Ralph Urbatzka

Endocrine disruption in aquatic vertebrates.

Environmental compounds can interfere with endocrine systems of wildlife and humans. The main sink of such substances, called endocrine disrupters (ED), are surface waters. Thus, aquatic vertebrates, such as fish and amphibians, are most endangered. ED can adversely affect reproductive biology and the thyroid system. ED act by (anti)estrogenic and (anti)androgenic modes of action, resulting in abnormal sexual differentiation and impaired reproduction. These effects are mainly driven by direct interferences of ED with sex steroid receptors rather than indirectly by impacting synthesis and bioavailability of sex steroids, which in turn might affect the hypothalamic–pituitary–gonadal axis. Recent findings reveal that, in addition to the human‐produced waste of ED, natural sources, such as parasites and decomposition of leaves, also might act as ED, markedly affecting sexual differentiation and reproduction in fish and amphibians. Although the thyroid system has essential functions in both fish and amphibians, amphibian metamorphosis has been introduced as the most sensitive model to detect thyroidal ED; no suitable fish model exists. Whereas ED may act primarily on only one specific endocrine target, all endocrine systems will eventually be deregulated as they are intimately connected to each other. The recent ecotoxicological issue of pharmaceutically active compounds (PhACs) present in the aquatic environment indicates a high potential for further endocrine modes of action on aquatic vertebrates by ED derived from PhACs, such as glucocorticoids, progestins, and ß‐agonists.

Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.

Testing the effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens as found in the Douro River (Portugal) on the maturation of fish gonads—A stereological study using the zebrafish (Danio rerio) as model

In natural environments fish populations are exposed to many potential xenoestrogens, whereby understanding the impacts of mixtures continue to be of great interest. The main objective of this study was, therefore, to understand whether and how an environmentally relevant mixture of xenoestrogens found in the Douro River estuary can disrupt the normal gametogenesis in fish. For this purpose, adult zebrafish of both sexes were exposed for 21 days to an environmental mixture (MIX) of 11 xenoestrogens from diverse sources. A 100 ng/L ethinylestradiol (EE2) positive control was added. A quantitative (stereological) analysis with systematic sampling was made in the gonads, and using light microscopy both the relative and the absolute volumes of the gametogenic stages were estimated. Data point that the EE2 stimulus induced changes in structural compartments; with decreasing trends for the advanced maturation stages both in males and females. There was also a trend for a greater amount of interstitial tissue in males. Along with an interstitial fibrosis increase detected, the presence of a proteinaceous fluid was observed in both sexes and experimental groups (EE2 and MIX). Other histopathologic alterations were observed in the EE2 female group, such as the presence of foci of granulomatous inflammation and follicular mineralization in the germinal parenchyma and luminal areas. The most interesting finding of this study was that the exposure to the MIX caused a decrease of the relative volume of spermatozoa in zebrafish. This kind of estrogenic effect has not earlier been structurally quantified in such a fine detail with unbiased stereology in fish gonads. Despite the ultimate consequences of such disruptions being unknown, it could be logically argued that reduction or slowing-down of the appearance of the most mature cohorts and/or eventual interstitial fibrosis and other pathologic changes can adversely affect breeding. The findings add further explanatory bases for understanding the negative impacts of xenoestrogens.

Mate calling behavior of male South African clawed frogs (Xenopus laevis) is suppressed by the antiandrogenic endocrine disrupting compound flutamide

Several environmental pollutants have been identified as antiandrogenic endocrine disrupting chemicals (EDC), with flutamide (FLU) being a model compound for this type of action. Despite impacts of EDC interfering with sexual differentiation and reproduction in amphibians, established information about suggested effects on sexual behavior is still lacking. In this study adult male Xenopus laevis were injected with human chorionic gonadotropin (hCG) to initiate mate calling behavior. After one day hCG-stimulated frogs were treated via aqueous exposure over three days without and with FLU at concentrations of 10(-8) and 10(-6) M in comparison to untreated frogs. Androgen controlled mate calling behavior was recorded during the 12h dark period. At the end of exposure circulating levels of testosterone (T) and 17beta-estradiol (E2) were determined and furthermore gene expression was measured concerning reproductive biomarkers such as hypophysial luteinizing hormone (LH), follicle-stimulating hormone (FSH), testicular aromatase (ARO), 5alpha reductase type 1 (SRD5alpha1) and 5alpha reductase type 2 (SRD5alpha2). Both concentrations of FLU caused a significant decrease in calling activity starting at the second day of exposure. HCG injected positive controls had elevated levels of T compared to negative control frogs while in parallel treatment with FLU did not affect significantly the hCG elevated sex steroid levels. Furthermore, hCG treatment led to significantly decreased levels of gene expression for ARO and SRD5alpha2 but no impacts were detected on LH, FSH or SRD5alpha1 mRNA levels compared to negative controls. In summary, the behavioral parameter mate calling is the most sensitive biomarker detecting antiandrogenic modes of action in this challenge-experiment indicating that this non-invasive method could markedly contribute for sensitive assessment of antiandrogenic EDC.

Expression profiles of LHβ, FSHβ and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles

The gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH), are important hormones regulating reproductive biology in vertebrates, especially the processes of steroidogenesis and gamete maturation. Despite the role of gonadotropins during the reproductive cycle in amphibians is well established, much less is known about the functional maturation of the hypothalamus-pituitary-gonad axis during larval development. Therefore, the present study aimed to analyze the expression profiles of hypophyseal LHbeta and FSHbeta mRNA and of their corresponding gonadal receptors (LH-R, FSH-R) in Xenopus laevis tadpoles during their ontogeny and sexual differentiation. The first significant elevation of LHbeta and FSHbeta mRNA was observed at late premetamorphosis. A clear raise of LHbeta mRNA was present during prometamorphic stages especially in males, while the LH-R only slowly increased during ontogeny with highest levels during metamorphic climax. In contrast, FSHbeta mRNA expression only slightly increased during ontogeny, however in both sexes the FSH-R mRNA was considerably elevated at prometamorphosis and further at metamorphic climax. Our results suggest that LHbeta and LH-R mRNA expression might be involved in initial maturation events of gametes, at least in males, while the gradually increase of FSH-R mRNA coincided with the advancing process of gamete maturation in both sexes. The present study provides for the first time evidence based on expression of gonadotropins and their corresponding gonadal receptors that the hypothalamus-pituitary-gonad axis evolves already at early stages of ontogeny and sexual differentiation in amphibians.

Exposure of Xenopus laevis tadpoles to finasteride, an inhibitor of 5-α reductase activity, impairs spermatogenesis and alters hypophyseal feedback mechanisms

Sexual steroids have major regulatory functions in gonadal development, maturation of gametes and sexual differentiation in vertebrates. Previous studies in amphibians provided evidence that dihydrotestosterone and activity of 5-alpha reductases might play a significant role in androgen-mediated reproductive biology. To test the involvement of 5-alpha reductases in maturation of gametes in amphibians, Xenopus laevis was exposed to finasteride (FIN), a known inhibitor of 5-alpha reductase enzyme activity. In a long-term exposure from stage 46 to 66, severe disruption of spermatogenesis was observed in histological analysis of testes as detected by occurrence of empty spermatocysts, while ovaries remained unaffected. Real-time PCR analyses of male and female brain revealed an increase of LHbeta mRNA and a decrease of FSHbeta mRNA in males, suggesting a signalling on testes that could result in increased steroidogenesis and reduced Sertoli cell proliferation. Accordingly, the mRNA expression of P450 side chain cleavage enzyme and 5-alpha reductase type 2 was increased in testes, while no effects could be observed on steroidogenic genes in ovaries. A short-term exposure to testosterone, FIN and testosterone+FIN showed that transient effects of FIN targeted males selectively and, in particular, interfered with the hypothalamus-pituitary-gonad axis. Furthermore, a negative feedback of testosterone on LHbeta was observed on males and females. This study provides evidence that exposure of X. laevis to FIN, an inhibitor of 5-alpha reductases, impaired spermatogenesis and involved sex-specific hypophyseal feedback mechanisms.

Does Atrazine Affect Larval Development and Sexual Differentiation of South African Clawed Frogs

The potential impact of atrazine (ATZ) on gonadal malformations in larval Xenopus laevis has been controversially discussed, and a hypothesis has been generated that ATZ might induce the estrogen‐synthesizing enzyme aromatase, leading to feminization or demasculinization. Recently, extensive long‐term studies clearly indicate that no adverse effect of ATZ on larval development and sexual differentiation could be found. Therefore, to determine potential transient impacts of ATZ on sexual differentiation processes, short‐term exposures were conducted using tadpoles treated for 4 days with ATZ at 25 μg/L. The expression levels of the key players for sexual differentiation in amphibians were determined in the brain, assessing aromatase, 5α‐reductase type 1 (S1) and type 2 (S2), and the gonadotropins luteinizing hormone and follicle‐stimulating hormone, and in the gonads, measuring aromatase, S1, and S2, by means of quantitative RT‐PCR. No significant changes in any of these parameters have been found, implicating, in accordance with recent long‐term exposures, that no aromatase induction by ATZ could be observed, and it seems likely that no further endocrine mechanism of ATZ affecting sexual differentiation in X. laevis exists.

Cytological, immunocytochemical, ultrastructural and growth characterization of the rainbow trout liver cell line RTL-W1.

Despite its wide use in toxicology, a detailed characterization of RTL-W1 cell line lagged behind leaving ambiguities about its cell origin. We aimed to better characterize the line regarding cell phenotype and tumorigenic state. We studied RTL-W1 cells in monolayers and in (4-22-week-old) aggregates considering: (a) morphology (light and electron microscopy); (b) immunophenotype using AE1/AE3, vimentin, Cam5.2, CK7 and CK19 and e-cadherin antibodies and (c) growth behavior. RTL-W1 organelle content is constituted basically by mitochondria and abundant free ribosomes, with no (cytochemically) detectable peroxisomes and lysosomes. Immunocytochemistry showed a strong marking for AE1/AE3 and vimentin (in a cell subset). Since AE1/AE3 stained biliary epithelial ducts in trout liver, and considering the morphological characteristics and long term culture, RTL-W1 cells seem more similar to bile preductular epithelial cells (considered as stem cells in teleost liver). Also, we observed abnormal nuclear features described for both malignant cell lines and stem cells, so we could not conclude about tumorigenicity. Cell aggregates had signs of hepatocytic differentiation, such as the development of RER and microvillus-like projections into intercellular spaces. The morphological resemblance to the original tissue suggests that aggregates could have an added value in metabolic as well as in cell-to-cell interaction studies.

Aqueous leaf extracts display endocrine activities in vitro and disrupt sexual differentiation of male Xenopus laevis tadpoles in vivo.

The occurrence of natural substances acting as endocrine disrupting compounds (EDC) in the environment is to date poorly understood. Therefore, (anti)androgenic and (anti)estrogenic activities of three different aqueous leaf extracts (beech, reed and oak) were analyzed in vitro using yeast androgen and estrogen screen. The most potent extract was selected for in vivo exposure of Xenopus laevis tadpoles to analyze the potential effects on development and reproductive biology of amphibians. Tadpoles were exposed from stage 48 to stage 66 (end of metamorphosis) to aqueous oak leaf extracts covering natural occurring environmental concentrations of dissolved organic carbon. Gene expression analyses of selected genes of the hypothalamus-pituitary-gonad and of the hypothalamus-pituitary-thyroid axis as well as histological investigation of gonads and thyroid glands were used to evaluate endocrine disrupting effects on the reproductive biology and development. Female tadpoles remained unaffected by the exposure whereas males showed severe significant histological alterations of testes at the two highest oak leaf extract concentrations demonstrated by the occurrence of lacunae and oogonia. In addition, a significant elevation of luteinizing hormone beta mRNA expression with increasing extract concentration in male tadpoles indicates an involvement of hypothalamus-pituitary-gonad axis mainly via antiandrogenic activity. These results suggest that antiandrogenic EDC of oak leaf extract are responsible for inducing the observed effects in male tadpoles. The present study demonstrates for the first time that in surface waters, natural occurring oak leaf compounds at environmentally relevant concentrations display antiandrogenic activities and have considerable effects on the endocrine system of anurans affecting sexual differentiation of male tadpoles.

Regulation of Ovarian Development and Function in Teleosts

Publisher Summary Fish reproduction is characterized by a puzzling diversity of strategies regarding how different species determine their sex, produce male and female fertile gametes, and mate. Maybe the success of the teleost group in conquering so many different habitats around the world are owed, at least in part, to these special features. Sex in fishes may be determined genetically, by environmental factors, or by social behavior. Temperature is an important environmental factor, and an influence on sex determination is observed in several groups of fishes. The genesis and development of oocytes in teleost fishes are a fascinating process with tight and complex physiological regulation mechanisms that ensure the organization of genetic and nutritional bases for the development of new individuals. Female gametogenesis comprises several developmental steps and consists of oogenesis, oocyte growth, maturation, and ovulation.