PROTPROTEOME AND CHARACTERIZATION OF GENES AND PROTEINS FUNCTIONALLY INVOLVED IN OOGENESIS OF THE BLACK TIGER SHRIMP Penaeus monodon

Abstract

Proteomics of ovarian proteins in domesticated and wild broodstock of the giant tiger shrimp Penaeus monodon were studied using GeLC-MS/MS. In total, 1638 proteins were identified and 1253 (76.50%) proteins matched known proteins. Of these, 514 proteins were differential expressed between groups of samples. To characterized proteins involved in GVBD, nuclear membrane and nuclear proteins were identified. In total, 724 proteins were identified and matched known proteins. Localization of these proteins were searched and 89 and 99 proteins were classified as those integrated to membrane and nuclear proteins. In this thesis, proteins involved in signal transduction pathways and cytoskeletal reorganization during GVBD were characterized. The full-length cDNA of Valosin-containing protein (PmVCP1, 2724 bp with an ORF of 2481 deducing to 826 amino acids), Thymosin beta (PmTmsb; 1084 bp, an ORF = 387 bp, 128 aa), Protein kinase C (PmPKC; 3404 bp, an ORF = 2235 bp, 744 aa), cyclic AMP-regulated protein like protein (PmcAMP-RLP; 1272 bp, an ORF = 435 bp, 144 aa), Nuclear pore complex protein NUP133 (PmNup133; 4130 bp, an ORF = 3228 bp, 1085 aa) were successfully characterized by RACE-PCR. In addition, the complete ORF of Rac GTPase activating protein 1 (PmRacgap1; 1881 bp, 626 aa) was also successfully isolated.

The expression levels of PmVCP were not significant different throughout ovarian development of intact broodstock (P > 0.05). However, PmVCP was up-regulated during vitellogenesis and final maturation of eyestalk-ablated broodstock (P < 0.05) and its expression in stage IV ovaries was greater than that of the same stage in intact broodstock (P < 0.05). The expression level of PmTmsb was not differential expressed in ovaries of wild intact broodstock but it was up-regulated in stages II and IV ovaries in eyestalk-ablated broodstock (P < 0.05). Eyestalk ablation resulted in the reduction of this transcript in stage III ovaries (P < 0.05). The expression of PmcAMP-RPL was not different during ovarian development of intact and eyestalk-ablated broodstock. Similarly, the expression of PmPKC was not significantly expressed during ovarian development of intact and eyestalk-ablated brooodstock. Nevertheless, eyestalk ablation resulted in the reduction of this transcript in stages I-IV ovaries. The expression level of PmRacgap1 was not differentially expressed during ovarian development of intact and eyestalk ablated broodstock (P > 0.05). In addition, effects of progesterone and serotonin (5-HT) administration in domesticated P. monodon were evaluated Results indicated that progesterone did not induce the expression of PmVCP. In contrast, the expression level of PmVCP, PmRacgap1, PmcAMP-RPL and PmPKC but not PmTmsb was stimulated by serotonin administration.

The expression profiles of ovarian PmVCP, PmTmsb and PmRacgap1 proteins were examined. PmVCP were observed in juvenile ovaries and at all stages of ovarian development in both intact and eyestalk-ablated broodstock of wild P. monodon. It seemed to be expressed at comparable levels for all stages of ovarian development in shrimp broodstock. Two immunoreactive bands (34 and 100 kDa) of PmRacgap1 was observed in ovarian membrane proteins. The expression level of PmRacgap1 reflected from a 34 kDa band seemed to be decreased in late stages of ovarian development (stages III and IV ovaries) in both intact and eyestalk-ablated broodstock. Anti-rPmTmsb PAb gave positive the immunoreactive signals of 22 and 28 kDa, respectively. The expression level of PmTmsb reflected from a positive 28 kDa band (thymosin-β-repeated protein 2) seemed to be decreased in mature (IV) ovaries in intact broodstock. In eyestalk-ablated broodstock, it was not expressed in late vitellogenic (III) and mature ovaries. Localization of PmVCP protein was observed in the ooplasm of previtellogenic oocytes and it was translocated into the nucleus of vitellogenic oocytes. Interestingly, it was found in nucleo-cytoplasmic compartments, the cytoskeletal architecture and the plasma membrane in mature oocytes of both intact and eyestalk-ablated broodstock. PmRacgap1 was observed in oogonia and ooplasm of all developmental stages of oocytes in both intact and eyestalk-ablated broodstock. During vitellogenesis, it was also observed in the nucleus of vitellogenic oocytes and subsequently, in nucleo-cytoplasmic compartments, the cytoskeletal architecture and in cortical rods of more mature oocytes of both intact and eyestalk-ablated broodstock.