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Inactivation of p34cdc2 kinase by the accumulation of its phosphorylated forms in porcine oocytes matured and aged in vitro

Published online by Cambridge University Press:  01 May 1999

Kazuhiro Kikuchi
Affiliation:
Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan.
Kunihiko Naito
Affiliation:
Department of Applied Genetics, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Junko Noguchi
Affiliation:
Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan.
Arata Shimada
Affiliation:
Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan.
Hiroyuki Kaneko
Affiliation:
Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan.
Masakane Yamashita
Affiliation:
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
Hideaki Tojo
Affiliation:
Department of Applied Genetics, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Yutaka Toyoda
Affiliation:
The Research Center for Protozoan Molecular Immunology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

Abstract

Culturing of matured porcine oocytes in vitro results in the enhancement of their cytoplasmic ability for oocyte activation (so-called ageing), although they are arrested at metaphase II. The enhanced ability for oocyte activation is related to decreased activity of the maturation promoting factor (MPF). In the present study we clarified the molecular mechanism of MPF inactivation during ageing, especially the changes in the phosphorylation status of p34cdc2, a catalytic subunit of MPF, compared with that in fertilised oocytes. The MPF activity decreased gradually when maturation culture was prolonged from 36 to 72 h, confirming the decreasing MPF activity in aged oocytes. The activity of 48 h matured oocytes also decreased after in vitro fertilisation. Immunoblotting of p34cdc2 with anti-PSTAIRE antibody revealed that the culturing of matured oocytes induces a gradual increase in pre-MPF, which is a p34cdc2 and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. In contrast, pre-MPF decreased after fertilisation, indicating the degradation of cyclin B. These results suggest that the molecular mechanisms of inactivation of MPF are different between oocyte activation and ageing, and that the mechanism during ageing might be based on the inhibitory phosphorylation of p34cdc2, whereas that of oocyte activation is based on the degradation of cyclin B.

Type
Research Article
Copyright
1999 Cambridge University Press

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