【作者】 王志成；

【导师】 陈仕毅； 张明； 张谊；

【作者基本信息】 四川农业大学 ， 农业硕士（专业学位）， 2022， 硕士

【摘要】 双链RNA病毒感染生殖道后,通过干扰卵巢功能和妊娠过程,从而影响雌性动物生殖潜能。然而病毒感染是否会直接影响早期胚胎发育过程仍有待深入探讨。本研究旨在利用双链RNA病毒病原相关分子模式物Poly(I:C)模拟病毒感染小鼠卵母细胞体外受精过程,探究病毒感染影响体外受精和早期胚胎发育的潜在作用机制。试验中,首先小鼠MII期卵母细胞随机分为6个组,分别用0,0.001,0.01,0.1,1,10μg/m L Poly(I:C)处理卵母细胞体外受精过程,并观察统计不同浓度Poly(I:C)对原核形成率、卵裂率以及囊胚率的影响,以此确定Poly(I:C)诱导浓度。然后在确定了10μg/m L Poly(I:C)作为诱导浓度后,设置对照组,检测Poly(I:C)诱导组和对照组早期胚胎发育过程中细胞免疫应激反应、DNA损伤,以及染色质重塑、纺锤体组装和囊胚增殖、分化相关基因或者蛋白的表达。结果如下:(1)1μg/m L Poly(I:C)显著降低小鼠胚胎发育过程中原核形成以及第一次卵裂(P<0.01),10μg/m L Poly(I:C)显著影响了原核形成、第一次卵裂和囊胚形成(P<0.01),因此后续试验选择10μg/m L Poly(I:C)进行诱导处理。(2)10μg/m L Poly(I:C)处理小鼠卵母细胞体外受精过程后,与对照组相比,第一次卵裂时TLR3和IFNα基因表达量上升(P<0.05),卵裂期的IFNα1蛋白表达量上升(P<0.01),说明Poly(I:C)能够引起小鼠早期胚胎产生免疫应激反应。(3)Poly(I:C)诱导了早期胚胎发育过程中二细胞胚胎的γ-H2AX表达量上调(P<0.01),原核期PARP1表达量下调(P<0.05),说明Poly(I:C)导致早期胚胎发育过程中DNA损伤上升,DNA修复能力下降。(4)试验组与对照组相比,第一次卵裂过程中异常纺锤体组装受精卵的比例显著上升(P<0.05),与纺锤体组装相关基因FILIA的表达量显著下降(P<0.05),同时,检测原核形成过程中与染色质重塑相关基因表达结果显示,Poly(I:C)处理后,CFP1和CHD1的表达量显著下降(P<0.05)。说明Poly(I:C)处理后影响早期胚胎发育过程中染色质重塑和卵裂过程中纺锤体组装过程。(5)检测Poly(I:C)对早期胚胎发育过程囊胚细胞增殖、分化的影响,结果表明,与对照组相比,Poly(I:C)处理后,与囊胚细胞增殖SOX2、OCT4表达分别显著(P<0.05)和极显著(P<0.01)下降,与囊胚分化相关的CDX2表达显著下降(P<0.05),但是囊胚细胞数目没有显著差异。说明Poly(I:C)会损伤囊胚增殖以及分化过程。(6)Poly(I:C)处理后,囊胚细胞凋亡率显著上升,同时促进凋亡基因CASPASE3和BAX的表达显著上升(P<0.05)。说明Poly(I:C)可能促进了囊胚细胞凋亡。综上,Poly(I:C)可能通过诱导第一次有丝分裂过程DNA损伤以及纺锤体组装异常,降低体外受精卵裂率。随后通过诱导免疫应激反应、抑制囊胚细胞增殖和分化,促进囊胚细胞凋亡影响早期胚胎的发育。更多还原

【Abstract】 Infections of female genital tract with double-stranded RNA(ds RNA)virus,impair the potential reproductivity by disturbing ovarian function and pregnancy process.However,whether virus infection directly affect the early embryonic development remains to be further explored.The purpose of this study was to use poly(I:C),the pathogen-associated molecular patterns of double-stranded RNA virus,to simulate the oocyte in the process of in vitro fertilization(IVF),and then determine the pronucleus formation rate,cleavage rate and blastocyst rate to explore the potential mechanism of the ds RNA viral infection affecting early embryonic development.In this experiment,mouse MII stage oocytes were firstly randomly divided into 6 groups,and exposure IVF medium containing 0,0.001,0.01,0.1,1,10 μg/m L Poly(I:C)respectively,and then pronucleus formation rate,cleavage rate and blastocyst rate were determined in different concentrations of Poly(I:C)to define an optimized concentration of Poly(I:C).Followingly,10 μg/m L Poly(I:C)was added in the treated group using subsequent experiments.The expression level of genes and protein related to immune response,DNA damage,and chromosome remodeling,spindle assembly and proliferation,differentiation and apoptosis of blastocyst cell were compared between the treated group and the control group.These results are as follows:(1)1 μg/m L Poly(I:C)can significantly impair the formation of pronucleus and the first cleavage during mouse embryonic development(P < 0.01),and 10 μg/m L Poly(I:C)significantly decreased the percentage of pronucleus formation,the first cleavage and blastocyst development(P < 0.01).Therefore,10 μg/m L Poly(I:C)was used in subsequent experiments.(2)After oocytes being exposure IVF medium with 10 μg/m L Poly(I:C),the expression levels of TLR3 and IFNα m RNA significantly increased(P < 0.05),and the expression of IFNα1 protein also significantly increased(P < 0.01)at the first cleavage compared with the control.The result suggests that Poly(I:C)could induce an immune response in the early embryo of mice.(3)10 μg/m L Poly(I:C)induced the up-regulation of γ-H2 AX in the two-cell embryo(P < 0.01),and down-regulation of PARP1(P < 0.05)at stage of pronucleus formation compared with the control group.The result suggests that Poly(I:C)leads to the increase in DNA damage and the decrease in DNA repair capacity during embryonic development.(4)Compared with the control group,Poly(I:C)significantly increased the proportion of abnormal spindle assembly at the first cleavage(P < 0.05),and significantly decreased the expression of FILIA,a gene related to spindle assembly(P < 0.05).Meanwhile,the expression level of CFP1 and CHD1 related to chromosome remodeling significantly decreased after Poly(I:C)treatment(P < 0.05).The result implies that Poly(I:C)damage chromosome remodeling and spindle assembly at the first cleavage.(5)The expression level of some genes related to proliferation and differentiation of blastocyst cells were detected.The results showed that the expression level of SOX2 and OCT4(proliferation of blastocyst cells),and CDX2(differentiation of blastocyst cells)was significantly inhibited after Poly(I:C)-induction.However,the number of blastocyst cells was no different between the Poly(I:C)and control group.The result suggests that Poly(I:C)could impair proliferation and differentiation of blastocyst cells.(6)Poly(I:C)significantly increased the apoptotic rate of blastocyst cell,and the expression level of CASPASE3 and BAX(P < 0.05),related to cell apoptosis.The result suggests that Poly(I:C)induce apoptosis of blastocyst cells.In conclusion,Poly(I:C)inhibit the first cleavage by inducing DNA damage,abnormal spindle assembly and immune response after IVF,and then damage early embryonic development by inhibiting proliferation and differentiation of blastocyst cell,and inducing apoptosis of blastocyst cells.更多还原