Indian Freshwater Murrel Research Articles

Structural modeling and gene expression analysis of phosvitinless vitellogenin (vgc) in the Indian freshwater murrel, Channa punctatus (Bloch, 1793)

Vitellogenin (Vg) is a female-specific egg-yolk precursor protein, synthesized in the liver of fish in response to estrogens. In the present study, complete gene of phosvitinless vitellogenin (vgc) was sequenced, its 3D structure was predicted and validated by web-based softwares. The complete nucleotide sequence of vgc was 4126 bp which encodes for 1272 amino acids and showed the presence of three conserved domains viz. LPD_N, DUF1943 and DUF1944. The retrieved amino acid sequence of VgC protein was subjected to in silico analysis for understanding the structural and functional properties of protein. mRNA levels of multiple vg genes have also been quantified during annual reproductive cycle employing qPCR. A correlation has been observed between seasonal changes in gonadosomatic index with estradiol levels and hepatic expression of three types of vg genes (vga, vgb, vgc) during ovarian cycle of murrel. During preparatory phase, when photoperiod and temperature are low; low titre of E2 in blood induces expression of vgc gene. A rapid increase in the levels of E2 favours induction of vgb and vga genes in liver of murrel during early pre-spawning phase when photoperiod is long and temperature is high in nature. These results suggest that among three vitellogenin proteins, VgC is synthesized earlier than VgA and VgB during oogenesis.

Molecular cloning and bioinformatic characterization of Gonadotropin Inhibitory Hormone (GnIH) and its receptors in the freshwater murrel, Channa punctatus (Bloch, 1793).

Gonadotropin inhibitory hormone belonging to the RFamide peptide family, a hypothalamic neuropeptide, regulates Hypothalamus-pituitary-gonadal (HPG) axis and inhibits gonadal development. GnIH polypeptide precursor has an Arg-Phe-NH2 (RFamide) motif at the C-terminal, which has LPXRF (X = Q or L) domain. The actions of GnIH are mediated through G-protein coupled receptors and upto threereceptors have been characterized in many teleosts. GnIH exerts its inhibitory effect on the HPG axis through direct interaction with GnRH and Kisspeptin neurons in the brain and acts directly on the pituitary gonadotrophs. To decipher the role of GnIH in Indian freshwater murrel, Channa punctatus, we sequenced the cDNA encoding GnIH and its two receptors. The identified GnIH mRNA encodes three RFamide peptides having -MPMRF, -MPQRF, and -LPQRFamide motifs. In silico analysis of the amino acid sequence of GnIH exhibits its molecular and functional properties and the protein-protein interaction with significant factors regulating the HPG axis. The 3-D structure of GnIH and its receptors, provides more relevant information about the active residues of these proteins which might be involved in their functioning and interaction with other proteins. Molecular dynamic simulation of GnIH protein has provided more insight into its dynamic behavior. The expression of GnIH and its receptors, shows an inverse correlation with gonadal development during the annual reproductive cycle.

Structural analysis and characterization of egg-envelope in the Indian freshwater murrel, Channa punctatus.

Egg-envelope, an acellular coat, surrounds the egg and is essential for vitellogenin incorporation. It also plays a pivotal role during fertilization and provides protection to the developing embryo. In the present study, scanning electron microscopy was used to elucidate the structural details of isolated egg-envelopes from the Indian freshwater murrel, Channa punctatus. Several pores and single micropyle were observed on outer surface, whereas inner layer indicated deposition of proteinaceous material. The constituent proteins of egg-envelope were further characterized by Fourier transform infrared (FT-IR) spectroscopy, and electrophoresis and mass-spectrometry (MALDI-TOF-MS/MS). The secondary structure of egg-envelope proteins showed the presence of antiparallel ß-pleated sheets and aromatic amino acids. These proteins resolved into two peptides (130kDa and 68kDa) under denaturing conditions, which exhibited glycoprotein nature. The peptide band with low molecular mass showed significant similarity with transmembrane protein, whereas peptide band with high molecular mass matched with choriogenin protein of other fishes. These results confirm that chorion is derived from precursor protein, Choriogenin, in murrel. Chemical composition of egg-envelope supports that chorion is responsible exchange material and chemical defence during embryogenesis.

Poisonous Effects of Cadmium Chloride On Histological Variations in The Liver of South Indian Fresh Water Murrel, Channa Striatus (Bloch-1793)

Poisonous Effects of Cadmium Chloride On Histological Variations in The Liver of South Indian Fresh Water Murrel, Channa Striatus (Bloch-1793)

Characterization of incomplete vitellogenin (VgC) in the Indian freshwater murrel, Channa punctatus (Bloch).

A novel incomplete vitellogenin (VgC) was purified from the plasma of estradiol-treated male murrel, Channa punctatus, by gel filtration chromatography. The native mass of VgC protein was 180 kDa, and it resolved as a single peptide of 100 kDa on SDS-PAGE. The peptide on subjecting to matrix-assisted laser desorption/ionization-time of flight produced a peptide mass fingerprint. On tandem mass spectrometry, some of these peptides showed mass to charge (m/z) ratio and amino acid sequence similarity with VgC peptides of other teleosts. Phylogenetic analysis revealed a similarity of murrel VgC with fish species of the order Perciformes. Semi-quantitative RT-PCR assay was developed to study expression of vgc gene at variable levels of estradiol exposure. Presence of VgC in males indicates that fish has been exposed to estrogens; hence, it can be used as a biomarker for estrogenic exposure.

Purification, characterization and expression of two vitellogenins in the Indian freshwater murrel Channa punctatus

The present study was undertaken to characterize different vitellogenins in Channa punctatus. Protein purification by gel chromatography followed by fast protein liquid chromatography (FPLC) revealed existence of two different Vg forms. Liquid chromatography tandem mass spectrophotometry (LC-MS/MS) suggested the existence of Vga and Vgb. Cloning of partial sequences of vga and vgb mRNA and phylogenetic analysis substantiated the existence of two vitellogenins. Real time PCR for vga and vgb genes from liver of estradiol-17β (E2) treated fish reveals difference in expression levels of transcripts of these two genes. vgb is expressed at lower dose of estradiol suggesting a higher sensitivity to estradiol. The present study thus proposes different regulatory control for the expression of these two genes and vgb as a superior biomarker than vga to assess exposure of C. punctatus to environmental estrogens.

Immunostimulatory response induced by supplementation of Ficus benghalensis root powder, in the artificial feed the Indian freshwater murrel, Channa punctatus

Methanol extract from the dried aerial root of Ficus benghalensis, was used to evaluate antibacterial activity on the bacterial strains of Aeromonas hydrophila and Escherichia coli, by disc diffusion method. In order to study, if there is any immunostimulatory response of F. benghalensis, immunized fish were fed with supplementary artificial feed containing 5% F. benghalensis dried root powder. There was no marked difference in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in control and treated fish, suggesting that the supplementary feed had no adverse effect on liver or kidney. Serum lysozyme, tissue super oxide dismutase (SOD), percentage phagocytosis, phagocytotic index, nitric oxide (NO), total serum protein and immunoglobulin increased significantly in the treated fish compared to control fish. Serum immunoglobulin levels were estimated by development of a sandwich ELISA, and levels were found to increase with successive immunizations of BSA.

Vitellogenin genes in fish: differential expression on exposure to estradiol

Three types of vitellogenins (Vgs) namely vitellogenin A (VgA), vitellogenin B (VgB) and vitellogenin C (VgC) have been identified in fishes. The existence of VgA and VgB is reported in the Indian freshwater murrel Channa punctatus. Gene-specific primers were designed using available nucleotide sequences in National Centre for Biotechnology Information (NCBI), for amplification of VgA and VgB cDNA. Differential processing of Vgs is evident in many fishes. Adult male murrel expressed both the VgA and VgB genes when estradiol-17β (E(2)) is injected in vivo and Vg levels in blood quantified by Enzyme linked immunosorbent assay (ELISA) showed a dose-related response in such treatments. Cultured hepatocytes on treatment with E(2), however, expressed only VgB as detected by RT-PCR, suggesting different regulatory mechanism for the VgA and VgB genes.

Estradiol Induced Mechanisms Regulating Vitellogenin (VG) and Choriogenin Synthesis and Expression of VG in the Cultured Hepatocytes of Indian Freshwater Murrel Channa punctatus

Introduction: Vitellogenin (Vg) and Choriogenin (Chg) are femalespecific proteins, synthesized in the hepatocytes of teleosts. Several in vivo studies have shown that it is difficult to distinguish the primary effects of a compound from those induced secondarily because liver functions are under the influence of multiple endogenous factors. Therefore, in vitro hepatocyte systems present a better experimental model to investigate mechanism(s) by which Vg and chg are synthesized. Primary cultures of fish hepatocytes have been used to study hepatic structure and function, viz. endocrine regulation, Vg synthesis, hormone receptor expression, and toxicology. The Vg and Chg induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature etc.) and culture system (hepatocyte monolayers, aggregates, liver slices etc.). The main advantages of the hepatocyte Vg and Chg assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. The aim of the present study was to understand the induction of Vg & Chg by estradiol (E2) and expression of Vg genes. Methods: Hepatocytes were isolated following the method of Rani et al.[1]. Hepatocytes were exposed to various doses of E2, optimum dose was selected and used for rest of experiments. Vg and Chg concentrations were determined in culture medium using standardized ELISAs [2] with few modifications. Cells exposed to a constant dose of E2 (500nM) in the presence of actinomycin D (3 and 6μg/ml) or cycloheximide (10 μg/ml). After 48 hrs media were collected from all the groups and processed for Vg and Chg estimation. Statistical Significance was calculated in between different doses of same treated groups by Mann-Whitney Wilcoxon test/student t-test; significance is given as p<0.005. Vg was extracted from the medium by PAGE and cut band was characterized by LC-MS/MS peptide mapping. Total RNA was isolated by TRIzol method from treated and non treated hepatocytes and processed for expression of Vg gene(s) by reverse transcription PCR (RT-PCR). E2 exposed hepatocytes were fixed and processed for localization of Vg and Chg in cytoplasm by immunocytochemistry and for ultrastructural analysis by TEM.

Establishment of ELISA for murrel vitellogenin and choriogenin, as biomarkers of potential endocrine disruption

Vitellogenin (Vg) and choriogenin (Chg) are sensitive biomarkers for testing endocrine disruption in fish. Therefore, we have developed immunoassays for Vg and Chg in the Indian freshwater murrel, Channa punctatus. Vg is a known precursor of egg-yolk proteins, whereas Chg contributes to the formation of egg-envelope. Vg and Chg were induced in male murrel by administration of estradiol-17β. Chg had an apparent native molecular mass of 180 kDa. It consisted of a single peptide with a molecular mass of 110 kDa, whereas native Vg protein (530 kDa) contained 175 kDa peptide. Highly specific polyclonal antibodies against purified plasma proteins, Vg and Chg, were employed for developing competitive enzyme linked immunosorbent assays (ELISAs). The sensitivity of Vg assay was 3.9 ng/mL (working range 15–500 ng/mL) and of Chg assay was 1.56 ng/mL (working range 6–200 ng/mL). The inter- and intra-assay variations were well within acceptable limits. The two antisera did not cross-react with male plasma proteins. Antiserum to Vg did not cross-react with Chg. Similarly, antiserum to Chg showed no correlation with Vg. Further, immunofluorescence and Western blotting confirmed the specificity of Vg and Chg antisera.

Vitellogenin exists as charge isomers in the Indian freshwater murrel, Channa punctatus (Bloch)

Three charge isomeric forms of vitellogenin are reported in the blood of estrogen-treated murrel, Channa punctatus on the basis of the results of native and SDS–PAGE, isoelectric focusing, and Ferguson plot analysis. Vitellogenin was induced in adult murrels by exogenous administration of estradiol-17β and labelled with 32P in vivo. Labelled vitellogenin isolated from other plasma proteins on Ultrogel AcA 34 columns resolve into three bands on native-PAGE. Ferguson plot analysis reveals free electrophoretic mobilities of the three bands as 6.0, 4.5, and 3.7, which are very similar to the isoelectric point values of 5.9, 4.6, and 3.8, respectively. The apparent molecular weight of native murrel vitellogenin is 530 kDa. It consists of a single peptide with a molecular weight of 175 kDa. All the three bands on native PAGE resolves into a single peptide on SDS–PAGE. N-terminal amino acid sequence for murrel vitellogenin peptide is MKAVVLALLL. The protein phosphorus:lipid phosphorus ratio for murrel vitellogenin is 0.9. The total lipid content is 32.8%, consisting of 45% neutral lipids and 30% phospholipids. Phosphatidyl choline is the major phospholipid whereas triglycerides are the major neutral lipids. Results of these analytical analyses indicate that murrel native vitellogenin circulates as three charge isomers; poor in phosphorus but rich in lipids content.

Biochemical changes in the liver of the Indian freshwater murrel, Channa punctatus (Bloch) during estradiol-induced vitellogenin synthesis

Estrogen administration in C. punctatus increases RNA: protein and RNA: DNA ratios and depletes glycogen in the liver. Increase in glucose-6-phosphatase activity accounts for glycogen depletion whereas high activity of pyruvate kinase suggests stimulation of the glycolytic pathway at the pyruvate step to generate ATP and to provide carbon skeleton for the vitellogenin molecule. Five-fold increase in the activity of NADP+-dependant malate dehydrogenase generates NADPH possibly for fatty acid synthesis. Additional energy requirement (NADPH) is met by stimulation of hexose monophosphate pathway as well as NADP+-dependant isocitrate dehydrogenase. Increase in the activity of NADPH-cytochrome c reductase in microsomal fraction suggests stimulation of electron transport chain. Increase in the activity of glutamate pyruvate transaminase can be directly related to the synthesis to specific amino acids needed for the formation of vitellogenin whereas reduction in glutamate oxaloacetate suggests extra hepatic source of amino acids. Reduction in the activity of glutamate dehydrogenase may be due to catabolism.

A thyrotropin-like molecule from the pituitary of an Indian freshwater murrel: comparison of its biological activity with other thyrotropins

Murrel pituitary thyrotropin-like molecule (mTSH) was purified to homogeneity with the help of a convenient and sensitive in vitro assay system where addition of this material to the thyroid follicle incubation stimulated thyroxine (T 4) secretion into the medium. Pituitary extract of a freshwater murrel, Channa punctatus, was solvent extracted to obtain glycoprotein enriched fraction. This was subjected to Sephadex G-100 gel filtration and eluate of void volume (peak I) showed strong TSH activity (as reflected from T 4 secretion) which was further purified by using concanavalin A-Sepharose, FPLC Mono Q and immunoaffinity chromatography. Purified mTSH gave a single band in PAGE, and SDS PAGE revealed two dissimilar subunits, α and β. Addition of increasing concentrations of mTSH, Indian carp TSH (cTSH) and bovine TSH (bTSH) to in vitro murrel thyroid follicle incubations caused a linear increase in thyroxine (T 4) release into the medium, effect was highest with mTSH and lowest with bTSH. However, in in vivo experiments, injections of increasing doses of mTSH to murrel elevated plasma T 4 level in a linear manner while bTSH gave a biphasic response. Addition of mTSH and bTSH to rat or goat thyroid epithelial cell incubations equally stimulated T 4 release into the medium, while cTSH had significantly less effect. Binding affinity ( K a) and receptor occupancy ( B max) of mTSH to murrel thyroid follicular membrane preparation was considerably higher in comparison to cTSH or bTSH whereas both mTSH and bTSH had nearly similar K a and B max with rat thyroid epithelial cell membrane preparation. Findings indicate that mTSH is a more potent TSH as compared to carp and bovine TSH in murrel and has equipotent biological activity as bTSH on rat and goat thyroid.

Trypanosoma punctati n. sp. from the fish Ophicephalus punctatus Bloch, common fresh-water murrel of India.

Blood of O. punctatus, the common Indian freshwater murrel, was found to contain trypanosomes. The rate of infection of this trypanosome was about 5% and mainly occurred in large sized fishes. These trypanosomes belong to a new species and have been given the name Trypanosoma punctati. The parasite measures 30–36 μ in length and 1.02–1.67 μ in width. It is characterized by a long free flagellum measuring about 3/4 of the body length. The nucleus and kinetoplast are relatively large and the cytoplasm is vacuolated with granules concentrated around the vacuoles. This is the first record of any trypanosome from this fish.