Table 1.
Number of embryos used for each experimental setting.
Fig 1.
A schematic representation of the nucleus.
The radial position of the studied genes was evaluated as a distance from the FISH signal to the nuclear border, divided by the distance from the FISH signal to the nucleus centre. Three locations were considered central (C), intermediate (I) and peripheral (P).
Table 2.
Primer pairs and annealing conditions used for gene expression analyses.
Fig 2.
Possible CT positions within the nuclei of bovine embryos.
(A) CT12 (red) with the CDX2 locus (green) present an example of peripheral location, (B) CT23 (red) with the OCT4 locus (green) indicate central to intermediate location from 8–16 cell stage onwards. (C) presents possible radial distribution of CTs within the 3D nuclear space, (D) CT5 (red) with the NANOG locus (green) show an example of intermediate location in 8–16, morula and ICM. Confocal sections were taken every 0.5μm (zygotes to 8–16 cell stage) and every 0.4μm for morulae and blastocysts. DAPI stains the chromatin.
Fig 3.
Chromosome territory shape variants (A) and observed locus positioning (B) within the CT. (C) presents partial 3D reconstruction of a morula stage embryo from images taken for CT23 (red) and the OCT4 locus (green). Confocal sections were taken every 0.4μm. DAPI labels chromatin.
Fig 4.
Summary of data obtained for CT23 and the OCT4 locus.
Top panel presents the analysed developmental stages, pink circles point to cells for which analyses were carried out at morula and blastocyst stages. (A) summarises the contribution of peripheral, intermediate and central location of CT23 within the analysed nuclei, (B) presents localisation of the OCT4 locus in relation to its CT, (C) indicates changes in CT23 shape related to embryonic stage. (D) presents OCT4 localisation (red immunofluorescence) in bovine embryos. The encircled region indicates the ICM, highlighted in the far right images. Chromatin was visualised by DAPI, confocal sections were taken every 4μm. Scale bar:100μm.
Fig 5.
Summary of data obtained for CT12 and the CDX2 locus.
Top panel presents the analysed developmental stages, pink circles point to cells for which analyses were carried out morula and blastocyst stages. (A) summarises the contribution of peripheral, intermediate and central location of CT12 within the analysed nuclei, (B) shows the localisation of the CDX2 locus in relation to its CT, (C) indicates the changes in CT12 shape related to embryonic stage. (D) presents immunofluorescent localisation of CDX2 (green) in bovine embryos. The encircled region indicates the ICM, highlighted in the far right images. Chromatin was visualised by DAPI, confocal sections were taken every 4μm. Scale bar: 100μm.
Fig 6.
Summary of data obtained for CT5 and the NANOG locus.
Top panel presents the analysed developmental stages, pink circles point to cells for which analyses were carried out at morula and blastocyst stages. (A) summarises the contribution of peripheral, intermediate and central location of CT5 within the analysed nuclei, (B) shows the localisation of the NANOG locus in relation to its CT, (C) indicates the changes in CT5 shape related to embryonic stage. (D) presents NANOG localisation (red immunofluorescence) in bovine embryos. The encircled region indicates the ICM, highlighted in the far right images. Arrows indicate NANOG positive cells in morula inner and outer cells. Chromatin was visualised by DAPI, confocal sections were taken every 4μm. Scale bar: 100μm.
Fig 7.
Gene expression profiles of lineage specific markers during bovine preimplantation development.
(A) indicates the relative changes in transcript abundance for OCT4, (B) for NANOG and (C) for CDX2. The bars (± SEM) labelled with different letters indicate stages which significantly differ in transcript abundance (P≤0.05).
Fig 8.
Changes in gene’s locus position correspond to its transcriptional activity.
Y axis (left) presents the incidence (%) of gene locus moving away from its CT, and Y axis (right) indicates the relative transcript abundance. Because the quantitative gene expression studies were done on whole embryos (morula and blastocyst), the CT movement for the morula stage embryos has been presented as an average result obtained for morula inner and outer cells, and an average for ICM an TE for the blastocyst stage embryos.