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a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269
Bovine oocytes matured for 24 h (young) or 40 h (aged) were treated with calcium ionophore (A23187) alone or followed with 6-dimethylaminopurine (6-DMAP), a protein phosphorylation inhibitor, and were then assayed for histone H1 kinase and mitogen-activated protein kinase (MAPK) activities. Additionally, the changes in chromatin, meiotic spindle, and microfilament were assessed by immunofluoresence microscopy. In both young and aged oocytes, treatment with 6-DMAP following A23187 treatment abolished the activities of both H1 and MAPKs; the decline of H1 kinase preceded the decline in MAPK activity. However, A23187 treatment alone caused a slower decrease in H1 kinase activity and no evident MAPK alteration in young oocytes. In contrast, activities of both kinases decreased in aged oocytes after A23187 treatment, similar to the response in the combined treatments. The inactivation of MAPK was caused by dephosphorylation of MAP42/extracellular signal-regulated kinase 2 (ERK2) as detected by gel mobility shift in the Western blot assay. A23187 treatment of young oocytes led to chromosome separation and second polar body extrusion, but not pronuclear development, with the majority of the oocytes arrested at a transitional stage of metaphase to anaphase known as metaphase III (MIII). However, most of the A23187-treated aged oocytes developed to the pronuclear stage. When oocytes, regardless of age, were treated by A23187 plus 6-DMAP, bivalent chromosomes were clumped into a single mass, the spindle was disassembled, microtubule networks were distributed in the cytoplasm, and a pronucleus appeared. It is suggested that the decrease in H1 kinase activity is involved in the initiation of oocyte activation, i.e., the exit from metaphase II, whereas the decrease in MAPK activity correlates with onset of pronuclear formation. In conclusion, inactivation of maturation-promoting factor and MAPKs probably occurs via two independent processes, and the inactivation of both kinases is required for the metaphase II oocytes to progress through interphase. High MAPK activity might contribute to spindle stabilization, and inactivation of MAPK is associated with microtubular network formation in the cytoplasm.
2 Correspondence. FAX: (860) 486–4375; xyang{at}ansc1.cag.uconn.edu
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