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蛋白激酶C#alpha#在小鼠孤雌及四倍体胚胎早期发育过程中的分布和作用

陈雅隽, 申景岭, 冯秀清, 单智焱, 阎晓飞, 董建将, 钟淑琦, 雷蕾

哈尔滨医科大学组织学与胚胎学教研室.黑龙江,哈尔滨 150081

摘要

为了观察蛋白激酶C#alpha#（proteinkinase C#alpha#，PKC#alpha#）在昆明白小鼠受精卵、孤雌激活和四倍体胚胎早期发育阶段的亚细胞定位和致密化进程中的表达变化，该实验利用免疫荧光化学染色与激光共聚焦显微镜观察相结合的方法，对受精卵、孤雌激活和四倍体胚胎早期发育阶段PKC#alpha#的表达进行了定位观察，并利用Western blot对三组胚胎致密化进程中PKC#alpha#的表达进行定量分析。结果显示，PKC#alpha#在上述三组胚胎发育的2--细胞期至囊胚期均有表达，虽然不同胚胎PKC#alpha#的分布在同一发育阶段存在差异，却表现出在各胚胎期主要分布于卵裂球核染色质内，以及在胚胎致密化开始，PKC#alpha#在卵裂球连接处发生重新分布的共同特点。此外，三组胚胎PKC#alpha#在致密化进程中的表达呈升高趋势，即致密化后的表达高于致密化前。结果表明，PKC#alpha#对胚胎致密化的调节具有重要作用，其在8--细胞，4--细胞期的重新分布是胚胎进入桑椹胚期的必然事件，是胚胎致密化的前提，同时伴随蛋白表达增多。此外，PKC#alpha#在囊胚期发生了植入前的第二次重新分布。PKC#alpha#在三组胚胎各发育阶段表达情况各不相同，它对小鼠胚胎发育的影响体现在整个早期发育阶段。PKC#alpha#在小鼠受精卵早期发育阶段的两次重新分布可能与在致密化开始时启动的细胞黏附事件存在某种必然联系。

关键词： 蛋白激酶C#alpha#; 孤雌胚胎; 四倍体胚胎; 致密化

Location and role of protein kinase C#alpha# in parthenogenetic and tetraploid preimplantation embryonic development in mouse

Chen Yajun, Shen Jingling, Feng Xiuqing, Shan Zhiyan, Yan Xiaofei, Dong Jianjiang, Zhong Shuqi, Lei Lei

Department of Histology and Embryology, Harbin Medical University.Harbin 150081,Heilongjiang

Abstract

Protein kinase C （PKC） is a critical molecule in cellular signal transduction in mammals. It is involved in many biological processes in embryonic development, including nuclear remodeling, cell cycle adjustment and cellular polarity regulation. The present study aimed to observe the location of PKC#alpha#, an important isozyme of PKC, in fertilized, parthenogenetic and tetraploid preimplantation embryos, and compare the expression of PKC#alpha# during embryonic compaction in Kunming mice. The location of PKC#alpha# was detected by immunochemistry and laser confocal microscopy. Western blot was performed to quantify PKC#alpha# expression during embryonic compaction in the three kinds of embryos. In the experiment, fertilized embryos were flushed from oviduct or uterus at 45, 52, 69, 76 and 93 h after injection of human chorionic gonadotrophin （hCG）; parthenogenetic embryos were collected by SrCl_(2) activation of oocytes for 6 h; and tetraploid embryos were produced by electrofusion of 2-cell embryos. Embryos were fixed at different developmental stages for immunofluorescent staining. 8-cell/4-cell embryos and morula were lysed for Western blot. The results showed that PKC#alpha# had similar location pattern in different embryos. It was distributed mainly in the nuclear aggregating around chromatin at different developmental stages. However, PKC#alpha# expressed strongly in the interphase than in mitotic blastomere. Before embryonic compaction, PKC#alpha# was localized at the blastomere boundary. At late blastocyst stage of fertilized embryos, PKC#alpha# was localized only in the polar trophoblast, but not in other trophoblast. At late stage of pathenogenetic blastocyst, there was no clear PKC#alpha# signal in the polar trophoblast. Tetraploid embryos had larger blastomere than other embryos and compacted after 4-cell stage, but not after 8-cell stage. Meanwhile, there was PKC#alpha# signal at the blastomere boundary at 4-cell stage. Our results showed that the expression of PKC#alpha# lasted through all the preimplantation stage. Although there were different expression levels among different stages, the expression increased around embryonic compaction. Quantification of expression of PKC#alpha# by Western blot demonstrated that the expression increased after compaction, indicating that the compaction was possibly dependent on the relocation of PKC#alpha#. Moreover, it was shown that the second relocation of PKC#alpha# occurred during the blastocyst formation. PKC#alpha# had different expression patterns in the three kinds of preimplantation embryos. However, the effects of PKC#alpha# on embryonic development started in early stage. There must be a necessary connection between PKC#alpha# relocation and cell adhesion starting at embryonic compaction.

Key words: protein kinase C#alpha#;parthenogenetic embryo;tetraploid embryo;compaction

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引用本文：

陈雅隽, 申景岭, 冯秀清, 单智焱, 阎晓飞, 董建将, 钟淑琦, 雷蕾. 蛋白激酶C#alpha#在小鼠孤雌及四倍体胚胎早期发育过程中的分布和作用[J]. 生理学报 2008; 60 (1): 105-112.

Chen Yajun, Shen Jingling, Feng Xiuqing, Shan Zhiyan, Yan Xiaofei, Dong Jianjiang, Zhong Shuqi, Lei Lei. Location and role of protein kinase C#alpha# in parthenogenetic and tetraploid preimplantation embryonic development in mouse. Acta Physiol Sin 2008; 60 (1): 105-112 (in Chinese with English abstract).