The importance of canonical and alternate androgen production pathways in masculinisation, fertility and lifelong male health

Androgens, such as testosterone and dihydrotestosterone (DHT), regulate male sexual development, masculinisation, spermatogenesis, and metabolism. In adults, androgens are primarily synthesised in the testes by the Leydig cells. Testosterone is synthesised via the canonical androgen biosynthesis pathway, whereby 17β-Hydroxysteroid dehydrogenase type 3 (HSD17B3) catalyses the conversion of the androgen precursor, androstenedione, into testosterone. Testosterone can act on the androgen receptor or converted to the more potent DHT via 5α-steroid reductases. DHT can also be produced via the alternate pathway, whereby testosterone is bypassed. In the adult testes, the alternate pathway of androgen biosynthesis utilises 5α-Steroid reductase type 1 (SRD5A1) to convert androgen precursors in the canonical pathway into the alternate pathway. Both androgen biosynthesis pathways are essential for human male sexual development, yet how these pathways interact remains unknown. In humans, perturbed androgen biosynthesis due to loss-of-function mutations in HSD17B3 results in a disorder of sexual development. 46,XY HSD17B3-deficient individuals retain internal Wolffian structures however external genitalia is undermasculinised, appearing as female or ambiguous. Two independent research groups have generated Hsd17b3-deficient mouse models which show similarities and discrepancies to human HSD17B3-deficiency. In contrast to human cases of HSD17B3-deficiency, male Hsd17b3 knockout mice are masculinised from birth and fertile in adulthood. Although Hsd17b3 knockout mice exhibit high androstenedione/testosterone ratios (indicative of HSD17B3 dysfunction), intratesticular testosterone levels remain normal, indicating compensatory mechanisms in mice allow for continued androgen action. This thesis investigated what compensatory mechanisms are present in Hsd17b3-deficienct mice. These studies addressed the overarching hypotheses: (i) mice have additional hydroxysteroid dehydrogenase enzymes that are capable of synthesising testosterone and (ii) the alternate pathway of androgen biosynthesis functions alongside the canonical pathway and is upregulated to compensate following HSD17B3 ablation. Mouse HSD17B12 has previously been suggested as a potential enzyme responsible for continued testosterone biosynthesis in Hsd17b3 knockout mice. This thesis validated that mouse HSD17B12 can convert androstenedione into testosterone. We have demonstrated that a key amino acid in mouse HSD17B12 allows for testosterone biosynthesis, in contrast to the human enzyme which has a different amino acid and is unable to produce testosterone. To model human androgen production in mice, a Hsd17b3-deficient mouse with an altered HSD17B12 (which expresses the human amino acid and is thus unable to produce testosterone) was generated. Analysis of the Hsd17b3 knockout mouse expressing a humanised Hsd17b12 demonstrated mouse HSD17B12 contributes to total testosterone produced by the testes, however, does not completely deplete testosterone levels, indicating other enzymes are capable of this conversion which remain unidentified. Whilst the canonical and alternate androgen production pathways have been investigated individually, it has previously been unknown how these pathways interact and if the alternate pathway is upregulated following disruption to the canonical pathway. To dissect the roles and interactions of these pathways, a double knockout mouse model was generated involving genes in the canonical (Hsd17b3) and alternate (Srd5a1) pathways. In-depth investigation of circulating steroids demonstrated that alternate pathway androgen precursors are increased in the circulation of mice following the ablation of HSD17B3 due to activity of both the SRD5A1 and SRD5A2 enzymes. Further, whole testis proteomics identified mouse HSD17B7 as a potential testosterone producing enzyme. In conclusion, experiments described have expanded our understanding of androgen biosynthesis in mice and has highlighted differences between mouse and human androgen production. Knowledge obtained will allow for the generation of better models which more accurately depict human androgen related disorders and will assist in developing therapies to support endogenous androgen production over the life course.