The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos.

In contrast to somatic cells, our results point to a specific role for Aurora C in the CPC during human preimplantation embryo development. Although, the presence of Aurora C in itself may not explain the high chromosome segregation error rate, the data presented here provide novel information regarding possible mechanisms.

This study was funded by the Portuguese Fundação para a Ciência e Tecnologia and the Netherlands Organization for Scientific Research. The authors have no conflicts of interest to declare.

Immunofluorescence has been widely used to study histone modification dynamics and chromosome-associated proteins that regulate the segregation of chromosomes during cell divisions. Since many of these regulatory proteins interact (in)directly to exert their proper function, it is of interest to detect these proteins simultaneously, to establish their spatiotemporal relation. However, the detection of multiple epitopes on the same material is limited by the availability of antibodies derived from different host species. For Western blot membranes, buffers were developed to remove antibodies after the first round of detection and enable a second round of detection. In this study, we establish that this "stripping" principle can also be applied for sequential immunofluorescence on chromosome preparations. We first adapted a drying down fixation technique for the use on cultured cells from different primary cells and cell lines. These chromosome spreads were subsequently used to optimize the stripping procedure for this application. We investigated feasibility and reliability of detection of histones and their posttranslational modifications as well as chromatin interacting proteins in two subsequent rounds of immunofluorescence. We conclude that this method is a reliable option when spatial resolution and co-expression need to be investigated and the material or the choice of antibodies is limited.

In mouse female preimplantation embryos, the paternal X chromosome (Xp) is silenced by imprinted X chromosome inactivation (iXCI). This requires production of the noncoding Xist RNA in cis, from the Xp. The Xist locus on the maternally inherited X chromosome (Xm) is refractory to activation due to the presence of an imprint. Paternal inheritance of an Xist deletion (XpΔXist) is embryonic lethal to female embryos, due to iXCI abolishment. Here, we circumvented the histone-to-protamine and protamine-to-histone transitions of the paternal genome, by fertilization of oocytes via injection of round spermatids (ROSI). This did not affect initiation of XCI in wild type female embryos. Surprisingly, ROSI using ΔXist round spermatids allowed survival of female embryos. This was accompanied by activation of the intact maternal Xist gene, initiated with delayed kinetics, around the morula stage, resulting in Xm silencing. Maternal Xist gene activation was not observed in ROSI-derived males. In addition, no Xist expression was detected in male and female morulas that developed from oocytes fertilized with mature ΔXist sperm. Finally, the expression of the X-encoded XCI-activator RNF12 was enhanced in both male (wild type) and female (wild type as well as XpΔXist) ROSI derived embryos, compared to in vivo fertilized embryos. Thus, high RNF12 levels may contribute to the specific activation of maternal Xist in XpΔXist female ROSI embryos, but upregulation of additional Xp derived factors and/or the specific epigenetic constitution of the round spermatid-derived Xp are expected to be more critical. These results illustrate the profound impact of a dysregulated paternal epigenome on embryo development, and we propose that mouse ROSI can be used as a model to study the effects of intergenerational inheritance of epigenetic marks.