Jihye Shin

Expression of gonadotropin genes in Manchurian trout Brachymystax lenok and production of recombinant gonadotropins

Manchurian trout Brachymystax lenok is an endangered fish species in East Asia including the Korean peninsula. To establish a method for artificial propagation and to improve our understanding of reproduction in the species, cloning of the pituitary gonadotropin (GTH) subunit cDNAs encoding the Manchurian trout gonadotropin common α (mtGTHα), follicle-stimulating hormone β (mtFSHβ), and luteinizing hormone β (mtLHβ) by polymerase chain reaction, were attempted. The open reading frames of the mtGTHα, mtFSHβ and mtLHβ encoded 114, 137, and 142 amino acid residues, respectively. The mature peptides showed strikingly high sequence identities to those of salmonid species GTH α2, FSHβ, and LHβ subunits (92–100%). In an examination of tissue distribution of the mtGTHα, mtFSHβ, and mtLHβ, it was revealed that all of the three subunit genes are specifically expressed in the pituitary by reverse transcription-polymerase chain reaction analysis. Next, single-chain recombinant FSH and LH constructs using the mtGTHα, mtFSHβ, and mtLHβ cDNAs were produced. In a transient transfection of the FSH and LH constructs into Chinese hamster ovary (CHO-K1) cells followed by Western blot analysis, the tethered FSH in cell lysates or LH secreted into a medium was detected from 48 to 72 h after transfection. The present study provides a possibility of bioactive GTH production for studies on the reproductive physiology of GTH in the Manchurian trout.

cDNA cloning of Japanese flounder stanniocalcin 2 and its mRNA expression in a variety of tissues

Stanniocalcin 1 (STC1) is a glycoprotein hormone important in the maintenance of calcium and phosphate homeostasis in both fishes and mammals. Although two related STC genes, STC1 and STC2, were found to be expressed in multiple tissues as paracrine regulators in mammals, spatial expression pattern of stc2 mRNA has not been elucidated in fishes in contrast to that of clearly described stc1. In the present study, we have cloned and characterized a full-length stc2 cDNA from Japanese flounder (Paralichyhus olivaceus) ovary and analyzed expression pattern of stc2 in both sexes. The flounder stc2 cDNA (1501 nucleotides) encoded a putative prehormone of 286 amino acids (aa) with a signal peptide of 20 aa and a mature protein of 266 aa. The deduced aa sequence of flounder stc2 showed high sequence identity with those of pufferfish, zebrafish, and human (57.7-89.0%), whereas it showed less identity with that of flounder stc1 (24.3%). RT-PCR analysis revealed that the flounder stc2 gene is expressed in all examined tissues including the pituitary, brain, heart, kidney, gills, stomach, spleen, skin, dorsal fin, skeletal muscle, liver, corpuscles of Stannius, intestine, ovary and testis. Our data indicate that fish stc2 gene, like stc1, is expressed in a wide variety of tissues.

Molecular Cloning of Stanniocalcin 1 and Its Extracorpuscular Regulation by Salinity and Ca2+ in the Japanese Flounder

Abstract Stanniocalcin 1 (Stc1) was originally identified as an anti-hypercalcemic hormone produced by the corpuscles of Stannius (CS) associated with the kidney in teleosts. While the stc1 gene is expressed in various tissues in fishes, its role and regulation in extra-CS tissues are unexplored. In the present study, we characterized a cDNA of stc1 in a euryhaline fish, the Japanese flounder (Paralichyhus olivaceus), and examined its expression in peripheral tissues in response to different salinities and Ca2+ ion concentrations. The Japanese flounder stc1 cDNA (1331 bp) encodes a preprohormone of 251 amino acids (aa), with a signal peptide of 17 aa and a pro-sequence peptide of 15 aa followed by the mature protein of 219 aa. The deduced aa sequence of Japanese flounder stc1 showed highest sequence identity (94.0%) with the European flounder Stc1 among fish and mammalian species, but lower identity to zebrafish, pufferfish, and human STC2 (23.1–25.4%). Lowered environmental salinity resulted in a decrease in stc1 mRNA expression in vivo in the gills, kidney, intestine, and CS glands of the Japanese flounder. Furthermore, we found that extracellular Ca2+ increased steady-state stc1 mRNA levels in gill and kidney cells as well as in the CS cells. Our findings suggest that Stc1 synthesis in the ionregulatory tissues is responsive to environmental salinity and Ca2+ level.

Molecular characterization of tripartite motif protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii.

Tripartite motif-containing 25 (TRIM25), also known as estrogen-responsive finger protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii, designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4-28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo. Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart.

Identification of Ran-binding protein M as a stanniocalcin 2 interacting protein and implications for androgen receptor activity.

Stanniocalcin (STC), a glycoprotein hormone originally discovered in fish, has been implicated in calcium and phosphate homeostasis. While fishes and mammals possess two STC homologs (STC1 and STC2), the physiological roles of STC2 are largely unknown compared with those of STC1. In this study, we identified Ran-binding protein M (RanBPM) as a novel binding partner of STC2 using yeast two-hybrid screening. The interaction between STC2 and RanBPM was confirmed in mammalian cells by immunoprecipitation. STC2 enhanced the RanBPM-mediated transactivation of liganded androgen receptor (AR), but not thyroid receptor β, glucocorticoid receptor, or estrogen receptor β. We also found that AR interacted with RanBPM in both the absence and presence of testosterone (T). Furthermore, we discovered that STC2 recruits RanBPM/AR complex in T-dependent manner. Taken together, our findings suggest that STC2 is a novel RanBPM-interacting protein that promotes AR transactivation. [BMB Reports 2014; 47(11): 643-648]

Transcriptional Activity and Expression of Liver X Receptor in the Ascidian Halocynthia roretzi

Liver X receptors, LXRs, are ligand-activated transcription factors that belong to the group H nuclear receptor (NR) superfamily. In this study, an LXR (HrLXR) cDNA was cloned from the ascidian Halocynthia roretzi hepatopancreas and characterized to examine the functional conservation of ancestral LXRs in chordates. A phylogenetic analysis of HrLXR showed that it belongs to the tunicate (urochordate) LXR subgroup, which is distinct from vertebrate LXRs. Quantitative real-time PCR analysis revealed that HrLXR mRNA was expressed predominantly in the gills, and highly expressed in unfertilized eggs followed by decrease at later embryonic and larval stages. Unexpectedly, HrLXR was not activated by GW3965, whereas a synthetic ligand for a farnesoid X receptor, GW4064, activated HrLXR. This activation was abolished by the deletion of 51 amino acids from the N-terminus. In a mammalian two-hybrid system, HrLXR interacted with HrRXR in the presence of GW4064 or 9-cis retinoic acid. The injection of GW3965 and GW4064 in vivo increased the ATPbinding cassette sub-family G member 4 and HrLXR mRNA levels in the hepatopancreas and gills. These results suggest that the mRNA expression and transcriptional properties of HrLXR are different from those of vertebrate LXRs, although HrLXR is likely responsive to the related NR ligand, GW4064.

Production and characterization of recombinant Manchurian trout thyrotropin

Thyrotropin (thyroid-stimulating hormone, TSH), a heterodimeric glycoprotein hormone produced in the pituitary, stimulates the thyroid gland and release of thyroid hormones. In contrast to a well-known efficacy of recombinant mammalian TSHs, there is no report about the production of teleost recombinant TSH and its biological activity. In this study, we report the production of a single-chain recombinant TSH (mtTSH) of Manchurian trout (Brachymystax lenok), by baculovirus in silkworm (Bombyx mori) larvae. The mtTSH was produced in silkworm larvae and characterized as a form of N-linked glycosylation. The cAMP signaling system in transiently transfected COS-7 cells revealed that the mtTSH was recognized by their cognate receptors, salmon TSHα and TSHβ receptors, but not LH receptor. The thyrotropic potency of the mtTSH was examined by rainbow trout basibranchial tissues containing thyroid follicles. The height of follicle epithelial cells was significantly increased by treatments of mtTSH in vivo and in vitro. In conclusion, the present study suggests that the mtTSH produced by baculovirus–silkworm larvae is a biologically active recombinant TSH.

Transcriptional regulation of 5′-flanking regions of stanniocalcin genes by estrogen receptor and estrogen receptor-related receptor

Stanniocalcin (STC), a glycoprotein hormone originally identified in bony fish, is a regulator of calcium and phosphate homeostasis. Two related STC genes, STC1 and STC2, are expressed in various tissues in vertebrates, but their transcriptional regulation and differences between the two genes are poorly understood. The aim of this study was to determine and compare estrogen-dependent transcriptional activities of the 5′-flanking regions of the mouse STC1 (mStc1) and STC2 (mStc2) genes. Using HEK293 cells and a transient transfection method, the transcriptional activity of the mStc1 and mStc2 genes was measured by a luciferase reporter assay. In this study, we have identified the existence of putative estrogen response elements (EREs) and estrogen receptor (ER)-related receptor (ERR) response elements (ERREs) in the 5′-flanking regions of the mStc1 gene, but the putative ERRE sites only within the mStc2. Luciferase reporter assays showed that ligand bound ERα and ERRγ increase the transcription of mStc1 but not mStc2. In addition, the ERα- and ERRγ-mediated transactivation of the 5′-flanking region of mStc1 was further enhanced by the coregulators, steroid receptor coactivator 1 and peroxisome proliferator-activated receptor γ coactivator 1 α. Taken together, our results suggest that the putative promoter region of mStc1, but not mStc2, is transcriptionally enhanced by ligand-activated ERα and ERRγ.

Changes in Prolactin and Growth Hormone Gene Expression of Rainbow Trout Oncorhynchus mykiss Adapted to Seawater

Prolactin (PRL) plays an important role in freshwater (FW) osmoregulation by preventing the loss of ions and the uptake of water in fish. Growth hormone (GH) promotes acclimation to seawater (SW) in several teleosts. We acclimated rainbow trout Oncorhynchus mykiss weighting 68.2±16.6, 138.3±24, and 287.5±42.1 g in separate experiments to SW under slow-acclimation (SSW) or acute-acclimation (ASW) conditions, and then examined the PRL and GH mRNA levels using the real-time quantitative polymerase chain reaction. The PRL mRNA levels in all three experimental groups decreased significantly with both the SSW and ASW treatments, as compared to a control group kept in FW for 30days. The GH mRNA levels increased with ASW in the largest fish, whereas the levels in the other groups did not change significantly. The mortality rate of the largest fish was lower than for the other groups, whereas the growth rate among the three experimental groups did not differ significantly. The growth rate of the ASW group was highest for the smallest fish. These results suggest that SW acclimation is associated with the gene expression levels of PRL and GH in relatively large rainbow trout. In addition, the fish mortality and growth rate on FW-SW transfer seem to be related to body weight, and the SW acclimation method may be applied to the hatcheries industry.