Urbatzka R et al. (2006), Luteinizing hormone, follicle stimulating hormo...

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Gen Comp Endocrinol 2006 Apr 01;1462:119-25. doi: 10.1016/j.ygcen.2005.10.005.

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Luteinizing hormone, follicle stimulating hormone, and gonadotropin releasing hormone mRNA expression of Xenopus laevis in response to endocrine disrupting compounds affecting reproductive biology.

Urbatzka R , Lutz I , Opitz R , Kloas W .

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Environmental pollutants can interfere with the endocrine system of a variety of animals and are suggested to contribute to the worldwide decline of amphibians. In this study, the effects of endocrine disrupting compounds (EDC) on the hypothalamus-pituitary-gonad axis, regulating reproduction, were investigated in Xenopus laevis by determining their potential impact on gene expression of gonadotropin releasing hormone (GnRH), luteinizing hormone beta-subunit (LHbeta) and follicle-stimulating hormone beta-subunit (FSHbeta) in brain and pituitary using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One environmental sample and four model compounds, ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), and flutamide (FLU), corresponding to (anti)estrogenic and (anti)androgenic modes of action were used at 10(-8)M during a four weeks exposure of adults of both sexes. In general, males had a higher LHbeta mRNA level compared to females, while the mRNA expression of FSHbeta and GnRH did not differ between both sexes. EE2 and MDHT treatment decreased LHbeta mRNA expression in the brain of male X. laevis, while only EE2 but not MDHT reduced LHbeta mRNA in females indicating classical negative feed-back mechanisms on hypophyseal gonadotropin expression. TAM increased LHbeta mRNA and FSHbeta mRNA expression in female X. laevis while none of the other treatments showed an effect on FSHbeta mRNA expression. GnRH expression was not changed by any treatment and exposure of X. laevis to Lambro river water had no significant effect on any of the genes examined. It is reported for the first time in amphibians that gonadotropin mRNA expression is differentially regulated by (anti)estrogenic and (anti)androgenic EDC and that gender-specific patterns of gene expression exist.

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Species referenced: Xenopus laevis
Genes referenced: fshb gnrh1 lhb

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	Fig. 1. PCR products of luteinizing hormone -subunit (LH), follicle stimulating hormone -subunit (FSH), gonadotropin releasing hormone (GnRH) and elongation factor 1- (EF-1) from brainpituitary samples of male (A) and female (B) X. laevis are shown on an agarose gel. Duplicate determinations of LH, FSH, GnRH, and EF-1 demonstrate-specific product lengths of 218, 391, 163, and 285 bp, respectively. The molecular weight marker indicates distances of 100 bp DNA.
	Fig. 2. Relative expression levels of mRNA of gonadotropin releasing hormone (GnRH), luteinizing hormone β-subunit (LHβ) and follicle-stimulating hormone β-subunit (FSHβ) were compared between females and males of X. laevis of the control group. Nonparametric Mann–Whitney U test were applied to determine significant differences between both sexes indicated by asterisks (p < 0.05). Values refer to means ± SD of the control group, normalized by the house-keeping gene elongation factor 1-α (EF 1-α). Means were derived from samples of eight individuals each.
	Fig. 3. Relative mRNA expression of luteinizing hormone β-subunit (LHβ), follicle-stimulating hormone β-subunit (FSHβ) and gonadotropin releasing hormone (GnRH) expression of male X. laevis in response to (anti)estrogenic, (anti)androgenic model compounds, and to Lambro river water. Ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), and flutamide (FLU) were used as estrogenic, antiestrogenic, androgenic, and antiandrogenic compound, respectively, at a concentration of 10−8 M. Co refers to the solvent control and Lam to Lambro river water. Results were expressed relative to control values as means ± SD, normalized by the house-keeping gene elongation factor 1-α (EF 1-α). Means were derived from samples of 8 individuals each. Significant differences between treatments and control groups were tested by ANCOVA followed by Dunnett’s test. Significant differences were indicated by asterisks (p < 0.01, *p < 0.001).
	Fig. 4. Relative mRNA expression of luteinizing hormone β-subunit (LHβ), follicle-stimulating hormone β-subunit (FSHβ), and gonadotropin releasing hormone (GnRH) expression of female X. laevis in response to (anti)estrogenic, (anti)androgenic model compounds, and to Lambro river water corresponding to Fig. 3. Significant differences were indicated by asterisks (*p < 0.05).