ESR1 mediates estrogen-induced feminization of genetic male Chinese soft-shelled turtle†.

Exogenous estrogen have shown their feminization abilities during the specific sex differentiation period in several reptiles. However, the specific regulatory mechanism and downstream regulatory genes of estrogen remain elusive. In the present study, 17ß-estradiol (E2), as well as drugs of specific antagonists and/or agonists of estrogen receptors, were employed to figure out the molecular pathway involved in the E2-induced feminization in Chinese soft-shelled turtles, an important aquaculture species in China. E2 treatment led to typical female characteristics in the gonads of ZZ individuals, including thickened outer cortex containing a number of germ cells and degenerated medullary cords, as well as the disappearance of male marker SOX9, and the ectopic expression of ovarian regulator FOXL2 at the embryonic developmental stage 27 and 1 month after hatching. The specific ESR1 antagonist or a combination of three estrogen receptor antagonists could block the sex reversal of ZZ individuals induced by estrogen. In addition, specific activation of ESR1 by agonist also led to the feminization of ZZ gonads, which was similar to the effect of estrogen treatment. Furthermore, transcriptome data showed that the expression level of FOXL2 was significantly upregulated, whereas mRNA levels of DMRT1, SOX9, and AMH were downregulated after estrogen treatment. Taken together, our results indicated that E2 induced the feminization of ZZ Chinese soft-shelled turtles via ESR1, and decrease of male genes DMRT1, SOX9, and AMH and increase of ovarian development regulator FOXL2 might be responsible for the initiation of E2-induced feminization.

Long-Term Exposure to Neonicotinoid Insecticide Acetamiprid at Environmentally Relevant Concentrations Impairs Endocrine Functions in Zebrafish: Bioaccumulation, Feminization, and Transgenerational Effects.

Neonicotinoid insecticides have attracted worldwide attention due to their ubiquitous occurrence and detrimental effects on aquatic organisms, yet their impacts on fish reproduction during long-term exposure remain unknown. Here, zebrafish (F0) were exposed to a neonicotinoid, acetamiprid, at 0.19-1637 µg/L for 154 d. Accumulation and biotransformation of acetamiprid were observed in adult fish, and the parent compound and its metabolite (acetamiprid-N-desmethyl) were transferred to their offspring. Acetamiprid caused slight survival reduction and significant feminization in F0 fish even at the lowest concentration. Hormone levels in F0 fish were remarkedly altered, that is, gonad 17ß-estradiol (E2) significantly increased, while androstenedione decreased. The corresponding transcription of steroidogenic genes (ar, cyp19b, fshß, gnrh2, gnrh3, and lhß) were significantly upregulated in the brain and gonad of the females but downregulated in the males. The vtg1 gene expression in the liver of male fish was also upregulated. In addition to F0 fish, parental exposure to acetamiprid decreased hatchability and enhanced malformation of F1 embryos. Chronic exposure to acetamiprid at environmentally relevant concentrations altered hormone production and the related gene expression of the hypothalamic-pituitary-gonad (HPG) axis in a sex-dependent way, caused feminization and reproductive dysfunction in zebrafish, and impaired production and development of their offspring.

Soy Isoflavones Induce Feminization of Japanese Eel (Anguilla japonica).

Under aquaculture conditions, Japanese eels (Anguilla japonica) produce a high percentage of males. However, females gain higher body weight and have better commercial value than males, and, therefore, a high female ratio is required in eel aquaculture. In this study, we examined the effects of isoflavones, genistein, and daidzein on sex differentiation and sex-specific genes of eels. To investigate the effects of these phytoestrogens on the gonadal sex, we explored the feminizing effects of soy isoflavones, genistein, and daidzein in a dose-dependent manner. The results showed that genistein induced feminization more efficiently than daidzein. To identify the molecular mechanisms of sex-specific genes, we performed a comprehensive expression analysis by quantitative real-time PCR and RNA sequencing. Phenotypic males and females were produced by feeding elvers a normal diet or an estradiol-17ß- or genistein-treated diet for 45 days. The results showed that female-specific genes were up-regulated and male-specific genes were down-regulated in the gonads, suggesting that genistein induces feminization by altering the molecular pathways responsible for eel sex differentiation.

Effects of adult male rat feminization treatments on brain morphology and metabolomic profile.

Body feminization, as part of gender affirmation process of transgender women, decreases the volume of their cortical and subcortical brain structures. In this work, we implement a rat model of adult male feminization which reproduces the results in the human brain and allows for the longitudinal investigation of the underlying structural and metabolic determinants in the brain of adult male rats undergoing feminization treatments. Structural MRI and Diffusion Tensor Imaging (DTI) were used to non-invasively monitor in vivo cortical brain volume and white matter microstructure over 30 days in adult male rats receiving estradiol (E2), estradiol plus cyproterone acetate (CA), an androgen receptor blocker and antigonadotropic agent (E2 + CA), or vehicle (control). Ex vivo cerebral metabolic profiles were assessed by 1H High Resolution Magic Angle Spinning NMR (1H HRMAS) at the end of the treatments in samples from brain regions dissected after focused microwave fixation (5 kW). We found that; a) Groups receiving E2 and E2 + CA showed a generalized bilateral decrease in cortical volume; b) the E2 + CA and, to a lesser extent, the E2 groups maintained fractional anisotropy values over the experiment while these values decreased in the control group; c) E2 treatment produced increases in the relative concentration of brain metabolites, including glutamate and glutamine and d) the glutamine relative concentration and fractional anisotropy were negatively correlated with total cortical volume. These results reveal, for the first time to our knowledge, that the volumetric decreases observed in trans women under cross-sex hormone treatment can be reproduced in a rat model. Estrogens are more potent drivers of brain changes in male rats than anti-androgen treatment.

2,4-Dichlorophenol induced feminization of zebrafish by down-regulating male-related genes through DNA methylation.

2,4-Dichlorophenol (2,4-DCP) is ubiquitous in aquatic environment and has potential estrogenic effect on fish. However, the effect of 2,4-DCP on sex differentiation of zebrafish (Danio rerio) and the underlying mechanism are largely unknown. To address these questions, zebrafish larvae at 20 or 30 days post fertilization (dpf) were exposed to 2,4-DCP (0, 80 and 160 µg L-1) with/without 5-aza-2'-deoxycytidine (5AZA, 50 µg L-1) for 10 days. The sex ratios and the expressions of male-related genes including amh, gata4, nr5a1a, nr5a2 and sox9a were analyzed. In addition, the DNA methylation levels of amh, nr5a2 and sox9a were examined. The results showed that 2,4-DCP exposure resulted in significant increase of female ratios both in 20-30 and 30-40 dpf groups. Correspondingly, the expressions of gata4, nr5a1a, nr5a2 and sox9a were decreased by 2,4-DCP exposure in two treatment periods. However, the transcript of amh was decreased by 2,4-DCP exposure only from 30 to 40 dpf. The DNA methylation levels of amh, nr5a2 and sox9a were increased following 2,4-DCP exposure. Moreover, the addition of 5AZA could counteract the effects including feminization, disturbance of gene expression and DNA hypermethylation caused by 2,4-DCP. These results indicated that the feminizing effect of 2,4-DCP was accomplished by regulating the expression of male-related genes through DNA methylation.

Feminization, altered gonadal development, and liver damage in least killifish (Heterandria formosa) exposed to sublethal concentrations of 17α-ethinylestradiol.

The widespread use of the synthetic estrogen 17 α-ethinylestradiol (EE2) has resulted in elevated levels in aquatic environments, where it is known to act as an endocrine disrupting chemical affecting fish and other aquatic organisms. Examining changes in the structure of the fish' gonads and liver has proven to be an effective approach for assessing these impacts. While changes have been reported for various fish species, it is not clear whether impacts are equally severe in live-bearing fishes. The present study looked at gonadal and liver development in EE2-exposed least killifish (Heterandria formosa), a live-bearing Poeciliid. Exposures to 0, 5, or 25 ng/L EE2 began within six days of birth and continued until fish became sexually mature 12-23 weeks later. Exposure to 5 ng/L EE2 resulted in severe intersex in fish with external male characteristics, a slowdown of spermatogenesis in these intersex fish and a slowdown of oogenesis in the female fish. Moreover, these fish had a variety of liver injuries. Fish exposed to 25 ng/L EE2 exhibited intersex but at a lower frequency than occurred at 5 ng/L. In contrast, liver damage and slowdown of both oogenesis and spermatogenesis exhibited the typical dose-dependence. These findings illustrate the importance of including histological analyses when assessing endocrine disruption in fish, demonstrate that the live-bearing mode of reproduction appears to provide limited protection from the effects of waterborne EE2, and provide further evidence that EE2 has multiple impacts on fish health and reproduction that are severe enough to potentially affect fish populations.

Endocrine care of transpeople part II. A review of cross-sex hormonal treatments, outcomes and adverse effects in transwomen.

The treatment of transwomen relies on the combined administration of anti-androgens or GnRH analogues to suppress androgen production and thereby reduce male phenotypic characteristics together with oestrogens to develop female characteristics. In transwomen, synthetic oestrogens such as ethinyl oestradiol, as well as conjugated equine oestrogens (CEE), should be avoided to minimize thromboembolic risks especially in older transwomen and in those with risk factors. Currently, available short- and long-term safety studies suggest that cross-sex hormonal therapy (CHT) can be considered safe in transwomen improving the well-being and quality of life of these individuals. Long-term monitoring should aim to decrease cardiovascular risks and should include prostate and breast cancer screenings.

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol.

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1-10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish.

Complete feminization of catfish by feeding Limnodilus, an annelid worm collected in contaminated streams.

BACKGROUND: Feminization of animals derived from areas polluted by endocrine disrupting chemicals (EDCs) has been observed in all classes of vertebrates. However, feminization of artificially reared offspring by feeding of specific living organisms has never been reported. METHODS: Different food (including Limnodilus spp collected from the wild) and time treatment were applied to southern catfish. In addition, EDCs in Limnodilus spp., an annelid worm collected from wild contaminated small streams, was detected by LC-MS (Liquid chromatography-mass spectrometry). Serum estradiol-17ß and vitellogenin (VTG) levels and gonadal Sf1, Dmrt1, Foxl2, Cyp19a1a expression levels in the catfish were measured through Estradiol/VTG EIA Kit and real-time PCR. RESULTS: Here we report that feeding of Limnodilus spp. resulted in complete feminization of southern catfish, which has a 1:1 sex ratio in wild conditions. Furthermore, HPLC analysis showed that the extraction of Limnodilus spp. contained EDCs, including bisphenol A (BPA), diethylstilbestrol (DES), 4-tert-octylphenol (4-t-OP) and 4-nonylphenol (4-NP), which were further confirmed by LC-MS. Feeding southern catfish using commercial diets sprayed with EDCs cocktail also resulted in 100% female, whereas the control fish displayed approximate 1:1 sex ratio. Limnodilus spp. fed fish displayed similar serum estradiol-17ß and VTG levels and gonadal Sf1, Dmrt1, Foxl2, Cyp19a1a expression levels to those of female control. CONCLUSION: These results demonstrated that EDCs in Limnodilus spp. cause southern catfish feminization by affecting aromatase expression and endogenous estrogen level. This is the first report showing that feeding of any living organism resulted in complete feminization of a vertebrate.

Long-term effects of intraperitoneal injection of estradiol-17ß on the growth and physiology of juvenile stellate sturgeon Acipenser stellatus.

Juvenile stellate sturgeon Acipenser stellatus were intraperitoneally injected with estradiol-17ß (E2; 0 and 5 mg/kg fish) to investigate the possibility of sex reversal and also determine the changes in biochemical parameters. Five-month-old fish (40.9 ± 1.1 g) were injected every 3-week interval during a 190-day trial. At the termination of the experiment, final weight and other growth parameters including weight gain and specific growth rate, hepatosomatic and viscerosomatic indices were not affected by repetitive injection of E2. Hematological features of E2-treated fish showed significant reductions in number of red blood cells, hemoglobin concentration, hematocrit value and mean corpuscular hemoglobin (P < 0.05), but no significant changes were observed in number of white blood cells, mean corpuscular volume and mean corpuscular hemoglobin concentration (P > 0.05). Calcium, phosphorus, glucose, triacylglycerol, cholesterol, total protein and estradiol concentrations were significantly increased in fish injected with E2 (P < 0.001). Plasma progesterone and testosterone levels were noticeably lower in fish injected with 5 mg/kg E2 rather than the control fish (P < 0.001). Histological observations of gonads showed that all fish injected with 5 mg/kg E2 apparently feminized, while 66.6 % of the control group was female. These results revealed that the injection of E2 is an effective method for feminization of stellate sturgeon without having significant inhibitory effects on growth and survival.

Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric Müllerian duct development by eliminating asymmetric gene expression in Japanese quail embryos.

In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3ß-HSD) and estrogen receptor-ß, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.

Neuroprotection with non-feminizing estrogen analogues: an overlooked possible therapeutic strategy.

Although many of the effects of estrogens on the brain are mediated through estrogen receptors (ERs), there is evidence that neuroprotective activity of estrogens can be mediated by non-ER mechanisms. Herein, we review the substantial evidence that estrogens neuroprotection is in large part non-ER mediated and describe in vitro and in vivo studies that support this conclusion. Also, we described our drug discovery strategy for capitalizing on enhancement in neuroprotection while at the same time, reducing ER binding of a group of synthetic non-feminizing estrogens. Finally, we offer evidence that part of the neuroprotection of these non-feminizing estrogens is due to enhancement in redox potential of the synthesized compounds.

Burrowing in the freshwater mussel Elliptio complanata is sexually dimorphic and feminized by low levels of atrazine.

The widely used herbicide atrazine (ATR) may have endocrine-associated adverse effects, including on behavior. In this study, 120 adult freshwater mussels, Elliptio complanata, were exposed to ATR at the environmentally relevant concentrations of 1.5, 15, or 150 µg/L. Burrowing depth was evaluated hourly for 6 h and at sacrifice animals were sexed by gonad smear. Female controls burrowed overall approximately 30% less than males, the first report of sexual dimorphism in this behavior. Atrazine at 15 µg/L feminized burrowing in both sexes, in that exposed animals burrowed 20% less than their same-sex controls. Males treated with 1.5 µg /L ATR displayed approximately 20-fold higher vitellogenin (VTG) levels than same-sex controls. Higher concentrations of ATR were not associated with increasing effects. A scatterplot showed a weak binomial curve associating low burrowing with high VTG levels. Taken together, these data suggest a nonlinear dose response in behavioral and physiological feminization produced by ATR and support the need to reconsider the widespread use of this compound.

Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis).

The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines.

The effect of estrogen in a man with Parkinson's disease and a review of its therapeutic potential.

Estrogen has been implicated in controlling the pathogenesis and symptoms of Parkinson's disease (PD) in women. Here, we report a 53-year-old male with PD who underwent estrogen therapy with estradiol (E2). Within a month, he exhibited increased dyskinesias. His medication was reduced by 35% from a levodopa equivalent dose (LED) of 820-535 over three months, which overall improved his motor fluctuations and dyskinesias. Therefore, E2 therapy could have therapeutic potential in males with PD.

Levonorgestrel causes feminization and dose-dependent masculinization in medaka fish (Oryzias latipes): Endocrine-disruption activity and its correlation with sex reversal.

The effect of a synthetic progestin, levonorgestrel (LNG), on the sex of exposed embryos was examined in medaka fish (Oryzias latipes). The aims of this study are to clarify the dual effect of LNG on sex and the correlation with its androgenic/estrogenic potential in medaka. LNG exposure causes significant dose-dependent masculinization (0.1-100 µg/L), whereas a decrease in the masculinization ratio is observed at 100 µg/L. LNG also causes significant feminization at 1-100 µg/L, but not in a dose-dependent manner. Exposure of estrogen-responsive gene (choriogeninH-EGFP) transgenic embryos to 100 µg/L LNG produced significant fluorescent signals in hatched fry. In vitro transcriptional assays indicated that LNG at 10-7-10-5 M induced significant activity for estrogen receptor (ESR)2a and ESR2b, but not for ESR1. In pre-self-feeding fry at 5 days post hatching (dph), 1-100 µg/L LNG caused a significant increase in the mRNA of choriogeninH, irrespective of genetic sex. Moreover, LNG (10-10-10-5 M) also caused a significant increase in the transcriptional activity of androgen receptor (AR) α and ARß in vitro, and 0.1 µg/L LNG significantly increased the mRNA levels of a testis-differentiation initiation factor, gonadal soma-derived factor (gsdf), as an androgen-upregulated and estrogen-downregulated gene, in 5 dph XX fry to levels similar to those in the control XY fry. However, 100 and 10 µg/L LNG suppressed or did not induce gsdf mRNA expression in XY and XX fry, respectively. Together, these findings show that LNG exerts estrogenic and androgenic activities in different concentration ranges, which correlate with the ratio of LNG-induced sex reversal. These results suggest for the first time, that medaka exposure to LNG can induce masculinization and feminization, based on the balance between androgenic and estrogenic activities, and the protocol applied in this study represents an alternative to the traditional animal model used to screen for endocrine-disrupting potential.

Masculine epigenetic sex marks of the CYP19A1/aromatase promoter in genetically male chicken embryonic gonads are resistant to estrogen-induced phenotypic sex conversion.

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

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).

Monocrotophos is a highly toxic organophosphorus pesticide that has been confirmed to be an endocrine-disrupting chemical. To evaluate the influence of this pollutant on the reproductive system of male fish, we studied the sex steroid levels, reproductive traits, sex ratio, and reproductive success in male guppies (Poecilia reticulata) exposed to 40% monocrotophos pesticide at the nominal concentrations of 0.01, 0.10, and 1.00 mg/L for 90 days from birth to adulthood in a semi-static exposure system. Radioimmunoassay and western blot analyses demonstrated that the long-term exposure to monocrotophos pesticide during the sexual development of male guppies caused a significant increase in 17ß-estradiol levels and consequently induced vitellogenin synthesis, suggesting the feminization of the males. Monocrotophos pesticide also caused a significant decrease in testosterone levels, which consequently inhibited testis growth and reduced the sperm count and the area and intensity of their sexually attractive orange spots, which collectively indicated the significant demasculinization of the male sexual characteristics. Furthermore, these changes in the sexual characteristics at the cellular and organ levels translated into ecologically important effects on the reproductive success at the individual level, as measured by a decrease in offspring production and survival rate. The present study provides the first evidence that monocrotophos pesticide can cause severe reproductive abnormalities in fish due to its endocrine-disrupting action.

An integrated assessment of estrogenic contamination and feminization risk in fish in Taihu Lake, China.

An integrated approach, combining biological and chemical methods, was used to assess potential exposure to exogenous estrogens and their possible interference with the endocrine system of male goldfish (Carassius auratus) in Taihu Lake. A suite of biomarkers in caged fish after in situ exposure for 28 days, coupled with six selected exogenous estrogens in water, were determined at eight biomonitoring stations. The compounds estrone, 17ß-estradiol (E2), estriol, 17α-ethinylestradiol (EE2), bisphenol-A and diethylstilbestrol were detected in most of the samples and the concentrations of total estradiol equivalent (EEQ) ranged between 5.69 and 17.8 ng/L. E2 and EE2 were thought to be the major causal agents responsible for the estrogenic activities. Elevated serum vitellogenin and E2, gonadal DNA damage and reduced gonadosomatic index were observed in fish collected from most stations. The integrated biomarker response (IBR) index showed good agreement with the observed total EEQ levels in water, and feminization risk in fish may be present in northern Taihu Lake, especially in Meiliang Bay and Zhushan Bay.