Vassilios Papadopoulos

In Utero Exposure to Di-(2-Ethylhexyl) Phthalate Decreases Mineralocorticoid Receptor Expression in the Adult Testis

In utero exposure to di-(2-ethylhexyl) phthalate (DEHP) has been shown to result in decreased androgen formation by fetal and adult rat testes. In the fetus, decreased androgen is accompanied by the reduced expression of steroidogenic enzymes. The mechanism by which in utero exposure results in reduced androgen formation in the adult, however, is unknown. We hypothesized that deregulation of the nuclear steroid receptors might explain the effects of in utero DEHP exposure on adult testosterone production. To test this hypothesis, pregnant Sprague Dawley dams were gavaged with 100-950 mg DEHP per kilogram per day from gestational d 14-19, and testes were collected at gestational d 20 and postnatal days (PND) 3, 21, and 60. Among the nuclear receptors studied, the mineralocorticoid receptor (MR) mRNA and protein levels were reduced in PND60 interstitial Leydig cells, accompanied by reduced mRNA expression of MR-regulated genes. Methylation-sensitive PCR showed effects on the nuclear receptor subfamilies NR3A and -3C, but only MR was affected at PND60. Pyrosequencing of two CpG islands within the MR gene promoter revealed a loss of methylation in DEHP-treated animals that was correlated with reduced MR. Because MR activation is known to stimulate Leydig cell testosterone formation, and MR inhibition to be repressive, our results are consistent with the hypothesis that in utero exposure to DEHP leads to MR dysfunction and thus to depressed testosterone production in the adult. We suggest that decreased MR, possibly epigenetically mediated, is a novel mechanism by which phthalates may affect diverse functions later in life.

Translocator protein (18 kDa): an update on its function in steroidogenesis

Translocator protein (18 kDa) (TSPO) is a ubiquitous mitochondrial protein. Studies of its responses to drug and endogenous ligands have shown TSPO to be involved either directly or indirectly in numerous biological functions, including mitochondrial cholesterol transport and steroid hormone biosynthesis, porphyrin transport and heme synthesis, apoptosis, cell proliferation, and anion transport. Localised to the outer mitochondrial membrane of steroidogenic cells, TSPO has been shown to associate with cytosolic and mitochondrial proteins as part of a large multiprotein complex involved in mitochondrial cholesterol transport, the rate‐limiting step in steroidogenesis. There is general agreement as to the structure and pharmacology of TSPO. Stimulation of TSPO has been shown to have therapeutic use as anxiolytics by inducing allopregnanolone production in the brain, and also potentially for re‐establishing androgen levels in hypogonadal ageing animals. Until recently, there has been general agreement regarding the role of TSPO in steroidogenesis. However, recent studies involving genetic depletion of TSPO in mice have created controversy about the role of this protein in steroid and heme synthesis. We review the data on the structure and function of TSPO, as well as the recent results obtained using various genetic animal models. Taken together, these studies suggest that TSPO is a unique mitochondrial pharmacological target for diseases that involve increased mitochondrial activity, including steroidogenesis. Although there is no known mammalian species that lacks TSPO, it is likely that, because of the importance of this ancient protein in evolution and mitochondrial function, redundant mechanisms may exist to replace it under circumstances when it is removed.

TSPO mutations in rats and a human polymorphism impair the rate of steroid synthesis

The 18u2005kDa translocator protein (TSPO) is a ubiquitous conserved outer mitochondrial membrane protein implicated in numerous cell and tissue functions, including steroid hormone biosynthesis, respiration, cell proliferation, and apoptosis. TSPO binds with high affinity to cholesterol and numerous compounds, is expressed at high levels in steroid-synthesizing tissues, and mediates cholesterol import into mitochondria, which is the rate-limiting step in steroid formation. In humans, the rs6971 polymorphism on the TSPO gene leads to an amino acid substitution in the fifth transmembrane loop of the protein, which is where the cholesterol-binding domain of TSPO is located, and this polymorphism has been associated with anxiety-related disorders. However, recent knockout mouse models have provided inconsistent conclusions of whether TSPO is directly involved in steroid synthesis. In this report, we show that TSPO deletion mutations in rat and its corresponding rs6971 polymorphism in humans alter adrenocorticotropic hormone-induced plasma corticosteroid concentrations. Rat tissues examined show increased cholesteryl ester accumulation, and neurosteroid formation was undetectable in homozygous rats. These results also support a role for TSPO ligands in diseases with steroid-dependent stress and anxiety elements.

CRISPR/Cas9‒Mediated Tspo Gene Mutations Lead to Reduced Mitochondrial Membrane Potential and Steroid Formation in MA-10 Mouse Tumor Leydig Cells

The outer mitochondrial membrane translocator protein (TSPO) binds cholesterol with high affinity and is involved in mediating its delivery into mitochondria, the rate-limiting step in hormone-induced steroidogenesis. Specific ligand binding to TSPO has been shown to initiate steroid formation. However, recent studies of the genetic deletion of Tspo have provided conflicting results. Here, we address and extend previous studies by examining the effects of Tspo-specific mutations on steroid formation in hormone- and cyclic adenosine monophosphate (cAMP)-responsive MA-10 cells, using the CRISPR/Cas9 system. Two mutant subcell lines, nG1 and G2G, each carrying a Tspo exon2-specific genome modification, and two control subcell lines, G1 and HH, each carrying a wild-type Tspo, were produced. In response to dibutyryl cAMP, the nG1 and G2G cells produced progesterone at levels significantly lower than those produced by the corresponding control cells G1 and HH. Neutral lipid homeostasis, which provides free cholesterol for steroid biosynthesis, was altered significantly in the Tspo mutant cells. Interestingly, the mitochondrial membrane potential (ΔΨm) of the Tspo mutant cells was significantly reduced compared with that of the control cells, likely because of TSPO interactions with the voltage-dependent anion channel and tubulin at the outer mitochondrial membrane. Steroidogenic acute regulatory protein (STAR) expression was induced in nG1 cells, suggesting that reduced TSPO affected STAR synthesis and/or processing. Taken together, these results provide further evidence for the critical role of TSPO in steroid biosynthesis and suggest that it may function at least in part via its regulation of ΔΨm and effects on STAR.

Functional TSPO polymorphism predicts variance in the diurnal cortisol rhythm in bipolar disorder

INTRODUCTIONnPsychosocial stress contributes to onset/exacerbation of mood episodes and alcohol use, suggesting dysregulated diurnal cortisol rhythms underlie episodic exacerbations in Bipolar Disorder (BD). However, mechanisms underlying dysregulated HPA rhythms in BD and alcohol use disorders (AUD) are understudied. Knowledge of associated variance factors have great clinical translational potential by facilitating development of strategies to reduce stress-related relapse in BD and AUD. Evidence suggests structural changes to mitochondrial translocator protein (TSPO) (a regulator of steroid synthesis) due to the single nucleotide polymorphism rs6971, may explain much of this variance. However, whether rs6971 is associated with abnormal HPA rhythms and clinical exacerbation in humans is unknown.nnnMETHODSnTo show this common TSPO polymorphism impacts HPA rhythms in BD, we tested whether rs6971 (dichotomized: presence/absence of polymorphism) predicted variance in diurnal cortisol rhythm (saliva: morning and evening for 3u202fdays) in 107 BD (50 with and 57 without AUD) and 28 healthy volunteers of similar age and ethno-demographic distribution.nnnRESULTSnRepeated measures ANOVA confirmed effects BD (F5,525u202f=u202f3.0, pu202f=u202f0.010) and AUD (F5,525u202f=u202f2.9, pu202f=u202f0.012), but not TSPO polymorphism (pu202f>u202f0.05). Interactions were confirmed for TSPOu202f×u202fBD (F5,525u202f=u202f3.9, pu202f=u202f0.002) and for TSPOu202f×u202fAUD (F5,525u202f=u202f2.8, pu202f=u202f0.017).nnnDISCUSSIONnWe identified differences in diurnal cortisol rhythm depending on presence/absence of common TSPO polymorphism in BD volunteers with or without AUD and healthy volunteers. These results have wide ranging implications but further validation is needed prior to optimal clinical translation.

Leydig cells: formation, function, and regulation†

Abstract Herein we summarize important discoveries made over many years about Leydig cell function and regulation. Fetal Leydig cells produce the high levels of androgen (testosterone or androstenedione, depending upon the species) required for differentiation of male genitalia and brain masculinization. Androgen production declines with loss of these cells, reaching a nadir at postpartum. Testosterone then gradually increases to high levels with adult Leydig cell development from stem cells. In the adult, luteinizing hormone (LH) binding to Leydig cell LH receptors stimulates cAMP production, increasing the rate of cholesterol translocation into the mitochondria. Cholesterol is metabolized to pregnenolone by the CYP11A1 enzyme at the inner mitochondrial membrane, and pregnenolone to testosterone by mitochondria and smooth endoplasmic reticulum enzymes. Cholesterol translocation to the inner mitochondrial membrane is mediated by a protein complex formed at mitochondrial contact sites that consists of the cholesterol binding translocator protein, voltage dependent anion channel, and other mitochondrial and cytosolic proteins. Steroidogenic acute regulatory protein acts at this complex to enhance cholesterol movement across the membranes and thus increase testosterone formation. The 14-3-3γ and ϵ adaptor proteins serve as negative regulators of steroidogenesis, controlling the maximal amount of steroid formed. Decline in testosterone production occurs in many aging and young men, resulting inmetabolic and quality-of-life changes. Testosterone replacement therapy is widely used to elevate serum testosterone levels in hypogonadal men. With knowledge gained of the mechanisms involved in testosterone formation, it is also conceivable to use pharmacological means to increase serum testosterone by Leydig cell stimulation. Summary Sentence A summary of important discoveriesmade over the course of many years about Leydig cell function and regulation, and discussion of important issues that remain to be understood.

Leydig Cell Development and Aging in the Brown Norway Rat

Abstract Hypogonadism affects about 5xa0million American men and is associated with reduced lean body mass, bone mineral density, muscle mass, and libido. As in men, serum testosterone (T) declines in Brown Norway rats as a consequence of reduced Leydig cell T production in response to luteinizing hormone (LH) rather than from reduced LH. Reduced T is associated with reductions in Leydig cell cyclic adenosine monophosphate production, cholesterol transfer from intracellular sources into the mitochondria (the rate-determining step in T formation), and downstream steroidogenic enzymes. There is strong evidence for the involvement of altered balance between reactive oxygen production and the antioxidant defense system in age-related T reduction. These findings suggest that manipulation of the Leydig cell redox environment and/or the stimulation of cholesterol transfer into mitochondria may represent novel targets for the prevention or treatment of age-related reductions in T.

Disruption of ergosterol and tryptophan biosynthesis, as well as cell wall integrity pathway and the intracellular pH homeostasis, lead to mono-(2-ethylhexyl)-phthalate toxicity in budding yeast

Endocrine disrupting chemicals (EDCs) are substances in the environment, food, and consumer products that interfere with hormone homeostasis, metabolism or reproduction in humans and animals. One such EDC, the plasticizer di-(2-ethylhexyl)-phthalate (DEHP), exerts its function through its principal bioactive metabolite, mono-(2-ethylhexyl)-phthalate (MEHP). To fully understand the effects of MEHP on cellular processes and metabolism as well as to assess the impact of genetic alteration on the susceptibility to MEHP-induced toxicity, we screened MEHP-sensitive mutations on a genome-scale in the eukaryotic model organism Saccharomyces cerevisiae. We identified a total of 96 chemical-genetic interactions between MEHP and gene mutations in this study. In response to MEHP treatment, most of these gene mutants accumulated higher intracellular MEHP content, which correlated with their MEHP sensitivity. Twenty-seven of these genes are involved in the metabolism, twenty-two of them play roles in protein sorting, and ten of them regulate ion homeostasis. Functional categorization of these genes indicated that the biosynthetic pathways of both ergosterol and tryptophan, as well as cell wall integrity and the intracellular pH homeostasis, were involved in the protective response of yeast cells to the MEHP toxicity. Our study demonstrated that a collection of yeast gene deletion mutants is useful for a functional toxicogenomic analysis of EDCs, which could provide important clues to the effects of EDCs on higher eukaryotic organisms.

Leydig Cells: Fetal to Aged Testes

There are two distinct periods of testosterone production in males, one in the fetus and the other in the adult. The testosterone produced by fetal Leydig cells declines with the loss of these cells postnatally. Testosterone then gradually increases to high levels with the development of adult Leydig cells. Testosterone production is regulated by luteinizing hormone (LH) and involves cholesterol transport into the mitochondria, pregnenolone formation in the mitochondria, and conversion of pregnenolone into testosterone by enzymes of the smooth endoplasmic reticulum. Cholesterol transport, the rate-determining step in steroidogenesis, involves the actions of steroidogenic acute regulatory protein (STAR), translocator protein (18xa0kDa; TSPO), and other proteins of the transduceosome. With aging, there is a progressive decline in testosterone production and thus reduced serum levels of testosterone (hypogonadism). Although testosterone replacement therapy (TRT) can be used to raise serum testosterone levels, recent studies suggest increased risk of cardiovascular disease, and in most men TRT will result in reduced intratesticular testosterone concentration and thus spermatogenesis suppression. Better understanding of the mechanisms mediating testosterone formation might lead to the possibility of increasing serum (and intratesticular) testosterone by stimulating the Leydig cells themselves.

Leydig Cell Androgen Synthesis

Testosterone and its metabolite dihydrotestosterone (DHT) are the main androgens driving male sexual differentiation and masculinization, and supporting spermatogenesis. They are synthesized by the Leydig cells of the testis from cholesterol transferred from intracellular stores into mitochondria; the hormone-sensitive and rate-limiting step in androgen biosynthesis. In mitochondria, cholesterol is metabolized by the cytochrome CYP11A1 into pregnenolone, which is further metabolized to androgens in the endoplasmic reticulum by the 3β-HSD, CYP17A1, and 17β-HSD3 enzymes. Testosterone is metabolized to estradiol and DHT by CYP19A1 and 5α-reductase, respectively. Deficiencies in androgen biosynthesis are relatively rare and lead to male pseudohermaphroditism.