All Mixed Up: Phenotypic Plasticity in a Genotypic World

Transcription

1 All Mixed Up: Phenotypic Plasticity in a Genotypic World Amber Tompsett, Steve Wiseman, ric Higley, Sara Pryce, John P. Giesy, and Markus Hecker STAC urope Annual Meeting and World Congress May 21, 2012 Berlin, Germany

2 Sexual Determination and Differentiation Genotype Binary systems of genetic sex determination (GSD) Chromosome dependent XX/X, ZZ/ZW Phenotype Sexual characteristics Normal is a spectrum xtreme outliers are rare Athletes (i.e. Caster Semenya)

3 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

4 Objectives and xperimental Design 17α-ethynylestradiol (2) and X. laevis used as model systems Determine the biological and phenotypic effects of 2 exposure Determine the molecular mechanisms driving these effects Dosing Regime* 0.1, 1, and 10 µg/l 2 LC-MS/MS verification xposure through 89 d Time to metamorphosis Gross gonadal morphology Gonadal histology *strogen equivalent concentrations in surface water normally range from 3-30 ng/l

5 Genetic Sex Ratios 1:1 sex ratio overall 49% ZZ (male) 51% ZW (female) No treatment differences Replicate variability Focus on genetic males 80% 70% 60% 50% 40% 30% 20% 10% 0% Male Female Control Solvent µg/l 0.84 µg/l 8.81 µg/l 17α-ethynylestradiol Concentration

6 Normal Female Phenotype Normal Male Phenotype

7 Abnormal Male Phenotype

8 Mixed Sex Phenotype

9 Phenotype: Genetic Males xposed to 2 100% 90% 80% 70% 60% 50% 40% Female Mixed Sex Abnormal Male Male 30% 20% 10% 0% Control Solvent µg/l 0.84 µg/l 8.81 µg/l 17α-ethynylestradiol Concentration *Fisher s xact Test: p<0.0001

10 Molecular sex determination Sexual determination and differentiation Sexual development Metamorphic climax Maturation and Growth Increasing xpression Molecular and Biochemical Activity Cell and Tissue Level Development Pre /52 53/54 55/56 57/ Froglet Nieuwkoop-Faber Stage

11 Molecular sex determination Sexual determination and differentiation Sexual development Metamorphic climax Maturation and Growth DM-W CP19 CP19 Increasing xpression DM-W DMRT1 DMRT1 CP19 CP19 CP19 CP19 CP19 VTG VTG Primordial Gonad ZW Genotype Ovarian Differentiation Pre /52 53/54 55/56 57/ Froglet Nieuwkoop-Faber Stage Ovarian Development

12 Molecular sex determination Sexual determination and differentiation Sexual development Metamorphic climax Maturation and Growth Increasing xpression X DM-W DMRT1 DMRT1 CP19 X Primordial Gonad ZZ Genotype 5αreductase? DHT? Testicular Differentiation Pre /52 53/54 55/56 57/ Froglet Nieuwkoop-Faber Stage Testicular Development X VTG

13 Molecular sex determination Sexual determination and differentiation Sexual development Metamorphic climax Maturation and Growth Increasing xpression X DM-W DMRT1 DMRT1 ALL MIXD 5α- DHT? reductase? UP VTG VTG Primordial Gonad ZZ Genotype Altered Differentiation Pre /52 53/54 55/56 57/ Froglet 2 xposure Nieuwkoop-Faber Stage Abnormal, Intersex, or Ovarian Development

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15 Vitellogenesis Genotype Male and female genomes contain the vitellogenin gene Phenotype Normally, only females express vitellogenin gg yolk protein Vitellogenin expression is inducible in males strogen exposure

16 Immunochemical Detection of Vitellogenin 100 µm 100 µm 100 µm VTG A B H & stain Negative control Vitellogenin stain C

17 Molecular sex determination Increasing xpression Sexual determination and differentiation Sexual development Metamorphic climax Maturation and Growth DLAD VTG VTG Primordial Gonad Differentiation Pre /52 53/54 55/56 57/ Froglet 2 xposure Nieuwkoop-Faber Stage Gonadal Development

18 Conclusions Molecular pathways can help explain effects Molecular changes drive biologically relevant effects Abnormal sexual development Delayed metamorphosis

19 What s Next? Analysis of transcript abundances Tadpole (Illumina sequencing) Froglet (RT-PCR) Parallel experiment with wood frogs Histology RT-PCR

20 Acknowledgements Toxicology Centre Jon Doering ATRF/Jason Raine Shawn Beitel Brett Tendler Veterinary Biomedical Sciences Histology Laboratory Darren Nesbitt