Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

Corticosteroid Biosynthesis Revisited: No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase.

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.

Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB-. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3ß-hydroxysteroid dehydrogenase (3ß-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3ß-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- had an opposite effect on 3ß-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11ß1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.

Effects of defined mixtures of POPs and endocrine disruptors on the steroid metabolome of the human H295R adrenocortical cell line.

The presence of environmental pollutants in our ecosystem may impose harmful health effects to wildlife and humans. Several of these toxic chemicals have a potential to interfere with the endocrine system. The adrenal cortex has been identified as the main target organ affected by endocrine disrupting chemicals. The aim of this work was to assess exposure effects of defined and environmentally relevant mixtures of chlorinated, brominated and perfluorinated chemicals on steroidogenesis, using the H295R adrenocortical cell line model in combination with a newly developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method. By using this approach, we could simultaneously analyze 19 of the steroids in the steroid biosynthesis pathway, revealing a deeper insight into possible disruption of steroidogenesis. Our results showed a noticeable down-regulation in steroid production when cells were exposed to the highest concentration of a mixture of brominated and fluorinated compounds (10,000-times human blood values). In contrast, up-regulation was observed with estrone under the same experimental condition, as well as with some other steroids when cells were exposed to a perfluorinated mixture (1000-times human blood values), and the mixture of chlorinated and fluorinated compounds. Interestingly, the low concentration of the perfluorinated mixture alone produced a significant, albeit small, down-regulation of pregnenolone, and the total mixture a similar effect on 17-hydroxypregnenolone. Other mixtures resulted in only slight deviations from the control. Indication of synergistic effects were noted when we used a statistical model to improve data interpretation. A potential for adverse outcomes of human exposures is indicated, pointing to the need for further investigation into these mixtures.

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse-chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography.

11-Oxygenated Androgens Are Biomarkers of Adrenal Volume and Testicular Adrenal Rest Tumors in 21-Hydroxylase Deficiency.

Context: Patients with 21-hydroxylase deficiency (21OHD) have long-term complications, resulting from poor disease control and/or glucocorticoid overtreatment. Lack of optimal biomarkers has made it challenging to tailor therapy and predict long-term outcomes. Objective: To identify biomarkers of disease control and long-term complications in 21OHD. Setting and Participants: Cross-sectional study of 114 patients (70 males), ages 2 to 67 years (median, 15 years), seen in a tertiary referral center. Methods: We correlated a mass-spectrometry panel of 23 steroids, obtained before first morning medication, with bone age advancement (children), adrenal volume (adults), testicular adrenal rest tumors (TART), hirsutism, menstrual disorders, and pituitary hormones. Results: Total adrenal volume correlated positively with 18 steroids, most prominently 21-deoxycortisol and four 11-oxygenated-C19 (11oxC19) steroids: 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11ketoA4), 11ß-hydroxytestosterone (11OHT), and 11-ketotestosterone (11ketoT) (r ≈ 0.7, P < 0.0001). Nine steroids were significantly higher (P ≤ 0.01) in males with TART compared with those without TART, including 11OHA4 (6.8-fold), 11OHT (4.9-fold), 11ketoT (3.6-fold), 11ketoA4 (3.3-fold), and pregnenolone sulfate (PregS; 4.8-fold). PregS (28.5-fold) and 17-hydroxypregnenolone sulfate (19-fold) levels were higher (P < 0.01) in postpubertal females with menstrual disorders. In males, testosterone levels correlated positively with all 11oxC19 steroids in Tanner stages 1 and 2 (r ≈ 0.7; P < 0.001) but negatively in Tanner stage 5 (r = -0.3 and P < 0.05 for 11ketoA4 and 11ketoT). In females, testosterone level correlated positively with all four 11oxC19 steroids across all Tanner stages (r ≈ 0.8; P < 0.0001). Conclusion: 11oxC19 steroids and PregS might serve as clinically useful biomarkers of disease control and long-term complications in 21OHD.

Unveiling the crucial intermediates in androgen production.

Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17-C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.

Adrenal and Ovarian Phenotype of a Tissue-Specific Urocortin 2-Overexpressing Mouse Model.

Urocortin 2 (UCN2) is a neuropeptide of the CRH family, involved in homeostatic mechanisms, the stress response, and control of anxiety. To elucidate the effects of UCN2 on steroidogenesis, we developed a mouse model that allows a Cre recombinase-determined conditional overexpression of UCN2 (UCN2-COE). In these mice SF1-Cre-driven overexpression of UCN2 was restricted to the adrenal glands, gonads, and parts of the hypothalamus. UCN2-COE animals of both sexes revealed significantly higher plasma UCN2 levels and significantly higher UCN2 expression levels in the adrenals and ovaries. In contrast, the baseline expression of UCN2 was already high in the testes of control mice with no further increase achievable in UCN2-COE animals. Adrenal steroidogenesis of UCN2-COE animals was investigated under baseline conditions, upon an ACTH stimulation test, and following a restraint stress test. A tendency toward lower expression of steroidogenic enzymes was detectable in UCN2-COE animals of both sexes with slight differences between males and females. A similar reduction in the expression levels of the final steps of ovarian steroidogenesis, accompanied by reduced plasma estradiol levels, was observed in female UCN2-COE animals. Thus, adrenal UCN2 overexpression resulted in down-regulation of adrenal steroidogenesis, suggesting a reduction in the stress response in the mouse (stress coping behavior). Similarly, UCN2 overexpression in the ovaries caused a decrease in steroidogenesis and reduction of follicles that had undergone ovulation. Nevertheless, this finding was not associated with reduced fertility.

The non-benzodiazepine anxiolytic drug etifoxine causes a rapid, receptor-independent stimulation of neurosteroid biosynthesis.

Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.

[The alternative androgen synthesis pathway in humans]. / Der alternative Androgensyntheseweg des Menschen.

During the last year, alternative androgen synthesis pathways have been discovered in humans. This review article highlights these new concepts of androgen synthesis.We performed a selective literature research using PubMed.After the discovery of a new androgen synthesis pathway in marsupials, this new path-way of androgen synthesis could be established in humans during the last year from two independent studies. One of them could demonstrate that two pathways of androgen synthesis are needed for male sexual differentiation in humans; the other study established that the new pathway is an important source of androgen synthesis in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Additionally, it has been shown that an alternative androgen synthesis pathway that bypasses testosterone drives castration resistant prostate cancer.New and alternative androgen path-ways occur in humans. Importantly, these path-ways remain cryptic for the clinician, because the androgen synthesis circumvents classical intermediates like dehydroepiandrosterone, androstenedione and testosterone.

Cortisol production in sheep is influenced by the functional expression of two cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) isoforms.

In commercial production systems, the full expression of the genetic potential of an animal is limited by its intrinsic and extrinsic environment. It is therefore necessary to include robustness as a breeding goal because robustness is defined as the ability of an animal to express a high production potential in a wide variety of environmental conditions. The ability of mammals to produce sufficient cortisol on stimulation of the hypothalamic-pituitary-adrenal (HPA) axis is vital in its adaptation to stress. The biosynthesis of cortisol is dependent on the enzymatic activity of the microsomal enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17). Two isoforms for sheep (Ovis aries) CYP17, previously identified in 2 independent studies, differ by 2 nucleotides, resulting in 2 AA differences (Ser210Gly and Tyr464Asn). The present study investigates the effect of these differences on cortisol production as a function of the HPA axis activity by comparing the catalytic activities of these isoforms. The activities of the CYP17 isoforms were compared by expressing the enzymes in vitro. The kinetic constants, Vmax and Km, which were determined for pregnenolone and progesterone (in the absence of cytochrome b(5)), showed no significant difference (P > 0.05) between the CYP17 isoforms. In contrast, a time course of the metabolism of pregnenolone, 17-hydroxypregnenolone, and progesterone, assayed in the presence and absence of ovine cytochrome b(5) overexpression, showed significant differences (P < 0.05) between the isoforms. Wild-type 1 CYP17 (WT1, GenBank accession number L40335) yielded more cortisol precursors than wild-type 2 (WT2, GenBank accession number AF251388). Site-directed mutagenesis indicated that a tyrosine residue at position 464 of WT1 increased the 17α-hydroxylation of progesterone compared with an asparagine residue at that position of WT2. In a subsequent insulin-induced hypoglycemic stress test, the presence of WT1 resulted in a greater cortisol output from the sheep adrenal than the presence of WT2, as homozygous WT1/WT1 sheep produced more cortisol than heterozygous WT1/WT2 sheep. The SNP located within the WT1 allele may therefore have a potential application in marker-assisted selection of sheep exhibiting a greater release of cortisol from the adrenal gland in response to stressors.

7α-Hydroxypregnenolone, a new key regulator of amphibian locomotion: discovery, progress and prospect.

Seasonally-breeding amphibians have served as excellent animal models to investigate the biosynthesis and biological actions of neurosteroids. Previous studies have demonstrated that the brain of amphibians possesses key steroidogenic enzymes and produces pregnenolone, a precursor of steroid hormones, and other various neurosteroids. We recently found that the brain of seasonally-breeding newts actively produces 7α-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. This novel amphibian neurosteroid acts as a neuronal modulator to stimulate locomotor activity in newts. Subsequently, the mode of action of 7α-hydroxypregnenolone has been demonstrated in the newt brain. 7α-Hydroxypregnenolone stimulates locomotor activity through activation of the dopaminergic system. To understand the functional significance of 7α-hydroxypregnenolone in the regulation of locomotor activity, diurnal and seasonal changes in synthesis of 7α-hydroxypregnenolone have also been demonstrated in the newt brain. Melatonin derived from the pineal gland and eyes regulates 7α-hydroxypregnenolone synthesis in the brain, thus inducing diurnal locomotor changes. Prolactin, an adenohypophyseal hormone, regulates 7α-hydroxypregnenolone synthesis in the brain, and also induces seasonal locomotor changes. In addition, 7α-hydroxypregnenolone mediates corticosterone action to increase locomotor activity under stress. This review summarizes the discovery, progress and prospect of 7α-hydroxypregnenolone, a new key regulator of amphibian locomotion.

Clinicopathological features, biochemical and molecular markers in 5 patients with adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a very rare malignant tumor with poor prognosis. To gain insight into the pathogenic significance of ACC, we studied clinicopathological features and gene expression profile in ACC. We analyzed five ACC cases (two men and three women) with the median age of 45-year-old who underwent adrenalectomy at our institute. Endocrine studies revealed that two cases had subclinical Cushing's syndrome (SCS) and one with concomitant estrogen-secreting tumor, while the rest of three cases had non-functioning tumors. Analysis of urinary steroids profile by gas chromatography/mass spectrometry showed increased metabolites of corticosteroid precursors, such as 17-OH pregnenolone, 17-OH progesterone, dehydroepiandorosterone (DHEA), and 11-deoxycortisol in all five cases. The pathological diagnosis of ACC was based on Weiss's criteria with its score ≥ 3. The mean size of the resected tumors was 87 mm and Ki67/MIB1 labeling index, a proliferative marker, was 3-27%. Immunohistochemical analysis revealed a disorganized expression of several steroidogenic enzymes, such as 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase, and DHEA-sulfotransferase. Among several genes determined by RT-PCR, insulin-like growth factor (IGF)-II mRNA was consistently and abundantly expressed in all 5 tumor tissues. Postoperatively, two cases with SCS developed local recurrence and liver metastasis. The present study suggests that the disorganized expression of steroidogenic enzymes and the overexpression of IGF-II by the tumor are hallmarks of ACC, which could be used as biochemical and molecular markers for ACC.

Simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by semi-micro high-performance liquid chromatography with an immobilized cholesterol oxidase as a pre-column reactor: application to bovine adrenal fasciculata cells.

A method for the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by high-performance liquid chromatography with an immobilized cholesterol oxidation enzyme reactor was developed. Pregnenolone and 17α-hydroxypregnenolone were converted to progesterone and 17α-hydroxyprogesterone, respectively, by the immobilized enzyme packed into the reactor column, and could thus be monitored by UV absorption at 240 nm. The calibration curves for pregnenolone and 17α-hydroxypregnenolone were linear in the range of 0.4-10 and 0.3-10 µg/ml with a correlation coefficient of 0.9993 and 0.9998, respectively. The detection limit at a signal-to-noise ratio of 3 was 0.12 and 0.08 µg/ml for pregnenolone and 17α-hydroxypregnenolone, respectively. The conversion rate of pregnenolone to progesterone and 17α-hydroxypregnenolone to 17α-hydroxyprogesterone was 90.6% and 99.3%, respectively. Intra-day and inter-day precision (in terms of percentage coefficient of variation) were less than 9.3%, with accuracy greater than 94.8%. This method was successfully applied to the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone secreted into the culture medium of bovine adrenal fasciculata cells and of both analytes produced within the cells.

Prolactin increases the synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male Newts.

We recently found that the Japanese red-bellied newt, Cynops pyrrhogaster, actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. 7alpha-Hydroxypregnenolone stimulates locomotor activity of male newts. Locomotor activity of male newts increases during the breeding period as in other wild animals, but the molecular mechanism for such a change in locomotor activity is poorly understood. Here we show that the adenohypophyseal hormone prolactin (PRL) stimulates 7alpha-hydroxypregnenolone synthesis in the brain, thus increasing locomotor activity of breeding male newts. In this study, cytochrome P450(7alpha) (CYP7B), a steroidogenic enzyme catalyzing the formation of 7alpha-hydroxypregnenolone, was first identified to analyze seasonal changes in 7alpha-hydroxypregnenolone synthesis. Only males exhibited marked seasonal changes in 7alpha-hydroxypregnenolone synthesis and CYP7B expression in the brain, with a maximum level in the spring breeding period when locomotor activity of males increases. Subsequently we identified PRL as a key component of the mechanism regulating 7alpha-hydroxypregnenolone synthesis. Hypophysectomy decreased 7alpha-hydroxypregnenolone synthesis in the male brain, whereas administration of PRL but not gonadotropins to hypophysectomized males caused a dose-dependent increase in 7alpha-hydroxypregnenolone synthesis. To analyze the mode of PRL action, CYP7B and the receptor for PRL were localized in the male brain. PRL receptor was expressed in the neurons expressing CYP7B in the magnocellular preoptic nucleus. Thus, PRL appears to act directly on neurosteroidogenic magnocellular preoptic nucleus neurons to regulate 7alpha-hydroxypregnenolone synthesis, thus inducing seasonal locomotor changes in male newts. This is the first report describing the regulation of neurosteroidogenesis in the brain by an adenohypophyseal hormone in any vertebrate.

7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms.

Melatonin regulates diurnal changes in locomotor activity in vertebrates, but the molecular mechanism for this neurohormonal regulation of behavior is poorly understood. Here we show that 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid, mediates melatonin action on diurnal locomotor rhythms in quail. In this study, we first identified 7alpha-hydroxypregnenolone and its stereoisomer 7beta-hydroxypregnenolone in quail brain. These neurosteroids have not been described previously in avian brain. We then demonstrated that 7alpha-hydroxypregnenolone acutely increased quail locomotor activity. To analyze the production of 7alpha-hydroxypregnenolone, cytochrome P450(7alpha), a steroidogenic enzyme of this neurosteroid, was also identified. Subsequently, we demonstrated diurnal changes in 7alpha-hydroxypregnenolone synthesis in quail. 7Alpha-Hydroxypregnenolone synthesis and locomotor activity in males were much higher than in females. This is the first demonstration in any vertebrate of a clear sex difference in neurosteroid synthesis. This sex difference in 7alpha-hydroxypregnenolone synthesis corresponded to the sex difference in locomotion. We show that only males exhibited marked diurnal changes in 7alpha-hydroxypregnenolone synthesis, and these changes occurred in parallel with changes in locomotor activity. Finally, we identified melatonin as a key component of the mechanism regulating 7alpha-hydroxypregnenolone synthesis. Increased synthesis of 7alpha-hydroxypregnenolone occurred in males in vivo after melatonin removal via pinealectomy and orbital enucleation (Px plus Ex). Conversely, decreased synthesis of this neurosteroid occurred after melatonin administration to Px plus Ex males. This study demonstrates that melatonin regulates synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, thus inducing diurnal locomotor changes in male birds. This is a previously undescribed role for melatonin.

Structure-activity relationships of a series of analogs of the endozepine octadecaneuropeptide (ODN(11)(-)(18)) on neurosteroid biosynthesis by hypothalamic explants.

We have previously shown that the endozepine octadecaneuropeptide (ODN) stimulates the biosynthesis of neurosteroids from frog hypothalamic explants. In the present study, we have investigated the structure-activity relationships of a series of analogs of the C-terminal octapeptide of ODN (OP) on neurosteroid formation. We found that OP and its cyclic analog cyclo1-8OP stimulate in a concentration-dependent manner the synthesis of various steroids including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and dehydroepiandrosterone. Deletion or Ala-substitution of the Arg1 or Pro2 residues of OP did not affect the activity of the peptide. In contrast, deletion or replacement of any of the amino acids of the C-terminal hexapeptide fragment totally abolished the effect of OP on neurosteroid biosynthesis. The present study indicates that the C-terminal hexapeptide of ODN/OP is the minimal sequence retaining full biological activity on steroid-producing neurons.

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17.

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme.

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.

Lunar synchronization of in vitro steroidogenesis in ovaries of the golden rabbitfish, Siganus guttatus (Bloch).

To assess the relationship between lunar cycle and steroidogenesis in the ovaries of the golden rabbitfish, Siganus guttatus, the intact follicles of oocytes were incubated in vitro with human chorionic gonadotropin (hCG) and seven steroid hormones, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), 17alpha-hydroxyprogesterone (17alpha-OHP), progesterone (P), cortisol, estradiol-17beta (E2) and testosterone, during the two lunar phases, the new moon (1 week before spawning) and the first lunar quarter (just before spawning). Around the new moon, germinal vesicle breakdown (GVBD) could not be induced by addition of hCG or any steroid hormones. Around the first lunar quarter, GVBD was induced by addition of hCG, DHP, 20beta-S, 17alpha-OHP, P, and cortisol. DHP was the most potent steroid hormone. When the intact follicles of oocytes were incubated with hCG in both lunar phases, the production of E2 and DHP measured by enzyme immunoassay decreased and increased significantly from the new moon to the first lunar quarter, respectively. These results suggest that the ovarian follicles produce E2 around the new moon and DHP around the first lunar quarter and that the production/conversion of the steroid hormones is under the influence of gonadotropin(s). The synchronous increase in ovarian activity supports the hypothesis that lunar periodicity is a major factor for the ovarian development of S. guttatus.