Development of quantitative real-time PCR assays for fathead minnow ( Pimephales promelas) gonadotropin β subunit mRNAs to support endocrine disruptor research

Abstract

Fathead minnows ( Pimephales promelas) are a widely-used small fish model for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). Quantitative real-time PCR assays for measuring fathead minnow gonadotropin (GtH) β subunit transcripts were developed and “baseline” transcript levels in pituitary tissue were examined over a range of age classes and spawning states. Among females, GtHβ transcripts did not vary significantly with gonadal–somatic index or gonad stage. However, in males, follicle-stimulating hormone β subunit transcripts decreased significantly with increasing gonad stage, while mean luteinizing hormone β subunit expression trended in the opposite direction. GtHβ transcript levels measured in pituitaries from fish that had spawned within the preceding 24 h were not significantly different from those from fish that were 2–3 days post-spawn. Exposure to the fungicide ketoconazole, a known steroidogenesis inhibitor, for 21 days significantly affected the abundance of GtHβ transcripts in pituitary tissue in males, but not females. This study provides critical data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression. Results of such experiments should facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain–pituitary–gonadal axis to stressors including EDCs.