Transcription of Key Genes Regulating Gonadal Steroidogenesis in Control and Ketoconazole- or Vinclozolin-Exposed Fathead Minnows

Abstract

This study evaluated changes in the expression of steroidogenesis-related genes in male fathead minnows exposed to ketoconazole (KTC) or vinclozolin (VZ) for 21 days. The aim was to evaluate links between molecular changes and higher level outcomes after exposure to endocrine-active chemicals (EACs) with different modes of action. To aid our analysis and interpretation of EAC-related effects, we first examined variation in the relative abundance of steroidogenesis-related gene transcripts in the gonads of male and female fathead minnows as a function of age, gonad development, and spawning status, independent of EAC exposure. Gonadal expression of several genes varied with age and/or gonadal somatic index in either males or females. However, with the exception of aromatase, steroidogenesis-related gene expression did not vary with spawning status. Following the baseline experiments, expression of the selected genes in male fathead minnows exposed to KTC or VZ was evaluated in the context of effects observed at higher levels of organization. Exposure to KTC elicited changes in gene transcription that were consistent with an apparent compensatory response to the chemical's anticipated direct inhibition of steroidogenic enzyme activity. Exposure to VZ, an antiandrogen expected to indirectly impact steroidogenesis, increased pituitary expression of follicle-stimulating hormone beta-subunit as well as testis expression of 20beta-hydroxysteroid dehydrogenase and luteinizing hormone receptor transcripts. Results of this study contribute to ongoing research aimed at understanding responses of the teleost hypothalamic-pituitary-gonadal axis to different types of EACs and how changes in molecular endpoints translate into apical outcomes reflective of either adverse effect or compensation.

Evidence suggesting that di‐n‐butyl phthalate has antiandrogenic effects in fish

“…Previous studies with fathead minnows (Pimephales promelas) found that the expression of both StAR and 3β-HSD were significantly altered by both age and GSI [34]. Even when these factors were taken into account, the exposure of fathead minnows to the antiandrogen ketoconazole (4, 25, 100, and 400 µg/L) resulted in significantly increased expression of StAR in the testes of male fathead minnows exposed to concentrations of 6 and 400 µg/L.…”

Dynamic Nature of Alterations in the Endocrine System of Fathead Minnows Exposed to the Fungicide Prochloraz

“…Assays for most of the genes noted above have been described elsewhere (Villeneuve et al, 2006(Villeneuve et al, , 2007aMartinovic et al, 2008). However, QPCR assays for fathead minnow gnrhr1, gnrhr3, and cyp51 are reported here for the first time.…”

“…Samples were amplified over 40 cycles (melt 94°C for 20 s, anneal and extend, 58°C for 60 s) using a 7500 RT-PCR System. As above, relative transcript abundance was estimated based on a standard curve generated by analyzing multiple dilutions of a gene-specific amplicon, without correction for amplification efficiency (for details on preparation of gene-specific standards for Taqman assays, see Villeneuve et al, 2007a).…”

Influence of ovarian stage on transcript profiles in fathead minnow (Pimephales promelas) ovary tissue

“…A standard curve of known quantities of a gene-specific mRNA standard (10-fold dilution series; generally 20-2 × 10 7 copies) was used to calibrate the QPCR data and estimate the number of gene-specific transcripts per ng total RNA. Details regarding the specific primer and probe sequences used and the methods for preparing gene-specific mRNA standards have been reported previously (Villeneuve et al, 2006(Villeneuve et al, , 2007(Villeneuve et al, , 2009aMartinovic et al, 2008). Due to potential differences in amplification and probe binding efficiencies between total RNA samples and purified standards, the number of copies per ng total RNA is regarded as an approximate estimate of the number of transcripts.…”