309 CHROMOSOME CONDENSATION IS CORRELATED WITH HISTONE H3 PHOSPHORYLATION WITHOUT CDC2 KINASE AND MAP KINASE ACTIVITIES IN PIG OOCYTES

Abstract

Chromosome condensation is the first step of oocyte maturation. When the oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine (1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during meiotic maturation of pig oocytes, and (2) the effects of protein phosphatase 1/2A (PP1/PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase and histone H3 kinase in this process. Oocyte-cumulus-granulosa cell complexes (OCGCs) were collected from follicles of 4–6 mm in diameter. OCGCs were cultured in modified TCM 199 for different periods of time to obtain oocytes at the germinal vesicle (GV, 0 h), diakinesis (18 h), metaphase I (24–27 h), anaphase I to telophase I (30–33 h), and metaphase II (42 h) stages. To examine the effects of PP1/PP2A inhibitors on the chromosome condensation, oocyte-cumulus-complexes (OCCs) were cultured in modified TCM 199 with either 2.5 μM okadaic acid (OA) or 50 nM calyculin A (CL-A) for 0.5, 1, 2, 3, 4 and 6 h. To inhibit the MAP kinase activity in the oocytes treated with the PP1/PP2A inhibitor, OCCs were cultured in medium containing CL-A and the MEK inhibitor, U0126 (0.1 mM). Morphology of the chromosome and nuclear membrane, and phosphorylation of histone H3 were examined by the immunofluorescent microscopy. In each group 30 oocytes were examined for OA or CL-A and 60 oocytes for CL-A + U0126 treatments. Activities of Cdc2 kinase, MAP kinase and histone H3 kinase were also examined. Phosphorylation of histone H3 (Ser10) was not detected in the oocytes at the GV stage. The phosphorylation was first detected in the clump of condensed chromosomes at the diakinesis stage of prophase I and maintained until metaphase II. The kinase assay also showed that histone H3 kinase activity was low in GV oocytes, increased at the diakinesis stage, and then maintained high activity until metaphase II. PP1/PP2A inhibitors induced rapid chromosome condensation in pig oocytes. Histone H3 phosphorylation (Ser10) became detectable together with the chromosome condensation in the treated oocytes after 2 h. After 6 h, oocytes had highly condensed chromosomes with phosphorylated histone H3 (81% in CL-A- and 71% in OA-treated oocytes). Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and U0126, neither Cdc2 kinase nor MAP kinase were activated, although histone H3 kinase was still activated and chromosomes condensed. These results suggest that phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Futhermore, the chromosome condensation is correlated with histone H3 kinase activity, but not with Cdc2 kinase and MAP kinase activities.