Protein Modification by Phosphorylation during the Process of Nuclear Membrane Dissolution in Puromycin-Treated Mouse Oocytes1

Abstract

The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.

The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes

“…When metaphase I oocytes are cultured in cycloheximide-supplemented media, nuclei are formed and the first polar body is extruded (Clarke and Masui, 1983;Kang et al, 1991;Szollosi et al, 1991). These findings suggest that the capacity to support cell cycle progression is probably present during the metaphase I stage and only falls at the transition to anaphase.…”

“…Mouse oocytes treated with protein synthesis inhibitors at the first metaphase extrude the first polar body and form nuclei (Clarke and Masui, 1983;Kang et al, 1991). As shown by Szollosi et al (1991), these oocytes do not, however, proceed to second metaphase.…”

“…In oocytes of lower species, the rise in MPF a t metaphase I1 is dependent on protein synthesis and can readily be blocked by protein synthesis inhibitors (Picard et al, 1985;Gerhart et al, 1984). Mouse oocytes treated with protein synthesis inhibitors at the first metaphase extrude the first polar body and form nuclei (Clarke and Masui, 1983;Kang et al, 1991). As shown by Szollosi et al (1991), these oocytes do not, however, proceed to second metaphase.…”

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

“…A majority of the oocytes with multiple female pronuclei had no polar bodies or only one and this observation suggests that the high incidence of multiple female pronuclear formation in the oocytes exposed to an electrical pulse was due to unsuccessful exocytosis of polar bodies. It has been shown that treatment of mouse oocytes with protein synthesis inhibitors at metaphase I stage induces the release of the first polar body and nuclear formation (Clarke and Masui, 1983;Kang et al, 1991;Szollosi et al, 1991). However, a polypeptide profile from pig oocytes activated by a n electrical pulse was very similar to that from pig zygotes penetrated by sperm (Sun et al, 1992), and inhibition of protein synthesis of IVM pig oocytes did not affect sperm penetration or male and female pronuclear formation (Ding et al, 199213).…”