Contribution of high p34cdc2 kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes.
The present study was undertaken to clarify the relationship between the p34cdc2 kinase activity of in vitro-aged or enucleated rat oocytes and the premature chromosome condensation (PCC) of microinjected cumulus cell nuclei. Wistar rat oocytes were placed in vitro up to 120 min after the animal was killed. The p34cdc2 kinase activity of the oocytes decreased in a time-dependent manner. The incidence of PCC was higher when nuclear injection into intact oocytes was completed in 15-45 min rather than 46-120 min. When rat oocytes were enucleated for subsequent nuclear injection, the p34cdc2 kinase activity transiently increased soon after enucleation but drastically decreased after 30 min. Removal of the cytoplasm instead of the meta-phase-plate did not affect the p34cdc2 kinase activity even after 60 min. PCC occurred in intact and cytoplasm-removed oocytes but not in enucleated oocytes. In contrast, oocytes from BDF1 mice exhibited a p34cdc2 kinase level twice that of rat oocytes and supported PCC despite enucleation. The p34cdc2 kinase level of intact rat oocytes was reduced to the equivalent level of aged (120 min) or enucleated (+60 min) oocytes by a 45 min treatment with roscovitine, an inhibitor of p34cdc2 kinase. None of the roscovitine-treated oocytes supported PCC while half of the control oocytes did. When rat oocytes were treated with MG132, a proteasome inhibitor, delayed inactivation of the p34cdc2 kinase was observed in the MG132-treated oocytes. A significantly higher proportion of the MG132-treated oocytes supported PCC when compared with the control oocytes. Moreover, a higher proportion of MG132-treated and enucleated oocytes carried two pseudo-pronuclei after cumulus cell injection and developed to the two-cell stage when compared with the enucleated oocytes at the telophase-II stage. These results suggest that the decreased level of p34cdc2 kinase activity in aged or enucleated rat oocytes is responsible for their inability to support PCC of microinjected donor cell nuclei and that inhibition of p34cdc2 kinase inactivation by chemicals such as MG132 is in part effective for rat oocytes to promote PCC and further development.
Ito
Junya
J
National Institute for Physiological Sciences, Okazaki 444-8787, Japan.
Hirabayashi
Masumi
M
Kato
Megumi
M
Takeuchi
Ayumu
A
Ito
Mayumi
M
Shimada
Masayuki
M
Hochi
Shinichi
S
D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000201:enzymology; D002843:Chromatin; D023902:Chromosome Pairing; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008845:Microinjections; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000201:enzymology / Q000502:physiology*; D009866:Oogenesis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D000077546:Roscovitine
D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D016203:CDC2 Protein Kinase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde