Catecholamines modulate differentially nonapeptide precursor mRNA expression in the preoptic area and ovary of the catfish Heteropneustes fossilis: An in vitro study.

In the catfish Heteropneustes fossilis, three nonapeptide hormone genes were identified in the brain preoptic area (POA) and ovary: a pro-vasotocin (pro-vt) and two isotocin gene paralogs viz., a novel pro-ita and conventional pro-itb. In the present study, the regulatory role of catecholamines [CA: dopamine (DA), noradrenaline (NA), adrenaline (AD)] on the expression of these genes were investigated in vitro. DA (1, 10, and 100 ng/mL) inhibited significantly the mRNA expression in both the POA and ovary. NA upregulated the POA mRNA expression in a biphasic manner, the lower concentrations (1 ng and 10 ng) scaled up and the higher concentration (100 ng) scaled down the expression of pro-vt and pro-itb, while only the 1 ng NA scaled up the pro-ita expression. In the ovary, NA upregulated the mRNA expressions at all concentrations; the pro-vt expression was stimulated only at 10 and 100 ng. AD stimulated pro-vt and pro-ita expression in the POA at all concentrations but the pro-itb expression was inhibited at 1 and 10 ng, and stimulated at 100 ng concentrations. In the ovary, AD elicited varied effects; no significant change in pro-vt, a stimulation of pro-ita, and an inhibition of pro-itb at 1 ng, and stimulation of pro-itb at the 10 and 100 ng. The incubation of the POA and ovary with α-methylparatyrosine (MPT, 250 µg/mL, a tyrosine hydroxylase inhibitor) for 8 h downregulated the mRNA expression in the POA but unaltered the expression in the ovary. Pre-incubation with MPT for 4 h, followed by co-incubation with DA, NA or AD for 4 h elicited varied effects. In the POA, the co-incubations with the CAs rescued the inhibition due to MPT. The MPT + DA and MPT + AD treatments reduced the magnitude of the inhibition of pro-vt and pro-itb by MPT. But the pro-ita expression was modestly stimulated in the MPT + AD group. On the other hand, the MPT + NA treatment rescued the MPT effect and elicited 10-folds increase in the expression levels. In the ovary, the changes were: an inhibition in the MPT + DA group, no significant alteration in the MPT + NA group, and a mild stimulation in the MPT + AD group. The results suggest that CAs modulate brain and ovarian nonapeptide gene expression differentially, which is important in the neuroendocrine/endocrine integration of reproduction in the catfish.

Molecular cloning and characterisation of an isotocin paralogue ([V8] isotocin) in catfishes (superorder Ostariophysi): Origin traced likely to the fish-specific whole genome duplication.

The present study reports the molecular cloning of a previously uncharacterised neurohypophyseal nonapeptide precursor cDNA in two catfish species: Heteropneustes fossilis and Clarias batrachus. The deduced nonapeptide is CYISNCPVG ([V8] isotocin), which has not been reported in any vertebrate till date. Phylogenetic and conserved synteny analyses showed the gene to have originated from the isotocin precursor (pro-it) gene by fish-specific whole genome duplication (3R). The two isotocin lineages have been designated as pro-ita (new gene) and pro-itb (conventional it gene). All teleost groups may not possess both pro-ita and pro-itb and the pattern of losses/retention was found to be lineage-specific. Quantitative reverse transcriptase-polymerase chain reaction studies showed the expression of the pro-ita gene in the brain and ovary of H. fossilis. In situ hybridisation studies localised the pro-ita transcripts in the nucleus preopticus of the hypothalamus and the follicular layer (theca-granulosa) of oocytes, comprising tissues in which pro-itb and vasotocin precursor (pro-vt) mRNA expression was previously reported. The transcript levels varied with the reproductive stage and a high abundance was found in both brain and ovary during the breeding phase. The substitution of valine in place of isoleucine at the eighth position in Ita may have modified the ligand-receptor interaction, leading to sub-functionalisation and the retention of the gene in catfishes.

Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor genes from Ostariophysian catfishes: phylogeny and evolutionary considerations in teleosts.

Basic and neutral neurohypophyseal (NH) nonapeptides have evolved from vasotocin (VT) by a gene duplication at the base of the gnathostome lineage. In teleosts, VT and IT are the basic and neutral peptides, respectively. In the present study, VT and IT precursor genes of Heteropneustes fossilis and Clarias batrachus (Siluriformes, Ostariophysi) were cloned and sequenced. The channel catfish Icatalurus punctatus NH precursor sequences were obtained from EST database. The catfish NH sequences were used along with the available Acanthopterygii and other vertebrate NH precursor sequences to draw phylogenetic inference on the evolutionary history of the teleost NH peptides. Synteny analysis of the NH gene loci in various teleost species was done to complement the phylogenetic analysis. In H. fossilis, the NH transcripts were also sequenced from the ovary. The cloned genes and the deduced precursor proteins showed conserved characteristics of the NH nonapeptide precursors. The genes are expressed in brain and ovary (follicular envelope) of H. fossilis with higher transcript abundance in the brain. The addition of the catfish sequences in the phylogenetic analysis revealed that the VT and IT precursors of the species-rich superorders of teleosts have a distinct phylogenetic history with the Acanthopterygii VT and IT precursors sharing a less evolutionary distance and the Ostariophysi VT and IT having a greater evolutionary distance. The genomic location of VT and IT precursors, and synteny analysis of the NH loci lend support to the phylogenetic inference and suggest a footprint of fish- specific whole genome duplication (3R) and subsequent diploidization in the NH loci. The VT and IT precursor genes are most likely lineage-specific paralogs resulting from differential losses of the 3R NH paralogs in the two superorders. The independent yet consistent retention of VT and IT in the two superorders might be directed by a stringent ligand-receptor selectivity.