Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells

<正>Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction.However, its expression and function in chickens are still unclear.In this study, RNA-seq was performed on ovarian tissues from chickens aged 30 and 15 weeks to identify the differentially expressed gene TFPI2.The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs.In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT-PCR) and Western blotting.Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at differentdevelopmentalstates,suchasprimordialfollicles,theovarianstroma,andthegranulosaandtheca layers of prehierarchical follicles (6–8 mm) and preovulatory follicles (F1).In vitro, follicle stimulating hormone orluteinizinghormone(FSH/LH)stimulatedtheexpressionofTFPI2 in chicken granulosa cells.FSH-/LHinduced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways.Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes(STAR,Cyp17a1,Cyp19a1and3B-HSD)aswellasMMPs(MMP2,7,9and11).Mechanistically,TFPI2inhibitedtheexpressionofMMP7via the Wnt signaling pathway.These findings indicate thatTFPI2mayplay an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.