ORIGINAL RESEARCH

Early Embryonic Androgen Exposure Induces

Transgenerational Epigenetic and Metabolic Changes

Ning Xu, Angela K. Chua, Hong Jiang, Ning-Ai Liu, and Mark O. Goodarzi
Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Cedars-Sinai Medical
Center, Los Angeles, California 90048

Androgen excess is a central feature of polycystic ovary syndrome (PCOS), which affects 6% to 10%
of young women. Mammals exposed to elevated androgens in utero develop PCOS-like pheno-
types in adulthood, suggesting fetal origins of PCOS. We hypothesize that excess androgen
exposure during early embryonic development may disturb the epigenome and disrupt me-
tabolism in exposed and unexposed subsequent generations. Zebrafish were used to study the
underlying mechanism of fetal origins. Embryos were exposed to androgens (testosterone and
dihydrotestosterone) early at 26 to 56 hours post fertilization or late at 21 to 28 days post
fertilization. Exposed zebrafish (F0) were grown to adults and crossed to generate unexposed
offspring (F1). For both generations, global DNA methylation levels were examined in ovaries
using a luminometric methylation assay, and fasting and postprandial blood glucose levels
were measured. We found that early but not late androgen exposure induced changes in
global methylation and glucose homeostasis in both generations. In general, F0 adult ze-
brafish exhibited altered global methylation levels in the ovary; F1 zebrafish had global
hypomethylation. Fasting blood glucose levels were decreased in F0 but increased in F1;
postprandial glucose levels were elevated in both F0 and F1. This androgenized zebrafish
study suggests that transient excess androgen exposure during early development can result
in transgenerational alterations in the ovarian epigenome and glucose homeostasis. Current
data cannot establish a causal relationship between epigenetic changes and altered glucose
homeostasis. Whether transgenerational epigenetic alteration induced by prenatal androgen
exposure plays a role in the development of PCOS in humans deserves study. (Molecular
Endocrinology 28: 1329 –1336, 2014)

ndrogen excess affects around 7% of reproductive- adulthood (5, 6). Although the phenomenon of fetal an-
A aged women, and 80% to 85% of women with hy-
perandrogenism have polycystic ovary syndrome (PCOS)
drogen exposure leading to PCOS has been established in
animal models, little is known of the underlying molecu-
(1, 2). Patients with PCOS have an increased risk of in- lar mechanisms.
fertility, obesity, insulin resistance, diabetes, and cardio- Studies of common adult diseases with fetal origins
vascular disease (2, 3). The pathogenesis of PCOS is not suggest that intrauterine environmental factors can re-
well understood. It is thought that both genetic and early- program early development via disturbance of the epig-
life environmental factors in utero may contribute to the enome (7–11). Epigenetic modifications may be a key
development of PCOS (4). Animal models of PCOS sug- link between early intrauterine events and adult dis-
gest that it may have fetal origins. Prenatally androg- eases (9, 12), and hormonal insults in utero can result
enized (PA) rhesus monkeys, sheep, mice, or rats, after in epigenetic alterations with persistent changes in life
exposure to increased androgens during early or late ges- (13, 14). Epigenetic changes induced by environmental
tation, develop PCOS-like phenotypes once they reach factors not only perturb the somatic cells but also affect

ISSN Print 0888-8809 ISSN Online 1944-9917 Abbreviations: DME, dimethoxyethane; DHT, dihydrotestosterone; dpf, days postfertiliza-
Printed in U.S.A. tion; E, early treatment; EDC, endocrine-disrupting chemical; F, female; hpf, hours post-
Copyright © 2014 by the Endocrine Society fertilization; L, late treatment; M, male; PA, prenatally androgenized; PCOS, polycystic
Received February 4, 2014. Accepted June 25, 2014. ovary syndrome; T, testosterone; WT, wild type.
First Published Online July 3, 2014

doi: 10.1210/me.2014-1042 Mol Endocrinol, August 2014, 28(8):1329 –1336 mend.endojournals.org 1329

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1330 Xu et al Effects of Embryonic Androgenization in Zebrafish Mol Endocrinol, August 2014, 28(8):1329 –1336

the gametes that give rise to the next generation (15, Materials and Methods
16). Therefore, we and others (5) hypothesized that
excess androgen exposure during early development Maintenance of Zebrafish
Zebrafish strain AB (wild type [WT]) was used. Embryos
may disturb the epigenome and result in persistent ab-
were maintained and raised as described previously (20). In
normalities in reproductive and metabolic systems not brief, zebrafish embryos were collected after natural spawning
only in the exposed generation but also in subsequent and incubated in egg water in dishes (1.5-mL stock salts added
generations. to 1 L of distilled water). Zebrafish after 15 days postfertiliza-
To test this hypothesis, zebrafish were used as an tion (dpf) were maintained in system tank water. At 5 dpf,
animal model to study androgen effects. Zebrafish have zebrafish were fed with powdered food and then were fed with
brine shrimp twice daily from 9 dpf. Fish were maintained at
been used to study vertebrate reproduction and endo- 28°C on a 14-hour light, 10-hour dark cycle. All animal exper-
crine systems (17, 18). Their rapid embryonic develop- imentation was conducted in accord with accepted standards of
ment and short life cycle make zebrafish an ideal model humane animal care. This study was conducted with institu-
to examine transgenerational epigenetic effects. Previ- tional animal care and use committee approval.
ously, we did not find significant methylation changes
in peripheral blood from patients with PCOS compared Androgen exposure in vivo
with that from control subjects (19). Given that DNA Testosterone (T) (T5411; Sigma-Aldrich), and dihydrotes-
tosterone (DHT) (730637; Sigma-Aldrich) were prepared in the
methylation is tissue specific and that the ovary is one vehicle dimethoxyethane (DME) (E27408; Sigma-Aldrich), and
of the most relevant tissues in PCOS, we studied an- diluted in egg water at 50, 500, or 1000 ng/L concentrations (all
drogen effects on global methylation status in zebrafish in 2% DME). Exposure doses were lower than or similar to the
ovary in the present study. Circulating fasting and post- physiologic concentrations of T in adult zebrafish (600 ng/L for
prandial glucose levels were measured in both andro- female zebrafish to 2000 ng/L for male zebrafish) (21). Vehicle
groups were prepared using the same concentrations of DME
gen-exposed zebrafish and their unexposed offspring to
alone.
assess androgen impacts on metabolic systems. Our To mimic the early and late treatments in PA rhesus monkeys
findings that androgen exposure during early develop- (6), we selected 2 specific time periods for embryonic androgen
ment alters the epigenome and glucose homeostasis exposure in zebrafish (Figure 1). F0 zebrafish were exposed to
may have implications for the pathogenesis of PCOS in nothing, vehicle (DME), or androgens (T or DHT) at 2 different
humans. time windows: the early treatment (E) was from 26 to 56 hours
postfertilization (hpf) for 30 hours (during which GnRH neu-
rons appear and migrate to their desti-
nation), and the late treatment (L) was
from 21 to 28 dpf for 7 days (during
ovarian development). After treatment,
F0 zebrafish were removed from the
treated medium and grown in regular
egg water (early treatment) or tank wa-
ter (late treatment) till adulthood (3
months postfertilization). We selected
the most informative groups among F0
zebrafish to generate F1. F1 zebrafish
were generated either by in-crossing F0
zebrafish androgenized with 50 ng/L
DHT (DHT50 ⫻ DHT50) or testoster-
one at 50 ng/L (T50 ⫻ T50) or 1000
ng/L (T1000 ⫻ T1000), or by out-cross-
ing F0 females androgenized with 500
ng/L testosterone with unexposed males
[T500(female [F]) ⫻ WT(male [M])].
Tables 1 and 2 list all of the experimen-
tal zebrafish groups generated for this
study.
Figure 1. Breeding scheme for androgenized zebrafish. Parent individuals (P) were crossed to
generate embryos, which were split into 4 groups, exposed to nothing or to DME, T, or DHT at Ovary isolation
different doses (50, 500, and 1000 ng/L) during early or late time windows. The embryos were To obtain the zebrafish ovary, adult
grown to the F0 zebrafish. The F0 individuals were then crossed with early androgen-exposed
female zebrafish (5–10 months old) were
zebrafish or unexposed controls to generate 4 groups of F1 individuals: T500(F) ⫻ WT(M), T50 ⫻
T50, T1000 ⫻ T1000 and DHT50 ⫻ DHT50. Unexposed controls were included for both F0 and
killed by prolonged immersion in 0.2%
F1 generations. tricaine methanesulfonate for 5 minutes

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doi: 10.1210/me.2014-1042 mend.endojournals.org 1331

rosequencing reaction. The overall DNA methylation percent-

Table 1. Global Methylation Percentage in the Ovary
age of a DNA sample was calculated as 1 ⫺ the HpaII/MspI
for F0 Zebrafish Compared With That in Unexposed
ratio (which is percent unmethylated) (22).
Controls
Global Meal tolerance tests
Groups Methylation, % P As described previously (23), male and female zebrafish were
Unexposed controls 71.3 (3.2) Reference given ad libitum feeding of a regular diet (live adult brine
F0 early treatment shrimp) for 1 hour after 20 hours of fasting. Adult zebrafish
E-DME-500 71.6 (7.1) .76 (8 –12 months old) were killed in 0.2% tricaine methanesulfo-
E-DME-1000 71.2 (16.2) .99 nate before tail section. Blood was collected from the tail vein at
E-T-50 78.6 (1.1) .019a fasting and at 1, 2, 4, 6, 30, and 48 hours after feeding. At least
E-T-500 68.4 (7.1) .016a
E-T-1000 68.7 (11.4) .21 0.3 ␮L of blood was applied to a glucometer test strip (FreeStyle
E-DHT-50 74.9 (3.3) .032a InsuLinx; Abbott Laboratories). Exposed groups and their off-
E-DHT-500 74.6 (10.0) .061 spring (except E-T-50 and E-DHT-50) were available for meal
E-DHT-1000 67.5 (8.9) .067 tolerance tests (3– 6 adult fish per exposure group/time point).
F0 late treatment Three groups (L-DME-500, L-T-500, and DHT50 ⫻ DHT50)
L-DME-500 73.1 (6.8) .92 did not have fish for all time points of the meal tolerance tests.
L-DME-1000 63.6 (14.4) .13 Twelve to 19 unexposed control adult fish served as the refer-
L-T-500 60.0 (23.7) .075 ence group for each time point.
L-T-1000 66.2 (9.8) .16
L-DHT-500 73.0 (4.8) .42
L-DHT-1000 68.2 (2.3) .051 Statistical analysis
Mann-Whitney tests were used for comparison of global
Adult zebrafish were 5 to 10 months old. Data are medians
(interquartile ranges). P values were derived from the Mann-Whitney methylation levels or blood glucose at each time point between
test. unexposed controls and androgen-treated groups or their prog-
a
Significant P value (P ⬍ .05). eny. Data are expressed as medians (interquartile ranges). Sig-
nificance was taken at a value of P ⬍ .05.
before the ovaries were removed manually using forceps and
scissors. Each experimental (F0 or F1) group contained 5 ova-
ries (except for 2 F0 groups, early exposed T50 and early ex-
posed DHT50, in which only 3 individuals were available per
Results
group). The reference group consisted of 8 ovaries from unex-
posed control adult fish.
Global methylation analysis
Table 1 compares global methylation in ovaries be-
DNA extraction and global methylation analysis tween F0 androgenized zebrafish and unexposed con-
DNA was extracted from ovaries using the AllPrep DNA/ trols. At a low dose of 50 ng/L, both early T (E-T-50)– and
RNA/Protein Mini Kit (QIAGEN) and then was subjected to the early DHT (E-DHT-50)–treated zebrafish ovaries had in-
luminometric methylation assay (EpigenDx, Inc). The lumino- creased global methylation (78.6%, P ⫽ .019; 74.9%,
metric methylation assay is a quantitative method to estimate
P ⫽ .032, respectively, compared with that of the unex-
overall DNA methylation across the genome; it does not provide
gene-specific methylation. DNA was first digested with methy- posed controls, 71.3%).
lation-sensitive (HpaII) and methylation-insensitive (MspI) re- In contrast, early treatment of higher dose T at 500
striction enzymes. HpaII digests only the unmethylated se- ng/L significantly decreased global methylation in adult
quence, whereas MspI digests all the sites. Only digested sites zebrafish (68.4%) compared with that in unexposed con-
were subjected to single nucleotide extension followed by a py-
trols (71.3%) (P ⫽ .016). Global methylation of ovaries
with early T treatment of 1000 ng/L, although similar to
Table 2. Global Methylation Percentage in the Ovary that with E-T-500 treatment, did not statistically differ
for F1 Zebrafish Compared With That for Unexposed from that of unexposed controls. Zebrafish treated early
Controls
with DHT at higher doses (500 or 1000 ng/L) exhibited
Global no difference in global methylation levels compared with
Groups Methylation, % P those of untreated controls.
Unexposed controls 71.3 (3.2) Reference In the late treatment groups (T or DHT, 500 or 1000
T50 ⫻ T50 52.8 (24.5) .0085a
T1000 ⫻ T1000 64.8 (5.3) .0043a ng/L exposure), no statistically significant differences in
T500(F) ⫻ WT(M) 72.0 (27.2) .94 global methylation compared with that in controls were
DHT50 ⫻ DHT50 67.5 (13.7) .40 observed.
Adult zebrafish were 5 to 10 months old. Data are medians Next, we assessed whether global methylation status
(interquartile ranges). P values were derived from the Mann-Whitney
test. changed in ovaries of F1 zebrafish (unexposed offspring
a
Significant P value (P ⬍ .05). of F0 fish exposed early to androgens) compared with

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1332 Xu et al Effects of Embryonic Androgenization in Zebrafish Mol Endocrinol, August 2014, 28(8):1329 –1336

that of unexposed controls (Table 2). F1 T50 ⫻ T50 during development. The findings suggest that (1) early
female offspring had lower global methylation levels than but not late androgen exposure can induce global meth-
controls (52.8% vs 71.3%, P ⫽ .0085). F1 T1000 ⫻ ylation changes in the ovaries and alter glucose homeo-
T1000 female offspring also had lower global methyl- stasis and (2) these effects on the epigenetic state and
ation levels than controls (64.8% vs 71.3%, P ⫽ .0043). glucose levels are transmitted from the exposed gener-
ation to the next unexposed generation. Although DHT
Meal tolerance test is the more active androgen, we found that T had stron-
To determine the impact of excess androgen exposure ger effects, suggesting that estrogen might play a role in
on glucose homeostasis, we measured fasting and post- reprogramming during early development due to
prandial blood glucose in zebrafish (Table 3 and Figure aromatization.
2). Compared with unexposed controls, F0 zebrafish with
early androgen treatment had significantly lower glucose Epigenetic alterations induced by excess
at fasting (E-T-1000 and E-DHT-500) (P ⫽ .013 and androgens
.041, respectively) or 48 hours after a meal (E-DHT- Given that little is known about which genes may be
1000) (P ⫽ .035). Both E-T-500- and E-T-1000-treated
affected by androgen exposure, the global methylation
groups exhibited higher glucose at 4 hours after a meal
status was first assessed to determine whether the epig-
(P ⫽ .0044 and .032, respectively). In late treatment, no
enome was altered in androgenized zebrafish. Global
significant differences in glucose levels were observed in
methylation changes induced by androgen exposure sug-
any group at fasting or after feeding. In the F1 groups,
gest that excess androgens can disrupt epigenetic patterns
both fasting and postprandial glucose levels were signifi-
during early development, and epigenetic marks can be
cantly higher than those of the controls at several time
transmitted to the next generation. Patients with andro-
points (P ⫽ .0006 –.025) (Table 3 and Figure 2).
gen insensitivity syndrome had differential methylation
marks compared with normal control subjects, suggesting
that androgens can alter patterns of the methylome in
Discussion
humans (24).
Our study introduced a novel zebrafish model to discover We observed nonmonotonic changes in global methyl-
the transgenerational effects of excess androgen exposure ation status with T exposure, comprising hypermethyl-

Table 3. Fasting and Postprandial Glucose Levels of Zebrafish With and Without Androgen Exposure
Glucose Level, mg/dL

Groups Fasting 1H 2H 4H 6H 30 H 48 H
Unexposed controls 42.5 (6.3) 77.0 (28.0) 97.0 (49.0) 69.0 (16.5) 57.5 (43.5) 52.0 (48.0) 43.0 (16.0)
F0 early treatment
E-DME-500 33.0 (13.0) 70.5 (28.8) 94.5 (13.8) 67.5 (33.0) 80.5 (62.8) 44.0 (18.5) 48.0 (27.0)
E-DME-1000 32.0 (26.5) 82.0 (43.3) 92.0 (56.5) 79.0 (85.0) 57.0 (25.0) 37.5 (6.0) 35.5 (15.3)
E-T-500 40.0 (48.0) 68.0 (34.0) 87.5 (22.0) 132.0 (81.8)a 95.0 (62.0) 29.5 (11.8) 39.0 (5.5)
E-T-1000 32.0 (13.0)b 61.0 (22.0) 84.0 (29.0) 103.5 (44.0)c 95.0 (73.5) 38.0 (18.5) 35.5 (31.0)
E-DHT-500 28.5 (18.0)d 79.0 (26.0) 107.0 (23.8) 77.0 (110.0) 95.5 (63.8) 38.5 (41.3) 41.5 (27.3)
E-DHT-1000 37.5 (19.8) 67.5 (20.9) 85.5 (33.0) 75.5 (42.5) 68 0.0 (72.3) 40.5 (8.25) 30.0 (11.8)e
F0 late treatment
L-DME-500 45.5 (14.5) 79.5 (47.0) 111.0 (124.0) 84.0 (53.0) 73.0 (14.0) 72.0 (46.0) NA
L-DME-1000 31.5 (13.0) 97.0 (26.3) 89.0 (30.0) 73.0 (45.8) 61.0 (44.0) 26.0 (9.0) 46.0 (9.0)
L-T-500 44.0 (8.5) 63.5 (15.3) 108.0 (32.5) 91.0 (52.5) 60.0 (59.5) 39.0 (83) NA
L-T-1000 45.5 (21.3) 55.0 (57.0) 97.0 (75.5) 94.5 (123.0) 66.5 (25.0) 49.0 (21.0) 39.0 (21.0)
L-DHT-500 43.0 (8.3) 84.0 (22.0) 73.0 (23.5) 53.0 (22.5) 68.5 (120.5) 46.0 (30.0) 39.0 (23.0)
L-DHT-1000 37.0 (12.5) 65.5 (38.0) 109.0 (64.5) 81.0 (122.0) 44.5 (33.5) 31.0 (14.5) 48.0 (7.0)
F1 (offspring)
T50 ⫻ T50 47.0 (12.0) 69.0 (60.5) 139.0 (100.8) 96.5 (90.8) 104.0 (61.5) 40.5 (50.3) 55.5 (39.8)
T1000 ⫻ T1000 47.0 (38.0) 125.0 (36.0)f 119.0 (104.0) 121.5 (97.0) 138.0 (88.0)g 84.0 (55.0) 87.0 (17.0)h
T500(F) ⫻ WT(M) 58.0 (14.0)i 108.5 (51.8) 134.0 (118.0) 126.0 (76.5)j 103.5 (42.8)k 128.0 (65.0)l 67.5 (21.8)h
DHT50 ⫻ DHT50 40.5 (29.0) NA NA 86.0 (22.5) NA NA NA

Abbreviation: NA, fish not available; H, hours after a meal. Adult zebrafish were 8 to 12 months old. Data are medians (interquartile ranges). To
convert glucose from milligrams per deciliter to millimoles per liter, multiply by 0.05551.
Glucose levels with significant P values compared with those for unexposed controls: aP ⫽ .0044; bP ⫽ .012; cP ⫽ .032; dP ⫽ .041; eP ⫽ .035; fP ⫽
.025; gP ⫽ .020; hP ⫽ .00060; iP ⫽ .00070; jP ⫽ .011; kP ⫽ .015; lP ⫽ .0023.

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doi: 10.1210/me.2014-1042 mend.endojournals.org 1333

33). Human studies attempting to measure fetal androgen

exposure used blood obtained at term, which may be
inappropriate timing. The critical time of exposure may
be early in gestation, as observed in PA monkeys (6). In
the PA rhesus monkey model, the pregnant females were
given testosterone daily at 40 to 80 days of gestation for
the early treatment. This is the period during which
GnRH neurons migrate from the olfactory placode region
to their destination in the hypothalamus. For the late
treatment, monkeys were exposed to androgens at 100 to
140 days of gestation, which is the time window of devel-
opment of primordial follicles and appearance of multi-
layered antral follicles in the ovaries (6). To mimic the
exposure times in PA monkeys, zebrafish embryos were
exposed early from the time of appearance of GnRH-
expressing cells (26 hpf) to the time (52–54 hpf) at which
they reach their final destination (34) or exposed late
when the ovary appears during the larval stage (21–28
dpf) (35). Given conservation in vertebrate development,
findings from these 2 exposure times in zebrafish may
provide insights regarding the effects of prenatal androg-
enization on GnRH neuronal cells and ovaries during
embryogenesis in humans. We observed abnormal glu-
Figure 2. Exposed groups and their offspring exhibit altered fasting cose tolerance only in the early treated zebrafish. It is
and postprandial glucose levels. A, Glucose homeostasis for E-T-500
and T500(F) ⫻ WT(M) groups compared with that for unexposed possible that androgen penetration in late treatment is
controls, B, Glucose homeostasis for E-T-1000 and T1000 ⫻ T1000 lower than in early treatment, resulting in weaker expo-
groups compared with those for unexposed controls. Median glucose sure. However, our results are similar to the findings of
levels are displayed. Significant differences between groups at each
time point: *, F0 vs controls; †, F1 vs controls.
metabolic derangements found only in the early androg-
enized monkeys (6). We hypothesize that early androgen
exposure may disrupt pancreatic development, resulting
ation at low dose exposure and hypomethylation at in ␤-cell dysfunction, because the dorsal and ventral buds
higher dose exposure compared with changes in unex- fuse at around 50 hpf to form a mature pancreas (36). It
posed controls. This pattern has been described for many is also possible that androgen exposure has an effect on
studies of endocrine-disrupting chemicals (EDCs), which, insulin sensitivity.
similar to hormones, have displayed biphasic dose re-
sponses for various endpoints (25), which may be ex- Transgenerational metabolic derangements in
plained by the down-regulation of receptors at higher zebrafish programmed by androgen exposure
exposure levels or adaptive responses through complex PA animals often develop insulin resistance, ␤-cell dys-
signaling pathways (26). function, and increased visceral fat, free fatty acids, total
cholesterol, and triglycerides (27, 37, 38). Consistent
Critical timing of androgen exposure during with the metabolic alterations present in other PA animal
development models, we also found that disturbed glucose homeostasis
Our androgenized zebrafish model provides support- is passed down to the next generation in zebrafish. The
ive evidence that excess androgens induce reprogram- first epigenome-wide association study between DNA
ming of the fetus, possibly via epigenetic alteration as methylation and metabolic traits in human blood sug-
described previously in PA animals (27, 28). Whether gested that DNA methylation may play an important role
reprogramming occurs in humans in the etiology of PCOS in metabolism (39). Increased global methylation in pe-
is controversial. Some human studies suggest that prena- ripheral blood was associated with insulin resistance, in-
tal androgen excess may predispose the fetus to develop creased fasting glucose, and dyslipidemia (40, 41). In vis-
PCOS phenotypes in adulthood (29, 30). Studies of op- ceral adipose tissue of obese individuals, lower global
posite sex twins or measurement of androgen levels in methylation was associated with metabolic syndrome and
umbilical cord blood argue against reprogramming (31– increased fasting glucose (42). We found that F1 exhib-

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1334 Xu et al Effects of Embryonic Androgenization in Zebrafish Mol Endocrinol, August 2014, 28(8):1329 –1336

ited global hypomethylation in the ovary and higher fast- drogens. In addition to genetic predisposition, epigenetic
ing and postprandial glucose levels than the controls. transgenerational effects from environmental factors
Given the association of DNA methylation with meta- might contribute to the pathogenesis of human diseases.
bolic abnormalities, we hypothesize that DNA methyl-
ation alterations induced by excess androgens may be a Limitations
molecular basis underlying metabolic abnormalities and We performed a pilot study to test excess androgen
transgenerational effects in androgenized zebrafish. effects on zebrafish. Although the sample size in each
Interestingly, we found decreased fasting glucose levels treatment group was relatively small, in our study we
in certain F0 groups but increased glucose at both fasting were able to detect differences in global methylation sta-
and multiple time points after a meal in the F1 fish. Sim- tus and glucose tolerance in exposed and subsequent gen-
ilarly, in certain groups ovarian methylation was discor- erations, suggesting that transgenerational effects from
dant between generations. Because F0 fish experienced androgens were significant. Experiments were performed
androgen exposure as diploid embryos, whereas F1 fish once without replication; however, conclusions are par-
experienced the same exposure as haploid gametes, fol- tially supported by similar results from different doses.
lowed by development within androgen-exposed parents, For example, F1 zebrafish with both parents exposed to T
the environments experienced by F0 and F1 are quite (T50 ⫻ T50 or T1000 ⫻ T1000) had decreased ovarian
different and may result in different phenotypes. Elucida- methylation, and in meal tolerance tests, 4-hour post-
tion of these complex factors will require future prandial glucose levels were increased for both E-T-500
experimentation. and E-T-1000 fish. We targeted female zebrafish first, and
only ovaries were subjected to methylation analysis. We
Environmental androgen exposure and potential did not study methylation status in the pancreas or in
transgenerational effects insulin-responsive tissues. Global methylation analysis
Our results raise the possibility that environmental an- and glucose measurements were not performed in the
drogens (either endogenous androgens such as those in same individual zebrafish; therefore, we were unable to
PCOS or exogenous androgens such as androgenic EDCs) test the correlation between global methylation level and
may potentially disturb the epigenome and metabolic sys- glucose levels. Each fish used for glucose measurement at
tems in exposed and subsequent generations. EDCs can each single time point was killed during tail blood collec-
disrupt neuroendocrine systems that control reproduc- tion, so the area under the curve could not be used to
tion and metabolism, reprogram the epigenome, and af- measure the glucose response to a meal for each individ-
fect subsequent generations (43). Environmental andro- ual zebrafish. Androgen effects on methylation were dif-
gens have been identified in surface water, sediment pore ferent among F1 groups (T ⫻ T vs T(F) ⫻ WT). Without
water, and the effluent in estuaries (44). Growth promot- having performed crosses of androgen-exposed males to
ers given to farm animals such as trenbolone acetate are control females, we cannot determine whether transgen-
androgenic, and androgen activity has been found in beef erational methylation is dependent on androgen-treated
cattle runoff and feedlot surface soils (45). Exposure to males. Last, our results suggest that T had stronger effects
androgenic EDC can impair female reproductive behav- on methylation and glucose homeostasis than DHT, de-
ior and fin morphology in freshwater fish (46). spite the fact that DHT is a stronger androgen receptor–
Environmentally induced changes can be passed to activating ligand. Because T can be aromatized to estra-
subsequent generations (16). Paternal high-fat diet expo- diol, the current data cannot exclude estrogenic
sure disrupted ␤-cell function in rat female offspring, re- contributions to reprogramming. Further to this point,
sulting in increased body weight, adiposity, insulin resis- DHT can be converted to the estrogenic metabolite beta-
tance, and impaired glucose tolerance (47). In utero diol in the zebrafish brain (50). It is also possible that T
methyl donor supplementation during gestation in mice may have greater penetration than DHT into zebrafish
can induce heritable germline epigenetic changes in the embryos, resulting in stronger effects. These possibilities
Avy allele not only in the exposed fetuses but also in the deserve future study.
offspring (48). Exposure to environmental toxins (eg, a In conclusion, our results suggest that transient excess
fungicide, a pesticide mixture, and a plastic mixture con- androgen exposure during early development can induce
taining bisphenol A) during embryonic gonadal sex de- persistent changes in methylation status and disturb glu-
termination caused transgenerational changes in the epig- cose homeostasis in zebrafish. These effects can be passed
enome and ovarian morphology in rats (49). These down through generations. However, these experiments
observations and our zebrafish data highlight the need to do not definitively prove that androgen-mediated changes
study the transgenerational effects of environmental an- of the epigenome are the cause of changes in glucose ho-

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doi: 10.1210/me.2014-1042 mend.endojournals.org 1335

meostasis. Future studies will focus on the molecular basis 13. Ho SM, Tang WY, Belmonte de Frausto J, Prins GS. Developmental
exposure to estradiol and bisphenol A increases susceptibility to
of transgenerational epigenetic inheritance in androg-
prostate carcinogenesis and epigenetically regulates phosphodies-
enized zebrafish, such as identifying specific genes with terase type 4 variant 4. Cancer Res. 2006;66:5624 –5632.
altered methylation. Whether the epigenetic changes are 14. Li S, Hansman R, Newbold R, Davis B, McLachlan JA, Barrett JC.
directly correlated with glucose levels or other metabolic Neonatal diethylstilbestrol exposure induces persistent elevation of
c-fos expression and hypomethylation in its exon-4 in mouse
phenotypes deserves investigation. Key genes or path- uterus. Mol Carcinog. 2003;38:78 – 84.
ways whose epigenetic alterations due to androgen expo- 15. Skinner MK. What is an epigenetic transgenerational phenotype?
sure persist in generations represent worthy candidates F3 or F2. Reprod Toxicol. 2008;25:2– 6.
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for studies to elucidate the pathophysiology of human netic inheritance via the gametes in mammals. Nat Rev Genet.
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Acknowledgments 18. Lieschke GJ, Currie PD. Animal models of human disease: zebrafish
swim into view. Nat Rev Genet. 2007;8:353–367.
We thank Dr Daniel A. Dumesic for insightful comments on the 19. Xu N, Azziz R, Goodarzi MO. Epigenetics in polycystic ovary syn-
manuscript. drome: a pilot study of global DNA methylation. Fertil Steril. 2010;
Address all correspondence and requests for reprints to: 94:781–783.e1.
20. Westerfield M. The Zebrafish Book. A Guide for the Laboratory
Mark O. Goodarzi, Division of Endocrinology, Diabetes and
Use of Zebrafish (Danio rerio). 5th ed. Eugene, OR: University of
Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Bou- Oregon Press; 2007.
levard, Room B-131, Los Angeles, CA 90048. E-mail: 21. Liu C, Deng J, Yu L, Ramesh M, Zhou B. Endocrine disruption and
mark.goodarzi@cshs.org. reproductive impairment in zebrafish by exposure to 8:2 fluorote-
This work was supported by internal funds and by the Ce- lomer alcohol. Aquat Toxicol. 2010;96:70 –76.
22. Karimi M, Johansson S, Ekström TJ. Using LUMA: a luminomet-
dars-Sinai Winnick Clinical Scholars Award (to M.O.G.). ric-based assay for global DNA-methylation. Epigenetics. 2006;1:
Disclosure Summary: The authors have nothing to disclose. 45– 48.
23. Liu NA, Jiang H, Ben-Shlomo A, et al. Targeting zebrafish and
murine pituitary corticotroph tumors with a cyclin-dependent ki-
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