Once the prostanoid pathway had been established in P. monodon, we examined whether there is a correlation between prostanoid biosynthesis and shrimp ovary development. In wild P. monodon broodstock, the concentrations of PGE2 decreased from stage I to stage III, but abruptly increased in stage IV ovaries (). This is inconsistent with the trend found in domesticated shrimp, in which the amounts of PGE2 gradually increased during ovary development . The discrepancy between the two studies may be the result of different shrimp genetic background and/or dietary intake. Interestingly, the PGE2 concentrations were highest at stage IV ovaries in both wild and domesticated shrimp, but the significance of this observation has yet to be explored. When the gene expression of each PmPGES isoform was compared to the concentration of PGE2 found in each ovary stage, we observed no correlation between the two parameters. Furthermore, the lowest amount of PmPGES transcripts was found in stage IV ovaries, making it unlikely that the PGE2 biosynthesis is regulated at the transcriptional level in this organ.
The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development.
Basic Implications of Clinical Observations
In this study, the characterization of the P. monodon prostanoid pathway reveals that P. monodon contains the same types and number of prostaglandin synthase isoforms as those found in mammals (Fig, 1-4; ). In addition to the three PmPGES isoforms shown in the Results section, P. monodon also encodes two isoforms of prostaglandin D synthase that matched the glutathione-dependent prostaglandin D synthase and hematopoietic prostaglandin D synthase genes originally identified in mammals. Interestingly, the proposed prostanoid pathway in another crustacean Daphnia pulex contains only one isoform of each prostanoid biosynthesis gene , suggesting that the organization of the P. monodon prostanoid pathway is more conserved with those in mammals, or other divergent prostanoid biosynthetic genes are present in D. pulex that are not annotated. To compare if the P. monodon prostanoid protein sequence is more closely related to its homologs in D. pulex or mammals, phylogenetic analysis of PGES1 was performed, revealing that PmPGES1 is more closely related to its crustacean homologs, including D. pulex, than to its mammalian homologs ().