Sex Reversal TestUsing a New Quail Model,

F1(AWE x WE), is Useful to Assess Estrogenic Endocrine Activity

as Tier 1 in vivo Screening

K. Shibuya1, K. Sato1, M. Mizutani1, M. Wada2, K. Shimada3, and T. Nunoya1

1Nippon Institute for Biological Science, Tokyo, Japan2Tokyo Medical and Dental University, Chiba, Japan3Nagoya University, Aichi, Japan

IntroductionTier 1 screening battery in birds for the assessment of endocrine disrupting effects of

chemicals has not been completed. There are a few candidates of in vitro and in vivoscreening methods such as estrogen receptor binding assay, mRNA detections ofvetellogenin (VTG) and very low density lipoprotein, and monitoring of blood VTG level. We have developed a new in vivo screening method, which is rapid and cost-effective, for evaluating the estrogenic endocrine activities of chemicals using F1(AWE x WE) Japanese quail (Coturnix japonica) embryos. F1(AWE x WE) Japanese quail have been produced by mating between male AWE

(albino plumage color) and female WE (wild plumage color) strains. The male and female F1 quail exhibit wild and albino phenotypes, respectively, which are ruled by acriss-cross inheritance. Therefore, it is easy to determine genetic sex in the embryonic stage.

ObjectivesTo evaluate feminization effects and potential of natural and synthetic estrogens in the gonads of male Japanese quail embryos using the sex reversal test.

To investigate estrogenic endocrine disrupting effects of nonylphenol (NP) and bisphenol A (BPA) in Japanese quail embryos.

Female F1(AWE x WE) embryo(AL*A/W phenotype) with albino plumage at 16 days of incubation

Male AWE quail (AL*A/AL*A phenotype) with albino plumage

Female WE quail (AL*N/W phenotype) with wild plumage

Male F1(AWE x WE) embryo(AL*N/AL*A phenotype) with wild plumage at 16 days of incubation

Schema of Criss-cross Inheritance

IncubationDay-0

Administration Necropsy

▼

IncubationDay-16

▼

Materials & Methods

●Test substances:17 beta-estradiol (E2), Sigma Chemical Co., U.S.A.Diethylstilbestrol (DES), ICN Biomedicals Inc., U.S.A.Ethynylestradiol (EE2), Wako Pure Chemical Industries, Ltd., Japan Nonylphenol (NP), Kanto Chemical Co., Inc., JapanBisphenol A (BPA), Kanto Chemical Co., Inc., Japan

●Vehicle:Corn oil, Wako Pure Chemical Industries, Ltd., Japan

●Experiment design

40200 ngBPA 200 ng

50200000 ngNP 200000 ng

402000 ngBPA 2000 ng4020000 ngBPA 20000 ng

40Corn oilBPA control4020 ngBPA 20 ng

5020000 ngNP 20000 ng502000 ngNP 2000 ng50200 ngNP 200 ng50Corn oilNP control

No. ofeggs

Dose(20μL/egg)

Group

4020 ngEE2 20 ng40200 ngEE2 200 ng402000 ngEE2 2000 ng

4020 ngDES 20 ng

40200 ngDES 200 ng402000 ngDES 2000 ng

442000 ngE2 2000 ng45200 ngE2 200 ng4520 ngE2 20 ng44Corn oilControl

No. ofeggs

Dose(20μL/egg)

Group

Experiment-1 Experiment-2

●Measurements and observationsFertilityEmbryo viabilitySex differentiation (plumage color, gonadal appearance)Histopathology of the gonads

●Groups and doses

●Methods of morphometric analysis

Graphic analysisGraphic analysis system by ATTO Corp.

Area (%)

Transfer

×

100

Extraction of whole testis

Transfer

÷Extraction of ovarian tissue

〓

Testis

Ovarian tissues in the testis

Groups Control 63.6 (28/44) a 92.9 (26/28) b

E 2 20 ng 71.1 (32/45) 90.6 (29/32) E 2 200 ng 66.7 (30/45) 83.3 (25/30) E 2 2000 ng 70.5 (31/44) 83.9 (26/31) DES 20 ng 75.0 (30/40) 70.0 (21/30) c

DES 200 ng 72.5 (29/40) 79.3 (23/29) DES 2000 ng 87.5 (35/40) c 85.7 (30/35) EE 2 20 ng 77.5 (31/40) 80.6 (25/31) EE 2 200 ng 70.0 (28/40) 92.9 (26/28) EE 2 2000 ng 77.5 (31/40) 77.4 (24/31) a: Numbers of fertile eggs/numbers of eggs set. b: Numbers of viable embryos/numbers of fertile eggs. c: p < 0.05 from the control group.

Fertility(%)

Viability (%) of incubationday-16 embryos

Fertility and Viability of Embryos Treated with E 2 , DES and EE 2

Conformability Groups Male Female % of male Male Female % of male (%) Control 17 a 9 65.4 17 9 65.4 100.0 E 2 20 ng 15 14 51.7 15 14 51.7 100.0 E 2 200 ng 15 10 60.0 15 10 60.0 100.0 E 2 2000 ng 11 15 42.3 11 15 42.3 100.0 DES 20 ng 13 8 61.9 13 8 61.9 100.0 DES 200 ng 12 11 52.2 12 11 52.2 100.0 DES 2000 ng 20 10 66.7 20 10 66.7 100.0 EE 2 20 ng 15 10 60.0 15 10 60.0 100.0 EE 2 200 ng 11 15 42.3 11 15 42.3 100.0 EE 2 2000 ng 14 10 58.3 8 16 33.3 b 57.1 b

a: Numbers of embryos examined. b: p < 0.05 from the control group.

Conformability in Sex Difference between Plumage Color and Gross Gonad Appearance ofEmbryos Treated with E 2 , DES and EE 2

Plumage Gonad

Groups NP control 72.0 (36/50) a 86.1 (31/36) b

NP 200 ng 70.0 (35/50) 82.9 (29/35) NP 2000 ng 80.0 (40/50) 82.5 (33/40) NP 20000 ng 80.0 (40/50) 77.5 (31/40) NP 200000 ng 82.0 (41/50) 85.4 (35/41) BPA control 90.0 (36/40) 83.3 (30/36) BPA 20 ng 92.5 (37/40) 89.2 (33/37) BPA 200 ng 90.0 (36/40) 86.1 (31/36) BPA 2000 ng 90.0 (36/40) 86.1 (31/36) BPA 20000 ng 92.5 (37/40) 91.9 (34/37) a: Numbers of fertile eggs/numbers of eggs set. b: Numbers of viable embryos/numbers of fertile eggs.

Fertility(%)

Viability (%) of incubationday-16 embryos

Fertility and Viability of Embryos Treated with NP and BPA

Conformability Groups Male Female % of male Male Female % of male (%) NP control 15 a 16 48.4 15 16 48.4 100.0 NP 200 ng 14 15 48.3 14 15 48.3 100.0 NP 2000 ng 17 16 51.5 17 16 51.5 100.0 NP 20000 ng 16 15 51.6 16 15 51.6 100.0 NP 200000 ng 23 12 65.7 23 12 65.7 100.0 BPA control 14 16 46.7 14 16 46.7 100.0 BPA 20 ng 17 16 51.5 17 16 51.5 100.0 BPA 200 ng 21 10 67.7 21 10 67.7 100.0 BPA 2000 ng 15 16 48.4 15 16 48.4 100.0 BPA 20000 ng 22 12 64.7 22 12 64.7 100.0a: Numbers of embryos examined.

Conformability in Sex Difference between Plumage Color and Gross Gonad Appearance ofEmbryos Treated with NP and BPA

Plumage Gonad

Incidence of Ovotestis in Male Embryos Treated with E2, DES, and EE2

0

20

40

60

80

100In

cide

nce

(%)

Con

trol

E2

20 n

g

E2

200

ng

E2

2000

ng

DE

S 2

0 ng

DE

S 2

0 ng

DE

S 2

0 ng

EE

2 20

ng

EE

2 20

ng

EE

2 20

ng

**

****

**

****

**

**** : p < 0.01

Area Proportion of Ovarian Tissues in the Testis of Male Embryos Treated with E2, DES, and EE2

0

20

40

60

80

100A

rea

prop

ortio

n (%

)

Con

trol

E2

20 n

g

E2

200

ng

E2

2000

ng

DE

S 2

0 ng

DE

S 2

0 ng

DE

S 2

0 ng

EE

2 20

ng

EE

2 20

ng

EE

2 20

ng

**

**

**

**

** ****

**

** : p < 0.01

Incidence of Ovotestis in Male Embryos Treated with NP and BPA

Inci

denc

e (%

)

NP

con

trol

NP

200

ng

NP

200

0 ng

NP

200

00 n

g

NP

200

000

ng

BP

A c

ontro

l

BP

A 2

0 ng

BP

A 2

00 n

g

BP

A 2

000

ng

BP

A 2

0000

ng

0

20

40

60

80

100

**

* **

***** *

* : p < 0.05** : p < 0.01

Area Proportion of Ovarian Tissues in the Testis of Male Embryos Treated with NP and BPA

Are

a pr

opor

tion

(%)

NP

con

trol

NP

200

ng

NP

200

0 ng

NP

200

00 n

g

NP

200

000

ng

BP

A c

ontro

l

BP

A 2

0 ng

BP

A 2

00 n

g

BP

A 2

000

ng

BP

A 2

0000

ng

0

10

20

30

40

50

****

** ******

* : p < 0.05** : p < 0.01

Area Proportion of Ovarian Tissues in the Testis of MaleEmbryos Treated with E2, DES, EE2, NP, and BPA

0

20

40

60

80

100

20 200 2000 20000 200000

E2DESEE2NPBPA

Are

a pr

opor

tion

(%)

Dose (ng/egg)

0

20

40

60

80

100

10 100 1000 10000 1000000

20

40

60

80

100

100 1000 10000 100000 1000000

y = -1E - 0.6x + 5.0868R2 = 0.0003r = -0.0168468

y = -1E -0.5x + 5.6596R2 = 0.0002r = -0.0139722

NP BPA

Dose (ng/egg) Dose (ng/egg)

Are

a pr

opor

tion

(%)

Correlation Between Area Proportion of Ovarian Tissues in the Testis and Treatment Doses of E2, DES, EE2, NP, and BPA

0

20

40

60

80

100

1 10 100 1000 100000

20

40

60

80

100

1 10 100 1000 100000

20

40

60

80

100

1 10 100 1000 10000

y = 0.0205x + 8.7061R2 = 0.5306r =0.728397

y = 0.0241x + 25.721R2 = 0.5797r = 0.7613901

DES EE2

Dose (ng/egg)

E2

Dose (ng/egg) Dose (ng/egg)

Are

a pr

opor

tion

(%)

y = 0.0117x + 3.071R2 = 0.3817r = 0.6177859

E2 2000 ng/egg

EE2 2000 ng/eggDES 2000 ng/eggControl

NP 20000 ng/egg BPA 20 ng/egg

Histopathology of the Ovotestis Treated with E2, DES, EE2, NP, and BPA

Moderate degree

Severe degree Severe degree

Slight degree Slight degree

Results

●A newly developed sex reversal test using F1(AWE x WE) Japanese quail embryos found adverse estrogenic endocrine disrupting effects, such as feminization of male gonads, of E2, DES, EE2, NP, and

BPA.

●Dose-dependent increases in the incidence and degree of feminization effects in male gonads treated with E2, DES, and EE2 were disclosed.

●Treatment with NP and BPA showed significant feminized changes in male gonads with a dose-independent manner.

Discussions●A newly developed sex reversal test using F1(AWE x

WE) Japanese quail embryos proves useful to evaluate the effects of estrogenic endocrine disrupting chemicals in avian embryos.

●The dose-independent feminization effects of NP and BPA may be related to other unknown mechanisms without estrogen receptor binding.

ReferencesBerg C, Halldin K, and Brunstrom B. Environmental Toxicolology and Chemistry, 20: 2836-2840. 2001.

Berg C, Halldin K, Fridolfsson A-K, Brandt I, and Brunstrom B. Science of Total Environment, 223: 57-66. 1999. Gildersleeve RP, Tilson HA,

Mitchell CL. Teratology, 31: 101-109. 1985.

Halldin K, Berg C, Btandt I, Brunstrom B. Environmental Health Perspectives, 107: 861-866. 1999.

Perrin FMR, Stacy S, Burgess AMC, and Mittwoch U. Journal of Reproduction and Fertility, 103: 223-226. 1995. Romanoff AJ. The Avian

Embryo. New York: Macmillan. 1960.

Scheib D and Reyss-Brion M. Archives of Anatomy Microscopy and Morphological Experiment, 68: 85-98. 1979.

Shibuya K, Mizutani M, Wada M, Sato K, and Nunoya T. Journal of Toxicologic Pathology, in press, 2004.

SummaryThe feminization effects of 17 beta-estradiol (E2), ethynylestradiol (EE2), diethylstilbestrol (DES), nonylphenol (NP), and bisphenol A (BPA) on male gonads were assessed using fertile eggs of F1(AWE x WE) Japanese quail. Twenty micro-L of corn oil containing various doses of the test substances were

injected into egg whites just before incubation. Control eggs received the same volume of corn oil. At 16 days of incubation, embryos were grossly observed and the gonads were processed for histological examinations. The incidence and degree ofovotestis were histopathologically evaluated as an endpoint. Exposure of the eggs to E2, EE2 and DES resulted in a dose-dependent increase in

incidence and degree of ovotestis. Both NP and BPA also induced ovotestis with significantly higher incidence and degree when compared with the controls. NP and BPA were not dependent with dose levels. This in vivo screening using F1(AWE x WE) eggs is an inexpensive, simple and

time-saving procedure to achieve accurate results. This screening method is a promising candidate to evaluate feminization effects of estrogenic endocrine disrupters in birds.