卵母细胞敲除Rictor对纺锤体和早期胚胎发育的影响

Effects of knockout Rictor in oocyte on spindle and early embryo development

目的探讨Rictor对卵母细胞及早期胚胎发育的影响。方法获得卵母细胞条件性敲除(Rictor-cKO)小鼠(KO组),取同窝未敲除雌性小鼠作为对照组(WT组),腹腔注射促性腺激素后收集卵母细胞,观察条件性敲除小鼠排出的卵母细胞的形态及排卵的数量;免疫荧光化学结合激光共聚焦技术观察卵母细胞条件性敲除Rictor对染色体数量、纺锤体形态的影响;采用卵胞浆内单精子注射(ICSI)技术向敲除鼠卵母细胞内注射野生型雄鼠的精子头,观察胚胎发育情况。结果卵母细胞条件性敲除Rictor不显著影响小鼠的排卵数量(P>0.05)及卵母细胞的基本形态(P>0.05);然而早期胚胎发育出现明显迟缓,同时段内观察到敲除鼠的2PN率、发育到2-细胞期胚胎数量均显著低于对照组(P<0.05),发育到4-细胞期胚胎数量亦减少(44.4%)。免疫荧光化学结果显示,条件性敲除Rictor后,卵母细胞的染色体数量正常,但部分卵母细胞的纺锤体微管异常比例升高,对照组小鼠仅有少部分卵母细胞出现异常(11.57%),差异有统计学意义(P<0.05)。结论Rictor可能通过影响卵母细胞纺锤体的排列进而影响早期胚胎发育。

Objective:To explore the effect of Rictor on oocyte and early embryo development.Methods:The conditional knockout Rictor in oocytes of mice(KO group)were acquired,and the female mice without knockout Ricto from the same nest as the control group(WT group).The oocytes after intraperitoneal injection of gonadotropin were collected.The morphology of oocytes and the number of ovulated oocytes in Rictor-KO mice were observed.The effect of conditional knock out Rictor on chromosome number and spindle morphology in oocytes were observed by immunofluorescence combined with laser confocal technique.Intracytoplasmic sperm injection(ICSI)was used to inject sperm head of wild type male mice into oocyte of Rictor-KO mice to observe the embryonic development.Results:Conditional knockout Rictor in oocyte did not affect the number of ovulated oocytes(P>0.05)and the basic morphology of oocytes(P>0.05).The development of early embryos was obviously retarded.The 2PN rate and the number of 2-cell embryos in Rictor-KO mice were significantly lower than those in the control group in the same period(P<0.05).The number of 4-cell embryos also decreased(44.4%).The results of immunofluorescence showed that the number of chromosomes of oocytes was normal,but the abnormal proportion of spindle microtubules in some oocytes was significantly increased in Rictor-KO mice,and,only a few oocytes(11.57%)were abnormal in the control group(P<0.05).Conclusions:Rictor may affect early embryonic development by affecting the arrangement of oocyte spindles.