During the first month after birth, synchronous follicular growth occurs in the ovaries of immature mice (the first wave). Previously, we showed that mouse oocytes during the first wave were more competent developmentally in older females, although the numbers of mature oocytes did not differ with female age (17, 18, and 24 days old). In this study, we profiled the proteins of oocytes matured in vitro during the first wave using two-dimensional electrophoresis (2-DE) as a first step in search of proteins involved in the maturation of mouse oocytes. Oocytes collected from ovarian follicles of B6D2F1 females at nine ages (from 17 to 25 days of age) without hormonal treatment were matured in Waymouth medium (Sigma) supplemented with pyruvate (0.23 mM), antibiotics, polyvinylpyrrolidone (3 mg mL−1), and recombinant human FSH (0.5 iu mL−1, Serono). After culturing for 17 h at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2, oocytes whose germinal vesicles had broken down (mature oocytes) were dissolved in rehydration/sample buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3/10, and 0.001% BPB; Bio- Rad). In the first dimension, 4–10 μg of proteins from 200–400 mature oocytes were separated by isoelectric focusing using IPG ReadyStrip pH 3– 10 (7 cm in length, Bio-Rad) with a Protean IEF Cell (Bio-Rad). After alkylation with iodoacetamide, the proteins were separated in the second dimension using SDS-PAGE gradient gels (ReadyGel J 10–20%, gel size: 80[W] x 73[L] mm, Bio-Rad). The protein spots on the gels were visualized using SYPRO Ruby stain (Invitrogen). Approximately 200 protein spots were detected on each gel. We identified six commonly detected proteins on 2-DE gels throughout the first wave using peptide mass fingerprinting and a Mascot search against the NCBI nonredundant protein sequence database to identify protein spots that can be used as protein landmarks for future 2-DE analyses. The most prominent proteins were lactate dehydrogenase B (37 kDa, pI=5.70) and ubiquitin carboxy-terminal hydrolase L1 (25.2 kDa, pI=5.33). The other proteins were tumor rejection antigen gp96 (92.7 kDa, pI=4.74), a fragment of zona pellucida glycoprotein 2 (~18 kDa, pI ~4; the amino acid sequence was confirmed by MS/MS analysis), pyruvate kinase M (58.4 kDa, pI=7.58), and peroxiredoxin 1 (22.4 kDa, pI=8.26). Our results provide a basis for further 2-DE analyses of the proteins involved in the maturation of mouse oocytes. This work was supported by a grant from the Ministry of Health, Labor, and Welfare of Japan. (poster)