Research Of WT1 Gene On Bovine Granulosa Cells And Theca Cells Steroidogenesis

WT1 encodes a zinc finger transcription factor that plays important roles in tumorigenesis,the maintenance of tissue homeostasis,the regulation of organ development and so on.Steroidogenesis is a key biological process for determining the development and atresia of mammalian follicles,and it can effect follicular development and the quality of oocyte.As the main steroid secreting cells in the follicle,granulosa cells(GCs)and theca cells(TCs),which synthesize estrogen,progesterone and androgens,have key roles during follicular growth and atresia.WT1(+KTS)and WT1(-KTS)are two alternative splicing variants of WT1,and the variants play different roles in gonad development.It has been known that the downregulation of WT1 can regulate the early differentiation of GCs in murine preantral follicles and cause follicular dysplasia.However,the relationship between the alternative splicing isoforms of WT1,WT1(+KTS)and WT1(-KTS),and the regulation of steroidogenesis in GCs and TCs is not clear.In addition,the influence of WT1 on follicle development in domestic animals remains unclear.In this study,we revealed the expression patterns of WT1 in GCs and TCs at different stages of bovine antral follicle development.In addition,we studied the effects of WT1 on GCs and TCs steroidogenesis and further elaborated on PI3K/AKT and MAPK/ERK signaling pathways involved in this process in GCs.The main results are listed as follows:(1)We detected the expression of WT1 and steroidogenic genes in healthy antral follicles of small(2-4 mm),middle(4-8 mm),large(>8 mm)and in small atretic antral follicles.In addition,small healthy follicular GCs and TCs were used as in vitro culture models,and we examined the localization of WT1 in GCs and TCs.The results indicate that the expressions of WT1 in healthy small and middle follicular GCs were higher than in large follicular GCs;the expression of WT1 in healthy large follicular TCs was higher than in middle follicular TCs,and the expression of WT1 was the lowest in small follicular TCs;the expression of WT1 in GCs in healthy follicles was higher than that in atretic follicles;The WT1(+KTS)and WT1(-KTS)ratio was unchanged.The WT1 protein is expressed in the nucleus and cytoplasm of GCs and TCs but is mainly expressed in the nucleus in GCs;however,it is uniformly expressed in the nucleus and cytoplasm in TCs.The levels of estradiol(E2)and progesterone(P4)in healthy large follicular fluid,the mRNA expression levels of the steroidogenic genes CYP19A1,3?-HSD,CYP11A1,and the gonadotropin receptors LHR and FSHR in healthy large follicular GCs were higher than in small and middle follicle,the mRNA expression of STAR was lower than in small and middle follicle.The levels of E2 in the follicular fluid and the mRNA expression of CYP19A1 and FSHR in the GCs of healthy follicles were higher than those in atretic follicles,and the levels of P4 in the follicular fluid and the mRNA expression of STAR and 3?-HSD in GCs in healthy follicles were significantly lower than those in atretic follicles.(2)Healthy small follicular GCs were used as in vitro culture models,and we studied the effect of the downregulation of WT1 or the upregulation of WT1(+/-KTS)on steroidogenesis in GCs.The results show that downregulation of WT1 promoted the basal and FSH-induced secretion of P4 and the mRNA expression of 3?-HSD and LHR,and the downregulation of WT1 decreased E2 secretion and CYP19A1 mRNA expression in the absence of FSH.The overexpression of +KTS and –KTS decreased the basal and FSH-induced secretion of E2 and P4.The upregulation of-KTS decreased the basal and FSH-induced mRNA expression of CYP19A1,3?-HSD,CYP11A1,FSHR and LHR but promoted STAR mRNA expression.The upregulation of +KTS decreased the basal and FSH-induced mRNA expression of CYP19A1,3?-HSD and LHR but promoted STAR mRNA expression.Compared with the effect of +KTS,the effect of –KTS was more distinct.(3)We used healthy small follicular TCs as in vitro culture models to study the effect of the upregulation of WT1(+/-KTS)on steroidogenesis in TCs.The results show that the overexpression of +KTS and –KTS decreased the secretion of P4.The upregulation of-KTS decreased the mRNA expression of 3?-HSD and CYP11A1 but promoted the mRNA expression of CYP17A1 and STAR.The upregulation of +KTS decreased the mRNA expression of CYP11A1 but promoted the mRNA expression of CYP17A1 and STAR.Compared with the effect of +KTS,the effect of –KTS was more distinct.(4)Using healthy small follicular GCs as in vitro culture models,we studied the effect of the downregulation of WT1 and the upregulation of WT1(-KTS)on the AKT and ERK signaling pathways in GCs.The results show the upregulation of WT1(-KTS)in GCs promoted AKT phosphorylation and MKP3 expression but inhibited ERK phosphorylation.The downregulation of WT1 in GCs inhibited AKT and ERK phosphorylation but promoted MKP3 expression.PI3K/AKT-specific inhibitor LY294002 inhibited the mRNA expression of STAR and CYP19A1,but promoted the mRNA expression of CYP11A1,3?-HSD,MKP3 and the secretion of P4;MAPK/ERK-specific inhibitor U0126 promoted the mRNA expression of STAR,CYP11A1,3?-HSD,CYP19A1 and the secretion of E2 and P4.In conclusion,WT1 is expressed in antral follicular bovine GCs and TCs.During follicular development and atresia,the expression of WT1 in GCs is related to the production of steroid hormones and the expression of related genes.Compared with that of +KTS,the effect of –KTS was more distinct.WT1 inhibits the secretion of P4 by inhibiting the expression of CYP11A1 and 3?-HSD in GCs and TCs,and WT1 regulates E2 secretion by influencing CYP19A1 expression,at least in part,by modulating AKT and ERK1/2 signaling.