2/9/23

Session 6: The effect of an abnormal sperm epigenome on embryo development.

Effects of altering sperm DNA methylation on development of the embryo and

placental and on their gene expression.

R. Lambrot, C. Xu1, S. Saint-Phar, G. Chountalos, T. Cohen, M. Paquet, M.

Suderman, M. Hallett & S Kimmins (2013) Low paternal dietary folate alters the
mouse sperm epigenome and is associated with negative pregnancy outcomes.
NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889

Reducing histone methylation in sperm of one generation affects both

development and gene expression by embryos of subsequent generations.

K. Siklenka, S, Erkek, M, Godmann, R. Lambrot, S. McGraw, C. Lafleur, T. Cohen, J.

Xia, M. Suderman, M. Hallett, J. Trasler, A.H.F.M.Peters, S. Kimmins (2015).
Disruption of histone methylation in developing sperm impairs offspring health
transgenerationally. Science 350: 652-665.

R. Lambrot, C. Xu1, S. Saint-Phar, G. Chountalos, T. Cohen, M. Paquet, M.

Suderman, M. Hallett & S Kimmins (2013) Low paternal dietary folate alters the
mouse sperm epigenome and is associated with negative pregnancy outcomes.
NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889

Hypothesis Normal sperm DNA methylation is essential for proper embryonic

development.

DNA methylation of sperm was manipulated by subjecting animals in utero and

after birth to a diet that was deficient in folate.

Folate metabolism generates the methyl group used in the biochemical

synthesis of methionine from homocysteine.

Methionine donates methyl group to DNA, proteins and lipids.

Folate-deficient animals have other sources of methionine: diet and the

methionine salvage pathway.

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Biochemical Pathways Involved in Methylation of Cytosine Residues in DNA

Folic Acid
Diet. methylene

Folate

NADPH is the H donor

for Folate metabolism. Folate*
Serine is the methylene
(CH2:) donor, which
metabolism
upon reduction becomes
the methyl group (CH3-)
that is subsequently
used in methionine
synthesis.

5’ methyltetrahydrofolate (5-MTHF)

S-adenosyl-
methionine
cycle
Homocysteine

DNA, Protein and

Lipid Methylation
Methionine

S-Adenosylmethionine Cycle: Enzymatic Steps That Result in the

Transfer a Methyl Group from Methonine to Cytosine

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Dietary manipulation of sperm DNA methylation.

Males on a normal diet bred to

females on folate-deficient diet.
Controls: Experimental

Male offspring kept on folate-

deficient diet.

Examine DNA methylation

of sperm and testis morphology of
folate-deficient males.

Breed folate-deficient and sufficient

males to folate-sufficient females.

Day 18.5 embryos: examine

Morphology and gene

.NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889

Testicular histology of folate-sufficient and folate-deficient male mice.

Histology is normal in folate-deficient mice

NATURE COMMUNICATIONS | 4:2889 | DOI: 10.1038/ncomms3889

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Abnormal DNA Methylation in Gene Promoters in Sperm of Folate-Deficient Mice

Analysis of 5’mC in gene promoters. Data are Expressed as the
Ratio of 5 mC DNA to Total Input DNA.
Mir715 (microRNA) gene RNA helicase Centrosome-associated protein

Folate Sufficient

Folate Deficient
Hypermethylation
Hypomethylation

Analytical Approach:
DNA isolated from sperm, sheared to ~ 300-500 bp and denatured with heat. Sample split into 2 aliquots.
Immunoprecipitatation of DNA containing 5 methyl C residues
Quantification of selected promoter sequences in immunoprecipitated and total DNA by use of promoter arrays.
Folate deficiency causes hypomethylation of some promoters, but hypermethylation of others.

Gene Ontology Analysis:

Genes Involved in Development Are Differentially
Methylated in Sperm of Folate-Deficient Mice

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Embryo Viability was Markedly Reduced When

Folate Deficient Males Were Mated.

Who Gave Birth to Live Pups

Percent of Mated Females

That Were Resorbed

Percent of Embryos
Data collected at e18.5

Embryos (dpc 18.5) conceived by sperm from folate-sufficient

and folate-deficient males.
All embryos developed in a folate-sufficient female.

Normal Grossly Abnormal

Sperm from Folate- Hydrocephaly Abnormal Limbs Spine Malformation Dorsal Malformation
Sufficient Males
Sperm from Folate-Deficient Males;
27% of embryos were grossly abnormal
97% of embryos
were normal
Authors did not report analysis of embryonic gene expression

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Grossly Abnormal Fetuses Sired by Folate-Deficient Males

Had Abnormal Skulls
The FD skull lacks two bones.

Folate-Sufficient Male Folate-Deficient Male

* FS FD
*

* Interperital (IP) and Supraoccipital (SO)

bones missing in FD mice.
Red-Purple = Bone
Blue = Cartilage

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380 Genes are Mis-expressed in the Placentas of e18.5 Embryos Sired by Folate-Deficient Males.
Data for the top 39 genes are shown here.
Folate
Placentas not
Deficient Sufficient
s

selected for
Validation by RT-PCR
ne
Ge

morphological
abnormalities.
Whole transcriptome analysis by use of microarrays

N=4/group

Some of these genes regulate development.

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Sperm from 2 Folate-Deficient Males Sired Offspring

with Grossly Abnormal, Fused Placentas

Folate sufficient sired

Folate deficient sired
Fused Placenta
GiC
b Remodel uterus and
Folate sufficient
Folate Deficient sired
Sired
Layers of Placenta
Missing or Reduced
In Folate-Deficient
La. Sired Fetuses:
Sp.
Dec. GC=Giant Cells.
produce hormones

SP=Spongio-
Trophoblast
Runt La.
a
Dec:= Maternal
Dec. Decidua
c

La=Labyrinth;
Lack of SP in folate-deficient mice
Site of nutrient
Indicative of abnormal differentiation
of placental stem cells. exchange
Supplementary Figure S6. Placental abnormalities in pregnancies sired by a folate deficient father.
(a) The 18.5 day-post-conception (dpc) fetuses shared a fused placenta (arrowhead). The arrow indicates
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the runt fetus. Example of a 18.5dpc placenta cross sections from a fetus sired by a folate sufficient (FS)
(b) and one by a folate deficient (FD) father (c). Placenta cross-sections stained with haematoxylin and
eosin. The giant cell (GiC) layer indicated by an arrow in an FS-sired placenta was absent in the placenta
of the fetus sired by an FD male. The spongiotrophoblast (Sp.), situated in-between the labyrinth (La.) and
the maternal decidua (Dec.), was abnormally thin. Bars represent 500µm.

Summary
Lambrot, et. al. (2013) Low paternal dietary folate alters the mouse sperm epigenome and
is associated with negative pregnancy outcomes. NATURE COMMUNICATIONS | 4:2889

1. Sperm DNA methylation and thus their epigenome, is abnormal in mice fed a
folate-deficient diet.
2. Many of the genes associated with the abnormal epigenome regulate development.
3. Sperm from folate-deficient mice give rise to embryos that are at a significantly higher
risk of embryonic death and development of major birth defects.
4. Placental gene expression is aberrant with folate-deficient fathers. There were
2 instances of morphologically abnormal, fused placentas.

A normal sperm epigenome is important for embryo viability, fetal development and
placental gene expression.

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K. Siklenka, S, Erkek, M, Godmann, R. Lambrot, S. McGraw, C. Lafleur, T. Cohen, J. Xia,

M. Suderman, M. Hallett, J. Trasler, A.H.F.M.Peters, S. Kimmins (2015). Disruption of
histone methylation in developing sperm impairs offspring health
transgenerationally. Science 350: 652-665.

Hypothesis. Reduced sperm levels of histone 3 lysine 4 dimethyl increases

incidence of major birth defects. The effect of resulting epimutations are
transgenerational.

Siklenka et al generated mice that carried one copy of a transgene that drove
overexpression of histone 3, lysine 4 demethylase (KDM1A) via a spermatogenic
cell-specific promoter. (Expression of the KDM1A transgene significantly reduced
sperm levels of HeK4me2.)

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Intergenerational and Transgenerational Effects of Reducing Histone 3 Lysine 4

Methylation in Sperm:
Reduced histone methylation in sperm was the result of transgenic expression of a
histone demethylase specifically in spermatogenic cells

Founder female is heterozygous for the

KDM1A transgene (TG), which is only
expressed in male germ cells. Wild-type
females are used for all subsequent
breeding.

Males bred
to wild-type (Father but not son express TG)
females.

(Neither father nor son

Express TG)

Effect of transgene expression on epigenome of sperm.

Analysis of sperm nucleosomes from TG3 mice showed that overexpression of the demethylase resulted
in reduced H3K4me2 in nucleosomes associated with over 2,300 genes. Many of these genes regulate
development.

DNA. methylation was not affected.

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Chromatin H2K4me2 Occupancy is Reduced and Positioning

Altered in the the Sperm Genome of KDM1A Transgenic Mice

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TG4: Fathers and sons were transgenic

Intergenerational and Transgenerational
for Histone Lysine 4 demethylase.
nonTG4
Control

effects of reducing histone 3 lysine 4 Sons are bred.

methylation in sperm: Effects on
TG4

Effects on embryonic gene expression are

transcriptome of 2 cell embryo.
intergenerational.

Sperm from control mice, from nonTG4: Grandfathers but neither fathers
nor sons were transgenic.
TG4 mice and from nonTG4 mice Sons are bred.
fertilized wild-type eggs via IVF.
Transcriptomes of 2 cell embryos Effects on embryonic gene expression are
transgenerational.
were analyzed.

TG4: Expression of 874

RNAs differed from control

nonTG4: 123 RNAs differed

from control.

Heat map shows data for

36 mRNAs.
Embryonic gene expression
begins in S phase of 1 cell
embryo (PNAS 115, E6780,
K. Siklenka, et. al. (2015).
2018)
Science 350: 652-665.

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Intergenerational and Transgenerational Effects reducing histone 3 lysine 4 methylation

in sperm: Increased incidence of major birth defects in day 18.5 embryos.

Craniofacial
Normal Abnormalities

Group Inheritance % Abnormal Fetuses % Pregnancies Lost

Control NA 1.68 10.8
TG3 Intergenerational 7.61* 19.6*
Non-TG3 Intergenerational 11.3* 20.2*
Non-TG4 Transgenerational 6.42* 20.7*
* Significantly different from control

TG3 male mice carry the histone 3 lysine 4 demethylase

transgene.

Non-TG3 mice. Fathers carry transgene but not the son.

Sons are bred.

Non-TG4 mice. The grandfather carries the transgene.

Neither the father nor the son carries the transgene.
Sons are bred.

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Common Transgenerational Effects of Reduction of in Levels of Histone 4 Lysine 4 me2 .

Grandfather but not father or son transgenic for Histone 3 Lysine 4 Demethylase.

Analysis of day 18.5 embryos

Red/Purple: Bone
Green: Cartilage

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Authors evaluated placentas of e18.5 embryos sired by sperm from TG or

nonTG mice.

They did did not report any morphological abnormalities.

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Summary
K. Siklenka (2015). Disruption of histone methylation in developing sperm impairs
offspring health transgenerationally. Science 350: 652-665.
.
1. Overexpression of a histone 3 lysine 4 demethylase in germ cells results in
reduction in H3K4me2 at many genes in the sperm genome.
2. Fertilization by sperm from TG or non-TG4 mice resulted in altered gene
expression by 2 cell embryos (874 and 123 genes for TG and non-TG4,
respectively.
3. Sperm with demethylated histones sire offspring with a higher incidence of
both embryonic death and birth defects.
4. The effect of loss of histone 3 lysine 4 dimethyl is transgenerational.
This effect persists even when when the demethylase transgene is
expressed by neither the father nor the son and the son is the breeder.

Major conclusion from Labrot et al and Siklenka et al:

Decreased levels of histone 3 lysine 4 in sperm nucleosomes affect
embryonic development. These effects are transgenerational.

Why is this paper important to public health?

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