Investigating TCDD

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Investigating TCDDs Effects on Clock Genes in
Chicken Granulosa Layer Explant

• Becky Howell

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Outline

• TCDD
• Clock mechanisms (BMAL1, Per2, Cry1)
• Model
• Hypothesis and questions
• Experimental design
• Expected results

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TCDD

• 2,3,7,8-tetrachloro-dibenzo-p-dioxin
• Polyhalogenated polyaromatic hydrocarbon
• Industrial by-product
• Environmental toxin
• Seveso, Italy
• Woonasquatucket River
• Experimental evidence
• - Disrupts estrous cycle, developmental problems

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TCDDs Effects via AhR

TCDD
TCDD
AhR
AhR
Definitions AhR Aryl hydrocarbon Receptor ARNT
Aryl hydrocarbon Nuclear Translocator
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In the Nucleus
Definitions AhR Aryl hydrocarbon Receptor ARNT
Aryl hydrocarbon Nuclear Translocator Per2
Period BMAL1- Brain Muscle ARNT-Like
Promiscuous
TCDD
AhR
ARNT
Per2
BMAL1
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Also Tim and Crys
Okamura, Yamaguchi Yagita, Cell Tissue Res,
30947-56, (2002)
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• BMAL1 Brain-muscle-ARNT-like protein
• - homologue of ARNT, cycle driver
• CRY1 Cryptochrome
• - negative regulator, conserved, directly
  sensitive to light
• PER2 Period
• - negative regulator, more specific

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Why the Chicken?

• Ovulation cycle near 24hrs
• Follicular Hierarchy / Follicular Tissue
• Clock gene homology to mammals
• Dioxin response - similar to mammals

Artistic talent by Dr. S. Tischkau
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Hypothesis

• Ho TCDD will alter the circadian rhythm of
  BMAL1, Cry1, Per2 in granulosa layer explants.

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Questions

• Does granulosa layer explants express BMAL1,
  Cry1, Per2 in a circadian rhythm?
• Does TCDD alter the expression of BMAL1, Cry1,
  Per2 in granulosa layer explants?

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Experimental Design 1- Validation

• F1 Granulosa layer explant expresses clock genes
  in a circadian pattern for 48hrs
• - qPCR 0, 12, 24, 36, 48
• F1 Granulosa layer explant is viable for 48hrs
  /- TCDD
• - DNA ladder and hormone assays
• Tissue collected 18hrs before ovulation.

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Hormonal Profile during Ovulatory Cycle
Start of Ovulatory cycle
Follicle is able to ovulate
Basal hormone levels
LH ovulatory surge
Ovulation
24 hrs 18 hrs 12 hrs 6hrs
0hrs
Oviposition
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Experimental Design 2

• Clock gene expression every 4hrs after 24hr
  incubation.
• 10 hens (n5 control n5 TCDD)

Culture punches for 24hrs Sample every 4
hours for 24 hour period

• - - - In culture - - - -
• /- TCDD 0 4
  8 12 16 20 24
  

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Data Collection

• Determine the expression of BMAL1, Per2, Cry1 via
  qPCR
• Compare oscillating patterns between /- TCDD
  treated granulosa layer explants.

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Expected Results - Control

0hrs 4hrs 8hrs
16hrs 20hrs 24hrs
BMAL1 in Green PER2 in Blue CRY1 in Orange
Expected results - TCDD treated oscillating
antiphase, dampened expression, shift in peak
expression
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Thanks!
Dr. Bahr and Lab Dr. Masaaki Nakai
Claire Zimmerman
Dave Boltz Dr. Tischkau and Lab Bethany
Klementiev Jason Hickock
Motoko Mukai Dr. Kathy Bottum Dr. D.
Bunick and Dr. C. Bunick Funding NIH
Toxicology Grant
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Questions?