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Hitting the mark: studies of alterations in behaviour and fertility in ethinyl estradiol-exposed zebrafish and search related biomarkers
Örebro University, School of Science and Technology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, we have analysed the effects of EE2 on non-reproductive behaviours and fertility. We have showed that two doses of EE2 in male adult short-term exposures evokes opposite behaviours in the novel tank test. A lower dose induced increased bottom-dwelling, a sign of increased anxiety and a higher dose increased surface-dwelling, which would likely expose themselves to predation in a natural environment. Increased shoaling was observed in both exposures, possibly affecting feeding and reproduction opportunities. Fertility analysis of these fish demonstrated a complete inhibition of spawning in the highest dose group. To investigate mechanisms behind the spawning failure, we examined expression levels of genes involved in zebrafish sex differentiation and maintenance of gonadal function. We found downregulated transcription levels of male-predominant genes, suggesting a demasculinization of the testes contributing to functional sterility in these fish. We have demonstrated that non-reproductive behaviour in zebrafish is highly sensitive to EE2 exposure during development. After exposing male and female zebrafish to low doses of EE2 followed by remediation in clean water until adulthood, the fish displayed increased anxiety and shoaling behaviour, demonstrating persistent effects of EE2. Furthermore, behavioural effects were transferred to their progeny. Decreased fertilisation success of the developmentally exposed fish was observed in both sexes when mated to untreated animals of the opposite sex. These fertility effects persisted although the fish had a long remediation period, implying likely reduced fitness of fish populations in aquatic environments. Based on our findings on non-reproductive behaviours and fertility, we performed RNAsequencing analysis of the brain and testes in order to investigate possible biological mechanisms behind the persistent effects. There is a need for biomarkers allowing detection of both reversible and irreversible effects in animals exposed to estrogenic substances, hopefully contributing to better risk assessments for EDCs. Results from RNA-sequencing would serve as a basis for continued studies in pursuit of potential biomarkers.

Place, publisher, year, edition, pages
Örebro: Örebro university , 2016. , 55 p.
Series
Örebro Studies in Biology, ISSN 1650-8793 ; 10
Series
Södertörn Doctoral Dissertations, ISSN 1652-7399 ; 115
Keyword [en]
Endocrine disrupting compounds, 17α-ethinylestradiol, fertility, anxiety, behaviour, zebrafish, biomarkers, stress
National Category
Other Biological Topics
Research subject
Biology
Identifiers
URN: urn:nbn:se:oru:diva-47393ISBN: 978-91-7529-115-4 (print)OAI: oai:DiVA.org:oru-47393DiVA: diva2:893771
Public defence
2016-02-25, Sal MA636, Södertörns högskola, Alfred Nobels allé 7, Huddinge, 13:00 (English)
Opponent
Supervisors
Available from: 2016-01-13 Created: 2016-01-13 Last updated: 2017-10-17Bibliographically approved
List of papers
1. 17 alpha-Ethinyl estradiol affects anxiety and shoaling behavior in adult male zebra fish (Danio rerio)
Open this publication in new window or tab >>17 alpha-Ethinyl estradiol affects anxiety and shoaling behavior in adult male zebra fish (Danio rerio)
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2011 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 105, no 1-2, 41-48 p.Article in journal (Refereed) Published
Abstract [en]

Ethinyl estradiol is a potent endocrine disrupting compound in fish and ubiquitously present in the aquatic environment. In this study, we exposed adult zebra fish (Danio rerio) males to 0,5 or 25 ng Ethinyl estradiol/L for 14 days and analyzed the effects on non-reproductive behavior. Effects of treatment of the exposed males was shown by vitellogenin induction, while brain aromatase (CYP 19B) activity was not significantly altered. Both concentrations of Ethinyl estradiol significantly altered the behavior in the Novel tank test, where anxiety is determined as the tendency to stay at the bottom when introduced into an unfamiliar environment. The effects were, however, opposite for the two concentrations. Fish that were exposed to 5 ng/L had longer latency before upswim, fewer transitions to the upper half and shorter total time spent in the upper half compared with control fish, while 25 ng Ethinyl estradiol treatment resulted in shorter latency and more and longer visits to the upper half. The swimming activity of 25, but not 5 ng-exposed fish were slightly but significantly reduced, and these fish tended to spend a lot of time at the surface. We also studied the shoaling behavior as the tendency to leave a shoal of littermates trapped behind a Plexiglas barrier at one end of the test tank. The fish treated with Ethinyl estradiol had significantly longer latency before leaving shoal mates and left the shoal fewer times. Further, the fish exposed to 5 ng/L also spent significantly less time away from shoal than control fish. Fertilization frequency was higher in males exposed to 5 ng/L Ethinyl estradiol when compared with control males, while no spawning was observed after treatment with 25 ng/L The testes from both treatment groups contained a normal distribution of spermatogenesis stages, and no abnormality in testis morphology could be observed. In conclusion, we have observed effects on two behaviors not related to reproduction in zebra fish males after treatment with Ethinyl estradiol, adding to the ecological consequences of contamination of aquatic environments with estrogenic substances. (C) 2011 Elsevier B.V. All rights reserved.

National Category
Biological Sciences
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-18647 (URN)10.1016/j.aquatox.2011.05.009 (DOI)000294317500005 ()
Available from: 2011-09-30 Created: 2011-09-29 Last updated: 2017-12-08Bibliographically approved
2. Short-term treatment of adult male zebrafish (Danio Rerio) with 17α-ethinyl estradiol affects the transcription of genes involved in development and male sex differentiation.
Open this publication in new window or tab >>Short-term treatment of adult male zebrafish (Danio Rerio) with 17α-ethinyl estradiol affects the transcription of genes involved in development and male sex differentiation.
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2014 (English)In: Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology, ISSN 1532-0456, E-ISSN 1878-1659, Vol. 164, 35-42 p.Article in journal (Refereed) Published
Abstract [en]

The synthetic estrogen 17α-ethinyl estradiol (EE2) disturbs reproduction and causes gonadal malformation in fish. Effects on the transcription of genes involved in gonad development and function that could serve as sensitive biomarkers of reproductive effects in the field is, however, not well known. We have studied mRNA expression in testes and liver of adult zebrafish (Danio rerio) males treated with 0, 5 or 25ng/L EE2for 14days. qPCR analysis showed that the mRNA expression of four genes linked to zebrafish male sex determination and differentiation, Anti-Mullerian Hormone, Double sex and mab-related protein, Sry-related HMG box-9a and Nuclear receptor subfamily 5 group number 1b were significantly decreased by 25ng/L, but not 5ng/L EE2 compared with the levels in untreated fish. The decreased transcription was correlated with a previously shown spawning failure in these males (Reyhanian et al., 2011. Aquat Toxicol 105, 41-48), suggesting that decreased mRNA expression of genes regulating male sexual function could be involved in the functional sterility. The mRNA level of Cytochrome P-45019a, involved in female reproductive development, was unaffected by hormone treatment. The transcription of the female-specific Vitellogenin was significantly induced in testes. While testicular Androgen Receptor and the Estrogen Receptor-alpha mRNA levels were unchanged, Estrogen receptor-beta was significantly decreased by 25ng/L EE2. Hepatic Estrogen Receptor-alpha mRNA was significantly increased by both exposure concentrations, while Estrogen Receptor-beta transcription was unaltered. The decreased transcription of male-predominant genes supports a demasculinization of testes by EE2 and might reflect reproductive disturbances in the environment.

Place, publisher, year, edition, pages
New York: Elsevier, 2014
Keyword
17 alpha-Ethinyl estradiol; Biomarker; Endocrine disruptors; Gene regulation; Gonads; Sex differentiation; Zebrafish
National Category
Natural Sciences
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-35475 (URN)10.1016/j.cbpc.2014.04.003 (DOI)000337769100005 ()24747828 (PubMedID)2-s2.0-84899872779 (Scopus ID)
Note

Funding Agencies:

Swedish Baltic Sea Foundation

Stockholm County Council

Available from: 2014-06-24 Created: 2014-06-24 Last updated: 2017-12-05Bibliographically approved
3. Developmental exposure of zebrafish (Danio rerio) to 17 alpha-ethinylestradiol affects non-reproductive behavior and fertility as adults, and increases anxiety in unexposed progeny
Open this publication in new window or tab >>Developmental exposure of zebrafish (Danio rerio) to 17 alpha-ethinylestradiol affects non-reproductive behavior and fertility as adults, and increases anxiety in unexposed progeny
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2015 (English)In: Hormones and Behavior, ISSN 0018-506X, E-ISSN 1095-6867, Vol. 73, 30-38 p.Article in journal (Refereed) Published
Abstract [en]

Exposure to estrogenic endocrine disruptors (EDCs) during development affects fertility, reproductive and nonreproductive behavior in mammals and fish. These effects can also be transferred to coming generations. In fish, the effects of developmental EDC exposure on non-reproductive behavior are less well studied. Here, we analyze the effects of 17 alpha-ethinylestradiol (EE2) on anxiety, shoaling behavior and fertility in zebrafish after developmental treatment and remediation in clean water until adulthood. Zebrafish embryos were exposed from day 1 to day 80 post fertilization to actual concentrations of 1.2 and 1.6 ng/L EE2. After remediation for 82 days non-reproductive behavior and fertilization success were analyzed in both sexes. Males and females from the 1.2 ng/L group, as well as control males and females, were bred, and behavior of the untreated F1 offspring was tested as adults. Developmental treatment with 12 and 1.6 ng/L EE2 significantly increased anxiety in the novel tank test and increased shoaling intensity in both sexes. Fertilization success was significantly reduced by EE2 in both sexes when mated with untreated fish of opposite sex. Progeny of fish treated with 1.2 ng/L EE2 showed increased anxiety in the novel tank test and increased light avoidance in the scototaxis test compared to control offspring. In conclusion, developmental exposure of zebrafish to low doses of EE2 resulted in persistent changes in behavior and fertility. The behavior of unexposed progeny was affected by their parents' exposure, which might suggest transgenerational effects.

Keyword
Endocrine disruptors, 17 alpha-Ethinylestradiol, Stress behavior, Developmental exposure, Zebrafish, Neuroendocrinology, Social behavior, Anxiety, F1 effects, Fertility
National Category
Biological Sciences
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-45884 (URN)10.1016/j.yhbeh.2015.05.014 (DOI)000360251800005 ()26072466 (PubMedID)2-s2.0-84934983120 (Scopus ID)
Note

Funding Agency:

Swedish Baltic Sea Foundation 1742/42/2008 1556/42/2011

Available from: 2015-09-23 Created: 2015-09-21 Last updated: 2017-12-01Bibliographically approved
4. Testis transcriptome alterations in zebrafish (Danio rerio) with reduced fertility due to developmental exposure to 17α-ethinyl estradiol
Open this publication in new window or tab >>Testis transcriptome alterations in zebrafish (Danio rerio) with reduced fertility due to developmental exposure to 17α-ethinyl estradiol
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(English)Manuscript (preprint) (Other academic)
National Category
Other Biological Topics
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-47887 (URN)
Available from: 2016-02-02 Created: 2016-02-02 Last updated: 2017-10-17Bibliographically approved
5. Persistent effects of developmental exposure to 17α-ethinylestradiol on the zebrafish (Danio rerio) brain transcriptome and stress behavior
Open this publication in new window or tab >>Persistent effects of developmental exposure to 17α-ethinylestradiol on the zebrafish (Danio rerio) brain transcriptome and stress behavior
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(English)Manuscript (preprint) (Other academic)
National Category
Zoology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-45958 (URN)
Available from: 2015-09-30 Created: 2015-09-30 Last updated: 2017-10-17Bibliographically approved

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