Environmentally Induced Epigenetic Transgenerational
Inheritance of Altered Sertoli Cell Transcriptome and
Epigenome: Molecular Etiology of Male Infertility
Carlos Guerrero-Bosagna, Marina Savenkova, Md. Muksitul Haque, Eric Nilsson, Michael K. Skinner*
Center for Reproductive Biology, School of Biological Sciences, Washington State University, Pullman, Washington, United States of America
Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease,
including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm
epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific
disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during
gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and
assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells
provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the
F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be
transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly
linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA
methylation regions (DMR) modified. The genomic features and overlap with the sperm DMR were investigated.
Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of
a specific somatic cell (Sertoli cell) epigenome and transcriptome that correlates with adult onset disease (male infertility).
The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the
molecular etiology of male infertility.
Citation: Guerrero-Bosagna C, Savenkova M, Haque MM, Nilsson E, Skinner MK (2013) Environmentally Induced Epigenetic Transgenerational Inheritance of
Altered Sertoli Cell Transcriptome and Epigenome: Molecular Etiology of Male Infertility. PLoS ONE 8(3): e59922. doi:10.1371/journal.pone.0059922
Editor: Toshi Shioda, Massachusetts General Hospital, United States of America
Received December 17, 2012; Accepted February 19, 2013; Published March 28, 2013
Copyright: ? 2013 Guerrero-Bosagna et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The research was supported by a grant from the National Institute of Health (NIH) NIEHS to MKS. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Environmentally induced epigenetic transgenerational inheri-
tance of adult onset disease  can be promoted by factors such as
toxicants [2,3] or nutrition [4,5,6,7]. Environmental chemicals
shown to promote transgenerational disease include the fungicide
vinclozolin [2,8], plastics (bisphenol A (BPA) and phthalates
DEHP and DBP) [3,9,10], pesticides (methoxychlor and permeth-
rin) [2,3], dioxin [3,11], and hydrocarbons . A number of
transgenerational diseases/abnormalities have been shown to be
induced such as testis disease [2,9,12], prostate disease [13,14],
kidney disease [7,14], ovarian disease [3,15], reproductive tract
abnormalities , brain and behavior abnormalities [10,16,17],
and immune abnormalities . Environmentally induced
transgenerational phenomena have been observed in plants ,
flies [19,20], worms [21,22], rodents [2,11], and humans [23,24].
The current study was designed to investigate the actions of a
specific toxicant (vinclozolin) to promote a transgenerational
alteration in a somatic cell (Sertoli) that correlates to the induction
of disease in the tissue (testis).
Transgenerational phenotypes involve the germline transmis-
sion of epigenetic alterations (e.g. DNA methylation) in the
absence of any direct environmental exposures [1,25]. Environ-
mental exposures during fetal gonadal sex determination modifies
the epigenetic (DNA methylation) programming of the germline to
induce permanently programmed differential DNA methylation
regions (DMR) that then transmit an altered epigenome to the
subsequent generation [1,26]. Normal primordial germ cell
development in the gonad requires the erasure and re-methylation
of the DNA to promote the development of a male (sperm) versus
female (egg) germline [26,27,28]. The somatic cells and tissues
derived from this epigenetically altered germline will promote all
somatic cells to develop a modified epigenome and transcriptome
. Each tissue will develop an organ specific transgenerational
transcriptome [29,30] that is associated with the disease/abnor-
mality of the tissue . An example provided is the vinclozolin
induced transgenerational ovarian disease that correlates with an
altered granulosa cell epigenome and transcriptome associated
with the development of polycystic ovarian disease . This
provides insights into the molecular etiology of disease develop-
ment within the tissue.
The testis is the site of spermatogenesis that occurs within
seminiferous tubules composed of Sertoli cells and an adjacent
basal layer of mesenchymal peritubular cells [31,32]. The
interstitial tissue between tubules is composed of Leydig cells,
the site of testosterone production, and testicular macrophages
[33,34]. All these somatic cells cooperate in testicular function to
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support germ cell development and production [31,32]. The most
critical cell for the support of the developing spermatogenic cells is
the Sertoli cell that provides the physical support, formation of the
blood testis barrier, and nutritional factors needed for spermato-
genesis . The Sertoli cells synthesize a number of transport
binding proteins (e.g. transferrin) to carry essential factors (e.g.
iron) to the developing germ cells . In addition, the Sertoli cells
produce pyruvate/lactate that is used as the primary energy
metabolite by the germ cells that are sequestered within the blood
testis barrier and not able to acquire glucose [35,36].
Testis disease is primarily associated with abnormal spermato-
genesis and reduced sperm counts leading to male infertility
[37,38]. This can be the result of developmental defects such as
cryptorchidism or hypospadias . A number of studies have
suggested environmental exposures promote testis abnormalities
and disease [3,12,40]. This includes endocrine disruptors and
environmental chemicals. A number of histopathologies have been
associated with testis disease and termed testicular dysgenesis
syndrome [38,39,40]. One of the primary abnormalities is
increased spermatogenic cell apoptosis resulting in reduced sperm
numbers [41,42]. The male infertility associated with testis disease
has increased dramatically over the past decades and now affects
over 10% of the human male population . Although abnormal
endocrinology and environmental factors have been associated
with testis disease , the specific molecular etiologies remain to
Previous studies have demonstrated that exposure of a gestating
female to the agriculturally used fungicide vinclozolin  during
gonadal sex determination promotes the epigenetic transgenera-
tional inheritance of adult onset testis disease [2,12,14,45]. In the
F3 generation 90% of the males had a transgenerational
spermatogenic cell defect of enhanced apoptosis and reduced
sperm count and motility [2,12]. The objective of the current
study was to utilize this model to elucidate components of the
molecular etiology of male infertility. Since the Sertoli cell is the
primary somatic cell that supports the development of the
spermatogenic cells, the role of the Sertoli cell in mediating the
transgenerational testis disease is investigated. Observations
demonstrate that the vinclozolin induced epigenetic transgenera-
tional inheritance of spermatogenic cell defects and testis disease is
associated with a transgenerational alteration in the Sertoli cell
epigenome and transcriptome.
Transgenerational Spermatogenic Cell Abnormality
The experimental design involved the exposure of a gestating
female (F0 generation) Sprague-Dawley rat to vinclozolin
(100 mg/kg/day) during days 8–14 (E8–14) of fetal development
[2,12]. The F1 generation progeny were bred to obtain the F2
generation and the F2 bred to obtain the F3 generation [2,12]. No
sibling or cousin breeding was used in order to avoid any
inbreeding artifacts. Corresponding control (vehicle DMSO)
lineage F1, F2 and F3 generations were obtained to compare to
the vinclozolin lineage males. Some of the direct exposure F1
generation and transgenerational F3 generation control and
vinclozolin lineage males were aged to 1 year and testes collected
to examine the number of apoptotic spermatogenic cells as
previously described [2,12]. The F1 and F3 generation vinclozolin
lineage males had an increased number of apoptotic spermato-
genic cells (p,0.05), Figure 1. As previously described [2,12], the
vinclozolin lineage animals had spermatogenic cell defects that
correlated to testis disease and male infertility .
Another set of F3 generation males from the control and
vinclozolin lineages were aged to 20 days of postnatal development
and testes collected to isolate purified populations of Sertoli cells.
At this age of development no spermatogenic cell apoptosis has
been observed, so avoid disease artifacts, and the highest purity
cell preparations are obtained [2,12]. The isolated Sertoli cell
populations from three different groups of animals and cell isolates
were used to obtain Sertoli cell RNA and DNA from F3
generation control and vinclozolin lineages.
Sertoli Cell Transgenerational Transcriptome
The Sertoli cell RNA was used in a microarray analysis and the
quality of the RNA and array monitored. The comparison of the
microarrays demonstrated nearly identical profiles on the arrays,
Supplemental Figure S1. No batch effects were observed due to
cell or RNA isolation dates. The differential gene expression
between the control and vinclozolin lineage F3 generation Sertoli
cell arrays was determined with a statistical difference (p,0.05),
fold change (.1.2 fold) and mean difference (.10) as cut off limits,
described in the Methods. The comparison identified 417
differentially expressed genes in the F3 generation Sertoli cell
control versus vinclozolin comparison, Supplemental Table S1.
The differentially expressed genes involved 198 genes with up-
regulation and 219 genes with down-regulation, Figure 2. The
functional gene categories of transcription, metabolism, signaling
and development were predominant.
A cellular signaling and process pathway analysis was performed
as previously described . Analysis of the 417 differentially
expressed genes identified 22 pathways with variable impact
factors and statistically significant over-representation, Table 1.
One of the top pathways most relevant to the Sertoli cell was the
pyruvate/lactate metabolism pathway, Figure 3. A number of the
key enzymes in pyruvate and lactate metabolism were modified
that influence pyruvate and lactate production by the Sertoli cell.
These are subsequently a required energy source for the
spermatogenic cells. The key enzymes that are all significantly
down regulated (.2 fold) were hydroxyacyl-glutathione hydrolase
(Hagh), pyruvate dehydrogenase beta (Pdhb), lactate dehydrogenase
A like bb (Ldnalbb), and dihydrollipoxamideS-acetyltransferase
(Dlat), Figure 3. Other prominent pathways affected, Table 1, were
the proteasome, nucleotide excision repair, RNA transport, p53
Figure 1. Spermatogenic cell apoptosis in 1-year-old males.
Relative apoptotic spermatogenic cell number per testis section is
presented. F1 and F3 generation control and vinclozolin lineage testis
were examined with the mean 6 SEM presented and asterisks (*)
indicating a statistically significant difference (p,0.05).
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signaling, and mTOR signaling (Supplemental Figure S2). The
pathway class most affected was Genetic Information Processing,
A gene network analysis was performed on the 417 differentially
expressed genes with an unbiased literature evaluation protocol
using the Pathway Studio software. The down or up regulated
genes associated and cellular localization are shown, Figure 4. The
most highly interconnected genes were the Igf1r, Jak2, Hsp90aa1,
Hif1a and Ccne1. As identified in the gene functional categories,
Figure 2, a number of cellular processes are influenced by the gene
The differentially regulated genes were mapped to the genome
and shown in Figure 5. All chromosomes contained differentially
expressed genes. The potential over-representation of gene clusters
in specific chromosomal regions was examined as previously
described . Potential 2–5 megabase regions were examined for
statistical over-representation of differentially expressed genes.
These gene clusters are identified in Figure 5 and will be correlated
to the epigenetic analysis described below.
Sertoli Cell Transgenerational Epigenome
The F3 generation control and vinclozolin lineage Sertoli cell
DNA was used in a methylated DNA immunoprecipitation
(MeDIP) and genome wide promoter tiling array (Chip) analysis
(MeDIP-Chip)  as described in the Methods. A comparative
hybridization of the F3 generation control and vinclozolin lineage
Sertoli cell MeDIP samples identified differential DNA methyla-
tion regions (DMR), as previously described . The MeDIP-Chip
analysis identified 101 DMR and the chromosomal locations of
the DMR are shown in Figure 5 and Supplemental Table S2. All
chromosomes contained DMR. Interestingly, none of the Sertoli
cell DMR identified were in common with the previously
identified sperm DMR . In the analysis of sperm DMR
previously reported , two genomic features were identified. The
first was a consensus DNA sequence localization in the ,600 bp
region of the DMR termed ‘‘environmental induced DNA
methylation motif 1’’ (EDM1) . This DNA sequence motif is
a .20 bp sequence motif associated with 68.8% of the DMR
identified in sperm [3,8], as described in the Methods. The EDM1
motif was found in 7.1% of the Sertoli cell DMR identified, which
was not statistically different from a computer generated random
promoter region set with a 16.1% incidence of occurrence.
Therefore, the EDM1 motif genomic feature found in sperm
DMR was not associated with the Sertoli cell DMR. The second
genomic feature previously identified in sperm DMR was a low
density CpG content of less than 10 CpG/100 bp [3,8]. The
number of CpG/100 bp is generally between 1–4 in the sperm
DMR. The Sertoli cell DMR were also found to contain a low
density of CpG (,10 CpG/100 bp), Figure 6. The majority of
DMR had 1 or 2 CpG/100 bp. No DMR was found to have a
CpG density greater than 10.6 CpG/100 bp. Therefore, the
Sertoli cell DMR are similar to the Sperm DMR in that a CpG
‘‘desert’’ of low density CpG is a genomic feature involved [3,8].
The MeDIP-Chip DMR data was confirmed with a select set of
Sertoli cell DMR using an MeDIP-quantitative PCR (QPCR)
analysis. The selected 26 DMR sites with 7 confirmations using
the MeDIP-QPCR analysis are presented in Supplemental Figure
S3. This analysis confirmed the MeDIP-Chip data for these DMR.
A technical limitation to this analysis is that only ,150 bp region
within the ,600–800 bp DMR can be examined, such that false
negatives are common due to the inability to interrogate the entire
DMR. Although the MeDIP-Chip data for a number of DMR
were confirmed, a better technology is needed for future studies.
A correlation of Sertoli cell differentially expressed genes with
the DMR identified only two differentially expressed genes that
also had a corresponding promoter DMR (Pdrx5 and Pole3),
Supplemental Table S3. Therefore, the majority of differentially
expressed genes did not contain a DMR for potential direct
Figure 2. Gene functional categories in F3 generation vinclozolin lineage Sertoli cells differentially expressed gene (417 genes). The
number of genes associated with the different functional categories are presented for up-regulated (black bar) or down-regulated (gray bar).
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regulation of gene expression. Previously, we identified the
potential presence of ‘‘epigenetic control regions’’ (ECR) that
contain a statistically significant over-representation of differential
expressed genes . Gene clusters within 2–5 megabase regions
that contain a statistically over-representation of differentially
expressed genes are shown in Figure 5 and Supplemental Table
S4. Six clusters had a DMR present within a 2 megabase window,
Figure 5 and Supplemental Table S3. These regions may act as an
ECR to regulate gene expression in the region as previously
described . An example of one potential ECR is presented in
Figure 7. This ECR contained two DMR (Rnase1a and Kctdll) and
had 10 differentially expressed genes within the approximately 5
megabase region shown. Therefore, 61 genes in the 16 potential
DMR may be regulated as an ECR.
A correlation of potential distal gene expression identified
31 DMR that were within 2 megabase of 38 differentially
regulated genes, Supplemental Table S3. Although the potential
mechanism involved are unclear, distal regulation through
mechanisms such as non-coding RNA have been demonstrated
. A final correlation used an unbiased literature based analysis
to identify indirect gene interactions between the DMR and
differentially expressed genes. The DMR associated genes that
have been shown to interact with the Sertoli cell differentially
expressed genes are shown in Figure 8. A potential correlation is
shown between 25 DMR and 21 differentially expressed genes.
Although a limited number of genes had direct regulation, a large
number of the differentially expressed genes correlate to potential
ECR, distal regulation sites or indirect gene interactions,
Supplemental Table S3 and S4.
The 101 DMR associated genes and the 417 Sertoli cell
differentially regulated genes were used to identify correlations
with genes previously associated with male infertility. This analysis
used a literature based procedure in the Pathway Studio software
and results are shown in Figure 9. The correlation demonstrated
one DMR and eight differentially expressed genes had a direct
relationship with previously associated male infertility genes.
Observations provide additional insights into the molecular
etiology of male infertility.
The ability of environmental toxicants such as vinclozolin have
previously been shown to promote the epigenetic transgenera-
tional inheritance of adult onset disease such as male infertility
[2,12,14]. Epigenetic transgenerational phenomena require the
germ line transmission of a permanently modified epigenome (e.g.
epimutation) [1,26]. How this modified germline epigenome is
translated into later life adult onset disease was investigated in the
current study with a focus on testis biology and disease.
Observations confirmed the ability of vinclozolin to promote F3
generation spermatogenic cell apoptosis, that has previously been
correlated to adult onset male infertility [2,12]. This transgenera-
tional model system was used to evaluate the molecular etiology of
male infertility. The Sertoli cell is the essential somatic cell known
to support spermatogenesis [31,32] and abnormal Sertoli cell
Table 1. Associated Functional Pathway Categories.
Functional Pathway CategoryPathway
Oxidative phosphorylation13 207NA
Metabolism Pyruvate metabolism4 73NA
Glycerophospholipid metabolism4 82NA
Proteasome5 51 7.5
Nucleotide excision repair4 435.8
Genetic Information ProcessingRNA transport10 134NA
Protein processing in endoplasmic reticulum7 137 NA
mTOR signaling pathway4 54 5.9
Phosphatidylinositol signaling system4 70 18.9
Signal TransductionErbB signaling pathway4 83 3.8
Jak-STAT signaling pathway5 136 4.5
MAPK signaling pathway 7253 1.8
p53 signaling pathway567 4.8
Cellular ProcessesCell cycle8 117 6.1
Oocyte meiosis5 109 NA
Focal adhesion4 187 3.2
Fc epsilon RI signaling pathway4704.2
Chemokine signaling pathway4 189 NA
Progesterone-mediated oocyte maturation5 88 NA
GnRH signaling pathway4 91 2.8
Insulin signaling pathway4 130 2.6
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function has been shown to correlate to testis disease [38,39,40].
Therefore, the Sertoli cell was the focus of the current study to
assess how a germline epimutation may translate to male
The Sertoli cell transgenerational transcriptome induced in the
F3 generation vinclozolin lineage identified 417 differentially
expressed genes. All somatic cell types derived from the gamete
having germline epimutations will have a transgenerational
alteration in the transcriptome, as previously shown for 11
different male and female tissues  and granulosa cells .
Each tissue and somatic cell was found to have a unique
transgenerational transcriptome. The Sertoli cell transcriptome
identified in the current study supports the concept of transge-
nerational alteration of individual somatic cell types. Cell types or
tissues sensitive to this alteration in the transcriptome will be
susceptible to develop disease [1,29]. The vinclozolin lineage
Sertoli cell transgenerational transcriptome was found to have
both up and down regulated genes with predominant cellular
pathways and gene functional categories affected. A gene network
analysis identified specific genes that influence a number of
different signaling pathways and processes.
Considering the role of Sertoli cells in the maintenance of
spermatogenic cell development, a critical pathway was identified
that directly correlates with germ cell viability. Sertoli cells acquire
glucose on their basal surface and convert this to pyruvate and
lactate that is then secreted and provided to germ cells,
sequestered within the blood-testis barrier, as a primary energy
source [35,36]. Previous studies have shown abnormal pyruvate or
lactate production or transport promotes the degeneration of
[36,47,48,49]. Therefore, the transgenerational alteration in
pyruvate or lactate production by the Sertoli cells provides a
direct mechanism for the induction of spermatogenic cell apoptosis
observed in the F3 generation vinclozolin lineage males. Although
other differentially expressed genes and cellular processes are likely
involved, the abnormal pyruvate or lactate production directly
the processof apoptosis
Figure 3. Pyruvate and lactate metabolic pathway with colored genes having significantly different altered expression in the F3
generation vinclozolin lineage Sertoli cells.
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correlates with the transgenerational adult onset male infertility
The F3 generation vinclozolin lineage Sertoli cells also had an
altered transgenerational epigenome. The genome-wide promoter
analysis identified 101 differential DNA methylation regions
(DMR) that previously have been termed epimutations [1,8,29].
The Sertoli cell transgenerational DMR were localized on the
genome and genomic features analyzed. Interestingly, the
previously identified EDM1 DNA sequence motif shown to be
associated with the sperm DMR , was not associated with the
Sertoli cell DMR. Observations suggest different molecular
mechanisms may be involved in the germline versus somatic cell
DMR generation and function. In contrast, the low density of
CpG (,10 CpG/100 bp) identified in the sperm DMR was also
observed in the Sertoli cell DMR. As previously discussed [3,8],
these ‘‘CpG deserts’’ may have evolved as critical regulatory
epigenetic sites. The high mutation rates observed in CpG sites
 suggests evolutionarily over time that regions of the genome
develop low density CpG (e.g. CpG deserts). In the event a CpG
cluster is regulatory in these deserts then evolutionary pressure
maintains the presence of the clusters in these desert regions.
Observations suggest that this same genomic feature observed in
sperm of a low density ,10 CpG/100 bp in the 600–800 bp
DMR also exists in somatic cells (Sertoli cells). Although this CpG
desert genomic feature was similar, the specific DMR in sperm
were found to have no overlap with the Sertoli cell DMR
identified. Therefore, the DMR in somatic cells are likely unique
to the cell type and generated from a specific cascade of epigenetic
alterations derived from the germline and early embryo.
A correlation of the transgenerational Sertoli cell transcriptome
with the epigenome identified potential distinct regulatory
mechanisms. Interestingly, only two genes (Prdx5 and Pole3) had
a direct correlation to suggest the potential for the DMR to
directly regulate the promoter and expression of the adjacent gene.
Therefore, the majority of the altered transcriptome and DMR act
through different mechanisms. Distal regulation was considered
and approximately 10% of the differentially expressed genes were
found within 2 megabases of a DMR. Such distal regulation could
involve mechanisms such as non-coding RNA [30,51]. Perhaps the
most interesting correlation is the role of epigenetic control regions
(ECR) . Previously we demonstrated that differentially
expressed genes can be statistically over-represented and localized
in gene clusters on the genome . When an epigenetic
regulatory site such as a DMR is also localized in the 2–5
megabase gene cluster then the region may be regulated through
an ECR. The initial epigenetic regulatory regions identified were
imprinting control regions (ICR) [51,52]. A defined DNA
methylation region was found to regulate a non-coding RNA that
distally for several megabase regulated the expression of multiple
genes . The ICR are likely a subset of a large number of such
ECR that allow for a limited number of epigenetic regulatory sites
to influence the expression of a large number of genes . The
Figure 4. Gene network of Sertoli cell differentially expressed genes. Genes having direct connectivity are presented with cellular
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F3 generation Sertoli cell differentially expressed genes clustered
into 16 different potential regions and a number of these directly
correlated to the location of DMR identified. A large number of
genes were regulated within these potential ECR. Therefore, a
combination of direct regulation, distal regulation, and ECR are
likely involved in the Sertoli cell transgenerational transcriptome
identified. In addition, indirect regulatory links between DMR
associated genes and the differentially expressed genes were
identified. The epigenetic regulation of somatic cell gene
expression and role of epigenetic transgenerational inheritance
needs to be further elucidated.
The current study used a transient exposure during the
developmental period of germ cell epigenetic programming and
gonadal sex determination [1,2,3]. The disease observed in the F1
generation offspring are due to direct exposure of the fetal somatic
cells and not to a germline mediated transgenerational mechanism
observed in the F3 generation. Although a comparison of the F1
and F3 generation control versus vinclozolin lineage Sertoli cells
would be interesting, there is no anticipated correlations due to the
distinct molecular mechanism involved. Similarly, direct exposure
of an adult male can alter the epigenetic programming in
spermatogenic cells to subsequently effect the disease in the F1
generation offspring , but these direct exposures have not
previously been shown to promote transgenerational effects to
subsequent generations. Therefore, the current study focused on
the F3 generation to investigate the epigenetic transgenerational
inheritance of disease mechanisms. Future studies to investigate
the differences with the direct exposure fetal or adult male effects
on the F1 generation will be interesting, but have distinct
mechanism with the observations presented in the current study.
Figure 5. Chromosomal localization of differential DNA methylation regions (DMR), differential expressed genes, and clusters of
gene expression. The chromosomal number and relative size are presented. The DMR (arrow), gene expression changes (line) and over-represented
clusters of gene expression (box) are indicated.
Figure 6. CpG density of the F3 generation vinclozolin lineage
Sertoli cell DMR. The number of DMR and CpG density (CpG/100 bp)
range are presented.
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Male infertility in the human population has now increased to
affect over 10% of the population . A number of studies
suggest environmental factors such as toxicants likely have a
significant role in the etiology of male infertility [53,54]. Direct
exposure toxicity of some of these agents for testis function has
been observed [2,3], but how such exposures may promote later
life disease has not been clarified. The current study used a rat
model involving an environmental toxicant (vinclozolin) induced
epigenetic transgenerational inheritance of testis disease, that
appears in the vast majority of F3 generation males, to study the
molecular etiology of male infertility. Observations demonstrate a
transgenerational effect on the Sertoli cell transcriptome and
epigenome that impacts critical cellular processes involved in testis
function. The pyruvate/lactate pathway is critical for spermato-
genesis and the transgenerational Sertoli cell transcriptome
suggests that abnormal pyruvate/lactate production could directly
promote the spermatogenic cell apoptosis observed. Further
studies are needed to directly test this mechanism, but previous
Figure 7. Example of an epigenetic control region (ECR) on
chromosome 10. The chromosomal location in megabases is
presented with all genes (horizontal lines) and regulated genes (gene
symbols) listed. The DMR are listed in arrow heads.
Figure 8. Gene interactions between DMR and differentially
expressed genes in F3 generation vinclozolin lineage Sertoli
cells. The white genes are DMR associated genes and the colored
genes differentially expressed genes.
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literature supports the critical role of pyruvate/lactate in
spermatogenic cell viability. A correlation of the transgenerational
Sertoli cell DMR and differentially expressed genes with
previously identified genes associated with male infertility
[55,56] identified nine correlated genes. Therefore, in addition
to the abnormal pyruvate/lactate metabolism the abnormal
expression of these genes is known to be associated with male
The molecular etiology of male fertility identified suggests
environmental toxicant exposure of a gestating female at the
critical period of gonadal sex determination promotes an
abnormal programming of the germ line epigenome (DNA
methylation) that is transmitted transgenerationally to subsequent
generations and promotes adult onset testis disease. This is
correlated to abnormal Sertoli cell function and reduction in
pyruvate/lactate production, as well as other critical gene
expression abnormalities, to promote spermatogenic cell apoptosis
and male infertility. The degree environmental induced epigenetic
transgenerational inheritance of testis disease is associated with
human male infertility now needs to be assessed. The rat model
used clearly demonstrates that these exposures and molecular
events are associated with the high incidence of male infertility
transgenerationally. Combined observations clearly establish the
role of transgenerational alterations in a somatic cells transcrip-
tome and epigenome as a likely component of the etiology of
environmentally induced epigenetic transgenerational inheritance
of adult onset disease.
Materials and Methods
Animals and Treatments
Hsd:Sprague DawleyHTMSDHTMfemale and male rats of an
outbred strain (Harlan) were maintained in ventilated (up to 50 air
exchanges/hour) isolator cages (cages with dimensions of 10 L’’
W619 J ‘‘D610 L’’ H, 143 square inch floor space, fitted in
Micro-vent 36-cage rat racks; Allentown Inc., Allentown, NJ)
containing Aspen Sani chips (pinewood shavings from Harlan) as
bedding, on a 14 h light: 10 h dark regimen, at a temperature of
70 F and humidity of 25% to 35%. Rats were fed ad libitum with
standard rat diet (8640 Teklad 22/5 Rodent Diet; Harlan) and ad
libitum tap water for drinking. At pro-estrus as determined by daily
vaginal smears, the female rats (90 days of age) were pair-mated
with male rats (120 days). On the next day, the pairs were
separated and vaginal smears were examined microscopically. If
sperm were detected (day 0) the rats were tentatively considered
pregnant. Vaginal smears were continued for monitoring diestrus
status in these rats until day 7. Pregnant rats for the treatment
group (six different gestating females for each group) were given
daily intraperitoneal injections of vinclozolin (100 mg/kg BW/d;
Chem Service, West Chester, PA) and an equal volume of sesame
oil (Sigma) on days E8 through E14 of gestation; Vinclozolin was
dissolved in DMSO (Sigma). Pregnant rats for the control group
were given daily intraperitoneal injections of DMSO (100 ul/kg
BW/d) and an equal volume of sesame oil (Sigma) on days E8
through E14 of gestation . The pregnant female rats treated
with vinclozolin were designated as the F0 generation. All
experimental protocols for the procedures with rats were pre-
approved by the Washington State University Animal Care and
Use Committee (IACUC approval # 02568-029).
Breeding for F1, F2, and F3 Generations
The offspring of the F0 generation were the F1 generation. The
F1 generation offspring were bred to other F1 animals of the same
treatment group to generate an F2 generation and then F2
generation animals bred similarly to generate the F3 generation
animals. No sibling or cousin breeds were performed so as to avoid
inbreeding. Note that only the original F0 generation pregnant
females were injected with vinclozolin or vehicle. The animals
within a group were bred to optimize the transgenerational
Measurement of Testicular Apoptotic Cells by TUNEL
Testis sections were examined by terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) assay (in
situ cell death detection kit, Fluorescein, Roche Diagnostics,
Mannheim, Germany) as per the manufacturer’s protocols.
Sections were deparaffinized and rehydrated through alcohol
series. They were deproteinized by Proteinase K (20 mg/ml;
Invitrogen, Carlsbad, CA), washed with PBS and then 25 ml of the
enzyme-label solution mix was applied and incubated at 37uC for
90 min. After PBS washes, slides were mounted and kept at 4uC
until examination with a fluorescent microscope using dark field.
Both testis sections of each slide were microscopically examined to
identify and to count apoptotic germ cells by the bright
Sertoli Cell Preparation
Sertoli cells were isolated from the testes of 20-day-old rats (P20)
using a sequential enzymatic digestion procedure previously
described . This pubertal period allows the optimum purity
cells prior to disease onset. Three pools of P20 Sertoli cells were
produced per treatment, with each pool containing cells from 2 to
6 animals. In brief, decapsulated testes were minced with razor
blades and then tissue fragments were digested with trypsin
(1.5 mg/ml, Life Technologies, Gaithersburg, MD) to remove the
interstitial cells. This was followed by incubation with collagenase
(1 mg/ml type I, Sigma) for removal of peritubular cells and then
hyaluronidase (1 mg/ml, Sigma) for removal of germ cells. The
purity of the Sertoli cell preparations were determined by
immunohistochemistry to be .98% . Final Sertoli cell pellets
Figure 9. DMR and differentially expressed genes that
correlate with male infertility/testis disease. The genes identified
in the literature associated with male infertility and testis disease that
correlate with F3 generation vinclozolin lineage DMR and differentially
expressed genes are presented.
Epigenetic Transgenerational Male Infertility
PLOS ONE | www.plosone.org9 March 2013 | Volume 8 | Issue 3 | e59922
were then resuspended in 1 ml TrizolTM(Invitrogen) for further
RNA and DNA extractions.
RNA Extraction and Microarray Transcriptome Analysis
Messenger RNA was isolated using the TrizolTM(Invitrogen)
method as per the manufacturer protocol. Messenger RNA was
independently extracted from 3 pools of Sertoli cells (i.e. 3
biological replicas) per treatment. The mRNA processing and
hybridizations were performed at the Genomics Core Laboratory,
Center for Reproductive Biology, Washington State University,
Pullman, WA using standard Affymetrix reagents and protocol.
Briefly, mRNA was transcribed into cDNA with random primers,
then cRNA was transcribed from the cDNA, and from that, single-
stranded sense DNA was synthesized which was fragmented and
labeled with biotin. Biotin-labeled fragmented ssDNA was then
hybridized to the Rat Gene 1.0 ST microarrays containing more
than 27,000 transcripts (Affymetrix, Santa Clara, CA, USA).
Hybridized chips were scanned on an Affymetrix Scanner 3000.
CEL files containing raw data were then pre-processed and
analyzed with Partek Genomic Suite 6.5 beta software (Partek
Incorporated, St. Louis, MO) using an RMA and GC-content
adjusted algorithm (Supplemental Figure S1). The signals from an
average of 28 different probes for each transcript were compared
to give a single value. Two-way ANOVA was performed between
the Sertoli cell transcriptomes from vinclozolin-lineage and
controls. One factor of variation was treatment and the other
was batch effect. Corrections were made for Sertoli cell
preparation date batch effect by the Partek software according
to the Methods of Moments . The selection of the gene
expression change was based on the expression change between
vinclozolin and control lineage Sertoli cells limited to p-values
,0.05, expression fold change .1.2, and the mean difference
between vinclozolin and control un-logged signals .10. A higher
stringency cut off (.1.5 fold change) was not utilized since many
biological effects are observed with 20% alterations in gene
expression so a more genome wide view of the transcriptome is
identified with the stringency utilized. CEL files from this study
have been deposited with the NCBI gene expression and
hybridization array data repository (GEO, http://www.ncbi.nlm.
nih.gov/geo, GEO # pending) and can be also accessed through
www.skinner.wsu.edu. For gene annotation, the Affymetrix
annotation file RaGene1_0stv1.na31.rn4.transcript.csv was used
unless otherwise specified.
Pathway and Gene Network Analysis
Known functional relationships among the F3 generation
differentially expressed genes were identified using the KEGG
pathways from the University of Kyoto (Japan) Encyclopedia for
Genes and Genome website (http://www.genome.jp/kegg/) and
Pathway Express (http://vortex.cs.wayne.edu) . Functional
relationships among the F3 generation differentially expressed
genes and genes with changes in DNA methylation were
interrogated using Pathway Studio software (Ariadne, Genomics
Inc. Rockville MD), using an unbiased, automated survey of
published scientific literature (Global Literature Analysis). This
analysis identifies functional relations among genes, such as direct
binding, up-regulation or down-regulation and also builds sub-
networks of genes and cellular processes based on their inter-
DNA Extraction and Methylated DNA
DNA was isolated using the TrizolTM(Invitrogen) method as
per the manufacturer protocol, from the same Sertoli cell TrizolTM
preparations that were used for RNA isolations. Therefore, three
independent DNA TrizolTMfractions from Sertoli cells per group
were used to obtain three different biological replicates of DNA
samples from each of the two treatment groups. Each of these
DNA samples were then used for methylated DNA immunopre-
cipitation (MeDIP). MeDIP was performed as follows: 6 mg of
genomic DNA was subjected to a series of three 20 pulse
sonications at 20% amplitude. The appropriate fragment size
(200–1000 ng) was verified through 2% agarose gels. The
sonicated genomic DNA was resuspended in 350 ul TE and
denaturated for 10 min at 95uC and then immediately placed on
ice for 5 min; 100 ul of 5X IP buffer (50 mM Na-phosphate pH7,
700 mM NaCl, 0.25% Triton X-100) was added to the sonicated
and denatured DNA. An overnight incubation of the DNA was
performed with 5 ug of antibody anti-5-methylCytidine monoclo-
nal from Diagenode S.A (Denville, NJ) at 4uC on a rotating
platform. Protein A/G beads from Santa Cruz (Santa Cruz, CA)
were prewashed on PBS-BSA 0.1% and resuspended in 40 ul 1X
IP buffer. Beads were then added to the DNA-antibody complex
and incubated 2 h at 4uC on a rotating platform. Beads bound to
DNA-antibody complex were washed 3 times with 1 ml 1X IP
buffer; washes included incubation for 5 min at 4uC on a rotating
platform and then centrifugation at 6000 rpm for 2 min. Beads-
DNA-antibody complex were then resuspended in 250 ul diges-
tion buffer (50 mM Tris HCl pH 8, 10 mM EDTA, 0.5% SDS)
and 3.5 ul of proteinase K (20 mg/ml) was added to each sample
and then incubated overnight at 55uC on a rotating platform.
DNA purification was performed first with phenol and then with
chloroform:isoamyl alcohol. Two washes were then performed
with 70% ethanol, 1 M NaCl and glycogen. MeDIP selected DNA
was then resuspended in 30 ul TE buffer. Whole-genome
amplification was then performed with the WGA2 kit (Sigma-
Aldrich) on each MeDIP sample to be used in the microarray
comparative hybridization analysis.
Tilling Array and MeDIP-Chip Bioinformatic and Statistical
Roche Nimblegen’s Rat DNA Methylation 36720 K CpG
Island Plus RefSeq Promoter Array was used, which contains three
identical sub-arrays, with 713,670 probes per sub-array, scanning
a total of 15,287 promoters (3,880 bp upstream and 970 bp
downstream from transcription start site). Probe sizes range from
50–75 mer in length with the median probe spacing of 100 bp.
Three different comparative (amplified MeDIP vs. amplified
MeDIP) hybridizations experiments included in three sub-arrays
were performed by Nimblegen. Each comparative hybridization
experiment contained one biological replicate of Sertoli cell Whole
Genome Amplified-MeDIP-DNA sample from each lineage-
treatment. Samples from experimental groups were labeled with
Cy3 and MeDIP DNA samples from the control groups were
labeled with Cy5. For each comparative hybridization experiment,
raw data from both the Cy3 and Cy5 channels were imported into
R (R Development Core Team (2010), R: A language for statistical
computing, R Foundation for Statistical Computing, Vienna,
Austria. ISBN 3-900051-07-0, URL http://www.R-project.org),
checked for quality and converted to MA values (M=Cy5-Cy3;
A=(Cy5+Cy3)/2). The following normalization procedure was
conducted. Within each array, probes were separated into groups
by GC content and each group was separately normalized,
Epigenetic Transgenerational Male Infertility
PLOS ONE | www.plosone.org10 March 2013 | Volume 8 | Issue 3 | e59922
between Cy3 and Cy5 using the loess normalization procedure.
This allowed for GC groups to receive a normalization curve
specific to that group. After each array had its CG groups
normalized within the array, the arrays were then normalized
across arrays using the A quantile normalization procedure.
Following normalization each probe within each array was
normalized and M values were replaced with the median value
of all probe normalized M values across all arrays within a 600 bp
window. If the number of probes present in the window was less
than 3, no value was assigned to that probe. Each probe’s A values
were likewise normalized using the same procedure. Following
normalization each probe’s M value represents the median
intensity difference between vinclozolin generation and control
generation of a 600 bp window. Significance (p,1025) was
assigned to probe differences between treatment-generation
samples and control generation samples by calculating the median
value of the intensity differences as compared to a normal
distribution scaled to the experimental mean and standard
deviation of the normalized M. A Z-score and P-value were
computed for each probe from that distribution. The statistical
analysis was performed in pairs of comparative IP hybridizations
between treatment-lineage (T) and control-lineage (C). T1-C1 and
T2-C2 gave 715 sites; T1-C1 and T3-C3 gave 633 sites; T2-C2
and T3-C3 gave 807 sites. In order to assure the reproducibility of
the candidates obtained, only the candidates showing significant
changes in all three of the paired comparisons were chosen as
having a significant change in DNA methylation between the
experimental group and controls. This is a very stringent approach
to select for changes, since it only considers those changes repeated
in all paired analyses.
Clustered Regions of interest were then determined by
combining consecutive probes within 600 bases of each other,
and based on whether their mean M values were positive or
negative, with significance p-values less than 1025. The statistically
significant differential DNA methylated regions were identified
and p-value associated with each region presented. Each region of
interest was then annotated for gene and CpG content. This list
was further reduced to those regions with an average intensity
value exceeding 9.5 (log scale) and a CpG density $1 CpG/
100 bp. The web-based tool FIMO (Found Individual Motifs
Occurrences) was used for determining the incidence of motifs in
sets of sequences .
Chromosomal Location of Gene Expression Clusters
An R-code was developed to find chromosomal locations of
ECRs (Figure 8). A 2 megabase sliding window with 50,000 bases
interval was used to find the associated genes in each window.
Then a Z-test statistical analysis with p,0.05 was used on these
windows to find the ones with over-representation of differentially
expressed genes. The consecutive windows with over-represented
genes were merged together to form clusters of genes which we
named ECR regions. Typical ECR regions ranged from 2–5
box plot for The F3 generation control and vinclozolin
lineage Sertoli cell samples. Array data was pre-processed
with RMA and GC-content adjusted algorithm in Partek GS
program. The y-axis presents the new hybridization signal and box
plots the mean 6 SEM.
Microarray histograms for each array and
impacted by differentially expressed genes from KEGG
(see Methods). a) Proteosome, b) Nucleotide excision repair, c)
MTOR signaling pathway, d) RNA transport.
Cellular signaling and process pathways
MeDIP for selected genes. The fold change (2‘-deltaC+)
between the control and vinclozolin lineage Sertoli cell MeDIP
samples is presented with the black bars indicating samples with a
significant difference (p,0.05).
Quantitative PCR of F3 generation Sertoli cell
ation vinclozolin lineage Sertoli cells as compared to
control lineage cells (416 genes & ESTs).
Differentially expressed genes from F3 gener-
in F3 generation vinclozolin lineage Sertoli cells.
Differential DNA methylation regions (DMR)
generational DMR and expression changes in Sertoli
Relations between vinclozolin-induced trans-
expression and their relation to DMR in vinclozolin-
lineage F3 Sertoli cells.
Clusters of transgenerational changes in gene
We thank the expert technical assistance of Dr. Ingrid Sadler-Riggleman,
Ms. Rebecca Tracey, and Dr. Mohan Manikkam. We thank Ms. Heather
Johnson for assistance in preparation of the manuscript.
Edited the manuscript: MKS CGB MS MH EN. Conceived and designed
the experiments: MKS. Performed the experiments: CGB MS MH EN.
Analyzed the data: MKS CGB MS MH EN. Wrote the paper: MKS CGB.
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