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Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5’s biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5’s mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5’s biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5–protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.

Sex steroid hormones are not only synthesized from the gonads but also by other tissues, such as the brain (i.e., neurosteroids) and colon (i.e., gut steroids). Gut microbiota can be shaped from sex steroid hormones synthesized from the gonads and locally interacts with gut steroids as in turn modulates neurosteroids. Type 1 diabetes mellitus (T1DM) is characterized by dysbiosis and also by diabetic encephalopathy. However, the interactions of players of gut-brain axis, such as gut steroids, gut permeability markers and microbiota, have been poorly explored in this pathology and, particularly in females. On this basis, we have explored, in streptozotocin (STZ)induced adult female rats, whether one month of T1DM may alter (I) gut microbiome composition and diversity by 16S next-generation sequencing, (II) gut steroid levels by liquid chromatography-tandem mass spectrometry, (III) gut permeability markers by gene expression analysis, (IV) cognitive behavior by the novel object recognition (NOR) test and whether correlations among these aspects may occur. Results obtained reveal that T1DM alters gut beta-, but not alpha-diversity. The pathology is also associated with a decrease and an increase in colonic pregnenolone and allopregnanolone levels, respectively. Additionally, diabetes alters gut permeability and worsens cognitive behavior. Finally, we reported a significant correlation of pregnenolone with Blautia, claudin-1 and the NOR index and of allopregnanolone with Parasutterella, Gammaproteobacteria and claudin-1. Altogether, these results suggest new putative roles of these two gut steroids related to cognitive deficit and dysbiosis in T1DM female experimental model.

Altres ajuts: "la Caixa" Foundation (ID100010434) under agreement LCF/PR/HR19/52160016., Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone's effects on cognitive function were mediated via an autocrine mechanism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnenolone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders. Ramírez et al. show that reduced hypothalamic pregnenolone levels, in the context of metabolic diseases, are associated with cognitive deterioration. POMC neuron-derived pregnenolone is a key mediator of cognition, but not metabolism, acting via hippocampal mechanisms. These results identify an unsuspected role for POMC neuron-derived pregnenolone in memory performance.

Paroxetine, a selective serotonin reuptake inhibitor (SSRI), is prescribed to treat psychiatric disorders, although an off-label SSRI use is also for functional gastrointestinal disorders. The mutual correlation between serotonin and peripheral sex steroids has been reported, however little attention to sex steroids synthesized by gut, has been given so far. Indeed, whether SSRIs, may also influence the gut steroid production, immediately after treatment and/or after suspension, is still unclear. The finding that gut possesses steroidogenic capability is of particular relevance, also for the existence of the gut-microbiota-brain axis, where gut microbiota represents a key orchestrator. On this basis, adult male rats were treated daily for two weeks with paroxetine or vehicle and, 24 h after treatment and at 1 month of withdrawal, steroid environment and gut microbiota were evaluated. Results obtained reveal that paroxetine significantly affects steroid levels, only in the colon but not in plasma. In particular, steroid modifications observed immediately after treatment are not overlap with those detected at withdrawal. Additionally, paroxetine treatment and its withdrawal impact gut microbiota populations differently. Altogether, these results suggest a biphasic effect of the drug treatment in the gut both on steroidogenesis and microbiota.

Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone’s effects on cognitive function were mediated via an autocrine mechanism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnenolone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders.

BackgroundPostmenopausal pregnenolone and/or progesterone levels in relation to endometrial and ovarian cancer risks have been infrequently evaluated. To address this, we utilized a sensitive and reliable assay to quantify prediagnostic levels of seven markers related to endogenous hormone metabolism.MethodsHormones were quantified in baseline serum collected from postmenopausal women in a cohort study nested within the Breast and Bone Follow-up to the Fracture Intervention Trial (B∼FIT). Women using exogenous hormones at baseline (1992-1993) were excluded. Incident endometrial (n = 65) and ovarian (n = 67) cancers were diagnosed during 12 follow-up years and compared with a subcohort of 345 women (no hysterectomy) and 413 women (no oophorectomy), respectively. Cox models with robust variance were used to estimate cancer risk.ResultsCirculating progesterone levels were not associated with endometrial [tertile (T)3 vs. T1 HR (95% confidence interval): 1.87 (0.85-4.11); P trend = 0.17] or ovarian cancer risk [1.16 (0.58-2.33); 0.73]. Increasing levels of the progesterone-to-estradiol ratio were inversely associated with endometrial cancer risk [T3 vs. T1: 0.29 (0.09-0.95); 0.03]. Increasing levels of 17-hydroxypregnenolone were inversely associated with endometrial cancer risk [0.40 (0.18-0.91); 0.03] and positively associated with ovarian cancer risk [3.11 (1.39-6.93); 0.01].ConclusionsUsing sensitive and reliable assays, this study provides novel data that endogenous progesterone levels are not strongly associated with incident endometrial or ovarian cancer risks. 17-hydroxypregnenolone was positively associated with ovarian cancer and inversely associated with endometrial cancer.ImpactWhile our results require replication in large studies, they provide further support of the hormonal etiology of endometrial and ovarian cancers.

Aim and Objective: The development of small molecules that can interact with key therapeutic target represents an active field of research. Therefore, new approaches for increasing the molecular diversity of a starting material, such as a natural product, are needed. Herein, the carbonyl group present on a pregnane scaffold, or easily obtained from the oxidation of the corresponding alcohol, was used to obtain a series of diversified steroidal morpholinone derivatives. Materials and Methods: Using chemical synthesis, two levels of molecular diversity were introduced at position 20 of a C21-steroid scaffold. Nuclear magnetic resonance (NMR) spectroscopy and x-ray analysis were next used to characterize the morpholinone derivatives. Results: The C-20 carbonyl of pregnenolone was first transformed into an oxirane that reacted with an amino acid and the resulting amino alcohol was then cyclized to generate different spiro-2-morpholinones. X-ray analysis of one representative compound confirmed the 3-dimensional representation of this new family of diversified steroid derivatives. NMR analysis supported the expected structure and identified key markers of the chiral center configuration found in the 2-morpholinone moiety. Finally, the NH of the morpholinone ring was alkylated, thus increasing structural diversity. Conclusion: Considering the huge amount of building blocks (amino acids, bromobenzyl derivatives and ketones) that are commercially available, the strategy reported herein opens the door to the synthesis of diversified libraries of new compounds.

Steroids are a group of organic compounds that include sex hormones, adrenal cortical hormones, sterols, and phytosterols. In mammals, steroid biosynthesis starts from cholesterol via multiple steps to the final steroid and occurs in the gonads, adrenal glands, and placenta. This highly regulated pathway involves several cytochrome P450, as well as different dehydrogenases and reductases. Steroids in mammals have also been associated with drug production. Steroid pharmaceuticals such as testosterone and progesterone represent the second largest category of marketed medical products. There heterologous production through microbial transformation of phytosterols has gained interest in the last couple of decades. Phytosterols being the plants sterols serve as inexpensive substrates for the production of steroid derivatives. Various genes and biochemical pathways involved in phytosterol degradation have been identified in many Rhodococcus and Mycobacterium species. Apart from an early investigation in mammals, presence of steroids such as androsteroids and progesterone has also been demonstrated in plants. Their main role is linked with growth, development, and reproduction. Even though plants share some chemical features with mammals, the biosynthesis is different, with the first C22 hydroxylation as an example. This is performed by CYP11A1 in mammals and CYP90B1 in plants. Moreover, the entire plant steroid biosynthesis is not fully elucidated. Knowing this pathway could provide new processes for the industrial biotechnological production of steroid hormones in plants.

Neuroactive steroids, molecules produced from cholesterol in steroidogenic cells (i.e., peripheral glands and nervous system) are physiological modulators and protective agents of nervous function. A possible role for neuroactive steroids in the sex-dimorphic clinical manifestation, onset and progression of Multiple Sclerosis (MS) has been recently suggested. To explore this possibility, we assessed the synthesis of the first steroidogenic product (pregnenolone; PREG) in the spinal cord of experimental autoimmune encephalomyelitis rats, a MS model. Data obtained indicate that the synthesis of PREG in the spinal cord is altered by the pathology in a sex-dimorphic way and depending on the pathological progression. Indeed, in male spinal cord the synthesis was already decreased at the acute phase of the disease (i.e., 14 days post induction - dpi) and maintained low during the chronic phase (i.e., 45 dpi), while in females this effect was observed only at the chronic phase. Substrate availability had also a role in the sex-dimorphic kinetics. Indeed, at the chronic phase, male animals showed a reduction in the levels of free cholesterol coupled to alteration of cholesterol metabolism into oxysterols; these effects were not observed in female animals. These findings suggest that the comprehension of the neurosteroidogenic processes could be relevant to better understand the sexual dimorphism of MS and to possibly design sex-oriented therapeutic strategies based on neuroactive steroids

Neuroactive steroids, molecules produced from cholesterol in steroidogenic cells (i.e., peripheral glands and nervous system) are physiological modulators and protective agents of nervous function. A possible role for neuroactive steroids in the sex-dimorphic clinical manifestation, onset and progression of Multiple Sclerosis (MS) has been recently suggested. To explore this possibility, we assessed the synthesis of the first steroidogenic product (pregnenolone; PREG) in the spinal cord of experimental autoimmune encephalomyelitis rats, a MS model. Data obtained indicate that the synthesis of PREG in the spinal cord is altered by the pathology in a sex-dimorphic way and depending on the pathological progression. Indeed, in male spinal cord the synthesis was already decreased at the acute phase of the disease (i.e., 14 days post induction - dpi) and maintained low during the chronic phase (i.e., 45 dpi), while in females this effect was observed only at the chronic phase. Substrate availability had also a role in the sex-dimorphic kinetics. Indeed, at the chronic phase, male animals showed a reduction in the levels of free cholesterol coupled to alteration of cholesterol metabolism into oxysterols; these effects were not observed in female animals. These findings suggest that the comprehension of the neurosteroidogenic processes could be relevant to better understand the sexual dimorphism of MS and to possibly design sex-oriented therapeutic strategies based on neuroactive steroids.

Pregnenolone sulfate (PregS) is the precursor to all steroid hormones and is produced in neurons in an activity dependent manner. Studies have shown that PregS production is upregulated during certain critical periods of development, such as in the first year of life in humans, during adolescence, and during pregnancy. Conversely, PregS is decreased during aging, as well as in several neurodevelopmental and neurodegenerative conditions. There are several known targets of PregS, such as a positive allosteric modulator NMDA receptors, sigma1 receptor, and as a negative allosteric modulator of GABA-A receptors. Recently a transient receptor potential channel, TrpM3 has been shown to be activated by PregS. TrpM3 is a heat sensitive (between 33-40oC), non-selective cation channel that is outwardly rectifying. PregS has been shown to increase the frequency of post-synaptic currents in the hippocampus and developing cerebellum, induce calcium transients in a subset of retinal ganglion cells, and enhance memory formation in rodents. Furthermore, PregS mediated TrpM3 activation induces calcium dependent transcription of early immediate genes, suggesting that activation of this channel may produce lasting effects on cells and systems in which it is activated. Because PregS is abundant during critical periods of development, we hypothesized that it may play a significant role during development. Furthermore, the role of PregS or its receptor TrpM3, has not previously been well characterized in the retina. To address this question, in this dissertation, we examine the role of the neurosteroid PregS and its receptor, TrpM3, on retinal waves, which are characteristic of specific stages of synaptic development and connectivity. Briefly, we show that PregS induces a TrpM3 dependent prolonged calcium transient, which is absent in the TrpM3-/- animals and increases the correlation of cell participation in waves. We also show that TrpM3 increases the frequency of post-synaptic current

BackgroundTransient hypothalamic-pituitary-adrenal axis dysfunction occurs in critically ill foals with sepsis and neonatal maladjustment syndrome (NMS). Cortisol is the most commonly measured steroid. However, a complex interaction of various steroid compounds might play a role in pathophysiology of this disorder.ObjectiveTo identify steroid compounds present at high concentrations at birth that rapidly and steadily decrease within the first 7 days of life in healthy foals and that might be supportive diagnosis of NMS and other neonatal disorders.AnimalsTen healthy neonatal Quarter Horse foals (5 females and 5 males).MethodsProspective study. Blood was collected in heparinized tubes within 30 minutes after birth, and at 12, 24, 48, 72, 96, 120, 144, and 168 hours of age. Plasma was separated and a panel of steroid compounds was analyzed using liquid chromatography-mass spectrometry. A nonlinear regression model was used to determine decay concentrations over time. Confidence intervals (CIs) were calculated and significance was set a P ≤ .05.ResultsFive compounds were identified: pregnenolone, progesterone, deoxycorticosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. Pregnenolone and progesterone concentrations rapidly decreased by 24 hours of age and remained low throughout the first 7 days of life. Their half-life (95% CI) was short at 3.7 (3.4, 4.0) and 4.5 (2.8, 6.1) hours, respectively. No statistical differences in the concentrations of these compounds were found between males and females.Conclusions and clinical relevanceProgesterone might be a useful marker for identifying continuous endogenous production of neuroactive steroids in foals with suspected NMS and other neonatal diseases.

Pregnenolone sulfate (PregS) is the precursor to all steroid hormones and is produced in neurons in an activity dependent manner. Studies have shown that PregS production is upregulated during certain critical periods of development, such as in the first year of life in humans, during adolescence, and during pregnancy. Conversely, PregS is decreased during aging, as well as in several neurodevelopmental and neurodegenerative conditions. There are several known targets of PregS, such as a positive allosteric modulator NMDA receptors, sigma1 receptor, and as a negative allosteric modulator of GABA-A receptors. Recently a transient receptor potential channel, TrpM3 has been shown to be activated by PregS. TrpM3 is a heat sensitive (between 33-40oC), non-selective cation channel that is outwardly rectifying. PregS has been shown to increase the frequency of post-synaptic currents in the hippocampus and developing cerebellum, induce calcium transients in a subset of retinal ganglion cells, and enhance memory formation in rodents. Furthermore, PregS mediated TrpM3 activation induces calcium dependent transcription of early immediate genes, suggesting that activation of this channel may produce lasting effects on cells and systems in which it is activated. Because PregS is abundant during critical periods of development, we hypothesized that it may play a significant role during development. Furthermore, the role of PregS or its receptor TrpM3, has not previously been well characterized in the retina. To address this question, in this dissertation, we examine the role of the neurosteroid PregS and its receptor, TrpM3, on retinal waves, which are characteristic of specific stages of synaptic development and connectivity. Briefly, we show that PregS induces a TrpM3 dependent prolonged calcium transient, which is absent in the TrpM3-/- animals and increases the correlation of cell participation in waves. We also show that TrpM3 increases the frequency of post-synaptic current

BackgroundTransient hypothalamic-pituitary-adrenal axis dysfunction occurs in critically ill foals with sepsis and neonatal maladjustment syndrome (NMS). Cortisol is the most commonly measured steroid. However, a complex interaction of various steroid compounds might play a role in pathophysiology of this disorder.ObjectiveTo identify steroid compounds present at high concentrations at birth that rapidly and steadily decrease within the first 7 days of life in healthy foals and that might be supportive diagnosis of NMS and other neonatal disorders.AnimalsTen healthy neonatal Quarter Horse foals (5 females and 5 males).MethodsProspective study. Blood was collected in heparinized tubes within 30 minutes after birth, and at 12, 24, 48, 72, 96, 120, 144, and 168 hours of age. Plasma was separated and a panel of steroid compounds was analyzed using liquid chromatography-mass spectrometry. A nonlinear regression model was used to determine decay concentrations over time. Confidence intervals (CIs) were calculated and significance was set a P ≤ .05.ResultsFive compounds were identified: pregnenolone, progesterone, deoxycorticosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. Pregnenolone and progesterone concentrations rapidly decreased by 24 hours of age and remained low throughout the first 7 days of life. Their half-life (95% CI) was short at 3.7 (3.4, 4.0) and 4.5 (2.8, 6.1) hours, respectively. No statistical differences in the concentrations of these compounds were found between males and females.Conclusions and clinical relevanceProgesterone might be a useful marker for identifying continuous endogenous production of neuroactive steroids in foals with suspected NMS and other neonatal diseases.

The nervous system is a target and a source of steroids. Neuroactive steroids are steroids that target neurons and glial cells. They include hormonal steroids originated in the peripheral glands, steroids locally synthesized by the neurons and glial cells (neurosteroids) and synthetic steroids, some of them used in clinical practice. Here we review the mechanisms of synthesis, metabolism and action of neuroactive steroids, including the role of epigenetic modifications and the mitochondria in their sex specific actions. We examine sex differences in neuroactive steroid levels under physiological conditions and their role in the establishment of sex dimorphic structures in the nervous system and sex differences in its function. In addition, particular attention is paid to neuroactive steroids under pathological conditions, analyzing how pathology alters their levels and their role as neuroprotective factors, considering the influence of sex in both cases.

N-methyl-d-aspartate receptors (NMDARs) have multiple prominent roles in CNS function but their excessive or insufficient activity contributes to neuropathological/psychiatric disorders. Consequently, a variety of positive and negative allosteric modulators (PAMs and NAMs, respectively) have recently been developed. Although these modulators bind to extracellular domains, in the present report we find that the NMDAR's intracellular C-terminal domains (CTDs) significantly influence PAM/NAM activity. GluN2 CTD deletion robustly affected NAM and PAM activity with both enhancing and inhibiting effects that were compound-specific and NMDAR subunit-specific. In three cases, individual PAMs became NAMs at specific GluN2-truncated receptors. In contrast to GluN2, GluN1 CTD removal only reduced PAM activity of UBP684 and CIQ, and did not affect NAM activity. Consistent with these findings, agents altering phosphorylation state or intracellular calcium levels displayed receptor-specific and compound-specific effects on PAM activity. It is possible that the GluN2's M4 domain transmits intracellular modulatory signals from the CTD to the M1/M4 channel gating machinery and that this site is a point of convergence in the direct or indirect actions of several PAMs/NAMs thus rendering them sensitive to CTD status. Thus, allosteric modulators are likely to have a marked and varied sensitivity to post-translational modifications, protein-protein associations, and intracellular ions. The interaction between PAM activity and NMDAR CTDs appears reciprocal. GluN1 CTD-deletion eliminated UBP684, but not pregnenolone sulfate (PS), PAM activity. And, in the absence of agonists, UBP684, but not PS, was able to promote movement of fluorescently-tagged GluN1-CTDs. Thus, it may be possible to pharmacologically target NMDAR metabotropic activity in the absence of channel activation.

Aim and Objective: The development of small molecules that can interact with key therapeutic target represents an active field of research. Therefore, new approaches for increasing the molecular diversity of a starting material, such as a natural product, are needed. Herein, the carbonyl group present on a pregnane scaffold, or easily obtained from the oxidation of the corresponding alcohol, was used to obtain a series of diversified steroidal morpholinone derivatives. Materials and Methods: Using chemical synthesis, two levels of molecular diversity were introduced at position 20 of a C21-steroid scaffold. Nuclear magnetic resonance (NMR) spectroscopy and x-ray analysis were next used to characterize the morpholinone derivatives. Results: The C-20 carbonyl of pregnenolone was first transformed into an oxirane that reacted with an amino acid and the resulting amino alcohol was then cyclized to generate different spiro-2-morpholinones. X-ray analysis of one representative compound confirmed the 3-dimensional representation of this new family of diversified steroid derivatives. NMR analysis supported the expected structure and identified key markers of the chiral center configuration found in the 2-morpholinone moiety. Finally, the NH of the morpholinone ring was alkylated, thus increasing structural diversity. Conclusion: Considering the huge amount of building blocks (amino acids, bromobenzyl derivatives and ketones) that are commercially available, the strategy reported herein opens the door to the synthesis of diversified libraries of new compounds.

The calcium channel of sperm (CatSper) is essential for sperm hyperactivated motility and fertility. The steroid hormone progesterone activates CatSper of human sperm via binding to the serine hydrolase ABHD2. However, steroid specificity of ABHD2 has not been evaluated. Here, we explored whether steroid hormones to which human spermatozoa are exposed in the male and female genital tract influence CatSper activation via modulation of ABHD2. The results show that testosterone, estrogen, and hydrocortisone did not alter basal CatSper currents, whereas the neurosteroid pregnenolone sulfate exerted similar effects as progesterone, likely binding to the same site. However, physiological concentrations of testosterone and hydrocortisone inhibited CatSper activation by progesterone. Additionally, testosterone antagonized the effect of pregnenolone sulfate. We have also explored whether steroid-like molecules, such as the plant triterpenoids pristimerin and lupeol, affect sperm fertility. Interestingly, both compounds competed with progesterone and pregnenolone sulfate and significantly reduced CatSper activation by either steroid. Furthermore, pristimerin and lupeol considerably diminished hyperactivation of capacitated spermatozoa. These results indicate that (i) pregnenolone sulfate together with progesterone are the main steroids that activate CatSper and (ii) pristimerin and lupeol can act as contraceptive compounds by averting sperm hyperactivation, thus preventing fertilization.

Neuroactive steroids regulate the physiology of the central and peripheral nervous system, exert neuroprotective actions and represent interesting tools for therapeutic strategies against neurodegenerative and psychiatric disorders. Sex differences in their levels are detected not only under physiological conditions but are also modified in a sex-dependent way in different pathological alterations such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, traumatic brain injury, spinal cord injury, stroke, diabetic encephalopathy, psychiatric disorders and peripheral neuropathy. Interestingly, many of these disorders show sex differences in their incidence, symptomatology and/or neurodegenerative outcome. The neuroprotective actions of neuroactive steroids, together with the sex specific regulation of its levels might provide the basis to design sex-specific neuroprotective therapies. Indeed, some experiments here discussed suggest the viability of this approach.

Synthesis of the androgen dehydroepiandrosterone (DHEA) by the fetal adrenal gland is important for placental estrogen production and may also be important for modulating the effects of glucocorticoids on the developing brain. The presence of cortisol in spiny mouse (Acomys cahirinus) blood led us to determine whether the adrenal gland of this precocial rodent also synthesized DHEA. Cytochrome P450 enzyme 17alpha-hydroxylase/17,20-lyase (P450c17), cytochrome-b5 (Cytb5), and 3beta-hydroxysteroid dehydrogenase (3betaHSD) were detected in the adrenal gland from 30 days gestation (term=39 days), and DHEA, cortisol, and aldosterone were detected in fetal plasma from this time. PlasmaDHEAconcentrations increased 4-fold, whereas cortisol concentrations decreased from day 30 of gestation until the day of birth. Explant culture of fetal adrenal tissue showed that DHEA was produced from exogenous pregnenolone, and thus, the DHEA in the fetal circulation is likely to be of fetal origin. Clear zonation of the fetal adrenal cortex was evident by 38 days gestation when expression of Cytb5 was present throughout the cortex, and coexpression of P450c17 and Cytb5 occurred in the zona reticularis and fasciculata. 3betaHSD was expressed in the cortex from at least 30 days gestation and decreased as term approached, consistent with the fall of cortisol in late gestation in this species. These results show that the spiny mouse adrenal gland, like that of the human fetus, can synthesize and secrete DHEA from at least 30 days (relative gestation length, 30 days of a 39-day gestation, 0.76) of gestation, and DHEA may have important roles in placental biosynthesis of estrogens and in modulating the actions of glucocorticoids in the developing brain in this species. Copyright © 2013 by The Endocrine Society.

Synthesis of the androgen dehydroepiandrosterone (DHEA) by the fetal adrenal gland is important for placental estrogen production and may also be important for modulating the effects of glucocorticoids on the developing brain. The presence of cortisol in spiny mouse (Acomys cahirinus) blood led us to determine whether the adrenal gland of this precocial rodent also synthesized DHEA. Cytochrome P450 enzyme 17alpha-hydroxylase/17,20-lyase (P450c17), cytochrome-b5 (Cytb5), and 3beta-hydroxysteroid dehydrogenase (3betaHSD) were detected in the adrenal gland from 30 days gestation (term=39 days), and DHEA, cortisol, and aldosterone were detected in fetal plasma from this time. PlasmaDHEAconcentrations increased 4-fold, whereas cortisol concentrations decreased from day 30 of gestation until the day of birth. Explant culture of fetal adrenal tissue showed that DHEA was produced from exogenous pregnenolone, and thus, the DHEA in the fetal circulation is likely to be of fetal origin. Clear zonation of the fetal adrenal cortex was evident by 38 days gestation when expression of Cytb5 was present throughout the cortex, and coexpression of P450c17 and Cytb5 occurred in the zona reticularis and fasciculata. 3betaHSD was expressed in the cortex from at least 30 days gestation and decreased as term approached, consistent with the fall of cortisol in late gestation in this species. These results show that the spiny mouse adrenal gland, like that of the human fetus, can synthesize and secrete DHEA from at least 30 days (relative gestation length, 30 days of a 39-day gestation, 0.76) of gestation, and DHEA may have important roles in placental biosynthesis of estrogens and in modulating the actions of glucocorticoids in the developing brain in this species. Copyright © 2013 by The Endocrine Society.

There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases-confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development.

The nervous system is a steroidogenic tissue and several steroids synthesized locally in the brain, such as pregnenolone, progesterone and estradiol, modulate neuronal and glial physiology and are neuroprotective. The brain upregulates steroidogenesis at sites of injury as part of a program triggered by neural tissue to cope with neurodegenerative insults. Pharmacological targets to increase brain steroidogenesis and promote neuroprotection include the molecules that transport cholesterol to the inner mitochondrial membrane, where the first enzyme for steroidogenesis is located. Furthermore, the human gene encoding aromatase, the enzyme that synthesizes estradiol, is under the control of different tissue-specific promoters, and it is therefore conceivable that selective aromatase modulators can be developed that will enhance the expression of the enzyme and the consequent increase in estrogen formation in the brain but not in other tissues.

Peripheral neuropathy represents an important complication of diabetes involving a spectrum of structural, functional and biochemical alterations in peripheral nerves. Recent observations obtained in our laboratory have shown that the levels of neuroactive steroids present in the sciatic nerve of rat raised diabetic by a single injection of streptozotocin (STZ) are reduced and that, in the same experimental model, treatment with neuroactive steroids, such as progesterone, testosterone and their derivatives show neuroprotective effects. On this basis, an interesting therapeutic strategy could be to increase the levels of neuroactive steroids directly in the nervous system. With this perspective, ligands of translocator protein-18 kDa (TSPO) may represent an interesting option. TSPO is mainly present in the mitochondrial outer membrane, where it promotes the translocation of cholesterol to the inner mitochondrial membrane, and, as demonstrated in other cellular systems, it allows the transformation of cholesterol into pregnenolone and the increase of steroid levels. In the diabetic model of STZ rat, we have here assessed whether treatment with Ro5-4864 (i.e., a ligand of TSPO) could increase the low levels of neuroactive steroids in sciatic nerve and consequently to be protective in this experimental model. Data obtained by liquid chromatography-tandem mass spectrometry show that treatment with Ro5-4864 was able to significantly stimulate the low levels of pregnenolone, progesterone and dihydrotestosterone observed in the sciatic nerves of diabetic rats. The treatment with Ro5-4864 also counteracted the impairment of NCV and thermal threshold, restored skin innervation density and P0 mRNA levels, and improved Na+,K+-ATPase activity. In conclusion, data here reported show for the first time that a TSPO ligand, such as Ro5-4864, is effective in reducing the severity of diabetic neuropathy through a local increase of neuroactive steroid levels

Neurosteroids are endogenously derived compounds, mediating rapid effects in the central nervous system. They participate in vital processes, including memory and learning, neuroplasticity, and neuroprotection in Alzheimer's disease. However, the mechanisms behind those effects remain to be elucidated. The neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3alpha5betaS) have recently been shown to allosterically alter the NMDA receptor in nanomolar concentrations. Those studies featured ifenprodil, which is a dirty drug, with affinity to many targets. In this study we compare the NMDA receptors in the hippocampus to recombinant NMDA receptors, using [3H]-MK-801 as radioligand. The results show that neurosteroids modulate the ifenprodil binding kinetics in a narrow concentration interval, addressing it to the NR2B subunit, since no effects were recorded at recombinant NR1/NR2A receptors. The effects were also seen as changes in the manner ifenprodil displaced or induced the dissociation of [3H]-MK-801. It indicates that the neurosteroidal effects indeed alter the ion pore of the NMDA receptor, why it is reasonable to believe that these findings have physiological relevance.

La transcription des symboles et des caractères spéciaux utilisés dans la version originale de ce résumé n’a pas été possible en raison de limitations techniques. La version correcte de ce résumé peut être lue en PDF. Le contrôle du dopage sportif par les stéroïdes pouvant potentiellement se retrouver dans le corps humain de façon naturelle est très compliqué. En effet, les concentrations de ces stéroïdes peuvent varier naturellement de façon substantielle d'un individu à l'autre rendant difficile l'utilisation d'une fourchette de valeurs normales à des fins de référence. De plus, la méthode de dépistage par chromatographie en phase gazeuse et spectrométrie de masse (CG/SM) ne permet pas de différencier l'origine endogène ou exogène d'un stéroïde urinaire. C'est ici qu'entre en jeu la méthode de confirmation par la spectrométrie de masse des rapports isotopiques (CG/C/SMRI). Cette méthode, dont l'utilisation dans le domaine du contrôle de dopage sportif a été proposée en 1994, permet la différenciation des stéroïdes de sources endogène et exogène par l'analyse de leur teneur en carbone 13, isotope naturel du carbone (12C) Une première partie du projet était de vérifier la possibilité d'analyser des norstéroïdes par CG/C/SMRI. Cette analyse est limitée due aux très petites concentrations des métabolites norandrostérone (NA) et norétiocholanolone (NE) d'origine endogène retrouvées parfois dans les urines. Afin de vérifier la sensibilité de notre méthode, nous avons utilisé les urines recueillies dans le cadre d'une étude d'excrétion réalisée avec une crème à base de norandrostènedione et de norandrostènediol vendue commercialement aux États-Unis. Bien que nous ayons réussi à identifier les pics correspondants à la NA et la NE exogènes, la méthode utilisée ne nous donne pas la sensibilité requise pour l'analyse de traces. Le même problème de sensibilité est rencontré lors des analyses de rapports isotopiques de la testostérone urinaire. Généralement, sa concentration d

La transcription des symboles et des caractères spéciaux utilisés dans la version originale de ce résumé n’a pas été possible en raison de limitations techniques. La version correcte de ce résumé peut être lue en PDF.La détection de stéroïdes anabolisants androgènes pouvant être présents naturellement chez l'humain représente un réel défi pour les laboratoires impliqués dans la lutte au dopage sportif. L'analyse des modifications du profil d'excrétion des métabolites urinaires doit être effectuée afin de conclure à une administration de testostérone ou de l'un de ses précurseurs disponibles commercialement au Etats-Unis ou via l'internet. Des travaux rapportés par Becchi et al. ont indiqué la possibilité d'utiliser la spectrométrie de masse d'isotopes stables du carbone pour déterminer l'origine, endogène ou exogène, de métabolites urinaires (Becchi, Aguilera, Farizon, Flament, Casabianca, James. 1994. Rapid Communication in Mass Spectrometry, vol. 8, p. 304-308). Nous avons décidé de valider des applications pratiques de la spectrométrie de masse d'isotopes stables du carbone à l'identification formelle de stéroïdes "naturels" d'origine synthétique précurseurs de la testostérone. Dans un premier temps, nous nous sommes attardés aux aspects de validation de l'utilisation de cette teclmique soit à déterminer les performances du spectromètre de masse couplé à un chromatographe à phase gazeuse (CG/C/SMRI) pour la précision et le domaine de linéarité des analyses. En vue d'améliorer l'analyse chromatographique et de diminuer le fractionnement isotopique, nous avons opté pour l'optimisation de la méthode d'extraction sélective rapportée par le groupe de Shackleton reposant sur la formation de dérivés hydrazones par l'emploi du réactif de Girard (Shackleton, Phillips, Chang et Li. 1997A. Steroids, vol. 62, p. 379-387). Les mises au point techniques nous ont permis d'obtenir une meilleure résolution chromatographique des métabolites urinaires ainsi qu'une amélior

La transcription des symboles et des caractères spéciaux utilisés dans la version originale de ce résumé n’a pas été possible en raison de limitations techniques. La version correcte de ce résumé peut être lue en PDF.La détection de stéroïdes anabolisants androgènes pouvant être présents naturellement chez l'humain représente un réel défi pour les laboratoires impliqués dans la lutte au dopage sportif. L'analyse des modifications du profil d'excrétion des métabolites urinaires doit être effectuée afin de conclure à une administration de testostérone ou de l'un de ses précurseurs disponibles commercialement au Etats-Unis ou via l'internet. Des travaux rapportés par Becchi et al. ont indiqué la possibilité d'utiliser la spectrométrie de masse d'isotopes stables du carbone pour déterminer l'origine, endogène ou exogène, de métabolites urinaires (Becchi, Aguilera, Farizon, Flament, Casabianca, James. 1994. Rapid Communication in Mass Spectrometry, vol. 8, p. 304-308). Nous avons décidé de valider des applications pratiques de la spectrométrie de masse d'isotopes stables du carbone à l'identification formelle de stéroïdes "naturels" d'origine synthétique précurseurs de la testostérone. Dans un premier temps, nous nous sommes attardés aux aspects de validation de l'utilisation de cette teclmique soit à déterminer les performances du spectromètre de masse couplé à un chromatographe à phase gazeuse (CG/C/SMRI) pour la précision et le domaine de linéarité des analyses. En vue d'améliorer l'analyse chromatographique et de diminuer le fractionnement isotopique, nous avons opté pour l'optimisation de la méthode d'extraction sélective rapportée par le groupe de Shackleton reposant sur la formation de dérivés hydrazones par l'emploi du réactif de Girard (Shackleton, Phillips, Chang et Li. 1997A. Steroids, vol. 62, p. 379-387). Les mises au point techniques nous ont permis d'obtenir une meilleure résolution chromatographique des métabolites urinaires ainsi qu'une amélioration du s

[ES]El sistema nervioso es una diana de las hormonas esteroides y también un tejido esteroidogénico, y produce esteroides que actúan de una forma paracrina o autocrina sobre neuronas y glía. Los esteroides formados en el tejido nervioso se denominan neuroesteroides, para diferenciarlos, por su origen, de los esteroides periféricos, aunque tienen la misma estructura molecular que éstos. Desarrollo. Se analiza la capacidad de las neuronas y las células de glía de sintetizar esteroides, y se describe el papel de algunas moléculas clave en la esteroidogénesis, como la proteína de regulación aguda de esteroidogénesis, el receptor periférico de benzodiacepinas y la enzima aromatasa, que cataliza la conversión de testosterona en estradiol. También se describen los diversos mecanismos de acción de los esteroides hormonales y los neuroesteroides en el sistema nervioso. Éstos incluyen, tanto la regulación de la síntesis de proteínas por neuronas y glía, mediante acciones sobre receptores nucleares, como efectos rápidos mediados por receptores de membrana o la modulación alostérica de receptores para neurotransmisores. Se revisan las evidencias clínicas y experimentales de los efectos neuroprotectores de los esteroides sexuales y neuroesteroides y las limitaciones de la terapia hormonal sustitutiva, tras la menopausia. Conclusiones. Dada la limitación que plantea el uso sistémico de hormonas como terapia neuroprotectora, se necesitan encontrar estrategias alternativas que aprovechen las propiedades neuroprotectoras de los esteroides, tales como aumentar localmente su síntesis en el cerebro o desarrollar moléculas que activen a los receptores de esteroides en el sistema nervioso y no en los órganos periféricos, [EN]The nervous system is a target for steroid hormones as well as a steroidogenic tissue, and it produces steroids that have a paracrine or autocrine effect on neurons and glial cells. Steroids formed in nervous tissue are called neurosteroids in order to differentiate them, in terms of their origin, from the peripheral steroids, although they both share the same molecular structure. Development. We analyse the capacity of neurons and glial cells to synthesise steroids and describe the role played in steroidogenesis by certain key molecules, such as steroidogenic acute regulatory protein, peripheral benzodiazepine receptor and aromatase enzyme, which acts as a catalyst in the conversion of testosterone into estradiol. We also provide a description of the different mechanisms of action of the hormonal steroids and neurosteroids in the nervous system. These include both the regulation of protein synthesis by neurons and glial cells, by acting on nuclear receptors, and rapid effects mediated by membrane receptors or the allosteric modulation of neurotransmitter receptors. We review the clinical and experimental evidence for the neuroprotective effects of sex steroids and neurosteroids, and the limitations of hormone replacement therapy following menopause. Conclusions. Given the restraints involved in the systemic use of hormones as neuroprotective therapy, alternative strategies that take advantage of the neuroprotective properties of steroids must be sought. These could involve locally increasing their synthesis inside the brain or developing molecules that activate the steroid receptors in the nervous system and not in the peripheral organs

It has been observed that testicular macrophages and testicular macrophage-conditioned medium reduce LH-stimulated, but not basal, testosterone production by purified Leydig cells in vitro. In order to determine how this inhibition occurs, we have examined the effects of testicular macrophages and testicular macrophage-conditioned medium at discrete stages of the steroidogenic pathway. The lesion in steroidogenesis is located at a step beyond cAMP formation, because the addition of dibutyryl cAMP or cholera toxin did not overcome the testicular macrophage-conditioned medium inhibition of LH-stimulated steroidogenesis by Leydig cells. This effect of testicular macrophage-conditioned medium on Leydig cell testosterone production is first observed at 18 h after initiation of culture. However, subsequent additions of 22R-hydroxycholesterol, pregnenolone, dehydroepiandrosterone, or androstenedione to the Leydig cell cultures can overcome the inhibition so that, after a further 6 h of incubation, testosterone production is not significantly different from that of control Leydig cells cultured in the absence of testicular macrophage- conditioned medium. These results suggest that the block in steroidogenesis is beyond cAMP production but prior to the formation of pregnenolone, dehydroepiandrosterone, and androstenedione. Since the medium for these cultures contained lipoprotein, it is possible that the testicular macrophage-conditioned medium metabolizes the lipoprotein, making it unavailable to the Leydig cells. However, our results show that preincubation of lipoprotein with testicular macrophage-conditioned medium does not significantly alter testosterone production by the Leydig cells in the culture. It is concluded that testicular macrophage-conditioned medium affects the transport or availability of cholesterol to mitochondria prior to further steps in the steroidogenic pathway.

It has been observed that testicular macrophages and testicular macrophage-conditioned medium reduce LH-stimulated, but not basal, testosterone production by purified Leydig cells in vitro. In order to determine how this inhibition occurs, we have examined the effects of testicular macrophages and testicular macrophage-conditioned medium at discrete stages of the steroidogenic pathway. The lesion in steroidogenesis is located at a step beyond cAMP formation, because the addition of dibutyryl cAMP or cholera toxin did not overcome the testicular macrophage-conditioned medium inhibition of LH-stimulated steroidogenesis by Leydig cells. This effect of testicular macrophage-conditioned medium on Leydig cell testosterone production is first observed at 18 h after initiation of culture. However, subsequent additions of 22R-hydroxycholesterol, pregnenolone, dehydroepiandrosterone, or androstenedione to the Leydig cell cultures can overcome the inhibition so that, after a further 6 h of incubation, testosterone production is not significantly different from that of control Leydig cells cultured in the absence of testicular macrophage- conditioned medium. These results suggest that the block in steroidogenesis is beyond cAMP production but prior to the formation of pregnenolone, dehydroepiandrosterone, and androstenedione. Since the medium for these cultures contained lipoprotein, it is possible that the testicular macrophage-conditioned medium metabolizes the lipoprotein, making it unavailable to the Leydig cells. However, our results show that preincubation of lipoprotein with testicular macrophage-conditioned medium does not significantly alter testosterone production by the Leydig cells in the culture. It is concluded that testicular macrophage-conditioned medium affects the transport or availability of cholesterol to mitochondria prior to further steps in the steroidogenic pathway.

In an attempt to delineate the effect of corticotropin (ACTH) on post-pregnenolone steroidogenesis, the activity of enzymatic systems operative in conversion of pregnenolone into glucocorticoids and androgens was studied in adrenocortical cells from control rabbits and from animals treated with ACTH for 12 days (ACTH 1-24, 200 micrograms s.c. daily). The cells from ACTH-treated rabbits exhibited an increased overall steroidogenic capacity and produced much more cortisol (P less than 0.0005) as well as other 17-hydroxylated steroids as a result of increased activity of 17 alpha-hydroxylase; corticosterone generation was concomitantly reduced. The increased conversion of pregnenolone or progesterone into androgens, as a result of previous treatment with ACTH, provides additional evidence for an effect of ACTH on 17 alpha-hydroxylase activity. A stimulatory effect of ACTH on 11 beta-hydroxylase was also evidenced by these cells, since conversion of 11-deoxycortisol into cortisol was enhanced (P less than 0.005). The increased production of androgens from 17-hydroxylated precursors by cells from ACTH-treated rabbits suggests that ACTH also exerts a prolonged stimulatory effect on 17,20-lyase. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was apparently not influenced by chronic treatment with ACTH, judged from unchanged conversion of dehydroepiandrosterone into androstenedione. The activity of 11 beta-dehydrogenase was likewise unchanged in these conditions.