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 Tompsett
Aquatic Toxicity Workshop
Halifax, Nova Scotia
October 3, 2007
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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 (receptormediated) and indirect effects through changes in signal
transduction pathways.
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Model System
Whole-animal tissue section in situ
hybridization
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
In situ hybridization
Antisense RNA
probe
Fixed RNA in
tissue
In situ
hybridization
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Probe-RNA
hybrid in tissue
In situ hybridization
Sense RNA
probe
Fixed RNA in
tissue
In situ
hybridization
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Fixed RNA in
tissue
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Target Genes Along the HPG-Axis
Liver
Brain
Feedback
GnRH
Sex Steroids
GRIF
Vitellogenin
Adrenals
Pituitary
Gonadotropins
Gonads
Tissue
Primary Gene Targets
Brain
CYP19B; GnRHs; ERα & β; AR
Pituitary
GnRH receptors; GtH I & II; ERα & β; AR; Activin/Inhibin
Adrenals
CYP11A & B; CYP17; 3β-HSD; 17β-HSD; StAR; AR; GtH receptors; Activin/Inhibin
Gonads
CYP11A & B; CYP17; CYP19A; 3β-HSD; 11β-HSD; 17β-HSD; StAR; ERα & β; AR; GtH
receptors; Activin/Inhibin
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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.
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Methods – What didn’t work
•
Cryosectioning
•
DIG-labeled probes
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
ISH CYP19a Scoring System
Category 0
Category 1
-undefined
staining
-some
definition
Category 2
Category 3
-definition
throughout
gonad
-definition
plus dark
staining
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Gene Expression Category
CYP19a Gene Expression - ISH
Female
Male
B
3
A
2
1
A
A
0
CTR
1
10
100
Fadrozole (μg/L)
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Fold-change
16.0
Female
Male
B
12.0
B
8.0
4.0
1.0
0.0
A
A
CTR
1
10
Fadrozole (μg/L)
100
Expression Category
CYP19a Expression – PCR Validation
Female
Male
3
A
2
1
0
B
A
A
CTR
1
10
Fadrozole (μg/L)
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
100
Histological Evaluation - Male
CTR
100μg/L
SC
SZ
SZ
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
SC
Histological Evaluation - Female
CTR
1μg/L
VO
MO
VO
MO
100μg/L
100μg/L
VO
VO
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Ongoing Research
• Fluorescent detection
method
-better resolution
-multiple possible labels
• Additional genes
-CYP19b, ER, AR,
vitellogenin
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Funding for this project was provided through an EPA
STAR Grant (EPA Project #RD831849601-0).
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
Contact Information
• Amber Tompsett
Environmental Toxicology Laboratory
University of Saskatchewan
Saskatoon, SK S7N 5B3
amber.tompsett@usask.ca
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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., 1996
Research needs for the assessment of environmental effects of
endocrine disruptors: a report of the USEPA-sponsored workshop
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
CYP19a Radiographs – Proof of
Concept
A
B
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.
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
CYP19a Radiographs
A
B
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.
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan
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
Department of Veterinary Biomedical Sciences and
Toxicology Centre, University of Saskatchewan