Phase-specific differential regulation of mechanical allodynia in a murine model of neuropathic pain by progesterone.

Progesterone has been shown to have neuroprotective capabilities against a wide range of nervous system injuries, however there are negative clinical studies that have failed to demonstrate positive effects of progesterone therapy. Specifically, we looked into whether progesterone receptors or its metabolizing enzymes, cytochrome P450c17 and 5α-reductase, are involved in the effects of progesterone on neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve in mice. Intrathecal progesterone administration during the induction phase of chronic pain enhanced mechanical allodynia development and spinal glial fibrillary acidic protein (GFAP) expression, and this enhancement was inhibited by administration of ketoconazole, a P450c17 inhibitor, but not finasteride, a 5α-reductase inhibitor. Furthermore, phospho-serine levels of P450c17 in the spinal cord were elevated on day 1 after CCI operation, but not on day 17. In contrast, intrathecal progesterone administration during the maintenance phase of chronic pain decreased the acquired pain and elevated GFAP expression; this inhibition was restored by finasteride administration, but not by ketoconazole. The modification of mechanical allodynia brought on by progesterone in CCI mice was unaffected by the administration of mifepristone, a progesterone receptor antagonist. Collectively, these findings imply that progesterone suppresses spinal astrocyte activation via 5α-reductase activity during the maintenance phase of chronic pain and has an analgesic impact on the mechanical allodynia associated with the growing neuropathy. Progesterone, however, stimulates spinal astrocytes during the induction stage of peripheral neuropathy and boosts the allodynic impact caused by CCI through early spinal P450c17 activation.

17α-hydroxylase/17,20 carbon chain lyase deficiency caused by p.Tyr329fs homozygous mutation: Three case reports.

BACKGROUND: p.Tyr329fs is a cytochrome P450c17 mutation among Chinese individuals. However, data on 17-α-hydroxylase deficiency caused by cytochrome P450c17 p.Tyr329fs homozygous mutation are lacking. This paper is a case report of three patients homozygous for p.Tyr329fs who were diagnosed with 17-α-hydroxylase deficiency between 2005 and 2019. CASE SUMMARY: Case 1 presented with hypertension, hypokalemia, sexual infantilism and delayed bone age. The patient had a 46, XY karyotype, was homozygous for p.Tyr329fs and was recently treated with dexamethasone 0.375 mg qn. Case 2 presented with hypokalemia, sexual infantilism, osteoporosis and delayed bone age. The patient had a 46, XY karyotype, was homozygous for p.Tyr329fs and was treated with dexamethasone 0.75 mg qn at the last follow-up. Serum potassium and blood pressure could be maintained within normal range for cases 1 and 2. Case 3 presented with amenorrhea, sexual infantilism, osteopenia and delayed bone age. The patient had a 46, XX karyotype, was homozygous for p.Tyr329fs and was treated with dexamethasone 0.75 mg qn and progynova 1 mg qd. Outpatient follow-up revealed an adrenocorticotropic hormone (8 AM) of < 5.00 pg/mL. CONCLUSION: The homozygous p.Tyr329fs mutation usually manifests as a combined deficiency, and definitive diagnosis depends primarily on genetic testing.

miR-142-5p/DAX1-dependent regulation of P450c17 contributes to triclosan-mediated testosterone suppression.

Triclosan (TCS) is a potent antibacterial and antifungal compound that is extensively used in various daily products. TCS is also considered as an underlying endocrine disruptor and has anti-androgenic effects. In our previous work, we found that TCS suppressed testicular steroidogenesis via the miR-6321/JNK/Nur77 cascade, but roles of the abnormal expression of miR-142-5p and P450c17 in this molecular event were still unknown. Therefore, to verify the hypothesis that miR-142-5p and P450c17 might significantly function in other manner in testosterone decline after TCS exposure, Sprague-Dawley rats and the rat Leydig cell line were used in this study. Results showed that after TCS exposure, testicular histomorphology was abnormally changed and testosterone level was declined. Overexpressed miR-142-5p by TCS directly targeted the JAK1/STAT1 pathway. Bidirectional Co-IP assays and the use of STAT1 activator demonstrated that STAT1 could interact with and regulate Sp1. The activity, mRNA level, and protein expression of DNMT1 and DNMT3ß were all decreased after TCS treatment. Sp1 silencing, ChIP, and qPCR assays showed that Sp1 regulated DNMT1 expressions by directly binding to the promoter region of DNMT1. Though the DNA methylation status of the DAX1 promoter was not affected, TCS induced the transcription and translation of DAX1 by DNMT1, in turn leading to the inhibition of steroidogenic P450c17. Taken together, TCS-induced miR-142-5p inhibits P450c17 by the JAK1/STAT1 pathway and downstream Sp1/DNMT1/DAX1 cascade, finally facilitating the decrease in testosterone levels.

CYP17A1 exhibits 17αhydroxylase/17,20-lyase activity towards 11ß-hydroxyprogesterone and 11-ketoprogesterone metabolites in the C11-oxy backdoor pathway.

Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) plays a pivotal role in the regulation of adrenal and gonadal steroid hormone biosynthesis. More recent studies highlighted the enzyme's role in the backdoor pathway leading to androgen production. Increased CYP17A1 activity in endocrine disorders and diseases are associated with elevated C21 and C19 steroids which include 17α-hydroxyprogesterone and androgens, as well as C11-oxy C21 and C11-oxy C19 steroids. We previously reported that 11ß-hydroxyprogesterone (11OHP4), 21-deoxycortisol (21dF) and their keto derivatives are converted by 5α-reductases and hydroxysteroid dehydrogenases yielding C19 steroids in the backdoor pathway. In this study the 17α-hydroxylase and 17,20-lyase activity of CYP17A1 towards the unconventional C11-oxy C21 steroid substrates and their 5α- and 3α,5α-reduced metabolites was investigated in transfected HEK-293 cells. CYP17A1 catalysed the 17α-hydroxylation of 11OHP4 to 21dF and 11-ketoprogesterone (11KP4) to 21-deoxycortisone (21dE) with negligible hydroxylation of their 5α-reduced metabolites while no lyase activity was detected. The 3α,5α-reduced C11-oxy C21 steroids-5α-pregnan-3α,11ß-diol-20-one (3,11diOH-DHP4) and 5α-pregnan-3α-ol-11,20-dione (alfaxalone) were rapidly hydroxylated to 5α-pregnan-3α,11ß,17α-triol-20-one (11OH-Pdiol) and 5α-pregnan-3α,17α-diol-11,20-dione (11K-Pdiol), with the lyase activity subsequently catalysing to conversion to the C11-oxy C19 steroids, 11ß-hydroxyandrosterone and 11-ketoandrosterone, respectively. Docking of 11OHP4, 11KP4 and the 5α-reduced metabolites, 5α-pregnan-11ß-ol-3,20-dione (11OH-DHP4) and 5α-pregnan-3,11,20-trione (11K-DHP4) with human CYP17A1 showed minimal changes in the orientation of these C11-oxy C21 steroids in the active pocket when compared with the binding of progesterone suggesting the 17,20-lyase is impaired by the C11-hydroxyl and keto moieties. The structurally similar 3,11diOH-DHP4 and alfaxalone showed a greater distance between C17 and the heme group compared to the natural substrate, 17α-hydroxypregnenolone potentially allowing more orientational freedom and facilitating the conversion of the C11-oxy C21 to C11-oxy C19 steroids. In summary, our in vitro assays showed that while CYP17A1 readily hydroxylated 11OHP4 and 11KP4, the enzyme was unable to catalyse the 17,20-lyase reaction of these C11-oxy C21 steroid products. Although CYP17A1 exhibited no catalytic activity towards the 5α-reduced intermediates, once the C4-C5 double bond and the keto group at C3 were reduced, both the hydroxylation and lyase reactions proceeded efficiently. These findings show that the C11-oxy C21 steroids could potentially contribute to the androgen pool in tissue expressing steroidogenic enzymes in the backdoor pathway.

The 11ß-hydroxyandrostenedione pathway and C11-oxy C21 backdoor pathway are active in benign prostatic hyperplasia yielding 11keto-testosterone and 11keto-progesterone.

In clinical approaches to benign prostatic hyperplasia (BPH) and prostate cancer (PCa), steroidogenesis or the disruption thereof is the main thrust in treatments restricting active androgen production. Extensive studies have been undertaken focusing on testosterone and dihydrotestosterone (DHT). However, the adrenal C11-oxy C19 steroid, 11ß-hydroxyandrostenedione (11OHA4), also contributes to the active androgen pool in the prostate microenvironment, and while it has been shown to impact castration resistant prostate cancer, the C11-oxy C19 steroids together with the C11-oxy C21 steroids have not been studied in BPH. The study firstly investigated the metabolism of these adrenal steroids in the BPH-1 model. Comprehensive profiles identified 11keto-testosterone as the predominant active androgen in the metabolism of the C11-oxy C19 steroids, and we identified, for the first time, 11ß-hydroxy-5α-androstane-3α,17ß-diol, a novel steroid in the 11OHA4-pathway. Analysis of the inactivation and reactivation of the metabolites showed that DHT is more readily inactivated than 11keto-dihydrotestosterone (11KDHT). The conversion of 11ß-hydroxyprogesterone (11ßOHPROG) yielded 11keto-progesterone (11KPROG), while the latter yielded 11keto-dihydroprogesterone (11KDHPROG). BPH tissue analysis identified high levels of 11ß-hydroxyandrosterone (4-14 ng/g) and 11keto-androsterone (9-160 ng/g), together with androstenedione (A4; ∼7.5 ng/g). The major C11-oxy C21 steroids detected were 11ßOHPROG (∼46 ng/g), 11KPROG (∼130 ng/g) as well as 11KDHPROG (∼282 ng/g). While circulatory 11ßOHPROG was detected below the limit of quantification, 11KPROG and 11KDHPROG were detected at 6 and 8.5 nmol/L, respectively. Glucuronide derivatives of both 11KPROG and pregnanetriol were also detected. 11OHA4 was the major free androgen in circulation at 85.9 nmol/L, ±12-fold higher than A4, together with 5α-androstane-3α,17ß-diol quantified at 69.3 nmol/L. Circulatory C11-oxy C19 steroids levels were also significantly higher (8-fold) than the C11-oxy C21 steroid levels, while the former were similar to the C19 steroid levels, in contrast to levels in PCa. The study highlights the contribution of adrenal C11-oxy steroids to the androgen pool in BPH underscoring their limited reactivation and elimination, and significant inter-individual variations regarding steroid levels and conjugation. Targeted steroid metabolome analysis is critical to understanding prostate steroidogenesis and disease progression, and analysis of circulatory C11-oxy C19 and C11-oxy C21 steroids, together with intraprostatic levels, add to our current understanding of BPH.

Spinal Nitric Oxide Synthase Type II Increases Neurosteroid-metabolizing Cytochrome P450c17 Expression in a Rodent Model of Neuropathic Pain.

We have previously demonstrated that the neurosteroid dehydroepiandrosterone sulfate (DHEAS) induces functional potentiation of N-methyl-D-aspartate (NMDA) receptors via increases in phosphorylation of NMDA receptor GluN1 subunit (pGluN1). However, the modulatory mechanisms responsible for the expression of the DHEA-synthesizing enzyme, cytochrome P450c17 following peripheral nerve injury have yet to be examined. Here we determined whether oxidative stress induced by the spinal activation of nitric oxide synthase type II (NOS-II) modulates the expression of P450c17 and whether this process contributes to the development of neuropathic pain in rats. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of NOS-II in microglial cells and NO levels in the lumbar spinal cord dorsal horn at postoperative day 5. Intrathecal administration of the NOS-II inhibitor, L-NIL during the induction phase of neuropathic pain (postoperative days 0~5) significantly reduced the CCI-induced development of mechanical allodynia and thermal hyperalgesia. Sciatic nerve injury increased the expression of PKCand PKA-dependent pGluN1 as well as the mRNA and protein levels of P450c17 in the spinal cord at postoperative day 5, and these increases were suppressed by repeated administration of L-NIL. Co-administration of DHEAS together with L-NIL restored the development of neuropathic pain and pGluN1 that were originally inhibited by L-NIL administration alone. Collectively these results provide strong support for the hypothesis that activation of NOS-II increases the mRNA and protein levels of P450c17 in the spinal cord, ultimately leading to the development of central sensitization and neuropathic pain induced by peripheral nerve injury.

Inhibition of cytochrome P450c17 reduces spinal astrocyte activation in a mouse model of neuropathic pain via regulation of p38 MAPK phosphorylation.

We have recently demonstrated that the neurosteroid-metabolizing enzyme, cytochrome P450c17 is increased in spinal astrocytes contributing to the development of mechanical allodynia in chronic constriction injury (CCI)-induced neuropathic mice. However, the mechanisms by which spinal P450c17 modulates pathological changes in astrocytes remain unclear. In this study we investigated whether P450c17 modulates astrocyte activation and whether this process is mediated by spinal p38 mitogen-activated protein kinase phosphorylation ultimately leading to the development of mechanical allodynia in CCI mice. Sciatic nerve injury induced a significant increase in glial fibrillary acidic protein (GFAP) expression in the superficial dorsal horn (SDH, laminae I-II) and nucleus proprius (NP, laminae III-IV) regions of the spinal cord dorsal horn. Repeated daily (from days 0-3 post-surgery) intrathecal administration of the P450c17 inhibitor, ketoconazole (10 nmol) significantly inhibited the CCI-induced increase in GFAP-immunoreactivity, but had no effect on the CCI-induced increase in Iba-1-immunoreactivity. In addition, intrathecal administration of ketoconazole significantly inhibited the CCI-induced increase in p38 phosphorylation, while the levels of ERK and JNK phosphorylation remained unchanged. The CCI-induced development of mechanical allodynia was attenuated by administration of either ketoconazole (10 nmol) or the p38 MAPK inhibitor, SB203580 (5 nmol). Administration of a sub-effective dose of SB203580 (0.5 nmol) potentiated the pharmacological effect of ketoconazole (1 nmol) on spinal GFAP-immunostaining, as well as, the development of mechanical allodynia following CCI. Collectively these data suggest that spinal P450c17 activates astrocytes via p38 phosphorylation, ultimately leading to the development of mechanical allodynia in a model of peripheral neuropathy.

Spinal Interleukin-1ß Inhibits Astrocyte Cytochrome P450c17 Expression Which Controls the Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain.

We have recently demonstrated that sciatic nerve injury increases the expression of spinal cytochrome P450c17, a key neurosteroidogenic enzyme, which plays a critical role in the development of peripheral neuropathic pain. However, the modulatory mechanisms responsible for the expression of spinal P450c17 have yet to be examined. Here we investigated the possible involvement of interleukin-1ß (IL-1ß) in altering P450c17 expression during the induction phase of neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in mice and mechanical allodynia was evaluated in the hind paws using a von-Frey filament (0.16 g). Western blotting and immunohistochemistry were performed to assess the expression of spinal IL-1ß, interleukin-1 receptor type 1 (IL-1R1), P450c17, and GFAP. Spinal IL-1ß was significantly increased on day 1 post-surgery and its receptor, IL-1R1 was expressed in GFAP-positive astrocytes. Intrathecal administration of the recombinant interleukin-1 receptor antagonist (IL-1ra, 20 ng) on days 0 and 1 post-surgery enhanced GFAP expression on day 1 post-surgery and induced an early increase in P450c17 expression in astrocytes, but not in neurons. Administration of IL-1ß (10 ng) on days 0 and 1 post-surgery blocked the enhancement of both spinal P450c17 and GFAP expression induced by IL-1ra (20 ng) administration. Intrathecal administration of IL-1ra (20 ng) on days 0 to 3 post-surgery also facilitated the CCI-induced development of mechanical allodynia, and this early developed pain was dose-dependently attenuated by the administration of the P450c17 inhibitor, ketoconazole (1, 3, or 10 nmol) or the astrocyte metabolic inhibitor, fluorocitrate (0.01, 0.03, or 0.1 nmol). These results demonstrate that early increases in spinal IL-1ß temporally inhibit astrocyte P450c17 expression and astrocyte activation ultimately controlling the development of mechanical allodynia induced by peripheral nerve injury. These findings imply that spinal IL-1ß plays an important role as an early, but transient, control mechanism in the development of peripheral neuropathic pain via the inhibition of astrocyte P450c17 expression and astrocyte activation.

11α-Hydroxyprogesterone, a potent 11ß-hydroxysteroid dehydrogenase inhibitor, is metabolised by steroid-5α-reductase and cytochrome P450 17α-hydroxylase/17,20-lyase to produce C11α-derivatives of 21-deoxycortisol and 11-hydroxyandrostenedione in vitro.

11α-Hydroxyprogesterone (11αOHP4) and 11ß-hydroxyprogesterone (11ßOHP4) have been reported to be inhibitors of 11ß-hydroxysteroid dehydrogenase (11ßHSD) type 2, together with 11ß-hydroxytestosterone and 11ß-hydroxyandrostenedione, and their C11-keto derivatives being inhibitors of 11ßHSD1. Our in vitro assays in transiently transfected HEK293 cells, however, show that 11αOHP4 is a potent inhibitor of 11ßHSD2 and while this steroid does not serve as a substrate for the enzyme, the aforementioned C11-oxy steroids are indeed substrates for both 11ßHSD isozymes. 11ßOHP4 is metabolised by 11ßHSD2 yielding 11-ketoprogesterone with 11ßHSD1 catalysing the reverse reaction, similar to the reduction of the other C11-oxy steroids. In the same model system, novel 11αOHP4 metabolites were detected in its conversion by steroid-5α-reductase (SRD5A) types 1 and 2 yielding 11α-hydroxydihydroprogesterone and its conversion by cytochrome P450 17A1 (CYP17A1) yielding the hydroxylase product, 11α,17α-dihydroxyprogesterone, and the 17,20 lyase product, 11α-hydroxyandrostenedione. We also detected both 11αOHP4 and 11ßOHP4 in prostate cancer tissue- ∼23 and ∼32 ng/g respectively with 11KP4 levels >300 ng/g. In vitro assays in PC3 and LNCaP prostate cancer cell models, showed that the metabolism of 11αOHP4 and 11ßOHP4 was comparable. In LNCaP cells expressing CYP17A1, 11αOHP4 and 11ßOHP4 were metabolised with negligible substrate, 4%, remaining after 48 h, while the steroid substrate 11ß,17α-dihydroxyprogesterone (21dF) was metabolised to C11-keto C19 steroids yielding 11-ketotestosterone. Despite the fact that 11αOHP4 is not metabolised by 11ßHSD2, it is a substrate for SRD5A and CYP17A1, yielding C11α-hydroxy C19 steroids as well as the C11α-hydroxy derivative of 21dF-the latter associated with clinical conditions characterised by androgen excess. With our data showing that 11αOHP4 is present at high levels in prostate cancer tissue, the steroid may serve as a precursor to unique C11α-hydroxy C19 steroids. The potential impact of 11αOHP4 and its metabolites on human pathophysiology can however only be fully assessed once C11α-hydroxyl metabolite levels are comprehensively analysed.

Spinal cytochrome P450c17 plays a key role in the development of neuropathic mechanical allodynia: Involvement of astrocyte sigma-1 receptors.

While evidence indicates that sigma-1 receptors (Sig-1Rs) play an important role in the induction of peripheral neuropathic pain, there is limited understanding of the role that the neurosteroidogenic enzymes, which produce Sig-1R endogenous ligands, play during the development of neuropathic pain. We examined whether sciatic nerve injury upregulates the neurosteroidogenic enzymes, cytochrome P450c17 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which modulate the expression and/or activation of Sig-1Rs leading to the development of peripheral neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of P450c17, but not 3ß-HSD, in the ipsilateral lumbar spinal cord dorsal horn at postoperative day 3. Intrathecal administration of the P450c17 inhibitor, ketoconazole during the induction phase of neuropathic pain (day 0 to day 3 post-surgery) significantly reduced the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw. However, administration of the 3ß-HSD inhibitor, trilostane had no effect on the development of neuropathic pain. Sciatic nerve injury increased astrocyte Sig-1R expression as well as dissociation of Sig-1Rs from BiP in the spinal cord. These increases were suppressed by administration of ketoconazole, but not by administration of trilostane. Co-administration of the Sig-1R agonist, PRE084 restored the development of mechanical allodynia originally suppressed by the ketoconazole administration. However, ketoconazole-induced inhibition of thermal hyperalgesia was not affected by co-administration of PRE084. Collectively these results demonstrate that early activation of P450c17 modulates the expression and activation of astrocyte Sig-1Rs, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.

Oxidative stress increases the 17,20-lyase-catalyzing activity of adrenal P450c17 through p38α in the development of hyperandrogenism.

Unexplained hyperandrogenic oligoanovulation is a main feature of polycystic ovary syndrome (PCOS). P450c17 phosphorylation selectively increases 17,20-lyase activity and androgen biosynthesis but minimally affects 17α-hydroxylase. Studies have recently identified mitogen-activated protein kinase 14 (MAPK14, p38α) as the kinase responsible for enhancing 17,20-lyase activity through P450c17 phosphorylation. We investigated whether oxidant-induced oxidative stress increases 17,20-lyase activity through oxidant-sensitive p38α signaling pathways. NCI-H295R adrenal cells were treated with three oxidants, palmitate, H2O2 and 4-hydroxy-2-nonenal (HNE), to simulate the excessive oxidative stress of PCOS. Oxidant exposure significantly induced dehydroepiandrosterone production and increased p38α phosphorylation and activation, but the effect on 17α-hydroxyprogesterone production was far less clear. None of the treatments altered the expression of P450c17 or its necessary factors POR and b5. LC-MS/MS revealed increased DHEA production in NCI-H295R cells. Both p38α inhibition and siRNA-mediated silencing attenuated H2O2- or 0.45-0.75 mM PA-mediated augmentation of DHEA production with relatively stable 17OHP levels, indicating that activated p38α mediates oxidative stress-induced 17,20-lyase activation and androgen synthesis stimulation, which may underlie hyperandrogenism in PCOS.

Mechanism of the Dual Activities of Human CYP17A1 and Binding to Anti-Prostate Cancer Drug Abiraterone Revealed by a Novel V366M Mutation Causing 17,20 Lyase Deficiency.

The CYP17A1 gene regulates sex steroid biosynthesis in humans through 17α-hydroxylase/17,20 lyase activities and is a target of anti-prostate cancer drug abiraterone. In a 46, XY patient with female external genitalia, together with a loss of function mutation S441P, we identified a novel missense mutation V366M at the catalytic center of CYP17A1 which preferentially impaired 17,20 lyase activity. Kinetic experiments with bacterially expressed proteins revealed that V366M mutant enzyme can bind and metabolize pregnenolone to 17OH-pregnenolone, but 17OH-pregnenolone binding and conversion to dehydroepiandrosterone (DHEA) was impaired, explaining the patient’s steroid profile. Abiraterone could not bind and inhibit the 17α-hydroxylase activity of the CYP17A1-V366M mutant. Molecular dynamics (MD) simulations showed that V366M creates a “one-way valve” and suggests a mechanism for dual activities of human CYP17A1 where, after the conversion of pregnenolone to 17OH-pregnenolone, the product exits the active site and re-enters for conversion to dehydroepiandrosterone. The V366M mutant also explained the effectiveness of the anti-prostate cancer drug abiraterone as a potent inhibitor of CYP17A1 by binding tightly at the active site in the WT enzyme. The V366M is the first human mutation to be described at the active site of CYP17A1 that causes isolated 17,20 lyase deficiency. Knowledge about the specificity of CYP17A1 activities is of importance for the development of treatments for polycystic ovary syndrome and inhibitors for prostate cancer therapy.

Seasonal expression of P450c17 and 5α-reductase-2 in the scented gland of male muskrats (Ondatra zibethicus).

Cytochrome P450 17A1 (P450c17) is the key enzyme required for the production of androgenic sex steroids by converting progestogens to androgens. 5α-reductases are enzymes that convert testosterone (T) to dihydrotestosterone (DHT), which has a greater affinity for androgen receptors (AR) and stronger action than T. Our previous studies revealed that the scented glands of male muskrats expressed AR during the breeding and nonbreeding seasons. To further seek evidence of the activities of androgens in scented glands, the expression patterns of P450c17 and 5α-reductase 2 were investigated in the scented glands of male muskrats during the breeding and nonbreeding seasons. The weight and size of scented glands in the breeding season were significantly higher than those of the nonbreeding season. Immunohistochemical data showed that P450c17 and 5α-reductase 2 were presented in the glandular cells and epithelial cells of scented glands in both the seasons. The protein and mRNA expression of P450c17 and 5α-reductase 2 were significantly higher in the scented gland during the breeding season than those during the nonbreeding season. In addition, the levels of DHT and T in the scented gland were remarkably higher during the breeding season. Taken together, these results suggested that the scented glands of male muskrats were capable of locally synthesizing T and DHT, and T and DHT might play an important role in the scented glandular function via an autocrine or paracrine manner.

Association of polycystic ovary syndrome with metabolic syndrome and gestational diabetes: Aggravated complication of pregnancy.

Polycystic ovary syndrome (PCOS) affects 5-20% of the reproductive age women globally. PCOS is diagnosed by the presence of hyperandrogenism, oligo-anovulation, and polycystic morphology of at least one ovary. Insulin resistance (IR), hyperinsulinemia and associated metabolic abnormalities including metabolic syndrome play a significant role in the development of PCOS. The chances of developing MS in PCOS women was shown to increase by almost 14-fold in patients with increasing body mass index. Even in the absence of overt obesity, a preferential deposition of intra-abdominal fat is noted in PCOS women and this intra-abdominal fat leads to impaired insulin action and functional IR and hyperandrogenism. Functional ovarian hyperandrogenism of ovaries was suggested to be a consequence of IR, which activates androgen synthesizing enzyme, cytochrome p450-c17α-hydroxylase, in ovarian theca cells and causes elevated oxidative stress accompanied by lower antioxidant status in ovaries, which contribute to PCOS pathogenesis. The elevated levels of luteinizing hormone that accompany the early stages of hyperandrogenemia, accelerate ovarian functional deterioration, which is further aggravated by hyperinsulinemia, in PCOS women. The risk of developing gestational diabetes in PCOS women is approximately three times greater, as compared to non-PCOS women, due to IR and hyperinsulinemia. Typical insulin-sensitizing drugs such as metformin, have been used to curtail IR and hyperinsulinemia in pregnant PCOS women, with varying results indicating the complexity of the disease and the need for better controlled studies and additional efforts for PCOS-specific drug discovery.

Effects of different dietary DHA:EPA ratios on gonadal steroidogenesis in the marine teleost, tongue sole (Cynoglossus semilaevis).

The present study was conducted to investigate the effects of dietary DHA and EPA on gonadal steroidogenesis in mature females and males, with a feeding trial on tongue sole, a typical marine teleost with sexual dimorphism. Three experimental diets differing basically in DHA:EPA ratio, that is, 0·68 (diet D:E-0·68), 1·09 (D:E-1·09) and 2·05 (D:E-2·05), were randomly assigned to nine tanks of 3-year-old tongue sole (ten females and fifteen males in each tank). The feeding trail lasted for 90 d before and during the spawning season. Fish were reared in a flowing seawater system and fed to apparent satiation twice daily. Compared with diet D:E-0·68, diet D:E-1·09 significantly enhanced the oestradiol production in females, whereas diet D:E-2·05 significantly enhanced the testosterone production in males. In ovaries, diet D:E-1·09 induced highest mRNA expression of follicle-stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein, 17α-hydroxylase (P450c17) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). In testes, diet 2·05 resulted in highest mRNA expression of FSHR, cholesterol side-chain cleavage enzyme, P450c17 and 3ß-HSD. Fatty acid profiles in fish tissues reflected closely those of diets. Female fish had more gonadal EPA content but less DHA content than male fish, whereas there was a reverse observation in liver. In conclusion, the dietary DHA:EPA ratio, possibly combined with the dietary EPA:arachidonic acid ratio, differentially regulated sex steroid hormone synthesis in mature female and male tongue soles. Females seemed to require more EPA but less DHA for the gonadal steroidogenesis than males. The results are beneficial to sex-specific nutritive strategies in domestic teleost.

Estrogen receptor-related receptor γ regulates testicular steroidogenesis through direct and indirect regulation of steroidogenic gene expression.

Biosynthesis of testosterone, which mainly occurs in testicular Leydig cells, is controlled by steroidogenic proteins, such as StAR and P450c17. Although estrogen-related receptor gamma (ERRγ), an orphan nuclear receptor, is expressed in the testis, its role is not well understood. In this study, we investigated the expression of ERRγ in Leydig cells and its molecular action on testicular steroidogenesis. ERRγ is expressed in mouse Leydig cells from pre-pubertal stages. ERRγ overexpression in primary Leydig cells elevated the production of testosterone with a marked increase of P450c17 expression at both mRNA and protein levels, albeit decreased expression of StAR. Promoter-reporter analyses showed that ERRγ directly regulated the P450c17 promoter. Further deletion mutant analyses of the P450c17 promoter revealed that ERRγ activated expression of the P450c17 gene by binding to an ERRγ response element within the P450c17 promoter. Meanwhile, ERRγ suppressed cAMP-induced activation of the StAR promoter, which was likely due to ERRγ-mediated inhibition of the transcriptional activity of Nur77, which is induced by cAMP and regulates StAR gene expression in Leydig cells. Interestingly, ERRγ coexpression also decreased the protein level of Nur77, which occurred through proteasomal degradation, suggesting ERRγ-mediated regulation of steroidogenesis at another level. Taken together, these findings suggest that ERRγ regulates testicular steroidogenesis, both directly controlling and indirectly fine-tuning the expression of steroidogenic genes.

Electrochemistry of cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17).

Within the superfamily of cytochrome P450 enzymes (P450s), there is a small class which is functionally employed for steroid biosynthesis. The enzymes in this class appear to have a small active site to accommodate the steroid substrates specifically and snuggly, prior to the redox transformation or hydroxylation to form a product. Cytochrome P450c17 is one of these and is also a multi-functional P450, with two activities, the first 17α-hydroxylation of pregnenolone is followed by a subsequent 17,20-lyase transformation to dehydroepiandrosterone (DHEA) as the dominant pathways to cortisol precursors or androgens in humans, respectively. How P450c17 regulates these two redox reactions is of special interest. There is a paucity of direct electrochemical studies on steroidogenic P450s, and in this mini-review we provide an overview of these studies with P450c17. Historical consideration as to the difficulties in obtaining reliable electrochemistry due to issues of handling proteins on an electrode, together with advances in the electrochemical techniques are addressed. Recent work using Fourier transformed alternating current voltammetry is highlighted as this technique can provide both catalytic information simultaneously with the underlying redox transfer with the P450 haem.

A Homodimer Model Can Resolve the Conundrum as to How Cytochrome P450 Oxidoreductase and Cytochrome b5 Compete for the Same Binding Site on Cytochrome P450c17.

Cytochrome P450 17α-hydroxylase, 17,20-lyase (P450c17) is a key enzyme in the synthesis of cortisol in the zona fascicula of the adrenal cortex, and the synthesis of androgen precursors in the adrenal zona reticularis and the gonads. Each of these reactions require electrons transferred by the electron donor cytochrome P450 oxidoreductase. The 17α-hydroxylation of its substrate occurs in all cells where P450c17 is expressed. Remarkably, a second, subsequent reaction, namely the 17,20-lyase activity, only occurs in the zona reticularis and gonads. The specificity of the second reaction is due to the interaction with the haem-protein cytochrome b5. Surprisingly, cytochrome b5 and cytochrome P450 oxidoreductase have overlapping sites of interaction on the surface of P450c17. This poses the question as to how cytochrome b5 and cytochrome P450 oxidoreductase interact with P450c17 structurally, functionally and physiologically? This conundrum can be resolved based on the observation that P450c17 can homo-dimerise. A homodimer would allow cytochrome P450 oxidoreductase to bind to one P450c17 monomer of the P450c17 homodimer whilst cytochrome b5 could bind to the other P450c17 monomer simultaneously at the surfaces distal to the dimer interface. This structure is likely to be dynamic in vivo. Our modelling predicts that the proteins can assemble as a stable tetramer and is fully consistent with extensive experimental data that have been published over the last two decades. Predictions derived from this structural model are currently being tested by a range of in vitro and in vivo experimental approaches.

Clinical, hormonal, ovarian, and genetic aspects of 46,XX patients with congenital adrenal hyperplasia due to CYP17A1 defects.

OBJECTIVE: To perform a clinical, biochemical, and molecular evaluation of patients with CYP17A1 defects, including ovarian imaging. DESIGN: Retrospective study. SETTING: Tertiary care center. PATIENT(S): Sixteen patients with congenital adrenal hyperplasia due to CYP17A1 defects with a median chronological age of 20 years and belonging to 10 unrelated families. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical and biochemical parameters, molecular diagnosis, ovarian imaging, and therapeutic management. RESULT(S): Seventy-one percent of patients presented with primary amenorrhea, 50% had no breast development, and pubic hair was absent or sparse in all patients; 88% had high blood pressure at diagnosis. Basal LH and P levels were high, and androgen levels were low in all patients. Ultrasound revealed ovarian enlargement in 68.7% and ovarian macrocysts in 62.5% of patients before treatment; three patients had a previous surgical correction of ovarian torsion or rupture. Molecular analysis revealed inactivating CYP17A1 mutations in all patients. The most prevalent mutation was p.W406R, and one patient bore a novel p.G478S/p.I223Nfs*10 compound heterozygous mutation. Treatment with dexamethasone, estrogen, and P resulted in reduction of ovarian volume. CONCLUSION(S): Amenorrhea, absent/sparse pubic hair, hypertension, and ovarian macrocysts, whichincrease the risk of ovarian torsion, are important elements in the diagnosis of 46,XX patients with CYP17A1 defects. High basal P levels in patients with hypergonadotropic hypogonadism point to the diagnosis of CYP17A1 defects. Fertility can be achieved in these patients with novel reproductive techniques.

The feto-placental unit, and potential roles of dehydroepiandrosterone (DHEA) in prenatal and postnatal brain development: A re-examination using the spiny mouse.

Synthesis of dehydroepiandrosterone (DHEA) by the fetal adrenal gland is important for placental oestrogen production, and may also be important for modulating the effects of glucocorticoids on the developing brain. We have preciously shown that the enzymes and accessory proteins needed for DHEA synthesis-cytochrome P450 enzyme 17α-hydroxylase/17,20 lyase (P450c17), cytochrome-b5 (Cytb5), 3ß-hydroxysteroid dehydrogenase (3ßHSD)-are expressed in the adrenal gland from 30 days gestation, and DHEA, cortisol and aldosterone are present in fetal plasma from this time. Explant culture of fetal adrenal tissue showed that the spiny mouse adrenal gland, can synthesize and secrete DHEA from at least 0.75 of gestation, and suggest that DHEA may have an important role(s) in placental biosynthesis of oestrogens and in modulating the actions of glucocorticoids in the developing brain in this species. Post-natally, increased immuno-expression of P450c17 and Cytb5 expression in the zona reticularis of the adrenal gland and a significant increase in the synthesis and secretion of DHEA in plasma from 8 to 20 days of age in the spiny mouse, are representative of a period of high adrenal androgen production consistent with the human phenomenon of adrenarche. The studies summarised in this review also show that DHEA is produced de novo in the developing brain of the spiny mouse. These results showed that the spiny mouse brain can indeed produce DHEA from pregnenolone in a time-dependant manner, and coupled with the identification of P450c17 and Cytb5 protein in several regions of the brain, support the idea that DHEA is an endogenous neuro-active steroid in this species. Together, the studies outlined in this review indicate that the androgen DHEA is an important hormone of adrenal and Central Nervous System (CNS) origin in the fetal and postnatal spiny mouse. Disturbance of the development of these fetal tissues, and/or of the relationship between the fetal adrenal gland and placenta during pregnancy, may have significant consequences for fetal development, placental function, and maturation of the brain. It is proposed that such disturbances of normal adrenal function could account for some of the neuropathologies that arise in juvenile and adult offspring following illness and stress experienced by the mother during pregnancy.