Transcriptional networks associated with 5-alpha-dihydrotestosterone in the fathead minnow (Pimephales promelas) ovary.

Androgens play a significant role in regulating oogenesis in teleost fishes. The androgen dihydrotestosterone (DHT) is a potent non-aromatizable androgen involved in sexual differentiation in mammals; however, its actions are not well understood in teleost fish. To better characterize the physiological role of DHT in the fathead minnow (FHM) ovary on a temporal scale, in vitro assays for 17ß-estradiol (E2) production were conducted in parallel with microarray analysis. Ovarian explants were incubated at different concentrations of DHT (10(-6), 10(-7), and 10(-8)M DHT) in three separate experiments conducted at 6, 9, and 12h. DHT treatment resulted in a rapid and consistent increase in E2 production from the ovary at all three time points. Therefore, DHT may act to shift the balance of metabolites in the steroidogenic pathway within the ovary. Major biological themes affected by DHT in the ovary in one or more of the time points included those related to blood (e.g. vasodilation, blood vessel contraction, clotting), lipids (e.g. lipid storage, cholesterol metabolism, lipid degradation) and reproduction (e.g. hormone and steroid metabolism). Gene networks related to immune responses and calcium signaling were also affected by DHT, suggesting that this androgen may play a role in regulating these processes in the ovary. This study detected no change in mRNA levels of steroidogenic enzymes (cyp19a1, star, 11ßhsd, 17ßhsd, srd5a isoforms), suggesting that the observed increase in E2 production is likely more dependent on the pre-existing gene or protein complement in the ovary rather than the de novo expression of transcripts. This study increases knowledge regarding the roles of DHT and androgens in general in the teleost ovary and identifies molecular signaling pathways that may be associated with increased E2 production.

Classifying chemical mode of action using gene networks and machine learning: a case study with the herbicide linuron.

The herbicide linuron (LIN) is an endocrine disruptor with an anti-androgenic mode of action. The objectives of this study were to (1) improve knowledge of androgen and anti-androgen signaling in the teleostean ovary and to (2) assess the ability of gene networks and machine learning to classify LIN as an anti-androgen using transcriptomic data. Ovarian explants from vitellogenic fathead minnows (FHMs) were exposed to three concentrations of either 5α-dihydrotestosterone (DHT), flutamide (FLUT), or LIN for 12h. Ovaries exposed to DHT showed a significant increase in 17ß-estradiol (E2) production while FLUT and LIN had no effect on E2. To improve understanding of androgen receptor signaling in the ovary, a reciprocal gene expression network was constructed for DHT and FLUT using pathway analysis and these data suggested that steroid metabolism, translation, and DNA replication are processes regulated through AR signaling in the ovary. Sub-network enrichment analysis revealed that FLUT and LIN shared more regulated gene networks in common compared to DHT. Using transcriptomic datasets from different fish species, machine learning algorithms classified LIN successfully with other anti-androgens. This study advances knowledge regarding molecular signaling cascades in the ovary that are responsive to androgens and anti-androgens and provides proof of concept that gene network analysis and machine learning can classify priority chemicals using experimental transcriptomic data collected from different fish species.

The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas).

Linuron is a widely used urea-based herbicide that has anti-androgenic activity in both fish and rodents. To further elucidate the potential mode of action (MOA) of linuron on the vertebrate endocrine system, adult male and female fathead minnows were exposed for 21 days to dechlorinated water, a solvent control, 17ß-estradiol (E2; 0.1 µg/L), dihydrotestosterone (DHT; 100 µg/L), linuron (1, 10, 100 µg/L) and one co-treatment of DHT (100 µg/L) and linuron (100 µg/L). There were no effects of linuron on egg hatching, 7 day egg survival, nuptial tubercle formation or gonadal histopathology. Administration of DHT and 1 and 100 µg/L linuron reduced plasma vitellogenin in females, while male plasma vitellogenin were induced after E2 exposure and co-exposure of DHT and linuron. Ovarian mRNA levels were examined for several genes involved in steroidogenesis (e.g. p450scc, cyp19a, star, tspo, hsd17b and hsd11b) and estrogen-mediated responses (esr1, esr2b, esr2a). Only p450scc mRNA was significantly decreased with DHT+linuron co-treatment. Clustering of steroidogenic mRNA transcript expression patterns revealed that patterns for linuron were more similar to E2 compared to DHT. Collectively, this study supports the hypothesis that linuron may not be a pure anti-androgen and may have multiple MOAs that affect vertebrate reproduction.