TP63 gain-of-function mutations cause premature ovarian insufficiency by inducing oocyte apoptosis

Abstract

The transcription factor p63 guards genome integrity in the female germline, and its mutations have been reported in patients with premature ovarian insufficiency (POI). However, the precise contribution of the TP63 gene to the pathogenesis of POI needs to be further determined. Here, in 1,030 Chinese patients with POI, we identified 6 heterozygous mutations of the TP63 gene that impaired the C-terminal transactivation-inhibitory domain (TID) of the TAp63α protein and resulted in tetramer formation and constitutive activation of the mutant proteins. The mutant proteins induced cell apoptosis by increasing the expression of apoptosis-inducing factors in vitro. We next introduced a premature stop codon and selectively deleted the TID of TAp63α in mice and observed rapid depletion of the p63+/ΔTID mouse oocytes through apoptosis after birth. Finally, to further verify the pathogenicity of the mutation p.R647C in the TID that was present in 3 patients, we generated p63+/R647C mice and also found accelerated oocyte loss, but to a lesser degree than in the p63+/ΔTID mice. Together, these findings show that TID-related variants causing constitutive activation of TAp63α lead to POI by inducing oocyte apoptosis, which will facilitate the genetic diagnosis of POI in patients and provide a potential therapeutic target for extending female fertility.

Keywords: Apoptosis; Fertility; Genetic diseases; Genetics; Reproductive Biology.

Figure 1. Analysis of human TAp63α-mutant pathogenicity.

(A) Schematic diagram showing the 5 key domains of TAp63α: the TAD, the DBD, the oligomerization domain (OD), the SAM, and the TID. The positions of the variants identified in this study and reported in the literature are indicated in red and blue, respectively. Circles signify isolated POI; squares signify syndromic POI. The amino acid sequence of the EAO (504~550) is indicated. (B) After transfection with WT and mutant TAp63α plasmids in SAOS-2 cells, the intracellular protein level of TAp63α was detected by Western blotting. β-Actin was used as the loading control. (C) Oligomeric state analysis of WT and mutant TAp63α by BN-PAGE. The oligomeric conformation is indicated by T (tetramer), D (dimer), and M (monomer). In the protein samples of mutant TAp63α, no WT protein was present. (D) TAp63αΔTID and WT or mutant GFP-TID plasmids were cotransfected into HEK293 cells. Cells were harvested for co-IP assays and were immunoprecipitated with anti-p63 antibody, and then WT and mutant GFP-TID protein were detected by GFP antibody by Western blotting. IgG was used as the negative control. (E) Quantitative analysis of the amount of co-IP between TAp63αΔTID and WT or mutant GFP-TID. The immunoprecipitated GFP-TID was compared with the input. Data are presented as the mean ± SD of 3 independent experiments. **P < 0.01 and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s test.

Figure 2. TAp63α mutants induce cell apoptosis.

(A) Relative transcriptional activity of TAp63α mutants in SAOS-2 cells on NOXA, PUMA, and BAX promoters. The activity of the WT and mutant TAp63α is shown in blue and white, respectively. The activity of the WT group was set to 1. LUC, luciferase. (B) Western blot analysis of BAX expression after transfection with WT and mutant TAp63α plasmids in SAOS-2 cells. β-Actin was used as the loading control. (C) Quantification of BAX protein expression. (D) TUNEL staining of SAOS-2 cells after transfection with WT and mutant TAp63α plasmids. TUNEL-positive signals are indicated by arrows. Cell nuclei were counterstained with DAPI (blue). Scale bars: 50 μm. (E) Quantitative analysis of TUNEL-positive SAOS-2 cells after transfection. In panel A, C and E, data are presented as the mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s test.

Figure 3. Generation and characterization of p63^+/ΔTID mice.

(A) Strategy for the generation of p63^+/ΔTID (referred to in the figures as HET) mice. The c.1828_1829insGA (NM_001127259.1) mutation was introduced into exon 14, leading to the formation of a stop codon and loss of the TID. The primers used for genotyping the WT and p63^+/ΔTID mice are shown. For, forward; Rev, reverse. (B) Agarose gel electrophoresis of the PCR products obtained from genomic DNA of WT and p63^+/ΔTID mice. Sanger sequencing confirmed the creation of the insert mutation. (C) Western blot analysis of p63 expression in protein extracts from P1 WT and p63^+/ΔTID mouse ovaries. β-Actin was used as the loading control. (D) IF staining for DDX4 (green) and p63 (red) in ovary sections from P1 WT and p63^+/ΔTID mice. Cell nuclei were counterstained with DAPI (blue). Scale bars: 50 μM. (E) Gross morphology of 4M WT and p63^+/ΔTID females. (F) No significant difference in body weights was observed between 4M WT and p63^+/ΔTID mice. n = 6 per group. An unpaired, 2-tailed Student’s t test was used for the comparison of the 2 groups. (G) Number of pups obtained by crossing p63^+/ΔTID males (green line) and p63^+/ΔTID females (red line) with WT mice for a period of 6 months. n = 6 per group.

Figure 4. p63^+/ΔTID mice show rapid oocyte loss.

(A) The size of ovaries from 4M WT and p63^+/ΔTID females. (B) H&E staining of ovary sections from WT and p63^+/ΔTID mice at P1, P5, P21, and 4M. Scale bars: 50 μM. (C) IF staining for DDX4 (green) in ovary sections from E18.5, P1, P5, and P10 WT and p63^+/ΔTID mice. Cell nuclei were counterstained with DAPI (blue). Scale bars: 50 μM. (D) Quantitative analysis of DDX4-expressing oocyte numbers per ovary in each group. n = 5. Data are presented as the mean ± SD. ***P < 0.001, by unpaired, 2-tailed Student’s t test.

Figure 5. The oocytes in p63^+/ΔTID mouse ovaries died by apoptosis.

(A) IF staining for DDX4 (green) and cleaved-PARP1 (red) in ovary sections from P1 WT and p63^+/ΔTID mice. Cell nuclei were counterstained with DAPI (blue). Scale bars: 50 μM. (B) Quantitative analysis of cleaved-PARP1–positive oocytes in each group (n = 5). (C) Western blot analysis of BAX expression in P1 WT and p63^+/ΔTID ovaries. β-Actin was used as the loading control. (D) Quantitative RT-PCR analysis of Puma and Noxa gene expression in P1 ovaries of WT and p63^+/ΔTID mice. Gapdh was used as the internal control. Three mice for each genotype were used for each independent experiment, and 3 independent experiments were conducted. (E) The p63 and p63ΔTID plasmids were transfected into SAOS-2 cells, and protein levels were detected by Western blotting. β-Actin was used as the loading control. (F) Oligomeric state analysis of p63 and p63ΔTID using BN-PAGE. The oligomeric state is indicated by T, D, and M. (G) Transcriptional activity of p63ΔTID in SAOS-2 cells on the NOXA, PUMA, and BAX promoters. The activity of the p63 group was set to 1, and 3 independent experiments were conducted. In B, D and G, data are shown as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test.

Figure 6. Accelerated oocyte loss in p63^+/R647C mice by apoptosis.

(A) Validation of the genotype of the p63^+/R647C mouse by Sanger sequencing. (B) Gross morphology of 2M WT and p63^+/R647C females. (C) Sizes of ovaries from 2M WT and p63^+/R647C females. (D) H&E staining of ovary sections from P10 and 2M WT and p63^+/R647C mice. Scale bars: 50 μM. (E) IF staining for DDX4 (green) in P10 WT and p63^+/R647C ovaries. Cell nuclei were counterstained with DAPI (blue). Scale bars: 50 μM. (F) Quantitative analysis of DDX4-expressing oocytes from mice in each group. Data are presented as the mean ± SD. n = 5. ***P < 0.001, by unpaired, 2-tailed Student’s t test. (G) IF staining for DDX4 (green) and cleaved-PARP1 (red) in ovary sections from P1 WT and p63^+/R647C mice. Cell nuclei were counterstained with DAPI (blue). Scale bars: 50 μM. (H) Quantitative analysis of cleaved-PARP1–positive oocytes from mice in each group. Data are shown as the mean ± SD. n = 5. ***P < 0.001, by unpaired, 2-tailed Student’s t test.

Figure 7. Evaluation of fertility and oocyte quality in p63^+/R647C mice.

(A) Cumulative number of pups obtained from WT females (n = 5) and p63^+/R647C females (n = 5) crossed with WT males for a period of 3 months. (B and C) The numbers of pups per litter and the number of litters per mouse were recorded for each group (n = 5) in the fertility test. (D) Number of superovulated oocytes per mouse obtained from the 2 groups (n = 5 for each genotype). (E) Morphology of superovulated oocytes obtained from 3-week-old WT and p63^+/R647C mice. Scale bars: 100 μm. (F) Percentages of MII oocytes with PB1 emission in WT and p63^+/R647C oocytes. Three mice for each genotype were used for each independent experiment, and 3 independent experiments were conducted. (G) The morphology of spindle and chromosome organization in WT and p63^+/R647C oocytes. Anti–α-tubulin antibody (green) was used to stain the spindles. Chromosomes were counterstained with DAPI (blue). Scale bars: 20 μm. (H) Percentages of oocytes with spindle/chromosome defects in WT and p63^+/R647C oocytes. Three mice of each genotype were used for each independent experiment, and 3 independent experiments were performed. (I) Oocyte MMP shown by JC-1 staining in the 2 groups. Red fluorescence indicates JC-1 aggregates with higher MMP, and green fluorescence indicates JC-1 monomers with lower MMP. Scale bars: 20 μm. (J) Quantification of the ratio of red to green fluorescence intensity in the 2 groups. Three mice for each genotype were used for each independent experiment, and 3 independent experiments were performed. (A–D, F, H, and J) Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, by unpaired, 2-tailed Student’s t test.