Spatial and temporal distribution of Oct-4 and acetylated H4K5 in rabbit embryos

Abstract

Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB. Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.

Figure 1

(A–J3) Oocytes and embryos at different stages doubly labelled for DNA (4(,6-diamidino-2-phenylindole); DAPI; blue) and Oct-4 (green). Oct-4 staining of metaphase-II oocytes (MII) showed visible but diffuse signals distributed throughout the cytoplasm (A–A3). Oct-4 signals were detected both in the cytoplasm and the nuclei of zygotes (B–B3) and 2-cell (C–C3) and 4-cell (D–D3) embryos. A very weak Oct-4 signal was found in the cytoplasm and diminished in the nuclei of 8-cell embryos (E-E3). Nuclear Oct-4 staining was evident again at the 16-cell stage (F–F3) and became more intense at the compact morula stage (CM; G–G3). A wave of Oct-4 signal intensity change was observed in blastocyst embryos (H-J3): the average intensity of Oct-4 signal decreased at the early blastocyst stage (EB; H–H3), reached a minimum at the expanded blastocyst stage (EXPB; I–I3) and spiked at the hatching blastocyst stage (HB; J–J3). Bars = 100 μm (A–J3). (K) Quantification of Oct-4 intensity at different stages of in-vitro cultured rabbit embryos. Values are mean _± SEM. Numbers of embryos used for each stage are indicated above each bar. Different letters (a–c) indicate a statistically significant difference (P < 0.05).

Figure 2

(A–C3) Inner cell mass (ICM) and trophectoderm (TE) at the early blastocyst (EB), expanded blastocyst (EXPB) and hatching blastocyst (HB) stages doubly labelled for DNA (4(,6-diamidino-2-phenylindole); DAPI; blue) and Oct-4 (green). Oct-4 fluorescence associated with the ICM was strong in EB (A–A3), dramatically declined after expansion (B–B3), but then elevated to a higher level in HB (C–C3). The Oct-4 signal was detectable in TE cells throughout the blastocyst stages (arrows, A2–C2). Bars = 100 μm (A–C3). (D) Quantification of Oct-4 intensity in the nuclei of ICM and TE cells at different blastocyst stages. The Oct-4 signal in ICM cells at the HB stage was much higher than that in the EB and EXPB stages, whereas the signal intensity was similar in TE cells of all three stages. Oct-4 intensity of the ICM versus the TE regions was also analysed. Significantly higher intensity of the Oct-4 signal was found in ICM cells than in TE cells in both EB- and the HB-stage embryos. The Oct-4 signal was similarly low between these two groups of cells in EXPB-stage embryos. Values are mean ± SEM. Numbers of embryos used for each stage are indicated above each bar. Asterisks indicate statistically significant differences (P < 0.05).

Figure 3

(A–J3) Oocytes and embryos at different stages doubly labelled for DNA (4(,6-diamidino-2-phenylindole); DAPI; blue) and acetylated H4K5 (H4K5ac; red). A visible but weak H4K5ac signal was detected on MII chromosomes (A–A3). Strong signals associated with nuclei were observed in zygotes (B–B3) and 2-cell embryos (C–C3). Declined intensity of H4K5ac was shown in 4-cell embryos (D–D3). Low fluorescence with various staining intensity on nuclei was detected at the 8-cell (E–E3), 16-cell (F–F3), compact morula (CM; G–G3) and early blastocyst (EB; H–H3) stages. The average H4K5ac signal of the whole embryo started to increase at the expanded blastocyst stage (EXPB; G–G3) and peaked at the hatching blastocyst stage (HB; J–J3). Bars = 100 μm (A–J3). (K) Quantification of H4K5ac intensity at different in-vitro cultured stages. Values are mean ± SEM. Numbers of embryos used for each stage are indicated above each bar. Different letters (a–d) indicate a statistically significant difference (P < 0.05).

Figure 4

(A–C3) Inner cell mass (ICM) and trophectoderm (TE) at the early blastocyst (EB), expanded blastocyst (EXPB) and hatching blastocyst (HB) stages doubly labelled for DNA (4(,6-diamidino-2-phenylindole); DAPI; blue) and acetylated H4K5 (H4K5ac; red). Similar levels of H4K5ac signals were observed in ICM cells of EB (A–A3) and EXPB (B–B3). The H4K5ac signal in ICM cells increased at the HB stage (C–C3). On the other hand, the H4K5ac signal in TE cells remained similar throughout these three stages (arrows, A2–C2). Bars = 100 μm (A– C3). (D) Quantification of H4K5ac intensity in the nuclei of ICM and TE cells at different blastocyst stages. The H4K5ac signal was higher in TE cells than in ICM cells at the EB and EXPB stages. The trend reversed at the HB stage, where the H4K5ac signal was higher in ICM cells than in TE cells. Values are mean ± SEM. The number of embryos used for each stage is indicated above each bar. Asterisks indicate statistically significant differences (P < 0.05).