Gremlin1(GREM1) Affects Follicular Development By Regulating Cumulus Expansion

Ovarian reserve is a complex phenomenon refers to the quality and quantity of oocytes.Diminished ovarian reserve(DOR)is characterized by poor fertility outcomes,and it represents a major challenge in reproductive medicine.DOR may be a precursor to premature ovarian failure and menopause,however,the underlying mechanisms have not identified.Growth differentiation factor 9(GDF9)is an oocyte-expressed member of the transforming growth factor? superfamily and is required for cumulus expansion,normal ovarian follicle development and female fertility.As the downstream gene of GDF9,Gremlin1(GREM1)is a member of the family of bone morphogenetic protein(BMP)antagonists,and secreted by granulosa cells.GREM1 is an important regulatory molecule in the interaction between GDF9 and BMP signaling pathway.In young women with diminished ovarian reserve,the mRNA expression of GREM1 was down-regulated.For the study on the alterated expression of GREM in granulosa cells of DOR patients and the underlying mechanisms,the level of GREM1 protein level in granulosa cells of young women with DOR was detected and the effect of GREM1 on the expression of cumulus cells expansion-related genes was investigatedMethods:Clinical samples were collected from simple tubal infertility who received in-vitro fertilization(IVF)treatment in Women Hospital School Of Medicine Zhejiang University.According to the diagnostic criteria of American Society Of Reproductive Medicine(ASRM),these infertile women were divided into diminished ovarian reserve(DOR)group and normal ovarian reserve(NOR)group.Western blot and immunofluorescence were used to detect the GREM1 protein expression in the granulosa cells from these women.the primary granulosa cells were extracted and cultured from follicular fluid of these tubal infertility.siRNA and overexpression plasmid targeting GREM1 were designed and transfected into primary granulosa cells The protein expression of pentraxin 3(PTX3)?tumor necrosis factor alpha-induced protein 6(TNFAIP6)?prostaglandin synthase 2(PTGS2)and GREM1 in granulosa cells was measured by Western blot,to detect the effects of GREM1 silencing and overexpression on the cumulus expansion.Then we treated the primary granulosa cells with follicle-stimulating hormone(FSH)and GREM1 in the concentration of 0?0.05?0.5 and 1?g/mL to evaluate the effects of exogenous gremlin on the mRNA expression of PTX3,TNFAIP6 and PTGS2Result:The protein expression of GREM1 in DOR group was significantly down-regulated compared with NOR group.GREM1 silencing by siRNA transfection significantly decreased the protein expression of cumulus cells expansion-related genes.GREM1 overexpression by plasmid transfection significantly up-regulated the protein expression of cumulus cells expansion-related genes.Exogenous GREM1 protein induced PTX3,TNFAIP6 and PTGS2 mRNA expression,and the effect was does-dependent.Conclusion:The protein expression of GREM1 protein level in granulosa cells is related to the ovarian reserve.GREM1 is an essential positive regulator of follicular development and ovarian reserve.In this study,we revealed the key role of GREM1 in cumulus expansion during follicular development.GREM1 affects cumulus expansion as the downstream effector of GDF9 could be the potential mechanism of DOR.