Cytoplasmic changes in relation to nuclear maturation and early embryo developmental potential of porcine oocytes: Effects of gonadotropins, cumulus cells, follicular size, and protein synthesis inhibition

Morphological and biochemical changes indicative of cytoplasmic maturation in relation to nuclear maturation progression and early embryo developmenta l potential was studied. Fluorescently labeled microfilaments and cortical granules were visualized by using laser scanning confocal microscopy. The m itogen-activated protein (MAP) kinase phosphorylation and cyclin B1 levels were revealed by Western blot. With the maturation of oocytes, cortical gra nules and microfilaments were localized at the cell cortex. A cortical gran ule-free domain (CGFD) and an actin-thickening area were observed over both the MII spindle of a mature oocyte and chromosomes of a nocodazole-treated oocyte, suggesting that chromosomes, but not the spindle, determined the l ocalization of CGFD and actin-thickening area. In oocytes that are incompet ent to resume meiosis, as indicated by the failure of germinal vesicle brea kdown (GVBD), peripheral localization of cortical granules and microfilamen ts, phosphorylation of MAP kinase and synthesis of cyclin B1 did not occur after 44 hr in vitro. These cytoplasmic changes were also blocked when GVBD of meiotically competent oocytes was inhibited by cycloheximide. Culture o f oocytes in a chemically defined medium showed that biological factors suc h as gonadotropins, cumulus cells and follicle size affected both nuclear a nd cytoplasmic maturation as well as embryo developmental potential. Absenc e of gonadotropins or removal of cumulus cells alone did not significantly influence GVBD or cyclin B1 levels, but decreased the final maturation and developmental ability of oocytes. A combination of gonadotropin absence and cumulus removal decreased GVBD, MAP kinase phosphorylation and embryo deve lopment. A high proportion of oocytes derived from small follicles were abl e to resume meiosis, synthesize cyclin B1, phosphorylate MAP kinase and tra nslocate CGs, but their maturation and embryo developmental ability were li mited. Removal of cumulus cells from small follicle-derived oocytes severel y affected their ability to undergo cytoplasmic and nuclear maturation. Mol . Reprod. Dev. 59:192-198, 2001. (C) 2001 Wiley-Liss, Inc.