Showing papers on "Steroid biosynthesis published in 2009"

Anti-androgens and androgen-depleting therapies in prostate cancer: new agents for an established target

Abstract: Activation of the androgen receptor is crucial for prostate cancer growth at all points of the illness. Current therapies targeting the androgen receptor, including androgen-depletion approaches and anti-androgens, do not completely inhibit the receptor activity. Prostate cancer cells develop resistance to castration by acquiring changes that include androgen-receptor overexpression and overexpression of enzymes involved in androgen biosynthesis, which result in reactivation of the receptor. Based on an understanding of these resistance mechanisms and androgen biosynthesis pathways, new anti-androgens and androgen-depleting agents have been developed. Notably, promising activity has been shown in early phase trials by MDV3100, a new anti-androgen designed for activity in prostate cancer model systems with overexpressed androgen receptor, and by abiraterone acetate, a CYP17A inhibitor that blocks steroid biosynthesis in the adrenal gland and possibly within the tumour. Both agents are undergoing phase 3 testing. Here, we review the basic science and clinical development of these and other agents.

Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives

Abstract: Steroid hormones are synthesized in the adrenal gland, gonads, placenta and brain and are critical for normal reproductive function and bodily homeostasis. The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroid biosynthesis, i.e. the delivery of cholesterol from the outer to the inner mitochondrial membrane. The expression of the StAR protein is predominantly regulated by cAMP-dependent mechanisms in the adrenal and gonads. Whereas StAR plays an indispensable role in the regulation of steroid biosynthesis, a complete understanding of the regulation of its expression and function in steroidogenesis is not available. It has become clear that the regulation of StAR gene expression is a complex process that involves the interaction of a diversity of hormones and multiple signaling pathways that coordinate the cooperation and interaction of transcriptional machinery, as well as a number of post-transcriptional mechanisms that govern mRNA and protein expression. However, information is lacking on how the StAR gene is regulated in vivo such that it is expressed at appropriate times during development and is confined to the steroidogenic cells. Thus, it is not surprising that the precise mechanism involved in the regulation of StAR gene has not yet been established, which is the key to understanding the regulation of steroidogenesis in the context of both male and female development and function.

Gene expression profiling of lymphoblasts from autistic and nonaffected sib pairs: Altered pathways in neuronal development and steroid biosynthesis

Abstract: Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present “case-control” study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects ∼4 times as many males as females. Preliminary metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism.

Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor.

Abstract: In a recent report, Toxicity Testing in the 21st Century, the National Research Council Committee on Toxicity Testing and Assessment of Environmental Agents (2007) proposed that improved scientific understanding of toxicity pathways was central to the expanded use of predictive, pathway-based bioassays in risk assessment. Toxicity pathways can be viewed as the series of biological changes, spanning across multiple levels of biological organization, that lead from some molecular initiating event (perturbation) to an adverse outcome. A major challenge associated with dose–response modeling and extrapolation from laboratory to real-world conditions has been to understand under what conditions an organism may compensate for, or recover from, a given perturbation and under what conditions the perturbation will lead to an adverse outcome (Andersen et al. 2005). Thus, in developing useful predictive models of toxicity, we need to understand not only the direct effects of a chemical and how they translate into adverse effect, but also the potential mechanisms for compensation and recovery and how they may intersect with other biological pathways and processes. Previous studies with the fathead minnow (Pimephales promelas) have suggested potential compensatory responses to the direct effects of chemicals whose primary mode of action was inhibition of one or more enzymes involved in steroid biosynthesis. For example, the chemical fadrozole (FAD) inhibits aromatase, the enzyme that catalyzes the rate-limiting conversion of testosterone (T) to 17β-estradiol (E2) (Miller 1988). Villeneuve et al. (2006) observed significant, concentration-dependent up-regulation of transcripts coding for the ovarian isoform of aromatase (CYP19A) in female fathead minnows exposed to FAD for 7 days. The increased CYP19A gene expression was associated with an inverted U-shaped concentration–response profile for ovary aromatase activity. Although that study did not examine effects on plasma E2 concentrations, it was noted that the responses would favor increased synthesis of E2 to potentially offset the effect of FAD on aromatase. In another study, Ankley et al. (2007) exposed fathead minnows to the steroidogenesis inhibitor ketoconazole for 21 days. Testosterone production by testis or ovary tissue collected from ketoconazole-exposed fish was significantly reduced compared with control fish. However, there was significant up-regulation of genes coding for cytochrome P450 cholesterol side-chain cleavage (P450scc, CYP11A) and cytochrome P450 c17αhydroxylase, 17,20-lyase, and concentration-dependent proliferation of steroid-producing interstitial cells in the testis of exposed males. As a result, plasma T and E2 concentrations in exposed fish were similar to those of controls, despite the decreased rate of steroid production per unit mass of tissue, suggesting a compensatory response to ketoconazole (Ankley et al. 2007). Both the FAD and ketocona zole studies suggest that fish have the capacity to adapt to and potentially recover from the direct effects of steroidogenesis inhibitors. Aromatase is a key steroidogenic enzyme shown to be subject to inhibition, at least in vitro, by a variety of chemicals present in the environment, including certain pesticides, organochlorines, and organotins (Sanderson 2006). The aim of this study was to develop a more comprehensive understanding of molecular and biochemical responses of fathead minnows to aromatase inhibition, including direct effects, compensation, and/or recovery. We examined a time course of selected gene expression and biochemical responses over the course of an 8-day waterborne exposure to two concentrations of FAD, followed by an 8-day recovery period in control water. The experiment was designed to test several hypotheses: FAD would elicit direct effects consistent with its presumptive mode of action Over time, there would be compensatory molecular responses in females, consistent with an attempt to increase E2 synthesis Compensatory effects at the molecular level would correspond to changes in circulating E2 and/or rates of ex vivo E2 production Effects would be time and concentration dependent There would be recovery after cessation of FAD exposure. Although FAD is a drug with no direct environmental relevance, its specificity makes it a useful model chemical for studying this mode of action. Results of this study provide an improved understanding of the dynamics of biological response to this chemical, and its removal. This knowledge will contribute to formulation of a robust, biologically based toxicity pathway model for the effects of estradiol synthesis inhibitors.

Mechanisms of protein kinase C signaling in the modulation of 3',5'-cyclic adenosine monophosphate-mediated steroidogenesis in mouse gonadal cells.

Abstract: The protein kinase C (PKC) signaling pathway plays integral roles in the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. PKC can modulate the activity of cAMP/protein kinase A signaling involved in steroidogenesis; however, its mechanism remains obscure. In the present study, we demonstrate that activation of the PKC pathway, by phorbol 12-myristate 13-acetate (PMA), was capable of potentiating dibutyryl cAMP [(Bu)2cAMP]-stimulated StAR expression, StAR phosphorylation, and progesterone synthesis in both mouse Leydig (MA-10) and granulosa (KK-1) tumor cells. The steroidogenic potential of PMA and (Bu)2cAMP was linked with phosphorylation of ERK 1/2; however, inhibition of the latter demonstrated varying effects on steroidogenesis. Transcriptional activation of the StAR gene by PMA and (Bu)2cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of the cAMP response element binding protein (CREB...

Role of Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X Chromosome, Gene 1 in Protein Kinase A- and Protein Kinase C-Mediated Regulation of the Steroidogenic Acute Regulatory Protein Expression in Mouse Leydig Tumor Cells: Mechanism of Action

Abstract: Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) is an orphan nuclear receptor that has been demonstrated to be instrumental to the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. However, its mechanism of action remains obscure. The present investigation was aimed at exploring the molecular involvement of DAX-1 in protein kinase A (PKA)- and protein kinase C (PKC)-mediated regulation of StAR expression and its concomitant impact on steroid synthesis using MA-10 mouse Leydig tumor cells. We demonstrate that activation of the PKA and PKC pathways, by a cAMP analog dibutyryl (Bu)2cAMP [(Bu)2cAMP] and phorbol 12-myristate 13-acetate (PMA), respectively, markedly decreased DAX-1 expression, an event that was inversely correlated with StAR protein, StAR mRNA, and progesterone levels. Notably, the suppression of DAX-1 requires de novo transcription and translation, suggesting that the effect of DAX-1 in regulating StAR expression is dynamic. Chromatin immunoprecipitation studies revealed the association of DAX-1 with the proximal but not the distal region of the StAR promoter, and both (Bu)2cAMP and PMA decreased in vivo DAX-1-DNA interactions. EMSA and reporter gene analyses demonstrated the functional integrity of this interaction by showing that DAX-1 binds to a DNA hairpin at position -44/-20 bp of the mouse StAR promoter and that the binding of DAX-1 to this region decreases progesterone synthesis by impairing transcription of the StAR gene. In support of this, targeted silencing of endogenous DAX-1 elevated basal, (Bu)2cAMP-, and PMA-stimulated StAR expression and progesterone synthesis. Transrepression of the StAR gene by DAX-1 was tightly associated with expression of the nuclear receptors Nur77 and steroidogenic factor-1, demonstrating these factors negatively modulate the steroidogenic response. These findings provide insight into the molecular events by which DAX-1 influences the PKA and PKC signaling pathways involved in the regulation of the StAR protein and steroidogenesis in mouse Leydig tumor cells.

Investigations on the use of C-21-steroids as precursors for placental oestrogen synthesis in the cow

Abstract: Cotyledonary homogenates from 220 and 270 day pregnant and term cows were incubated (NADPH- and NAD(+)-regenerating system) with 3H-pregnenolone and 3H-progesterone, respectively. Identification of metabolites was based on separation on HPLC and the respective retention times. On days 220/270 the major metabolite formed after incubation with 3H-pregnenolone was progesterone, followed by 17 alpha-hydroxyprogesterone and dehydroepiandrosterone/17 alpha-hydroxypregnenolone; the formation of estrone was low (up to 6%), while it was the major metabolite after incubation of pregnenolone with term placenta. At all stages of pregnancy investigated, the only metabolite found after incubation with 3H-progesterone was 17 alpha-hydroxyprogesterone. These data provide evidence that 17 alpha-hydroxyprogesterone is the endpoint of steroid biosynthesis in the bovine placenta along the delta 4-route and that oestrogen synthesis follows the delta 5-pathway. Based on the high activity of 3 beta-hydroxysteroiddehydrogenase/delta 5/4-isomerase also found on days 220/270, the key enzyme allowing for placental oestrogen synthesis in the cow seems to be cytochrome P450c17 alpha. Thus the situation in the cow is similar to that in the sheep and the increased turnover of pregnenolone into estrone may explain the decrease of placental progesterone production in the cow towards the end of gestation.

Translocator protein blockade reduces prostate tumor growth.

Abstract: Purpose: The transmembrane molecule, translocator protein (TSPO), has been implicated in the progression of epithelial tumors. TSPO gene expression is high in tissues involved in steroid biosynthesis, neurodegenerative disease, and in cancer, and overexpression has been shown to contribute to pathologic conditions including cancer progression in several different models. The goal of our study was to examine the expression and biological relevance of TSPO in prostate cancer and show that the commonly prescribed benzodiazepine lorazepam, a ligand for TSPO, exhibits anticancer properties. Experimental Design: Immunohistochemical analysis using tissue microarrays was used to determine the expression profile of TSPO in human prostate cancer tissues. To show the effect of TSPO ligands (lorazepam and PK11195) in prostate cancer, we used cell proliferation assays, apoptosis ELISA, prostate cancer xenograft study, and immunohistochemistry. Results: TSPO expression is increased in prostatic intraepithelial neoplasia, primary prostate cancer, and metastases compared with normal prostate tissue and benign prostatic hyperplasia. Furthermore, TSPO expression correlates with disease progression, as TSPO levels increased with increasing Gleason sum and stage with prostate cancer metastases demonstrating the highest level of expression among all tissues examined. Functionally, we have shown that lorazepam has antiproliferative and proapoptotic properties in vitro and in vivo . Additionally, we have shown that TSPO overexpression in nontumorigenic cells conferred susceptibility to lorazepam-induced growth inhibition. Conclusion: These data suggest that blocking TSPO function in tumor cells induces cell death and denotes a survival role for TSPO in prostate cancer and provides the first evidence for the use of benzodiazepines in prostate cancer therapeutics. (Clin Cancer Res 2009;15(19):6177–84)

Construction of a polycystic ovarian syndrome (PCOS) pathway based on the interactions of PCOS-related proteins retrieved from bibliomic data

Abstract: Polycystic ovary syndrome (PCOS) is a complex but frequently occurring endocrine abnormality. PCOS has become one of the leading causes of oligo-ovulatory infertility among premenopausal women. The definition of PCOS remains unclear because of the heterogeneity of this abnormality, but it is associated with insulin resistance, hyperandrogenism, obesity and dyslipidaemia. The main purpose of this study was to identify possible candidate genes involved in PCOS. Several genomic approaches, including linkage analysis and microarray analysis, have been used to look for candidate PCOS genes. To obtain a clearer view of the mechanism of PCOS, we have compiled data from microarray analyses. An extensive literature search identified seven published microarray analyses that utilized PCOS samples. These were published between the year of 2003 and 2007 and included analyses of ovary tissues as well as whole ovaries and theca cells. Although somewhat different methods were used, all the studies employed cDNA microarrays to compare the gene expression patterns of PCOS patients with those of healthy controls. These analyses identified more than a thousand genes whose expression was altered in PCOS patients. Most of the genes were found to be involved in gene and protein expression, cell signaling and metabolism. We have classified all of the 1081 identified genes as coding for either known or unknown proteins. Cytoscape 2.6.1 was used to build a network of protein and then to analyze it. This protein network consists of 504 protein nodes and 1408 interactions among those proteins. One hypothetical protein in the PCOS network was postulated to be involved in the cell cycle. BiNGO was used to identify the three main ontologies in the protein network: molecular functions, biological processes and cellular components. This gene ontology analysis identified a number of ontologies and genes likely to be involved in the complex mechanism of PCOS. These include the insulin receptor signaling pathway, steroid biosynthesis, and the regulation of gonadotropin secretion among others.

Congenital Adrenal Hyperplasia: Diagnosis, Evaluation, and Management

Abstract: 1. Zoltan Antal, MD* 2. Ping Zhou, MD† 1. *Fellow 2. †Assistant Professor, Division of Pediatric Endocrinology, Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY After completing this article, readers should be able to: 1. Describe the pathophysiology of congenital adrenal hyperplasia (CAH). 2. Characterize the signs and symptoms of CAH. 3. Describe the appropriate laboratory evaluation of CAH. 4. Know that CAH can be diagnosed prenatally. 5. Recognize adrenal insufficiency by laboratory and clinical evaluation. 6. Anticipate and plan treatment for both acute adrenal crisis and long-term therapy for a patient who has CAH. 7. Discuss the value of newborn screening for salt-losing CAH in male infants. Congenital adrenal hyperplasia (CAH) refers to a family of inherited disorders of adrenal steroidogenesis. The common functional defect in each disorder is impaired cortisol secretion, resulting in hypersecretion of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) and consequent hyperplasia of the adrenal glands. More than 90% of cases of CAH are caused by a defect in the enzyme 21-hydroxylase (21-hydroxylase deficiency [21OHD]). Four other enzyme deficiencies in the steroid biosynthesis pathway, along with one cholesterol transport protein defect, account for the remaining cases. Depending on the severity of the enzyme deficiency, 21OHD is defined as classic (severe form) or nonclassic (mild form). Approximately 75% of patients who have the classic form also have salt wasting due to inadequate aldosterone production, further subdividing the classification into classic simple virilizing and classic salt-wasting forms. This review highlights the diagnosis and treatment of 21OHD, with a brief discussion of the other forms of CAH. A Hispanic baby girl is born at term, has a birthweight of 3.1 kg, and has ambiguous genitalia (Fig. 1). During the first week after birth, the baby is clinically stable but demands frequent feeding. On the sixth postnatal day, serum electrolyte measurements are normal. Additional evaluation reveals female internal organs on pelvic ultrasonography and female karyotype. Newborn screening results show 17-hydroxyprogesterone (17-OHP) values of 180 and 679 ng/mL 2 and 5 days …

Computational model of steroidogenesis in human H295R cells to predict biochemical response to endocrine-active chemicals: model development for metyrapone.

Abstract: Background: An in vitro steroidogenesis assay using the human adrenocortical carcinoma cell line H295R is being evaluated as a possible screening assay to detect and assess the impact of endocrine-active chemicals (EACs) capable of altering steroid biosynthesis. Data interpretation and their quantitative use in human and ecological risk assessments can be enhanced with mechanistic computational models to help define mechanisms of action and improve understanding of intracellular concentrationresponse behavior. Objectives: The goal of this study was to develop a mechanistic computational model of the metabolic network of adrenal steroidogenesis to estimate the synthesis and secretion of adrenal steroids in human H295R cells and their biochemical response to steroidogenesis-disrupting EAC. Methods: We developed a deterministic model that describes the biosynthetic pathways for the conversion of cholesterol to adrenal steroids and the kinetics for enzyme inhibition by metryrapone (MET), a model EAC. Using a nonlinear parameter estimation method, the model was fitted to the measurements from an in vitro steroidogenesis assay using H295R cells. Results: Model-predicted steroid concentrations in cells and culture medium corresponded well to the time-course measurements from control and MET-exposed cells. A sensitivity analysis indicated the parameter uncertainties and identified transport and metabolic processes that most influenced the concentrations of primary adrenal steroids, aldosterone and cortisol. Conclusions: Our study demonstrates the feasibility of using a computational model of steroidogenesis to estimate steroid concentrations in vitro. This capability could be useful to help define mechanisms of action for poorly characterized chemicals and mixtures in support of predictive hazard and risk assessments with EACs.

Translocator protein ligands as promising therapeutic tools for anxiety disorders.

Abstract: The Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is an 18 kDa mitochondrial protein primarily involved in steroid biosynthesis in both peripheral and glial cells. It has been extensively reported that TSPO regulates the rate-limiting translocation of cholesterol from the outer to the inner mitochondrial membrane before its transformation by cytochrome P450scc into pregnenolone, which is further converted into an array of different steroids. In the brain, neurosteroids such as allopregnanolone and pregnenolone, acting as positive modulators of γ-aminobutyric type A (GABAA) receptors, exert anxiolytic activity. Specific ligands targeting TSPO increase neurosteroid production and for this reason they have been suggested to play an important role in anxiety modulation. Unlike benzodiazepines (Bzs), which represent the most common anti-anxiety drugs administered around the world, selective TSPO ligands have shown anxiolytic effects in animal models without any of the side effects associated with Bzs. Therefore, specific TSPO ligands that are able to promote neurosteroidogenesis may represent the future of therapeutic treatment of anxiety disorders. Furthermore, TSPO expression levels are altered in several different psychiatric disorders in which anxiety is the main symptom. This article reviews the primary and patent literature over the last decade concerning the development of novel TSPO ligands that have resulted effective in various models of anxiety, taking into special consideration their structure-activity relationships.

Steroid biosynthesis within the frog brain: a model of neuroendocrine regulation.

Abstract: There is now clear evidence that the brain, similar to the adrenal gland, gonads, and placenta, is a steroidogenic organ Notably in the frog brain, the presence of various steroidogenic enzymes has been detected by immunohistochemistry in specific populations of neurons and/or glial cells These steroidogenic enzymes are biologically active, as shown by the ability of brain tissue explants to convert [(3)H]pregnenolone into various radiolabeled steroids The frog brain has also been extensively used as a model to study the mechanism of regulation of neurosteroidogenesis by neurotransmitters and neuropeptides It has been demonstrated that the biosynthesis of neurosteroids is inhibited by gamma-aminobutyric acid (GABA), acting through GABA(A) receptors, and neuropeptide Y, acting through Y1 receptors, and is stimulated by the octadecaneuropeptide (ODN), acting through central-type benzodiazepine receptors, triakontatetraneuropeptide (TTN), acting through peripheral-type benzodiazepine receptors, and vasotocin, acting through V1a-like receptors These data indicate that some of the neurophysiological effects of neurotransmitters and neuropeptides may be mediated through modulation of neurosteroid biosynthesis

Prednisone reduces ketoconazole-induced skeletal defects in rat fetuses.

Abstract: Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not specific, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also affected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic effects remains to be elucidated. When considering the potential inhibitory effect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis

Infertility in the Male: Microsurgical treatment of male infertility

Abstract: Apoptosis is a highly regulated form of cell death, involving a cascade-like activation of a series of catabolic processes that progressively disassemble the cell. High levels of endogenous maternal estrogens are associated with increased risks of testicular cancer and cryptorchidism in male offspring. In males, smoking negatively affects sperm production, motility, and morphology. Enzymes involved in the steroid biosynthesis pathway are beginning to be recognized as targets for the action of a number of endocrine-disrupting chemicals (EDCs). The blood-testis barrier (BTB) serves to protect spermatogenesis from many potentially cytotoxic drugs and other chemicals. Drugs and medications may have an adverse impact on fertility through a variety of mechanisms including direct toxicity to the testicular germ cells and supporting cells, alterations of the hypothalamic-pituitary-gonadal (HPG) axis, or effects on sexual performance by impairment of libido, erections, or ejaculation.

Parallel evolution between aromatase and androgen receptor in the animal kingdom

Abstract: There are now many known cases of orthologous or unrelated proteins in different species that have undergone parallel evolution to satisfy a similar function. However, there are no reported cases of parallel evolution for proteins that bind a common ligand but have different functions. We focused on two proteins that have different functions in steroid hormone biosynthesis and action but bind a common ligand, androgen. The first protein, androgen receptor (AR), is a nuclear hormone receptor and the second one, aromatase (cytochrome P450 19 [CYP19]), converts androgen to estrogen. We hypothesized that binding of the androgen ligand has exerted common selective pressure on both AR and CYP19, resulting in a signature of parallel evolution between these two proteins, though they perform different functions. Consistent with this hypothesis, we found that rates of amino acid change in AR and CYP19 are strongly correlated across the metazoan phylogeny, whereas no significant correlation was found in the control set of proteins. Moreover, we inferred that genomic toolkits required for steroid biosynthesis and action were present in a basal metazoan, cnidarians. The close similarities between vertebrate and sea anemone AR and CYP19 suggest a very ancient origin of their endocrine functions at the base of metazoan evolution. Finally, we found evidence supporting the hypothesis that the androgen-to-estrogen ratio determines the gonadal sex in all metazoans.

Co-induction of CYP3A12 and 3A26 in dog liver slices by xenobiotics: species difference between human and dog CYP3A induction.

Abstract: The induction of dog CYP3A12 and CYP3A26 mRNA levels was evaluated in liver slices after treatment with 22 xenobiotics. Eleven of the 22 xenobiotics increased 3A12 mRNA by more than four-fold, while nine did the same for 3A26 mRNA. A four-fold increase in the mRNA level was used as the cut-off for indication of induction based on the noise level of the real time-PCR. A good correlation was found between the mRNA levels for 3A12 and 3A26 after treatment with compounds, suggesting that these two CYPs may be co-induced. Induction of CYP3A4 in human hepatocytes was evaluated after treatment with the same 22 compounds. Thirteen out of the 22 compounds increased the 3A4 mRNA levels by more than four-fold. When the mRNA levels of 3A4 and 3A12 were compared after treatment with compounds, no correlation was found. The regulation of CYP3A expression has been demonstrated to be controlled by pregnane X receptor (PXR). Upon examination of the sequence homology and the three-dimensional structures of human PXR and a dog PXR model, only two different amino acids (met323/val and arg410/lys) were found in the ligand-binding domain. This finding suggests that these two amino acids may play a role in the binding specificity of ligands.

Substitution with testosterone as aromatizable substrate for induction of follicular maturation, estradiol production and ovulation in a patient with 17 alpha-hydroxylase deficiency.

Abstract: 17 alpha-Hydroxylase Deficiency (17 alpha-OHDS) is a rare defect of steroid biosynthesis, characterized by the inability to synthesize cortisol, androgens or estrogens, by the complete absence of follicular maturation, hypergonadotropic hypogonadism, primary amenorrhea and hypertension. Since the ovaries of such patients contain numerous primordial follicles, we hypothesized that the absence of spontaneous follicular maturation could be due to a lack of aromatizable substrate. To provide this substrate, testosterone was administered either by intra-ovarian injection or by vaginal administration. Ovarian stimulation was performed with human urinary gonadotropins. Follicular maturation and ovulation could be induced by this treatment, as determined from ultrasonography, the analysis of LH, estradiol and progesterone serum levels and the aspiration of oocytes from the mature follicles. Fertilization of these oocytes in vitro, however, did not succeed. We conclude that follicular maturation can be induced in 17 alpha-OHDS by gonadotropins when testosterone is provided as an aromatizable substrate and that estrogens are a necessary component of follicular maturation.

Serum levels of steroid hormones in men with varicocele and oligospermia as compared to normozoospermic men.

Abstract: In order to assess the contribution of individual steroidogenic organs to over-all steroid biosynthesis, the basal plasma levels of six hormonal steroids, reflecting the function of gonads and adrenals, as well as both gonadotropins and prolactin, have been determined in 63 oligo- and/or asthenozoospermic men with palpable varicocele, confirmed in each case by sonography. The values obtained were compared with the results of a group of age-matched normospermic men without endocrine disorders. Insignificantly lower levels of testosterone accompanied by significantly lower levels of dihydrotestosterone, slightly decreased LH but increased FSH undistinguishable concentrations of cortisol in patients with varicocele were in agreement with previous findings. Higher levels of an exclusively adrenal androgen 11 beta-hydroxyandrostenedione in men with varicocele indicate increased activity of the adrenal cortex. Significantly higher basal levels of 17 alpha-hydroxyprogesterone in the patient group (p < 0.001), so far not unequivocally proved, evidence for suggested decreased enzyme activity of C17-20 lyase in the testis in at least some of men with varicocele.

Detection of a mitochondrial kinase complex that mediates PKA-MEK-ERK-dependent phosphorylation of mitochondrial proteins involved in the regulation of steroid biosynthesis.

Abstract: In order to achieve the goal of this article, as an example we will describe the strategies followed to analyze the presence of the multi-kinase complex at the mitochondria and the posttranslational modification of two key mitochondrial proteins, which participate in the regulation of cholesterol transport across the mitochondrial membranes and in the regulation of steroid biosynthesis. Hormones, ions or growth factors modulate steroid biosynthesis by the posttranslational phosphorylation of proteins. The question still remains on how phosphorylation events transmit a specific signal to its mitochondrial site of action. Cholesterol transport requires specific interactions in mitochondria between several proteins including a multi-kinase complex. The presence of this multi-kinase complex at the mitochondria reveals the importance of the posttranslational modification of mitochondrial proteins for its activity and functions. The activation of PKA triggers the posttranslational modification of the mitochondrial acyl-CoA thioesterase (Acot2), which releases arachidonic acid (AA) in the mitochondria, and the activation of a kinase cascade that leads to the phoshorylation of the steroidogenic acute regulatory (StAR) protein. The function of StAR is to facilitate the access of cholesterol to the first enzyme of the biosynthesis process and its induction is dependent on Acot2 and intramitochondrial AA release. Truncation of the StAR protein is associated with the steroid deficiency disease, congenital lipoid adrenal hyperplasia.