International Journal of Marine Science 2016, Vol.6, No.4, 1-12
3
and physiological function of a fetus were investigated
by using zebrafish embryos exposure to 4-t-OP.
2 Materials and Method
2.1 Experimental animals and compound
Adult AB-strain zebrafish and transgenic zebrafish Tg
(fil-1: EGFP) were acquired from the Taiwan
Zebrafish Core Facility at Academia Sinica (Taipei,
Taiwan). The fish were acclimatized in the laboratory
culture condition and observed for clinical health for
at least one week prior to experiments. The fish were
raised in 10-L tanks and maintained at 28 °C in
recirculating freshwater with a controlled light cycle
(14 h light/10 h dark), and fed daily with commercial
pellet. A pair-wise breeding instead of group-breeding
was used for breeding of zebrafish in this study to
have a better interpretation of the effects. Fertilized
embryos generated by pair-wise breeding was used for
immersion treatment of 4-tert-octylphenol (4-t-OP).
All zebrafish were handled in compliance with the
local animal welfare regulations. The alkylphenol
4-t-OP with 97% purity (CAS No. 140-66-9) was
purchased from Sigma-Aldrich. The 4-t-OP was
dissolved in absolute ethanol as 6 mM stock solution
and then diluted in embryos medium for immersion
treatment of zebrafish embryos.
2.2 Immersion experimental design
Gastrulation is a key event during embryonic
morphogenesis and therefore zebrafish embryos with
gastrulation stage (5 hour post-fertilization) were used
for our exposure studies. Wild type zebrafish embryos
at 5 hours post-fertilization (hpf) were collected and
put in 12-well microplate for immersion treatment of
4-t-OP. One hundred embryos in each well were
immersed with 3 ml of embryos medium (14 mM
NaCl, 0.54 mM KCl, 0.026 mM Na
2
HPO
4
, 0.3 mM
K
2
HPO
4
, 0.1 mM CaCl
2
and 0.1 mM MgSO
4
·7H
2
O in
deionized
water)
containing
0.2
mM
1-phenyl-2-thiourea
(PTU)
and
a
various
concentration of 4-t-OP, and then incubated at 28°C
for 67 h. PTU added in embryos medium was used to
prevent pigmentation. Embryos immersed with
embryos medium containing 0.2 mM PTU was used
as control group. The embryo medium was renewed
daily to maintain the water quality and 4-t-OP
concentration. Survival rate, hatching rate and
malformation
were
evaluated
at
3
days
post-fertilization (dpf). The experiment was performed
in triplicate for each condition and repeated by three
times.
2.3 Heart rate determination and morphological
analysis
Tg (fil-1: EGFP) zebrafish embryos, which enhanced
green fluorescent protein (EGFP) was specifically
expressed in heart and blood vessel, were used to
evaluate the phenotypes of cardiovascular defects
resulting from 4-t-OP treatment. Immersion treatment
of Tg (fil-1: EGFP) zebrafish embryos with 4-t-OP
were carried out as follows: One hundred embryos in
each well of 12-well microplate was exposed to 0.5
M or 1.0
M of 4-t-OP from 5 hpf until to the end of
embryogenesis (96 hpf). The experiment was
performed in triplicate. Twenty Tg (fil-1: EGFP)
zebrafish embryos were picked into a petri dish
containing 15 ml of embryos medium at 48, 72 and 96
hpf, and heart rate of each zebrafish embryos were
calculated under microscopy (Leica Z16 APO). Ten
embryos were collected at 48 and 72 hpf for real-time
PCR. To observe the morphological defects of heart
and blood vessel, live control and 4-t-OP treated
embryos
were
anesthetized
with
tricaine
methanesulfonate (MS222) before mounting in 3%
methyl-cellulose (Sigma M-0387) and examined
under a Leica stereomicroscope. Digital images or
video was acquired using a Leica camera (Leica
DFC310 FX).
2.4 Gene expression detected by real-time PCR
The total RNA was isolated from the Tg (fil-1:EGFP)
zebrafish embryos with or without 4-t-OP treatment
(control group). The expression levels of estrogen
receptor (ER, ER 1, ER 2, Nk2 homeobox 5 (Nkx2.5),
Nkx2.7, heart and neural crest derivatives expressed 2
(Hand2), GATA-binding protein 4 (GATA-4),
GATA-5, GATA-6, fibroblast growth factor 1a
(FGF1a), T-box 2a (Tbx2a), Tbx2b, Tbx5a and
elongation factor 1-α (ef1-α) were determined using
quantitative PCR. The ef1-α was used as an internal
control. The specific PCR primers used in this study
are listed in Table 1. Real-time PCR was performed
using SYBR Green PCR reagents and an Applied
Biosystems StepOnePlus Real-Time PCR system. The
cycling profile was as follows: 60 °C for 2 min, 95 °C
for 10 min followed by 40 cycles of denaturing at 95 °C
for 15 s, and annealing and primer extension at 60 °C
