Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes.
Intracellular localization of maturation/M-phase promoting factor (MPF) and mitogen activated protein (MAP) kinase in mature oocytes has been examined by immunocytochemical methods and the authors of these studies have reported that they are localized on spindles during M-phase. Although these reports showed the relative localization of MPF and MAPK on spindles, it has never been shown whether these kinases are present in the cytoplasm and, if they are present, how many parts of the kinases are localized on the metaphase spindle. In the present study, we made quantitative analyses of MPF and MAP kinase localized on oocyte spindles by kinase assays and immunoblotting after removal of the spindles from porcine mature oocytes. First, we certified their intracellular distribution by immunocytochemical methods and observed sharp signals of cyclin B1 on spindle poles and MAP kinase signals on the microtubule of metaphase spindles. In contrast to these results by immunostaining, the amounts of MPF and MAP kinase localized on spindles examined by immunoblotting and kinase assays were undetectable and less than 20%, respectively. These results indicate that the immunocytochemical technique is a powerful method for showing relative localization, but it is not suitable for quantitative analysis, and that the removal of metaphase spindles from mature oocytes does not have a severe negative impact on the subsequent MPF and MAP kinase activity and on the cell cycle progression in early embryo development.
Goto
Seitaro
S
Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
Naito
Kunihiko
K
Ohashi
Satoshi
S
Sugiura
Koji
K
Naruoka
Haruna
H
Iwamori
Naoki
N
Tojo
Hideaki
H
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D005260:Female; D007150:Immunohistochemistry; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*; D013552:Swine / Q000378:metabolism*
D019926:Cyclin B; D056744:Cyclin B1; D016200:Maturation-Promoting Factor; D020928:Mitogen-Activated Protein Kinases