👤 Peter J O'Shaughnessy

Testosterone and dihydrotestosterone (DHT) are essential for male development and fertility. In the canonical androgen production pathway, testosterone is produced in the testis by HSD17B3; however, adult male Hsd17b3 knockout (KO) mice continue to produce androgens and are fertile, indicating compensatory mechanisms exist. A second, alternate pathway produces DHT from precursors other than testosterone via 5α-reductase (SRD5A) activity. We hypothesized that the alternate pathway contributes to androgen bioactivity in Hsd17b3 KO mice. To investigate contributions arising from and interactions between the canonical and alternate pathways, we pharmacologically inhibited SRD5A and ablated Srd5a1 (the predominant SRD5A in the testis) on the background of Hsd17b3 KO mice. Mice with perturbation of either the canonical or both pathways exhibited increased LH, testicular steroidogenic enzyme expression, and normal reproductive tracts and fertility. In the circulation, alternate pathway steroids were increased in the absence of HSD17B3 but were reduced by co-inhibition of SRD5A1. Mice with perturbations of both pathways produced normal basal levels of intratesticular testosterone, suggesting the action of other unidentified hydroxysteroid dehydrogenase(s). Strikingly, testicular expression of another SRD5A enzyme, Srd5a2, was markedly increased in the absence of Hsd17b3, suggesting a compensatory increase in SRD5A2 to maintain androgen bioactivity during HSD17B3 deficiency. Finally, we observed elevated circulating concentrations of the 11-keto-derivative of DHT, suggesting compensatory extra-gonadal induction of bioactive 11-keto androgen production. Taken together, we conclude that, in the absence of the canonical pathway of androgen production, multiple intra- and extra-gonadal mechanisms cooperate to maintain testosterone and DHT production, supporting male development and fertility. Show less

Male development, fertility, and lifelong health are all androgen-dependent. Approximately 95% of circulating testosterone is synthesized by the testis and the final step in this canonical pathway is controlled by the activity of the hydroxysteroid-dehydrogenase-17-beta-3 (HSD17B3). To determine the role of HSD17B3 in testosterone production and androgenization during male development and function we have characterized a mouse model lacking HSD17B3. The data reveal that developmental masculinization and fertility are normal in mutant males. Ablation of HSD17B3 inhibits hyperstimulation of testosterone production by hCG, although basal testosterone levels are maintained despite the absence of HSD17B3. Reintroduction of HSD17B3 via gene-delivery to Sertoli cells in adulthood partially rescues the adult phenotype, showing that, as in development, different cell-types in the testis are able to work together to produce testosterone. Together, these data show that HS17B3 acts as a rate-limiting-step for the maximum level of testosterone production by the testis but does not control basal testosterone production. Measurement of other enzymes able to convert androstenedione to testosterone identifies HSD17B12 as a candidate enzyme capable of driving basal testosterone production in the testis. Together, these findings expand our understanding of testosterone production in males. Show less

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place. Show less

During mammalian testis development distinct generations of fetal and adult Leydig cells arise. Luteinising hormone (LH) is required for normal adult Leydig cell function and for the establishment of normal adult Leydig cell number but its role in the process of adult Leydig cell differentiation has remained uncertain. In this study we have examined adult Leydig cell differentiation in gonadotrophin-releasing hormone (GnRH)-null mice which are deficient in circulating gonadotrophins. Adult Leydig cell differentiation was assessed by measuring expression of mRNA species encoding four specific markers of adult Leydig cell differentiation in the mouse. Each of these markers (3beta-hydroxysteroid dehydrogenase type VI (3betaHSD VI), 17beta-hydroxysteroid dehydrogenase type III (17betaHSD III), prostaglandin D (PGD)-synthetase and oestrogen sulphotransferase (EST)) is expressed only in the adult Leydig cell lineage in the normal adult animal. Real-time PCR studies showed that all four markers are expressed in adult GnRH-null mice. Localisation of 3betaHSD VI and PGD-synthetase expression by in situ hybridisation confirmed that these genes are expressed in the interstitial tissue of the GnRH-null mouse. Treatment of animals with human chorionic gonadotrophin increased expression of 3betaHSD VI and 17betaHSD III within 12 hours further indicating that differentiated, but unstimulated cells already exist in the GnRH-null mouse. Thus, while previous studies have shown that LH is required for adult Leydig cell proliferation and activity, results from the present study show that adult Leydig cell differentiation will take place in animals deficient in LH. Show less

The final step in the biosynthesis of testosterone is the reduction of androstenedione to testosterone catalysed by the enzyme 17beta-hydroxysteroid dehydrogenase (17betaHSD). Five isoforms of the enzyme have been identified in the mouse and the type 3 isoform has been shown to be the predominant reductive form present in the adult human and mouse testis. In this study the regulation of 17betaHSD type 3 isoform mRNA levels and the cellular localisation of the enzyme mRNA have been studied in the mouse testis. To examine regulation of 17betaHSD type 3 mRNA expression in the testis, mRNA levels were measured during development in normal mice and in mice lacking circulating gonadotrophins (hpg) or functional androgen receptors (Tfm). In these mutants testicular descent does not occur at the normal time (25 days) and control animals were, therefore, rendered cryptorchid at 19 days. In neonatal mice, it has been shown a peak of type 3 expression occurs around day 5 and this was found to be normal in all groups in the current study. In normal animals there was a marked increase in type 3 isoform expression between 25 and 30 days and this continued into adulthood. In cryptorchid animals the increase in type 3 mRNA levels after 25 days was less marked than in untreated controls and by 90 days was about 15% of normal animals. In Tfm mice, levels of 17betaHSD type 3 mRNA failed to show any increase around puberty (25 days) and in adult Tfm mice, levels were less than 1% of cryptorchid controls. In hpg mice, levels of type 3 mRNA increased slowly after puberty and were about 30% of cryptorchid controls by 90 days. Studies using in situ hybridisation showed that the type 3 isoform was expressed only in the interstitial tissue of the adult normal mouse testis. No specific hybridisation could be determined in adult hpg or Tfm testes. Results show that 17betaHSD type 3 is an interstitial enzyme in the testis and is, probably, localised in the Leydig cells. During neonatal development expression of 17betaHSD type 3 is independent of gonadotrophin action while the increase in type 3 expression at puberty is primarily dependent upon androgen action although testicular descent and gonadotrophins are also required. Show less

The enzyme 17beta-hydroxysteroid dehydrogenase (17betaHSD) interconverts 17-ketosteroids and 17beta-hydroxysteroids. Five isoforms of the enzyme have been identified in the human, two of which (types 1 and 3) have been shown to catalyse the reduction reaction preferentially. The cDNA encoding mouse 17betaHSD type 3 was isolated from testis cDNA using the RACE technique and primers based on the human sequence. The mouse protein is 305 amino acids in length which is five short of the human protein with four of these amino acids missing at the N-terminus. The predicted amino acid sequence is 72.5% identical and 94.8% similar to the human sequence. Tissue distribution of mRNA encoding both types 1 and 3 17betaHSD was studied using reverse transcription and the polymerase chain reaction (RT-PCR). Highest levels of type 1 mRNA were found in the ovary whereas highest levels of type 3 were in the testis. All other tissues tested contained mRNA encoding both isoforms of the enzyme although levels were markedly lower than in the gonads. Show less