259 INVOLVEMENT OF Ca²⁺/CALMODULIN-DEPENDENT PROTEIN KINASE II IN THE SPONTANEOUS ACTIVATION IN RAT OOCYTES

Abstract

Immediately after recovery from the oviduct, rat metaphase-arrested oocytes are very sensitive to spontaneous activation (SA), which is characterized by resumption of meiotic division followed by the cytoplasmic scattering of chromosomes. Although it has been shown that proteasome inhibitor is effective in stopping SA, it is likely that an upstream event is controlling SA. Constitutively active Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) triggers cyclin B destruction and release from MII arrest. To investigate the kinetics and mechanism of rat spontaneous oocyte activation, we (1) compared SA status after different periods of aging in oviducts and examined the effects of hyalurondiase treatment, (2) compared SA between Ca²⁺-free and Ca²⁺-containing conditions, (3) examined the pattern of SA after using CaMII inhibitor and proteasome inhibitor, and (4) analyzed the activity of CaMKII at different times after oocyte collection. Oocytes were collected from three 4-wk-old PMSG-primed Sprague Dawley rats. ANOVA Tukey-Kramer HSD was used for statistical analysis using a P value of 0.05. Each experiment contained at least four replicates with at least 100 oocytes. Experiment 1: Oocytes were collected at different times post-hCG injection (14, 18, 22 h) and cumulus cells were removed before or after 2 h of in vitro culture with hyaluronidase. There was no difference in SA due to oocyte aging in oviducts or hyalurondiase treatment. Experiment 2: Oocytes were cultured in Ca²⁺-containing and Ca²⁺-free KSOM for 2 h after collection at different times (14, 18, 22 h) post hCG injection. Although there were no differences among Ca²⁺-containing KSOM culture groups (91%, 82%, and 90%, respectively), Ca²⁺-free KSOM significantly decreased SA at 14, 18, and 22 h post-hCG (19%, 43%, and 51%, respectively). Experiment 3: Oocytes were treated with different doses of inhibitor of CaMKII, myr-AIP (10, 20, 50, 100 μM), and proteosome inhibitor MG132 (10, 20, 50, 100 μM) after oocyte collection. Fifty and 100 μM myr-AIP induced significantly lower SA (36%, and 17%) than 0, 10, 20 μM (91%, 65%, and 53%, respectively). Moreover, higher concentrations of MG132 (50 and 100 μM) induced significantly lower SA rates (6% and 5%) than 0, 10, 20 μM (91%, 41%, and 22%, respectively). We also investigated the effect of short exposures to myr-AIP and MG132 during the first 2 min post-recovery. The myr-AIP group produced significantly lower SA compared to control and MG132-treated groups. Experiment 4: Oocytes collected at different times (0, 10, 20, 30, 60 and 90 min) were used for CaMKII activity assay. CaMKII activity increased at 20 min and remained high for 30 min followed by decreased activity by 60 min. In conclusion, CaMKII seems to be one of the upstream signals that causes rat oocytes to spontaneously activate after recovery.

Financial support was provided by NSERC and Canada Research Chairs.