Dynamic events are differently mediated by microfilaments, microtubules, and mitogen-activated protein kinase during porcine oocyte maturation and fertilization in vitro

The role of microfilaments, microtubules, and mitogen-activated protein (MA P) kinase in regulation of several important dynamic events of porcine oocy te maturation and fertilization is described. Fluorescently labeled microfi laments, microtubules, and cortical granules were visualized using either e pifluorescence microscopy or laser scanning confocal microscopy. Mitogen-ac tivated protein kinase phosphorylation was revealed by Western immunoblotti ng. We showed that 1) microfilament disruption did not affect meiosis resum ption and metaphase I meiotic apparatus formation but inhibited further cel l cycle progression (chromosome separation) even though MAP kinase was phos phorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized be neath the oolemma their anchorage to the cortex was independent of either m icrofilaments or microtubules; 3) neither microfilaments nor microtubules w ere involved in CG exocytosis during oocyte activation; 4) sperm incorporat ion was mediated by microfilaments, while pronuclear (PN) syngamy was contr olled by microtubules rather than microfilaments; 5) spindle microtubule or ganization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activa tion; and 6) MAP kinase phosphorylation did not change either when microtub ules were disrupted by nocodazole or when cytoplasmic microtubule asters we re induced by taxol. The present study suggests that the role of the cytosk eleton during porcine oocyte maturation is similar to that of rodents, whil e the mechanisms of fertilization in pig resemble those of lower vertebrate s.