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存活素蛋白在牛卵泡卵母细胞中的表达及其体外发育能力

Survivin protein expression in bovine follicular oocytes and their in vitro developmental competence.

作者信息

Jeon Kilsoo, Kim Eun Young, Tae Jin Cheol, Lee Chang Hyun, Lee Keum Sil, Kim Yeon Ok, Jeong Dong Kee, Cho Somi K, Kim Jae Hoon, Lee Hyo Yeon, Riu Key Zung, Cho Ssang Goo, Park Se Pill

机构信息

Mirae Biotech./Cheju National University Stem Cell Research Center, Seoul 143-854, South Korea.

出版信息

Anim Reprod Sci. 2008 Nov;108(3-4):319-33. doi: 10.1016/j.anireprosci.2007.09.003. Epub 2007 Sep 19.

Abstract

This study examined the relationship between survivin expression and the stage of development of in vitro cultured bovine oocytes and embryos; and whether survivin expression is affected by the quality of cumulus-oocyte complexes (COCS) or the quality of pre-implantation embryos. A polyclonal antibody was prepared using recombinant bovine survivin protein. Expression of survivin mRNA and protein was analyzed by real-time quantitative RT-PCR and immunocytochemistry. In the first experiment, survivin mRNA expression was examined at developmental stages from germinal vesicle (GV) oocyte to blastocyst, it was significantly decreased after fertilization of matured oocytes (P<0.05), then increased slightly to the 8-cell stage followed by rapid increases at the morula and blastocyst stages (P<0.05). In the second experiment, the effect of oocyte quality on survivin protein, pro-apoptotic (bax, caspase-3) and anti-apoptotic (survivin, bax inhibitor) mRNA expression was examined. Survivin protein was more strongly expressed in good quality COCS than in poor quality COCS. The expression of the anti-apoptotic genes, survivin and bax inhibitor, was significantly higher (P<0.05) and that of the pro-apoptotic genes, bax and caspase-3, was significantly lower (P<0.05) in good compared to poor quality COCS. The developmental competence of good quality COCS (30.4% blastocysts) was significantly better than that of poor quality COCS. In the last experiment also, we confirmed that significantly higher expression of survivin and bax inhibitor genes and significantly lower expression of bax and caspase-3 genes was resulted in good quality blastocysts than in poor quality blastocysts (P<0.05). It was concluded that the expression of survivin was related to the quality of COCS, their developmental competence and the quality of in vitro produced blastocysts. Consequently, survivin may be a good candidate marker for embryo quality.

摘要

本研究检测了生存素表达与体外培养的牛卵母细胞和胚胎发育阶段之间的关系;以及生存素表达是否受卵丘-卵母细胞复合体(COCs)质量或植入前胚胎质量的影响。使用重组牛生存素蛋白制备了多克隆抗体。通过实时定量逆转录-聚合酶链反应(RT-PCR)和免疫细胞化学分析生存素mRNA和蛋白的表达。在第一个实验中,检测了从生发泡(GV)卵母细胞到囊胚发育阶段的生存素mRNA表达,成熟卵母细胞受精后其表达显著降低(P<0.05),然后在8细胞阶段略有增加,随后在桑葚胚和囊胚阶段迅速增加(P<0.05)。在第二个实验中,检测了卵母细胞质量对生存素蛋白、促凋亡(bax、caspase-3)和抗凋亡(生存素、bax抑制剂)mRNA表达的影响。优质COCs中生存素蛋白的表达比劣质COCs中更强。与劣质COCs相比,优质COCs中抗凋亡基因生存素和bax抑制剂的表达显著更高(P<0.05),而促凋亡基因bax和caspase-3的表达显著更低(P<0.05)。优质COCs的发育能力(30.4%囊胚)显著优于劣质COCs。在最后一个实验中,我们也证实,与劣质囊胚相比,优质囊胚中生存素和bax抑制剂基因的表达显著更高,而bax和caspase-3基因的表达显著更低(P<0.05)。得出的结论是,生存素的表达与COCs的质量、它们的发育能力以及体外产生的囊胚质量有关。因此,生存素可能是胚胎质量的一个良好候选标志物。

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