Protein phosphatase 2A regulatory subunit B55α functions in mouse oocyte maturation and early embryonic development

Abstract

Protein phosphatase 2A regulatory subunit B55α (PP2A-B55α) has been studied in mitosis. However, its functions in mammalian meiosis and early embryonic development remain unknown. Here, we report that PP2A-B55α is critical for mouse oocyte meiosis and preimplantation embryo development. Knockdown of PP2A-B55α in oocytes led to abnormal asymmetric division, disordered spindle dynamics, defects in chromosome congression, an increase in aneuploidy, and induction of the DNA damage response. Moreover, knockdown of PP2A-B55α in fertilized mouse zygotes impaired development to the blastocyst stage. The impairment of embryonic development might have been due to induction of sustained DNA damage in embryos, which caused apoptosis and inhibited cell proliferation and outgrowth potential at the blastocyst stage. Overall, these results provide a novel insight into the role of PP2A-B55α as a novel meiotic and embryonic competence factor at the onset of life.

Keywords: PP2A-B55α; cytokinesis; oocyte maturation; preimplantation development; reproduction.

Figure 1. Localization and expression patterns of PP2A-B55α in mouse oocytes and preimplantation embryos

(A) Subcellular localization of PP2A-B55α from the GV stage to the MII stage of mouse oocyte meiotic maturation. PP2A-B55α mainly localized in the nucleus at the GV stage. After the GVBD stage, PP2A-B55α was distributed throughout the oocyte. (B) Subcellular localization of PP2A-B55α during mouse embryonic development. Blue, DNA; red, PP2A-B55α. Bar = 20 μm. (C) PP2A-B55α transcript levels determined by real-time RT-PCR at different stages of mouse oocyte meiotic maturation and embryonic development. 2C: 2-cell; 8C: 8-cell; BL: blastocyst.

Figure 2. Effects of PP2A-B55α knock down on mouse oocyte maturation

(A) Endogenous PP2A-B55α was knocked down by injecting PP2A-B55α-targeting dsRNA. The PP2A-B55α levels after dsRNA microinjection are shown and were confirmed by western blot analysis (B). Percentages of cultured, dsRNA-injected, GV-stage oocytes that underwent GVBD (C) and PBE (D). Meiotic resumption was investigated at 0, 1, 2, 3, and 4 h. PBE was investigated at 8, 9, 10, 11, and 12 h. The data are the mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (***p < 0.001).

Figure 3. Knock down of PP2A-B55α impairs spindle assembly and chromosome alignment and increases the incidence of aneuploidy during oocyte meiosis

(A) Representative confocal images of control and PP2A-B55α-KD oocytes at 12 h after release from milrinone are shown. Control oocytes (left) exhibited normal spindles and well-aligned chromosomes at the metaphase equator, whereas PP2A-B55α-KD oocytes displayed various spindle defects and misaligned chromosomes (arrows). Percentages of abnormal oocytes displaying aberrant spindles (B) and misaligned chromosomes (C) after PP2A-B55α-KD. Blue, DNA; green, α-tubulin. Bar = 20 μm. (D) Chromosome spreads (stained with Hoechst 33342, blue) and kinetochores (stained with anti-centrosome antibody [ACA], white) of MII-stage oocytes. Representative confocal images are shown. Bar = 10 μm. (E) Quantification of aneuploidy in control and PP2A-B55α-KD oocytes. The data are the mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (*p < 0.05).

Figure 4. PP2A-B55α knock down triggers the DNA damage response in oocytes

(A) Localization of γH2AX in nuclei of GV-stage oocytes. Blue, DNA; green, γH2AX. Bar = 20 μm. (B) Quantification of γH2AX levels in nuclei of control and PP2A-B55α-KD oocytes. The numbers of oocytes examined in each experimental group are shown in the bars. The data are mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (**p < 0.01).

Figure 5. Knock down of PP2A-B55α impairs embryonic development to the blastocyst stage

(A) Representative images of control and PP2A-B55α-KD blastocysts at 4.5 days. Bar = 50 μm. (B) Endogenous PP2A-B55α mRNA expression levels analyzed by real-time RT-PCR at the blastocyst stage after injection of PP2A-B55α-targeting dsRNA. Expression was normalized to that in the control group. Ppia was used as an internal standard because it was not affected by PP2A-B55α-targeting dsRNA injection. (C) Embryonic development rates in the control and PP2A-B55α-KD groups. (D) The number of cells in blastocysts was determined by counting cells in Hoechst 33342-stained embryos. The numbers of embryos examined in each experimental group are shown in the bars. The data are mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (*p < 0.05; **p < 0.01).

Figure 6. Knock down of PP2A-B55α affects the level of DNA damage in mouse embryos

(A) Representative fluorescence images showing the presence of γH2AX139ph protein in nuclei of embryos at different developmental stages. Nuclei in embryos were stained blue and γH2AX139ph foci were stained green. Bar = 20 μm. The average numbers of γH2AX139ph foci in nuclei of PP2A-B55α-KD embryos are shown at the 2-cell (B), 4-cell (C), and blastocyst (D) stages. The numbers of embryos examined in each experimental group are shown in the bars. The data are the mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (*p < 0.05, **p < 0.01).

Figure 7. Knock down of PP2A-B55α affects the levels of apoptosis and cell proliferation in mouse embryos

(A) Representative images of embryos at the blastocyst stage in the TUNEL assay. Bar = 50 μm. (B) The percentage of apoptotic cells in blastocysts that developed in vitro. (C) Immunofluorescence staining of BrdU in mouse embryos at the blastocyst stage. Bar = 50 μm. (D) Percentages of BrdU-positive cells in blastocysts. (E) Immunofluorescence staining of OCT4 in mouse embryos at the blastocyst stage. Bar = 50 μm. (F) ICM rate of blastocysts. The numbers of blastocysts examined in each experimental group are shown in the bars. The data are the mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (*p < 0.05, **p < 0.01).

Figure 8. Knock down of PP2A-B55α affects the implantation potential of mouse embryos

(A) Representative images showing blastocyst outgrowth at 7.5 dpc in the control (A and A') and PP2A-B55α-KD (B and B') groups. Bar = 100 μm. (C) The total areas of outgrowth and ICM/TE areas in the control and PP2A-B55α-KD groups. The numbers of blastocysts examined in each experimental group are shown in the bars. The data are the mean ± SD of three independent experiments. Statistically significant differences are indicated by asterisks (*p < 0.05).