Feminizing/demasculinizing effects of polychlorinated biphenyls on

Aquatic Toxicology 84 (2007) 321–327
Feminizing/demasculinizing effects of polychlorinated biphenyls
on the secondary sexual development of Xenopus laevis
Zhan-Fen Qin ∗ , Xiao-Fei Qin, Lei Yang, Li Han-Ting, Xing-Ru Zhao, Xiao-Bai Xu
The State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences,
The Chinese Academy of Sciences, Beijing, China
Received 29 January 2007; received in revised form 15 June 2007; accepted 15 June 2007
Abstract
We have previously demonstrated that polychlorinated biphenyls (PCBs) have caused phenotypic feminization/demasculinization of gonadal
development in Xenopus laevis. Whether PCBs affect secondary sexual development has remained unknown. In this study, X. laevis tadpoles were
exposed to Aroclor1254 and PCB3 from stage 46/47 (system of Nieuwkoop and Faber) for up to 1 month postmetamorphosis. After 24 months
postmetamorphosis, the degree of secondary sexual development was examined. Male oviducts were observed in some of the PCB-exposed male
frogs, but not in control males. These male oviducts had not completely developed in histological structure when compared with mature female
oviducts. Larynx weight and width of PCB-exposed males were significantly less than those of control males. Laryngeal histology showed that PCBs
inhibited cartilaginous and muscular development of male frogs, i.e. elastic cartilages had not completely developed and laryngeal muscle fibers
were smaller. In a further study on adult male frogs, a decrease in serum testosterone level was found in PCB-exposed frogs compared with controls,
but serum estradiol level was not significantly affected. Our study suggests that PCBs can cause phenotypic feminization/demasculinization of
male genital ducts and larynges, and these effects may, in part, result from the decrease in serum testosterone level in X. laevis.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Polychlorinated biphenyls; Secondary sexual development; Xenopus laevis; Feminization/demasculinization
1. Introduction
Amphibians are unique sentinels for environmental health
because of their permeable skins and biphasic life-history
strategies (van der Schalie et al., 1999). Xenopus laevis is
an amphibian species widely used as a model for developmental biology in laboratory studies. Especially with regard
to endocrine disruptors, X. laevis has increasingly aroused
the interest of ecotoxicologists in recent years. Since the
Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommended the Amphibian Development and
Reproduction Test as a method to assay endocrine disruptors
(US EPA, 1998), there have been numerous studies of endocrine
disruptors using X. laevis as a model animal species (Lutz and
Kloas, 1999; Hurter et al., 2002; Crump et al., 2002; Bevan et al.,
∗ Corresponding author at: P.O. Box 2871, Research Center for EcoEnvironmental Sciences, The Chinese Academy of Sciences, Beijing 100085,
China. Tel.: +86 10 62919177; fax: +86 10 62923563.
E-mail address: [email protected] (Z.-F. Qin).
0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2007.06.011
2003; Qin et al., 2003; Klann et al., 2005; Huang et al., 2005).
Several endpoints such as gonadal development, vitellogenin
expression, and tail resorption in X. laevis have been used to
assay endocrine disruptors. In particular, gonadal development,
which is sensitive to estrogens, has been used as an endpoint for
assaying endocrine disruptors with estrogenic activity (Witschi
and Allison, 1950; Gallien, 1974; Chang and Witschi, 1956;
Villalpando and Merchant-Larios, 1990; Kloas et al., 1999;
Miyata and Kubo, 2000). Some endocrine disruptors such as
atrazine, polychlorinated biphenyls (PCBs) and bisphenol A
have been reported to have feminizing effects on gonadal development of X. laevis (Kloas et al., 1999; Hayes et al., 2002; Qin et
al., 2003; Levy et al., 2004) although contradictory results have
also been reported (Pickford et al., 2003; Carr et al., 2003).
PCBs are persistent environmental pollutants with a wide
range of toxicities (Hansen, 1998). Although commercial production of PCBs has been banned since the 1970s and 1980s,
PCBs are still ubiquitous in the environment because of their
stability and lipophilicity (Ross et al., 2004; Wong et al., 2004).
Due to improper treatment of PCB-containing appliances in
some countries, PCBs are released into the environment and
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result in seriously PCB-polluted sites (Zhang et al., 2003; Mai et
al., 2005). In recent years, the endocrine disrupting activities of
PCBs have drawn considerable attention. In particular, a number
of reports have demonstrated that PCBs can alter the sex steroid
level and affect gonadal development in several species (Matta et
al., 1998, Willingham et al., 2000; Kaya et al., 2002). In the past
few years, we have been studying endocrine disruption by PCBs
using X. laevis. In previous studies, we described the feminizing
effects of PCBs on gonadal development in X. laevis (Qin et al.,
2003, 2005). Feminizing and demasculinizing effects are possibly similar in phenotypic characteristics, because females are
the default with respect to phenotypic sex in X. laevis (Witschi,
1971). Therefore, we hypothesize that the phenotypic feminization of gonadal development caused by PCBs reported in our
previous study may also result from demasculinizing effects. In
this study, we examined the effects of PCBs on the secondary
sexual development of X. laevis.
shape (30 cm × 20 cm × 25 cm). The experimental water was
changed every 4 days. It took approximately 2 months from the
beginning of exposure for X. laevis to complete metamorphosis.
Exposure continued until 1 month postmetamorphosis. During
the PCB-exposure period, tadpoles and frogs were collected to
analyze the accumulation of PCBs. At the end of PCB exposure,
froglets with Aroclor1254 exposure contained approximately
64 ␮g/g PCBs (fresh weight); the PCBs level in froglets with
PCB3 exposure was not analyzed. Twenty of the remaining frogs
in each treatment at the end of PCB exposure were randomly chosen to be maintained in the experimental water without PCBs.
Naturally, although the frogs were moved to water not containing PCBs, some exposure continues during the continued
development, including secondary sexual development, owing
to bioaccumulated PCBs.
2. Materials and methods
At 24 months postmetamorphosis, the secondary sexual
development of frogs was examined. Frogs were sacrificed by
immersion in MS222 (3-aminobenzoic acid ethyl ester methanesulfonate) at 3 g/l and weighed. Frogs were sexed by cloacal
and gonadal examination. For determination of the percentage
of each sex, intersexuals with testes but some ovarian characteristics were regarded as males. Larynges and genital ducts
with gonads were examined using a dissecting microscope after
fixation with Bouin’s fixative. Larynges were weighed and measured. Fixed samples were then embedded in paraffin, sectioned
at 5 ␮m, and stained with hematoxylin and eosin (HE). Sections
were examined with a light microscope.
2.1. Chemicals
Aroclor 1254 was obtained from Supelco (Bellefonte, PA,
USA). PCB3 , one of two main industrial PCB mixtures produced
in China, was obtained from the Institute for Environmental
Chemistry of the Chinese Academy of Sciences. PCB3 contains
relatively abundant low-chlorinated congeners, and has been
identified to be close to Aroclor 1242 in composition (Wang
et al., 1981). PCB3 has been approved as a standard by the Environment Science Council of the Chinese Academy of Sciences
(Beijing, China).
2.2. Breeding and housing
Mature female and male X. laevis were separately maintained
in glass tanks containing dechlorinated water at 22 ± 2 ◦ C with
a 12-h light:12-h dark cycle and fed once a week on chopped
pork liver. Breeding was induced by injection of human chorionic gonadotrophin (Sive et al., 2000). After eggs were laid, the
females and males were taken out of the breeding tank. Fertilized eggs were incubated at 22 ± 2 ◦ C with a 12-h light:12-h
dark cycle. From the 5th day after fertilization, tadpoles were
fed daily on Daphnia.
2.3. Exposure of tadpoles to chemicals
On the 6th day after fertilization, healthy tadpoles at NF stage
46/47 (system of Nieuwkoop and Faber, 1956) were randomly
selected from the offspring of a pair of parental frogs for the
exposure experiment. PCBs were dissolved in ethanol to produce
stock solutions (10 mg/ml). Experimental water was prepared by
adding the stock solution to dechlorinated water. The nominal
concentration of PCBs was 10 ␮g/l in water (Qin et al., 2005).
The control group received the same amount of ethanol used as
solvent for PCBs (18 ␮l ethanol in each tank). Each treatment
comprised a series of replicated glass tanks with 30 tadpoles per
tank containing 18 l water. All tanks were the same size and
2.4. Examination of secondary sexual development
2.5. Exposure of adult frogs to chemicals and hormone
assay
In X. laevis, secondary sexual development, which is controlled by sex hormones, begins at the end of metamorphosis
(Kelley, 1992). At this stage, gonads of froglets, as those of
adult frogs, can secrete sex hormones. To some degree, adult
frogs can reflect froglets with respect to the responsiveness of
sex hormones to PCBs. Hayes et al. (2002) studied effects of
atrzine on sex hormones of X. laevis tadpoles using adult frogs.
Therefore, we exposed adult frogs to PCBs and examined their
serum sex hormones in order to explore the possible mechanism by which PCBs affect secondary sexual development.
Forty-five adult male frogs from the same clutch were divided
into three tanks; each tank contained 15 frogs in 18 l water. All
tanks were the same size and shape (30 cm × 20 cm × 25 cm).
Exposure of frogs to PCBs was carried out similarly as the tadpole exposure. The experimental water was changed following
feeding once a week on chopped pork liver. After exposure lasting 3 months, frogs were sacrificed by immersion in MS222.
Blood was collected and serum was obtained. To reduce the
costs of the assay, serum from two frogs was combined into one
sample. In addition, one serum sample was derived from three
frogs exposed to Aroclor1254. Some individual serum samples
from frogs in the PCB3 -exposed and control groups were also
assayed. In total, eight, seven and nine serum samples from the
Z.-F. Qin et al. / Aquatic Toxicology 84 (2007) 321–327
control frogs, PCB3 -exposed frogs and Aroclor 1254-exposed
frogs were obtained, respectively. Serum testosterone and estradiol (E2 ) levels were determined by chemiluminescence assay
using the Automated Chemiluminescence System 180 and corresponding reagent kits in accordance with the manufacturer’s
instructions (Bayer Healthcare). All procedures were conducted
in accordance with the National Institute of Health Guide for the
Care and Use of Laboratory Animals (NRC, 1996).
2.6. Statistical analyses
Within the corresponding sexes, data were pooled by treatment for statistical analysis. Body weight and larynx weight of
the two sexes, and hormone levels, were analyzed using one-way
analysis of variance. A value of α = 0.05 was chosen to give a
significant difference.
3. Results
During the period of development from tadpoles to frogs,
mortality rates in all groups were less than 25%. None
of the frogs died during this experiment. At 24 months
postmetamorphosis, the body weights of frogs exposed to Aroclor1254 (females: 11.51 ± 2.44 g; males: 10.86 ± 1.45) and
PCB3 (females: 12.77 ± 2.79 g; males: 11.79 ± 1.25 g) showed
no significant difference from controls (females: 13.93 ± 2.30 g;
males: 12.1 ± 1.43 g) of the corresponding sex. Frogs in this
study weighed less than adult females and males reported in the
literature (Tobias et al., 1991). Tobias et al. reported that body
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weight may not be well correlated with age because the growth
of X. laevis may be affected by breeding conditions (Tobias et
al., 1991). The lower body weight of frogs in this study was
possibly related to the small aquarium.
Sexes distinguished by cloacal examination were consistent with gonadal examination. Based on the literature, normal
females at 24 months postmetamorphosis have sufficiently
developed ovaries and oviducts, while normal males at this age
have mature testes and Wolffian ducts as urogenital ducts. In this
study, the percentages of females in PCB-exposed groups (55%)
were not different from that of controls (60%). Two frogs with
intersexual gonads, which appeared to be testes in gross morphology but had some ovarian characteristics, were observed
in the Aroclor 1254- and PCB3 -exposed groups, respectively.
However, no intersexuality was observed in the controls. In addition, oviducts appeared in these abnormal males. Oviducts were
also found in three and two males with normal gross testicular
morphology in the Aroclor 1254- and PCB3 -exposed groups,
respectively. Thus, male oviducts appeared in 5 of 11 males
(45%) exposed to Aroclor 1254 and 4 of 11 males (36%) exposed
to PCB3, while no male oviducts were observed in controls.
Most of the male oviducts were straight and thin, while several
oviducts were convoluted like mature female oviducts (Fig. 1).
Testicular and genital duct abnormalities induced by PCBs were
also demonstrated by histological examination (Fig. 2). A normal testis consists of well-developed seminiferous tubes with
multiple stages of spermatocysts and spermatozoa. Seminiferous tubules from some testes of PCB-exposed frogs were
undeveloped and a lack of spermatozoa, and even the presence
Fig. 1. Male oviducts and ovotestes caused by polychlorinated biphenyls (PCBs) (exposed from stage 46/47) compared with normal oviducts and gonads in Xenopus
laevis. (A) Normal male with testes and Wolffian duct. (B) Normal female with ovary and oviduct. (C) Abnormal male with testes and an oviduct caused by PCB3.
(D) Abnormal male with ovotestes and oviducts induced by Aroclor1254.
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Fig. 2. Photomicrographs of gonads and oviducts from polychlorinated biphenyl-exposed Xenopus laevis (exposed from stage 46/47) compared with controls at
24 months postmetamorphosis. (A) Well-developed female oviduct with numerous secretory products. (B) Normal testis consisting of well-developed seminiferous
tubes with multi-stage spermatocysts and spermatozoa (). (C) Developing female oviduct with a thin layer of serosa and mucous. (D–F) Normal testicular tissue
with numerous spermatozoa and oviduct at the development stage from a frog exposed to PCB3 . (E and F) are magnified testis and oviduct. (G–I) Abnormal testis
with fewer spermatozoa and oviducts at the development stage from a frog exposed to Aroclor1254. (H and I) are magnified testis and oviduct. (A, D and G) scale
bar: 100 ␮m, (B, C, E, F, H and I) scale bar: 50 ␮m.
of oocytes, was found in testicular tissue. At 24 months postmetamorphosis, the developed female oviduct is composed of a
thin serosal layer, a thick mucosal layer that produces copious
secretions, and numerous epithelial ridges, which are formed of
ciliated and secretory cells. Both straight and convoluted male
oviducts were undeveloped, i.e. the mucosa was composed of a
single layer of columnar epithelium. In comparison, the mucosa
of developing female oviducts was composed of multiple or
single layers of round or columnar epithelium. These results
have shown that PCBs feminized/demasculinized genital duct
development in X. laevis.
Larynx weight and width of PCB-exposed males were significantly less than those of control males, while those of females
had no significant difference among all groups (Fig. 3, larynx
width not shown). Laryngeal histological structure showed that
PCBs inhibited the development of cartilages and muscles in
male X. laevis (Fig. 4). Elastic cartilage, which contains very
large lacunae and a thin extracellular matrix, and exhibits a
characteristic “Swiss cheese” appearance, is a sexually dimorphic tissue, i.e. it occurs in the adult male frog but not in the
female (Fisher et al., 1995). Elastic cartilages of the larynx of
male PCB-exposed frogs did not differentiate like that of normal males but like that of females. Females do not have elastic
cartilages, but mesenchymal tissues appear in the elastic precartilage zone (EPZ), a zone corresponding to the elastic cartilages
in males. Sassoon et al. (1987) used histochemical techniques
to show that male laryngeal muscle is made of medium-sized
Fig. 3. Effects of polychlorinated biphenyl exposure (from stage 46/47) on
larynx weight in Xenopus laevis. Data are means ± S.E.M. Asterisks indicate
significant differences (P < 0.05) in larynx weight between controls and PCBexposed frogs. The larynx weight of females among all groups showed no
significant differences.
Z.-F. Qin et al. / Aquatic Toxicology 84 (2007) 321–327
325
Fig. 4. Effects of polychlorinated biphenyl exposure (from stage 46/47) on elastic cartilages and laryngeal muscle fibers compared with controls in Xenopus laevis.
(A) Elastic cartilage of normal male larynx contains very large lacunae and a thin extracellular matrix and exhibits a characteristic “Swiss cheese” appearance.
(B) EPZ in the female larynx contains mesenchymal tissue (mt). (C) EPZ in the male larynx of PCB-exposed frog contains developing elastic cartilage (ec) and
mesenchymal tissue (mt). (D) Normal male laryngeal muscle fibers (↑) are larger than female laryngeal muscle fibers (E), but laryngeal muscle fibers of PCB-exposed
males (F) are similar to female laryngeal muscle fibers. (A–C) scale bar: 100 ␮m, (D–F) scale bar: 20 ␮m.
fibers (∼9 ␮m2 ), and female laryngeal muscle has more small
fibers (∼6 ␮m2 ) and fewer medium-sized fibers (∼13 ␮m2 ) and
large fibers (∼15 ␮m2 ). In this study, we used HE staining to
demonstrate that male laryngeal muscles were made of mediumsized fibers and female laryngeal muscle comprised small fibers
and very few medium-sized or large fibers. Laryngeal muscles
of PCB-exposed males, like female laryngeal muscles, were
composed almost entirely of small fibers. These characteristics
suggest that PCBs cause phenotypic demasculinization of male
larynges in X. laevis.
Hormone assays of adult frogs showed that serum testosterone levels of male frogs were significantly reduced by exposure to Aroclor1254 (236.7 ± 30.2 ng/dl) and PCB3 (273.3 ±
21.0 ng/dl) compared with controls (459.5 ± 51.3 ng/dl). However, serum E2 levels of male frogs exposed to Aroclor1254
(12.5 ± 2.0 ng/dl) and PCB3 (11.2 ± 0.4 ng/dl) showed no significant difference from controls (11.2 ± 0.4 ng/dl) (Fig. 5). The
results suggest that phenotypic feminization/demasculinization
of male genital ducts and larynges in X. laevis caused by PCBs
might, in part, result from a decrease in the serum testosterone
level.
Fig. 5. Effects of polychlorinated biphenyl (PCB) exposure of adult male
Xenopus laevis on sex hormone levels compared with controls. Data are
means ± S.E.M. Asterisks indicate significant differences (P < 0.05) in serum
testosterone (T) levels between controls and PCB-exposed frogs. Serum estradiol
(E2 ) levels showed no difference between PCB-exposed frogs and controls.
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4. Discussion
In X. laevis, as in other vertebrates, secondary sexual development is controlled by sex hormones secreted by the gonads
(Kelley, 1992). These secondary sexual characteristics, such as
the oviducts, the cloacal labia and the larynx, exhibit feminization at the end of metamorphosis. The oviducts begin to develop
during the late tadpole stages and continue to grow for the next
4–6 months postmetamorphosis. At about 7 months postmetamorphosis, the oviducts of females become convoluted and their
walls start to thicken while those of males begin to regress and
are lost (Kelley, 1992). The development of oviducts depends
on estrogens. Ovariectomy before 7 months postmetamorphosis results in undeveloped oviducts (Kelley, 1992). However,
oviduct regression in the male could be due to testicular secretion of anti-Mullerian hormone, which is responsible for the loss
of Mullerian ducts. If males are castrated before 7 months postmetamorphosis, the oviducts are maintained (Kelley, 1992). In
this study, some of the 2-year-old male frogs exposed to PCBs
still had oviducts. The result suggests that male oviducts may
result from the feminizing/demasculinizing effects of PCBs in
X. laevis. Clark et al. (1998) used an amphibian (the tiger salamander) to study the effects of some pollutants on genital duct
growth, and found that p,p -DDE as well as estrogens promote
the development of the Mullerian ducts of females. However,
male oviducts in amphibians caused by endocrine disruptors
have been found for the first time in our study.
Masculinization of the male X. laevis larynx relies absolutely
on androgen secretion (Tobias et al., 1991). At the end of metamorphosis, the gross morphology of the larynx is female-like
in both sexes (Sassoon and Kelley, 1986). The male larynx then
undergoes an androgen-driven program, which results in the formation of the complex laryngeal cartilage skeleton and robust
musculature required for song production (Marin et al., 1990).
In the male cartilage, cells proliferate markedly in the perichondrium of the hyaline cartilage and in a zone of precartilage,
which later differentiates into the elastic cartilage. In male muscle, myoblasts proliferate and then fuse; the fiber number rapidly
increases. In this study, the control male larynges had welldeveloped elastic cartilages and typical male laryngeal muscle
fibers. However, the larynxes of male frogs exposed to PCBs
had female-like laryngeal muscle fibers, i.e. PCBs demasculinized male larynxes. Hayes et al. (2002) reported that atrazine, as
an endocrine disruptor, demasculinized the larynxes of exposed
males. These studies have demonstrated that larynxes of X. laevis can be a target of endocrine disruptors, despite contradictory
results (Carr et al., 2003; Coady et al., 2005).
There is a great deal of evidence that PCBs can result in
abnormalities of gender-specific characteristics and disrupt sex
hormone homeostasis in animals (Hany et al., 1999; Kaya et al.,
2002). Some studies have concluded that sex hormone disruption
might be one of causes of abnormal gender-specific characteristics. Similar effects of PCBs have been reported to occur
in human beings. For example, Vreugdenhil et al. (2002) have
reported the gender-specific neurobehavioral effects of perinatal exposure to PCBs and dioxins in humans. A recent study
on Yucheng boys suggested that prenatal exposure to PCBs
may have implications for sex hormone homeostasis at puberty
(Hsu et al., 2005). In this study, exposure to PCB3 and Aroclor 1254 caused a decrease in the serum testosterone level
of male X. laevis frogs. We suggest that the decrease in the
androgen level may be one of the causes of PCB-induced feminization/demasculinization of secondary sexual development in
X. laevis.
Gonadal development of X. laevis as an endpoint for assaying endocrine disruptors has gained increasing attention in recent
years. In this study, it is suggested that the development of genital
ducts may be a sensitive endpoint for assaying endocrine disruptors. In addition, our study suggests that X. laevis might also
be an alternative for conventional model animal species (such
as rats/mice and birds) for study of the effects of the endocrine
system on genital duct structure and function, due to the sensitivity of its genital ducts to endocrine disruptors (Hendry et al.,
2004; Okada et al., 2004).
Acknowledgements
This work was supported by grants from the National Science Foundation of China (20377044 and 20437020), the 973
Plan of Ministry of Science and technology of the Peopole’s
Republic of China (2003CB415005) and the 863 Plan of Ministry of Science and technology of the Peopole’s Republic of
China (2003AA646010).
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