in vitro assays with rock shell rxr · in vitro assays with rock shell rxr 0510 [3h] - 9-cis ra...

In Vitro Assays with Rock Shell RXR

0 5 10[3H] - 9-cis RA (nM)

Spec

ific

bind

ing

(dpm

x 10

-3)

0 0.2 0.4 0.6 0.8 1bound (nM)

Bou

nd/fr

ee Kd = 15.2 nM

-10 -9 -8 -7 -6 -5 -4log dose (M)

Bin

ding

rate

(%)

0

40

80

120

160

0

0.04

0.08

0.12

0.16

9-cis RAATRATBTTPT

0

20

40

60

80

100

Nishikawa, J., Mamiya, S., Kanayama, T., Nishikawa, T., Shiraishi, F., Horiguchi, T.: Environ. Sci & Technol. 38: 6271-6276, 2004.

A: Binding assayB: Scatchard analysisC: Competition assay

A

B

C

Effects of 9CRA to the Development of Imposex in Thais clavigera (1)

Incidence of Imposex

010

2030

4050

6070

8090

100

Control RA TPT

Inci

denc

e (%

)

**

***


9CRA


Female Penis Length

0

1

2

3

4

5

6

Control RA TPT

Peni

s Le

ngth

(mm

)

*****


9CRA


ov

cg

ov

cg p

ov

cg p

Control 9CRA TPTPL=6.06 mm PL=6.50 mm



Incidence of Imposex

0

10

20

30

40

50

60

70

80

90

100

Control RA 0.1ppm RA 1ppm RA 5ppm TBT TPT

Inci

denc

e (%

)

* *** *** ***

******* **

Horiguchi, T., Ohta, Y., Nishikawa, T., Shiraishi, F., Shiraishi, H., Morita, M.: In press Cell Biol. Toxicol.

Penis and vas deferens developed in female T. clavigera which received 9CRA injection (PL: 7.00 mm) Horiguchi, T., Ohta, Y., Nishikawa, T., Shiraishi, F., Shiraishi, H., Morita, M., In press CBTO.

p

vdp

vdHistology of Penis Developed in Female T. clavigera

0

0.5

1

1.5

2

2.5

3

3.5

RXR

/ 16

S rR

NA

* **

(n=3, *; p<0.05, **; p<0.01)

ctenidium

ctenidium

ctenidium

testis

ovary

ovary

digestiv

e glan

d

digestiv

e glan

d

digestiv

e glan

dpen

is

penis

forming ar

eapen

is

head gan

glia

head gan

glia

head gan

glia

male female imposex-exhibiting femaleRXR gene expression in various tissues of T. clavigera

Horiguchi et al., Aquat. Toxicol. 84: 379-388, 2007.

Vas deferens (Male) Vas deferens (Imposex)

Penis (Imposex) Ganglion (Female)Horiguchi et al., Aquat. Toxicol. 84: 379-388, 2007.

Penis forming area

Ovary

Head Ganglia

Penis

differentiationand

proliferation

ovarianspermatogenesis

?

?

?

Other tissue(s)

RXR

RXR RXR

RXR

RXR

Chemical messengers responsible for inducingspermatogenesis in the ovary might be secreted from other tissue(s) whereRXR is expressed.

Chemical messengers responsible for spermatogenesis in the ovary or the development of a penis (as well as a vas deferens) might be secreted from the head ganglia, because high concentrations of organotins were accumulated in, and RXRwas expressed in, the head ganglia of wild imposex rock shells.

TBT and/orTPT

It is uncertain whether RXR plays important roles in ovarian spermatogenesis caused by exposure to organotincompounds, because there were only trace amounts of RXR gene expression and protein production in the ovariesof normal and slightly affected imposex-exhibiting females.

?

Horiguchi et al., Aquat. Toxicol. 84: 379-388, 2007.

Speculative mechanism of induction of imposex in gastropods

ConclusionsPhysiological regulatory system of reproduction Physiological regulatory system of reproduction may be different in gastropods, compared to that of may be different in gastropods, compared to that of vertebrates.vertebrates.Retinoid X receptor (RXR) has an important role in Retinoid X receptor (RXR) has an important role in the development of the development of imposeximposex caused by TBT and/or caused by TBT and/or TPT in gastropods.TPT in gastropods.

Future StudiesNatural ligand for RXR in Thais clavigeraHeterodimer, homodimer or homotetramer?Target gene(s) of RXR in T. clavigeraGenes and their functions in T. clavigera

AcknowledgementCollaboration:Collaboration: Prof.Prof. EmerEmer. M. Shimizu, Prof. . M. Shimizu, Prof. EmerEmer. S. Yamazaki,. S. Yamazaki,

Prof. H.S. Prof. H.S. ChoCho, Prof. A. Okubo, Prof. E. Yoshimura, , Prof. A. Okubo, Prof. E. Yoshimura, Prof. O. Matsushima, Prof. F. Prof. O. Matsushima, Prof. F. MorishitaMorishita, Prof. T. Nagao,, Prof. T. Nagao,Prof. T. Iguchi, Prof. J. Nishikawa, Dr. Y. Prof. T. Iguchi, Prof. J. Nishikawa, Dr. Y. KatsuKatsu,,Dr. T. Imai, Mr. N. Dr. T. Imai, Mr. N. TakiguchiTakiguchi, Mr. M. , Mr. M. TeruiTerui,,Mr. A. Mr. A. KajikawaKajikawa, Mr. F. Hamada, , Mr. F. Hamada, Dr. H. Dr. H. ShiraishiShiraishi, Dr. F. , Dr. F. ShiraishiShiraishi, Dr. M. Morita, Dr. M. MoritaDr. T. Nishikawa, Dr. M. Lu, Dr. A. Dr. T. Nishikawa, Dr. M. Lu, Dr. A. TreunerTreuner,,Ms. A. Sugimoto, Ms. M. Shibuya,Ms. A. Sugimoto, Ms. M. Shibuya,Mr. T. Sakuma, Mr. S.W. Mr. T. Sakuma, Mr. S.W. SeolSeol & Mr. J.H. Lee& Mr. J.H. Lee

Assistance:Assistance: Mr. M. Kojima, Ms. M. Mr. M. Kojima, Ms. M. KayaKaya, Ms. M. Higashiyama,, Ms. M. Higashiyama,Ms. Y. Ms. Y. TezukaTezuka, Ms. K. Miyoshi, Ms. N. Hamada,, Ms. K. Miyoshi, Ms. N. Hamada,Ms. A. Ms. A. NabataNabata, Ms. T. Kinoshita, Ms. A. Honda,, Ms. T. Kinoshita, Ms. A. Honda,Ms. K. Ms. K. StockwellStockwell, Ms. S. , Ms. S. NagumoNagumo, Ms. Y. , Ms. Y. KinumiKinumi,,Mr. T. Matsuo, Mr. S. Mr. T. Matsuo, Mr. S. OchiaiOchiai, Mr. M. , Mr. M. OoyamaOoyama,,Mr. S. Hamada & Mr. A. TokugawaMr. S. Hamada & Mr. A. Tokugawa

Pictures of several shells were cited from Pictures of several shells were cited from ‘‘Gakken Illustrated Nature Gakken Illustrated Nature Encyclopedia THE MOLLUSKS OF JAPAN (SNAILS)Encyclopedia THE MOLLUSKS OF JAPAN (SNAILS)’’ ((HabeHabe, T. & , T. & OkutaniOkutani, T. eds.), Gakken, Tokyo, Japan, 294p. (1983), T. eds.), Gakken, Tokyo, Japan, 294p. (1983)

Funding

Ministry of the Environment, JapanMinistry of the Environment, JapanMinistry of Education, Culture, Sports, Ministry of Education, Culture, Sports, Science and Technology, JapanScience and Technology, JapanNihonseimeiNihonseimei--ZaidanZaidan Foundation, JapanFoundation, JapanNational Institute for Environmental Studies, National Institute for Environmental Studies, JapanJapan

in vitro assays with rock shell rxr · in vitro assays with rock shell rxr 0510 [3h] - 9-cis ra...

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