The orphan nuclear receptor small heterodimer partner mediates male infertility induced by diethylstilbestrol in mice.
ABSTRACT Studies in rodents have shown that male sexual function can be disrupted by fetal or neonatal administration of compounds that alter endocrine homeostasis, such as the synthetic nonsteroidal estrogen diethylstilbestrol (DES). Although the molecular basis for this effect remains unknown, estrogen receptors likely play a critical role in mediating DES-induced infertility. Recently, we showed that the orphan nuclear receptor small heterodimer partner (Nr0b2), which is both a target gene and a transcriptional repressor of estrogen receptors, controls testicular function by regulating germ cell entry into meiosis and testosterone synthesis. We therefore hypothesized that some of the harmful effects of DES on testes could be mediated through Nr0b2. Here, we present data demonstrating that Nr0b2 deficiency protected mice against the negative effects of DES on testis development and function. During postnatal development, Nr0b2-null mice were resistant to DES-mediated inhibition of germ cell differentiation, which may be the result of interference by Nr0b2 with retinoid signals that control meiosis. Adult Nr0b2-null male mice were also protected against the effects of DES; however, we suggest that this phenomenon was due to the removal of the repressive effects of Nr0b2 on steroidogenesis. Together, these data demonstrate that Nr0b2 plays a critical role in the pathophysiological changes induced by DES in the mouse testis.
- SourceAvailable from: Fabiano Nino
Article: Spermatogenesis and Cryptorchidism.[Show abstract] [Hide abstract]
ABSTRACT: Cryptorchidism represents the most common endocrine disease in boys, with infertility more frequently observed in bilateral forms. It is also known that undescended testes, if untreated, lead to an increased risk of testicular tumors, usually seminomas, arising from mutant germ cells. In normal testes, germ cell development is an active process starting in the first months of life when the neonatal gonocytes transform into adult dark (AD) spermatogonia. These cells are now thought to be the stem cells useful to support spermatogenesis. Several researches suggest that AD spermatogonia form between 3 and 9 months of age. Not all the neonatal gonocytes transform into AD spermatogonia; indeed, the residual gonocytes undergo involution by apoptosis. In the undescended testes, these transformations are inhibited leading to a deficient pool of stem cells for post pubertal spermatogenesis. Early surgical intervention in infancy may allow the normal development of stem cells for spermatogenesis. Moreover, it is very interesting to note that intra-tubular carcinoma in situ in the second and third decades have enzymatic markers similar to neonatal gonocytes suggesting that these cells fail transformation into AD spermatogonia and likely generate testicular cancer (TC) in cryptorchid men. Orchidopexy between 6 and 12 months of age is recommended to maximize the future fertility potential and decrease the TC risk in adulthood.Frontiers in Endocrinology 05/2014; 5:63.
- [Show abstract] [Hide abstract]
ABSTRACT: Bile acids (BAs) are signaling molecules that are involved in many physiological functions, such as glucose and energy metabolism. These effects are mediated through the activation of the nuclear and membrane receptors FXRα and TGR5. Although both receptors are expressed within the testes, the potential impact of BAs on testis physiology and male fertility has not been explored thus far. Here, we demonstrate that mice fed a diet supplemented with cholic acid have reduced fertility subsequent to testicular defects. Initially, germ cell sloughing and rupture of the blood-testis barrier occur and are correlated with decreased protein accumulation of CONNEXIN43 and N-CADHERIN, whereas at later stages, apoptosis of spermatids is observed. These abnormalities are associated with increased intra-testicular BA levels in general and deoxycholic acid, a TGR5 agonist, in particular. We demonstrate here that Tgr5 is expressed within the germ cell lineage where it represses Cx43 expression through the regulation of the transcriptional repressor Tbx2. Consistent with this finding, mice deficient for Tgr5 are protected against the deleterious testicular effects of BA exposure. Conclusions: These data identify the testis as a new target of BAs and emphasize TGR5 as a critical player in testicular pathophysiology. This work may open new perspectives on the potential impact of BAs on testis physiology during liver dysfunction. (Hepatology 2014)Hepatology 05/2014; · 12.00 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Methoxychlor (MXC), an organochlorine pesticide, has adverse effects on male reproduction at toxicological doses. Humans and wild animals are exposed to MXC mostly through contaminated dietary intake. Higher concentrations of MXC have been found in human milk, raising the demand for the risk assessment of offspring after maternal exposure to low doses of MXC. In this study, pregnant mice (F0) were given intraperitoneal daily evening injections of 1 mg/kg/d MXC during their gestational (embryonic day 0.5, E0.5) and lactational periods (postnatal day 21.5, P21.5), and the F1 males were assessed. F1 testes were collected at P0.5, P21.5 and P45.5. Maternal exposure to MXC disturbed the testicular development. Serum testosterone levels decreased, whereas estradiol levels increased. To understand the molecular mechanisms of exposure to MXC in male reproduction, the F1 testes were examined for changes in the expression of steroidogenesis- and spermatogenesis- related genes. RT-PCR analysis demonstrated that MXC significantly decreased Cyp11a1 and increased Cyp19a1; furthermore, it downregulated certain spermatogenic genes (Dazl, Boll, Rarg, Stra8 and Cyclin-a1). In summary, perinatal exposure to low-dose MXC disturbs the testicular development in mice. This animal study of exposure to low-dose MXC in F1 males suggests similar dysfunctional effects on male reproduction in humans.PLoS ONE 01/2014; 9(7):e103016. · 3.73 Impact Factor
3752? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 119? ? ? Number 12? ? ? December 2009
The orphan nuclear receptor small
heterodimer partner mediates male infertility
induced by diethylstilbestrol in mice
David H. Volle,1,2 Mélanie Decourteix,1,2 Erwan Garo,3 Judy McNeilly,4 Patrick Fenichel,1,2
Johan Auwerx,3,5 Alan S. McNeilly,4 Kristina Schoonjans,3,5 and Mohamed Benahmed1,2
1INSERM U895, Centre Méditerranéen de Médecine Moléculaire, Hôpital l’Archet 2, Nice, France.
2Faculty of Medicine, University of Nice/Sophia-Antipolis, Nice, France. 3Institut de Génétique et de Biologie Moléculaire et Cellulaire,
CNRS/INSERM/ULP, Illkirch, France. 4MRC Human Reproductive Sciences Unit, The Queen’s Medical Research Institute,
Centre for Reproductive Biology, Edinburgh, United Kingdom. 5Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
The small heterodimer partner (Shp; referred to herein as Nr0b2)
is mainly known for its role in the liver, where it is involved in the
feedback inhibition of bile acid synthesis (1–4). The functions of
Nr0b2 have been linked to its ability to repress the transcriptional
activity of other nuclear receptors (NRs) such as the liver homolog-1
(Lrh-1; referred to herein as Nr5a2; refs. 1, 3) and the estrogen
receptors (Erα/β; referred to herein as Nr3a1/2; ref. 5). In addition
to the liver, Nr0b2 has also been shown to be expressed in the testis
(6–8). We have recently demonstrated that Nr0b2 interacts with
retinoid signaling in the testis, which leads to germ cell entry in
meiosis (8). Moreover, Nr0b2 controls testosterone synthesis inde-
pendently of the hypothalamus/pituitary axis (8). These results
suggest that in the testis, Nr0b2 interferes with several signaling
pathways important for reproductive biology.
In recent years, a causal link between in utero and/or neonatal
exposure to molecules that alter endocrine functions and the devel-
opment of genital tract abnormalities, such as cryptorchidism,
hypospadias, and impaired spermatogenesis, has emerged from
studies in rodents. Most of these endocrine disrupters (EDs) exert
some estrogenic and/or antiandrogenic activities (9). However, these
molecules are rarely pure agonists or antagonists and can induce sev-
eral signaling pathways. A good example is diethylstilbestrol (DES),
known to induce reproductive disorders (10). DES has been shown
to activate several members of the NR family, such as the Er and
the estrogen-related receptors α/β/γ (Errα/β/γ; referred to herein as
Nr3b1, Nr3b2, and Nr3b3, respectively; refs. 11–14). Even though
the exact molecular basis remains unknown, the use of transgenic
models suggests that Ers are involved (15–18). Moreover, some stud-
ies show similar changes in testicular gene expression induced by
estradiol and DES (15). Other data, however, suggest differences in
the molecular targets introduced by estradiol and DES (19).
Interestingly, Nr0b2 is a direct target gene of both the Ers (20) and
the Errs (21) and inhibits their transcriptional activity (6, 22, 23). We
hence hypothesized that part of the testicular effects of DES, both
estrogen dependent and independent, could be mediated through
Nr0b2. Using exposure to DES, to the pure estrogen agonist estradiol
benzoate (EB), and to Er antagonist ICI 182,780 (referred to herein as
ICI), we demonstrate here that Nr0b2 deficiency protects male mice
against the harmful estrogenic and nonestrogenic effects of DES.
During postnatal development, Nr0b2-null mice were more resistant
to the DES-mediated inhibition of germ cell differentiation, which is
explained by our finding that Nr0b2 interfered with retinoid signals
that control meiosis. In adult Nr0b2-null male mice, however, protec-
tion against the negative effects of DES was caused, at least in part,
by the removal of Nr0b2-repressive effects on steroidogenesis. These
data demonstrate that Nr0b2 plays a critical role in the pathophysi-
ological changes induced by EB and DES in the testis.
Estrogenic compounds induce testicular expression of Nr0b2. In order to
identify a potential molecular link between Nr0b2 and the estab-
lished negative effects of DES on male fertility, we monitored
Nr0b2 expression at different time points during which treat-
ment with DES is known to induce reproductive abnormalities
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 119:3752–3764 (2009). doi:10.1172/JCI38521.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
(P1–P5). mRNA analyses demonstrated that Nr0b2 was expressed
in the whole testis at these ages (Supplemental Figure 1A; supple-
mental material available online with this article; doi:10.1172/
JCI38521DS1). Moreover, the mRNA of the NRs known to be tar-
gets of DES, such as Nr3a1, Nr3a2, and Nr3b1/2/3, were expressed
in the testis (Supplemental Figure 1A). Treatment with DES caused
higher mRNA accumulation of Nr0b2 in testes of P10 Nr0b2 wild-
type (Nr0b2+/+) male mice (Figure 1A). In the adult testis, a dose-
dependent increase in Nr0b2 mRNA was also observed upon DES
administration. Interestingly, similar effects on Nr0b2 mRNA
levels were observed using the pure estrogen EB (Supplemental
Figure 1B), which suggests that the regulation of Nr0b2 gene was
caused, at least in part, by the estrogenic effect of DES.
DES has a weak impact on the fertility of Nr0b2-knockout mice. We then
treated neonatal Nr0b2+/+ and knockout (Nr0b2L–/L–) male mice with
increasing doses of DES (0–5 μg). Neonatal exposure of Nr0b2+/+
males to DES caused a significant 80% decrease in the number
of pups per litter, whereas it had a lesser impact on litters from
Nr0b2L–/L– males (Figure 1B). These data were consistent with the
decreased number of spermatozoa in the epididymis tails of treat-
ed Nr0b2+/+ males, which was much less pronounced in Nr0b2L–/L–
males (Figure 1C). The weights of testis, epididymis, and semi-
nal vesicles were also more reduced in Nr0b2+/+ than in Nr0b2L–/L–
male mice exposed to DES (Figure 1D). Consistent with these
protective effects, the minimal dose of DES at which testis weight
started to decrease was between 0.75 and 1.5 μg in Nr0b2+/+ males,
whereas there was almost no effect in Nr0b2L–/L– males (Figure 1D).
The protection against DES caused by Nr0b2 gene inactivation was
not a general or systemic effect, as body and liver weights were sim-
ilarly affected by DES in both genotypes (Figure 1D).
Even though DES belongs to the group of EDs with estrogen
activity, it can also activate other signaling pathways (24). Neo-
natal administration of the pure estrogen, EB, to Nr0b2+/+ males
decreased the adult weights of the testis, epididymis, and seminal
vesicles (Supplemental Figure 1C). As for DES administration,
the Nr0b2L–/L– males were significantly protected from the unde-
sired effects of EB compared with Nr0b2+/+ males. The number of
spermatozoa decreased in the epididymis tails of treated Nr0b2+/+
Nr0b2 deficiency protects male mice against DES-induced abnormalities. (A) Nr0b2 mRNA levels in whole testes of P10 Nr0b2+/+ and Nr0b2L–/L–
males exposed to 0.75 μg DES (n = 6), and in whole testes of 10-week-old males neonatally exposed to 0, 0.75, and 5 μg DES (n = 3–5). Values
were normalized to β-actin. (B) Each male was bred with 5 C57BL/6J females to analyze the number of pups per litter. (C and D) Spermatozoa
count in the tail of epididymis (C) and whole body weight as well as weights of testis, epididymis, seminal vehicles, and liver normalized to body
weight (D) of 10-week-old Nr0b2+/+ and Nr0b2L–/L– males neonatally exposed to 0, 0.35, 0.5, 0.75, 1.5, and 5 μg DES (n = 6–14 per group).
*P < 0.05 versus vehicle; #P < 0.05 versus Nr0b2+/+ given the same DES dose.
3754?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
males, whereas no effect of EB was observed in Nr0b2L–/L– males
(Supplemental Figure 1D). These effects of DES might be in
part mediated by the Ers, as the use of the Er antagonist ICI on
Nr0b2+/+ males was able to reverse part of the impact of DES on the
decreased count of spermatozoa as well as on the decrease in organ
weights (Supplemental Figure 1, E and F).
Nr0b2 deficiency protects mice from DES-induced germ cell death. In
10-week-old adult Nr0b2+/+ mice exposed to 5 μg DES, the testes
showed major morphological alterations, as reflected by the absence
of germ cells in several tubules (Figure 2A). Conversely, even at this
high dose, Nr0b2L–/L– males did not show apparent abnormalities.
The use of cell type–specific markers (25) allowed us to determine
that postmeiotic cells were the most affected cell population in
DES-treated Nr0b2+/+ mice, as reflected by the decreased number
of Smad6-positive cells (Figure 2B and Supplemental Figure 2A).
On the contrary, no difference was observed in the number of Ser-
toli and premeiotic/early meiotic germinal cells (as determined by
staining for Tubulin3 and Cyclin-A1, respectively; Figure 2B).
We then assessed potential changes in the rate of proliferation. No
significant change in the expression of the proliferation marker Ki-67
or in the accumulation of PCNA was observed between untreated
or DES-treated Nr0b2+/+ and Nr0b2L–/L– males (Supplemental Figure
2B). However, DES induced a significant increase in apoptotic cells
in the testes of Nr0b2+/+ mice starting at the 0.75-μg dose, as deter-
mined by TUNEL analysis (Figure 3, A and B). The increased apopto-
sis in Nr0b2+/+ testis corroborated with the increased accumulation of
active caspase-3 (Figure 3C). On the contrary, Nr0b2L–/L– males were
protected from the increased apoptosis induced by DES (Figure 3).
In a similar experiment, neonatal exposure of mice to EB resulted
in germ cell loss caused by an increased number of apoptotic cells
in adult Nr0b2+/+ mice, whereas Nr0b2L–/L– males were also protected
from apoptosis induced by EB (Supplemental Figure 2, C and D).
Nr0b2 reduces testosterone levels after DES exposure. Germ cell death
has previously been associated with androgen withdrawal (26, 27).
Consistent with previous data (28, 29), DES induced a decrease in
testosterone concentrations in Nr0b2+/+ males at as little as 0.75 μg
(Figure 4A). This effect was further pronounced at 5 μg DES. The
inhibition of testosterone was associated with a decrease in mRNA
levels of Star, Cyp11a1, and Cyp17a1, which are critically involved in
steroidogenesis (Figure 4B). Interestingly, Nr0b2L–/L– males seemed
to be protected from these effects of DES, as testosterone concen-
trations as well as Star, Cyp11a1, and Cyp17a1 mRNA levels were
not affected (Figure 4, A and B). No effect of DES was observed on
mRNA accumulation of the androgen receptor Nr3c4 in Nr0b2+/+ or
Nr0b2L–/L– testes (Figure 4C). The decrease in testosterone was also
consistent with the reduction in mRNA abundance of the andro-
gen-dependent genes Pem and Osp in Nr0b2+/+ males. In addition to
the impact of DES on the androgen pathway, we analyzed its impact
on Er target genes. The mRNA accumulation of Nr3a1 and insulin-
like–3 (Insl3) decreased in Nr0b2+/+ testes, as expected, whereas this
effect of DES was not observed in Nr0b2L–/L– testes (Figure 4C). Also
as expected, DES had no effect on the accumulation of Nr3a2.
We next analyzed the expression of known regulators of ste-
roidogenesis to identify how Nr0b2 could control steroid synthe-
sis. Regarding the inducers of steroidogenesis, Nr5a2 and the ste-
roidogenic factor–1 (Sf-1, referred to herein as Nr5a1), both known
targets of Nr0b2, only Nr5a2 expression decreased at 0.75 μg DES
(Figure 4C). DES had no effect on the expression of dosage-sensi-
DES-induced histological abnormalities caused by loss of postmeiotic cells in Nr0b2+/+ mice. (A) Representative micrographs of H&E-stained
testes of 10-week-old Nr0b2+/+ and Nr0b2L–/L– mice exposed to vehicle or 5 μg DES (n = 6 per group). The arrow indicates tubules with a slight
loss of germ cells; the arrowhead indicates tubes with complete loss of germ cells. Original magnification, ×100. (B) Quantification of cells per
100 seminiferous tubules (n = 4–6) positively stained for markers Smad6 (postmeiotic germ cells), Tubulin3 (Sertoli cells), and Cyclin-A1 (pre-
and meiotic germ cells). Vehicle-treated mice were arbitrarily set at 100%. *P < 0.05 versus vehicle.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
tive sex reversal, adrenal hypoplasia congenita, critical region on
the X chromosome, gene 1 (Dax-1, referred to herein as Nr0b1), a
negative regulator of steroidogenesis (Figure 4C). The use of the Er
agonist EB, or the antagonist ICI, suggests a significant contribu-
tion of the estrogenic effects of DES on the repression of steroido-
genesis (Supplemental Figure 2, E and F). Indeed, EB exposure
resulted in a decrease of the intratesticular concentration of tes-
tosterone in Nr0b2+/+ mice. Similar to what we observed after DES
treatment, we observed no effect on testosterone level in Nr0b2L–/L–
males after EB exposure. Moreover, ICI exposure reversed the effect
of DES on testosterone in Nr0b2+/+ males.
To test whether the DES-induced effects on the testis are reg-
ulated at the level of the hypothalamus/pituitary axis, we first
measured LH and FSH plasma levels. Interestingly, no impact of
DES on LH or FSH level was observed, which suggests that the
primary effect of DES occurs at the testicular level (Figure 4D).
Several studies have demonstrated that estrogen can regulate ste-
roidogenesis directly on the level of the Leydig cell. To confirm this
hypothesis, we performed some in vitro analyses using the Ma-10
Leydig cell line. EB and DES increased Nr0b2 mRNA accumulation
at 12 and 24 hours after treatment, whereas the decrease in the
mRNA levels of steroidogenic genes such as Star was observed only
after 24 hours (Figure 4E). Consistent with our previous report
(8), these results suggest that Nr0b2 might directly repress steroid
synthesis after DES and/or estrogen treatment.
DES exposure alters neonatal germ cell differentiation through Nr0b2.
DES was administered close to the beginning of germ cell differen-
tiation (30, 31). To determine an eventual impact of DES exposure
on germ cell differentiation, we analyzed the testis at P6 or P10.
At P6, there was no alteration in the expression of genes specific
for undifferentiated spermatogonia (Oct3/4, Nanos3, and Cyclin-d2;
Figure 5A). However, in Nr0b2+/+ mice treated with DES, expression
of transcripts involved in germ cell differentiation (Stra8 and Dmc1)
was significantly lower. This effect was not observed in the Nr0b2L–/L–
males. At P10, the accumulation of mRNAs involved in germ cell dif-
ferentiation was still reduced in the testes of Nr0b2+/+ mice exposed
to DES, an effect that was accompanied by the robust induction of
specific transcripts of undifferentiated spermatogonia (Figure 5B),
suggesting an alteration of the relative proportion of undifferenti-
ated versus differentiating spermatogonia following DES admin-
istration in Nr0b2+/+ mice. Moreover, at P10, the meiotic marker
Cyclin-a1 was found to be decreased by DES treatment, which is con-
Nr0b2 controls DES-induced adult germ cell apoptosis through regulation of testosterone synthesis. (A) Apoptosis in 10-week-old Nr0b2+/+ and
Nr0b2L–/L– mice exposed to vehicle or 5 μg DES (n = 6 per group), as analyzed by TUNEL staining. Arrowheads denote TUNEL-positive cells. Rep-
resentative micrographs are shown. Original magnification, ×100. (B) Quantification of TUNEL analyses. Shown is the number of positive cells per
100 seminiferous tubules (n = 4–6). (C) Immunoblot of activate caspase-3 performed on testicular protein extracts of Nr0b2+/+ and Nr0b2L–/L– mice
exposed to 0 or 5 μg DES (n = 6 per group). Quantification of activated caspase-3 protein accumulation relative to total caspase-3 is shown below;
vehicle-treated mice were arbitrarily fixed at 100%. *P < 0.05 versus vehicle; #P < 0.05 versus Nr0b2+/+ given the same DES dose.
3756?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
sistent with a decreased germ cell differentiation. Again, Nr0b2L–/L–
testes were not affected. Similar studies using EB instead of DES
showed that part of the effect of DES is likely mediated by Ers, as
several genes, including Nanos3, Stra8, Dmc1, Cyclin-a1, Cyclin-d2,
appeared to be regulated in a similar way (Supplemental Figure 3,
A and B). Interestingly, some other genes, such as Oct3/4, showed a
different expression pattern in mice treated with EB compared with
those treated with DES (compare Figure 5B and Supplemental Fig-
ure 3B). Interestingly, this effect of DES on Oct3/4 expression was
also inhibited in Nr0b2L–/L– males, which suggests that Nr0b2 is a
major component of the signaling pathways activated by DES.
Neonatal effect of DES exposure involves apoptosis and estrogenic signal-
ing, but is independent of androgen status. To understand the molecular
pathways underlying the alteration in germ cell maturation, we ana-
lyzed both proliferation and apoptotic processes in P10 testis. No
effect was observed on cell proliferation, as suggested by the analysis
of PCNA protein accumulation (Supplemental Figure 3C). In P10
mice, DES induced apoptosis, as revealed by increased accumulation
of the active caspase-3 protein, in Nr0b2+/+ mice (Figure 5C). On the
contrary, the impact of DES on apoptosis was not observed in the
testes of Nr0b2L–/L– males. Interestingly, the changes in the number
of apoptotic cells did not seem to be linked to the androgen status,
as intratesticular testosterone decreased in both genotypes (Figure
5D). In fact, DES treatment induced a drastic decrease of the intra-
testicular testosterone in both Nr0b2+/+ and Nr0b2L–/L– males. We also
analyzed mRNA accumulation of Star and of the androgen-depen-
dent genes Pem and Osp. The expression of these genes was decreased
following DES exposure in both genotypes (Figure 5E), reflecting the
decrease of intratesticular androgen levels. As for DES, EB adminis-
tration resulted in a decreased expression of the steroidogenic genes
Star, Cyp11a1, and Cyp17 (Supplemental Figure 3D). This result sug-
gests that neonatal steroidogenesis, in contrast to that of adult mice,
is not controlled by Nr0b2, since Nr0b2L–/L– mice showed a profile
similar to that of Nr0b2+/+ mice (Figure 5, D and E).
Nr0b2 controls DES-induced repression of testosterone synthesis. (A) Plasma and intratesticular testosterone levels in 10-week-old Nr0b2+/+
and Nr0b2L–/L– mice exposed to 0, 0.75, or 5 μg DES (n = 10–15 per group). (B) Testicular mRNA expression of Star, Cyp11a1, Hsd3b1, and
Cyp17a1, normalized to β-actin levels, in whole testes of 10-week-old Nr0b2+/+ and Nr0b2L–/L– mice exposed to 0 or 0.75 μg DES (n = 10–15
per group). (C) Testicular mRNA expression of Nr3c4, Pem, Osp, Nr3a1, Nr3a2, Insl3, Nr5a2, Nr5a1, and Nr0b1, normalized to β-actin levels,
in whole testes of 10-week-old Nr0b2+/+ and Nr0b2L–/L– mice exposed to 0 or 0.75 μg DES (n = 10–15 per group). (D) Plasma LH and FSH con-
centration in 10-week-old Nr0b2+/+ and Nr0b2L–/L– mice exposed to 0, 0.75, or 5 μg DES (n = 10–15 per group). (E) mRNA expression of Nr0b2
and Star normalized to β-actin levels in MA-10 Leydig cells exposed to vehicle, EB, or DES for 12 or 24 hours (n = 6 per group). Vehicle-treated
mice were arbitrarily fixed at 100%. *P < 0.05 versus vehicle; #P < 0.05 versus Nr0b2+/+ given the same DES dose.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
The impact of DES on the estrogenic pathway was then ana-
lyzed in the testes of Nr0b2L–/L– mice and Nr0b2+/+ littermates.
No effect of DES was observed on the expression of Nr3a2 (Fig-
ure 5F). However, consistent with the estrogenic effect of DES,
the expression of known target genes of the Ers (16) — including
Nr3a1, Insl3, and renin-1 (Ren1) — decreased, whereas the expres-
sion of gene regulated in breast cancer 1 protein (Greb1) increased
(Figure 5, F and G). For Insl3 and Ren1, the effect of DES was
similar in Nr0b2+/+ and Nr0b2L–/L– males. On the contrary, the
impact of DES on the expression of Nr3a1 and Greb1 was lost
or diminished in Nr0b2L–/L– versus Nr0b2+/+ mice. Similarly, the
estrogenic target genes Nr3a1, Insl3, and Ren1 were affected by EB
(Supplemental Figure 3E). These data demonstrated that Nr0b2
is not involved in the regulation of all the Er target genes, which
suggests that there might be some compensatory mechanisms
involving other cofactors of Nr3a1/2.
Nr0b2 mediates part of the neonatal effects of DES through modifica-
tion of histone methylation marks. Previous studies have shown that
mice lacking the H3K9 histone methyltransferase G9a are sterile,
with germ cells undergoing apoptosis during the pachytene stage
(32). G9a is reported to perform H3K9 mono- and dimethylation
(marked by H3K9me1 and H3K9me2, respectively; refs. 33, 34).
In view of the Nr0b2-dependent proapoptotic effects of DES on
germ cells, we wondered whether these effects could be medi-
ated by factors affecting histone methylation, such as G9a (31).
Interestingly, in testes of DES-treated P10 Nr0b2+/+ animals, the
H3K9me1/2 marks decreased, whereas they were unaffected in
Nr0b2L–/L– mice (Figure 6, A and B). This suggests that DES might
affect G9a expression. Consistent with this hypothesis, testicular
G9a mRNA and G9a protein expression decreased in Nr0b2+/+ mice
treated with DES, whereas no effect was observed in the Nr0b2L–/L–
mice (Figure 6, C and D). The decrease of the transcriptional
Nr0b2 inhibits neonatal germ cell differentiation in response to DES. Nr0b2+/+ and Nr0b2L–/L– mice were exposed to 0 or 0.75 μg DES. (A and B)
Testicular mRNA expression of Oct3/4, Nanos3, Cyclin-d2, Stra8, Dmc1, and Cyclin-a1, normalized to β-actin levels, in P6 (A) or P10 (B) mice
(n = 5–10 per group). (C) Immunoblot of activate caspase-3 on testicular protein extracts (n = 6 per group). Quantification of activate capase-3
protein accumulation relative to total caspase-3 is also shown. (D) Intratesticular testosterone levels in P10 mice (n = 10–15 per group). (E–G)
mRNA expression of Star, Pem, and Osp (E); Nr3a1, Nr3a2, Nr3c4, Nr5a2, and Nr0b1 (F); and Insl3, Ren1, and Greb1 (G) in whole testes of
P10 mice (n = 10–15 per group). Values were normalized to β-actin levels. Vehicle-treated mice were set at 100%. *P < 0.05 versus vehicle;
#P < 0.05 versus Nr0b2+/+ given the same DES dose.
3758? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
repressor G9a following DES administration in Nr0b2+/+ mice was
also associated with an increase of several G9a target genes (32,
35), including Akr1c13, Akr1c12, Chst11, 290, 291, 110, Wfdc15a, and
Iap, as a result of relief of repression (Figure 6E). Conversely, and
in line with previous studies describing a downregulation of Mei1
in G9a-null mice (32), mRNA levels of this gene were decreased in
the Nr0b2+/+ mice treated with DES (Figure 6E). In the Nr0b2L–/L–
mice, neither a significant effect nor an opposite effect of DES was
observed on the expression of these genes compared with Nr0b2+/+
mice. In Nr0b2+/+ males, the use of EB also induced a clear decrease
in G9a mRNA levels at P6 and P10, which translated to a robust
decrease in G9a protein levels (Supplemental Figure 4, A and B).
The H3K9me2 mark was also decreased by EB in Nr0b2+/+ testes,
whereas no effect was observed in those of Nr0b2L–/L– mice (Supple-
mental Figure 4C). This effect was consistent with the deregulation
of G9a targets genes in the testes of Nr0b2+/+, but not Nr0b2L–/L–,
mice (Supplemental Figure 4D).
To corroborate the effect of histone modifications directly
on the G9a target genes, we performed H3K9me1 or H3K9me2
IP on chromatin extracted from vehicle- or DES-treated mice at
P10. DES induced a decrease in both H3K9me1 and H3K9me2
marks on the DNA sequences of G9a target genes 290, 291, 110,
and Wfdc15a, whereas no effect was observed on the negative con-
trol locus 282 (Figure 7). Surprisingly, for some G9a target genes,
such as 290 and 110, we noticed a significant increase of H3K9me2
in Nr0b2L–/L– mice treated with DES compared with vehicle treat-
ment. This effect was opposite to what was observed in Nr0b2+/+
mice. In Nr0b2L–/L– mice treated with DES, the higher recruitment
of H3K9me2 was consistent with the lower expression of some
G9a target genes, such as 290, 291, 110, and Iap (Figures 6 and 7).
Nr0b2 induces alterations of histone H3K9 marks through the inhibition of G9a, in response to DES. Nr0b2+/+ and Nr0b2L–/L– mice were exposed
to 0 or 0.75 μg DES. (A and B) Immunoblots of H3K9me1 and H3K9me2 performed on testis (A), and quantification of H3K9me2 and H3K9me2
accumulation, relative to total H3 (B), of P10 mice (n = 9 per group). (C) Testicular mRNA expression of G9a, normalized to β-actin levels, in
whole testes of P6 and P10 mice (n = 10–15 per group). (D) Immunoblot of G9a performed on P10 mice (n = 9 per group). Quantification of
G9a protein accumulation, relative to actin, is also shown. (E) Testicular mRNA expression of G9a target genes Akr1c13, Akr1c12, Chst11,
Wfdc15a, Mei1, 290, 110, 291, and Iap, normalized to β-actin levels, in whole testes of P10 mice (n = 10–15 per group). Vehicle-treated mice
were fixed at 100%. *P < 0.05 versus vehicle.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
Nr0b2 is responsible for the cross-talk between DES and the retinoid pathway.
The analysis of G9a expression in the testes of untreated P10
Nr0b2+/+ and Nr0b2L–/L– mice showed higher protein accumulation
in Nr0b2L–/L– mice (Figure 8A), which suggests that G9a is a tar-
get gene of Nr0b2. This was consistent with the higher levels of
H3K9me1/2 marks and the deregulation of the G9a target genes
observed in Nr0b2L–/L– mice (Figure 8, B and C).
Retinoids (i.e., all-trans-retinoic acid; RA) are key components that
induce the entry of germ cells into meiosis (30, 31, 36). We hence
evaluated the interplay among RA signaling, Nr0b2, and G9a using
the embryonic teratocarcinoma-derived F9 cell line. This cell line is
a well-established cell-autonomous model for investigating retinoid
signaling in vitro, as RA can induce the cells’ differentiation (37). In
F9 cells, RA treatment led to an increase in G9a mRNA accumula-
tion, which was inhibited by Nr0b2 in a dose-dependent manner
(Figure 8D). These results were confirmed at the protein level (Fig-
ure 8E), which suggests that Nr0b2 could regulate the expression
of G9a by inhibiting the RA receptor (Rar) pathway. Using an in
silico approach based on the Genomatix program (see Methods),
we mapped a putative Rar response element (RARE) in the promoter
of G9a. To validate the relevance of this RARE in the regulation of
G9a expression in response to retinoids, we performed chromatin
IP (ChIP) experiments using either wild-type or Rarg–/– F9 cells (38).
Treatment with RA led to a higher level of Rarγ IP on the RARE in
wild-type cells, whereas no difference was observed in the Rarg–/– F9
cells (Figure 8F). The potential interaction of Nr0b2 with Rar on the
promoter of the G9a gene was further substantiated by ChIP experi-
ments using wild-type or Rarg–/– F9 cells transfected with pCMV or
pCMV-Nr0b2 expression vectors. Consistent with our expression
data, we found specific enrichment of Nr0b2 on the DNA sequences
surrounding the RARE of the G9a promoter in wild-type cells, but
not Rarg–/– F9 cells (Figure 8G), further underscoring the impor-
tance of the Nr0b2/Rar complex in the regulation of G9a.
Here, we demonstrated that DES induced abnormalities in the
male genital tract upon fetal and/or neonatal exposure in mice. In
animal models, neonatal administration of DES decreased fertility
in adult male mice as a result of altered morphology of the male
genital tract, with decreased relative weights of epididymis, vesicle
seminals, and testis. This was associated with a decrease in epididy-
mis sperm count, which was caused by the increased germ cell death
we observed in male mice exposed to DES. Interestingly, for all these
parameters, deficiency for Nr0b2 protected the males exposed to
DES, which suggests that Nr0b2 plays a critical role in the testicular
pathophysiology induced by DES. However, we have demonstrated
that DES acts via several signaling pathways. Through the use of
estradiol and/or of the Er antagonist ICI, we showed that Nr0b2
protected the male mice against both estrogenic and nonestro-
genic effects of DES. Even though it is not established in humans,
these rodent data are the basis of the hypothesis of a potential link
between these environmental factors (25), including the exposure
to EDs (39–41), and the fast increase of the incidence of male repro-
ductive disorders (42, 43). Our data could potentially establish a link
between EDs and male reproductive disorders in humans.
High doses of EDs induce a severe decrease in testicular weight
caused by germ cell death, which leads to important modifica-
tions in the relative cell type proportions. Such changes could lead
to false interpretations, because the observed differences in gene
expression are the result of changes in testicular cell content rather
Nr0b2 mediates DES-induced alterations of histone H3K9 marks on the promoter of G9a target genes. Shown are results of ChIP of crosslinked
DNA from testes of P10 Nr0b2+/+ and Nr0b2L–/L– mice using H3K9me1 or H3K9me2 antibodies. The loci studied as G9a targets (290, 291, 110,
Wfdc15a, and 282) were previously defined and cover at least 1 gene (35). The 282 locus was used as a negative control of H3K9 methylation.
IgG was used as a negative internal control. For each condition, 6 ChIPs were performed. *P < 0.05 versus vehicle.
3760?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
than of the real regulation of gene expression. Through a dose-
effect experiment of DES, we first determined the concentration
that did not dramatically affect testicular weight. This allowed us
to assume that at the doses used for the molecular analyses, the
modifications of gene expression were significant, not the conse-
quences of altered cell content.
Unexpectedly, we observed higher levels of active caspase-3 in
Nr0b2L–/L– mice compared with Nr0b2+/+ littermates. This increase
in basal levels of active caspase-3 in Nr0b2L–/L– mice was significant
in P10 animals (Figure 5C), which could reflect the earlier entry
in meiosis. In adult mice, we also detected a significant difference
between active caspase-3 levels in Nr0b2+/+ and Nr0b2L–/L– mice
(Figure 3C). Interestingly, and in line with our previous studies
(8), there was no difference in the number of TUNEL-positive
cells between vehicle-treated Nr0b2+/+ and Nr0b2L–/L– males. The
increase in active caspase-3 was not consistent with the unchanged
results of the TUNEL assay and requires further study; this find-
ing reflects the complexity of apoptosis, which can be regulated at
both activation and inhibition levels.
Even though the present report showed that Nr0b2L–/L– mice were
protected against the deleterious effect of DES and EB on the male
reproductive tract, basal estrogen metabolism was similar between
Nr0b2+/+ and Nr0b2L–/L– males (Supplemental Figure 5, A–C). These
data suggested that the role of Nr0b2 on the estrogenic pathway is
only revealed under pathophysiological conditions when exposed
to estrogenic endocrine disrupters. However, the lack of difference
between the genotypes under basal condition was surprising and
difficult to explain; it suggests the existence of some yet-unidenti-
fied compensatory mechanisms. One could speculate that Nr0b1,
which is very close to Nr0b2 at the structural level, could compen-
sate for the lack of Nr0b2.
Nr0b1 and Nr0b2 are closely related nuclear receptors. More-
over, the impact of Nr0b1 on steroidogenesis has been well dem-
onstrated. In our experiments, however, we have not shown a sig-
Nr0b2 controls G9a expression. (A) G9a immunoblot from whole testes of P10 Nr0b2+/+ and Nr0b2L–/L– untreated mice (n = 5–7 per group).
Lanes were run on the same gel but were noncontiguous (white line). (B) Immunoblots of H3K9me1 or H3K9me2 performed on P10 Nr0b2+/+ and
Nr0b2L–/L– mice (n = 6–7 per group). (C) mRNA levels of G9a target genes Defb42, Chst11, 110, 290, Mei1, and Akr1c13 in whole testes of P10
Nr0b2+/+ and Nr0b2L–/L– mice exposed to 0 or 0.75 μg DES (n = 10–15 per group). (A–C) Levels were normalized to actin, and normalized values
of Nr0b2+/+ were set at 100%. (D) mRNA expression of Nr0b2 and G9a in F9 cells transfected with 0–800 ng pCMV-Nr0b2 (n = 3). (E) Protein
accumulation of G9a in F9 cells transfected with pCMV-Nr0b2 in the absence or presence of RA (n = 3). (F and G) ChIP of crosslinked DNA from
F9 wild-type or Rarg–/– cells using an anti-Rarγ antibody (F) or from F9 wild-type or Rarg–/– cells transfected with pCMV or pCMV-Nr0b2 using
an anti-Flag antibody (G). Inset: A DNA sequence of ±100 bp covering either the RARE (i) or a sequence 3.0 kb upstream of the RARE (ii) was
amplified. Results are expressed as fold enrichment over wild-type vehicle-treated cells (F) or over pCMV cells (G) and represent amplification
variability (n = 4). *P < 0.05 versus vehicle; #P < 0.05 versus next-smallest transfected pCMV-Nr0b2 amount.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
nificant effect of DES treatment on Nr0b1 expression in P10 pups
(Figure 5F) or in adult males (Figure 4C). These results suggest
that Nr0b1 is not involved in the DES-induced phenotypes. Sev-
eral studies have shown that Nr0b1 KO mice have normal levels of
testosterone (44, 45). The Nr0b1 KO mouse phenotype can mostly
be explained by the altered expression of aromatase, the enzyme
that synthesizes estrogens, whereas other steroidogenic genes, like
Star, were not affected (45). Together, these observations imply a
converging role of both Nr0b members to control full testicular
steroid metabolism, in which Nr0b2 would predominantly regu-
late steroidogenesis up to the level of testosterone synthesis, and
Nr0b1 would instead control aromatization.
In adult mice, Nr0b2 was responsible for the DES-induced decrease
in testosterone production. The absence of a decrease of steroido-
genic genes in Nr0b2L–/L– males after DES exposure is suggestive of
a major role for Nr0b2 in the DES-induced repression of steroido-
genesis. This hypothesis is further corroborated by the induction
of Nr0b2 mRNA in response to DES. This effect was also observed
with EB, which suggests that it might be driven by Nr3a1/2 signal-
ing. This is consistent with previous studies showing that estrogen
can directly regulate steroidogenesis at the Leydig cell levels (46).
The ability of estrogen to decrease steroid synthesis in the mouse
Leydig cell line MA-10, highlighted by the decrease in the mRNAs of
Star and Cyp11a1, has been described previously (47). To identify the
possible direct effect of Nr0b2 in Leydig cells following DES admin-
istration, we also performed experiments on MA-10 cells. Similar
to our in vivo results, EB and DES resulted in an increase of Nr0b2
mRNA 12 hours after treatment. This increase in Nr0b2 mRNA was
followed at 24 hours by a significant decrease in Star mRNA. The
kinetics of the mRNA increase of Nr0b2, followed by the decrease
of Star mRNA, is consistent with Nr0b2 being a major inhibitor of
steroidogenesis in Leydig cells. This is consistent with our previous
work demonstrating that overexpression of Nr0b2 in a Leydig cell
line represses Star and Cyp11a1 expression and that Nr0b2 binds to
the promoter of these 2 genes (8). Moreover, our conclusion that
Nr0b2 controls steroidogenesis directly at the testicular level after
DES exposure was confirmed by the finding that plasma LH and
FSH levels were both unaltered by DES as well as Nr0b2 genotype.
At the molecular level, we have previously shown that Nr0b2
regulates testicular androgen synthesis through repressed mRNA
expression of 2 activators of steroidogenesis, Nr5a2 and Nr5a1,
and/or through inhibited transcriptional activities of the same (8).
Here, the repression of steroidogenesis by Nr0b2 at the lower 0.5-μg
dose is likely caused by protein-protein interaction, as mRNA
expression of both Nr5a1 and Nr5a2 was not affected, whereas Star
accumulation was already reduced at this DES concentration, in
Nr0b2+/+ mice (data not shown). At the dose of 0.75 μg DES, this
effect was further amplified, probably by the decreased mRNA
expression of Nr5a2. Even though Nr5a1 was defined as a target
gene of Nr0b2, its expression was not affected by DES administra-
tion. The absence of any effect of DES was consistent with previ-
ous studies (15). These data demonstrate the complexity of the
regulation of Nr5a1, which is known to be controlled by multiple
factors (48); furthermore, it would be too simplistic to assume that
the mRNA expression of Nr5a1 is only regulated by Nr0b2.
In contrast to our findings in adult mice (Figure 4), neonatal ste-
roidogenesis seemed not to be controlled by Nr0b2, as Nr0b2L–/L–
and Nr0b2+/+ mice showed similar profiles (Figure 5, D and E). This
difference in Leydig cell response seems to occur in parallel with
the cells’ transition from the fetal to the adult population and is
in line with the finding that from P20 onward, Nr0b2 becomes
expressed only in interstitial cells, where it controls steroidogen-
esis (8). Moreover, P10 testis samples showed that Nr0b2 was main-
ly expressed in the tubular compartment of the testis (data not
shown). This suggests that, at P10, the impact of Nr0b2 following
DES exposure occurs in the intratubular compartment.
In addition to its role on androgen synthesis in adult mice after
neonatal DES exposure, Nr0b2 has other functions during postna-
tal testicular development. We administered DES during early post-
natal development, close to the beginning of germ cell differentia-
tion. Indeed, the first spermatocytes are seen at P5 (31). Neonatal
administration of either DES or EB induced modified expression
of genes associated with differentiated or undifferentiated cells,
such as Nanos3, Stra8, and Dmc1. We demonstrated here that DES
induced accumulation of specific transcripts of undifferentiated
spermatogonia, specifically in treated Nr0b2+/+ testis, and reduced
the expression of transcripts involved in germ cell differentiation.
These results suggest a change of the relative proportion of undiffer-
entiated versus differentiating spermatogonia after DES exposure.
We thus hypothesize that Nr0b2 mediates the effect of DES on germ
cell differentiation, which is in line with our previous report that
Proposed model for the role of Nr0b2 in DES-induced testicular abnor-
malities. Our results indicate that Nr0b2 is a major actor in DES-induced
testicular pathophysiology. Nr0b2 deficiency counteracts the negative
effects of DES. In P10 mice, DES induces a blockage in meiosis entry
and/or progression, which is characterized by the higher expression
of genes of undifferentiated spermatogonia (Nanos3) and a decrease
of meiotic genes (Stra8). The effect is stronger in DES-treated males
compared with males with 1 EB treatment, which could be explained, at
least in part, by the specific induction of Oct3/4 expression by DES. EB
and DES treatment alters H3K9me1 and H3K9me2, which are essen-
tial for meiosis progression (29). The impact on histones is driven by
the lower accumulation of G9a mRNA after DES exposure. This effect
on meiosis is explained, at least in part, by the lack of the repressive
activity of Nr0b2 on Rar and retinoid signaling. Finally, in adults, Nr0b2
inhibits testicular steroidogenesis, on the one hand by inhibiting the
expression of Nr5a2, which controls the expression of the steroido-
genic genes, and on the other hand by repressing the transcriptional
activity of Nr5a2 and/or Nr5a1. All these data explain how Nr0b2 plays
a major role in the subfertility induced by DES exposure.
3762? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
Nr0b2L–/L– mice showed earlier differentiation of germ cells than did
control littermates (8). To understand the alteration of these rela-
tive cell proportions, we analyzed both proliferation and apoptotic
processes in P10 mouse testes. In contrast to adult mice, this differ-
ential effect observed between Nr0b2+/+ and Nr0b2L–/L– males in early
postnatal development did not seem to be linked with androgenic
status. The increased apoptosis observed in Nr0b2+/+ males is most
likely caused by other alterations. DES treatment clearly affected
the meiotic cells. In oocytes, DES induced a severe, yet reversible,
deterioration of meiotic spindle microtubule organization during
maturation (49). DES reduces viability of Caenorhabditis elegans and
its fertility, associated with the production of aberrant gametes, as a
result of nuclear abnormalities and loss of synaptonemal complexes
(50). Moreover, recent studies have demonstrated the importance
of germ cell–specific epigenetic processes in the initiation and early
progression of meiosis (51). Interestingly, mice with a loss-of-func-
tion mutation for H3K9 histone methyltransferases are sterile, with
germ cells undergoing apoptosis during the pachytene stage (32, 52).
Several proteins possess H3K9 methyltransferase activity. Suv39h1,
Suv39h2, and G9a are able to perform H3K9 dimethylation (33),
whereas only G9a performs H3K9 monomethylation (34). In tes-
tes of DES-treated P10 Nr0b2+/+ mice, we observed decreases in the
H3K9me1 and H3K9me2 marks, which suggests that DES might
affect G9a expression and/or activity in the testis. Consistent
with this hypothesis, G9a mRNA and G9a protein expression was
decreased in Nr0b2+/+ mice treated with DES. Because of its histone
methyltransferase activity, G9a acts as a repressor of transcription.
The decrease of G9a after DES exposure was confirmed in Nr0b2+/+
mice by higher mRNA accumulation of known G9a target genes,
such as Akr1c13 and Chst11 (32). We observed no significant effect
in the Nr0b2L–/L– mice. It has been recently demonstrated that Nr0b2
physically interacts with G9a to induce repression of gene expression
(53, 54). Here, we demonstrated another form of cross-talk between
Nr0b2 and G9a, in which Nr0b2 inhibited the mRNA expression of
G9a. It is possible that both coexist and are part of a feedback loop in
which Nr0b2 inhibits G9a gene expression to attenuate the repres-
sion of their common target genes. The effect of DES on the histone
methylation marks was also found when we used EB, highlighting
the involvement of the estrogenic part of DES in this pathway.
If the signaling of G9a was decreased in Nr0b2+/+ mice treated
with DES, as evidenced by decreased expression of G9a, decreased
H3K9 methylation, and increased expression of G9a target genes, we
unexpectedly observed an opposite effect of DES in the Nr0b2L–/L–
males. Indeed, the expression of some target genes was found more
repressed in the Nr0b2L–/L– males exposed to DES compared with
vehicle. This effect, observed in Nr0b2L–/L– males, was confirmed by
a higher level of methylated histones on the DNA sequences of these
G9a target genes. This effect is surprising and to date remains unex-
plained. However, this is in line with our conclusion that Nr0b2 par-
ticipates to control G9a signaling. The observed data in Nr0b2L–/L–
mice treated with DES suggest that the lack of Nr0b2 induced an
increase in the G9a pathway, or a compensatory pathway through
other histone methyltransferases. The exact molecular mechanisms
involved are not established yet and will require further studies.
The molecular mechanisms triggering the initiation of germ cell
differentiation, in particular the mitotic/meiotic transition, are not
completely understood. However, retinoids are key components to
induce entry of germ cells in meiosis (30, 31, 36). Most intriguing-
ly, retinoids have also been shown to induce an increase in H3K9
methylation during differentiation (55). Based on these reports and
our above-described findings, we hypothesized that Nr0b2, via the
control of G9a expression, is the link between RA and DES path-
ways in the control of the meiotic process. This was confirmed by
the induction of G9a expression following RA administration in
different cell lines. Moreover, we found a specific enrichment of a
Rar/Nr0b2 complex on the DNA sequences surrounding the RARE
of the G9a promoter. Our results therefore show, for the first time
to our knowledge, a potential interaction between the retinoid sig-
naling pathway and the expression of the histone methyltransferase
G9a, which could explain, at least in part, the impact of G9a and
H3K9 methylation in germ cell differentiation.
Interestingly, this effect of DES on germ cell differentiation
seemed to persist in adult mice, as we observed a clear decrease
in mRNA accumulation of Stra8 (Supplemental Figure 5D). We
have indeed demonstrated that the deregulation of G9a was still
observed at the mRNA and protein levels in Nr0b2+/+ adult males
treated neonatally with DES (Supplemental Figure 5, E and F). This
perpetuation of G9a deregulation in adult testis, combined with the
altered testosterone synthesis, might cooperate to decrease germ
cell survival and lead to subfertility after DES administration.
It has been previously demonstrated that DES can have estrogenic
and nonestrogenic effects (15–18). Compared with EB, DES appeared
to have a stronger effect. To determine the estrogenic part of DES
activity, we used either a pure estrogenic compound, EB, or the Er
antagonist ICI. Most of the macroscopic phenotypes observed with
DES were also obtained using EB (e.g., organ weight, apoptotic pro-
cess), and the Nr0b2L–/L– mice were also protected against the delete-
rious effects of EB (Supplemental Figure 1, B–D, and Supplemental
Figure 2, B and E). ICI has previously been demonstrated to have del-
eterious effects on the male genital tract (56), with loss of germ cells
and decrease of fertility. Here, we also observed a significant altera-
tion in sperm count induced by ICI alone (Supplemental Figure 2E).
In adult rats and mice, treatment with ICI induced effects similar to
those previously observed in the male reproductive tract of Nr3a1
KO mice (56–59). These findings suggest that, following ICI treat-
ment, the decrease in sperm concentration in the cauda epididymis
could be explained, at least in part, by the fact that Nr3a1 is required
for normal fluid reabsorption, as concluded from studies of Nr3a1
KO males (60, 61). Most interestingly, ICI partially reversed the effect
of DES on all the macroscopic testicular abnormalities it induced
(Supplemental Figure 1F and Supplemental Figure 2F). Notably, we
used only 1 dose of ICI (50×) to compete with DES; perhaps a higher
dose would produce even more pronounced competition with DES.
Most of the molecular pathways altered by DES were also found
to be affected by EB (e.g., testosterone, retinoid pathway). However,
some of our results highlighted differential effects of DES and EB
at the dose we used: at P10, the expression of Oct3/4 was changed by
DES, not by EB. However, this is an important difference, as Oct3/4
plays an important role in the determination of the pluripotency of
cells, which could explain why the effect of DES was more potent
compared with EB. Indeed, the altered expression of Oct3/4 by DES
might more robustly inhibit germ cell differentiation than that
by EB. At the molecular level, Nr5a2 has been described to induce
Oct3/4 expression (62). At P10, the expression of Nr5a2 was not
affected by DES treatment. This result demonstrated that the effect
on Oct3/4 expression at P10 age does not seem to be related to the
status of Nr5a2. On the one hand, differences in dosing and rela-
tive receptor affinity could contribute to the difference in Oct3/4
expression between DES and EB. Indeed, both compounds have a
different affinity to either Nr3a1 or Nr3a2, as DES was shown to
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
bind to Ers with a higher affinity than did estradiol (63, 64). On
the other hand, it could be hypothesized that in response to DES,
Oct3/4 expression might be regulated by Nr3b1/2/3. DES has pre-
viously been demonstrated to activate Nr3b1/2/3 (13), and these
receptors positively regulate Oct3/4 expression (14). Even though
we did not determine the underlying molecular mechanisms, the
effect of DES on Oct3/4 expression was also inhibited in Nr0b2L–/L–
mice; therefore, Nr0b2 deficiency might also protect against the
nonestrogenic effects of DES.
In conclusion, our results demonstrated that Nr0b2 plays a major
role in the DES signaling pathway, which affects the development and
function of the male reproductive system. We showed that Nr0b2L–/L–
male mice were protected against the deleterious effects of DES, as
they were still able to reproduce even when exposed to high doses of
DES. This is caused by the multiple actions of Nr0b2 during testicular
development (Figure 9). First, in neonatal animals, Nr0b2 controls
germ cell differentiation through inhibition of the retinoid pathway.
Nr0b2 regulates the expression of genes involved in the entry and pro-
gression of meiosis, such as Stra8 and Nanos3. It also affects meiosis
through regulation of the expression of the histone methyltransferase
G9a and the subsequent modification of H3K9 methylation marks.
These alterations in methylation upon DES exposure induce abnor-
mal chromosomal complexes favoring germ cell apoptosis and could
affect the meiosis process. Next to these effects, which appear to be
mediated through the estrogenic pathway, DES seems to inhibit germ
cell differentiation at P10 through estrogen-independent pathways,
as shown by Oct3/4 deregulation specifically in DES-treated mice.
Second, in adult animals, the effect of Nr0b2 was dependent on the
inhibition of testosterone production, leading to germ cell death.
Together, our present data define Nr0b2 as one of the major actors in
the molecular events leading to DES-mediated male infertility.
Animals. The Nrb02L–/L– mice used were previously described (8) and
maintained on a mixed background (C57BL/6J/129sv). Mouse treat-
ment protocols are detailed in Supplemental Methods. This study was
approved by the Institut National de la Santé et de la Recherche Médi-
cale Animal Care Committee.
Histology and immunohistochemistry. H&E and immunohistochemistry
stainings were performed as described previously (27). See Supplemental
Methods for details.
Endocrine investigations. Plasma and intratesticular concentrations were
measured as described previously (27). Testosterone level is expressed as a
percentage of the vehicle-treated mice for each genotype. See Supplemental
Methods for details.
Plasma LH and FSH measurements. Plasma concentrations of LH and
FSH were measured as previously described (65). See Supplemental
Methods for details.
Real-time PCR. Following testis RNA extraction (TRIzol; Invitrogen)
and cDNA synthesis (SuperScript II First-Strand Synthesis System; Life
Technologies), real-time PCR measurement of individual cDNAs was per-
formed using SYBR green dye to measure duplex DNA formation. Primer
sequences are shown in Supplemental Tables 1 and 2. Results were ana-
lyzed using the Ct method. Quantitative PCR experiments were performed
as previously described (35). See Supplemental Methods for details.
Western blot. Proteins were extracted using SDS lysis buffer described in
the ChIP protocol from Upstate (Upstate Biotechnology Inc). See Supple-
mental Methods for details.
Transient transfection. F9 cells (provided by P. Chambon, Institut de Géné-
tique et de Biologie Moléculaire et Cellulaire, Strasbourg, France), were trans-
fected with lipofectamine (Invitrogen). Rar (obtained from C. Rochette-Egly,
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg,
France) was transfected with increasing amounts of pCMV-Nr0b2 plasmid
(8). The quantity of DNA was maintained constant by the addition of empty
pCMV vector. After 24 hours, either 10–6 M RA or vehicle (1:1,000) was added
to the cells. Cells were harvested 24 hours later, and mRNA or protein extrac-
tions were performed. See Supplemental Methods for details.
TUNEL analysis and Ki-67 staining. TUNEL and Ki-67 experiments were
performed as described previously (27) on 5 μm of testis fixed in 4% PFA.
Results are expressed as the number of TUNEL-positive or Ki-67 positive
cells per 100 seminiferous tubules. See Supplemental Methods for details.
ChIP. In vitro and in vivo ChIP experiments were respectively performed
from 106 cells, or P10 testis, of 3 mice. ChIP assays were carried out fol-
lowing the protocol provided by the manufacturer (Upstate Biotechnology
Inc.). See Supplemental Methods for details.
Promoter analysis. To analyze the promoter of G9a, we used the Genomatix
MatInspector program, which identifies transcription factor binding sites
in nucleotide sequences using a large library of weight matrices. The analy-
sis of the mouse G9a promoter sequence predicted a potential binding site
(599–623 nt from the transcription initiation site). Sequence identity was
Statistics. For statistical analysis, 2-way ANOVA was performed. When
significant effects of treatment or genotype or their interactions were
obtained, multiple comparisons were made with Tukey’s test. All numerical
data are mean ± SEM. A P value less than 0.05 was considered significant.
This work was supported by grants from Institut National de la
Santé et de la Recherche Médicale, the Centre National de Recher-
che Scientifique, the Université Louis Pasteur, the Agence Natio-
nale de la Recherche (ANR R06116AA, ANR R07023AA, and ANR
R08008AA), the Ecole Polytechnique Fédérale de Lausanne, the
Swiss National Science Foundation, the ERC, the NIH, and the
Plan National de Recherche en Reproduction et Endocrinologie (to D.H.
Volle). The authors also thank Pierre Chambon for the wild-type
and Rarg–/– F9 cells; Cécile Rochette-Egly for the Rar expression
vector; the members of the Benahmed and Auwerx laboratories
for scientific discussions and support; Jean-Marc Lobaccaro, Fran-
çoise Senegalas-Balas, and Georges Pointis for critically reading
the manuscript; and Geoffroy Marceau for his help on the intra-
testicular testosterone extractions.
Received for publication January 9, 2009, and accepted in revised
form September 9, 2009.
Address correspondence to: David H. Volle, Unité INSERM U895,
Centre Méditerranéen de Médecine Moléculaire, C3M, Hôpital
l’Archet 2, bâtiment Archimed, 151 route Saint-Antoine de Gin-
estière BP 2 3194, 06204 Nice Cedex 3, France. Phone: 33-4-89-06-
42-52; Fax: 33-4-89-06-42-60; E-mail: firstname.lastname@example.org.
1. Goodwin, B., et al. 2000. A regulatory cascade of the
nuclear receptors FXR, SHP-1, and LRH-1 represses
bile acid biosynthesis. Mol. Cell. 6:517–526.
2. Kerr, T.A., et al. 2002. Loss of nuclear receptor
SHP impairs but does not eliminate negative
feedback regulation of bile acid synthesis. Dev Cell.
3. Lu, T.T., et al. 2000. Molecular basis for feedback reg-
ulation of bile acid synthesis by nuclear receptors.
Mol. Cell. 6:507–515.
4. Wang, L., et al. 2002. Redundant pathways for neg-
ative feedback regulation of bile acid production.
Dev. Cell. 2:721–731.
5. Brendel, C., Schoonjans, K., Botrugno, O.A.,
Treuter, E., and Auwerx, J. 2002. The small het-
3764? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
erodimer partner interacts with the liver X receptor
alpha and represses its transcriptional activity. Mol.
6. Johansson, L., et al. 1999. The orphan nuclear
receptor SHP inhibits agonist-dependent tran-
scriptional activity of estrogen receptors ERalpha
and ERbeta. J. Biol. Chem. 274:345–353.
7. Lu, T.T., Repa, J.J., and Mangelsdorf, D.J. 2001.
Orphan nuclear receptors as eLiXiRs and FiXeRs of
sterol metabolism. J. Biol. Chem. 276:37735–37738.
8. Volle, D.H., et al. 2007. The small heterodimer part-
ner is a gonadal gatekeeper of sexual maturation in
male mice. Genes Dev. 21:303–315.
9. Toppari, J. 2008. Environmental endocrine disrupters.
Sex Dev. 2:260–267.
10. Bullock, B.C., Newbold, R.R., and McLachlan, J.A.
1988. Lesions of testis and epididymis associated
with prenatal diethylstilbestrol exposure. Environ.
Health Perspect. 77:29–31.
11. Greschik, H., Flaig, R., Renaud, J.P., and Moras, D.
2004. Structural basis for the deactivation of the
estrogen-related receptor gamma by diethylstilbes-
trol or 4-hydroxytamoxifen and determinants of
selectivity. J. Biol. Chem. 279:33639–33646.
12. Greschik, H., et al. 2002. Structural and functional
evidence for ligand-independent transcriptional
activation by the estrogen-related receptor 3. Mol.
13. Nam, K., Marshall, P., Wolf, R.M., and Cornell, W.
2003. Simulation of the different biological activi-
ties of diethylstilbestrol (DES) on estrogen recep-
tor alpha and estrogen-related receptor gamma.
14. Tremblay, G.B., et al. 2001. Diethylstilbestrol regu-
lates trophoblast stem cell differentiation as a
ligand of orphan nuclear receptor ERR beta. Genes
15. Cederroth, C.R., et al. 2007. Estrogen receptor alpha
is a major contributor to estrogen-mediated fetal
testis dysgenesis and cryptorchidism. Endocrinology.
16. Prins, G.S., et al. 2001. Estrogen imprinting of the
developing prostate gland is mediated through
stromal estrogen receptor alpha: studies with
alphaERKO and betaERKO mice. Cancer Res.
17. Rivas, A., et al. 2003. Neonatal coadministration of
testosterone with diethylstilbestrol prevents dieth-
ylstilbestrol induction of most reproductive tract
abnormalities in male rats. J. Androl. 24:557–567.
18. Singh, J., and Handelsman, D.J. 1999. Morphomet-
ric studies of neonatal estrogen imprinting in the
mature mouse prostate. J. Endocrinol. 162:39–48.
19. Ofner, P., Bosland, M.C., and Vena, R.L. 1992. Dif-
ferential effects of diethylstilbestrol and estradiol-17
beta in combination with testosterone on rat pros-
tate lobes. Toxicol. Appl. Pharmacol. 112:300–309.
20. Lai, K., Harnish, D.C., and Evans, M.J. 2003. Estro-
gen receptor alpha regulates expression of the
orphan receptor small heterodimer partner. J. Biol.
21. Sanyal, S., et al. 2002. Differential regulation of the
orphan nuclear receptor small heterodimer partner
(SHP) gene promoter by orphan nuclear receptor
ERR isoforms. J. Biol. Chem. 277:1739–1748.
22. Johansson, L., et al. 2000. The orphan nuclear
receptor SHP utilizes conserved LXXLL-related
motifs for interactions with ligand-activated estro-
gen receptors. Mol. Cell. Biol. 20:1124–1133.
23. Klinge, C.M., Jernigan, S.C., and Risinger, K.E.
2002. The agonist activity of tamoxifen is inhibited
by the short heterodimer partner orphan nuclear
receptor in human endometrial cancer cells. Endo-
24. Shigeta, H., Zuo, W., Yang, N., DiAugustine, R.,
and Teng, C.T. 1997. The mouse estrogen receptor-
related orphan receptor alpha 1: molecular clon-
ing and estrogen responsiveness. J. Mol. Endocrinol.
25. Maire, M., et al. 2005. Alteration of transforming
growth factor-beta signaling system expression in
adult rat germ cells with a chronic apoptotic cell
death process after fetal androgen disruption.
26. El Chami, N., et al. 2005. Androgen-dependent
apoptosis in male germ cells is regulated through
the proto-oncoprotein Cbl. J. Cell Biol. 171:651–661.
27. Volle, D.H., et al. 2007. Multiple roles of the nuclear
receptors for oxysterols liver X receptor to maintain
male fertility. Mol. Endocrinol. 21:1014–1027.
28. Goyal, H.O., et al. 2003. Neonatal estrogen expo-
sure of male rats alters reproductive functions at
adulthood. Biol. Reprod. 68:2081–2091.
29. Guyot, R., et al. 2004. Diethylstilbestrol inhibits the
expression of the steroidogenic acute regulatory
protein in mouse fetal testis. Mol. Cell. Endocrinol.
30. Bowles, J., et al. 2006. Retinoid signaling deter-
mines germ cell fate in mice. Science. 312:596–600.
31. Bowles, J., and Koopman, P. 2007. Retinoic acid,
meiosis and germ cell fate in mammals. Development.
32. Tachibana, M., Nozaki, M., Takeda, N., and Shinkai,
Y. 2007. Functional dynamics of H3K9 methylation
during meiotic prophase progression. EMBO J.
33. Peters, A.H., et al. 2003. Partitioning and plasticity
of repressive histone methylation states in mam-
malian chromatin. Mol. Cell. 12:1577–1589.
34. Tachibana, M., et al. 2002. G9a histone methyl-
transferase plays a dominant role in euchromatic
histone H3 lysine 9 methylation and is essential for
early embryogenesis. Genes Dev. 16:1779–1791.
35. Ikegami, K., et al. 2007. Genome-wide and locus-
specific DNA hypomethylation in G9a deficient
mouse embryonic stem cells. Genes Cells. 12:1–11.
36. Koubova, J., et al. 2006. Retinoic acid regulates sex-
specific timing of meiotic initiation in mice. Proc.
Natl. Acad. Sci. U. S. A. 103:2474–2479.
37. Rochette-Egly, C., and Chambon, P. 2001. F9
embryocarcinoma cells: a cell autonomous model
to study the functional selectivity of RARs and
RXRs in retinoid signaling. Histol. Histopathol.
38. Boylan, J.F., Lohnes, D., Taneja, R., Chambon, P.,
and Gudas, L.J. 1993. Loss of retinoic acid recep-
tor gamma function in F9 cells by gene disruption
results in aberrant Hoxa-1 expression and differ-
entiation upon retinoic acid treatment. Proc. Natl.
Acad. Sci. U. S. A. 90:9601–9605.
39. Delbes, G., Levacher, C., and Habert, R. 2006. Estro-
gen effects on fetal and neonatal testicular develop-
ment. Reproduction. 132:527–538.
40. Olesen, I.A., et al. 2007. Environment, testicular
dysgenesis and carcinoma in situ testis. Best Pract.
Res. Clin. Endocrinol. Metab. 21:462–478.
41. Sikka, S.C., and Wang, R. 2008. Endocrine disrup-
tors and estrogenic effects on male reproductive
axis. Asian J. Androl. 10:134–145.
42. Sharpe, R.M., and Skakkebaek, N.E. 2008. Tes-
ticular dysgenesis syndrome: mechanistic insights
and potential new downstream effects. Fertil Steril.
43. Skakkebaek, N.E., Rajpert-De Meyts, E., and Main,
K.M. 2001. Testicular dysgenesis syndrome: an
increasingly common developmental disorder with
environmental aspects. Hum. Reprod. 16:972–978.
44. Jeffs, B., et al. 2001. Blockage of the rete testis and
efferent ductules by ectopic Sertoli and Leydig
cells causes infertility in Dax1-deficient male mice.
45. Wang, Z.J., et al. 2001. Aromatase (Cyp19) expres-
sion is up-regulated by targeted disruption of
Dax1. Proc. Natl. Acad. Sci. U. S. A. 98:7988–7993.
46. Delbes, G., et al. 2005. Endogenous estrogens inhib-
it mouse fetal Leydig cell development via estrogen
receptor alpha. Endocrinology. 146:2454–2461.
47. Houk, C.P., Pearson, E.J., Martinelle, N., Donahoe,
P.K., and Teixeira, J. 2004. Feedback inhibition of
steroidogenic acute regulatory protein expression
in vitro and in vivo by androgens. Endocrinology.
48. Manna, P.R., Wang, X.J., and Stocco, D.M. 2003.
Involvement of multiple transcription factors in
the regulation of steroidogenic acute regulatory
protein gene expression. Steroids. 68:1125–1134.
49. Can, A., and Semiz, O. 2000. Diethylstilbestrol (DES)-
induced cell cycle delay and meiotic spindle disrup-
tion in mouse oocytes during in-vitro maturation.
Mol. Hum. Reprod. 6:154–162.
50. Goldstein, P. 1986. Nuclear aberrations and loss of
synaptonemal complexes in response to diethylstil-
bestrol (DES) in Caenorhabditis elegans hermaph-
rodites. Mutat. Res. 174:99–107.
51. Matsui, Y., and Hayashi, K. 2007. Epigenetic regula-
tion for the induction of meiosis. Cell. Mol. Life Sci.
52. Peters, A.H., et al. 2001. Loss of the Suv39h
histone methyltransferases impairs mamma-
lian heterochromatin and genome stability. Cell.
53. Boulias, K., and Talianidis, I. 2004. Functional role of
G9a-induced histone methylation in small heterodi-
mer partner-mediated transcriptional repression.
Nucleic Acids Res. 32:6096–6103.
54. Fang, S., et al. 2007. Coordinated recruitment of
histone methyltransferase G9a and other chroma-
tin-modifying enzymes in SHP-mediated regula-
tion of hepatic bile acid metabolism. Mol. Cell. Biol.
55. Feldman, N., et al. 2006. G9a-mediated irrevers-
ible epigenetic inactivation of Oct-3/4 during early
embryogenesis. Nat. Cell Biol. 8:188–194.
56. Cho, H.W., et al. 2003. The antiestrogen ICI
182,780 induces early effects on the adult male
mouse reproductive tract and long-term decreased
fertility without testicular atrophy. Reprod. Biol.
57. Lee, K.H., et al. 2000. Estrogen receptor alpha has a
functional role in the mouse rete testis and efferent
ductules. Biol. Reprod. 63:1873–1880.
58. Oliveira, C.A., Carnes, K., Franca, L.R., and Hess,
R.A. 2001. Infertility and testicular atrophy in the
antiestrogen-treated adult male rat. Biol. Reprod.
59. Oliveira, C.A., et al. 2002. ER function in the adult
male rat: short- and long-term effects of the anties-
trogen ICI 182,780 on the testis and efferent duct-
ules, without changes in testosterone. Endocrinology.
60. Hess, R.A., et al. 1997. A role for oestrogens in the
male reproductive system. Nature. 390:509–512.
61. Zhou, Q., et al. 2001. Estrogen action and male fertil-
ity: roles of the sodium/hydrogen exchanger-3 and
fluid reabsorption in reproductive tract function.
Proc. Natl. Acad. Sci. U. S. A. 98:14132–14137.
62. Gu, P., et al. 2005. Orphan nuclear receptor LRH-
1 is required to maintain Oct4 expression at the
epiblast stage of embryonic development. Mol. Cell
63. Kuiper, G.G., et al. 1997. Comparison of the ligand
binding specificity and transcript tissue distribu-
tion of estrogen receptors alpha and beta. Endocri-
64. Petersen, D.N., Tkalcevic, G.T., Koza-Taylor, P.H.,
Turi, T.G., and Brown, T.A. 1998. Identification of
estrogen receptor beta2, a functional variant of estro-
gen receptor beta expressed in normal rat tissues.
65. McNeilly, J.R., et al. 2000. Loss of oocytes in
Dazl knockout mice results in maintained ovar-
ian steroidogenic function but altered gonado-
tropin secretion in adult animals. Endocrinology.