Epigenetics and chromosome segregation in human pre-implantation embryos

Abstract Chapter 1 Currently, the average pregnancy rate per embryo transfer after in vitro fertilization (IVF) is around 32%. In order to achieve better results in the future, we need to gain knowledge on all aspects of the treatment, including pre-implantation embryo development. In this thesis, we describe the research we performed into epigenetics and chromosome segregation in human pre-implantation embryos derived from IVF. The term ‘epigenetics’ refers to heritable marks on the genome, such as DNA methylation and histone modifications. These marks are essential for chromosome structure, chromosome segregation and gene expression. Chromosome segregation is the process in which duplicated chromosomes are equally separated over two cells during cell division. Chromosomal abnormalities are detected at high frequencies in human pre-implantation embryos. This suggests that mechanisms regulating chromosome segregation are less functional during the first cell divisions of an embryo. Next to that, epigenetic marks, which are also important for correct chromosome segregation, are different in oocytes and spermatozoa and need to be re-established in early embryos. The research described in this thesis aimed to investigate both the mechanisms regulation chromosome segregation and the re-establishment of epigenetics marks in human pre-implantation embryos, in order to shed light on the causes of chromosomal abnormalities. Chapter 2 DNA is wrapped around histones, together forming chromatin. Epigenetic marks like histone modifications determine the structure of chromatin and define certain chromatin domains. Oocytes and spermatozoa have a very different chromatin structure, both from each other and from somatic cells, and after fertilization, canonical chromatin domains need to be re-established. In this chapter we investigated the re-establishment of a chromatin domain important for chromosome segregation, constitutive heterochromatin (cHC), in human pre-implantation embryos derived from IVF. We describe that human spermatozoa carry histones with canonical cHC modifications (H3K9me3, H4K20me3, H3K64me3). After fertilization, these modified histones contribute to the formation of paternal embryonic cHC. The histone modifications are recognized by maternal chromatin regulators (e.g. HP1, SUV39H) and propagated over the embryonic cell divisions. These results indicate transgenerational epigenetic inheritance of cHC structure in human embryos and show that there is an important contribution of the spermatozoon to embryo development. Until now, this process was studied only in mouse embryos, in which paternal cHC in spermatozoa and embryos lacks canonical modifications and is transiently established by other mechanisms, present in the oocyte. Often these results are assumed to be applicable for all mammalian species. However, we now show that the mechanism in human embryos differs significantly from what has been described for mice. This points out that mouse embryos are not a representative animal model and underlines the need for research on human embryos. Chapter 3 One of the most important players in the regulation of chromosome segregation is a protein complex named chromosomal passenger complex (CPC). The CPC consists of the proteins Survivin, Borealin, INCENP and Aurora kinase B in somatic cells divisions (mitotis). In this chapter we investigated presence and composition of the CPC in human pre-implantation embryos derived from IVF. We describe that the composition of the CPC in the first embryonic cell divisions is different from what has been described for somatic cells. In somatic cells, Aurora B is the kinetic subunit of the CPC. In pre-implantation embryos, from the zygote (day 1 of embryonic development) up to the 8- to 16-cell stage (day 3), we found Aurora C to be the main kinetic subunit present in the CPC. Until now, Aurora C has been described only in the cell divisions of germ cells, which result in oocytes and spermatozoa (meiosis). Around the morula stage (day 4), Aurora C levels decreased and at the blastocyst stage (day 5) Aurora C was completely replaced by Aurora B again. As subtle differences between the Aurora kinases might lead to less accurate regulation of chromosome segregation, the different composition of the CPC may contribute to chromosome segregation errors, which lead to chromosomal abnormalities, in the first cell divisions of human embryos. Chapter 4 Immunofluorescence has been widely used to study histone modifications and proteins involved in the regulation of chromosome segregation. Although it is important to study co-localization of these modifications and proteins, this is very difficult due to the limited availability of antibodies derived from different host species. For Western blot membranes, buffers were developed to remove antibodies after the first round of detection, called ‘stripping’, and thereby enable a second round of detection. In this chapter, we describe that we were able to apply the stripping principle for sequential immunofluorescence on chromosome preparations of diverse cell types and human embryos. We show feasibility and reliability of detection of histone modifications and proteins in two rounds of immunofluorescence. This method is a reliable option when co-localization needs to be investigated and the choice of antibodies or the material, for example in case of human embryos, is limited. Chapter 5 Precise localization of the CPC at the inner centromeric area of chromosomes is crucial for accurate regulation of chromosome segregation. This localization is regulated by two histone modifications, H2ApT120 and H3pT3, which are catalyzed by the proteins Bub1 and Haspin respectively. In this chapter we investigated the localization of the CPC during the first cell divisions of human pre-implantation embryos derived from IVF. We describe that CPC localization is less restricted to the inner centromere in the first embryonic cell division and that during the subsequent cell divisions, it becomes increasingly restricted, comparable with its localization in somatic cells. Of the two pathways that regulate inner centromeric localization of the CPC, we found the Bub1-H2ApT120 pathway to be comparable with what has been described for somatic cells. However, the Haspin-H3pT3 pathway was different in zygotes, during the first cell division of the embryo; instead of only centromeric, it was detected along the whole chromosome. This difference in the regulation of CPC localization may explain the less restricted CPC localization during the first cell divisions of embryos, which might be an explanation for chromosome segregation errors. We also describe that the (partly altered) distribution of histone modifications involved in CPC localization and function, such as H3pT3 and H3pS10, in human pre-implantation embryos seems to be related to the epigenetic asymmetry between maternal and paternal chromosomes. Possibly, all these differences together lead to less accurate chromosome segregation and thereby may be a cause of chromosomal abnormalities in human pre-implantation embryos. Chapter 7 Our results show altered, and thereby probably less accurate, mechanisms that are involved in the prevention of chromosome segregation errors in human pre-implantation embryos. Also, our results show that human spermatozoa have an epigenetic contribution to embryo development. The altered error-correction mechanism and the epigenetic asymmetry between maternal and paternal chromosomes might together explain the high rates of chromosome segregation errors in human pre-implantation embryos. In our future research, we want to focus on the possible contribution of the spermatozoon to chromosome abnormalities in embryos. Knowledge of the influence of sperm quality on embryo developmental potential may lead to the optimization of IVF procedures in the future.