自噬调节因子Atg5和Beclin1在不同来源小鼠胚胎早期发育过程中的表达分析

doi:10.11843/j.issn.0366-6964.2020.12.015

摘要/Abstract

摘要： 旨在探究自噬调节因子Atg5和Beclin1在胚胎早期发育过程中的表达模式及胚胎的不同生产方式对两种因子表达的影响。本研究将6~8周龄雌性小鼠进行超数排卵，分为2组，一组收集小鼠卵母细胞，孤雌激活处理后进行体外培养；另一组超排小鼠与公鼠1：1合笼，第2天收集小鼠受精卵进行体外培养；分别在2细胞期、4~8细胞期、桑葚胚期和囊胚期收集不同阶段小鼠孤雌激活胚胎和自然受精胚胎。提取RNA和蛋白，通过实时荧光定量PCR、Western blot等方法检测自噬关键因子Atg5和Beclin1的表达，通过间接免疫荧光法检测Atg5和Beclin1在小鼠囊胚中的表达定位。结果显示，小鼠自然受精和孤雌激活胚胎在发育各时期均可表达Atg5和Beclin1，表达量在胚胎发育的早期呈现出较高的水平，其中二者的表达在小鼠自然受精胚胎中从2细胞期起逐渐降低，而在孤雌激活胚胎的4~8细胞阶段表达量最高，与同期自然受精胚胎差异极显著（P<0.01）；从4细胞期开始，各时期孤雌激活胚胎中Atg5和Beclin1蛋白表达水平均高于自然受精胚胎，差异极显著（P<0.01）；在囊胚中，滋养层细胞和内细胞团中均可检测到Atg5和Beclin1蛋白的荧光，但内细胞团中的荧光强度高于滋养层细胞，且Beclin1蛋白在孤雌激活胚胎囊胚内细胞团中荧光强度高于自然受精胚胎。自噬关键因子Atg5和Beclin1在不同来源小鼠胚胎早期发育各时期均有不同程度的表达，提示自噬对早期胚胎发育的调控作用与胚胎的生产方式存在一定关联，研究结果为进一步探索细胞自噬参与哺乳动物胚胎发育的生理调控提供理论依据。

关键词: 胚胎发育, 孤雌激活, 自然受精, 自噬

Abstract: This study aimed to explore the expression patterns of autophagy regulators Atg5 and Beclin1 in the early embryonic development and the effects of different embryonic production methods on the expression of the two factors. Female mice aged 6-8 weeks were subjected to superovulation and divided into 2 groups. The mouse oocytes of one group were collected, and cultured in vitro after parthenogenetic activation. The other group of female mice were caged with male mice (1:1), and the next day, the mouse fertilized eggs were collected for in vitro culture. Parthenogenetic activated embryos and naturally fertilized embryos were collected at 2 cell stage, 4-8 cell stage, mulberry embryo stage and blastocyst stage, respectively. RNA and protein were extracted, real-time fluorescence quantitative PCR, Western blot and other methods were used to detect the expression of key autophagy factors Atg5 and Beclin1. And indirect immunofluorescence was used to detect the expression and location of Atg5 and Beclin1 in mouse blastocysts. The results showed that Atg5 and Beclin1 were expressed in all development stages of naturally fertilized and parthenogenetic activated embryos in mice, and showed a high level in the early stage of embryonic development. The expression of Atg5 and Beclin1 were gradually reduced from the 2 cell stage in mouse naturally fertilized embryos. The expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were the highest in the 4-8 cell stage, which was extremely significantly different from the naturally fertilized embryos of the same period (P<0.01). From the 4 cell stage, the expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were higher than naturally fertilized embryos at all subsequent stages, the difference was extremely significantly different (P<0.01). In mouse blastocysts, the fluorescence of Atg5 and Beclin1 protein could be detected in the trophoblast cells and the inner cell mass, but the fluorescence intensity in the inner cell mass was higher than that in the trophoblast cells. In addition, the fluorescence intensity of Beclin1 protein in the inner cell mass of parthenogenetic activated embryos was higher than that in naturally fertilized embryos. Atg5 and Beclin1, the key autophagy factors, are expressed at different levels in the early development of mouse embryos from different sources. It is suggested that the regulation of autophagy on early embryonic development is related to embryo production modes. The results will provide a theoretical basis for further exploring the role of autophagy in the physiological regulation of mammalian embryo development.

Key words: embryonic development, parthenogenetic activation, natural fertilization, autophagy

中图分类号:

S814

马睿, 王萌, 孙莹, 芮弦, 王靖雷, 付延, 余四九, 王立斌, 崔燕, 潘阳阳. 自噬调节因子Atg5和Beclin1在不同来源小鼠胚胎早期发育过程中的表达分析[J]. 畜牧兽医学报, 2020, 51(12): 3057-3067.

MA Rui, WANG Meng, SUN Ying, RUI Xian, WANG Jinglei, FU Yan, YU Sijiu, WANG Libin, CUI Yan, PAN Yangyang. Expression Analysis of Autophagy Regulators Atg5 and Beclin1 during Early Embryonic Development of Mouse (Mus musculus) from Different Sources[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3057-3067.

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