Fig. 3: Age-dependent subfertility in Mlh1 mutant females and increased aneuploidy in oocytes.

a Litter sizes of mutant females over their reproductive lifespans. Eight-week old wild-type and littermate mutant females were mated with wild-type males, and the number of pups born per litter are plotted. For all panels, the following abbreviations apply to genotypes: E268G, Mlh3^E268G/E268G; K618E, Mlh1^K618E/K618E; K618T, Mlh1^K618T/K618T; V326A, Mlh1^V326A/V326A; V716M, Mlh1^V716M/V716M. Numbers of animals examined for each genotype are shown in parentheses. b Total pups delivered by females during their reproductive lifespan (~2 to 10 months, see Methods). c Follicle counts (all stages; the great majority were primordial follicles) in three-week old females. p-values < 0.05, in comparisons of mutant to WT, are indicated. Dpp, days post-partum. N indicates animals/group. One ovary from each animal was prepared for histological quantification. d Number of total germinal vesicle (GV) stage oocytes collected from superovulated WT and mutant females (3–4 month old). p-values < 0.05 relative to WT (+/+) are shown. Horizontal lines are mean and whiskers are ± Std. dev. e Percent of GV oocytes (shown in d) that matured to metaphase meiosis II (Met II). p-values < 0.05 relative to WT (+/+) are shown. f In situ metaphase chromosome spreads showing abnormalities in mutants. Oocytes were stained to detect centromeres (ACA, red) and DNA (DAPI, gray). Representative confocal z projections are shown. Sister chromatid pairs are numbered and insets represent each sister chromatid pair in MII. The red squares indicate prematurely separated sister chromatid pairs. Scale Bar = 10 μm in main panels, and 2 µm in insets; g Percentage of aneuploid metaphase II oocytes from (E). p-values are shown above bars. h Violin plots of embryo loss estimates in Mlh1 mutants. Females homozygous for the indicated mutations (3–4 mth old) were mated to C57BL/6 J (WT) males, sacrificed at E13.5, and the number of viable embryos scored, as well as ovulation sites on the ovary. The difference between the two were presumed to have died during gestation at various stages and are plotted on the Y axis. The control females (“WT”) were +/+ or heterozygous littermates. p-values are shown and were calculated from the Student’s two-tailed t-test. Box limits in c and d indicate the 25th and 75th percentiles. Whiskers in h extend 1.5 times the interquartile range of the 25th and 75th percentiles; polygons in h represent density estimates of data and extend to extreme values. N values are number of pregnancies examined. Data in b–e, g and h were analyzed using Unpaired Student’s t-test from multiple biological replicates shown as ‘N’.