PP6c-deficient oocytes are susceptible to induced DNA damage.

<p>(A) Morphology of ovaries from <i>Ppp6c</i>^<i>F/F</i> and <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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 <i>Ppp6c</i>^<i>F/F</i> and <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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 <i>Ppp6c</i>^<i>F/F</i> and <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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.</p

Premature ovarian failure in <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> mice.

<p>(A-L) Histology of ovarian sections from <i>Ppp6c</i>^<i>F/F</i> and <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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 <i>Ppp6c</i>^<i>F/F</i> and <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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.</p

<i>Ppp6c</i> deletion results in increased level of γH2AX and abolished DNA damage response pathway in oocytes.

<p>(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 <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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 <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> oocytes. PD35 GV oocytes were isolated and matured <i>in vitro</i>, 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 <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> oocytes at 8 h and 13 h, respectively. Bar = 10 μm. <i>In vitro</i> maturation experiments were repeated at least three times.</p

<i>Ppp6c</i> deletion results in POF independent of AKT/mTOR but partially dependent on LKB1/AMPK pathway activity.

<p>(A-B) Western blots showing up-regulated AKT/mTOR and AMPK signaling in <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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 <i>Lkb1</i>^<i>F/F</i><i>;GCre+</i>, <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> and <i>Lkb1</i>^<i>F/F</i><i>;Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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.</p

DNA damage response pathway activity is enhanced in PP6c-deficient oocytes after zeocin treatment.

<p>(A) Western blots showing up-regulated CHK1/2-p53 pathway activity in zeocin-treated <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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 <i>in vitro</i>. 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 <i>Ppp6c</i>^<i>F/F</i><i>;GCre+</i> 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 <i>in vivo</i>. For each lane, 30 μg proteins were loaded. For each experiment, at least 3 mice of each genotype were used.</p

Effects of different carcass preservation temperatures on oocyte meiotic process.

<p>(A) The number of GV oocytes collected from mouse carcass after preserved for 4 hours at different temperatures. (B) The percentage of GVBD oocytes and PBE in PMI 4 h group at different preservation temperatures. Each bar represents mean ±SEM (n = 3). * P<0.05.</p

Effects of different preservation periods and temperatures on oocyte spindle configuration after IVM.

<p>Oocytes cultured in M2 medium for 8h (MI) and 12h (MII), spindle was stained with FITC-α-tubulin and DNA was stained with PI. (A, B,C) the normal spindle in mouse MI and MII oocyte in control group and experimental group at different preservation temperatures.</p

Effects of preservation time and temperature on mitochondrial distribution and GSH level in mouse oocytes.

<p>(A) Mitochondrial distribution was evaluated by fluorescence microscopy. Three localization patterns were observed: perinuclear, homogenous and clustered. (B) The percentage of abnormal mitochondrial distribution in GV oocytes in the control group (n = 279) and preservation group at different temperatures. * indicate statistically significant differences (P<0.05). (C) The GSH concentrations in GV oocytes (n = 30) in different groups.</p

Generation of <i>Tsc1^cko</i> mutant mice and characterization of <i>Tsc1</i> disruption by western blot and PCR analysis.

<p>(A) Schematic representation of deletion of <i>Tsc1</i> exon17 and exon18 by cyp19-cre mediated recombination in granulosa cells. (B) Granulosa cells were collected from COC of both <i>Tsc1^flox/flox</i> mice and <i>Tsc1^cko</i> mutant mice and lysed for western blot: <i>Tsc1</i> was almost absent in <i>Tsc1^cko</i> granulosa cells, β-Tubulin was used as an internal control. (C) PCR analysis indicated that cyp19-cre mediated recombination of <i>Tsc1</i> exclusively occurred in <i>Tsc1^cko</i> ovary.</p

Evaluation of reproductive activity of <i>Tsc1^flox/flox</i>;cyp19-cre mice.

<p>(A) <i>Tsc1^flox/flox</i>; cyp19-cre mice produced more pups than wild type mice. (B) The average litter size of <i>Tsc1^flox/flox</i>; cyp19-cre mice was bigger than that of the wild type. (C) <i>Tsc1^flox/flox</i>; cyp19-cre mice ovulated more oocytes than wild type mice in superovulation analysis. (D) A little more oocytes were collected from the oviduct of <i>Tsc1^flox/flox</i>; cyp19-cre mice than that from wild mice after natural ovulation and copulation.WT, wild type. *, <i>P</i><0.05.</p