Study On The Regulatory Mechanism Of N-acetylcysteine On Uterine Receptivity In Early Pregnancy Of Goats

The uterus is an important place for mammals to give birth to new life,and improving the physiological function of the uterus is important for improving the reproductive efficiency of mammals.It was found that N-acetylcysteine(NAC),a natural small molecule antioxidant,could improve reproductive function,promote embryonic development,maintain normal pregnancy and suppress inflammation in animals by increasing the antioxidant activity of the animal organism.Preliminary research by our group found that NAC was beneficial in improving the reproductive performance of goats.Therefore,in this study,we used RNA-Seq technology to construct gene expression profiles and screen differentially expressed genes and signaling pathways in the uterine of Qianbei Ma goat in early pregnancy.In-depth analysis of the regulatory mechanism of NAC and differentially expressed genes on the reproductive performance of goat uterus using cell culture technology.The effects of NAC on the reproductive performance of Qianbei Ma goat and its molecular regulatory mechanism were investigated in vivo and in vitro,respectively.The main findings are as follows:1.Endometrial Stromal Cells(ESCs)of Qianbei Ma goat were successfully isolated and purified,and ESCs of Qianbei Ma goat were cultured using different concentrations of NAC.A combination of CCK-8,q RT-PCR,ELISA and flow cytometry were used to investigate the effects of NAC on the biological functions of ESCs in Qianbei Ma goat.The results showed that 200 μmol/L NAC may promote the proliferation of ESCs in Qianbei Ma goat by increasing the expression of PCNA,Cyclin D2(P < 0.01)and Cyclin E(P < 0.05)m RNAs.It also increased the cell viability(P <0.01)and SOD and GR activities(P < 0.01)of Qianbei Ma goat ESCs.In addition,200μmol/L NAC increased the mitochondrial membrane potential(P < 0.01)and the m RNA expression of HOXA10,PTGS2,PGR(P < 0.01)and LIF(P < 0.05)genes in ESCs.2.To investigate the effect of NAC on the expression pattern of related genes in the uterine horn of Qianbei Ma goat in early gestation.In this experiment,RNA-seq was performed after feeding Qianbei Ma goat with 0.07% NAC for 35 days.The results showed that 531 and 787 differentially expressed genes were enriched in the control and experimental groups,respectively.GO functional analysis showed that the identified differential genes were mainly involved in cell activation,cytokine production,cell mitotic processes and angiogenesis pathways.KEGG enrichment analysis revealed that these differential genes were enriched in signaling pathways related to reproductive regulation,immune regulation,anti-oxidative stress,and cell adhesion.In addition,we found that the most differentially expressed genes were enriched in the PI3K/Akt signaling pathway.We also screened FN1,CSF-1,CSF-1R,BDNF,EREG,IFNAR1,IGF1,ITGAM,ITGAV,ITGA1,ITGA5,ITGB1,ITGB2,KRAS,LAMC1,and VEGFC genes are expected to be key genes for NAC regulation of early gestation in Qianbei Ma goat.3.To further understand the regulatory mechanism of the differentially expressed genes screened by RNA-seq on the reproductive performance of the uterus of Qianbei Ma goat.We selected colony-stimulating factor 1(CSF-1)for our study.The q RT-PCR results showed that the expression of CSF-1 gene was higher in hypothalamus,pituitary and uterus in the test group than in the control group,except for ovary and fallopian tube(P < 0.01).Bioinformatics analysis revealed that goat CSF-1 protein is a relatively unstable and signal peptide hydrophilic secretory protein mainly composed of irregularly coiled and α-helices,which has multiple interactions with its receptor protein CSF-1R.The amino acid sequence encoded by the CSF-1 gene of goat was more than 65% homologous to the remaining nine species;it was the closest genetic distance to sheep and cattle,and relatively distant from the remaining species.In addition,both the double digestion and sequencing results indicated that the p EGFPC1-CSF-1 eukaryotic expression vector was successfully constructed,which laid the foundation for further investigation of CSF-1 gene function in cells.4.To elucidate the specific regulatory mechanism of the cytokine CSF-1 on the reproductive performance of the uterus of the Qianbei Ma goat.The recombinant plasmids were introduced into the ESCs of Qianbei Ma goat as a model to detect their effects on cell proliferation and gene expression.The results showed that overexpression of CSF-1 gene in the ESCs of Qianbei Ma goat may promote cell proliferation(P < 0.01)by increasing the expression levels of PCNA,Cyclin D2,Cyclin E and Cyclin D1 m RNA(P < 0.01).In addition,the expression levels of HOXA10,LIF and PGR gene m RNAs were increased(P < 0.01)after upregulating the expression level of CSF-1 gene in the ESCs of Qianbei Ma goat.In summary,NAC may establish and maintain pregnancy by regulating signalling pathways and gene expression associated with the goat uterus in early gestation,and may mediate the establishment of endometrial receptivity in goats by increasing the antioxidant capacity and cell viability of goat ESCs,improving mitochondrial function and promoting cell proliferation.