Isolation, characterization and expression of genes and proteins in testes of the giant tiger shrimp penaeus monodon

Abstract

Proteomic analysis based on two-dimensional gel electrophoresis (2-DE) was carried out to identify reproduction-related proteins in testes of wild and domesticated 14-month-old broodstock of the giant tiger shrimp (Penaeus monodon). Total proteins extracted from testes of wild (group A, GSI = 1.08 ± 0.18%, sperm sac/testis = 0.26 ± 0.06,) and domesticated broodstock (group B, GSI = 0.37 ± 0.05%, sperm sac/testis = 0.22 ± 0.01 and group C, GSI = 0.31 ± 0.05%, sperm sac/testis = 0.52 ± 0.02) were further analyzed by 2-DE. In total, 640 protein spots were characterized including 394 spots from wild broodstock (group A), 120 spots from domesticated broodstock group B and 126 spots from domesticated broodstock group C, respectively. Interesting proteins such as farnesoic acid-O-methyltransferase (FAMeT), progesterone receptor-related protein p23, receptor activating protein kinase C (RACK), 14-3-3-like protein and NADP-dependent leukotriene B4 12-hydroxydehydrogenase (LTB4DH) were identified. In addition, a new proteomic analysis based on one-dimensional gel electrophoresis (1-DE) of total proteins in testes of wild (group A; GSI 0.66 ± 0.18%, sperm sac/testis = 0.51 ± 0.09 and group B; GSI = 0.68 ± 0.09%, sperm sac/testis = 0.49 ± 0.66), and domesticated 14-month-old (group C; GSI = 0.37 ± 0.03%, sperm sac/testis = 0.5 ± 0.02) and 18-month-old (group D; GSI = 0.37 ± 0.01%, sperm sac/testis = 0.44 ± 0.01) broodstock of P. monodon was also carried out. Approximately 50 proteins that showed large differential (up-regulation and down-regulation) expression profiles among sample groups were further annotated. In total, 345 differentially expressed proteins were identified. Interestingly, 1 (0.29%) and 18 (5.22%) proteins were found in only group A and in both groups of wild broodstock, respectively whereas 231 (66.96%) proteins were commonly found in all groups of samples. Reproduction-related proteins such as vasa-like protein, Ran GTPase activating protein 1 and seven transmembrane helix receptor were identified. The full length cDNA of ubiquitin specific peptidase 14 (ORF of 1524 bp corresponded to a polypeptide of 507 amino acids), ubiquitin carboxyl-terminal hydrolase 5 (ORF of 2442 bp corresponded to a polypeptide of 813 amino acids), Cdk17 (ORF of 1470 bp corresponded to a polypeptide of 489 amino acids) and proteasome alpha subunit (ORF of 765 bp corresponded to a polypeptide of 254 amino acids) of P. monodon was successfully identified and reported for the first time in this species. Tissues distribution analysis was examined. These genes were constitutively expressed in all examined tissues of P. monodon broodstock. Quantitative real-time PCR indicated that the expression levels of testicular serine/threonine-protein kinase 23 and ubiquitin carboxyl-terminal hydrolase 14 between juveniles and domesticated and wild broodstock of male P. monodon were not significantly different (P > 0.05). In contrast, the expression levels of proteasome alpha subunit and proteasome delta in testes of 10- and 14-month-old shrimp were not significantly different from those of wild broodstock (P < 0.05) but significantly greater than those of 6-month-old juveniles and 18-month-old broodstock (P > 0.05). In addition, the expression levels of 26S proteasome regulatory subunit S3 in testes of domesticated 14-month-old broodstock and wild broodtock were not different (P > 0.05) but its expression level in 6-month-old juveniles and domesticated 10- and 18-month-old broodstock were significantly lower than that of 14-month-old shrimp (P < 0.05). Taken together, the expression profiles of proteasome alpha subunit, proteasome delta and 26S proteasome regulatory subunit S3 indicated that domesticated male P. monodon possibly reached the initial maturation period at 10 months old, attained the maximal maturation peak at 14 months old and reduced the reproductive maturation at 18 months old.