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Keywords:

  • Endocrine disruptors;
  • Sex differentiation markers;
  • Ethinylestradiol;
  • Pejerrey

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In pejerrey (Odontesthes bonariensis), ovarian differentiation has been associated with gonadal aromatase expression. It is also known that exposure of pejerrey larvae to estradiol (E2) produces all female populations. During the last few years, the presence of ethinylestradiol (EE2), a synthetic E2 analogue, has been reported in water reservoirs of different parts of the world. In the present study, the effects of EE2 were assessed on sex ratio bias and gene expression levels of gonadal aromatase (cyp19a1a), 11β-hydroxysteroid dehydrogenase type 2 (hsd11b2), estrogens (erα, erβ1), and androgen receptors (arα, arβ). Pejerrey larvae were fed with commercial food containing EE2 (0.1 and 1 µg/g) and E2 (50 µg/g) as a positive control for six weeks after hatching. The gonadal histological analysis showed that 42 to 46% of the fish had clearly differentiated ovaries in both the EE2- and E2-treated groups, compared with 27% in the control group. Moreover, in the EE2- (1 µg/g) and E2-treated groups, no fish presented signs of testicular development compared with controls. In addition, expression of cyp19a1a and hsd11b2 was significantly up- and downregulated, respectively, by EE2 and E2. The authors' results suggested that the feminization process driven by EE2 depends on the positive balance of cyp19a1a in relation to hsd11b2. Thus, these genes can be used as early indicators of exposure to xenoestrogens in this species. Environ. Toxicol. Chem. 2012; 31: 941–946. © 2012 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In the last several decades, the effects of environmental endocrine-disrupting chemicals (EDCs) on human health and wildlife have been a growing concern among researchers and policy makers 1–3. These substances can be defined as hormonally active xenobiotics that can alter the homeostasis of the endocrine system, causing adverse effects on the exposed organisms or their progeny. In contrast to other pollutants, some unique characteristics of the endocrine system (i.e., cyclicity) require understanding the sensitivity window and delayed effects after EDC exposure 4. In particular, EDCs that possess the ability to mimic or antagonize endogenous steroid hormones have been shown to affect sexual differentiation and reproduction in vertebrates 5–7.

The main estrogenic component of oral contraceptives, 17α-ethinylestradiol (EE2), is a synthetic steroid whose presence in sewage treatment works (STW) effluents and receiving waters has been documented widely. This is also true for other natural estrogens such as 17β-estradiol (E2) and estrone (E1) 8–10. Considering that the estrogenic potency of EE2 is 10 to 50 times higher than that of other natural estrogens 11, 12, the fact that EE2 is able to bioconcentrate to a certain degree in fish 13, and that the half-life and toxicological action of this xenoestrogen is magnified because of enterohepatic recirculation 14, the ecotoxicological risks of EE2 are high for these aquatic vertebrates.

The biological effects exerted by EE2 on fish have been studied intensively in the last decade, and such effects have been documented on survival, growth, hatching success, sex ratios, the sex differentiation process, liver and gonadal histology, plasma vitellogenin, and steroid levels. In the last few years in particular, effects on different tissues and biofluids have also been described at the transcriptomic, proteomic, and metabolomic levels 15–19.

Historically, fish have been used in aquatic toxicology for evaluating endocrine disruption not only because they are vertebrates with a full aquatic life cycle, but also because their endocrine system has close similarities with that of higher vertebrates, including humans 20. In addition, fish represent an interesting group for studying the effect of EDCs, because they have very diverse mechanisms of sex determination and gonadal differentiation. Some species show a labile genetic control, and they are susceptible to the influence of factors such as environmental cues (e.g., temperature, pH, social context, and behavior) 21. Steroid hormones play a key role in these processes; for example, 11-ketotestosterone (11-KT) promotes testicular development, and E2 is primarily responsible for the induction and maintenance of the ovary 21. In particular, the biosynthesis of 11-KT is mediated by 11β-hydroxysteroid dehydrogenase 22, and synthesis of E2 is catalyzed by cytochrome P450 aromatase 7.

Pejerrey (Odontesthes bonariensis) is a characteristic fish of the southern sector of the Rio de la Plata Basin (South America) and is highly appreciated as a game fish and for the quality of its flesh 23. This fish has been demonstrated to be very sensitive to pollutants and has been previously used for ecotoxicological studies 24–26. It has been particularly abundant in inland water bodies of the pampas region in Argentina. A decline in the pejerrey population has been reported in the shallow Lake Chascomús since the 1960s, a decline that shows a good association with the increase in the human population of the city with the same name 27. Chascomús City is located beside the lake, and domestic effluents are discharged next to this water body. A recent study revealed that the concentrations of natural and synthetic estrogens in Chascomús effluents and receiving waters are relatively high, with values of E2 between 369 and 361 ng/L and EE2 between 43 and 65 ng/L (P. Carriquiriborde, Universidad Nacional de La Plata, La Plata, Argentina, personal communication).

Pejerrey has a strong thermolabile sex determination, and the proportion of females has been shown to be 100% at 19°C, 50% at 25°C, and 0% at 29°C. The temperature-sensitive window was estimated to be three to five weeks, two to four weeks, and one to four weeks after hatching at 17, 19, and 27°C, respectively 28. In this species, it has also been demonstrated that administering E2 at early developmental stages can produce 100% females 29, 30. Some genes involved in the gonadal sex differentiation related to steroid hormones have been characterized in pejerrey: cyp19a1a (codifying for gonadal aromatase), which is related to the ovarian differentiation process 31, cyp11b1 (codifying for P45011β, 11β-hydroxylase), and hsd11b2 (codifying for 11β-hydroxysteroid dehydrogenase type 2), which is associated with testicular differentiation 32, 33.

Estrogens mimicking EDCs can act on estrogen receptors and/or modify gonadal aromatase expression and activity 34. Thus, exposure to xenoestrogens during a critical period of early development can lead to alterations in the sex differentiation process. The aim of the present study was to evaluate the effects of EE2 on sex differentiation in early stages of pejerrey development by analyzing sex ratio bias, the expression of some selected genes related to the synthesis of gonadal androgens and estrogens, and their receptors.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Chemicals

Both 17α-ethinylestradiol (Parafarm, Saporiti, Argentina, purity 99%) and E2 (Sigma-Adrich, E8875, purity ≥ 98%) were diluted in absolute ethanol (Biopack, Argentina, purity 99–100%). The purities of these compounds were determined by their manufacturers.

Test organisms, experimental design, and test conditions

Newly hatched larvae were obtained from the IIB-INTECH brood stock. Two experiments were conducted using static conditions. Water volume was partially renewed (25%) every day after cleaning food debris and dirt from the bottom of the aquaria. In the first experiment, considered as a preliminary trial, five groups of 100 pejerrey larvae each were fed with commercial fish food (Shulet SRL) with 0 (negative control), 0.1, 0.5, and 1.0 µg of EE2/g and 50 µg of E2/g (positive control). Ethanol was used as a solvent vehicle, and the same volume (700 µl/g of food) was added to all treatments and evaporated in an oven at 30°C. In the second experiment, reported here, four groups of 400 pejerrey larvae each were fed with commercial fish food fortified with 0 (negative control), 0.1, and 1.0 µg of EE2/g and 50 µg of E2/g as a positive control.

In both experiments, larvae were fed to satiation three times a day with fortified commercial fish food, supplemented with Artemia nauplii in the evening, from hatching to the end of the sixth week. After that, fish were fed with commercial fish food and Artemia until the end of the experiment four times a day. Each treatment was performed in duplicate tanks. Sampling was conducted at two, four, and six weeks after hatching for molecular analysis. The remaining larvae were then kept until the end of the experiment, nine weeks after hatching in the preliminary experiment and 11 weeks after hatching in the experiment reported in the present study, for sex analysis by gonadal histology.

In the case of the experiment described here, the larvae were maintained in 46-L glass aquaria, at 25 ± 0.5°C under a 16:8-h L:D photoperiod with gentle aeration during the whole experimental protocol. In both experiments, the mortality rate did not differ between treatments, and it was as expected for these developmental stages 35, ruling out any treatment effect.

Gene expression analysis

Individual trunks (n = 5 for aquarium, n = 10 for each treatment, nT  = 40) were sampled separately from each fish for gene expression analysis at two, four, and six weeks after hatching. Total RNA was extracted using TRIzol reagents (Life Techologies) according to the manufacturer's protocol. Extracts were then treated with Deoxyribonuclease I Amplification Grade (Life Technologies) and reverse-transcribed using SuperScript II RT (Life Technologies) with oligo(dT)12-18 following the manufacturer's instructions. Selected genes for real-time quantitative polymerase chain reaction were cyp19a1a (EF030342), hsd11b2 (HM755974), estrogen receptor α (erα, EU284021), estrogen receptor β1 (erβ1, EU284021), androgen receptor α (arα, HM755973), and androgen receptor β (arβ, HM755974). The oligonucleotides were designed using Beacon Designer 7; (Scorpions; primers and probes; Biosearch Technologies) with annealing temperature setting at 60.0 ± 0.5°C (Table 1). All reactions were performed in 12.5 µl final volume reaction, containing 2 × FastStart Universal SYBRH Green Master (ROX), 1 µl of first strand cDNA, and 0.001 pmol of each primer in a Applied Biosystems (Life Techologies). Transcript abundance was quantified using the relative standard curve method and normalized against a reference gene, β-actin, in each sample, as already described 36.

Table 1. Oligonucleotides used for real-time quantitative polymerase chain reaction
NameSequence (5′ to 3′)
RQbactinFwCTCTGGTCGTACCACTGGTATCG
RQbactinRvGCAGAGCGTAGCCTTCATAGATG
RQaromFwGCGAGCTGTCTGGCTGAGAA
RQaromRvAGGAGCAGCAGCATGAAGAAGA
RQ11betahsd2FwCGAGCTGTCTCTGATGTCCAAC
RQ11betahsd2RwTGCTCAGAGTGCCGAAGAAGT
RQeralphaFWGAGAGCCGGGGGTCTGGAGG
RQeralphaRvGGGCCGCTGTGTTTCCTCCC
RQerbetaFWTGCGATGGCCTCTGCCTCCT
RQerbetaRvCGCCTTGCAGGCATGCTCCT
RQaralphaFWCCAGGCGTGTTCTTGTCAGA
RQaralphaRvTGACAACCCGAGGCATCAT
RQarbetaFWGCTCGTGCTACCTCCTACCCTTA
RQarbetaRvCATCCGTATGGCCGAAGTGT

Histological sex ratio determination

Larval trunks were fixed in Bouin's fluid overnight and then subjected to dehydration and embedding. They were then transversally sectioned at 6 µm, mounted on glass slides, and stained with hematoxylin–eosin. The samples were analyzed with a light microscope (Nikon E600) for determination of gonadal sex, according to Ito et al. 37.

Statistical analysis

Differences between control and treated groups were determined by a one-way analysis of variance nested blocks design, followed by Bonferroni's multiple comparison test. Data were log-transformed prior to statistical analysis to ensure normality and variance homogeneity. Data on gene expression are presented as mean and SEM in all treatments. A χ2 contingency test was used to compare sex ratios of each treatment against control. In all cases, differences were considered statistically significant at p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Sex ratios

Histological analysis of the gonads indicated that treatment with EE2 and E2 was able to shift sex ratios toward the female (Table 2). The gonadal histology of females, males, and undifferentiated fish are shown in Figure 1. In the preliminary experiment, both EE2 (0.1, 0.5, and 1 µg/g) and E2 (50 µg/g) produced a female-shifted sex ratio compared with the control group. In the experiment described here, an increase in the proportion of females was observed, with no signs of males, in the groups treated with EE2 (1 µg/g) and E2 (50 µg/g). Sexually undifferentiated fish were found in all experimental groups (Table 2).

Table 2. Sex ratio observed in experiments 1 and 2
TreatmentFemalesMalesUndifferentiatednχ2
(%)(%)(%)
  • *

    p < 0.05.

  • **

    p < 0.001.

    E2 = estradiol; EE2 = ethynilestradiol.

Exp 1
 Control2366126 
 EE2 0.1 µg/g100006**
 EE2 0.5 µg/g100006**
 EE2 1.0 µg/g670336**
 E2 50 µg/g100006**
Exp 2
 Control27185511 
 EE2 0.1 µg/g46233113*
 EE2 1.0 µg/g4605413**
 E2 50 µg/g4205812**
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Figure 1. Histological cross section of the gonads of pejerrey larvae at 11 weeks after hatching. (A) Ovary, (B) testis, and (C) undifferentiated gonad. Scale bars = 10 µm. CSC = clusters of somatic cells; SD = sperm duct; GC = germ cell.

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Effects of EE2 on gene expression

A significant overexpression of cyp19a1a was induced by EE2 (1 µg/g) and E2 (50 µg/g) at six weeks after hatching compared with the control group. No differences were observed at two and four weeks after hatching (Fig. 2).

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Figure 2. Expression of cyp19a1a in larval trunks at different developmental time points. Results are presented as means ± SEM and are expressed as normalized expression in relation to β-actin. Different letters indicate differences between experimental groups at six weeks after hatching (wah) (p < 0.05). E2 = estradiol; EE2 = ethinylestradiol.

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Relative expression of hsd11b2 began to be significantly downregulated at four weeks after hatching. At this time, a significant decrease in the expression of this gene was observed for EE2 (0.1 µg/g) and E2 (50 µg/g), but not for EE2 (1 µg/g) (Fig. 3). Nevertheless, all treatments showed a statistically significant inhibition at six weeks after hatching, showing a clear decrease in fish exposed to EE2 and E2 (Fig. 3).

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Figure 3. Expression of hsd11b2 in larval trunks at different developmental time points. The results are presented as means ± SEM and are expressed as normalized expression in relation to β-actin. Different letters indicate significant differences between experimental groups at four and six weeks after hatching (wah). Both times were analyzed independently (p < 0.05). E2 = estradiol; EE2 = ethinylestradiol.

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Because the effects of estrogens on both cyp19a1a and hsd11b2 were statistically significant at six weeks after hatching, this sampling point was selected to follow the expression of estrogen and androgen receptors. Analysis of estrogen receptors showed that erα expression was not affected by any of the treatments (Fig. 4). A significant increase was only observed in erβ1 relative expression in the group treated with E2 (50 µg/g) (Fig. 5). In contrast, expression analysis of both androgen receptors showed that they were significantly affected by EE2 and E2 treatments. Specifically, expression of arα was reduced by 3.4- and 8.5-fold in the groups treated with 1 µg of EE2/g and 50 µg of E2/g, respectively (Fig. 6). A similar pattern was observed for arβ, with a 35.9- and 12.7-fold reductions in groups treated with 1 µg of EE2/g and 50 µg of E2/g, respectively (Fig. 7).

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Figure 4. Expression of erα in larvae trunks at six weeks after hatching. The results are presented as means ± SEM and expressed as normalized expression in relation to β-actin. No significant differences were detected among experimental groups. E2 = estradiol; EE2 = ethinylestradiol.

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thumbnail image

Figure 5. Expression of erβ in larvae trunks at six weeks after hatching. The results are presented as means ± SEM and expressed as normalized expression in relation to β-actin. Different letters indicate significant differences between experimental groups (p < 0.05). E2 = estradiol; EE2 = ethinylestradiol.

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thumbnail image

Figure 6. Expression of arα in larvae trunks at six weeks after hatching. The results are presented as means ± SEM and expressed as normalized expression in relation to β-actin. Letters indicate significant differences between experimental groups (p < 0.05). E2 = estradiol; EE2 = ethinylestradiol.

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thumbnail image

Figure 7. Expression of arβ in larvae trunks at six weeks after hatching. The results are presented as means ± SEM and expressed as normalized expression in relation to β-actin. Letters indicate significant differences between experimental groups (p < 0.05). E2 = estradiol; EE2 = ethinylestradiol.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In the present study, the exposure of pejerrey larvae to dietary EE2 during the sex determination window was evaluated. The results showed that EE2 can induce a feminization process when administered during early stages of development in this species. This feminization process can be observed at two different levels, the morphological differentiation of an ovary, and at a molecular level using the expression of selected genes: cyp19a1a, hsd11b2, arα, and, arβ. Treatment with E2 (50 µg/g) was used as a positive control because its feminizing effects had been reported previously at morphological 29 and molecular (cyp19a1a expression) levels in this species 30.

In pejerrey, Karube et al. 31 established that cyp19a1a began to be expressed just before the appearance of the first histological signs of ovarian differentiation at a feminizing temperature (17°C), but remained at low levels at a masculinizing temperature (29°C). In the same species it was also established that the exogenous administration of E2 rescued the female phenotype in larvae raised at a “sexually neutral” temperature (25°C) 30. The present results also reinforce the data on the effects of E2 on cyp19a1a expression and the induction of ovarian differentiation. An analogous process was induced by EE2 but at lower concentrations compared with E2. Interestingly, hsd11b2 expression began to be downregulated by EE2 (0.1 µg/g) and E2 at four weeks after hatching, two weeks before the stimulation of cyp19a1a. This gene encodes for the enzyme responsible for the synthesis of 11-KT, a major fish androgen 38 that has recently been associated with testicular development in the African catfish Clarias gariepinus39. Also, in the adult roach, Rutilus rutilus, exposure to EE2 caused a significant reduction in the testicular concentrations of 11-oxygenate androgens 17, and thus it is possible that EE2 could induce a significant inhibition of hsd11b2 in adult stages.

The effects of EE2 on the androgen pathway were accompanied by downregulation of the two androgen receptors in pejerrey. However, the effects on estrogen receptors were not clearly evident. Even though a significant increase was detected in the E2-treated group compared with control, a similar profile could not be seen in the EE2-treated groups, suggesting a differential response for both estrogens. Different fish species have different expression profiles of ers before, during, and after sex differentiation 17, 40. Although it is not clear whether estrogens can downregulate the expression of ars, the present study reinforces previous data on adult fathead minnow (Pimephales promelas) showing that ars could be downregulated by EE2 treatments 16.

Similarly to other teleosts fish species, a trend toward the increase in female frequency was observed in the EE2-treated larvae 41. In the present study, undifferentiated larvae were observed at 11 weeks after hatching, a time by which gonadal differentiation is usually completed in this species 37. Although a delay in gonadal development in zebrafish has been shown to be caused by chronic exposure to EE242, this effect could not be shown in this experiment. Further experiments are needed to see the long-term effects of EE2 in pejerrey.

The EE2-induced feminization under experimental conditions or in natural fish populations has been the classic observed effect, showing the hazards of continuous or long-term exposure to this synthetic estrogen (for review, see Scholz and Klüver 41 and Tyler et al. 5). Teleost fish have a highly labile genetic sex determination, and in many fish, sex differentiation is influenced by environmental factors and steroid hormones 21. Thus any alteration in the hormonal balance induced by EDCs can alter this process, even when these substances are in low concentrations in the environment 43. The present results suggest that a balance between the expression of cyp19a1a, an enzyme involved in ovarian differentiation, and hsd11b2, an enzyme involved in testicular differentiation, at early stages of development is essential to drive the process of gonadal differentiation in this species. Thus the feminization effects could be produced not only by an increase in the expression of cyp19a1a, but also by inhibition of hsd11b2, which is related to 11-oxygenated androgen production.

The ubiquous presence of EE2 in surface waters has been demonstrated worldwide, and concentrations up to 42 ng/L have been shown 44, 45. Moreover, recent studies have reported bioaccumulation factors of EE2 higher than 450 ng/L in phytoplankton 46 and residue levels in aquatic organisms between 2.71 and 78.15 ng/g (lipid wt) 45, 47, indicating that food could be an important route of exposure in the aquatic ecosystem. Pejerrey is a characteristic fish of the southern sector of the Del Plata River Basin (South America) 23. A recent survey of STW effluents and receiving waters in the region have found levels of EE2 up to 43 ng/L in small streams receiving STW discharges (P. Carriquiriborde, Universidad Nacional de La Plata, La Plata, Argentina, personal communication). When the bioaccumulation factor of EE2 and the zooplanktivorous habit of pejerrey larvae during the first stages of life are considered, together with the observed feminization effect, ingestion appears to be an environmentally relevant route of exposure to EE2.

According to the present study, an imbalance in sex ratios driven by EE2 exposure would be expected in populations of pejerrey inhabiting aquatic ecosystems receiving STW effluents. In addition, the present results indicate that the balance between the expression levels of cyp19a1a and hsd11b2 in early life stages of pejerrey could be used as a marker of sex ratio deviation induced by EDCs before gonadal histological changes could be observed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank the following supporting scientific institutions: the National Agency for the Promotion of Science and Technology (ANPCYT), Argentina (PICT2008-1383 to G.M. Somoza and PICT2008-1598 to P. Carriquiriborde) and the National Council for Scientific and Technical Research (CONICET), Argentina.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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