| AMH(anti-mullerian hormone)belongs to the TGF-? superfamily and currently it has been well established that TGF-?/Smad signalling pathway plays a crucial role in the regulation of ovarian follicular development.In the ovary,AMH is mainly produced by follicular granulosa cells.It functions as the only in vivo cytokine to inhibit the growth of primordial follicles and is also involved in regulating follicular development through inhibition of either follicular recruitment or FSH sensitivity.However,until now nothing is known about its functions and regulatory mechanisms during geese follicular development.The objectives of the present study were:1)to clone and analyze the full-length coding and 5'flanking sequences of the geese AMH gene;2)to predict thecore regulatory regionand key transcription factors in the geese AMH promoter through construction of the promoter luciferase plasmids,transient transfection and luciferase acitivity assay;3)to determine the expression patterns of both AMH and its key transcription factors in granulosa cells of follicles at different developmental stages as well as their underlying relationships and 4)to investigate the transcriptional regulation of AMH by GATA-4 via co-transfection of GATA-4 eukaryotic expression vector and AMH site-directed mutagenic promoter reporter vector.The main results were listed as follows:(1)We obtained 2013bp of the coding sequence of the geese AMH gene by RT-PCR,molecular cloning and sequencing analysis.Its putative amino acid sequence showed 96%,95%,85%and 47%homology with predicted sequence of goose AMH,that of duck,chicken and human,respectively.Two structural domains were identified for the geese AMH protein,with the TGF_beta domain being highly conversed among species.However,there weremore unique mutations in the geese AMH_N domainthan the other species.Thus,we speculated that goose AMH may act differently in regulation of sex differentiation and gonadal development from the other species.(2)After analyzing the promoter sequenceof goose AMH,several transcription-factor binding sites were identified,including the typical TATA-box,SF-1SF-1 WT1 and three GATA-4 binding sites.(3)Seven promoter deletion mutants were directly sub-cloned into the pGL4.10 vector using the PCR-based genomic DNA walking method and were named pGL4.10-AMHl~pGL4.10-AMH7,respectively.After transient transfection into the CHO cell lines and Dual-Glo luciferase activity assay,it was found that only pGL4.10-AMH6 has certain activity after 24 hours,whereas,the others showed much lower activity than pGL4.10-basic.These data suggested that there were important cis-acting elements between pGL4.10-AMH6(-370bp--39bp)and pGL4.10-AMH7(-126bp--39bp),which might be responsible for the transcriptional activity of AMH promoter.(4)Our qPCR results showed that levels of AMH/mRNA decreased in granulosa cells during follicular development.Specifically,AMH had significant higher mRNA levels in granulosa cells of prehierarchical follicles than in those of hierarchical follicles.It could be speculated that goose AMH may have a similar function to chicken in prehierarchiacl follicles.In particular,AMH may have different functions at different developmental stages of follicle development because its mRNA levels fluctuated during the growing period from the 6-8 mm,8-10 mm to F4 follicles.(5)Our results alsoshowed that expression of the transcription factor GATA-4 had a significantly positive correlation with that ofAMH(P=0.026).However,there was no significant correlation between expression of AMH and WTI(P=0.458).These results suggested that GATA-4 instead of WT1 may participate in the transcriptional regulation of AMH.(6)The GATA-4 transcription factor binding site mutation vectors were successfully constructed using thesite-directed mutation method and were named pGL4.10-AMH2-GATA-4-M778,pGL4.10-AMH2-GATA-4-M1399 and pGL4.10-AMH2-GATA-4-M1477.The recombinant pEGFP-GATA-4 eukaryotic expression vector was co-transfected with each GATA-4 site-directed mutagenic vector into the CHO cell lines.Dual-Glo luciferase activity assay showed that over-expression of GATA-4 significantly(P<0.05)increased the activity of AMH promoter by 14.3-,9.26-,9.04-and 5.31-folds,respectively.These results suggestedthat the transcription factor GATA-4 could bind to three binding sites in the AMH promoter region,with higher binding activitv at-1477hn than that at either-778 or -1399bp.In summary,AMH is involved in regulating goose follicular developmentand is transcriptionally regulated by GATA-4. |
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