Selective Estrogen Receptor Modulators Cause Abnormal Skeletal Development in the Zebrafish Danio re

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Selective Estrogen Receptor Modulators Cause
Abnormal Skeletal Development in the Zebrafish
(Danio rerio)
Joshua Simulcik 05 VMI Departments of Biology
and Chemistry Center for Molecular, Cellular, and
Biological Chemistry Advisor Dr. Turner 65
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Why Zebrafish?

• Humans and zebrafish share 90 of their genes.
• Individual gene location and resultant protein
  function is conserved in most species.
• Models have been created in zebrafish that mimic
  symptoms of human disease.

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Estrogen Basics

• Estrogen is not a single hormone, but a family of
  hormones produced mainly in the bodies of women,
  but also in men.
• Originally, estrogen was thought to be simply a
  female sex hormone, but studies have shown that
  it is also located in the brain and demonstrates
  developmental and neuroprotective effects in the
  nervous system.

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SERMs

• SERMs are selective estrogen receptor modulators
  one example is tamoxifen.
• SERMs are non-steroidal, but have elements that
  are structurally similar to estrogen.
• Tamoxifen blocks the uptake of estrogen by taking
  its place on the cell receptor, but not sending
  the signal that estrogen would.
• Some prescribed breast cancer and osteoporosis
  drugs are SERMs Tamoxifen (Nolvadex), and
  Raloxifene (Evista).

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Purpose

• The purpose of my research is to develop a model
  in the embryonic zebrafish (Danio rerio) that
  shows the importance of estrogen in skeletal
  development.
• Preliminary studies have shown SERMs have an
  effect on bone tissue. This study focuses on
  cartilaginous tissue, especially the effects of
  tamoxifen on head and spine development.

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Hypothesis

• SERMs have a negative effect on the maturing of
  skeletal cartilage by blocking the uptake of
  estrogen at critical points during development.

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Materials and Methods

• Embryonic zebrafish were shipped from Oregon and
  dechorinated (removal of the egg sac) at
  approximately 36 hours post-fertilization (hpf).
• The embryos were randomly separated into three
  groups controls in egg rearing solution (ERS),
  experimentals in 10-8 M estrogen, or
  experimentals in a SERM (tamoxifen, 10-7 M).

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Materials and Methods, 2

• At various points in development, a sample of the
  population was taken for fixing and staining to
  observe the effects of the medium at that stage
  of development.
• The technique used was a whole-mount cartilage
  preparation in alcian blue developed by
  Schilling, et al.

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Results

• A noticable difference in cartilage maturation
  occurred between the control embryos and the SERM
  group.
• Visual differences in the retardation of
  cartilage development by tamoxifen treatment were
  noted as early as 72hpf, and the change only
  became greater over time.

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Results, 2

• No difference was notable between the controls
  and the experimental group treated with estrogen.
• This suggests that pharmacological levels of
  estrogen have no greater effect than
  physiological levels on the developmental process
  at this stage.
• The naturally occurring levels in the zebrafish
  are more than adequate during development, and
  extra estrogen has no additional benefits.

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Results, 3
Visible bands of cartilage between vertebra of
the spinal column.
No pattern to staining of the spinal column.
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Conclusions

• A successful model of estrogen mediated skeletal
  cartilage development in zebrafish embryos has
  been created.
• Estrogen is important to skeletal development in
  the zebrafish.
• Pharmacological levels of estrogen are not needed
  in this model, as the zebrafish has naturally
  adequate levels to stimulate skeletal development.

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Future Work

• A time assay of the staining procedure will
  produce more contrast in the final tissues, thus
  allowing for more accurate comparison.
• Other SERMs and Aromitase Inhibitors (AIs) may
  block Estrogen differently, and thus have
  different results.
• The effects of estrogen replacement therapy on
  cartilage development.

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Acknowledgements/Works Cited

• Ng JK, et al. The limb identity gene Tbx5
  promotes limb initiation by interacting with
  Wnt2b and Fgf10. Development. 2002
  Nov129(22)5161-70.
• Schilling, et al. Jaw and brachial arch
  mutations in zebrafish I brachial arches.
  Development., 1996. 123 329-344.

• Special Thanks to
• Dr. Turner 65
• VMI Biology and Chemistry Departments
• The Center for Molecular, Cellular, and
  Biological Chemistry
• SURI / VMIRL