Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion

Abstract

We have examined the response of bovine oocytes matured in vitro for 24 hr to parthenogenic activation using compounds that increase intracellular calcium (ionomycin) or inhibit protein phosphorylation (6-dimethylaminopurine, DMAP). Treatment with ionomycin alone caused resumption of meiosis (57.8 +/- 7.8%) but not pronuclear formation (8.9 +/- 7.3%). DMAP alone did not cause resumption of meiosis or pronuclear formation. Sequential treatment with ionomycin (5 microM for 4 min) immediately followed by DMAP (1.9 mM for 5 hr) resulted in activation that led to pronuclear formation (80.5 +/- 13.1%). Completion of meiosis, however, was bypassed as evidenced by only one polar body and one pronucleus present in activated parthenogenones. It was necessary to incubate the oocytes for at least 3 hr in DMAP to obtain high rates of activation (76.6 +/- 9.8%) and development to blastocysts (21.1 +/- 1.5%). Temporal separation of the two treatments resulted in a decrease in oocytes with one pronucleus and one polar body (uniformly diploid parthenogenones) and an increase in a mixture of diploid and haploid parthenogenones since DMAP was capable of causing transition to interphase of all chromatin configurations after anaphase commenced and prior to metaphase arrest. Parthenotes produced with ionomycin and DMAP that developed to the blastocyst stage had high cell numbers (70 to 88 cells) and were able to cause extended cycles in 33.3% of recipient cattle after nonsurgical transfer to the uterus. Response of the bovine oocyte arrested in metaphase II to different activation stimuli was also found to show age-dependent changes in pattern of activation response and developmental competence.