Study of newly synthesized proteins during bovine oocyte maturation in vitro using image analysis of two‐dimensional gel electrophoresis

Abstract

To investigate protein synthesis during bovine oocyte maturation in vitro, oocytes were put in culture with 35S-methionine for 4 hr periods from time zero to 28 hr. Pools of 10 oocytes were then prepared for two-dimensional gel electrophoresis (2-DE). For each time interval, three gels were obtained, digitalized, and analyzed to detect proteins. Then, the gel containing the most proteins was chosen as the reference gel and compared with the others. An averaged gel was created with proteins present in at least two gels of the three. Our results indicate that the rate of protein synthesis is higher at the beginning of maturation until the appearance of metaphase I (MI, 8-12 hr) and then it decreases and stays relatively constant. Percentages of initial proteins (0-4 hr) and remaining present during the progression decrease progressively from 100% to 53%. In contrast, when we compare proteins synthesized from the 4 to 8 hr period with proteins from the 8 to 12 or the 12 to 16 hr intervals, percentages of overall protein matching are stable with values of 81 and 79%, respectively. Comparison of proteins from 20 to 24 hr with proteins from 16 to 20 or 24 to 28 hr intervals also gives stable percentages of overall protein matching with values of 83 and 84%, respectively. Furthermore, a higher number of new proteins is observed at 4-8 hr (n=130) and 16-20 hr (n=136) of maturation. Thus, three major patterns of protein synthesis were observed during bovine oocyte maturation in vitro: one at the beginning of maturation (0-4 hr), another one in the middle (4-16 hr), and the last one after the completion of MI stage (16-28 hr).

ATP content and maturational/developmental ability of bovine oocytes with various cytoplasmic morphologies

“…It was reported that the rate of protein synthesis in bovine oocytes was higher at the beginning of IVM culture until the appearance of MI and then decreased (Coenen et al, 2004). Oocytes may use a larger amount of ATP for production of proteins and these proteins may be necessary for the progression of GVBD.…”

Bovine germinal vesicle oocyte and cumulus cell proteomics

“…Here, we used the bovine system because it is important for both agricultural and biomedical studies. Coenen et al (2004) pioneered proteomics of bovine female gametogenesis. Using radio labeling and two-dimensional gel electrophoresis, they demonstrated three major patterns of translational activity during bovine oogenesis (one at the initiation of maturation, 0-4 h; one in the middle, 4-16 h; and one after completion of MI, 6-28 h) suggesting a developmentally regulated series stage-specific protein synthesis.…”

“…In meiotic development, nuclear maturation is manifest by GV breakdown (GVBD), condensation of chromosomes, realization of first meiosis (MI), and another arrest of development at the metaphase of the second meiosis (MII). In addition, these events related to nuclear maturation, significant changes occur in the cytoplasm including structural changes of organelles, major translational activity in which while many new proteins are synthesized, synthesis of others is terminated (Moor et al 1990, Coenen et al 2004. Developmentally competent MII oocytes require four periods of protein synthesis; namely, synthesis required for GVBD, MI, MII, and maintenance of MII (Khatir et al 1998).…”

Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation

“…In bovine oocytes, protein neosynthesis is indispensable for meiosis resumption and progression. A significant increase in overall protein synthesis has been reported in cattle oocytes at 6-10 h after beginning of IVM (Tomek et al 2002), and numerous proteins were translated de novo (Coenen et al 2004). Coincidently, GSK3B was shown to be progressively inactivated, i.e.…”