Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos
Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos
复制标题
重组人骨形态发生蛋白 6 增强卵母细胞重编程潜力和克隆牦牛胚胎的后续发育
DOI：
10.1089/cell.2015.0049
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发表时间：
2015
影响因子：
1.6
通讯作者：
Yu Sijiu
中科院分区：
文献类型：
--
作者：
Pan Yangyang;He Honghong;Cui Yan;Baloch Abdul Rasheed;Li Qin;Fan Jiangfeng;He Junfeng;Yu Sijiu
本研究探讨了体外成熟（IVM）过程中在培养基中添加骨形态发生蛋白6（BMP 6）对卵母细胞发育潜力以及克隆牦牛胚胎后续发育的影响。从牦牛卵巢有腔卵泡中吸出卵丘-卵母细胞复合体（COCs），在卵母细胞成熟培养液中加入不同浓度的重组人骨形成蛋白6（BMP 6）进行培养。成熟后，中期II（MII）卵母细胞用于体细胞核移植（SCNT），并在体外培养。在第8天检测囊胚发育和细胞数。采用相对定量RT-PCR和免疫荧光技术检测牦牛克隆囊胚细胞凋亡相关基因和蛋白（Bax、Bcl-2、H3K9ac、H3K18ac和H3K9me3）的表达，分析牦牛克隆囊胚细胞凋亡和组蛋白修饰情况。100 ng/mL的BMP 6显著提高了牦牛克隆胚胎的卵母细胞成熟率（66.12 ± 2.04% vs. 73.11 ± 1.38%）、卵裂率（69.40 ± 1.03% vs. 78.16 ± 0.93%）和囊胚形成率（20.63 ± 1.32% vs. 28.16 ± 1.67%）。囊胚总数（85.24 ± 3.12 vs.103.36 ± 5.28）、内细胞团（ICM）细胞数（19.59 ± 2.17 vs.32.20 ± 2.61）和ICM/滋养外胚层（TE）比值（22.93 ± 1.43% vs.31.21 ± 1.62%）也有所增加（p <0.05）。SCNT + BMP6组中Bcl-2基因表达的比例最低（p <0.05）。在SCNT + BMP 6组中H3K9ac和H3K18ac水平增加（p <0.05），而H3K9me3水平降低;囊胚中的差异不显著（p> 0.05）。这些研究结果表明，在卵母细胞成熟培养基中添加重组BMP 6可以增强牦牛卵母细胞的发育潜力和随后的SCNT胚胎的发育能力，并证明BMP 6是哺乳动物卵母细胞发育重编程的重要决定因素。
This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus–oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were culturedin vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax,Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p< 0.05). The ratio of theBaxto theBcl-2gene was lowest in the SCNT + BMP6 groups (p< 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p< 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p> 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.