In the present study, we tested the hypothesis that the brain of the black porgy fish, Acanthopagrus schlegeli, has the capacity for de novo steroidogenesis and that these neurosteroids may impact sex differentiation. Gonadal histology and Dmrt1 gene expression revealed that the fish were not sex differentiated until 155 dah (days after hatching). We further demonstrated the developmental expressions of the mRNAs encoding for four key neurosteroidogenic enzymes, namely, the cytochrome P450 side chain cleavage (CYP11A1), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3betaHSD), cytochrome P450c17 (CYP17) and aromatase (CYP19b) in the brain at different post-hatching developmental ages. The results indicated that steroidogenic genes are expressed in brain from the earliest sampling time, 60 dah. Quantitative real-time polymerase chain reaction analysis demonstrated significantly higher expression levels of these enzymes at 120 dah compared to 60 dah in all the brain regions. However, the increase for 3betaHSD was significant only in hypothalamus and midbrain, whereas it was significant only in forebrain and hypothalamus for CYP19b. A decline in mRNA levels were observed for all the genes at 155 dah except in midbrain for CYP11A1 and in hindbrain for CYP19b. Analysis of aromatase enzyme activity showed a significant increase in aromatase activity in the forebrain at 120 dah. Thus, the present study demonstrated for the first time an age- and/or region dependent expression of the mRNAs encoding the steroidogenic enzyme genes in the brain of black porgy. The presence of key steroidogenic enzymes as early as 60 dah, before gonadal sex differentiation, demonstrates that steroid biosynthetic capacity in brain precedes histological gonad differentiation. The mRNA transcripts of these genes showed a synchronous peak at 120 dah, suggesting that oestradiol may be locally formed in most parts of the brain. The study suggests an important role for brain aromatase in male black porgy brain sex differentiation, and considers the possibility of a role for this enzyme in neurogenesis.

ABSTRACT:The mechanisms underlying brain sex differentiation in animals are poorly understood. In the present study, using black porgy, Acanthopagrus schlegeli, as primary experimental model, we investigated the temporal expression patterns of receptors for androgen (ar) and estrogen (esr1 and esr2a) in the brain during posthatching ages and analyzed them against the timing of gonadal germ cell development. We hypothesized that endogenous estrogens naturally masculinize the brain of black porgy. The expression of sex steroid receptors was studied in relation to a wider suite of other related genes (nr5a2, nr0b1, star, and cyp19a1b) to provide some insight into the monomale sex differentiation pattern observed in this species. Our results revealed a highly significant increase in esr1 together with the increase in esr2a at 120 dph (days posthatching), suggesting a significant role for esr in sex differentiation in this species. Temporal expression patterns of nr5a2, nr0b1, star, sex steroid receptors, and cyp19a1b in the brain provided evidence for their physiological roles in the monomale sex differentiation in this species. The expression of nr5a2, star, ar, esr1, esr2a, and cyp19a1b increased at 120 dph, a period when brain sex differentiation probably occurs in this species. The study also suggests that neurosteroidogenesis in black porgy may be regulated by both nr5a2-dependent and nr5a2-independent mechanisms. The results demonstrated striking differences in the abundance of the gene transcripts in discrete brain region throughout ontogeny. In addition, the sex steroid hormone levels and aromatase activity in brain at different developmental states and the changes in the gene expression patterns in response to aromatase inhibitor treatment are also discussed. ' 2009 Wiley Periodicals, Inc. Develop Neurobiol