The role of RhoA in the germinal vesicle breakdown of mouse oocytes

We have investigated a new role of RhoA in the germinal vesicle breakdown ( GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and AD P-ribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly loca lized in the ooplasmic area, but rarely detected in germinal vesicle. Incub ation of oocyte extract with C3 transferase induced a strong ADP-ribosylati on at about 25 kDa, Incubation of GV-stage oocytes in culture medium induce d the spontaneous maturation to GVBD by about 78 and 87% of total oocytes a t 1 and 3 h, respectively, However, microinjection of C3 transferase into G V-stage oocytes significantly inhibited GVBD at 1 (GVBD = 29%) and 3 h (GVB D = 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra oocyte ROS was measured using a ROS-specific fluorescent dye H(2)DCFDA during the oocyte maturation, Spontaneous matura tion of GV-stage oocytes induced a significant increase of ROS at 3 h by ab out twofold over the control level and then the increased level was maintai ned until. 6 h, However, microinjection of C3 transferase inhibited the pro duction of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-L-cys teine and catalase, blocked the ROS increase. The ROS scavengers also signi ficantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse o ocytes, (C) 2000 Academic Press.