The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17β-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 μM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 μM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development.
Keywords: cloning; expression; flatfish; regulation; sex control; steroidogenic genes.