RNA interference analysis of the potential functions of cyclin-dependent kinase 2 in sexual reproduction of male oriental river prawns (Macrobrachium nipponense)

Cyclin-dependent kinase 2 ( Cdc2 ) is involved in the metabolic pathways of the cell cycle and plays essential roles in the regulation of cell proliferation in eukaryotic cells. Previous research predicted that Cdc2 is involved in regulating sexual reproduction in male oriental river prawns ( Machrobrachium nipponense ). In the present study, we investigated the potential functions of Cdc2 in male sexual reproduction in this species. The full-length cDNA sequence of Mn-Cdc2 is 1541 base pairs long, and it encodes 299 amino acids. Phylogenetic tree analysis revealed that the amino acid sequence of Mn-Cdc2 has the closest evolutionary distance with the Cdc2 s of freshwater prawns. Quantitative PCR analysis showed that Mn-Cdc2 mRNA expression was highest in the testis and ovary of male and female prawns, respectively, which suggests that Cdc2 promotes gonad reproduction in M. nipponense . In situ hybridization revealed that Cdc2 plays essential role in sperm proliferation. RNA interference analysis showed that synthesized dsRNA of Mn-Cdc2 efficiently knocked down the expression of Mn - Cdc2 in male prawns. Additionally, the expression of Mn-insulin-like androgenic gland hormone decreased as Mn-Cdc2 expression decreased, indicating that Cdc2 positively regulates the expression of this hormone in M. nipponense . Histological observations showed significant morphological differences between testis samples taken from dsCdc2 -injected and dsGFP -injected prawns. Sperm were rarely observed at day 14 after dsCdc2 injection, indicating that Cdc2 is involved in regulating testis reproduction in M. nipponense . This study highlighted the functions of Cdc2 in M. nipponense , and the results provide a basis for further studies of the regulation of male sexual reproduction in other crustacean species and for the development of an artificial technique to regulate the process of testis reproduction in M. nipponense .