Endocrine disrupting chemicals (EDC) with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: II. Effects on gonad histomorphology

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Abstract

A number of man-made chemicals has been shown to mimic endogenous hormones and to induce alterations of reproductive physiology in wild populations. Of particular importance are compounds that mimic estrogens and androgens (and their antagonists), because of their central role in reproductive function. In this study, male and female adult South African clawed toads (Xenopus laevis) were exposed to ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT) and flutamide (FLU) as (anti)estrogenic and (anti)androgenic model compounds, respectively, at a concentration of 10 8 M, and to water from the river Lambro (LAM), a contaminated watercourse from Northern Italy. Potential disrupting effects on reproduction were studied by histological analyses of gonads. The strongest adverse effects were observed in EE2 and LAM exposed males, e.g. tubule mean diameter reduction, spermatogenic nest breakdown and interlobular wall thickening. In both groups, the occurrence of small oocytes within the seminiferous tubules was observed. In TAM and MDHT exposed females slight oocyte atresia and occurrence of spermatogenic nests were observed. In contrast to previous studies addressing the alteration of molecular biomarkers in the same experimental setup, histological analyses of gonads were very sensitive and indicated an adverse effect of water from Lambro River on reproductive physiology of X. laevis.

Introduction

Many environmental contaminants either of natural (e.g. phytoestrogens) or anthropogenic (e.g. industrial by-products) origin are described to affect reproductive biology of vertebrates by mimicking or antagonizing the action of hormones. The environmental presence of these substances, called Endocrine Disrupting Chemicals (EDCs), have been only rarely related to reproductive disturbances in wild mammals (Facemire et al., 1995, Harding et al., 1999, Vos et al., 2000, Fossi and Marsili, 2003), birds (Fry and Toone, 1981, Giesy et al., 1994), reptiles (Guillette et al., 1994, Guillette and Iguchi, 2003) and fish (Jobling et al., 1998, Jobling et al., 2002, Van Aerle et al., 2001). Nevertheless EDCs are found almost everywhere in the environment and they mainly accumulate in surface waters and sediments. For this reason, permanent or occasional inhabitants like fish and amphibians face an increased risk of being harmed by EDCs. Amongst aquatic species, amphibians represent a unique target in the environment. They are either important predator or prey components of aquatic and terrestrial ecosystems, because of their aquatic way of living during breeding, their sensitive phase of sexual differentiation, and their terrestrial adult life phase (Carey and Bryant, 1995, Fort et al., 2004). Several studies indicate that, amongst a variety of physical and chemical stressors, EDCs may contribute to the general decline of amphibians (Weller and Green, 1997, Houlahan et al., 2000, Stuart et al., 2004). In fact, in response to EDC a number of reproductive abnormalities (Bögi et al., 2002, Bögi et al., 2003, Hayes et al., 2002a, Hayes et al., 2002b, Hayes et al., 2003, Mosconi et al., 2002, Pickford et al., 2003), increased mortality and embryo malformation (Burkhart et al., 1998, Fort et al., 2001); altered gonad differentiation and development and sex ratio (Kloas et al., 1999, Ohtani et al., 2000, Ohtani et al., 2001, Qin et al., 2003, MacKenzie et al., 2003), impaired spermatogenesis (Lee and Veeramachaneni, 2005), gonadal dysgenesis (Fort et al., 2004, Hecker et al., 2005), hermaphroditism (Reeder et al., 1998, Hayes et al., 2003), feminization (Palmer and Palmer, 1995, Hayes and Menendez, 1999, Levy et al., 2004a, Kloas and Lutz, 2006) and inhibition of ovarian steroidogenesis (Pickford and Morris, 2003) have been described in amphibians. Effects of EDC on the reproductive system of animals are often mediated via steroid receptors. Thereby chemicals may act as agonists or antagonists of the estrogen or androgen receptor.

The South African clawed toad Xenopus laevis is an amphibian species consolidated as an in vitro (Kloas et al., 1999, Fort et al., 2002, Lutz et al., 2005) and in vivo (Bögi et al., 2002, Kloas, 2002, Lutz and Kloas, 1999, Levy et al., 2004b, Urbatzka et al., 2006, Urbatzka et al., 2007b) model for the screening of EDCs affecting reproductive biology, thyroid system (Opitz et al., 2005, Opitz et al., 2006) and neural development (Bevan et al., 2000). In this study X. laevis was used as an in vivo model to study the effects of model EDCs with (anti)estrogenic and (anti)androgenic modes of action on gonad morphology. Adult X. laevis were exposed to ethinylestradiol (EE2) as estrogenic compound, tamoxifen (TAM) as anti-estrogenic compound, methyldihydrotestosterone (MDHT) as androgenic compound and flutamide (FLU) as anti-androgenic compound in a four week exposure at a concentration of 10 8 M. Moreover, aiming to increase the knowledge about the environmental hazard, a group of X. laevis was exposed to Lambro river water, a well known polluted source of EDs of Northern Italy (Viganò et al., 1994, Viganò et al., 1998, Fattore et al., 2002) able to induce intersexuality in cyprinids (Viganò et al., 2001). More recently, estrogenic, androgenic and anti-androgenic activities have been demonstrated in vitro both in water and sediment fractions from the Lambro river (Urbatzka et al., 2007b). The present paper focuses attention on establishing morphological endpoints for the detection of effects of EDCs on the gonads of X. laevis, such as testis length and width, seminiferous tubule diameter, relative abundance of spermatogenic nests and occurrence of oocytes in males and oocyte stage distribution and occurrence of oocyte atresia in females.

Section snippets

In vivo exposure

Adult Xenopus laevis (3–4 years old) were taken from the breeding stock of the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin. The frogs were fed twice a week and kept on a 12:12 h light : dark cycle.

For the exposure, adult male and female X. laevis were transferred to aerated 10 L tanks containing reconstituted tap water using distilled water supplemented with 2.5 g marine salt (Tropic Marin Meersalz, Tagis, Dreieich, Germany). X. laevis were exposed to tamoxifen

Gross abnormalities

No mortality and gross abnormalities of gonads were observed for males and females during the exposure period. Additionally, the weight of X. laevis did not differ between the treatment groups.

Testis morphometry

In control males, the testis length and width were 4.32 ± 0.19 and 1.55 ± 0.07, respectively. The average testis length was significantly shorter in EE2 exposed males compared to the control group and increased in MDHT treated males. No significant differences for testis length were observed for the other

Discussion

Histological analyses were conducted by using gonads of male and female adult X. laevis exposed to (anti)estrogenic and (anti)androgenic model compounds (EE2, TAM, MDHT and FLU) and to Lambro river water, a known polluted river of Italy. This watercourse was chosen since several authors described both the presence of high levels of contaminants (Viganò et al., 1994, Barghigiani et al., 2001, Fattore et al., 2002) and the occurrence of their biological effects such as intersexuality in barbels

Acknowledgements

This research was funded by the European Union within the EU-project EASYRING, contract No. QLK4-CT-2002-02286.

Authors thank Dott. Luigi Viganò and Mr. Luciano Previtali (IRSA-CNR, Milan, Italy) for their kind help.

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