Fig 1.
Schematic overview of the experimental design.
Schematic overview of the experimental design. This study comprised two study arms. Left: Mice assays for age-related reproductive changes. Four groups of female ICR mice (young [4–5 weeks], 30 weeks, 45 weeks, and 60 weeks) were used for whole ovarian tissue collection for qRT-PCR and p16 ELISA (n = 6–8 animals/group) and immunohistochemistry (n = 3 animals/group) (Fig 2), in vitro fertilization using female mice from groups 1–4 (n = 10–15 animals/group) with male sperm donors (n = 4 animals) (Fig 5), and embryo transfer to pseudopregnant foster mothers (n = 10–15 animals/group) (Fig 6). Right: Assays for young versus aged mice comparative study. Two groups (young [4–5 weeks] and aged [50–55 weeks]) were used for ovarian cell specific p16 levels (ovarian cells obtained from 8 animals, 6–8 samples/group) (Fig 3), oocyte/embryo specific p16 levels (n = 5 oocytes or embryos/sample, 6–8 samples/group) (Fig 3), and organ specific p16 levels (n = 8 animals/group) (Fig 4).
Fig 2.
Age-dependent increase in p16 levels in mouse ovary.
Gene expression analysis of p16 in mouse ovaries by quantitative real-time PCR (qPCR). p16 mRNA levels in the ovaries of young (4–5 weeks of age), 30-, 45-, and 60-weeks of age mice were normalized for GAPDH (n = 6–8 per group). Data were analyzed by one-way ANOVA followed by Tukey#39;s post-hoc test (*p < 0.05). (B) p16INK4a protein concentration (pg/ml) measured by ELISA (n = 6–8 per group; < LQQ, below the limit of quantification). Data were analyzed by one-way ANOVA followed by Tukey#39;s post-hoc test (*p < 0.05). (C) Representative immunohistochemical staining for p16 in ovarian sections of young, 30-, 45- and 60- week of age mice (n = 3 per group, scale bar = 1 mm). (D) Representative higher-magnification images of follicles showing p16 staining in young and 60-week-old follicles (scale bar = 100 μm). Black arrowheads indicate granulosa cells (GC); white arrowheads indicate theca cells (TC). wks: weeks.
Fig 3.
Cell-type specific p16 level changes with aging.
mRNA levels increased with age in somatic cells but did not change in oocytes. Changes in mRNA levels in ovarian component cells in young (4–5 weeks of age) and aging (50–55 weeks of age) mice. (A) Cumulus cell (CC), granulosa cell (GC) and theca cell (TC). (B) Corpus luteum (CL). (C) corpus albicans (CA). (D) MII oocyte. (E) blastocyst-stage embryo (BL). For all samples, n = 6–8 per group. Values are normalised by GAPDH. Data are presented as mean ± SEM. Statistical analysis was performed using Student#39;s t-test (*p < 0.05 compared to young control).
Fig 4.
Tissue-specific patterns of age-related p16 levels.
Comparison of p16 mRNA levels in different tissues in young (4–5 weeks of age) and aging (50–55 weeks of age) mice. mRNA levels of p16 normalised by GAPDH (n = 8 per group). Data are presented as mean ± SEM and analyzed using Student#39;s t-test (*p < 0.05, young vs aging).
Fig 5.
Age-dependent decline in ovulation with preserved fertilization capacity.
Mice were treated with hCG to induce ovulation, followed by in vitro fertilization (n = 10–15 / group). This figure shows fertility parameters in young (4–5 weeks of age), 30, 45, and 60 weeks of age mice: (A) Number of ovulated oocytes after hCG injection. (B) Degeneration rate of ovulated oocytes (%). (C) Fertilization rate (2-cell stage/MII oocytes). (D) Embryo development rate from 2-cell stage to 4-cell stage (4 cells/2 cells); (E) Development rate from 2-cell stage to 8-cell stage (8 cells/2 cells); (F) Development rate to morula stage (morula/2 cells); (G) Development rate to blastocyst stage (BL/2 cells) BL: blastocyst. wks: weeks. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey#39;s post-hoc test (*p < 0.05 compared to young control).
Fig 6.
Impaired reproductive outcomes with advancing maternal age.
Fertilized eggs obtained from young mice (4–5 weeks old), 30-week-old mice, 45-week-old mice, and 60-week-old mice were transferred as blastocysts into pseudopregnant surrogate mothers (n = 10–15 / group). (A) Implantation rate (implantation site/transferred blastocyst embryo), (B) Live pups rate (live pups/implantation site), (C) Miscarriage rate (miscarriages/implantation site), (D) Pup body weight (g), (E) Placental weight (mg). Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey#39;s post-hoc test. wks: weeks. *p < 0.05 compared to young control.