Search

Background The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) determines prereceptor metabolism and activation of glucocorticoids within peripheral tissues. Its dysregulation has been implicated in a wide array of metabolic diseases, leading to the development of selective 11β-HSD1 inhibitors. We examined the impact of the reversible competitive 11β-HSD1 inhibitor, AZD4017, on the metabolic profile in an overweight female cohort with idiopathic intracranial hypertension (IIH). Methods We conducted a UK multicenter phase II randomized, double-blind, placebo-controlled trial of 12-week treatment with AZD4017. Serum markers of glucose homeostasis, lipid metabolism, renal and hepatic function, inflammation and androgen profiles were determined and examined in relation to changes in fat and lean mass by dual-energy X-ray absorptiometry. Results Patients receiving AZD4017 showed significant improvements in lipid profiles (decreased cholesterol, increased high-density lipoprotein [HDL] and cholesterol/HDL ratio), markers of hepatic function (decreased alkaline phosphatase and gamma-glutamyl transferase), and increased lean muscle mass (1.8%, P < .001). No changes in body mass index, fat mass, and markers of glucose metabolism or inflammation were observed. Patients receiving AZD4017 demonstrated increased levels of circulating androgens, positively correlated with changes in total lean muscle mass. Conclusions These beneficial metabolic changes represent a reduction in risk factors associated with raised intracranial pressure and represent further beneficial therapeutic outcomes of 11β-HSD1 inhibition by AZD4017 in this overweight IIH cohort. In particular, beneficial changes in lean muscle mass associated with AZD4017 may reflect new applications for this nature of inhibitor in the management of conditions such as sarcopenia.

The roles of androgens in male reproductive development and function in zebrafish are poorly understood. To investigate this topic we employed CRISPR/Cas9 to generate cyp11c1 (11β-hydroxylase) mutant zebrafish lines. Our study confirms recently published findings from a different cyp11c1-/- mutant zebrafish line, and also reports novel aspects of the phenotype caused by loss of Cyp11c1 function. We report that Cyp11c1-deficient zebrafish display predominantly female secondary sex characteristics, but may possess either ovaries or testes. Moreover, we observed that cyp11c1-/- mutant male zebrafish are profoundly androgen- and cortisol-deficient. These results provide further evidence that androgens are dispensable for testis formation in zebrafish, as has been demonstrated previously in androgen-deficient and androgen-resistant zebrafish. Herein, we show that the testes of cyp11c1-/- mutant zebrafish exhibit a disorganised tubular structure; and for the first time demonstrate that the spermatic ducts, which connect the testes to the urogenital orifice, are severely hypoplastic in androgen-deficient zebrafish. Furthermore, we show that spermatogenesis and characteristic breeding behaviours are impaired in cyp11c1-/- mutant zebrafish. Expression of nanos2, a type A spermatogonia marker, was significantly increased in the testes of Cyp11c1-deficient zebrafish, whereas expression of markers for later stages of spermatogenesis was significantly decreased. These observations indicate that in zebrafish, production of type A spermatogonia is androgen-independent, but differentiation of type A spermatogonia is an androgen-dependent process. Overall, our results demonstrate that whilst androgens are not required for testis formation, they play important roles in determining secondary sexual characteristics, proper organisation of seminiferous tubules, and differentiation of male germ cells.

Cytochrome P450 side-chain cleavage enzyme, encoded by the CYP11A1 gene, catalyzes the first and rate-limiting step of steroid hormone biosynthesis. Previous morpholino knockdown studies in zebrafish suggested cyp11a2 is a functional equivalent of human CYP11A1 and is essential for interrenal steroidogenesis in zebrafish larvae. The role of Cyp11a2 in adult zebrafish, particularly in gonadal steroidogenesis, remains elusive. To explore the role of Cyp11a2 in adults, we developed zebrafish mutant lines by creating deletions in cyp11a2 using the CRISPR/Cas9 genomic engineering approach. Homozygous cyp11a2 mutant zebrafish larvae showed an upregulation of the hypothalamic–pituitary–interrenal axis. Furthermore, these Cyp11a2-deficient zebrafish demonstrated profound glucocorticoid and androgen deficiencies. Cyp11a2 homozygotes only developed into males with feminized secondary sex characteristics. Adult cyp11a2^-/- mutant fish showed a lack of natural breeding behaviors. Histological characterization revealed disorganized testicular structure and significantly decreased numbers of mature spermatozoa. These findings are further supported by the downregulation of the expression of several pro-male genes in the testes of cyp11a2 homozygous zebrafish, including sox9a, dmrt1 and amh. Moreover, the spermatogonia markers nanos2 and piwil1 were upregulated, while the spermatocytes marker sycp3 and spermatids marker odf3b were downregulated in the testes of cyp11a2 homozygous mutants. Our expression analysis is consistent with our histological studies, suggesting that spermatogonia are the predominant cell types in the testes of cyp11a2 homozygous mutants. Our work thus demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroidogenesis in zebrafish larvae and adults.

The roles of steroids in zebrafish sex differentiation, gonadal development and function of the adult gonad are poorly understood. Herein, we have employed a ferredoxin 1b (fdx1b) mutant zebrafish to explore such processes. Fdx1b is an essential electron-providing cofactor to mitochondrial steroidogenic enzymes, which are crucial for glucocorticoid and androgen production in vertebrates. Fdx1b-/- zebrafish mutants develop into viable adults, in which concentrations of androgens and the glucocorticoid, cortisol, are significantly reduced. Adult fdx1b-/- mutant zebrafish display predominantly female secondary sex characteristics but may possess either ovaries or testes, confirming that androgen signaling is dispensable for testicular differentiation in this species, as previously demonstrated in androgen receptor mutant zebrafish. Adult male fdx1b-/- mutant zebrafish do not exhibit characteristic breeding behaviors, and sperm production is reduced, resulting in infertility in standard breeding scenarios. However, eggs collected from wild-type females can be fertilized by the sperm of fdx1b-/- mutant males by IVF. The testes of fdx1b-/- mutant males are disorganized and lack defined seminiferous tubule structure. Expression of several pro-male and spermatogenic genes is decreased in the testes of fdx1b-/- mutant males, including pro-male transcription factor SRY-box 9a (sox9a) and spermatogenic genes insulin-like growth factor 3 (igf3) and insulin-like 3 (insl3). This study establishes an androgen- and cortisol-deficient fdx1b zebrafish mutant as a model for understanding the impacts of steroid deficiency on sex development and reproductive function. This model will be particularly useful for further investigation of the roles of steroids in spermatogenesis, gonadal development and regulation of reproductive behavior, thus enabling further elucidation of the physiological consequences of endocrine disruption in vertebrates.

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterized. Since zebrafish are increasingly employed in endocrine and stress research, a better characterization of steroidogenic pathways is required to target specific steps in the biosynthetic pathways in the future. Here, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing co-factors ferredoxin (fdx1, fdx1b) during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative RT-PCR and whole-mount in situ hybridization in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid THDOC in the adult zebrafish brain from radiolabeled pregnenolone. Taken together, our study is a comprehensive characterization of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesizing in the brain of adult zebrafish facilitated by enzymes involved on glucocorticoid biosynthesis. Our work provides a valuable source for establishing the zebrafish as a translational model to understand the roles of the genes of glucocorticoid biosynthesis and fdx co-factors during embryonic development, stress and in brain homeostasis and function.

Two CYP17 genes, located on different loci and expressing enzymes with significantly different activities, have been identified in the South African Angora goat population. Three unique genotypes (named He, Hu, and Ho), which differed not only in the genes encoding CYP17, but also in copy number were subsequently identified in the Angora goat. The aim of this study was to evaluate the production and reproduction performance of these three genotypes. Bodyweight, fleece and reproduction data, and blood samples from 466 Angora ewes from three flocks were obtained from the GADI-Biobank. Data had been collected on Flock 1 from 2000 to 2015, Flock 2 from 2000 to 2014 and Flock 3 from 2000 to 2010. Bodyweight data included birth weight, weaning weight, 8-, 12-, and 16-month bodyweight, as well as bodyweight recorded annually for the ewe flock before mating. Fleece data included fleece weight and fibre diameter recorded at the second and third shearings at 12 and 18 months old, respectively. Fleece weight, fibre diameter, style and character were also recorded annually for the ewe flocks during the winter shearing. Individual reproduction records included information on whether the ewe had kidded, whether the ewe had aborted, number of kids born, stillborn kids, kids that died soon after birth, kids reared by a foster mother, kids reared as orphans, number of kids weaned and total weight of kids weaned. Total lifetime reproductive performance of genotyped ewes was calculated for number of kids born, number of kids weaned, and total weight of kids weaned. Blood samples were also collected from 100 sexually active Angora rams from four sources. CYP17 genotyping was carried out using an ARMS-qPCR (amplification refractory mutation system qPCR) assay. Serum testosterone was quantified using high performance liquid chromatography mass spectrometry. The distribution of the ewes across the three CYP17 genotypes was 36.7% He, 51.5% Hu, and 11.8% Ho, and was in accordance with the distribution of the Angora veld rams (38.0% He, 46.4% Hu, and 15.6% Ho). In this study, animals of the Hu genotype were heavier from weaning age onwards, although this difference in bodyweight was significant only at 8 months old and in the adult ewes. No differences were observed between the He and Ho animals. Adult ewes of the He genotype (1.35 kg) produced heavier (P 0.05). No significant differences were recorded in reproductive performance among the genotypes, although the Ho ewes had the lowest (1.03 and 0.89) and the He ewes the highest (1.07 and 0.93) number of kids born and weaned per year respectively. Results on the rams indicated that the CYP17 genotype had no effect on testosterone production by Angora rams. From the results of this study no evidence could be found that selection for any of the three genotypes would adversely affect any growth, mohair production or reproduction function of Angora ewes. A breeding strategy incorporating selection for productive traits and the CYP17 genotype, aimed at increasing the frequency of the ACS+ gene and thus the He genotype in the population, could be followed without having a negative effect on the genetic progress of productive traits.Keywords: Bodyweight, fleece weight, number of kids weaned, testosterone, weight of kids weaned

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we have developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the Cyp21a2 protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatio-temporal expression patterns of cyp21a2 by whole mount in situ hybridisation and RT-PCR throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21-hydroxylase deficiency we created several cyp21a2 null-allele zebrafish lines employing a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrrenal axis and interrenal hyperplasia. Furthermore, Cyp21A2-deficient larvae had a typical steroid profile with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the hypothalamic-pituitary-interrrenal axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes a novel in vivo model allowing for studies of systemic consequences of altered steroid hormone synthesis.

Context: The classic androgen synthesis pathway proceeds via dehydroepiandrosterone, androstenedione, and testosterone to 5α-dihydrotestosterone. However, 5α-dihydrotestosterone synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens, and in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid (GC) therapy is unknown. Objective: We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation, and their response to conventional GC therapy and modified-release hydrocortisone. Methods: We used urinary steroid metabolome profiling by gas chromatography–mass spectrometry for 24-hour steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, and dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excretion by comparing 8-hourly collections (23:00–7:00, 7:00–15:00, and 15:00–23:00) in 16 patients with CAH taking conventional GCs and during 6 months of treatment with modified-release hydrocortisone, Chronocort. Results: Patients with CAH taking conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11β-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near-normal levels more consistently than other GC preparations. Conclusions: Alternative pathway-mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP.

The ability of animals to adapt to stress is not only an animal health and welfare concern, but also influences reproduction potential and robustness. An important pathway involved in the stress response is the hypothalamic-pituitary-adrenal axis (HPAA) that results in the release of cortisol from the adrenal gland. In this study the cortisol responses of South African Merinos were measured to assess HPAA responsiveness to stress and relate it to behavioural stress responses to\ud flock-isolation. The experiment was structured according to a 2×2 statistical design, with CYP17 genotype (WT1/WT1 vs. WT1/WT2) and selection line (H-line vs. L-line) as factors. Selection line criteria was based on divergent selection for (H-line) or against (L-line) maternal multiple rearing ability, where the H-line generally outperformed the L-line in terms of reproduction, animal\ud welfare and resistance to certain pathogens. The CYP17 genotype is involved in the biosynthesis pathway of cortisol. In the present study the CYP17 genotype showed a significant influence on behavioural stress responses, where three parameters of the flock-isolation test were affected (P

South African Merino sheep were selected divergently from the same base population for their ability to rear multiples. Two distinct populations were formed over a period of more than 20 years of selection. Reproduction (and therefore presumably fitness) in the line selected in the upward direction (H-line) was substantially improved compared to the line selected in the downward direction (L-line). In the present study, it was demonstrated that the H-line was more stresstolerant\ud than the L-line in terms of their glucose and cortisol response when challenged with insulin. Sheep from the breeding program were genotyped according to one of two cytochrome P450 17α-hydroxylase/17-20 lyase (CYP17) alleles, as these genotypes were previously linked to the ability of Angora goats to cope with external stressors. However, no association was found between CYP17 genotype and selection line. The difference in insulin induced stress response between the H- and the L-line can therefore not be attributed to CYP17 genotype.