Kwang Wook An

Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus)

Glutathione peroxidase (GPX) and glutathione S-transferase (GST) are key enzymes of cellular detoxification systems that defend cells against reactive oxygen species (ROS). In this study, we isolated the GPX and GST full-length cDNA and investigated the expression of these mRNAs from livers of olive flounder during salinity changes (35, 17.5, 8.75, 4 and 0 psu) by quantitative PCR (QPCR). GPX cDNA consists of 429 base pairs (bp) and encodes a protein of 142 amino acids. GST cDNA consists of 663 bp and encodes a protein of 220 amino acids. Both of GPX and GST mRNA expressions were the highest in 4 psu and then decreased in 0 psu. Also, the levels of Na(+) and Cl(-) decreased, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased during the experimental period. These findings provide molecular characterization of GPX and GST in olive flounder and suggest that GPX and GST play important roles in detoxification of ROS, thereby these maybe indicators of oxidative stress responses by salinity changes in olive flounder.

Profiles of antioxidant gene expression and physiological changes by thermal and hypoosmotic stresses in black porgy (Acanthopagrus schlegeli).

We determined oxidative stress by measuring the expression and activity of 3 antioxidant enzymes [Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPX)] in black porgy exposed to thermal (20 degrees C-->30 degrees C) and hypoosmotic (35 psu-->10 psu and 0 psu) stresses. The expression and activity of antioxidant enzymes were significantly higher after exposure to 30 degrees C, 10 psu, and 0psu. Furthermore, we measured H(2)O(2) and lipid peroxidation (LPO) levels. As a result, H(2)O(2) and LPO levels were significantly increased after exposure to thermal (20 degrees C-->30 degrees C) and hypoosmotic stress (35 psu-->10 psu and 0 psu) stress. These results indicate that thermal and hypoosmotic stress induces oxidative stress in black porgy. Additionally, we investigated the changes due to thermal and hypoosmotic stress by measuring plasma cortisol and ion (Na(+) and Cl(-)) levels. Plasma cortisol levels increased at 30 degrees C and at 10 psu and then decreased at 0 psu. However, plasma Na(+) and Cl(-) levels did not change after exposure to thermal stress (30 degrees C), and decreased at 10 psu and 0 psu. In conclusion, thermal and hypoosmotic environments increase oxidative stress, thereby these results may be indicators of oxidative stress in black porgy.

Physiological responses and expression of metallothionein (MT) and superoxide dismutase (SOD) mRNAs in olive flounder, Paralichthys olivaceus exposed to benzo[a]pyrene

We cloned complementary DNA (cDNA) encoding metallothionein (MT) and superoxide dismutase (SOD) from the liver of olive flounder, Paralichthys olivaceus. The full-length MT cDNA consists of 183 base pairs (bp) and encodes a protein of 60 amino acids; partial SOD cDNA consists of 326 bp and encodes a protein of 109 amino acids. We investigated the dose- and time-related effects of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) on MT and SOD mRNA using quantitative polymerase chain reaction (QPCR). The expression levels of MT mRNA were highest at 24 h (about five times) in 10 microg/L BaP, and at 6 h (about twelve times) in 30 microg/L BaP. The expression levels of SOD mRNA were highest at 12 h (about three times) in 10 microg/L BaP, and at 6 h (about six times) in 30 microg/L BaP, and then decreased toward the end of the experiment. We also measured plasma glucose and cortisol, all of which increased with BaP exposure. These results suggest that MT and SOD play an important role in the detoxification of reactive oxygen species (ROS) caused by BaP exposure, and thus may be indicators of oxidative stress responses.

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy, Acanthopagrus schlegeli: effects of estradiol-17β on mRNA expression profiles.

The cDNAs of three gonadotropin (GTH) subunits (GTHalpha, FSHbeta, and LHbeta) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHalpha, FSHbeta, and LHbeta cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17beta (E(2)) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E(2) level when E(2) treatment was applied to immature black porgy. E(2) treatment increased mRNA expression levels of the genes and plasma E(2) levels, indicating that E(2) stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E(2) plays an important regulatory role in the brain-pituitary-gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.

Characterization and mRNA expression of Mn-SOD and physiological responses to stresses in the Pacific oyster Crassostrea gigas.

Abstract Superoxide dismutases (SODs) are metalloenzymes that play an important role in mollusc immune defence systems by eliminating oxidative stress to reactive oxygen species. We investigated physiological changes in the Pacific oyster Crassostrea gigas caused by exposure to pollutants (cadmium, tributyltin) and acute water temperature change. We analysed mRNA expression of Mn-SOD in gills using a quantitative polymerase chain reaction (QPCR), and measured the glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and hydrogen peroxide (H2O2) levels in haemolymph based on time- and dose-related effects of pollutants and acute water temperature treatments. We cloned a cgMn-SOD full-length cDNA that included an open reading frame of 675 nucleotides that was predicted to encode proteins of 225 amino acids. BLAST analysis of other species indicated that residues essential to the enzymatic functions of Mn-SOD proteins are highly conserved. Levels of Mn-SOD mRNA expression, GOT and GPT were gradually increased and then decreased during the experimental periods. On the other hand, H2O2 levels increased continuously during the exposure periods. These results suggest that Mn-SOD plays an important role in the physiological changes related to metabolism and cell protection that occur in C. gigas when exposed to oxidative stress by pollutants and fluctuations in water temperature.

Molecular characterization and expression of three GnRH forms mRNA during gonad sex-change process, and effect of GnRHa on GTH subunits mRNA in the protandrous black porgy (Acanthopagrus schlegeli).

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in control of reproduction and gonadal maturation in teleost fish. To investigate the action GnRH in black porgy (Acanthopagrus schlegeli), we examined the mRNA expression of GTH subunits (GTHalpha, FSHbeta, and LHbeta) in the pituitary as well as plasma estradiol-17beta (E(2)) level following treatment with a GnRH analog (GnRHa) in immature fish. The expression levels of GTH subunits mRNA and plasma E(2) level were increased after GnRHa injection. We were also able to identify three GnRH forms: salmon GnRH (sGnRH), seabream GnRH (sbGnRH) and chicken GnRH-II (cGnRH-II) by cDNA cloning in the ovary of the black porgy. Black porgy gonadal development is divided into seven stages, involving sex change from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary, and mature ovary). In the present study, we investigated the expression pattern of three GnRH molecular forms in the black porgy gonads at different stages of gonadal development by quantitative polymerase chain reaction (QPCR). The mRNA expressions of sGnRH, sbGnRH and cGnRH-II were found to be higher in mature testis and ovary, compared to gonads at different stages of maturity. The findings support the hypothesis that the three forms of GnRH play important roles in the regulation of hypothalamic-pituitary-gonadal axis, and are likely involved also in gonadal development and sex change in black porgy.

Expression of warm temperature acclimation‐related protein 65‐kDa (Wap65) mRNA, and physiological changes with increasing water temperature in black porgy, Acanthopagrus schlegeli

We isolated the warm temperature acclimation-related protein 65-kDa (Wap65) cDNA from the liver of black porgy and investigated the expression by increasing water temperature in black porgy, Acanthopagrus schlegeli. Black porgy Wap65 full-length cDNA consists of 1,338 nucleotides, including an open reading frame, predicted to encode a protein of 425 amino acids and showed high homology to pufferfish (79%), Medaka (73%), carp (70%), and goldfish (68%) Wap65. Increase in water temperature (20 degrees C --> 30 degrees C; 1 degrees C/day) induced the rise of Wap65 mRNA expression in liver of black porgy. Also, the levels of cortisol and glucose in plasma were significantly higher at 30 degrees C than at 20 degrees C. To determine the high water temperature stressor specificity of the induction of Wap65, black porgy were transferred from seawater (SW) to freshwater (FW) for 24 hr. Wap65 expression was not detected when the fish were transferred from SW to FW (in fish transferred from SW to FW), although the levels of cortisol and glucose in plasma were increased. These results suggest that increase in Wap65 gene is related to high water temperature stress and play important roles in high water temperature environment of black porgy.

Gender-related expression of TRα and TRβ in the protandrous black porgy, Acanthopagrus schlegeli, during sex change processes

We cloned the thyroid hormone receptor alpha (TRalpha) and beta (TRbeta) cDNAs from the ovaries of the protandrous black porgy and compared the expression levels of TRalpha and TRbeta mRNA during the sex change in black porgy. We observed that the TRalpha mRNA by quantitative real-time PCR and protein levels by Western blot were highest in the mature ovaries. Additionally, TRbeta mRNA levels were only expressed highly in the mature ovaries when compared to any other gonadal stages. Then, we injected gonadotropin-releasing hormone analogue (GnRHa) to know the effects on TRs mRNA in immature black porgy. Injection with GnRHa resulted in a significant increase in TRalpha level while significantly reducing TRbeta level after 12h. We concluded that TRalpha was related in testicular development as well as ovarian development and TRbeta was only affect to ovarian development in black porgy. These results will provide a framework for better understanding of the role of TRs during sex change processes in this fish.

Quantitative mRNA expression of sox3 and DMRT1 during sex reversal, and expression profiles after GnRHa administration in black porgy, Acanthopagrus schlegeli.

We cloned full-length sox3 cDNA from testis of black porgy, Acanthopagrus schlegeli. Black porgy sox3 cDNA consists of 897 base pairs (bp) and encodes a protein of 298 amino acids. We have investigated the expression pattern of sox3 and DMRT1 mRNA during the sex-reverse process from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary and ovary). The expression of sox3 and DMRT1 mRNA was high in mature testis of black porgy during sex-reverse process. In a histological analysis, testicular portion of gonad was degenerated and the ovary portion was increased during sex reversal from male to female, and then oocytes were increased in ovary. Also we examined the expression of sox3 and DMRT1 mRNA after gonadotropin-releasing hormone analogue (GnRHa) treatment in immature black porgy. The expression of sox3 and DMRT1 mRNA was increased after GnRHa treatment (in vivo and in vitro experiment) in immature black porgy. Therefore, we concluded that sox3 and DMRT1 were involved in the development of testis than ovary in black porgy.

Characterization of estrogen receptor β2 and expression of the estrogen receptor subtypes α, β1, and β2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.