Expression Regulation And Function Of TFPI2 Gene In Chicken Ovarian Granulosa Cells

The chicken ovary is a unique and dynamic organ in the reproductive system.The degree of tissue development and remodeling is rapid and extensive,and repeats periodically.The development and ovulation of the ovary are also affected by follicular inner membrane cells and granular cells(Granulosa Cells,GCs)and other related secretory factors.Tissue factor path way inhibitor-2(TFPI2)was originally a 30-36 kDa glycoprotein isolated from human placental tissue.Early studies found that it is abundantly present in the placenta,seminal plasma,seminal vesicles and follicular fluid,and can affect the extracellular matrix(ECM)metalloproteinases(MMPs)to affect the remodeling of tissues,which indicates that TFPI2 is involved in the reproductive development of animals.It plays an important role in chicken ovarian GCs,but its expression regulation in chicken ovarian GCs is still unclear.According to the preliminary project team’s RNA-seq data of ovarian tissues at different physiological stages in the pre-laying period(15W),pre-laying period(20W),peak period(30W)and late-laying period(68W),the TFPI2 gene was found.There are significant differences in expression at different physiological stages during the reproductive period.Therefore,this study mainly used Hy-Line laying hens as the research object to carry out molecular biology research on the TFPI2 gene,and explored the expression patterns of TFPI2 in the follicles of different developmental states of sexually mature chickens.Research on its regulation and function in ovarian GCs.Mainly obtained the following research results:Experiment 1.Tissue expression analysis,full-length cloning and bioinformatics analysis of TFPI2 geneqRT-PCR was used to verify the data,and the results were consistent with RNA-seq data;tissue expression results showed that TFPI2 gene was highly expressed in ovarian tissue.The full-length sequence of TFPI2 gene was obtained by race technology,including 55 bp 5‘untranslated region(5’ UTR),975 bp 3’ untranslated region(3’ UTR),720 bp protein coding region(CDS)and 240 amino acid residues.The phylogenetic tree showed that TFPI2 was conserved in poultry.Experiment 2.The expression and distribution of TFPI2 in different developmental follicles of ovaries of sexually mature layersqRT-PCR was used to detect the expression of TFPI2 mRNA in different grades of follicles.The results showed that the expression of TFPI2 mRNA in granular layer was significantly higher than that in membranous layer(P<0.05).and the difference of TFPI2 mRNA in 4-6 mm and 9-12 mm granular layer before follicle selection was significant(P<0.05);in post ovulation(POF)follicles,the expression of TFPI2 mRNA in granular layer was significantly higher than that in membranous layer(P<0.05).The expression of TFPI2 mRNA in healthy follicles was significantly higher than that in atresia follicles(P<0.05).The polyclonal antibody against TFPI2 protein was successfully prepared and Western blotting was performed The expression of TFPI2 protein in different developmental follicles was detected by blot.It was found that the expression of TFPI2 protein in granulosa layer was higher than that in membranous layer in pre grade follicles.The results of cellular immunofluorescence showed that TFPI2 protein was located in cytoplasm.The results of immunohistochemistiy showed that TFPI2 protein was located in ovarian stroma,primordial follicles,membranous and granular layers of 6-8mm and FI follicles,post ovulatory follicles,healthy and atresia follicles in stromal cells.Experiment 3.Study on the regulation of TFPI2 on the function of chicken ovarian granulosa cellsIn order to study the biological function and expression regulation mechanism of TFPI2 gene,we successfully constructed the overexpression vector TFPI2-pCDNA3.1-EGFP containing the target gene TFPI2,and designed the siRNA interference fragment according to the CDS sequence of TFPI2 gene.Overexpression of TFPI2 in Prehierarchical granulosa cells(PhGCs)significantly decreased the expression of FSHR(P<0.05),promoted cell proliferation,inhibited cell apoptosis,and inhibited the expression of MMPs.Overexpression of TFPI2 in Preovulatory granulosa cells(PoGCs)significantly decreased the expression of FSHR(P<0.05),but had no effect on cell proliferation,apoptosis and MMPs.However,TFP12 overexpression significantly decreased steroid hormone synthesis in both PhGCs and PoGCs(P<0.05).The TFPI2 mRNA expression in ovary of 28w,36W and 43W local layers(Gushi layers)in Henan Province was detected.The results showed that the TFP12 mRNA expression in ovary of 28w,36W and 43W local layers was significantly higher than that in high yield group(P<0.05),36W and 43W local layers was significantly higher than that in high yield group(P<0.01),and 43W local layers was significantly higher than that in low yield group(P<0.01).The mRNA expression in low yield group was higher than that in high yield group,but the difference was not significant.Experiment 4.Effect of exogenous hormone(FSH/LH)on TFPI2 gene expression in chicken ovarian granulosa cellsFSH and LH(dose:0,5,10,25,50.100 ng/ml)could significantly promote the expression of TFPI2 gene and protein in PhGCs and PoGCs(P<0.05),but there was no dose-dependent effect.When granulosa cells were treated with intermediate concentration of 25 ng/ml at different time gradients(3,6,12.24 h),the results showed that TFPI2 gene and protein expression in PhGCs was significantly increased.The expression of TFPI2 mRNA increased at first(the highest at 6h)and then decreased,and there was no regular change in PhGCs;the expression of TFPI2 gene and protein in PoGCs could be significantly inhibited by LH treatment before grade(P<0.05),but it could be significantly promoted in PoGCs(P<0.05);The middle concentration(25ng/ml)was used to treat granulosa cells at different time gradients.The expression of TFPI2 mRNA in PhGCs and PoGCs increased at first(the highest at 6 h)and then decreased.FSH+LH at an intermediate concentration(25ng/ml)had no effect on TFPI2 mRNA expression in pre grade granulosa cells after 24h.The expression of TFPI2 mRNA was significantly increased in PoGCs(P<0.05),while the expression of TFPI2 mRNA was first increased(the highest at 6h)and then decreased at different time gradients in PhGCs and PoGCs.FSH alone and FSH+LH co-treatment of granulosa cells can promote the expression of steroid hormone synthesis related genes(STAR,CYP17A1,CYP11A1,3B-HSD,etc.).In addition,FSH and LH can regulate TFPI2 gene expression by inducing different pathways(PKA,PKC,PI3K,mTOR,etc.).These results indicate that TFPI2 is expressed in different follicles,and the main target of TFPI2 is granulosa cells.TFPI2 can affect follicular development by regulating the proliferation and differentiation of granulosa cells,the synthesis of steroid hormones and reducing the dissolution of ECM.In addition,FSH and LH can induce different pathways to regulate the expression of TFPI2.These results provide a basis for the identification of TFPI2 gene in chicken.It provides theoretical and experimental basis for follicular development and molecular regulation mechanism of follicular maturation and ovulation.