AcknowledgmentsThis research is supported by NSF CAREER Award 1253126 to SR.

Authors thank Sean Stackhouse, Jessica L Dyck and Jessica M Evans for fish care.

Transgenerational effects of Bisphenol A on embryogenesis, GnRH3 neural systems and locomotor behavior in Japanese medaka

T. INAGAKI1,2, N. L. SMITH 2,3, K.M.SHERVA 2,3, S. RAMAKRISHNAN1,2 Dept. of Biology1, Neuroscience program2, Dept. of Chemistry/Biochemistry3, University of Puget Sound, Tacoma, WA

Introduction

Methods

Figure 1 Figure 3

Figure 2

Movement (20 dpf) Terminal Nerve (TN) GnRH3 neural population

Trigeminal (TG) GnRH3 neural population

Parental BPA exposure alters F2 fluorescence of GnRH3-GFP neurons

BPA exposure in F1 generation decreased F2 locomotor activity at 20 dpf

Figure 4 SV2 expression was reduced only in G1 larvae

Speed (20 dpf)

• BPA exposure to F1 generation (parents) results in significant alternations in the development of GnRH3 neurons, embryonic mortality, time to hatch, synaptic connectivity and locomotor behavior of F2 generation.

• Transgenerational BPA effects were more potent in G1 fish whose parents were exposed to BPA for life-long.

• Some effects were different from BPA effects in F1 generation reported in the previous study.

Timeline/Experimental Design

Testing:- GnRH3-GFP

intensity - Survival rates- Time to hatch- Locomotor

behavior- Synaptic

connectivity

100 μm

Discussion/Summary

• Bisphenol A (BPA) is a synthetic non-steroidal environmental chemical. Evidence suggests that chronic exposure to low-dose BPA may induce a variety of adverse effects in developing brain.

• Our previous study shows that BPA exposure (200 ng/ml) during embryogenesis disrupts normal development of Gonadotropin-Releasing-Hormone 3(GnRH3) neurons on 3 days post fertilization (dpf), alters physiological development, and decreases larval locomotor activity on 20 dpf. Yet, transgenerational impacts of BPA are not understood.

W21 431.02

Sv2 expression (20 dpf)

Survival rates Time to hatch

3 dpf GnRH3-GFP

intensity

4 dpf GnRH3-GFP

intensity

Normalized Fluorescence at 3 dpf. n=15 (G0), 5 (G1), 8 (G2), 12 (G3) and 15 (G4).

Normalized Fluorescence at 4 dpf. n=12 (G0), 15 (G1), 10 (G2), 18 (G3) and 11 (G4).

• Using a transgenic medaka fish model with GnRH3 neurons tagged with GFP, this study examined if BPA produces transgenerational alternations in GnRH3 neural systems, embryonic mortality, and larval locomotor behavior.

Subjects were F2 transgenic Japanese medaka embryos/larvae that have never been exposed to BPA (F2) , but their parents (F1) were exposed to BPA for different durations in their life span.

General Methods

• GnRH3-GFP neurons at the terminal nerve (TN) and the trigeminal nerve (TG) were visualized from 3 and 4 dpf F2 embryos, and fluorescence intensity of each neural population was quantified.

• For behavior tests, 20 dpf F2 larvae were placed into the 6-well plates, and locomotor activity was tracked using the Noldus EthoVision .

• Immunohistchemical staining with SV2 antibody was performed at 20 dpf.

• Raw data were analyzed on ANOVAs and independent t-tests. Values are shown as mean± SEM. p<0.05, 2-tailed is considered as significance.

G0 G1 G2 G3 G4Mean 8.91 9.89 8.37 8.24 7.47SEM 0.30 0.23 0.11 0.16 0.17

n 44 87 74 79 49

Time to hatch (dpf)G0 G 1 G 2 G 3 G 4

Mean 93.72 60.65 89.40 86.48 81.05SEM 3.15 8.14 5.34 4.96 1.93

n 72 90 75 80 50

Embryonic survival rates (%)

G0 G1 G2 G3 G4Mean 17.94 13.19 18 17.49 22.51SEM 1.30 1.45 1.54 1.39 4.13

n 15 17 11 17 12

SV2/GnRH3 area ratios (%)

G0 G1 G2 G3 G4 Mean 193.5 110 92.36 70.66 115.5 SEM 31.19 18.31 19.02 19.43 28.17

n 17 15 15 14 17

20 dpf Movement (mm)G0 G1 G2 G3 G4

Mean 3.23 2.88 1.74 1.56 2.02 SEM 0.52 0.39 0.31 0.36 0.41

n 17 18 15 14 16

20 dpf Velocity (mm/s)

-100

-50

0

50

100

******, P<0.001

G0G1G2G3G4

TN TG

% c

ontr

ol (

G0)

-100

-50

0

50

100

150 ***

*, P<0.05

**, P<0.01

***,P<0.001

TN TG

*

**

G0G1G2G3G4

% c

ontr

ol (

G0)

-50

-25

0

25

50

*G0 G1 G2 G3 G4

*, P<0.05

% c

ontr

ol (

G0)

-60

-40

-20

0

20

**G0 G1 G2 G3 G4

**, P<0.01,

% c

ontr

ol (

G0)

-30

-20

-10

0

10

20**

G0 G1 G2 G3 G4

**, P<0.01,

***, P<0.001

***

% c

ontr

ol (

G0)

-100

-50

0

50

G0 G1 G2 G3 G4

*

**, p<0.01, *, p<0.05

* ** *% c

on

tro

l (G

0)

-100

-50

0

50

G0 G1 G2 G3 G4

*, p<0.05

* *

% c

on

tro

l (G

0)

100 μm100 μm

G0

Offspring :no BPA exposure

G1

G2

G3

G4

F2

F 1 treatment and groups

(No BPA exposure)

(BPA life-long exposure)

(BPA 2 weeks developmental exposure)

(BPA early development only)

(BPA during sex differentiation)

G 1

G 2

G 3

G 4

G 0

( BPA exposu re)

dpf 0 1 2 3 4 5 6 7 8 9 10 - - - - 15 - - - - 20 - - - - 25 - - - - - 90

Parents : BPA exposureF1

GnRH3-GFP medakaF0

dpf 0 1 2 3 4 5 6 7 8 9 10 - - - -15 - - - - 20 - - - - 90

75 μm

50 μm