Fig. 1: Spermatogenesis defects in mutant mice.

a Overview of process for selecting human MLH1 and MLH3 variants for mouse modeling. gnomAD (https://gnomad.broadinstitute.org) was the source of Allele Frequencies (AF). MMR, mismatch repair; CO, crossover. The SNPs tested are listed in Table S1. b Testis weights of mutants. The Mlh1 and Mlh3 samples were taken from 6 month and 2–3 month old mice, respectively, hence the difference between WT samples. For this and panel C, numbers are p values relative to corresponding WT of that cohort; only genotypes with values <0.05 are shown. Genotype abbreviations: WT, wild-type E268G, Mlh1^E268G/E268G; K618E, Mlh1^K618E/K618E; K618T, Mlh1^K618T/K618T; V326A, Mlh1^V326A/V326A; V716M, Mlh1^V716M/V716M. A1394T, Mlh3^{A1394T/ A1394T}; R93Q, Mlh3^R93Q/R93Q; R1230H, Mlh3^{R1230H/R1230H}. c Epididymal sperm counts. All were from 6-month old mice, except Mlh3^{A1394T/A1394T} and Mlh3^R93G/R93G, which were from 3 month old animals). Controls were from pooled littermates. Genotype abbreviations are as in “b.” d Testis histology. Examples of H&E-stained seminiferous tubule cross sections of homozygotes for the indicated genotypes. Asterisk (*) represents Stage XI–XII tubules with metaphase cells. Arrowheads represent pyknotic or multinucleated degenerating metaphase cells. Red arrows indicate normal metaphases in WT sections, while black arrows indicate abnormal metaphases with or without lagging chromosomes. Scale Bar = 50 μm. e Testis sections immunolabeled by phosphorylated histone H3 (PH3, red), and also TUNEL (green)-stained for apoptosis counterstained with DAPI (for DNA, blue). Mlh1/3 testes are from homozygous mutants. Scale Bar = 50 μm. Genotypes are indicated. f, g Quantification of TUNEL data from samples in “E.” Plotted are overall percentages of TUNEL⁺ cells (f) and also tubules containing >4 TUNEL⁺ cells (g). This number was chosen because no WT tubule sections had >4 TUNEL⁺ cells. There are 2 N/n values: n = Total numbers of tubules sections analyzed for each genotype, and N = number of mice (biological replicates from which the tubules came. At least >25 Ph3⁺ tubules from each mouse/genotype were analyzed. *p ≤ 0.0003. h Fertility trials of homozygous males and control littermates. *p ≤ 0.0001. The X-axis values are in 21-day intervals, which approximates litter intervals for fertile females housed continuously with a male. Number of animals that underwent fertility trials (N) are shown in parentheses. All p-values were from Student’s two-tailed t-test. Data are presented as mean values ± SEM. In b, c, f, g, each plot is defined as follows: Horizontal line in box = median; box width = data points between the first and third quartiles of the distribution; whiskers = minimum and maximum values; ‘+’ sign = mean value. N represents number of biologically independent animals/genotype examined in each experiment.