褪黑素对牛卵母细胞体外成熟及孤雌胚胎发育的影响

Influence of melatonin on oocyte in vitro maturation and parthenogenetic embryo development in bovine

褪黑素对生殖系统发挥着重要调节作用,本研究旨在探究褪黑素对牛卵母细胞体外成熟和孤雌胚胎发育的影响及作用机制。本试验用化学激活法对牛卵母细胞孤雌激活,且在卵母细胞体外成熟液和胚胎体外培养液中添加不同浓度(0 pmol/ml、10 pmol/ml、20 pmol/ml、30 pmol/ml)的褪黑素,以检测牛卵母细胞成熟率和孤雌胚胎发育效果,同时利用DCHF-DA染色方法检测卵母细胞中ROS水平,采用免疫荧光技术检测褪黑素受体MT1在牛早期胚胎发育过程中的表达。结果表明,外源褪黑素可促进卵母细胞体外成熟,卵母细胞成熟率随褪黑素浓度的增加而升高。褪黑素显著降低了卵母细胞中的ROS水平(P<0.05),且卵母细胞中的ROS水平随着褪黑素浓度增加而降低。褪黑素可显著提高孤雌胚胎的卵裂率、桑葚胚率和囊胚率(P<0.05),促进胚胎发育。相比对照组,10pmol/ml褪黑素剂量更有利于胚胎发育,桑葚胚率和囊胚率都显著高于其他试验组。另外,本研究首次探究了褪黑素受体MT1在早期胚胎发育过程中的表达分布。MT1在未卵裂的激活卵母细胞中主要分布于细胞膜上,随着孤雌胚胎卵裂的进行,受体表达量逐渐增加,主要表达定位于细胞膜上,随着胚胎发育的进行,MT1在卵裂球内部开始表达和分布,退化胚胎中MT1表达水平较低。可见,褪黑素可促进卵母细胞成熟和胚胎发育,且褪黑素受体MT1在胚胎发育早期已开始表达。

Melatonin plays an important role in the pathogenesis of many reproductive processes.The study aimed to investigate the effects of melatonin on in vitro maturation of bovine oocytes and in vitro parthenogenetic embryos development.The oocytes collected from bovine ovaries were cultured in in vitro maturation medium.The parthenogenetic embryos were cultured in vitro after the chemical activation of oocytes.The different concentrations of melatonin were added to in vitro maturation medium and embryo culture medium (0 pmol/ml, 10 pmol/ml, 20 pmol/ml, 30 pmol/ml).In vitro maturation rates of bovine oocytes and the parthenogenetic embryos development were detected.The levels of reactive oxygen species(ROS) were detected in bovine oocytes by DCHF-DA staining.The expression of melatonin receptor MT1 was detected in bovine early embryo.The results showed that melatonin improved in vitro maturation of oocytes, and in vitro maturation rates of oocytes were increased with increased concentration of melatonin.Meanwhile, the levels of ROS were decreased with increased concentration of melatonin in oocytes.And the rates of cleavage, morula and blastocyst in parthenogenetic embryos were significantly increased, indicating that melatonin promoted the development of embryo.Compared with the control group, 10pmol/ml melatonin was more conducive to the development of parthenogenetic embryos, reflected by higher rates of morula and blastocyst.Melatonin receptor MT1 was expressed in the cell membrane of noncleved activated oocytes, and the expression level was lower than that in 16 cells embryos.The expression of MT1 was increased with the development of embryos, and mainly in the cell membrane and blastomeres of embryos.However, MT1 reduced its expression in degenerated embryos.It was inferred that melatonin could induce the level of ROS to improve in vitro maturation of bovine oocytes, the development of embryo, and melatonin receptor MT1 was expressed in early embryo.