Regulation of human (adrenal) androgen biosynthesis-New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Relationships of dehydroepiandrosterone sulfate in the elderly with functional, psychological, and mental status, and short-term mortality: A French community-based study

In human beings of both sexes, dehydroepiandrosterone sulfate (DHEAS) circulating in blood is mostly an adrenally secreted steroid whose serum concentration (in the micromolar range and 30–50% higher in men than in women) decreases with age, toward ≈20–10% of its value in young adults during the 8th and 9th decades. The mechanism of action of DHEA and DHEAS is poorly known and may include partial transformation into sex steroids, increase of bioavailable insulin-like growth factor I, and effects on neurotransmitter receptors. Whether there is a cause-to-effect relationship between the decreasing levels of DHEAS with age and physiological and pathological manifestations of aging is still undecided, but this is of obvious theoretical and practical interest in view of the easy restoration by DHEA administration. Here we report on 622 subjects over 65 years of age, studied for the 4 years since DHEAS baseline values had been obtained, in the frame of the PAQUID program, analyzing the functional, psychological, and mental status of a community-based population in the south-west of France. We confirm the continuing decrease of DHEAS serum concentration with age, more in men than in women, even if men retain higher levels. Significantly lower values of baseline DHEAS were recorded in women in cases of functional limitation (Instrumental Activities of Daily Living), confinement, dyspnea, depressive symptomatology, poor subjective perception of health and life satisfaction, and usage of various medications. In men, there was a trend for the same correlations, even though not statistically significant in most categories. No differences in DHEAS levels were found in cases of incident dementia in the following 4 years. In men (but not in women), lower DHEAS was significantly associated with increased short-term mortality at 2 and 4 years after baseline measurement. These results, statistically established by taking into account corrections for age, sex, and health indicators, suggest the need for further careful trials of the administration of replacement doses of DHEA in aging humans. Indeed, the first noted results of such “treatment” are consistent with correlations observed here between functional and psychological status and endogenous steroid serum concentrations.

Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation.

Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases.

The mechanisms underlying the menstrual lysis leading to shedding of the human endometrium and its accompanying bleeding are still largely unknown. In particular, whether breakdown of the endometrial fibrillar extra-cellular matrix that precedes bleeding depends on aspartic-, cysteine-, serine-, or metalloproteinases remains unclear. In the present study, menstrual regression of the human endometrium was mimicked in organ culture. Whereas sex steroids could preserve tissue integrity only in nonperimenstrual explants, matrix breakdown upon sex steroid deprivation was completely and reversibly inhibited at all stages of the menstrual cycle by specific inhibitors of matrix metalloproteinases, but not by inhibitors of the other classes of proteinases. Matrix metalloproteinases are thus identified as the key class of proteinases involved in the initiation of menstruation.

A possible role of vitamin D receptors in regulating vitamin D activation in the kidney.

The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases. Previously, we reported that renal proximal convoluted tubules, the major site of 1 alpha, 25-dihydroxyvitamin D3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1 alpha, 25-dihydroxyvitamin D3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities. In rat models of enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3, expression of vitamin D receptors and 25-hydroxyvitamin D3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts. In vitamin D-deficient rats with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity, expression of vitamin D receptor mRNA in renal proximal convoluted tubules was also down-regulated, indicating that the down-regulation of vitamin D receptor mRNA is not the result of the enhanced production of 1 alpha, 25-dihydroxyvitamin D3. In Japanese quail models with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity by sex steroids, expression of vitamin D receptor mRNA was also down-regulated in the kidney but not in the duodenum. These results suggest that the down-regulation of vitamin D receptors plays a critical role in production of 1 alpha, 25-dihydroxyvitamin D3 in renal proximal convoluted tubules.

Pseudopregnancy in the bitch

Pseudopregnancy in the bitch is a normal phenomenon. It refers to the dioestrus phase of the bitch's reproductive cycle. It can range in severity from covert pseudopregnancy, in which the signs are barely noticeable, to severe clinical or overt pseudopregnancy. It occurs six to eight weeks following oestrous. The bitch may present with pregnancy-like behaviour including nesting and aggression, excessive mammary enlargement and contractions. These signs are an exaggerated version of the normal signs shown in a normal entire cycling bitch. The exact aetiology behind pseudopregnancy is not known. It is believed to be associated with a rapid decline in serum progesterone concentrations and the resulting surge in prolactin. Treatment may involve conservative management, medical therapy or ovariectomy/ovariohysterectomy. In the past medical therapy has included sex steroids including oestrogens, androgens and progestins. However, due to the frequent incidence and severity of side effects their use is contraindicated. Recommended forms of management incorporate the use of prolactin inhibitors such as cabergoline, metergoline and bromocriptine. These drugs reduce serum levels of prolactin and therefore reduce the development of mammary glands and decrease the behavioural signs associated with overt pseudopregnancy.

Physiological evidence for reproductive suppression in the introduced population of brown tree snakes (Boiga irregularis) on Guam

Introduced species are an increasingly pervasive problem. While studies on the ecology and behavior of these pests are numerous, there is relatively little known of their physiology, specifically their reproductive and stress physiology. One of the best documented introduced pest species is the brown tree snake, Boiga irregularis, which was introduced onto the Pacific island of Guam sometime around World War II. The snake is responsible for severely reducing Guam's native vertebrates. We captured free-living individuals throughout the year and measured plasma levels of stress and sex hormones in an effort to determine when they were breeding. These data were compared to reproductive cycles from a captive population originally collected from Guam. Free-living individuals had chronically elevated plasma levels of the stress hormone corticosterone and basal levels of sex steroids and a remarkably low proportion were reproductively active. These data coincide with evidence that the wild population may be in decline. Captive snakes, had low plasma levels of corticosterone with males displaying a peak in plasma testosterone levels during breeding. Furthermore, we compared body condition between the free-living and captive snakes from Guam and free-living individuals captured from their native range in Australia. Male and female free-living snakes from Guam exhibited significantly reduced body condition compared to free-living individuals from Australia. We suggest that during the study period, free-living brown tree snakes on Guam were living under stressful conditions, possibly due to overcrowding and overexploitation. of food resources, resulting in decreased body condition and suppressed reproduction. (C) 2004 Elsevier Ltd. All rights reserved.

The environmental control of reproduction in the female dace, Leuciscus leuciscus

An investigation was made into the nature and control of the annual reproductive cycle of the dace, Leuciscus leuciscus. It includes 1) a study of the natural reproductive cycle, 2) the use of Carp Pituitary Extract (CPE) to induce final maturation and ovulation in captive fish, 3) the effect of artificial light treatments on ovarian development and 4) the measurement of serum melatonin levels under different photoperiod regimes. Ovarian development was monitored by endocrinological data, notably serum cycles of 17-oestradiol (E2), testosterone (T), and calcium (as an index of vitellogenin), oocyte diameter, the gonadosomatic index and histological studies of the ovary. Under natural conditions, ovarian development can broadly be divided into 4 stages: 1) oogenesis which occurs immediately after spawning; 2) a primary growth phase (previtellogenic growth) prevalent between spawning and June; 3) a secondary growth phase (yolk vesicle plus vitellogenic growth) occurring between June and December and 4) final maturation and ovulation which occurs in mid-March. During the annual ovarian cycle, the sex steroids E2 and T showed two clear elevations. The first occurred initially in April followed by a rise in serum calcium levels. This subsequently initiated the appearance of yolk granules in the oocytes in June. The second rise occurred in September and levels were maintained until December, after which there was a decline in serum E2 levels. It is proposed that in the dace, high serum E2 levels between September and December were required to maintain vitellogenin production and therefore its uptake into the developing oocytes which occurred during this time, albeit at a slower rate than in the summer months. After December, prior to final maturation, whereas serum E2 and calcium levels declined, serum T levels remained elevated. In captivity, final maturation beyond the germinal vesicle migration stage failed to occur suggesting that the stimuli required for these events were absent. However ovulation could be induced by a single injection of CPE, which induced ovulation between 6 and 14 hours after treatment. Endocrine events associated with the artificial induction of spawning included a rise in serum levels of E2, T and the maturation inducing steroid 1720-dihydroxy progesterone. Photoperiodic manipulation demonstrated that whereas short or increasing daylengths were stimulatory to ovarian development, long days delayed development. Changes from long to short and constant short daylengths early in the reproductive cycle advanced maturation (up to 5 months), suggesting that the stimulus for ovarian development and maturation was a short day. However, experiments conducted later in the reproductive cycle demonstrated that only a simulated ambient photoperiod could induce final maturation. It is proposed therefore that under natural conditions the environmental stimulus for ovarian development and final maturation are short and increasing daylengths respectively. Further support that photoperiod is the dominant timing cue in this species was provided by the pattern of serum melatonin levels. Under different photoperiod treatments, serum melatonin, which is believed to be the chemical transducer of photoperiodic information (similar to other photoperiodic species) was elevated for the duration of the dark phase, indicating that the dace at least has the ability to `measure' changes in daylength.

Changes in peak expiratory flow and respiratory strength during the menstrual cycle

This study evaluated the spirometry and respiratory static pressures in 17 young women, twice a week for three successive ovulatory menstrual cycles to determine if such variables changed across the menstrual, follicular, periovulatory, early-tomid luteal and late luteal phases. The factors phases of menstrual cycle and individual cycles had no significant effect on the spirometry variables except for peak expiratory flow (PEF) and respiratory static pressures. Significant weak positive correlations were found between the progesterone:estradiol ratio and PEF and between estrogen and tidal volume (r = 0.37), inspiratory time (r = 0.22), expiratory time (r = 0.19), maximal inspiratory pressure (r = 0.25) and maximal expiratory pressure (r = 0.20) and for progesterone and maximal inspiratory pressure (r = 0.32) during the early-to-mid luteal phase. Although most parameters of the spirometry results did not change during the menstrual cycle, the correlations observed between sexual hormones and respiratory control variables suggest a positive influence of sexual female hormones controlling the thoracic pump muscles in the luteal phase

Étude de l'expression des microARNs et des enzymes de synthèse des corticostéroïdes dans le développement pulmonaire

Le syndrome de détresse respiratoire du nouveau-né (SDR) est l’une des pathologies les plus fréquentes dont souffrent les bébés prématurés. Le SDR est causé par un déficit dans la synthèse du surfactant pulmonaire en raison de l’immaturité du poumon lors d’une naissance prématurée. Plusieurs éléments régulent le développement pulmonaire notamment les stéroïdes sexuels et les corticostéroïdes. Le sexe est aussi un élément régulateur du développement pulmonaire. En effet, les garçons sont plus atteints que les filles par le SDR. Ce dimorphisme sexuel est attribué aux androgènes. Le traitement anténatal aux glucocorticoïdes est prescrit aux femmes qui sont à risque d’accoucher prématurément. En effet, les corticostéroïdes favorisent la maturation pulmonaire anténatale. Également, il a été démontré que les microARNs sont primordiaux pour le développement pulmonaire. Ceci nous a conduit à étudier l’impact des androgènes sur le profil d’expression des microARNs lors de la transition du stade canaliculaire au stade sacculaire (jour gestationnel (JG)17.0 au JG18.0), période qui coïncide avec la montée de la synthèse et de la sécrétion du surfactant chez la souris. Tout d’abord, nous avons étudié la stabilité des gènes de normalisation (snoRNAs) afin de quantifier les microARNs par qPCR. Cette analyse a été effectuée avec 3 logiciels différents et sur plusieurs stades du développement notamment de la période pseudoglandulaire jusqu’au stade alvéolaire chez les deux sexes. On a identifié les meilleures combinaisons de gènes de normalisation les plus stables pour chaque stade du développement étudié ainsi que pour la période couvrant tous les stades étudiés. Ensuite nous avons analysé à GD17.0 et GD18.0 le profil d’expression des microARNs chez des fœtus mâles dont les mères ont été traitées au flutamide (anti-androgènes pure). Les résultats ont montré que 43 microARNs matures sont modulés par les androgènes à GD17.0 et 35 microARNs à GD18.0. Pour certains microARNs, nous avons identifié des cibles potentielles qui sont inversement modulées par les androgènes par rapport aux microARNs. Ces cibles sont impliquées dans plusieurs processus biologiques tels que le métabolisme des lipides et la prolifération cellulaire ainsi que dans des fonctions moléculaires tels que la liaison des facteurs de transcription. Des expériences de validation ont été effectuées par qPCR. Nos résultats ont montré que les androgènes régulent des processus qui peuvent être impliqués dans la maturation pulmonaire via la régulation des microARNs. En plus de l’intérêt porté aux androgènes dans la maturation pulmonaire, nous avons analysé l’expression d’enzymes de synthèse des corticostéroïdes dans le poumon fœtal humain. L’expression de l’enzyme 21-hydroxylase a été étudiée par qPCR et par immunobuvardage. Également la localisation de l’ARNm de cette enzyme clé de la synthèse des glucocorticoïdes, a été effectuée par hybridation in situ. L’ARNm de CYP21A2 a été détecté par qPCR dans les 34 échantillons analysés et dont les âges variaient entre 17 et 40 semaines de grossesse. Aucune corrélation, avec l’âge gestationnel ou le sexe, n’a été observée. Des niveaux significatifs de la protéine 21-hydroxylase ont été détectés dans nos échantillons. Nous avons investigué l’expression d’autres enzymes impliquées dans la voie de synthèse des glucocorticoïdes notamment CYP11B1, CYP11B2 et CYP17A1. Les ARNm des gènes CYP11B1, CYP11B2 n’ont pas été détectés dans nos échantillons, contrairement à CYP17A1 dont l’ARNm a été détecté dans tous nos tissus fœtaux analysés. La protéine de la 17α-hydroxylase a été détectée à de faibles niveaux. Nos résultats d’hybridation in situ ont montré que l’expression de CYP21A2 est localisée presqu’exclusivement dans l’épithélium pulmonaire distal. Nos résultats suggèrent que les produits de la 21-hydroxylase agiront via une action intracrine sur l’épithélium distal en activant le récepteur des glucocorticoïdes (GR). L’activation du récepteur des minéralocorticoïdes (MR) ne semble pas dépendre de produits de la 21-hydroxylase en raison des quantités importantes d’aldostérone circulante.

Changes in peak expiratory flow and respiratory strength during the menstrual cycle

This study evaluated the spirometry and respiratory static pressures in 17 young women, twice a week for three successive ovulatory menstrual cycles to determine if such variables changed across the menstrual, follicular, periovulatory, early-tomid luteal and late luteal phases. The factors phases of menstrual cycle and individual cycles had no significant effect on the spirometry variables except for peak expiratory flow (PEF) and respiratory static pressures. Significant weak positive correlations were found between the progesterone:estradiol ratio and PEF and between estrogen and tidal volume (r = 0.37), inspiratory time (r = 0.22), expiratory time (r = 0.19), maximal inspiratory pressure (r = 0.25) and maximal expiratory pressure (r = 0.20) and for progesterone and maximal inspiratory pressure (r = 0.32) during the early-to-mid luteal phase. Although most parameters of the spirometry results did not change during the menstrual cycle, the correlations observed between sexual hormones and respiratory control variables suggest a positive influence of sexual female hormones controlling the thoracic pump muscles in the luteal phase

Involvement of pituitary gonadotropins, gonadal steroids and breeding season in sex change of protogynous dusky grouper, Epinephelus marginatus (Teleostei: Serranidae), induced by a non-steroidal aromatase inhibitor

Two experiments were performed using the aromatase inhibitor (AI) letrozole (100mg/kg) to promote sex change, from female-to-male, in protogynous dusky grouper. One experiment was performed during the breeding season (spring) and the other at the end of the breeding season (summer). During the spring, AI promoted sex change after 9weeks and the sperm produced was able to fertilize grouper oocytes. During the summer, the sex change was incomplete; intersex individuals were present and sperm was not released by any of the animals. Sex changed gonads had a lamellar architecture; cysts of spermatocytes and spermatozoa in the lumen of the germinal compartment. In the spring, after 4weeks, 11ketotestosterone (11KT) levels were higher in the AI than in control fish, and after 9weeks, coincident with semen release, testosterone levels increased in the AI group, while 11KT returned to the initial levels. Estradiol (E2) levels remained unchanged during the experimental period. Instead of decreasing throughout the period, as in control group, 17 α-OH progesterone levels did not change in the AI-treated fish, resulting in higher values after 9weeks when compared with control fish. fshβ and lhβ gene expression in the AI animals were lower compared with control fish after 9weeks. The use of AI was effective to obtain functional males during the breeding season. The increase in androgens, modulated by gonadotropins, triggered the sex change, enabling the development of male germ cells, whereas a decrease in E2 levels was not required to change sex in dusky grouper. © 2013 Elsevier Inc.

New analytical methodologies for doping control – detection of anabolic androgenic steroids in human urine

Dissertation submitted to Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa in fulfilment of the requirements for the degree of Doctor of Philosophy (Biochemistry - Biotechnology)

Sumoylated PPARalpha mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice.

As most metabolic studies are conducted in male animals, understanding the sex specificity of the underlying molecular pathways has been broadly neglected; for example, whether PPARs elicit sex-dependent responses has not been determined. Here we show that in mice, PPARalpha has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and immunity. In male mice, this effect was reproduced by the administration of a synthetic PPARalpha ligand. Using the steroid oxysterol 7alpha-hydroxylase cytochrome P4507b1 (Cyp7b1) gene as a model, we elucidated the molecular mechanism of this sex-specific PPARalpha-dependent repression. Initial sumoylation of the ligand-binding domain of PPARalpha triggered the interaction of PPARalpha with GA-binding protein alpha (GABPalpha) bound to the target Cyp7b1 promoter. Histone deacetylase and DNA and histone methylases were then recruited, and the adjacent Sp1-binding site and histones were methylated. These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1. Physiologically, this repression conferred on female mice protection against estrogen-induced intrahepatic cholestasis, the most common hepatic disease during pregnancy, suggesting a therapeutic target for prevention of this disease.