23 Dec

Involvement of Histone H3 (Ser10) Phosphorylation in Chromosome: RESULTS

RESULTS

Phosphorylation of Histone H3 Takes Place During Oocyte Maturation

Phosphorylated histone H3 at Ser10 was detected in pig maturing oocytes using an antibody against histone H3 with a single phosphorylated Ser10. Histone H3 phosphorylation was not detected in any oocytes at the GV stage before culture (0%, 0/41 oocytes; Fig. 1A”). Phosphorylation of histone H3 was first detected in the clump of condensed chromosomes (green color; Fig. 1B”) in all of the examined oocytes (100%, 38/38 oocytes) at the diakinesis stage after 18 h of culture and thereafter it was maintained during oocyte maturation (Fig. 1, C”-F”). The integrity of the nuclear envelope was monitored by immunostaining with an anti-lamin A/C antibody. At the GV stage, the lamin A/C antibody recognized the entire contour of the nuclear envelope (Fig. 1A). At the diakinesis stage, chromosomes were frequently in contact with the envelope and lamin A/C staining was closely associated with the chromosome (Fig. 1, B and B’). The nuclear membrane breakdown initiated during the diakinesis stage and the nuclear membrane was completely disassembled when the oocytes entered metaphase I (Fig. 1, C-F). The control oocytes did not show any fluorescence at any stages.

The changes in the activities of Cdc2 and the histone H3 kinases during oocyte maturation were measured after determination of the meiotic stage of each oocyte by Hoechst 33342 (Fig. 2). Histone H3 kinase activity was lowest in the GV-stage oocytes, increased at the diakinesis stage, reached a peak at metaphase I, and was maintained until metaphase II (Fig. 2C). Cdc2 kinase was inactive at the GV and diakinesis stages. Thereafter, the activity increased and reached a peak at metaphase I. The activity fell during anaphase I and telophase I and increased again at metaphase II (Fig. 2B).

PP1/PP2A Inhibitors Induce Rapid Chromosome Condensation with Histone H3 Phosphorylation

The effects of CL-A and OA on histone H3 phosphorylation, chromosome condensation, and nuclear membrane morphology were examined. Both CL-A (Fig. 3 and Table 1) and OA (Fig. 4 and Table 2) induced rapid chromosome condensation in a similar manner. Chromosomes started to condense after 2 h of treatment, but most of the chromosome configuration was still at the GV I-IV stages, which were described by Motlik and Fulka for normal maturation of pig oocytes. Some oocytes had a chromosome configuration that looked like GV III or GV IV, but the chromosomes were much more condensed (GV’) (Figs. 3C’ and 4C’). Chromosome condensation progressed throughout the treatment. After 6 h, in most of the oocytes, the condensation of the chromosomes was like that at metaphase I (81% in the CL-A and 71% in the OA-treated oocytes). Histone H3 started to be phosphorylated after 2 h in 56% and 93% of the CL-A- and OA-treated oocytes, respectively. After 3 h, most of the treated oocytes showed histone H3 phosphorylation, which was maintained during the course of treatment. In the nontreated oocytes, the chromosomes stayed in the GV and no histone H3 phosphorylation was observed during the culture. In all of the CL-A- and OA-treated oocytes, an intact nuclear membrane was observed around the condensed chromosomes until 4 h. At 6 h, 66% of the CL-A-treated oocytes underwent GVBD, while no OA-treated oocytes underwent GVBD (Tables 1 and 2). However, 100% of the OA-treated oocytes underwent GVBD after 8 h (data not shown).

Change in The Activities of Cdc2 Kinase, MAP Kinase, and Histone H3 Kinase During Chromosome Condensation in CL-A- and OA-Treated Oocytes

When the oocytes were treated with CL-A, both histone H3 kinase (shown by phosphorylation of histone H3 in Fig. 5A) and MAP kinase (shown by phosphorylation of MBP in Fig. 5A) were activated after 2 h, and the activities increased during the time of treatment. Cdc2 kinase (shown by phosphorylation of H1 in Fig. 5A) was not activated until 6 h. In the control nontreated oocytes, Cdc2 kinase, MAP kinase, and histone H3 kinase were not activated. The oocytes treated with OA also showed a similar result to the CL-A-treated oocytes (data not shown). These results indicate that the PP1/PP2A inhibitors induce activation of both MAP kinase and histone H3 kinase, although they have no effect on Cdc2 kinase. To investigate whether MAP kinase activation is required for the phosphorylation of histone H3, the oocytes were treated with CL-A in the presence of the MAP kinase inhibitor U0126. In the oocytes, MAP kinase was not activated, but the histone H3 kinase was still activated after 2 h of treatment (Fig. 5A). There were no differences between the histone H3 kinase levels of oocytes treated with CL-A and CL-A+U0126 (Fig. 5B). Histone H3 phosphorylation and chromosome condensation in the oocytes were also examined after 6 h of treatment (Table 3). The histone H3 phosphorylation and chromosome condensation in the oocytes treated with CL-A and CL-A+U0126 was similar However, no GVBD was observed in the CL-A+U0126-treated oocytes.
Fig1Involvement of Histone-1
FIG. 1. Immunofluorescent localization of phosphorylated histone H3 (Ser10) during maturation of pig oocytes. Anti-lamin A/C- and Alexa 350-labeled antibodies stained the nuclear membrane (blue), PI stained the chromosome (red), and anti-phospho-histone H3- (Ser10) and Alexa 488-labeled antibodies stained phosphorylated histone H3 (green) in the same oocyte. The meiotic stages are (A) germinal vesicle stage (GV), (B) diakinesis (D), (C) metaphase I (MI), (D) anaphase I (AI), (E) telophase I (TI), (F) metaphase II (MII). Scale bar = 10 ^m.

Fig2Involvement of Histone-2
FIG. 2. A) An autoradiograph showing the changes in Cdc2 kinase and histone H3 kinase activities during maturation of pig oocytes. Histone H1 (H1) and histone H3 (H3) were used for the substrates of Cdc2 kinase and histone H3 kinase, respectively. B, C) The intensity of each phosphorylated histone H1 and histone H3 were measured by densitometry analysis of blots. The data are represented as the mean ± SEM. The lower figures of the graphs show the meiotic stages of oocytes as presented in the legend for Figure 1. The experiments were conducted three times. Values with different superscripts were significantly different (P < 0.05).

Fig3Involvement of Histone-3
FIG. 3. Effect of calyculin A (CL-A) on histone H3 phosphorylation, chromosome configuration, and nuclear membrane morphology of pig oocytes. Each treated oocyte with different times was stained with anti-lamin A/C- and Alexa 350-labeled antibodies for nuclear membrane (blue), PI for chromosome (red), and anti-phospho-histone H3- (Ser10) and Alexa 488-labeled antibodies for phosphorylated histone H3 (green). A, B) germinal vesicle stage (GV). C) Chromosomes started to condense with phosphorylated histone H3 (GV’). D) Early diakinesis stage (ED). E) Late diakinesis stage (LD). F) Chromosomes condensed like in metaphase I (MI). Scale bar = 10 ^m.

Table 1.
table1Involvement of Histone-4
* Oocytes at the GV stage were cultured with or without calyculin A (CL-A) for different times. Histone H3 phosphorylation, chromosome configuration, and nuclear membrane morphology were evaluated by fluorescent microscopy. GV I-IV, germinal vesicle I to IV stages as defined by Motlik and Fulka. GV’, chromosomes start to condense with phosphorylated histone H3. ED, Early diakinesis stage; LD, late diakinesis stage; MI, chromosomes condensed like those at metaphase I.
a-c Values with different superscripts in the same column differ significantly (P < 0.05).

Figf4Involvement of Histone-5
FIG. 4.Effect of okadaic acid (OA) on histone H3 phosphorylation, chromosome configuration, and nuclear membrane morphology of pig oocytes. Each treated oocyte with different times was stained with anti-lamin A/C- and Alexa 350-labeled antibodies for nuclear membrane (blue), PI for chromosome (red), and anti-phospho-histone H3- (Ser10) and Alexa 488-labeled antibodies for phosphorylated histone H3 (green). Panels show the identical stages of oocytes as presented in the legend for Figure 3. Scale bar = 10^m.

Table 2.
table2Involvement of Histone-6
* Oocytes at the GV stage were cultured with or without okadaic acids (OA) for different times. Histone H3 phosphorylation, chromosome configuration, and nuclear membrane morphology were evaluated by fluorescent microscopy. The meiotic stages are the same as presented in Table 1. a-c Values with different superscripts in the same column differ significantly (P < 0.05).

Fig5Involvement of Histone-7
FIG. 5. A) Autoradiographs showing effects of calyculin A (CL-A) and MAP kinase inhibitor U0126 on the activities of Cdc2 kinase, MAP kinase, and histone H3 kinase in pig oocytes. Histone H1 (H1), myelin basic protein (MBP), and histone H3 (H3) were used for the substrates of Cdc2 kinase, MAP kinase, and histone H3 kinase, respectively. Samples were subjected to a double kinase assay of H1/MBP or H1/H3. Change in the activities of Cdc2 kinase, MAP kinase, and histone H3 kinase in oocytes treated with (+) or without (—) the CL-A and CL-A and MAP kinase inhibitor U0126. B) The intensity of phosphorylated histone H3 was measured by densitometry analysis of blots. The activities of histone H3 kinase in MI oocytes were set to 100%. The data represent the histone H3 kinase activities as the mean ± SEM. Values with different superscripts are significantly different (P < 0.05). The experiments were conducted three times with similar results.

Table 3.
table3Involvement of Histone-8
* Oocytes at the GV stage were treated with calyculin A (CL-A) and U0126 (U) for 6 h. Histone H3 phosphorylation, chromosome configuration, and nuclear membrane morphology were evaluated by fluorescent microscopy. The meiotic stages are the same as presented in Table 1. a-b Values with different superscripts in the same column differ significantly (P < 0.05).

Fig6Involvement of Histone-9
FIG. 6. A hypothetical model showing how PP1/PP2A inhibition induces histone H3 phosphorylation leading to chromosome condensation in pig oocytes.

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