Supattra Treerattrakool

Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference

One of the important peptide hormones that control reproduction in crustaceans is gonad‐inhibiting hormone (GIH). GIH is known to modulate gonad maturation by inhibiting synthesis of vitellogenin (Vg), the precursor of yolk proteins. In this study, a cDNA encoding a GIH (Pem‐GIH) from the eyestalk of Penaeus monodon was cloned using RT‐PCR and RACE techniques. Pem‐GIH cDNA is 861 bp in size with a single ORF of 288 bp. The deduced Pem‐GIH consists of a 17‐residue signal peptide and a mature peptide region of 79 amino acids with features typical of type II peptide hormones from the CHH family. Pem‐GIH transcript was detected in eyestalk, brain, thoracic and abdominal nerve cords of adult P. monodon. The gonad‐inhibiting activity of Pem‐GIH was investigated using the RNA interference technique. Double‐stranded RNA, corresponding to the mature Pem‐GIH sequence, can trigger a decrease in Pem‐GIH transcript levels both in eyestalk ganglia and abdominal nerve cord explant culture and in female P. monodon broodstock. The conspicuous increase in Vg transcript level in the ovary of GIH‐knockdown shrimp suggests a negative influence for Pem‐GIH on Vg gene expression, and thus implies its role as a gonad‐inhibiting hormone. This is the first report to demonstrate the use of double‐stranded RNA to elucidate the function of GIH in P. monodon.

Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of Penaeus monodon in Pichia pastoris

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the α-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.

Potential roles of transglutaminase and thioredoxin in the release of gonad-stimulating factor in Penaeus monodon: implication from differential expression in the brain during ovarian maturation cycle.

The synthesis of vitellogenin during ovarian maturation in crustacean is induced by gonad-stimulating factor(s) that are synthesized in the brain and thoracic ganglia. This process is negatively regulated by a gonad-inhibiting hormone (GIH) from the eyestalk. This study utilized differential-display RT-PCR technique to identify putative genes in brain and thoracic ganglia that may be involved in ovarian maturation of the black tiger shrimp, Penaeus monodon under the condition in which the expression of GIH was suppressed by GIH-specific dsRNA. After excluding redundant clones and subsequent verification by RT-PCR, 10 and 5 transcripts exhibited up-regulated and down-regulated expressions, respectively, in the GIH-dsRNA injected shrimp when compared with the Tris/NaCl injected shrimp. Among the up-regulated genes, a full sequence of thioredoxin cDNA was cloned, and nucleotide sequence analysis showed that it was highly similar to other crustacean thioredoxin. The thioredoxin gene as well as the other four genes including transglutaminase and three unknowns; U10-11, U10-15 and U13-11 that were up-regulated upon GIH-knockdown exhibited similar expression profile in the brain during ovarian maturation cycle. The highest expression level was detected in the brain of early-vitellogenic female shrimp suggesting that they are required for an initial stage of vitellogenesis. Our results posted for the first time a possible function of transglutaminase and thioredoxin in regulating the gonad-stimulating pathway in the brain of the shrimp.