Molecular characterization, expression, and H2O2 induction of p53 and mdm2 in the ricefield eel, Monopterus albus

Abstract

The proteins P53 and Mdm2 (murine double minute 2) play a prominent role in preventing the induction of uncontrolled apoptosis in numerous cell types. To better understand their potential roles during gonadal development in Monopterus albus, p53 and mdm2 homologues were isolated and characterized, and their mRNA expression patterns in developing gonadal and ovarian tissue incubated with H2O2in vitro were analysed. The open reading frames (ORFs) of p53 and mdm2 consisted of 1125 and 1497 base pairs (bp), respectively. A multiple amino acid residue alignment analysis showed that p53 and Mdm2 harboured the typical conserved domains of their gene families. Phylogenetic analysis also demonstrated that the Mdm2 of M. albus exhibited high sequence similarity with Lates calcarifer; however, the p53 sequences of the Neoteleostei, including M. albus, were distinct from those of the Euteleostei and other vertebrates. Quantitative real-time PCR revealed that p53 and mdm2 were mainly expressed in the ovary; their expression was significantly increased in the previtellogenic stage, was maintained at the increased level thereafter, showed a second increase in the early intersexual stage, and then decreased with the natural sex change from female to male. After incubation with H2O2, the expression patterns of p53 and mdm2 in ovarian fragments were similar to that of the apoptotic molecular marker caspase3, showing a significant increase at 1 h post incubation (hpi), a decrease after 2 hpi, and then a return to significantly upregulated levels at 4 hpi and 8 hpi. In addition, p53, mdm2 and caspase3 were strongly correlated. These results will be useful for further studies on the function and regulation of p53 and Mdm2 during gonadal development in Monopterus albus, particularly in the ovary.