Fig 1.
PP6c is essential for female fertility.
(A) Schematic representation of deletion of Ppp6c exons and creation of Ppp6c Δ allele by Gdf9-Cre-mediated recombination in oocytes. (B) Western blots showing the absence of PP6c protein expression in Ppp6cF/F;GCre+ oocytes. The amount of β-actin was used as an internal control. Molecular mass is given in kilodaltons. Oocytes were isolated from ovaries of PD35 mice and used for western blotting. For each lane, 200 GV oocytes were used. For each experiment, at least 5 mice of each genotype were used. (C) Infertility of the female F/F;GCre+ mice. Continuous breeding showed the cumulative number of progeny per female mouse for 6 months. At least 6 mice of each genotype were used. (D) Anovulation of Ppp6cF/F; GCre+ female mice. Fertilized eggs were collected and counted from females with vaginal plugs after mating. At least 6 mice of each genotype were used.
Fig 2.
Premature ovarian failure in Ppp6cF/F;GCre+ mice.
(A-L) Histology of ovarian sections from Ppp6cF/F and Ppp6cF/F;GCre+ females of 2 months, 3 months, 4 months and 6 months-of-age, respectively, stained with hematoxylin and eosin. White arrowheads in C point to primordial follicles; yellow arrows in F show activated follicles; yellow arrowheads in I and L indicate atretic follicles. Panels C’, F’, I’ and L’ are magnified images of rectangular areas marked with a solid line in panels C, F, I and L, respectively. Bars: 100 μm in C, F, I and L; 50 μm in C’, F’, I’ and L’; 500 μm in the others. For each time point, at least 3 mice of each genotype were used for analysis, and representative images are shown. (M-N) Numbers of primordial follicles (M) and activated follicles (N) in ovaries of 1-month (1 mo), 2-month (2 mo), 3-month (3 mo), 4-month (4 mo) and 6-month (6 mo)-old Ppp6cF/F and Ppp6cF/F;GCre+ females. For each time point, at least 3 mice of each genotype were used for analysis. Data are shown as mean ± SEM.*P< 0.05; **P< 0.01.
Fig 3.
Ppp6c deletion results in POF independent of AKT/mTOR but partially dependent on LKB1/AMPK pathway activity.
(A-B) Western blots showing up-regulated AKT/mTOR and AMPK signaling in Ppp6cF/F;GCre+ oocytes. Each sample (200 GV oocytes) was collected from PD35 ovaries and immunoblotted for p-AKT, p-mTOR, p-S6K, p-rpS6, p-AMPK and β-actin. For each experiment, at least 5 mice of each genotype were used. Molecular mass is given in kilodaltons. (C-E) Histology of ovarian sections from 2-month-old Lkb1F/F;GCre+, Ppp6cF/F;GCre+ and Lkb1F/F;Ppp6cF/F;GCre+ females stained with hematoxylin and eosin. Magnified images of rectangular areas marked with a solid line are shown in H&E staining and TUNEL immunofluorescence staining. Yellow arrowheads point to atretic follicles. Green: TUNEL positive signal; Blue: DAPI. At least 3 mice of each genotype were used for analysis, and representative images are shown. Bar = 500 μm.
Fig 4.
Ppp6c deletion results in increased level of γH2AX and abolished DNA damage response pathway in oocytes.
(A) Western blots showing up-regulated level of γH2AX and down-regulated CHK1/2-p53 pathway. Level of GAPDH was used as internal controls. Molecular mass is given in kilodaltons. Oocytes were isolated from ovaries of PD35 mice and used for western blot. For each lane, 200 GV oocytes were used. For each experiment, at least 5 mice of each genotype were used. (B) Immunofluorescent staining of 2-month-old ovarian sections showing increased γH2AX in Ppp6cF/F;GCre+ oocytes. Green: γH2AX; Red: MVH; Blue, DAPI. White arrows point to nucleus of control oocytes; yellow arrows point to nucleus of mutant oocytes. Bar = 20 μm. At least 3 mice of each genotype were used for analysis, and representative images are shown. (C) Decreased incidence of GVBD and PBE of Ppp6cF/F;GCre+ oocytes. PD35 GV oocytes were isolated and matured in vitro, oocytes that resumed meiosis I (GVBD) and extruded the first polar body (PBE) were counted at 4 h and 13 h, respectively. Data are shown as mean ± SEM. *P< 0.05; **P< 0.01. Representative images of immunostaining for DNA (red) and α-tubulin (green) showing abnormal spindle assembly and aberrant chromosome alignment in Ppp6cF/F;GCre+ oocytes at 8 h and 13 h, respectively. Bar = 10 μm. In vitro maturation experiments were repeated at least three times.
Fig 5.
PP6c-deficient oocytes are susceptible to induced DNA damage.
(A) Morphology of ovaries from Ppp6cF/F and Ppp6cF/F;GCre+ mice treated with zeocin or vehicle. At least 3 mice of each genotype were used for analysis, and representative images are shown. (B) Numbers of follicles including activated follicles and primordial follicles in ovaries from 2-month-old Ppp6cF/F and Ppp6cF/F;GCre+ mice treated with zeocin or vehicle. For each group, at least 3 mice were used for analysis. Data are shown as mean ± SEM. **P< 0.01. (C) Histology of ovaries from 2-month-old Ppp6cF/F and Ppp6cF/F;GCre+ mice after zeocin treatment. For each group, at least 3 mice were used for analysis. White arrows show healthy growing follicles, white arrowheads show healthy primordial follicles; yellow arrows show atretic growing follicles, yellow arrowheads show atretic primordial follicles. Bar = 50 μm.
Fig 6.
DNA damage response pathway activity is enhanced in PP6c-deficient oocytes after zeocin treatment.
(A) Western blots showing up-regulated CHK1/2-p53 pathway activity in zeocin-treated Ppp6cF/F;GCre+ oocytes. Level of β-actin was used as internal controls. Molecular mass is given in kilodaltons. GV oocytes were isolated from ovaries of PD35 mice and treated with zeocin in vitro. For each lane, 200 GV oocytes were used. For each experiment, at least 5 mice of each genotype were used. (B) Western blots showing up-regulated CHK2-p53 pathway activity in zeocin-treated Ppp6cF/F;GCre+ ovaries. Level of β-actin was used as internal controls. Molecular mass is given in kilodaltons. Ovary lysates were prepared from ovaries of PD35 mice after zeocin treatment in vivo. For each lane, 30 μg proteins were loaded. For each experiment, at least 3 mice of each genotype were used.