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Growth hormone (GH), in the recent past, has been recognized as a potent steroid stimulating hormone independent of gonadotropin (GtH). However, the mode and mechanism of its steroidogenic action in the testis is not yet elucidated, particularly in fish. The present study was designed to understand the mode and mechanism of steroidogenic action of growth hormone in testis of the catfish, Clarias batrachus through in vivo and in vitro Leydig cell culture studies using the signaling molecule inhibitors. Exogenous administration of GtH, GH and insulin to the male catfish increased testicular and circulating testosterone level. In vitro treatment of Leydig cells with these hormones also increased testosterone production. The steroidogenic action of GH appeared to be indirect and mediated through Leydig cell produced insulin-like growth factor I (IGF-I), as the treatments with actinomycin D, cycloheximide and anti-IGF-I abolished the GH-induced testosterone production by Leydig cells. The GH-induced stimulation in IGF-I production by the isolated Leydig cells further substantiates this notion. GH appears to employ cAMP/PKA and tyrosine kinase signaling pathways to induce IGF-I production, as the adenylyl cyclase inhibitor (SQ 22,536), cAMP-dependent protein kinase (PKA) blocker (H-89) and tyrosine kinase inhibitor (lavendustin A) abolished the GH-induced IGF-I production and in turn testosterone by the Leydig cells. This study suggests that GH exerts independent androgenic effect in the catfish testis indirectly through augmenting the Leydig cell production of IGF-I., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.

The present in vitro study for the first time demonstrates the role of extragonadal hormones in regulation of NO production by testicular macrophages in vertebrates and paracrine role of NO in Leydig cell steroidogenesis in fishes. N-nitro L-arginine methyl ester (L-NAME - a NOS inhibitor) treatment substantially reduced NO production by testicular macrophages suggesting that testicular macrophages are one of the sources of testicular NO in the catfish, Clarias batrachus. Significant decline in NO production was also recorded following treatment of macrophages with the gonadotropin (GtH), growth hormone (GH) and insulin indicating that macrophage-produced NO is under endocrine inhibitory control. Treatment of Leydig cells with sodium nitroprusside (SNP) decreased testosterone (T) production. SNP treatment also remarkably suppressed the GtH, GH and insulin-stimulated T production by Leydig cells indicating that Leydig cell steroidogenesis is sensitive to exogenous NO. Further, effect of conditioned medium of testicular macrophages incubated with medium alone (non-treated TMCM) or GtH (GtH-treated TMCM) or GH (GH-treated TMCM) or insulin (insulin-treated TMCM) were also observed on Leydig cell T production. Non-treated TMCM as well as hormone-treated TMCM stimulated T production by Leydig cells; hormone-treated TMCM were more effective in stimulating T production than non-treated TMCM and/or hormones alone. These experiments altogether suggest that testicular macrophage secrete some factors, which influence Leydig cell steroidogenic activity through paracrine mechanism, and these paracrine secretions are under the endocrine control. Decline in NO in hormone-treated TMCM might also be one of the reasons for more stimulation in T production than that of hormones alone.

Nitric oxide has been recognized as an important inter- and intra-cellular modulator of testicular steroidogenesis in higher vertebrates with conflicting results. Moreover, its role in regulation of testicular steroidogenesis in ectothermic vertebrates is non-existent. The present study was, therefore, undertaken to examine whether Leydig cells of a freshwater catfish, Clarias batrachus produce nitric oxide (NO), if so, does it regulate its steroidogenic activity? The purified Leydig cells were stained histochemically for NADPH-diaphorase (NADPH-d) activity, and immunocytochemically for neuronal and inducible nitric oxide synthase (nNOS and iNOS) like molecules. Leydig cells were also incubated with NOS inhibitor, N-nitro-l-arginine methyl ester (l-NAME), and NO donor, sodium nitroprusside (SNP). NO and testosterone released in incubation medium were analyzed. A distinct positive NAPDH-d staining was observed in Leydig cells. These cells also exhibited immunoprecipitation of variable intensity with nNOS and iNOS antibodies. Further, l-NAME treatment caused significant suppression in NO production and elevation in testosterone secretion by Leydig cells. On the contrary, exposure of Leydig cells to SNP resulted in increased NO production with concomitant decline in testosterone level. Thus, the present study reports NO production by Leydig cells in fish for the first time, which appears to inhibit its own androgen production.

Asprosin is an orexigenic adipokine that regulates appetite and glucose homeostasis in mammals. To date, only fragmentary findings are reported regarding its role in testicular activities. In the current investigation, immunolocalization and direct action of asprosin in adult mice testis was evaluated. Immunohistochemical and immunoblot studies were performed to analyse the testicular expression of asprosin. Intratesticular treatment of asprosin (0.1 μg and 1.0 μg per testis) was given to evaluate its direct action on testicular functions. Sertoli and Leydig cells were found to be immuno‐positive for asprosin. Intratesticular administration of asprosin resulted into a significant increase in glucose and lactate levels along with enhanced expression of asprosin receptor OLFR734, insulin receptor (IR), glucose transporter 8 (GLUT 8), lactate dehydrogenase (LDH) activity and monocorboxylate transporters (MCT2 and 4). In addition, asprosin administration increased the testicular expression of cell proliferation (proliferating cell nuclear antigen: PCNA), cell survival (B cell lymphoma 2: Bcl2) and decreased germ cell apoptosis (Cysteine aspartic acid protease 3: Caspase 3) leading to increased sperm counts. Further, asprosin treatment resulted into increased level of total cholesterol, testosterone and steroidogenic markers (steroidogenic acute regulatory protein: StAR; 3beta‐hydroxysteroid dehydrogenases: 3β HSD and 17beta‐hydroxysteroid dehydrogenases: 17β HSD). Asprosin treatment promotes testicular glucose uptake and lactate synthesis to provide energy for steroidogenesis and spermatogenesis. The significant correlation between the asprosin‐induced increased IR expression and increased testosterone, glucose and lactate levels suggests its role in increased survival and proliferation but decrease in germ cell apoptosis. This study proposed asprosin's role as an autocrine/paracrine regulator of testicular functions in adult mice. [ABSTRACT FROM AUTHOR]

In an earlier study we have demonstrated reproductive-stage dependent, cell specific existence of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS)/NO system in testis of the catfish, Clarias batrachus . The present study is an extension to examine the role of NO in steroidogenesis and spermatogenesis through in vivo administration of a NO donor, sodium nitroprusside (SNP) and a NOS inhibitor, N-nitro- l -arginine methyl ester ( l -NAME) during the quiescence and recrudescence phase of the reproductive cycle of the catfish. Effects of these chemicals were assessed on the gonadosomatic index (GSI), levels of circulating & testicular testosterone, NO, activities of 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) in testis, expression of different NOS isoforms and testicular morphology in relation to spermatogenesis. SNP treatment increased the GSI, testicular and circulating testosterone & NO, activities of testicular 3β-HSD & 17β-HSD, and expression of NOS isoforms. It also increased the area and perimeters of interstitium and seminiferous tubules in the testis. It accelerated the spermatogenesis, as was evident from the large number of spermatids/spermatozoa in seminiferous tubules and very few spermatogonial cells/primary spermatocytes in comparison to the control testis. On the contrary, l -NAME significantly suppressed GSI, testosterone & NO levels in serum and testis, and activities of testicular 3β-HSD & 17β-HSD. It also suppressed the expression of NOSs in testis. Though l -NAME did not alter the spermatogonial mitotic proliferation with the advancement of testicular recrudescence, it halted the progression of spermatogenesis (meiotic division and spermatozoa formation) as was clear from the increase in spermatogonial cells and very few advanced germ cells in the seminiferous tubules in l -NAME treated testis, compared to the control testis. The above noted effects were highly pronounced in the recrudescing catfish. Their effects were very marginal and at a particular dose levels of SNP and l -NAME in the quiescent testis. This study distinctly provides evidence of pro-steroidogenic and pro-spermatogenic role of NO. This study also demonstrates the existence of eNOS in fish testis for the first time. The positive feedback control of expression of all isoform of NOS in testis by NO is also noteworthy. [ABSTRACT FROM AUTHOR]

Abstract: Many hormones are known for their role in the regulation of metabolic activities and somatic growth in fishes. The present study deals with the effects of malathion (an organophosphorous pesticide) on the levels of metabolic hormones that are responsible for promotion of somatic and ovarian growth of the freshwater catfish, Clarias batrachus. Malathion treatment for thirty days drastically reduced the food intake and body weight of fish. These fish also exhibited a great avoidance to food. Exposure of catfish to malathion reduced the levels of thyroxine (T4), triiodothyronine (T3), growth hormone (GH), insulin like growth factor-I (IGF-I), testosterone (T) and estradiol-17β (E2) in a dose dependent manner during all the studied reproductive phases, in general, except that malathion increased the level of GH during the quiescence phase. Significant reduction in muscle and hepatic protein content also occurred in the malathion-treated fish. Malathion exposure induced lipolysis too in the liver and muscle. The results thus support that malathion treatment disrupts the endocrine functions and the olfactory sensation responsible for food intake and gustatory feeding behavior, which ultimately leads to retardation of fish growth. [Copyright &y& Elsevier]

In the present investigation, daily variations in plasma levels of melatonin,testosterone and estradiol-17β were analysed in the tropical freshwatercatfish, Clarias batrachus during early and late-preparatory, pre-spawning,late-spawning and post-spawning phases of its annual reproductive cycle. Plasmamelatonin levels exhibited a significant circadian rhythm during all the reproductivephases but peaks were invariably at night. The amplitude of melatonin washigh during the early-preparatory phase and low in the pre-spawning phase.Testosterone also displayed significant rhythm during all the studied phasesexcept post spawning phase. Estradiol-17β revealed diurnal rhythm onlyduring preparatory and pre-spawning phases. The peak in the levels of plasmatestosterone and estradiol-17β were recorded during the photophase. Duringthe late-preparatory phase, the rhythm of testosterone coincided with therhythm of estradiol-17β. [ABSTRACT FROM AUTHOR]

Highlights: [•] Somatic as well as reproductive actions of GH is temperature dependent in the catfish, Clarias batrachus. [•] GH stimulates spermatogonial proliferation and accelerates spermatogenic processes. [•] GH promotes testosterone and estradiol production in the catfish. [•] Reproductive action of GH action is mediated through IGF-I in the catfish. [Copyright &y& Elsevier]