CYP17A1 Activities Research Articles

Chemicals of environmental concern as inhibitors of human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase: Screening and docking analysis

Many environmental pollutants act as endocrine-disrupting compounds by inhibiting human placental 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase type 1 (HSD3B1) and aromatase (CYP19A1) activities. In this study, we screened 13 chemicals of environmental concern for their ability to inhibit human HSD3B1 and CYP19A1 by measuring the conversion of pregnenolone to progesterone for HSD3B1 activity and the conversion of testosterone to 17β-estradiol for CYP19A1 activity in human JEG-3 choriocarcinoma cell microsomes. HSD3B1 had an apparent Km of 0.323 μM and an apparent Vmax of 0.111 nmol/mg/min and CYP19A1 had an apparent Km of 56 nM and an apparent Vmax of 0.177 nmol/mg protein/min. 17β-Estradiol, bisphenol A, and bisphenol AF competitively inhibited HSD3B1 with Ki values of 0.8, 284.1, and 141.2 μM, respectively, while diethylstilbestrol had a mixed inhibition on human HSD3B1 with the Ki of 8.0 μM. Ketoconazole, bisphenol A, and bisphenol AF noncompetitively inhibited CYP19A1 with Ki values of 10.3, 54.4, and 45.7 μM, respectively, while diethylstilbestrol and zearalenone competitively suppressed CYP19A1 with Ki values of 63.0 and 16.6 μM, respectively. Docking analysis showed that 17β-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF bound the steroid binding pocket facing the catalytic residues Y155 and K159 of HSD3B1, and that ketoconazole, bisphenol A, and bisphenol AF bound heme binding pocket while diethylstilbestrol and zearalenone bound the steroid binding site of CYP19A1. In conclusion, 17β-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF are human HSD3B1 inhibitors, and ketoconazole, zearalenone, diethylstilbestrol, bisphenol A, and bisphenol AF are human CYP19A1 inhibitors.

Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels.

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Androgens Tend to Be Higher, but What about Altered Progesterone Metabolites in Boys and Girls with Autism?

Background: Evidence exists that steroid hormones are altered in individuals with autism, especially androgens. Despite lower prevalence in girls than boys, evidence of potential alterations in progesterone metabolites is sparse, so the aim of this study was to elucidate different progesterone metabolites in affected children with autism versus healthy controls. Material and Methods: Circadian urine samples from 48 boys and 16 girls with autism spectrum disorders and a matched case–control group were analysed for progesterone metabolites by gas chromatography–mass spectrometry and normalised for creatinine excretion. Results: In boys with autism, the majority of progesterone metabolites were reduced, such as progesterone, 6a-OH-3a5b-TH-progesterone, or 20a-DH-progesterone (p < 0.01 for all). In girls with autism, a similar pattern of reduction in progesterone metabolites was detected; however, potentially due to the relatively small sample, this pattern was only detectable on the level of a trend. Discussion: As stated, androgen levels are higher in boys and girls with autism, but evidence for progesterone metabolites is much sparser. The pattern of a decrease in progesterone metabolites suggests the existence of an altered routing of steroid metabolites, probably in combination with a dysregulation of the HPAG axis. As, recently, increased CYP17A1 activity has been suggested, the stronger routing towards androgens is further implied in line with our findings of lower progesterone concentrations in boys and girls with autism than healthy controls.

Differences in the spectrum of steroidogenic enzyme inhibition between Osilodrostat and Metyrapone in ACTH-dependent Cushing syndrome patients.

Osilodrostat is a new 11β-hydroxylase inhibitor with a mode of action analogous to Metyrapone. The objective of this study was to compare steroidogenic profiles in patients treated with either Osilodrostat or Metyrapone for adrenocorticotrophic hormone (ACTH)-dependent Cushing's syndrome (CS). Patients followed up at Cochin hospital Endocrinology department between March 2019 and December 2021 for an ACTH-dependent CS, controlled by either Osilodrostat or Metyrapone, were included. A serum profile of five steroids (cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione and testosterone) was determined using UPLC- tandem mass spectrometry (UPLC-MS/MS). Nineteen patients treated with Osilodrostat, eight patients treated with Metyrapone and six patients treated with consecutive Metyrapone then Osilodrostat were included. Hypocortisolism (basal cortisol <100 nmol/L) was found in 48% of patients treated with Osilodrostat and 7% of patients treated with Metyrapone. 11-deoxycortisol and androstenedione levels were higher in patients treated with Metyrapone (80.9 (2.2-688.4) and 14.9 (2.5-54.3) nmol/L, respectively) than in patients treated with Osilodrostat (10.3 (0.5-71.9) and 4.0 (0.3-13.3) nmol/L) (P = 0.0009 and P = 0.0005). Testosterone level in women was also higher in Metyrapone group (3.3 (0.93-4.82) nmol/L vs 1.31(0.13-5.09) nmol/L, P = 0.0146). CYP11B1 activity (11-deoxycortisol/cortisol) was not significantly different between the two groups. CYP21A2 activity (17OHprogesterone/11-deoxycortisol) and CYP17A1 activity (17OHprogesterone/androstenedione) were significantly decreased in Osilodrostat group (P < 0.0001). In patients with ACTH-dependent CS, the use of CYP11B1 inhibitors in routine care suggests that Osilodrostat has a less specific effect on the inhibition of steroidogenic enzymes than Metyrapone. This might explain a smaller increase in 11-deoxycortisol and androgen levels in patients treated with Osilodrostat.

Essential oil metabolites can regulate adrenal androgen production by inhibition of CYP17A1 activities

Endocrine‐disrupting chemicals (EDCs) can affect human steroid metabolism. Previous clinical case reports have shown that some Essential Oils (EOs) like lavender oil and tea tree oil may act as potential EDCs and are linked to prepubertal gynecomastia in boys and premature thelarche in girls due to regular exposure to lavender based fragrances among Hispanic population. These studies suggested role of EOs in steroid metabolism in humans. We have screened a range of EO metabolites for effects on androgen production by CYP17A1.For preliminary screening, human adrenal NCI H295R cells were treated with 10 µM of test compounds for 24 hours. The test compounds had been extracted and purified from natural resources and are found as major components in EOs. For CYP17A1 activity, the conversion of radiolabelled substrate, 17‐Hydroxy‐pregnenolone to Dehydroepiandrosterone was determined using tritiated water release assay.Eucalyptol, Dihydro‐β‐Ionone, (‐)‐α‐pinene were extracted from eucalyptus, rose and pine resin. Out of about 50 test compounds, eucalyptol, Dihydro‐β‐Ionone &amp; (‐)‐α‐pinene showed 20% to 40% inhibition of DHEA production. Rest of the compounds showed either no or low inhibition. Some compounds were also tested for effects on CYP19A1 (aromatase) activity where upto 30% inhibition was observed.EOs are often used in various beauty and hygiene products as they have few known side‐effects. However, prolonged exposure to these products may result in steroid imbalance. Due to their anti‐androgenic activity, these compounds should be studied further as chemical leads for the treatment of hyperandrogenic disorders such as Prostate cancer and Polycystic ovary syndrome.

High intratumoral dihydrotestosterone is associated with antiandrogen resistance in VCaP prostate cancer xenografts in castrated mice

SummaryAntiandrogen treatment resistance is a major clinical concern in castration-resistant prostate cancer (CRPC) treatment. Using xenografts of VCaP cells we showed that growth of antiandrogen resistant CRPC tumors were characterized by a higher intratumor dihydrotestosterone (DHT) concentration than that of treatment responsive tumors. Furthermore, the slow tumor growth after adrenalectomy was associated with a low intratumor DHT concentration. Reactivation of androgen signaling in enzalutamide-resistant tumors was further shown by the expression of several androgen-dependent genes. The data indicate that intratumor DHT concentration and expression of several androgen-dependent genes in CRPC lesions is an indication of enzalutamide treatment resistance and an indication of the need for further androgen blockade. The presence of an androgen synthesis, independent of CYP17A1 activity, has been shown to exist in prostate cancer cells, and thus, novel androgen synthesis inhibitors are needed for the treatment of enzalutamide-resistant CRPC tumors that do not respond to abiraterone.

Exploration of structural requirements for azole chemicals towards human aromatase CYP19A1 activity: Classification modeling, structure-activity relationships and read-across study

Human aromatase, also called CYP19A1, plays a major role in the conversion of androgens into estrogens. Inhibition of aromatase is an important target for estrogen receptor (ER)-responsive breast cancer therapy. Use of azole compounds as aromatase inhibitors is widespread despite their low selectivity. A toxicological evaluation of commonly used azole-based drugs and agrochemicals with respect to CYP19A1 is currently requested by the European Union- Registration, Evaluation, Authorization and Restriction of Chemicals (EU-REACH) regulations due to their potential as endocrine disruptors. In this connection, identification of structural alerts (SAs) is an effective strategy for the toxicological assessment and safe drug design. The present study describes the identification of SAs of azole-based chemicals as guiding experts to predict the aromatase activity. Total 21 SAs associated with aromatase activity were extracted from dataset of 326 azole-based drugs/chemicals obtained from Tox21 library. A cross-validated classification model having high accuracy (error rate 5%) was proposed which can precisely classify azole chemicals into active/inactive toward aromatase. In addition, mechanistic details and toxicological properties (agonism/antagonism) of azoles with respect to aromatase were explored by comparing active and inactive chemicals using structure-activity relationships (SAR). Lastly, few structural alerts were applied to form chemical categories for read-across applications.

High incidence of hypertension-mediated organ damage in a series of Chinese patients with 17α-hydroxylase deficiency.

To analyze the prevalence of hypertension-mediated organ damage (HMOD) and its relationship with enzyme activity of mutant CYP17A1 and other risk factors in patients with 17α-hydroxylase/17,20-lyase deficiency (17-OHD). A total of 68 patients with 17-OHD were recruited in the Peking Union Medical College Hospital from 2003 to 2021. The incidence of hypertension and HMOD was respectively analyzed. CYP17A1 sequencing was performed and the enzyme activity of mutant CYP17A1 was determined by analyzing the characteristics of mutation itself and the functional data reported previously. A logistic regression model was employed to analyze the factors related to HMOD and the specific damaged organs in 17-OHD patients. Sixty-five patients (95.6%) exhibited hypertension, 32 of whom were diagnosed with HMOD. c.985_987delTACinsAA (p.Y329KfsX418) (53.8%) and c.1459_1467del (p. del D487_F489) (11.4%) were the top two mutations, and no correlation was found between enzyme activity of mutant CYP17A1 and HMOD. The risk of HMOD increased by 32% for each additional year of hypertension duration, 10.2-fold for each one-grade increase in hypertension level, 2.3-fold for each grade of exacerbation of hypokalemia. Patients with 17-OHD experience a high incidence of HMOD. There was no correlation between the HMOD occurrence and enzyme activity of mutant CYP17A1. Longer duration of hypertension, more severe hypertension, and hypokalemia were independent risk factors for the occurrence of HMOD in patients with 17-OHD.

Altered Steroidome in Women with Gestational Diabetes Mellitus: Focus on Neuroactive and Immunomodulatory Steroids from the 24th Week of Pregnancy to Labor.

Gestational diabetes mellitus (GDM) is a complication in pregnancy, but studies focused on the steroidome in patients with GDM are not available in the public domain. This article evaluates the steroidome in GDM+ and GDM− women and its changes from 24 weeks (± of gestation) to labor. The study included GDM+ (n = 44) and GDM− women (n = 33), in weeks 24–28, 30–36 of gestation and at labor and mixed umbilical blood after delivery. Steroidomic data (101 steroids quantified by GC-MS/MS) support the concept that the increasing diabetogenic effects with the approaching term are associated with mounting progesterone levels. The GDM+ group showed lower levels of testosterone (due to reduced AKR1C3 activity), estradiol (due to a shift from the HSD17B1 towards HSD17B2 activity), 7-oxygenated androgens (competing with cortisone for HSD11B1 and shifting the balance from diabetogenic cortisol towards the inactive cortisone), reduced activities of SRD5As, and CYP17A1 in the hydroxylase but higher CYP17A1 activity in the lyase step. With the approaching term, the authors found rising activities of CYP3A7, AKR1C1, CYP17A1 in its hydroxylase step, but a decline in its lyase step, rising conjugation of neuroinhibitory and pregnancy-stabilizing steroids and weakening AKR1D1 activity.

Aberrant H19 Expression Disrupts Ovarian Cyp17 and Testosterone Production and Is Associated with Polycystic Ovary Syndrome in Women.

As one of the most common endocrine disorders affecting women, polycystic ovary syndrome (PCOS) is associated with serious conditions including anovulation, endometrial cancer, infertility, hyperandrogenemia, and an increased risk for obesity and metabolic derangements. One contributing etiology to the pathophysiology of hyperandrogenemia associated with PCOS is an intrinsic alteration in ovarian steroidogenesis, leading to enhanced synthesis of androgens including testosterone. Studies have suggested that the increased testosterone synthesis seen in PCOS is driven in part by increased activity of CYP17A1, the rate-limiting enzyme for the formation of androgens in the gonads and adrenal cortex, which represents a critical factor driving enhanced testosterone secretion in PCOS. In this work, we evaluated the hypothesis that dysregulation of the noncoding RNA H19 results in aberrant CYP17 and testosterone production. To achieve this, we measured Cyp17 in ovarian tissues of H19 knockout mice, and quantified serum testosterone levels, in comparison with wild-type controls. We also evaluated circulating and ovarian H19 expression and correlated results with the presence or absence of PCOS in a group of women undergoing evaluation and treatment for infertility. We found that the loss of H19 in a mouse model results in decreased ovarian Cyp17, along with decreased serum testosterone in female mice. Moreover, utilizing serum samples and cumulus cells from women with PCOS, we showed that circulating and ovarian levels of H19 are increased in women with PCOS compared to controls. Findings from our multimodal experimental strategy, involving both a mouse model of dysregulated H19 expression and clinical serum and ovarian cellular samples from women with PCOS, suggest that the loss of H19 may disrupt androgen production via a Cyp17-mediated mechanism. Conversely, excess H19 may play a role in the pathogenesis of PCOS-associated hyperandrogenemia.

ABERRANT H19 EXPRESSION DISRUPTS OVARIAN Cyp17 AND TESTOSTERONE PRODUCTION AND IS ASSOCIATED WITH POLYCYSTIC OVARY SYNDROME

One contributing factor to the pathophysiology of hyperandrogenemia associated with PCOS is an intrinsic alteration in ovarian steroidogenesis, leading to enhanced synthesis of androgens including testosterone (T). Increased activity of CYP17A1, the rate-limiting enzyme for the formation of androgens in the gonads, is thought to be a critical factor driving enhanced T secretion in PCOS. Given their diverse roles in biological process, interest has grown in the role of noncoding RNAs (ncRNAs) in the pathogenesis of PCOS.

Rutin inhibits androgen synthesis and metabolism in rat immature Leydig cells in vitro.

In the early stage of androgen-sensitive prostate cancer, cancer cells require androgens to grow. Hormone therapy that lowers androgen output or blocks androgen receptor can suppress the growth of this type of prostate cancer. Rutin, a flavonoid derivative of many plants, has numerous pharmacological effects. The objective of this study was to investigate the effect of rutin on androgen biosynthesis in Leydig cells isolated from the testes of pubertal rats. Immature Leydig cells isolated from 35days-old male Sprague-Dawley rats were cultured in vitro with 0.5-50μM rutin for 3hr. Rutin significantly inhibited androgen secretion at 0.5, 5 and 50μM under basal condition (medium only). At 50μM, rutin also markedly compromised androgen secretion stimulated by 10ng/ml luteinising hormone and 10mM 8-bromoadenosine 3', 5'-cyclic monophosphate. Further analysis demonstrated that rutin compromised the transcript levels of Scarb1, Cyp11a1 and Hsd3b1 and their proteins expression. Rutin directly inhibited rat testicular CYP17A1, HSD17B3 and AKR1C14 activities at 50μM. Rutin did not alter mitochondrial membrane potential at up to 50μM. In conclusion, rutin suppresses androgen biosynthesis in Leydig cells through multiple mechanisms, thereby having benefits for the treatment of androgen-sensitive prostate cancer.

Identification of novel COX-2 / CYP19A1 axis involved in the mesothelioma pathogenesis opens new therapeutic opportunities

BackgroundBased on previous studies highlighting that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of malignant pleural mesothelioma (MPM), and that aromatase (CYP19A1), an enzyme that plays a key role in estrogen biosynthesis, along with estradiol (E2) were expressed in MPM, this study aimed to investigate the possible interplay between COX-2 and CYP19A1 in the pathogenesis of mesothelioma, as well as the underlying mechanism.MethodsThe interaction between COX-2 and CYP19A1 was first investigated on different MPM lines upon PGE2, and COX-2 inhibitor (rofecoxib) treatment by western blot, RT-PCR. The key regulatory pathways involved in the COX-2 and CYP19A1 axis were further studied in MPM cells, after rofecoxib and exemestane (CYP19A1 inhibitor) treatment in monotherapy and in combination, by cell cycle distribution, western blot, and combination index analysis. To explore the role of COX-2/CYP19A1 axis in 3D preclinical models of MPM cells, we analyzed the effect of combination of COX-2 and CYP19A1 inhibitors in mesosphere formation. Immunohistochemical analysis of MPM mesosphere and specimens was utilized to evaluate the involvement of COX-2 on the CYP19A1 activity and the relationship between E2 and COX-2.ResultsPGE2 or rofecoxib treatment caused in MPM cells an increased or decreased, respectively, CYP19A1 expression at mRNA and protein levels. The effect of rofecoxib and exemestane combination in MPM cell proliferation was synergistic. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for MPM cell lines. Increased expression levels of p53, p21, and p27, downregulation of cyclin D1 and inhibition of Akt activation (pAKT) were also found. The antagonistic effect of rofecoxib and exemestane combination found only in one cell line, was reverted by pretreatment with MK2206, a pAKT inhibitor, indicating pAKT as an actionable mediator in the COX-2-CYP19A1 axis. Reduction of size and sphere-forming efficiency in MPM spheres after treatment with both inhibitor and a decrease in COX-2 and E2 staining was found. Moreover, immunohistochemical analysis of 46 MPM samples showed a significant positive correlation between COX-2 and E2.ConclusionsCollectively, the results highlighted a novel COX-2/CYP19A1 axis in the pathogenesis of MPM that can be pharmacologically targeted, consequently opening up new therapeutic options.

Association of Genetically Predicted Serum Estradiol With Risk of Thromboembolism in Men: A Mendelian Randomization Study

ContextAn association was recently reported between genetic markers related to high testosterone and increased risk of thromboembolism in men, but a possible causal role of estradiol for risk of thromboembolism in men remains unknown.ObjectiveThis work aimed to determine whether endogenous estradiol has a causal role in thromboembolism in men.MethodsA 2-sample mendelian randomization study using gene-based genetic instruments assessed the association between endogenous estradiol genetically predicted by 22 variants in the aromatase CYP19A1 gene region and the risk of thromboembolism (5815 cases) in 170 593 unrelated men of White ancestry in the UK Biobank. The main outcome measure included thromboembolism based on self-reports, hospital episodes, and death.ResultsEndogenous estradiol genetically predicted by variants in the CYP19A1 gene region was inversely associated with the risk of thromboembolism (odds ratio per SD increase in estradiol 0.74; 95% CI, 0.62-0.90). In contrast, genetic variants in the JMJD1C gene, used as a predictor of high endogenous testosterone, were associated with an increased risk of thromboembolism (odds ratio per SD increase in testosterone 1.39; 95% CI, 1.12-1.72). Subsequent explorative analyses evaluating potential repercussions of thromboembolism revealed that endogenous estradiol genetically predicted by variants in the CYP19A1 gene region was inversely associated with the risk of ischemic stroke (0.68; 95% CI, 0.49-0.95) but not myocardial infarction (0.97; 95% CI, 0.84-1.13).ConclusionGenetically predicted estradiol was inversely associated with the risk of thromboembolism and ischemic stroke in men. The ratio between testosterone and estradiol, determined by CYP19A1 activity, may contribute to the overall impact of sex steroids on thromboembolism in men.

Alteration of the steroidogenesis in boys with autism spectrum disorders

The etiology of autism spectrum disorders (ASD) remains unknown, but associations between prenatal hormonal changes and ASD risk were found. The consequences of these changes on the steroidogenesis during a postnatal development are not yet well known. The aim of this study was to analyze the steroid metabolic pathway in prepubertal ASD and neurotypical boys. Plasma samples were collected from 62 prepubertal ASD boys and 24 age and sex-matched controls (CTRL). Eighty-two biomarkers of steroidogenesis were detected using gas-chromatography tandem-mass spectrometry. We observed changes across the whole alternative backdoor pathway of androgens synthesis toward lower level in ASD group. Our data indicate suppressed production of pregnenolone sulfate at augmented activities of CYP17A1 and SULT2A1 and reduced HSD3B2 activity in ASD group which is partly consistent with the results reported in older children, in whom the adrenal zona reticularis significantly influences the steroid levels. Furthermore, we detected the suppressed activity of CYP7B1 enzyme readily metabolizing the precursors of sex hormones on one hand but increased anti-glucocorticoid effect of 7α-hydroxy-DHEA via competition with cortisone for HSD11B1 on the other. The multivariate model found significant correlations between behavioral indices and circulating steroids. From dependent variables, the best correlation was found for the social interaction (28.5%). Observed changes give a space for their utilization as biomarkers while reveal the etiopathogenesis of ASD. The aforementioned data indicate a direction of the future research with a focus on the expression and functioning of genes associated with important steroidogenic enzymes in ASD patients from early childhood to adrenarche.

&lt;i&gt;In silico&lt;/i&gt; analysis of the phosphorylation effect on the structure of the human sterol-hydroxylases CYP17A1 AND CYP19A1

The trajectories of molecular dynamics simulation of phosphorylated S258 (CYP17A1), T162 and Y361 (CYP19A1) were analyzed to understand a possible mechanism of influence of post-translational modification (PTM) on the structure and functions of human sterol-hydroxylases CYP17A1 and CYP19A1. It was found that PTM has no dramatic influence on the structures of the enzymes but stabilizes them. According to our data, the phosphorylation of S258, T162 and Y361 influences the interface of interaction between human sterol-hydroxylases and the corresponding electron donors by decreasing the mobility of amino acids that take part in forming molecular complexes of the enzymes and the corresponding redox-partners. The phosphorylation of T162 (CYP19A1) decreases the mobility of amino acids forming access channel. The obtained results can shed light on the mechanism of fast regulation of human CYP17A1 and CYP19A1 activity by PTM.

Insulin-like growth factors 1 and 2 regulate gene expression and enzymatic activity of cyp17a1 in ovarian follicles of the yellowtail, Seriola quinqueradiata

There is accumulating evidence that insulin-like growth factors (IGFs), primary mediators of somatic growth, play an important role in fish reproduction. Previously, we showed that IGF-1 and IGF-2 are expressed in the ovarian follicle cells of the yellowtail (Seriola quinqueradiata) during the vitellogenic phase, suggesting that IGFs may be involved in ovarian steroidogenesis. In this study, we examined the effects of IGF-1 and IGF-2 on gene expression and activity of steroidogenic enzymes in yellowtail ovary in vitro. IGF-1 and IGF-2 had no effect on mRNA levels of several steroidogenesis-related genes (star, cyp11a1, hsd3b, cyp17a2, and cyp19a1). However, both IGFs enhanced the transcription of cyp17a1 in vitellogenic ovaries, although such up-regulation was not found in the ovary at the pre-vitellogenic stage. The stage-dependent effects of IGFs were correlated with changes in ovarian cyp17a1 mRNA levels during the reproductive cycle: transcript abundances increased in conjunction with ovarian development. In addition, IGF-induced cyp17a1 gene expression was significantly inhibited by wortmannin, suggesting that PI3 kinase plays an essential role in IGF-mediated ovarian steroidogenesis. Furthermore, IGF-1 and IGF-2 promoted the conversion of both progesterone and 17α-hydroxyprogesterone to androstenedione in vitellogenic ovaries, suggesting that both IGFs stimulated 17α-hydroxylase and C17-20 lyase activities. Taken together, these findings suggest that IGF-1 and IGF-2 act directly on follicle cells to stimulate steroid production through an increase in gene expression and enzymatic activity of cyp17a1 via induction of PI3 kinase.

Non-classic cytochrome P450 oxidoreductase deficiency strongly linked with menstrual cycle disorders and female infertility as primary manifestations.

Can cytochrome P450 oxidoreductase deficiency (PORD) be revealed in adult women with menstrual disorders and/or infertility? PORD was biologically and genetically confirmed in five adult women with chronically elevated serum progesterone (P) who were referred for oligo-/amenorrhea and/or infertility. PORD is an autosomal recessive disease typically diagnosed in neonates and children with ambiguous genitalia and/or skeletal abnormalities. It is responsible for the decreased activity of several P450 enzymes, including CYP21A2, CYP17A1 and CYP19A1, that are involved in adrenal and/or gonadal steroidogenesis. Little is known about the optimal way to investigate and treat patients with adult-onset PORD. In this series, we report five adult females who were evaluated in three tertiary endocrine reproductive departments between March 2015 and September 2018. Five women aged 19-38years were referred for unexplained oligo-/amenorrhea and/or infertility. Genetic testing excluded 21-hydroxylase deficiency (21OH-D), initially suspected due to the increased 17-hydroxyprogesterone (17-OHP) levels. Extensive phenotyping, steroid profiling by mass spectrometry, pelvic imaging and next-generation sequencing of 84 genes involved in gonadal and adrenal disorders were performed in all patients. IVF followed by frozen embryo transfer (ET) under glucocorticoid suppression therapy was performed for two patients. All patients had oligomenorrhea or amenorrhea. None had hyperandrogenism. Low-normal serum estradiol (E2) and testosterone levels contrasted with chronically increased serum P and 17-OHP levels, which further increased after adrenocorticotrophic hormone (ACTH) administration. Despite excessive P, 17OH-P and 21-deoxycortisol rise after ACTH stimulation suggesting non-classic 21OH-D, CYP21A2 sequencing did not support this hypothesis. Basal serum cortisol levels were low to normal, with inadequate response to ACTH in some women, suggesting partial adrenal insufficiency. All patients harbored rare biallelic POR mutations classified as pathogenic or likely pathogenic according to the American College of Medical Genetics and Genomics standards. Pelvic imaging revealed bilateral ovarian macrocysts in all women. IVF was performed for two women after retrieval of a normal oocyte number despite very low E2 levels during ovarian stimulation. Frozen ET under glucocorticoid suppression therapy led to successful pregnancies. The number of patients described here is limited and these data need to be confirmed on a larger number of women with non-classic PORD. The diagnosis of PORD must be considered in infertile women with chronically elevated P and 17OH-P levels and ovarian macrocysts. Differentiation of this entity from non-classic 21OH-D is important, as the multiple enzyme deficiency requires a specific management. Successful fertility induction is possible by IVF, providing that P levels be sufficiently suppressed by glucocorticoid therapy prior to implantation. No specific funding was used for this study. There are no potential conflicts of interest. N/A.

CYP11B and CYP17A1 in the Biosynthesis and Metabolism of C11‐oxy Steroids

Cytochrome P450 (CYP11B1) and cytochrome 17α‐hydroxylase (CYP17A1) play pivotal roles in the biosynthesis of adrenal glucocorticoids and androgens. It is generally accepted that CYP11B1 catalyses only the production of cortisol and corticosterone while CYP17A1 catalyses the hydroxylation of pregnenolone and progesterone at C17 and, in humans, the 17,20‐lyase of 17‐hydroxypregnenolone to produce DHEA. CYP11B1, however, also catalyses the biosynthesis of C11‐oxy C21 steroids, 11β‐hydroxyandrostenedione, 11β‐hydroxyprogesterone (11OHPROG) and 21‐deoxycortisol (21dF) in the adrenal [1, 2].We investigated the potential role of CYP17A1 in the metabolism of these C11‐oxy C21steroids leading to the production of 11keto‐dihydrotestosterone since 17‐hydroxyprogesterone has been shown to lead to the production of dihydrotestosterone in the backdoor pathway.The 17α‐hydroxylase/17,20‐lyase activity of CYP17A1 towards 11OHPROG and 21dF was assayed as well as the catalytic activity of the enzyme towards the downstream C11‐oxy C21steroid products. In vitro conversions were carried out with 1 μM steroid substrate in HEK293 and in LNCaP cells transiently expressing CYP17A1 and metabolites analysed using UPC2‐MS/MS. Both 11OHPROG and 11keto‐progesterone (11KPROG) were hydroxylated at C17 producing 21dF, 0.27 μM and 21‐deoxycortisone, 0.65 μM, respectively while no lyase activity was detected. It was only once reduced by 5α‐reductase and 3α‐hydroxysteroid dehydrogenase that the aforementioned steroids were also lyased to produce the C11‐oxy C19steroids, 11‐hydroxyandrosterone, 0.6 μM and 11‐ketoandrosterone, 0.43 μM. In LnCaP cells 11OHPROG and 11KPROG were metabolised to C11‐oxy C19steroids indicative of the C11‐oxy backdoor pathway. In addition, in HEK293 cells transiently transfected with 11β‐hydroxysteroid dehydrogenase (11βHSD) type 1 and 11βHSD2 the isozymes readily catalysed the interconversion of all the C11‐keto C21steroid intermediates produced in the pathway with the production of the keto intermediates being favoured.Although the C11‐oxy C21steroids produced in the adrenal may serve as ligands for the progesterone receptor they also potentially add to the pool of potent androgens via the C11‐oxy backdoor pathway. These C11‐oxy steroids would thus further contribute to the androgen pool in adrenal disorders and diseases characterised by androgen excess in which these steroids have been identified. We recently reported 11OHPROG, 11keto‐dihydroprogesterone and pregnanetriol in circulation in BPH patients [3]. The C11‐oxy C21steroids and C11‐oxy C19steroids, however, remain to be fully explored –not only in pathophysiology but also in sexual differentiation and development.Support or Funding InformationThe South African National Research Foundation (IFR170125217588, CSUR160414162143)

Clinical, Molecular, Functional, and Structural Characterization of CYP17A1 Mutations in Brazilian Patients with 17-Hydroxylase Deficiency.

17-Hydroxylase-deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The aim of the work was to study clinical, biochemical, and the follow up of 17OHD patients and evaluate the function and structure of CYP17A1 mutations. Brazilian patients (three 46, XX and four 46, XY; 17±1.9 years) with combined 17-hydroxylase/17,20-lyase deficiency were evaluated. CYP17A1 gene was sequenced. Functional analysis was performed transfecting COS7 cells, which were exposed to progesterone or 17α-hydroxypregnolone substrates. Hormones were determined by RIA or LC-MS/MS. Three-dimensional structural modeling was performed by Modeller software. All patients presented prepubertal female external genitalia, primary amenorrhea, hypergonadotrophic hypogonadism, hypokalemic hypertension, decreased cortisol, and increased ACTH and corticosterone levels. Five patients presented previously described mutations: p.W406R/p.W406R, IVS2-2A>C/p.P428L, and p.P428L/p.P428L. Two patients presented the compound heterozygous p.G478S/p.I223Nfs*10 mutations, whose CYP17A1 activity and the three dimensional structural modeling are originally studied in this paper. CYP17A1 activity of p.G478S was 13 and 58% against progesterone and 17-hydroxypregnenolone, respectively. The p.I223Nfs*10 caused a truncated inactive protein. Three-dimensional p.G478S structural modeling showed different internal hydrophobic interaction with W313 and created an additional chain side contact with L476 residue. Due to the rarity of 17OHD, the long term follow up (15.3±3.1 years) of our patients will help endocrinologists on the management of patients with 17OHD. The mutation p.G478S/pI223Nfs*10 led to severe 17OHD and impaired CYP17A1 structure and function. The integration of in silico and in vitro analysis showed how the amino acid changes affected the CYP17A1 activity and contributed to clarify the molecular interactions of CYP17A1.