Nuclear remodeling in bovine somatic cell nuclear transfer embryos using MG132-treated recipient oocytes.
The early events in the nuclear reprogramming process during somatic cell nuclear transfer (SCNT) consist of morphological remodeling of the donor nucleus including premature chromosome condensation (PCC). In the present study, the objective was to increase oocyte M-Phase Promoting Factor (MPF) kinase activity and to examine the fate of the donor nucleus and the development of SCNT embryos thereafter. Indeed, in controls, recipient oocytes activated upon nuclear transfer, undergo a decrease in MPF activity, responsible for the inability to promote PCC in 77.8% of reconstituted embryos. Here we showed that exposure of the recipient oocyte to the proteasome inhibitor MG132 prior to fusion inhibited the degradation of cyclin B, which normally occurred immediately after activation by electro stimulation, and therefore sustained a high level of MPF. Treatment with MG132 also significantly increased the percentage of SCNT embryos with PCC when compared to the nontreated SCNT control embryos (94.1 vs. 22.2%, respectively, p < 0.01). The frequency of development to the blastocyst stage did not differ between MG132-treated or untreated recipient oocytes. However, we observed a significant increase of the total cells number in embryos produced after MG132 treatment. Investigation of the global nuclear organization by immunodetection of heterochromatin protein 1 (CBX1) showed that SCNT embryos derived from MG132-treated recipient oocytes displayed organization patterns similar to the ones observed in IVF embryos in contrast to the nontreated SCNT controls. Taken together, these results suggest that the PCC induced by MG132 treatment allows reorganization of the chromatin at an appropriate time potentially, leading to better reprogramming.
INRA, UMR 1198 Biologie du Développement et Reproduction, Jouy en Josas, France.
Daniel
D
Le Bourhis
Nathalie
N
Beaujean
Sylvie
S
Ruffini
Xavier
X
Vignon
Laurence
L
Gall
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D002843:Chromatin / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology*; D047108:Embryonic Development / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology; D007976:Leupeptins / Q000494:pharmacology*; D016200:Maturation-Promoting Factor / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology
D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D016200:Maturation-Promoting Factor; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde