Wild-type zebrafish were crossed with transgenic zebrafish containing a fluorescent marker in oxytocin cells. Their offspring were treated with estrogen at the shield stage of development and the number of fluorescent oxytocin cells were later counted. Embryos treated with estrogen had more oxytocin cells than controls, and embryos treated and then removed from estrogen had the highest number, suggesting estrogen levels impact oxytocin cell growth.

1. Introduction
Procedure
Breeding the zebrafish: Wild-type fish, which do not
contain any transgenes, were crossed with transgenic
eg1 fish. 50% of offspring were expected to contain
the transgene, which were retrieved under a
florescent microscope and chosen for experimental
data.
Making the estrogen solution: 36.7µM stock solution
of E2 was diluted to 36.7nM solution using fish water
and phenylthiourea (PTU).
Estrogen treatment: Transgenic eg1 zebrafish
embryos were divided into 3 groups: control (without
estrogen or inhibitor), embryos treated at shield
stage, and embryos treated with estrogen at shield
stage and removed from after 24 hours. The
experimental groups received 5 mL of estrogen
solution and the control group received 5 ml of fish
water (plus PTU). All groups were incubated at
27.9℃.
Results
Acknowledgements
References
1. Champagne, Frances, Josie Diorio, et al. "Naturally occurring variations in
maternal behavior in the rat are associated with differences in estrogen inducible
central oxytocin receptors." PNAS. 98.22 (2001): 12736-41.
2. Choleris, Elena, Jan-Ake Gustafsson, et al. "An estrogen-dependent four-gene
micronet regulating social recognition: A study with oxytocin and estrogen
receptor-a." PNAS. 100.10 (2003): 6192-97.
3. Gorelick, Daniel, and Marnie Halpern. "Visualization of Estrogen Receptor
Transcriptional Activation in Zebrafish." General Endocrinology. 152.7 (2011):
2690-2703.
Discussion
I would like to thank my mentor, Dr. Eric Glasgow at
oncology department for his guidance and support. I would
also like to thank Allyson Raymond and Matt Swift for their
help.
Hormones play critical roles in the
development and function of the nervous system.
Alteration of these important signaling systems
either by genetic abnormalities or environmental
insults have adverse consequences for brain
development and subsequent behavior.
Estrogen (or estradiol; E2), a steroid hormone,
has several physiological functions, such as
increasing fat stores, stimulating endometrial
growth, increasing uterine growth, maintaining
vessels and skin, and producing female secondary
sex characteristics. In addition, estrogen affects
brain development.
Oxytocin (oxt), a peptide hormone, influences
several behavioral and physiological processes
such as social, sexual, and maternal behaviors,
learning and memory, parturition, and milk
letdown. The interaction between estrogen and
oxytocin is critical for the proper development and
function of the brain. How these interactions
influence brain development, however, is poorly
understood. The zebrafish embryo provides a
productive system in which to study these
interactions.
Zebrafish (Danio rerio) have become a major
animal model because they lay large numbers of
externally fertilized eggs, which develop rapidly
and are transparent. In addition, zebrafish have a
similar body plan and most of the same organs as
humans. Further, 70% of their genes are similar to
ours.
The transparent zebrafish embryos are
utilized here
to determine if estrogen exposure influences the
development of oxytocin producing cells. In order
to visualize oxytocin cellular development in living
embryos we previously generated a transgenic
zebrafish line that produces a green fluorescent
protein in oxytocin cells, called Tg(oxtl:GFP)eg1.
The experiments presented here test whether
exposure to E2 during early embryonic
development alters the development of oxytocin
expressing cells. Embryos were placed in E2
solutions at the beginning of gastrulation (shield
stage). The effect on oxytocin cell development
was determined by counting the numbers of green
fluorescent oxytocin cells at 2 dpf.
Based on the experimental results, estrogen
treatment increases the number of oxytocin
expressing cells, compared to the control group
(no estrogen).
Although the data show that estrogen
increases the number of oxytocin cells, the
embryos treated with estrogen and then removed
from solution after 24 hours had the highest
number of oxytocin cells. This result could be
due to the fact that too much estrogen does not
allow the cells to function properly.
This suggests a relationship between
estrogen levels and oxytocin cell growth.
However small sample size precludes statistical
analysis.
Future research is necessary to explain the
effects of estrogen on transcription and early
development. This is especially important
because there is no scientific literature
addressing this subject yet. However there is
significant evidence that estrogen and oxytocin
affect interpersonal relationships, stress, and
anxiety. A simple system, such as zebrafish,
where we have direct control and access to brain
development, will enable the study of these
complex relationships.
Eryn Terry1 and Eric Glasgow2
1 The Holton-Arms School, 7303 River Rd Bethesda, MD 20817
2 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC
Georgetown University
The Effect of Estrogen on Oxytocin Gene Expression Evaluated
in Living Transgenic Zebrafish Embryos
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30
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45
Control Shield 24hrs at Shield
NumberofGFPCells
The Effect of Estrogen on GFP Cell Numbers
Tg(oxtl:GFP)eg1 embryo at 1 day post fertilization (dpf)
oxytocin expressing cells glow green in these embryos
Adult zebrafish
Early zebrafish development
ControlE2shield24hrE2shield
DIC Fluorescence Merge
Figure 1
Fig. 1 Control and experimental embryos were visualized at 2 dpf using
differential interference contrast (DIC) and fluorescent microscopy. The
numbers of green fluorescent cells were counted while focusing through
the brain
Figure 2
Fig. 2 The effect of estrogen treatment on oxytocin cell
development was quantified by counting the numbers of green
fluorescent cells in each treatment group. The average number
of fluorescent cells per embryos in each group are shown in the
graph.
Red outline indicates the developmental stage at estrogen treatment.