Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies

Abstract

Increased meiotic spindle abnormalities and aneuploidy in oocytes of women of advanced maternal ages lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Despite the significance of the problem, strategies to sustain oocyte quality with age have remained elusive. Here we report that adult female mice maintained under 40% caloric restriction (CR) did not exhibit aging-related increases in oocyte aneuploidy, chromosomal misalignment on the metaphase plate, meiotic spindle abnormalities, or mitochondrial dysfunction (aggregation, impaired ATP production), all of which occurred in oocytes of age-matched ad libitum-fed controls. The effects of CR on oocyte quality in aging females were reproduced by deletion of the metabolic regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Thus, CR during adulthood or loss of PGC-1α function maintains female germline chromosomal stability and its proper segregation during meiosis, such that ovulated oocytes of aged female mice previously maintained on CR or lacking PGC-1α are comparable to those of young females during prime reproductive life.

Fig. 1.

CR prevents the aging-related decline in ovulated oocyte numbers. (A) Yield and morphology of oocytes obtained after induced ovulation of 3-mo-old (3M) AL-fed (n = 6), 12-mo-old (12M) AL-fed (n = 12), and 12M CR–AL-fed (n = 6) mice (mean ± SEM; *P < 0.05 versus 3M AL-fed females). (B) Number of in vitro fertilized MII oocytes that developed to blastocysts per induced ovulation cycle per female (n = 11–16 mice per group; mean ± SEM; *P < 0.05 versus 3M AL-fed females). (C) Number of nonatretic immature follicles per ovary in 3M AL-fed, 12M AL-fed, and 12M AL–CR-fed mice (mean ± SEM, n = 9–14 mice per group; *P < 0.05 versus 3M AL-fed females; **P < 0.05).

Fig. 2.

Aging-associated aneuploidy in MII oocytes is prevented by CR. (A) Example of a typical chromosome spread of an MII oocyte containing 20 chromosomes (DAPI staining of DNA shown in white). (B) Incidence of hyperploidy, hypoploidy, and PSCS (and total chromosomal defects from all three endpoints combined) in MII oocytes of 3M AL-fed, 12M AL-fed, and 12M CR–AL-fed females (mean ± SEM, n = 18–23 mature oocytes analyzed per group in each experiment replicated four times using a total of 20–34 mice per group; *P < 0.05 versus 3M AL-fed females; nd, none detected).

Fig. 3.

CR prevents spindle and chromosomal alignment defects in oocytes of aged females. (A and B) Incidence of spindle abnormalities (A) and chromosomal misalignment on the metaphase plate (B) in MII oocytes of 3M AL-fed, 12M AL-fed, and 12M CR–AL-fed mice (mean ± SEM, n = 3–20 oocytes analyzed per group in each experiment replicated four to seven times using a total of four to eight mice per group; *P < 0.05 versus 3M AL-fed females). (C) Representative examples of meiotic spindles in MII oocytes from the indicated mice (n = 22–72 oocytes analyzed per group), after labeling with α-tubulin antibody (green) and counterstaining of DNA with PI (red).

Fig. 4.

CR maintains normal mitochondrial dynamics in oocytes of aged females. (A) Representative mitochondrial distribution in MII oocytes from 3M AL-fed, 12M AL-fed, and 12M CR–AL-fed mice (MitoTracker staining shown in red). (B) Incidence of abnormal mitochondrial aggregation in MII oocytes from 3M AL-fed, 12M AL-fed, and 12M CR–AL-fed mice (mean ± SEM, n = 23–46 oocytes analyzed per group from three independent experiments using 4–11 mice per group; *P < 0.05 versus 3M AL-fed females). (C) Cytoplasmic ATP levels in individual MII oocytes from 3M AL-fed, 12M AL-fed, and 12M CR–AL-fed mice (mean ± SEM, n = 38–145 total oocytes analyzed per group from five to seven independent experiments using a total of 5–21 mice per group; *P < 0.05 versus 3M AL-fed females).

Fig. 5.

Loss of PGC-1α improves oocyte yield and quality in aging females. (A) RT-PCR analysis of Pgc-1α and Pgc-1β mRNA levels in isolated MII oocytes of 3M AL-fed wild-type (WT) mice, 12M AL-fed or CR–AL-fed WT mice, or 12M AL-fed or CR–AL-fed Pgc-1α–null mice (Actin, control gene for sample loading; size, molecular size marker; Ov, adult ovary RNA used as a positive control; −RT, RT-PCR analysis of ovary RNA without reverse transcriptase as a negative control). (B−E) Effects of PGC-1α deficiency in AL-fed and CR-AL-fed females on oocyte yield following superovulation (B), meiotic spindle formation (C), chromosomal alignment on the metaphase plate (D), and mitochondrial distribution (E). Definitions for D and E are the same as C. Data are the mean ± SEM (n = 20–117 oocytes analyzed per group for each endpoint from three independent experiments using a total of 3–14 mice per group; *P < 0.05 versus all other groups).