Identification and characterization of genes functionally related to reproduction of the giant tiger shrimp Penaeus monodon

Abstract

Low degrees of reproductive maturation of the giant tiger shrimp (Penaeus monodon) in captivity have limited the ability to genetically improve this important species effectively. Therefore, mechanisms governing gonadal maturation of P. monodon at the molecular level are important and can be directly applied to the shrimp industry. Genes expressed in testes of P. monodon were identified and characterized by EST analysis. A total of 896 clones from the conventional testis cDNA library were sequenced and 606 ESTs (67.6%) significantly matched sequences in the GenBank (E-value < 1e-04). In addition, 178 clones from the forward and 187 clones from the reverse SSH libraries between cDNA in testes of broodstock and juvenile P. monodon were also constructed and sequenced. Of which, 67 ESTs (37.1%) and 104 ESTs (54.0%) significantly matched known genes. Several genes functionally involved in testicular development were found such as small ubiquitin-like modifier (SUMO-1), cyclophilin A and dynactin subunit 5. In addition, transformer-2 (Tra-2), a gene involving sex determination cascades, was also found. Apart from the full length cDNA that found in the established libraries, additional 16 functionally important gene homologues including low molecular weight neurofilament protein XNF-L (termed P. monodon testis-specific transcript 1, PMTST1), multiple inositol polyphosphate phosphatase 2 (MIPP2), prohibitin-2, cell division kinase 7 (cdk7), flotillin 2, growth factor receptor-bound protein, innexin 1, innexin 2, Rac-GTPase activating protein 1, transformer 2 (Tra-2), meiotic recombination protein DMC1/LIM15 homolog isoform 1 (Dmc1), progestin membrane receptor component 1 (PGMRC1), saposin, troponin T isoform 3, Ero1L CG1333-PB isoform B, and dihydrolipoamide dehydrogenase were successfully characterized by RACE-PCR. Expression patterns of 59 gene homologues in testes and ovaries of juvenile and broodstock P. monodon (N = 4 for each group) were non-quantitatively examined by RT-PCR. PMTST1 was only expressed in testes (N = 8) but not ovaries (N = 8) whereas MIPP, MIPP2, Dmc1, and HSP70-2 exhibited a trend of preferential expression in testes of P. monodon. Thirty-six genes showed a trend of greater expression levels in ovaries than testes. Semiquatitative RT-PCR and quantitative real-time PCR were carried out to examine expression levels of 12 gene homologues in different groups of shrimp. Testis-specific expression of PMTST1 was confirmed. CYA and Trap240 were more abundantly expressed in ovaries than testes (P <0.05). Dmc1, saposin, spermatogonial stem-cell renewal factor, MIPP and HSP70-2 were preferentially expressed in testes to ovaries (P <0.05). Expression levels of SUMO-1, Tra-2 and prohibitin2 in ovaries and testes of P. monodon were not significantly different (P >0.05). PMTST1 was up-regulated but that of the remaining genes in testes of P. monodon broodstock was down-regulated after shrimp were molted (P <0.05). Significant reduction of SUMO-1, Dmc1, and spermatogonial stem-cell renewal factor and increment of prohibitin2 transcripts in domesticated broodstock (P <0.05) suggested that these reproductively related genes may be used as biomarkers to evaluate reduced degrees of the reproductive maturation in domesticated P. monodon. In addition, effects of dopamine on expression of PGMRC1 and Dmc1 in testes of juvenile P. monodon (3, 6, 12, and 24 hr post injection) were examined. Dopamine administration (10-6 mol/shrimp) resulted in up-regulation of PGMRC1 in testes of juvenile P. monodon at 3 h post treatment (P < 0.05) but had no effect on Dmc1 (P > 0.05). Recombinant proteins of Dmc1, spermatogonial stem-cell renewal factor, SUMO-1, and CYA were successfully expressed in vitro. The polyclonal antibody was produced from recombinant Dmc1, spermatogonial stem-cell renewal factor, and CYA proteins for further functional analysis of these genes at the translational level