Regulatory Mechanisms Of Interleukin-6 On Ovulation-related Genes Expression In Ovarian Granulosa Cells Of Dairy Cows

Fertility in mammals depends on the coordinated execution of multiple events within the fully grown ovarian follicle at the time of ovulation.Inflammatory cytokines play an important regulatory role in follicular development and ovulation.In addition,inflammatory cytokines expression or changes in activity can lead to prolonged ovarian lifespan and ovulation failure when interfere with specific cytokines antagonists or in gene knockout animals model.Regulation of the expression of ovulation-related genes in granulosa cells is the primary pathway of inflammatory cytokines to regulate ovulation.However,the current research mainly focused on IL-1? and TNF-?.Many studies have found that IL-6 plays a more complex role than other inflammatory cytokines in the ovulation.We hypothesize that this is related to the biological characteristics of IL-6.It also suggests that it may play a central role in regulating inflammation-related ovulation.However,little is known about the biological regulation of IL-6 during ovulation.In this study,we investigated the regulatory effect of IL-6 on the expression of EGFR,VEGF,NOS,StAR and TIMPs in primary cultured ovarian granulosa cells of dairy cows by using RT-qPCR,western blotting,ELISA and immunofluorescence technique.In addition,we also identify the mechanisms underlying the effect of IL-6 on the expression of these ovulation-related genes.The results showed that:(1)EGFR is highly expressed in granulosa cells derived from large follicular(diameter>8mm).The expression level of EGFR mRNA was higher after treatment with FSH than that of LH.It not only significantly reduced the expression of EGFR mRNA and protein,but also inhibited the phosphorylation of ERK1/2 protein induced by BTC in granulosa cells after treatment with high concentration of IL-6(10ng/ml).The inhibitory effect of IL-6 on EGFR expression is mainly achieved by activating ERK1/2 and PI3K/AKT signaling pathways,which can be reversed by ERK1/2 and AKT inhibitors.This part of the study show that IL-6 inhibits the expression and function of EGFR in mRNA transcription,protein translation and biological activity levels,respectively.This inhibitory effect of IL-6 is mediated predominantly through the ERK1/2 and PI3K/AKT pathways.(2)High concentration of IL-6 can significantly up-regulate FSH-induced VEGF gene and protein expression levels in granulosa cells,and also promote the VEGF upstream regulators HIF-1? and COX2 mRNA expression.VEGF gene and protein expression levels were significantly decreased after specifically blocking HIF-1? and COX2 by using inhibitors.The up-regulation effect of IL-6 on expression of VEGF in granulosa cells mainly through activating the JAK/STAT3 signaling pathway,which can be impaired by JAK inhibitors.This part of the study shows that IL-6 can promote the synthesis of VEGF in granulosa cells by increasing the expression of HIF-1? and COX2.This promoting effect is related to activation of JAK/STAT3 signaling pathway.(3)IL-6 and its receptor IL-6R? are highly expressed in granulosa cells derived from large follicles.High concentration of IL-6 could down-regulate the StAR mRNA expression and progesterone production levels in granulosa cells,but had no significant effects on P450 scc and 3?-HSD expression.IL-6 down-regulates StAR expression by activating the ERK1/2 signaling pathway,which is reversed after ERK1/2 inhibitors treatment.This part of the study shows that IL-6 may inhibit the progesterone synthesis by down-regulating the expression of StAR.ERK1/2 signaling pathway involved in the regulation of IL-6 on StAR expression in granulosa cells.(4)High concentration of IL-6 can significantly increase the expression of iNOS and NO synthesis in granulosa cells,but had no significant effects on eNOS expression.The NO synthesis level was significantly reduced after specific blocking iNOS.IL-6 can up-regulate iNOS expression by activating the JAK/STAT3 pathway,which can be attenuated by the use of JAK inhibitors.This part of the study shows that IL-6 can promote the synthesis of NO in granulosa cells by up-regulating iNOS gene and protein expression.The JAK/STAT3 signal transduction pathway mediates this biological effect of IL-6.(5)High concentration of cryptotanshinone(10?M)can significantly down-regulate IL-6-induced expression of TIMP2 in granulosa cells.IL-6-induced phosphorylation of ERK1/2 and STAT3 proteins in granulosa cells were significantly reduced after the intervention of cryptotanshinone at 5,15 and 30 min.In addition,cryptotanshinone can also down-regulate the activation level of STAT3 pathway upstream protein JAK2.Cryptotanshinone reduces the expression of TIMP2 mainly by inhibiting the activation of JAK/STAT3 pathway,and the JAK/STAT3 inhibitor can reverse this inhibitory effect.This part of the study shows that cryptotanshinone play a regulatory role mainly through the inhibition the phosphorylation of downstream proteins of IL-6 receptor signaling pathway.Cryptotanshinone inhibits IL-6-induced expression of TIMP2 in granulosa cells by inhibiting the phosphorylation of STAT3 protein.This can not only clarify its role in the treatment of ovulation disorders at the molecular level,but also further validates the important roles of IL-6 in regulating ovulation process.In conclusion,this study reveals the biological function at the different levels and aspects of IL-6,which is an important inflammatory cytokine present in the local follicles.It is not only conducive to in-depth understanding of the intrinsic mechanism of ovarian cycle activity,but also provides a new way of intervention and theoretical basis for clinical to control and treatment of ovulatory disorders.