Gonadotropin-releasing hormone plays a crucial role in vertebrate reproduction through the hypothalamo-hypophyseal-gonadal axis. While its functional effects are well understood, the transcriptional regulation of gnrh1 remains poorly characterized in teleosts. This study investigates the promoter regulation of catfish gnrh1, i.e., cfgnrh1, in the brain of the Asian stinging catfish, Heteropneustes fossilis. To accomplish this, the 5'-upstream promoter fragments of cfgnrh1 were isolated and characterized using in silico motif analysis, luciferase reporter assays, chromatin immunoprecipitation, and site-directed mutagenesis. The results identified Pax6 as a key regulator of cfgnrh1 transcription. Immunolocalization of Pax6 and Gnrh in the preoptic area and hypothalamus of the H. fossilis brain indicates possible interaction. Polyethylenimine-mediated siRNA transient gene silencing of pax6 confirmed its regulatory importance, as evidenced by the downregulation of cfgnrh1. Collectively, these findings highlight and validate a clear transcriptional interaction between pax6 and cfgnrh1, emphasizing their significant roles in catfish reproduction.

Teleost species display a diverse range of reproductive strategies, consisting mainly of gonochorism and hermaphroditism, either sequentially or simultaneously. This leads to species-specific sex determination processes. Owing to the vast range of teleost species, sex determination and differentiation mechanisms vary in their sexual patterns; therefore, sexual plasticity in gonadal development is inevitable. Under certain environmental conditions, such as fluctuations in temperature, pH, density, social interaction, and hypoxia, gonadal fate is liable to change through a phenomenon referred to as gonadal transdifferentiation. This is a reprogrammable mechanism that can occur either naturally or be induced artificially in certain species at various life stages. Transdifferentiation results from the differential expression of steroidogenic enzyme genes or transcription factors that confer phenotypic, molecular, or morphological changes, such as sexual dimorphism. In addition, many endocrine disruptors manipulate the endocrine system of various fish species by interfering with their genetic pathways or with the profiles of endogenous steroid hormones, causing a shift in the sex ratio of a given population. In the current modern chemical environment that includes endocrine disrupting chemicals, fish can undergo gonadal transdifferentiation, which can lead to functional sex reversal. This review summarizes gonadal transdifferentiation and provides evidence of diverse strategies found in teleosts, from molecular-level changes to feminization or masculinization of gonads to sex reversal, induced either naturally or unnaturally. We end with a discussion of the adverse effects of endocrine disruptors and summarize areas for future research.

Steroidogenic acute regulatory protein (Star) is a key factor that predominantly mediates the rate-limiting step of steroidogenesis which involved in the translocation of cholesterol from the outer to inner mitochondrial membrane in most of the vertebrates. To understand the transcriptional regulation of star (star1) in fish, the 5' upstream region of the star was cloned from both common carp and catfish genomic libraries and in silico promoter motif analysis predicted putative binding sites of C-jun, AP-1, Gata2, and Oct1 in common carp while C-jun, Gata1, Gata2, and Oct1 binding sites were predicted in star promoter of catfish. Luciferase reporter assay through sequential deletion constructs in TM3 cells revealed significantly high promoter activity (P < 0.001) having C-jun site in both common carp and catfish but not with other motifs largely. Increased activity of star promoter was observed upon hCG induction, in vitro, indicating gonadotropin dependency. Site-directed mutagenesis and chromatin immunoprecipitation assays further confirmed the binding of C-jun to the upstream promoter region of star in both species. Expression of c-jun was significantly (P < 0.05) higher during the spawning phase of the testicular cycle in both species. Both c-jun and star were found to be gonadotropin inducible and androgen-dependent in both species. Further, transient gene silencing through c-jun-siRNA in both the species decreased expression of c-jun, star, and other AP-1 factors such as c-fos, junB, sox family genes, steroidogenic enzyme genes, sperm-related factor, testis-related genes, along with reduced testosterone and 11-ketotestosterone levels. The protein level of C-jun was decreased upon transient gene silencing in both species. This was the first report to show that C-jun binds to the upstream promoter motif of stars in both common carp and catfish and also adds valuable new insights that define the molecular mechanisms of c-jun regulating star transcription during steroidogenesis in teleost and probably testicular function.

Testicular function in fish is mediated by steroids, with cytochrome P450 11B1, mitochondrial, or 11β-hydroxylase, encoded by cyp11b1, and cytochrome P450 17A1, or 17α-hydroxylase/17,20-lyase, encoded by cyp17a1, being key steroidogenic enzymes. However, the regulation of cyp11b1 and cyp17a1 has not yet been studied in fish. To address this, the 5' upstream regions of cyp11b1 and cyp17a1 from catfish, Clarias batrachus, were cloned and sequenced. Predicted putative transcription factor binding sites included Ad4bp/sf-1, Foxp1, Pax1, Pax2, Gata1, and Oct1 in both promoter fragments. Luciferase reporter assays in TM3 mouse Leydig cells, with and without human chorionic gonadotropin (hCG) induction, showed significant promoter activity in constructs containing ad4bp/sf-1 and foxp1, but not with the other transcription factors. Site-directed mutagenesis and chromatin immunoprecipitation further confirmed the binding of Ad4bp/sf-1 to the promoters of both cyp11b1 and cyp17a1, while Foxp1 binding was observed only in the cyp11b1 promoter. Immunolocalization of Ad4bp/sf-1 revealed its presence in interstitial/Leydig cells and also in the testicular lumen. The expression of ad4bp/sf-1 increased after hCG induction in vivo and following 11-ketotestosterone (11-KT)/methyl testosterone stimulation in vitro, indicating gonadotropin- and androgen-dependent regulation. Additionally, transient gene silencing of ad4bp/sf1 using small interfering RNA (siRNA) decreased the expression of cyp11b1, cyp17a1, and foxp1, suggesting co-regulation. Finally, decreased serum testosterone and 11-KT levels upon ad4bp/sf-1 siRNA silencing further support its role in regulating male steroidogenesis in males.

Induction of spawning in catfish is well demonstrated in females while stimulation of spermiation in males seems difficult. This has led to least understanding of gene profile changes during testicular maturation. Expression of the factors after hormonal induction that control reproductive processes has become an intense research area in fish endocrinology. In view of this, de novo assemblies of testicular transcriptomes of the Asian catfish, Clarias batrachus and the African catfish, C. gariepinus, were performed to identify genes/factors involved in testicular maturation. For testicular induction, human chorionic gonadotropin (hCG) was administered in vivo to both the catfish species during active phase of reproduction. 1,68,071 and 1,26,232 assembled transcripts were obtained from C. batrachus and C. gariepinus testicular transcriptomes and were annotated using different databases. Further, in silico analysis suggested the presence of several transcripts that were involved in steroidogenesis and male reproduction. Comparison of transcriptomes of both species revealed the presence of certain unique genes related to reproduction differentially. Expression profiles after hCG induction in testis showed higher expression of certain steroidogenesis related genes such as star, cyp11b1, cyp17a and cyp21a in both the species. Further, expression levels of crucial factors related to testis, such as dmrt1/2/3, were also found to be significantly higher after gonadotropin induction. However, amh, tgfα and dmrt4/5 expression levels were significantly low. Factors related to male reproduction i.e., tekt1, tekt2, sox9, spag1, spata2 and spata7 were found to be differentially upregulated in hCG treated testicular tissues of both the species. Histology analysis indicated that the gonadotropin induction either short or long term is a better strategy to highlight expression profile changes during testicular maturation in teleost.

Peptide YY (PYY) is an anorectic brain-gut pancreatic peptide that helps in feeding regulation by reducing appetite and is well characterized in mammals. The role of PYY in relation to brain is least studied in mammals as well as in lower vertebrates including fish, however high expression was evident in male reproductive tissue. In this regard, this study attempts to evaluate the significance of PYY in the brain of common carp, Cyprinus carpio. As a first step, the cDNA of PYY from brain of adult male carp was cloned. Following which expression analysis was performed using juvenile and adult fish. The differential distribution pattern in various regions of brain and ontogeny expression analysis indicated that PYY may involve in physiological processes related to brain-pituitary axis. In addition, a significant decrease in neuropeptide Y expression was observed upon PYY- endoribonuclease-prepared small interfering RNA transfection in brain cells, in vitro indicating plausible PYY-NPY interaction in brain-pituitary axis of common carp.

Tyrosine hydroxylase (Th) is an allosteric rate-limiting enzyme in catecholamine (CA) biosynthesis. The CAs, dopamine (DA), norepinephrine (NE), and epinephrine are important neurotransmitters wherein DA contributes a key role in the central nervous system of vertebrates. The present study evaluated DA and Th's significance in DA-ergic activity and neurodegeneration upon 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure in catfish. Further, the expression of certain brain-and ovary-related genes measured through qPCR were downregulated upon MPTP treatment which is in accordance with the decreased levels of L-Dopa, DA, and NE levels estimated through HPLC-ECD. Additionally, TEM analysis depicted structural disarray of brain upon MPTP exposure and also decreased serum levels of testosterone, 11-ketotestosterone, and estradiol-17β. MPTP treatment, in vitro, using primary brain cell culture resulted in diminished cell viability and increased ROS levels leading to elevated apoptotic cells significantly. Consequently, the study highlights the MPTP-induced neurodegeneration of the Th and DA-ergic activity in corroboration with female brain-related genes downregulation, also gonadal function as evidenced by depleted sex steroids level and low expression of ovary-related genes.

Occurrence of neurotoxic chemicals in the aquatic environment is on the rise posing a potential threat to aquatic biota including fish. In teleosts, zebrafish has become a popular model organism for toxicological studies and testing strategies. However, over the decade, siluriformes (catfish) are also finding ever increasing application being robust as well as their adaptability to adverse ecological conditions, surgical interventions, and tolerant models for toxicity studies and manipulations. Such information can infer potential effects occurring to other species exposed to neurotoxins in their aquatic environment and predicting potential risks of a chemical for the aquatic ecosystem. The aim of this review is to compare and interpret recent results published concerning neuro-behavioral and morphological disturbances caused by toxicants/pollutants providing a holistic view of potential neurotoxic outcomes in catfish. Overall, this review summarizes various effects of toxicants/pollutants in terms of neurotoxicity and neurodegeneration associated with behavioral phenotypes.

Background: Cytochrome P450 11?-hydroxylase (H11b) converts testosterone (T) into 11-hydroxytestosterone. In Clarias batrachus (catfish), H11b occurs in regular form and has three variants as well. The variant forms contain the complete steroid binding site, though they lack other regions. Thus, they are suggested to be involved in regulating steroid concentration. Method: The structure of H11b and its three variants, Var1, Var2 and Var3 were modeled by homology modeling using human cholesterol side-chain cleavage enzyme, CYP11A1 as a template. The binding of T with the H11b in holo and apo (non-heme state) forms and with all three variants was studied using docking. Furthermore, molecular dynamics simulations of the protein-ligand complexes were carried out and the binding energies of T were calculated using the molecular mechanics-Poisson?Boltzmann surface area (MM-PBSA) method. Results: The analysis showed that the regular protein in its apo-form had the largest binding affinity with T followed by holo-form and Var1. The higher binding affinity was found to be driven by van der Waals interactions. Var3 had the least binding affinity possibly due to the high polar solvation energy. Conclusion: The differences in the binding affinities suggest that the competitive binding of T between H11b and its variants could be the key factor in regulating the synthesis of 11-hydroxytestosterone.

Two putative boar pheromones (Androstenone and Androstenol) were discovered in endangered mouse deer during captive breeding program. This study further examined the molecular characteristics, pheromone synthesis pathway, and the functional role of these pheromones in reproduction of mouse deer. CYP17A1 and CYB5 genes were cloned and expressed in HEK-293, COS-7 cell lines and gonads of mouse deer to investigate CYP17A1 gene&rsquo;s andien-&beta;-synthase activity towards synthesis of sex pheromones in mouse deer. An enzyme immunoassay was also developed and standardized to measure the fecal androstenone during reproductive cycles of mouse deer. Results showed that mouse deer&rsquo;s CYP17A1 gene possesses andien-&beta;-synthase activity and could transform pregnenolone into 5,16-androstadien-3&beta;-ol. The expression of CYP17A1 gene upregulated in the testis and ovary, compared to other tissues in mouse deer. Significantly elevated pheromones and estrogens were recorded prior to delivery and postpartum estrus / mating in mouse deer. Further, there were weak correlations between fecal pheromones and estrogens/ androgens in mouse deer during breeding season. The findings suggest that the boar pheromones might play a direct role in the reproductive activities of mouse deer which might be used for breeding of mouse deer elsewhere.

In the present study, effects of synthetic human Kisspeptin1 (hKiss1) and catfish Kisspeptin2 (cfKiss2) on the hypothalamic - pituitary - ovarian (HPO) axis, and induction of final oocyte maturation and ovulation were investigated in prespawning female stinging catfish (Heteropneustes fossilis) under in vivo and in vitro conditions. Gene expression was quantified by real time quantitative PCR and steroids were measured by specific enzyme linked immunoassays. Intraperitoneal (ip) injections (1, 2, 3 ng/g body weight, BW) of synthetic hKiss1 and cfKiss2 stimulated hypothalamic, pituitary and ovarian gnrh1 and gnrh2 expression at 24 h and the effect was higher after the cfKiss2 treatment. In vitro incubation of hypothalamus, pituitary and ovary pieces with the Kiss peptides (5, 10, 20 nM) produced a similar effect. GPR54 (Kiss1 receptor) antagonist peptide234, when given ip (5, 10, 20 ng/g BW) or incubated in vitro (5, 10, 20 nM), inhibited the gnrh1 and gnrh2 expression at 24 h. The supplementation with hKiss1 or cfKiss2 restored the inhibition due to peptide234 in vivo and in vitro and the effect was higher in the cfKiss2 combination group. Both hKiss1 and cfKiss2 altered the expression of pituitary gonadotropin (Gth) subunit genes follicle-stimulating hormone beta (fsh beta), luteinizing hormone beta (lh beta) and glycoprotein alpha (gp alpha) in vivo and in vitro. The expression of fsh beta was more sensitive to the treatments than lh beta expression and the effect was greater in the cfKiss2 groups. Peptide234 in vivo and in vitro inhibited the expression of the Gth genes and the effect was reversed and restored in the hKiss1 and cfKiss2 combination groups. The Kiss peptide treatments in vivo or in vitro stimulated both plasma and ovarian levels of estradiol-17 beta, progesterone and 17,20 beta-dihydoxy-4-pregnen-3-one levels. The peptide234 treatment inhibited, or elicited a decreasing trend on the steroid levels both in vivo and in vitro, and the inhibition was reversed by the hKiss1 and cfKiss2 combination treatments. Incubation of post vitellogenic follicles with hKiss1 or cfKiss2 stimulated germinal vesicle breakdown (GVBD) and ovulation. The inhibition due to peptide234 was reversed in the combination groups. Ovulation was not elicited or unaffected in the peptide234 treated groups. The data show that the Kiss peptides act downstream the HPO axis to stimulate oocyte maturation and ovulation, and cfKiss2 peptide is functionally more effective than hKiss1.

Identification of germ cell markers in fishes is crucial to track the germ cell differentiation and migration for manipulation of the cells to study sexual differentiation as well as to carry out transgenic transplantation techniques. Several germ cell-specific markers such as vasa, cnbp, dnd, nanos3, cbx2, amh, dmrt1, Ly75/CD205 have been characterized so far in fishes using localization and expression analysis, which have highlighted the spatio-temporal pattern of expression in early gonadal development. Incidentally, seasonal breeders show dramatic changes during gonadal recrudescence, which might also influence germ cell differentiation and growth to entrain the reproductive cycle. Hence, an in-depth analysis of the gonadal cycle is required to delineate germ cell progress, differentiation, and maturation explicitly. In this context, fishes undergoing gonadal recrudescence for the seasonal cycle show germ cell proliferation differentially. Most of these germ cell markers belong to the DEAD-box protein family of ATP-dependent RNA helicases sharing consensus motifs and clustering in phylogenetic analysis. These markers were found to be well-conserved throughout evolution. In situ hybridization approaches confirmed the germ cell specific distribution of these molecular markers. In addition, several genes such as fgf and gsdf seem to facilitate germ cell development and differentiation. Hence, more detailed studies on these factors will facilitate a better understanding of germ cell development. This review highlights various germ cell markers in fishes and their immense potential to use these cells for germ cell transplantation. The extensive knowledge of the germ cell markers can also be exploited to carry out other biotechnological experiments aiming at the preservation of genetic information of endangered species or the analytical study of gonadogenesis.

In teleost, ovarian steroidogenesis governed by the neuroendocrine system is also regulated by several transcription factors of gonadal origin. Investigating the synchronized interactions between the transcriptional and the hormonal factors is vital to comprehend the mechanisms that lead to gonadal differentiation. This study signifies the role of sry-related box (sox) 19 in ovarian steroidogenesis regulation of the common carp, Cyprinus carpio. Analysis of tissue distribution displayed higher sox19 expression in brain and ovary, and gonadal ontogeny showed higher expression of sox19 at 80 days post hatch (dph). Higher sox19 mRNA expression during spawning and increase of sox19 post human chorionic gonadotropin induction substantiate gonadotropin dependency. Estradiol-17β treatment but not 17α-methyl-di-hydroxy-testosterone to 50 dph common carp for inducing mono-sex, elevated sox19 expression substantially. Sox19 protein was observed in granulosa cells of the follicular layer in common carp ovary. Higher sox19 expression was detected in isolated granulosa and theca cells, in vitro. Transient gene silencing with sox19-siRNA caused downregulation of various ovary-related genes including those specific to activator protein-1 factors, fibroblast growth factors, wnt-signaling, steroidogenic genes along with certain transcription factors. Serum 17α, 20β-dihydroxy-4-pregnen-3-one and estradiol-17β reduced significantly post sox19 silencing, in vivo. Concomitantly, a decrease in aromatase activity was detected post sox19-siRNA treatment, in vivo. This study demonstrates the impact of sox19 in the regulation of common carp ovarian growth and steroidogenesis.

Sex reversal is one of the characteristic properties of sexual plasticity in bony fishes wherein both natural and induced sex change happens at various stages of life cycle in different species. Sex determination in gonochoristic species is genetically regulated, wherein the same sex is retained throughout their life span whereas hermaphrodites change their sex during development or adulthood. In sequential hermaphrodites, serial sex change occurs at different points of life cycle. Concurrently, synchronous hermaphrodites function as both the sexes during spawning. Other variables like temperature, pH and social factors can trigger sex reversal in teleost. Sex reversal through gene mutations and chemicals/hormones, including sex steroids, can be induced mostly at early developmental stages but natural sex reversal can occur at any time. Sex reversal mechanism shows morphological to molecular changes, which are ideal for identification of sex-specific gene markers. In fact, gonadal transdifferentiation occurs at the molecular level through differential expression of transcription factors and steroidogenic enzyme genes vis-a-vis hormones, thereby imparting phenotypic or structural changes. In addition, brain shows sexual dimorphism which is mostly consequential to gonadal sex development and occasionally either causative. The major breakthrough in this line is the identification of sex determining genes such as dmy/dmrt1Yb, gsdfY, sox3 in the Japanese medaka and amhY in Patagonian pejerrey. Incidentally, the induction of mono-sex population by favouring one sex due to sex-specific differences in growth is an important economic boom for aquaculture. This review comprehensively highlights key molecular factors involved in natural and induced sex reversal conditions to illustrate teleostean sexual plasticity and its application perspectives.

We discovered odorous 16-androstenes (Androstenone and Androstenol) in endangered mouse deer during a captive breeding program. This study examined the molecular characteristics, their synthesis pathway, and the possible functional role of these compounds in the reproduction of mouse deer. CYP17A1 and CYB5 genes were cloned and expressed in HEK-293, COS-7 cell lines, and gonads of mouse deer to investigate the CYP17A1 gene’s andien-β-synthase activity towards the synthesis of 16-androstenes in mouse deer. An enzyme immunoassay was further developed and standardized to measure fecal androstenone during the reproductive cycles of mouse deer. Results showed that the mouse deer CYP17A1 gene possesses andien-β-synthase activity and could transform pregnenolone into 5,16-androstadien-3β-ol. The expression of the CYP17A1 gene upregulated in the testis and ovary compared to other tissues in mouse deer. Significantly elevated androstenone and estrogens were recorded prior to delivery and postpartum estrus/mating in mouse deer. Further, there were weak correlations between fecal androstenone and estrogens/androgens in mouse deer during the breeding season. These findings suggest that androstenone probably plays a role in the reproductive activities of mouse deer. This knowledge can be used for captive breeding programs of mouse deer in India and elsewhere.

Carbon nanotubes production has been rapidly increasing for many potential applications, however, the environmental impact of this nanomaterial needs to be comprehended. The present work focused on unraveling the effects of single-walled carbon nanotubes (SWCNT) in the common carp, Cyprinus carpio. The physicochemical properties of SWCNT were analyzed with X-ray diffraction, Fourier transforms infra-red, UV-Vis absorption, transmission electron microscopy (TEM), and Raman spectroscopy before testing for exposure impact. The effects of SWCNT, were investigated by exposing to two doses viz., 10 and 50 μg/L, for 7 days in adult common carp, in vivo. Expression of key steroidogenic and transcription factor genes related to testis and brain were downregulated after the treatment. The concomitant decreases in serum testosterone and 11-ketotestosterone levels revealed the impact of SWCNT after exposure. Further, SWCNT exposure induced antioxidant enzymes namely glutathione-S-transferases, superoxide dismutase, and catalase in both testis and brain. Concurrently, histological and TEM analysis of testis revealed structural disarray. In addition, SWCNT treatment, in testicular and brain primary cell cultures decreased cell viability with an increase of reactive oxygen species levels, leading to a significant elevation of apoptotic cells. In line with this, low mitochondrial membrane potential and DNA damage were also observed during post SWCNT treatment. Taken together, transient exposure of SWCNT causes toxic effects and alters testicular and brain function in the common carp. Thus, the discharge of carbon nanotubes poses a greater risk to the aquatic environment warranting regulatory measures.