Raju Murugananthkumar

Dimorphic Expression of Various Transcription Factor and Steroidogenic Enzyme Genes during Gonadal Ontogeny in the Air-Breathing Catfish, Clarias gariepinus

In the present study the expression of 13 genes known to be involved in sex differentiation and steroidogenesis in catfish was analyzed during gonadal ontogeny by quantitative real-time RT-PCR. Dmrt1 and sox9a showed exclusive expression in male gonads while ovarian aromatase (cyp19a1) and foxl2 were abundant in differentiating female gonads. Most of the genes related to steroidogenesis were expressed only after gonadal differentiation. However, genes coding for 3β-hydroxysteroid dehydrogenase (3β-hsd), 17α-hydroxylase/C17–20 lyase type 1 (cyp17) and steroidogenic acute regulatory protein (star) were barely detectable during gonadal differentiation. Ovarian aromatase, cyp19a1, which is responsible for estradiol-17β biosynthesis in females, was expressed very early in the undifferentiated gonads of catfish, around 30–40 days post hatch (dph). The steroidogenic enzyme, 11β-hydroxylase (cyp11b1) required for the production of 11-ketotestosterone (11-KT) was expressed only after differentiation of testis. These results suggest that estradiol-17β has a critical role in ovarian differentiation, while the role of 11-KT in testicular differentiation is doubtful. In conclusion, dimorphic expression of dmrt1 and sox9a in gonads during early development is required for testicular differentiation, and sex-specific expression of cyp19a1 and foxl2 in females plays a critical role in ovarian development. Our study reveals that the critical period of gonadal differentiation in catfish starts around 30–40 dph when sex-specific genes showed differential expression.

Endosulfan and flutamide impair testicular development in the juvenile Asian catfish, Clarias batrachus.

Endosulfan and flutamide, a widely used pesticide and a prostate cancer/infertility drug, respectively, have an increased risk of causing endocrine disruption if they reach water bodies. Though many studies are available on neurotoxicity/bioaccumulation of endosulfan and receptor antagonism of flutamide, only little is known about their impact on testicular steroidogenesis at molecular level. Sex steroids play an important role in sex differentiation of lower vertebrates including fishes. Hence, a small change in their levels caused by endocrine disruptors affects the gonadal development of aquatic vertebrates significantly. The aim of this study was to evaluate the effects of endosulfan and flutamide on testis-related transcription factor and steroidogenic enzyme genes with a comparison on the levels of androgens during critical period of catfish testicular development. We also analyzed the correlation between the above-mentioned genes and catfish gonadotropin-releasing hormone (cfGnRH)-tryptophan hydroxylase2 (tph2). The Asian catfish, Clarias batrachus males at 50 days post hatch (dph) were exposed to very low dose of endosulfan (2.5 μg/L) and flutamide (33 μg/L), alone and in combination for 50 days. The doses used in this study were far less than those used in the previous studies of flutamide and reported levels of endosulfan in surface water and sediments. Sampling was done at end of the treatments (100 dph) to perform testicular germ cell count (histology), measurements of testosterone (T) and 11-ketotestosterone (11-KT) by enzyme immunoassay and transcript quantification by quantitative real-time PCR. In general, treatments decreased the expression of several genes including testis-related transcription factors (dmrt1, sox9a and wt1), steroidogenic enzymes (11β-hsd2, 17β-hsd12 and P450c17), steroidogenic acute regulatory protein and orphan nuclear receptors (nr2c1 and Ad4BP/SF-1). In contrast, the transcripts of cfGnRH and tph2 were elevated in the brain of all treated groups with maximum elevation in the endosulfan group. However, combination of endosulfan and flutamide (E+F) treatment showed minor antagonism in a few results of transcript quantification. Levels of T and 11-KT were elevated after flutamide and E+F treatments while no change was seen in the endosulfan group signifying the effect of flutamide as an androgen receptor antagonist. All the treatments modulated testis growth by decreasing the progression of differentiation of spermatogonia to spermatocytes. Based on these results, we suggest that the exposure to endosulfan and flutamide, even at low doses, impairs testicular development either directly or indirectly at the level of brain.

Two-dimensional proteomic analysis of gonads of air-breathing catfish, Clarias batrachus after the exposure of endosulfan and malathion.

Endocrine disrupting chemicals have raised public concern, since their effects have been found to interfere with the physiological systems of various organisms, especially during critical stage of development and reproduction. Endosulfan and malathion, pesticides widely used for agricultural purposes, have been known to disrupt physiological functions in aquatic organisms. The current work analyzes the effects of endosulfan (2.5 parts per billion [ppb]) and malathion (10 ppb) on the reproductive physiology of catfish (Clarias batrachus) by evaluating protein expression profiles after 21 days of exposure. The proteomic profile of testis and ovary after exposure to endosulfan showed downregulation of proteins such as ubiquitin and Esco2, and upregulation in melanocortin-receptor-2 respectively. Malathion exposed ovary showed upregulated prolactin levels. Identification of proteins differentially expressed in gonads due to the exposure to these pesticides may serve as crucial indications to denote their disruptive effects at the level of proteins.

Gene expression analysis in gonads and brain of catfish Clarias batrachus after the exposure of malathion.

Pesticides like malathion have the potential to disrupt development and reproduction of aquatic organisms including fishes. To investigate the likely consequences of malathion exposure at low doses in juvenile catfish, Clarias batrachus, we studied the expression pattern of genes encoding certain transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes which are known to be involved in gonadal development along with histological changes. To compare further, we also analyzed certain brain specific genes related to gonadal axis. Fifty days post hatch catfish fingerlings were exposed continuously to 1 and 10 µg/L of malathion for 21 days. Results from these experiments indicated that transcript levels of various genes were altered by the treatments, which may further affect the gonadal development either directly or indirectly through brain. Histological analysis revealed slow progression of spermatogenesis in testis, while in ovary, the oil droplet oocytes were found to be higher after treatment (10 µg/L). Our findings revealed that the exposure of malathion, even at low doses, hinder or modulate early gonadal development differentially by targeting gene expression pattern of transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes with an evidence on histological changes. Further, some of the genes showed differential expression at the level of brain in male and female sex after the exposure of malathion.

In vivo induction of human chorionic gonadotropin by osmotic pump advances sexual maturation during pre-spawning phase in adult catfish.

Gonadal maturation is a critical event wherein gonads, under the influence of several hormones and factors, undergo cyclic morphological and physiological changes to produce functional gametes during the spawning phase. However, artificial induction can be effectively used to advance the maturation of gonad vis-à-vis spawning like behavior in seasonal breeders during the off-breeding season. In the present study, osmotic pumps loaded with 5000IU of human chorionic gonadotropin (hCG) or saline as control were implanted intraperitoneally for 21days during the pre-spawning phase (May-June) in catfish Clarias batrachus and C. gariepinus. Significant increase in gonado-somatic index and sperm motility, and in the levels of certain sex steroids were observed in the hCG treated catfish when compared to control while estradiol-17β (E2) was low. Histological analysis in hCG treated testis revealed densely packed sperm and/or spermatids inside the lumen wherein the control testis displayed normal characteristics of the pre-spawning phase. In females, histological analysis showed a significant increase in post-vitellogenic full-grown immature follicles as seen in the spawning phase. In accordance with this, the steroid hormone profile correlated well with steroidogenic shift from E2 to 17α,20β-DP indicating oocyte maturation. However, in the control ovaries of C. batrachus, perinucleolar and pre-vitellogenic oocytes were seen to be predominant. In addition, when compared with the control, the hCG treated group displayed a significant increase in the transcripts of several genes associated with gonadal growth. Taken together, artificial induction by slow release of hCG is an effective strategy to advance sexual maturation in catfish in a programmed manner.

Expression analysis and localization of wt1, ad4bp/sf-1 and gata4 in the testis of catfish, Clarias batrachus: Impact of wt1-esiRNA silencing.

In teleosts, a comprehensive role or interaction of wt1, ad4bp/sf-1 and gata4 genes in relation to gonadal development and/or recrudescence was never attempted. Present study aimed to identify the involvement of these genes during testicular development of catfish, Clarias batrachus. Dominant expression of wt1 and gata4 was observed in developing and adult testis, while ad4bp/sf-1 showed steady expression. Localization of these genes in adult testis revealed their presence in spermatogonia, spermatocytes and interstitial/Leydig cells. Significant high expression during pre-spawning and spawning phases, and upregulated levels of these genes after hCG induction authenticated gonadotropic regulation. Transient silencing of wt1-esiRNA displayed decrease in wt1 expression, which further downregulated the expression of ad4bp/sf-1 and gata4, and certain steroidogenic enzyme genes related to androgen production. These results suggest that wt1 might target ad4bp/sf-1 and gata4 expression, and also have regulatory influence either indirectly or directly on the steroidogenic enzyme genes of catfish.

Cloning, localization and differential expression of Neuropeptide-Y during early brain development and gonadal recrudescence in the catfish, Clarias gariepinus

Neuropeptide-Y (NPY) has diverse physiological functions which are extensively studied in vertebrates. However, regulatory role of NPY in relation to brain ontogeny and recrudescence with reference to reproduction is less understood in fish. Present report for the first time evaluated the significance of NPY by transient esiRNA silencing and also analyzed its expression during brain development and gonadal recrudescence in the catfish, Clarias gariepinus. As a first step, full-length cDNA of NPY was cloned from adult catfish brain, which shared high homology with its counterparts from other teleosts upon phylogenetic analysis. Tissue distribution revealed dominant expression of NPY in brain and testis. NPY expression increased during brain development wherein the levels were higher in 100 and 150days post hatch females than the respective age-matched males. Seasonal cycle analysis showed high expression of NPY in brain during pre-spawning phase in comparison with other reproductive phases. Localization studies exhibited the presence of NPY, abundantly, in the regions of preoptic area, hypothalamus and pituitary. Transient silencing of NPY-esiRNA directly into the brain significantly decreased NPY expression in both the male and female brain of catfish which further resulted in significant decrease of transcripts of tryptophan hydroxylase 2, catfish gonadotropin-releasing hormone (cfGnRH), tyrosine hydroxylase and 3β-hydroxysteroid dehydrogenase in brain and luteinizing hormone-β/gonadotropin-II (lh-β/GTH-II) in pituitary exhibiting its influence on gonadal axis. In addition, significant decrease of several ovary-related transcripts was observed in NPY-esiRNA silenced female catfish, indicating the plausible role of NPY in ovary through cfGnRH-GTH axis.

Copper Nanoparticles Differentially Target Testis of the Catfish, Clarias batrachus: In vivo and In vitro Study

Present study examined the impact of copper (Cu) as nanoparticle (Cu-NPs) and soluble form (CuSO4) in adult male catfish Clarias batrachus. Following treatment, upregulation of transcripts of several testis-related genes and increased androgen levels were evident. Though profound promotional effect was seen at gene and androgen levels, structural analysis revealed completely different scenario. Histological observations of testis showed disruption of basal lamina, distinct spermatogonium and enlarged spermatocytes in the treated groups. Quite alarmingly, transmission electron microscopic analysis showed damage of testicular lumen with irregular basal lamina and distinctive spermatocytes. In addition, Sertoli cells and erythrocytes exhibited damage after Cu treatments. Cytotoxic effects of Cu in catfish primary testis and TM3 Leydig cells showed cytotoxicity which is correlated well with morphological and flow cytometric analysis. Taken together, the results demonstrated that low dose of Cu/Cu-NPs can be detrimental to catfish testicular recrudescence vis-a-vis reproduction and the toxic effect manifest drastically in in vitro cultures.

Enterotoxic effects of Aeromonas hydrophila infection in the catfish, Clarias gariepinus: Biochemical, histological and proteome analyses

Aeromonas hydrophila is considered as a potential risk to fish populations in the aquaculture industry and could also pose a serious threat to humans. In this study, the impact of A. hydrophila infection in the air-breathing catfish, Clarias gariepinus was analyzed using a multidimensional approach. Aeromonas hydrophila (1 × 107 cells) was injected into C. gariepinus intraperitoneally and maintained at an ambient temperature and photoperiod with periodical monitoring for morphological changes. After 7 days post-infection, tissue samples of the gills, liver, intestine, and kidney were subjected to biochemical, histological, transmission electron microscope (TEM) and proteomic analyses. Observed results indicated distinct morphological changes with the significant increase of ROS and oxidative stress enzymes (CAT and SOD) in tissues of the infected group when compared to the control. Histological analysis in infected fish revealed the presence of pyknotic nuclei, early stages of necrosis in the liver, degradation of renal tubules and widened sinusoidal space in kidneys along with enlargement of the epithelial region in the intestine. TEM analysis of the infected intestine showed degeneration of villi and the presence of multinucleated erythrocytes. Two-dimensional proteomic and mass spectrometry analysis of intestine and liver displayed up-regulation of several immune regulatory proteins such as proteasome subunit 3 protein, prolactin and intermediated filament protein; and down-regulation of proteins including actin, serine/arginine-rich splicing factor and carbonic anhydrase. Taken together, these results suggest that the identified proteins may have a role in immune regulation against A. hydrophila infection in C. gariepinus and support further investigations of host-pathogen interactions.

Molecular cloning, expression analysis and transcript localization of testicular orphan nuclear receptor 2 in the male catfish, Clarias batrachus.

Testicular receptor 2 (TR2; also known as Nr2c1) is one of the first orphan nuclear receptors identified and known to regulate various physiological process with or without any ligand. In this study, we report the cloning of full length nr2c1 and its expression analysis during gonadal development, seasonal testicular cycle and after human chorionic gonadotropin (hCG) induction. In addition, in situ hybridization (ISH) was performed to localize nr2c1 transcripts in adult testis and whole catfish (1day post hatch). Tissue distribution and gonadal ontogeny studies revealed high expression of nr2c1 in developing and adult testis. Early embryonic stage-wise expression of nr2c1 seems to emphasize its importance in cellular differentiation and development. Substantial expression of nr2c1 during pre-spawning phase and localization of nr2c1 transcripts in sperm/spermatids were observed. Significant upregulation after hCG induction indicate that nr2c1 is under the regulation of gonadotropins. Whole mount ISH analysis displayed nr2c1 expression in notochord indicating its role in normal vertebrate development. Taken together, our findings suggest that nr2c1 may have a plausible role in the testicular and embryonic development of catfish.