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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., (© 2022 Wiley-VCH GmbH.)

Unlike mammals, two kisspeptins genes encoding, kiss1 and kiss2 are detected in fishes with highly varied and contradictory difference in their reproductive activities. The present study was undertaken to examine the direct action of kisspeptin-10 and its role in gonadal activities in the gonadally quiescent Asian catfish using native mammalian kisspeptin decapeptide (KP-10) involving in vivo and in vitro approaches. The in vivo KP-10 treatment caused precocious onset of gametogenesis and its rapid progression, as was evident from the appearance of advanced stages of ovarian follicles in ovary, and advanced germ cells (spermatocytes/ spermatids) in the testis of the treated Clarias batrachus in comparison to the control gonads. It also elevated the steroid levels in gonads of the catfish in vivo and in vitro conditions. Simultaneously, it increased the expressions of key steroidogenic enzymes like 3β-HSD, 17β-HSD, and StAR protein, responsible for transfer of cholesterol from outer to inner membrane of the mitochondria of steroidogenic cells. Concurrently, it augmented the activities of 3β-HSD and 17β-HSD in the ovarian explants. The expressions of MAPK component (pERK1/2 and ERK1/2) were also up-regulated by KP-10 in gonadal explants. Thus, the data suggest that kisspeptin-10 stimulates gametogenesis by enhancing gonadal steroid production. The study also describes the putative mechanistic cascade of steroidogenic actions of kisspeptin-10 in the catfish so much so in teleost fish. The study also suggests that, kisspeptin may act locally to regulate gonadal activities in an autocrine/paracine manner, independent of known extra-gonadal factors in the catfish., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The central role of kisspeptin (kiss) in mammalian reproduction is well established; however, its intra-gonadal role is poorly addressed. Moreover, studies investigating intra-gonadal role of kiss in fish reproduction are scanty, contradictory and inconclusive. The expression of kiss1 mRNA has been detected in the fish brain, and functionally attributed to the regulation of reproduction, feeding and behavior. The kiss1 mRNA has also been demonstrated in tissues other than the brain in some studies, but its cellular distribution and role at the tissue level have not been adequately addressed in fish. Therefore, an attempt was made in the present study to localize kiss1 in gonadal cells of the freshwater catfish, Clarias batrachus. This study reports the presence of kiss1 in the theca cells and granulosa cells of the ovarian oocytes and interstitial cells in the testis of the catfish. The role of kiss1 in the ovary and testis of the catfish was also investigated using kiss1 receptor (kiss1r) antagonist (p234). The p234 treatment decreased the production of 17β-estradiol in ovary and testosterone in the testis by lowering the activities of 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase under both, in vivo as well as in vitro conditions. The p234 treatment also arrested the progression of oogenesis, as evident from the low number of advancing/advanced oocytes in the treated ovary in comparison to the control ovary. It also reduced the area and perimeter of the seminiferous tubules in the treated catfish testis. Thus, our findings suggest that kiss is involved in the regulation of gonadal steroidogenesis, independent of known endocrine/ autocrine/ paracine regulators, and thereby it accelerates gametogenic processes in the freshwater catfish., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The present study demonstrates seasonal variation in the cellular expression of neuropeptide Y (NPY), a known orexigenic neuropeptide, in the testis of the catfish, Clarias batrachus and its relation with testicular steroids. In vitro effects of NPY on androgen production and activities of steroidogenic enzymes were also analyzed to reaffirm the relation between NPY and steroids. NPY-immunoprecipitation was observed in Sertoli cells, interstitial cells and germ cells in recrudescing testis. Intensity of NPY-immunoreaction in the interstitial cells increased steadily with initiation of spermatogenesis and reached maximal in fully grown testes, and then decreased suddenly in the spermiating/spent testis. NPY was also expressed considerably in Sertoli cells in recrudescing testis, but disappeared in the fully grown testis. A moderate NPY-immunoreactivity was also seen in spermatogonial cells in recrudescing testis, but intense NPY-immunoprecipitation was detected in advanced germ cells (spermatids/spermatozoa) in fully mature testis. NPY-immunoreation intensity in interstitial cells showed positive correlation with increasing levels of testicular testosterone and 11-ketotestosterone, and with activities of 3β-HSD & 17β-HSD coinciding with advancing testicular activities. NPY treatment of testicular fragments in vitro stimulated the activities of 3β-HSD & 17β-HSD and increased testosterone & 11-ketotestosterone levels. This study for the first time demonstrates the existence of NPY peptide at cellular levels in fish testis, which stimulates androgen production by acting directly at testicular level., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Considering the absence of information on testicular growth factors in fishes, present study was aimed to elucidate the existence of BMP15, an important member of TGF-β superfamily, in the testis of a seasonally breeding freshwater catfish, Clarias batrachus and its role in regulation of testicular activities. The study demonstrated the expression of BMP15 in the somatic cells (Sertoli and interstitial cells) in fish testis. The expression varied with changing testicular activity; the expression was very high in the quiescent and early recrudescing testis coinciding with the renewal of spermatogonial cells. Expression then declined gradually with progression of spermatogenesis and steroidogenesis. Expression of BMP15 showed positive correlation with seasonally changing testicular 17β-estradiol but negatively with testicular testosterone and 11-ketotestosterone. In vitro treatment of testis with recombinant human BMP15 enhanced the production of estradiol-17β but concurrently suppressed the production of testosterone and 11-ketotestosterone in testis. Though BMP15 did not alter the expression of StAR protein in the testis, it promoted the expression of 3β-hydroxysteroid dehydrogenase and aromatase in fish testis. Thus the present study for the first time demonstrates that fish testis is capable of producing BMP15 and is expressed by the somatic cells unlike mammals wherein it is produced exclusively by germ cells. Study also suggests that BMP15 may modulate the testicular steroidogenesis by altering the expression of steroidogenic enzymes. BMP15 also appears to play crucial role in renewal of spermatogial cells by augmenting the testicular production of 17β-estradiol., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Effects of growth hormone on somatic growth and testicular activities were studied during late quiescence and early recrudescence phases of the reproductive cycle of the catfish, Clarias batrachus. The administration of exogenous growth hormone (GH) during the late quiescence phase (December-January; ambient water temperature-15.2±1°C) did not influence the somatic growth as well as the testicular activity, as no change in body weight, testis weight, plasma level of insulin-like growth factor I (IGF-I) and testicular morphology was detected following GH treatment, though the plasma testosterone was marginally increased. While during the early recrudescence phase (March-April; ambient water temperature-28.1±2°C), GH treatment promoted the production of insulin like growth factor-I and testicular steroidogenic activity in a dose dependent manner, as was evident from the significant increase in the circulating levels of testosterone and estradiol-17β. GH treatment also increased body weight, testicular weight and gonadosomatic index, suggesting its involvement in testicular development. The GH treatment promoted spermatogonial proliferation and accelerated the spermatogenic process in the present catfish. These results, thus, suggest that GH influences the somatic growth and testicular activities depending on the temperature of the rearing water; warmer temperature and longer photoperiod promote testicular steroidogenic and spermatogenic activities in fish. This study has immense practical use in fisheries science., (Copyright © 2013 Elsevier Inc. 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.