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. 2013;4:2889.
doi: 10.1038/ncomms3889.

Low Paternal Dietary Folate Alters the Mouse Sperm Epigenome and Is Associated With Negative Pregnancy Outcomes

Free PMC article

Low Paternal Dietary Folate Alters the Mouse Sperm Epigenome and Is Associated With Negative Pregnancy Outcomes

R Lambrot et al. Nat Commun. .
Free PMC article


Epidemiological studies suggest that a father's diet can influence offspring health. A proposed mechanism for paternal transmission of environmental information is via the sperm epigenome. The epigenome includes heritable information such as DNA methylation. We hypothesize that the dietary supply of methyl donors will alter epigenetic reprogramming in sperm. Here we feed male mice either a folate-deficient or folate-sufficient diet throughout life. Paternal folate deficiency is associated with increased birth defects in the offspring, which include craniofacial and musculoskeletal malformations. Genome-wide DNA methylation analysis and the subsequent functional analysis identify differential methylation in sperm of genes implicated in development, chronic diseases such as cancer, diabetes, autism and schizophrenia. While >300 genes are differentially expressed in offspring placenta, only two correspond to genes with differential methylation in sperm. This model suggests epigenetic transmission may involve sperm histone H3 methylation or DNA methylation and that adequate paternal dietary folate is essential for offspring health.


Figure 1
Figure 1. Meiotic onset is delayed in FD mice but adult spermatogenesis is normal.
(a) C57BL/6 females were fed either a FS or a FD diet, (n=64 for each) 2 weeks before breeding with a C57BL/6 male fed with control mouse chow. Females were maintained on the experimental diet through pregnancy and lactation. From weaning until throughout life, male pups received the same experimental diet as their mother. PND12 testis cross-sections from FS (b) and FD (c) testis cross-sections were stained using anti-histone H3 monomethylation at lysine 4 (H3K4-me1), a marker of early meiotic spermatocytes. Fewer meiotic cells (M, brown staining) were present in FD tubules (c) in comparison with FS mice (b), whereas spermatogonia (SG) and sertoli cells (SC) were not affected. (d) The ratio of meiotic tubules (>10 meiotic germ cells) to total number of tubules was quantitated. Means±s.e.m. of three determinations are shown. *P<0.05 by the Mann–Whitney U-test. Sperm counts were performed in adult FS and FD mice (e). Means±s.e.m. of five determinations are shown. *P<0.05 by Student’s t-test. Spermatogenesis was assessed in adult FS (f) and FD mice (g) by haematoxilin/eosin staining. Scale bars, 10 μm (b,c) and 100 μm (d,e).
Figure 2
Figure 2. Paternal folate deficiency increases birth defects in offspring.
Fetuses sired by FD males displayed increased developmental abnormalities indicated by arrows (be) than those sired by FS males (a). Shown are the following: (b) hydrocephalus and associated craniofacial defects, (c) limb hyperextension with dysgenesis of digits, (d) spine malformation and (e) dorsal malformations. Histopathological analysis was carried out on selected FS- (f) and FD- (g) sired fetuses. (g) In this thoracic transverse section of an FD-sired fetus, the arrow indicates an imbalance between the right and left side muscular and bone tissues indicated muscular dysplasia. Scale bars, 1 mm (f,g). Skeletal staining was performed in both FS- (h,j) and FD- (i,k,l) sired fetuses. Bone is stained purple and cartilage blue. (i) FD-sired fetus lacking interparietal (IP) and supraoccipital (SO) bones and had underdeveloped frontal (Fr), parietal (Pr) bones and digits. FD-sired fetuses, with misaligned (k), or incomplete development (l) of the sternebrae plates.
Figure 3
Figure 3. Folate deficiency alters sperm DNA methylation.
Changes in DNA methylation are illustrated by smoothed MeDIP over input log2 ratios of individual oligonucleotides for the folate-sufficient (FS) and the folate-deficient (FD) animals. The gene is indicated at the bottom of the graph and the arrow represents the transcription start site (TSS).
Figure 4
Figure 4. Folate deficiency alters sperm DNA methylation at genes implicated in development and metabolic processes.
Sequenom MassARRAY methylation analysis was performed on selected targets of interest identified by MeDIP-chip as altered in FD versus FS sperm. FD sperm had significantly reduced 5-methylcytosine at CpG locations of Rfwd2 (a), Sfi1 (b), Kdm3b (c), Gm52 (d) and Rbks (e). Means±s.e.m. of five determinations are shown. *P<0.05 by Student’s t-test.
Figure 5
Figure 5. Differential gene expression in placenta of offspring sired by FD versus FS males.
(a) Heat-map showing the expression levels of 39 genes in four placentas of 18.5 dpc fetuses sired by either an FS (n=4) or FD (n=4) male. Placentas analysed were from unique litters. (b) Validation of array results by real-time PCR on an extended group of samples (n=8, FS and n=8, FD). (c) Selected array targets Cav1 and Txndc16 showed altered gene expression and were differentially methylated in sperm of FD sires. Data are expressed as a percentage of the control β-actin, with the value of the FS at 100%. Means±s.e.m. of eight determinations are shown. For b and c, *P<0.05, **P<0.01 by Student’s t-test.
Figure 6
Figure 6. Genomic regions located near Txndc16 and Cav1 that are differentially methylated in FD sperm are not differentially methylated in 18.5 dpc placentas.
Pyrosequencing analysis after bisulfite conversion was carried out on 18.5 dpc placentas sired by FS and FD males (FS sired, n=4; FD sired, n=4). The values are means ±s.e.m.

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