Transcriptional and epigenetic regulation of steroid 5alpha-reductase type 2 and androgen action in epididymal principal cells

Androgens, in particular dihydrotestosterone (DHT), are crucial for maintaining the structure and functions (sperm maturation and storage) of the epididymis. DHT is synthesized from testosterone by steroid 5alpha-reductase (Srd5aR) type 1 and type 2. Srd5aR1 and srd5aR2 have different tissue-specific distribution patterns and distinct genetic and biochemical properties. Srd5aR2 mRNA is more abundant than that of srd5aR1 in several sex accessory tissues, most dramatically in the epididymis, where it is predominantly expressed in the caput region.;In the first objective, the 5'upstream region of srd5aR2 was cloned and analyzed. The gene's transcriptional start site was localized, regulatory elements and the core promoter were mapped, and the transcription factors SP1, and to a lesser extent SP3, were found to interact with srd5aR2 in the rat caput epididymidis and mouse proximal caput epididymidis (PC-1) cell line. The epigenetic regulation of srd5aR2 by DNA methylation was subsequently examined and contrasted to that of srd5aRl in tissues where the isozymes are differentially expressed. DNA methylation levels fluctuated along the 5'upstream region of both genes. Tissue-specific variations in adenine and cytosine methylation were also identified for srd5aR1 and srd5aR2, respectively. These studies constitute the first analysis of the 5'upstream region of srd5aR2, at both the transcriptional and epigenetic levels.;In the second objective, the direct effects of androgens on principal cell gene expression were examined in PC-1 cells. The majority of androgen-regulated genes displayed an early or late transient increase in expression levels following androgen withdrawal. When DHT was added back to the media, a differential ability of rescue was seen among androgen-regulated genes depending on time of supplementation. This rescue ability was severely compromised after 4 days of androgen deprivation. These results provide novel insights into the response of principal cells to androgens.;Together, the two objectives of this thesis impart greater understanding into the mechanisms of androgen action in the epididymis.;The two main objectives of this thesis are to elucidate the transcriptional and epigenetic regulation of srd5aR2 in the rat epididymis and to identify androgen-dependent genes in epididymal principal cells.