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Testis transcriptome alterations in zebrafish (Danio rerio) with reduced fertility due to developmental exposure to 17α-ethinyl estradiol
Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Environmental Science.ORCID iD: 0000-0001-7239-7121
Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology. Örebro universitet.
Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology. Örebro universitet.
Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
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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
Resource type
Text
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.

Place, publisher, year, edition, pages
2018. Vol. 262, p. 44-58
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:sh:diva-29441DOI: 10.1016/j.ygcen.2018.03.011ISI: 000430995100006PubMedID: 29526718Scopus ID: 2-s2.0-85044314644Local ID: 1556/42/2011OAI: oai:DiVA.org:sh-29441DiVA, id: diva2:900442
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
In thesis
1. Hitting the mark: Studies of alterations in behaviour and fertility in ethinyl estradiol-exposed zebrafish and search related biomarkers
Open this publication in new window or tab >>Hitting the mark: Studies of alterations in behaviour and fertility in ethinyl estradiol-exposed zebrafish and search related biomarkers
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. p. 55
Series
Örebro Studies in Biology, ISSN 1650-8793 ; 10Södertörn doctoral dissertations, ISSN 1652-7399 ; 115
Keywords
Endocrine disrupting compounds, 17α-ethinylestradiol, fertility, anxiety, behaviour, zebrafish, biomarkers, stress
National Category
Other Biological Topics
Identifiers
urn:nbn:se:sh:diva-29440 (URN)978-91-7529-115-4 (ISBN)
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 Studies
Available from: 2016-02-04 Created: 2016-02-04 Last updated: 2016-02-04Bibliographically approved
2. Endocrine disruption in fish: Effects of 17α-ethinylestradiol exposure on non-reproductive behavior, fertility and brain and testis transcriptome
Open this publication in new window or tab >>Endocrine disruption in fish: Effects of 17α-ethinylestradiol exposure on non-reproductive behavior, fertility and brain and testis transcriptome
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Aquatic environments are the end recipient for most anthropogenic chemical pollutants. One common chemical pollutant found in the aquatic environment is 17α-ethinylestradiol (EE2), a synthetic estrogen used in contraceptive pills. EE2 is found in sewage treatment plant effluents and surface waters in concentrations from non-detectable up to 300 ng/L. EE2 has the ability to bioaccumulate and is more than 10 fold more potent in fish than the natural counterpart estradiol. Exposure has led to skewed sex ratios, decreased egg and sperm production, and altered reproductive behavior.  The aim of this thesis was to investigate the effects of EE2 exposure on non-reproductive behavior and fertility in fish. We found that zebra fish exposed to low concentrations of EE2 during development showed increased anxiety-like behavior and decreased fertility that were persistent in adulthood, even after a long remediation period in clean water. The altered behavior and lowered fertility were accompanied by alterations in the testis and brain transcriptome of possible significance for the behavior and fertility effects. The zebrafish was also used in adult exposures of EE2 and citalopram, alone and in combination to investigate if behavioral effects can be detected at very low concentrations, and if so, if the two compounds would interact and affect the behavioral outcome. Anxiety-like behavior was altered by EE2 and the two compounds in combination affected the outcome of each other. Further, when developmental exposure of progeny to wild caught three spined stickleback was used as a link between laboratory fish and natural fish populations, EE2 was found to decrease the anxiety-like behavior in the adult stickleback as well as cause ovotestis and intersex, feminization and sex reversal of genetic males. In conclusion, fertility and non-reproductive behaviors in the zebrafish and three spined stickleback are sensitive to EE2 exposure and effects from developmental exposures seem to be persistent.  Fertility and behavior are of high ecological significance for fish and alterations due to EE2 exposure might have negative effects on population fitness. The persistent alterations in the transcriptome of the zebrafish testis and brain lead to generation of hypotheses of mechanisms involved in the behavior and reproductive phenotypes caused by developmental exposure to EE2.

Abstract [sv]

Den akvatiska miljön är ofta särskilt utsatt för miljöföroreningar då de flesta ämnen förr eller senare sprids dit genom bland annat markavrinning, nedfall från luften och/eller från vattenreningsverk. Syntetiskt östrogen som används i p-piller, 17α-etinylestradiol (EE2), sprider sig ofta till vattenmiljön från avloppsvatten, då det bara delvis renas bort i vattenreningsprocessen. EE2 är ett hormonstörande ämne med förmågan att ansamlas i organismer och mer än tio gånger så verksamt i fisk som det naturliga hormonet estradiol (E2). Man har tidigare sett att exponering för EE2 även i de låga halter som påvisas vid vattenreningsverk bland annat har lett till minskade produktion av ägg och spermier samt förändrade parningsbeteenden.

I denna avhandling undersöks effekterna av EE2 på fiskars icke-reproduktiva beteende och fertilitet. Dessa faktorer är av hög ekologisk relevans för fisk då förändringar på dessa kan ge negativa effekter på populationsnivå. Resultaten visar att zebrafisk som exponeras för låga halter av EE2 under utvecklingen uppvisar ett mer ängsligt beteende och har lägre fertilitet när de blir vuxna även efter en lång återhämtningsperiod i rent vatten. Det förändrade beteendet uppvisas även hos avkomman till dessa fiskar.  Förändringarna i beteende och fertilitet åtföljdes av förändringar i hjärnans och testikelvävnadens transkriptom, dvs. förändringar i vilka gener som uttrycks. I zebrafiskens testikelvävnad hittades bland annat en skillnad i uttryck av gener kopplade till könsdifferentiering och utveckling samt spermatogenesen, gener som kan ha betydelse för den nedsatta fertiliteten. I zebrafiskens hjärna hittades ingen skillnad i uttryck på gener direkt kopplade till stressaxeln men däremot på flera andra gener i nätverk som indirekt kan kopplas till det ängsliga beteendet som dygnsrytm och kolesterolsyntes. I naturen exponeras organismer ofta för en blandning av föroreningar. Därför undersöktes också effekter av EE2 i kombination med citalopram, ett antidepressivt läkemedel som ofta påvisas i vattenmiljön, för att undersöka om förändringar i beteende kan påvisas även vid väldigt låga koncentrationer av varje ämne och, om så är fallet, vilka effekter en kombination av de två ämnena ger. Vuxna zebrafiskar uppvisade beteendeförändringar även vid dessa låga koncentrationer efter två veckors exponering och de två ämnena påverkade beteendet olika var för sig och i kombination. Vidare, användes vildfångad storspigg i ett exponeringsexperiment för att undersöka om resultaten från zebrafisken, som är en avlad laboratoriefisk, kan ses också i vilda fiskpopulationer. Även storspiggar som exponerades för EE2 under utveckling fick ett modifierat beteende samt en skev könsfördelning som vuxna: fler honor fanns hos de storspiggar som exponerats och vidare undersökningar visade att dessa var genetiska hanar som utvecklats som honor.

Den här avhandlingen visar beständiga förändringar i zebrafiskens transkriptom i hjärna och testikelvävnad efter exponering för EE2 i låga halter under utveckling. Både icke-reproduktiva beteenden och fertilitet visade sig känsliga för EE2 i såväl zebrafisk som storspigg och effekterna verkar vara permanenta. Sammanfattningsvis har EE2 effekter på ekologiskt relevanta faktorer hos fisk i de halter som förekommer i vattenmiljön och hormonstörande föroreningar som EE2 kan ge långvariga effekter som syns senare i livet även vid låga koncentrationer.

Place, publisher, year, edition, pages
Huddinge: Södertörns högskola, 2018. p. 165
Series
Södertörn Doctoral Dissertations, ISSN 1652-7399 ; 155
Keywords
17α-ethinylestradiol, endocrine disrupting chemical, fish, behavior, fertility, transcriptome, RNA sequencing
National Category
Environmental Sciences
Research subject
Environmental Studies; Baltic and East European studies
Identifiers
urn:nbn:se:sh:diva-34913 (URN)978-91-88663-43-6 (ISBN)978-91-88663-44-3 (ISBN)
Public defence
2018-06-01, MA624, Alfred Nobels allé 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Funder
The Foundation for Baltic and East European Studies
Available from: 2018-05-08 Created: 2018-04-30 Last updated: 2018-05-08Bibliographically approved

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Porseryd, ToveReyhanian Caspillo, NasimVolkova, KristinaElabbas, LubnaDinnétz, PatrikPorsch Hällström, Inger

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