In silico analysis, temporal expression and gonadotropic regulation of receptors for follicle‐stimulating hormone and luteinizing hormone in testis of spotted snakehead Channa punctata

Abstract

This study in spotted snakehead Channa punctata was aimed to develop a comprehensive understanding of testicular gonadotropin receptors, from their sequence characterization, temporal expression to gonadotropic regulation, in seasonally breeding teleosts. A single form of follicle-stimulating hormone receptor (cpfshra) and luteinizing hormone/choriogonadotropin receptor (cplhcgr), was identified from testicular transcriptome data of C. punctata. Although deduced full-length protein sequence for cpFshra (694 amino acids) and cpLhcgr (691 amino acids) showed homology with their counterparts of other vertebrates, multiple insertion-deletion-substitution of residues suggest marked alterations in their structure and ligand specificity. The absolute quantification of testicular cpfshra and cplhcgr was estimated along the reproductive cycle following real-time PCR. The temporal expression profile showed highest testicular expression of both the gonadotropin receptors during resting phase. Their expression progressively decreased during preparatory and spawning phases concomitant with spermatogonial proliferation and differentiation and spermiogenesis. However, levels of cpfshra and cplhcgr sharply increased during post-spawning when seminiferous lobules were largely devoid of germ cells. To explore gonadotropic regulation of testicular cpfshra and cplhcgr, one group of fish of resting phase was administered with single dose of human chorionic gonadotropin (hCG; 5,000 IU/kg body mass) on day 0 and sacrificed on day 3 and day 5, while another group receiving two injections of hCG (day 0 and day 7) was sacrificed on day 14. The expression pattern of testicular gonadotropin receptors in hCG-treated fish sacrificed after 3, 5 and 14 days was similar to that of preparatory, spawning and postspawning phases, respectively. Likewise, testicular histology of hCG-treated fish sacrificed on day 3, day 5 and day 14 was comparable with that of preparatory, early spawning and late spawning phases, respectively. In light of the fact that gonadotropin receptors are largely expressed on somatic cells, an apparent decrease in testicular cpfshra and cplhcgr levels during preparatory and spawning phases or after 3 and 5 days from first hCG injection might not be due to downregulation of their expression. Rather, this could be due to dilution of somatic cell mRNA by large amount of germ cell mRNA. To verify this assumption, effect of hCG on plasma level of androgens was investigated employing enzyme-linked immunosorbent assay. A marked increase in plasma level of testosterone and 11-ketotestosterone was observed after hCG treatment in C. punctata. This would have been possible only when hCG upregulated the expression of testicular gonadotropin receptors.