Based on our previous work, EaCyp19a1a gene was cloned from the SMART cDNA library of red-spotted grouper oocytes, and the corresponding anti-EaCyp19a1a antiserum was generated for the detection of expression pattern and regulation mechanism in adults.
Multiple amino acid sequence alignment of the deduced EaCyp19a1a and other known Cyp19 aromatase proteins revealed high homology among the teleost ovarian isoform, including the common functional domains of aromatases. Moreover, some specific residues were found to be conserved among the Cyp19a1a proteins of teleosts.
The detailed localization and dynamic expression change were observed in the gonads of protogynous hermaphrodite red-spotted grouper by utilizing the advantages that gonad development undergoes transition from ovary to intersexual gonad and then to testis. Studies revealed ovary-specific expression pattern of EaCyp19a1a in adults. And EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues.
Another significant progress in this study was to isolate and sequence the promoter region of EaCyp19a1a. Sequence analysis showed twenty-seven potential binding sites were on the promoter for some transcriptional factors, such as SF-1, CREB, SOX5, and so on. luciferase assays in vitro indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter. Moreover, specific green fluorescence was observed mainly in primordial gonadal cells of the zebrafish embryos after microinjection in vivo, which indicated that some cis-acting elements were binding to EaCyp19a1a promoter and had the ability to regulate its expression specifically in gonad.
Moreover, we found a high degree of conserved region about 300 bp existed in the proximal Cyp19a1a promoters of teleost fishes. And the motifs of TATA box, SF-1, SOX5, and CREB were most notably well-conserved in this region. In view of the Cyp19 studies in human, we suggested the conserved promoter region among teleosts might be important for the ovary-specific expression of Cyp19a1a.