INFLUENCE OF TESTOSTERONE METABOLIC PATHWAYS ON DEVELOPMENT AND REPRODUCTION IN AMPHIBIANS

Abstract

Amphibian populations are declining worldwide partly due to environmental pollution. Certain chemicals (e.g., pesticides, pharmaceuticals, and plasticizers) present in our ecosystem have been shown to disrupt the endocrine system in vertebrates by interfering with normal steroidogenesis. Steroidogenesis includes the synthesis of androgens, which are essential hormones involved in vertebrate development and reproduction. Androgens are synthesized from the precursor testosterone (T) by specific enzymes (e.g., steroid 5-reductases) and need to be present in a balanced ratio for an organism to function normally. Three types of 5α-reductases (Srd5α) and one type of 5β-reductase (Srd5β) are involved in the conversion of T into 5α-dihydrotestosterone (5α-DHT) and 5β-dihydrotestosterone (5β-DHT), respectively. 5α-DHT is a potent androgen regulating male fertility, whereas 5β-DHT and other 5β-metabolites synthesized by Srd5β have been shown to be involved in bile acid synthesis, initiation of hormone clearance, drug metabolism, erythropoiesis, vasorelaxation, parturition, and synthesis of neuroactive steroids. Given the vital functions of steroid metabolites produced by Srd5α and Srd5β, it is not surprising that a reduced synthesis and functionality of these enzymes can cause substantial health issues. In amphibians, the inhibition of the activity of Srd5α2 leads to feminization and decreased spermatogenesis. However, little is known about the biological functions and regulation of Srd5 in this taxon and how endocrine disrupting chemicals affect these enzymes. Hence, the main objective of this thesis was to further our understanding on Srd5 in developing and adult Silurana tropicalis frogs. First, I confirmed that endocrine disrupting chemicals (i.e., phthalates) modify srd5 mRNA levels during embryogenesis in S. tropicalis. Then, to gain a better understanding of the tissue distribution of srd5 (srd5α1, srd5α2, srd5α3, and srd5β) during embryogenesis, whole mount in situ hybridization was performed and showed a unique expression pattern for all srd5. Data suggested that metabolites synthesized by Srd5 are required in the central nervous, sensory, cardiac, respiratory, and detoxifying systems aside from reproduction in early anuran development. Complementary hormonal and specific DNA methylation assays were conducted during anuran embryogenesis and adulthood. Experiments demonstrated that specific DNA methylation of srd5α1 and srd5α3 is involved in regulation of the gene expression during embryogenesis as well as in mature gonads. In addition, exposure to T modified srd5 mRNA levels in gonads in a sex-specific manner demonstrating that androgens also regulate srd5 in frog gonads. Taken together, my thesis confirms the importance of Srd5 in biological functions related to anuran reproduction, but also provides evidence that this enzyme family is crucial in other biological functions for proper development.