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

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). Copyright 2004 Wiley-Liss, Inc.