The Role Of TGF-?1 In The Regulation Of Ovulation-related Genes In Humuan Granulosa Cells

Objective Ovulation is a LH-induced,precisely and neatly controlled process,allowing the release of a fertilizable oocyte.After LH surge,the cumulus oocyte complex(COC)undergoes a dramaticly change including restart of oocyte meiosis,reconstruction of the extracellular matrix of COC,and expansion of COC.In addition to gonadotropins,local autocrine/paracrine factors also play an important regulatory role during this process.Transforming growth factor ?1(TGF-?1)belongs to the transforming growth factor-? superfamily.It is highly expressed in human oocytes and granulosa cells and is involved in the regulation of follicular development and ovulation.It is an important autocrine,paracrine factor.There are few studies on the regulation role of TGF-?1 on ovulation-related genes,but largely still are unknown.The objective of our study is to explore the regulatory effects and mechanisms of TGF-?1 on ovulation-related genes,especially those related to cumulus expansion in human granulosa cells.Therefore,to provide more information for understanding the physiological regulation of ovulation in human.Methods In the first part,human primary luteinized granulosa cells(hGL)and human immortalized granulosa cell lines(SVOG cell lines)were used to study the mechanism of TGF-?1 in regulating hyaluronan synthesis.We use RT-qPCR,Western Blot,siRNA to investigate the expression of target gene or protein.The level of hyaluronan in the culture medium was detected by enzyme-linked immunosorbent assay.In the second part,hGL and SVOG cell lines were used to study the mechanism of TGF-?1 in regulating the expression of vitamin D receptor(VDR).The same cell models were usd to study the role of vitamin D in regulation of cyclooxygenase 2(COX-2)and prostaglandin E2(PGE2)synthesis.We further studied the synergistic effect of TGF-?1 and vitamin D on PGE2 production in human ovarian granulosa cells.Results Part 1:TGF-?1 promoted hyaluronan synthesis by upregulating hyaluronan synthases 2(HAS2)in human granulosa cells.Using both primary and immortalized hGL cells as study models,we provided the first data showing that TGF-?1 significantly promoted the synthesis of hyaluronan by upregulating the expression of HAS2 in these cells.Additionally,using dual inhibition approaches,we showed that the TGF-? type ?(T?RI? receptor and TGF-? type ?(ALK5)receptor are functional receptors that mediate stimulatory effects in response to TGF-?1.Moreover,we found that the canonical SMAD2/SMAD3-SMAD4 signaling pathway is the principal intracellular signaling pathway that upregulates the expression HAS2 and subsequent hyaluronan synthesis.Notably,we showed that SNAIL transcription factor is a critical molecule mediating the TGF-? signaling,which contributes to the increase in hyaluronan synthesis.These results of our in vitro studies suggest that intraovarian TGF-?1 plays a functional role in the local regulation of hyaluronan synthesis in hGL cellsPart 2:In this study,we showed that TGF-?1 significantly promoted the expression of VDR,and this stimulatory effect occurred through the ALK5 type I receptor-mediated SMAD3 and ERK1/2 signaling pathways in hGL cells.Additionally,we showed that Vit D increased the expression of COX-2 and the synthesis of PGE2 in a time-and dose-dependent manner.Furthermore,we demonstrated a synergistic effect of TGF-?1 and Vit D on the expression of COX-2 and synthesis of PGE2,and this effect could be attenuated by silencing the expression of VDR.Conclusion TGF-?1 promotes hyaluronan synthesis by upregulating the expression of HAS2 in human ovarian granulosa cells through SMAD2,SMAD3 and SNAIL pathways.TGF-?1 regulates VDR expression in human ovarian granulosa cells through SMAD3 and ERK1/2 signaling pathways.Vitamin D induces COX-2 expression in ovarian granulosa cells and promotes PGE2 synthesis.TGF-?1 can synergistically promote vitamin D-induced COX-2 expression and PGE2 production by up-regulating VDR expression.TGF-?1 plays an important role as a autocrine/paracrine factor in regulation of COC expansion in the ovary.