The Effect Of SPP1 Gene And Its DNA Methylated Modification On Embryo Implantation And Placental Development At Early Pregnancy In Goats

Embryonic implantation is an vital process of placenta formation,attaching with endometrium and establishing structural connection,achieved material exchange between fetus and mother.In ruminant,their placenta showed the multiple cotyledons component of villus trophoblast and caruncle of uterus and belong to prolapsed placenta,in other words,the trophoblastic epithelium will not invade the uterine stroma,only with the endometrial epithelial cells fusion to achieve maternal-fetal material exchange.Embryonic implantation is the precondition for maternal-fetal material exchange,placental development and high quality fetuses,which has become the focus of current research.In the early stage,we sequenced the placental transcriptome sequencing at the 20 d and 30 d pregnancy of Dazu Black Goat,and screened out the gene Secreted phosphoprotein 1(SPP1),which plays a crucial part in the immune regulation,bone development,embryo attachment,placental development and other physiological processes.In our study,we also found that the polymorphism of the SPP1 gene is related to the placental efficiency and lambing number of goats.However,the role of SPP1 in embryo implantation and placental development at early pregnancy in goat is still unclear.In human and mouse studies,it has been found that DNA methylation and DNA methylation-related enzymes play a crucial epigenetic part in immune response,embryo implantation and placental development,and the degree of methylation in gene promoter regions leads to transcriptional changes,thereby affecting gene expression and function.However,the regulation of SPP1 gene by DNA methylation embryo implantation and placental development at early pregnancy of goat has not been reported.5.In this study,the early pregnancy placentas of Dazu Black Goat were used as the research object.(1)Through the hematoxylin-eosin staining(H&E)to test the structure of the goat early pregnancy placentas and periodic acid-Schiff staining(PAS)was used to detect the goat placental trophoblastic cells at early pregnancy type and distribution,so as to explore the structure of goat placenta tissue and the degree of maternal-fetal attachment at early pregnancy;(2)SPP1 gene of Dazu Black Goat was cloned,and the gene was analyzed and predicted by comprehensive bioinformatics;(3)Immunohistochemistry,RT-qPCR and Western blot were used to detect the distribution and expression pattern of SPP1 protein in the placenta of goats during early pregnancy to explore the biological characteristics and expression pattern of SPP1;(4)The expression patterns of major SPP1 receptor integrin families(ITGB1,ITGB3,ITGB5,ITGA2,ITGA3,ITGA5,ITGAV),CD44 and Wnt5 a were detected by RT-qPCR,and the dynamic expression patterns of related genes at early pregnancy of goat were constructed;(5)MS-PCR was used to detect the methylation status of the promoter of SPP1 gene in the placenta of goats at different pregnancy periods,and the expression levels of SPP1 and its receptor genes were correlated with the expression levels of DNA methylation related genes DNMTs and TETs,to explore the correlation between SPP1 and its receptor and DNA methylation related genes;(6)The effects of DNA methylation on SPP1 gene in trophoblast cells were further investigated by treating immortalized trophoblast cells with DNA methylation transferase inhibitor 5-Aza,and the effects of 5-Aza inducing SPP1 expression in trophoblast cells on cell function were analyzed,so as to explore the mechanism of DNA methylation regulating SPP1 gene in the early pregnancy placentas of goat.The conclusions of this study were as follows:1.At the beginning of embryo attachment,trophoblast giant cells form and continue to exist in the trophoblast epithelium,and participate in placental cell fusion,invasion and migration.2.Successfully cloned the Dazu Black Goat SPP1 gene sequence,which has a high level of transcription and translation in the placenta tissue,and is expressed in the nucleus and cytoplasm.3.SPP1 protein is widely distributed in fetal placental trophoblast and maternal placental uterine caruncle at early pregnancy.Its expression is regulated by DNA methylation and could affect the expression of integrin family-related receptor genes.4.The SPP1 promoter region in goat placenta was partially methylated at early pregnancy.5-Aza inhibition of DNMT1 and DNMT3 b activity could regulate the methylation level of SPP1 promoter region,thereby affecting trophoblast cells invasion or placental development.