Gender differences in tryptophan hydroxylase-2 mRNA, serotonin, and 5-hydroxytryptophan levels in the brain of catfish, Clarias gariepinus, during sex differentiation

Tryptophan hydroxylase (tph) is the key regulator in serotonin (5-HT) biosynthesis that stimulates the release of GnRH and gonadotropins by acting at the level of hypothalamo–hypophyseal axis. In brain, 5-HT is expressed predominantly in preoptic area–hypothalamus (POA–HYP) region in teleosts. Therefore, in the present study we isolated tph2 from catfish brain to evaluate its expression pattern in male and female brains during early development. Tph2 cloned from catfish brain is 2.768 Kb in length which encodes predicted protein of 488 amino acid residues. The characterization of recombinant tph2 was done by transient transfection in CHO cells. Tissue distribution of tph2 revealed ubiquitous expression except ovary. Real time PCR analysis in discrete regions of adult male brain revealed that tph2 mRNA was abundant in the POA–HYP and optic tectum + cerebellum + thalamus (OCT) regions. Differential expression of tph2 was observed at mRNA and protein levels in the POA–HYP and OCT regions of male and female brains during development that further correlate with the 5-hydroxytryptophan (5-HTP) and 5-HT levels measured using HPLC method in these regions of male and female brains. Tph2 immunoreactive neurons were observed in different regions of brain at 50 days post hatch using catfish specific tph2 antibody. Changes in tph2 mRNA expression, 5-HTP, and 5-HT levels in the POA–HYP + OCT region of brains of methyltestosterone and para-chlorophenylalanine treated fishes during development further endorse our results. Based on our results, we propose that the serotonergic system is involved in brain sex differentiation in teleosts.

Research highlights
► Cloning of full-length cDNA of tph2 from catfish brain by cDNA library screening.
► Characterization of recombinant tph2 by transient transfection in CHO cells.
► Tph2 expression was abundant in the pre-optic area–hypothalamus of adult brain.
► Gender differential expression of tph2 in catfish brain during development.
► Serotonergic system is involved in male brain sex differentiation in teleosts.