Browsing by Author "S.G. Bhat"

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Expression profile of kisspeptin2 and gonadotropin-releasing hormone2 mRNA during photo-thermal and melatonin treatments in the female air-breathing catfish Heteropneustes fossilis
(Springer Science and Business Media B.V., 2020) R. Chaube; S. Sharma; B. Senthilkumaran; S.G. Bhat; K.P. Joy
In seasonally breeding vertebrates, extrinsic factors like photoperiod and temperature are major determinants, controlling the annual reproductive cycle. In teleosts, kisspeptin, which occurs in two molecular forms: kisspeptin1 (Kiss1) and kisspetin2 (Kiss2), has been reported to alter gonadotropin (Lh and Fsh) secretion but its effect on gonadotropin-releasing hormone (Gnrh) secretion is not unequivocally proved. In the catfish Heteropneustes fossilis, we isolated and characterized kiss2 and gnrh2 cDNAs and the present work reports effects of altered photo-thermal conditions and melatonin (MT, a pineal hormone) on their expressions in the brain. The exposure of the catfish to long photoperiod (LP, 16 h light) at normal temperature (NT) or high temperature (HT, 28 °C) at normal photoperiod (NP) for 14 or 28 days stimulated both kiss2 and gnrh2 expression in both gonad resting and preparatory phases with the combination of LP + HT eliciting maximal effects. Short photoperiod (SP, 8 h light) under NT or HT altered the gene expression according to the reproductive phase and temperature. MT that mediates photo-thermal signals to the brain inhibited brain kiss2 and gnrh2 gene expression in the NP + HT, LP + NT, and SP + NT groups. The altered photo-thermal conditions elicited changes in steroidogenic pathway as evident from changes in plasma E2, progesterone, and testosterone levels. The results show that brain kiss2-gnrh2 signaling is involved in photo-thermal-mediated mechanisms controlling reproduction. © 2020, Springer Nature B.V.
Identification of kisspeptin2 cDNA in the catfish Heteropneustes fossilis: Expression profile, in situ localization and steroid modulation
(Academic Press Inc., 2020) R. Chaube; S. Sharma; B. Senthilkumaran; S.G. Bhat; K.P. Joy
Kisspeptin (Kiss) is considered an upstream regulator of gonadotropin-releasing hormone in mammals but its role in non-mammalian vertebrates is not unequivocally established. In the catfish Heteropneustes fossilis, a 605 bp long cDNA was identified from the brain by cloning as well as by retrieving from the catfish transcriptome database. The open reading frame (ORF, 93–405 bp) codes for a 113 amino acids long precursor protein. Homology and phylogenetic analyses showed that the predicted protein belongs to the vertebrate Kiss2 type with a high degree of conservation in the Kiss2-10 region (FNFNPFGLRF). The kiss2 transcripts were expressed highly in the brain and gonads in a dimorphic manner with a female bias. In the brain, kiss2 transcripts showed regional differences with higher expression in the medulla oblongata and forebrain regions. The kiss2 transcripts showed significant seasonal variations with the highest expression in the brain in spawning phase and in the gonads in prespawning phase. The kiss2 transcripts were localized in the brain (nucleus preopticus, habenular nucleus, nucleus recessus posterioris, nucleus recessus lateralis) and stratum periventriculare (radial glial cells) of optic tectum, pituitary and ovary (follicular layer and germinal vesicle). Ovariectomy (1, 2, 3 and 4 weeks) decreased brain kiss2 mRNA levels and a single injection of estradiol-17β (E2; 0.5 μg/g body weight) in 3- week ovariectomized (OVX) and sham operated fish resulted in an increase in the transcript levels after 24 h. The E2 receptor antagonist Tamoxifen (TMX) produced biphasic effects on the kiss2 expression in the dose- response study. TMX inhibited the expression in the OVX fish, but elicited a stimulatory effect in the OVX + E2-treated fish. Testosterone (T) decreased, and progesterone (P4) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E2 increased, and TMX, T and P4 inhibited the kiss2 transcript levels. The results suggest that Kiss2 is an important regulator of the brain- pituitary- gonadal- endocrine axis, and in habenular and optic tectum functions. © 2020 Elsevier Inc.
Kisspeptins stimulate the hypothalamus - pituitary - ovarian axis and induce final oocyte maturation and ovulation in female stinging catfish (Heteropneustes fossilis): Evidence from in vivo and in vitro studies
(Elsevier B.V., 2022) R. Chaube; S. Sharma; B. Senthilkumaran; S.G. Bhat; K.P. Joy
In the present study, effects of synthetic human Kisspeptin1 (hKiss1) and catfish Kisspeptin2 (cfKiss2) on the hypothalamic - pituitary - ovarian (HPO) axis, and induction of final oocyte maturation and ovulation were investigated in prespawning female stinging catfish (Heteropneustes fossilis) under in vivo and in vitro conditions. Gene expression was quantified by real time quantitative PCR and steroids were measured by specific enzyme-linked immunoassays. Intraperitoneal (ip) injections (1, 2, 3 ng/g body weight, BW) of synthetic hKiss1 and cfKiss2 stimulated hypothalamic, pituitary and ovarian gnrh1 and gnrh2 expression at 24 h and the effect was higher after the cfKiss2 treatment. In vitro incubation of hypothalamus, pituitary and ovary pieces with the Kiss peptides (5, 10, 20 nM) produced a similar effect. GPR54 (Kiss1 receptor) antagonist peptide234, when given ip (5, 10, 20 ng/g BW) or incubated in vitro (5, 10, 20 nM), inhibited the gnrh1 and gnrh2 expression at 24 h. The supplementation with hKiss1 or cfKiss2 restored the inhibition due to peptide234 in vivo and in vitro and the effect was higher in the cfKiss2 combination group. Both hKiss1 and cfKiss2 altered the expression of pituitary gonadotropin (Gth) subunit genes follicle-stimulating hormone β (fshβ), luteinizing hormone β (lhβ) and glycoprotein α (gpα) in vivo and in vitro. The expression of fshβ was more sensitive to the treatments than lhβ expression and the effect was greater in the cfKiss2 groups. Peptide234 in vivo and in vitro inhibited the expression of the Gth genes and the effect was reversed and restored in the hKiss1 and cfKiss2 combination groups. The Kiss peptide treatments in vivo or in vitro stimulated both plasma and ovarian levels of estradiol-17β, progesterone and 17,20β-dihydoxy-4-pregnen-3-one levels. The peptide234 treatment inhibited, or elicited a decreasing trend on the steroid levels both in vivo and in vitro, and the inhibition was reversed by the hKiss1 and cfKiss2 combination treatments. Incubation of post vitellogenic follicles with hKiss1 or cfKiss2 stimulated germinal vesicle breakdown (GVBD) and ovulation. The inhibition due to peptide234 was reversed in the combination groups. Ovulation was not elicited or unaffected in the peptide234 treated groups. The data show that the Kiss peptides act downstream the HPO axis to stimulate oocyte maturation and ovulation, and cfKiss2 peptide is functionally more effective than hKiss1. © 2021 Elsevier B.V.
Molecular cloning and characterization of a gonadotropin-releasing hormone 2 precursor cDNA in the catfish Heteropneustes fossilis: Expression profile and regulation by ovarian steroids
(Academic Press Inc., 2019) R. Chaube; A. Rawat; S. Sharma; B. Senthilkumaran; S.G. Bhat; K.P. Joy
Gonadotropin-releasing hormone 2 (Gnrh2) is one of the three classes of Gnrh distributed in vertebrates and is highly conserved. In the present study, the cDNA encoding Gnrh2 was isolated and characterized in the ostariophysan catfish Heteropneustes fossilis (hf). The cDNA is 611 bp long with an open reading frame (ORF) of 261 bp that encodes a highly conserved protein of 86 amino acids. The deduced Gnrh2 precursor protein clustered with the vertebrate Gnrh2 type. The sequence identity of hfgnrh2 is 94% with African catfish (Clarias gariepinus) gnrh2 mRNA (accession no. X78047). The hfgnrh2 transcripts were expressed only in the brain and gonads with a higher expression in the female brain and ovary in both resting and prespawning phases. The expression was higher in the prespawning phase than the resting phase. The gnrh2 expression in the brain and ovary showed significant seasonal variations but with opposite patterns. In the brain, the expression was the highest in the preparatory phase, decreased progressively to low levels in the postspawning and resting phases. In the ovary, the transcript level was low in the resting and preparatory phases, increased sharply in the prespawning phase reaching the peak level in the spawning phase and declined sharply in the postspawning phase. The gnrh2 mRNA showed the highest expression in the hind brain-medulla oblongata and moderate to low expression in forebrain regions and pituitary. Ovariectomy resulted in a duration-dependent inhibition of hfgnrh2 mRNA levels in the resting and prespawning phases. Steroid (E2, testosterone and progesterone) replacement treatments (0.5 μg/g body weight) in the 3- week ovariectomized fish restored the inhibition due to ovariectomy, elevated the expression over and above the sham level in the resting phase (E2 group), and raised the levels almost to that of the sham group (testosterone and progesterone groups) in the prespawning phase. In the sham control groups, the steroid replacement resulted in a significant reduction in the mRNA levels. The expression of the gnrh2 mRNA in the brain-pituitary-gonadal axis and its regulation by gonadal steroids suggest that Gnrh2 may have a reproductive role in the catfish. © 2019 Elsevier Inc.