ARTICLE
Human MLH1/3 variants causing aneuploidy,
pregnancy loss, and premature reproductive aging
Priti Singh1,2, Robert Fragoza3,4, Cecilia S. Blengini 5, Tina N. Tran1, Gianno Pannafino4, Najla Al-Sweel4,
Kerry J. Schimenti1, Karen Schindler5, Eric A. Alani4, Haiyuan Yu 3,6 & John C. Schimenti 1,4✉
Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy
loss. Proper disjunction of homologous chromosomes requires the mismatch repair
(MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can
increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if
MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive
abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR
and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice
via genome editing. We identify seven alleles causing reproductive defects in mice including
female subfertility and male infertility. Remarkably, in females these alleles cause agedependent
decreases in litter size and increased embryo resorption, likely a consequence of
fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that
hypomorphic alleles of meiotic recombination genes can predispose females to increased
incidence of pregnancy loss from gamete aneuploidy.
https://doi.org/10.1038/s41467-021-25028-1 OPEN
1 Dept of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY, USA. 2 Preclinical Modeling Core Lab, Fred Hutchinson Cancer
Research Center, Seattle, WA, USA. 3 Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA. 4 Department of Molecular Biology
and Genetics, Cornell University, Ithaca, NY, USA. 5 Rutgers University, Dept. of Genetics, Piscataway, NJ, USA. 6 Department of Computational Biology,
Cornell University, Ithaca, NY, USA. ✉email: jcs92@cornell.edu
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T
he essential function of meiotic recombination is to drive
pairing of homologous chromosomes, ensuring proper
disjunction of homologs to daughter cells at the first
meiotic division. Failure of a chromosome pair to undergo at least
one crossover per chromosome arm predisposes to formation of
aneuploid gametes that, if fertilized, leads to pregnancy loss.
Approximately 50–70% of early miscarriages are associated with
chromosome abnormalities, with most having aneuploidy, primarily
trisomies1,2. It is well recognized that the incidence of
aneuploidy in abortuses increases with female age and has been
hypothesized to be attributable to non-genetic factors such as
decay of chromosome cohesion3,4. However, there are indications
that parental genetic factors can predispose to generation of
aneuploid conceptuses, and may contribute to cases of recurrent
pregnancy loss (RPL; defined as three or more consecutive
miscarriages)5,6. For example, much attention has focused on the
potential effects of variants/mutations in the synaptonemal
complex protein SYCP3 in human RPL7,8, because SYCP3deficient
female mice experience loss of embryos resulting from
aneuploid oocytes9. However, it is unclear whether, or to what
extent, mutations or deleterious variants in SYCP3 or other genes
cause miscarriages or RPL.
Chromosome mis-segregation during meiosis can occur at
Meiosis I (MI) or Meiosis II (MII), though a lack of recombination
on extra/missing chromosomes has been associated with
inefficient crossover maturation before MI10. The resulting
gametes, if fertilized, can lead to aneuploid embryos that are
almost universally incompatible with viability, thus causing
miscarriage. Several genes are required for crossover (CO)
recombination (and thus fertility) in mice, including the mismatch
repair (MMR) proteins MLH1 and MLH3, which form a
heterodimeric endonuclease required to resolve double Holliday
junction recombination intermediates11–19. Null alleles of Mlh1
or Mlh3 cause meiotic arrest and sterility in mice because they are
needed for ~90% of all crossovers (those known as interferencedependent
Class I crossovers)11,20–25. The absence of COs leads
to univalent chromosomes that fail to align properly while
attached to microtubules at the metaphase plate, thus triggering
the spindle assembly checkpoint (SAC) and blocking anaphase
progression26. However, recombination defects affecting bivalent
formation of only one or a few chromosome pairs is inefficient at
triggering the SAC, allowing such gametes to survive and thus
predisposing to aneuploidy27–30.
We hypothesized that human population variants in MLH1/3
might cause defects in chromosome segregation during MI or MII,
leading to infertility, miscarriage, and/or developmental defects.
To test this and gain a comprehensive understanding of the
functional impact of such variants, we systematically evaluated
human missense single nucleotide polymorphisms (SNPs) curated
in the ExAC and (subsequently) gnomAD databases31,32. Minor
alleles predicted to be deleterious in silico by various algorithms,
and which deviated from normal function in biochemical assays
or genetic assays in yeast, were selected for modeling in mice.
Most of the mutant mouse models were fertile but exhibited
crossing over defects that led to age-related decreases in female
fecundity, elevated aneuploidy, and consequent increases in
pregnancy loss. These results have implications especially for older
couples, or those experiencing RPL, who may be at increased risk
for having conceptuses or children with trisomies as a result of
bearing variants in these recombination genes.
Results
Strategy and selection of candidate pathogenic MUTL homolog
variants. To test the possibility that segregating infertility variants
of MLH1 or MLH3 exist in human populations, we identified
potentially pathogenic missense SNPs in these genes using
methods and criteria outlined in Fig. 1a, including: (1) functional
prediction algorithms scoring the variant as deleterious; (2) has
an allele frequency (AF) within the range of 0.02 to 2% in any
gnomAD-listed population; (3) disruptive to binary proteinprotein-interactions
in yeast two-hybrid (Y2H) assay; or (4)
causes an MMR and/or recombination phenotype in baker’s yeast
(S. cerevisiae) when the orthologous amino acid is altered. These
steps were used to prioritize a subset of MLH1 and MLH3 variants
for subsequent modeling in mice. In particular, steps 3 and 4
were explored as a means to potentially improve the success rate
of identifying functionally consequential infertility variants for
mouse modeling, compared to our previous studies that utilized
only computational predictions33,34.
Disease-associated mutations in MLH1 often function molecularly
by disrupting corresponding protein-protein
interactions35,36. As such, we searched for all potentially
functional MLH1 missense variants that matched our defined
0.02% < AF < 2% criteria range. These MLH1 variants were
introduced into Y2H bait and prey vectors, then tested for
whether they disrupted known interactions with 8 proteins that
were available as full-length clones in the hORFeome v8.1 or the
Mammalian Genome Collection (MGC). Overall, 13 MLH1
variants were tested by Y2H across 68 total SNP-interaction pairs.
We then further filtered these variants for SNPs that scored as
deleterious across multiple functional prediction algorithms. This
resulted in five MLH1 predicted deleterious variants that
disrupted 27 out of 37 (73%) of their corresponding SNPinteractions
pairs (Table 1; Supplementary Data 1).
For MLH3, we modeled eleven human SNPs in yeast by
constructing strains bearing the cognate amino acid (AA) changes
in the endogenous MLH3 gene, then measuring MMR using a lys2
frameshift reversion assay37. Most of the AAs are located in
conserved endonuclease and ATP binding domains (Table 2;
Supplementary Fig. 1). To measure meiotic crossing over, these
strains were mated to an mlh3Δ strain so that the diploids
contained fluorescence markers separated by ~20 cM on chromosome
VIII38. The diploids were sporulated to identify, in complete
tetrads, presence or absence of crossovers (Supplementary Fig. 2).
Three of the MLH3 SNPs (rs781779034, rs138006166 and
rs781739661) had null-like or intermediate phenotypes for
MMR, and null-like crossing over defects (Tables 1 and 2;
Supplementary Table 1). Additionally, all three were classified as
deleterious by functional prediction algorithms (Table 1). Overall,
nine of the eleven alleles modeled in yeast conferred null-like or
intermediate phenotypes in either or both tested metrics (Table 2).
Mouse modeling of nsSNPs and reproductive phenotypes.
Based on the bioinformatic, biochemical and genetic data
described above, we initially modeled five MLH1 and three MLH3
nsSNPs in mice via CRISPR/Cas9-mediated genome-editing
(Table 1, Supplementary Fig. 2). These eight mutant mouse
lines with ‘humanized’ alleles were phenotyped in several ways,
beginning with male reproductive parameters. Two mutants had
obvious phenotypes. Mlh3R1230H/R1230H males exhibited a ~4 fold
reduction in testes size, and an absence of epididymal sperm
(Fig. 1b, c). Consistent with the absence of sperm, histological
sections of mutant testes revealed a lack of haploid spermatids,
and the most advanced spermatocytes appeared to be at meiotic
metaphase I or anaphase I (Fig. 1d), reminiscent of null animals
for both Mlh1 and Mlh311,14,21. The phenotypes of Mlh1K618T/
K618T males were less severe; their testes weighed 80% of WT, and
there were ~35% fewer epididymal sperm (Fig. 1b, c). Histology
also revealed a substantial number of spermatocytes arrested in
metaphase I (Fig. 1d). Fertility trials of homozygous mutant males
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confirmed that Mlh3R1230H/R1230H males were completely sterile,
whereas Mlh1K618T/K618T males were subfertile, producing 40%
fewer pups over a duration 10 months of fertility trials (Fig. 1h).
In contrast to Mlh1K618T and Mlh3R1230H, there were no obvious
reproductive defects in males homozygous for the remaining 6
alleles that were modeled. Sperm counts were normal and litter
sizes sired by these mutant males were not lower than controls
(Fig. 1c, h). Testis weights were also normal, though modestly
lower in Mlh3R93G/R93G homozygotes.
Lack of COs between homologs at meiotic metaphase I in Mlhnull
mutants causes a failure of proper alignment at the
metaphase plate, leading to anaphase block11,20–25. Despite the
absence of reproductive defects in the remaining 6 mutant alleles,
testis sections revealed abnormal pyknotic-appearing metaphase
spermatocytes with disorganized metaphase plates and misaligned
chromosomes (Fig. 1d; explored in more detail below).
This was more directly assessed by TUNEL staining of
seminiferous tubule cross-sections positive for the metaphase
marker phospho-histone H3 (Ser10)39 which showed marked
increases in apoptotic cells compared to WT (Fig. 1e–g). Confocal
analysis readily revealed misaligned chromosomes at the
metaphase plates of MI spermatocytes in the less severe mutants
(Supplementary Fig. 3). These data suggested that the allelic
variants caused severe-to-subtle defects in chromosome
ATPase
Trans
ducer
Mutl_CLinker
• 68 SNP-interaction pairs tested
Y2H screen
WT
SNP1
SNP2
SNP3
SNP20
...
• 27 disrupted interactions
d
*
*
*
*
*
*
*
PH3/TUNEL/DAPI
WT
EG
Mlh1K618E
Mlh1K618T
Mlh1V326A
Mlh1V716M
Mlh1E268G
Spo11-/-
Mlh3A1394T
e
+
+
+
+
+
+ +
+
+
+
+ +
+
+
+ +
+
+
+ +
Sperm(x107
/ml)
0
1
2
3
4
5
Mlh1Mlh3
40
100
120
20
Testisweight(mg)
60
80
WT
E268G
K618E
V326A
V716M
A1394T
Mlh1Mlh3
WT
R93Q
R1230H
K618T
0
5
10
15
20
25
30
TUNEL+
cells/Ph3+
tubule
Mlh1
E268G
Mlh1
K618E
Mlh1
V326A
Mlh1
V716M
Mlh3
A1394T
WT
Mlh1
K618T
f
g
0
10
20
30
40
50
60
%Ph3+
TUNEL+
tubules
0 2 4 6 8 10
0
20
40
60
80
WT (6)
E268G (3)
K618E (5)
K618T (3)
V326A (3)
V716M (3)
Litters (21 d interval)
Cumulativepups
h
.03
.08
CRISPR mice
Pathogenicity
algorithms
0.02% < AF < 2%
13 SNPs
GnomAD
MLH1 MLH3a
Conserved
in yeast
MMR; CO
assays on
11 variants
3 SNPs
5 SNPs
b
c
<.0001
<.0001
.0005
.0005
*
*
*
*
*
*
*
*
*
*
.003 .003
Mlh3A1394T Mlh3R1230H
Mlh1E268G
Mlh1K618E
Mlh1K618T
Mlh1V326A
Mlh1V716M
WT
Missense SNPs
Mlh1
E268G
Mlh1
K618E
Mlh1
V326A
Mlh1
V716M
Mlh3
A1394T
WT
Mlh1
K618T
Lorem ipsum
R1230H (2)
ATPase endonucleaselinker
WT
E268G
K618E
V326A
V716M
A1394TWT
R93Q
R1230H
K618T
<0.0001
N= 14 4 6 3 12 3 3 5 3 3
N=17 3 4 3 9 3 3 5 3 3
N= 5 4 3 5 4 3 3
N= 5 4 3 5 4 3 3
Mean±SEM)
90 135 70 95 59 26 37
+
+
+
+
+
+
+
=n
gnomADgnomAD
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alignment at the metaphase plate at MI, and/or unrepaired
recombination intermediates, leading to apoptosis.
When this work began, the two K618 variants in MLH1
(K618E and K618T) were (and are still) present in dbSNP as
rs35001569 and rs63750449, respectively. These amino acid
changes are caused by AAG>GAG and AAG>ACG nucleotide
changes, respectively. However, after those mouse models were
created and phenotype analyses were performed (including
female data presented below), the gnomAD database (v2.1) was
revised to indicate that these two variants co-occur (are in phase)
nearly perfectly (963/973 and 963/971 occurrences, respectively),
encoding a K618A AA change as a result. This explains why both
variants are annotated to occur at essentially identical frequencies
in populations (Table 1). We therefore generated an Mlh1K618A
allele and found that both male and female homozygotes are
fertile. Thus, this allele is either a neutral variant or has subtle
defects that may be revealed with more comprehensive studies
such as those described below.
Meiotic recombination defects in mutants. To determine if
defects in crossing-over might underlie the metaphase I
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, Mlh1E268G/E268G; K618E, Mlh1K618E/K618E; K618T, Mlh1K618T/K618T; V326A, Mlh1V326A/V326A; V716M, Mlh1V716M/V716M. A1394T, Mlh3A1394T/
A1394T; R93Q, Mlh3R93Q/R93Q; R1230H, Mlh3R1230H/R1230H. c Epididymal sperm counts. All were from 6-month old mice, except Mlh3A1394T/A1394T and Mlh3R93G/
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&Estained
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.
Table 1 SNPs modeled in mice.
Gene SNP Human allele Benign/Delet. MAF (Population) Functional assay Protein domain
MLH3 rs28756978 R93G None/S,P,R,M 0.076% (EAS) CO- ATPase domain
MLH3 rs781739661 R1230H None/S,P,R,M 0.025% (EAS) MMR- CO- MutL C-ter dimer;
endo. domain
MLH3 rs138006166 A1394T None/S,P,R,M 0.039% (NFE) MMR+/− CO- MutL C-ter dimer;
endo. domain
MLH1 rs63750650 E268G None/S,P,R,M 0.16% (Fin) Y2H (3/8) Transducer domain
MLH1 rs35001569 K618E None/S,P,R,M 0.57% (NFE) Y2H (7/8) Exo1 Interaction domain
MLH1 rs63750449 K618T None/ S,P,R,M 0.57% (NFE) Y2H (8/8) Exo1 Interaction domain
MLH1 rs3550531 K618A None/S,P 0.34%* (overall) ND Exo1 Interaction domain
MLH1 rs63751049 V326A P**/S,R,M 0.11% (SAS) Y2H (4/5) Transducer domain
MLH1 rs35831931 V716M None/S,P,R,M 0.20% (NFE) Y2H(5/8) C-terminal
gnomAD (http://gnomad.broadinstitute.org) allele frequencies (version 2.1.1) correspond to the population with the highest allele frequency (AF).
EAS East Asian, NFE European (non-Finnish), Fin Finnish, SAS South Asian, S SIFT, P Polyphen2, REVEL R, M Mutation assessor, ND no data, MMR mismatch repair, CO meiotic crossing over phenotypes
(+, wildtype, −, null), dimer dimerization, endo endonuclease, “Y2H” yeast two-hybrid disruption, with the values in parentheses indicating the fraction of known interactions with proteins disrupted by
the corresponding MLH1 mutation (see details in Table S1).
*This allele was generated near the end of the study, and consists of a dinucleotide change, each corresponding to one of the nucleotides altered in the K618E and K618T alleles. REVEL, Mutation
Assessor and CADD scores were not obtained for K618A because this allele is not a recognized population variant. gnomAD lists overall AF for K618A, but its NFE-specific AF is likely identical to K618E
and K618T, which almost always co-occur (see text). For functional assays in yeast, see details in Table S2.
**This pathogenicity score is classified as “possibly” damaging.
Table 2 Analysis summary of yeast equivalents to human
MLH3 SNPs for defects in crossing over and mismatch
repair.
Variant SNP ID Yeast Equiv MMR CO Domain
G62R rs761501352 T65R + + ATP binding
R93Q rs781779034 R96Q +/− − ATP binding
R93G rs28756978 R96G ND − ATP binding
P183L rs759067670 P199L +/− − ATP binding
N291K rs767413852 L313K + + Conserved
amino acid
F390I rs61752721 R407I +/− + undefined
R1230C rs746431837 R530C − − Endonuclease
R1230H rs781739661 R530H − − Endonuclease
R1232C rs550698696 R532C +/− + Endonuclease
A1394T rs138006166 A702T +/− − Endonuclease
G1396W rs368876363 G704W +/− +/− Endonuclease
“Variant” refers to amino acid change conferred by the indicated SNP. “Yeast equiv” is the
orthologous amino acid in Saccharomyces cerevisiae. MMR mismatch repair phenotype, CO
crossing over phenotype. For these phenotypes, “+” is WT level, “−” is null, and “+/−” is
intermediate. ND no data. See Supplementary Table 1 for the complete dataset.
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aberrations, we immunolabeled pachytene spermatocyte chromosome
spreads to quantify MLH1 foci, a proxy for “Type 1”
crossovers (COs), which are subject to interference and constitute
~90% of all CO40. In the two most severe mutants Mlh3R1230H/
R1230H and Mlh1K618T/K618T, chromosomes exhibited normal
homolog pairing and synapsis as reported for knockouts11,14,21,
but had markedly reduced MLH1 foci (Fig. 2a, b; 2.9 and 7.7 per
cell, respectively, vs. 21.4 foci in WT). Such a severe reduction in
‘obligate’ class I COs might either trigger apoptosis at metaphase
I, or prevent zygotic progression after fertilization21,26.
The Mlh1V716M and Mlh1E268G mutants, though less affected,
also had significantly fewer total MLH1 foci per spermatocyte on
average. Since even small reductions in CO recombination might
lead to some chromosomes lacking an obligate CO (chiasma), we
quantified MLH1 focus-deficient chromosomes. Five of the seven
mutants examined exhibited a significant increase in MLH1
focus-deficient chromosomes (Fig. 2c). This suggests an increase
in spermatocytes bearing one or more achiasmate chromosomes.
Consistent with this hypothesis, Mlh1K618T/K618T spermatocytes
exhibited an average of ~2 fewer metaphase I chiasmata than
Mlh1K618T/K618T
Mlh1+/+
MLH1/SYCP3
Mlh1K618T/K618T
Mlh3R1230H/R1230H
WT
MLH1foci/cell
WT
0
5
10
15
20
#homologswithout
MLH1foci
a
b c
d
*
*
*
*
*
*
*
*
0.02
+
+ +
+
Chiasmata Bivalents
p=0.0012 p=0.022
14
16
18
20
22
24
26
Number/cell
N= 8 3 2 5 3 5 4 4 N= 8 3 2 5 3 5 4 4
E268G
K618E
V326A
V716M
A1394T
K618T
R1230H
Mlh1Mlh3
WT
E268G
K618E
V326A
V716M
A1394T
K618T
R1230H
Mlh1Mlh3
+/+ +/+K618T K618T
e
6 month old males
0
5
10
15
20
25
30
2776 30 83 46 32 71 100 = n
57 30 39 45 43 24 40 42 = n
+
+
+
+
+
+
+ +
Fig. 2 Recombination in mutant spermatocytes. a MLH1 immunolocalization in spermatocyte nuclei. Shown are pachytene spermatocyte chromosome
surface spreads from 6 month old males, immunolabeled with SYCP3 (red) and MLH1 (green). White arrows indicate MLH1. Note weak and nearly absent
staining in the mutants. b MLH1 focus quantification in spermatocytes. *p-value <0.0001. Others do not have p-values < 0.05 for decreased foci. Over 30
cells from at least 3 animals were quantified, except for Mlh3R1230H, where 27 cells from two animals were scored. c Violin plots illustrating the
chromosome pairs in a spermatocyte nucleus lacking an MLH1 focus. White circles show the medians; box limits indicate the 25th and 75th percentiles;
whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme
values. For mutants with more MLH-deficient pairs, *p-value <0.0001 unless otherwise indicated; comparisons to WT with p-values >0.05 are not shown.
d Example of spermatocyte metaphase spread from the Mlh1K618T mutant and control. e Quantification of chiasmata and bivalents in Mlh1K618T and WT.
Twenty or more cells from three animals were analyzed. Scale Bar = 5 μm. Data in b and e are plotted as follows: Boxes indicate data points between the
first and third quartiles of the distribution, whiskers show minimum and maximum values, black horizontal lines and ‘+’ sign indicate the median and mean
values respectively. Data in b, c and e were analyzed using Unpaired Student’s t-test from the total number of spermatocytes (shown as ‘n’) from multiple
animals indicated as ‘N’.
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controls, leading to a significant decrease in bivalent chromosomes
(increase in univalents; Fig. 2d, e).
Age-dependent decline in reproductive capacity of Mlh1/3
mutant females associated with increased oocyte aneuploidy. In
Mlh1 or Mlh3 knockouts, females are sterile because their oocytes
cannot form a normal spindle and chromosomes do not properly
align to the meiotic metaphase I plate30,41. This near complete
absence of bi-oriented, CO-tethered bivalents at diakinesis can
activate quality control mechanisms, namely the Spindle
Assembly Checkpoint (SAC), causing meiotic arrest26,28. The
SAC appears to be more stringent in spermatocytes than
oocytes27,29,42,43, thus eliminating defective spermatocytes
whereas oocytes attempt to complete gametogenesis. To test
potential impacts on fertility of females bearing the humanized
Mlh1/3 alleles, we evaluated fecundity of the Mlh1E268G,
Mlh1V326A, Mlh1K618E, Mlh1K618T, and Mlh1V716M mutants.
Interestingly, litter sizes of all mutant females declined over time
compared to controls (Fig. 3a) and overall, they had only ~ half of
the total offspring as controls over the 36 weeks they were housed
with WT sires (Fig. 3b). The overall shortfall became dramatic
when mutant dams were 5–6 months old, and this shortfall was
largely attributable to the following: (i) decrease in fecundity after
the first ~six litters (Fig. 3a); (ii) longer intervals between litters;
and (iii) earlier cessation of litters, despite evidence for continued
ovulation (Supplementary Fig. 4). Collectively, these resulted into
f
edc
a b
%oocytestoMII
Mean±SEM
GVoocytes/female
60
40
20
Follicles/ovary(21dpp)
1000
1600
2000
Cumulativepups
Mean±SEM
50
100
21d intervals
1 3 6 9 12
V326A (4)
V716M (4)
WT (6)
E268G (5)
K618E (4)
K618T (3)
Mlh1 Mlh1
h
%Lost/resorbed
embryos
60
40
20
.04
.03
.08
.8
.03
N=6 6 4 5 3 3
Mlh1
Mlh1
%aneuploideggs
Mean±SEM
40
20
0
g
.04
.72
.08
.06
.7
Mlh1
60
40
20
80
100
Mlh1
TotalPups
Mean±SEM
0
50
100
.008 .01 .01 .01 .008
N 3 2 3 3 3 3 N 3 3 3 4 3 3
1200
0
4 4 3533
4 4 3445 =N
=N
N 4 3 3 3 4 3
0.0005
0.007
0.01
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1.6–2-fold fewer pups from all 5 mutant dams compared to WT
(Fig. 3b). The most severe phenotypes were displayed by
Mlh1K618T and Mlh1V716M dams. After their first five litters, these
dams produced 2.2- fold and 6.5-fold fewer pups than WT,
respectively, (22 ± 6.1; 7.7 ± 2.6; 49.8 ± 8 SEM).
Whereas homozygotes of the aforementioned five alleles
showed age-related decrease in litter sizes, only Mlh1K618T/K618T
females had dramatically fewer (~half) follicles (combined
primordial and developing follicles, Fig. 3c) at three weeks of
age. While we did not perform longitudinal studies on the
Mlh1K618A allele, female homozygotes were fertile. In preliminary
crosses, two young homozygous females had apparently normal
litter sizes—an average of 7.5 pups/litter (N = 5 litters)—when
crossed to heterozygous males.
It is possible that in the Mlh1K618T mutants with reduced
follicles at three weeks of age (and which also had the greatest
reduction in COs in males), oocyte loss was triggered by failure to
repair CO-designated DSBs, and subsequent activation of the
meiotic DNA damage checkpoint44. Alternatively, the smaller
litter sizes from mutant dams might be attributable decreased
COs and thus increased aneuploidy in ovulated eggs with age,
although it is unclear how this would occur. An increase in
aneuploidy could lead to increased embryonic lethality as with
Sycp3 null females9.
To test the hypothesis that reduced litter sizes are attributable
to aneuploidy-induced embryo loss, we first examined the
fraction of aneuploid eggs produced by mutant females
(3–4 months of age). Following hormone stimulation, oocytes
were matured in vitro to MII, then spread to visualize
chromosomes. All mutants examined except Mlh1K618T and
Mlh1V716M, both of which had smaller ovarian reserves (see
above), contained similar numbers of prophase I arrested oocytes
as WT females (Fig. 3d). About 70% of the oocytes from all
genotypes matured to MII after 15 h (Fig. 3e), although
maturation, as marked by polar body extrusion, was ~20% less
efficient for the Mlh1K618E and Mlh1V326A genotypes. Next, we
evaluated the ability of these females to produce euploid eggs (20
pairs of sister chromatids), and then we quantified the number of
sister chromatid pairs in eggs from each mutant. The Mlh1E268G,
Mlh1K618T and Mlh1V326A alleles presented marked increases in
aneuploid eggs, detected as abnormal number of pairs of sister
chromatids (Fig. 3f and insets, g; 20.9 ± 7.1%, 25.8 ± 12.9% and
12.7 ± 2.3, respectively, compared to 4.5 ± 2.7% in WT). Moreover,
eggs from Mlh1E268G and Mlh1V326A alleles presented an
abnormal phenotype of prematurely separated sister chromatids
(Fig. 3f insets, outlined red).
We next explored whether the high levels of aneuploidy in
eggs from mutant females led to increased embryonic death of
embryos. Control and mutant females that were 3–4 months old
(similar to the age of females used in the ploidy experiments)
were mated with wild-type males, dissected at E13.5,
and the numbers of viable embryos and corpora lutea (CL;
reflecting the number of ovulated oocytes) were counted. The
fraction of embryonic lethality was inferred as: [CL-embryos]/
CL45. Whereas lethality was only 3.4% in WT mothers, it was
several fold higher in the mutants, ranging from 11.4% in
Mlh1V326A to 34.5% in Mlh1E268G (Table 3). These results suggest
that mutations in MLH1/3 can elevate the incidence of aneuploid
oocytes, leading to a corresponding increase in spontaneous
pregnancy loss.
Discussion
The process of meiotic recombination, specifically crossing over,
is essential for fertility and the production of viable, healthy
offspring. A severe reduction in COs will prevent proper alignment
of meiotic chromosomes at the metaphase I plate, leading to
arrest or failed fertilization. However, if only one or a very few
chromosomes lack a chiasma, viable gametes can still form but
will be prone to mis-segregation of parental homologs; this can
lead to aneuploid embryos. A recent study of recombination in
human fetal oocytes revealed that having 10–20% fewer COs
(MLH1 foci) overall was associated with such oocytes having 1–2
chromosomes completely lacking a CO46. Given that the most
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, Mlh3E268G/E268G; K618E, Mlh1K618E/K618E; K618T, Mlh1K618T/K618T; V326A, Mlh1V326A/
V326A; V716M, Mlh1V716M/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’.
Table 3 Embryo loss during development.
Genotype Pregs CL (Avg) Embryos (Avg) % embryo loss
WT 6 59 (9.8) 57 (9.5) 3.4
Mlh1E268G 6 58 (9.7) 38 (6.3) 34.5
Mlh1K618E 3 37 (12.3) 29 (9.7) 21.6
Mlh1K618T 5 46 (9.2) 39 (7.8) 15.2
Mlh1V326A 4 35 (8.8) 31 (7.8) 11.4
Mlh1V716M 3 33 (11) 23 (7.7) 30.3
All mutant genotypes are homozygous. Pregs pregnancies dissected, CL Corpora lutea, Avg
average. The embryos counted were viable when dissected at E13.5. The % embryo loss = (#CL#embryos)/#CL
* 100.
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common cause of miscarriage (~60%) is embryonic aneuploidy,
primarily trisomies1, it is of interest whether the frequency of socalled
“exchangeless” chromosomes is genetically controlled.
There are indications that parental genetic factors can predispose
to generation of aneuploid conceptuses, and may contribute to
cases of RPL5,6. In this respect, variants impacting genes important
for recombination, including the MUTL homologs, are
potential candidates for influencing aneuploidy rates.
SNPs in MLH1 and MLH3 have been statistically associated
with male infertility in some studies focusing on candidate genes
(i.e., not GWAS), but there have not been validation studies47–49.
For female fertility, GWAS for phenotypes such as age to
menopause, polycystic ovary syndrome, and endometriosis have
been performed, but only a few associations have been reported
for genes involved in oogenesis per se50. In this study, we focused
on human Variants of Unknown Significance (VUS) in MLH1/3,
with the goal of determining possible effects on fertility. Because
our earlier studies of VUS in essential fertility genes indicated
that functional prediction algorithms alone overpredicted
deleteriousness33, we applied additional functional criteria to
select variants for mouse modeling, namely, studies of analogous
mutations in yeast or disruption of protein-protein interactions
in Y2H assays. Given that most of the alleles reported here
that exhibited defects in these assays caused phenotypes in
mice, we believe that this approach is valuable for prioritizing
experimentation.
In our opinion, the most intriguing result from this study is
that female fecundity in several mutants declined with age and
reproduction ceased prematurely (Fig. 3a, b), despite having no
apparent shortfall (compared to WT) in the ovarian reserve at
wean age (Fig. 3c). One possibility is that after wean age
(~3 weeks), oocyte attrition accelerated in the mutants, such that
they exhausted their ovarian reserve. However, histology of these
older females revealed numerous corpora lutea, indicative of
continued ovulation in these aged females (Supplementary Fig. 5).
Future experiments aimed at quantifying the reserve, numbers of
ovulated eggs, the ability of these eggs to become productively
fertilized, and other markers of ovarian aging could reveal the
basis of this phenomenon.
An alternative explanation is that the quality of oocytes
recruited later in life might be lower than at earlier stages in the
mutants. We can only speculate as to the nature of such quality
differences, but they might be related to increased aneuploidy
caused by prolonged periods in primordial follicles with achiasmate
chromosomes. It is also possible that these mice might
represent an example of the “production line” hypothesis that was
originally proposed as an explanation for increased aneuploidy in
women as they age51. This hypothesis posits that eggs are ovulated
in the order that they originally proceed through meiosis in utero,
and that the “later” oocytes suffer from deficient recombination,
and thus higher aneuploidy at MI. While this hypothesis has been
refuted in humans52, there is evidence that in mice, the first
oogonia to enter meiosis are the first to be ovulated53. Nevertheless,
there is no evidence relating crossover rates to time of
meiotic entry. Interestingly, a similar phenomenon was observed
in Sycp3 null mice, where an age-correlated decrease in litter sizes
was attributable to increased embryonic death from aneuploidy9.
Additionally, a small scale study implicated mutations in SYCP3 as
being responsible for RPL in two women7. It has been hypothesized
that women with smaller ovarian reserves have an increased
chance of having aneuploid abortuses54. In future work, our
models may be useful in exploring this possible relationship in
aging mice, and to better understand sexually dimorphic consequences
upon gamete quality and checkpoint mechanisms.
Inherited mutations in MLH1 can cause Lynch Syndrome,
sometimes referred to as hereditary non-polyposis colorectal
cancer (HNPCC), although patients are also subject to a variety of
cancers including endometrial55. Lynch Syndrome exhibits a
dominant mode of inheritance, and the tumors are characterized
by microsatellite instability (MSI). Tumor initiation is attributable
to either epigenetic silencing or mutation of the normal MLH1
allele inherited from one parent55–57. Mice null for Mlh1 exhibit
MSI, increased morbidity (moribund at an average age of
7 months), and develop lymphoma and various types of tumors
including gastrointestinal58. However, we did not observe an
increased incidence of tumors in any lines or microsatellite
instability in the mutants tested (data not shown). Mutations in
MLH3 are not associated with Lynch Syndrome, but there is one
report that null mice are predisposed (50%) to gastrointestinal
tumors with a long latency59. Although the orthologous yeast
mlh3 alleles have MMR defects (Table 1), we did not observe
tumors or premature morbidity in our Mlh3 mouse mutants.
However, we did not age them beyond 1 year. It is also possible
that for both sets of models (Mlh1/3), that the genetic background
we used was not as tolerant to transformation (at least before 1
year of age) as those in published studies. Because of these
caveats, we cannot conclude that these alleles do not cause Lynch
Syndrome in humans.
As mentioned in the Results, when this project began, we
selected the two variants at K618 (K618E and K618T) that remain
present in dbSNP as rs35001569 and rs63750449, respectively.
However, after the mouse models were created, the gnomAD
database was revised to indicate that these two variants co-occur
(are in phase), encoding a K618A amino acid change at essentially
identical frequencies in populations (Table 1). The question
remains as to whether the MLH1K618E and MLH1K618T exist at all
in populations, or at very low frequencies (approximately 100fold
lower based on the numbers cited above). Notably, the
MLH1K618E and MLH1K618T alleles are listed in the ClinVar
database, and reported in patient samples60–62. It is unclear if the
three alleles arose independently, or if they were derived from one
another. Regarding the MLH1K618A allele and its relationship to
Lynch Syndrome, which is somewhat controversial, a recent large
study found no evidence for this allele’s involvement, and thus
concluded that MLH1K618A is benign63. Interestingly, a computational
and experimental study of MLH1 variants suggested that
many variants that cause Lynch Syndrome do so by thermodynamically
de-stabilizing the MLH1 protein, leading to
increased degradation64. Abildgaard and colleagues64 tested four
of the alleles modeled here (MLH1E268G, K618T, K618A and V716M),
and each were classified as being “likely benign” by virtue of the
proteins having substantially normal stability.
In sum, these studies indicate that variants in the MUTLγ
(MLH1/3) complex genes can have subtle-to-severe effects on
gametogenesis that may go unnoticed without rather detailed
phenotyping, including aging studies. Homozygosity for most of
the alleles did not cause infertility or drastic reductions in ovarian
reserve or sperm counts, although it is conceivable that some of
these alleles in trans to a null allele, or in the context of a
variant in the other member of the MUTLγ complex, could
cause synthetic phenotypes. Nevertheless, the phenotypes of
increased aneuploidy in oocytes, and reduced reproductive lifespan
of females are highly relevant to the human condition,
and thus these mouse models can be valuable for assessing risk
at an early age in women, and to dissect the biology of these
phenomena.
Methods
Construction of baker’s yeast strains to measure meiotic crossing over and
DNA mismatch repair. The SK1 background baker’s yeast strain EAY3255 was
constructed for the analysis of MMR and meiotic crossing over phenotypes65.
These and all yeast strains generated/used are listed in Supplementary Table 2.
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EAY3255 contains a spore autonomous fluorescent protein (Tomato) marker
located near the centromere on chromosome VIII38 and a lys2::InsE-A14 cassette to
measure reversion to Lys+ 37. mlh3 alleles cloned into pEAI plasmids (mlh3X::KANMX)
were transformed into EAY3255 by digesting each plasmid with
BamHI and SalI to generate an mlh3-X::KANMX fragment. Approximately 1 μg of
each digested plasmid was mixed with 40 μg of salmon sperm carrier DNA
(Invitrogen, San Diego, CA, boiled just before addition) and added to ~108 yeast
that had been grown to mid-log in yeast extract-peptone-dextrose (YPD) media.
The cell-DNA mixture was suspended in a solution containing 0.1 M lithium
acetate, 10 mM Tris 8.0, 1 mM EDTA, 40% polyethylene glycol66. The resulting
mixture was incubated for 40 min at 42 °C then allowed to recover overnight in
5 ml of YPD media and then plated onto a plate containing geneticin (G418;
Invitrogen) at 200 μg/ml. At least two independent transformants for each genotype
(verified by DNA sequencing) were made.
EAY3255 and derivatives were used to measure the effect of mlh3 mutations on
lys2::InsE-A14 reversion rate (see below). To measure meiotic crossing over, strains
from the EAY3255 background were each mated to EAY3486, an SK1 mlh3Δ strain
containing the m-Cerulean fluorescence marker located at THR1 on chromosome
VIII, ~20 cM away from the Tomato marker present at CEN8 in EAY3255.
Diploids were selected on yeast synthetic minimal media lacking tryptophan and
leucine and maintained as stable strains. Diploids were then patched onto YPD
media for growth for two days at 30 °C. Meiosis was induced upon patching the
diploid strains from YPD onto sporulation media (1% potassium acetate; 0.1%
yeast extract; 0.05% glucose; 0.021% complete amino acids (2% Bacto Agar (Difco))
and incubated at 30 °C for 48–72 h prior to analysis. Wild-type strains carrying the
fluorescent protein markers used to make the above test strains were obtained from
Scott Keeney38.
Spore autonomous fluorescent protein expression assay to measure meiotic
crossing over. Tetrads obtained from EAY3255/EAY3486 background diploids
were treated with 0.5% NP40 and briefly sonicated before analysis by fluorescence
microscopy. Tetrads were analyzed using a Zeiss AxioImager M2 microscope
equipped with RFP and CFP filters. At least 250 tetrads for each mlh3 allele were
counted to determine the % tetratype (two spores show the parental genotype and
two show a configuration consistent with a crossover event between the Tomato
and m-Cerulean markers located on Chromosome VIII; Supplementary Table 1).
Two independent transformants were measured per allele, measured on at least two
days with a similar amount analyzed to avoid batch effects. A statistically significant
difference (p<0.002) from wild-type and mlh3Δ controls, based on χ2
analysis (Pearson χ2 contingency test, with a Bonferonni correction for 26 comparisons),
was used to classify each allele as exhibiting a wild-type (+), intermediate
(+/−, +/−), or null (-) phenotype.
lys2A14 reversion assay to monitor MMR. The EAY3255 derived haploid strains
described above were analyzed for reversion to Lys+. Briefly, strains described in
Supplementary Table 2 were freshly struck from frozen stocks and grown in
complete synthetic minimal media to ~2 mm in diameter colonies (~3 days growth
at 30 °C) and then individual colonies were inoculated in 3 ml of yeast synthetic
minimal media and grown overnight at 30 °C (~16 h). Overnight cultures were
plated with appropriate dilutions onto plates with complete synthetic minimal
media and synthetic minimal media lacking lysine to measure lys2::insE-A14
reversion. Colonies were counted after 3 days of incubation at 30 °C. Rates of
lys2::insE-A14 reversion were calculated as μ=f/ln(N·μ), where f is reversion frequency
and N is the total number of revertants in the culture37. 11 to 16 independent
cultures were analyzed for each mutant allele as well as wild-type and
mlh3Δ controls. Two independently constructed transformants for each allele were
analyzed on at least two days to avoid batch effects, with a similar number of
repetitions performed each day. Reversion rates (Supplementary Table 1) were
measured and the median rate for each genotype was normalized to the wild-type
median rate (1X) to calculate fold increase. Alleles were classified as wild-type (+),
intermediate (+/−), or null (-) based on the 95% confidence intervals (CI)67. If the
95% CI for the allele overlapped with the 95% CI for one of the controls (wild-type
or null) they were considered statistically equivalent to the respective control.
Variant selection for MLH1. To select MLH1 SNPs for cloning and subsequent
Y2H testing, we filtered gnomAD v2.1 for missense mutations with overall AF
between 0.02% and 2%. 15 missense SNPs fell within this range, of which 13 were
successfully cloned and tested by Y2H.
Generating mutant MLH1 clones. Clones for MLH1 and all Y2H-tested interaction
partners were obtained from hORFeome v8.168 or v5.169. The MLH1 alleles
were generated by site-directed mutagenesis. Primers for mutagenesis were
designed using the webtool primer.yulab.org. To minimize sequencing artifacts,
PCR was limited to 18 cycles using Phusion polymerase (New England Biolabs,
M0530). Mutagenesis PCR product was then digested overnight using DpnI (New
England Biolabs, R0176) followed by bacterial transformation into competent cells.
Single colonies were recovered on LB agar plates containing spectinomycin. Four
colonies were picked per mutagenesis attempt and then were validated to contain
the mutation of interest through Sanger sequencing using primers designed to
cover the entire MLH1 sequence.
Profiling disrupted protein interactions through Y2H. Wild-type and successfully
mutated MLH1 clones were transferred into Y2H vectors pDEST-AD and
pDEST-DB by Gateway LR reactions then transformed into MATa Y8800 and
MATα Y8930, respectively. Testable Y2H interaction partners were identified by
surveying a Y2H reference interactome comprised of interactions reported in four
publications70–73. All ORFs corresponding to interactions with MLH1 were then
selected for Y2H testing. MLH1 interaction partners, transformed into MATa
Y8800 and MATα Y8930 were then mated against AD- and DB-MLH1 transformed
yeast of the opposite mating type in a pairwise orientation by co-spotting
yeast inoculants onto YEPD agar plates. To screen for autoactivators, DB-MLH1
yeast cultures were also mated against MATa yeast transformed with empty
pDEST-AD vector. Mated yeast were incubated overnight at 30 °C and then
replica-plated onto dropout Synthetic Complete agar media lacking leucine and
tryptophan (SC-Leu-Trp) to select diploid yeast. After overnight incubation at
30 °C, diploid yeast were then replicate-plated onto dropout SC agar media lacking
leucine, tryptophan, histidine, and supplemented with 1 mM of 3-amino-1,2,4triazole
(SC-Leu-Trp-His+3AT). Diploid yeast were also replica-plated onto SC
agar media lacking leucine, tryptophan, and adenine (SC-Leu-Trp-Ade). After
overnight incubation at 30 °C, plates were replica-cleaned and incubated again for
three days at 30 °C. An interaction was scored as disruptive only if (1) mutant
MLH1 reduces growth by at least 50% relative to its corresponding wild-type
interaction, and (2) neither wild-type nor mutant DB-MLH1 scored as an
autoactivator.
Production of ‘humanized’ mouse-lines and mouse breeding. All ‘humanized’
alleles were generated by CRISPR/Cas9-mediated genome editing33,74. In particular,
high-quality gRNA seed sequences were selected using the Benchling
(https://benchling.com) guide design tool, and sgRNAs were produced via cloningfree
in vitro transcription75. Cas9 protein, sgRNA and single stranded oligodeoxynucleotides
(ssODN) bearing the desired sequence changes (oligos listed in
Supplementary Table 3) were microinjected into single cell zygotes produced from
matings between FVB/NJ and B6(Cg)-Tyrc-2J/J) inbred mice. Correctly edited
founder (F0) animals were backcrossed to B6(Cg)-Tyrc-2J/J for two generations,
then maintained in a mixed strain background (C57BL/6 J x FVB/NJ).
Fertility tests and embryo loss. Control and experimental animals (8–10 weeks
old) were housed with age-matched fertile mates (C57BL/6J) for up to 10–12 months
of age. Litter sizes were determined by counting pups on the day of birth.
For embryo loss analyses, homozygous females (12–16 weeks old) were mated
to WT males and the presence of a copulation plug in the morning was recorded as
0.5 dpc. Females were sacrificed at 13.5 dpc for counting of both implantation sites
and corpora lutea (CL; indicating the numbers of ovulated oocytes). Graphs and
statistical analyses were performed with GraphPad Prism5.
Ovary histology and follicle quantification. Ovaries were collected from 3-week
females and were fixed in Bouin’s for 24 h, washed in 70% ethanol, paraffin
embedded, and further serial sectioned at 6 µm followed by staining with hematoxylin
and eosin (H&E). Every fourth section was counted under light microscopy.
Though we reported the combined number of follicles in Fig. 3, we maintained
records of the follicle subtypes (e.g., primordial, primary, secondary, antral) and
can provide them upon request.
Surface spread preparation and immunocytology. To prepare prophase I surface
spreads76, mouse seminiferous tubules were incubated in a buffer consisting of
30 mM Tris pH7.2, 50 mM sucrose, 17 mM trisodium dehydrate, 5 mM EDTA,
0.5 mM DTT, 0.1 mM PMSF, pH8.2-8.4) at room temperature for 1 h. Small sections
of testis tubule were dissected in 100 mM sucrose and spread onto slides
coated with 1% paraformaldehyde and 0.15% Triton X, then incubated in a humid
chamber for 2 h at room temperature. For all experiments, at least 3 males from
each genotype were evaluated. Following final washing of chromosome slides in
0.4% Photo-Flo 200 (Kodak 1464510) for 2 × 5 min, slides were air dried for
~10 min and stored in −80 °C or used immediately for staining.
For staining, slides were washed in 1xPBS, 0.4% Photoflo for 10 min, followed
by 2 × 10 min. washes in 1x PBS, 0.1% Triton X and 1 hr incubation in blocking
buffer (3% BSA, 10% Goat Serum, 0.0125% Triton X, 1 x PBS), at 37 °C. Finally, all
antibodies were diluted in blocking buffer, and slides were incubated for
12 hr at RT.
Primary antibodies used were: rabbit anti-SYCP3 (1:500, ab15093; Abcam),
mouse anti-SYCP3 (1:500, ab97672; Abcam), and mouse anti-MLH1 (1:50, 550838;
BD Pharmingen). Slides were washed and incubated for 1 hr with the following
secondary antibodies: goat anti rabbit-IgG 488 (1:2,000, A11008; Molecular
Probes), goat ant-rabbit IgG 594 (1:1,000, A11012; Molecular Probes), and goat
anti-mouse IgG 594 (1:1,000, A11005; Molecular Probes). Nuclei were
counterstained with DAPI, and slides were mounted with ProLong Gold antifade
reagent (P36930; Molecular Probes).
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Sperm counting. Cauda epididymides were dissected and minced into 2 mL of
PBS. Sperm were allowed to swim into the buffer via 15 min incubation at 37 °C.
The sperm were diluted PBS, incubated for 1 min at 60 °C, and then counted with a
hematocytometer.
Testes histology and Immunohistochemistry. For histological analyses, testes
were fixed for 24 h at room temperature (RT) in Bouin’s solution. Tissue was
further paraffin-embedded, sectioned (7 μm), and stained with H&E. For immunohistology,
testes were essentially fixed in 4% paraformaldehyde for ∼24 h.
Paraffin-embedded tissues were further sectioned at 7 μm. For TUNEL and PH3
double immunostaining, sections were deparaffinized followed by antigen retrieval
using Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0).
Sections were blocked in PBS containing 5% goat serum for 1 h at RT, followed by
TUNEL staining, performed following instructions manufacturer’s instruction
(Invitrogen, Click-iT™ TUNEL kit). This was followed by 1° antibody incubation
overnight at 4 °C (anti-pH3 (Ser10), 1:100, Millipore) and anti-rabbit AF488
(Invitrogen, 1:1000) 2° antibody incubation for 1 h, RT. Slides were counterstained
using DAPI, mounted in Vectashield and imaged as described in below.
Imaging and analysis of testes cross sections. Testes were fixed in Bouin’s,
embedded in paraffin and stained with hematoxylin and eosin (H&E) in Cornell’s
Diagnostic Pathology Lab. For TUNEL labeling, testes cross sections were double
immunolabeled with TUNEL and anti-pH3 (see above) antibody and were imaged
using a confocal microscope (u880, Carl Zeiss, Germany) with a Plan Apo 40×
water immersion objective (1.1 NA) and Zen black software. The following lasers
were used: argon laser-488 nm, blue-diode-405 nm, DPSS laser—561 nm. Following
identical background adjustments for all images, cropping, color, and
contrast adjustments were made with Adobe Photoshop CC 2017.
Oocyte in vitro maturation and in situ chromosome counting. Prophase
I-arrested oocytes (GV) were collected following standard methods77 as follows.
Forty-eight hours prior to collection, females were injected intraperitoneally with
CARD HyperOva (KYD-010-EX-X5; CosmoBio USA) consisting of Inhibin antiserum
and equine chorionic gonadotrophin (Sigma, 230734). Further, ovaries from
12–16 weeks old females were dissected and GV oocytes were released from the
follicles by puncturing the ovaries several times with 27-gauge sewing needles in
MEM/polyvinylpyrrolidone media containing 2.5 μM milrinone (Sigma-Aldrich;
M4659) to prevent oocyte maturation after the release from cumulus cells. Then,
cumulus cells were removed by gentle pipetting and fully-grown oocytes were
matured in vitro in Chatot, Ziomek, and Bavister (CZB) media without milrinone
in a humidified incubator programmed to 5% CO2 and 37 °C for 14 h until they
arrived to metaphase II. They were cultured for 2 h in 100 µM Monastrol (Sigma,
M8515) to disorganize the spindle and separate the chromosomes. Then, eggs were
fixed in 2% paraformaldehyde in PBS for 20 min and permeabilized in PBS containing
0.2% Triton X-100 (Sigma-Aldrich, 900-93-1) for 20 min. Eggs were stained
with the anticentromere antibody (ACA) to detect centromeres (Antibodies
Incorporated; #15-234; 1:30) and DAPI to detect DNA. Chromosome numbers
were quantified in metaphase II eggs78. Mouse eggs should have 20 pairs of sister
chromatids; any deviation of this number was considered aneuploid. Eggs were
imaged with the i880 (Zeiss) confocal at 0.5 µm z-intervals. Chromosome counting
was performed with NIH Image J software, using cell counter plugins.
Animals. All animal strains were maintained in a mixed strain background
(C57BL/6 J x FVB/NJ). We have complied with all relevant ethical regulations for
animal experiments, and these experiments were performed under a protocol
(2004-0038) approved by Cornell’s Institutional Animal Care and Use Committee
(IACUC), which ensures ethical animal research use. Mice were housed at 23 °C
with a light cycle of 10 h dark and 14 h light.
Statistics and reproducibility. All data presented in this manuscript is generated
from at least 3 or more biological replicates, unless otherwise noted. Statistics is
performed on datapoints with ≥3 biological replicates. Each mouse is considered a
biological replicate.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
All relevant data such as litter sizes and sperm numbers are present in the manuscript.
Fluorescent images (immunolabelings) from which numbers were generated (such as
MLH1 foci) are stored by the authors and primary images can be provided upon request.
Source data are included with this manuscript. All unique materials used are readily
available from the authors, with the exception of mouse strains, which may require
extended periods of time for reanimation and/or breeding before providing to
requestors. Source data are provided with this paper.
Received: 22 January 2021; Accepted: 20 July 2021;
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Acknowledgements
We thank S. Keeney for yeast strains, R. Munroe and C. Abratte in Cornell’s transgenic
core for making mice, and P. Cohen for critical reading of the manuscript in advance of
submission. This work was supported by grants R01HD082568 to J.S. and H.Y.,
R01HD091331 to K.S., and R35 GM134872 to E.A.
Author contributions
P.S. characterized all Mlh1 and the Mlh3A1394T mouse mutants and contributed to writing the
manuscript. R.F. performed the Y2H experiments and wrote relevant sections of the manuscript;
H.Y. supervised R.F. and was involved in SNP selection metrics; C.S.B. (with P.S.)
performed the oocyte development and ploidy experiments presented in Fig. 3d–g, was
supervised by K.Schindler, and C.S.B. and K.S. wrote relevant parts of the manuscript. T.N.T.
analyzed Mlh3 mouse mutants not done by P.S. G.P. and N.A-S. performed the yeast studies
of mismatch repair and recombination, and were supervised by E.A.A., who wrote relevant
parts of the manuscript and design/interpretation of the yeast experiments. K.J.S. assisted with
mouse husbandry. J.C.S. oversaw the overall project, its design, and had a major role in writing
and revising the manuscript.
Competing interests
The authors declare no competing interests.
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