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Phosphorylation of histone H3 at SerlO is one of the signalling events involved in chromosome condensation and is probably mediated by kinases of the Aurora family, namely Aurora kinase B (Giet and Glover, 2001; Hsu et al., 2000 ), reviewed by (Cheung et al., 2000). In Xenopus there are 2 members of the Aurora kinase family, termed pEg2 (Aurora A) and AIRK B (Aurora B), which have both been implicated in phosphorylation of histone H3 at SerlO (Mumion et al., 2001; Scrittori et al., 2001). However, it remains to be determined how these kinases are activated at the onset of mitosis.

Using a anti phospho-histone H3 rabbit polyclonal antibody, which specifically recognises phosphorylated SerlO by in the immunofluorescent stainings of Xenopus oocyte spindles, allowed me to monitor histone H3 phosphorylation during meiosis. I found a striking dependence on protein synthesis of the phosphorylation of Histone H3 at SerlO. Both Xenopus Aurora kinases contain a sequence which loosely resembles a destruction box in their C-terminus (RXXLXG), and Aurora B is a target of the APC/C in human cells (Honda et al., 2000); (Walter et al., 2000). Moreover both pEG2 and AIRK B are unstable in G1 extracts of Xenopus embryonic fibroblasts (C. Prigent, personal communication). It is thus conceivable that, similar to cyclins, Aurora kinases are destabilised by the APC/C during Xenopus oocyte maturation. I investigated this point by studying the stability of both Aurora kinases during meiosis. Figure 8.2a shows that, in contrast to cyclin B l, Aurora B is a stable protein even in the presence of CHX. Similarly, endogenous pEG2 is stable in CHX treated oocytes after GVBD (Figure 8.2b). These data suggest that in Xenopus

(a) Aurora kinase B destruction assay in oocytes

Labelled protein

Bl wt AIRKB AIRKB

-CHX -CHX +CHX

0 20 40 60 0 20 40 60 0 20 40 60 min after injection

(b) Immunoblot

-CHX -kCHX

Eg2

0 20 40 60 0 20 40 60 min after GVBD

Figure 8.2 Stability of aurora kinases in Xenopus oocytes

(a) Destruction assays in oocytes using [35S]-labelled cyclin Bland AIRK B injected into oocytes at GVBD. Chx was added prior injection in the left most panel (+CHX). (b) Immoblot with monoclonal eg-2 antibodies of oocytes collected at indicated timepoints after GVBD. CHX was added just after white spot formation .

that histone H3 phosphorylation is contingent on a protein synthesis-dependent activity, other then the Aurora kinases.

One possible explanation of this observation is that activation of the Aurora family may depend indirectly on protein synthesis. The CHX rescue experiments presented in chapter 7 provide some evidence to support this hypothesis. Whereas the signal for phosphorylated histone H3 disappeared rapidly in oocytes treated with CHX, the expression of stable cyclin B was sufficient to maintain chromosome condensation and histone H3 phosphorylation even in the absence of protein synthesis. This result suggests that MPF directly or indirectly activates the kinase (possible AIRK B (Mumion et al., 2001)) that phosphorylates histone H3 in Xenopus oocytes.

In the absence of B-type cyclin synthesis, on the other hand, chromosome

condensation and histone H3 phosphorylation are maintained during M il (Figure 5.3). This suggests that besides cyclin B/Cdkl there is another protein synthesis dependent kinase that is able to keep AIRK B active. Cyclin A 1/Cdkl, which starts to accumulate during Mil, is a possible candidate for this alternative activator.

Taken together these results suggest that phosphorylation of histone H3 during meiotic maturation depend on a protein synthesis dependent activity, which at least partly consists of MPF.

Summary of this thesis

In this thesis I present a characterisation of the 4 B-type cyclins present in Xenopus

oocytes, eggs, and embryos. I studied the expression patterns of these cyclins during early embryonic cell cycles and found that cyclins B4 and B5 are expressed from maternal mRNAs and disappear after MBT. Analysing the timing of their synthesis and destruction during oocyte maturation 1 found that cyclins B 1 and B4 are newly synthesised in response to progesterone and that their synthesis is enhanced after GVBD and significantly

upregulated during Mil. In the light of the discovery of novel B-type cyclins B4 and B 5 ,1 reassessed the question of cyclin synthesis requirements during oocyte maturation using antisense oligonucleotides. The data presented here suggest that cyclin synthesis is not required for MPF activation at 0 2 phase, but essential for MPF reactivation at the onset of M il and for the establishment of CSF at metaphase 11. These data suggest that a previous unsuspected step of translational control of cyclin B mRNA after M l is essential for meiotic cell cycle control.

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