Metabolomics reveals a role of betaine in prenatal DBP exposure-induced epigenetic
transgenerational failure of spermatogenesis in rats
Beilei Yuan, ‡,§, ¶, † Wei Wu, ‡,§,llll, †, * Minjian Chen, ‡,§, † Hao Gu, ‡,§ Qiuqin Tang, || Dan Guo, ‡,§
Ting Chen, lll Yiqiu Chen, ‡,§ Chuncheng Lu, ‡,§ Ling Song, ‡,§ Yankai Xia, ‡,§ Daozhen Chen, llll
Virender K. Rehan, # Jiahao Sha, ** and Xinru Wang‡,§, *
‡
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical
University, Nanjing 211166, China
§
Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health,
Nanjing Medical University, Nanjing 211166, China
¶
College of Safety Science & Engineering, Nanjing Tech University, Nanjing 210009, China
||
State Key Laboratory of Reproductive Medicine, Department of Obstetrics, Obstetrics and
Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing 210004, China
lll
Nanjing Maternal and Child Health Medical Institute, Obstetrics and Gynecology Hospital
Affiliated to Nanjing Medical University, Nanjing 210004, China
State Key Laboratory of Reproductive Medicine, Wuxi Maternal and Child Health Care
llll
Hospital Affiliated to Nanjing Medical University, Wuxi 214002, China
#
Department of Pediatrics, Los Angeles BioMedical Research Institute at Harbor-UCLA
Medical Center, Torrance, CA 90502-2006, USA
**
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 211166,
China
†
These authors contributed equally to the study and they should be regarded as joint first authors.
*
To whom correspondence should be addressed:
Wei Wu Ph.D. and Xinru Wang Ph.D.
© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.
All rights reserved. For permissions, please email: [email protected]
State Key Laboratory of Reproductive Medicine, Institute of Toxicology,
Nanjing Medical University, 101 Longmian Avenue, Nanjing 211166, China.
Tel: +86-25-86868425 Fax: +86-25-86868499
Email: [email protected]; [email protected]
2
ABSTRACT
There is increasing concern that early-life exposure to endocrine disruptors affects male
offspring reproduction. However, whether di-n-butyl phthalate (DBP), a widely used endocrine
disruptor, has transgenerational effects and, if so, the exact underlying molecular mechanisms
involved remain unknown. In our study, five of time-mated pregnant SD rats were exposed to 0
and 500 mg/kg DBP with corn oil as the vehicle via oral gavage from embryonic days (E8 to
E14). Epigenetic and metabolomic of testis were analyzed after postnatal 60 days. Sperm and
testicular cell functions were examined to confirm the transgenerational effects. DBP exposure
significantly decreased the sperm counts in F1 through F3 generation. We found distinct
metabolic changes in the testis of both F1 and F3 generation offspring, specifically, a
significantly increased level of betaine, which is an important methyl donor. In contrast, the
expression of betaine homocysteine S-methyltransferase (BHMT), which catalyzes the transfer
of methyl moiety from betaine to homocysteine, significantly decreased. There was
accompanying global DNA hypomethylation, along with a reduction in follistatin-like 3 (Fstl3)
promoter hypomethylation, which is a known modulator of Sertoli cell number and
spermatogenesis. In summary, we conclude that metabolomic and epigenetic changes induced by
the aberrant expression of BHMT represent a novel mechanism linking in utero DBP exposure to
transgenerational spermatogenesis failure.
Key words: Di-n-butyl phthalate; Spermatogenesis; Metabolomics; Betaine; Betaine
homocysteine S-methyltransferase.
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INTRODUCTION
Di-n-butyl phthalate (DBP) is a ubiquitous chemical that is widely used in the manufacture of
polyvinyl chloride plastics, latex adhesives, cosmetics, personal care products, cellulose plastics,
and solvent for dyes (Heudorf et al., 2007;
Jia et al., 2016). Widespread use of DBP-containing
consumer products has led to omnipresent exposure to DBP in general population. It can be
detected in air, soil and aquatic ecosystems (Shen et al., 2011). DBP is one of the endocrine
disruptors released into the environment that can interfere with reproductive and neural
development in individuals exposed in utero (Lien et al., 2015;
Rodriguez-Sosa et al., 2014).
In utero exposure to high doses of DBP (greater than 500 mg/kg/day) in the developing rat
causes reduced fetal survival, low birth weight, and impaired steroidogenesis by the fetal testis
and reproductive abnormalities in the surviving offspring (Drake et al., 2009;
2002;
Mylchreest et al.,
Wilson et al., 2009). Environmental exposures during embryonic gonadal development
and sex determination periods are capable of inducing adult onset disease states that can be
perpetuated across multiple generations (Martinez et al., 2014;
Taki et al., 2014). Previous
studies with DBP have primarily focused on F0 and F1 generations (Aly et al., 2015;
Heng et
al., 2012). However, since DBP affects germ cell number, differentiation, and aggregation in the
human and rat testis, it has potential implications for intergenerational effects (van den Driesche
et al., 2015). Transgenerational phenotypes by definition exclude direct exposure and need to
transmitted to at least F3 generation (Skinner, 2011). During the F0 exposure, F1 generation
fetus and the germ line which will develop into F2 generation are both exposed. However, F3
generation eliminates the possibility of any direct exposure effects. Therefore, a phenotype in the
F3 generation is required to have a transgenerational phenomenon (Skinner, 2008;
Skinner et
al., 2010). Whether DBP exposure results in transgenerational spermatogenesis failure and, if so,
4
the potential underlying molecular mechanisms involved remain unknown.
Metabolomics is an emerging “omics” technology which allows the simultaneous, analysis of
various metabolites within a given sample (Stewart and Bolt, 2011), providing unique insights to
health and disease at both the cellular and organismal levels (Weiss and Kim, 2012). It aims at
identifying unique fingerprints of specific disturbances, for example, diseases or effects of
exposure to toxic compounds. The metabolome has been claimed to be the ‘best indicator of an
organism’s phenotype’ (Blow, 2008). Our previous work has highlighted a clear relationship
between metabolites of environmental chemicals and male infertility (Xia et al., 2013), further
highlighting metabolomics to be a promising tool to study the effects of various chemical
exposures on spermatogenesis (Shen et al., 2013;
Xu et al., 2014). Here, we rationalized that
metabolomics might hold a key to accurately study the transgenerational spermatogenesis failure
caused by DBP.
Betaine is critical for embryonic and fetal development. Betaine functions primarily as a
methyl donor substrate in one-carbon metabolism to convert homocysteine to methionine by
betaine homocysteine methyltransferase (BHMT). Methionine then converts to
S-adenosylmethionine (SAM), the universal methyl donor for DNA and protein methylation
processes that are essential for epigenetic gene regulation (Zhao et al., 2017). Restriction of
methyl donors in pregnant animals can change offspring’s DNA methylation (Sinclair et al.,
2007). Epigenetic phenomenon such as DNA methylation is a potential vector between the
environment and multigenerational/transgenerational effects (Zhao et al., 2016). In particularly,
during the embryonic gonadal development period, germ cells undergo remethylation, rendering
this stage to be especially vulnerable to environmental influences (Rodgers and Bale, 2015). In
male rodent models, exposure to nutritional challenges, drugs of abuse, or social stresses support
5
the transmission of paternal experiences to offspring through epigenetic marks in germline
(Lambrot et al., 2013;
Skinner et al., 2013). Spermatogenesis is essential for the transmission
of parental information and the maintenance of species continuity. Sertoli Cells is a major target
for hormonal signaling and provides physical and nutritional support to developing germ cells
(Reis et al., 2015). The Sertoli cells cytoplasm extends around germ cells, providing physical
support and creating a suitable ionic (Alves et al., 2014) and metabolic environment for
spermatogenesis occurrence (Rato et al., 2012). Thus, the Sertoli cell injury is a key point of the
molecular mechanisms that regulate spermatogenesis.
In this study, we aimed to identify potential metabolic and epigenetic changes in testis across
three generations following DBP exposure to only F0 generation dams. To our knowledge, this is
the first study demonstrating metabolomic and epigenetic mechanisms to explain DBP-induced
transgenerational spermatogenesis failure.
MATERIALS AND METHODS
Experimental Animal. Adult Sprague-Dawley rats were purchased from Vital River
Laboratory Animal Technology Co. Ltd. All animals were housed in temperature-controlled
rooms with 12-h light, 12-h dark cycles and given free access to water and feed. After two weeks,
the female was mated with males, and the pregnant dams were administered daily by oral gavage
500 mg/kg of DBP (99% pure; Sigma-Aldrich) or vehicle (corn oil) from E8-E14. DBP was
dissolved in corn oil (Sigma, USA) at 100 mg/ml. We gave 1 ml stock solution per 200g weight
of rat to make sure the oral gavage concentration is 500 mg/kg. The DBP solution was stored at
room temperature in the dark until it was ready to use. The number of animal in each F0 group
was five. Gestating rat dams (designated F0 generation) were transiently exposed to DBP during
the embryonic gonadal sex determination period (E8-E14). Subsequently, F1, F2, and F3
6
generation progeny from the control and DBP-treated F0 mothers were produced. The control
and DBP group animals were maintained under similar conditions (i.e., feeding, housing, room
temperature, and other environmental conditions), but were kept in different cages. F1 control (C)
and DBP-treated (T) males were bred with females from the same group before sperm isolation.
Offspring of these pregnancies were designated as F2C (F2 offspring of control males) and F2T
(F2 offspring of treated males). In addition, DBP F2 generation were crossed with wild-type
untreated control, the descendant of wild type mated with F2 treated males called TM, which
mated with treated F2 females called TF (Figure 1A). All tissues of male rats from control and
treated groups were collected after postnatal 60 for analyses. Of note, only the F0 generation
dams were treated with DBP. No inbreeding or sibling crosses were generated. This study was
carried out strictly in accordance with the international standards on animal welfare and the
guidelines of the Institute for Laboratory Animal Research of Nanjing Medical University.
Epididymal sperm collection, testis histology and serum hormone level determination. The
cauda epididymis was collected from male rats after postnatal 60 days. The tissue was placed in
1 ml M199 culture medium containing 0.1% bovine serum albumin for 5 minutes at 37°C to
release the sperm, and then measured by IVOS sperm Analyzer (HamiltonThorne Biosciences).
Testis was collected and fixed in 4% paraformaldehyde, then processed for paraffin embedding
by standard procedures for histopathology examination. Then stained with hematoxylin and
eosin and examined for histopathology. Blood samples were collected at the time of dissection,
allowed to clot, centrifuged and serum samples stored for steroid hormone assays. The hormone
radioimmunoassays (RIA) were performed by Beijing north institution of biological technology.
Immunohistochemistry analysis. IHC analysis of tissues from at least three rat for each group
was performed using an UltraSensitiveTM SAP (Mouse/Rabbit) IHC Kit (Fuzhou Maixin
7
Biotech. Co., Ltd.), as recommended, using antibodies to WT1 (Abcam, ab89901). We randomly
chose 10 tubules of the section, counted the sum of Sertoli cells and then counted the average
number of each tubule. The IHC procedure was performed as described previously (Xu, Chen, Ji,
Mao, Zhang, Wang and Xia, 2014).
Metabolomic profiling. The testes of F1 and F3 generation rats were stored at -80°C until
analysis. The sample preparation for liquid chromatography–high resolution mass spectrometry
(LC-HRMS)-based metabolomic analysis was conducted according to our previous report (Chen
et al., 2012). Every 100 mg of the testis was mixed with 300 µl cold double-distilled water and
1200 µl methanol. Then the mixture was ultrasonicated at 50% power for 6 minutes, and next
was centrifugalized at 16,000 g/min at 4°C for 15 min. Then 200 µl supernatant was mixed with
20 µl internal standards Progesterone-2,2,4,6,6,17α,21,21,21-d9 solution. After dryness, the
sample was reconstituted in 20 µl 50/50 methanol/water (V/V). Quality control (QC) samples
were prepared by mixing equal volumes (100 µl) from each original sample and were analyzed
in parallel with the testis samples. The metabolomic analysis was conducted according to our
previous report (Xu et al., 2015). Briefly, it was performed on a UPLC Ultimate 3000 system
(Dionex, Germering, Germany), coupled to an Orbitrap HRMS (Thermo Fisher Scientific,
Bremen, Germany) in both positive and negative modes simultaneously. The chromatographic
separation was performed on a 1.9 µm Hypersile Gold C18 column (100mm×2.1mm) (Thermo
Fisher Scientific), and the column was maintained at 40°C. A multistep gradient had mobile
phase A of 0.1% formic acid in ultra-pure water and mobile phase B consisting of acetonitrile
(ACN) acidified with 0.1% formic acid; the gradient operated at a flow rate of 0.4 ml/min over a
run time of 15 min. The UPLC autosampler temperature was set at 4°C and the injection volume
for each sample was 5 µl. All samples were analyzed in a randomized fashion to avoid
8
complications related to the injection order. MS data were collected by the HRMS with a heated
electrospray source at the resolution of 700000. For both positive and negative modes, the
operating parameters were as follows: a spray voltage of 3.5 kV for positive, 2.5 kV for negative,
the capillary temperature of 300°C, sheath gas flow of 50 arbitrary units, auxiliary gas flow of 13
arbitrary units, sweep gas of 0 arbitrary units and S-Lens RF level of 60. In the full scan analysis
(70 to 1,050 amu) with an automatic gain control (AGC) target of 3×106 charges and a maximum
injection time (IT) of 120 ms. The mass spectrometry was calibrated according to manufacturer’s
instructions to ensure the mass accuracy. The metabolite identification was based on searching
against in-house generated authentic standard library, which includes retention time and accurate
mass. We found the >70% of differential metabolites in QC sample had a %RSD of less than 30%
and the internal standard had a %RSD of 28.5%, indicating the metabolomic analysis was
reliable (Gika et al., 2008).
RNA extraction and qRT-PCR. Total RNA was extracted from testis using TRIzol Reagent
(Invitrogen Life Technologies Co, USA), following manufacturer's protocol, as described
previously (Wu et al., 2012). Three steps of phenol/chloroform purification were added to get rid
of proteins. UV-absorbance of RNA solutions was measured at 260 and 280 nm to measure RNA
content and quality of each sample and its quality was further verified by denaturing agarose gel
electrophoresis. qRT-PCR was carried out using manufacturer's recommendation, as described
previously (Wu, Hu, Qin, Dong, Dai, Lu, Zhang, Shen, Xia and Wang, 2012). The primer
sequences of mRNAs are shown in Supplementary Table 1.
RNA sequencing. The testis of the F3 generation rats were flash-frozen in liquid nitrogen and
then stored at -80°C after about 4 h. Total RNA was extracted and treated with the Ribo-Zero™
rRNA Removal Kit (Cat. No. RZH1046- 6 Reactions) to remove all rRNAs. The remaining
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RNAs were processed using the TruSeq RNA Sample Prep Kit v2 according to the Illumina
protocol. RNA-Seq and bioinformatics analysis to identify candidate fusion transcripts were
performed on three independent samples per group. Libraries were prepared according to
RiboZero library and sequenced on HiSeq 2500 (Illumina). RNA-Seq data were analyzed using
TopHat and Cufflinks (Ghosh and Chan, 2016). Sequences were identified by comparison
against the positions of mRNA from Ensembl (Rno5.0), and also protein-coding and noncoding
genes and repeats using annotation from the Gene ontology (http://geneontology.org/). Pathway
analysis was based on KEGG database (http://www.kegg.jp/).
DNA extraction, bisulphite treatment and methylation-specific PCR. DNA was isolated from
testicular tissues using Phenol-Chloroform following manufacturer’s recommendations (Shen,
Wu, Du, Liu, Yu, Sun, Han, Jiang, Shi, Hu, Song, Xia, Wang and Wang, 2011). Bisulfite
conversion of 1 µg of all genomic DNA was achieved using a kit (EpiTect Bisulfite Kits; Qiagen.
USA) following the manufacturer’s recommendations. Thereafter, 1 µl was amplified in a
methylation specific PCR (MSP). The following Fstl3 primers were used to detect the
methylated (M) or unmethylated (U) alleles of Fstl3 promoter: for methylated alleles, Fstl3-MF
5ʹ-GTATTTTTTAGAATATGTTTTTCGG-3ʹ and Fstl3-MR
5ʹ-GAAAACTCAACGAATACAAACG-3ʹ; for unmethylated alleles, Fstl3-UF
5ʹ-GTATTTTTTAGAATATGTTTTTTGG-3ʹ and Fstl3-UR
5ʹ-CAAAAACTCAACAAATACAAACACC-3ʹ. Each of the DNA samples was amplified by
PCR as follows: a PCR reaction mix containing 5µl of the bisulfite-treated DNA,0.5 mM each of
forward and reverse primers, 200 mM dNTPs, 1 × PCR buffer, 1.25 U of Ex Taq Hot Start DNA
Polymerase (Takara Bio, Tokyo, Japan) in a total volume of 25µl. After activation of the
polymerase at 95°C for 10 min, followed by 40 cycles of the following sequence: 30 s at 95°C,
10
30 s at 51.9°C, 1 min at 72°C and final extension at 72°C for 10 min. The amplified PCR product
was then run on a 2% low-range ultra-agarose gel with ethidium bromide and then visualized
using ultraviolet light.
Western blot. Western blotting was performed according to standard methods. Protein
concentrations were estimated using a BCA Protein Assay Kit (Beyotime, China). Equal
amounts (80 µg) of total protein were applied to SDS-PAGE. After electrophoresis, transferred to
polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk powder for 1
hour, the membranes were then probed with specific primary antibodies and incubated at 4°C
overnight: BHMT (1:500, Santa Cruz, sc-69708), GAPDH (1:1000, AG019). After washing three
times, the membranes were incubated with a HRP-conjugated secondary antibody diluted in 5%
milk in PBS with 0.05% Tween-20, and labelling was detected using enhanced
chemiluminescence.
Enzyme-linked immunosorbent assay (ELISA). Testis tissue was homogenized in nine
volumes of cold phosphate-buffered saline (PBS; 0.01M, pH = 7.4) with a homogenizer on ice.
To further break the cells, the homogenate was sonicated with an ultrasonic cell disruptor. The
homogenate was then centrifuged at 5000g for 5 min to get the supernatant. The levels of BHMT
(CSB-EL002693RA), FSTL3 (CSB-EL009026RA) (Elabscience Biotechnology, Wuhan, China)
in the testis homogenate supernatants were assayed using commercially available ELISA kit
according to the manufacturer’s protocol. The 5-mC DNA ELISA Kit (ZYMO RESEARCH,
USA) was used to measure the percentage of 5-mC. The procedures were performed according
to the protocol provided by the manufacturer. Average 5-mC of each sample was used in
subsequent analyses.
Cell culture and treatment. TM-4 Sertoli cells were purchased from ATCC (Manassas, VA,
11
USA) and were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C
and 5% CO2 in a humidified incubator. The cells were plated onto 6-, or 96-well plates, treated
for 24h and subsequently incubated with different concentrations of 5-Aza-dC (Sigma-Aldrich) 0,
0.1, 0.5, 1 µM for 48 h.
Statistical analysis. Statistical analysis of the data was performed using SPSS version 15.0
(SPSS, Inc., Chicago, IL). Results are expressed as mean ± SEM. Significance was evaluated
using the unpaired Student t test. P value <0.05 was considered to be a statistically significant
difference. The normalized metabolomic data were imported into SIMCA-P software (Umetrics,
Sweden) for multivariate analysis. All the data were UV (unit variance)-scaled and auto
log-transformed when appropriate by the SIMCA-P software. Principal component analysis
(PCA) was applied to find out the difference between sample groups.
RESULTS
Prenatal DBP exposure leads to spermatogenic failure in offspring.
Compared to control rats, the body weights of TM and TF F3 generation rats were
significantly decreased. However, surprisingly, prenatal DBP exposure did not alter the body
weights of the F1 and F2 generation offspring (Supplementary Figure 1 A, B and C).
Spermatogenesis defects were found in DBP exposed F1 to F3 lineages. The number of sperms
deceased to about 50% in lineages of DBP-treated dams compared with the controls (Figure 1B).
However, no significant changes in testis histology were observed between the lineages of
DBP-treated and control rats (Figure 1C). The progeny of DBP-treated dams, mated with wild
type F2 generation dams (TF and TM), revealed no significant changes in sperm count, although
showed a trend towards a slightly decrease (Figure 1B). The amplitude of sperm lateral head
displacement was reduced in DBP-treated F2 and F3 generation animals. The sperm velocity of
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F3 generation was decreased (Supplementary Figure 1D, E and F). In addition, the levels of
serum hormones were evaluated in the adult offspring of different generations. The levels of FSH
changed only in TM dams of F3 generations, and E2 changed only in F2 generation
(Supplementary Figure 2 A and B). When compared with controls, the treated group displayed a
significant decrease in serum concentrations of testosterone in F1 through F3 generation
(Supplementary Figure 2 C). Wt1 (Wilms tumor 1) is specifically expressed in Sertoli cells of the
testis and Wt1 antibody can be employed to identify Sertoli cells. Wt1 positive Sertoli cells were
localized at the periphery of the seminiferous tubules in both treated and control’s testes (Figure
2A). However, the number of Sertoli cells in the control group was twice as much as in the
treated group (Figure 2B). Significant reduction in both testis and epididymis relative weights in
the F2 generation. The epididymis relative weights were slightly lower than the control and testis
relative weights were no significant differences in F1 and F3 generations (Figure 2C, D).
Prenatal DBP exposure alters metabolites in both F1 and F3 generations.
To determine the likely mechanism underlying DBP-induced transgenerational
spermatogenesis failure, we performed a non-targeted metabolomic analysis of F1 and F3
generation testes of the control and DBP exposed animals. In the first stage of analysis, 226
metabolites were profiled in rat testis by using the LC-HRMS platform. A FDR-corrected
P-value threshold of 0.05 was used to account for multiple testing across the inter-correlated
metabolites. The metabolic fingerprints acquired in both F1 and F3 generations were subjected to
PCA analysis and a good discrimination between the control and DBP-treated groups was
observed (Figure 3A and B, Supplementary Table 2), indicating significant metabolic differences
between the two groups. A total of 13 and 56 metabolites showed differences between the groups
in F1 and F3 generations, respectively. Among the 13 altered metabolites in F1 generation, 8
13
were up-regulated and 5 were down-regulated, while 32 were up-regulated and 24 were
down-regulated in F3 generation in DBP-treated groups (vs. controls) (Figure 3C,
Supplementary Table 3 and 4). Of the altered metabolites, 4 were found to be common in F1 and
F3 generations (Table 1); these were 5-Aminovaleric acid, Betaine, Salicylic acid and
Ureidopropionic acid (Figure 3D).
Prenatal DBP exposure disturbs betaine metabolic pathway, including an associated reduction
in global DNA methylation.
Secondary analyses were performed to identify the key enriched metabolic pathway using
MetaboAnalyst (http://www.metaboanalyst.ca/faces/ModuleView.xhtml). Results of the main
analysis identified that betaine metabolism was one of the most over-represented metabolic
pathway that was altered in the experimental group, i.e., DBP exposure in F0 gestation
(Supplementary Figure 3). Betaine promotes homocysteine remethylation, acting as a methyl
donor for the formation of methionine (Figure 4A) (Cai et al., 2014;
Cordero et al., 2013;
Lever and Slow, 2010). We focused on the betaine metabolism since metabolomic analysis
showed notable increase in betaine levels in both F1 and F3 generation experimental (T) animals
(Figure 4B). Nevertheless, no significant difference in betaine levels were observed in TM and
TF groups (Table 1). To evaluate the changes in DNA methylation pattern between the control
and treated offspring, we analyzed the 5-methylcytosine (5-mC) content of genomic DNA as a
percent of 5-mC level by the enzyme-linked immunosorbent assay (ELISA). The level of 5-mC
was significantly decreased in DBP-treated testes of F1 (5.48 ± 0.29 % and 4.70 ± 0.15% (C vs.
T), respectively, P < 0.05) and F3 (5.55 ± 0.09 % and 5.14 ± 0.19% (C vs. T) offspring,
respectively, P < 0.05) generations. However, F2 generation only had a trend towards a decrease
in the treated rats (Figure 4C).
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Prenatal DBP exposure decreases BHMT expression.
To explore the mechanism underlying 5-mC reduction, we examined Bhmt expression in testis
by quantitative real time PCR (qRT-PCR), western blot, and ELISA. Compared with the controls,
DBP-treated group showed significantly down-regulated Bhmt mRNA expression in F2
generation (Figure 4D). The western blotting and ELISA analyses demonstrated significantly
decreased levels of Bhmt in F1-F3 generation of the DBP-treated exposed group (Figure 4E, G).
Prenatal DBP exposure alters transcriptome in offspring testis.
To gain molecular insight into the pathogenesis of DBP-induced transgenerational effects on
spermatogenesis in rats, we examined the mRNA expression profile in the testis of the F3
generation rats using RNA-seq technique. We generated an average of 90, 922, 455 (range: 85
156 370-95 177 358) reads and 7.26 (range: 6.77-7.65) gigabases of sequenced nucleotides per
sample. An average of 84.79% (range: 84.42-85.21%) of the reads mapped to the rat genome
(Ensemble version Rnor_5.0). We identified a range of protein coding RNAs, ncRNAs, and
others that were aberrantly expressed in testes of the DBP-treated F3 generation rats (Figure 5A).
Compared with the transcriptome sequences of the controls, a total of 549 genes changed in the
DBP treated offspring (316 up-regulated and 233 downregulated, P < 0.05) (Figure 5B and D).
Prenatal DBP exposure affects spermatogenesis pathways, with Fstl3 changing significantly
from F1 to F3 generation.
Pathway-based analyses are helpful in identifying biological functions and gene interactions
involved in a pathway affected by an experimental intervention. Based on RNA-seq, the top ten
biological pathways of aberrantly expressed genes are: both negative and positive regulations of
transcription from RNA polymerase II promoter, heart development, positive regulation of
transcription (DNA-dependent), regulation of transcription (DNA-dependent), regulation of
15
transcription from RNA polymerase II promoter, skeletal system development, transcription
(DNA-dependent), spermatogenesis, and in utero embryonic development (Figure 5C). Based on
the enrichment analyses, 11 genes (Apob, Ar, Brca2, Cyp26b1, Foxa3, Fstl3, Lhcgr, Notch1,
Piwil1, Sox9, and Wipf3) were identified to be affected in the spermatogenesis pathway. Six out
of the eleven genes were significantly changed in F3 generation (Figure 6A, Supplementary
Figure 4). The mRNA expression levels of Foxa3 and Fstl3 in treated groups were greater than
two times in testis of control groups from F1, F2 and F3 generations (Figure 6B and C). A
previous study has demonstrated that Fstl3 plays a crucial role in maintaining Sertoli cell
numbers and spermatogenesis (Oldknow et al., 2013). Our results further showed that FSTL3
protein levels were also up-regulated in F1, F2, and F3 generation rats exposed to DBP in F0
gestation (Figure 6D).
Methylation-specific PCR (MSP) analysis showed that there were indeed distinct DNA
methylation in testes of F1 generation at the Fstl3 promoter CpG islands. Compared with
controls, the Fstl3 promoter region were significantly hypomethylated in F1 generation
DBP-treated rats. However, no significant change in Fstl3 promoter region methylation was
observed in the testis of F2 and F3 generations (Figure 6E).
Global methylation results in reduction of TM-4 cell growth.
To determine the consequence of reduction in global DNA methylation on cell proliferation,
we performed an in vitro experiment using a demethylating agent (5-Aza-dC) to inhibit the
global methylation in TM-4 cells, which is an immortalized non-tumorogenic cell line derived
from mouse testis. After 5-Aza-dC treatment, the proliferation of TM-4 cells decreased more
than 20% (Figure 4F).
DISCUSSION
16
In the present study, we focused on how DBP affects the metabolic pathways and epigenetic
inheritance of reduced sperm and Sertoli cell across multiple generations. A general scheme for
these results is summarized in Figure 7. Pregnant dams were exposed to DBP from E8-E14,
which resulted in reduced sperm and Sertoli cell in F1 through F3 generations. Metabolomics
analysis revealed that betaine, a methyl donor, increased in testes of F1 and F3 generations.
BHMT, a methyl donor and a facilitator for converting homocysteine to methionine decreased.
The BHMT reduction likely also explains the observed genome-wide hypomethylation, as well
as the promoter hypomethylation of Fstl3, a key gene that regulates spermatogenesis. These data
for the first time provide the possible metabolic and epigenetic explanation for the
transgenerational effects following in utero DBP exposure.
The most dramatic increase in the incidence of adult onset disease observed was reduced
sperm and Sertoli cell number but otherwise normal histology from F1 to F3 generations.
Toxicological effects following direct DBP exposure have been documented in numerous studies
(Alam et al., 2010;
Moody et al., 2013). Other studies show that a mixture of plastic-derived
compounds, including BPA and phthalates (DBP, DEHP) can promote epigenetic
transgenerational transmission of adult-onset diseases, characterized by an altered
spermatogenesis. The differentially-methylated regions (DMR) can provide potential epigenetic
biomarkers for transgenerational diseases and ancestral environmental exposures (Manikkam et
al., 2013). For example, environmental exposures have been shown to induce an epigenetic
transgenerational phenotype through the male germ line. Intriguingly, our results show the
abnormal phenotype only in the offspring of DBP exposure to both parents, indicating that the
non-exposed parents might have a protective effect, preventing the transgenerational
transmission of DBP effects across generations. In eliciting a transgenerational phenotype, an
17
endocrine disrupting chemical (EDC) such as DBP may affect the germ cell either directly or
indirectly, e.g., by altering the function of supporting cells (Xin et al., 2015). Previous results
showed testicular damage caused by DEHP treatment in neonatal and adult rats, all ages showed
decreased cell numbers at the 2000 mg/kg of DEHP. In the neonatal rats, Sertoli cell numbers
were decreased, giving further evidence of a direct effect of the active metabolite of DEHP on
the Sertoli cell (Dostal et al., 1988). Although neonatal exposure of DEHP did not appear to have
any significant effects on the subsequent fertility of male rats, may have caused subtle effects on
sperm production in adulthood. We observed the same failure in adult rats from fetal life exposed
to DBP. In our present study, we show that the sperm counts decreased in F1 through F3
generation. During fetal life, Sertoli cells are the first somatic elements to differentiate in testis
and are thought to coordinate the differentiation of other testis cell-types (Capel, 2000;
DiNapoli and Capel, 2008), as well as provide an environment conducive for spermatogenesis
(Hess and Renato de Franca, 2008). Most importantly, Sertoli cell metabolism is central to the
maintenance of spermatogenesis and male fertility (Rato, Alves, Socorro, Duarte, Cavaco and
Oliveira, 2012).
It is becoming increasingly clear that metabolomics approaches can reveal global changes in
molecular signatures. Since disease-related metabolic patterns are the consequence of aberrant
biological function, these can provide a direct diagnostic and mechanistic information, e.g.,
underlying toxin-induced infertility (Zhang et al., 2014). DBP can potentially affect offspring’s
metabolism via a number of mechanisms. BHMT catalyzes homocysteine methylation using
betaine as a methyl donor (Zhang et al., 2015). Betaine can transfer the methyl moiety to
homocysteine in the process of forming methionine. BHMT catalyzes homocysteine methylation
using betaine as a methyl donor, though the reduced BHMT levels can theoretically reduce
18
global DNA methylation. Betaine is an important nutrient for humans; it’s obtained mainly from
seafood, wheat, bran, and spinach, and its principal physiologic roles include being an osmolyte
and a methyl donor (Craig, 2004). Several studies indicate that a higher betaine levels can
effectively protect against kidney and liver injuries caused by ethanol, high-fructose and other
toxins (Fan et al., 2014;
Varatharajalu et al., 2014), even protecting against cancer (Nitter et al.,
2014). However, in marine gastropods (abalone), on exposure to DBP, the most obvious
metabolic change was an increase in intracellular betaine levels (Zhou et al., 2015). Our data
indicate that after DBP exposure to only the F0 generation dams, betaine levels increased in both
F1 and F3 generation offspring, possibly secondary to reduced BHMT activity. The reduced
BHMT levels can theoretically explain the reduced global DNA methylation found in our study.
Spermatogenesis is a complex process highly regulated by genes and the abnormality of these
genes may lead to spermatogenesis impairment and male infertility(Massart et al., 2012). Fstl3,
which plays a critical role in regulating spermatogenesis, deletion has been shown to increase
both Sertoli cell and germ cell numbers, along with an associated increase in testicular size
(Oldknow, Seebacher, Goswami, Villen, Pitsillides, O'Shaughnessy, Gygi, Schneyer and
Mukherjee, 2013). Fstl3 binds and alters activities of the transforming growth factor-β (TGF-β)
family of molecules (Brandt et al., 2015;
Hill et al., 2002;
Sidis et al., 2006), suggesting it to
be a local regulator of activin, which in turn is known to be critical in gonadal development and
spermatogenesis (Xia et al., 2004). This is highly relevant since DBP is a known modulator of
TGF-β signaling (Lee et al., 2014).
Methylation of cytosine residues globally and in the promoter regions of specific genes plays a
crucial role in many vital biological processes, in particular, during the embryonic development.
A previous study suggested that changes in DNA methylation might at least partially regulate
19
cell proliferation (Lin et al., 2014). When TM-4 cells were treated with 5-Aza-dC, a DNMT
inhibitor, it decreased cell proliferation. Furthermore, a negative correlation between the
promoter methylation of Fstl3 and its expression in the testis of F1 generation rats was observed.
However, persistence of DBP-induced alteration in DNA methylation in subsequent generations
was not observed. Nonetheless altered gene expression was observed in all generations, i.e., F1
to F3. Such differences in gene expression could reflect the impact of altered methylation during
early development, with subsequent persistence of transcriptional patterns despite DNA
remethylation in later generations. Further investigation to better explain this intriguing
observation is needed.
CONCLUSIONS
In summary, using state-of-the-art metabolic and epigenetic approaches, this study
investigated F1 and F3 generation offspring testis metabolic and epigenetic patterns following in
utero (from E8 to E14) DBP exposure to F0 generation rat dams. Furthermore, several
biologically important observations were made and novel biomarkers of DBP-induced sperm and
Sertoli cell number were identified. Increased testicular betaine levels, but reduced BHMT
activity, accompanied by global and gene (Fstl3)-specific promoter hypomethylation, likely
explains the failure in F1 through F3 generation offspring following DBP exposure in only F0
gestation. These observations provide a novel metabolic and epigenetic mechanistic link for the
in utero DBP exposure-induced transgenerational effect of reduced sperm and Sertoli cell.
ACKNOWLEDGEMENTS
This work was supported by National Natural Science Foundation of China (No. 81673217,
81172696, 81302457, 81401213), and the Priority Academic Program for the Development of
Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).
20
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FIGURE LEGENDS
FIG. 1 Spermatogenesis and testis from F1-F3 generations. (A) Schema of study design. (B)
The number of sperms decreased about 50% in the DBP treated dams from F1-F3 generations.
F1 (control, n=11; treated, n = 12), F2 (control, n = 9; treated, n = 7), F3 (control, n=14; treated,
n = 15; TM, n = 5 TF, n = 5). (C) Morphology was assessed in adult testis by haematoxilin/eosin
staining. Scale bars represent 200 µm. Values are mean ± SEM. * P < 0.05. Student’s t test. See
also Supplementary Figure 1 and 2.
FIG. 2 Sertoli cells express in the testes. The Sertoli cells were labeled with the anti-Wt1
antibody. (A) The nuclei of the Wt1-positive Sertoli cells were localized to the peripheral regions
of the seminiferous tubules in the control and treated testes. (B) The number of Wt1-positive
Sertoli cells decreased in treated testes. The Wt1-positive cell number (mean ± SEM) per
seminiferous tubules was summarized in the graph. (C) The organ coefficient of epididymis (D)
The organ coefficient of testis. Scale bars represent 100 µm. * P < 0.05. *** P < 0.001 Student’s
t test.
FIG. 3 Metabolomics change in F1 and F3. (A) Metabolic profiles depicted by 3D-PCA scores
plots of LC-HRMS. F1 (control, n = 10; treated, n = 10). (B) Metabolic profiles depicted by
3D-PCA scores plots of LC-HRMS. F3 (control, n = 10; treated, n = 10). (C) The 65 metabolic
fingerprints acquired in both F1 and F3 generations, 4 metabolites emerged as particularly
significant changed in both F1 and F3 generations. F1 (control, n = 10; treated, n = 10), F3
(control, n = 10; treated, n = 10). (D) The structure of the four small molecule metabolites. See
also Supplementary Table 2, 3, 4 and 5.
FIG. 4 Metabolomics change in F1 and F3. (A) Metabolomic pathway topology analysis
highlighted the potential importance of distinct pathways that comprise the metabolome that
24
were represented by metabolites associated with DBP treated in the current study. (B) The most
important metabolic substances betaine differentially expressed in both F1 and F3 generations.
F1 (control, n = 10; treated, n = 10), F3 (control, n = 10; treated, n = 10; TM, n = 5 TF, n = 5). (C)
Global 5-methy-cytosine levels in testis of F1-F3 generation male offspring after DBP treatment
(control, n = 10; treated, n = 10) (D) The expression of Bhmt. F1 (control, n = 13; treated, n = 8),
F2 (control, n = 12; treated, n = 12), F3 (control, n = 12; treated, n = 12). (E) Protein expression
by ELISA in F1 (control, n = 10; treated, n = 10), F2 (control, n = 7; treated, n = 8), F3 (control,
n = 12; treated, n = 12). (F) Cell proliferation was analyzed using CCK8 cell proliferation assay
kits. (G) Protein expression by western blot. F1 (control, n = 5; treated, n = 5), F2 (control, n = 5;
treated, n = 5), F3 (control, n = 5; treated, n = 5). All results replicated in three independent
experiments. Values are the mean ± SEM. *P < 0.05, ***P < 0.001. Student’s t test. See also
Supplementary Figure 3.
FIG. 5 Transcriptome profiling analyzed in F3 generation. (A) Biotype distribution of the
different types of genes following treatment with DBP from 8 - 14 day in F3 generation testis
(control, n = 3; treated, n = 3). (B) Numbers distribution of the differentially expressed genes
(control, n = 3; treated, n = 3). (C) The top ten biological pathways of differently expression
genes (control, n = 3; treated, n = 3). (D) Supervised hierarchical clustering analysis using 549
mRNA that were consistently upregulated or down regulated (P ≤ 0.05). Shades of yellow and
blue are used to illustrate whether the expression value is above (yellow) or below (blue) the
mean expression value across all samples (control, n = 3; treated, n = 3).
FIG. 6 Transcriptome profiling identifies spermatogenesis pathway genes differentially
expressed from F1 to F3 generations. RT-PCR was used to analyze genes expression in testis.
(A) Six of eleven spermatogenesis pathway genes were significantly changed in F3 generation.
25
F1 (control, n = 11; treated, n = 10), F2 (control, n = 12; treated, n = 17), F3 (control, n = 12;
treated, n = 12). (B) Fstl3 and Foxa3 mRNA were as the same as F3 generation changed in F2
generations (control, n = 12; treated, n = 17). (C) Fstl3 and Foxa3 mRNA were as the same as
F3 generation changed in F1 generations (control, n = 11; treated, n = 10). (D) The expression of
FSTL3 changed in protein. F1 (control, n = 10; treated, n = 10), F2 (control, n = 7; treated, n = 8),
F3 (control, n = 12; treated, n = 12). (E) Representative results of the MSP analysis of Fstl3.
DNA obtained from the testis was amplified with primers specific to the unmethylated (U) or the
methylated (M) of Fstl3 after treatment with sodium bisulfite. C1-C5 represent control group,
T1-T5 represent treated group. F1 (control, n = 10; treated, n = 10), F2 (control, n = 10; treated, n
= 10), F3 (control, n = 10; treated, n = 10). Values are the mean ± SEM. *P < 0.05. Student’s t
test. See also Supplementary Figure 4.
FIG. 7 Schematic representation of the role of BHMT and betaine in spermatogenesis
pathology of DBP induced transgenerational effects. Pregnancy rats received Di-n-butyl
phthalate (DBP) exposure from E8-E14, causing spermatogenesis persistent changed from F1 to
F3 generation. Metabonomics analysis found betaine which was the donor of the methylation
reaction increased both in F1 and F3 generations. BHMT as a facilitator of methyl group
donation for remethylation of homocysteine into methionine decreased, and reduced functioning
of BHMT could result in decreasing genome-wide total.
Table 1: Metabolites significantly changed both in F1 and F3 generations.
Supplementary Figure 1: Body weight and sperm motility from F1 to F3 generation. (A)
The body weight of F1 generation. (B) The body weight of F2 generation. (C) The body weight
of F3 generation. (D) The sperm motility of F2 generation. (E) The sperm motility of F3
generation. (F) The sperm motility of F3 generation. Amplitude of lateral head displacement
26
(ALH), Beat cross-frequency (BCF), Linearity (LIN), Straightness (STR), Average path velocity
(VAP), Curvilinear velocity (VCL), Straight-line velocity (VSL).
Supplementary Figure 2: Levels of serum hormones from F1 to F3. (A) The level of FSH
from F1 to F3 generation. (B) The level of E2 from F1 to F3 generation. (C) The level of T from
F1 to F3 generation.
Supplementary Figure 3: The metabolism sets enrichment overview. The analyses showed
the most over-represented pathway by using MetaboAnalyst
(http://www.metaboanalyst.ca/faces/ModuleView.xhtml).
Supplementary Figure 4: Expression of spermatogenesis pathway genes in F3 generation.
(A) The expression of Apob. (B) The expression level of Ar. (C) The expression level of Brca2.
(D) The expression level of Foxa3. (E) The expression level of Fstl3. (F) The expression level of
Sox9. (G) The expression level of Cyp26b1. (H) The expression level of Hcgr. (I) The
expression level of Notch1. (J) The expression level of Piwil1. (K) The expression level of
Wipf3.
Supplementary Table 1: List of primers used to test the expression of the genes and the
reference genes.
Supplementary Table 2: Metabolites used for metabolomics analysis.
Supplementary Table 3: Metabolites significantly changed in F1 generation.
Supplementary Table 4: Metabolites significantly changed in F3 generation.
27
Spermatogenesis and testis from F1-F3 generations
88x66mm (300 x 300 DPI)
Sertoli cells express in the testes.
88x54mm (300 x 300 DPI)
Metabolomics change in F1 and F3
88x60mm (300 x 300 DPI)
Metabolomics change in F1 and F3.
177x99mm (300 x 300 DPI)
Transcriptome profiling analyzed in F3 generation
177x94mm (300 x 300 DPI)
Transcriptome profiling identifies spermatogenesis pathway genes differentially expressed from F1 to F3
generations.
177x106mm (300 x 300 DPI)
Schematic representation of the role of BHMT and betaine in spermatogenesis pathology of DBP induced
transgenerational effects.
77x67mm (300 x 300 DPI)
Table 1 | Metabolites significantly changed both in F1 and F3 generations.
Betaine
5-Aminopentanoic acid
Salicylic acid
Ureidopropionic acid
Note: a P < 0.05, b P < 0.01
F1 (n = 10)
fold change
F3 (n = 10)
fold change
TM (n = 5)
fold change
TF (n = 5)
fold change
1.24 a
1.19 a
0.82
0.92
a
a
0.80
0.89
1.53
1.36
11.12
17.18 a
1.25
14.74
20
b
1.24
a
100
b
12.50
a