2012 The Journal of reproduction and development Vol.58 No.5
<P>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.</P>