Article | Open

Vitellogenin induction in caudal fin of guppy (Poecilia reticulata) as a less invasive and sensitive biomarker for environmental estrogens

  • Scientific Reportsvolume 7, Article number: 7647 (2017)
  • doi:10.1038/s41598-017-06670-6
  • Download Citation
Published online:


Guppy (Poecilia reticulata) is an ideal model for studying environmental estrogens, and its large caudal fin has a high capacity to regenerate. This study analyzed the feasibility of caudal fin for detecting vitellogenin (Vtg), the most commonly used biomarker of environmental estrogens. Firstly, a sandwich ELISA for guppy Vtg was developed using purified lipovitellin and its antibody and it had a working range of 7.8–1000 ng/mL and detection limit of 3.1 ng/mL. The ELISA was used to detect tissue distribution of Vtg. In male guppy exposed to 50 and 100 ng/L 17β-estradiol (E2), Vtg concentration in caudal fin was higher than that in whole fish, brain, eyes, gonad, and skin, and was close to that in the liver. Furthermore, male guppies were exposed to environmental concentrations of 17a-ethinylestradiol (EE2) and bisphenol S (BPS) to validate the utility of caudal fin Vtg for detecting estrogenic activities. The lowest observed effect concentration of EE2 and BPS were lower than 2 ng/L and 1 μg/L, which were below or equal to the values reported for other species, demonstrating that caudal fin Vtg was highly sensitive to estrogenic chemicals. Therefore, caudal fins of guppies are suggested as alternative samples for Vtg biomarker detection.


Environmental estrogens have aroused great concern worldwide owing to their adverse effects on wildlife health, including altered sex hormone levels1, gonadal abnormalities, reduced fertility2, and feminization of males3. To screen chemicals with estrogenic activity, US Environmental Protection Agency and Organization for Economic Cooperation Development (OECD) have developed test guidelines using small fish species as model organisms and vitellogenin (Vtg) as a core biomarker4, 5. Currently, plasma and whole-body homogenates (WBH) are normally used for Vtg detection, which involves killing experimental organisms6, 7. In some cases, such as field sampling of natural populations and serial monitoring of the effects of estrogenic chemicals on experimental organisms, a less invasive sampling method for repeated Vtg measurements is needed. Several studies reported that skin mucus was an alternative choice for Vtg quantification8, 9. However, Maltais et al.10 and our previous study11 found that Vtg levels in fish skin mucus were considerably lower than those in plasma, demonstrating that surface mucus Vtg induction exhibited a lower sensitivity to estrogenic chemicals. Recently, Zhong et al.12 reported that Vtg could be detected in various extrahepatic tissues of 17α-ethinylestradiol (EE2)-exposed male zebrafish (Danio rerio), and recommended skin and eye tissues as for Vtg analysis. Sampling of these tissues would kill the fish, thus it was unable to measure Vtg induction at a later point in time. By contrast, caudal fin has a high capacity to regenerate, and zebrafish caudal fin could quickly regenerate in approximately two weeks after 95% excision13. We speculated that using caudal fins as the sampling tissue could be helpful for continuous measurement of Vtg biomarker. However, it is still unclear whether caudal fin contains Vtg and whether the sensitivity of Vtg induction in caudal fin is high enough to detect weak estrogenic activity.

The guppy (Poecilia reticulata) has a large caudal fin, which is almost half of their total length and can regenerate quickly and reliably after amputation, restoring both size and shape14, 15. Thus, it provides a good model to evaluate the feasibility of caudal fin Vtg as a less invasive biomarker for detecting environmental estrogens. Guppy is easily cultured in the laboratory and easy to identify gender due to its obvious secondary sexual characteristics (Fig. 1). Moreover, it was reported that guppy was more sensitive to exogenous estrogens than zebrafish and rainbow trout16, 17. Guppy was therefore considered an ideal model to study environmental estrogens18, 19. However, Vtg in guppy is still detected using qualitative methods, such as SDS-PAGE and Western blot analysis, and its quantitative tool has not been developed owing to the lack of anti-guppy Vtg antibody until now17, 20.

Figure 1
Figure 1

Photograph of male and female guppy (Poecilia reticulata).

In the present study, guppy lipovitellin (Lv), the main egg yolk protein derived from Vtg, was purified and used to prepare antibodies. Using the purified Lv and its antibody, a sandwich ELISA for measuring guppy Vtg was established. Subsequently, Vtg induction in the WBH, caudal fin, liver, brain, eye, gonad, and skin tissues of male guppy exposed to different concentrations of 17β-estradiol (E2) were measured by Western blot and the sandwich ELISA to evaluate the possibility of using caudal fin for Vtg detection. Furthermore, the reliability of Vtg in caudal fin as a biomarker of estrogenic contamination was validated by quantifying Vtg levels in WBH, liver, and caudal fin of male guppy exposed to environmental concentrations of two different exogenous estrogens, EE2 and bisphenol S (BPS), which are two commonly detected estrogenic chemicals in aquatic environments.


Purification of Lipovitellin

Ovarian homogenate yielded two peaks in gel filtration chromatography and the first peak appeared clear protein bands (Fig. 2). Thus fractions of this peak were collected and subjected to anion exchange chromatography. The main peak was found in 25 mM Tris-HCl buffer containing 0.1 M NaCl and it showed a single band in Native-PAGE (Fig. 3).

Figure 2
Figure 2

Elution profiles of ovarian homogenates on a Sephacryl S-300 column (A) and Native-PAGE (4–7.5%) detection of peak elution (B). lane 1, Peak P1; lane 2, Peak P2.

Figure 3
Figure 3

DEAE anion exchange column of guppy Lv (A) and Native-PAGE (4–7.5%) of the peak eluted with buffer containing 0.1 M NaCl (B).

Characterization of Lipovitellin

The purified protein was stained positively with Schiff reagent, methyl green, and Sudan black B (Fig. 4), confirming that it was a phospholipoglycoprotein.

Figure 4
Figure 4

Specific staining of carbohydrate (lane 2), phosphorus (lane 3) and lipid components (lane 4) for the purified protein. Lane 1 was stained with CBB.

The apparent molecular weight of guppy Lv was estimated to be approximately 480 kDa by native-PAGE (Fig. 5A). On SDS-PAGE, guppy Lv resolved into a major band at 112 kDa and a minor band at 83 kDa (Fig. 5B).

Figure 5
Figure 5

Native PAGE (4–7.5%) (A) and SDS-PAGE (B) electrophoretic patterns of purified guppy Lv. lane 1, protein maker; lane 2, the purified Lv.

Specificity of the antiserum to vitellogenin

Western blot results showed that anti-Lv antibody detected several bands in WBH from E2-exposed male guppy and the purified Lv, while no visible bands were detected in WBH from male fish (Fig. 6).

Figure 6
Figure 6

Western blot analysis of WBH from male (lane 1), E2-exposed male guppy (lane 2), and purified Lv (lane 3) using anti-Lv antiserum as primary antibody.

Development and validation of an ELISA for guppy vitellogenin

Serial dilutions of HRP-labeled anti-Lv antibody were used as detecting antibodies to determine the optimal condition of the assay (Fig. 7A). When the detecting antibody was diluted 1:1250 times, the curve showed a wide linear work range with high maximum absorbance of about 2.6. Under this condition, the working range of this assay was 7.8–1000 ng/mL (y = 1.1747x − 1.0473, R2 = 0.992), and the detection limit was estimated as 3.1 ng/mL (Fig. 7B). Moreover, the Lv standard curve was parallel to dilutions of WBH from E2-exposed male guppy over the entire working range of the assay, while control male WBH showed no reaction in the ELISA (Fig. 7C). The matrix effects for caudal fin and WBH were found to be reduced to acceptable levels when they were diluted 20-fold and 40-fold, respectively (Figs S1 and S2). At these levels of dilution, the matrix interferences were similar to those observed in the case of the matrix free buffer (PBST). Therefore, the practical detection limit for Vtg in caudal fin and WBH samples were 62 and 124 ng/mL, respectively.

Figure 7
Figure 7

Determination of optimal dilution of HRP-labeled anti-Lv IgG (A), and a representative standard curve obtained for guppy Lv (B) and WBH dilution curves of control male and E2-exposed male guppy in sandwich ELISA (C).

The intra- and inter-assay CVs of the ELISA within the working range were 0.73~3.85% and 0.36~7.43%, respectively (Tables 1 and 2).

Table 1 Precision tests of the sandwich ELISA.
Table 2 Lowest observed effect concentration (LOEC) of Vtg induction in different fish exposed to waterborne 17a-ethynylestradiol.

Distribution of Vitellogenin in male guppy exposed to E2

After a 21-day exposure, the distribution of Vtg in male guppy was qualitatively detected by Western blot analysis. Prior to E2 exposure, there was no positive signal for Vtg in WBH of control male fish, while strong signal was found in WBH of ovariectomized female fish. After exposure to 50 ng/L E2 for 21 days, three clear positive bands were detected in the liver and caudal fin, which had the similar position to that of WBH from the female guppy (Fig. 8A). In 100 ng/L E2 exposure group, male fish showed detectable Vtg signals in skin, caudal fin, gonad, liver, eye, and brain tissues, and the strongest signal was detected in the liver (Fig. 8B). Positive bands were observed in all tissues of male fish exposed to 100 ng/L E2 and the number of bands was more than the fish exposed to 50 and 100 ng/L E2 (Fig. 8C).

Figure 8
Figure 8

Western blot analysis of Vtg induction in different tissues of adult male guppy exposed to 50 (A), 100 (B), and 200 ng/L E2 (C) for 21 days. Lane 1, WBH of ovariectomized female fish; lane 2, WBH of control male fish; lane 3, skin; lane 4, caudal fin; lane 5, gonad; lane 6, liver; lane 7, eye; lane 8, brain.

Vtg concentrations in each tissue and WBH of male guppy in control and exposure groups were quantified by ELISA (Fig. 9). In control male guppies, Vtg values were all below the detection limit of ELISA, while concentration-dependent increases of Vtg concentrations were observed in all tissues of male guppies after 21 day exposure to E2. In 200 ng/L E2-exposed male fish, Vtg concentrations in brain, eye, testis, skin, caudal fin, whole body, and liver tissues showed an increasing tendency, and concentrations of Vtg in caudal fin, whole body, and liver were above 7 μg/g. Similarly, Vtg concentrations in caudal fin, whole fish, and liver of male fish exposed to 100 ng/L E2 were 2.42 ± 0.13 μg/g, 2.05 ± 0.14 μg/g, 2.73 ± 0.20 μg/g, significantly higher than other tissues in the same group (P < 0.01). For 50 ng/L E2 group, Vtg concentrations in caudal fin, whole fish and liver were 1.31 ± 0.10 μg/g, 0.71 ± 0.06 μg/g, and 1.56 ± 0.15 μg/g. Moreover, Vtg concentration in caudal fin was significantly higher than in the whole fish (P < 0.05).

Figure 9
Figure 9

Concentrations of Vtg in guppy exposed to 50, 100, and 200 ng/L E2 for 21 days. Values are means ± S.D and asterisks indicate statistically significant difference from the control group (**P < 0.01).

Caudal fin vitellogenin induction in male guppy exposed to environmental concentrations of EE2 and BPS

Vtg concentrations in caudal fin of male guppy exposed to 2, 10, and 50 ng/L EE2 for 21 days were 0.35 ± 0.08 μg/g, 0.55 ± 0.08 μg/g, and 2.18 ± 0.37 μg/g, respectively (P < 0.01, Fig. 10A). In 2 and 10 ng/L EE2 exposure groups, there was no difference in Vtg concentrations between these sampled tissues. Exposure to 1, 10, and 100 μg/L BPS also increased the concentration of caudal fin Vtg significantly compared to solvent control group (P < 0.01, Fig. 10B). Furthermore, 10 μg/L BPS exposure induced the highest caudal fin Vtg concentration, while 100 μg/L BPS dose resulted in less induction of Vtg, which showed the same trend as Vtg in the liver and whole body.

Figure 10
Figure 10

Concentrations of Vtg in guppy exposed to 2, 10, and 50 ng/L EE2 (A) and 1, 10, and 100 μg/L BPS (B) for 21 days. Values are means ± S.D. and asterisks indicate statistically significant difference from the control group (**P < 0.01).


In the present study, Vtg in caudal fin of guppy was found to be highly sensitive to exogenous estrogens, and it could be used as a less invasive biomarker for measuring estrogenic activity. As the most commonly used biomarker for environmental estrogens, Vtg was normally quantified by ELISA21, 22. Our previous study confirmed that Lv was very stable and showed the same binding efficiency to anti-Lv antibody as did Vtg. In addition, the ELISA developed using Lv as the antigen could quantify Vtg more accurately23. Therefore, in the present study, we chose Lv to develop the ELISA for quantification of guppy Vtg. Guppy Lv purified by a two-step chromatographic method was characterized as a phospholipoglycoprotein with an apparent molecular weight of approximately 480 kDa and produced a major band of approximately 112 kDa in SDS-PAGE, which was similar to that in other teleosts11, 24. Western blot elucidated that the polyclonal antibody raised against purified Lv reacted with WBH from E2-exposed male guppy, whereas no positive reaction occurred in control male WBH, indicating that the anti-Lv antibody was highly specific to guppy Vtg25. The sandwich ELISA developed using purified Lv and anti-Lv antibody had a working range of 7.8–1000 ng/mL and a detection limit of 3.1 ng/mL, which was consistent with the vaules reported for Vtg ELISA of tilapia (Sarotherodon melanotheron)26 and rare minnow (Gobiocypris rarus)27. In order to avoid the matrix effect, all tissues except caudal fin were diluted at least 1:40 in the routine assay. The average intra-assay and inter-assay CVs of Lv-based sandwich ELISA were 2.1% and 4.1%, respectively, which were lower than those of ELISAs developed using Vtg as antigen28, 29, demonstrating that the established ELISA had high precision. Moreover, the parallelism observed between the Lv standard curve and WBH dilution curves of E2-exposed male guppy demonstrated that anti-Lv antibody recognized Lv and Vtg similarly. In addition, the ELISA did not detect positive signal in control male WBH, indicating that this assay was very specific to Vtg30, 31. The above results confirmed that the sandwich ELISA established in the present study could accurately quantify guppy Vtg.

Guppy is a new promising model organism for studying environmental estrogens32, 33. Male guppies have a large caudal fin that is easily regenerated15. If the sensitivity of Vtg induction in caudal fin is close to that in WBH, caudal fin could replace the whole fish as the preferred sample for Vtg detection, which will be more suitable for field investigations of wild populations and for repeated vtg measurements. In the present study, Vtg concentrations in caudal fin, liver, brain, eyes, gonads, skin of male guppy exposed to E2 were quantified using the developed sandwich ELISA developed. It was found that exposure to 50, 100, and 200 ng/L E2 significantly increased Vtg concentrations in all tissues. The amount of Vtg in all exposure groups was greatest in the liver tissue, which was consistent with the tissue distribution of Vtg reported in zebrafish12. In the 100 ng/L E2-exposed group, the concentration of Vtg in caudal fin was significantly higher than that in eyes, skin, other tissues, and the whole fish. Vtg concentration in caudal fin of male fish exposed to 50 ng/L E2 was 1.85 times that of whole fish, and it was much closer to Vtg concentration in the liver than that in the 100 ng/L E2-exposed group. Evidently, Vtg induction in caudal fin had equal or even higher sensitivity than Vtg in whole fish at lower E2 concentrations (<100 ng/L). Owing to the ease of sampling and lower damage to fish, caudal fins can be good alternatives to WBH for the detection of Vtg in guppy.

To further test the reliability of caudal fin Vtg for the detection of estrogenic activity, the present study used two common exogenous estrogens to carry out exposure experiments. EE2 is the most detected synthetic estrogen that is not broken down in surface and sewage waters. Concentrations of EE2 in surface water of the UK, Japan, and Canada usually ranged from 1 to 20 ng/L34, 35, 36, although a maximum of 273 ng/L was found in US streams37. Many studies have reported that Vtg induction in various fish species after waterborne exposure to EE2 (Fig. 2). The lowest observed effect concentration (LOEC) of Vtg induction was normally 5–20 ng/L for zebrafish, medaka (Oryzias latipes), and fathead minnow (Pimephales promelas)38, 39, 40, except one study reported the LOEC of Vtg induction in male fathead minnow for EE2 was 1 ng/L41. For three-spined stickleback (Gasterosteus aculeatus), Sheepshead minnow (Cyprinodon variegatus), and Mummichog (Fundulus heteroclitus), the LOEC of Vtg induction for EE2 was higher than 50 ng/L42, 43, 44. In the present study, we found that 2 ng/L EE2 exposure significantly induced caudal fin Vtg, indicating that the LOEC of Vtg induction in guppy caudal fin was lower than 2 ng/L EE2, which was lower than the values reported for most tested species. Moreover, Vtg concentration in caudal fin was not significantly different from that in the liver and whole fish. Thus, the results demonstrated that Vtg in caudal fin of male guppy could detect weak estrogenic activity of EE2. In addition, this study tested the sensitivity of guppy caudal fin Vtg to BPS, which has emerged as a potential bisphenol A replacement and has been frequently detected in aquatic environments of various countries in recent years. A maximum of 7.2 μg/L of BPS was found in Adyar River of India45. Naderi et al.46 reported that 10 μg/L BPS exposure could significantly increase the plasma Vtg concentration in male zebrafish. In this study, 1 μg/L BPS exposure for 21 days was found to significantly increase Vtg concentration in caudal fin, indicating that caudal fin Vtg in male guppy had an equal or even higher sensitivity to BPS than plasma Vtg in zebrafish. The above results confirmed that Vtg induction in caudal fin of guppy was highly sensitive to estrogenic chemicals and could be used as a reliable biomarker for the detection of estrogenic activity in the aquatic environment, though the stress of handling fish repeatedly should then be taken into account.

In summary, a sandwich ELISA for accurate quantification of guppy Vtg was established using purified Lv and its polyclonal antibody. Subsequently, this ELISA was used to detect tissue distribution of Vtg in male guppy exposed to exogenous estrogens. Caudal fin produced Vtg in response to low concentrations of exogenous estrogens, and its Vtg concentration was significantly higher than that in the whole fish at low E2 concentration. Further, we found that caudal fin Vtg of guppy was capable of detecting estrogenic activities of EE2 and BPS at environmentally relevant concentrations. Therefore, we suggest guppy caudal fin with a high capacity to regenerate as a potential alternative sample for repeated Vtg analysis.


Experimental fish

Sexually mature red albino guppies (Poecilia reticulata) (wet mass, 0.32 ± 0.10 g; standard length, 2.2 ± 0.4 cm) were mainatined in 50-L aquaria filled with 30-L dechlorinated tap water at 26 ± 1 °C, with 7.0 ± 0.1 mg/L dissolved oxygen and 14 h:10 h light-dark cycle. The fish were fed with newly hatched brine shrimp twice and half of the aquaria water was replaced daily. In addition, the fish were handled according to the National Institute of Health Guidelines for the handling and care of experimental animals and the animal utilization protocol was approved by the Institutional Animal Care and Use Committee of the Ocean University of China.

Lipovitellin purification

Ovaries were removed from adult female guppies and homogenized in ice-cold homogenate buffer (20 mM Tris-HCl, pH 7.5, containing 100 mM NaCl and 10 mM EDTA) using a glass homogenizer. After centrifugation at 8,000 × g for 10 min at 4 °C. Lv was purified from ovarian homogenates by gel filtration (Sephacryl S-300 HR 16/60 column; GE Healthcare, Uppsala, Sweden) and anion-exchange chromatography (DEAE-Sepharose FF 12/20 column; GE Healthcare, Uppsala, Sweden) according to our previously reported methods62. After determination of protein concentration by the Bradford assay using bovine serum albumin (BSA) as the standard, the purified Lv was stored at −80 °C.

Lipovitellin characterization

The purified protein was analyzed using native polyacrylamide gel electrophoresis (PAGE; 4–7.5%), and the gels were stained with Coomassie Brilliant Blue (CBB), Schiff reagent, methyl green, and Sudan black B following the methods as describe by Pan et al.63.

The molecular mass of purified Lv was estimated by HMW native marker Kit (GE Healthcare, USA) according to the method described by Sun and Zhang64. Polypeptide components of Lv were analyzed by sodium dodecyl sulfate (SDS)-PAGE. After electrophoresis, gels were stained with CBB, and molecular weight of polypeptide units was estimated using an unstained protein ladder (20–200 kDa, Thermo Scientific, Waltham, MA, USA).

Antibody production and label

Polyclonal antiserum against guppy Lv was raised in rabbits following routine methods. Briefly, rabbits were injected subcutaneously with 1 mL of Lv solution (800 μg) emulsified in complete Freund’s adjuvant followed by another three boosts of Lv solution (500 μg) in incomplete Freund’s adjuvant at 2-week intervals. Blood was collected, centrifuged, and anti-Lv immunoglobulin G (IgG) was purified from the supernatant by affinity chromatography on a HiTrap Protein G column (GE Healthcare). The purified IgG was labeled with horseradish peroxidase (HRP, Sigma, USA) by an improved sodium periodate-oxidation method25.

Western blot of Vitellogenin

Western blot analysis was performed to check the specificity of anti-Lv antibody to guppy Vtg. The WBH of control male and E2-exposed male guppy, and the purified Lv were electrophoresed on SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. After incubation with anti-Lv antibody at a 1:1000 dilution, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Solarbio, Beijing, China) at a 1:2000 dilution. Finally, the membranes were visualized with freshly prepared DAB substrate.

Sandwich ELISA

A sandwich ELISA was developed by the procedure of Mitsui et al.65 with minor modification. Microtiter plates (Costar, Cambridge, MA) were coated with 100 μL of anti-Lv IgG diluted in 0.05 M sodium carbonate overnight at 4 °C and washed three time with 200 μL/well of PBST (10 mM PBS containing 0.05% Tween-20). The wells were then incubated with 200 μL PBST containing 1% BSA for 1 h at 37 °C. After three washes with PBST, 100 μL of samples and standard serially diluted with PBST were added to each well and incubated at 37 °C for 1 h. The wells were washed five times and received 100 μL/well of HRP-labeled anti-Lv antibody at serial dilutions (1:1250, 1:2500, 1:5000, 1:10 000). After incubation at 37 °C for 1 h, the color was developed with 100 μL of tetramethylbenzidine enzyme substrate (Solarbio, China) and stopped by adding 100 μL of 2 M sulfuric acid. Absorbance values were measured at 450 nm in a plate reader. Standards and samples were run in duplicate.

The reliability of ELISA assay were evaluated by measuring its precision, sensitivity, and specificity30. Briefly, precision was evaluated using various purified Lv concentrations by measuring the intra- and inter-assay coefficients of variation (CV%), which were defined as the standard deviation devided by the mean and mutiplied by 100. The specificity was assessed by comparing curves of serial dilutions of WBH from E2-treated male guppy and the Lv standard curve. The limit of detection was defined as the concentration corresponding to the mean of the absorbance values for 12 replicates of the zero standards plus two times the standard deviation. Additionally, the matrix effect of caudal fin and WBH samples were evaluated by two different methods30, 38.

Tissue distribution of Vitellogenin in male guppy exposed to E2

Adult male guppies (n = 16) were exposed to nominal concentrations of 50, 100, and 200 ng/L E2 in 5-L aquaria. The stock solution of E2 was prepared in ethanol and kept at 4 °C. The ethanol concentration in each group was below 0.001%. The exposure solution was renewed daily. After 21 days of exposure, fish were anesthetized in a bath of tricane methane sulfate, body lengths and weights were measured. The liver, eyes, testis, brain, and skin of each fish were separated with sterilized scissors and tweezers. Stainless steel blades were used to cut one third of caudal fins with an approximate weight of 0.02 g. Each tissue was weighed, diluted 1:4 (v/v) in 10 mM PBS containing 0.02% aprotinin, and homogenized by an automatic grinding machine (ShangHai Jingxin industrial development CO., LTD) with a homogeneous velocity of 50 HZ for 2 min. After centrifugation (8000 g, 10 min) at 4 °C, the supernatant was transferred to a clean tube for Vtg detection by Western blot and sandwich ELISA assay. All assays were carried out in duplicate.

Vitellogenin induction in caudal fin of male guppy exposed to EE2 and BPS

Adult male guppies (n = 20) were exposed to 2, 10, and 50 ng/L EE2 and 1, 10, 100 μg/L BPS, respectively. After 21 days, caudal fin, liver tissue, and whole body were homogenated and centrifugated as described above. Vtg in the supernatant were quantified by the developed sandwich ELISA.


Vtg induction data are presented as the mean ± standard deviation, and the differences between the control and exposed groups were assessed by one-way analysis of variance followed by a Tukey’s post hoc tests. Prior to parametric analysis, data were log-transformed to achieve variance homogeneity. All statistical tests were conducted using SPSS 18.0 software (SPSS Inc., USA), and values were determined as significant when P < 0.05.

Data Availability

All data has been included in the manuscript. The primary data is available to all interested researchers by contacting

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Zoeller, R. T. et al. Endocrine-disrupting chemicals and public health protection: a statement of principles from The Endocrine Society. Endocrinology 153, 4097–4110 (2012).

  2. 2.

    Tetreault, G. R. et al. Intersex and reproductive impairment of wild fish exposed to multiple municipal wastewater discharges. Aquatic Toxicology 104, 278–290 (2011).

  3. 3.

    Thorpe, K. L., Maack, G., Benstead, R. & Tyler, C. R. Estrogenic wastewater treatment works effluents reduce egg production in fish. Environmental science & technology 43, 2976–2982 (2009).

  4. 4.

    US EPA. Fish Screening Assay Discussion Paper Available from: (accessed 10.10.10) (2006).

  5. 5.

    OECD, OECD Guideline for the Testing of Chemicals. Test No. 229: Fish Short Term Reproduction Assay. Available from: (2012).

  6. 6.

    Bourrachot, S. et al. Effects of depleted uranium on the reproductive success and F1 generation survival of zebrafish (Danio rerio). Aquatic Toxicology 154, 1–11 (2014).

  7. 7.

    Peng, S. et al. Effect of dietary n−3 LC-PUFAs on plasma vitellogenin, sex steroids, and ovarian steroidogenesis during vitellogenesis in female silver pomfret (Pampus argenteus) broodstock. Aquaculture 444, 93–98 (2015).

  8. 8.

    Arukwe, A. & Røe, K. Molecular and cellular detection of expression of vitellogenin and zona radiata protein in liver and skin of juvenile salmon (Salmo salar) exposed to nonylphenol. Cell and tissue research 331, 701–712 (2008).

  9. 9.

    Dzul-Caamal, R. et al. Multivariate analysis of biochemical responses using non-invasive methods to evaluate the health status of the endangered blackfin goodeid (Girardinichthys viviparus). Ecological Indicators 60, 1118–1129 (2016).

  10. 10.

    Maltais, D. & Roy, R. L. Effects of nonylphenol and ethinylestradiol on copper redhorse (Moxostoma hubbsi), an endangered species. Ecotoxicology and environmental safety 108, 168–178 (2014).

  11. 11.

    Wang, J. et al. Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus). Ecotoxicology and environmental safety 120, 80–87 (2015).

  12. 12.

    Zhong, L. et al. Distribution of vitellogenin in zebrafish (Danio rerio) tissues for biomarker analysis. Aquatic Toxicology 149, 1–7 (2014).

  13. 13.

    Poss, K. D., Keating, M. T. & Nechiporuk, A. Tales of regeneration in zebrafish. Developmental Dynamics 226, 202–210 (2003).

  14. 14.

    Hopper, A. F. & Wallace, E. Anal fin regeneration in thiouracil-treated guppies. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 173, 251–261 (1970).

  15. 15.

    Kolluru, G. R., Ruiz, N. C., Del Cid, N., Dunlop, E. & Grether, G. F. The effects of carotenoid and food intake on caudal fin regeneration in male guppies. Journal of fish biology 68, 1002–1012 (2006).

  16. 16.

    Gallo, D., Merendino, A., Keizer, J. & Vittozzi, L. Acute toxicity of two carbamates to the guppy (Poecilia reticulata) and the zebrafish (Brachydanio rerio). Science of the total environment 171, 131–136 (1995).

  17. 17.

    Li, M. H. & Wang, Z. R. Effect of nonylphenol on plasma vitellogenin of male adult guppies (Poecilia reticulata). Environmental toxicology 20, 53–59 (2005).

  18. 18.

    Cardinali, M. et al. Temporary impairment of reproduction in freshwater teleost exposed to nonylphenol. Reproductive Toxicology 18, 597–604 (2004).

  19. 19.

    Heintz, M. M., Brander, S. M. & White, J. W. Endocrine Disrupting Compounds Alter Risk-Taking Behavior in Guppies (Poecilia reticulata). Ethology 121, 480–491 (2015).

  20. 20.

    Tian, H., Li, Y., Wang, W., Wu, P. & Ru, S. Exposure to monocrotophos pesticide during sexual development causes the feminization/demasculinization of the reproductive traits and a reduction in the reproductive success of male guppies (Poecilia reticulata). Toxicology and applied pharmacology 263, 163–170 (2012).

  21. 21.

    Nilsen, B. M. et al. Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening. Analytical and bioanalytical chemistry 378, 621–633 (2004).

  22. 22.

    Fujiwara, Y., Fukada, H., Shimizu, M. & Hara, A. Purification of two lipovitellins and development of immunoassays for two forms of their precursors (vitellogenins) in medaka (Oryzias latipes). General and comparative endocrinology 143, 267–277 (2005).

  23. 23.

    Wang, J. et al. Lipovitellin as an antigen to improve the precision of sandwich ELISA for quantifying zebrafish (Danio rerio) vitellogenin. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 185, 87–93 (2016).

  24. 24.

    Li, Y. P. et al. Purification and characterization identification of lipovitellin from Pelteobagrus vachelli and preparation of anti-serum. J. Fish. China 34, 116–125 (2010).

  25. 25.

    Garnayak, S. K., Mohanty, J., Rao, T. V., Sahoo, S. K. & Sahoo, P. K. Vitellogenin in Asian catfish, Clarias batrachus: Purification, partial characterization and quantification during the reproductive cycle by ELISA. Aquaculture 392, 148–155 (2013).

  26. 26.

    Okoumassoun, L. E. et al. Vitellogenin in tilapia male fishes exposed to organochlorine pesticides in Ouémé River in Republic of Benin. Science of the Total Environment 299, 163–172 (2002).

  27. 27.

    Liao, T., Jin, S., Yang, F. X., Hui, Y. & Xu, Y. An enzyme-linked immunosorbent assay for rare minnow (Gobiocypris rarus) vitellogenin and comparison of vitellogenin responses in rare minnow and zebrafish (Danio rerio). Science of the Total Environment 364, 284–294 (2006).

  28. 28.

    Roy, R. L., Morin, Y., Courtenay, S. C. & Robichaud, P. Purification of vitellogenin from smooth flounder (Pleuronectes putnami) and measurement in plasma by homologous ELISA. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 139, 235–244 (2004).

  29. 29.

    Holbech, H. et al. Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 130, 119–131 (2001).

  30. 30.

    Luo, W., Zhou, Q. & Jiang, G. Development of enzyme-linked immunosorbent assays for plasma vitellogenin in Chinese rare minnow (Gobiocypris rarus). Chemosphere 84, 681–688 (2011).

  31. 31.

    Peck, K. A. et al. Development of an enzyme-linked immunosorbent assay for quantifying vitellogenin in Pacific salmon and assessment of field exposure to environmental estrogens. Environmental Toxicology and Chemistry 30, 477–486 (2011).

  32. 32.

    Hallgren, S. & Olsén, K. H. Effects on guppy brain aromatase activity following short-term steroid and 4-nonylphenol exposures. Environmental toxicology 25, 261–271 (2010).

  33. 33.

    Volkova, K. et al. Developmental exposure of zebrafish (Danio rerio) to 17α-ethinylestradiol affects non-reproductive behavior and fertility as adults, and increases anxiety in unexposed progeny. Hormones and behavior 73, 30–38 (2015).

  34. 34.

    Ternes, T. A. et al. Behavior and occurrence of estrogens in municipal sewage treatment plants-I. Investigations in Germany, Canada and Brazil. Science of the Total Environment 225, 81–90 (1999).

  35. 35.

    Liney, K. E., Jobling, S., Shears, J. A., Simpson, P. & Tyler, C. R. Assessing the sensitivity of different life stages for sexual disruption in roach (Rutilus rutilus) exposed to effluents from wastewater treatment works. Environmental health perspectives 1299–1307 (2005).

  36. 36.

    Komori, K., Tanaka, H., Okayasu, Y., Yasojima, M. & Sato, C. Analysis and occurrence of estrogen in wastewater in Japan. Water Science and Technology 50, 93–100 (2004).

  37. 37.

    Kolpin, D. W. et al. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: A national reconnaissance. Environmental science & technology 36(6), 1202–1211 (2002).

  38. 38.

    Van den Belt, K., Verheyen, R. & Witters, H. Reproductive effects of ethynylestradiol and 4t-octylphenol on the zebrafish (Danio rerio). Archives of environmental contamination and toxicology 41, 458–467 (2001).

  39. 39.

    Seki, M. et al. Effect of ethinylestradiol on the reproduction and induction of vitellogenin and testis-ova in medaka (Oryzias latipes). Environmental Toxicology and Chemistry 21, 1692–1698 (2002).

  40. 40.

    Salierno, J. D. & Kane, A. S. 17α-Ethinylestradiol alters reproductive behaviors, circulating hormones, and sexual morphology in male fathead minnows (Pimephales promelas). Environmental toxicology and chemistry 28, 953–961 (2009).

  41. 41.

    Pawlowski, S., Van Aerle, R., Tyler, C. R. & Braunbeck, T. Effects of 17α-ethinylestradiol in a fathead minnow (Pimephales promelas) gonadal recrudescence assay. Ecotoxicology and environmental safety 57, 330–345 (2004).

  42. 42.

    Andersson, C., Katsiadaki, I., Lundstedt-Enkel, K. & Örberg, J. Effects of 17α-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback (Gasterosteus aculeatus). Aquatic toxicology 83, 33–42 (2007).

  43. 43.

    Folmar, L. C. et al. Comparative estrogenicity of estradiol, ethynyl estradiol and diethylstilbestrol in an in vivo, male sheepshead minnow (Cyprinodon variegatus), vitellogenin bioassay. Aquatic Toxicology 49, 77–88 (2000).

  44. 44.

    Peters, R. E., Courtenay, S. C., Cagampan, S., Hewitt, M. L. & MacLatchy, D. L. Effects on reproductive potential and endocrine status in the mummichog (Fundulus heteroclitus) after exposure to 17α-ethynylestradiol in a short-term reproductive bioassay. Aquatic Toxicology 85, 154–166 (2007).

  45. 45.

    Yamazaki, E. et al. Bisphenol A and other bisphenol analogues including BPS and BPF in surface water samples from Japan, China, Korea and India. Ecotoxicology and environmental safety 122, 565–572 (2015).

  46. 46.

    Naderi, M., Wong, M. Y. & Gholami, F. Developmental exposure of zebrafish (Danio rerio) to bisphenol-S impairs subsequent reproduction potential and hormonal balance in adults. Aquatic toxicology 148, 195–203 (2014).

  47. 47.

    Rose, J. et al. Vitellogenin induction by 17β-estradiol and 17α-ethinylestradiol in male zebrafish (Danio rerio). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 131, 531–539 (2002).

  48. 48.

    Petersen, G. I., Andersen, L., Holbech, H. & Pedersen, K. L. Baseline test studies and development of ELISA. Zebrafish for Testing Endocrine Disrupting Chemicals. TemaNord 555, 35–46 (2000).

  49. 49.

    Orn, S., Yamani, S. & Norrgren, L. Comparison of vitellogenin induction, sex ratio, and gonad morphology between zebrafish and Japanese medaka after exposure to 17alpha-ethinylestradiol and 17beta-trenbolone. Archives of environmental contamination and toxicology 51, 237–243 (2006).

  50. 50.

    Fenske, M., van Aerle, R., Brack, S., Tyler, C. R. & Segner, H. Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals. Comp. Biochem. Physiol. C 129, 217–232 (2001).

  51. 51.

    Van den Belt, K., Verheyen, R. & Witters, H. Effects of 17α-ethynylestradiol in a partial life-cycle test with zebrafish (Danio rerio): effects on growth, gonads and female reproductive success. Science of the Total Environment 309(1), 127–137 (2003).

  52. 52.

    Scholz, S., Kordes, C., Hamann, J. & Gutzeit, H. O. Induction of vitellogenin in vivo and in vitro in the model teleost medaka (Oryzias latipes): comparison of gene expression and protein levels. Marine environmental research 57, 235–244 (2004).

  53. 53.

    Japanese Ministry of Environment, Available from: (accessed 10.10.10) (2006).

  54. 54.

    Tilton, S. C., Foran, C. M. & Benson, W. H. Relationship between ethinylestradiol-mediated changes in endocrine function and reproductive impairment in Japanese medaka (Oryzias latipes). Environmental toxicology and chemistry 24, 352–359 (2005).

  55. 55.

    Scott, P. D. et al. An assessment of endocrine activity in Australian rivers using chemical and in vitro analyses. Environmental Science and Pollution Research 21, 12951–12967 (2014).

  56. 56.

    Ekman, D. R. et al. Profiling lipid metabolites yields unique information on sex-and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol. Metabolomics 5, 22 (2009).

  57. 57.

    Panter, G. H. et al. Utility of a juvenile fathead minnow screening assay for detecting (anti−) estrogenic substances. Environ Toxicol Chem 21, 319–326 (2002).

  58. 58.

    Purdom C. E. et al. Estrogenic effects of effluents from sewage treatment works Chem. Ecol. pp. 275–285 (1994).

  59. 59.

    Van den Belt, K., Verheyen, R. & Witters, H. Comparison of vitellogenin responses in zebrafish and rainbow trout following exposure to environmental estrogens. Ecotoxicology and environmental safety 56, 271–281 (2003).

  60. 60.

    Woods, M. & Kumar, A. Vitellogenin induction by 17β-estradiol and 17α-ethynylestradiol in male Murray rainbowfish (Melanotaenia fluviatilis). Environmental Toxicology and Chemistry 30, 2620–2627 (2011).

  61. 61.

    Mortensen, A. S. & Arukwe, A. Effects of 17α-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar). Aquatic Toxicology 85, 113–123 (2007).

  62. 62.

    Wang, J. et al. Preparation of a polyclonal antibody against goldfish (Carassius auratus) vitellogenin and its application to detect the estrogenic effects of monocrotophos pesticide. Ecotoxicology and environmental safety 111, 109–116 (2015).

  63. 63.

    Pan, Z., Tian, H., Wang, W., Wang, J. & Ru, S. Identification, purification, and immunoassay of stone flounder (Kareius bicolouratus) vitellogenin. Journal of the Korean Society for Applied Biological Chemistry 55, 219–227 (2012).

  64. 64.

    Sun, X. & Zhang, S. Purification and characterization of a putative vitellogenin from the ovary of amphioxus (Branchiostoma belcheri tsingtaunese). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 129, 121–127 (2001).

  65. 65.

    Mitsui, N., Tooi, O. & Kawahara, A. Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17β effects on whole animals and primary-cultured hepatocytes. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 135, 305–313 (2003).

Download references


This research was supported by the National Natural Science Foundation of China (21607144) and General Administration of Quality Supervision, Inspection and Quarantine of China (2015IK202).

Author information

Author notes

  1. Jun Wang and Shuwei Ma contributed equally to this work.


  1. Marine Life Science College, Ocean University of China, Qingdao, 266003, China

    • Jun Wang
    • , Shuwei Ma
    • , Zhenzhong Zhang
    • , Mingyi Zheng
    • , Yifei Dong
    •  & Shaoguo Ru


  1. Search for Jun Wang in:

  2. Search for Shuwei Ma in:

  3. Search for Zhenzhong Zhang in:

  4. Search for Mingyi Zheng in:

  5. Search for Yifei Dong in:

  6. Search for Shaoguo Ru in:


Professor S.R. and associate professor J.W. designed the project. S.M. performed the experiment and conducted data analysis with the help of Z.Z. and M.Z. J.W. wrote the manuscript with the help of Y.D. and S.R. Y.D. revised the manuscript according to the reviewers’ comments. All authors reviewed and approved the final manuscript.

Competing Interests

The authors declare that they have no competing interests.

Corresponding authors

Correspondence to Yifei Dong or Shaoguo Ru.

Electronic supplementary material


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Creative Commons BY

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit