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(438) CaMKII ACTIVITY INCREASES IN FERTILIZED MOUSE EGGS. Markoulaki, Styliani2, Abbott, Allison2, Ducibella, Tom1,2, 2 Dept of Anatomy and Cellular Biology, Boston, MA1 Dept of Obstetrics and Gynecology, Boston, MA ABSTRACT- Although it is widely appreciated that fertilization and the initiation of development are dependent upon the increase in egg intracellular calcium, less is known about the biochemical mechanisms by which calcium stimulates the events of egg activation. Because mammalian eggs undergo calcium oscillations for several hours, we investigated if the calcium-dependent and oscillation-sensitive enzyme, CaMKII, undergoes an increase in activity upon fertilization. After 30 min, aliquots of inseminated or control zona-free ovulated mouse eggs were either frozen (SignaTECT/Promega CaMKII assay), or fixed for immunofluorescence analysis of fertilization and anaphase onset (both >90%). Eggs were assayed for CaMKII activity without added CaM and calcium. Uninseminated eggs had a basal level of activity that was removed by the addition of competing autocamtide-2 CaMKII substrate. Pooled samples of 4 eggs/determination had variable levels of CaMKII enzyme activity with an average increase in inseminated eggs of 50% compared to uninseminated eggs. This average increase is likely to be an underestimate of that during an actual calcium rise, because eggs oscillate asynchronously with respect to each other, calcium is elevated only ~20% of the entire period of time in which calcium oscillations occur, and CaMKII activity is predicted to oscillate. Single egg assays, made possible by increasing the specific activity of labeled ATP, indicated an estimated 0.01-0.1 ng of CaMKII per egg. Extending the work of others, we show that ionomycin-induced anaphase onset is reduced from 80% to 26% by the CaMKII antagonist, myristoylated autoinhibitory peptide (ionomycin treatment: 4 KEY WORDS: fertilization, calcium, kinase, egg |
M, 2 min, in calcium-free medium). Our results demonstrate a fertilization-associated increase in CaMKII activity that appears to be part of the mechanism to transduce the calcium signal and cause meiotic resumption in mammalian eggs. (support: NIH HD24191).