牛体内外植入前胚胎IFNτ的差异表达及对子宫上皮细胞ISG15和Wnt7a表达的体外诱导

IFNτof the pre-implantation bovine embryos, and its induction and regulation of the expression of ISG15 and Wnt7a on their uterine epithelium endormetrium, are the key pre-requests for regulating their uterine receptivity and initiating their successful imlantation. Although it has been reported that some gene expression differences shown on the in-vitro cultured bovine uterine epithelium cells by adding with the recombinant IFNτ,the correlation between embryonic IFNτfrom those bovine embryos by in-vitro manipulation,and its induction to expression of ISG15 and Wnt7a on their uterine epithelium cells cultured in vitro is still kept unknown. Meanwhile, it is obvious that only those in vitro manipulated or produced bovine embryos cultured in vitro on their uterine epithelium cells, can most similarly mimic their in vivo early implantation processes,and provide an appropriate new way to judge the quality of bovine embryos exactly. For investigating this bovine embryonic IFNτexpression tendency and its in-vitro induction to the expression of ISG15 and Wnt7a on their uterine epithelium cells further, here, the comparision of IFNτexpression tendency among these different in-vivo and in-vitro early bovine embryos, and its appropriate induction to the ISG15 and Wnt7a on their uterine epithelium cells co-cultured with them in vitro, was initiated and analyzed by in-situ hybridization, semi-quantative RT-PCR, CONFOCAL and Western Blot. Collectively, it will be helpful as a potential reference for digging out the correlation between the potential unnormal expression of IFNτfrom thses different types of early bovine embryos and their pregnancy failure.

牛早期胚胎分泌的IFNτ及其对ISG15和Wnt7a蛋白的诱导是调控其子宫容受态、保障胚胎正常着床的关键因素。虽然添加重组IFNτ同样可以体外诱导牛子宫上皮细胞部分基因发生差异变化，但只有通过直接研究不同胚胎体外诱导子宫上皮细胞ISG15和Wnt7a蛋白差异表达,才能更贴近模拟其体内同类过程变化,为体外研究胚胎与子宫在着床过程中的变化及据此更准确地判断胚胎质量奠定良好基础。本研究拟采用原位杂交，半定量PCR，CONFOCAL和Western检测等方法，以体内发育的牛早期囊胚、扩张囊胚中IFNτ基因在转录和翻译水平上的变化为对照，系统比较经IVF、孤雌激活和体细胞克隆等体外操作获得的相应发育阶段胚胎中IFNτ基因的表达；并通过与子宫上皮细胞共培养来检测它们对子宫上皮细胞分泌ISG15和Wnt7a蛋白的诱导效果，为分析牛体外操作胚胎中IFNτ的异常表达与其早期妊娠失败之间的潜在联系提供参考。

研究表明：牛早期胚胎分泌的IFNτ及其对ISG15和Wnt7a蛋白的诱导是调控其子宫容受态、保障胚胎正常着床的关键因素。只有直接研究不同胚胎体外诱导子宫上皮细胞ISG15和Wnt7a蛋白差异表达,才能更贴近模拟其体内同类过程变化,为此，本研究采用了定量PCR，CONFOCAL和Western检测等方法，以体内发育的牛大腔囊胚中IFNτ基因在转录和翻译水平上的变化为对照，系统比较经体外受精（IVF）、孤雌激活（PA）和体细胞克隆（SCNT）等体外操作获得的牛囊胚中IFNτ基因的表达变化；并通过与其子宫上皮细胞体外共诱导培养发现：①牛体内囊胚IFNτ的表达，显著高于其IVF，SCNT和PA囊胚，而IVF和SCNT囊胚IFNτ的表达，也显著高于PA囊胚，但是它们两者之间差异不显著。②牛体内大腔囊胚诱导子宫上皮细胞中ISG15蛋白表达，显著高于其它类型的牛体外制备胚胎，但是这些胚胎各自单独与子宫上皮细胞体外共诱导培养时，ISG15蛋白表达在共培养的子宫上皮细胞组间差异不显著；而Wnt7a蛋白在经过这些同样的体外共诱导培养后，每枚胚胎共诱导的子宫上皮细胞之间都存在显著差异，提示Wnt7a蛋白可以作为一种牛胚胎体外诱导模型的检测Marker，为建立全新的无损伤牛胚胎质量精准评定方法，以及牛的早期妊娠失败原因分析等提供了一条全新思路。