Gonadotropin inhibitory hormone receptors (GnIHRs): Molecular characterization and synergistic effect of different drugs in Indian major carp, Labeo catla.

After the discovery of Gonadotropin-inhibitory hormone (GnIH) in birds in 2000, it showed different roles in different vertebrate classes and even in different species of same classes. In birds and mammals, GnIH inhibits the expression of gonadotropins during reproduction, while in fishes it exerts both inhibitory and stimulatory effect on reproduction. The current study evaluates the role of GnIH during reproduction in Labeo catla. The partial cDNA sequence of GnIHR1 and GnIHR3 receptor genes was identified by degenerate PCR. The mRNA expression analysis of GnIHRs during different reproductive phases showed that the expression of all three GnIH receptor genes is highest during spawning phase. The expression of GnIH receptors is detected in both brain and gonads except for GnIHR3 which only expressed in gonads. The in vivo experiments with GnIH antagonist, RF313 drastically reduced the expression level of reproduction related genes like LH, FSH, and GnRH at 1 h post-injection. In another experiment the surge induced by cGnIH-III peptide on gonadotropins gene expression is further increased when co-injected with LHRHa. However, co-injection of melatonin along with cGnIH-III peptide had opposite effects. These results showed that the GnIH/GnIHRs system has positive effect on reproduction in L. catla.

Characterization, Docking and Molecular Dynamics Simulation of Gonadotropin-Inhibitory Hormone Receptor (GnIHR2) in Labeo Catla.

BACKGROUND/AIMS: GnIH receptors (GnIHRs) belong to the family of G-protein coupled receptors (GPCRs) and play a key role in the regulation of reproduction from fishes to mammals, either by inhibiting or stimulating the expression of gonadotropins. The aim of this study was to characterize GnIH receptor (GnIHR2) from Indian Major Carp, Labeo catla and its docking and simulation with GnIH antagonist RF313. METHODS: The full length sequence of GnIHR2 was obtained with RACE PCR. The docking analysis of RF313 with GnIHR2 receptor was performed with AutoDock v. 4.2.6 and molecular dynamics (MD) simulation with GROMACS 5.0. RESULTS: In the present study, we cloned full-length cDNA (1733 bp) of GnIHR2 from the brain of L. catla. The phylogenetic analysis showed clustering of catla GnIHR2 with goldfish and zebrafish in the GPR147 group. L. catla GnIHR2 receptor comprised seven transmembrane domains and the 3D-structure was predicted by I-TASSER tool. The docking analysis revealed high binding affinity (-11.6 kcal/mol) of GnIH antagonist, RF313 towards GnIHR2 receptor. The primary bonds involved were alkyl and hydrogen bonds while the amino acids participated were proline 43, 210, 339, cysteine 214, leucine 211, serine 213 and phenylalanine 338. The MD simulation analysis of docked complex for 100 nano-seconds (ns) in the lipid membrane environment showed the stability of the complex with time. CONCLUSION: Our study showed that GnIH antagonist, RF313 interact tightly with the GnIH receptor, GnIHR2 of L. catla. To the best of our knowledge, this is the first report on computational modelling and MD simulation of GnIH receptor in fishes. This will help in functional characterization studies of GnIH/GnIHR system in vertebrates.

Ontogenetic and tissue-specific expression of gonadotropin-inhibitory hormone (GnIH) and its receptors in Catla catla.

Gonadotropin-inhibitory hormone (GnIH) is an RFamide peptide, and its role in reproduction is well studied from fish to mammals, but very few reports are available about the function of GnIH during larval development. In this study, we examined the GnIH and GnIH receptors (GnIHRs) expression from embryogenesis to adult stage and tissue-specific expression in adult Catla catla using quantitative real-time (qRT) PCR. The qRT PCR analysis of GnIH mRNA during ontogenetic development showed the increasing trend from early developmental stages to the adult stage with the highest expression in 24 months fish. However, the expression of two GnIH receptors, GnIHR1 and GnIHR2 also increased from larval stages to the adults with a peak at 17 days post-hatching, while GnIHR3 showed the higher mRNA expression during embryogenesis and then decreasing gradually. Tissue distribution analysis of GnIH showed the highest mRNA expression of GnIH in the brain, followed by gonads of both the sexes. GnIHR1 and GnIHR2 were also highly expressed in the brain and gonads of both the sexes, while GnIHR3 showed the highest expression in gonads of both the sexes without any expression in the brain. These results suggest that the brain is the primary site of action for GnIH, GnIHR1 and GnIHR2, while gonads for GnIHR3.

Molecular characterization of gonadotropin-inhibitory hormone (GnIH) gene and effect of intramuscular injection of GnIH peptide on the reproductive axis in Catla catla.

Gonadotropin-inhibitory hormone (GnIH) plays an important role in reproduction by inhibiting the expression of gonadotropins in birds and mammals, but in fishes, it is ambiguous. In this study, we cloned 606 bp long cDNA of GnIH from Catla catla brain (cGnIH). The encoded preproGnIH peptide generated three putative peptides (cGnIH-I, -II, -III) of different size. Phylogenetic analysis of GnIH showed clustering of different peptide sequence with its orthologs in separate clades. The real-time PCR analysis showed the expression of cGnIH in brain, gonads, intestine, stomach, heart, gill and liver with the highest expression in the brain and gonads of both sexes. The basal GnIH mRNA expression was higher in spawning and spent phase of the male brain and spawning phase of the female brain. In testis, the expression was highest in spent phase, while in ovary the expression did not change significantly during reproductive phases. The in vivo experiment of cGnIH-III peptide exhibited the higher expression of HPG axis genes, lhb, fshb, cgnrh, kiss2 and kiss1r and serum hormone level of LH and FSH as soon as 3 h after the intramuscular delivery. These results suggest that the GnIH is positively involved in regulation of reproduction in HPG axis of C. catla.

Effects of Water pH on Life History Parameters of a New Bosminid Cladocera: Bosmina (Bosmina) Tripurae (Korinek, Saha and Bhattachaya, 1999) in Laboratory Condition.

The effects of water pH on life history parameters of Bosmina tripurae have been studied to determine the most suitable water pH desired for the maximum growth and development of this newly discovered cladoceran species. The study was carried out under the laboratory condition at 20 ± 2°C. Five pH ranges 5.0 ± 0.2, 6.0 ± 0.2, 7.0 ± 0.2, 8.0 ± 0.2 and 9.0 ± 0.2 with six replicates for each pH consisting of one animal in each Petri dish (80 × 15 mm) were used for the study. 20 mL of respective test medium was maintained with Chlorella sp. (2 × 104 ± 0.03 cells mL-1) in each Petri dish throughout the experiment. Thirty (30) animals were observed daily to investigate different life history parameters like total life span, age at maturity, number of eggs, neonates and egg batches etc. at different condition. From the study it was found that acidic water (pH 5 ± 0.2) is more suitable for the culture of Bosmina tripurae in laboratory condition.

Teleost Nonapeptides, Isotocin and Vasotocin Administration Released the Milt by Abdominal Massage in Male Catfish, Clarias magur.

In the present work the nonapeptides i.e., isotocin and vasotocin alone or in a combination were tested in C. magur to evaluate their effect on stripping by abdominal massage. Also, we used chitosan-carbon nanotube nanocomposites to conjugate the nonapetides isotocin (abbreviated as COOH-SWCNTCSPeP) and isotocin and vasotocin (COOH-SWCNTCSPePs) with the aim of sustaining the effect for a longer duration. The conjugation of nonapeptides with nanocomposites was confirmed by Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). Two experiments were conducted to study the effect of naked (without nanoparticles) and conjugated nonapeptides on the milt release by stripping. Both the experiments consisted of eight treatments which included four naked groups two nanoconjugated groups and two controls. Both naked and nonconjugated formulations were successful in stripping the male catfish. The mRNA expression of selected reproductive genes was analysed to decipher the effect of nanopeptides at the molecular level. Nonapeptide treatment either naked or nanoconjugated, resulted in the upregulation of the transcript level of genes. Histological analysis revealed the concentration of spermatozoa was more in peptide injected groups than in the controls. The synergistic effects of nonapeptides and Ovatide had a positive impact on GSI. Thus, the present formulations were successful in stripping the male catfish to obtain the milt with significant reproductive success. Even though the naked groups perform better but the number of males required to fertilize the eggs in nanoconjuagted groups was smaller making it worth using for the delivery of nonapeptides.

Chitosan grafting onto single-walled carbon nanotubes increased their stability and reduced the toxicity in vivo (catfish) model.

The present work was aimed to develop the tissue benign, modified acid-functionalized single-walled carbon nanotube (COOH-SWCNT) chitosan (CS) hybrid (COOH-SWCNT-CS). Chitosan-nanotube hybrids were characterized by Fourier-transform infrared spectroscopy (FTIR), Thermogravimetry Analysis (TGA), Raman spectroscopy, Emission Gun-Scanning Electron Microscopes (FEG-SEM), Transmission Electron Microscopy (TEM) and Energy-dispersive X-ray spectroscopy (EDS). Micronuclei test of blood cells, comet assay of liver tissue and histological analysis of liver and kidney tissues were conducted after different treatments to evaluate the toxicity. Fish receiving COOH-SWCNT developed more numbers of micronuclei than COOH-SWCNT-CS treatments. Similarly, more DNA damage was observed in fish injected with nanotubes alone than chitosan hybrid groups. Histological observations revealed severe liver cell damage at higher concentrations of COOH-SWCNT whereas, in COOH-SWCNT-CS, no such damage was observed. However, kidney tissue remained unaffected in all groups. The study suggests that the nanohybrid developed will be safe and useful for delivery of micro or macro biomolecules in fish and higher animals.