The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET.

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG‐binding domain. Four MBD proteins (MeCP2 and MBDs 1–3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species‐specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species‐specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis. Mol. Reprod. Dev. 75: 48–59, 2008. © 2007 Wiley‐Liss, Inc.

The Cbx gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation, and chromatin remodeling. In Drosophila embryos, the single family member HP1 localizes to the nuclei from the blastoderm stage, nuclear division cycle 10, suggesting that heterochromatin formation occurs around the time of embryonic transcriptional activation (James et al. 1989 Eur. J. Cell Biol. 50, 170–180). In mice, HP1� is absent in the first cell cycle in both parental nuclei following fertilization (van der Heijden et al. 2005 Mech. Dev. 122, 1008–1022). We report the first study of gene expression, mRNA and protein localization, of the Cbx gene HP1� in in vitro-produced bovine embryos. Expression of the HP1� gene was assessed non-quantitatively in amplified cDNA from 3 individual oocytes or embryos for each stage. HP1� (Cbx5) transcripts were detected in all samples except one oocyte, three 4-cell embryos, and one Day 7 blastocyst. The lack of transcripts at the 4-cell stage may reflect an early degradation of maternal transcripts, or indicate that transcript levels were too low to be detected with this assay. Immuno-localization of HP1� was then performed by using an anti-HP1� monoclonal antibody (Upstate, Auspep Pty., Ltd., Parkville, Victoria, Australia), revealing HP1� to be localized evenly in the cytoplasm of oocytes, and then progressing to both cytoplasmic and nuclear staining at the 2-cell stage. This was followed by nuclear staining from the 4-cell stage onward. Higher power investigation of the subnuclear localization of HP1� showed a diffuse type staining pattern within the nuclei of 2- and 4-cell embryos, followed by punctate staining within the euchromatin in the 8-cell embryos, and within the heterochromatin by the 16-cell stage. At the blastocyst stage, staining appeared more diffuse, but localized to the heterochromatin. This suggests that HP1� is localized to the euchromatin early, followed by subsequent localization to the heterochromatin following the MET. HP1� localization to the euchromatin was recently described (Grigoryev et al. 2004 J. Cell Sci. 117, 6153–6162) during a differentiation event in murine lymphocytes. It is evenly distributed throughout the nucleus in quiescent lymphocytes, but localizes to the vicinity of centromeric chromatin during lymphocyte reactivation. HP1-TIF1α interaction was also recently shown to be required during a short period of time within primitive endoderm-like cells for terminal differentiation (Cammas et al. 2004 Genes Dev. 18, 2147–2160). The results thus indicate that HP1� behaves similarly in embryonic differentiation and lymphocyte reactivation, implying a common mechanism of chromatin remodeling in the 2 cellular systems. Collectively, the results suggest that dynamic changes of the nuclear–cytoplasmic and subnuclear distribution of HP1� may play a key role in the bovine MET.