Rat Ovarian Function Is Impaired during Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease affecting the CNS and occurring far more prevalently in women than in men. In both MS and its animal models, sex hormones play important immunomodulatory roles. We have previously shown that experimental autoimmune encephalomyelitis (EAE) affects the hypothalamic-pituitary-gonadal axis in rats of both sexes and induces an arrest in the estrous cycle in females. To investigate the gonadal status in female rats with EAE, we explored ovarian morphometric parameters, circulating and intraovarian sex steroid levels, and the expression of steroidogenic machinery components in the ovarian tissue. A prolonged state of diestrus was recorded during the peak of EAE, with maintenance of the corpora lutea, elevated intraovarian progesterone levels, and increased gene and protein expression of StAR, similar to the state of pseudopregnancy. The decrease in CYP17A1 protein expression was followed by a decrease in ovarian testosterone and estradiol levels. On the contrary, serum testosterone levels were slightly increased. With unchanged serum estradiol levels, these results point at extra-gonadal sites of sex steroid biosynthesis and catabolism as important regulators of their circulating levels. Our study suggests alterations in the function of the female reproductive system during central autoimmunity and highlights the bidirectional relationships between hormonal status and EAE.

Agmatine Mitigates Inflammation-Related Oxidative Stress in BV-2 Cells by Inducing a Pre-Adaptive Response.

Neuroinflammation and microglial activation, common components of most neurodegenerative diseases, can be imitated in vitro by challenging microglia cells with Lps. We here aimed to evaluate the effects of agmatine pretreatment on Lps-induced oxidative stress in a mouse microglial BV-2 cell line. Our findings show that agmatine suppresses nitrosative and oxidative burst in Lps-stimulated microglia by reducing iNOS and XO activity and decreasing O2- levels, arresting lipid peroxidation, increasing total glutathione content, and preserving GR and CAT activity. In accordance with these results, agmatine suppresses inflammatory NF-kB, and stimulates antioxidant Nrf2 pathway, resulting in decreased TNF, IL-1 beta, and IL-6 release, and reduced iNOS and COX-2 levels. Together with increased ARG1, CD206 and HO-1 levels, our results imply that, in inflammatory conditions, agmatine pushes microglia towards an anti-inflammatory phenotype. Interestingly, we also discovered that agmatine alone increases lipid peroxidation end product levels, induces Nrf2 activation, increases total glutathione content, and GPx activity. Thus, we hypothesize that some of the effects of agmatine, observed in activated microglia, may be mediated by induced oxidative stress and adaptive response, prior to Lps stimulation.

Testicular steroidogenesis is suppressed during experimental autoimmune encephalomyelitis in rats.

Multiple sclerosis (MS) is an autoimmune disease that usually occurs during the reproductive years in both sexes. Many male patients with MS show lower blood testosterone levels, which was also observed in male rats during experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To better understand the causes of decreased testosterone production during EAE, we investigated the expression status of genes and proteins associated with steroidogenesis in the testes. No changes in the number of interstitial cells were observed in EAE animals, but the expression of the insulin-like 3 gene was reduced at the peak of the disease, implying that the Leydig cell functional capacity was affected. Consistent with this finding, the expression of most steroidogenic enzyme genes and proteins was reduced during EAE, including StAR, CYP11A1, CYP17A1 and HSD3B. No signs of testicular inflammation were observed. Recovery of steroidogenesis was observed after injection of hCG, the placental gonadotropin, or buserelin acetate, a gonadotropin-releasing hormone analogue, at the peak of EAE. Together, our results are consistent with the hypothesis that impaired testicular steroidogenesis originates upstream of the testes and that low serum LH is the main cause of decreased testosterone levels during EAE.

Expression and Role of Thyrotropin Receptors in Proopiomelanocortin-Producing Pituitary Cells.

Background: Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through Gs and Gq/11 protein coupled TSH receptors (TSHRs). Methods: In this study, we examined whether the functional TSHRs are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Results: Double immunocytochemistry revealed the expression of TSHRs in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in folliculostellate cells. The functional coupling of these receptors to the Gq/11 signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP3-dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the Gs signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and ß-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. Conclusions: These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner.

The sex-specific patterns of changes in hypothalamic-pituitary-gonadal axis during experimental autoimmune encephalomyelitis.

Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here aimed to determine the extent and sex specificity of alterations in the hypothalamic-pituitary-gonadal axis in the rat model of multiple sclerosis named experimental autoimmune encephalomyelitis. During the disease course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh1 (in males only) expression. In females, the expression of gonadotrope-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone levels and a transient arrest of the estrous cycle. In contrast, Fshb expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr expression and basal and stimulated serum luteinizing hormone and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh1 expression in females and males, respectively, leading to sex-specific changes downstream the axis.

Divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo.

Continuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment.

Distinct Expression Patterns of Osteopontin and Dentin Matrix Protein 1 Genes in Pituitary Gonadotrophs.

Cell-matrix interactions play important roles in pituitary development, physiology, and pathogenesis. In other tissues, a family of non-collagenous proteins, termed SIBLINGs, are known to contribute to cell-matrix interactions. Anterior pituitary gland expresses two SIBLING genes, Dmp1 (dentin matrix protein-1) and Spp1 (secreted phosphoprotein-1) encoding DMP1 and osteopontin proteins, respectively, but their expression pattern and roles in pituitary functions have not been clarified. Here we provide novel evidence supporting the conclusion that Spp1/osteopontin, like Dmp1/DMP1, are expressed in gonadotrophs in a sex- and age-specific manner. Other anterior pituitary cell types do not express these genes. In contrast to Dmp1, Spp1 expression is higher in males; in females, the expression reaches the peak during the diestrus phase of estrous cycle. In further contrast to Dmp1 and marker genes for gonadotrophs, the expression of Spp1 is not regulated by gonadotropin-releasing hormone in vivo and in vitro. However, Spp1 expression increases progressively after pituitary cell dispersion in both female and male cultures. We may speculate that gonadotrophs signal to other pituitary cell types about changes in the structure of pituitary cell-matrix network by osteopontin, a function consistent with the role of this secretory protein in postnatal tissue remodeling, extracellular matrix reorganization after injury, and tumorigenesis.

Intrinsic and Regulated Gonadotropin-Releasing Hormone Receptor Gene Transcription in Mammalian Pituitary Gonadotrophs.

The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH), acting via its receptors (GnRHRs) expressed in pituitary gonadotrophs, represents a critical molecule in control of reproductive functions in all vertebrate species. GnRH-activated receptors regulate synthesis of gonadotropins in a frequency-dependent manner. The number of GnRHRs on the plasma membrane determines the responsiveness of gonadotrophs to GnRH and varies in relation to age, sex, and physiological status. This is achieved by a complex control that operates at transcriptional, translational, and posttranslational levels. This review aims to overview the mechanisms of GnRHR gene (Gnrhr) transcription in mammalian gonadotrophs. In general, Gnrhr exhibits basal and regulated transcription activities. Basal Gnrhr transcription appears to be an intrinsic property of native and immortalized gonadotrophs that secures the presence of a sufficient number GnRHRs to preserve their functionality independently of the status of regulated transcription. On the other hand, regulated transcription modulates GnRHR expression during development, reproductive cycle, and aging. GnRH is crucial for regulated Gnrhr transcription in native gonadotrophs but is ineffective in immortalized gonadotrophs. In rat and mouse, both basal and GnRH-induced Gnrhr transcription rely primarily on the protein kinase C signaling pathway, with subsequent activation of mitogen-activated protein kinases. Continuous GnRH application, after a transient stimulation, shuts off regulated but not basal transcription, suggesting that different branches of this signaling pathway control transcription. Pituitary adenylate cyclase-activating polypeptide, but not activins, contributes to the regulated transcription utilizing the protein kinase A signaling pathway, whereas a mechanisms by which steroid hormones modulate Gnrhr transcription has not been well characterized.

The relationship between basal and regulated Gnrhr expression in rodent pituitary gonadotrophs.

Hypothalamic GnRH together with gonadal steroids and activins/inhibin regulate its receptor gene (Gnrhr) expression in vivo, which leads to crucial changes in GnRHR numbers on the plasma membrane. This is accompanied by alterations in the gonadotroph sensitivity and responsiveness during physiologically relevant situations. Here we investigated basal and GnRH-regulated Gnrhr expression in rodent pituitary gonadotrophs in vitro. In pituitary cells from adult animals cultured in the absence of GnRH and steroid hormones, the Gnrhr expression was progressively reduced but not completely abolished. The basal Gnrhr expression was also operative in LßT2 immortalized gonadotrophs never exposed to GnRH. In both cell types, basal transcription was sufficient for the expression of functional GnRHRs. Continuous application of GnRH transiently elevated the Gnrhr expression in cultured pituitary cells followed by a sustained fall without affecting basal transcription. Both basal and regulated Gnrhr transcriptions were dependent on the protein kinase C signaling pathway. The GnRH-regulated Gnrhr expression was not operative in embryonal pituitary and LßT2 cells and was established neonatally, the sex-specific response patterns were formed at the juvenile-peripubertal stage and there was a strong correlation between basal and regulated gene expression during development. Thus, the age-dependent basal and regulated Gnrhr transcription could account for the initial blockade and subsequent activation of the reproductive system during development.

Characterization of GPR101 transcript structure and expression patterns.

We recently showed that Xq26.3 microduplications cause X-linked acrogigantism (X-LAG). X-LAG patients mainly present with growth hormone and prolactin-secreting adenomas and share a minimal duplicated region containing at least four genes. GPR101 was the only gene highly expressed in their pituitary lesions, but little is known about its expression patterns. In this work, GPR101 transcripts were characterized in human tissues by 5'-Rapid Amplification of cDNA Ends (RACE) and RNAseq, while the putative promoter was bioinformatically predicted. We investigated GPR101 mRNA and protein expression by RT-quantitative PCR (qPCR), whole-mount in situ hybridization, and immunostaining, in human, rhesus monkey, rat and zebrafish. We identified four GPR101 isoforms characterized by different 5'-untranslated regions (UTRs) and a common 6.1kb long 3'UTR. GPR101 expression was very low or absent in almost all adult human tissues examined, except for specific brain regions. Strong GPR101 staining was observed in human fetal pituitary and during adolescence, whereas very weak/absent expression was detected during childhood and adult life. In contrast to humans, adult monkey and rat pituitaries expressed GPR101, but in different cell types. Gpr101 is expressed in the brain and pituitary during rat and zebrafish development; in rat pituitary, Gpr101 is expressed only after birth and shows sexual dimorphism. This study shows that different GPR101 transcripts exist and that the brain is the major site of GPR101 expression across different species, although divergent species- and temporal-specific expression patterns are evident. These findings suggest an important role for GPR101 in brain and pituitary development and likely reflect the very different growth, development and maturation patterns among species.

Melatonin replacement restores the circadian behavior in adult rat Leydig cells after pinealectomy.

Melatonin actions on oscillators in reproductive organs are poorly understood. Here we analyzed melatonin effects on rhythmic expression of clock and steroidogenesis-related genes in adult rat Leydig cells (LCs). The effect of melatonin was tested both in vivo using pinealectomized and melatonin-substituted rats and in vitro on isolated LCs. Data revealed 24-h-rhythmic expression of clock genes (Bmal1, Per1,2,3, Rev-erba,b, Rorb), steroidogenic genes (Star, Cyp11a1, Cyp17a1), and genes of steroidogenic regulators (positive-Nur77, negative-Arr19). Pinealectomy increased 24-h-oscillations of serum testosterone and LC's cAMP levels, expression of Insl3, Per1, Star/StAR, Hsd3b1/2, Nur77, decreased Arr19 and canceled Per2 oscillatory expression pattern. At hypothalamic-pituitary level, pinealectomy increased mesor of Gnrh, Lhb and rhythm robustness of Mntr1a expression. All parameters disturbed were restored by melatonin-replacement. In vitro studies did not confirm direct melatonin effects on neither clock nor steroidogenic genes. Accordingly, melatonin influence 24-h-rhythmic LC-function likely through hypothalamic-pituitary axis and consequently cAMP-signaling in LCs.

Cell Type-Specific Sexual Dimorphism in Rat Pituitary Gene Expression During Maturation.

The most obvious functional differences between mammalian males and females are related to the control of reproductive physiology and include patterns of GnRH and gonadotropin release, the timing of puberty, sexual and social behavior, and the regulation of food intake and body weight. Using the rat as the best-studied mammalian model for maturation, we examined the expression of major anterior pituitary genes in five secretory cell types of developing males and females. Corticotrophs show comparable Pomc profiles in both sexes, with the highest expression occurring during the infantile period. Somatotrophs and lactotrophs also exhibit no difference in Gh1 and Prl profiles during embryonic to juvenile age but show the amplification of Prl expression in females and Gh1 expression in males during peripubertal and postpubertal ages. Gonadotrophs exhibit highly synchronized Lhb, Fshb, Cga, and Gnrhr expression in both sexes, but the peak of expression occurs during the infantile period in females and at the end of the juvenile period in males. Thyrotrophs also show different developmental Tshb profiles, which are synchronized with the expression of gonadotroph genes in males but not in females. These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes.

Stress triggers mitochondrial biogenesis to preserve steroidogenesis in Leydig cells.

Adaptability to stress is a fundamental prerequisite for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored. Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP, and ß-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage. Given the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies.

Purinergic signaling pathways in endocrine system.

Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling.

Intratesticular alpha1-adrenergic receptors mediate stress-disturbed transcription of steroidogenic stimulator NUR77 as well as steroidogenic repressors DAX1 and ARR19 in Leydig cells of adult rats.

The aim of the present study was to define the role of testicular α1-adrenergic receptors (α1-ADRs) in stress-triggered adaptation of testosterone-producing Leydig cells of adult rats. Results showed that in vivo blockade of testicular α1-ADRs prevented partial recovery of circulating androgen levels registered after 10× repeated immobilization stress (10 × IMO). Moreover, α1-ADR-blockade diminished 10 × IMO-triggered recovery of Leydig cell androgen production, and abolished mitochondrial membrane potential recovery. In the same cells, 10 × IMO-induced increase in Star transcript was abolished, Lhcgr transcript decreased, while transcription of other steroidogenic proteins was not changed. α1-ADR-blockade recovered stress-induced decrease of Nur77, one of the main steroidogenic stimulator, while significantly reduced 10 × IMO-increased in the transcription of the main steroidogenic repressors, Arr19 and Dax1. In vitro experiments revealed an adrenaline-induced α1-ADR-mediated decrease in Nur77 transcription in Leydig cells. Adrenaline-induced increase of repressor Dax1 also involves ADRs in Leydig cells. Accordingly, α1-ADRs participate in some of the stress-triggered effects on the steroidogenic machinery of Leydig cells.

Prolonged in vivo administration of testosterone-enanthate, the widely used and abused anabolic androgenic steroid, disturbs prolactin and cAMP signaling in Leydig cells of adult rats.

This study was designed to systematically analyze and define the effects of 1-day, 2-weeks, 10-weeks intramuscular administration of testosterone-enanthate, widely used and abused anabolic androgenic steroid (AAS), on main regulators of steroidogenesis and steroidogenic genes expression in testosterone-producing Leydig cells of adult rats. The results showed that prolonged (10-weeks) intramuscular administration of testosterone-enanthate, in clinically relevant dose, significantly increased prolactin, but decreased Prlr2 and Gnrhr in pituitary of adult rat. The levels of testosterone, Insl3, cAMP and mitochondrial membrane potential of Leydig cells were significantly reduced. This was followed by decreased expression of some steroidogenic enzymes and regulatory proteins such as Lhcgr, Prlr1/2, Tspo, Star, Cyp11a1, Cyp17a1, Dax1. Oppositely, Hsd3b1/2, Hsd3b5, Hsd17b4, Ar, Arr19 increased. In the same cells, transcriptional milieu of cAMP signaling elements was disturbed with remarkable up-regulation of PRKA (the main regulator of steroidogenesis). Increased prolactin together with stimulated transcription of Jak2/Jak3 could account for increased Hsd3b1/2 and Hsd3b5 in Leydig cells following 10-weeks in vivo treatment with testosterone-enanthate. In vitro studies revealed that testosterone is capable to increase level of Prlr1, Prlr2, Hsd3b1/2, Hsd3b5 in Leydig cells. Accordingly, testosterone-induced changes in prolactin receptor signaling together with up-regulation of PRKA, Hsd3b1/2, Hsd3b5, Ar in Leydig cells, could be the possible mechanism that contribute to the establishment of a new adaptive response to maintain homeostasis and prevent loss of steroidogenic function. Presented data provide new molecular insights into the relationship between disturbed testosterone homeostasis and mammalian reproduction and are important in terms of wide use and abuse of AASs and human reproductive health.

Loss of basal and TRH-stimulated Tshb expression in dispersed pituitary cells.

This study addresses the in vivo and in vitro expression pattern of three genes that are operative in the thyrotroph subpopulation of anterior pituitary cells: glycoprotein α-chain (Cga), thyroid-stimulating hormone ß-chain (Tshb), and TRH receptor (Trhr). In vivo, the expression of Cga and Tshb was robust, whereas the expression of Trhr was low. In cultured pituitary cells, there was a progressive decline in the expression of Cga, Tshb, and Trhr. The expression of Tshb could not be reversed via pulsatile or continuous TRH application in variable concentrations and treatment duration or by the removal of thyroid and steroid hormones from the sera. In parallel, the expression of CGA and TSHB proteins declined progressively in pituitary cells from both sexes. The lack of the effect of TRH on Tshb expression was not related to the age of pituitary cultures and the presence of functional TRH receptors. In cultured pituitary fragments, there was also a rapid decline in expression of these genes, but TRH was able to induce transient Tshb expression. In vivo, thyrotrophs were often in close proximity to each other and to somatotroph and folliculostellate cell networks and especially to the lactotroph cell network; such an organization pattern was lost in vitro. These observations suggest that the lack of influence of anterior pituitary architecture and/or intrapituitary factors probably accounts for the loss of basal and TRH-stimulated Tshb expression in dispersed pituitary cells.

Molecular adaptations of testosterone-producing Leydig cells during systemic in vivo blockade of the androgen receptor.

This study systematically evaluates the effects of androgen receptor (AR) blockade on molecular events in Leydig cells. Results showed that intramuscular administration of testosterone-enanthate, at clinically relevant dose, decreased testosterone in interstitial fluid and Leydig cells from adult rats. AR-blocker (Androcur) prevented this effect and testosterone-reduced Leydig cells steroidogenic capacity/activity. Testosterone-reduced expression of some steroidogenic enzymes/proteins (Tspo,StAR,Hsd3b1/2) and transcription factors (Nur77,Gata4,Dax1) was completely abrogated, while decreased expression of Star,Cyp11a1,Cyp17a1,Hsd17b4,Creb1a was partially prevented. In the same cells, increased expression of Hsd3b5/HSD3B and Ar/AR was abolished. Androcur-treatment abolished testosterone-reduced cAMP, coupled with a changed expressional milieu of cAMP signaling elements. Results from in vitro experiments suggest that some of these effects are testosterone-AR dependent, while others could be due to disturbed LH and/or other signals. Presented data provide new molecular insight into Leydig cells function and are important in terms of human reproductive health and the wide-spread use of Androcur as well as use/abuse of testosterone-enanthate.

In vivo blockade of α1-adrenergic receptors mitigates stress-disturbed cAMP and cGMP signaling in Leydig cells.

The molecular mechanism of stress-associated reproductive dysfunction is complex and largely unknown. This study was designed to systematically analyze molecular effects of systemic in vivo blockade of α1-adrenergic receptors (α1-ADRs) on stress-induced disturbance of cAMP/cGMP signaling in testosterone-producing Leydig cells using the following parameters (i) level of circulating stress hormones, LH and testosterone; (ii) level of main molecular markers of Leydig cell functionality (testosterone, Insl3, cAMP); (iii) expression of cAMP signaling (cAMP 'producers'/'effectors'/'removers') and (iv) expression of NO-cGMP signaling (NO-cGMP 'producers'/'effectors'/'removers'). The results showed that oral administration of α1-ADR blocker before stress increased cGMP and diminished stress-reduced cAMP production in Leydig cells. In the same cells, stress-induced effects on cAMP/cGMP signaling pathways elements were changed. Sustained in vivo α1-ADR blockade completely abolished stress-increased transcription of most abundantly expressed phosphodiesterase that remove cAMP (Pde4b) and potentiated stress-increased expression of PRKA, the main stimulator of Leydig cell steroidogenesis. In the same Leydig cells, stress-decreased NOS3 expression was abolished, while stress-increased GUCY1 (cGMP 'producer') and PRKG1 (cGMP 'effector') were potentiated. It is possible that all molecules mentioned could contribute, at least in part, in recovery of Leydig cell testosterone production. Presented data provide new role of α1-ADRs in stress-triggered disturbance of cAMP/cGMP signaling, and new molecular insights into the relationship between stress and mammalian reproduction. Regardless of whether the effects of α1-blocker + stress are direct or indirect, the results are important in terms of human reproductive health and the wide use of α1-ADR antagonists, alone or in combination, to treat post-traumatic stress disorders, hypertension, benign prostatic hyperplasia symptoms and potential drugs for prostate cancer prevention/treatment.

The opposing roles of nitric oxide and cGMP in the age-associated decline in rat testicular steroidogenesis.

The molecular mechanism of the aging-associated dysfunction of Leydig cells (LCs) is complex and poorly understood. In this study, we analyzed the contribution of nitric oxide (NO) and cGMP signaling to the age-dependent decline in LC function. Significant (>50%) decreases in serum, intratesticular, and LC androgens in aging rats (15-24 months) were accompanied by a proportional increase in NO production, an up-regulation of cGMP levels, and the expression of soluble guanylyl cyclase-1B and protein kinase G1 in LCs. In contrast, LC cAMP levels decreased with age, most likely reflecting the up-regulation of cAMP-specific phosphodiesterase expression. Moreover, the expression of genes encoding enzymes responsible for cholesterol transport and its conversion to T were reduced. Exposing LCs from aged animals to NO further increased cGMP levels and decreased cAMP and androgen production, whereas the addition of cell-permeable 8-bromoguanosine-cGMP alone had the opposite effect. In vivo inhibition of cGMP-specific phosphodiesterase-5 for 3 and 6 months in aged rats led to a partial restoration of androgens, NO, and cyclic nucleotide levels, as well as the expression of steroidogenic and NO/cGMP signaling genes. These results indicate that a progressive increase in NO production contributes to the age-dependent decrease in steroidogenesis in a cGMP-independent manner, whereas the sustained elevation in cGMP levels significantly slows the decline in LC function.