Molecular cloning of Daphnia magna catalase and its biomarker potential against oxidative stresses.

Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to oxygen and water. Catalase mRNAs have been cloned from many species and employed as useful biomarkers of oxidative stress. In the present study, we cloned the cDNA from the catalase gene in Daphnia magna, analyzed its catalytic properties, and investigated mRNA expression patterns after the exposure to known oxidative stressors. The catalase proximal heme-ligand signature sequence, FDRERISERVVHAKGSGA, and the proximal active site signature, RLFSYTDTH, are highly conserved. The variation of catalase mRNA expression in D. magna was quantified by real-time PCR, and the results indicated that catalase expression was up-regulated after exposure to UV-B light or cadmium (Cd). The activity of catalase enzyme also showed a similar increasing pattern when exposed to these model stressors. The full-length catalase cDNA of D. magna was cloned using mixed primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1515 nucleotides, encoding 504 amino acids. Sequence comparison showed that the deduced amino acid sequence of D. magna shared 73%, 72%, 71% and 70% identity with that of Chlamys farreri, Fenneropenaeus chinensis, Litopenaeus vannamei and Anopheles gambiae, respectively. This study shows that the catalase mRNA from D. magna could be successfully employed as a biomarker of oxidative stress, which is a common mode of toxicity for many water contaminants.

Expression of three gonadotropin subunits and gonadotropin receptor mRNA during male-to-female sex change in the cinnamon clownfish, Amphiprion melanopus.

To quantify the sex-change progression from male to female in the cinnamon clownfish, Amphiprion melanopus, we divided gonadal development into three stages (I, mature male; II, male at 90 days after removal of the female; and III, mature female), and the expression of GTH subunits and GTH receptors during each of these stages was investigated. The mRNA of the three GTH subunits and their receptors increased with progression from male to female. To understand the effect of gonadotropin-releasing hormone (GnRH) on this progression, we examined expression of genes encoding the GTH subunit mRNA in the pituitary and the GTH-receptor mRNA in the gonads in addition to investigating changes in plasma E(2) levels after GnRH analogue (GnRHa) injection. GnRHa treatment increased mRNA expression levels of these genes, as well as plasma E(2) levels, indicating that GnRH plays an important regulatory role in the brain-pituitary-gonad axis of immature cinnamon clownfish.

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.

Gender-related expression of TRalpha and TRbeta 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.

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy, Acanthopagrus schlegeli: effects of estradiol-17beta 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.

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.

Cloning and expression of aquaporin 1 and arginine vasotocin receptor mRNA from the black porgy, Acanthopagrus schlegeli: effect of freshwater acclimation.

We cloned complementary DNA (cDNA) encoding aquaporin 1 (AQP1) and arginine vasotocin receptor (AVT-R) from gill and kidney tissue of the black porgy (Acanthopagrus schlegeli), respectively. Black porgy AQP1 cDNA consists of 786 base pairs (bp) and encodes a protein of 261 amino acids, and AVT-R partial cDNA consists of 606 bp. To investigate the osmoregulatory abilities of black porgy in different salinities (35 per thousand seawater, SW, 10 per thousand SW, freshwater, FW), we examined the expression of AQP1 and AVT-R mRNA in osmoregulatory organs using the reverse transcription polymerase chain reaction (RT-PCR). AQP1 mRNA levels increased in the gill and intestine during FW acclimation, and the mRNA expression in the kidney was greatest in 10 per thousand SW and then decreased in FW. On the other hand, AVT-R mRNA was expressed in the gill only in 10 per thousand SW, while it increased in the kidney in 10 per thousand SW and then decreased in FW. Thus, the expression of these mRNAs increased in hypoosmotic environments. These results suggest that AQP1 and AVT-R genes play important roles in hormonal regulation in osmoregulatory organs, thereby improving the hyperosmoregulatory ability of black porgy in hypoosmotic environments.

Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 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.

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.

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.

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.

Cloning and expression of Na+/K+ -ATPase and osmotic stress transcription factor 1 mRNA in black porgy, Acanthopagrus schlegeli during osmotic stress.

We cloned complementary DNA (cDNA) encoding the Na(+)/K(+)-ATPase (NKA) and the osmotic stress transcription factor 1 (OSTF1) from the kidney and gill, respectively, of the black porgy, Acanthopagrus schlegeli. Black porgy NKA full-length cDNA consists of 3078 base pairs (bp) and encodes a protein of 1025 amino acids; OSTF1 partial cDNA consists of 201 bp. To investigate the osmoregulatory ability of black porgy when black porgy were transferred to freshwater (FW), we examined the expression of NKA and OSTF1 mRNA in osmoregulatory organs, i.e., gill, kidney and intestine, using quantitative polymerase chain reaction (QPCR). To determine the hypoosmotic stressor specificity of the induction of NKA and OSTF1, black porgy were exposed to 30 degrees C water temperature for 24 h. In the gill, NKA mRNA was 4.2 times higher in FW, its expression in the kidney was 5.7 times higher in 10 per thousand seawater (10 per thousand SW) than in SW. In contrast, OSTF1 mRNA in the gill was 3.7 times higher in FW than in SW. The expression of heat shock protein 90 (HSP90) mRNA occurred not only during transfer to FW, but also in high-temperature water in all tested tissues, although the mRNA levels were not significantly different. Plasma osmolality level was decreased and cortisol level was increased when the fish were transferred from SW to FW. These results suggest that NKA and OSTF1 genes play important roles in hormonal regulation in osmoregulatory organs and that these genes are specific to hypoosmotic stress, improving the hyperosmoregulatory ability of black porgy in hypoosmotic environments.

Cadmium affects the expression of metallothionein (MT) and glutathione peroxidase (GPX) mRNA in goldfish, Carassius auratus.

Cadmium (Cd) is a widespread non-essential heavy metal that enters the aquatic environment as a result of natural and human-caused activities, including industrial effluent, mining, and agricultural runoff. In the present study, we investigated time and dose-related effect of CdCl(2) on metallothionein (MT) and glutathione peroxidase (GPX) mRNA levels in a number of goldfish tissues, in vivo. Basal MT and GPX mRNA levels remained unchanged in the tissues tested throughout the experiment. Injection with CdCl(2) significantly increased MT mRNA levels in the brain, liver, kidney and intestine in a dose-dependant manner at all time tested (6, 12, 24 and 36 h). We isolated the full length GPX cDNA from goldfish kidneys, and found it to contain 785 nucleotides, including an open reading frame, predicted to encode a protein of 142 amino acids. In contrast, injection with CdCl(2) significantly decreased GPX mRNA levels in the liver and kidney in a time-, and dose-, dependant, and became undetectable after 12, 24 and 36 h. The findings provide molecular characterization of MT and GPX in goldfish and suggest that exposure to Cd results in significant physiological changes in goldfish.

Effects of gonadotropin-releasing hormone analog (GnRHa) on steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) gene expression in the black porgy (Acanthopagrus schlegeli).

We examined effects of GnRHa on expression of steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) in the pituitary and gonad of protandrous black porgy (Acanthopagrus schlegeli). Fish were intraperitoneally injected with 0.2 microg GnRHa/g fish and then pituitary, gonad and plasma were sampled at 0, 6, 12, 24 and 48 h after injection. In gonad, the mRNA levels of the SF-1 were high at 6 h post injection, and then continuously decreased until 24 h; high expression of ERbeta mRNA levels was only observed at 12 h. In contrast, pituitary SF-1 mRNA levels were very low during the experimental period. GnRHa stimulation caused a significant increase of plasma testosterone (T) and estradiol-17beta (E(2)) after 24 h. We suggest that SF-1 and ERbeta play an important role in the development of gonad and these genes are involved with sex change in fish.