J. Biol. Macromol. 2 (3), 95-102 (2002)

Estradiol-induced expression of a protein in male medaka fish Oryzias latipes :Ⅰ.

Induction test for bisphenol A and tributyltin (ⅳ) chloride

Akiko Nochide, Mitsuru Sugiura, Masaomi Saka, and Masatake Ohnishi

Research Laboratory of Cellular Macromolecules, Graduate School of Agriculture,
The Prefecture University of Kyoto(1-5, Nakaragi-cho, Shimogamo, Sakyo-ward, Kyoto City, Kyoto 606-8522, Japan)
Received August 26, 2002, accepted September 10, 2002
A protein, here called protein X, was found in blood plasma and liver of male medaka fish Oryzias

latipes, which was kept in the natural water containing a female hormone 17β-estradiol.
Protein X, which is ordinary expressed in female fish by the hormone, is supposed to be vitellogenin, however, we do not identify what is protein X.
index of water-environmental quality examination.

Nevertheless, protein X will be useful for an
In this point of view, bisphenol A andtributyltin (ⅳ) chloride, which are suggested to be a kind of endocrine disruptors (hormonal
action-disturbing chemicals), were examined for expression of protein X in male medaka fish.
Bisphenol A was confirmed to little effect on the expression, whereas tributyltin chloride wassuggested to induce the expression under the experimental conditions employed.
Keywords
Medaka fish Oryzias latipes, estradiol-induced expression, bisphenol A, tributyltin (ⅳ) chloride,
Recently quality of natural water isscience and environmental technology.

Inatypical case of the most important and serious

fact, very intensive studies have been carriedenvironmental problems, on the other hand aout on mechanism of development, reprod-

fresh-water fish medaka (Oryzias latipes) isuction, and other biological functions withappointed to a class of VU, which means

some kinds of fishes including medaka [1-4].

vulnerable (extermination-feared) species.

Using electron microscopic observation

Quality of the natural water could be intim-

techniques mainly, it has been found that aately related with existence of the fish life.

protein is expressed by a female hormone 17β

On this viewpoint, cellular macromolecule--estradiol [5] and identified to be vitellogeninmechanism on fertilization of the fish is veryin medaka fish Oryzias latipes [6].

interesting theme for basic and practicalfindings are very interesting and suggestive forstudies in the field of molecular biologicalinvestigation of cellular macromolecule-

These

96

A. Nochide et al.

mechanism on the fertilization of the fish.

temperature 26 ℃, fed with a commercial

Thereafter, some important investigations on

fishfood twice a day [18].

the points of mechanism, gene, and bio-

17β-estradiol, tributyltin (ⅳ) chloride, bis-

response on the hormone-induced expression ofphenol A, dimethyl sulfoxide (DMSO), bovine

the protein vitellogenin have been intensively

serum albumin (BSA), and other chemicals, of

carried out with adult female and maleguaranteed grade, were purchased frommedaka fishes [7-10] and other fishes [11-12].

Nacalai Tesque Inc., Kyoto and used without

It should be considered to one of the mostfurther purification.

A female hormone

interesting findings that vitellogenin has been
expressed in male zebrafish (Danio rerio) by
induction with the female hormone estradiol[12].

Thus, estradiol-induced expression of

Methods
For examination of the estradiol-induced
expression of a protein, adult male fishes

the protein vitellogenin can be effectively used

(usually 2 fishes) were kept in a 200 ml glass-for evaluation of endocrine disruptors

made beaker filled with the natural water(hormonal action- disturbing chemicals).

In

containing 17β-estradiol at concentration 1 tofact, some chemical substances have been

100 ppb under the controlled envir- onment.

examined for their effect on the endocrine

Estradiol, tributyltin (ⅳ) chloride anddisruption (hormonal action disturbance)bisphenol A were dissolved in 0.1% DMSO and[13-17].

diluted with water to the final concentrations

In this study, expression of a protein was
examined with male medaka fishes, which

employed.
Tail or head was cut off from fish body andwere kept in natural water containing theblood exuded was put into microtubes filled

female hormone 17β-estradiol, bisphenol A ,with a buffer prepared for protein extraction.

and tributyltin (ⅳ) chloride.

A microsyringe was employed to take out blood

The SDS- PAGE

chromatograms clearly indicate estradiol-from the fish body without head.

Supernatantinduced expression of the protein, which is

was obtained by centrifugation of blood at 3000

normally found in the female fishes.

rpm and 4 ℃ for 15 min, and employed forprotein analysis as a sample of blood plasma.

EXPERIMENTAL

In another way for sampling, liver took out

Materials

from fish body was put into microtubes filled

Medaka fishes were purchased fromwith the buffer and homogenized by a

Shimizu Suisan Co., Kyoto and usually kept inhomoginizer for microtubes.

the glass-made 57 liter aquarium (vessel) filled

obtained by centrifugation of the homogenate

with natural water (tap water, after leaving forat 15,000 rpm and 4 ℃ for 20 min, and used

2 days) under a controlled environment as

for protein analysis as the liver sample.

follows: light 14 hr, dark 10 hr, water

buffer prepared for protein extraction consists

Supernatant was

TheProtein expression in medaka

97

of 25 mM Tris-HCl, 150 mM NaCl, and 5 mM

M

1

2

3

4

EDTA, pH 7.2 [19].
Determination of protein quantity was
carried out using the Bradford method withbovine serum albumin as a standard.

200,000

The

SDS-PAGE chromatography and CBB116,250

staining were employed for analysis on the
97,400

estradiol-induced expression of protein.

45,000RESULTS AND DISCUSSION

Protein X expression in blood plasma of female
and male fishes
Medaka fishes were kept in natural water

31,000at 26 ℃ (bleeding state) and 4 ℃ (nonbleeding state) for 15days.

Blood plasma wereobtained from female and male medaka fish
bodies, and proteins in blood plasma were

Fig. 1.

separated using the SDS-PAGE techniques andplasma proteins, which are expressed in medaka

stained with CBB as the results are illustrated

kept in natural water.

in Fig. 1, where lanes 1 and 3 are for female,

Medaka fishes were kept in the natural water atand 2 and 4 are for male. The SDS-PAGE

26 ℃ (lanes 1 and 2) and 4 ℃ (lanes 3 and 4).

chromatogram shows some interesting protein

Lanes 1 and 3 are for female, lanes 2 and 4 are forbands, however there is a critical evidence at amale. M: Molecular weight protein standards;

protein band having MW 200,000, thus thismyosin:protein is called “protein X” in this paper.

phosphorylase: 97,400, serum albumin: 45,000Innatural water, it is concluded that protein X is

The SDS-PAGE chromatogram of blood

200,000,

β –galactosidase:

116,250,and ovalbumin: 31,000

expressed in female medaka fish but not
expressed in male.

PAGE techniques as the results are illustrated

Estradiol-induced expression of protein X inblood plasma of male fish
Male fishes were kept in the natural watercontaining a female hormone estradiol, of
which concentration in the water is 1ppb, for 1
to 5 days at 26 ℃.

Expression of protein X inblood plasma was examined using the SDS-

in Fig. 2, clearly showing that protein X, at
MW 200,000 in the PAGE chromatogram, isfound in male medaka fish, which was kept in
the presence of the hormone estradiol.

The

protein bands in the chromatogram are clear
and large (increase in intensity) together withkeeping day.

These results conclude that

protein X in male fish is expressed by the effect98

A. Nochide et al.

C

1

2

3

4

M

M

C

C

1

2

3

4

200,000
116,250
97,40045,00031,000Fig. 2.

Estradiol-induced expression of protein X

Fig. 3.

The SDS-PAGE chromatogram of bloodin blood plasma.

plasma proteins.

With 1 ppb 17β-estradiol, male fishes were kept

With 10ppt estradiol, male fishes were kept for 5

for 1, 2, 3 and 5 days (lanes 1, 2, 3 and 4,(lane 1) and 20 days (2). With 100ppt estradiol

respectively).

for 5 (3) and 10 days (4).

C: control, M; MW standard

proteins.

M; MW standard

proteins, C; control.

of the hormone estradiol.

illustrated in Fig. 4.

Estradiol-induced expression of protein X in

Estradiol-induced expression of protein X inblood plasma of male fish was examined at low

liver of male fishconcentration of the hormone, 10 and 100 ppt
for 5 to 20 days.

The results are shown in Fig.

The hormone estradiol-induced expression
was analyzed in liver of male fish using the

3, indicating that concentration of the hormone

SDS-PAGE techniques as the chromatogram is

100 ppt for 5 to 10 days and 10 ppt for 5 to 20

shown in Fig. 5.

days has no effect for the expression of protein

natural water containing the hormone in the

X in the male fish.

concentration of 1 ppb for 1, 2, and 5 days at

In the presence of 100 ppt, the estradiol-

26 ℃.

Fishes were kept in the

It is clearly shown that the hormone-

induced expression of protein X was confirmed

induced expression of protein X was confirmed

at keeping of the male fish for 15 days as

in liver of male fish within 2 days after keep
under the experimental conditions employed.

Protein expression in medaka

C

1

99

M

1
200,000M

116,250

97,40097,40045,00045,00031,000Estradiol-induced expression of protein X

3200,000

116,250

Fig. 4.

2

31,000Fig. 5. The SDS-PAGE chromatogram of proteinsin blood plasma.

in liver.

Male fishes were kept in the water with 100 ppt

Male fishes were kept in the water with 1 ppb 1717β-estradiol for 15 days (lane 1). C; control. M;

β-estradiol for 1, 2 and 5 days (lanes 1, 2 and 3,

MW standard proteins.

respectively), M; MW standard proteins.

Examination of protein X expression withbisphenol A and tributyltin chloride
Protein X expression in male medaka fish
was suggested to be a useful indicator for thewater-environmental quality examination.
Recently, a great of attention is gathered tosome chemicals; thus we selected two sample
chemicals, tributyltin (ⅳ) chloride and bisphenol A, for the test of protein X expression
here.

Male fishes were kept in the natural

water containing bisphenol A in the concentration of 1 ppm for 5 and 15 days and protein X
in blood plasma was analyzed using the SDSPAGE techniques.

The chromatogram is

illustrated in Fig. 6, showing that no band isfound for protein X under the experimental
conditions.

In this experiment, it will beconcluded that bisphenol A has not so muchinfluence on the protein X expression in fish.
Male medaka fishes were kept in thenatural water containing tributyltin chloride in
the concentration of 1 or 10 ppt for 7 days, andprotein X in blood plasma of the fish wasanalyzed using the same procedures as described above. As an example of the chromatograms are shown in Fig. 7, suggesting that
tributyltin chloride induced the expression ofprotein X under the experimental conditions
employed.

100

A. Nochide et al.

1

2

M

M
200,000
116,250
97,40045,00031,000Fig. 6.

The SDS-PAGE chromatogram of blood

C

1

2

200,000
116,250
97,40045,00031,000Fig. 7.

Tributyltin chloride-induced expression ofplasma proteins.

protein X in blood plasma.

Male fishes were kept in the water with 1 ppm

Male fishes ware kept with 10 ppt tributyltin

bisphenol A for 5 and 15 days (lanes 1 and 2,

chloride (lanes 1 and 2, respectively) for 7 days,

respectively), M; MW standard proteins.

M; MW standard proteins, C; control.

Tributyltin chloride is known to effect on

SDS-PAGE techniques and confirmed to havesome shellfishes and to convert female to male

MW 200,000, of which protein corresponds to[21-22].

In this study, tributyltin chloride wasvitellogenin, which is found ordinary in female

suggested to effect on the protein expression infish [6]. Thus, protein X could be supposed tomale medaka fish.

vitellogenin. In this stage of studies, it is not

However, it must be conf-

irmed that tributyltin chloride induces themade clear that protein X is vitellogenin,

expression of protein X in male medaka fishes

nevertheless, for the purpose of this research it

and effects or not on female medaka fishes.

is not essential to confirm what protein X is.

These are now under investigation intensively

However, identification of protein X is now

and will be reported soon.

under investigation using the procedures ofprotein-sequence analysis.

Characterization of protein X
Protein X produced by the hormoneinduced expression was observed using theProtein expression in medaka

101

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Communicated by Matsumoto Tadashi