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.

[1]  O. Carnevali,et al.  Gonadotropins and reproductive function in the anuran amphibian, Rana esculenta. , 1998, Biology of reproduction.

[2]  S. Chattopadhyay,et al.  Antiandrogenic effects of bisphenol A and nonylphenol on the function of androgen receptor. , 2003, Toxicological sciences : an official journal of the Society of Toxicology.

[3]  S. Morris,et al.  Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback. , 2009, Aquatic toxicology.

[4]  D. Kelley,et al.  Androgen and gonadotropin effects on male mate calls in South African clawed frogs, Xenopus laevis , 1983, Hormones and Behavior.

[5]  C. Porte,et al.  Effects of endocrine disrupters on sex steroid synthesis and metabolism pathways in fish , 2004, The Journal of Steroid Biochemistry and Molecular Biology.

[6]  I. Lutz,et al.  Retinol-binding protein as a biomarker to assess endocrine-disrupting compounds in the environment , 2004, Analytical and bioanalytical chemistry.

[7]  M. Carrillo,et al.  Regulation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gene expression by gonadotropin-releasing hormone (GnRH) and sexual steroids in the Mediterranean Sea bass. , 2002, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[8]  I. Lutz,et al.  Amphibians as model to study endocrine disrupters. , 2006, Journal of chromatography. A.

[9]  S. Ishii,et al.  Annual cycle of pituitary and plasma gonadotropins and plasma sex steroids in a wild population of the toad, Bufo japonicus. , 1990, General and comparative endocrinology.

[10]  I. Lutz,et al.  Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny. , 2007, General and comparative endocrinology.

[11]  P. Licht,et al.  Effects of gonadectomy and steroids on pituitary gonadotropin secretion in a frog, Rana pipiens. , 1989, Biology of reproduction.

[12]  P. Tsai,et al.  Alteration of the hypothalamic-pituitary-gonadal axis in estrogen- and androgen-treated adult male leopard frog, Rana pipiens , 2005, Reproductive biology and endocrinology : RB&E.

[13]  B. Palmer,et al.  Vitellogenin induction by xenobiotic estrogens in the red-eared turtle and African clawed frog. , 1995, Environmental health perspectives.

[14]  W. Kloas,et al.  Endocrine disrupting chemicals (EDC) with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: II. Effects on gonad histomorphology. , 2008, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[15]  J. M. Torres,et al.  Semiquantitative RT-PCR method coupled to capillary electrophoresis to study 4α-reductase mRNA isozymes in rat ventral prostate in different androgen status , 2003, Molecular and Cellular Biochemistry.

[16]  C. Tohyama,et al.  Effects of vinclozolin administration on sperm production and testosterone biosynthetic pathway in adult male rat. , 2003, The Journal of reproduction and development.

[17]  J. Hutchison,et al.  A Neurological Study of the Clasp Reflex in Xenopus Laevis (Daudin)1) , 1963 .

[18]  Terry J. Smith,et al.  17β-Estradiol Treatment Decreases Steroidogenic Enzyme Messenger Ribonucleic Acid Levels in the Rainbow Trout Testis. , 2001, Endocrinology.

[19]  D. Pfaff,et al.  Hormone effects on male sex behavior in adult South African clawed frogs, Xenopus laevis , 1976, Hormones and Behavior.

[20]  D. Melton,et al.  The mRNA encoding elongation factor 1-α (EF-1α) is a major transcript at the midblastula transition in Xenopus , 1989 .

[21]  W. Kloas.  Amphibians as a model for the study of endocrine disruptors. , 2002, International review of cytology.

[22]  R. Opitz,et al.  Luteinizing hormone, follicle stimulating hormone, and gonadotropin releasing hormone mRNA expression of Xenopus laevis in response to endocrine disrupting compounds affecting reproductive biology. , 2006, General and comparative endocrinology.

[23]  M. Murakami,et al.  The Complete Sequence of Mitochondrial Genome from a Gynogenetic Triploid “Ginbuna” (Carassius auratus langsdorfi) , 1998, Zoological science.

[24]  I. Huhtaniemi,et al.  Biphasic effect of exogenous testosterone on follicle-stimulating hormone gene expression and synthesis in the male rat , 1993, Molecular and Cellular Endocrinology.

[25]  R. Einspanier,et al.  Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo. , 1999, The Science of the total environment.

[26]  S. Miyata,et al.  Hormonal Reversal and the Genetic Control of Sex Differentiation in Xenopus , 1999 .

[27]  J. H. Wyk,et al.  The Effects of Anti-Androgenic and Estrogenic Disrupting Contaminants on Breeding Gland (Nuptial Pad) Morphology, Plasma Testosterone Levels, and Plasma Vitellogenin Levels in Male Xenopus laevis (African Clawed Frog) , 2003, Archives of environmental contamination and toxicology.

[28]  R. Opitz,et al.  Endocrine Disruption in Aquatic Vertebrates , 2009, Annals of the New York Academy of Sciences.

[29]  D. Kelley,et al.  Temporal constraints on androgen directed laryngeal masculinization in Xenopus laevis. , 1991, Developmental biology.

[30]  I. Lutz,et al.  Functional genomics and sexual differentiation in amphibians. , 2002, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[31]  H. Habibi,et al.  Molecular characterization of LH-β and FSH-β subunits and their regulation by estrogen in the goldfish pituitary , 2002, Molecular and Cellular Endocrinology.

[32]  Ioanna Katsiadaki,et al.  Use of the Three-Spined Stickleback (Gasterosteus aculeatus) As a Sensitive in Vivo Test for Detection of Environmental Antiandrogens , 2005, Environmental health perspectives.

[33]  P. Licht,et al.  The role of androgen in the development of sexual differences in pituitary responsiveness to gonadotropin releasing hormone (GnRH) agonist in the bullfrog, Rana catesbeiana. , 1984, General and comparative endocrinology.

[34]  E. Baatrup,et al.  Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy Poecilia reticulata. , 2001, Environmental health perspectives.

[35]  S. Miyata,et al.  Analysis of genes related to expression of aromatase and estradiol-regulated genes during sex differentiation in Xenopus embryos. , 2004, General and comparative endocrinology.

[36]  I. Lutz,et al.  Endocrine disrupters with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: I. Effects on sex steroid levels and biomarker expression. , 2007, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[37]  E. Baatrup,et al.  The Effects of Vinclozolin, an Anti-Androgenic Fungicide, on Male Guppy Secondary Sex Characters and Reproductive Success1 , 2003, Biology of reproduction.

[38]  D. Kelley,et al.  Testicular masculinization of vocal behavior in juvenile female Xenopus laevis reveals sensitive periods for song duration, rate, and frequency spectra , 1992, Journal of Comparative Physiology.

[39]  P. Licht,et al.  Seasonal and stress related changes in plasma gonadotropins, sex steroids, and corticosterone in the bullfrog, Rana catesbeiana. , 1983, General and comparative endocrinology.