169 CHANGES OF PROTEIN PROFILES DURING FOLLICLE DEVELOPMENT AND IN VITRO OOCYTE MATURATION IN THE PIG

Abstract

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro conditions is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. In the first study, to investigate protein patterns during oocyte maturation in vitro, immature oocytes (germinal vesicle stage; GV stage) as control and oocytes matured (M-II phase) in vitro were analysed. The porcine oocytes in the GV stage were put in culture with TCM-199 for 44 h for M-II-stage oocytes. Total proteins were extracted from 1200 oocytes for GV and M-II stages, separated on 2-D gels, and stained with silver. In general, the overall protein staining pattern between the 2 gels was remarkably similar for most protein spots. Analysis of the gels identified proteins that were up- or down-regulated between GV and M-II stages. Up-regulated proteins were identified as PDE4D, GPKOW, PGM5, HSP70, ZPG4, galK1, GST-β, PDX1, PDX2, and PDX3. In contrast, down-regulated proteins were identified as PRKAB1, GRP78, TD-pozl, ERP57, MPP1, DTNA, ZP3B, HSP90, HSP86, and HSP27. This study has identified a novel protein, named myomegalin, that interacts with cyclic nucleotide phosphodiesterase (PDE4D). The second study analysed changes in proteins in follicular fluids during porcine follicular development. Follicular fluids were collected from follicles of 1- to 2-, 2- to 6-, and 6- to 10-mm diameter from ovaries of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. Differentially expressed proteins were analysed by MALDI mass spectrometry and searched on NCBInr. As a result, Spot No. 28 from the 2- to 6-mm follicle was Ig lambda chain C region, and Spot No. 32 and 33 from 6- to 10-mm were Apolipoprotein A-IV (APOA4). Increases of those proteins were correlated with follicular development. These results indicate that in vitro maturation changes the protein profile of porcine oocytes, which play important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.