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Doi:10.1016/j.cca.2004.09.004

Comparative Biochemistry and Physiology, Part C 139 (2004) 47 – 55 Life-stage-dependent sensitivity of zebrafish (Danio rerio) to estrogen exposure Gerd Maack*, Helmut Segner1 UFZ Centre for Environmental Research, Department of Chemical Ecotoxicology, Permoserstr. 15, D-04318 Leipzig, Germany Received 11 March 2004; received in revised form 6 September 2004; accepted 9 September 2004 The aim of this study was to identify periods in zebrafish (Danio rerio) development when estrogen exposure has long-term consequences on reproductive capabilities at the adult stage. To this end, zebrafish were exposed to 10 ng/L ethynylestradiol (EE2) during three stages ofgonadal differentiation: (i) the juvenile hermaphroditic stage when gonads display the morphology of an immature ovary (in our zebrafishcolony this lasted from 15 to 42 days post-fertilization [dpf]), (ii) the gonad transition stage when the hermaphroditic gonad differentiates intoeither testes or ovary (from 43 to day 71 dpf), and (iii) the premature stage of testicular and ovarian development (from 72 to 99 dpf). Theconsequences of stage-specific exposure to EE2 were assessed by determining time to first spawning, fecundity (number of eggs per femaleper day), fertilization success (percentage of fertilized eggs) and sex ratio of the adults. Exposure during the gonad transition period induced adelay in the onset of spawning and a significant reduction of fecundity and fertilization success, whereas exposure during the hermaphroditicstage or during the premature stage had no significant impact on the reproductive parameters of adult fish. The results from this experimentpointed to the gonad transition stage as being most susceptible to persistent effects of developmental estrogen exposure. In a secondexperiment, the concentration dependency of the EE2effects was evaluated by exposing zebrafish during the gonad transition stage (43–71dpf) to 1.67, 3 or 10 ng EE2/L. Significant effects of EE2 on adult reproduction were found with 3 and 10 ng EE2/L, but not with 1.67 ng/L.
Histological examination of the gonads revealed that at termination of EE2 exposure (71 dpf), all individuals in the 3 and 10 ng EE2/Ltreatment possessed ovaries. However, this feminising effect appeared to be reversible since at the adult stage (190 dpf), both fish withovaries and with testes were found. Thus, EE2 exposure during the gonad transition stage seems to have no persistent effect on gonadhistology but on reproductive capabilities.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Zebrafish; Danio rerio; 17a-ethynylestradiol; Gonadal development; Gonad histology; Reproduction; Sexual differentiation; Stage-specific modulate or disrupt developmental and reproductive pro-cesses (Among these so-called There is considerable concern about the potential of both endocrine-disrupting compounds (EDCs), substances with natural and man-made environmental substances to interfere estrogenic activity have attracted most attention. Particularly with the endocrine system of vertebrates and thereby to for fish, which show high plasticity in phenotypic sexualdifferentiation (a number oflaboratory and field studies have demonstrated that estro- * Corresponding author. Current address: Department of Biological gen-active compounds are able to disturb sexual develop- and Chemical Sciences, Hatherly Laboratories, University of Exeter, Prince ment and reproductive capabilities ( of Wales Road, Exeter, EX4 4PS, United Kingdom. Tel.: +44 1392 263796; Tyler et al., 1998; L7nge et al., 2001; Segner et al., 2003).
fax: +44 1392 263700.
The developmental and reproductive effects of estrogen- E-mail address: [email protected] (G. Maack).
1 active substances are depending on the precise time in the Current address: Centre for Fish and Wildlife Health, University of Bern, L7nggass-Str. 122, CH-3012 Bern, Switzerland.
life cycle when the endocrine signal is being released at a 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cca.2004.09.004 G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 specific dosage (In many teleost fish surface waters at concentrations able to affect reproductive species, sex steroids are considered to have organizational parameters of fish ( effects on sex differentiation, i.e. to direct the developing gonad into the male or female direction (Piferrer, 2001; Devlin and Nagahama, 2002; Strqssmannand Nakamura, 2002). The ontogenetic period during which 2. Materials and methods the undifferentiated gonads are susceptible to the organiza-tional effects of steroids is the critical or labile period 2.1. Culture of fish (In gonochor-istic fish, this period covers the developmental window prior Fertilised eggs for the tests were obtained from parental to or concomitant with the histological differentiation of the fish reared in the laboratory of the UFZ, Leipzig, Germany.
indifferent gonad into either ovary or testis. The chrono- The zebrafish strain was a descendant of progeny from West logical timing of this period can vary strongly among fish Aquarium, Germany, a commercial breeder of ornamental species, from the embryo/hatching stage until the juvenile fish, and have been bred at the UFZ laboratory for 3 years.
The fishes were devoid of obvious disease and were free of The critical window is of particular relevance for tuberculosis. Fish were kept in filtered tap water, which was understanding the effects of EDCs on fish, since even purified with activated charcoal and aerated. The water short-term exposure during this life stage may have lasting, temperature was maintained at 26 8C F 1 8C. Light/dark irreversible effects on phenotypic sex and reproductive cycle was 12 h/12 h. Animals were fed twice a day with function. From a practical point of view, knowledge on the TetraMinR dry flake food (Tetra Werke, Melle, Germany) ad critical window will help to establish short-term tests for libitum and once a day with nauplii of the crustacean assessing the consequences of developmental EDC expo- sure on adult reproductive capabilities (Benson, 1998: Papoulias et al., 1999; Koger et al., 2000).
2.2. Test substance and chemical analysis Zebrafish (Danio rerio) has been suggested as an experimental model in assessing developmental and The tests were performed under semi static conditions, reproductive effects of estrogen-active compounds (see with a total exchange of the test solution every third day.
In a number of studies, zebrafish 17a-ethynylestradiol (MM 296.4 g, CAS 57-63-6) was were exposed to estrogens and estrogen mimics either obtained from Sigma (Deisenhofen, Germany) with a purity during early life stages or during the adult stage and the of 98%. A stock solution of 10 mg EE2/10 mL acetone was effects on vitellogenin, gonad histology, or reproductive diluted with double autoclaved distilled water to a concen- parameters were assessed at the end of the exposure tration of 1 mg EE2/L. The nominal test concentrations were 1.67, 3 and 10 ng EE2/L. Analysis of the actual EE2 ¨ rn et al., 2003; Fenske et al., in press). The aim of the concentration in the test water was performed on extracts present study is to compare the consequences of estrogen using gas chromatographic separation with ion trap GC/MS/ exposure of zebrafish during specific stages of gonad MS (GC Varian Saturn 3400, MS Varian Saturn 4D). The development on reproductive capabilities at the adult method is described in detail by stage. To this end, zebrafish were exposed to the synthetic estrogen, 17a-ethynylestradiol (EE2), during the following The actual test concentrations of EE2 in the aquaria were periods: (i) stage of juvenile hermaphroditism or non- in good agreement with the nominal concentrations. The functional protogyny when all individuals, irrespective of measured values of EE2 varied between 80% and 120% of their genetic sex, display the morphology of an immature the nominal concentrations of 3 and 10 ng EE2/L, ovary (in our zebrafish colony: from 15 to 42 days post- respectively. Only for the 1.67-ng EE2/L treatment the fertilization, [dpf]; (ii) the average concentration of EE2 accounted for only 71% (1.14 gonad transition stage when the hermaphroditic ovary ng EE2/L) with a variation from 15 to 110% of the nominal either proceeds to develop as ovary or resolves into a testis (43 to 71 dpf), and (ii) the premature stage whenthe early ovary or testis complete developmental differ- 2.3. Exposure experiments: rearing conditions entiation (72 to 99 dpf). The developmental stage that wasmost susceptible to persisting effects of EE2 on adult The exposure experiments started with larvae at an age of reproduction was the gonad transition stage. In a second 15 dpf. 80 individuals of that age were kept per one glass experiment, therefore, we further explored EE2 effects on aquarium of 202015 cm (lengthdepthheight; total this particular life stage by studying the concentration- volume=6 L) containing 5 L of water. In order to maintain dependency of the EE2 effects as well as the associated stable water temperature, these aquaria were placed into gonad histological changes. The test compound EE2 is a larger glass aquaria with a total volume of 29 L heated water component of oral contraceptives and can be present in (402727 cm; lengthdepthheight). At 43 dpf the G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 number of zebrafish per vessel was reduced to 60 tions—see above). The experiment was conducted with 3 individuals and the fish were transferred into larger aquaria replicates per concentration and 3 additional control containing 20 L of water. At 71 dpf, the number of fish per replicates. The experiment started with 960 zebrafish aquarium was reduced to 40 individuals per 20-L aquarium.
larvae in 12 aquaria.
For the measurement of the reproductive parameters, the gives an overview of the design of both experi- fishes were grouped at 99 dpf, shortly before the onset of ments. Effect endpoints included the reproductive parame- spawning, in breeding groups of four females and eight ters: onset of spawning, number of eggs per female males per vessel.
(fecundity) and fertilization success and, additionally in Oxygen concentration, pH value and temperature in the experiment 2, gonad histology (in order to assess effects on aquaria were measured twice a week. The mean temper- gonadal sex ratio and on the morphological differentiation ature F S.D. (standard deviation) for all test vessels was of ovaries and testes).
26.0 F 0.6 8C. Single values ranged from 24.5 to 27.5 8C.
The mean pH-value for the test vessels was 8.2F0.2, with 2.4. Effect endpoints: reproductive parameters single values ranging from 7.7 to 8.4. The oxygen saturationvalues ranged from 70.9% to 94.6% with a mean value of At 99 dpf, spawning groups comprising four females and 87.2F4.6% (equivalent to 6.96 mg dissolved oxygen/L).
eight males were placed in each test vessel. Spawning trays Nitrite was measured weekly and concentrations were were placed at the bottom of the aquaria. The trays were mostly below the detection limit, with a maximum of 0.3 covered by lattice lids (stainless steel). Artificial bspawning- mg/L. The carbonate hardness was 2.5–2.8 mmol/L.
treesQ (i.e. plastic water plants for breeding stock aquariaand glass plants during exposure), were attached to the lids 2.3.1. Exposure experiment 1: exposure to 10 ng EE2/L to enhance the spawning (modified method according to during different developmental stages When mating behaviour and spawning were The test concentration of 10 ng EE2/L (nominal) was observed regularly (i.e. 3 to 4 days in a row), reproductive selected on the basis of the results from previous full life parameters were measured over a 25-day-period. After cycle experiments (EC50, fertility: 1.1 ng EE2/L) and of a completing the reproduction assessment, fish from the 28-day prolonged toxicity test with juvenile and adult Experiment 1 were sexed by macroscopic examination of zebrafish (EC50, lethality: 100 ng EE2/L) (C. Sch7fers, FhG the gonads to check the male/female ratio that was Institute for Molecular and Applied Ecology, Schmallen- established at the beginning of the spawning period.
berg, Germany: unpublished data).
The fish of experiment 2 were sexed by histological Zebrafish were exposed to 10 ng EE2/L during the analysis. The number of eggs per tank was related to the following developmental stages: number of females in order to calculate the fecundity (i.e.
number of eggs per female and day). Fertilization per tank day 15 to day 42 pf (juvenile hermaphroditic stage), was determined by the percentage of fertilized eggs day 43 to day 71 pf (transition stage), compared to the total amount of eggs laid.
day 72 to day 99 pf (stage of pre-mature gonads).
2.5. Effect endpoints: histological analysis of gonads For each period, four replicates of EE2 treatment and four replicates of control fish were used. The experiment started Histological analysis was done on fish from experiment 2 with 1280 zebrafish larvae in 16 test vessels each containing At each sampling point, a maximum of 20 fish were To examine whether zebrafish were able to recover from taken per concentration for histological analysis. In total, possible developmental effects of EE2 exposure, reproduc- 229 zebrafish from the second experiment, were examined tive parameters were not only measured in 150–180-day-old histologically (.
fish (see experiments 1 and 2), but were measured a second For sampling, fish were anaesthetized in ice water and time 3 weeks later, between days 213 and 250 pf. Assess- were then fixed in Bouin' s fluid for 24 h. Young stages ment of reproductive parameters was performed as were fixed entirely, larger fish were first decapitated. To described below.
facilitate infiltration of the fixative, a ventral incision intothe body of the fish was made. Fixed tissues were 2.3.2. Exposure experiment 2: exposure to different EE2 dehydrated through a graded series of ethanol and embed- concentrations during the gonad transition stage (43–71 ded in paraffin wax (Leica Histowax). Serial sections were cut at 7 Am and collected onto glass slides, stained with For the period of the transition of the hermaphroditic Periodic-Acid Schiff's (PAS) and analysed by light micro- gonad into testis or ovary (43–71 dpf), the concentration scopy. Seventy-one-day-old fishes were dehydrated and dependence of the EE2 effects was examined by infiltrated with Technovit 7100R (hydroxyethylmethacry- exposing zebrafish to 1.67, 3 and 10 ng EE2/L, late) according to the manufacturer's instructions (Kulzer, respectively (nominal concentrations; actual concentra- Germany) and sectioned (2–5 Am). Technovit sections were G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 Fig. 1. Design of the zebrafish exposure experiments. Both experiments were started with 15 dpf larvae. (A) Experiment 1; exposure to 10 ng EE2/L duringdifferent stages of gonadal development: hermaphroditic stage, gonad transition stage, and premature gonad stage. The different endpoints measured atdifferent times are indicated by arrows. Four replicates per treatment were used. (B) Experiment 2; exposure to 1.7, 3, or 10 ng EE2/L during the gonadtransition stage. The different endpoints measured at different times are indicated by arrows. Three replicates per treatment were used.
stained in toluidin–methylene blue, dried overnight at 60 8C and mounted with EntellanR.
4.1. Experiment 1: exposure during different developmentalstages: effects on adult reproduction Developmental exposure to 10 ng EE2/L had no effect on Mean values and standard deviation of the total number the time of first spawning, regardless at which develop- of eggs per female and day, and of the fertilization success mental stage the estrogen was administered. In the control per day (n=20–25 counts) were calculated. To create a replicates, first spawning was observed at 138F6.4 dpf, normal distribution, fertilization values (0–100%) were z- while first spawning occurred at 126F1.2 dpf in fish transformed (arc sin (square root x)), and egg numbers were exposed between 15 and 42 dpf, at 150F2.0 dpf in fish log-transformed. With the transformed data, ANOVA was exposed between 43 and 71 dpf, and at 138F4.1 dpf in fish performed. In case of significant differences between the exposed between 77 and 99 dpf.
mean values, Scheffle's Test was used to analyse which The effects of developmental exposure to 10 ng EE2/L mean values were significantly lower than the mean value of on fecundity and fertilization success of adult zebrafish the control group.
was determined at two time periods: from 155 to 180 dpf, G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 The results of this experiment indicated that devel- opmental EE2 exposure has significant long-term conse-quences on reproductive capabilities of adult zebrafishwhen exposure occurs during the period between day 42and 71 pf, i.e. during the gonad transition stage. Therefore,we focused the further work on this developmental periodand studied in a second experiment the concentrationdependency of the estrogenic effect as well as gonaddifferentiation.
4.2. Experiment 2: exposure to graded concentrations ofEE2 during the gonad transition stage: effects on adult Fig. 2. Mean number of eggs per adult female per day (bars; left y-axis) of reproduction and on gonadal differentiation zebrafish treated with 10 ng/L EE2 during different developmental stages(x-axis), and mean percentage of fertilized eggs (points/right y-axis). Datarepresent average values of 25 daily counts, undertaken from 155 to 180 4.2.1. Adult reproduction dpf; (*significant pb0.05, following Sheffle's Test; one way ANOVA).
Juvenile zebrafish were exposed to 1.67, 3 or 10 ng EE2/ Sexing of fish was done after finalisation of the breeding experiment by L between 43 and 71 dpf, and effects on time of first macroscopical examination of the gonads.
spawning, fecundity and fertility were assessed at the adultstage. Fish from the control replicates started to spawn on and from 213 to 250 dpf. The spawning groups were 137F9.8 dpf, while fish exposed to 10 ng EE2/L showed composed of four females and eight males (selected on the first spawning at 160F9.0 dpf. Fish exposed to the two basis of external morphology; with the classification being lower EE2 concentrations (1.67 and 3 ng EE2/L) started to confirmed or rejected after termination of the spawning spawn at an earlier age than the control: at 123F4.0 dpf in experiment by means of gonad histology). During the the group treated with 1.67 ng EE2/L, and at 124F5.4 dpf in measurement periods, the fish showed normal mating the 3 ng EE2/L-group.
behaviour, and daily spawning. Fecundity of developmen- Fecundity and fertilization success were measured in tally exposed fish, however, was reduced regardless at adult zebrafish between 165 and 190 dpf. The number of which developmental stage the fish had been exposed. In eggs per female per day did not differ significantly between the period between 155 and 180 dpf, female zebrafish controls and the EE2 treatments (Fertilization from the control group produced 11.3F0.6 eggs per day success, however, showed a concentration-dependent and female. These numbers were 6.9F0.6 for fish exposed reduction: It decreased from 88.2% in control fish to from 15 to 42 dpf, 3.7F0.7 for fish exposed between 43 76.3% in fish treated with 1.67 ng EE2/L, to 62.7% in fish and 71 dpf, and 7.6F0.7 for fish exposed between 72 and exposed to 3 ng EE2/L and to 48.6% in the group exposed 99 dpf, respectively (left y-axis). Compared to the to 10 ng EE2/L (The regression coefficient for the control group, a significant ( pb0.05) decrease occurred concentration-dependent decline of fertilization success was only in fish treated during the gonad transition stage (between 43 and 71 dpf).
A similar response as described for fecundity was observed for the fertilization success (right y-axis in 2). During the period between 155 and 180 dpf, fertilizationsuccess in the control treatment was 76.8F1.7% (Fish exposed from 15 to 42 dpf or from 72 to 99 dpf showeda slight, but non-significant reduction (64.3F2.2% and67.8F1.7%, respectively), while zebrafish developmentallyexposed during the gonad transition stage (43–71 dpf)showed a significant ( pb0.05) decline of fertilizationsuccess (51.8F2.8%).
Assessment of reproductive parameters in 213- to 250- day-old adult zebrafish revealed that fecundity was notdifferent between control and fish exposed between 43 and71 dpf (11.0F1.3 eggs per day and female in both Fig. 3. Mean number of eggs per adult female per day (bars; left y-axis) of treatments). Fertilization success, however, still differed zebrafish treated with different concentrations of EE2 (x-axis) during 43–71 significantly: Zebrafish exposed to 10 ng EE dpf, and mean percentage of fertilized eggs (points/right y-axis). Data represent average values of 25 daily counts undertaken, undertaken from 43 and 71 dpf showed a fertilization success of 165–190 dpf (*significant pb0.05, following Sheffle's Test; one-way 62.7F2.1%, while in controls, the fertilization success ANOVA). Sexing of fish was done after finalisation of the breeding was 76.8F1.7%.
experiment by histological examination of the gonads.

G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 ing 12 fish showed the morphology of immature testes, withspermatogonia, spermatocytes, spermatids and no or veryfew spermatozoa (In the groups exposed to 3 or 10ng EE2/L, all individuals had gonads with ovarian morphol-ogy, and no single fish possessed a testis. In the 1.67-nggroup, 11 out of 13 fish had ovaries while the remaining twoindividuals contained testes. The histological structure ofovaries and testes from EE2-treated fish was not different tothat found in control fish.
4.2.3. Gonad histology of 190-day-old zebrafish After termination of the spawning measurements, fish were sacrificed and the gonad morphology was examinedhistologically. Although at 71 dpf, we observed all-female Fig. 4. Gonadal sex ratio (determined by histological analysis) of 71-dpf- cohorts in the 3 and 10 ng/L treatments, at the adult stage old zebrafish exposed from 43 to 71 dpf to different concentration of EE2(Experiment 2) (*significant p both fishes with testes and ovaries were present in these b0.05; following chi2 test). White bars indicate the number of fish with ovaries, dark bars the number of fish with treatments. A sex ratio could be not determined due to the non-arbitrary composition (four females, eight males; seeabove) of the spawning groups. The testes of 190-day-old 4.2.2. Gonad histology of 71-dpf-old zebrafish control fish contained the full range of sperm maturation In zebrafish exposed to EE2 between 43 and 71 dpf, the testis/ovary ratio at 71 dpf differed significantly (chi2-test;pb0.05) to the ratio found in the control treatment (The effect was concentration-dependent. Among the 25 fishof the control group, the gonads of 13 fish possessed themorphology of an immature ovary, containing oogonia andprimary growth stage oocytes (while the remain- Fig. 6. Gonads of 190-dpf old zebrafish. (A) Ovary of a 190-dpf-controlfish: the gonad contains oocytes of all different maturation stages: primary Fig. 5. Gonads of 71-dpf-old zebrafish, control treatment. (A) Immature growth stage oocytes (poc), cortical alveolus stage oocytes (coc) ovary containing densely packed oocytes of the primary growth stage vitellogenic oocytes (voc), mature oocytes (moc). (B) Ovary of a 190- (poc). (3 Am section). (B) Early testis containing spermatogonia, dpf-fish exposed to 10 ng EE2/L during day 43 to day 71 pf with increased spermatocytes and few spermatids (white arrow) (5 Am section). Bar: number of degenerating oocytes (DO). poc: primary growth stage oocytes; coc: cortical alveolus stage oocytes; voc: vitellogenic oocytes. Bar 100 Am.
G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 stages, with the relative frequency of the spermatogonia, included all stages of gonad development, from the gonad spermatocytes, spermatids and spermatozoa differing anlage to the (pre)mature stage. A more restricted exposure between individual fish. The testis histology of fish exposed period was chosen by who treated to EE2 between days 43 and 71 pf showed no deviations zebrafish from 21 to 42 dpf to 17h-estradiol at concen- compared to testis of control fish; this was the case with all trations of 25 ng/L and higher. In the zebrafish colony used three EE2 concentrations.
by this period covered gonad develop- The females of the control group had mature ovaries ment from the indifferent through the hermaphroditic to the containing varying numbers of primary growth stage early transition stage. Estradiol treatment during these stages oocytes, cortical alveolar oocytes, vitellogenic oocytes and led to a reduction of adult fecundity. In our study, the use of mature oocytes (Female zebrafish developmen- stage-specific exposure identifies the gonad transition stage tally exposed to EE2 displayed mature ovaries as well.
to be most susceptible to persistent effects of estrogens.
However, compared to the ovaries of control females, the Remarkably, the persistent effects were not evident at the number of atretic oocytes (primarily of the vitellogenic or level of gonad histology but only at the level of reproductive mature stage) appeared to be increased. (. Aug- mented oocyte atresia was observed in six out of 12 females Why is the gonad transition stage more sensitive to exposed to 10 ng EE2/L during 43–71 dpf, and in 2 fish out estrogen exposure than the hermaphroditic or the premature of 13 females exposed to 3 ng/L EE2. No alterations of stage? Gonad differentiation of (teleost) fish is considered to ovarian morphology were observed in fish developmentally be organized by the sex steroids: during the critical stage of exposed to 1.67 ng EE2/L.
development, when the presence of high estrogen levels willinduce the differentiation of ovaries, while androgens willlead to the differentiation of testes ( Piferrer, 2001; Devlin and Nagahama, 2002). Disturbanceof the endogenous androgen/estrogen ratio during the In this study, we examined the consequences of exposure critical period, for instance by exposing the fish to to EE2 during particular stages of gonad differentiation on exogenous steroids, can therefore lead to altered phenotypic reproductive capability of the adult zebrafish. The selected sex. This knowledge is practically applied in aquaculture exposure periods were the hermaphroditic stage (when all where sex steroid treatment during critical developmental individuals display an ovarian morphology), the transition stages is used to produce monosex populations of fish stage (when the hermaphroditic gonad develops into the sexually dimorphic gonad), and the premature gonad stage we could demonstrate that manipulation of the endogenous steroid production by the application of aromatase inhibitors 2003). The results from the present study suggest the gonad during the gonad transition stage, leads to an irreversible transition stage to be a critical period of estrogen sensitivity: masculinisation of the developing fish ( exposure during that stage led to significant reduction of 2004). We speculate that the gonad transition stage is the adult egg production and fertilization success, while labile period of phenotypic sex differentiation in zebrafish, exposure during the hermaphroditic or the premature stage when exogenous EE2 can direct gonad development into the had no significant effect on adult reproductive capability.
female phenotype. This shift in gonadal sex could then be Previous studies have shown that developmental expo- responsible for the reduction of reproductive capabilities at sure of zebrafish can have lasting effects on adult the adult stage.
reproductive capabilities ( In order to verify this hypothesis, we performed in the Belt et al., 2003; Brion et al., 2004), however, those studies second experiment a histological examination of the gonads, did not relate the reproductive effects to a specific stage of both at the end of the exposure period (71 dpf) and at the gonad development. treated zebrafish adult stage, after reproductive parameters had been meas- with estrogens over the complete period of gonad develop- ured (190 dpf). At 71 dpf, all individuals in the 3 and 10 ng/ ment until the appearance of mature testes and ovaries, what L treatments displayed ovaries. This finding seems to lasted in their zebrafish colony until 60 days post-hatch confirm that estrogen treatment induces phenotypic femini- (dph). When zebrafish were exposed to 10 ng EE2/L during sation of developing zebrafish. Feminisation of zebrafish by these initial 60 dph, and reared thereafter for grow-out in developmental estrogen exposure has been also reported by clean water, a significant reduction of adult breeding success other authors. For instance, (egg number, hatchability, swim-up survival) was observed.
developing zebrafish with concentrations of 1 to 25 ng Similarly, found that treatment of EE2/L from 20 to 60 dpf, which in their zebrafish colony zebrafish during the initial 3 months of life with 10 or 25 ng included all three stages of gonad development. Histological EE2/L resulted in reduced egg production. Although the examination of the gonads at 60 dpf showed that in the authors provided no information on the stage of gonad groups exposed to 2, 5 or 10 ng EE2/L, no single fish had differentiation, it is likely that the 3-month-period, com- male gonads, but the fish showed either female gonads or parable to the 60 dph in the study of gonads that could not be sexed. In the 1 ng/L group, a small G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 percentage of males was found. Interestingly, in the 25 ng/L the developmental exposure period ( group 22% of the fish possessed testes. Thus, both literature et al., 2003, this study), followed by the appearance of testes and the present findings indicate that developmental EE2 after depuration in clean water (this treatment leads to phenotypic feminisation of zebrafish.
The gonadal feminisation observed at termination of Contrary to the reversibility of gonad changes, repro- estrogen exposure at 71 dpf was not persisting until the ductive capabilities of zebrafish were permanently altered adult stage. At 190 dpf, both fish with ovaries and with by developmental estrogen exposure ( testes were observed. The testes had a normal histological this study). The findings of the histological examination do appearance, with no pathological deviations; only the not support our initial assumption that the reduced ovaries showed some increase in the frequency of oocyte reproductive output is due to permanent gonadal feminisa- atresia. Obviously, the feminisation observed at 71 dpf tion. What is indicated from the histological diagnosis is the was reversible. Reversibility of estrogen-induced gonad presence of moderate pathological alterations of the ovaries changes in zebrafish have been reported by other authors (increased atresia) in the adult fish. If this morphological as well. after exposing zebrafish change is responsible for the impaired reproductive function from hatch to 60 dph to 15.4 ng EE2/L, found a 100% remains to be demonstrated.
feminisation. However, when shorter exposure periods Given the findings of the present study, could a stage were used, e.g. from hatch until 10 dph, or from 20 to 40 specific exposure test in which zebrafish is exposed to dph, both males and females as well as a variable EDCs only during the gonad transition stage replace a full percentage of intersex fish were present at 60 dph. life cycle test? In principle, the effects in adult zebrafish and Janz (2003) exposed zebrafish to 10 ng EE2/L from 2 induced by the stage-specific exposure to EE2 are in to 60 dph, which included in their zebrafish colony all agreement to the effects induced by full life cycle three stages of gonad development. At the end of the exposure period, 80% of the fish had undeveloped Life cycle exposure of zebrafish to EE2 resulted in gonads, 20% had female gonads, but no fish had male reduced fertilization success at a concentration as low as gonads. However, after rearing this cohort in clean water 1.7 ng EE2/L and complete inhibition of spawning at until 160 dph, a 1:1 ratio of male and female fish was concentrations of 3 ng EE2/L and higher ( found. Histological examination of the gonads of those press). Estrogen exposure during the gonad transition fishes at 300 dph showed normal testicular histology, but stage, as done in the present study, led to a significant increased atresia of ovarian follicles ( decline of fertilisation success for 3 and 10 ng EE2/L but In addition, adult breeding success was reduced. The not to complete abolition. The 1.7 ng EE2/L concentration observations of on the effects of did not result in a significant effect. Thus, the two exposure of zebrafish over several developmental stages exposure treatments evoked congruent response patterns, are perfectly in agreement with our own findings on but the life cycle test appears to be more sensitive. The zebrafish exposed to EE2 specifically during the gonad blower sensitivityQ of the life stage-specific exposure may transition stage.
be due to the fact that after termination of exposure at 71 The obvious reversibility of gonad phenotypic femini- dpf, arrested male gonadal development can be re-initiated sation in zebrafish after estrogen exposure during the and the delay in sexual differentiation might be caught up critical period is different to results obtained, for instance, until adulthood, while under the conditions of life cycle with medaka. In the latter species, estrogen treatment treatment, the arrestment of male differentiation can be during the critical period results in permanent feminisation not overcome due to permanent exposure.
When developing a standardized protocol for a life species difference may be related to the different patterns stage-specific test on estrogen-active substances, the of sexual development in zebrafish and medaka: zebrafish apparently large variation in the timing of the gonad develops as undifferentiated gonochorist (i.e passing transition period among zebrafish colonies from different through an all-ovarian stage) while medaka develops as laboratories has to be taken into account. For instance, the differentiated gonochorist (i,.e developing directly male or zebrafish colony studied by went female gonads). Estrogen treatment of genetically male through the gonad transition stage between 22 and 34 days zebrafish during the sensitive period of sexual differ- post-hatching, in the study of this entiation possibly does not re-direct testicular development period lasted beyond 42 dpf, and in our zebrafish colony it to an female gonad, but may arrest male gonad differ- lasted until 71 dpf. Currently, we have no explanation for entiation at the transition from the ovarian-like hermaphro- these differences.
ditic stage into the testis stage. If, estrogen administration is In conclusion, the results from this study indicate that stopped, then the endogenous genetic program can be the gonad transition stage of zebrafish is critical with realized and the arrested hermaphroditic gonad resumes respect to persisting effects of estrogen exposure on differentiation into a testis. This hypothesis would explain reproduction. Estrogen exposure during the gonad tran- the observation of 100% gonadal feminisation at the end of sition stage leads to significant alterations of adult G. Maack, H. Segner / Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 reproduction, which is not the case when estrogen Fenske, M., Maack, G., Sch7fers, C., Segner, H., 2004. An environ- exposure takes place during the hermaphroditic or the mentally relevant concentration of oestrogen induces arrest of malegonadal development in zebrafish, Danio rerio. Environ. Toxicol.
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