Wang L M, Feng H L, Ma Y Zh, Cang M, Li H J, Yan Zh, Zhou P, Wen J X, Bou Shorgan, Liu D J
Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology of Inner Mongolia University, Huhhot 010021, PR China.
Anim Reprod Sci. 2009 Aug;114(1-3):99-108. doi: 10.1016/j.anireprosci.2008.09.019. Epub 2008 Oct 8.
The objectives of this study were to assess the mRNA expression and protein location of IGF receptors and its ligands in bovine oocytes and different stages of preimplantation embryos, and then evaluate the effect of different concentrations of IGF-II when added to either the maturation or culture medium on in vitro embryo development. For the assessment of mRNA expression by RT-PCR three replicates each of 100 oocytes, and 60 embryos at each of the 2-cell, 8-cell, morula and blastocyst stages of development were used. Immunocytochemical techniques were used to study the location of IGFs and their receptors for COC, oocytes, and embryos at the same stages of development (n=25). The effect of supplementing maturation medium with IGF-II was examined using groups of 20 oocytes exposed to 0 (control), 10, 20, 50 or 100 ng IGF-II/ml medium. Each treatment was replicated five times. To study the effect of IGF-II added to culture medium, groups of 10 zygotes were cultured in the presence of 0 (control), 50, 100 or 150 ng IGF-II/ml medium and the treatments replicated four times. The results showed that IGF-I mRNA could not be detected but IGF-II, IGF-IR and IGF-IIR mRNA existed in bovine preimplantation embryos. Proteins for IGF-II, IGF-IR and IGF-IIR were detected on the cell plasma membrane of cumulus cells of COC, immature and mature oocytes, and 2-cell stage embryos. They were observed in blastomere cytoplasm of 8-cell and morula stage embryos. In blastocysts, the IGF proteins were distributed in the trophectoderm but not in the inner cell mass. Adding 20 ng/ml IGF-II to maturation medium resulted in higher rates of post-fertilization development than control at 8-cell (58.2% versus 44.5%; p<0.05) and blastocyst (37.0% versus 25.0%; p<0.05) stages of development; and the number of viable cells per blastocyst were significantly higher (126+/-6 versus 103+/-5; p<0.05). When IGF-II was added to the culture medium, no significant treatment differences were observed at 8-cell embryo stage but the development rate of zygotes cultured in the presence of 100 ng IGF-II/ml medium to blastocysts was significantly higher than that of control (30.0% versus 19.2%; p<0.05). It was concluded that supplementation of in vitro maturation or culture media with IGF-II affects the development of bovine embryos and could be used to improve in vitro embryo production.
本研究的目的是评估胰岛素样生长因子（IGF）受体及其配体在牛卵母细胞和植入前胚胎不同阶段的mRNA表达及蛋白定位，进而评估在成熟或培养液中添加不同浓度IGF-II对体外胚胎发育的影响。通过逆转录聚合酶链反应（RT-PCR）评估mRNA表达时，使用了100个卵母细胞的三个重复样本，以及发育至2细胞、8细胞、桑葚胚和囊胚阶段的各60个胚胎。采用免疫细胞化学技术研究了处于相同发育阶段的卵丘-卵母细胞复合体（COC）、卵母细胞和胚胎中IGF及其受体的定位（n = 25）。使用20个暴露于0（对照）、10、20、50或100 ng IGF-II/ml培养液中的卵母细胞组，检测在成熟培养液中添加IGF-II的效果。每个处理重复五次。为研究向培养液中添加IGF-II的效果，将10个合子培养组置于0（对照）、50、100或150 ng IGF-II/ml培养液中，处理重复四次。结果显示，在牛植入前胚胎中未检测到IGF-I mRNA，但存在IGF-II、IGF-IR和IGF-IIR mRNA。在COC的卵丘细胞、未成熟和成熟卵母细胞以及2细胞期胚胎的细胞质膜上检测到了IGF-II、IGF-IR和IGF-IIR蛋白。在8细胞和桑葚胚阶段胚胎的卵裂球细胞质中也观察到了这些蛋白。在囊胚中，IGF蛋白分布于滋养外胚层，而在内细胞团中未分布。在成熟培养液中添加20 ng/ml IGF-II，导致在8细胞（58.2%对44.5%；p<0.05）和囊胚（37.0%对25.0%；p<0.05）发育阶段的受精后发育率高于对照；每个囊胚的活细胞数也显著更高（126±6对103±5；p<0.05）。当向培养液中添加IGF-II时，在8细胞胚胎阶段未观察到显著的处理差异，但在含有100 ng IGF-II/ml培养液中培养的合子发育至囊胚的发育率显著高于对照（30.0%对19.2%；p<0.05）。得出的结论是，在体外成熟或培养液中添加IGF-II会影响牛胚胎的发育，可用于提高体外胚胎生产效率。
