289 THE EFFECTS OF MEIOSIS BLOCKING BY CDK INHIBITORS ON THE ACTIVITY OF MATURATION PROMOTING FACTOR, ERK1 AND 2, AND THE DISTRIBUTION OF CYTOPLASMIC ORGANELLES IN IN VITRO-MATURED BOVINE OOCYTES

Abstract

Studies have suggested that the prematuration with meiotic blockers can improve oocyte quality promoting embryonic development; however, its exact effects on cytoplasmic characteristics remain unclear. Thus, this study aimed to evaluate the effects of meiosis block of bovine oocytes with the CDK inhibitors roscovitine (ROS) and butyrolactone (BL-I) on nuclear maturation, expression, and localization of ERK 1 and 2, cyclin B1, and p34cdc2 proteins and the ultrastructure of oocytes. Immature oocytes from the slaughterhouse were divided into the following groups: (1) control, in vitro maturated (IVM) in TCM 199 for 24 h; (2) ROS 12.5 µM; (3) BL-I 50 µM; and (4) ROS (6.25 µM) + BL-I (25 µM) treatment for 6 h followed by IVM in CDK inhibitor-free medium for 18 h. Immature oocytes and IVM oocytes in each group were then fixed stained by immunofluorescence for nuclear visualisation (n = 600), localization, and expression of ERK1 and 2 proteins, cyclin B1 and p34cdc2 protein (n = 350), and prepared for evaluation of the ultrastructure by electron microscopy (n = 100). Data were analysed using the ANOVA test (nuclear visualisation), Student-Newman-Keuls test (expression of ERK1 and 2 proteins, cyclin B1 and p43cdc2) by the PROC GLM procedure of SAS (SAS Inst. Inc., Cary, NC, USA). At 6 h of IVM, a lower (P < 0.05) percentage of oocytes were at the metaphase I (MI) stage in the control group (C = 18.2 ± 5.4%) compared with other groups and a higher percentage of oocytes were degenerated in the ROS group (16.3 ± 5.6%) compared with other groups (C = 13.6 ± 4.6%, BL-I = 10.0 ± 4.5%, BL-I+ROS = 8.0 ± 5.6%). At 24 h of IVM, higher (P < 0.05) percentages of oocytes were at the MI stage in the control and ROS group (8.3 ± 5.9% and 6.8 ± 6.4%, respectively). There was no difference (P > 0.05) in percentage of metaphase II (MII) oocytes among the groups. Only the ROS group showed lower fluorescence intensity of ERK1 and 2 proteins in the ooplasma (P < 0.05). Immature oocytes showed higher expression of cyclin B1 and p34cdc2 (P < 0.05). There was no difference in the localization of these proteins in the ooplasm and there was no difference in the oocyte ultrastructure (mitochondria, cortical granules, endoplasmic reticulum, microvilli, zona pellucida, lipid granules) among treatments (P > 0.05). The results suggest that a temporary blocking of oocyte maturation by CDK inhibitors affect neither the expression and distribution of MPF components (cyclin B1/cdc2) nor the distribution of cytoplasmic organelles in IVM oocytes. However, the expression of ERK 1 and 2 in mature oocytes may be reduced by pre-IVM with ROS.