👤 Sean Curley

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

High-fat diet (HFD)-induced obesity impairs macrophage-to-feces reverse cholesterol transport (RCT). It is hypothesized that dietary supplementation with the polyunsaturated fatty acids conjugated linoleic acid (CLA) or alpha linolenic acid (ALA) would prevent HFD-impaired RCT by modulating hepatic protein pathways. ApoE3L.CETP mice are fed a HFD supplemented ± CLA or ALA for 12 weeks and in vivo macrophage-to-feces RCT is determined. Hepatic cholesterol transporters and the hepatic proteome are assessed by immunoblotting and mass spectrometry, respectively. Mice fed HFD alone, but not ALA-HFD or CLA-HFD, exhibit increased systemic cholesterol levels, increased ALA and CLA exert distinct mechanistic advantages on cholesterol homeostasis and RCT in obesity. 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

Assessing whether next-generation DNA sequencing (NGS) can be used to screen prostate cancer for multiple gene alterations in men routinely diagnosed with this disease and/or who are entered into clinical trials. Previous studies are limited and have reported only low success rates. We marked areas of cancer on H&E-stained sections from formalin-fixed needle biopsies, and used these as templates to dissect cancer-rich tissue from adjacent unstained sections. DNA was prepared using a Qiagen protocol modified to maximise DNA yield. The DNA was screened simultaneously for mutations in 365 cancer-related genes using an Illumina HiSeq 2000 NGS platform. From 63 prostate cancers examined, 59(94%) of the samples yielded at least 30 ng of DNA, the minimum amount of DNA considered suitable for NGS analysis. Patients in the D'Amico high-risk group yielded an average of 1033 ng, intermediate-risk patients 401 ng, and low-risk patients 97 ng. NGS of eight samples selected from high-risk and intermediate-risk groups gave a median exon read depth of 962 and detected TMPRRS2-ERG fusions, as well as a variety of mutations including those in the SPOP, TP53, ATM, MEN1, NBPF10, NCOR2, PIK3CB and MAP2K5 (MEK5) genes. Using the methods presented here, NGS technologies can be used to screen a high proportion of patients with prostate cancer for mutations in cancer-related genes in tissue samples opening up its general use in the context of clinical trials or routine diagnosis. Show less