Rice field eel (Monopterus albus), a protogynous hermaphrodite fish, is a good model for the research of sex determination and gonadal differentiation in teleosts. In this study, we cloned the full-length cDNA sequence of trh, which encoded a predicted protein with 270 amino acids. Trh mainly expressed in the brain, followed by the ovary, testis, muscle and pituitary, and had low levels in other peripheral tissues. During natural sex reversal, trh mRNA expression levels exhibited a significant increase at the late intersexual stage in the hypothalamus. In the gonad, trh mRNA expression levels showed a trend of increase followed by decrease, and only increased significantly at the middle intersexual stage. No matter static incubation or intraperitoneal (IP) injection, TRH had no significant effect on trh and thyroid-stimulating hormone βsubunit (tshβ) mRNA expression levels, and serum T3, T4 and TRH release. After static incubation of ovarian fragments by TRH, the expression of gonadal soma derived factor (gsdf) was up-regulated significantly at both the doses of 10 and 100 nM. IP injection of TRH stimulated the expression of gsdf, and inhibited the expression of ovarian aromatase gene (cyp19a1a), accompanied by the increase of serum 11-KT levels. The results indicated that TRH may play a novel role in gonadal differentiation by the regulation of gonadal differentiation-related gene expression and sex steroid hormone secretion in rice field eel.

Cortisol is the major glucocorticoid, which is considered to be a key factor linking the environmental stress and sex differentiation in teleosts. In the present study, we explored the possible role of cortisol in sex differentiation in a protogynous hermaphroditic swamp eel, Monopterus albus. We cloned and characterized the full-length cDNA sequences of glucocorticoid receptor genes, gr1 and gr2. A novel alternative splicing variant (gr1b) was also discovered, which lacked a 27-bp nucleotide insertion in gr1, resulting in a nine-amino-acid deletion in GR1. Real-time PCR indicated that swamp eel gr1a mainly expressed in the brain; gr1b mainly in the spleen, head kidney, kidney and muscle; gr2 mainly in the muscle. During female-to-male sex reversal, the expression levels of gr1a and gr1b decreased significantly at the stage of ovotestis III, and gr2 showed a trend of increase, and increased significantly in the testis. In the brain, acute heat stress up-regulated significantly the expression of gr1a and gr1b, while had no significant effect on gr2. However, in the ovary, heat stress only up-regulated significantly the expression of gr1b at 12 h. Serum cortisol concentration increased significantly by heat stress at 1 h, and returned to normal levels at 6 and 12 h. Cortisol injection up-regulated significantly the expression levels of gr1b and gr2, while had no significant effect on gr1a in the ovary. At the same time, cortisol injection down-regulated significantly the expression of cyp19a1a, and only decreased significantly the expression of foxl2 at 6 and 12 h. Cortisol injection up-regulated significantly the expression of dmrt1a at 6, 24 and 48 h, while only increased the expression of gsdf at 6 h. The results indicate that cortisol and GRs may play roles in sex differentiation by the regulation of gonadal differentiation-related gene expression in swamp eel.