Cloning and characterisation of mitochondrial manganese superoxide dismutase (mtMnSOD) from the giant freshwater prawn Macrobrachium rosenbergii.

A cDNA encoding a mitochondrial manganese superoxide dismutase (mtMnSOD) was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription polymerase chain reaction (RT-PCR) by degenerate primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) PCR method. Analysis of nucleotide sequence revealed that the mtMnSOD full-length cDNA consists of 1202bp containing an open reading frame of 654bp, which encodes a protein consisting of 218 amino acids including a signal peptide of 16 amino acid residues. The calculated molecular mass of the mature proteins (202 amino acids) is 24kDa with an estimated pI of 7.12. Two putative N-glycosylation sites, NXT and NXS were observed in the mtMnSOD. Manganese superoxide dismutase signatures from 180 to 187 (DVWEHAYY), and four conserved amino acids responsible for binding manganese were observed (H48, H96, D180 and H184). Sequence comparison showed that the mtMnSOD deduced amino acid sequence of Macrobrachium rosenbergii has similarity of 88%, 78%, 56%, 54% and 46% to that of blue crab Callinectes sapidus, crucifix crab Charybdis feriatus, brown shrimp Farfantepenaeus aztecus, European lobster Palinurus vulgaris, and grass shrimp Palaemontes pugio, respectively, and has similarity of 45%, 44%, 43%, 26% and 25% to cytMnSOD (cytosolic MnSOD) deduced amino acid sequence of blue crab C. sapidus, prawn M. rosenbergii, tiger shrimp Penaeus monodon, grass shrimp P. pugio and brown shrimp F. aztecus, respectively. Quantitative real-time RT-PCR analysis showed that levels of mtMn-SOD transcripts in hepatopancreas and haemocytes were not significantly different between the M. rosenbergii injected with Lactococcus garvieae, and that injected with saline after 3h to 24h.

Hormones and reproduction in scleractinian corals.

Most broadcast spawning scleractinian corals synchronously release gametes during a brief annual spawning period. In southern Taiwan, the mass spawning of scleractinians occurs in lunar mid-March. The exact cues triggering this annual phenomenon remain unclear. A scleractinian coral, Euphyllia ancora has been selected as a model for the hormones and reproduction studies. Testosterone (T) and estradiol (E2) in free and glucuronided forms were identified and consistently detected in coral polyps throughout the year. Peak levels of free E2, glucuronided E2 and T were obtained in the coral tissue just prior to spawning. The presence of specific aromatase activity was demonstrated in coral tissue. Higher concentrations of free E2 than glucuronided E2 were detected in the coral tissue throughout the year. In contrast, higher levels of glucuronided E2 than free E2 and glucuronided T were found in seawater during mass coral spawning. Furthermore, immunoreactivity and biological activity of immunoreactive gonadotropin-releasing hormone (irGnRH) was detected and quantified in coral tissue. Coral extracts (irGnRH) and mammalian (m)GnRH agonist had a similar dose-dependent effect on luteinizing hormone (LH) release in black porgy fish pituitary cells (in vitro). Peak levels of irGnRH were detected during the spawning period. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity, as well as the levels of T and E2 in free and glucuronided forms in coral. In conclusion, our data suggest that irGnRH and glucuronided E2 may play important roles in the control of reproduction and mass spawning in corals.

Molecular cloning and characterisation of cytosolic manganese superoxide dismutase (cytMn-SOD) from the giant freshwater prawn Macrobrachium rosenbergii.

A cytosolic manganese superoxide dismutase (cytMn-SOD) cDNA was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription polymerase chain reaction (RT-PCR) by degenerate primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cytMn-SOD cDNA clone consists of 1339 bp with an open reading frame of 858 bp encoding a protein of 286 amino acids. The calculated molecular mass of the mature proteins (286 amino acids) is 31 kDa with an estimated pI of 5.52. Two putative N-glycosylation sites, NXT and NXS were observed in the cytMn-SOD. Four conserved amino acids responsible for binding manganese were observed (H110, H158, D243 and H247). Sequence comparison showed that the cytMn-SOD deduced amino acid sequence of M. rosenbergii has an overall similarity of 77% and 54% to that of blue crab Callinectes sapidus and tiger shrimp Penaeus monodon, respectively. Quantitative real-time RT-PCR analysis showed that cytMn-SOD transcript in hepatopancreas of M. rosenbergii decreased 3h after Lactococcus garvieae injection, but no significant change in cytMn-SOD transcript was observed in the haemocytes 3-24 h after L. garvieae injection.

Molecular cloning and characterisation of copper/zinc superoxide dismutase (Cu,Zn-SOD) from the giant freshwater prawn Macrobrachium rosenbergii.

A copper/zinc superoxide dismutase (Cu,Zn-SOD) cDNA was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription-polymerase chain reaction (RT-PCR) by degenerate primers. Both 3'- and 5'-regions were isolated by the rapid amplification of cDNA ends method. Analysis of nucleotide sequence revealed that the Cu,Zn-SOD cDNA clone consists of 845 bp with an open reading frame of 603 bp encoding a protein of 201 amino acids with a 22 amino acid signal peptide. The calculated molecular mass of the mature proteins (179 amino acids) is 21 kDa with an estimated pI of 4.75. Two putative N-glycosylation sites, NXT and NXS, were observed in the Cu,Zn-SOD. Four conserved amino acids responsible for binding copper (H86, H89, H106 and H163) and four conserved amino acids responsible for binding zinc (H106, H114, H123 and D126) were observed. Sequence comparison showed that the Cu,Zn-SOD deduced amino acid sequence of M. rosenbergii has similarity of 60% and 64% to that of freshwater crayfish Pacifastacus leniusculus ecCu,Zn-SOD and blue crab Callinectes sapidus ecCu,Zn-SOD, respectively. Quantitative real-time RT-PCR analysis showed that Cu,Zn-SOD transcripts in haemocytes of M. rosenbergii increased 3h and 6h after injection of Lactococcus garvieae, whereas Cu,Zn-SOD transcripts decreased in the hepatopancreas 3h after L. garvieae injection.

The presence and ancestral role of gonadotropin-releasing hormone in the reproduction of scleractinian coral, Euphyllia ancora.

The objectives of this study were to investigate the presence of immunoreactive GnRH (irGnRH) in scleractinian coral, Euphyllia ancora, study its seasonal variation, and evaluate its biological activity. irGnRH was detected and quantified in coral polyps. The biological activity of coral irGnRH was tested on pituitary cells from black porgy by evaluating its ability to stimulate LH release. Coral extracts (10(-9)-10(-5) M irGnRH) as well as mammalian (m) GnRH agonist (10(-10)-10(-6) M) had a similar dose-dependent effect on LH release. Furthermore, GnRH receptor antagonist dose-dependently inhibited the stimulation of LH release in response to coral extracts (10(-5) M irGnRH) and mGnRH agonist (10(-6) M). Peak levels of irGnRH (10-fold increase) were observed during the spawning period in a 3-yr investigation. Significantly higher aromatase activity and estradiol (E2) levels were also detected during the period of spawning compared with the nonreproductive season. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity as well as the concentrations of testosterone and E2 in free and glucuronided forms in coral. In conclusion, our data indicate that irGnRH does exist in coral, with its ability to stimulate LH release in fish. Seasonal variations of coral irGnRH, with a dramatic increase during the spawning period, concomitant to that in aromatase and E2, as well as the ability of mGnRH agonist to stimulate coral aromatase, steroidogenesis, and steroid glucuronization suggest that irGnRH plays an important role in the control of oocyte growth and mass spawning in corals.