41 NUCLEAR REMODELING IS AFFECTED BY TREATMENT OF BOVINE OOCYTES WITH A PROTEASOME INHIBITOR BEFORE NUCLEAR TRANSFER

Abstract

Complete reprogramming of somatic cell nuclei after nuclear transfer (NT) depends on extensive remodeling of chromatin by factors present in the recipient cytoplast. M-Phase Promoting Factor (MPF) activity, responsible for nuclear remodeling in metaphase II recipients, may be lowered by oocyte enucleation and handling prior to NT. Then, a partial nuclear envelope breakdown or incomplete premature chromosome condensation (PCC) may be, in turn, associated with an inefficient reprogramming. The aim of the present study was to maintain the bovine recipient cytoplast at a high level of MPF activity during the fusion procedure by using a proteasome inhibitor, MG132, and to assess the consequences on nuclear remodeling and developmental potential. Bovine COCs were in vitro-matured for 23 h. Matured oocytes were denuded, and then incubated in TCM-199 for 45 min and enucleated in the presence (treated group) or absence (control group) of 5 µm MG132. Embryos were reconstructed by fusion with adult fibroblasts and activated in 10 µg mL^–1 cycloheximide and 5 µg mL^–1 cytochalasin B. In Experiment 1, MPF activity was analyzed immediately after fusion/activation by measuring the phosphorylation of exogenous histone H1, and Cyclin B expression was assessed by Western blotting. In Experiment 2, microtubules revealed by immunofluorescense with anti-tubulin antibody and chromatin stained with 10 µg mL^–1 propidium iodide were analyzed by confocal microscopy 1 h after fusion/activation. In Experiment 3, NT embryos activated for 5 h were cultured in vitro for 7 days. Rate of development and cell counts in both groups were then recorded at the blastocyst stage. Remarkably, in Experiment 1, a high MPF activity was found in only 50% of the control oocytes, but MG132 treatment did not enhance this rate. On the other hand, cyclin B persisted for 2 h after activation in treated oocytes whereas it had dropped in controls. Experiment 2 revealed a higher rate of PCC in the treated embryos (n = 51) than in control embryos (n = 54): 96.0% v. 24.0% (chi-square, P < 0.001). Moreover, microtubules reorganized in a metaphasic spindle in embryos undergoing PCC, whereas cytoplasmic microtubules were observed in the others. In Experiment 3, cleavage and blastocyst rates were not significantly different between the treated (n = 92) and the control groups (n = 105): 83.7% and 53.3% v. 78.1% and 50.5%, respectively. However, the mean cell number in treated embryos (n = 27) was significantly higher than in controls (n = 20): 134 ± 25 v. 109 ± 43 (P < 0.05). This study suggests that MG132 treatment improved the maintenance of oocyte factors responsible for PCC in bovine NT embryos, although it did not modify MPF activity, thus questioning the role of MPF in the induction of PCC. Accordingly, PCC may be important for blastocyst quality and nuclear reprogramming in NT embryos. Full-term development of MG132-derived embryos is under investigation.