Characterisation of the hypoxia-responsive leptin receptor gene (omLepRL) of the marine medaka Oryzias melastigma

Abstract

Aquatic hypoxia is a common phenomenon that causes great stress to marine fauna. Aside from causing significant changes in energy metabolism and growth, it also evokes abnormalities in reproductive development in fish. Meanwhile, leptin and its receptor have been implied in energy homeostasis and reproductive functions in mammals. As many of the physiological functions in fish that are affected by hypoxia are controlled by the leptin system in mammals, it is hypothesised that the leptin system in fish may be involved in mediating many of the physiological and biochemical responses of fish to acute and chronic hypoxia. This study aims to provide further insights into the expression of the leptin receptor (a leptin signal elucidator) in the marine medaka fish Oryzias melastigma in response to hypoxia. A 4450-bp full-length cDNA encoding the long form of the leptin receptor was isolated from the marine medaka O. melastigma using degenerate genomic PCR and RACE PCR. The open reading frame of the omLepRL cDNA is 3300 bp in length and is predicted to encode a 125-kDa protein of 1109 amino acid residues. Sequence analysis showed that the predicted omLepRL protein carries all of the signature structural domains and functionally important signalling motifs present in the mammalian long-form leptin receptor. Pairwise sequence comparison also indicated that the omLepRL shares low but significant sequence identity (23.5–24.9%) with the long-form leptin receptor of mammals, birds and Xenopus, moderate identities with the zebrafish (31.8%) and Fugu (45.4%) leptin receptors and high sequence similarity (78.9%) with the leptin receptor of the Japanese medaka (O. laptipes). Quantitative RT-PCR analysis indicated that omLepR mRNA is expressed ubiquitously in both male and female fish and is highly abundant in the kidney, spleen, gill and muscle. In situ hybridisation analysis of the medaka fish brain showed that omLepR mRNA is localised in specific regions of the hypothalamus and in the entire pituitary. Overall, quantitative RT-PCR and in situ hybridisation analyses showed that omLepR is differentially expressed along the hypothalamus-pituitary-gonadal (HPG) axis in response to hypoxia, which suggests that the leptin signalling system may have a role in regulating reproductive functions in fish. Quantitative RT-PCR showed that omLepR expression was markedly induced in various fish tissues by 2.2- to 11.1-fold under hypoxia in both male and female fish along with the hypoxia marker gene heme oxygenase (omHo). Analysis of the omLepR promoter with gene transfection and luciferase reporter assays indicated that the 1.2 kb 5'-flanking sequence of omLepR was transcriptionally activated by the hypoxia-inducible transcription factors omHIF-1, -2 and -4 (yielding 3.4-, 2.0- and 3.6-fold increases in relative luciferase activity, respectively, as compared to the control). Detection of the three omHIF-α proteins in hypoxic tissues of the marine medaka in vivo by Western blot analysis strongly suggests that hypoxic induction of omLepR expression is mediated through the activation of the omLepR promoter by one or more of the omHIF transcription factors. Overall, the results of this study suggest that unique molecular mechanisms operate through the omLepR receptor and that these mechanisms may underlie certain physiological responses to hypoxia along the HPG axis in the marine medaka fish. This is the first report demonstrating regulation of the leptin receptor gene by HIF transcription factors in fish.

Date of Award	2 Oct 2009
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Yuen Chong Richard KONG (Supervisor)