239 OOCYTE MATURATION IN VITRO: PROTEOME ALTERATIONS ANALYZED BY ULTRA-SENSITIVE FLUOROCHROME LABELING AND NANO-ESI-MS-MS

Abstract

In mammals, oocytes are arrested in the diplotene stage of the first prophase until the beginning of the estrous cycle. Directly before ovulation, the oocyte completes the first meiotic division and progresses to metaphase II. This process is mimicked by oocyte maturation in vitro, which is critical in assisted reproduction techniques in humans and animals. A sensitive 2-dimensional (2D) difference gel electrophoresis (DIGE) saturation labeling approach including an internal pooled standard was used for quantitative proteome profiling of immature v. in vitro-matured bovine oocytes. A mixture of 0.25 µg of oocyte sample and 0.25 µg of the internal pooled standard was separated on each analytical 2D gel, corresponding to the protein content of approximately five oocytes. In total, the study comprised 48 2D gel images representing 24 DIGE experiments [6 independent samples × 2 gels per stage (immature/mature) × 2 pH gradients]. DeCyder differential in-gel analysis (DIA; Amersham Biosciences, Piscataway, NJ, USA) detected 2244 spots (SD = 100) in pH 4–7 images, and 1291 spots (SD = 65) in pH 6–9 images. In total, 38 spots with different intensity were detected (abundance ratio ≥2; Student's t-test, P ≤ 0.01). Differently abundant spots were identified by nano-LC-MS/MS analysis of matched spots in a preparative gel of saturation-labeled protein extract from 2200 immature oocytes. Ten spots could be unambiguously identified and comprised, e.g., interesting proteins such as clusterin, 14-3-3 ε, redox enzymes, and new polymorphic forms of glutathione-S-transferases. Clusterin is suggested to interact with components of the complement membrane attack complex, and 14-3-3 ε is a mediator of the activation of the maturation promoting factor, a key enzyme of the meiosis. Our study demonstrates the feasibility of differential oocyte proteomics using saturation labeling. Several of the proteins identified in this study are already known to play an important role in oocyte maturation, which reflects the relevance and reliability of our approach. Furthermore, we found proteins which are so far unknown in the context of oocyte development. A more detailed knowledge of the role of these candidates during oocyte maturation may help to optimize the in vitro-maturation process in order to increase the rate of successful in vitro fertilization and other assisted reproduction techniques in cattle and other mammals.

This work was supported by grant Deutsche Forschungsgemeinschaft (FOR 478, GRK 1029).