Dynamic Events Are Differently Mediated by Microfilaments, Microtubules, and Mitogen-Activated Protein Kinase During Porcine Oocyte Maturation and Fertilization In Vitro1

Abstract

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization 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 activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.

In vitro fertilization and polyspermy in the pig: Factors affecting fertilization rates and cytoskeletal reorganization of the oocyte

“…These observations suggest that the cytoskeletal architecture of the oocyte may alter irreversibly during the intrafollicular development, affecting the ability of the oocyte to become mature and fertilized, although gilts of a similar age were immature. Further, the high incidence of decreased cortical microfilaments could account for the decreased rate of PBII expulsion in the denuded (grade 4) oocytes, because microfilaments are required for cytokinesis (Sun et al, 2001).…”

“…Sperm aster enlarged during sperm head decondensation and extended throughout the cytoplasm during PN formation (Kim et al, 1996c(Kim et al, , 1997b. Recent reports describe the origin of the active microtubule-organizing center as paternally derived and its role in PN migration and apposition (Kim et al, 1996c(Kim et al, , 1997bSun et al, 2001). In monospermic zygotes, the male and female PN were included in two different domains of microtubules, respectively.…”

“…Our previous studies showed that cytoskeletal alteration is involved in the dynamic change of cumulus-oocyte cell communication during oocyte maturation in the pig (Suzuki et al, 2000). Cytoskeletal reorganization is important during oocyte maturation and fertilization in mammals (Kim et al, 1996b(Kim et al, ,c, 1997bLong et al, 1993;Maro et al, 1986;Navara et al, 1994;Schatten et al, 1985;Sun et al, 2001;Wang et al, 2000). However, little information is available on a relationship between the oocyte quality and cytoskeletal distribution.…”

Comparing Pig and Amphibian Oocytes: Methodologies for Aneuploidy Detection and Complementary Lessons for MAPK Involvement in Meiotic Spindle Morphogenesis

“…Similarly, it was reported that the microfilament modulator JAS inhibited sperm incorporation and prevented cortical granules (CG) exocytosis in murine oocytes (Terada et al, 2000). In murine oocytes, pronucleus migration is blocked upon inhibition of microfilament assembly (Schatten et al, 1989;Terada et al, 2000), while in pigs (Sun et al, 2001a) or urchins (Schatten et al, 1992), microtubule assembly is not required for pronucleus formation. During fertilization in sea urchins, pigs, cattle and humans, sperm introduces the centrosome into the egg.…”

“…Several studies have reported that fertilization of pig oocytes resembles more that of lower vertebrates than mice (Long et al, 1993;Sun et al, 2001a). In sea urchin, starfish, porcine or bovine oocytes, sperm interaction with the oocyte or their penetration is impaired by microfilament inhibitors (Schatten et al, 1982).…”