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. 2012 Oct 11;87(4):85.
doi: 10.1095/biolreprod.112.101014. Print 2012 Oct.

Aurora kinase A drives MTOC biogenesis but does not trigger resumption of meiosis in mouse oocytes matured in vivo

Petr Solc  1 , Vladimir Baran, Alexandra Mayer, Tereza Bohmova, Gabriela Panenkova-Havlova, Adela Saskova, Richard M Schultz, Jan Motlik
Affiliations

Aurora kinase A drives MTOC biogenesis but does not trigger resumption of meiosis in mouse oocytes matured in vivo

Petr Solc et al. Biol Reprod. .

Abstract

Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.

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Figures

FIG. 1
FIG. 1
Activation of AURKA during resumption of meiosis induced by hCG in 0–4 h intervals. A) The time course of the AURKA activating T288 phosphorylation on MTOC and in the cytoplasm. At 0-, 1-, and 2-h intervals, pericentrin (MTOC marker) and pT288 AURKA are shown in a single confocal section through the MTOC whereas at the 3- and 4-h intervals. the pictures represent maximum projections of 9–11 and 12–19 sections, respectively, to cover MTOC multiplication. DNA is always presented as a maximum projection. Note that pericentrin staining also nonspecifically detected the zona pellucida and precluded using a maximum projection of the entire stack. Entire stacks are available in Supplemental Movie S1. Original magnification ×63, zoom ×3. B) Quantification of AURKA phosphorylation. Data are means with 95% confidence intervals in arbitrary units.
FIG. 2
FIG. 2
Conditional expression of human wild-type (WT) and kinase-dead (KD) AURKA transgenes in mouse oocytes. A) RT-PCR analysis of single oocytes using primers detecting human AURKA mRNA but not the endogenous mouse mRNA. Mouse NIH3T cells were used as negative (NC) and human HeLa cells as positive (PC) controls. B) Immunoblot of samples with 20 oocytes. Note that to detect endogenous mouse AURKA, about 200 oocytes were required. PC, HeLa cells. C) Premature T288 AURKA activating phosphorylation in GV-stage oocytes (time = 0 h) from WT-AURKA but not KD-AURKA and control mice. Note MTOC multiplication in WT-AURKA oocyte. Pericentrin (red) was used as MTOC marker. Both pT288 and pericentrin are shown in a single confocal section for control and KD-AURKA, and as maximum projection of 8-22 sections for WT-AURKA, to cover MTOC multiplication. DNA is always presented as a maximum projection. Entire stacks are available in Supplemental Movie S2. Original magnification ×63, zoom ×3.
FIG. 3
FIG. 3
Meiotic maturation and fertility of AURKA transgenic mice. A) Resumption of meiosis (GVBD) at 3 and 7 h after hCG administration. The number of oocytes examined was 262 for controls (CTRL), 195 for WT-AURKA, and 136 for KD-AURKA. B) Incidence of maturation 12 h after hCG administration. The number of oocytes examined was 49 for controls, 49 for WT-AURKA, and 92 for KD-AURKA. Error bars in both graphs represent 95% confidence intervals. Differences between groups are statistically significant at P < 0.05. C) Fertility expressed as cumulative numbers of pups per litter during a 3–12 mo breeding trial. Data are expressed as mean from four mice per each group.
FIG. 4
FIG. 4
WT-AURKA overexpression induces a transient and premature increase in MTOC number in prophase I arrested oocytes. A) MTOC biogenesis of control and KD- and WT-AURKA oocytes. Images are presented as maximum projections for γ-tubulin and DNA staining. All the MTOCs are also positive for pericentrin that is clearly visible; see Supplemental Movies S3–S5 showing entire stacks. The zona pellucida is nonspecifically stained, which precludes using a maximum projection of the entire stack of the pericentrin channel. Original magnification ×63, zoom ×3. B) Quantitative data regarding MTOC(s) number at 0, 3, and 4.5 h after hCG administration and sorted according to GV and GVBD stages. The number of oocytes examined was 146 for controls, 119 for WT-AURKA, and 110 for KD-AURKA. C) Quantification of total MTOCs volume per oocytes, the total of γ-tubulin associated with MTOCs and γ-tubulin density in MTOCs in control and KD- and WT-oocytes at 0 h.
FIG. 5
FIG. 5
Full activation of AURKA is essential for the increase in MTOCs number and depends on CDC25B-CDK1 activity. A) Partial activation of AURKA in Cdc25b−/− oocytes at 4 and 7 h after hCG administration is not sufficient to induce the increase in MTOC number. Images show confocal sections through chromosomes and MTOCs. For 4-h interval, detailed pseudocolored (fire) images of pAURKA MTOC-associated signal are provided. All the MTOCs are clearly visible on complete stacks in Supplemental Movie S6. Original magnification ×63, zoom ×3. B) Quantification of partial activation of AURKA on MTOCs in Cdc25b+/+ and Cdc25b−/− oocytes. There are not significant changes in pT288 AURKA in the cytoplasm. The number of oocytes examined was 14 for Cdc25b+/+ and 19 for Cdc25b−/− oocytes. The asterisk indicates P < 0.001. C) Overexpression of GFP-AURKA in Cdc25b−/− oocytes restores MTOCs multiplication in the absence of CDK1 activity. Control oocytes were microinjected with water. Maximum projections are shown. The entire Z-stacks are available as Supplemental Movie S7. Original magnification ×63, zoom ×3.
FIG. 6
FIG. 6
Spindle length, volume, and γ-tubulin expression in metaphase I oocytes. A) Images of control, KD-AURKA, and WT-AURKA oocytes stained for tubulin and DNA. Original magnification ×63, zoom ×2.6. B) Quantification of the effect of AURKA on spindle length and volume. The number of oocytes examined was 19 for controls, 13 for WT-AURKA, and 11 for KD-AURKA. DNA staining (blue) and γ-tubulin staining (green) are shown as maximum projections of the entire stacks, but γ-tubulin staining are shown as a maximum projection of confocal sections that contain the spindle. The entire stacks are available as Supplemental Movie S8. The asterisk indicates P = 0.017. C) Quantification of the total number of MTOCs per oocytes, total MTOCs volume, the total of γ-tubulin associated with MTOCs, and γ-tubulin density in MTOCs in control and KD- and WT-oocytes in KD- and WT-AURKA oocytes. The number of oocytes examined was 10 for controls, 10 for WT-AURKA, and 7 for KD-AURKA. All the data are expressed with 95% confidence intervals. The asterisk indicates P < 0.05.
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