Higher intake of ß-glucan impairs reproduction in a female teleost, Tor putitora (Hamilton, 1822).

Although the immuno-modulatory and stress-relieving properties of ß-glucan is well elucidated in humans and other animal models, including fish, its role as a dietary supplement on reproduction is extremely scarce. Therefore, in this study, adult female fish were fed one of four test diets having 0 (control), 0.5, 1, and 1.5% ß-D-glucan for 130 days and its effect on reproductive performance, ovarian and liver histology, sex hormones, and transcript abundance of selected reproduction-related genes was assessed. Low dietary intake of ß-glucan improved fertilization and hatching rates (p<0.05). The relative fecundity and percentage of spawning females were higher (non-significant) in 0.5% ß-glucan-fed groups. Surprisingly, even after 130 days, spawning did not occur in 1.5% ß-glucan-fed individuals. Irrespective of ß-glucan intake, all the brooders recorded similar plasma 17ß-estradiol and maturation-inducing hormone (p>0.05). Higher intake of ß-glucan (1.5%) upregulated aromatase genes without a parallel increase in 17ß-estradiol. However, plasma vitellogenin increased with increasing ß-glucan up to 1.0% then declined at 1.5% (p<0.05). The fish that received control, 0.5, and 1.5% ß-glucan recorded similar vitellogenin levels in their plasma. Significantly higher plasma cortisol was evidenced in 1.5% ß-glucan fed brooders (p<0.05). Histologically, higher follicular atresia and leaking of yolk material was evidenced in 1.5% ß-glucan-fed group. Liver histology revealed the highest nutrient/lipid accumulation in fish that received 1.0% and 1.5% ß-glucan. This study demonstrated the stimulatory effect of ß-glucan intake at a lower dose (0.5%) on reproduction. However, higher intake (1.5%) could perturb normal reproductive function in a fish model and caused an increased number of atretic follicles leading to spawning/reproductive failure.

Quantitative proteomic analysis of GnRH agonist treated GBM cell line LN229 revealed regulatory proteins inhibiting cancer cell proliferation.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) receptor, a rhodopsin-like G-protein coupled receptor (GPCR) family member involved in GnRH signaling, is reported to be expressed in several tumors including glioblastoma multiforme (GBM), one of the most malignant and aggressive forms of primary brain tumors. However, the molecular targets associated with GnRH receptor are not well studied in GBM or in other cancers. The present study aims at investigating the effect of GnRH agonist (Gosarelin acetate) on cell proliferation and associated signaling pathways in GBM cell line, LN229. METHODS: LN229 cells were treated with different concentrations of GnRH agonist (10-10 M to 10-5 M) and the effect on cell proliferation was analyzed by cell count method. Further, total protein was extracted from control and GnRH agonist treated cells (with maximum reduction in cell proliferation) followed by trypsin digestion, labeling with iTRAQ reagents and LC-MS/MS analysis to identify differentially expressed proteins. Bioinformatic analysis was performed for annotation of proteins for the associated molecular function, altered pathways and network analysis using STRING database. RESULTS: The treatment with different concentrations of GnRH agonist showed a reduction in cell proliferation with a maximum reduction of 48.2% observed at 10-6 M. Quantitative proteomic analysis after GnRH agonist treatment (10-6 M) led to the identification of a total of 29 differentially expressed proteins with 1.3-fold change (23 upregulated, such as, kininogen-1 (KNG1), alpha-2-HS-glycoprotein (AHSG), alpha-fetoprotein (AFP), and 6 downregulated, such as integrator complex subunit 11 (CPSF3L), protein FRG1 (FRG1). Some of them are known [KNG1, AHSG, AFP] while others such as inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), ITIH4, and LIM domain-containing protein 1 (LIMD1) are novel to GnRH signaling pathway. Protein-protein interaction analysis showed a direct interaction of KNG1, a hub molecule, with GnRH, GnRH receptor, EGFR and other interactors including ITIH2, ITIH4 and AHSG. Overexpression of KNG1 after GnRH agonist treatment was validated using Western blot analysis, while a significant inhibition of EGFR was observed after GnRH agonist treatment. CONCLUSIONS: The study suggests a possible link of GnRH signaling with EGFR signaling pathways likely via KNG1. KNG1 inhibitors may be investigated independently or in combination with GnRH agonist for therapeutic applications.

Transgenerational effects of ß-glucan on thermal tolerance, growth performance, and immune gene expression of endangered cyprinid Tor putitora progeny.

Nutritional programming signifies a process in which broodstock feeding approaches have long-term effects on the subsequent progeny. The present study aimed to elucidate whether supplementing golden mahseer, Tor putitora broodstock diets with ß-glucan affects progeny growth performance, survival, thermal tolerance, and non-specific immunity. Initially, the growth performance of progeny produced from brooders fed with different levels of ß-glucan was non-significant. However, on the 15th and 35th DPH, the maximum weight was observed in fry obtained from the brooders fed with 0.5% followed by 1.0% ß-glucan. Furthermore, on 50th DPH, significantly higher weight was registered in the fry from the 0.5% ß-glucan fed group while 1.0% ß-glucan group had no transgenerational effect on growth. The condition factor of fry obtained from golden mahseer brooders fed with a 0.5% ß-glucan diet was greater than the control and 1.0% ß-glucan fed group. On the other hand, we did not find any significant transgenerational influence of ß-glucan on the survival of the progeny. The thermal tolerance of fry produced from brooders fed with ß-glucan was significantly modulated at both end-points (CTmax and CTmin). Expression of interleukin-1ß was significantly up-regulated in fry obtained from ß-glucan fed brooders. In contrast, the expression level of tumor necrosis factor-α was significantly higher only in fry produced from 1.0% ß-glucan fed brooders. The expression of immunoglobulin light chain and serum amyloid A gene was significantly higher in fry produced from 0.5% ß-glucan fed brooders. Overall results suggest that the dietary provisioning of ß-glucan in golden mahseer brooders can be a strategy to produce healthy and robust fry in captivity for stock enhancement and conservation programs.

Molecular characterization of non-specific immune genes of endangered golden mahseer (Tor putitora) and their expression during embryonic and larval development.

The present study was undertaken to characterize and analyze the expression of non-specific immune genes to get an insight into the early immune status of endangered golden mahseer. In this study, the full-length mRNA sequence of IFNγ, TNFα, C3, and IL10 was 927, 1409, 5125 and 1177 bp with an ORF of 558, 765, 4938, and 540 bp, encoding a putative protein of 185, 254, 1645, and 179 amino acid residues, respectively. The deduced amino acid sequences of these genes shared highly conserved structures with those from other cyprinids. Ontogenic real-time qPCR results indicated that expression of IFNγ and TNFα was lower until the morula stage and increased from blastula stage and found maximum at the organogenesis stage. Expression of the C3 gene was lower until the gastrula stage, followed by a linear increase from organogenesis to the pre-metamorphosis stage. The expression of IL10 was significantly lower during early developmental stages (till gastrula stage) and reached maximum at organogenesis. The level of IL1ß was found maximum in unfertilized eggs and remained elevated till the morula stage. TLR4 expression remained lower during the initial developmental stages and reached the maximum at the organogenesis stage. The expression level of defensin1 was substantially low until the organogenesis stage. In comparison, hepcidin1 was found considerably high until the blastula stage and remained significantly lower during later stages of development. Overall, the data generated improves knowledge on the immune status of endangered golden mahseer during embryonic and larval development, which may help develop effective immunomodulatory interventions during nursery rearing of golden mahseer to produce fry with better fitness.