The Role Of TGF-?1 In The Regulation Of Extracellular Matrix Associated Genes Expression In Human Granulose Cells

Transforming growth factor? 1(TGF-? 1)is indicated to play important roles in mammalian reproduction,such as affecting the maintenance of primordial follicle,the early folliculogenesis,granulose cell proliferation and differentiation,oocyte maturation and embryo implantation and the maintenance of pregnancy.Whether TGF-? 1 participates the regulation of proliferation and differentiation of human pre-ovulatory granulose cell is not clear.Human granulose cell is one of the important cell types in supporting the oocyte development by effect the process of follicle development,mature,releasing and atresia.In present study,we want to determine whether TGF-? 1 was involved in the proliferation of human granulose cell and the ovulation process.TGF-? 1 promoted the expression of PTX3 and TNFAIP6,which were human granulose cell matrix related gene,and the reflect an time-dependent and concentration-dependent attitude.AD AMTS-1 is a recently discovered a distintegrin and metalloprotease with thrombospondin motifs.ADAMTS-1 is found to take part in the ovulation process by cleavage of versican,AD AMTS-1 deficient can result in ovulation disorders.So we want to determine whether TGF-(3 1 participate the process of ovulation by regulate the expression of ADAMTS-1 and versican.Our study found that TGF-? 1 down regulated the expression of ADAMTS-1 in human granulose cells,and exhibited a concentration-dependent and time-dependent attitude.The inhibitory effect of TGF-(3 1 to ADAMTS-1 can partially restored when the human granulose cells cultured with LY364947,which was the inhibitor of TGF-? 1.TGF-? 1 up-regulated the expression of versican in human granulose in a concentration-dependent and time-dependent attitude,and the effect can be inhibited by the use of LY364947.In conclusion,our study proved that TGF-? 1 promoted the proliferation of human granulose cell and participated ovulation by regulating the expression of AD AMTS-1 and versican.Part I.TGF-? 1 promoted the expression of PTX3 andTNFAIP6 in human granulose cells.Objective:To research the regulation effect of TGF-? 1 on PTX3 and TNFAIP6 in human granulose cells.Methods:Twelve women underwent IVF-ET were chosen,granulose cells were extracted from follicular fluid and cultured in vitro.The granulose cell cultures were then treated with different concentrations of TGF?1(0,1,5 or 10ng/ml)or LY364947(luM)for 24h or TGF-? 1(5ng/ml)for 0,6,12,24 or 48h.The expression levels of PTX3 and TNFAIP6 were compared to determine whether there were differences between groups.Results:The expression levels of PTX3?TNFAIP6 mRNA and protein were increased in human granulose cells after con-cultured with TGF-? 1 for 24h,and revealed a concentration-dependent attitude.Compared with control group,the expression levels of PTX3?TNFAIP6 mRNA and protein of human granulose cells con-cultured with LY364947 for 24h were significantly decreased.The expression levels of PTX3?TNFAIP6 mRNA and protein in human granulose cells con-cultured with a fixed-concentration of TGF-? 1 at 5ng/ml were up-regulated in a time-dependent attitude.Conclusion TGF-? 1 signal pathway was involved in the regulation of the expression of PTX3 and TNFAIP6,and revealed concentration-dependent and time-dependent attitudes.Part ?.TGF-? 1 down-regulated the expression of ADAMTS-1 in human granulose cells.Objective:To research the regulation effect of TGF-? 1 on ADAMTS-1 in human granulose cells.Methods:Twelve women underwent IVF-ET were chosen,granulose cells were extracted from follicular fluid and cultured in vitro.The granulose cell cultures were then treated with different concentrations of TGF?1(0,1,5 or lOng/ml)or LY364947(luM)for 24h or TGF-?1(5ng/ml)for 0,6,12,24 or 48h.The expression levels of ADAMTS-1 were compared to determine whether there were differences between groups.Results:The results of western blotting analysis showed that the expression levels of ADAMTS-1 were significantly decreased when human granulose con-cultured with TGF-? 1,and exhibited a concentration-dependent and time-concentration attitude.The result of RT-PCR analysis showed that the ADAMTS-1 mRNA were down-regulated by TGF-? 1,also exhibited a concentration-dependent and time-concentration attitude.Conclusion TGF-? 1 signal pathway was involved ovulation process by the regulating the expression ADAMTS-1.Part ?.TGF-? 1 up-regulated the expression of versican in human granulose cells.Objective:To research the regulation effect of TGF-? 1 on versican in human granulose cells.Methods:Twelve women underwent IVF-ET were chosen,granulose cells were extracted from follicular fluid and cultured in vitro.The granulose cell cultures were then treated with different concentrations of TGF?1(0,1,5 or 10ng/ml)or LY364947(luM)for 24h or TGF-?1(5ng/ml)for 0,6,12,24 or 48h.The expression levels of versican were compared to determine whether there were differences between groups.Results:The results of western blotting analysis showed that the expression levels of versican were significantly increased when human granulose con-cultured with TGF-? 1,and exhibited a concentration-dependent and time-concentration attitude.The result of RT-PCR analysis showed that the versican mRNA were up-regulated by TGF-?1,also exhibited a concentration-dependent and time-concentration attitude.Conclusion TGF-? 1 up-regulated the expression of versican in human granulose cell and hence involved in ovulation process.