Molecular Cloning,Tissue Expression And Promoter Activity Analysis Of The Geese (Anser Cygnoides) CYP17/19 Genes

CYP17 and CYP19,two members of the P450 superfamily,are known as the key rate-limiting enzymes in the biosynthesis of androgen and estrogen,respectively,thus both play important roles in the regulation of ovarian follicular growth and development.In contrast to the mammals where the functions of CYP17 and CYP19 during follicular development have been extensively studied,the mechanisms regulating the expression of the CYP17 and CYP19 genes during geese follicular development are poorly understood and remain to be further investigated.In this study,female geese from the maternal line of Tianfu Meat Goose(an indigenous goose breed)during the peak laying period were used as experimental animals.The objectives of this study were:1)to obtain the coding and 5'flanking sequences of the geese CYP17 and CYP19 genes;2)to examine their expression patterns in theca and granulosa layers of developing follicles;3)to explore the regulatory mechanisms controlling the promoer activity of the CYP19 gene.The main results were listed as follows:(1)We first obtained the full-length cDNA sequences of the geese CYP19 and CYP17 genes,with the length of 1521 bp and 1512bp,respectively.The putative amino acid sequences of the geese CYP17/19 proteins had a more than 90%homology with those of other avian species.Moreover,phylogenetic analysis showed that both genes were firstly clustered with chickens(gallus gallus).These results suggest that the geese CYP17/19 genes have a close evolutionary relationship with that of chickens than other species examined,which is in accord with the organims' phylo-genetic relationship.(2)During ovarian follicular development,the mRNA expression levels of CYP17 and CYP19 in theca cells were significantly higher than those in granular cells(P<0.05).Immunohistochemical analysis showed that CYP17 and CYP19 were mainly located in goose theca interna and externa,respectively,suggesting that the former may play an important role in theca interna while the latter functions in theca externa.(3)Levels of CYP17 mRNA were higher in theca cells of preovulatory F4-F2 follicles than those of prehierarchical follicles(P>0.05)and were also significantly higher than those of the F1 follicle(P<0.05),indicating that CYP17 has a different role in different sized follicles.In contrast,CYP19 had higher mRNA levels in theca cells of the F6-F1 follicles than those of all prehierarchical follicles(P>0.05)but had significantly higher levels than those of the 2-6 mm follicles(P<0.05),which suggested that CYP19 might have a different role between prehierarchical and hierarchical follicles.(4)We successfully obtained 1924bp of the 5'flanking sequence of the geese CYP19 gene and constructed the p4.10-CYP19M1-p4.10-CYP19M6 vectors using the DNA walking method.These vectors were subsequently transfected into CHO cells to compare the promoter activity of different deletion fragments,and we founded that the luciferase activity of pGL4.10-CYP19M1 was significantly higher than that of other deletion promoter reporter vector(P<0.05),indicating that the-118bp-Obp region of the CYP19 gene promoter was the core transcriptionally regulatory region.Since the activity of different deletion fragments of the CYP19 promoter were reduced as the length of vectors increased,potentially negative regulatory elements may exist in the promoter-1924bp?119bp.(5)The qPCR results in theca layers showed that expression of CYP19 was clustered with the transcription factors AR,GATA4 and GATA6 but not with SREBP and C/EBP,showing that AR,GATA4 and GATA6 rather than SREBP and C/EBP may participate in the transcriptional regulation of CYP19 in geese theca layers.Taken together,these results showed that CYP17 and CYP19 had different roles during follicular development,and AR,GATA4 and GATA6 were involved in regulating the expression of CYP19.