Irfan Ahmad Bhat

Expression analysis of Sox9 genes during annual reproductive cycles in gonads and after nanodelivery of LHRH in Clarias batrachus

Transcription factor Sox9 plays a crucial role in determining the fate of several cell types and is a primary factor in regulation of gonadal development. Present study reports full-length cDNA sequence of Sox9a gene and partial coding sequence (cds) of Sox9b (two duplicate orthologs of Sox9 gene) from Clarias batrachus. The coding region of Sox9a gene encoded a peptide of 460 amino acids. The partial cds of Sox9b with the length of 558bp was amplified that codes for 186 amino acids. Quantitative Real-time PCR (qRT-PCR) analysis revealed that Sox9a and Sox9b mRNA expression was significantly higher in gonads and brain tissues. Furthermore Sox9a and Sox9b mRNA expression levels were high during preparatory and pre-spawning phases and decreased gradually with onset of spawning and post-spawning phases of reproductive cycles in gonads. Chitosan nanoconjugated sLHRH (CsLHRH) of particle size 133.0nm and zeta potential of 34.3mV were synthesized and evaluated against naked sLHRH (salmon luteinizing hormone-releasing hormone). The entrapment efficiency of CsLHRH was 63%. CsLHRH nanoparticles increased the expression level of Sox9 transcripts in gonads and steroid hormonal levels in blood of male and female. Thus, our findings clearly indicate that Sox9 genes play essential role during seasonal variation of gonads. Besides, the current study reports that sustained release delivery-system will be helpful for proper gonadal development of fish. To the best of our knowledge, till date no study has been reported on nanodelivery of sLHRH and their effect on reproductive gene expression in fish.

In silico analysis and expression studies of kisspeptin gene in C. catla

We report the characterization of kisspeptin gene which is considered to be essential for successful animal reproduction. The full-length cDNA sequence of kiss2 was 583 bp, consisted of 11 bp 5′-UTR (untranslated region) and 194 bp 3′-UTR, respectively. Open reading frame of 378 bp encoding a putative protein of 125 amino acids. The Catla catla kiss2 protein was having a molecular weight of 14.51 kDa and isoelectric point (pI) of 8.46. There were four serine (Ser), four threonine (Thr) and two tyrosine (Tyr) phosphorylation sites and no N-glycosylation sites on the predicted protein. The amino acids on positions 8, 11, 24, 80 and 114 were detected to be ligand binding sites. The signal peptide analysis predicted that C. catla kiss2 is a secretory protein. Kiss2 protein is localized in nuclear region (49.7%) and the extracellular region (38.3%) of the cell. Analysis of tissue distribution revealed that, kiss2 transcripts were predominantly expressed in the brain and gonads, with expression levels in female higher than those of male. Ontogenetic analysis of kiss2 demonstrated that expression level was low during early phase of development stages and more expression was observed during mature stage. Overall present results lay a strong basis for understanding the role of kisspeptin in the neuroendocrine system in teleosts.

Steroidogenic acute regulatory protein (StAR) gene expression construct: Development, nanodelivery and effect on reproduction in air-breathing catfish, Clarias batrachus

Steroidogenic acute regulatory protein (StAR) is responsible for the relocation of cholesterol across mitochondrial membrane in vertebrates and is, therefore, a key factor in regulating the rate and timing of steroidogenesis. In the present study, we developed chitosan nanoparticle (CNP) conjugated StAR gene construct (CNP-pcDNA4-StAR) in a eukaryotic expression vector, pcDNA4/HisMax A. CNPs of 135.4nm diameter, 26.7mV zeta potential and 0.381 polydispersity index were used for conjugation. The loading efficiency (LE) of pcDNA4-StAR construct with CNPs was found to be 86%. After the 24h of intramuscular injection, the CNP-pcDNA4-StAR plasmid could be detected from testis, brain, kidney and muscle tissues of Clarias batrachus. The transcript levels of important reproductive genes viz. cyp11a1, cyp17a1, 3β-hsd, 17β-hsd and cyp19a1 in CNP-pcDNA4-StAR treated group were initially low up to 24h, but significantly increased subsequently up to 120h. In naked pcDNA4-StAR treated group, the mRNA level of 3β-hsd, 17β-hsd and cyp19a1 increased initially up to 24h, while cyp11a1 and cyp17a1 increased up to 48h and then started declining. Similar results were obtained for 11-Ketotestosterone and 17β-estradiol. The results indicate relatively long lasting effects of nano-conjugated construct compared to the construct alone. Furthermore, the histopathology of gonads and liver authenticates its possible role in the gonadal development in fish without any adverse effect.

Fabrication and characterization of chitosan conjugated eurycomanone nanoparticles: In vivo evaluation of the biodistribution and toxicity in fish

Chitosan nanoparticles (CNPs) have been proven considerable delivery agents due to their remarkable physicochemical properties. Present study reports the fabrication of CNPs by ionic gelation process and their characterization by different approaches. The constructed nanoparticles were successfully conjugated with eurycomanone with significant entrapment efficiency. Particle size of chitosan and chitosan conjugated eurycomanone nanoparticles were 126.2nm and 130nm respectively. Scanning electron microscopy showed that the particles were spherical in shape and well dispersed. Cross-linking between CNPs and eurycomanone (CENPs) were confirmed by Fourier-transform infrared (FTIR) spectroscopy. Fluorescent nanoparticles were prepared by using Rhodamine-6G dye, characterised by SEM and confirmed for conjugation by FTIR. Biodistribution of CENPs showed the presence of fluorescent nanoparticles in liver, kidney, testes and brain of C. magur. The toxicity of CENPs was evaluated by comparing the histological sections of catfish testes collected from treated and control group. No signs of toxicity were seen in testes after the delivery of CENPs. Molecular docking study revealed high spontaneous binding ability of chitosan with eurycomanone and aromatase enzyme. The study reports that CNPs can act as a stabilizing agent for eurycomanone formulation and could be a promising approach to increase the reproductive performance of the fishes.

Chitosan-eurycomanone nanoformulation acts on steroidogenesis pathway genes to increase the reproduction rate in fish

The study was undertaken to explore the molecular mechanism of eurycomanone, a major compound of Eurycoma longifolia plant in increasing the reproductive processes in the male fish model. Chitosan-nanoconjugated eurycomanone nanoparticles with a significant particle size [130 nm (CED1); 144.1 nm (CED2)] and stable zeta potentials (+49.1 mV and +30 mV) were synthesized and evaluated against naked eurycomanone (ED1 and ED2). In present study, short-term and long-term experiments were conducted to evaluate the effect of nano-formulation on expression of endocrine-related genes, circulating hormone concentrations (Follicle stimulating hormone, FSH; luteinizing hormone, LH; progesterone, testosterone and 17-β estradiol) and reproductive capacity of male Clarias magur. In short-term experiment, the sampling of tissues was done on hourly basis after injection of eurycomanone either alone or with chitosan and long-term experiment was carried for 21 days and in this the injection was repeated after 7 days and 14 days. Treatments CED1 and CED2 showed controlled and sustained surge of the transcript level of selected genes (except aromatase) and serum hormones (except 17β-estradiol) compared to ED1 and ED2 groups. The transcript levels of aromatase and serum 17β-estradiol hormone showed the declining trend in the chitosan conjugated groups. The gonadosomatic index (GSI), reproductive capacity, intracellular calcium and selenium and cellular structure of testes were improved in CED1 and CED2 groups compared to other treatments. Furthermore, the effect of chitosan conjugated eurycomanone was evaluated in primary testicular cells and an increase in the mRNA expression level of endocrine-related genes was detected. This is the first report of the use of chitosan conjugated eurycomanone and present study elucidates the molecular mechanism of eurycomanone in increasing the reproductive output in animals.

Molecular and Cellular Toxicology of Nanomaterials with Related to Aquatic Organisms

The increasing application of nanomaterials both in commercial and industrial products has led their accumulation in the aquatic ecosystem. The rapid development and large scale production of nanomaterials in the last few decades have stimulated concerns regarding their potential environmental health risks on aquatic biota. Inorganic nanoparticles, due to their unique properties and associated material characteristics resulted in toxicity of these nanomaterials in aquatic organisms. Understanding their novel properties at nanoscale has established to be a significant aspect of their toxicity. Unique properties such as size, surface area, surface coating, surface charge, aggregation of particles and dissolution may affect cellular uptake, molecular response, in vivo reactivity and delivery across tissues of living organism. Already lot of research in the past three or four decades within the nano-ecotoxicology field had been carried out. However, there is not any standard technique yet to assess toxicity of nanoparticles (NPs) on different biological systems such as reproductive, respiratory, nervous, gastrointestinal systems, and development stages of aquatic organisms. Specific toxicological techniques and quantification of nanoparticles are vital to establish regulations to control their impact on the aquatic organism and their release in the aquatic environment. The main aim of this chapter is to critically evaluate the current literature on the toxicity of nanomaterials on aquatic organism.

Inhibin anti-peptide antibody macromolecule: An approach to improve fecundity in Clarias batrachus

Inhibins are members of the transforming growth factor beta (TGFβ) superfamily known to regulate ovarian functions through stimulation of the hypothalamo-pituitary-gonadal axis. In the present study, we aimed to design a species-specific inhibin-α chimeric peptide (ICP) and evaluate the effect of immunoneutralization using anti-ICP antisera to enhance the reproductive performance in female Clarias batrachus. Injection of anti-ICP antisera caused a significant increase in the number of oocytes at a medium dose (200 μl) in comparison to high dose (400 μl) and control (normal rabbit serum). Histological observations confirmed the dose-dependent advancement in oocyte maturation. Furthermore, anti-ICP antisera treated groups exhibited a significant increase in the serum concentrations of follicle stimulating hormone (FSH) and 17β-estradiol (E2) hormones. The anti-ICP antisera decreased the mRNA expression levels of inhibin-α while stimulated the transcript levels of inhibin-βA, FSHβ, CYP 19a1, 3β-HSD and StAR respectively in a dose-dependent manner. Collectively, these findings indicate that anti-ICP antibody macromolecules modulate the endogenous reproductive hormonal secretion and enhance oocyte quality and quantity in female C. batrachus. This is the first report wherein antibodies against inhibins were used to promote reproductive performances and investigate the underlying molecular mechanisms in fishes.