PLCγ1基因对绵羊早期胚胎体外发育的影响

Effect of PLCγ1Gene on the Development of Sheep Early Embryo in vitro

旨在探究磷脂酶Cγ1(phospholipase Cgamma 1,PLCγ1)基因对绵羊早期胚胎体外发育的影响,为揭示PLCγ1基因在早期胚胎发育过程中的作用机制奠定基础。本研究选取包裹3层以上颗粒细胞的绵羊卵母细胞为试验材料,体外培养成熟后将其分为两组,利用显微注射技术将PLCγ1基因导入MⅡ期(减数第二次分裂中期)卵母细胞(n=127)中为试验组,以显微注射空载体pcDNA3.1-EGFP作为对照组(n=120),经48h后统计其卵裂率;并利用Ca^(2+)探针Rhod 2-AM监测MⅡ卵母细胞和显微注射后16、48、72、96、120h时各时期卵母细胞以及早期胚胎内Ca^(2+)波动。结果显示,PLCγ1基因显微注射后卵母细胞被激活,卵裂率为((19.67±0.2)%,P<0.01),桑椹胚发育率为((9.00±0.17)%,P<0.05);PLCγ1基因注射后,卵母细胞和早期胚胎不同发育时期有Ca^(2+)浓度变化,可观察发现Ca^(2+)主要分布在胞质中且注射48h时可达到峰值(P<0.01)。结果表明,PLCγ1基因可以引起绵羊卵母细胞发育过程中发生Ca^(2+)振荡,启动其卵裂,并促使早期胚胎的继续发育。

The purpose of this study was to investigate the effect of PLCγ1on the development of sheep early embryo in vitro,and to lay a foundation for revealing the action mechanism of PLCγ1 in the development of sheep early embryo.In this study,sheep oocytes with more than 3layers of granulosa cells were selected as experimental materials,and they were divided into two groups after mature invitroculture.PLCγ1was introduced into MⅡoocytes(n=127)by microinjection as experimental group.Microinjection of empty vector pcDNA3.1-EGFP was used as the control group(n=120),and after 48hthe cleavage rate were measured.The Ca^(2+)probe Rhod 2-AM was used to monitor the Ca^(2+)fluctuation of MⅡoocytes and early embryos at 16,48,72,96and 120hafter microinjection.The results showed that the cleavage rate was((19.67±0.2)%,(P<0.01))and morula development rate was((9.00±0.17)%,(P<0.05))after microinjection of PLCγ1.After injection of PLCγ1,Ca^(2+)concentration in oocytes and early embryos varied at different developmental stages.It was observed that Ca^(2+)was mainly distributed in the cytoplasm and reached the peak at 48hafter injection(P<0.01).The results showed that PLCγ1gene could induce Ca^(2+)oscillation during ovine oocyte development,initiate cleavage,and promote the development of early embryo.