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

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.

In silico analysis, seasonal variation and gonadotropic regulation of jag1 and its receptor notch1 in testis of spotted snakehead Channa punctatus.

The present study in seasonally breeding spotted snakehead Channa punctatus, for the first time in nonmammalian vertebrates, demonstrated correlation between reproductive phase-dependent testicular expression of ligand Jag1/receptor Notch1 and spermatogenic events. Testicular transcriptome sequencing data from our earlier study in C. punctatus was used in the present study to select the best transcript for jag1 (cpjag1) and notch1 (cpnotch1). The transcripts cpjag1 and cpnotch1 encoded full-length putative proteins of 1215 (cpJag1) and 2475 (cpNotch1) amino acids, respectively. A marked homology in the extracellular domains of Jag1 and Notch1 was observed following their alignment with respective proteins from different vertebrates, suggesting conservation in ligand-receptor interaction in C. punctatus. Both cpJag1 and cpNotch1 showed phylogenetic closeness with their teleostean counterparts, especially with that of Perciformes. Temporal expression of cpjag1 and cpnotch1 in testis depending on reproductive phases showed an appreciably high expression during spermatogenically inactive resting and postspawning phases when seminiferous lobules consisted of spermatogonial stem cells and undifferentiated spermatogonia. Their expression sharply declined during spermatogenically active preparatory and spawning phases. It appears that involvement of cpjag1/cpnotch1 is restricted to inactive phases when spermatogonial stem cells renew themselves and replenish undifferentiated spermatogonia. This assumption is ascertained by an experimental study in which high level of testicular cpjag1/cpnotch1 expression in control fish of resting phase markedly decreased after administration of human chorionic gonadotropin that is known to induce proliferation and differentiation of spermatogonia and spawning of spermatozoa.

De novo sequencing and comparative analysis of testicular transcriptome from different reproductive phases in freshwater spotted snakehead Channa punctatus.

The spotted snakehead Channa punctatus is a seasonally breeding teleost widely distributed in the Indian subcontinent and economically important due to high nutritional value. The declining population of C. punctatus prompted us to focus on genetic regulation of its reproduction. The present study carried out de novo testicular transcriptome sequencing during the four reproductive phases and correlated differential expression of transcripts with various testicular events in C. punctatus. The Illumina paired-end sequencing of testicular transcriptome from resting, preparatory, spawning and postspawning phases generated 41.94, 47.51, 61.81 and 44.45 million reads, and 105526, 105169, 122964 and 106544 transcripts, respectively. Transcripts annotated using Rattus norvegicus reference protein sequences and classified under various subcategories of biological process, molecular function and cellular component showed that the majority of the subcategories had highest number of transcripts during spawning phase. In addition, analysis of transcripts exhibiting differential expression during the four phases revealed an appreciable increase in upregulated transcripts of biological processes such as cell proliferation and differentiation, cytoskeleton organization, response to vitamin A, transcription and translation, regulation of angiogenesis and response to hypoxia during spermatogenically active phases. The study also identified significant differential expression of transcripts relevant to spermatogenesis (mgat3, nqo1, hes2, rgs4, cxcl2, alcam, agmat), steroidogenesis (star, tkt, gipc3), cell proliferation (eef1a2, btg3, pif1, myo16, grik3, trim39, plbd1), cytoskeletal organization (espn, wipf3, cd276), sperm development (klhl10, mast1, hspa1a, slc6a1, ros1, foxj1, hipk1), and sperm transport and motility (hint1, muc13). Analysis of functional annotation and differential expression of testicular transcripts depending on reproductive phases of C. punctatus helped in developing a comprehensive understanding on genetic regulation of spermatogenic and steroidogenic events in seasonally breeding teleosts. Our findings provide the basis for future investigation on the precise role of testicular genes in regulation of seasonal reproduction in male teleosts.

Seasonality of reproduction in male spotted murrel Channa punctatus: correlation of environmental variables and plasma sex steroids with histological changes in testis.

The present study was undertaken to develop a comprehensive understanding of how environmental cues and sex steroids relate with cyclic changes in spermatogenesis in freshwater spotted snakehead Channa punctatus that is nutritious and economically important. The seasonal histological changes in testis and annual profile of gonadosomatic index (GSI) of C. punctatus delineated the testicular cycle into four phases: regressed (December-March), preparatory (April-June), spawning (July and August) and postspawning (September-November). Among environmental variables, correlation and regression analyses exhibited an important relationship between photoperiod and testicular weight while role of rainfall was seen confined to spawning. The seasonal profile of plasma sex steroids when correlated with cyclic changes in spermatogenesis in spotted snakehead, testosterone (T) seems to be involved in controlling the major events of spermatogenesis from renewal of stem cells to spawning of spermatozoa. Another important androgen prevalent in teleosts, 11-ketotestosterone (11-KT), was high during preparatory phase, suggesting that 11-KT in addition to T plays an important role in progression of spermatogenesis and spermiation in C. punctatus. However, 11-KT was not seen to be associated with milt production and release of spermatozoa during spawning. Plasma profile of estradiol-17ß (E2) during different reproductive phases revealed the involvement of E2 in repopulation of stem cells during postspawning phase and in maintaining quiescence of testis during regressed phase.