Diethylstilbestrol induces fish oocyte maturation
Toshinobu Tokumoto*†‡, Mika Tokumoto*†, Ryo Horiguchi§¶, Katsutoshi Ishikawa*, and Yoshitaka Nagahama†§
*Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan; †CREST Research Project, Japan Science and
Technology Corporation, Kawaguchi 332-0012, Japan; §Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan;
and ¶Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki 444-8585, Japan
Communicated by Howard A. Bern, University of California, Berkeley, CA, January 6, 2004 (received for review August 6, 2003)
An endocrine-disrupting chemical, diethylstilbestrol (DES), a non-
steroidal estrogen, triggers oocyte maturation in fish. The mor-
phology (the time course of the change in germinal vesicle break-
down) and an intracellular molecular event (the de novo synthesis
of cyclin B) induced by DES were indistinguishable from those
induced by a natural maturation-inducing hormone, 17␣,20␤-di-
hydroxy-4-pregnen-3-one (17,20␤-DHP). A synergistic action of
DES on 17,20␤-DHP-induced oocyte maturation was observed.
Both 17,20␤-DHP- and DES-induced oocyte maturation was inhib-
ited by an antibody against the maturation-inducing hormone
receptor. The structural requirement for the action of DES is
discussed based on results obtained with DES analogs.
maturation-inducing hormone 兩 endocrine-disrupting chemical 兩
goldfish 兩 zebrafish
O ocyte maturation in lower vertebrates is triggered by mat-
uration-inducing hormone (MIH), which acts on receptors
located on the oocyte membrane and induces the activation of
maturation-promoting factor in the oocyte cytoplasm (1–4).
During the course of maturation, oocytes undergo drastic mor-
phological changes associated with progression of the meiotic
cell cycle, in which breakdown of the oocyte nuclear envelope
[germinal vesicle breakdown (GVBD)] occurring at the
prophase兾metaphase transition is usually regarded as a hallmark
of the progress of oocyte maturation. Two MIHs, 17␣,20␤-
dihydroxy-4-pregnen-3-one (17,20␤-DHP) and 17␣,20␤,21-
trihydroxy-4-pregnen-3-one (20␤-S), have been identified in
several fish species (5, 6). In goldfish, 17,20␤-DHP has been
shown to induce oocyte maturation by stimulating the de novo
synthesis of cyclin B, a regulatory subunit of maturation-
promoting factor (7). Although progestins including 17,20␤-
DHP and 20␤-S are the most potent steroid inducers of oocyte
maturation in fish, other hormones such as deoxycorticosterone
and testosterone, but not estradiol or its analogs, are also
effective (8).
Several endocrine-disrupting chemicals, Kepon and dichlo-
rodiphenyldichloroethane, have been reported to antagonize
MIH-induced meiotic maturation of fish oocytes in vitro (9).
One of the environmental endocrine-disrupting chemicals
(EEDCs), diethylstilbestrol (DES) is a nonsteroidal substance
that was prescribed from the late 1940s to the early 1970s to
pregnant women to prevent abortion, preeclampsia, and other
complications of pregnancy. Male and female offspring ex-
posed in utero to DES may develop multiple and neoplastic
lesions of the reproductive tract, along with other changes,
during development (10). Here we show that exposing fish
oocytes to DES at a dose within a range similar to that used
in experimental exposure to 17,20␤-DHP induces oocyte
maturation. Estradiol-17␤ has been reported to be ineffective
in inducing fish oocyte maturation (11, 12) and even inhibitory
in several teleost species (13–15). Thus, the stimulatory effect
of DES to induce fish oocyte maturation observed in this study
has not been published previously. This report shows that
EEDC can potentially induce oocyte maturation like an en-
dogenous MIH, 17,20␤-DHP.
3686 –3690 兩 PNAS 兩 March 9, 2004 兩 vol. 101 兩 no. 10
Materials and Methods
Materials. Goldfish were purchased from a local supplier and
maintained at 15°C until used. Zebrafish were maintained at
28.5°C on a 14-h light兾10-h dark cycle (16). 17,20␤-DHP, DES,
DES dimethyl ether (DM-DES), DES dipropionate (DP-
DES), and 17␤-estradiol were purchased from Sigma. Di-
methylstilbestrol (DMS) was a generous gift from J. Katzenel-
lenbogen (University of Illinois, Urbana). 17␣-Estradiol,
ethynylestradiol, butyl benzyl phthalate, di(2-ethylhexyl)-
phthalate, and pentachlorophenol were obtained from Wako
Pure Chemical (Osaka). Other chemicals were purchased as
follows: hexestrol (HEX; ICN); trans,trans-dienestrol( ␣ -
dienestrol) (DIES; U.S. Pharmacopeia, Rockville, MD); res-
veratorol (Calbiochem); DDTs (AccuStandard, New Haven,
CT); bisphenol A (Nacalai Tesque, Kyoto); p-nonylphenol
(Kanto Chemical, Tokyo); 4-octylphenol (Aldrich).
Oocyte Preparation and in Vitro Culture. Ovaries of goldfish were
isolated from killed females and placed in fresh goldfish
Ringer’s solution (125 mM NaCl兾2.4 mM KCl兾0.28 mM
MgSO4兾0.89 mM MgCl2兾2.4 mM CaCl2兾2 mM Hepes兾5.6 mM
glucose兾100 units/ml penicillin兾0.2 mg/ml streptomycin, pH
7.5) and washed three times with the same solution. Full-grown
immature oocytes were exposed in vitro by incubating ovarian
fragments (each containing 5–20 oocytes) in 4 ml of goldfish
Ringer’s solution containing each agent (from a 1,000-fold
stock in ethanol) at room temperature with gentle agitation (40
rpm). To assess maturation processes, germinal vesicles in
full-grown oocytes were examined under a binocular micro-
scope (SMZ645, Nikon) after placing the oocytes in clearing
solution (17). The nuclear state (%) at each time point was
determined in 40 oocytes. The morphology of oocytes was
photographed with a digital microscope (VH8000, Keyence,
Osaka).
Ovaries of zebrafish were isolated from killed females and
placed in fresh zebrafish Ringer’s solution (116 mM NaCl兾2.9
mM KCl兾1.8 mM CaCl2兾5 mM Hepes, pH 7.2) and washed three
times with the same solution. Immature oocytes were exposed in
vitro by incubating ovarian fragments (each containing 2–10
oocytes) in 4 ml of zebrafish Ringer’s solution containing each
agent (from a 1,000-fold stock in ethanol) at room temperature
with gentle agitation (40 rpm). To assess maturation processes,
germinal vesicles in full-grown oocytes were examined under a
binocular microscope (SMZ645, Nikon) after placing the oo-
cytes in clearing solution (17). The nuclear state (%) at each time
point was determined in ⬎20 oocytes.
Preparation of Oocyte and Egg Extracts. Groups of 20 oocytes were
washed in extraction buffer (0.1 M sodium ␤-glycerophos-
Abbreviations: 17,20␤-DHP, 17␣, 20␤-dihydroxy-4-pregnen-3-one; MIH, maturation-
inducing hormone; EEDC, environmental endocrine-disrupting chemical; DES, diethyl-
stilbestrol; HEX, hexestrol; DIES, trans,trans-dienestrol(␣-dienestrol); DMS, dimethyl-
stilbestrol;, DM-DES, DES dimethyl ether; DP-DES, DES dipropionate; GVBD, germinal
vesicle breakdown; DDT, dichlorodiphenyltrichloroethane; mPR␣, membrane progestin
receptor ␣.
‡To whom correspondence should be addressed. E-mail: sbttoku@ipc.shizuoka.ac.jp.
© 2004 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0400072101
Table 1. In vitro induction of GVBD in goldfish oocytes
GVBD, % at concentration of

Treatment 0.1 ␮M 1 ␮M 2 ␮M

EtOH 0
17,20␤-DHP 83.3 ⫾ 6.3 98.3 ⫾ 2.9 98.3 ⫾ 2.9
DES 0 18.3 ⫾ 6.3 85.0 ⫾ 8.7
␤-Estradiol 0 0 0
17␣-Estradiol 0 0 0
Ethynylestradiol 0 0 0
Resveratorol 0 0 0.8 ⫾ 1.4
o,p⬘-DDT 0 0 0
p,p⬘-DDT 0 0 0
Butyl benzyl phthalate 0 0 0
Di(2-ethylhexyl)phthalate 0 0 0
Bisphenol A 0 0 0
p-Nonylphenol 0 0 0
4-Octylphenol 0 0 0
Pentachlorophenol 0 0 0

The percentage of GVBD was calculated by determining the percentage of

oocytes that had undergone GVBD in a group of 40 oocytes cultured in parallel
for 6 h. Each value is the mean (⫾ SD) of three separate experiments with
ovaries of three separate females.

phate兾15 mM MgCl2兾5 mM EGTA兾20 mM Hepes兾1 mM DTT,

pH 7.5) and transferred to a 1.5-ml Eppendorf microcentrifuge
tube. After the excess buffer was removed, 200 ␮l of buffer was
added. The samples were crushed with five strokes of a plastic
pestle and centrifuged at 13,500 rpm for 10 min at 4°C in a
fixed-angle rotor (MX-300 microcentrifuge, Tomy, Tokyo). The
clear supernatant (100 ␮l) was collected for electrophoresis and
immunoblotting.
SDS兾PAGE and Immunoblotting. Proteins were separated by
PAGE under denaturing conditions (SDS兾PAGE with 10%
gel) by the method of Laemmli (18) and transferred to
Immobilon membrane (Millipore). Membranes were blocked
in 5% nonfat powdered milk and incubated with primary
antibodies for 1 h at room temperature. Immunocomplexes
Fig. 1. DES induces genuine oocyte maturation. (A) The morphology of
oocytes after 6 h of each treatment was photographed. Germinal vesicles were
seen near the center of oocytes after EtOH treatment, whereas they disap-
peared after 17,20␤-DHP and DES treatments. (B) Extracts were prepared from
20 oocytes after incubation with EtOH, 17,20␤-DHP, or DES. Extracts of each
treatment were electrophoresed under denaturing conditions (10.0% gel)
and stained with Coomassie brilliant blue (CBBR) or immunostained with
anti-goldfish cyclin B polyclonal antibody after electroblotting (␣-cycB). The
48-kDa band of cyclin B is indicated by an arrow. Molecular masses of standard
proteins are indicated on the left.

were visualized by using the ECL detection kit (Amersham
Biosciences).
cDNA Cloning and Production of Recombinant Proteins. Recently, a
strong candidate for an MIH membrane receptor has been
identified and characterized in spotted seatrout (19). We cloned
cDNA for this membrane progestin receptor ␣ (mPR␣) from
goldfish. Details of cDNA cloning of a homologue of mPR␣ will
be described elsewhere. GST-tagged recombinant protein of a
fragment of 78 N-terminal amino acids of goldfish mPR␣ was
produced. 35S-Labeled mPR␣ was produced by using a TNT
T7-coupled Reticulocyte Lysate System (Promega) according to
the manufacturers instructions.
Antibody Production. Full-length goldfish cyclin B was produced
in Escherichia coli BL21 (DE3) and 78 N-terminal amino
acids of goldfish mPR␣ was produced in E. coli XL1Blue.
These were purified by SDS兾PAGE, and subsequently ob-
tained from the gel by electroelution (20). Polyclonal antibod-
ies specific for cyclin B and the receptor protein were raised
against purified recombinant proteins according to a proce-
dure described by using guinea pigs (21). Antisera that rec-
ognize the 48-kDa band of cyclin B and the 40-kDa band of
mPR␣ were obtained.
Results
Relative Potency of Various Substances in Inducing Fish Oocyte
Maturation. The relative effectiveness of MIH and 13 other
agents, including EEDCs and several steroid hormones, in
PHYSIOLOGY
inducing GVBD was investigated by using goldfish oocytes
(Table 1). Of the 13 agents tested, only DES was effective in
inducing GVBD. As in the previous report (8), two other
estrogens (␤-estradiol and ethynylestradiol) and 17␣-estradiol
did not induce GVBD at the concentration tested. Eight kinds
of EEDCs (DDTs, phthalates, and phenols) did not induce
GVBD at the doses tested.
DES Induces Natural Oocyte Maturation. Fig. 1A shows the mor-
phology of oocytes after 6 h of EtOH, 17,20␤-DHP, and DES
treatment. Germinal vesicles were seen near the center of
oocytes after EtOH treatment, whereas they disappeared after
the 17,20␤-DHP and DES treatments. Also, 17,20␤-DHP- and
DES-treated oocytes became transparent and changed color to
a bright yellow. DES also induced the translation of cyclin B
protein (Fig. 1B), a well characterized intracellular molecular
event that results in an elevation of the kinase activity of
maturation-promoting factor. These results show that the
DES-induced maturation was identical with the physiological
maturation of oocytes as judged by several criteria.
Possible Target of DES in Inducing GVBD. To address the target of
DES in the signal transduction pathway to induce GVBD, as
a first step, the time-course changes of GVBD induced by DES
were compared with those of GVBD induced by 17,20␤-DHP.
The time course of oocyte maturation induced by DES was the

Tokumoto et al. PNAS 兩 March 9, 2004 兩 vol. 101 兩 no. 10 兩 3687

Fig. 2. Time-course change of GVBD induced by 17,20␤-DHP and DES. Oocyte
maturation induced by 0.01% EtOH (E), 1 ␮M 17,20␤-DHP (F), or 2 ␮M DES (‚).
The percentage of GVBD was calculated by determining the percentage of
oocytes that had undergone GVBD in a group of 40 oocytes cultured for 6 h.
Each value is the mean of three separate experiments with ovaries from three
separate females. Vertical lines indicate standard deviation.
same as that induced by 17,20␤-DHP (Fig. 2), suggesting that
the most likely candidate for the target of DES is a membrane
receptor of 17,20␤-DHP. To clarify whether DES induces
oocyte maturation through interaction with the MIH receptor,
the combined effect of DES and 17,20␤-DHP on the induction
of GVBD was examined (Fig. 3). When oocytes were treated
with a mixture of DES and 17,20␤-DHP at concentrations at
which each agent could not induce GVBD alone, GVBD was
induced: 20 nM 17,20␤-DHP and 0.5 ␮M DES or 10 nM
17,20␤-DHP and 0.5 ␮M DES (Fig. 3A). Also, the magnitude
of GVBD was significantly elevated at concentrations at which
DES and 17,20␤-DHP induced GVBD to only a minor extent
(⬍10%): 50 nM 17,20␤-DHP and 1 ␮M DES or 20 nM
17,20␤-DHP and 1 ␮M DES. These results strongly suggest
that DES and 17,20␤-DHP are agonists to induce goldfish
oocyte maturation.
Effect of DES Analogs on Inducing Oocyte Maturation. In further
analysis, we checked the potency of DES in inducing oocyte
maturation in another species of fish, the zebrafish. Although the
goldfish is suitable for biochemical analyses, its spawning season
is limited to the spring. In zebrafish, full-grown oocytes that can
be induced to undergo maturation by MIH are collectable in all
seasons.
Zebrafish oocytes were found to be more sensitive to 17,20␤-
DHP and DES than goldfish oocytes (Table 2). Zebrafish
oocytes can be induced to undergo maturation at concentrations
approximately one order of magnitude lower than those needed
for goldfish oocytes. We therefore decided to use zebrafish in
further analyses.
The relative effectiveness of DES analogs in inducing
GVBD was determined (Table 2). We examined two groups of
DES derivatives. The compounds in the first group had a
change in the center part of DES (olefinic double bond and
ethyl groups), whereas those in the second group had a
hydroxyl changed. Among the first group, HEX induced
GVBD almost the same as DES. DIES induced GVBD at a
concentration one order of magnitude higher than HEX or
DES. These compounds are altered at the olefinic double bond
compared with DES. Another compound in the first group,
DMS, induced GVBD only at 10% even at concentrations 100
times greater than DES. The ethyl groups in DES are changed
3688 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0400072101
Fig. 3. Synergistic action of DES on 17,20␤-DHP-induced oocyte maturation.
(A) Oocytes were incubated with various concentrations of 17,20␤-DHP and
DES. The percentage of GVBD was assessed after 6 h. (B) Oocytes were
incubated with (F) or without (E) 20 nM 17,20␤-DHP for 6 h in the presence
of increasing concentrations of DES. GVBD was 0% at 20 nM 17,20␤-DHP
alone. Each value is the mean of three separate experiments with ovaries from
three separate females. Vertical bars show standard deviation.
to methyl groups in DMS. The results suggest that ethyl groups
are important for interaction between DES and the MIH
receptor and the length of the ethyl group is necessary. They
also suggest that the f lexibility of side chains is important for
appropriate interaction. In the second group, although DP-
DES induced GVBD at 50%, DM-DES did not have any effect.
These results suggest that hydroxides also contribute to the
interaction of DES with the MIH receptor.
Inhibition of DES-Induced Oocyte Maturation by Anti-mPR Antibody.
We cloned a cDNA for mPR␣ from goldfish showing overall
80% homology with spotted sea trout. A polyclonal antibody
against its N-terminal fragment (79 amino acids), which is
predicted as an external cellular region, was prepared. The
antibody cross-reacted with the 40-kDa band of goldfish and
zebrafish extracts from immature oocytes (Fig. 4A). Then, the
effects of the antibody on the 17,20␤-DHP- and DES-induced
oocyte maturation were examined. Immature oocytes were
treated with the antibody before addition of 17,20␤-DHP and
DES. The percentage of GVBD was lowered to ⬇50% in both
17,20␤-DHP and DES groups, compared with that of the control
IgG group (Fig. 4B). These results strongly suggested that
DES-induced oocyte maturation is by way of the MIH-mediated
pathway.
Discussion
In this study, the effects of endocrine-disrupting chemicals on
the maturation of fish oocytes were examined. We found that
Tokumoto et al.
 Table 2. In vitro induction of GVBD in zebrafish oocytes
GVBD, % at concentration, ␮M of

Treatment 0.001 0.01 0.1 1 2

EtOH 0

17,20␤-DHP 28.1 ⫾ 17.4 86.5 ⫾ 11.9 92.6 ⫾ 4.1 92.1 ⫾ 6.9 –

DES – 2.9 ⫾ 2.6 72.5 ⫾ 10.3 84.9 ⫾ 9.3 91.2 ⫾ 6.4

DMS – 0 ⫾ 0.0 1.8 ⫾ 3.1** 7.9 ⫾ 6.3** 11.5 ⫾ 8.1**

HEX – 0 ⫾ 0.0 38.3 ⫾ 21.2** 82.4 ⫾ 5.8 86.3 ⫾ 8.2

DIES – 0 ⫾ 0.0 4.5 ⫾ 3.8** 49.2 ⫾ 3.4** 73.2 ⫾ 2.2**

DM-DES – 0 ⫾ 0.0 0 ⫾ 0.0** 0 ⫾ 0.0** –

DP-DES – 5.0 ⫾ 0.5 16.7 ⫾ 7.1** 63.5 ⫾ 4.5** 74.4 ⫾ 2.8*

The percentage of GVBD was calculated by determining the percentage of oocytes that had undergone GVBD in a group composed
of ⬎20 oocytes cultured in parallel for 3 h. Each value in the mean (⫾ SD) of three separate experiments with ovaries of three separate
females. ** and * indicate statistically significant differences between the percentage of GVBD induced by the same concentration of
DES and DES analogues at the P ⬍ 0.01 and P ⬍ 0.05 levels, respectively.

treatment of oocytes with a nonsteroidal substance, DES, alone
induces maturation. The morphology and an intracellular mo-
lecular event induced by DES and 17,20␤-DHP were indistin-
guishable and suggest that, at least qualitatively, DES and
17,20␤-DHP induced the same type of maturation. However, a
difference occurred between the relative potency of DES to
induce oocyte maturation and that of 17,20␤-DHP. Other
EEDCs did not induce GVBD at the doses examined in this
study, although we cannot rule out the possibility that these
agents might have effects at higher doses.
In general, the potency and biological properties of a
compound can be predicted from its structure. It is possible,
therefore, to dissect the contribution of a particular structural
modification of a compound that binds to the 17,20␤-DHP
receptor to the biological response of a target. To study the
structural requirement for the MIH action of DES, the relative
potency of DES analogs to induce GVBD was compared. All
of the compounds except DM-DES induced a dose-related
stimulation of oocyte maturation similar to that induced by
17,20␤-DHP, although potencies were significantly different
(17,20␤-DHP ⬎ DES, HEX ⬎ DIES, and DP-DES ⬎ DMS).
This analytical approach to the structure–activity relationships
of compounds revealed that ethyl groups and hydroxyls of DES
contribute to the interaction between DES and the MIH
receptor. Other compounds, such as 4-hydroxytamoxifen and
bisphenol, have structural features similar to DES necessary
PHYSIOLOGY
for their potential interaction with the MIH receptor. These
compounds might also have the ability to induce oocyte
maturation in fish.
To understand whether DES acts by means of the MIH
receptor specific for 17,20␤-DHP, we used indirect strategy by
raising an antibody against goldfish mPR␣. Both 17,20␤-DHP-
and DES-induced oocyte maturation was inhibited by this anti-
body. These findings, together with data from time-course
studies and synergistic effects, suggest that DES may interact
with the MIH receptor. Further evidence to support these
mechanisms of DES action is required.
It is well known that DES is a ligand for estrogen receptors (22,
23). However, interaction between DES and other physiological
receptors has been reported recently (24–26). Orphan nuclear
receptors are members of the nuclear receptor family that lack
identified ligands (27). Recent studies have identified several
natural and synthetic ligands for orphan nuclear receptors, which
led to new hormone-response systems implicated in the control
of various aspects of development and adult physiology (28, 29).
Among orphan nuclear receptors, the estrogen-related receptors
have been reported to be inhibited in their binding with coac-
tivator protein by DES. In trophoblast stem cells, DES promotes
coactivator release from estrogen-related receptor ␤ and inhibits
its transcriptional activity (25). By using fluorescence resonance
energy transfer assay, it has also been shown that DES binds to
estrogen-related receptor ␥ (26). These findings suggest a path-
way for DES action as a ligand for receptors besides estrogen

Tokumoto et al. PNAS 兩 March 9, 2004 兩 vol. 101 兩 no. 10 兩 3689

Fig. 4. Inhibition of 17,20␤-DHP- and DES-induced oocyte maturation by anti-mPR␣ antibody. (A) Extracts were prepared from 20 oocytes of goldfish (lane 1)
and zebrafish (lane 3). Extracts were electrophoresed under denaturing conditions (10.0% gel) and immunostained with anti-goldfish mPR␣ polyclonal antibody
after electroblotting. The 35S-labeled goldfish mPR␣ was produced in vitro in rabbit reticulocyte lysate (lane 2). After the translation, 35S-labeled proteins were
resolved by SDS兾PAGE followed by autoradiography on imaging plates (Fuji). The 40-kDa band of mPR␣ is indicated by an arrow. Molecular masses of standard
proteins are indicated on the left. (B) Oocytes were incubated with 100 ␮g兾ml anti-goldfish mPR␣ polyclonal antibody (Anti) or control IgG (Cont) for 1 h at 25°C,
then oocytes were treated with 0.1 ␮M 17,20␤-DHP or 1 ␮M DES for 3 h. The percentage of GVBD was calculated by determining the percentage of oocytes that
had undergone GVBD in a group of ⬎20 oocytes. Each value is the mean of three separate experiments with ovaries from three separate females. Vertical bars
show standard deviation. **, statistically significant differences between the percentage of GVBD induced in control and anti-mPR␣ antibody-treated oocytes
at the P ⬍ 0.01 level.

receptors. Likewise, this study shows that DES may interact with
the MIH receptor.
Many EEDCs, including DES, may mimic the action of the sex
hormone estradiol and are therefore referred to as xenoestro-
gens. We show here the possibility that they mimic other steroids.
Our findings emphasize the need for studies to examine more
widely the various nonestrogenic effects of endocrine disrupters.
We thank Dr. J. A. Katzenellenbogen for the gift of DMS. This work was
supported by the CREST Research Project of Japan Science and
Technology Corporation (to Y.N.) and by grants-in-aid for Scientific
Research on Priority Areas from the Ministry of Education, Culture,
Sports, Science and Technology of Japan. Part of this study was
performed as the National Institute for Basic Biology Cooperative
Research Program (00-121 and 01-112 to T.T). M.T. is a Research Fellow
of the Japan Science and Technology Corporation.

1. Ishikawa, K., Hanaoka, Y., Kondo, Y. & Imai, K. (1977) Mol. Cell. Endocrinol.
9, 91–100.
2. Godeau, J. F., Schorderet-Slatkine, S., Hubert, P. & Baulieu, E. E. (1978) Proc.
Natl. Acad. Sci. USA 75, 2353–2357.
3. Yoshikuni, M., Shibata, N. & Nagahama, Y. (1993) Fish Physiol. Biochem. 11, 15–24.
4. Masui, Y. & Clarke, H. J. (1979) Int. Rev. Cytol. 57, 185–282.
5. Nagahama, Y. & Adachi, S. (1985) Dev. Biol. 109, 428–435.
6. Patino, R. & Thomas, P. (1990) Gen. Comp. Endocrinol. 78, 474–478.
7. Yamashita, M., Kajiura, H., Tanaka, T., Onoe, S. & Nagahama, Y. (1995) Dev.
Biol. 168, 62–75.
8. Nagahama, Y., Hirose, K., Young, G., Adachi, S., Suzuki, K. & Tamaoki, B.
(1983) Gen. Comp. Endocrinol. 51, 15–23.
9. Thomas, P. (1999) in Endcrine Disruptors: Effects on Male and Female
Reproductive Systems, ed. Naz. R. K. (CRC, Boca Raton, FL), pp. 3–38.
10. Bern, H. A. (1992) in Chemically Induced Alterations in Sexual and Functional
Development: The Wildlife兾Human Connection, eds. Collborn. T. & Clement. C.
(Princeton Scientific Publishing, Princeton, NJ.), pp. 9–15.
11. Young, G., Kagawa, H. & Nagahama, Y. (1982) J. Exp. Zool. 224, 265–275.
12. Trant, J. M. & Thomas, P. (1988) Gen. Comp. Endocrinol. 71, 307–317.
13. Pandey, S. & Hoar, W. S. (1972) Can. J. Zool. 50, 1679–1680.
14. Jalabert, B. (1975) C. R. Hebd. Seances Acad. Sci. D 281, 811–814.
15. Sundararaj, B. I., Goswami, S. V. & Lamba, V. (1979) J. Steroid Biochem. 11,
701–707.
16. Westerfield, M. (1995) in The Zebrafish Book: A Guide for the Laboratory Use
of Zebrafish (Danio rerio) (Univ. of Oregon Press, Eugene).
17. Lessman, C. A. & Kavumpurath, S. (1984) Gamete Res. 10, 21–29.
18. Laemmli, U. K. (1970) Nature 227, 680–685.
19. Zhu, Y., Rice, C. D., Pang, Y., Pace, M. & Thomas, P. (2003) Proc. Natl. Acad.
Sci. USA 100, 2231–2236.
20. Hirai, T., Yamashita, M., Yoshikuni, M., Lou, Y. H. & Nagahama, Y. (1992)
Mol. Reprod. Dev. 33, 131–140.
21. Tokumoto, T., Tokumoto, M., Seto, K., Horiguchi, R., Nagahama, Y., Yamada,
S., Ishikawa, K. & Lohka, M. J. (1999) Exp. Cell Res. 247, 313–319.
22. Katzenellenbogen, B. S., Iwamoto, H. S., Heiman, D. F., Lan, N. C. &
Katzenellenbogen, J. A. (1978) Mol. Cell. Endocrinol. 10, 103–113.
23. Prins, G. S., Birch, L., Couse, J. F., Choi, I., Katzenellenbogen, B. & Korach,
K. S. (2001) Cancer Res. 61, 6089–6097.
24. Lu, D., Kiriyama, Y., Lee, K. Y. & Giguere, V. (2001) Cancer Res. 61, 6755–6761.
25. Tremblay, G. B., Kunath, T., Bergeron, D., Lapointe, L., Champigny, C., Bader,
J. A., Rossant, J. & Giguere, V. (2001) Genes Dev. 15, 833–838.
26. Coward, P., Lee, D., Hull, M. V. & Lehmann, J. M. (2001) Proc. Natl. Acad.
Sci. USA 98, 8880–8884.
27. Giguere, V. (1999) Endocr. Rev. 20, 689–725.
28. Blumberg, B. & Evans, R. M. (1998) Genes Dev. 12, 3149–3155.
29. Kliewer, S. A., Lehmann, J. M. & Willson, T. M. (1999) Science 284, 757–760.

3690 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0400072101 Tokumoto et al.