Doctor of Philosophy (PhD)
Biological and Chemical Sciences
Faculty of Science
Dr. Deborah MacLatchy
Ph. D. Supervisor
Androgens are a recognized class of endocrine disrupting compounds that are introduced into the aquatic environment through a variety of anthropogenic sources. Exogenous androgens can interact with the hypothalamus-pituitary-gonadal (HPG) axis in fish and alter its normal function, resulting in effects such as skewed sex ratios, intersex individuals, reduced fecundity, or altered mating behaviours. In fish, these apical effects are commonly correlated to depression of the terminal sex hormones testosterone, 17β-estradiol and 11-ketotestosterone (11KT). It is currently thought that the reduction in plasma sex hormones is an effect of negative feedback inhibition of steroid synthesis within the HPG axis after androgen receptor agonism. The specific point(s) of this inhibition are currently unknown. To begin to identify specific areas of the steroidogenic pathway which may be altered during exposure to exogenous androgens, I utilized male mummichog (Fundulus heteroclitus), an estuarine species inhabiting the eastern coast of North America. Using model androgens 5α-dihydrotestosterone (DHT) and 17α-methyltestosterone, I conducted a series of gonadal exposures to determine if specific genes, coding for critical enzymes within the steroidogenic pathway, fluctuate after incubation with exogenous androgens. Tissue processed through in vitro incubation expressed gene response profiles that were inconsistent and counterintuitive to established effects of plasma hormone depression. A series of follow-up exposures was conducted, with fish injected with 1 pg/g, 1 ng/g or 1 µg/g body weight DHT to induce plasma hormone depression and quantify steroidogenic gene transcript levels at the same time period. Testis tissue was sectioned upon excision with one section immediately frozen and a second section processed through in vitro incubation before subsequently being frozen. Results from DHT injected fish indicate a correlation between gene expression in testis tissue frozen immediately and plasma hormone depression, with a decrease in transcript levels of steroidogenic acute regulatory protein (star), cytochrome P450 17a1 (cyp17a1), 3β-hydroxysteroid dehydrogenase (3βhsd), 11β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase occurring at the same time as plasma hormone depression. Response amplitudes were greatest for 3βhsd and cyp17a1, with both of these genes being depressed immediately prior to plasma hormone depression and their recovery correlating to plasma hormone recovery. Gene responses following incubation did not display specific response patterns, indicating that analysis of gene transcript changes from in vitro samples is not reflective of actual in vivo responses. To test if the observed correlation between plasma hormone depression and gene transcript reduction in DHT injected fish was applicable to other androgens, I injected male mummichog to 11KT to induce plasma hormone depression and quantify steroidogenic genes. Incorporating various steroidogenic substrates into this protocol allowed me to test whether 3βhsd and/or cyp17a1 are responsible for depressed gonadal hormone production in 11KT-injected fish. Overall, steroidogenic genes correlated to plasma hormone depression in 11KT-injected fish, with 3βhsd and cyp17a1 still being the two most responsive genes. Use of precursors did not increase hormone production rate at any timepoint where transcripts of 3βhsd and cyp17a1 were depressed, confirming the important role these two genes play in steroid synthesis during exposure to exogenous androgens. This is the first work to clearly demonstrate the correlation between these genes and depression of plasma sex hormones, on an hourly scale. The data from this thesis will strengthen mechanistic understanding of molecular perturbation resulting in changes at higher levels of biological organization in androgen exposed fish.
Rutherford, Robert, "Effects and modes of action of exogenous androgens on gonadal steroidogenesis in male mummichog (Fundulus heteroclitus)" (2020). Theses and Dissertations (Comprehensive). 2273.