Characterization and expression of o-methyltransferase and broad complex genes and proteins in the giant tiger shrimp penaeus monodon

Abstract

Identification and characterization of genes and proteins involved in ovarian development are the initial step necessary for understanding molecular mechanisms of reproductive maturation in the giant tiger shrimp (Penaeus monodon). The full length cDNAs of catechol-O-methyltransferase (PmCOMT), farnesoic-O-methyltransferase (PmFAMeT), broad comples Z1 (PmBr-cZ1) and broad comples Z4 (PmBr-cZ4) were successfully characterized. Only a single form of PmFAMeT and PmBr-cZ4 was found but two isoforms were observed in PmFAMeT (PmFAMeT-l and PmFAMeT-s) and PmBr-cZ1 (PmBr-cZ1-l and PmBr-cZ1-s). In addition, genomic organization of PmCOMT (3 exons of 194, 111 and 361 bp and 2 introns of 143 and 147 bp) was also isolated. Quantitative real-time PCR indicated that the expression level PmCOMT was not significantly different during ovarian development in both intact and eyestalk-ablated P. monodon broodstock (p>0.05). PmFAMeT mRNA was significantly up-regulated at stage IV ovaries in intact wild broodstock (p<0.05). In contrast, its expression level was not significantly different during ovarian development of eyestalk-ablated broodstock (P > 0.05). In intact wild broodstock, PmBr-cZ1 was significantly down-regulated at stages II and III ovaries (p<0.05) and returned to the basal level at stage IV ovaries and after spawning. Eyestalk ablation resulted in its up-regulation at stage IV ovaries of P. monodon broodsotck. The level of PmBr-cZ4 mRNA was down-regulated at stage IV ovaries of intact P. monodon broodstock (p<0.05). Nevertheless, this transcript was up-regulated at stage IV ovaries of eyestalk-ablated broodstock (p>0.05). Effects of serotonin (5-HT), progesterone (P4) and 20-hydroxysteriod (20E) on expression of these genes in domesticated shrimp were examined. Serotonin administration immediately elevated the expression level of FAMeT approximately 50 fold at 1 hpi (p<0.05). In contrast, progesterone had no effects on expression of these genes (P > 0.05). The expression levels of PmCOMT (at 6 hpi), PmBr-cZ1 (at 168 hpi) and PmBr-cZ4 (at 168 hpi) in ovaries of juvenile P. monodon was significantly decreased following 20E treatment (p<0.05). In situ hybridization revealed that PmFAMeT, PmBr-cZ1 and PmBr-cZ4 transcripts were localized in cytoplasm of previtellogenic oocytes while PmCOMT mRNA was clearly observed in the cytoplasm of follicular cells, oogonia and previtellogenic oocytes. Recombinant protein of PmCOMT, PmFAMeT-l and PmFAMeT-s were successfully expressed in vitro. The polyclonal antibody against each recombinant protein was produced. Western blot analysis indicated more preferentially expressed of PmCOMT in previtellogenic and vitellogenic ovaries than that in early cortical rod and mature ovaries of P. monodon. PmFAMeT was found in ovaries of juveniles and stages I and II ovaries of broodstock. Interestingly, juvenile shrimp possessed either 32 kDa, 37 kDa or both positive bands whereas only a 37 kDa band owing to posttranslational modifications of ovarian FAMeT was observed in stages I and II ovaries of shrimp broodstock. Immunohistochemistry revealed the positive signals of the PmCOMT protein in cytoplasm of previtellogenic and vitellogenic oocytes. Subsequently, the positive signals were observed in cortical rods of stages III and IV oocytes in both intact and eyestalk-ablated broodstock. Interestingly, the PmFAMeT protein was detected in stages III and IV oocytes but not in stage I and II oocytes of P. monodon broodstock. Taken the information together PmCOMT, PmFAMeT, PmBr-cZ1 and PmBr-cZ4 gene products seem to play the important role on ovarian development and PmFAMeT, PmBr-cZ1 and PmBr-cZ4, in particular, may be used as the bioindicators for monitoring progression of oocyte/ovarian maturation in P. monodon.