Effects Of 5-hydroxytryptamine On Porcine Early Embryo Development And Epigenetic Modification

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.