All Mixed Up:
Phenotypic Plasticity in
a Genotypic World
Amber Tompsett, Steve Wiseman, Eric Higley, Sara Pryce,
John P. Giesy, and Markus Hecker
SETAC Europe Annual Meeting
and World Congress
May 21, 2012
Berlin, Germany
Sexual Determination and Differentiation
• Genotype
– Binary systems of genetic sex determination (GSD)
• Chromosome dependent
• XX/XY, ZZ/ZW
• Phenotype
– Sexual characteristics
– ‘Normal’ is a spectrum
– Extreme outliers are rare
• Athletes (i.e. Caster Semenya)
Sex Determination in Xenopus laevis
• ZW system of genetic sex determination
– ZW female; ZZ male
– Sex-linked gene (DM-W) on W chromosome
• Simple PCR assay to determine genetic sex
• Sensitive to estrogenic exposure around the time of
sexual determination and differentiation
– Feminization/demasculinization
– Male-to-female phenotypic sex reversal
Objectives and Experimental Design
• 17α-ethynylestradiol (EE2) and X. laevis used as model systems
– Determine the biological and phenotypic effects of EE2 exposure
– Determine the molecular mechanisms driving these effects
• Dosing Regime*
– 0.1, 1, and 10 µg/L EE2
– LC-MS/MS verification
• Exposure through 89 d
– Time to metamorphosis
– Gross gonadal morphology
– Gonadal histology
*Estrogen equivalent concentrations in surface water normally range from 3-30 ng/L
Genetic Sex Ratios
• 1:1 sex ratio overall
– 49% ZZ (male)
– 51% ZW (female)
• No treatment differences
– Replicate variability
80%
70%
Male
Female
60%
50%
40%
30%
20%
• Focus on genetic males
10%
0%
Control
Solvent
0.092 µg/L
0.84 µg/L
17α-ethynylestradiol Concentration
8.81 µg/L
Normal Female Phenotype
Normal Male Phenotype
Abnormal Male Phenotype
Mixed Sex Phenotype
Phenotype: Genetic Males Exposed to EE2
100%
90%
80%
70%
Female
60%
Mixed Sex
50%
Abnormal Male
Male
40%
30%
20%
10%
0%
Control
Solvent
0.092 µg/L
0.84 µg/L
17α-ethynylestradiol Concentration
*Fisher’s Exact Test: p<0.0001
8.81 µg/L
Sexual determination
and differentiation
Increasing Expression
Molecular sex
determination
Sexual development
Metamorphic
climax
Maturation
and Growth
59-65
Froglet
Molecular and Biochemical
Activity
Cell and Tissue Level
Development
Pre-50
50
51/52
53/54
55/56
Nieuwkoop-Faber Stage
57/58
Increasing Expression
Molecular sex
determination
Sexual determination
and differentiation
Sexual development
DM-W
DMRT1
CYP19
Y
Y
CYP19
DM-W
CYP19
E
E
CYP19
Y Y Y
E
CYP19
Y
E
E
E
DMRT1
Primordial Gonad
ZW Genotype
Pre-50
50
Maturation
and Growth
CYP19
CYP19
Y
Y
E
E
VTG
VTG
Ovarian
Development
Ovarian
Differentiation
51/52
Metamorphic
climax
53/54
55/56
Nieuwkoop-Faber Stage
57/58
59-65
Froglet
Increasing Expression
Molecular sex
determination
Sexual determination
and differentiation
DMRT1
X
Sexual development
Metamorphic
climax
Maturation
and Growth
DHT?
5αreductase?
DM-W
E
E
Y Y
DMRT1
X Y
VTG
CYP19
Primordial Gonad
ZZ Genotype
Pre-50
X
E
50
Testicular
Development
Testicular
Differentiation
51/52
53/54
55/56
Nieuwkoop-Faber Stage
57/58
59-65
Froglet
Increasing Expression
Molecular sex
determination
Sexual determination
and differentiation
Sexual development
Metamorphic
climax
E
ALL
MIXED
Y
X
Y
UP
Y Y Y Y
DMRT1
DM-W
E E E
E
5αDHT?
reductase?
E
E
E
E
E
E
E
E
E
E
E
E
E
Pre-50
50
Altered
Differentiation
51/52
EE2 Exposure
E E E
Y
VTG
E
DMRT1
Primordial Gonad
ZZ Genotype
E
Y
E
Maturation
and Growth
53/54
VTG
Abnormal, Intersex, or Ovarian
Development
55/56
Nieuwkoop-Faber Stage
57/58
59-65
Froglet
Vitellogenesis
• Genotype
– Male and female genomes contain the
vitellogenin gene
• Phenotype
– Normally, only females express vitellogenin
• Egg yolk protein
– Vitellogenin expression is inducible in males
• Estrogen exposure
Immunochemical Detection of Vitellogenin
100 µm
100 µm
100 µm
VTG
A
B
H & E stain
C
Negative control
Vitellogenin stain
Increasing Expression
Molecular sex
determination
Sexual determination
and differentiation
Sexual development
Metamorphic
climax
E
E
E
E E E
E
E
E
E
Y Y Y
E
Primordial Gonad
Pre-50
50
E
E
E
Y
E
E
Y
E
EE2 Exposure
53/54
Y
E
E E E
Y
VTG
DELAYED
VTG
Gonadal
Development
Differentiation
51/52
E
E
Y
E
Maturation
and Growth
55/56
Nieuwkoop-Faber Stage
57/58
59-65
Froglet
Conclusions
• Molecular pathways can help explain effects
• Molecular changes drive biologically relevant
effects
– Abnormal sexual development
– Delayed metamorphosis
What’s Next?
• Analysis of transcript abundances
– Tadpole (Illumina sequencing)
– Froglet (RT-PCR)
• Parallel experiment with wood frogs
– Histology
– RT-PCR
Acknowledgements
• Toxicology Centre
–
–
–
–
Jon Doering
ATRF/Jason Raine
Shawn Beitel
Brett Tendler
• Veterinary Biomedical Sciences
– Histology Laboratory
– Darren Nesbitt