Jean Luc Do Rego

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17α-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17‐hydroxypregnenolone (17OH‐Δ5P), 17‐hydroxyprogesterone (17OH‐P), dehydroepiandrosterone (DHEA) and androstenedione (Δ4). However, the presence, distribution and activity of cytochrome P450 17α‐hydroxylase/C17, 20‐lyase (P450C17), a key enzyme required for the conversion of pregnenolone (Δ5P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450C17‐like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450C17‐containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450C17‐like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450C17‐positive cell bodies also contained 3β‐hydroxysteroid dehydrogenase‐like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [3H]Δ5P resulted in the formation of several tritiated steroids including 17OH‐Δ5P, 17OH‐P, DHEA and Δ4. De novo synthesis of C21 17‐hydroxysteroids and C19 ketosteroids was reduced in a concentration‐dependent manner by ketoconazole, a P450C17 inhibitor. This is the first detailed immunohistochemical mapping of P450C17 in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.

Molecular Coevolution of Neuropeptides Gonadotropin-Releasing Hormone and Kisspeptin with their Cognate G Protein-Coupled Receptors

The neuropeptides gonadotropin-releasing hormone (GnRH) and kisspeptin (KiSS), and their receptors gonadotropin-releasing hormone receptor (GnRHR) and kisspeptin receptor (KiSSR) play key roles in vertebrate reproduction. Multiple paralogous isoforms of these genes have been identified in various vertebrate species. Two rounds of genome duplication in early vertebrates likely contributed to the generation of these paralogous genes. Genome synteny and phylogenetic analyses in a variety of vertebrate species have provided insights into the evolutionary origin of and relationship between paralogous genes. The paralogous forms of these neuropeptides and their receptors have coevolved to retain high selectivity of the ligand–receptor interaction. These paralogous forms have become subfunctionalized, neofunctionalized, or dysfunctionalized during evolution. This article reviews the evolutionary mechanism of GnRH/GnRHR and KiSS/KiSSR, and the fate of the duplicated paralogs in vertebrates.

Immunohistochemical detection and biological activities of CYP17 (P450c17) in the indifferent gonad of the frog Rana rugosa.

Sex steroids play a crucial role in the gonad differentiation in various species of vertebrates. However, little is known regarding the localization and biological activity of steroid-metabolizing enzymes during gonadal sex differentiation in amphibians. In the present study, we showed by real-time RT-PCR analysis that the expression of CYP17, one of the key steroidogenic enzymes, was higher in the indifferent gonad during sex differentiation in male than in female tadpoles of Rana rugosa but that there was no difference detected in the 3betaHSD mRNA level between the male and female gonads. We next examined the localization of CYP17, 3betaHSD and 17betaHSD in the indifferent and differentiating gonads by using three kinds of antibodies specific for CYP17, 3betaHSD and 17betaHSD, respectively. Positive signals for CYP17, 3betaHSD and 17betaHSD were observed in somatic cells of the indifferent gonad of males and in the interstitial cell of the testis. The enzymatic activity of CYP17 was also examined in the gonad during sex differentiation in this species. [(3)H]Progesterone (Prog) was converted to [(3)H]androstenedione (AE) in the indifferent gonad in males and females, but the rate of its conversion was higher in males than in females. Moreover, fluorescence in situ hybridization (FISH) analysis revealed that the CYP17 gene was located on the q arm of chromosome 9, indicating that CYP17 was autosomal in R. rugosa. Taken together, the results demonstrate that the CYP17 protein is synthesized in somatic cells of the indifferent gonad during gonadal sex differentiation in R. rugosa and that it is more active in converting Prog to AE in males than in females. The data suggest that CYP17 may be involved in testicular formation during sex differentiation in this species.

Regulation of neurosteroid biosynthesis by neurotransmitters and neuropeptides

The enzymatic pathways leading to the synthesis of bioactive steroids in the brain are now almost completely elucidated in various groups of vertebrates and, during the last decade, the neuronal mechanisms involved in the regulation of neurosteroid production have received increasing attention. This report reviews the current knowledge concerning the effects of neurotransmitters, peptide hormones, and neuropeptides on the biosynthesis of neurosteroids. Anatomical studies have been carried out to visualize the neurotransmitter- or neuropeptide-containing fibers contacting steroid-synthesizing neurons as well as the neurotransmitter, peptide hormones, or neuropeptide receptors expressed in these neurons. Biochemical experiments have been conducted to investigate the effects of neurotransmitters, peptide hormones, or neuropeptides on neurosteroid biosynthesis, and to characterize the type of receptors involved. Thus, it has been found that glutamate, acting through kainate and/or AMPA receptors, rapidly inactivates P450arom, and that melatonin produced by the pineal gland and eye inhibits the biosynthesis of 7α-hydroxypregnenolone (7α-OH-Δ5P), while prolactin produced by the adenohypophysis enhances the formation of 7α-OH-Δ5P. It has also been demonstrated that the biosynthesis of neurosteroids is inhibited by GABA, acting through GABAA receptors, and neuropeptide Y, acting through Y1 receptors. In contrast, it has been shown that the octadecaneuropetide ODN, acting through central-type benzodiazepine receptors, the triakontatetraneuropeptide TTN, acting though peripheral-type benzodiazepine receptors, and vasotocin, acting through V1a-like receptors, stimulate the production of neurosteroids. Since neurosteroids are implicated in the control of various neurophysiological and behavioral processes, these data suggest that some of the neurophysiological effects exerted by neurotransmitters and neuropeptides may be mediated via the regulation of neurosteroid production.

Acute Stress Increases the Synthesis of 7α-Hydroxypregnenolone, a New Key Neurosteroid Stimulating Locomotor Activity, through Corticosterone Action in Newts

7α-Hydroxypregnenolone (7α-OH PREG) is a newly identified bioactive neurosteroid stimulating locomotor activity in the brain of newt, a wild animal, which serves as an excellent model to investigate the biosynthesis and biological action of neurosteroids. Here, we show that acute stress increases 7α-OH PREG synthesis in the dorsomedial hypothalamus (DMH) through corticosterone (CORT) action in newts. A 30-min restraint stress increased 7α-OH PREG synthesis in the brain tissue concomitant with the increase in plasma CORT concentrations. A 30-min restraint stress also increased the expression of cytochrome P450(7α) (CYP7B), the steroidogenic enzyme of 7α-OH PREG formation, in the DMH. Decreasing plasma CORT concentrations by hypophysectomy or trilostane administration decreased 7α-OH PREG synthesis in the diencephalon, whereas administration of CORT to these animals increased 7α-OH PREG synthesis. Glucocorticoid receptor was present in DMH neurons expressing CYP7B. Thus, CORT appears to act directly on DMH neurons to increase 7α-OH PREG synthesis. We further investigated the biological action of 7α-OH PREG in the brain under stress. A 30-min restraint stress or central administration of 7α-OH PREG increased serotonin concentrations in the diencephalon. Double immunolabeling further showed colocalization of CYP7B and serotonin in the DMH. These results indicate that acute stress increases the synthesis of 7α-OH PREG via CORT action in the DMH, and 7α-OH PREG activates serotonergic neurons in the DMH that may coordinate behavioral responses to stress. This is the first demonstration of neurosteroid biosynthesis regulated by peripheral steroid hormone and of neurosteroid action in the brain under stress in any vertebrate class.

Identification of 7α-hydroxypregnenolone, a novel bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion

We now know that steroids can be synthesized de novo by the brain and the peripheral nervous system. Such steroids are called neurosteroids and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. Our studies over the past decade have demonstrated that the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids in sub-mammalian species. However, neurosteroid biosynthetic pathways in amphibians, as well as other vertebrates may still not be fully mapped. We first found that the newt brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. We then demonstrated that 7alpha-hydroxypregnenolone acts as a novel bioactive neurosteroid to stimulate locomotor activity of newt by means of the dopaminergic system. Subsequently, we analyzed the physiological roles of 7alpha-hydroxypregnenolone in the regulation of locomotor activity of newt. This paper summarizes the advances made in our understanding of 7alpha-hydroxypregnenolone, a newly discovered bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion in newt.

Comparative aspects of neurosteroidogenesis: From fish to mammals.

It is now clearly established that the central and peripheral nervous systems have the ability to synthesize de novo steroids referred to as neurosteroids. The major evidence for biosynthesis of neuroactive steroids by nervous tissues is based on the expression of enzymes implicated in the formation of steroids in neural cells. The aim of the present review is to summarize the current knowledge regarding the presence of steroidogenic enzymes in the brain of vertebrates and to highlight the very considerable contribution of Professor Kazuyoshi Tsutsui in this domain. The data indicate that expression of steroid-producing enzymes in the brain appeared early during vertebrate evolution and has been preserved from fish to mammals.

The Non-Benzodiazepine Anxiolytic Drug Etifoxine Causes a Rapid, Receptor-Independent Stimulation of Neurosteroid Biosynthesis

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

Steroid biosynthesis within the frog brain: A model of neuroendocrine regulation

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 [3H]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 γ‐aminobutyric acid (GABA), acting through GABAA 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.

Maintaining physical activity during refeeding improves body composition, intestinal hyperpermeability and behavior in anorectic mice

A role of gut-brain axis emerges in the pathophysiology of anorexia nervosa and maintaining adapted physical activity during refeeding remains discussed. We aimed to assess gastrointestinal protein metabolism and investigate the contribution of physical activity during refeeding in C57BL/6 mice with activity-based anorexia (ABA). ABA mice exhibited lower body weight and food intake with increase of lean mass/fat mass ratio and fat oxidation. Colonic permeability was increased in ABA. Ad libitum food access was then restored and ABA group was divided into two subgroups, with access to running wheel (ABA-PA) or not (ABA-NPA). After refeeding, fat free mass was completely restored only in ABA-PA. Colonic permeability was enhanced in ABA-NPA. Finally, muscle kynurenine conversion into kynurenic acid was lower in ABA-NPA who also exhibited altered behavior. Maintaining physical activity during refeeding may thus limit colonic hyperpermeability and improve behavior in anorectic mice.