【作者】 韩宇；

【导师】 唐博；

【作者基本信息】 吉林大学 ， 基础兽医学， 2021， 硕士

【摘要】 5-羟色胺对猪早期胚胎发育及表观遗传修饰的影响5-羟色胺(5-HT)是一种神经递质和生长因子,其发挥作用通常依赖于与其受体的结合。当机体受到刺激,出现应激状态时,会造成体内5-HT分泌紊乱,影响生长发育。近年来,诸多证据表明5-HT在雌性生殖系统中广泛表达,促进细胞增殖及分化,并在卵泡及胚胎发育过程中扮演着重要的角色。5-HT参与调控小鼠早期胚胎发育过程,且这一研究已取得显著进展。在猪这一物种中,雌性生殖系统中是否存在5-HT和受体,5-HT对胚胎发育、卵泡发育是否有影响,5-HT与胚胎发育的表观遗传是否有相关性,这些都还未见报道。为解决以上问题,本研究以猪体外培养的孤雌激活早期胚胎为主要研究对象,进行了如下实验。首先,在实验组胚胎培养液中添加5-HT,检测了5-HT对早期胚胎发育的影响;通过荧光定量PCR技术,检测了5-HT对不同阶段早期胚胎中各受体表达的影响。研究发现实验组胚胎的卵裂率及囊胚率下降,囊胚的细胞总数减少,凋亡细胞数增加,说明5-HT抑制了胚胎的发育能力及发育质量。经5-HT处理早期胚胎,5-HT2A、5-HT4及5-HT7受体在猪早期胚胎的表达出现了明显的变化,5-HT2A及5-HT4受体的表达水平在2细胞期显著升高,在4细胞及囊胚阶段显著降低;5-HT7受体的表达则完全相反,在2细胞显著降低,而在4细胞及囊胚期显著升高,表明5-HT可能通过5-HT受体影响猪的早期胚胎发育。接下来,经5-HT处理早期胚胎,运用荧光定量PCR技术,检测了早期胚胎中多能性和凋亡相关基因的表达,结果显示,5-HT处理显著降低了多能性基因NANOG的表达,增加多能性基因SOX2及促凋亡基因BAX的表达,表明5-HT能够通过调控基因表达影响猪的早期胚胎发育。最后,利用免疫荧光染色及荧光定量PCR技术,检测了5-HT对猪早期胚胎DNA甲基化和组蛋白修饰水平的影响,结果显示,5-HT处理后,DNA甲基化未发生变化,H3K4me2及H3K4me3水平在2细胞期显著降低,在4细胞期显著增加,最后在囊胚期显著降低;H3K9Ac水平在2细胞期无显著差异,但在4细胞期显著增强,在囊胚期显著降低;H4K16Ac水平在2细胞期和4细胞期显著升高,但在囊胚期显著降低,表明5-HT处理会影响猪早期胚胎发育的组蛋白修饰水平。通过上述结果,我们推测5-HT可能通过受体介导调控基因表达,通过改变组蛋白修饰来影响早期胚胎的表观遗传,进而影响猪的早期胚胎发育过程。作为影响胚胎发育能力关键因素之一,卵母细胞的质量好坏尤为重要。哺乳动物卵母细胞位于卵泡内,其成熟伴随着卵泡的生长发育。颗粒细胞的发育及卵母细胞的成熟在卵泡发育过程中占据了主要地位。基于以上研究发现,5-HT对猪早期胚胎发育是有影响的,那对胚胎形成的重要参与者---卵母细胞是否有影响呢?5-HT是否参与调控猪卵泡发育和卵母细胞成熟?这需要我们进一步探究。通过上述实验发现,5-HT会影响猪早期胚胎发育,因此我们进行如下实验,首先,用5-HT处理卵母细胞,检测卵母细胞的第一极体排出情况,发现5-HT能够在1000μM时抑制猪卵母细胞成熟。接下来,用ELISA方法检测了猪体内三种不同发育阶段大、中、小卵泡分泌的5-HT含量,结果显示,大中小不同卵泡内分泌的5-HT含量差异显著,大卵泡内5-HT含量约为50 ng/m L,此结果表明,猪卵泡内分泌5-HT,且不同发育阶段卵泡内分泌的5-HT含量不同,推测5-HT可能与卵泡发育具有相关性。最后,用荧光定量PCR方法检测猪颗粒细胞的增殖相关基因,和雌二醇合成相关基因,结果显示,5-HT能够促进猪颗粒细胞的增殖,抑制雌二醇的合成。经以上实验结果,推测5-HT可能通过降低颗粒细胞的雌二醇的合成进而抑制猪卵母细胞的成熟,进而推测5-HT会影响猪的卵母细胞成熟及颗粒细胞雌二醇的分泌,进而调控了猪的卵泡发育过程。以上结果为进一步研究5-HT与胚胎发育及卵泡发育的相关性奠定了基础,也为5-HT、雌性生殖及表观遗传三者的关系提供了一个新的视角。更多还原

【Abstract】 Effects of 5-hydroxytryptamine on porcine early embryodevelopment and epigenetic modification5-hydroxytryptamine(5-HT)is a neurotransmitter and growth factor,which functions by binding to its receptor.When mammals are stimulated and stressed,the secretion of 5-HT is disordered,which affects growth and development.In recent years,it has been found that 5-HT is widely distributed in mammalian female reproductive tissues,and plays important roles in cell proliferation,differentiation,follicle development and embryo development.Researches on 5-HT’s involvement in the regulation of mouse early embryo development has made significant progress.Are there 5-HT and receptors in porcine female reproductive system? Does 5-HT have an effect on embryo development and follicular development? Does 5-HT have a correlation with epigenetic modification of embryo development? These questions have not yet been explored.In order to solve the above problems,this study took porcine parthenogenetic embryos cultured in vitro as the main research object,and conducted the following experiments.Firstly,5-HT was added to porcine embryo culture medium to examine the effect of 5-HT on embryo development.And q PCR(fluorescent quantitative PCR technology)was used to explore the influence of 5-HT on the expression of 5-HT receptors during embryo developmental stages.The results showed that 5-HT decreased cleavage rate and blastocyst rate,reduced the total cell number of blastocyst and increased the number of apoptotic cells.It indicated that 5-HT inhibited the developmental ability and quality of porcine embryos.After 5-HT treatment,the expression of 5-HT2 A,5-HT4 and 5-HT7 receptors in porcine early embryos showed significant changes.The expression of 5-HT2 A and5-HT4 receptors was significant increased at the 2-cell stage,and markedly decreased at 4-cell and blastocyst stage,which was completely opposite to the expression of5-HT7 receptor.The m RNA level of 5-HT7 was significantly decreased at 2-cell and obviously increased at 4-cells and blastocyst stage.These results indicated that 5-HT may affect porcine early embryo development through 5-HT receptors.Secondly,q PCR was used to detect the expression of pluripotency genes and apoptosis genes in early embryos treated with 5-HT.The results showed that 5-HT treatment significantly reduced the expression of pluripotency gene NANOG and increased the expression of pluripotency gene SOX2 and the pro-apoptotic gene BAX,which indicated that 5-HT can affect porcine early embryo development by regulating gene expression.Finally,the effects of 5-HT on DNA methylation and histone modification were detected by immunofluorescence staining and q PCR.The results showed that 5-HT treatment did not influence DNA methylation.After 5-HT treatment,the levels of H3K4me2 and H3K4me3 were significantly decreased at the 2-cell stage,markedly increased at the 4-cell stage,and finally decreased at the blastocyst stage;H3K9Ac levels had no significant difference at 2-cell stage,but it was significantly increased at the 4-cell stage and obviously decreased at the blastocyst stage;H4K16Ac levels were significantly increased in the 2-cell and 4-cell stage,but it was significantly decreased at blastocyst stage.These results indicated that 5-HT treatment would affect the histone modification in the porcine early embryo development.Based on the above results,we speculate that 5-HT may regulate gene expression through 5-HT receptors,and affect epigenetic modification by influencing histone modifications,which in turn interferes porcine early embryo development.During embryo development,oocyte quality determines the developmental ability of embryos.In mammals,oocytes develop in follicle,and it gradually mature as the follicle grows and develops.During the development of follicles,the development and maturation of granulosa cells and oocytes occupies a major position.Based on the above results,5-HT affects porcine early embryo development.Consequently,whether oocyte,an important role in embryo development,participate in the regulation of porcine follicle development and oocyte maturation need us to further explore.To solve this problem,we conduct the following experiments.Firstly,oocytes were treated by 5-HT to detect the discharge of first polar body.The results showed that 5-HT could inhibit oocyte maturation at 1000 μM.Secondly,ELISA was used to examine the content of 5-HT secreted by large,medium and small follicles at three different developmental stages.The results suggested that there were significant differences in the content of 5-HT secreted by different follicles,and the content of5-HT in large follicles is about 50 ng/m L.Thus,porcine follicles can secret 5-HT,and the content of 5-HT in follicles at different developmental stages is significantly different,which made us speculate that 5-HT may be related to follicle development.Finally,q PCR was used to detection the genes related to the proliferation of porcine granulosa cells and estradiol synthesis.The results showed that 5-HT can promote the proliferation of porcine granulosa cells and inhibit the synthesis of estradiol.Based on the above experimental results,it is speculated that 5-HT may inhibit the maturation of porcine oocytes by reducing the synthesis of estradiol in granulosa cells.And we further speculate that 5-HT may affect the maturation of porcine oocytes and the secretion of estradiol in granulosa cells to regulate porcine follicle development.This study lays the foundation for further research on the correlation between5-HT,embryo development and follicle development,and also provides a new perspective for the relationship between 5-HT,female reproduction and epigenetics.更多还原