| Abstract:Transcription factor GATA family are common to the nucleic acid sequence (A/T) GATA (A/G), which is composed of six members (GATA-1,-2,-3,-4,-5 and-6). Among these factors, only GATA-4 and GATA-6 mRNAs and/or proteins have been detected in the ovary. Studies have shown that GATA-4 related on the proliferation, differentiation, apoptosis of human, mouse, pig granulosa cells and follicularatresia. However, little is known about the possible roles of GATA-6. A clear understanding of the localization and possible functions of GATA-4 and GATA-6 during goose follicle development remain elusive. In this paper, Sichuan goose as experimental material, from the molecular level to study the structural differences between the goose GATA-4 and GATA-6. In addition, we examined the mRNA expression levels of GATA-4 and GATA-6 in three types of follicles (healthy follicles, atretic follicles and postovulatory follicles) and in the granulosa and theca cells at different stages during follicledevelopment. The results should help to further elucidate the functional roles of the GATA-4 and GATA-6 during goose follicle development. Moreover, we constructing a eukaryotic expression vector of GATA-4 and transfected into cultured granulosa cells of goose, analyzed the overexpression of GATA-4 gene, the target genes, cholesterol synthesis, proliferation of granulosa cells, initially identified the functions of GATA-4 in goose. Finally we utilize granulosa cell apoptosis model by drugs treatment, exploring GATA-4 overexpression protects goose granulosa cells from drug-induced apoptosis and improve the ability of cholesterol synthesis, determine the role of GATA-4 in the process of follicular development. The main findings are as follows:(1) By sequencing and assembling the data, we obtained the full-length coding sequences of goose GATA-4 and GATA-6, which consist of 1236 and 1104 nucleotides encoding proteins with 411 and 367 amino acids, respectively. These sequence data have been submitted to the GenBank database under accession numbers KC454275 and KC493562, respectively.(2) Bioinformatics indicate:sequences of both GATA-4 and GATA-6 proteins include two adjacent zinc finger domains. The the zinc finger domain located at position 188-275 of GATA-4 amino acid sequence; located at position 161-248 of GATA-6 amino acid sequence, respectively. Both zinc finger domains with the distinctive form (CVNC-X17-CNAC)-X29-(CANC-X17-CNAC) and share 84.76% identity within this domain. In silico prediction together with matching of the high affinity RRXS(T)Y motif revealed that the GATA-4 protein might be phosphorylated predominantly at S233, but no phosphorylation site was found in the GATA-6 protein.(3) It has been well established that GATA-6 has an extra amino-terminal extension from the initiator methionine and can use two Met-codons in frame as translational initiation codons, forming a short open reading frame (uORF) in the entire upstream sequence; therefore, this led to the long-type and short-type GATA-6 proteins. Through multiple amino acid sequence alignments of GATA-6, we found that the GATA-6 protein in the goose, Gallus gallus and Meleagris gallopavo is the short type, while the GATA-6 protein in Taeniopygia guttata has an extra amino-terminal extension from the initiator methionine and forms the long type. To date, the functional significance of the two forms of GATA-6 remains to be investigated.(4) The expression level of GATA-4 mRNA in healthy follicles was significantly higher than in atretic follicles or postovulatory follicles (P<0.01), and the expression level of GATA-6 mRNA in healthy follicles was significantly lower than in atretic follicles or postovulatory follicles (P<0.01). The expression level of GATA-4 mRNA in granulosa cells was downregulated during follicle development; the peak of expression occurred in the 8-10 mm follicles, and the lowest expression was in the Fl follicles. GATA-6 was upregulated and reached its peak expression in the F1 follicles. These results indicate that the molecular structural differences in goose GATA-4 and GATA-6 may be related to their different roles during follicle development.(5) we constructed the eukaryotic expression recombinant plasmid pEGFP-N1-GATA-4, for the first time and then transfected it into goose GCs. We found that the expression level of GATA-4 mRNA was significantly increased compared to pEGFP-N1 and a control group (P<0.01) and that the mRNA expression levels of GATA-dependent genes, including StAR, Cyp11al, Cypl9al, and bcl-2, significantly increased several-fold (P<0.01) in GCs overexpressing GATA-4. A BrdU assay showed that overexpression of GATA-4 improves GC proliferation.(6) The apoptosis assay showed that 25HC can induce cultured GCs from geese to undergo apoptosis, nd as the concentration of 25HC increased, the number of apoptotic cells increased. Among the potential mechanisms involved in 25HC-induced apoptosis, the inhibition of sterol synthesis could be suggested. We examined mRNA expression levels of steroidogenesis genes including StAR, Cyp11al, and Cypl9al, all of which decreased with increasing concentrations of 25HC (P<0.05).(7) Overexpressing GATA-4 efficiently protected GCs from 25HC-induced apoptosis and 25HC-inhibited sterol synthesis, Overexpression of GATA-4 could up-regulate progesterone and estradiol expression in the presence of a high dose of 25HC (100 μM) after 24 h of stimulation (P<0.01) the expression levels of 3β-HSD and Cyp19al were increased (P<0.01). |
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