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Hitting the mark: Studies of alterations in behaviour and fertility in ethinyl estradiol-exposed zebrafish and search related biomarkers
Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology. Örebro universitet.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Resource type
Text
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. , p. 55
Series
Örebro Studies in Biology, ISSN 1650-8793 ; 10Södertörn doctoral dissertations, ISSN 1652-7399 ; 115
Keywords [en]
Endocrine disrupting compounds, 17α-ethinylestradiol, fertility, anxiety, behaviour, zebrafish, biomarkers, stress
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:sh:diva-29440ISBN: 978-91-7529-115-4 (print)OAI: oai:DiVA.org:sh-29440DiVA, id: diva2:900395
Public defence
2016-02-25, Sal MA636, Alfred Nobels allé 7, Huddinge, 13:00 (English)
Opponent
Supervisors
Funder
The Foundation for Baltic and East European StudiesAvailable from: 2016-02-04 Created: 2016-02-04 Last updated: 2016-02-04Bibliographically approved
List of papers
1. 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, p. 35-42Article 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.

Keywords
landscape scars, industrial heritage, waste, nuclear power, mining, Malmberget, Ignalina, Sweden, Lithuania
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-23362 (URN)10.1016/j.cbpc.2014.04.003 (DOI)000337769100005 ()24747828 (PubMedID)2-s2.0-84899872779 (Scopus ID)
Funder
The Foundation for Baltic and East European Studies
Available from: 2014-04-25 Created: 2014-04-25 Last updated: 2017-12-05Bibliographically approved
2. Developmental exposure of zebrafish (Danio rerio) to 17α-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 17α-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, p. 30-38Article in journal (Refereed) Published
Abstract [en]

Exposure to estrogenic endocrine disruptors (EDCs) during of development affects fertility, reproductive and non-reproductive 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 is less well studied. Here, we analyze the effects of 17α-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 1.6ng/L EE2. After remediation for 82days non-reproductive behavior and fertilization success were analyzed in both sexes. Males and females from the 1.2ng/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 1.2 and 1.6ng/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 1.2ng/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 were affected by their parents' exposure, which might suggest transgenerational effects.

Keywords
17α-Ethinylestradiol; Anxiety; Developmental exposure; Endocrine disruptors; F1 effects; Fertility; Neuroendocrinology; Social behavior; Stress behavior; Zebrafish
National Category
Biological Sciences Environmental Sciences
Research subject
Environmental Studies; Baltic and East European studies
Identifiers
urn:nbn:se:sh:diva-27795 (URN)10.1016/j.yhbeh.2015.05.014 (DOI)000360251800005 ()26072466 (PubMedID)2-s2.0-84934983120 (Scopus ID)1556/42/2011 (Local ID)1556/42/2011 (Archive number)1556/42/2011 (OAI)
Projects
Endocrine disruption in fish
Funder
The Foundation for Baltic and East European Studies, 1742/42/2008The Foundation for Baltic and East European Studies, 1556/42/2011
Available from: 2015-06-18 Created: 2015-06-18 Last updated: 2018-05-18Bibliographically approved
3. 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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2018 (English)In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 262, p. 44-58Article in journal (Refereed) Published
Abstract [en]

17α-Ethinylestradiol (EE2) is a ubiquitous aquatic contaminant shown to decrease fish fertility at low concentrations, especially in fish exposed during development. The mechanisms of the decreased fertility are not fully understood. In this study, we perform transcriptome analysis by RNA sequencing of testes from zebrafish with previously reported lowered fertility due to exposure to low concentrations of EE2during development. Fish were exposed to 1.2 and 1.6 ng/L (measured concentration; nominal concentrations 3 and 10 ng/L) of EE2 from fertilization to 80 days of age, followed by 82 days of remediation in clean water. RNA sequencing analysis revealed 249 and 16 genes to be differentially expressed after exposure to 1.2 and 1.6 ng/L, respectively; a larger inter-sample variation was noted in the latter. Expression of 11 genes were altered by both exposures and in the same direction. The coding sequences most affected could be categorized to the putative functions cell signalling, proteolysis, protein metabolic transport and lipid metabolic process. Several homeobox transcription factors involved in development and differentiation showed increased expression in response to EE2 and differential expression of genes related to cell death, differentiation and proliferation was observed. In addition, several genes related to steroid synthesis, testis development and function were differentially expressed. A number of genes associated with spermatogenesis in zebrafish and/or mouse were also found to be differentially expressed. Further, differences in non-coding sequences were observed, among them several differentially expressed miRNA that might contribute to testis gene regulation at post-transcriptional level. This study has generated insights of changes in gene expression that accompany fertility alterations in zebrafish males that persist after developmental exposure to environmental relevant concentrations of EE2 that persist followed by clean water to adulthood. Hopefully, this will generate hypotheses to test in search for mechanistic explanations.

National Category
Other Biological Topics
Identifiers
urn:nbn:se:sh:diva-29441 (URN)10.1016/j.ygcen.2018.03.011 (DOI)000430995100006 ()29526718 (PubMedID)2-s2.0-85044314644 (Scopus ID)1556/42/2011 (Local ID)1556/42/2011 (Archive number)1556/42/2011 (OAI)
Funder
The Foundation for Baltic and East European Studies, 1556/42/2011Stockholm County Council, 806/3.1.1/2014
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2016-02-02 Created: 2016-02-04 Last updated: 2018-05-16Bibliographically approved
4. 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:sh:diva-28425 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2015-09-30 Created: 2015-10-01 Last updated: 2017-10-24Bibliographically approved

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Caspillo, Nasim Reyhanian

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