| GATA gene family are transcription factors which can be combined with common sequence (A/T) GATA (A/G) of target gene nucleic acid. GATA-4 and GATA-6, the main members of the GATA gene family, are mainly expressed in the animal ovariangranulosa cells and theca cells. GATA-4 and GATA-6 are proved to participate in synthesis process of steroid hormones in human, mouse and pig. Until now, the mechanism of GATA-4 regulating Steroidsynthesis of ovarian granulosa cells has been deeply researched, while the studies of functional single GATA-6 are rarely reported. Therefore, in our studies, the laying maternal line of Tianfu goose (anser cygnoides) are applied as experimental materials. Firstly we constructed the GATA-6 eukaryotic expression and shRNA interference vectors. Then we analyzed how GATA-6 genes regulategranulosa cell steroid synthesis and related genes through interfering and over-expressing GATA-6 genes. Finally, we discussed GATA-4 and GATA-6’s different roles and their relationships ingranulosa cell by co-transfecting GATA-4 and GATA-6 overexpression vectors. See the results as follows.(1) We are informed that the complete coding region sequence of GATA-6 is 1173bp through RT-PCR, clone sequencing, sequence assembly and then predict that GATA-6 encodes 390 amino acids in total with an accession No. of KP723851.(2) Based on result of CCK-8, overexpression of GATA-6 leads to the rising of the proliferation rate ingranulosa cells, while interfering of GATA-6 leads to the falling of the proliferation rate ingranulosa cells. The results suggest that GATA-6 can regulate the proliferation and differentiation of granulosa cells.(3) Compared with the control group and the negative control group, we found that after interfering GATA-6 gene expression, the secretion of cholesterol and progesterone increased dramatically (p<0.05), while, for its downstream target genes, the expression level of the 3β-HSD, Cyp17, P450scc,17β-HSD decreased significantly (p<0.05 and Cyp19, StAR increased significantly (p<0.05), and FSHR had no significant change. The results suggest that 3β-HSD, Cyp19, Cypl7, P450scc,17β-HSD, and StAR are downstream target genes involved in regulation and transcription during the follicular development of GATA-6 and indirectly regulate the synthesis of steroid hormones by regulating relevant target genes transcription.(4) Compared with the control group and the negative control group, we found that after over-expressing GATA-6 gene, progesterone secretion increased (p>0.05), cholesterol secretion decreased (p>0.05), testosterone secretion decreased significantly (p<0.01), while, for its downstream target genes, the expression level of 3/3-HSD, Cypl9, and FSHR decreased significantly (p<0.01),17β-HSD decreased significantly (p<0.05), the expression level of Cypl 7 and StAR increased significantly (p<0.01), and the expression level of P450scc increased a little (p>0.05). In addition, the expression level of GATA-4 gene had no significant change. Thus, there is little difference between the control group and the negative control group, (p>0.05). Compared with the results of interference and overexpression experiments, it is clear that GATA-6 has different control modes for regulating the downstream target genes, but GATA-6 has no different impact on expression of GATA-4.(5) Compared with the control group and the negative control group, we found that afterco-transfeeting eukaryotic overexpression vectors of GATA-4 and GATA-6, cholesterol secretion increased significantly (p<0.05), progesterone secretion increased a little (p>0.05), testosterone secretion decreased significantly (p<0.05), while, for its downstream target genes, the expression level of 3β-HSD, Cyp19, and FSHR increased significantly (p<0.05), 17β-HSD increased a little (p>0.05), the expression level of Cyp17 and P450scc decreased significantly (p<0.01), and the expression level of StAR decreased significantly (p<0.05). Thus, there is little difference between the control group and the negative control group, (p>0.05). The results suggest that the regulation of GATA-4 and GATA-6 for related target genes synthesized in steroid of follicle is featured with antagonism and Compensation metabolic mechanism. |
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