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Endocrine disruption in fish: Effects of 17α-ethinylestradiol exposure on non-reproductive behavior, fertility and brain and testis transcriptome
Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Environmental Science.ORCID iD: 0000-0001-7239-7121
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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 [en]
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: urn:nbn:se:sh:diva-34913ISBN: 978-91-88663-43-6 (print)ISBN: 978-91-88663-44-3 (electronic)OAI: oai:DiVA.org:sh-34913DiVA, id: diva2:1202713
Public defence
2018-06-01, MA624, Alfred Nobels allé 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Funder
The Foundation for Baltic and East European StudiesAvailable from: 2018-05-08 Created: 2018-04-30 Last updated: 2018-05-08Bibliographically approved
List of papers
1. 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: 2019-04-18Bibliographically approved
2. 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: 2019-04-18Bibliographically approved
3. Persistent Effects of Developmental Exposure to 17α-Ethinylestradiol on the Zebrafish (Danio rerio) Brain Transcriptome and 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 Behavior
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2017 (English)In: Frontiers in Behavioral Neuroscience, ISSN 1662-5153, E-ISSN 1662-5153, Vol. 11, article id 69Article in journal (Refereed) Published
Abstract [en]

The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting compound of concern due to its persistence and widespread presence in the aquatic environment. Effects of developmental exposure to low concentrations of EE2 in fish on reproduction and behavior not only persisted to adulthood, but have also been observed to be transmitted to several generations of unexposed progeny. To investigate the possible biological mechanisms of the persistent anxiogenic phenotype, we exposed zebrafish embryos for 80 days post fertilization to 0, 3 and 10 ng/L EE2 (measured concentrations 2.14 and 7.34 ng/L). After discontinued exposure, the animals were allowed to recover for 120 days in clean water. Adult males and females were later tested for changes in stress response and shoal cohesion, and whole-brain gene expression was analyzed with RNA sequencing. The results show increased anxiety in the novel tank and scototaxis tests, and increased shoal cohesion in fish exposed during development to EE2. RNA sequencing revealed 34 coding genes differentially expressed in male brains and 62 in female brains as a result of EE2 exposure. Several differences were observed between males and females in differential gene expression, with only one gene, sv2b, coding for a synaptic vesicle protein, that was affected by EE2 in both sexes. Functional analyses showed that in female brains, EE2 had significant effects on pathways connected to the circadian rhythm, cytoskeleton and motor proteins and synaptic proteins. A large number of non-coding sequences including 19 novel miRNAs were also differentially expressed in the female brain. The largest treatment effect in male brains was observed in pathways related to cholesterol biosynthesis and synaptic proteins. Circadian rhythm and cholesterol biosynthesis, previously implicated in anxiety behavior, might represent possible candidate pathways connecting the transcriptome changes to the alterations to behavior. Further the observed alteration in expression of genes involved in synaptogenesis and synaptic function may be important for the developmental modulations resulting in an anxiety phenotype. This study represents an initial survey of the fish brain transcriptome by RNA sequencing after long-term recovery from developmental exposure to an estrogenic compound.

National Category
Biological Sciences
Research subject
Environmental Studies; Baltic and East European studies
Identifiers
urn:nbn:se:sh:diva-32462 (URN)10.3389/fnbeh.2017.00069 (DOI)000400103600001 ()28473760 (PubMedID)2-s2.0-85018300638 (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, 1556/42/2011Stockholm County Council, 806/3.1.1/2014
Available from: 2017-05-03 Created: 2017-05-03 Last updated: 2019-04-18Bibliographically approved
4. Combinatory effects of low concentrations of 17α-etinylestradiol and citalopram on non-reproductive behavior in adult zebrafish (Danio rerio)
Open this publication in new window or tab >>Combinatory effects of low concentrations of 17α-etinylestradiol and citalopram on non-reproductive behavior in adult zebrafish (Danio rerio)
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2017 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 193, p. 9-17Article in journal (Refereed) Published
Abstract [en]

Sewage treatment plant effluents contain a complex mixture of pharmaceuticals, personal care products and industrial chemicals, thus exposing aquatic organisms. Still, the consequences of exposure to combinations of different classes of drugs is largely unknown. In this study, we expose adult zebrafish (Danio rerio) males and females to low, environmentally relevant concentrations of the endocrine disrupting chemical 17α-ethinyl estradiol (EE2) and the selective serotonin re-uptake inhibitor (SSRI) citalopram, alone and in combination, and analyse three non-reproductive behaviours of importance for population fitness.

Two weeks exposure to 0.1 and 0.5 ng/LEE2 resulted in increased anxiety in males in the scototaxis (light/dark preference) test. Significantly longer latency periods before entering the white zone and fewer visits in the white zone were observed in males exposed to both 0.1 and 0.5 ng/LEE2 compared to unexposed males. No significant effects of citalopram alone (0.1 and 0.5 µg/L) were observed in the scototaxis test. The combined exposures (0.1 ng/L EE2 + 0.1 µg/L citalopram and 0.5 ng/L EE2 + 0.5 µg/L citalopram) resulted in abolishment of the anxiogenic effects of EE2, with significantly shorter latency period (low dose) and more transitions to white (high and low dose) than in fish exposed to EE2 alone. No significant effects of either EE2, citalopramor the combination of the two were observed in females. In the novel tank test, significantly more transitions to the upper half of the tank were observed in males exposed to 0.1 µg/L citalopram alone compared to unexposed males while males exposed to 0.1 ng/lEE2 had significantly shorter latency period to enter the upper half. Exposure to the combination of the two low concentrations did, however, result in a significantly longer latency and fewer transitions to upper half compared to both control, EE2- and citalopram-exposed males. These males also spent significantly less time in the upper half than the fish exposed to 0.1 ng/l EE2 or 0.1 µg/l citalopram alone. No significant effects on novel tank behaviour were observed in females or males exposed to the higher concentrations. In the shoaling test, males exposed to 0.1 µg/L citalopram and females exposed to 0.5 ng/l EE2 made significantly fewer transitions away from peers while males exposed to 0.1 µg/L citalopram + 0.1 ng/l EE2 performed significantly more transitions than the fish exposed to 0.1 µg/L citalopram alone.

In conclusion, this study shows that very low concentrations ofEE2, at or slightly above the predicted noeffect concentration (NOEC), affects anxiety in zebrafish males. Furthermore, citalopram, in spite of marginal effect of its own at such low levels, counteracts the response to EE2. This study represents an initial effort to understand the effects on water-living organisms of the cocktails of anthropogenic substances contaminating aquatic environments.

National Category
Environmental Sciences
Research subject
Environmental Studies
Identifiers
urn:nbn:se:sh:diva-32427 (URN)10.1016/j.aquatox.2017.10.001 (DOI)000417658800002 ()29017090 (PubMedID)2-s2.0-85030678324 (Scopus ID)
Funder
Stockholm County Council, 806/3.1.1/2014The Foundation for Baltic and East European Studies, 1352/3.1.1/2015
Note

As manuscript in dissertation with title: Combinatory effects of low-dose 17alpha-etinyl estradiol and citalopram on behavior in adult zebrafish (Danio rerio)

Available from: 2017-04-19 Created: 2017-04-19 Last updated: 2019-04-18Bibliographically approved
5. Altered non-reproductive behavior and feminization caused by developmental exposure to 17α-ethinylestradiol persist to adulthood in three-spined stickleback (Gasterosteus aculeatus)
Open this publication in new window or tab >>Altered non-reproductive behavior and feminization caused by developmental exposure to 17α-ethinylestradiol persist to adulthood in three-spined stickleback (Gasterosteus aculeatus)
Show others...
2019 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 207, p. 142-152Article in journal (Refereed) Published
Abstract [en]

The synthetic estrogen 17α-ethinylestradiol (EE2), ubiquitous in the aquatic environment and commonly detected in sewage effluents, interferes with the endocrine system in multiple ways. Exposure during sensitive windows of development causes persistent effects on fertility, reproductive and non-reproductive behavior in mammals and fish. In the present study, three-spined stickleback (Gasterosteus aculeatus) were exposed to nominal 0 and 20 ng/L EE2 from fertilization to 7 weeks post-hatch. After 8 months of remediation in clean water three non-reproductive behaviors, not previously analyzed in developmentally EE2-exposed progeny of wild-caught fish, were evaluated. Chemical analysis revealed that the nominal 0 and 20 ng/L exposure contained 5 and 30 ng/L EE2, respectively. Therefore, the use of control fish from previous experiments was necessary for comparisons. Fish exposed during development showed significant concentration-dependent reduction in anxiety-like behavior in the scototaxis (light/dark preference) test by means of shorter latency to first entrance to the white compartment, more visits in white, and longer total time in white compared to unexposed fish. In the novel tank test, developmental exposure significantly increased the number of transitions to the upper half of the aquaria. Exposure to EE2 during development did not alter shoal cohesion in the shoaling test compared with unexposed fish but fish exposed to 30 ng/L EE2 had significantly longer latency to leave the shoal and fewer transitions away from the shoal compared to fish exposed to 5 ng/L EE2. Skewed sex ratio with more females, sex reversal in genetic males as well as intersex in males was observed after exposure to 30, but not 5 ng/L EE2. In conclusion, EE2 exposure during development in three-spined stickleback resulted in persistent effects on anxiety-like behaviors. These long-term effects from developmental exposure are likely to be of higher relevance for natural populations than are short-term effects from adult exposure.

Keywords
Endocrine disruption, 17α-ethinylestradiol, fish, estrogens, developmental exposure, behavior, intersex
National Category
Environmental Sciences
Research subject
Environmental Studies; Baltic and East European studies
Identifiers
urn:nbn:se:sh:diva-34932 (URN)10.1016/j.aquatox.2018.11.024 (DOI)000457659300016 ()30572174 (PubMedID)2-s2.0-85058462347 (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, 1556/42/2011
Note

As manuscript in dissertation.

Available from: 2018-05-04 Created: 2018-05-04 Last updated: 2019-04-18Bibliographically approved

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