Two distinct cytochrome P450 aromatases in the orange-spotted grouper (Epinephelus coioides): cDNA cloning and differential mRNA expression.

The cDNA sequences encoding two distinct cytochrome P450 aromatases, namely P450aromB and P450aromA, were isolated from brain and ovary cDNA libraries of the orange-spotted grouper, respectively. The P450aromB cDNA consists of 1892 bp, and the open reading frame (ORF) encodes a putative protein of 506 amino acids. The P450aromA cDNA consists of 1836 bp, and the ORF encodes a putative protein of 518 amino acids. Northern blot analysis revealed a transcript of about 1.9 kb for P450aromB in the brain and kidney, and 2.1 kb for P450aromA in the ovary. The expression of both P450aromB and P450aromA genes in different tissues was further examined using one-step RT-PCR followed by Southern blot analysis. High levels of P450aromB mRNA expression were detected in the olfactory bulb, forebrain, midbrain, hypothalamus, medulla, pituitary, gill filament, gill arch, kidney, muscle, adipose tissue, and blood cells, but low levels in the hindbrain and ovary. High levels of P450aromA mRNA expression were detected in the ovary, pituitary, gill filament, gill arch, and spleen, but low levels in the forebrain, hindbrain, hypothalamus, and blood cells. In addition, the expression of P450arom genes in the orange-spotted grouper of different gonadal stages as induced by 17 alpha-methyltestosterone (MT) was investigated. The mRNA expression of P450aromB in the hypothalamus was highest in the intersexual stage, whereas the mRNA expression of P450aromA in the gonads was highest in the female stage, decreased in the intersexual stage, and lowest in the male stage. Results from current study indicate that P450aromB and P450aromA genes of the orange-spotted grouper have distinct tissue patterns of mRNA expression, and both of them may be involved in the MT-induced sex change.