35 DYNAMICS OF PERICENTRIC REPETITIVE SEQUENCES IN PREIMPLANTATION RABBIT EMBRYOS UNDERLINES INADEQUATE SPATIO-TEMPORAL REORGANIZATION AFTER NUCLEAR TRANSFER

Abstract

Nowadays, a critical question in the epigenetic field is how chromatin is organised within the cell nucleus and how it affects gene expression. In this study we hypothesise that nuclear structure could be involved in the control of gene expression during early rabbit embryonic development. We focused on pericentric/centric heterochromatin, a peculiar region within nuclei known to form higher-order chromatin structures. We therefore performed immunostaining of associated proteins (HP1 and CENP) as well as FISH (fluorescent in situ hybridization) with probes corresponding to these genomic regions. Fertilized embryos were collected from New Zealand white rabbit female, cultured in vitro and fixed at different developmental stages up to 16-cell. Nuclear transfer of rabbit fetal skin fibroblasts was performed by electrofusion and electroactivation (Chesne et al., 2002). Immunostaining experiments were performed as previously described (Martin et al., 2006) and FISH experiments were performed using DNA probes specific to Rsat I and Rsat II sequences (Ekes et al., 2004) according to our published protocol (Maalouf et al., 2010). Three-dimensional images were acquired by confocal laser microscopy (each group included 20 to 40 embryos) and then analysed through automated 3D image processes (according to Ballester et al., 2008; Pichugin et al., 2010). Briefly, after segmentation of the nuclei, immunostaining signals were analysed using an automatic threshold segmentation procedure and FISH spots distributions were analysed with the eroded volume fraction (EVF) method. In in vivo fertilized rabbit embryos, we observed that during the 1- and 2-cell cycles pericentric heterochromatin was dispersed throughout the embryonic nuclei. Large foci of pericentric heterochromatin then progressively appeared at the 4-cell stage and increased in frequency and size by the 8/16-cell stage [∼75% of the embryos, n = 40; i.e. the stage of major transcriptional activation also called MET (maternal to embryonic-transition)]. This suggested that dramatic aggregation of pericentric heterochromatin is associated to the onset of transcription. Interestingly, after nuclear transfer, the donor nuclear organisation was quickly reverted into an early embryonic dispersed form in most 1-cell embryos (80%, n = 35). Subsequently, a somatic-like nuclear reorganization with large heterochromatin foci was re-established in 86% of the 4-cell stage cloned embryos (n = 35). This pattern was similar to the one observed in fertilized embryos at MET; it also appeared one stage earlier (4-cell vs 8-cell). Coincidently, most cloned embryos stopped developing at that stage (85% of cleavage until the 4-cell but only 38% at morula, n = 173). Together, the results suggested that inadequate spatio-temporal reorganization of the donor nucleus could be deleterious during early development in rabbit clones.

The present work was supported by INRA « Jeune Equipe » funding, the European CLONET (MRTN-CT-2006-035468) grant and the Ambassade de France en Chine.