Effects Of WT1 On Progesterone Synthesis Of Hen Granulosa Cells From Pre-ovulatory Follicles

WT1,encoding a nuclear transcription factor containing four zinc fingers,is expressed in various issues and organs of animals and plays an important role in many physiologic processes.It not only regulates tumorigenesis but also mediates normal development of issues or organs.There are two isoforms(+KTS,-KTS)that execute different functions in dissimilar cells or development stages.Progesterone(P4),secreted by granulosa cells(GCs),takes part in ovarian physiologic processes,and is indispensable for females to maintain normal reproductive performance.But effects of WT1(+/-KTS)on P4 biosynthesis of hen GCs remain unclear.The purpose of this research is to descript the influences of WT1(+/-KTS)on P4 synthesis of GCs from hen pre-ovulatory follicles.We detected the WT1 m RNA expression in GCs from different stage follicles,and evaluated its expression tendency by RT-q PCR.GCs from pre-ovulatory follicles firstly transfected with WT1(+/-KTS)overexpression vectors,then incubated with recombinant human follicle-stimulating hormone(rh FSH).Cell supernatants were collected to measure P4 concentrations by radioimmunoassay(RIA),and the m RNA expression of follicle-stimulating hormone receptor(FSHR)and steroidogenic enzyme(STAR,HSD3B1 and CYP11A1)was detected using RT-q PCR.The protein activities of ERK1/2,BRAF and CREB were determined using western blotting(WB).The major work and results are as follow:(1)The amounts of WT1 m RNA were great higher in pre-hierarchical follicles than in pre-ovulatory follicles,and maintained at a very high levels in pre-hierarchical follicles.The m RNA expression of WT1 increased first and then decreased,and was up to maximum at stage of large white follicles(LWF).(2)GCs from pre-ovulatory follicles treated with 0,30,50,100,150 ng/m L rh FSH,respectively.P4 concentrations dramatically increased at 50 ng/m L,so it was applied to further experiments.(3)GCs were treated with pc-DNA3.1,WT1(+KTS)or WT1(-KTS)overexpression vectors,respectively,and then incubated with 50 ng/m L rh FSH.Overexpression efficiencies were detected by RT-q PCR and WB.Results indicated that the overexpression efficiencies were enough for the further experiments,and WT1(+KTS)and WT1(-KTS)are remarkably reduced FSH-induced P4 secretion.(4)Without treatment with FSH,WT1(+KTS)remarkably decreased m RNA expression of STAR,HSD3B1 and CYP11A1,but had no significant effects on basal m RNA expression FSHR.WT1(-KTS)had no notable effects on basal m RNA expression of FSHR,STAR and HSD3B1,but significantly decreased basal m RNA expression of CYP11A1.Treating with FSH,WT1(+KTS)dramatically declined FSH-induced m RNA expression of FSHR,STAR and CYP11A1,but apparently increased FSH-induced m RNA expression of HSD3B1.WT1(-KTS)dramatically reduced FSH-induced m RNA expression of FSHR and CYP11A1,but not apparently provoked the changes of FSH-induced m RNA expression of STAR and HSD3B1.(5)The phosphorylation levels of ERK1/2 were suppressed by WT1(+/-KTS),but the m RNA and total protein levels of ERK1/2 were not significant changed.At the same time,the phosphorylation levels of BRAF were also declined.(6)WT1(+/-KTS)overexpression suppressed total CREB protein expression,the phosphorylation levels of FSH-induced CREB were simultaneously decreased in various degree.Collectively,WT1(+/-KTS)suppressed CREB activity via ERK1/2 signaling,which significantly changed expression of FSHR and steroidogenic genes,and finally inhibited P4 synthesis of granulosa cells from pre-ovulatory follicles.