PLCγ1对绵羊早期胚胎超微结构的影响

Effects of PLCγ1 on Ultrastructure of Early Embryo in Sheep

为了探究绵羊磷脂酶Cγ1(PLCγ1)基因对绵羊早期胚胎发育的影响,从微观角度解释胚胎的内部发育规律。卵母细胞分离培养至成熟,显微注射同等浓度的pcDNA3.1-EGFP-PLCγ1重组质粒后随机分成两组,PLCγ1处理组在注射后培养24 h、48 h;U73122处理组在显微注射后12 h向培养液中添加最优抑制PLCγ1的U73122浓度0.5μmol/L,两组分别在24 h、48 h固定胚胎做超薄切片,电镜观察透明带、脂滴、线粒体及胚胎发育情况。结果表明,显微注射重组质粒后能够促进胚胎发育,并且随着胚胎的发育,透明带呈不规则性疏松,PLCγ1处理组与U73122处理组的不同时间段以及相同时间段之间,透明带厚度均存在极显著差异(P<0.01)。线粒体在胞质内呈均匀分布,PLCγ1处理组线粒体横脊少且形状规则,成熟的线粒体含量较多;U73122处理组线粒体嵴深且长,出现空泡化异常形态,线粒体异常率(31.72%±3.316%)极显著高于PLCγ1处理组(22.01%±6.652%)。脂滴随机分布于各卵裂球中,U73122处理组脂滴形态出现异常,数量多于PLCγ1处理组。以上结果初步证明,绵羊早期胚胎发育过程中,PLCγ1基因可通过影响透明带厚度、线粒体及脂滴等改变细胞代谢水平从而调整胚胎发育。

In order to investigate the effects of phospholipase Cγ1(PLCγ1)gene on early embryos development in sheep,and to explain the internal development of embryo from a microscopic perspective,the cumulus-oocyte complex was isolated from the ovaries and cultured to maturity.After microinjection of pcDNA3.1-EGFP-PLCγ1 recombinant plasmid with the same concentration,they were randomly divided into two groups.One group(PLCγ1 treatment group)was cultured for 24 h and 48 h after injection,another group(U73122 treatment group),the optimal concentration of U731220.5μmol/L was added to the culture medium 12 hours after microinjection.The embryos were fixed for ultra-thin sections at 24 h and 48 h,respectively,and the development of zonal pellucida,lipid droplets,mitochondria and embryos were observed by transmission electron microscopy.The results showed that microinjection of recombinant plasmid could promote the development of embryos,and the zona pellucida showed irregular porosity with the development of embryos.The thickness of zona pellucida in PLCγ1 treatment group and U73122 treatment group was significantly different at different time and between the same time(P<0.01).Mitochondria were evenly distributed in the cytoplasm,and the mitochondria crists were deep and long in U73122 treatment group,and vacuolated abnormal morphology was observed.The abnormal rate of mitochondria(31.72%±3.316%)was significantly higher than that of PLCγ1 group(22.01%±6.652%).Lipid droplets were randomly distributed in each blastomere,and the number of lipid droplets in U73122 treatment group was more abnormal than that in PLCγ1 treatment group.These results suggest that the PLCγ1 gene can regulate embryo development by changing cell metabolism by affecting zona pellucida thickness,mitochondria and lipid droplets during early embryo development.