348 INACTIVATION OF MAPK AFFECTS CENTROSOME ASSEMBLY, BUT NOT ACTIN FILAMENT ASSEMBLY, IN MOUSE OOCYTES MATURING IN VITRO

Abstract

Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, γ-tubulin and NuMA distribution, and actin filament assembly in mouse oocytes maturing in vitro. Significant differences between control and treatment were determined using Tukey's multiple range test; P < 0.05 was considered significant. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 h after the initiation of maturation cultures, was fully active at 6 h, and remained active until metaphase II (MII). When GV oocytes were cultured in medium containing 20 µM U0126 for 9 h, the oocytes underwent geminal vesicle breakdown (GVBD), but were significantly inhibited in progression to the MI stage as compared to controls (control: 76.7 ± 5.66%; 10 µM U0126: 29.0 ± 7.35%; 20 µM U0126: 26.4 ± 5.74%). Polar body extrusion was observed in most oocytes when cultured in the presence of 20 µM U0126 from 4 h after initiation of in vitro oocyte maturation to 14 h into the in vitro maturation incubation (control: 76.6 ± 4.21%; 20 µM U0126: 70.7 ± 4.61%). However, there was a decrease in normal MII-stage oocytes when cultured for 4 to 14 h (control: 87.7 ± 5.12%; 20 µM U0126: 32.8 ± 3.79%); abnormality included, for example, a polar body lacking a nucleus, as well as an abnormal spindle or metaphase plate. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of γ-tubulin and NuMA, but did not affect actin filament assembly. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly.