Effects of waterborne exposure of octylphenol and oestrogen on pregnant viviparous eelpout (Zoarces viviparus) and her embryos in ovario
Tina H. Rasmussen1,*,
Thomas K. Andreassen1,
Søren N. Pedersen1,
Leo T. M. Van der Ven2,
Poul Bjerregaard1 and
Bodil Korsgaard1
1 Institute of Biology, University of Southern Denmark, Odense,
Denmark
2 Lab. of Pathology and Immunobiology, National Institute of Public Health
and the Environment (RIVM), Bilthoven, The Netherlands
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Fig. 1. Actual concentrations of 4-tert-octylphenol (4-tOP) in the aquaria
during the experiment from 11 October to 15 November. Broken lines indicate
nominal water concentrations. To the water was added isopropanol only
(control) or 4-tOP dissolved in isopropanol to nominal concentrations of 25
µg l-1 (OP25) or 100 µg l-1 (OP100). Values are
means of concentration of each group in 2-3 aquaria ± S.E.M.
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Fig. 2. Induction of oestrogenic biomarkers in pregnant eelpout:
17ß-oestradiol (E2)-binding activity, vitellogenin (Vtg) in
plasma and Vtg mRNA in liver. (A) E2-binding capacity in hepatic
cytosolic extracts. E2-binding capacity was estimated by
single-point assays. (B) Vtg in plasma. (C) Vtg mRNA in liver tissue.
Ethidium-bromide-stained agarose gels showing amplified RT-PCR products using
Vtg-specific primers (215 bp). As an internal control for the RT-PCR reaction,
eelpout ß-actin mRNA was amplified (band at 340 bp) using
ß-actin-specific primers. Fish were exposed to isopropanol (C; control),
25 µg l-1 4-tert-octylphenol (4-tOP) (OP25), 100 µg
l-1 4-tOP (OP100) or 0.5 µg l-1 E2 for 17
days. Values are means ± S.E.M. Significant difference from control is
indicated by asterisks (*P<0.05;
**P<0.01; ***P<0.001).
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Fig. 3. The relationship between actual plasma concentration of
4-tert-octylphenol (4-tOP) and plasma vitellogenin concentration of
pregnant eelpout exposed to isopropanol (control), 25 µgl-1
4-tOP or 100 µgl-1 4-tOP for 35 days.
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Fig. 4. Changes in five different ovarian indices and in the hepatosomatic index of
pregnant eelpout after 35 days water exposure to isopropanol (control, C), 25
µgl-1 4-tert-octylphenol (4-tOP) (OP25), 100
µgl-1 4-tOP (OP100) or 0.5 µgl-1
17ß-oestradiol (E2). (A) Ovarian fluid index (OFI). (B) Embryo
somatic index (ESI). (C) Ovarian sac somatic index (OSSI). (D) Ovarian sac
mass percentage (OSM). (E) Gonadosomatic index (GSI). (F) Hepatosomatic index
(HSI). OFI, ESI, OSSI, GSI and HSI are somatic indices calculated in relation
to the total mass of pregnant eelpout (excluding ovary). Values are means
± S.E.M. Significant difference from control is indicated by asterisks
(*P<0.05; **P<0.01;
***P<0.001).
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Fig. 5. The relationship between concentrations of calcium in ovarian fluid and in
plasma of pregnant eelpout after 35 days water exposure to isopropanol
(control, C), 25 µgl-1 4-tert-octylphenol (4-tOP)
(OP25), 100 µgl-1 4-tOP (OP100) or 0.5 µgl-1
17ß-oestradiol (E2). Values are means ± S.E.M.
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Fig. 6. The relationship between actual concentration of
4-tert-octylphenol (4-tOP) in ovarian fluid and the percentage of
dead embryos found in the ovary of pregnant eelpout after 35 days water
exposure to isopropanol (control), 25 µgl-1 4-tOP (OP25) or 100
µgl-1 4-tOP (OP100).
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Fig. 7. Effects of treatment on embryonic growth. (A) Mass and (B) length of
embryos at day 0 (11 October) and day 35 (15 November) after water exposure of
pregnant mother fish to isopropanol (control, C), 25 µgl-1
4-tert-octylphenol (4-tOP) (OP25), 100 µgl-1 4-tOP
(OP100) or 0.5 µgl-1 17ß-oestradiol (E2). Values
are means ± S.E.M. Significant difference from the respective control
is indicated by asterisks (*P<0.05,
**P<0.01, ***P<0.001).
Significant difference of 35-day control from the 0-day control is indicated
by a dagger (P<0.05).
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Fig. 8. Induction of vitellogenin (Vtg) mRNA in eelpout embryos exposed to
4-tert-octylphenol (4-tOP) in ovario for 35 days.
Ethidium-bromide-stained agarose gel showing the amplified RT-PCR products
using Vtg-(215 bp) or ß-actin- (340 bp) specific primers on total RNA
extracted from embryos dissected out of mother fish exposed to isopropanol
(control), 25 µgl-1 4-tOP (OP25), 100 µgl-1 4-tOP
(OP100) or 0.5 µgl-1 17ß-oestradiol (E2). Three
embryos were pooled per mother fish. Numbers indicate different mother fish.
M, 100 bp marker; Neg, negative control (no RT-enzyme).
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Fig. 9. Immunohistochemical localisation of vitellogenin (Vtg) in the liver and
hindgut of eelpout embryos exposed to 4-tert-octylphenol (4-tOP)
in ovario for 35 days. (A) Control liver: no Vtg-staining. (B) 100
µgl-1 4-tOP (OP100)-treated liver: strong Vtg-specific staining
in cytoplasm of hepatocytes (arrow) and blood capillaries (b). (C) Control
hindgut: no Vtg-staining. (D) OP100-treated hindgut: Vtg-staining in cellular
debris (cd) in the lumen (L) and the periphery of epithelial cells of the
hindgut (arrow). Scale bars, A and B=20 µm; C and D=200 µm.
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Fig. 10. In situ hybridisation analysis of oestrogen receptor (ER)
expression in gonads of control eelpout embryos. Bright- and dark-field
micrographs of representative transverse sections from the gonadal regions of
embryos hybridised with anti-sense (A,B,D,E) or sense (C,F) eelpout ER
RNA probe. (A-C) Presumptive male gonad at the beginning of the experiment (11
October). (D-F) Normal female gonad (control) at the end of the experiment (15
November). Arrows indicate the position of gonads.
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Fig. 11. Light micrographs of an undifferentiated gonad (A) and early
differentiating gonads from control eelpout embryos at day 0 (11 October)
(B-G), showing transverse sections from the anterior end, central part and
posterior end of the gonad. Scale bars, 100 µm. Paraffin section.
Haematoxylin-eosin staining. (A) Undifferentiated gonad (20 September). (B-D)
Early differentiating female gonad (B = anterior end; C = central part; D =
posterior end). (E-G) Early two-lobed differentiating presumptive male gonad
(E = anterior end; F = central part; G = posterior end). GC, germ cell; I,
intestine; K, kidney; M, mesentery; OC, ovarian cavity; Og, oogonia.
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Fig. 12. Light micrographs of gonads from control eelpout embryos at day 35 (15
November) showing transverse sections from the anterior end, central part and
posterior end of the gonad. Scale bars, 100 µm. Paraffin section.
Haematoxylin-eosin staining. (A—C) Female gonad with a single hollow
ovary containing oocytes (A = anterior end; B = central part; C = posterior
end). (D—F) Presumptive male gonad, two-lobed. GC, germ cell; I,
intestine; K, kidney; M, mesentery; Oc, oocyte (primary); OC, ovarian cavity;
Og, oogonia.
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Fig. 13. Light micrographs of abnormal gonads of eelpout embryos from the OP100 (100
µgl-1 4-tert-octylphenol) group at day 35 (15 November)
showing transverse sections from the anterior end, central part and posterior
end of the gonad. Scale bars, 100 µm. Paraffin section. Haematoxylin-eosin
staining. (A—D) and (E—H) Abnormal gonad of embryos from two
different mother fish. The abnormal gonads have structures resembling both
male (two-lobed organ) and female (endo-ovarian cavity) gonads. (A,E) Anterior
end; (B,C,F,G) central part; (D,H) posterior end. GC, germ cell; I, intestine;
K, kidney; M, mesentery; OC, ovarian cavity.
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© The Company of Biologists Ltd 2002
