水研センター研報，第36号，1−56，平成23年
Bull. Fish. Res. Agen. No. 36, 1‑56, 2012

博士号論文

Studies on the evaluation of the effect of endocrine disrupting
chemicals using transgenic see-through medaka (),＊/STII-YI strain＊

Takeshi HANO

Abstract：In the past few years, general concerns about the release of endocrine disrupting
chemicals (EDCs) into the environment have increased because of their potential to cause adverse physiological effects in wildlife. In particular, estrogenic potential of certain chemicals
have been shown to interfere with the sexual development and reproduction of fish. Evaluation of chemicals using intact fish is an important and available approach for monitoring andassessing the risks of chemical exposure, however, it requires labor-intensive, expensive, andtime-consuming work. To solve this problem, an approach using transgenic fish has been
proposed as a facilitated screening test model to evaluate the effect of EDCs because it may
have the potential to provide
biomonitoring and on-time and on-site evaluation of theeffects of EDCs. This study was performed to evaluate the effects of estrogenic chemicals
using transgenic see-through medaka (),/STII-YI strain, which contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka
gene, and germ cell-specific expression of GFP can be visualized in living individuals.
We exposed transgenic medaka to known doses of estrogen-mimicking chemicals and examined whether the effects on sexual differentiation including germ cell proliferation and matured gonad could be detected using GFP fluorescence.
Prior to the chemical exposure, we had observed that the number of GFP-fluorescent
germ cells and area of GFP fluorescence in normal XX females was about 10 times that innormal XY males at 10 days posthatch (dph). These results showed that sexual dimorphism
could be detected on approximately the day of hatching using GFP fluorescence, and further
indicated that alterations in sexual differentiation by EDCs could be determined by just witha single slice of image of gonads as early as 10days.
In the first chemical exposure, we examined the effect of 17α-ethinyletradiol (EE2), a representative estrogen-mimicking chemical, on sexual differentiation after
and waterborne exposures.
exposure was performed using a nanoinjection method in which
chemicals were directly injected into the embryo. Embryos (within 8 h after fertilization)
were nanoinjected with 0 1, 0 5, 2 5, or 5 0 ng of EE2 and results indicated that some 10-dph,
XY males from embryos injected with 0 5 ng EE2 showed a larger fluorescent area andmore germ cells than those of control. At 100 dph, complete male to female sex reversal occurred in >
̲ 0 5 ng treatments. In waterborne exposure experiment, from 0 dph onwards, juveniles were exposed to graded concentrations of EE2 (25 2, 45 1, 80 1, 158, 447, 880, or 1710ng/L) for 35 days. The gonadal size of 10 dph males that had been exposed to 158 ng/L of
EE2 significantly increased up to twice the size of control males. At 35dph, male to female
sex reversal occurred at EE2 exposure >̲ 45 1 ng/L. These results suggest that enhanced
germ cell proliferation in males indicated the occurrence of abnormal sexual differentiation
2011年 月27日受理（Received. May 27. 2011）＊
九州大学審査学位論文（Ph.D. Thesis, Kyushu University）（掲載に際し投稿規定に沿って一部修正した）＊
瀬戸内海区水産研究所 〒739-0452 広島県廿日市市丸石2-17-5(National Research Institute of Fisheries and Inland Sea, 2-17-5, Maruishi, Hatukaichi, Hiroshima 739-0452, Japan)

Takeshi HANO

toward females and that observation of proliferative activity of germ cells by GFP fluorescence can be applied to facilitated screening fish model to detect adverse effects on sexual
differentiation as early as 10 dph juveniles.
To assess further potential of the/STII-YI strain,mature medaka at 60 dph
were exposed to EE2 (47 8, 94 8, 216, or 522 ng/L) for 4 weeks. The gonads showed a significant reduction of the GFP-fluorescent area in males exposed to EE2 at >216 ng/L andhistologically, high connective tissue prevalence were observed at >
̲ 216 ng/L. Next, mature
male medaka were exposed to EE2 (43 7 85 8, 215, or 473 ng/L) for 3 weeks and allowed todepurate for 6 weeks, to investigate persistent effects of EE2. Continuous gonad observation
showed that GFP began to decline 3 weeks after initial exposure to >
̲ 215 ng/L. After depuration, the gonad s fluorescent areas gradually recovered, with no statistical difference atthe end of the depuration period; normal spermatogenesis was present in these individuals.
These results showed that the condition of the gonad can clearly be described by GFP fluorescence and that abnormal changes of the gonad can be detected by just with a single sliceof images of the gonad even in 60dph adult medaka..
Overall, the present study clearly showed that the transgenic line test model can provide
a more practical choice of the evaluation for the effect of EDCs and could be a promisingnoninvasive approach to identify the effects of chemicals on the gonad. Although we investigated a few chemicals only, further studies to investigate a variety of chemicals will
elucidate the usefulness of/STII-YI strain as a facilitated screening fish model todetect EDC-induced abnormalities.
Key words：transgenic medaka, endocrine disrupting chemicals, germ cell, Green fluorescent protein, sexual differentiation

Contents

3 4 Discussion
Chapter 4 Effect ofChapter 1 General Introduction

exposure of nonylphenol

on sexual differentiation in the

Chapter 2 Detection of sexual dimorphism as/ STII-YI strain

estimated by proliferative activity of

4 1 Introduction

germ cells in the

4 2 Materials and Methods/ STII-YI

4 3 Results

strain2 1 Introduction

4 4 Discussion
Chapter 5 Effect of 17α-ethinylestradiol on matured

2 2 Materials and Methods

gonad in the

2 3 Results and Discussion
Chapter 3 Effect of 17α-ethinylestradiol on sexual
differentiation in thestrain3 1 Introduction
3 2 Materials and Methods
3 2 1 Nanoinjection exposure
3 2 2 Waterborne exposure
3 2 3 Statistical analysis
3 3 Results
3 3 1 Nanoinjection exposure
3 3 2 Waterborne exposure/ STII-YI/STII-YI strain

5 1 Introduction
5 2 Materials and Methods
5 2 1 Direct waterborne exposure
5 2 2 Waterborne exposure followed bydepuration
5 2 3 Statistical analysis
5 3 Results
5 3 1 Direct waterborne exposure
5 3 2 Waterborne exposure followed bydepuration
5 4 Discussion

Evaluation of the effects of chemicals using transgenic fish
Chapter 6 General Discussion

fish species exposed to effluents from pulp and paper

Referencemills (Howell

Acknowledgement

endocrine disruption in wildlife is the induction

1980; Hegrenes, 1999). Anotherof imposex in gastropods, which is defined as an
Chapter 1 General Introduction

irreversible syndrome imposing male-type genitalorgans upon females, collected from tributyltin and

For decades, human beings have been generating

triphenyltin polluted sites (Horiguchi1997).

abundant synthetic chemicals. These chemicals have

Reduced serum concentrations of thyroxine werebeen discharged into the environment, reachingreported in fish sampled from sites contaminatedwater bodies and becoming concentrated in thewith polychlorinated biphenyl (PCB) congeners intissues of aquatic organisms, including fish. The

the San Francisco Bay (Brar2010).

lethal, sublethal, and other physiological effects

EDCs have been studied extensively in theof these compounds endanger the survival oflaboratory for their estrogenic potencies in standard

these organisms. Although the use and disposal

test fish including medaka (of chemicals has been regulated, concern over the

minnow (), and sheepshead

potential impact of natural steroid hormones andminnow (Lacépède). For

endocrine disrupting chemicals (EDCs) on aquatic

example, deleterious effects of EE2 and NP include

organisms has been heightened after finding thatskewed sex ratio toward females (Langethese compounds can adversely affect sexual

2001; Scholz and Gutzeit, 2000; Yokota

development and reproduction in wildlife (Colborn

Zillioux

1993; Stone, 1994). The evaluation of chemicals

2001;2001), induction of intersex gonad

and Vtg (Harries

using fish is an important and available approach), fathead

2000; Seki

2002; Kang

2003), reproductive impairment (Sekifor monitoring and assessing the risks of chemical

2002; Kang

exposure (Guilletteand failed swim-up of hatched larvae (Yokota

1994; Jobling1998).

2003), and decreased hatchability

Field studies have examined estrogenic and2001). In addition to estrogen-related chemicals,

androgenic response in aquatic organisms, alteredother compounds are known to show binding

hormone levels in fish, and induction of imposex

affinity to the androgen receptor and act as bothin gastropods. Estrogen-related responses inandrogen agonist (e.g., methyltestosterone; Seki

male fish include altered serum steroid levels

2004; Kangand delayed gonadal maturation in lake whitefish

DDT metabolite(

fungicide vinclozolin (Gray) living near pulp mills(Munkittrick1992); abnormally elevated2008) and antagonists (e.g.,-DDE (Kelce

1995), the1994), and theanticancer agent flutamide (Jensen

2004)).

vitellogenin (Vtg; a precursor of egg yolk protein)

TBT was also reported to cause masculinization inconcentrations and reduced serum testosterone inJapanese flounder, (male carp () captured near sewage

treatment plants (Folmar

1996); and a highprevalences of intersex gonads in roach () and gudgeon () collected near

sewage treatment plants (Jobling
Aerle

1998; Van

2001) and male flounder (Shimasaki2003) and abnormal development in medaka
embryos (Hano2007).

A variety ofandtests have beendeveloped for evaluating the effects of EDCs. Mostof themethods are cell-based, including

receptor binding assays (Folmar) from contaminated estuaries (Hashimoto2000). These abnormalities in wild fish mightproliferation assays (Jones

2002), cell
1998; Metcalfe2001), and reporter gene assays (Kojima

be caused by natural (17β-estradiol and estrone)2004). They are relatively simple, high-throughput,and synthetic estrogens (17α-ethinlyestradiol (EE2),

sensitive, and quantifiable (Zengnonylphenol (NP), and bisphenol-A (Desbrow

However, the effects of metabolic conversion,

1998; Körner

which occurs with NP (Gigar

2000; Thomas

2001).

Masculinization has also been reported in some2005).
1984), and/or

bioconcentration are not considered; therefore,

Takeshi HANO
these tests may fail to correlate

effects to

1990; Koopman

1991), and various

effects on whole organisms. Another problem is thefactors including

differential affinity of certain estrogen receptors for(Katoh-Fukui

the compounds of interest. Furthermore,2002), andassays could potentially result in false-negative

to be involved in sexual differentiation. As forevaluations of chemicals (Folmar

nonmammalian vertebrates, Matsuda

contrast, commonly used

2002). In

assays expose test

cloned(Swain1998),(Kitamura(Vidal(1998), M33
2001) were foundand(2002)

domain geneorganisms to known doses of chemicals to evaluate

on the Y chromosome) from the sex-determining

their effects at target physiological endpoints,

region on the Y chromosome of medaka andsuch as reproduction, sexual differentiation, andfurther established a transgenic medaka line

abnormal induction or suppression of sex-specificoverexpressing

proteins (e.g., Vtg) or endogenous hormones.

the cytomegalovirus promoter and demonstrated

assays are, however, generally low-throughput

that

cDNA under the control ofis sufficient for male development inbecause they require labor-intensive, expensive,

medaka and is an additional sex-determining gene inand time-consuming work (Zengvertebrates (Matsuda

2005). Thus,

novel fish test models that combine the advantagesof bothand2007).

Several GFP transgenic lines have been generated

assays are desirable.

using tissue-specific promoters. The fluorescence

Transgenic fish models have the potential to answer

is stable, and virtually no photobleaching occurs.

these demands because they can provide

Because GFP requires no exogenous substrates or

biomonitoring and on-time and on-site evaluation of

cofactors for fluorescence emission, it provides an

the effects of EDCs using reporter genes such as

excellent means for monitoring gene expression

green fluorescent protein (GFP) under the inducible

and protein localization in living organisms. Since

promoters of targeted, tissue-specific genes. GFP is athe cloning of GFP cDNA by Prasher

photoprotein of the bioluminescent jellyfish

the usefulness of GFP as a reporter gene has been

, which emits green fluorescence when
excited by blue light.

proved in transgenic animals such as(Chalfie1994),

Transgenic organisms are used in biological(Wang and Hazelriggs, 1994), mice (Ikawa

research, drug production, agriculture, and aqua1995), zebrafish (farming. In the mid 1970s, transgenic animals

1995), and medaka (Hamada

were first generated by adding foreign genes to
the genome of an animal (Jaenisch(1992),; Amsterdam
1998; Kinoshita

2000).

1974). In

Medaka is one of most popular fish species inmammals, transgenic mice were first developed

Japan and one of the most extensively studied

to study tissue-specific responses to disease, ̶because it matures and reproduces quickly

for example, limb deformity (Woychik1985),(approximately 3 months), is highly fecund (20‑30

embryonic lethal mutation (Harbers

1984),

eggs/day), exists in a variety of strains, has a small

inactivation of the X chromosome (Krumlauf

genome size, and requires only a small facility to1986), and carcinogenesis (Adams

1985) ̶atmaintain. These properties render medaka usefulan individual and molecular biological level. In fish,in many fields of study, including biology, genetics,

medaka has been first proven to be a useful model

radiology, and pathology. Medaka is also known

for studies of transgenic fish since the transgenic

to be a differentiated gonochorist (Aida, 1921) andmedaka expressing the chicken δ-crystallin geneis therefore not expected to show spontaneous

was produced in 1986 (Ozato

intersexuality. Sexual dimorphism first appears soon1986).

Molecular and biological approaches using

after hatching, when male and female phenotypes

transgenic organisms are also applied to elucidate

can easily be distinguished by the proliferative

the mechanisms of sexual differentiation. As a result

activity of germ cells (Satoh and Egami, 1972).

of studies in transgenic mice, thegeneGerm cells in the female gonads enter meiosis on

was identified as a sex-determining gene (Sinclair

approximately the day of hatching, whereas germ

Evaluation of the effects of chemicals using transgenic fish
cells in the male gonad undergo meiotic arrest(Satoh and Egami, 1972; Kobayashi

homolog of thegene (, from2004).

) (Fig. 1‑1), and demonstrated

This distinction facilitates sex determination in

GFP expression exclusively in the germ cells ofvery young fry as well as the detection of abnormal

medaka (Shinomiya

sexual differentiation caused by EDCs. Owing

model to visualize germ cells using GFP fluorescenceto these advantageous physiological and sexualand offer a useful model for analyzing gonadal

traits, medaka is an attractive model organism fordevelopment in a living vertebrate. A drawback of

toxicological testing of acute or chronic toxicity

this model, however, is that direct observation with

(Weber2002,

the naked eye is possible only during juvenile stages

2002), life cycle (Yokota

because the peritoneum is covered with pigments

2004), reproduction (Kang

2003, 2006, 2008; Seki
2001; Seki

2004), behavior (Oshima2002), early life (Yokota

2000; Sekiand nanoinjection (Edmunds

2000). This line is the firstsuch as iridophore and melanophore (Wakamatsu
2003),

2000; Papoulias

2001) during the later stages of life. To solve
the problem, Wakamatsu(2001) generated

2000a, 2000b). Hence, medaka is a proposeda see-through (STII) medaka that is transparent

standard test fish in the Organization for Economic

throughout its life (Fig. 1‑2), by genetically removing

Cooperation and Development (OECD) guidelines

most of the pigments from the entire body of the(1999).

organism using a combination of recessive alleles at

Transgenic fish models for the monitoring
environmental pollution have been developed using
zebrafish and medaka because of their excellent

4 loci (melanophores, iridophores, leucophores, andxanthophores).
The/STII-YI strain used in thisperformance in fish toxicological studies and theirstudy was produced by introducing

rich genome information. In zebrafish, Mattingly

into the genome of STII medaka. In this strain,(2001) reported a GFP transgenic line using

germ-cell-specific expression was confirmed by thethe humanpromoter that is capable ofobservation of GFP through the transparent body

responding to TCDD (2, 3, 7, 8-tetra-chlorodibenzo-

wall of the living fish throughout life. Furthermore,-dioxin) and thus may be used as a biomonitoring

matured testes and ovaries are easily distinguishable

system for detecting xenobiotic toxicants inwith the naked eye under fluorescence microscopy

the environment. Blechinger(2002) used a(Fig. 1‑3). The apparent sexual dimorphism ofheat-shock-inducible hsp70: GFP transgenic zebrafish

the gonads indicated by GFP fluorescence canline to study cadmium toxicity and demonstrated

also provide a useful indicator of the effect of

cadmium-inducible GFP expression that can beenvironmental substances on gonadal development.

used in biomonitoring systems. Kurauchi

This strain carries 2 genotypic sex markers:(2005) produced a transgenic medaka line harboring

leucophore (the GFP gene driven by a regulatory region of1(

the choriogenin H gene, one of the precursors toappears in the skin of 2-day-old males but not in

egg envelope proteins (Murata

that of females (Fig. 1‑4). The

1997). They

Wakamatsu

Matsuda2003) and sex-linked1997). The leucophore marker
DNA marker,

observed GFP expression in the liver of malewhich exists on sex chromosomes, is detected as

medaka in response to estrogens and indicated that

1 band in females and as 2 bands in males, using

the transgenic line was sufficient for application as

agarose electrophoresis after polymerase chain

an alternative model in monitoring environmental

reaction (PCR) (Fig. 1‑5). Therefore, we can use

water samples. Other transgenic lines for this use

these markers to discriminate genotypic males (XY)

have been established using choriogenin L (Uenofrom genotypic females (XX), which facilitates the2004) and medaka Vtg-1 (ZengIn 2001, Tanaka

2005).

established a transgenic

medaka line by microinjecting an

detection of abnormal sexual differentiation caused
by EDCs.
In this study, EE2 and NP were selected as test

construct containing the coding region of the GFPchemicals (Fig. 1‑6) due to their estrogenic potency

gene fused to the regulatory regions of the medaka(Folmar2002). Because they have been

Takeshi HANO

Fig. 1‑1. Establishment of theprotein; STII, see-through II.(Tanaka/STII-YI strain. GFP, green fluorescent
2001; Wakamatsu
2001)

Fig. 1‑2. The left (A) and right (B) sides of a female see-throughmedaka. The dark color of the gut comes from ingested food.
a, air bladder; b, brain; bv, blood vessels; f, fat tissue; g, gill; gu,
gut; h, heart; k, kidney; li, liver; o, ovary; s, spleen; sc, spinal cord.
Reprinted from Wakamatsu(2001).

Evaluation of the effects of chemicals using transgenic fish

Fig. 1‑3. Right lateral sides of gonads demonstrating green fluorescentprotein (GFP) in/STII-YI adults. (A) testis, and (B) ovary;arrow: leucophore, a male-specific pigment. STII, see-through II.

detected in aquatic environment, they are suspected

adult medaka at concentrations of 488 ng/L for 21

causes of estrogenic effects observed in wild fishdays and induced a high prevalences of connective(Holthaustissue in male testes (Seki2002).

2002).

EE2 is used in contraceptive formulations (Folmar

Nonylphenol ethoxylates (NPEOs) are widely

2002) and has been detected in surface

used as cleaning agents and nonionic surfactants

waters and sewage treatment plant effluents. The

in a variety of industrial and agricultural processes

concentrations of EE2 in river surface waters were(Tollsrelatively low ̶a few nanograms per liter (Belfoid

and nontoxic (Giger

1994). They are relatively hydrophilic1984), but their bacterial1999). Adverse effects of EE2 demonstrated

degradation product, nonylphenols, are toxic

in laboratory experiments include sex-reversal

and hydrophobic compounds produced mainly

followed by a skewed sex ratio toward females,

during sewage treatment. Nonylphenols have

Vtg induction, and intersex gonads in male fish

been detected at microgram-per-liter levels in theat extremely low concentrations. Skewing of theaquatic environment (Rudel

sex ratio toward females was observed in medaka

1999; Tsuda

1998; Blackburn2000). Several

exposed to 100 ng/L for 2 months (Scholz andstudies have confirmed that NP has physiologicalGutzeit, 2000), in fathead minnow exposed to >̲and developmental effects related to its estrogenic

4.0 ng/L for 56 days (Lange

2001), and inpotency. Reproductive failure and testis-ova

sheepshead minnow exposed to >̲200 ng/L for 73

induction as well as elevated Vtg have beendays (Zillioux

observed in NP-exposed medaka (Kang2001). Purdom(1994) found2003)

that a 10-day immersion exposure of male rainbow

and fathead minnow (Harries

trout () to EE2 induced theof medaka to NP for 60 days beginning at 0 days

dose-dependent Vtg response, ranging from 0 1 topost-fertilization resulted in a significant decrease

10 ng/L. Metcalfe

in hatchability and successful swim-up of hatched(2001) reported that the2000). Exposure

lowest observable effective concentration (LOEC) for

larvae at concentrations of 183μg/L (Yokota

testis-ova induction in male medaka during 100 days

2001) and skewed the sex ratio toward females atof exposure was 0 1 ng/L, which was well within the

23 5μg/L (Sekiconcentrations detected in many sewage treatmentusing medaka, Yokota

plant effluents. EE2 also impaired reproduction in

induced testis-ova in both parental medaka (2003). In a full life cycle test
(2001) showed that NP) at

Takeshi HANO

Fig. 1‑4. One day posthatch (A) genotypic male and (B) genotypic female juveniles of/STII-YI strain. Magnified image of (C) genotypic male and (D) genotypic female,arrow: leucophore, a male-specific pigment. GFP, green fluorescent protein; STII, see-throughII.

Fig. 1‑5. Identification of genotypic sex by detecting a sex-linked 1 (in the/STII-YI strain.

) marker sequenceEvaluation of the effects of chemicals using transgenic fish

Fig. 1‑6. Structural formula of test chemicals used in this study.

>
̲17 7μg/L and their progeny () at >
̲8 2μg/L.

cells in female fry enter meiotic cell division within

Given these findings, EE2 and NP could be major

24 h after hatching, whereas in males, germ cells

contributors to the estrogenic response observed inproliferate much later ̶15 days posthatch (dph)̶

fish exposed to sewage treatment plant effluents.

which facilitates sexing very young fry. However,In this study, we aimed to evaluate the application

detection of the sexual dimorphism in the normal/STII-YI strain to models for detecting

fish has the demand for preparaing the serial

the effect of EDCs. The transgenic medaka weresections to count the germ cell numbers, which

exposed to known doses of contaminants, and we

is generally labor-intensive and time-consuming

examined whether effects on sexual differentiation

work. Here, we established a facilitated method

including germ cell proliferation and matured

to detect sexual dimorphism by both calculating

gonad could be detected using GFP fluorescence.

GFP-fluorescent germ cells and measuring the area

To assess the potential of theof green fluorescent protein (GFP) fluorescence inof/STII-YI

strain, we used a four-part experiment: first, we

the/STII-YI strain.

established a method to monitor sexual dimorphism
on approximately the day of hatching using GFP

2 2 Materials and Methods

fluorescence. Second, we examined the effect of EE2
on sexual differentiation after
exposures.

and waterborne

exposure was performed using

2 2 1 Test organism
The/STII-YI strain was provided by Y.

a nanoinjection method in which chemicals were

Wakamatsu, Bioscience Center, Nagoya University,

directly injected into the embryo. Third, weJapan and was bred in our laboratory. Each pair was

examined the effect of NP on sexual differentiation

placed in a 2-L glass chamber (20 × 10 × 15 cm)

and embryonic development after via

filled with static water at 25‑27℃. The fish were

exposure. Finally, we examined the effects of EE 2

kept under a constant 16:8-h light:dark photoperiodon the gonads of mature, living medaka and theand were fed withrecovery of the fluorescent area in the testis after

hatching) twice a day. Half the water was replaced

the transfer of exposed males to clean water.

once a day with dechlorinated tap water.

Chapter 2 Detection of sexual dimorphism as

2 2 2 Determination of genotypic sex

estimated by proliferative activity of germ
cells in the/STII-YI strain

nauplii (< 24 h after

As mentioned in Chapter 1, this strain carries 2
genotypic sex markers: leucophore (2003) and sex-linked 1 (2 1 Introduction
In medaka (Wakamatsu
Matsuda1997). However, the recombination frequency), male and female

between thelocus, which determines leucophore

phenotypes can first be distinguished by theexpression in the skin, and sex-determinant ()

proliferative activity of germ cells on approximately

locus is about 1 4% in FLF stock and 4 2% in FLFII

the day of hatching (Satoh and Egami, 1972). Germ

stock (Wakamatsu

2003), that is, an error of

Takeshi HANO
1 4‑4 2% is unavoidable in sex identification basedof a 460‑490 nm excitation filter and a 510-nm

on this color marker. In contrast,

is known tobarrier filter. Fluorescent images of the gonad were2003).

recorded with a digital camera (Coolpix950, Nikon,closely link to thelocus (Wakamatsu

Because of the close linkage of thelocus toTokyo, Japan). We created image of extracted

locus, identification of genotypic sex in eachgreen fluorescent area from each image by green

individual was performed with polymerase chain

red blue (RGB) processing (Adobe systems Corp.,

reaction (PCR) analysis of.

Tokyo, Japan) and then manually outlined its shapesequence was amplified according to thethat corresponds to the gonad. Then the area ofthemethods of Matsuda(1997) with slightthe shape was measured with an image-processing

modifications. After removal of the gonad from eachsystem (Luminavision, Mitani Corp., Fukui, Japan).

specimen, part of the caudal fin (approximately 1

After recording the GFP fluorescence, each× 1 mm) was excised with a razor, and DNA was

specimen was dissected out its gonad with a razor

extracted from the piece of fin with a Dneasy

under a stereoscopic microscope (SZX12, Olympus

Tissue kit®

(QIAGEN GmbH, Hilden, Germany).

Amplification of thegene was carried out by

Co., Tokyo, Japan), and laid on a glass slide. Caudal
fin from each specimen was then removed and

PCR. PCR primers were sense (5́-CCT GCA ATG

was subjected to

extraction for determining

GGA AAT TAT TCT GCT C3́) and antisense (5́genotypic sex of each individual. Then 20μL of-CTT TTG TGT CTT TGG TTA TGA AAC GATtrypsin solution (0 2% trypsin, 0 02% EDTA in PBS

G-3́). The reaction mixture was composed of 25μ(‑)) was dripped onto the gonad; 10 min later, germ

L of 10× Ex Taq Buffer (Takara, Tokyo, Japan), 0 2

cells were completely dissociated, which enabled us

mM dNTPs, 0 2μM dNTPs, 0 2μM of each primer,

to count them (Fig. 2‑1). Counting the germ cells

1μL of template DNA, and 0 6 unit of Ex Taq

from the same gonad was repeated three times forpolymerase (Takara, Tokyo, Japan) in a final volume

accuracy.

of 25μL. The thermal cycling involved 30 repeatsof denaturation at 95 LC for 30 s, annealing at 57℃
for 60 s, and extension at 90℃ for 120 s. The

2 2 4 Statistical analysis
Statistical differences of germ cells number

genotype was identified by electrophoresis on a 2%

between male and females at 1, 5, 10 and 15

agarose gel.

dph juveniles were analyzed by a two-sample
t-test (parametric data) and a Mann-Whitney

2 2 3 Measurement of GFP area and counting thetest (nonparametric data). Correlation between

number of germ cells in GFP-positive juvenile

GFP-fluorescent area and germ cell number was

Embryos were collected from breeding females

assessed by Kendall s pie test. All statisticalin the morning, and healthy embryos were selected.

analyses were performed with the SPSS Base 10 0J

The embryos were sterilized in 0 9% H 2 O 2 for(SPSS Inc., Chicago, IL, USA). Differences were

10 min to minimize the likelihood of fungal andconsidered significant at< 0.05.

bacterial infections and washed with embryo rearing
medium (0 1% NaCl, 0 003% KCl, 0 004% CaCl 2・
2H2O, 0 008% MgSO4 adjusted to pH 7 0‑7 2 with

2 3 Results and Discussion
Changes in GFP-florescent area and number

NaHCO3 dissolved in deionized water) and hatchedof germ cells in/STII-YI strain arejuveniles were maintained in dechlorinated water

shown in Table 2‑1. In XX females at 15 dph, thefor 15 dph. At 1, 5, 10, and 15 dph, the juveniles

average fluorescent area dramatically increased

were anesthetized by cooling them in ice water for

approximately 10-fold and the averaged germ cell

10 to 15 s. Then each juvenile that showed GFP

numbers increased by almost 20-fold compared withfluorescence in its germ cells was selected andthose of 1 dph XX females. In contrast to female, thethe fluorescence was measured with an inverted

average fluorescent area and the average number

fluorescence microscope (Eclipse TE300; Nikon,of germ cells in XY males were constant and didTokyo, Japan) equipped with a filter set composed

not significantly change throughout the experiment

Evaluation of the effects of chemicals using transgenic fish

Fig. 2‑1. Procedures for measuring the green fluorescent protein (GFP) area and counting the numberof germ cells.

(duration, 15 dph;

= 0 114). Consequently, at 10

in both genotypic sexes, that developed from bothdph, the fluorescent area and germ cell number

treated and untreated embryos, even in juveniles as

in XX females were 10-fold the values in XY

young as 10 dph. This fact demonstrates that earlymales (Fig. 2‑2). The GFP fluorescent area waseffects of chemicals may be evaluated by measuringsignificantly correlated with germ cell number boththe GFP fluorescent area of gonads in this strain of

in XX females (R = 0 923,

medaka.

(R = 0 400,

< 0 01) and in XY males< 0 05).

Visual differences in germ cell proliferation

Chapter 3 Effect of 17α-ethinylestradiol on sexual

pattern between the sexes during development

differentiation in thefrom untreated embryos were detected bystrain/STII-YI

measuring GFP fluorescence. Dimorphism of gonad
differentiation between male and female in teleosts

3 1 Introduction

has been reported for medaka (Satoh and Egami,

In chapter 2, a novel method was established to1972) and tilapia (Nakamura and Takahashi, 1973).

determine the germ cell numbers by measuring

Satoh and Egami (1972) reported that the number of

the area of green fluorescent protein (GFP)

germ cells in female medaka was 5-fold that in males

fluorescence. This finding indicated that observation

at 10 dph. Our results, obtained from counting theof GFP-fluorescent gonad have a potential to detect

numbers of germ cells showing GFP fluorescence,the effects of endocrine disrupting chemicals (EDCs)

are consistent with this report. Furthermore, weon the sexual differentiation in the living organisms.

found an apparent correlation between the area

In chapter 3, we exposed ethinylestradiol (EE2) toof GFP fluorescence and the number of germ cells

the early-stages of/STII-YI strain in 2

Takeshi HANO
Table 2‑1. Area of green fluorescent protein (GFP) fluorescence and number of GFP-positive germ
cells in
/STII- YI strain at 1, 5, 10 and 15 days posthatch

Fig. 2‑2. Green fluorescent protein (GFP) fluorescent images captured from lateral side in the/STII-YI strain of (A, B) genotypic male and (C, D) genotypic female. (A, C) 1 day
posthacth and (B, D) 10 days posthatch. Outlined area in each of photo corresponds to the
gonad. Arrow: leucophore, a male specific pigment. Arrowhead: autologous red fluorescence ofbrine shrimp. R: residual food and/or discharges in the gut. STII, see-through II.

different designs and examined the effects on germfemale gonadal development is complete (Yamamoto,cell proliferation and sexual differentiation. First,

1963).

1 days post-fertilization (dpf) embryos collectedfrom parental fish were

exposed to EE2 with

exposure of EE 2 was performed withnanoinjection method. Nanoinjection into the fish

the nanoinjection technique. Second, the newly

embryos (Walker

1996) has been proposed ashatched fry were continuously exposed to different

a powerful tool for evaluating the effects of EDCs

concentrations of waterborne EE2 for 35 days, when

(Edmunds

2000; Papoulias

2000a, 2000b)

Evaluation of the effects of chemicals using transgenic fish
and crude extracts of chemical contaminated soil
(Wilson and Tillitt, 1996; Ishaq

3 2 Materials and Methods

1999), because

it allows researchers to assess the toxicity of

3 2 1 Nanoinjection exposure

lipophilic environmental contaminants to early life
stages of fish in a manner that realistically reflects

3 2 1 1 Preparation of microneedles andenvironmental exposure and simulates maternal

nanoinjection proceduretransfer with no need to chronically expose adult

Microneedles were prepared as based on thefish and also allows accurate quantitation of themethods of Walker(1996) and Papoulias

dose to the developing embryo including the germ(2000a) with slight modification. Briefly,

cell proliferation stage (Fig. 3‑1). This technique isaluminosilicate capillary tubes (1.0 mm o.d. andalso expected to provide a small-scale facility and

0 58 mm i.d; Sutter Instrument, Novato, CA,

experimental equipments and relatively shorter

USA) were drawn to 8 10μm diameter with a

exposure duration.

microelectrode puller (PN-30; Narishige, Tokyo,

Waterborne exposure to evaluate the effects

Japan) and then were siliconized (Sigmacote; Sigma,

of chemicals on aquatic organisms was a well

St Louis, MO, USA) and beveled at a 30 angle withestablished method proposed as standard

a beveler (EG-40, Narishige, Tokyo, Japan). The

screening testing methods in fish Organization for

microneedles were loaded with chemical solution

Economic Cooperation and Development (OECD)

via microloading Femtotips (Eppendorf, Madison,guidelines (1999). In chapter 3, we examined the

WI, USA). The injection volume of each microneedle

effects of EE2 on sexual differentiation by estimating

was adjusted to 0 5 nL (Villalobos

the proliferative activity of germ cells at 10 days

corresponds to 0 1% of medaka egg volume, andposthatch (dph) and further investigated the chroniccalibrated before use by measuring the diameter

effects on gonadal development in the medaka(approximately 100μm) of a droplet formed in(water and controlling the injection pressure).

1999), which(500‑1000 hPa) and/or injection duration (0 5‑1 0 s)

Fig. 3‑1. Schematic images of maternal transfer of lipophilic chemicals into eggs andnanoinjection technique.

Takeshi HANO
appropriately with the aid of a Femtojet (Eppendorf)

Hatched juvenile were transferred to 2-L square

and a micromanipulator (MN-151; Narishige). The

glass containers (20 individuals per container) anddiameter (approximately 100μm) of oil droplet that

kept in dechlorinated tap water at 25‑27℃ and werecorresponds to 0 5nL was calculated as follows;

fed granulated freeze-dried brine shrimp (Tetra,

As medaka egg radius is approximately 0 5 mm,
44πr3 = π(0 5 mm)33
3
= 0 5236 mm3 = 0 5236μL

assessed daily during the embryogenesis. Delay= 0 5236μL × 0 001

comparison of developmental stages according to

medaka egg volume =injection volume

Japan).

3

Mortality and abnormal development werein embryonic development was identified by= 0 52 nL = 0 0005 mm

Iwamatsu (2004). Hatchability, swim-up failure and

4
= πr3
3
r = 0 049237 mmtime-to-hatching were also assessed in the hatching

diameter of a droplet = 2r = 0 0985mm

and maintained in artificial seawater (salinity wasembryos. Hatched juvenile were transferred to 2-L
square glass containers (20 individuals per container)= 98 5μm = approx.100μm

adjusted at 0 1%) at 25‑27℃ Hatching rate was

In this condition, variation of nanoinjection was

calculated as the ratio of hatched embryos to theassessed to less than 20% by measurement of

total number of live embryo at day 1 post-injection.

tributyltin (TBT) in embryo nanoinjected with TBTSurvival rate was calculated as the ratio of live

(data not shown)embryos at 100 dph to the total number of live
embryo at day 1 post-injection. They were fed

3 2 1 2 Test organism
The

granulated freeze-dried brine shrimp (Tetra, Japan)/STII-YI strain was provided

around the onset of hatching and, as they grew up,

by Y. Wakamatsu, Bioscience Center, Nagoya

they were fed with

University, Japan and was bred in our laboratory.

hatching) twice a day.

We used a line heterozygous fornauplii (< 24 h afterin thisexperiment; the ratio of GFP-positive to negative

3 2 1 4 EE2 working solution(no fluorescence) embryos was theoretically 1 to

EE2 was obtained from Nacalai Tesque (Kyoto,

1. Parental fish were kept under the conditions asJapan) and prepared as a stock solution by dissolving

described in chapter 2 2 1.

in acetone (10 mg/ml). Triolein (99 0% purity) was
purchased from Wako Pure Chemical (Tokyo, Japan)

3 2 1 3 Embryo Nanoinjection
Embryos were collected from breeding females

and filter-sterilized before use through a 0 22μm
PTFE filter (Millipore, Bedford, USA).

in the morning, and healthy embryos were selected.

The EE 2 stock solution was diluted with 1 mL

The embryos were sterilized and washed (chapterof triolein, and acetone was evaporated under a2 2 3 in detail) and were embedded in 2 % agarose

stream of nitrogen at room temperature. The final

gel on glass slides just before nanoinjection. EE2

concentrations of EE2 were 0 2, 1, 5, 10 ng/nl. Thesein triolein was injected into 71, 159, 162 and 161

EE2 solutions were stored at 4℃ under nitrogen.

embryos (within 8 h after fertilization) in the 0 1, 0 5,
2 5 and 5 0 ng of EE2 per egg, respectively. EE2 wasnanoinjected directly into the oil globule with the

3 2 1 5 Measurement and quantification of the areaof GFP fluorescence at 10 dph juvenile

aid of a Femtojet and a micromanipulator. Embryos

At 10 dph, gonad development in GFP-positive

were also injected with EE 2-free triolein (triolein

juveniles was assessed by measuring the area ofcontrol) or not injected (uninjected control). The

GFP fluorescence and counting germ cell numbers.

treated embryos were washed and maintained in

Identification and record of GFP fluorescent image

10 mL of embryo rearing medium (chapter 2 2 3 inof the gonad were performed according to thedetail) in a 6-well multiwell plate (Becton Dickinson,

procedures in chapter 2 2 3 Caudal fin from eachFranklin Lakes, NJ, USA) at 25‑27℃ until hatching.

specimen was then removed and was subjectedEvaluation of the effects of chemicals using transgenic fish
to sex-linked 1 () extraction for determining

Kinder, Freiburg, Germany), and examined undergenotypic sex of each individual. GFP-negative

a light microscope. Inter sex (juveniles were continued to maintain in the artificial

combined into the male category.

) fish were

seawater at 25‑27℃ for 100 days; then their gonads
3 2 1 7 Determination of genotypic sex

were examined histologically (Fig. 3‑2).

Genotypic sex in each individual at 10 dph and3 2 1 6 Characterization of adult fish at 100 dph
At 100 dph, the fish were anesthetized in ice
water, blotted on filter paper, and measured for body

100 dph was determined by the polymerase chain
reaction (PCR) analysis ofaccording to theprocedures as described in chapter 2 2 2.

weight and total length. Then, their developmentof secondary sex characteristics (size and shape

3 2 2 Waterborne exposureof anal and dorsal fins, presence or absence ofpapillary processes on anal fin, dorsal fin notch,

3 2 2 1 Test organismand female-like urogenital papillae) were assessed.

Brood strain of/STII-YI strain was

Gonads were removed and weighed to calculate themaintained at Nagoya University. Healthy embryos

gonad somatic index (GSI), fixed in Bouin s solution,collected from breeding females were sterilized

and prepared for histological observation. Finally,

and maintained until hatching under the described

the caudal fin from each specimen was removed

conditions in chapter 2 2 3.

and subjected to extraction of themarker for

identification of the genotypic sex. The fixed gonads

3 2 2 2 Identification of GFP Fluorescencewere dehydrated and embedded in Technovit 7100

The hatched juveniles were first anesthetized(Heraeus Kulzer GmbH, Wehrheim, Germany).

in ice water in a petri dish (60 × 15 mm; Becton

Serial transverse sections (5μm thick) were cut

Dickinson and Company, Franklin Lakes, NJ, USA)with a microtome. The sections were stained withand GFP expression was detected in the germ cells

hematoxylin and eosin, mounted with Eukitt (O.

by using a stereoscopic fluorescence microscope

Fig. 3‑2. Experimental design of nanoinjection experiment using
green fluorescent protein; STII, see-through II.

/STII-YI strain. GFP,Takeshi HANO(MZFLIII; Leica, Heerbrugg, Switzerland) equipped

Throughout the experimental period, hardness

with a GFP2 filter set composed of a 480-nm / 40-nm

in the water was 20‑30 mg CaCO3/L and pH was

excitation filter and a 510-nm barrier filter.

6 7‑7 0. Water temperature remained at 24 ± 1℃.
The fish were observed daily to assess mortality and3 2 2 3 Special reagents

appearance.

EE2 was purchased from Nacalai Tesque (Kyoto,

EE 2 concentrations were measured four times

Japan) and a 100 mg/L stock solution was prepared(every 7‑8 days). The means (coefficients ofby dissolving EE2 in acetone. An aliquot was thenvariation) of measured EE2 concentrations in each

evaporated to dryness under N2 gas, diluted withof the test solutions during the exposure period

deionized water, and magnetically stirred for

were 25 2 (15 8), 45 1 (6 1), 80 1 (7 4), 158 (22 4),

approximately 24 h to obtain a final concentration

447 (28 8), 880 (18 3) and 1710 (11 4) ng/L. The EE2

of 1 mg/L of EE2 in water. This aqueous solution

concentration in the control treatment was less than

was diluted with dechlorinated water to prepare athe detection limit (2 0 ng/L) in all analyses.

working EE2 stock solution of 9μg/L.
3 2 2 5 Measurement and quantification of the GFP
3 2 2 4 Exposure conditions

fluorescent area at 10 dph juvenile

Normal and healthy 1 dph juveniles showing

At 10 dph, 10 leucophore-positive males and 10

intense GFP fluorescence in their gonads wereleucophore-free females were randomly selected

exposed to EE2 for 35 days. The sex of each fishfrom each treatment group, anesthetized on ice, andwas tentatively determined by the presence or

the GFP fluorescence in their gonads was visualized.

absence of leucophores. Each treatment consisted

Fluorescent images were then recorded by a color,of three glass chambers, holding a total of 30 males

digital, cooled, charge-coupled camera (C4742-95;(leucophore-positive) and 20 females (leucophore-free).

Hamamatsu Photonics, Hamamatsu, Japan) mounted

One chamber contained the 20 females and theon the microscope, and analyzed using IPLabother two chambers contained 15 males each,Spectrum 3 5 software (Scanalytics, Fairfax, VA).

because we had assumed higher mortality in males

After recording the GFP fluorescence intensity inthan in females according to the results reported

their gonads, the medaka were returned to their glassby an earlier study (Seki2002). As describedchambers to resume their continuous exposure to(1982), a flow-through mini-diluter

EE2. The area of GFP fluorescence in each specimen

system was used to provide exposure to EE 2 inwas determined by an image-processing system

two phases, namely, 0 (control), 25, 50, 100, and 200(Scion image 4 0 2, Scion Corporation, Washington,

ng/L in the first phase, and 0 (control), 400, 800,

D.C., USA).

by Benoitand 1600 ng/L in the second phase. The EE2 stock
solution was delivered by a mini-chemical pump

3 2 2 6 Characterization of fish at 35 dph(TOKYO RIKAKIKAI, Tokyo, Japan) and diluted

At 35 dph, exposure to EE 2 was terminatedwith dechlorinated tap water. Flow rates of theand each medaka was anesthetized on ice. Theirstock solution and dilution water were checked

fluorescent images were captured from the rightdaily. The 1 8-L of test solution in each chamber (235side of the fish to reduce measurement errors

× 110 × 70 mm) was replaced nine times per day(Wakamatsu

whereas the stock solution was changed every 2compared with the histological results. The medaka

days. Fish were maintained under 14:10-h light: dark

were blotted on filter paper, their body weights

photoperiod and fed granulated freeze-dried brine

and total lengths were measured, and parts ofshrimp (Tetra, Japan) until 10 dph from hatch andtheir caudal fins were excised with a razor for2001) and the images were

nauplii ad libitum from 11 dph to 35 dph

identification. Ten XY and 10 XX specimens weretwice daily. Residual food and feces were carefully

randomly selected from each treatment group on

removed each day; hardness, pH, and temperature

the basis ofof test solutions were monitored periodically.

subjected to histological examination. To compensate

identification and subsequentlyEvaluation of the effects of chemicals using transgenic fishfor the higher mortality due to higher EE 2

Tokyo, Japan) which yielded target ion values (m/z)

concentrations in the 880 and 1710 ng/L groups,

for EE2 and IS of 295 and 299, respectively.

gonads were obtained from 13 individuals (nine XY
and four XX individuals from the 880 ng/L group,3 2 2 8 Determination of genotypic sex

and eight XY and five XX individuals from the 1710

Identification of genotypic sex of each specimen

ng/L group; Table 3‑3). The entire bodies of theat 35 dph was performed with PCR analysis of

medaka were fixed in Bouin s solution, followed

according to the procedures described in chapter

by dehydration and embedding in paraffin wax.

2 2 2.

Serial longitudinal sections 5μm in thickness wereobtained and stained with hematoxylin and eosin

3 2 3 Statistical analysis

before being mounted with Eukitt and examined

In the nanoinjection experiment, to detect

under a light microscope. Gonad histology wasstatistical differences, other than differences

categorized as normal testis, testis-ova in XY fish,in mortality, between uninjected and triolein

and ovary in XX fish and undifferentiated in XY andcontrols, a two-sample t-test (parametric data) andXX fish. The results of the histological examinationa Mann‑Whitney test (nonparametric data) were

were used to determine the sex phenotype of eachperformed. When no difference was found between

specimen.

the triolein control and uninjected groups, thesegroups were pooled as a control for subsequent

3 2 2 7 Determination of EE2 concentration in thetest solution

analysis. Correlation between GFP-fluorescent area
and germ cell number was assessed by Kendall

The concentrations of EE2 in the test solutionss pie test. To compare the GSI among all treatment

were measured periodically during the entire

groups, gonad weight was log-transformed and thenexposure period. Aliquots of 250 ml of the test

analyzed by analysis of covariance (ANCOVA), using

solution were taken from all chambers of eachthe body weight of individuals as the covariate.

treatment group and pooled. After adding 30 ng of
13

In both nanoinjection and waterborne experiment,

C2D2-ethynylestradiol in methanol as an internal

the experimental data including growth (total length

standard (IS), each pooled water sample was passed

and body weight) and area of GFP fluorescencethrough a Sep-Pak PS2 solid-phase extraction

were checked for homogeneity of variances andcartridge column (Waters, Tokyo, Japan) pre-washedassumptions of normality across treatments bywith 10 ml of methanol and 20 ml of purified water.

Levene s test. When the assumptions were met,

Finally, the Sep-Pak PS2 column was washed withthe data were subjected to analysis of variance

20 ml of purified water and the captured EE2 was(ANOVA) followed by Dunnett s multiple

eluted with 3 ml of methanol. This eluate was thencomparison test. When the data failed to meet

concentrated to approximately 0 3 ml under a

the assumptions, a Kruskal‑Wallis test was used,stream of nitrogen at 40℃, after which the sample

followed by a Mann‑Whitney test with Bonferroni

was analyzed by using a high-performance liquids adjustment. A chi-square test was used to assess

chromatography (Shimadzu LC-10AD, Tokyo, Japan)

the difference in mortality. All statistical analyses,

equipped with an Inertsil ODS-3V column (length,

except for ANCOVA, were performed with the SPSS

250 mm; inner diameter, 4 0 mm; particle size, 5μBase 10.0J (SPSS Inc., Chicago, IL, USA). ANCOVA

m; GL Sciences, Tokyo, Japan) at 40℃. Each 20-μ

was performed with the SPSS Advanced Modell sample was injected into the chromatograph and

11 0J (SPSS). Differences were considered significant

eluted in the mobile phase. Gradient programs wereatperformed by using mobile phase gradients of A/B= 20/80 to A/B = 0/100 over seven minutes (A:100% acetonitrile; B: methanol in water) at a flowrate of 1 0 ml/min. After elution, the sample wasanalyzed with a mass spectrometer (JEOL LC Mate,< 0.05.

Takeshi HANO
3 3 Results

at 100 dph of fish further decreased to approx. 30%
in the EE2 treatment groups (Table 3‑1). However,3 3 1 Nanoinjection exposureno effect of EE2 on total length or body weight wasobserved among surviving adults (100 dph) from all

3 3 1 1 Survival and growth after treatment with EE2treatments (Table 3‑2).

Fig. 3‑3 shows the toxicity of EE2 nanoinjected
into embryos until the day of hatching. The majorityof embryos were dead on the next day of injection,
indicating the lethal toxicity of EE2. Survival rates

3 3 1 2 Effect of in ovo exposure of EE2 on germ
cell proliferation at 10 dph
Deleterious effects of EE2 on proliferation of germ

Fig. 3‑3. Cumulative mortality from nanoinjection (day 0) through to day 11 post-injection(around the day of hatching) in medaka juveniles that developed from embryos injected with
0 1‑5 0 ng/egg of 17α-ethinylestradiol (EE2). ** denotes significantly different from pooled
control groups at <0 01.
Table 3‑1. Survival, phenotypic sex and gonadal histology of adult medaka (100 days posthacth)injected
with 17α-ethinylestradiol (EE2)

Evaluation of the effects of chemicals using transgenic fish
Table 3‑2. Morphological characteristics of adult medaka (100 days posthatch) injected
17α-ethinylestradiol (EE2)

withcells were detected as early as 10 dph (Fig. 3‑4 and( < 0 01), but not significantly for treatments with5). In the uninjected and triolein treatment groups,<2 5 ng/egg ( = 0 0649). Significant relationships

sexual dimorphism of the gonad between sexes

between fluorescent area and germ cell number

was evident (Fig. 3‑4 A and C). The area of GFP

were also observed in EE 2 -treated XX females

florescence and the number of germ cells in XX(R = 0 632,

< 0 01) and EE 2-treated XY males

females that developed from embryos treated with

(R = 0 747,

< 0 01) at 10 dph (Fig. 3‑6).

5 0 ng EE2/egg were only 40 9% and 53 1% of pooled
control values, and the response was dose dependent

3 3 1 3 Effect of EE2 on sexual differentiation at 100
dph( = 0.048) (Fig. 3‑4 D and 5). In XY males, 1 out of
7, 2 out of 7, and 1 out of 3 males that developed

Complete male-to-female phenotypic sex reversal

from embryos injected with 0 5, 2 5, and 5 0 ng

occurred in genotypic male fish at 100 dph that

EE2/egg had a large fluorescent area (> 15 000μm2),

developed from eggs injected with 0 5, 2 5, and 5 0

accompanied by increased numbers of germ cells

ng EE2 (Table 3‑1). In all fish examined, the sexual(> 180 cells) (Fig. 3‑4B and 5); however, considering

phenotype determined by examining the external

the data from all genotypic males, no significant

secondary sex characteristics matched the sexual

difference was observed among treatment groups.

phenotype determined by gonad histology. XY males

Comparison of males with females in eachin all treatments had male-specific characters suchtreatment group at 10 dph (Fig. 3‑5) shows that theas papillary processes on the anal fin. The number

difference in fluorescent area and number of germof papillary processes in XY males did not differ

cells between males and females decreased withamong treatments (data not shown). In contrast,

increasing concentration of EE2. The area of GFP

sex-reversed XY females could be determined byfluorescence significantly differed between sexes forthe absence of papillary processes on the anal fin

the pooled control group and for fish that developed

and dorsal fin notch or the presence of urogenitalfrom embryos treated with 0 1, 0 5 ( < 0 01), and 2 5

papillae.

ng EE2/egg ( < 0 05), but not significantly for theHistological examination showed that both XY

5 0 ng EE2/egg treatment ( = 0 0924). The number

females and XX females had ovaries that activelyof germ cells significantly differed between sexes for

produced maturing oocytes at post-vitellogenic

the pooled control group and for fish that developed

phase. The GSI was the same in XY females andfrom eggs treated with 0 1 and 0 5 ng EE 2/egg

XX females, but was significantly lower in XY males

Takeshi HANO

Fig. 3‑4. Green fluorescent protein (GFP) fluorescent images captured from the lateral sideof XY (A, B) and XX (C, D) juveniles at 10 days posthatch. (A) uninjected XY juvenile, (B)XY juvenile injected with 0.5 ng of 17α-ethinylestradiol (EE2), (C) uninjected XX juvenile and(D) XX juvenile injected with 5.0 ng EE2. Outlined area in each of photo corresponds to theundifferentiated gonad. Arrow: leucophore, a male specific pigment. Arrowhead: autologous red
fluorescence of brine shrimp. R: residual food and/or discharges in the gut.

Fig. 3‑5. (A) Number of germ cells and (B) area of green fluorescent protein (GFP) fluorescence plotted forindividual fish at 10 days psthatch injected with the indicated amounts of 17α-ethinylestradiol (EE2).

Evaluation of the effects of chemicals using transgenic fish

Fig. 3‑6. Correlation between green fluorescent protein (GFP) fluorescent area and number ofgerm cells in (A) XX females and (B) XY males at 10 days posthatch for all treatment groups.
Significant relationships between them were observed in XX females (R=0 632, <0 01) and XY
males (R=0 747, <0 01).

Fig. 3‑7. Gonad somatic index (GSI) for XY males, XY females and XX females (mean G standard
deviation) at 100 days posthatch adult exposed
to 17α-ethinylestradiol (EE2). Numbers above
the bars indicate the number of individuals examined. ** denotes significant difference between XY
males and XY females ( < 0 01).

Takeshi HANO
(Fig. 3‑7). XY males, treated at EE2 concentrations in

3 3 2 Waterborne exposure

which sex reversal was induced in some individuals,
had normal GSI values, and no difference in GSI
was observed between them and the pooled control
groups.

3 3 2 1 Survival and growth after treatment with EE2
Fig. 3‑8 shows the effect of EE2 on the survival ofthe medaka during the exposure period. Mortality
in the 880 and 1710 ng/L groups began to increase

Fig. 3‑8. Cumulative mortality from hatch to 35 days posthatch in each 17α-ethinylestradiol (EE2)
treatment group (25 2-1710 ng/L). Concentrations in the figure are expressed as mean measured
concentrations (ng/L). **denotes significantly different from that of the control-2 ( <0 01).

Table 3‑3. Total length, body weight and gonad histology of medaka at 35 days posthatch exposed to 17α-ethinylestradiol (EE2)

Evaluation of the effects of chemicals using transgenic fishfrom the 7th day after the initiation of exposure,with cumulative mortality in these groups found to

3 3 2 2 Effect of EE2 on germ cell proliferation at 10
dph

be significantly higher than that of the control at

Deleterious effects on germ cell proliferation

35 dph ( <0 01) (Fig. 3‑8). Dying EE2-treated fish

were detected for EE 2 as early as 10 dph (Fig.

exhibited lightened body color and lay at the bottom

3‑9 and 10). In males receiving 158 ng/L EE2, theof the chambers, with little or no swimming.

area of GFP fluorescence showed an increase andNo significant differences were observed in either

was significantly different from that of the control

mean total length or body weight of the medaka at( <0 01). On the other hand, the GFP-fluorescent

35 dph in the 45 1, 80 1, or 158 ng/L EE2 treatmentarea was seen to gradually decrease in the >̲ 447groups (Table 3‑3). Growth promotion at 25 2 ng/L

ng/L treatment group, and this fluorescent area wasin XX female was due to low stock density ofsignificantly different in the >̲ 880 ng/L group whentest fish because of accidental mortality that wascompared to the GFP fluorescence in the control

associated with unrelated EE 2 effects. Exposure

fish ( ＝0 001 at 880ng/L and =0 009 at 1710ng/L,

to >̲447 ng/L EE2 was seen to result in a marked

respectively). In females whose sex genotype had

decrease in growth in both XY and XX fish in

been determined by the absence of leucophores,

comparison to the control (Table 3‑3). No obvious

the area of GFP fluorescence was seen to bedifference in growth parameters was detectedsignificantly decreased in the >̲ 880 ng/L treatment

between XY and XX fish in respective treatment.

groups ( =0 002 at both 880ng/L and 1710ng/L,
respectively). The area of GFP fluorescence between
XY and XX fish significantly differed in respective

Fig. 3‑9. Green fluorescent protein (GFP) fluorescent area plotted for individual fish at 10days posthatch exposed to 17α-ethinylestradiol (EE2). Genotypic sex of each individual
was determined by presence (male) or absence (female) of leuchophore in the skin, a malespecific pigment. *and ** denote significantly different from control at <0 05 and <0 01,
respectively.

Takeshi HANO

Fig. 3‑10. Green fluorescent protein (GFP) fluorescent images captured from the lateral side ofleucophore-positive male (A-C) and leucophore-negative female (D-F) at 10 days posthatch juvenile.
Males exposed to (A) 0 ng/L (control), (B) 158 ng/L of 17α-ethinylestradiol (EE2) and (C) 880ng/L.
Females exposed to (D) control, (E) 158 ng/L of EE2 and (F) 1710 ng/L. Outlined area in each photocorresponds to the undifferentiated gonad. Arrow: leucophore, a male specific pigment. Arrowhead:
autologous red fluorescence of brine shrimp.

treatment ( <0 05).

have indifferent gonads that were mostly composedof small numbers of germ cells showing no evidence

3 3 2 3 Histological examination and GFP
fluorescent area of the gonad at 35 dphof spermatogenesis or oogenesis (Table 3‑3 and Fig.
3‑11F).

The results of histological examination of XY fish

Fig. 3‑12 and 13 show the histology of theat 35 dph revealed that the induction of testis-ova

GFP-fluorescent area of the gonads in XY andwas observed in the medaka receiving <
̲ 158

XX

ng/L EE 2 (Table 3‑3 and Fig. 3‑11). In intersex

significant differences were detected in the areas

gonads, spermatocytes and spermatids could stillof GFP fluorescence in control testes and control

be identified but the incidence of previtellogenic

ovaries (Fig. 3‑12 and 13) or in those of the testes

oocytes in the testes was terrified with increasing

and ovaries of sex-reversed XY fish exposed to <̲concentrations of EE 2. Ovaries were observed in

447 ng/L EE2 (Fig. 3‑11AB and 12). The exception

XY fish at frequencies of 1 out of 10, 5 out of 10, 7

to this last observation was the significant decreaseout of 10, and 4 out of 10 in the 45 1, 80 1, 158, andin the GFP area of the intersex gonads in XY fish

447 ng/L treatment groups respectively (Table

exposed to 45 1 ng/L EE2 ( = 0.014) compared with

3‑3 and Fig. 3‑11E). The ovaries of sex-reversedthat of XY control (Fig. 3‑12). In the fish exposed

XY females, although they were immature, wereto >
̲ 447 ng/L EE2, GFP-area was also seen to haveoccupied with many previtellogenic oocytes anddecreased to only 3 to 25% of the control GFP area,

indistinguishable from that of XX control females

in addition to having weak fluorescence intensity,(Fig. 3‑11E). In XX fish, no obvious differences were

resulting in a significant difference when compared

observed between control and EE2-exposed ovary

with the controls (Fig. 3‑11C, 12 and 13).

/STII-YI strain, respectively. No

at <̲ 158ng/L. In contrast, XY and XX fish that had
been exposed to >
̲ 447 ng/L EE2 and one XY fishfrom the 80 1 ng/L treatment group were seen to

3 4 Discussion
EE2 is known to exert strong estrogenic activity

Evaluation of the effects of chemicals using transgenic fish

Fig. 3‑11. Green fluorescent protein (GFP) images from the right lateral aspect of the XY fish (A-C)
and transverse sections of XY fish at the end of 17α-ethinylestradiol (EE2) exposure at 35 days
posthatch stained with hematoxylin and eosin (D-E). (A) GFP image of 0 ng/L (control) XY male, (B)sex-reversed XY female exposed to 94 8 ng/L of EE2 and (C) XY fish exposed to 880 ng/L of EE2.
Histological section of testis of (D) control male, (E) sex-reversed ovary of a male exposed to 94 8
ng/L of EE2 and (F) unidentified gonad exposed to 880 ng/L of EE2. Gc, germ cells; Oc, oocytes; Sz,
spermatozoa. Bar = 1 0 mm (A-C) and 25 μm (D-F).

Fig. 3‑12. Green fluorescent protein (GFP) fluorescent area of the XY fish at 35 days posthatch
exposed to 17α-ethinylestradiol (EE2) categorized by the gonad histology. Error bar represents
standard deviation of the mean. Numbers in parenthesis denote number of fish examined.
One specimen possessing indifferent gonad in the 80 1ng /L could not be determined its
GFP-fluorescent area because of weak fluorescence intensity. *and ** denote significantly different
from control at <0 05 and <0 01, respectively.

Takeshi HANO

Fig. 3‑13. Green fluorescent protein (GFP) fluorescent area of the XX fish at 35 days posthatch
exposed to 17α-ethinylestradiol (EE2) categorized by the gonad histology. Error bar represents
standard deviation of the mean. Numbers in parenthesis denote number of fish examined. **denotes significantly different from control at <0 01.
and also to be lethal to fish (Folmar

2002). EE2

abnormal sexual differentiation could be detected as

was observed to cause pathological abnormalities

early as 10 dph juvenile because lowest observablesuch as lightened body color and increased

effective dose (LOED; 0 5 ng/egg) for inducing

immobility, finally leading to increased mortality atabnormal gonadal development in genotypic maleEE2 exposures of >
̲ 880 ng/L. Growth (total length

at 10 dph was the same as that for inducing sex

and body length) inhibition was also observed atreversal in 100 dph adult fish.

aqueous EE2 exposure of >
̲ 447 ng/L in both sexes.
Identification of

To detect abnormal changes in sexualand leucophore marker fromdifferentiation, we focused on the different maleeach individual have indicated that no significant

and female proliferation patterns of germ cells thatdifferences could be found in these parameters inoccur near the onset of gonadal differentiation. In

medaka of XY and XX genotypes in any of thethe present study, the average GFP area in control

treatment groups. Weber(2004) reported thatXX female medaka was about 10 times larger thanchronic exposure of medaka (ranging from the newlythat of control XY males, indicating that sexual

hatched to the adult fish) to 10 ng/L of EE2 resulted

dimorphism was significantly evident at 10 dph.

in toxicity in the form of increased interstitial cell

Aqueous exposure to 158 ng/L EE 2 was founddeath in head kidneys. This sex-specific toxicity

to abnormally enhance germ cell proliferation in

was manifested as severely adverse effects on

males at 10 dph. The average GFP area seen inhematopoiesis in growing medaka, especially in themales with 158 ng/L EE 2 treatment was 11376

male medaka. Thus, the findings of the study by

μm 2 , which corresponded to 137 germ cells

Weber(2004) indicated that sex-specific effects

calculated using a correlation equation betweenof EE2 can be seen at concentrations lower than that

GFP area and germ cell numbers (Table 2‑1); thiscausing lethal toxicity.

was about twice the number seen in control XY

Identification ofmarker facilitates theconfirmation of genotypic sex of each individual atmales. Sex-based differences in germ cell numbers
depend on the expression of(andany stages of development, including juveniles or

domain gene on the Y chromosome), which

adults. This made more efficient to detect abnormal

has been identified as a prime candidate for thesexual differentiation caused by EE2. For example,

sex-determining gene located on the Y chromosome

in nanoinjection experiment, it is demonstrated thatin medaka (Matsuda

2007) and which functions

Evaluation of the effects of chemicals using transgenic fish
as an inhibitory regulator in germ cell proliferation

no-observed effective dose (NOED) of 0 1 ng fromin normal XY fish (Paul(2006)

the EE2 nanoinjection experiment. Our results also

observed the meiotic activity of germ cells throughshow that male to female sex-reversal occurred at

the expression of the synaptonemal complex protein

35 dph at exposure levels of 45 1 and 80 1 ng/L

3 (Scp3) in E2 (17β-estradiol)-exposed medaka at 1

EE2, even though no effects were seen on germ celldph. They had demonstrated that E2 might mitigate

proliferation at 10 dph. These observations suggest

the action of2006). Paulto get rid of the blockadethat continuous EE 2 intake from <̲ 158 ng/L of

on germ cell proliferation and thus reverse theEE2-contaminated water over the period from 10

phenotypic sex of the XY medaka. To investigate

to 35 dph could have resulted in EE2 absorption to

how EE2 induces abnormal germ cell proliferationlevels above the threshold concentration necessary

in XY medaka, analysis of functional

to induce feminization of the fish.

proteinshould provide further insights into the mechanisms
responsible for estrogen s effects.

We investigated the availability of GFP
fluorescence as a means to detect adverse effects

It is noteworthy that the increase in germ cellon sexual differentiation, however, no significant

proliferation by EE2 in XY males at 10 dph after

differences were observed between the GFP

aqueous exposure and after direct nanoinjection

fluorescence in normal testes and ovaries at 35

was seen in a different manner. The present study

dph. This could be because of the difficulty inshowed that aqueous exposure of medaka juveniles

capturing the GFP images of the gonads in medaka

to 158 ng/L of EE2 enhanced germ cell proliferation

at 35 dph due to the randomly locating fatty tissues

to levels that were twice those seen in control males,surrounding the normally developed ovaries andalthough they were still only about 25% of the values

testes. The significant decrease observed in thein the control females. In contrast, nanoinjection

GFP-fluorescent area of the intersex gonads in 35

of >̲ 0.5 ng EE2 into the medaka embryos further

dph medaka exposed to 45 1 ng/L EE2 was moreenhanced germ cell proliferation in some XY males

due to the accidental localization of the gonads

to levels that were 60‑100% of those in the control

in the abdominal cavity of the fish rather thanfemales, indicating sexual differentiation towards

estrogenic effects of EE2 (Fig. 3‑14). These results

female characteristics. The dissimilar responses toindicate that the determination of sex phenotype

EE2 could be associated with the different amounton the basis of gonadal size through the calculationof EE2 intake at 10 dph. To further examine theseof the GFP-fluorescent areas in the fish can only beeffects, the bioconcentration factor (BCF) was usedperformed in the 10 dph juvenile medaka and not inas an estimate of the bioaccumulation potential of

the 35 dph medaka.

EE2. A positive relationship has been demonstrated

On the other hand, EE2 at levels of >
̲ 447 ng/L

between the log of the octanol-water partition

was seen to not only reduced germ cell numbers

coefficient (Kow value) and the log BCF value in fishat 10 dph but also reduce GFP-fluorescent area and(Geyerfluorescence intensity of the gonad at 35 dph. In

1984). Given that a log K ow value of

4 2 for EE2 (Schweinfurth1997), calculated

male fathead minnows exposed to 10 nM (27ng/L)

BCF value of EE2 is to be 741 on the basis of theof E 2 , Miles-Richardson

following linear relationship as described by Veith

increased number of sertoli cells, which play a role

(1980): log BCF = 0 85 log Kow ‑ 0.70. An

in phagocytosis, with distended cytoplasm filled

full life toxicity test in fathead minnow ((1999) observedwith degenerate spermatozoa and suggested an) also gave a BCF value of 660 for EE2 (Lange

increase in necrosis or apotosis of germ cells. The

2001). Considering the average weight of thelesions observed in the present study might havejuvenile medaka at 10 dph to be 3 mg, recalculation

occurred due to an increase in necrosis or apotosis

gave absorption values of approximately 0 31 toof germ cells and an arrest in germ cell maturation

0 35 ng of EE2 after exposure to 158 ng/L of EE2.

with subsequent degeneration of spermatozoa andThese absorption values were still lower thanoocytes. Meanwhile, the impairment of germ cellthe LOED of 0 5 ng of EE 2 but higher than the

proliferation in XX females at 10 dph was transientTakeshi HANO

Fig. 3‑14. Green fluorescent protein (GFP) images from the right lateral side of the fish andlongitudinal sections of ovaries stained with hematoxylin and eosin at the end of 35 days EE2
exposure. (A) Clearly identified GFP image of ovary. (B) Obscured GFP image of ovary surroundedwith fatty tissues. (C) Longitudinal sectioned ovary (GFP image corresponds to A) located in the rightlateral side in the abdomen cavity. (D) Longitudinal sectioned ovary (GFP image corresponds to B)
located in the central region of abdomen cavity. O, ovary; f, fatty tissues; gu, gut. Bar = 0 5 mm (A-D).

in nanoinjection experiment: at 100 dph, mature XX

the skewing of the sex ratios towards females at 0 1

females of all treatment groups had normal ovaries.

μg/L (100 ng/L) of EE2 at approximately 100 dph

They are explained by that EE2 might be eliminated(Metcalfe

from the body and germ cell proliferation recovered

60 days from 1 dpf (Sekias the fish continued to develop.

the orange-red strain of Japanese medaka. Taking

2001) and at >
̲ 27 9 ng/L of E2 for2005) onwards in

Sensitivity to EE2 was evaluated in the transgenic

all these findings together, if EE2 and E 2 can be/STII-YI strain to examine its suitability

considered to be of approximately equal potency inas a fish model to screen for EDCs effects. In

the reproduction test in medaka (Sekiexperimtnt-1, agreement of LOED of EE 2 (0 5Kangng/egg) to induce male-to-female sex reversal was

be predicted to show almost the same sensitivity toobserved in an earlier study of the d-rR strain of

estrogenic chemicals as Japanese medaka.

medaka reported by Papoulias

2002), the

2002;/STII-YI strain may(2000a). In

Overall, the present study showed that thewaterborne experiment, the lowest observablemeasurement of GFP-fluorescent area of the gonads

effective concentration (LOEC) to increase germ

could reveal abnormal germ cell proliferation by EE2,

cell proliferation in medaka at 10 dph and for thewith a reversal of the male sex phenotype towards

induction of male to female sex-reversal in medaka

the female phenotype at 10 dph. However, theat 35 dph were found to be 158 and 45 1 ng/L,

effects of EE2 on proliferative activity of germ cells

respectively. In an earlier study, Suzuki(2005)

were less dramatic with exposure to waterborne

had reported that 0 1μg/L (100 ng/L) of E2 was

EE2 than with direct nanoinjection of EE2. Future

seen to enhance germ cell proliferation at 0 dph instudies focusing on germ cell proliferation in thethe Hd-rR medaka strain. Other studies had reported/STII-YI strain can be a useful aid inEvaluation of the effects of chemicals using transgenic fish
elucidating not only unknown mechanism of sexual

4 2 Materials and Methods

differentiation but also effects of EDCs on early
development in fish.

4 2 1 Test organism

Chapter 4 Effect of in ovo exposure of nonylphenol

at Nagoya University. We used a line homozygous

on sexual differentiation in thethat almost 100% of embryos were theoretically

The/STII-YI strain/STII-YI strain was maintained

GFP-positive. Parental fish at 60 days posthatch (dph)were kept in 16-L tanks with a water circulating

4 1 Introduction

system (MH; Meito-Suien, Nagoya, Japan) at 25 to 27

Nonylphenol (NP), a bacterial degradation℃ under a constant 14:10-h light:dark photoperiod

product of Nonylphenol Ethoxylates (NPEOs), hasand were fed three times daily ad libitum with

been detected at microgram-per-liter levels in theaquatic environment (Rudel
1999; Tsuda

nauplii (<24 h after hatching).

1998; Blackburn
4 2 2 Embryo nanoinjection

2000). Several

studies have confirmed that NP has physiological

Preparation of microneedles and nanoinjection

and developmental effects related to its estrogenic

were conducted according to the procedures

potency, including reproductive failure, production

described in chapter 3 2 1 1. Embryos were collectedof testis-ova and vitellogenin (Vtg) in NP-exposedfrom breeding females in the morning, and healthy

medaka (embryos were selected. The embryos were sterilized; Kangfathead minnow (2003) and; Harries

and washed (chapter 2 2 3. in detail) and were2000). Exposure of medaka to NP for 60 days fromembedded in 2% agarose gel on glass slides just0 days post-fertilization (dpf) result in significant

before nanoinjection. Within 8 h after fertilization,

decrease of hatchability and successful swim-up of

NP in triolein was injected at 2 0, 10, 50, 125, or 250

hatched larvae at 183μg/L (Yokota

2001)

ng per egg, with about 80 embryos per dose group.

and skewed the sex ratio toward females at 23 5

NP was nanoinjected directly into the oil globule

μg/L (Sekiwith the aid of a Femtojet and a micromanipulator.

2003). In a full life cycle test

using medaka, Yokota(2001) showed that NP

induced testis-ova in both parental medaka (Embryos were also injected with NP-free triolein) at(triolein control) or were not injected (uninjected) at >
̲8 2μg/L.

control). The treated embryos were washed andThey suggested that exposure of NP beginningmaintained in 10 mL of embryo rearing medium

and continuing throughout the early life

(chapter 2 2 3. in detail) in a 6-well multiwell plate at>
̲17 7μg/L and their progeny (stage of thegeneration induced the formation

25‑27℃ until hatching.

of testis-ova at lower concentrations than in the

Mortality and abnormal development weregeneration. This finding suggests that hydrophobic

assessed daily during the embryogenesis.

NP accumulated in fish is maternally transferred

Hatchability, swim-up failure and time-to-hatching

to the next generation via the eggs, however, it iswere also assessed in the hatching embryos.

not well known effective concentration of NP that is

Hatched juveniles were transferred to 1 8-L squarematernally transferred into eggs.

glass containers (20 individuals per chamber) andIn chapter 4, we nanoinjected NP into the embryosof themaintained in dechlorinated tap water at 25‑27/STII-YI strain and observed its℃. Survival and growth were observed daily until

adverse effects on the development of embryo andthey sexually mature for approximately 100 days.

juveniles and also performed bioimaging analysis of

They were fed granulated freeze-dried brine shrimp

the gonads, using green fluorescent protein (GFP)(Tetra, Tokyo, Japan) around the onset of hatchingfluorescence to assess the effects of NP on sexual

and, as they grew up, they were fed withdifferentiation.

nauplii (< 24 h after hatching) twice a day.

Takeshi HANO
4 2 3 NP working solution

water, blotted on filter paper, and their body weightThe selected embryos were exposed via

and total length measured. Then their development

nanoinjection to NP (97 4% purity, consisting ofof secondary sex characteristics were assesseda mixtures of isomers, Kanto chemicals, Tokyo,(chapter 3 2 1 6,

Japan) dissolved in triolein (99 0% purity, Wako Pure

removed and weighed to calculate the gonad somatic

Chemical Industries, Osaka, Japan). Triolein was

index (GSI) and hepatosomatic index (HSI). Finally,

filter-sterilized by passage through a 0 22 μm PTFE

the caudal fin from each specimen was removed

filter (Millipore, Bedford, USA) before use.

and subjected to extraction of the). Gonads and livers weremarker for

Prior to experiment, we conducted a preliminary

identification of the genotypic sex. Removed gonads

toxicity test of NP. Thirty healthy eggs obtained

were prepared for histological observation and serial

from medaka stock were exposed to NP via

transverse sections (thickness, 5μm) were obtained

nanoinjection at 500 ng/egg, and the toxicity of NP

following previously outlined histological proceduresto the embryos was observed. Twenty-eight out

in chapter 3 2 2 6.

of 30 eggs died within 24 h after injection and noembryonic body could be seen in the embryo. On the

4 2 6 Determination of genotypic sex

basis of the results of this toxicity test, we chose NP

Genetic sex in each individual at 100 dph wasdoses of 2, 10, 50, 125, and 250 ng/egg for this study.

performed with polymerase chain reaction (PCR)

3 ml of NP stock solution (500 ng/nl =1500 mg/3 ml)

analysis of sex-linked 1 (was prepared by mixing the 1 579 ml of NP (1 500

procedures as described in chapter 2 2 2.

) according to themg, d=0 950 g/ml) and 1 421 ml of triolein (1 300 mg,
d=0 915 g/ml). NP working solutions (4, 20, 100 and
250 ng/nl) were prepared by serial dilution of the

4 2 7 Statistical analysis
Statistical analysis was conducted according to thestock solution with triolein.

procedures in chapter 3 2 3.

4 2 4 Measurement and quantification of GFP

4 3 Results

fluorescent area at 10 dph juvenile
At 10 dph, all specimens that showed GFP

4 3 1 Survival and growth

fluorescence in their germ cells were anesthetized

NP showed lethal toxicity to medaka embryos

by being cooled in iced water. GFP expression was(Table 4‑1). Since no significant difference in survival

detected using equipments as described in chapter

was found between the triolein (0 ng NP/egg) and3 2 2 2. GFP fluorescent images were recorded

uninjected groups, they were pooled for statisticalanalyzed and finally determined its area following

analysis. Survival at 1 dpf was significantly lower

previously outlined procedures in chapter 3 2 2 5.

than in the pooled controls at a dose of 50 ng/egg or

After we had measured this area, all specimens

more ( < 0 01).

were separated into three chambers per treatment

The number of hatched embryos was significantly

and maintained for 100 days under the conditions

reduced in all dose groups except the 10 ng/egg

described above. The first chamber held juveniles

group (Table 4‑1). The time to hatch was aboutthat had male-like gonad size (GFP area <15 000

8 days in all treatments. Swim-up failure, whichμm2;) and leucophore cells in their skin; the secondmeans that larvae were unable to survive for longer

held juveniles that had female-like gonad size (GFP

than 5 days, was significantly increased in the 50,

2area >15 000μm ) but no leucophore cells; and the125, and 250 ng/egg treatment groups. Tails of

third held juveniles that had male-like gonads but noaffected larvae in these groups were usually bent

leucophores in their skin or female-like gonads andat the tip, curled, shortened, or all three (Fig. 4‑1).

leucophores in their skin.

Affected larvae lay on their sides and swam little
or were immobile. Consequently, in the 250 ng/egg

4 2 5 Characterization of adult fish at 100 dph
At 100 dph, the fish were anesthetized in ice

treatment group, no individuals survived for longer
than 10 days after hatching.

Evaluation of the effects of chemicals using transgenic fish
Table 4‑1. Toxicity of nonylphenol (NP) to the medaka during embryogenesis and around the dayof hatching

Fig. 4‑1. Swim-up failed larvae at 1 day posthatch
juvenile injected with 250 ng of nonylphenol (NP).

At 100 dph, the survival rates of hatched larvae

NP-treated groups, sexual dimorphism of the gonad

were constant, with no significant differences

between sexes was evident: that is, the area of

between treatment groups and pooled controls

GFP fluorescence differed significantly between(Table 4‑2). Measurement of body weight and length

the sexes in the pooled control group and in fishat 100 dph gave no significant difference amongthat developed from embryos treated with 2, 10, 50,

treatment groups (Table 4‑3).

or 125 ng/egg ( < 0.01). Furthermore, the extent
of the GFP area did not differ significantly among

4 3 2 Effect of NP on germ cell proliferation at 10
dph

treatment groups in the two sexes.
The numbers of juveniles that had male-likeNo deleterious effects of NP on proliferation

gonads but no leucophores in their skin orof germ cells were detected as early as 10 dph

female-like gonads with leucophores in the skin were

(Fig. 4‑2). In each treatment, including in the

3, 1, 7, 3, 2, and 1 in the uninjected, triolein, 2, 10, 50,

Takeshi HANO
Table 4‑2. Survival, phenotypic sex and gonadal histology of adult medaka (100 days posthacth)injected
with nonylphenol (NP)

Table 4‑3. Morphological characteristics of adult medaka (100 days posthatch) injectednonylphenol (NP)withand 125 ng/egg groups, respectively. PCR analysis

NP-treated fish, genotypic sex determined byofamplification of

showed that recombination between thelocus and sex-determinant (matched phenotypic sex) locus occurred indetermined by analysis of gonadal sex and secondary

all of these individuals, indicating no impairment onsex characteristics (Table 4‑2). Histologicalgerm cell proliferation at 10 dph juvenile.

observation of the gonad in both sexes also showedno abnormalities. XY males and XX females had

4 3 3 Sexual development at 100 dph
Among the surviving adults, including thenormal GSI and HSI values, and no difference inthem was observed between the NP-treated and theEvaluation of the effects of chemicals using transgenic fish

Fig. 4‑2. Areas of green fluorescent protein (GFP) fluorescence plotted for individual fish (10 days posthatch)that developed from embryos injected with the indicated amounts of nonylphenol (NP).

pooled control groups (data not shown).

mortality rates can probably be explained bysuccessful metabolism and elimination of injected NP

4 4 Discussion

in surviving fish.

Our results clearly showed that NP injection
into medaka embryos of the

To detect changes in sexual differentiation, we/STII-YI

focused on the different proliferation pattern ofstrain adversely affected embryonic development

germ cells between sexes that occurs near the onset(Table 4‑1and Fig. 4‑1) but did not impair germ cellof gonadal differentiation. Although the reason for

proliferation by 10 dph and sexual differentiation inthis difference between sexes is not understood

adult fish by 100 dph (Table 4‑2 and Fig. 4‑2).

well, histological and ultrastructural studies ofNP exerts estrogenic activity (Folmar
and is also lethal to fish (Yokota
2003; Seki

2002)

2001; Kang

gonad anlage in medaka suggest that gonadal sex
differentiation does not depend on sex steroids in2003). We detected deleterious

this species (Satoh, 1974). However, nanoinjection

effects of NP with respect to survival, hatchabilityof EE2 causes abnormal sexual differentiation in 10

and swim-up failure. Its toxic effect was evident

dph juveniles of both sexes and in genotypic adultas early as 1 dpf. The number of embryos hatchedmales at 100 dph at the same initial EE2 levels (>0 5

decreased significantly in all treatment groups

ng/egg; Fig. 3‑4 and 5). Nevertheless, in the present

except the 10 ng/egg group. The lowest observablestudy we found no impairment by NP ̶an artificial

effective dose (LOED) of NP on embryo survival isestrogenic chemical̶ of germ cell proliferation

estimated to be between 0 and 2 ng/egg. Yokota

in medaka juveniles or of sexual differentiation in(2001) reported reduced embryo survival andadult medaka at any NP dose (2‑125 ng/egg). On

swim-up success in medaka treated with 183μg/L

the other hand, Seki

(2003) reported that NP

NP by waterborne exposure from 0 dpf; they also

at >23 5μg/L skewed the sex ratio toward females

found increased mortality at >
̲17 7μg/L NP fromin medaka exposed for 60 days from 0 dpf. Folmar

0 to 60 dph. In contrast, nanoinjected NP affected(2002) also demonstrated that the estrogenic

neither survival rates nor growth of surviving larvae

potency of NP was about 1% that of EE2 for inducing

up to 100 days. These differences in post-swim-up

Vtg production in sheepshead minnow (Takeshi HANO
Lacépède). From these findings we had(2006) reported the rapid decrease of NP

predicted that nanoinjected NP at 50 ng/egg ̶concentration from 2‑7 to <1μg/g in the eggs

100 times the EE2 dose that induced sex reversal

spawned from female medaka that were exposed toin the EE 2 nanoinjection study (>0 5 ng/egg;

NP at 61 2μg/L for 21 days and then transferred toTable 3‑1) and within the range of our test doses

dechlorinated tap water for 7 days. In the present(2‑250 ng/egg)̶ would be required to induce bothstudy, injected NP might be eliminated rapidly inabnormal sexual differentiation at 10 dph and sex

the surviving embryo so that NP failed to exert its

reversal at 100 dph in medaka. In addition, we also

estrogenic activity enough to induce sex reversal.

chose the amounts of NP in which were presumably

Second, both germ cell proliferation by 10 dph andenough to induce sex-reversal because the LOED for

sexual differentiation in adult medaka might not be

inducing sex reversal is generally lower than the dose

impaired by the only NP that maternally transferredthat causes acute toxicity to organisms (Papoulias

from parental fish in the aquatic environment. In

2000a; Edmunds

2000).

Two explanations for the failure of NP to impair

a full life cycle test using medaka, Yokota(2001) showed that NP skewed sex ratio toward

the germ cell proliferation and sexual differentiation

female in both parental medaka (are possible. First, NP might be metabolized

g/L and their progeny () at >
̲51 5μand/or excreted in the surviving embryos fasterdemonstrated that occurrence of biased sex ratio

than expected, which could have made the NP

at lower concentration inconcentration below the threshold concentrationby that NP might exert enhanced effects to thenecessary to influence sexual differentiation during

progeny generation through maternal transfer of NP

sensitive periods. Although the kinetics of NP

into thein the fish embryo is not well known, Ishibashi

showed that newly hatched fry, rather than intact) at >
̲17 7μg/L andthan

was explained

embryos. Kogar and Hinton (2000) also

Fig. 4‑3. Schematic images of nonylphenol (NP) exposure to medaka by nanoinjection, maternaltransference to the next generation and waterborne exposure.

Evaluation of the effects of chemicals using transgenic fish
chorionated embryos, are the most developmental

critical to aquatic organisms, previously reported assensitive stages to 17β-estradiol. Additionally,

impairment of reproductive capabilities in medaka

Ishibashi((2006) detected 2 7μg/g of NP from) exposed to 488 ng/L of EE2 for 3

the eggs that were spawned from females exposed

weeks (Sekito averaged measured concentrations of 61 2 μg/L

capacity derives from decreases in fertility, andof NP, which was about half of concentration thatlesioned gonad by estrogenic chemicals, in which

impairs the reproduction of adult medaka (Kangabnormal connective tissues was developed, was2003). When calculated based on average2002). Impairment of reproductive

found to be causative (Kang

2002; Seki

medaka egg weight of 1 mg, NP concentrations per2002). Considering that abnormal changes in fish

egg was 2‑7 ng, which was higher than estimatedtesticular structure have been found in the aquatic

LOED in this study and is much lower than the dose

environment (Gillthat we had predicted the sex reversal would be

develop a facilitated system to monitor the gonadal

induced (50 ng/egg). Together with these findings,

change adversely affected by temporarily highabsorption of NP via the NP-contaminated waterlevels of estrogenic chemicals released into theafter hatch as well as maternally transferred NP

environment. In addition, although many studies

might play a key role in the induction of abnormal

have focused on the effect of estrogenic compounds

sexual differentiation in the medaka (Fig. 4‑3).

on the testis, little is known whether testis lesions

In summary, our results clearly demonstrated
that a single

injection of NP into the medaka

embryo impaired embryonic development but2002), it is important toand impaired sparmatogenesis induced by estrogenic
chemicals can be reconstituted in adult medaka
transferred to clean water.

not germ cell proliferation by 10 dph and sexual

In the present chapter, we examined the effects

differentiation in adult fish by 100 dph. Althoughof different concentrations of EE2 on matured malefurther investigations might be needed to elucidate

and female gonads in thethe usefulness of nanoinjection of embryos ofand further investigated the chorological changes ofthetestis after the exposed males are transferred to clean/STII-YI strain in detecting the

estrogenic effects of other EDCs, the nanoinjection/STII-YI strainwater.

model has potential for use in evaluating the effects
of chemicals on early development and sexual

5 2 Materials and Methods

differentiation in fish.
5 2 1 Direct waterborne exposure
Chapter 5 Effect of 17α-ethinylestradiol on matured
gonad in the/STII-YI strain

5 2 1 1 Test organism/STII-YI strain were maintained at

5 1 Introduction

Nagoya University. Medaka selected for this study

As previously described in chapter 1, we selected

were approximately 60 days posthatch and were

17α-ethinylestradiol (EE2) as a test chemical because

fully mature (body weight, 334 ± 6 mg; total length,

it has strong estrogenic potency (Folmar

2002)

33 ± 2 mm). They were kept in 16-L tanks with aand has been detected in many sewage treatment

water circulating system (MH; Meito-suien, Nagoya,work effluents and in many surface waters. The

Japan) at 26℃ under 14:10-h light:dark photoperiod

concentrations of EE2 in river surface waters wereand fed three times daily ad libitum withrelatively low and reported to be a few nanograms

nauplii (<24 h after hatching).

per liter (Belfoid1999). Despite the frequently

low environmental concentrations of EE 2 , highlevels of EE2 concentrations to these lower values

5 2 1 2 Measurement and quantification of GFP
fluorescent area

is a recent survey of U.S. waters that reported EE2Prior to the initiation of exposure, test specimens

concentrations ranging 73 to 831 ng/L (Kolphin

whose gonads had intensive green fluorescent

2002). These high levels of concentrations were

protein (GFP) fluorescence were selected from

Takeshi HANO
stocks and anesthetized in ice water on a petri

and 4 females each, because we had assumed thatdish (60 × 15 mm). GFP expression was detected

higher mortality would occur in males than inusing equipments as described in chapter 3 2 2 2.

females according to previous study (Seki

GFP fluorescent images were recorded analyzed

2002), and our preliminary study had found that theand finally determined its area following previously

optimum ratio of male to female in each chambers

outlined procedures in chapter 3 2 2 5. All GFP

was 1 to 2, to maintain normal ovarian maturation inimages of the gonads were captured from thethis strain (data not shown). Fecundity and fertility

right lateral side of the fish, to reduce artificial

were not pre-assessed.

measurement errors.

At the end of the exposure period, each medaka

Prior to experimentation, the GFP fluorescent

was anesthetized on ice, and their fluorescentimage, and total length and body weight of each

image and area recorded. Average growth rate in

test specimen were recorded for each individual

each treatment was calculated by averaging theto observe the change between initiation and end

ratio of gonadal size of 4th week to that of day

of exposure. In males, the distribution pattern of

0 for each individual per treatment. Specimens

leucophore, a sex-specific pigment (Wakamatsu

were then blotted on filter paper and measured2003), was also used in identifying each individual.

for body weight and total length. Gonads andlivers were removed and weighed to calculate

5 2 1 3 Special reagents

the gonadosomatic index (GSI) and hepatosomatic

EE2 was purchased from Nacalai Tesque (Kyoto,index (HSI). Removed gonads were prepared forJapan). An EE 2 stock solution (100 mg/L) was

histological observation following previously outlined

prepared by dissolving the reagent in acetone. An

histological procedures in chapter 3 2 2 6. Serial

aliquot was then evaporated to dryness under N2transverse sections (thickness, 6μm) were obtainedgas, diluted with deionized water, and magneticallyfrom male fish gonads, and 6 total section slides

stirred for approximately 24 h to resolve the EE2 inwere prepared from the females. The determinationwater (1 mg/L). This aqueous solution was dilutedof testis-ova and connective tissue was based onwith dechlorinated water to prepare a working EE2

previous studies (Kangstock solution of 9μg/L.

2002; Seki

2002).

EE2 concentrations were measured every 2 weeks.
The means (coefficients of variation) of measured

5 2 1 4. Exposure design

EE2 concentrations in each of test solutions during

The selected fish were exposed to EE 2 in a

the exposure period were 47 8 (41 1), 94 8 (32 3), 216

flow-through mini-diluter system, as previously(8 6), and 522 (26 2) ng/L. The EE2 concentration indescribed in chapter 3 2 2 4. Fish were maintained

the control treatment was less than the detection

under 14:10-h light: dark photoperiod and fed

limit (2 0 ng/L) in all analyses.

nauplii ad libitum twice daily. Residual
food and feces were carefully removed each day;hardness, pH, and temperature of test solutions was

5 2 1 5 Determination of EE2 concentration in thetest solution

monitored periodically. Throughout the experimental

The concentrations of EE2 in the test solutions

period, hardness in the water was 20‑30 mg

were measured periodically during the exposure

CaCO3/L and pH was 6 7‑7 0. Water temperature

period according to the procedures described inremained at 24 ± 1℃. Medaka were observed daily

chapter 3 2 2 7.

to assess mortality and appearance.
Mature medaka were exposed to EE 2 for 4

5 2 2 Waterborne exposure followed by depuration

weeks in test glass chambers (235 × 110 × 70 mm)

In the same kind of flow-through systemthat contained about 1 8-L of test solution. Eachdescribed in chapter 5 2 1 4, mature medaka (6treatment consisted of 3 glass chambers holding amales per treatment) were exposed for 3 weeks tototal of 10 males and 8 females: 1 chamber had 6

EE2, followed by 6 weeks of culture in clean water,males and the other 2 chambers contained 2 males

in order to investigate the persistent effects of EE2.

Evaluation of the effects of chemicals using transgenic fish
One glass chamber was prepared per treatment.

individuals as the covariate. Correlations between

The experimental exposure period was shortened

the GFP-fluorescent area and male and female

to 3 weeks because of high early mortality in >
̲215

gonad weight were assessed by Kendall s test. All

ng/L treatments probably due to anesthesia instatistical analyses were completed using the 10.0J

ice-water.

SPSS Base System (SPSS Inc., Chicago, IL, USA).

fluorescent images of GFP wereobtained continuously, once a week through the

Differences were considered significant at< 0.05.

whole experimental period (0‑9 weeks) for the

In the depuration experiment, the log ofcontrol, 215, and 473 ng/L treatments and only ontransformed GFP fluorescence area values at

the 0, 1, 2, 3, 6 and 9th week for the 43 7 and 85 8

3 weeks were compared by Tukey s multiple

ng/L treatments, since dramatic changes were notcomparison test. The treatments were grouped into

expected at lower concentrations. At the end of

G1 (control, 43 7, and 85 8 ng/L) and G2 (215 and 473

the experiment, surviving specimens were sampledng/L), based on no significant differences among thefollowing previously outlined histological procedures

treatments forming these groups. The area values atin chapter 3 2 2 6.

9 wk were then analyzed by ANOVA. Differences

EE 2 concentrations in test solutions were

were considered significant at< 0 05. Assuming

measured once a week. The means (coefficients ofa second order spline function of time and dose, thevariation) of measured EE2 concentrations in test

best model that is fitted to the data was selected

solutions during the exposure period were 43 7

by evaluating AIC (Akaike s information criterion)(35 7), 85 8 (31 0), 215 (6 1), and 473 (26 9) ng/L.

using SAS (SAS Institute Inc, NC, USA).

5 2 3 Statistical analysis

5 3 Results

In the direct exposure experiment, theexperimental data were checked for homogeneity

5 3 1 Direct waterborne exposureof variances and assumptions of normality acrosstreatments by Levene s test. When the assumptions

5 3 1 1 Mortality and abnormal appearance inwere met, the data were subjected to analysis of

medaka during EE2 exposure.

variance (ANOVA) followed by Dunnet s Multiple

During the exposure period, adult medaka in thecomparison test. When the data failed to meet the>̲216 ng/L treatment groups exhibited pathologicalassumptions, the Kruskal Wallis test was usedlesions such as lightened body color, swollen

followed by a Mann Whitney test with Bonferroni

abdomen and subcutaneous hemorrhage (Table 5‑1).

s adjustment. To compare the GSI and HSI

Death occurred in 3 males and 4 females from theamong all treatment groups, gonad weight was>
̲216 ng/L treatment groups, showing these lesions

log-transformed and then analyzed by analysis of(Table 5‑1). In females with a swollen abdomen,

covariance (ANCOVA), using the body weight ofthe ovary was visibly shrunken and located in the

Table 5‑1. Mortality and pathological responses in medaka during the experimental period in direct
exposure.

Takeshi HANO

Fig. 5‑1. Appearance of a
/STII-YI strain medaka at the end of EE2 exposure.
(A) 0 ng/L (control) female. (B) Abnormal appearance of a female medaka that showedswollen abdomen exposed to 522 ng/L. a, air bladder; o, ovary; sw, swollen abdomen. STII,
see-through II.

Table 5‑2. Changes in area of green fluorescent protein (GFP) fluorescence and average growth rateduring the exposure periodEvaluation of the effects of chemicals using transgenic fish

Fig. 5‑2. Green fluorescent protein (GFP) images from the right lateral aspect of the male fish (A-C)
and transverse sections of testes at the end of 4 week 17α-ethinylestradiol (EE2) exposure period
stained with hematoxylin and eosin (D-E). (A) GFP image of a 0 ng/L(control), (B) 94 8 ng/L of EE2and (C) 522 ng/L of EE2. Histological section of (D) control testis, (E) testis-ova exposed to 94 8 ng/L of
EE2 (E), and (F) development of connective tissue exposed to 522 ng/L of EE2. Arrowhead: leucophore,a male specific pigment. CT, abnormal connective tissues; Oc, oocytes; Sz, spermatozoa. Bar = 1 0 mm
(A-C) and 100μm (D-F).

viscera around the liver (Fig. 5‑1). One male died inconstant and without significant differences amongthe 47 8 ng/L treatment group, but without lesions

groups ( =0 455). At the end of exposure, the GFP

or abnormal pathology. None of the control fish diedfluorescent area of specimens in only the 522 ng/Lor developed any lesions.

treatment group decreased by half and therefore
were significantly different from the controls

5 3 1 2 Changes in GFP fluorescent area between( =0 002) (Table 5‑2 and Fig. 5‑3B). Significant

the initiation and the end of EE2 exposure

correlations between fluorescent area and gonad

Table 5‑2 shows the changes and ratio in GFP

weight were detected in EE2-treated and control

fluorescent area in the individuals exposed to EE2. In

males (R=0 76, <0 01) and females (R=0 70, <0 01)

males, average GFP-fluorescent area at the initiation

at the end of exposure (Fig. 5‑4).

of exposure was constant, with no significant
differences among treatment groups ( =0 805).

5 3 1 3 GSI and HSI of fish exposed to EE2

However, by the end of exposure, GFP-fluorescent

The GSI of male medaka showed a significant

area in the >̲216 ng/L treatment groups had

decrease in the 216 ng/L and 522 ng/L ( <0 001,

declined to approximately 30% of the size at theboth) treatment groups, as compared with theinitiation of exposure ( <0 001), with a simultaneouscontrol group (Table 5‑3a). In contrast, the HSI

decrease in intensity of GFP fluorescence (Fig. 5‑2C).

of male medaka treated with 522 ng/L EE2 for 4

In contrast, no significant changes were observed

weeks was significantly higher than the controls

in both control and <̲ 94 8 ng/L treatment groups.

( =0 00974), but not significant in the <̲216 ng/L(Table 5‑2).

treatment groups. In female fish, there was a

The same trend was observed in females (Tablesignificant difference in GSI between control and

5‑2). Average GFP fluorescent area at initiation was

522 ng/L groups ( =0 013), but not in HSI ( =0 379)

Takeshi HANO

Fig. 5‑3. Green fluorescent protein (GFP) images from the right lateral aspectof the female fish (A,B) and transverse sections of ovaries at the end of 4
week 17α-ethinylestradiol (EE2) exposure period stained with hematoxylin
and eosin (C,D). (A) GFP image of a 0 ng/L(control) ovary and (B) regressedovary exposed to 522 ng/L of EE2. Histological section of (C) control ovary and(D) regressed ovary exposed to 522 ng/L of EE2. Oc, oocytes; CT, abnormal
connective tissues. Bar = 1 0 mm (A-D).

Fig. 5‑4. Correlation between green fluorescent protein (GFP) fluorescent area and gonad weight in males(A) and females (B) for all treatment groups. Significant relationships between them were observed in males(R=0 76, <0 01) and in females (R=0 70, <0 01).

(Table 5‑3a).

all EE 2 treatment groups (Table 5‑3a and Fig.
5‑2E). In the testis-ova gonads of the 47 8 and

5 3 1 4 Histological observation of the gonad at theend of exposure
Testis-ova were observed in male medaka from

94 8 ng/L treatment groups, spermatocytes andspermatids could still be identified, indicating
active spermatogenesis. However, in the >̲216 ng/L

Evaluation of the effects of chemicals using transgenic fish
Table 5‑3. Gonadosomatic index (GSI), hepatosomatic index (HSI) and results of histology in gonad of adultmedaka at the end of direct exposure (Table5-3a) and depuration experiment (Table5-3b)

treatment groups, few oocytes were observed insuch as lightened body color, swollen abdomen andthe testis and almost the whole area in the testis

subcutaneous hemorrhage (data not shown). Oneof each specimen was composed of abnormally

male each also died in the control and 47 8 ng/L

developed connective tissues, with only a few

treatment groups, in the fifth and ninth weeks,spermatozoa and spermatocytes present (Fig. 5‑2F).

respectively. After transfer to clean water, surviving

In ovaries, histological abnormalities were not foundspecimens seemed to recover well and appear

in control and <̲216 ng/L treatment groups (Fig.

normal.

5‑3C). However, in the 522 ng/L treatment, many
previtellogenic oocytes and abnormal connective

5 3 2 2 Changes in GFP fluorescent area during thetissue were observed in the ovary of all specimens,

EE2 exposure phase and clean water phaseindicating a regressed condition of the ovaries (Fig.

In the control and <̲ 85 8 ng/L treatment groups,

5‑3D).

the GFP fluorescent area was not significant
between the initial and the final value (Fig. 5‑5). In

5 3 2 Waterborne exposure followed by depuration

the third week, the GFP-fluorescent area in the 215
and 473 ng/L treatment groups had decreased to

5 3 2 1 Mortality and abnormal appearance in

29% and 19% of that in week 0, respectively, as did

medaka during the experimental period

the intensity of fluorescence (Fig. 5‑5, 5‑6B). Due to

Two weeks after the initial exposure, the 215

a lack of differences in areas for certain groups ofand 473 ng/L treatment groups had 2 and 1 deaddose levels, values for GFP-fluorescent area weremales, respectively. At three weeks, 4 males diedgrouped into G1 (control, 43 7, and 85 8 ng/L) and G2

in the 473 ng/L treatment group. Dead specimens(215 and 473 ng/L) for analysis (Fig. 5‑5). At week

displayed the characteristic antemortem symptoms

3, the area of GFP fluorescence in the G2 group had

Takeshi HANO

Fig. 5‑5. Changes in average green fluorescent protein (GFP) fluorescent area in the same individuals during
the experimental period. Values for GFP-fluorescent area were grouped into G1 (control, 43 7, and 85 8 ng/L)
and G2 (215 and 473 ng/L) for analysis (see Results for details). * denotes significant difference from G1groupat <0 05.

significantly decreased to 23 7% of G1 ( =0 033).

o b s e r v e d r e c o v e r y o f G F P - f l u o r e s c e n t area

These reductions in area were associated with a(Table 5‑3b), as significant differences amonghigh prevalence of connective tissue in the testis,the treatment groups disappeared ( =0 930 anda condition that was expected to be irreparable.

=0 10 for GSI and HSI, respectively). The final

Interestingly, the area of GFP fluorescence inhistological analysis also demonstrated a substantial

testis began to restitute its size soon after the EE2

reconstitution of the testis (Table 5‑3b). Males

exposure ceased. There were no differences infrom the 43 7 ng/L treatment group had no

GFP-fluorescent area among treatment groups by

testis-ova and normal-appearing testis with active

the end of the experiment (9 weeks) (Fig. 5‑5, 5‑6C).

spermatogenesis. In the >̲85 8 ng/L treatment

As a result of second order spline function modelgroups, some individuals still had testis-ova;

selection, we believe the best model for the area of

however, the occupancy of oocytes in the testis

GFP fluorescence is described in Fig. 5‑5.

had obviously declined. Moreover, not only had
abnormal connective tissues completely disappeared

5 3 2 3 GSI and HSI of fish, and comparison within the 215 and 473 ng/L treatment groups, but

histological observation of the gonad after

active spermatogenesis was evident, with manytransfer of exposed fish to clean water.

spermatocytes and spermatozoa observed (Fig.

GSI and HSI of exposed male fish after theirtransfer to clean water clearly reflected the5‑6D).

Evaluation of the effects of chemicals using transgenic fish

Fig. 5‑6. Chronological changes of green fluorescent protein (GFP) images in the same individuals
from the right lateral aspect of the male fish exposed to 522 ng/L of 17α-ethinylestradiol (EE2) for 3
weeks followed by culture in clean water for 6 weeks (A-C) and transverse sections of testis at theend of experiment stained with hematoxylin and eosin (D). GFP image of a male fish at the initiationof the exposure (0 wk., A), at the end of EE2 exposure (3rd week.,B) and after the transfer to cleanwater for 6 weeks (9th week., C). Histological section of reconstituted spermatogenesis in the testis(D). Arrow = leucophore, a male specific pigment. Bar = 1 0 mm (A-C) and 500μm (D).

5 4 Discussion

the anesthesia. Mortality in the control and 47.8

Present study clearly showed that observation ofng/L treatment groups may also be attributed tomatured gonad by GFP fluorescence could monitor

anesthesia in ice-water. An alternative anesthetizing

the gonadal changes adversely affected by EE2 andmethod that is less stressful to the specimens is

the recovery of testis after the exposed males arenecessary to be developed so that they can enduretransferred to clean water.

frequent observation.

EE 2 caused pathologic abnormalities such

Significant reductions were observed in theas lightened body color, swollen abdomen andgonad s GFP-fluorescent area and GSI of adult males

subcutaneous hemorrhage, and finally leading toin the 216 and 522 ng/L, and females in the 522 ng/L

lethality. Transparent body of this strain made ittreatment exposed to EE2 for 4 weeks. Development

much easier to observe these abnormal appearances.

of abnormal connective tissues in the testis induced

Significantly increased HSI in males might beby estrogens might be ascribed to the inhibition

associated with abnormal vitellogenin (Vtg) synthesisof spermatogenesis at the concentrations in whichin the liver (Sekireproduction failed (Seki2002; Kang

2002). Seki

2002). Greater(2002) suggested that excessive Vtg levels inconcentrations of E2 also resulted in preponderance

males exposed to estrogens may cause lethal andof primary follicles and in the presence of several

sublethal toxicities.

atretic follicles in fathead minnows (Miles-Richardson

The increase in mortality in depuration

1999). Scholz and Gutzeit (2000) reported aexperiment might be associated to the more

reduced GSI in female medaka exposed to 10 and

frequent anesthetizing in ice-water, that was

100 ng EE2/L for 6 weeks. Such lesions also mightnecessary to observe the GFP-fluorescent in living

have been caused by an alteration or disruption of

medaka. In the 473 ng/L treatment group, 3 of 5

the hypothalamic-pituitary axis with the subsequent

dead specimens showed pathologic abnormalities

suppression of follicle stimulating hormone (FSH)

and unfortunately died in the third week, just afterand luteinizing horumone (LH) (Miles-Richardson

Takeshi HANO
1999). In the present study, a sublethal EE2

exposure to estrogens have been conducted atconcentration in which abnormal connective tissue

the early life stages or juvenile fish during sex

and a significant reduction of GSI was induced,

determination. The functional and morphologicalcorresponded with a significant reduction in therecovery of the gonad was observed in

GFP-fluorescent area and average growth rate.

hermaphrodite species, zebrafish (These results confirm that the GFP-fluorescentFenskearea of the gonad does serve to indicate thegonochorist species, medaka (Sekihistological conditions of test specimens exposed tofindings and present study indicated that the ability

reproduction-impairing concentrations of toxicant.

to recover may depend on time, duration, species

Additionally, there were significant relationships

and concentration of estrogen exposure.

;2005), but not in differentiated2003). Thesefound between gonad weight and the area ofPossible mechanisms for the histological

GFP fluorescence in both sexes (Fig. 5‑4). This

reconstitution of exposed fish testis after transfer to

implies that there is a possibility of predicting theclean water might be explained by the contribution

gonad weight by only measuring the area of GFPof Leydig cells in testis. Yamamoto (1953)

fluorescence, which might obviate the need fordemonstrated the sexual biopotentiality of germ cells

dissecting specimens of this test organism.

in developing gonads after sexual differentiation

Adverse effects of EE 2 on the female gonad

persists. Germ cells differentiate into spermatocytes

were also observed. In the 522 ng/L treatment,

under the influence of androgen, which is typicallythe fluorescent area of atrophied ovary wassynthesized in the testis Leydig cells (Miura andsignificantly smaller than the untreated control and

Miura, 2001). During direct exposure experiment

the intensity of GFP fluorescence was quite strong

and the exposure portion of depuration experiment,

because the atrophied ovary was mostly composedand even among testis almost completely filled withof previtellogenic oocytes (Fig. 5‑3). Oocyte size or

abnormally developed connective tissues, the Leydig

intensity of GFP fluorescence facilitated detection

cells remained present. Counting the Leydig cellof the effects of EE 2 in females. This regressed

numbers and morphological examinations were notcondition of the ovary likely arose from compromised

conducted in this study, because their small size (<5ovarian development (Scholz and Gutzeit, 2000),μm) contributed to difficulties in their observationas EE2 might exert ovary-specific toxicity and/or

and identification under the light microscope.

interfere with the release of gonadotropins, which

However, Cavaco

have been shown to stimulate ovarian development

not induce morphological changes in Leydig cells(Chan, 1976).

in the juvenile African catfish ((1999) reported that E2 did).

After the removal of exposure to EE2, continuous

It is likely that androgen synthesized in the Leydig

observation of the gonad for GFP fluorescence

cells after the transfer of exposed fish to clean

revealed that there was a rapid reconstitution

water might play an important role in restoringof the normal testis tissue structure and somespermatogenesis.

spermatogenic ability, indicating that the effects

We have further elucidated the atrophied andof EE 2 on adult testis are reversible. Although

regressed condition of EE 2 -exposed testis andtestis-ova were still present in some specimens, their

its reconstitution after transfer from different

incidence was apparently lower than with directconcentrations of EE 2 solution to clean water.

exposure experiment. Similar results had beenThis fact implies that the testis might indeed bereported by Shibata and Hamaguchi (1988), and ourreparable even after pathological changes occurredfindings indicated that, EE2 influences spermatogonia

at the tissue level in response to the exposure

to develop into oocytes; however, when animals

with EE 2. Fish and other aquatic organisms are

were allowed to recover from EE 2 exposure, theinhabitants of rivers, estuaries, and bays, where

inhibition of spermatogenesis ceased and these

they may receive a large amount of industrial

oocytes disappeared.

and domestic sewage effluent. Their reproductive

Some studies on the recovery of fish afterorgans would be impaired by exposure to different

Evaluation of the effects of chemicals using transgenic fishlevels of environmental estrogens discharged by

0 to 15 days posthatch (dph), by measuring thethe effluent into their aquatic environment. Gillarea of GFP fluorescence in theME(2002) reported abnormal hypertrophystrain. Germ cell number and GFP area show anof connective tissues in testicular structure in wild

approximately 10-fold increase in female gonads

male flounders that were collected from the lower

when compared with the germ cell number andTyne estuary in northeast England, a heavily

GFP area in male gonads in 10 dph juveniles,

impacted site known to contain high levels of

which thus facilitates sexing of very young fry as

endocrine disrupting chemicals (EDCs). Hashimoto

early as 10 dph, as indicated by the appearance of(2000) also reported that some male flounders

GFP-fluorescent germ cells. Identification of germ(

/STII-YI) collected from Tokyo

cell number by either calculating GFP-fluorescent

Bay in Japan had testes containing small numbers

germ cells or measuring the GFP area is lessof oocytes. Our experimental findings indicate thatlaborious and time consuming than conventional

these observations may be explained by exposure tohistological techniques. Therefore, we set abnormal

estrogenic compounds. Our present study suggests

germ cell proliferation as a target endpoint, which isthat some males that show testicular lesions after

critical to organisms and may induce sex reversal.

exposure to estrogens would be able to regain their

In the EE 2 exposure experiment (chapter 3),

reproductive functions after cessation of exposure to

we quantitatively analyzed GFP fluorescenceenvironmental estrogens.

and detected abnormally enhanced germ cell

In summary, we have visualized the impairment

proliferation by visualizing GFP-positive germ cells.

to both gonads and the recovery of testis byThus, theGFP fluorescence, through the exposure and thecell proliferative activity is targeted, should haveremoval of EE2 in mature/STII-YI strain.

a potential for application in the detection of the

Observation of the gonad with GFP fluorescenceeffects of EDCs on the gonad. However, a drawback

enabled us to detect the regressed condition ofof this model to determine the target endpoint is

the gonad caused by EE 2 in living individuals.

less sensitive to chemicals than the other establishedFurthermore, continuous observation of the testis

transgenic lines. As we mentioned in chapter 3, thewith GFP fluorescence throughout the exposure

lowest observable effective concentration (LOEC)

and depuration period permitted the tracing ofof 17α-ethinylestradiol (EE 2) for enhancing germchronological changes from atrophy to restitution ofcell proliferation was 158 ng/L, whereas that in

the testis

the GFP transgenic medaka (, in the same individuals. This is the/STII-YI strain, in which germ) linefirst report of the effects of EDCs on the gonad inunder the medaka vitellogenin1 (mvtg1) promoter

a living model using mature transgenic organisms.

was 50 ng/L (Zeng

Overall, our findings showed that this strain could befurther established a highly sensitive transgenic

applied to facilitated screening fish model to detectzebrafish (the adverse effects on the gonad exposed estrogenic

response element zebrafish vitellogenin 1) thatchemicals.

showed GFP expression with as low as 0 1 ng/L

2005). Chen

(2010)) line (ere-zvtg1 gfp; estrogen

EE2. They suggested that the high sensitivity may
Chapter 6 General Discussion

be due to the addition of an estrogen response
element (ERE) upstream of the vitellogenin 1 (vtg1)

This study was performed to evaluate the effects
of endocrine disrupting chemicals (EDCs) in the

promoter. To improve the sensitivity to EDCs in
the/STII-YI strain, it might be sufficient/STII-YI strain by monitoring gonad

to establish transgenic fish under the control of a 2

development with green fluorescent protein (GFP)

or more different inducible promoters, for example,

fluorescence in a living organism.

both

In chapter 2, we described a novel method that(critical) and ere-vtg1 (sensitive).

In chapter 3, we described how we performed

was established to determine the germ cell number

exposure of chemicals with the nanoinjection

before the onset of sexual differentiation, at about

technique. Nanoinjection is an alternative exposure

Takeshi HANO
method that can directly expose developing

chemicals in the aquatic environment. Although

embryos to chemicals instead of exposing adult

NP did not affect either germ-cell proliferation orfish to waterborne chemicals; in this technique,sexual differentiation, the study of NP nanoinjection

the chemicals deposit naturally into the oocytes

in chapter 4 is of great importance in that NP(Walker

1996). In the environment, lipophilic

kinetics in the aquatic environment was elucidated

contaminants might readily bioaccumulate in fish andbetween maternally transferred NP doses fromtranslocate from adult female body stores into the

exposed breeding females and that of nanoinjection.

eggs during oocyte maturation (Niimi, 1983; Miller,

In addition, failure of NP to impair either germ

1993). Bioaccumulation of these chemicals in adultcell proliferation or sexual differentiation indicatedfish may have significant effects on the developmentthat NP were metabolized or excreted faster thanand survival of their offspring because early-stage

expected, and that maternally transferred NP alone

embryos are more sensitive than adults are tois not enough to induce abnormalities in germ cellchemical toxicants. Thus, the presence of persistent,

proliferation or sexual differentiation.

bioaccumulative contaminants in the environment

With regard to EE2, bothand waterborne

may pose a risk to the early-stage survival of fish

exposure of EE2 enhanced germ cell proliferation in

and ultimately limit their recruitment into adult

10 dph juveniles; however, germ cell proliferative

populations. The present study clearly demonstrated

activity showed dissimilar response to EE2 at 10that nanoinjection of EE 2 impaired survival anddph in the 2 experimental designs. Bioconcentration

normal hatching and also induced either abnormal

factor was used as an estimate of theenhancement of germ cell proliferation in 10 dph

bioaccumulation potential of EE2. The amount of EE2

XY fish or sex reversal in 100 dph XY medaka.

intake via waterborne exposure (0 31‑0 35 ng/fish)

Nanoinjection of nonylphenol (NP) and tributyltin

was predicted to be much lower than the lowest(TBT) into medaka embryos also impaired survival

observable effective dose (LOED) to induce germ celland hatching (Hanoproliferation in the

Nassef2007 and Chapter 4).

exposure experiment (0 5(2010) nanoinjected triclosan, diclofenac,

ng/egg). To the best of our knowledge, this is theand cabamazepine, which are compounds found in

first report to investigate the dissimilar responses

pharmaceuticals and personal-care products, into

to EDCs on germ cell proliferation observed in 2

medaka embryos and detected toxicity-induced

different exposure designs.

responses during embryonic development, including

The comparison of the concentration of EDCs

decreased hatchability and failed swim-up.

between maternally transferred into eggs and that

Furthermore, nanoinjection has been proposed asof nanoinjected EDCs has been studied in organotinsa useful exposure method to elucidate the joint

such as TBT and triphenyltin (TPT). Hano

toxicity of chemicals（Nassef

in preparation).

(2007) observed that nanoinjection of TBT induced

Talykina (2003) also nanoinjected genotoxins (TBT

embryologic abnormalities, including ocular defects

and mixtures of polychlorinated naphthalene)

and hemorrhage, and further indicated that the

into medaka embryos and applied the piscine

LOED for inducing decreased hatchability in medaka

micronuclear test to detect erythrocyte micronuclei.

(0.16 ng/egg) was around the concentration levels

Ekman (2003) presented a new approach toin the eggs of marine fish [0 091 ng/egg (Takahashi

nanoinjection by using it as a tool to mimic the1997) and 0 431 ng/egg (Suzuki

1992)].

vertical transmission of pathogens in rainbow

Furthermore, this value was in agreement with thetrout. With this approach, nanoinjection provides a

TBT concentrations required to induce decreased

large amount of information for environmental risk

hatchability in the eggs that were spawned fromassessments, and future investigations based on new

medaka pairs dietary exposed to dietary TBT

concepts will broaden the usefulness and potential of(>0 123 ng/egg; Nakayama

the nanoinjection method.

(2009) nanoinjected environmentally relevant levels

The present study offers an additional
consideration with regard to the behavior of2005). Huof TPT into the eggs of Siberian sturgeon () and observed both ocular and morphologicalEvaluation of the effects of chemicals using transgenic fish
malformations. These observations indicated thatof the testis in the same organisms. These results

the incidence of deformities in fish larvae is ansuggest that some males that show testicular

indicator of the overall population-level effects oflesions after exposure to estrogens would be able toorganotin on aquatic organisms, because EDCs,

regain their reproductive functions after cessation

including organotin, at environmentally relevantof exposure to environmental estrogens. This is theconcentrations can result in a significant decrease

first report to analyze the effects of EDCs on thein both the quality and quantity of eggs and the

gonad by monitoring chronological changes in the

spawning frequency of fish.

same and living model by using mature transgenic

In chapter 3, we discussed how the identification

organisms. Furthermore, monitoring GFP-fluorescentof leucophore and sex-linked 1 marker played an

gonads in the same specimen could be a promising

important role in the confirmation of abnormal

noninvasive approach to identify the effects ofsexual differentiation of the gonad caused by EE2 atchemicals on the gonad. For example, in depuration

10, 35 and 100 dph. In previous studies, prior to theexperiment of chapter 5, 30 males (6 males per

identification of genotypic sex markers, abnormal

treatment multiplied by 5 treatments) were

sexual differentiation had been assessed by the

prepared to monitor gonadal changes for 9 weeks

sex ratios of males to females between control

periodically, whereas 222 individuals were calculated

and chemical treatment groups. The sex of eachto be necessary if we were to use nontransgenic

individual was identified by gross examination

medaka for the same experimental design. Thisof secondary sex characteristics and by gonad

indicated that the use of thehistology. However, this traditional assessment has

strain reduced by 86% the need for animal sacrifice

the disadvantage of unavoidable uncertainty because

as well as the required time and labor.

/STII-YI

the sex ratios of control groups are unstable andRecently, a transgenic fish model has been

it is necessary to lengthen the exposure duration

generated for researches on evolution, organ

until specimens show secondary characteristics, fordevelopment, diseases, and aquaculture. Punnamoottil

example, 60 days or longer from hatching; it also(2010) used GFP reporter gene constructs indemands additional laborious and time-consuming

zebrafish to identify noncoding sequences conserved

work. Identification of sex markers allows foraround thedetecting the genotypic sex of each individual at any

anterior-posterior axis patterning and have a major

stage of development and provides a more practicalrole in hindbrain development, and characterized

choice; for example, it shortens the exposure

genes, which are key regulators of-regulatory sequences in the Hox clusters as

duration or requires less labor intensive work. In

expression enhancers ofgenes. The authors

addition to these sex markers, detection offurther elucidated that

-regulatory sequences(

are conserved during evolution, but their functions

anddomain gene on the medaka

Y chromosome) would be helpful to rapidly detect

have significantly evolved. McDermottabnormal sexual differentiation.

labeled hair cells with GFP in the inner ear andIn chapter 5, we have visualized the impairment(2010)

lateral line of the zebrafish and investigated auralof mature gonads and the recovery of testis indevelopment. Hu(2010) produced anotherthe same individuals through the exposure andzebrafish transgenic line in which thesubsequent removal of EE2. In normal gonads of 60

driven by the keratin 5 gene promoter is expresseddph medaka adults, the testis can be distinguishedin the tongue and may be used for future studies

from the ovary with the naked eye. Observation of

on oral development and carcinogenesis. In the field

the gonads with GFP fluorescence enabled detectionof aquaculture, Kobayashi

of the EE2-induced regression of the gonads in living

transgenic Nile tilapia (organisms. Furthermore, continuous observation

overexpressed the growth hormone gene throughoutof the testis with GFP fluorescence throughout thetheir bodies and that displayed high food-conversion

exposure and depuration periods allowed the tracing

efficiency, shorter rearing period, and reducedof chronological changes from atrophy to restitution

ammonia excretion than nontransgenic fish. The

gene(2007) produced) thatTakeshi HANO
present proposed approach has the potential to

XY gonad (Matsuda

reduce the amount of nitrogen pollution caused

mechanisms underlying2007). However, theby farmed fish and to further lessen the pollution

factors involved in sexual differentiation remain to

problem in the aquatic environment.

be determined. Analysis of functional

function and theproteinFurthermore, a biomonitoring system to detectshould provide further insights into the mechanisms

xenobiotic toxicants in the environment has beenof germ cell proliferation and induction of germdeveloped by inducing thecell proliferation by exogenous estrogens in malegene under thecontrol of the target gene promoter, and it can

medaka.

be a good candidate for detecting the modulating

In summary, the present study clearly showed

effects of chemicals in living organisms. In zebrafish,that monitoring germ cells with GFP fluorescence

Mattingly

by using the(2001) reported a GFP transgenic/STII-YI strain allows

line using the human CYP1A1 promoter, which is

rapid detection of the adverse effects of EE 2, acapable of responding to 10 nM TCDD (2 3 7 8-te

representative estrogenic chemical, on sexual

tra-chlorodibenzo- -dioxin). Blechinger(2002)

differentiation or mature gonads in intact living

used a heat-shock-inducible hsp70:GFP transgenic

fish. This result indicated that the transgenic line

line to detect cadmium toxicity and observed

test model can provide a more practical choice and

GFP expression in the gill, olfactory system, andhas the combined advantages of bothliver. In an experiment on medaka, Kurauchi

andassays: an on-line and on-site evaluation for(2005) produced a transgenic line harboring

the effect of EDCs. In other words, it is directly

gene driven by the regulatory region ofobservable, easy to analyze, rapid, and accurate.

choriogenin H gene. They observed GFP expression

Further studies to investigate a variety of chemicalsof the liver in the male medaka in response towill elucidate the usefulness of this transgenic

estrogens and showed that the transgenic line wasstrain as a facilitated screening fish model to detectsufficient for application as an alternative model in

EDC-induced abnormalities in sexual differentiation.

monitoring environmental water samples. Kinoshita

Although thethe/STII-YI strain was(2009) later produced transgenic medaka

designed for monitoring germ cells only, the same

whose oocytes were labeled with GFP using theconcept and technology would also be applicableregulatory region of thegene, which was

to other environmental pollutants by replacing the

expected to be abundantly expressed in the oocytes

inducible promoters. In the near future, it is possible(Kanamori, 2000). After treatment with 830 ng/L

to develop multicolor transgenic fish by using 2

estradiol-benzoate, testis-ova were also labeled withor more fluorescent protein reporter genes under

GFP in the matured testes in this transgenic medaka

different inducible promoters, and thus, the samestrain. Overall, the development of transgenic fishtransgenic fish can be used for monitoring different

will help in more efficiently meeting the demand fortypes of environmental pollutants.

human life and will greatly benefit various fields ofReferenceresearch.
In addition to the proposed toxicological model, the/STII-YI strain has potential to be used

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Increased kidney, liver, and testicular cell deathvaluable advices and corrections.
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Dr. Hisashi Hashimoto 10 for continuous advices

mixture extracted from Lake Michigan lake

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Dr. Masato Kinoshita12 and Dr. Minoru Tanaka13

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Dr. Takashi Yanagawa14 and Dr. Tetsuji Ohyama15,

Yamamoto M. 1963. Electron microscopy of fishfor their valuable and helpful advices.
Mr. Tsuruda 16 for his guidance and friendly

development. II. Occyte-follicle cell relationshipand formation of chorion inassistance.

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Finally, laboratory members11, 17 and my family for

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their dedicated help during my study period.

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1: Associate Professor of Kyushu University

Yokota H, Seki M, Maeda M, Oshima Y, Tadokoro H,

2: Associate Professor of Kyushu University,

Honjo T, Kobayashi K. 2001. Life-cycle toxicity

3: Associate Professor of Kumamoto Universityof 4-nonylphenol to medaka (4: Emeritus Professor of Kyushu University,

5: Associate Professor of Kyushu University,

Yokota H, Tsuruda Y, Maeda M, Oshima Y,

6: Assistant Professor of Kyushu University,

Tadokoro H, Nakazono A, Honjo T, Kobayashi

7:Associate Professor of Kyushu University

K. 2000. Effect of Bisphenol-A on the early life8

: Emeritus Professor of Nagoya University,

stage in Japanese medaka (9: Professor of Nagoya University, 10 : Assistant).

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).

19: 1925‑1930.
Zeng Z, Tong SY, Lam SH, Gong Z. 2005.

Professor of Nagoya University, 11: Laboratory ofFreshwater Fish Stocks, Nagoya University

Takeshi HANO
12
13

: Assistant Professor of Kyoto University,

Biology
14

This work was supported by a grant from the15: Professor of Kurume University, : Oita University

16

Kyushu University: Associate Professor of National Institute for Basic: Technical assistant of Kyushu University

17: Laboratory of Marine Environmental Science,

Research for Millennium Project by the Ministry of
Education, Culture, Sports, Science, and Technologyof Japan.