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Embryonic low dose BPA Exposure has transgenerational effects on
GnRH neurons in Japanese medaka
Natalie Smith1, Tomoko Inagaki2, Siddharth Ramakrishnan2,3
Department of Chemistry1, Neuroscience Program2 , Department of Biology3, University of Puget Sound, WA
INTRODUCTION
RESULTS
G1- Two week (0-14dpf) embryonic parental exposure to 200ng/ml BPA
• This study examines the transgenerational effects of the xenoestrogen Bisphenol A (BPA) on the
Gonadotropin-releasing hormone (GnRH) neural pathway.
• GnRH neurons responsible for reproductive and allied behaviors in most vertebrates.
• Previous studies in our lab on medaka embryonic BPA low-dose exposure showed a 23.7%
increase in the terminal nerve and a 14.3% decrease in trigeminal region at 3dpf.
G2: 0-6dpf embryonic parental exposure to 200ng/ml BPA
G1: 4.57
G2: 11.28
• Significance is reported as a two-tail t-test
4
G1: -32.39
G2: -28.92
G1: -28.04
G2: -43.03
• Significant difference in brain size shows differential effect of BPA on first
generation embryos
5
G1: -3.21
G2: -16.08
G1: 69.49
G2: -20.69
• DPF stands for Days post fertilization.
DPF 3 Normalized Fluorescence
DPF 4 Normalized Fluorescence
Parental exposure during
Embryonic development
A
G1
Three Different Groups (Fig. 1):
1. G1: Parental treatment for from egg laying until 2
B
2 week exposure from
weeks post fertilization (14dpf)
egg laying
2. G2: Parental treatment for 0-6 dpf
3. Control: Parental exposure to vehicle control
Parents are raised until maturity
(EtOH) only
in regular water
G2
Exposure from 0-6 dpf
• Fluorescent Intensity calculated as percentage
from control
Figure 2. Normalized Fluorescence at
DPF 3 for the terminal nerve (TN) and
trigeminal (TG) regions of interest (ROI).
G1 followed by G2 data are reported at
each ROI. For TN, G1 n=8; G2 n=12. For
TG, G1 n=7; G2 n=11.
%Control
TN
*
*
Figure 3. Normalized Fluore
oscence at DPF 4
for the terminal nerve (TN) and trigeminal
(TG) regions of interest (ROI). G1 followed
by G2 data are reported at each ROI. For
TN, G1 n=10; G2 n=17 (p<0.05). For TG,
G1 n=11; G2 n=18 (p<0.04).
0
-10
*
*
-20
*
*
DPF
*
50
0
-50
TG
TG
TN
Figure 4. Normalized Fluorescence at
DPF 5 for the terminal nerve (TN) and
trigeminal (TG) regions of interest (ROI).
G1 followed by G2 data are reported at
each ROI. For TN, G1 n=6; G2 n=4. For
TG, G1 n=6; G2 n=4.
Hatching Rate
16
12
8
4
0
7
8
DPF 5
Figure 5. Embryonic brain size as percentage from control on DPF
3-5. G1 data followed by G2 data are reported for each DPF. For
DPF 3: G1 n=8 (p<0.01), G2 n=13. For DPF 4: G1 n=10 (p<0.03),
G2 n=19 (p<0.01). For DPF 5: G1 n=6, G2 n=8 (p<0.03).
9
10
11
DPF
Control
G1
G2
Figure 6. Hatching rate as observed with collected embryos
inventoried each day after fertilization.
CONCLUSIONS & FURTHER STEPS
Bright-field and
fluorescent imagery of
embryos
• Hatching rate of each group measured
• Brain size measured with eye-to-eye distance
using bright-field microscopy
-25
-50
TG
DPF 3
B
• Fluorescence normalized per region area
TN
-30
A
1. Terminal Nerve (A)
2. Trigeminal Region (B)
0
0
10
Fluorescence imaging of GnRH3-GFP neuronsat
dpf 3-5
Two Regions of Interest examined:
35
Normalized Brain Size DPF 3-5
Fertilized embryos are collected
•
% Control
% Control
Parental group then raised to maturity,
fertilized embryos collected from non-treated
tank water.
100
Number of Hatchings
•
DPF 5 Normalized Fluorescence
25
-35
200 ng/mL
BPA treatment
TG
(% Control)
G1: 38.96
G2: 43.04
%Control
•
Chronic Low Does BPA Exposure (200 ng/mL)
Using GnRH3:GFP medaka (with GnRH3
neurons tagged with green fluorescent protein)
Embryonic only exposure of parental groups
TN
(% Control)
3
70
•
•
DPF
• All results are reported with standard error of mean
• Here we wanted to determine if these effects were carried into the next generation
• Parental medaka were exposed to a chronic low-dose of BPA ONLY during early embryonic
development.
• First generation fertilized embryos in untreated water were then examined for changes in the
GnRH neural system.
METHODS
Table 1. Fluorescent Intensity at DPF 3-5
Figure 1. Experimental overview of BPA treatment of
parental groups and fluorescent imagery of first
generation untreated embryos.
• Just parental exposure to chronic low dose BPA results significant changes in embryonic
development of GnRH neurons in F1 embryos
• Brain sizes also seem to vary initially in the embryo, but go back to normalcy with time
• These changes are different from previous studies seen in GnRH development in BPA exposed
parental embryos
• How does this affect behavior?
• Does it have impact on reproduction/development?
• How does it affect GnRH physiology?
Study supported by NSF CAREER Award 1253126 to SR