In somatic cells, the chromosomal passenger complex (CPC) plays a crucial role in the prevention of chromosome missegregation by ensuring accurate chromosome attachment and regulating mitotic checkpoint complex (MCC) activity (see chapter 1). In human pre-implantation embryos, chromosomal abnormalities are detected at high frequencies [1,4,17-26], which suggest impaired functionality of these mechanisms. Therefore, we investigated composition (chapter 3) and localization (chapter 5) of the CPC in human pre-implantation embryos derived from IVF.
In chapter 3, we describe a difference in composition of the CPC during the first embryonic cleavage divisions, as compared to somatic cells. In somatic cells, Aurora B is the kinetic subunit of the CPC. In pre-implantation embryos, from the zygote (embryonic day 1) up to the 8- to 16-cell stage (embryonic day 3 and 4), we found Aurora C to be the main kinetic subunit present in the CPC.
Around the morula stage (embryonic day 4), Aurora C levels at the inner centromere decrease and at the blastocyst stage (embryonic day 5) Aurora C is completely replaced by Aurora B.
Aurora C has been known as the meiotic counterpart of Aurora B and is thus present in oocytes.
Due to different mRNA and protein characteristics, Aurora C is less susceptible to degradation in comparison with Aurora B [27-29]. This might explain why Aurora C is still present after meiosis, whereas Aurora B is degraded. Possibly, this also explains why Aurora C is needed during the first mitotic divisions of the embryo; before embryonic genome activation, embryos rely on maternal transcripts, which are stored in the oocyte.
As Aurora C is present only during the first cleavage divisions of embryos, which are more prone to segregation errors than mitotic division later during development [4], it is tempting to speculate about a link between Aurora C and chromosome missegregation. Aurora C has been shown to compensate for the absence of Aurora B in HeLa cells [30-31] and mouse pre-implantation embryos [32], enabling normal progression of mitosis. Although possible subtle aneuploidies were not examined in these cells, there was no indication of reduced error-correction activity of Aurora C.
However, several studies have shown that overexpression of the Aurora kinases and an imbalance in expression of Aurora B and Aurora C lead to chromosome segregation errors [33-35]. In our study, we detected varying ratios of Aurora B and Aurora C in human oocytes. Therefore, we hypothesize that these differences in concentration, rather than the presence of Aurora C, might influence the accuracy of the error-correction mechanism. Investigation of the differences between the two Aurora kinases, for example of their substrates and binding partners, and the regulation of their expression might provide some clues to understand the function of these kinases in the regulation of chromosome segregation in pre-implantation embryos. Also, since it is know that female age is an important factor in oocyte quality [36-37], it would be of interest to determine whether the Aurora B/C ratio is influenced by maternal age.
Precise inner centromeric CPC localization is crucial for accurate function of the error-correction mechanism [38-39]. In chapter 5 we describe our investigation into the localization of the CPC in the first embryonic divisions. For this investigation, we made use of the technique we developed and described in chapter 4. With this ‘stripping’ technique, we were able to perform sequential immunofluorescent analysis on the same chromosome preparation, which allowed us to investigate co-localization of several proteins and histone modifications that play a role in CPC localization.
The ability to perform two immunofluorescent analyses on the same material is particularly useful in our studies on chromosome segregation and chromatin structure in human oocytes and pre-implantation embryos, since this material is rare and valuable.
Previously, it was hypothesized that the extremely high incidence of chromosomal abnormalities in human embryos were due to a lack of a functional mitotic checkpoint [21]. Using different kinase inhibitors in our investigation of CPC localization pathways (chapter 5), we demonstrate that feedback loops between CPC and MCC proteins, as described in somatic cells [40-43], are functional and that the checkpoint is active. Therefore, we hypothesize that chromosomal abnormalities in human embryos do not arise because of a total lack of mitotic checkpoint function activity. However, this does not rule out the possibility of more subtle differences in checkpoint functionality.
In chapter 5, we found CPC localization to be less confined to the inner centromeric region in zygotes than at later developmental stages. Also, we found that in the zygote stage, one of the pathways for CPC targeting (see chapter 1) is different; H3T3 phosphorylation failed to enrich at the
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centromeric region on metaphase chromosomes in human zygotes. From the 2-cell stage onwards, H3pT3 was detected in a normal pattern and towards the blastocyst stage, CPC localization slowly normalized too. These findings suggest that centromeric targeting of the CPC is controlled differently and possibly less accurate in zygotes and to a lesser extent also in cleavage stage embryos. It is tempting to speculate that altered CPC targeting and localization contribute to the occurrence of chromosomal abnormalities. Future research should elucidate if this is the case, for example by investigating the error-correcting ability of the CPC in the first mitotic division of embryos.
Despite abundant H3pT3 on chromosome arms in zygotes, the CPC still localizes to the centromeric area, suggesting that centromeric CPC localization is not completely dependent on centromeric enrichment of H3pT3. Still, CPC localization is less restricted to the centromere, so the different H3pT3 pattern may have an impact on the accuracy of the CPC targeting. Studies in somatic cells support this hypothesis, as induced abundant H3pT3 on the chromosome arms leads to CPC mislocalization [44]. However, the pattern of H3pT3 normalizes already in the 2-cell stage, whereas CPC localization normalizes slowly towards the blastocyst stage. Thus, more factors will probably underlie the less accurate CPC localization during the cleavage divisions. Differences in the pathways that regulate CPC targeting may be an explanation for chromosome missegregation in embryos, as subtle changes in CPC localization are known to affect chromosome alignment and the error-correction mechanism [38-39,43]. Next to this, high levels of H3pT3 on chromosome arms were shown to affect mechanisms responsible for cohesion resolution in somatic cells [45], which leads to chromosome segregation defects. Thus, the observed difference in H3pT3 localization in human zygotes may have different consequences for the regulation of chromosome segregation and might cause chromosome segregation to be more error prone.
Taken together, results described in chapter 3 and chapter 5 show that there are differences in both CPC composition and localization in the first mitotic divisions of embryos, compared with mitotic divisions in later embryonic stages and somatic cells. Presence of Aurora C as the main CPC kinase and different targeting and localization of the CPC might lead to a less accurate regulation of chromosome segregation. Further research is needed to determine if the differences we observed indeed affect the prevention and correction of erroneous chromosome segregation, for example by assessing if the error correcting ability of the CPC is different in the first cleavage divisions.
Our knowledge on the high rate of chromosomal abnormalities is almost exclusively derived from embryos generated by IVF. It is possible that our observations are induced by the IVF procedure, for example through influence of hormonal treatment or in vitro embryo culture. However, chromosomal abnormalities are also observed after natural cycle IVF and in in vivo fertilized porcine and bovine embryos, indicating that embryo’s of certain mammalian species are predisposed to chromosome segregation errors. Whether (suboptimal) IVF procedures increase the chance on these errors is very hard to examine, because of ethical and practical difficulties in obtaining human in vivo pre-implantation embryos. Still, our increasing knowledge on chromosome segregation in IVF-derived embryos is relevant for daily IVF practice and future optimization of IVF procedures.