development and validation of a novel in situ - usask.cas new/tompsett... · development and...

Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan

Development and validation of a novel in situ hybridization system to detect gene

expression and histological structure as biomarkers of chemical exposure in Japanese

medaka

Amber TompsettAquatic Toxicity Workshop

Halifax, Nova ScotiaOctober 3, 2007


Background• EDC’s hypothesized to elicit a wide variety of adverse effects:

- Promotion of hormone-dependent cancers - Reproductive tract disorders - Reduction in reproductive fitness

• Most screening for the effects of EDCs was limited to measuring direct effects- Receptor binding of steroid hormone receptors

• However, chemicals can cause both direct (receptor-mediated) and indirect effects through changes in signal transduction pathways.


Research Needs

• Methods are needed that:– Permit the analysis of multiple effects. – Can look at these effects simultaneously in a

number of tissues, including during critical windows of development

– Effects analyzed spatially


Model SystemWhole-animal tissue section in situ

hybridization


Fixed RNA in tissue

Probe-RNA hybrid in tissue

In situ hybridization

Antisense RNA probe




Fixed RNA in tissue

Fixed RNA in tissue


Sense RNA probe


Whole Animal In situ hybridization

• Compatible with histopathology and IHC methods

• Allows spatial analysis of gene expression

• Small samples are preferable

• Some drawbacks: time and labor intensive


CYP11A & B; CYP17; CYP19A; 3β-HSD; 11β-HSD; 17β-HSD; StAR; ERα & β; AR; GtHreceptors; Activin/Inhibin

Gonads

CYP11A & B; CYP17; 3β-HSD; 17β-HSD; StAR; AR; GtH receptors; Activin/InhibinAdrenals

GnRH receptors; GtH I & II; ERα & β; AR; Activin/InhibinPituitary

CYP19B; GnRHs; ERα & β; ARBrain

Primary Gene TargetsTissue

Gonads

LiverBrain

Pituitary

GnRH GRIF

Gonadotropins

Feedback

Sex SteroidsVitellogenin

Adrenals

Target Genes Along the HPG-Axis


Specific Objectives of this Study

To develop whole-animal tissue section ISH methods to examine the effects of fadrozole on aromatase gene expression along the HPG-axis in Japanese medaka, including designing and synthesizing RNA probes of interest.


Methods• Fixed and paraffin embedded samples

• 7μm tissue sections on slides-ISH with 35S-labeled CYP19a probe-H & E staining

• Detection of ISH signal-Radiography-Categorical classification system


Methods – What didn’t work

• Cryosectioning • DIG-labeled probes


Fadrozole Validation Exposure • 4 month old medaka

• Control and 1, 10, and 100 ug/L fadrozole treatments

• 2 replicate tanks of each treatment-10 fish per tank-5 male, 5 female

• 7 day static renewal


ISH CYP19a Scoring System

Category 0

-undefined staining

Category 3

-definition plus dark staining

Category 2

-definition throughout gonad

Category 1

-some definition


CYP19a Gene Expression - ISH

Fadrozole (μg/L)

Gen

e Ex

pres

sion

Cat

egor

y

CTR 1 10 1000

1

2

3

A A

A

BFemale Male


CYP19a Expression – PCR Validation

Fadrozole (μg/L)

Fold

-cha

nge

CTR 1 10 1000.0

4.0

8.0

12.0

16.0

1.0A

A

B

BFemale Male

Expr

essi

on C

ateg

ory

CTR 1 10 1000

1

2

3

A A

A

BFemale Male

Fadrozole (μg/L)


Histological Evaluation - Male

SC

SC

SZ SZ

CTR 100μg/L


Histological Evaluation - FemaleCTR 1μg/L

100μg/L 100μg/L

VOMO

MO

VO

VO

VO


Exposure Conclusions• Fadrozole increased expression of CYP19a in female

medaka gonads

• Induced morphological changes in female gonads

• No measurable effects on male gene expression or histology

• ISH useful for determining spatial aspects of gene expression


Overall Conclusions

• Successfully developed an ISH method that permitted the identification of changes in CYP19a gene expression along the HPG-axis in medaka

• Applied histological techniques to analyze gonadal morphology in the same fish


Ongoing Research

• Fluorescent detection method-better resolution-multiple possible labels

• Additional genes-CYP19b, ER, AR, vitellogenin


Funding for this project was provided through an EPA STAR Grant (EPA Project #RD831849601-0).


Acknowledgements

• Dr. John Giesy• June Woo Park• Dr. Markus Hecker• Dr. Paul Jones• Dr. John Newsted• Howard Zhang

• MSU students and staff• City U students and staff• ENTRIX staff• Committee Members:

-Dr. Norbert Kaminski-Dr. Steve Bursian


Contact Information

• Amber TompsettEnvironmental Toxicology LaboratoryUniversity of SaskatchewanSaskatoon, SK S7N [email protected]


Endocrine Disruption

“...an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior.”

Kavlock et al., 1996Research needs for the assessment of environmental effects of endocrine disruptors: a report of the USEPA-sponsored workshop


CYP19a Radiographs – Proof of Concept

CO EO

Radiographs of female fish from control (A) and 100 ug/L fadrozole (B) treatments. The control ovary (CO) shows no CYP19a expression; the exposed ovary (EO) shows CYP19a expression.

A B


CYP19a Radiographs

CT ET

Radiographs of male fish from control (A) and 100 ug/L fadrozole (B) treatments. Both the control testis (CT) and exposed testis (ET) show similar levels of CYP19a expression.

A B


Fadrozole Mechanism of Action• CYP19a expression increased in female gonads after

fadrozole exposure-linked to promoter control and signal transduction-SF-1 in promoter-Gonadotropin-mediated cAMP signal-altered expression of steroidogenic genes, including aromatase

• Fadrozole acts indirectly through modulating signal transduction pathways

• ISH a valuable tool for elucidating this type of response

development and validation of a novel in situ - usask.cas new/tompsett... · development and...

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