| In mammals,granulosa cells(GCs)are essential for follicular growth and ovulation.Interleukin(IL)-6 is a cytokine secreted by the ovary granulosa cells and participates in follicular development and ovulation through autocrine and paracrine forms.IL-11 belongs to the IL-6 cytokine family and plays a crucial role in the decidualization of the uterus through its receptor(IL-11Rα).However,the regulatory roles of IL-11/ IL-11Rα in bovine GCs are still unclear.Therefore,in this study,the effects of IL-11/IL-11Rα on the proliferation,steroidogenesis and prostaglandin synthesis of GCs were investigated by culturing bovine ovarian primary granulosa cells in vitro,flow cytometry,Immunofluorescence(IF),Immunohistochemistry(IHC),q RT-PCR,Western blot and ELISA,aiming to elucidate the regulation of IL-11/ IL-11Rα on follicular development and ovulation.The main results of this study are as follows:1.The expression of IL-11/IL-11Rα in bovine tissues and its localization in GCsThe heat,liver,spleen,lung,kidney,brain,stomach,intestine,uterus,ovary and muscle tissues of healthy dairy cows were collected for q RT-PCR analysis,the results showed that IL-11/IL-11Rα were high expression in ovary of dairy cows.Immunohistochemistry results showed that IL-11/IL-11Rα were present in all atages of follicular development,and highly expressed in GCs.Immunofluorescence results further showed that IL-11,IL-11Rα and glycoprotein 130(gp130)were localized in the cytoplasm of GCs.The IL-11 concentration and the m RNA expression levels of IL-11 and IL-11Rα in large follicles GCs were higher than that in small and medium follicles.2.Effects of IL-11Rα knockdown on proliferation,apoptosis and steroidogenesis of bovine GCs(1)Effects of IL-11Rα knockdown on proliferation and apoptosis in bovine GCsThe bovine GCs were transfected with IL-11Rα si RNA(si IL-11Rα)for 48 h.The results showed that compared with the negative control group(NC),the m RNA expression and protein expression of si IL-11Rα were decreased by 85.64 % and41.05 %,respectively.Transfection for 24 h and 48 h,CCK-8 results showed that the cell viability in si IL-11Rα group significantly decreased by 34.85% and 64.15%,respectively.Transfection for 48 h,q RT-PCR results showed that IL-11Rα knockdown induced G1 phase cell cycle arrest by significantly reducing the m RNA expression of cell cycle related genes CCND1 and CCNB1.The expressions of BAX and BAX/BCL-2 were significantly up-regulated,and the expression of BCL-2 was down-regulated,which significantly induced GCs apoptosis.Western blot results showed that IL-11Rα knockdown significantly reduced the protein expressions of PCNA and CCND1,as well as the phosphorylation levels of protein kinases of JAK1,STAT3 and m TOR related to cell proliferation and apoptosis.(2)Effects of IL-11Rα knockdown on steroidogenesis in bovine GCsThe bovine GCs were transfected with si IL-11Rα for 48 h,the results showed that IL-11Rα knockdown significantly reduced the secretion of estradiol(E2)and progesterone(P4)at the basal levels and forskolin(FSK)-induced levels,as well as the m RNA expression of CYP19A1,CYP11A1,St AR and HSD3B1 related to steroidogenesis.Meanwhile,the protein expressions of CYP19A1 and St AR were also significantly decreased and decreased the phosphorylation levels of PKA substrates,CREB,ERK1/2 and P38 MAPK.3.Effects of IL-11 on proliferation,apoptosis and prostaglandin synthesis in bovine GCs(1)Effects of IL-11 on proliferation and apoptosis in bovine GCsThe bovine GCs were treated with 0,10 ng/m L IL-11 for 48 h.CCK-8 results showed that the cell viability of IL-11 treatment group significantly increased by52.48%.q RT-PCR results showed that the IL-11 treatment group significantly up-regulated the m RNA expressions of PCNA,CCND1,CCNB1 and BCL-2,and significantly down-regulated the expressions of BAX and BAX/BCL-2.(2)Effects of IL-11 on prostaglandin synthesis in bovine GCsThe bovine GCs were treated with 0,1,5,10 ng/m L IL-11 and 100 ng/m L luteinizing hormone(LH)for 48 h.The results showed that IL-11 significantly increased the secretion of prostaglandin E2(PGE2)and the m RNA expressions of PLA2G4 A,PTGS2 and PTGES related to PGE2 synthesis in the basal group and LH-induced group,and significantly increased the protein expression of PTGS2.In addition,Western blot results showed that the phosphorylattion protein expression of JAK1 and STAT3 were the strongest at 30 min of IL-11 treatment.(3)The effects of STAT3 inhibitor on IL-11 mediated prostaglandin synthesisThe bovine GCs were pretreated with STAT3 inhibitor C188-9 for 2 h,and then treated with 0,10 ng/m L IL-11 and 100 ng/m L LH,the results showed that C188-9significantly reduced the phosphorylattion levels of STAT3 and attenuated IL-11-induced p-STAT3 nuclear translocation.Meanwhile,C188-9 significantly reduced PGE2 secretion and PTGS2 expression induced by IL-11.In summary,IL-11/IL-11Rα significantly promote the proliferation of bovine GCs and inhibit apoptosis through JAK1/STAT3 and m TOR pathways,regulate the expression of steroidogenesis through PKA-CREB,P38 MAPK and ERK1/2 signaling pathways,and active the JAK1-STAT3 pathway to promote PGE2 synthesis.This study preliminarily elucidates the role and mechanism of follicular development by IL-11/IL-11Rα,provides a new insight of folliculogenesis and ovulation of dairy cow,then,lay the foundation for the development of novel ovulatory induction technologies. |
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