Characterization and expression of cell division cycle 2 (Cdc2) mRNA and protein during ovarian development of the giant tiger shrimp Penaeus monodon

Abstract

The meiotic maturation of oocytes is regulated by the maturation-promoting factor (MPF), a complex of Cdc2 (Cdk1) and Cyclin B. Here, the complete open reading frame (ORF) of Cdc2 in Penaeus monodon was characterized. PmCdc2 were 900bp in length corresponding to a polypeptide of 299 amino acids with the conserved Thr14, Tyr15 and Thr161 residues. Quantitative real-time PCR indicated that the expression level of PmCdc2 in wild intact broodstock was significantly increased in stages II (vitellogenic) and III (early cortical rod) ovaries relative to stage I (previtellogenic) ovaries and peaked in stage IV (mature) ovaries (P<0.05). The expression level of PmCdc2 in stages I–IV ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian developmental stages in intact broodstock (P<0.05). Expression levels of PmCdc2 in ovaries of 18-month-old P. monodon upon 5-HT injection (50μg/g body weight) were significantly increased at 1hour post injection (hpi, P<0.05). Recombinant PmCdc2 protein and its polyclonal antibody were successfully produced. Western blot analysis revealed the expected 34kDa band (PmCdc2) along with a smaller band of 23kDa (ribosomal protein S3) in ovaries of juveniles and various ovarian stages of broodstock. Using phospho-Cdc2 (Thr161) polyclonal antibody, the positive signal of 34kDa was observed in all ovarian stages but the most intense signal was found in stage IV ovaries. Results in the present study indicated that PmCdc2 gene/protein plays an important role in the development and maturation of oocytes/ovaries in P. monodon.