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The increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored. Here, the stress-response of the testosterone-producing rat Leydig cells depending on the time of stressful events was studied. The study analyzes the effects of 3-h immobilization (IMO) applied at different periods during the day. The IMO performed once [1 time immobilization stress (1×IMO)] or repeated in 10 consecutive days [10 time repeated immobilization stress (10×IMO)]. Both types of IMO increased corticosterone and decreased testosterone blood level. However, the effect of 10×IMO occurring in the active phase on blood testosterone was less pronounced. This is related to different sensitivity to IMO-events depending on the diurnal time. Most steroidogenesis-related genes [gene encoding luteinizing hormone/choriogonadotropin receptor ( Lhcgr), gene encoding cytochrome P450, family 11, subfamily a, polypeptide 1 ( Cyp11a1), gene encoding hydroxy-δ-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 1 ( Hsd3b1/2), and gene encoding cytochrome P450, family 17, subfamily a, polypeptide 1 ( Cyp17a1)] were downregulated in the inactive phase but unchanged or even upregulated in the active phase of the day. Both types of IMO stimulated the expression of clock elements gene encoding aryl hydrocarbon receptor nuclear translocator-like ( Bmal1)/aryl hydrocarbon receptor nuclear translocator-like (BMAL1), gene encoding period circadian regulator 1 ( Per1) /period circadian regulator 1 (PER1) regardless of the day’s stage and reduced gene encoding nuclear receptor subfamily 1, group D, member 1 ( Rev-erba) in the inactive phase. The principal component analysis (PCA) confirmed a major shift, for both IMO-types, in the transcription of the genes across the passive/active stage. Further, 10×IMO changed a diurnal pattern of the glucocorticoid receptor [gene encoding nuclear receptor subfamily 3, group C, member 1 ( Nr3c1)/nuclear receptor subfamily 3, group C, member 1 (GR)] expression, whereas the observed time-dependent IMO-response of the Leydig cells correlated with different corticosterone engagements. Altogether, the Leydig cell’s stress response depends on the daytime of the stressful event, emphasizing the importance of the circadian system in supporting androgen homeostasis and male fertility.

Since mitochondria play an essential role in the testosterone biosynthesis, serve as power centers and are a source of oxidative stress, a possible mitochondrial dysfunction could be connected with decreased activity of Leydig cells and lowered testosterone production during aging. Here we chronologically analyzed age-related alterations of mitochondrial function in Leydig cells correlated by the progressive rise of cGMP signaling and with respect to testosterone synthesis. To target cGMP signaling in Leydig cells, acute or long-term in vivo or ex vivo treatments with sildenafil (phosphodiesterase 5 [PDE5] inhibitor) were performed. Aging-related accumulation of cGMP in the Leydig cells is associated with mitochondrial dysfunction illustrated by reduced ATP and steroid production, lowered O2 consumption, increased mitochondrial abundance and mtDNA copies number, decreased expression of genes that regulate mitochondrial biogenesis (Ppargc1a/PGC1a-Tfam-Nrf1/NRF1), mitophagy (Pink1), fusion (Mfn1, Opa1), and increased Nrf2/NRF2. Acute in vivo PDE5 inhibition overaccumulated cGMP and stimulated testosterone but reduced ATP production in Leydig cells from adult, middle-aged, and old rats. The increased ATP/O ratio observed in cells from old compared to adult rats was diminished after stimulation of cGMP signaling. Opposite, long-term PDE5 inhibition decreased cGMP signaling and improved mitochondrial function/dynamics in Leydig cells from old rats. Mitochondrial abundance in Leydig cells decreased while ATP levels increased. Chronic treatment elevated Tfam, Nrf1, Nrf2, Opa1, Mfn1, Drp1, and normalized Pink1 expression. Altogether, long-term PDE5 inhibition prevented age-related NO and cGMP elevation, improved mitochondrial dynamics/function, and testosterone production. The results pointed on cGMP signaling in Leydig cells as a target for pharmacological manipulation of aging-associated changes in mitochondrial function and testosterone production.

© 2019 Elsevier Ltd Leydig cell steroidogenesis is the complex process of biosynthesis of androgens (mainly testosterone)from cholesterol as a precursor, via steroidogenic machinery, which includes cholesterol transporters, steroidogenic enzymes, transcription factors, and wide variety of regulators. In this review, we will summarize important discoveries made over the past several years that have contributed to our current understanding of (1)a complex and intriguing network of plethora signaling pathways regulating Leydig cell steroidogenesis during prenatal and postnatal life, as well as the essential roles played by autocrine and paracrine regulation; (2)the importance of the mitochondrial signalosome, interactome, and dynamics; and (3)the regulation and adaptation of Leydig cell steroidogenesis during psychophysiological stress and aging. It is well known that neural, endocrine, paracrine (including cryptocrine and juxtacrine), and autocrine inputs activate complicated signaling networks targeting Leydig cell steroidogenic machinery. Besides the well-established, leading regulatory role of cAMP-PRKA signaling, which is followed by cGMP-PRKG, mitogen-activated protein kinase, PRKC, AMPK, progenitor adult Leydig cell, and phosphatase signaling, recent data have revealed other signaling pathways (desert hedgehog, nerve growth factor, neuregulin-1, notch, insulin/insulin-like growth factor 1 receptor, retinoic acid, Wnt/β-catenin, and Wilms tumor gene 1)and molecules (14-3-3γ-adapter protein, 14-3-3ε-adapter protein, clock genes, gonadotropin-regulated testicular RNA helicase, LIM-homeobox gene 9, and Smad ubiquitylation regulatory factor 1)as important regulators of Leydig cell steroidogenesis. Recent data have also emphasized the importance of mitochondria-associated membranes, mitochondria–peroxisome interactions, lipophagy/autophagy, and processes of mitochondrial dynamics for Leydig cell steroidogenesis. These processes not only correlate with but also are essential for testosterone production, being dependent on the same regulators. Furthermore, recent discoveries have highlighted that the structural complexity of the testis and interactions between Leydig and neighboring cells are essential for regulation, preservation, and maintenance of Leydig cell steroidogenesis. Because androgens play a crucial role in the masculinization of male fetuses, in secondary male-specific sexual maturation, as well as in the well-being and reproduction of male adults, all recent knowledge may help in the prevention and treatment of androgen-related disorders, both in males and females.

© 2019 The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. In mammals, circadian clock regulates concentration of many reproductive hormones including testosterone. Previously, we characterized pattern of circadian transcription of core clock genes in testosterone-producing Leydig cells. Here, the potential role of luteinizing hormone receptor (LHR)-cAMP signaling in synchronization of Leydig cell's circadian clock and rhythmic testosterone production were examined. Results showed that activation of LHR-cAMP signaling in primary rat Leydig cell culture increased Star/STAR and changed expression of many clock genes (upregulated Per1/PER1, Dec1/2, and Rorb, and downregulated Bmal1 and Rev-erba/b). Inhibition of protein kinase A prevented LHR-triggered increase in transcription of Per1 and Dec1. Effect of stimulated LHR-cAMP signaling on Leydig cell's clock transcription was also confirmed in vivo, using rats treated with single hCG injection. To analyze in vivo effect of low LH-cAMP activity on rhythmical Leydig cell function, rats with experimental hypogonadotropic hypogonadism were used. Characteristics of hypogonadal rats were decreased LH and testosterone secretion without circadian fluctuation; in Leydig cells decreased arrhythmic cAMP and transcription of steroidogenic genes (Cyp11a1 and Cyp17a1) were observed, while decreased Star/STAR expression retains circadian pattern. However, expression of clock genes, despite changes in transcription levels (increased Bmal1, Per2, Cry1, Cry2, Rora, Rorb, Rev-erba/b/REV-ERBB, Dec1, Csnk1e, and decreased Npas2 and PER1) kept circadian patterns observed in control groups. Altogether, the results strengthened the hypothesis about role of LH-cAMP signaling as synchronizer of Leydig cell's clock. However, clock in Leydig cells is not sufficient to sustain rhythmicity of testosterone production in absence of rhythmic activity of LH-cAMP signaling.

BackgroundThe increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored.ObjectiveThe purpose of the study was to analyze the stress-response of the testosterone-producing Leydig cells depending on the stressful event’s time.Materials and methodsThe study was designed to follow the effects of 3-hour immobilization (IMO) applied at different periods during the day. The IMO performed once (1xIMO) or repeated in 10 consecutive days (10xIMO). Principal-component-analysis (PCA) followed the expression study of the clock and steroidogenic-related genes in Leydig cells.ResultsBoth types of IMO in all investigated periods increased corticosterone and decreased testosterone blood level. Transcriptional analysis revealed different sensitivity to IMO events depending on the circadian time. The majority of steroidogenesis-related genes (Lhcgr, Cyp11a1, Cyp17a1, Hsd3b1/2) were down-regulated in the inactive but unchanged or even up-regulated in the active phase of the day. Both types of IMO potentiated the expression of clock elements Bmal1/BMAL1, Per1/PER1 regardless of the day’s stage and reduced Reverba in the inactive phase. The PCA confirmed a major shift, for both IMO-types, in the transcription of steroidogenesis and clock genes across the inactive/active phase. Further, the diurnal pattern of the glucocorticoid receptor (Nr3c1/GR) expression in Leydig cells was increased in the inactive phase due to 10xIMO. The observed time-dependent IMO-response of the Leydig cells correlated with different GR engagements.DiscussionStress- and the circadian-system coordinatively shape Leydig cell’s physiology, assuming diverse GR engagement as a possible factor in mediating the diurnal effect of stress.ConclusionThe Leydig cell’s stress-response depends on the time of the stressful situation, emphasizing the importance of circadian activity in supporting androgen homeostasis and male fertility.

The factors influencing Leydig cell maturity and the acquisition of functional capacity are incompletely defined. Here we analyzed the constant light (LL) influence on Leydig cells’ endocrine function during reproductive maturation. Rats were exposed to LL from P21 to P90. Data were collected at juvenile (P35), peri/pubertal (P42, P49), and adult (P90) stages of life. The results proved the effect of LL on rats’ physiology by changing of bimodal voluntary activity pattern into free-running. Additionally, the peripheral clock in Leydig cells changed in LL condition, indicating disturbed rhythm: the positive element (Bmal1) increased in pre-/pubertal but decreased in the adult period, while negative elements (Per2 and Reverba) were increased. The effects of LL were most prominent in puberty: pituitary genes encoding gonadotropic hormones (Cga, Lhb, Fshb) decreased; serum corticosterone increased, while serum androgens and mass of testicular and sex accessory organs reduced; markers of Leydig cells maturity/differentiation (Insl3, Lhcgr) and steroidogenesis-related genes (Scarb1, Star, Cyp11a1, Cyp17a1) decreased; the steroidogenic and energetic capacity of the Leydig cell mitochondria decreased; the mtDNA copy number reduced, and mitochondrial dynamics markers changed: fusion decreased (Opa1 and Mfn2), and mitophagy increased (Pink1). In adults, the negative effect of LL on mitochondrial function and steroidogenic capacity persists in adult Leydig cells while other parameters reached control values. Altogether, the results indicate that LL slows down Leydig cells’ maturation by reducing the endocrine and energy capacity of cells leading to the delay of reproductive development.

Here, we study possible mechanisms of (in/sub)fertility related to the acute or repeated psychological stresses (the most common stresses in human society) by following the transcriptional profile of 22 mitochondrial dynamics/function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality. An in vivo study mimicking acute (once for 3 h) and repeated (3 h for 10 consecutive days) psychophysical stress was performed on adult rats. The analysis of hormones, the number/functionality of spermatozoa, and 44 transcriptional markers were performed on individual samples from up to 12 animals per group. Results showed that both types of stress reduced spermatozoa functionality (acute by 4.4-fold, repeated by 3.3-fold) and ATP production (acute by 2.3-fold, repeated by 14.5-fold), while only repeated stress reduces the number of spermatozoa (1.9-fold). Stress significantly disturbed transcription of 34-out-of-44 markers (77%). Mitochondrial dynamics and functionality markers: 18-out-of-22 =&gt, 82% (mitochondrial-biogenesis-markers –&gt, 6-out-of-8 =&gt, 75%, mitochondrial-fusion-markers –&gt, 3-out-of-3 =&gt, 100%, mitochondrial-fission-markers –&gt, 1-out-of-2 =&gt, 50%, mitochondrial-autophagy-markers –&gt, mitochondrial-functionality-markers –&gt, 5-out-of-6 =&gt, 83%). Markers of signaling pathways regulating both mitochondrial dynamics/functionality and spermatozoa number/functionality important for male (in/sub)fertility –&gt, 16-out-of-22 =&gt, 73% (cAMP-signaling-markers –&gt, 8-out-of-12 =&gt, 67%, MAPK-signaling-markers –&gt, 8-out-of-10 =&gt, 80%). Accordingly, stress-triggered changes of transcriptional profile of mitochondrial dynamics/functionality markers as well as signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality represent adaptive mechanisms.

Knowledge about the relationship between steroidogenesis and the regulation of the mitochondrial bioenergetics and dynamics, in steroidogenic cells, is not completely elucidated. Here we employed in vivo and ex vivo experimental models to analyze mitochondrial physiology in Leydig cells depending on the different LH-cAMP environments. Activation of LH-receptor in rat Leydig cells ex and in vivo triggered cAMP, increased oxygen consumption, mitoenergetic and steroidogenic activities. Increased mitoenergetic activity i.e., ATP production is achieved through augmented glycolytic ATP production and a small part of oxidative phosphorylation (OXPHOS). Transcription of major genes responsible for mitochondrial dynamics was upregulated for Ppargc1a (regulator of mitogenesis and function) and downregulated for Drp1 (main fission marker), Prkn, Pink1 and Tfeb (mitophagy markers). Leydig cells from gonadotropin-treated rats show increased mitogenesis confirmed by increased mitochondrial mass, increased mtDNA, more frequent mitochondria observed by a transmission electron microscope and increased expression of subunits of respiratory proteins Cytc/CYTC and COX4. Opposite, Leydig cells from hypogonadotropic-hypogonadal rats characterized by low LH-cAMP, testosterone, and ATP production, reduced markers of mitogenesis and mitofusion (Mfn1/2, Opa1) associated with reduced mtDNA content. Altogether results underline LH-cAMP signaling as an important regulator of mitochondrial physiology arranging mitochondrial dynamics, bioenergetic and steroidogenic function in Leydig cells.

Here we investigate the stress-signaling responsible for the effects of acute/repeated psychological stresses (the most common stresses in human society) on spermatozoa number and functionality, as well as the transcriptional profile of mitochondrial dynamics markers by using the in vivo and ex vivo approaches. Acute and repeated stress inhibit spermatozoa functionality (acute –> 3.2-fold, repeated –> 2.5-fold), while only repeated stress reduces the spermatozoa number (1.7-fold). Stress hormones mimic these effects and decrease the spermatozoa functionality (adrenaline: 10 µM –> 2.4-fold, 100 µM – > 2.8-fold; hydrocortisone: 50 pM –> 2.7-fold, 500 pM –> 8.5-fold). They also significantly disturb the transcriptional profile of all main mitochondrial dynamics markers in spermatozoa. Ex vivo manipulation of stress signaling in spermatozoa reveals that most of these effects are mediated through ɑ1-and/or-β-adrenergic receptors. The transcription of these receptors and their kinases in the same samples is under the significant influence of adrenergic signaling. Our results are the first to show the importance of mitochondrial dynamics markers in spermatozoa since the transcriptional profiles of sixteen-out-of-ninteen are disturbed by manipulation of stress-hormones-signaling. This is a completely new molecular approach to assess spermatozoa functionality and it is important for a better understanding of the correlations between stress, environmental-life-style and other factors, and male (in)fertility.

Aim A growing body of evidence pointed correlation between insulin-resistance, testosterone level and infertility, but there is scarce information about mechanisms. The aim of this study was to identify the possible mechanism linking the insulin-resistance with testosterone-producing-Leydig-cells functionality. Methods We applied in vivo and in vitro approaches. The in vivo model of functional genomics is represented by INSR/IGF1R-deficient-testosterone-producing Leydig cells obtained from the prepubertal (P21) and adult (P80) male mice with insulin + IGF1-receptors deletion in steroidogenic cells (Insr/Igf1r-DKO). The in vitro model of INSR/IGF1R-deficient-cell was mimicked by blockade of insulin/IGF1-receptors on the primary culture of P21 and P80 Leydig cells. Results Leydig-cell-specific-insulin-resistance induce the development of estrogenic characteristics of progenitor Leydig cells in prepubertal mice and mature Leydig cells in adult mice, followed with a dramatic reduction of androgen phenotype. Level of androgens in serum, testes and Leydig cells decrease as a consequence of the dramatic reduction of steroidogenic capacity and activity as well as all functional markers of Leydig cell. Oppositely, the markers for female-steroidogenic-cell differentiation and function increase. The physiological significances are the higher level of testosterone-to-estradiol-conversion in double-knock-out-mice of both ages and few spermatozoa in adults. Intriguingly, the transcription of pro-male sexual differentiation markers Sry/Sox9 increased in P21-Leydig-cells, questioning the current view about the antagonistic genetic programs underlying gonadal sex determination. Conclusion The results provide new molecular mechanisms leading to the development of the female phenotype in Leydig cells from Insr/Igf1r-DKO mice and could help to better understand the correlation between insulin resistance, testosterone and male (in)fertility.

© 2018 Elsevier Inc. NO-cGMP signaling pathway has been implicated in reduction of testicular steroidogenesis during aging. Here we analyzed the effect of PDE5 inhibition on old testicular phenotype formation. The old phenotype exhibited low testosterone and increased nitrite levels in circulation, increased cGMP accumulation in testicular interstitial fluid (TIF), progressive atrophy of testicular seminiferous tubules and enlargement of interstitial area followed by rise in blood vessel density and slight increase in the number of Leydig cells and macrophages. Leydig cells have reduced steroidogenic capacity, increased MAP kinases expression (MEK, ERK1/2, JNK) and antiapoptotic PRKG1 and AKT, suggesting increased proliferation/survival and accumulation of senescent Leydig cells in testis. In 12 month-old rats, a long-term treatment with sildenafil (PDE5 inhibitor) normalized testosterone/nitrite levels in circulation and cGMP accumulation in TIF; improved Leydig cell steroidogenic capacity; decreased MEK, ERK1/2 and PRKG1 expression; prevented an increase in the Leydig cells number and atrophy of seminiferous tubules leading to histological appearance of young rat testes. In 18 month-old rats, long-term PDE5 inhibition partially recovered testosterone and nitrite levels in serum; normalized PRKG1 expression without effect on MEK and ERK1/2; and slowed down Leydig cell and macrophage accumulation and regressive tubular changes. Culturing of primary Leydig cells from aged rats in presence of PDE5-inhibitor stimulated steroidogenic and MAPK gene expression. Taking together, results indicate that cGMP targeting alter both steroidogenesis and signaling pathways associated with cell proliferation/survival. The long-term PDE5 inhibition improves testicular steroidogenesis and slows-down regressive changes in testes during aging.

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

The study aimed to analyze the time-dependent consequences of stress on gene expression responsible for diurnal endocrine Leydig cell function connecting them to the glucocorticoid-signaling. In the first 24h after the stress event, a daily variation of blood corticosterone increased, and testosterone decreased; the testosterone/corticosterone were lowest at the end of the stress session overlapping with inhibition of Leydig cells' steroidogenesis-related genes (Nr3c1/GR, Hsd3b1/2, Star, Cyp17a1) and changed circadian activity of the clock genes (the increased Bmal1/BMAL1 and Per1/2/PER1 and decreased Cry1 and Rev-erba). The glucocorticoid-treated rats showed a similar response. The principal-component-analysis (PCA) displayed an absence of significant differences between treatments especially on Per1 and Rev-erba, the findings confirmed by the in vivo blockade of the testicular glucocorticoid receptor (GR) during stress and ex vivo treatment of the Leydig cells with hydrocortisone and GR-blocker. In summary, stressful stimuli can entrain the clock in the Leydig cells through glucocorticoid-mediated communication.

In response to the Bologna Declaration and contemporary trends in Animal Physiology education, the Animal Physiology course at the Faculty of Sciences, University of Novi Sad, Serbia, has evolved over a 12-yr period (2001–2012): from a classical two-semester course toward a one-semester course utilizing computer simulations of animal experiments, continual assessment, lectures, and an optional oral exam. This paper presents an overview of student achievement, the impact of reforms on learning outcomes, and lessons that we as educators learned during this process. The reforms had a positive impact on the percentage of students who completed the course within the same academic year. In addition, the percentage of students who completed the practical exam increased from 54% to >95% following the transition to a Bologna-based approach. However, average final grades declined from 8.0 to 6.8 over the same period. Students also appear reluctant to take the optional oral exam, and 82−91% of students were satisfied with the lower final grade obtained from only assessments and tests administered during the semester. In our endeavor to achieve learning outcomes set during the pre-Bologna period, while adopting contemporary teaching approaches, we sought to increase students’ motivation to strive toward better performance, while ensuring that the increased quantity of students who complete the course is coupled with increased quality of education and a more in-depth understanding of animal physiology.

The Leydig cell physiology displays a circadian rhythm driven by a complex interaction of the reproductive axis hormones and circadian system. The final output of this regulatory process is circadian pattern of steroidogenic genes expression and testosterone production. Aging gradually decreases robustness of rhythmic testosterone secretion without change in pattern of LH secretion. Here, we analyzed effect of aging on circadian variation of cAMP and cGMP signaling in Leydig cells. Results showed opposite effect of aging on cAMP and cGMP daily variation. Reduced amplitude of cAMP circadian oscillation was probably associated with changed expression of genes involved in cAMP production (increased circadian pattern of Adcy7, Adcy9, Adcy10 and decreased Adcy3); cAMP degradation (increased Pde4a, decreased Pde8b, canceled rhythm of Pde4d, completely reversed circadian pattern of Pde7b and Pde8a); and circadian expression of protein kinase A subunits (Prkac/PRKAC and Prkar2a). Aging stimulates expression of genes responsible for cGMP production (Nos2, Gucy1a3 and Gucy1b3/GUCYB3) and degradation (Pde5a, Pde6a and Pde6h) but the overall net effect is elevation of cGMP circadian oscillations in Leydig cells. In addition, the expression of cGMP-dependent kinase, Prkg1/PRKG1 is up-regulated. It seems that aging potentiate cGMP- and reduce cAMP-signaling in Leydig cells. Since both signaling pathways affect testosterone production and clockwork in the cells, further insights into these signaling pathways will help to unravel disorders linked to the circadian timing system, aging and reproduction.

OBJECTIVES/GOALS: The aim of this study was to define the transcription profiles of the molecular markers of mitochondrial biogenesis and fusion/architecture, and the markers of mtDNA copy numbers in the peripheral blood mononuclear cells (PBMCs) from war veterans with/without post-traumatic stress disorder (PTSD). METHODS/STUDY POPULATION: The peripheral blood mononuclear cells (PBMCs) from war veterans with/without post-traumatic stress disorder (PTSD) were used to monitor transcription profile of the molecular markers of mitochondrial biogenesis and fusion/architecture, as well as the markers of mtDNA copy numbers. The human male immortalized monocytes were exposed in vitro to hormonal markers of PTSD in order to monitor the effects of each particular hormonal marker on the molecular markers of mitochondrial biogenesis and fusion/architecture, as well as the markers of mtDNA copy numbers. RQ-PCR analyses were used to define transcriptional profile of above mentioned markers. RESULTS/ANTICIPATED RESULTS: The transcription profiles of above mentioned markers were disturbed, with high individual variability within the groups. A significant increase in the expression of the PPARGC1A transcript was observed in a group of subjects with current PTSD, as well as in the subjects with “life-time” PTSD, compared to healthy controls. PPARGC1B, NRF2 and MFN2 transcripts increased only in PBMCs of “life-time"-PTSD, while the level of transcripts for other investigated genes and the ratio of markers of mtDNA copy numbers showed no significant difference between groups. The in vitro results showed parallelism with the results obtained using the PBMCs from the subjects of the PTSD study. DISCUSSION/SIGNIFICANCE OF IMPACT: Although preliminary (the analysis require a larger number of subjects), the results are first findings and a solid base for further extensive multidisciplinary research in order to clarify the molecular mechanisms for the prevention and treatment of trauma-induced pathological conditions.

Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian nature has not been analyzed. Here we describe changes in circadian behavior in Leydig cells isolated from adult (3-month) and aged (18- and 24-month) rats. The results showed reduced circadian pattern of testosterone secretion in both groups of aged rats despite unchanged LH circadian secretion. Although arrhythmic, the expression of Insl3, another secretory product of Leydig cells, was decreased in both groups. Intracellular cAMP and most important steroidogenic genes (Star, Cyp11a1 and Cyp17a1), together with positive steroidogenic regulator (Nur77), showed preserved circadian rhythm in aging although rhythm robustness and expression level were attenuated in both aged groups. Aging compromised cholesterol mobilization and uptake by Leydig cells: the oscillatory transcription pattern of genes encoding HDL-receptor (Scarb1), hormone sensitive lipase (Lipe, enzyme that converts cholesterol esters from lipid droplets into free cholesterol) and protein responsible for forming the cholesterol esters (Soat2) were flattened in 24-month group. The majority of examined clock genes displayed circadian behavior in expression but only a few of them (Bmal1, Per1, Per2, Per3 and Rev-Erba) were reduced in 24-month-old group. Furthermore, aging reduced oscillatory expression pattern of Sirt1 and Nampt, genes encoding key enzymes that connect cellular metabolism and circadian network. Altogether circadian amplitude of Leydig cell's endocrine function decreased during aging. The results suggest that clock genes are more resistant to aging than genes involved in steroidogenesis supporting the hypothesis about peripheral clock involvement in rhythm maintenance during aging.

© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. Controlled changes in mitochondrial biogenesis and morphology are required for cell survival and homeostasis, but the molecular mechanisms are largely unknown. Here, male and female prepubertal mice (P21) with insulin and IGF1 receptors deletions in steroidogenic tissues (Insr/Igf1r-DKO) were used to investigate transcription of the key regulators of mitochondrial biogenesis (Ppargc1a, Ppargc1b, Pparg, Nrf1, Tfam) and architecture in Leydig cells, ovaries, and adrenals. Results showed that the expression of PGC1, a master regulator of mitochondrial biogenesis and integrator of environmental signals, and its downstream target Tfam, significantly decreased in androgen-producing Leydig cells. This is followed by reduction of Mtnd1, a mitochondrial DNA encoded transcript whose core subunit belongs to the minimal assembly required for catalysis. The same markers remained unchanged in ovaries. In contrast, in adrenals, the pattern of transcripts for mitochondrial biogenesis markers was the same in both sexes, but opposite from that observed in Leydig cells. The level of transcripts for markers of mitochondrial architecture (Mfn1, Mfn2) significantly increased in Leydig cells from Insr/Igf1r-DKO, but not in ovaries. This was followed by mitochondrial morphology disturbance, suggesting that the mitochondrial phase of steroidogenesis could be affected. Indeed, basal and pregnenolone stimulated progesterone productions in the mitochondria of Leydig cells from Insr/Igf1r-DKO decreased more than androgen production, and were barely detectable. Our results are the first to show that INSR/IGF1R are important for mitochondrial biogenesis in gonadal steroidogenic cells of prepubertal males, but not females and they serve as important regulators of mitochondrial architecture and biogenesis markers in Leydig cells.

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.

Here, we chronologically analyzed age-associated changes of cAMP- and MAPK-signaling in Leydig cells (LCs) in relation with decreased testosterone (T) production. In Wistar rats, decreased serum T observed in 12 to 24-month-old rats was not related to decreased serum LH concentration but to reduced luteinizing hormone receptor (Lhr/LHR) and time-coordinated reduction of steroidogenic gene expression (decreased Cyp11a1, Cyp17a1 in 12-month-old rats followed by decreased Star/StAR, Hsd3b/HSD3B, Hsd17b4, and increased Cyp19a1 later in life). The predecessors of age-related changes noted in LCs from 6 to 12-month-old rats were increased level of soluble adenylate cyclase (Adcy/AC) 10, increased JNK phosphorylation but suppressed P38 MAPK. At approximately the same time changed mRNA abundance for transcription factors important for steroidogenesis was detected (increased Nur77 and decreased Sf1, Dax1). Aging caused biphasic expression pattern of ERK1/2 and Nur77: increased in 12-month but decreased in LCs from 24-month-old rats. Further, decreased basal cAMP level observed from 12 to 24th month coincidence with increased expression of cAMP-specific phosphodiesterase (Pde)4a, Pde4b and regulatory subunit of protein kinase A (Prkar/PKAR). Exposing of senescent LCs to permeable cAMP-analog improved transcription of Sf1, Nur77, Star, Cyp11a1,Cyp17a1, but without effect on aging pattern of Dax1, Pde4a/b, Prkar2a, Lhr and MAPK genes. Collectively, results indicated that age-related LC dysfunction is accompanied with changes in MAPK and cAMP signaling and coordinated reduction in the expression of many of the genes that participate in T synthesis. The predecessors of aged-related changes are increased ratio of pJNK/JNK, AC10 and decreased P38 level in LCs from 6-month-old rats.

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.

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.

This study was designed to systematically analyze and evaluate the effects of in vivo blockade of α1-adrenergic receptors (α1-ADRs) on the stress-induced disturbance of steroidogenic machinery in Leydig cells. Parameters followed 1) steroidogenic enzymes/proteins, transcription factors, and cAMP/testosterone production; 2) the main hallmarks of stress (epinephrine, glucocorticoids); and 3) transcription profiles of ADRs and oxidases with high affinity to inactivate glucocorticoids. Results showed that sustained blockade of α1-ADRs prevented stress-induced 1) decrease of the transcripts/proteins for main steroidogenic CYPs (CYP11A1, CYP17A1); 2) decrease of Scarb1 and Hsd3b1 transcripts; 3) decrease of transcript for Nur77, one of the main activator of the steroidogenic expression; and 4) increase of Dax1 and Arr19, the main steroidogenic repressors in Leydig cells. In the same cells, the expression of steroidogenic stimulatory factor Creb1, StAR, and androgen receptor increased. In this signaling scenario, stress-induced stimulation of Adra1a/Adra1b/Adrbk1 and Hsd11b2 (the unidirectional oxidase with high affinity to inactivate glucocorticoids) was not changed. Blockade additionally stimulated stress-increased transcription of the most abundantly expressed ADRs Adra1d/Adrb1/Adrb2 in Leydig cells. In the same cells, stress-decreased testosterone production, the main marker of Leydig cells functionality, was completely prevented, while reduction of cAMP, the main regulator of androgenesis, was partially prevented. Accordingly, the presented data provide a new molecular/transcriptional base for “fight/adaptation” of steroidogenic cells and new molecular insights into the role of α1-ADRs in stress-impaired Leydig cell steroidogenesis. The results are important in term of wide use of α1-ADR selective antagonists, alone/in combination, to treat high blood pressure, nightmares associated with posttraumatic stress disorder, and disrupted sexual health.

The stress-induced initiation of proapoptotic signaling in Leydig cells is relatively well defined, but the duration of this signaling and the mechanism(s) involved in opposing the stress responses have not been addressed. In this study, immobilization stress (IMO) was applied for 2 h daily, and animals were euthanized immediately after the first (IMO1), second (IMO2), and 10th (IMO10) sessions. In IMO1 and IMO2 rats, serum corticosterone and adrenaline were elevated, whereas serum androgens and mRNA transcription of insulin-like factor-3 in Leydig cells were inhibited. Reduced oxygen consumption and the mitochondrial membrane potential coupled with a leak of cytochrome c from mitochondria and increased caspase-9 expression, caspase-3 activity, and number of apoptotic Leydig cells was also observed. Corticosterone and adrenaline were also elevated in IMO10 rats but were accompanied with a partial recovery of androgen secretion and normalization of insulin-like factor-3 transcription coupled with increased cytochrome c expression, abolition of proapoptotic signaling, and normalization of the apoptotic events. Blockade of intratesticular glucocorticoid receptors diminished proapoptotic effects without affecting antiapoptotic effects, whereas blockade of intratesticular α1-adrenergic receptors diminished the antiapoptotic effects without affecting proapoptotic effects. These results confirmed a critical role of glucocorticoids in mitochondria-dependent apoptosis and showed for the first time the relevance of stress-induced upregulation of α1-adrenergic receptor expression in cell apoptotic resistance to repetitive IMOs. The opposite role of two hormones in control of the apoptotic rate in Leydig cells also provides a rationale for a partial recovery of androgen production in chronically stressed animals.

Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone (T) predominantly taken as drugs of abuse. Using in vivo treatment of adult male rats we investigated the effects of testosterone enanthate (TE) a widely abused AAS, on apoptosis of Leydig cells. Increased T and decreased luteinizing hormone levels in serum and decreased intra-testicular T values were found in 2 and 10 weeks treated groups. Two weeks of TE-treatment stimulated the expression of inducible nitric oxide synthase (NOS2) followed by increased NO production, decreased mitochondrial membrane potential and increased prevalence of Leydig cell apoptosis. This was prevented by in vivo administration of androgen receptor blocker. The induced NOS2 level and apoptosis returned to control levels after 10 weeks of TE-treatment but testes contained fewer Leydig cells. Overall, AAS in addition to reduced steroidogenesis induce transient increase of Leydig cells apoptotic rate through mechanism associated with androgen receptor, most likely involving NOS2 induction.

Doxazosin (Doxa) is an α1-selective adrenergic receptor (ADR) antagonist widely used, alone or in combination, to treat high blood pressure, benign prostatic hyperplasia symptoms, and recently has been suggested as a potential drug for prostate cancer prevention/treatment. This study was designed to evaluate the effect of in vivo Doxa po-application, in clinically relevant dose, on: (i) steroidogenic machinery homeostasis; (ii) cAMP/cGMP signalling; (iii) transcription profile of ADR in Leydig cells of adult rats. The results showed that po-application of Doxa for once (1×Doxa), or for two (2×Doxa) or 10 (10×Doxa) consecutive days significantly disturbed steroidogenic machinery homeostasis in Leydig cells. Doxa po-application significantly decreased circulating luteinizing hormone and androgens levels. The level of androgens in testicular interstitial fluid and that extracted from testes obtained from 1×Doxa/2×Doxa rats decreased, although it remained unchanged in 10×Doxa rats. Similarly, the ex vivo basal androgen production followed in testes isolated from 1×Doxa/2×Doxa rats decreased, while remained unchanged in 10×Doxa rats. Differently, ex vivo testosterone production and steroidogenic capacity of Leydig cells isolated from 1×Doxa/2×Doxa rats was stimulated, while 10×Doxa had opposite effect. In the same cells, cAMP content/release showed similar stimulatory effect, but back to control level in Leydig cells of 10×Doxa. 1×Doxa/2×Doxa decreased transcripts for cAMP specific phosphodiesterases Pde7b/Pde8b, whereas 10×Doxa increased Pde4d. All types of treatment reduced the expression of genes encoding protein kinase A (PRKA) regulatory subunit (Prkar2b), whereas only 10×Doxa stimulated catalytic subunit (Prkaca). Doxa application more affected cGMP signalling: stimulated transcription of constitutive nitric oxide synthases (Nos1, Nos3) in time-dependent manner, whereas reduced inducible Nos2. 10×Doxa increased guanylyl cyclase 1 transcript and PRKG1 protein in Leydig cells. Orally applied Doxa significantly disturbed the transcriptional 'signature' of steroidogenic machinery, cAMP/cGMP signalling and ADRs and β-ADRs kinases in Leydig cells, thus giving new molecular insights into the role of cAMP/cGMP/adrenalin signalling in Leydig cells homeostasis.

Introduction Phosphodiesterase type 5 (PDE5) inhibitors have been established in therapy for a variety of physiological disorders including erectile dysfunction. Despite its popularity and wide usage in erectile dysfunction treatment, the short‐term effect of PDE5 inhibition on Leydig cell functionality and testosterone dynamics is missing. Aim This study was designed to assess the acute in vivo effects of sildenafil citrate (Viagra) treatment on testosterone production. Methods Male adult rats were given sildenafil (1.25 mg/kg BW) per os, and testosterone production were analyzed 30, 60, 120, and 180 minutes after treatment. Additionally, in vitro effect of sildenafil extract on Leydig cell steroidogenesis was estimated. Main Outcome Measures The formation of testicular interstitial fluid (TIF), and testosterone, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) content was followed. Occurrence and phosphorylation of mature steroidogenic acute regulatory protein (StAR) and interaction with protein kinase G 1 (PRKG1) were assessed by immunoprecipitation and Western blot. Results Serum testosterone was increased 60 and 120 minutes after sildenafil treatment. In 60 minutes, TIF volume was doubled and stayed increased till the end of the experimental period. cGMP and testosterone content in TIF were increased 30 minutes after treatment, and cAMP decreased in 60 minutes. Further, sildenafil‐induced stimulation of testosterone production was abolished by ex vivo addition of PRKG1 inhibitor but not by protein kinase A inhibitor. Sildenafil treatment increased the level of phosphorylated and total StAR protein. Moreover, co‐immunoprecipitation of StAR and PRKG1 was increased following sildenafil treatment suggesting the active role of this kinase in initiation of testosterone synthesis. Additionally, sildenafil extract applied in vitro on primary Leydig cell culture increased cGMP accumulation and testosterone production in time‐ and dose‐dependent manner without effect on cAMP level. Conclusion Acute sildenafil treatment enlarged TIF volume but also stimulated testosterone production which may be significant considering the positive testosterone effect in regulation of sexual activity. Janjic MM, Stojkov NJ, Bjelic MM, Mihajlovic AI, Andric SA, and Kostic TS. Transient rise of serum testosterone level after single sildenafil treatment of adult male rats. J Sex Med **;**:**–**.

This study was designed to evaluate the effect of acute (2 h daily) and repeated (2 h daily for 2 or 10 consecutive days) immobilization stress (IMO) on: 1) the steroidogenic machinery homeostasis; 2) cAMP signaling; and the expression of receptors for main markers of 3) adrenergic and 4) glucocorticoid signaling in Leydig cells of adult rats. The results showed that acute IMO inhibited steroidogenic machinery in Leydig cells by downregulation of Scarb1 (scavenger receptor class B), Cyp11a1 (cholesterol side-chain cleavage enzyme), Cyp17a1 (17α-hydroxylase/17,20 lyase), and Hsd17b3 (17β-hydroxysteroid dehydrogenase) expression. In addition to acute IMO effects, repeated IMO increased transcription of Star (steroidogenic acute regulatory protein) and Arr19 (androgen receptor corepressor 19 kDa) in Leydig cells. In the same cells, the transcription of adenylyl cyclases (Adcy7, Adcy9, Adcy10) and cAMP-specific phosphodiesterases ( Pde4a, Pde4b, Pde4d, Pde7a, Pde8a) was stimulated, whereas the expression of the genes encoding protein kinase A subunits were unaffected. Ten times repeated IMO increased the levels of all adrenergic receptors and β-adrenergic receptor kinase ( Adrbk1) in Leydig cells. The transcription analysis was supported by cAMP/testosterone production. In this signaling scenario, partial recovery of testosterone production in medium/content was detected. The physiological significance of the present results was proven by ex vivo application of epinephrine, which increased cAMP/testosterone production by Leydig cells from control rats in greater fashion than from stressed. IMO did not affect the expression of transcripts for Crhr1/Crhr2 (corticotropin releasing hormone receptors), Acthr (adrenocorticotropin releasing hormone receptor), Gr (glucocorticoid receptor), and Hsd11b1 [hydroxysteroid (11-β) dehydrogenase 1], while all types of IMO stimulated the expression of Hsd11b2, the unidirectional oxidase with high affinity to inactivate glucocorticoids. Thus, presented data provide new molecular/transcriptional base for “fight/adaptation” of Leydig cells and new insights into the role of cAMP, epinephrine, and glucocorticoid signaling in recovery of stress-impaired Leydig cell steroidogenesis.

Anabolic androgenic steroids (AAS) are testosterone derivatives originally designed to enhance muscular mass and used for the treatment of many clinical conditions as well as in contraception. Despite popular interest and abuse, we still lack a broad understanding of effects of AAS on synthesis of steroid hormones on the molecular level. This study was designed to systematically analyze the effects of pharmacological/high doses of testosterone on steroidogenic machinery in Leydig cells. Two different experimental approaches were used: (1) In vivo experiment on groups of adult male rats treated with testosterone for 1 day, 2 weeks, and 2 months; (2) Direct in vitro testosterone treatment of Leydig cells isolated from intact rats. Result showed that prolonged in vivo treatment with testosterone decreased the expression of Scarb1 (scavenger receptor class B type 1), Tspo (translocator protein), Star (steroidogenic acute regulatory protein), Cyp11a1 (cholesterol side-chain cleavage enzyme), and Cyp17a1 (17α-hydroxylase/17, 20 lyase) in Leydig cells. Oppositely, the expression of Hsd3b (3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase), Ar (androgen receptor), and Pde4a/b (cyclic adenosine monophosphate-dependent phosphodiesterases) was increased. Androgenization for 2 weeks inhibited Cyp19 (aromatase) transcription, whereas 2-month exposure caused the opposite effect. Direct in vitro testosterone treatment also decreased the expression of Cyp11a1, Cyp17a1, and Cyp19a1, whereas Hsd3b was upregulated. The results of expression analysis were supported by declined steroidogenic capacity and activity of Leydig cells, although conversion of pregnenolone to progesterone was stimulated. The upregulation of AR and 3βHSD in testosterone-impaired Leydig cells steroidogenesis could be the possible mechanism that maintain and prevent loss of steroidogenic function.

Sildenafil citrate (Viagra), a cGMP-selective phosphodiesterase (PDE) inhibitor, is widely used to treat erectile dysfunction and pulmonary arterial hypertension. In contrast to its well established action on erectile dysfunction, little is known on the action of sildenafil on cGMP/cAMP signaling and testicular steroidogenesis. This study was designed to assess the effects of prolonged sildenafil treatment on NO synthase-dependent signaling and steroidogenic function of rat Leydig cells. Male adult rats were treated with Viagra (1.25 mg/kg body wt) daily for 30 days. In our studies, serum testosterone and ex vivo testosterone production significantly increased in sildenafil-treated animals. Human chorionic gonadotropin-stimulated testosterone production and cAMP accumulation were also significantly higher in Leydig cells obtained from sildenafil-treated rats. The expression of soluble guanylyl cyclase (GUCY1) subunits ( Gucy1a1, Gucy1b1) significantly increased; cAMP-specific Pde4a, cGMP-specific Pde6c, and dual Pde1c and Nos2 were inhibited and expression of Nos3, protein kinase G1 ( Pkg1), and Pde5 remained unchanged. Treatment of purified Leydig cells with NO donor caused a dose-dependent increase in both testosterone and cGMP production. Testosterone and cGMP production was significantly higher in Leydig cells obtained from sildenafil-treated animals. The stimulatory effect of NO donor was significantly enhanced by saturating concentrations of hCG in both Leydig cells obtained from control and sildenafil-treated animals. Occurrence of mature steroidogenic acute regulatory protein also increased in sildenafil treated animals in accord with increased cAMP and cGMP production. In summary, inhibition of PDE activity during prolonged sildenafil treatment increased serum testosterone level and Leydig cells' steroidogenic capacity by coordinated stimulatory action on cAMP and cGMP signaling pathway.

Testosterone, acting as a systemic and local factor, is one of the major regulatory molecules that initiate and maintain testicular function. In the present study, different experimental approaches were used to evaluate the role of testosterone in regulation of the nitric oxide (NO)-cGMP pathway in Leydig cells derived from normal and hypogonadotropic male rats treated with testosterone for 24 h and 2 wk. Real-time quantitative PCR and Western blot analysis revealed increased inducible NO synthase (NOS2) expression followed by increased NO secretion from Leydig cells ex vivo after continuous treatment with testosterone for 2 wk in vivo. The cGMP-specific phosphodiesterases Pde5, Pde6, and Pde9 were up-regulated, whereas PRKG1 protein was decreased after a 2-wk testosterone treatment. Induction of Nos2 and Pde5 in Leydig cells was blocked by androgen receptor antagonist. In experimental hypogonadotropic hypogonadism, expression of NOS2 was significantly reduced, and treatment with testosterone increased NOS2 expression above control levels. PDE5 protein level was unchanged in hypogonadal rats, whereas treatment of hypogonadal rats with testosterone significantly increased it. In contrast, hypogonadism and testosterone replacement reduced PRKG1 protein in Leydig cells. In vitro treatment with testosterone caused gradually increased Nos2 gene expression followed by increased nitrite and cGMP production by purified Leydig cells. In summary, testosterone up-regulated NO signaling via increased NOS2 expression and contributed to down-regulation of cGMP signaling in Leydig cells. Thus, testosterone-induced modulation of NO-cGMP signaling may serve as a potent autocrine regulator of testicular steroidogenesis.

The role of the structural complexity of the testis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway was analysed in adult male rats exposed to acute and repeated immobilization stress (IMO). In whole testis preparations, exposure to acute and repeated IMO caused an increase in NO production. In contrast, NO production was inhibited in interstitial cell preparations after exposure to all types of stress. In purified Leydig cell preparations, NO production was inhibited only after exposure to prolonged IMO. These findings indicate that biologically active compounds released from various testicular compartments exert both stimulatory and inhibitory effects on NO production. TaqMan Low Density Array of rat phosphodiesterases revealed a decrease in the expression of cGMP-specific phosphodiesterase 5 (PDE5) in Leydig cells of animals exposed to repeated IMO. In contrast, the expression of cGMP-dependent protein kinase type I (PKG I), total and phosphorylated steroidogenic acute regulatory protein (StAR), and PKG I/StAR immunoprecipitated complex was increased during repeated exposure to IMO. The increase in both total and phosphorylated StAR formation was effectively blocked by inhibition of PKG I in vitro. Thus, increased expressions of PKG I and StAR complex, accompanied by decreased PDE5 activity, suggest that the NO-cGMP signalling pathway and consequent activation of the StAR protein regulate the adaptive response of Leydig cells to repeated IMO stress.

G(i/o) protein-coupled receptors, signaling through G protein-dependent and protein-independent pathways, have prominent effects on secretion by modulating calcium signaling and regulating the size of the releasable secretory pool, the rates of exocytosis and endocytosis, and de novo synthesis. Pituitary cells fire action potentials spontaneously, and the associated calcium influx is sufficient to maintain prolactin (PRL) release but not gonadotropin release at high and steady levels for many hours. Such secretion, termed intrinsic, spontaneous, or basal, reflects fusion of secretory vesicles triggered by the cell type-specific pattern of action potentials. In lactotrophs, activation of endothelin ET(A) and dopamine D(2) receptors causes inhibition of spontaneous electrical activity and basal adenylyl cyclase activity accompanied with inhibition of basal PRL release. Agonist-induced inhibition of cAMP production and firing of action potentials is abolished in cells with blocked pertussis toxin (PTX)-sensitive G(i/o) signaling pathway. However, agonist-induced inhibition of PRL release is only partially relieved in such treated cells, indicating that both receptors also inhibit exocytosis downstream of cAMP/calcium signaling. The PTX-insensitive step in agonist-induced inhibition of PRL release is not affected by inhibition of phosphoinositide 3-kinase and glycogen synthase kinase-3 but is partially rescued by downregulation of the G(z)alpha expression. Thus, ET(A) and D(2) receptors inhibit basal PRL release not only by blocking electrical activity but also by desensitizing calcium-secretion coupling.

The ability of immobilization stress (IMO) to decrease Leydig cell steroidogenesis and serum androgen concentration has been previously observed, but the possible mechanism(s) involved in the adaptation to prolonged or repeated stress have not been identified. In this study, we investigated whether the Leydig cells obtained from adult rats subjected to acute (15 min, 30 min or 2 h) and repeated (2 or 10 days, 2 h daily) IMO show adaptive mechanism(s) in response to stress-impaired steroidogenesis. The results showed that basal and human chorionic gonadotropin-stimulated cAMP production by Leydig cells isolated from rats exposed to both acute and repeated IMO was significantly reduced. Despite the reduced cAMP production, immunoblot analysis revealed increased immunoreactivity for both protein kinase A (PKA) and steroidogenic acute regulatory (StAR) protein in Leydig cells obtained from rats repeatedly exposed to IMO. Also, the phosphorylation and production of mature StAR protein was evident during exposure of rats to repeated IMO treatment. Treatment with cholesterol, the steroid substrate transported into mitochondria by StAR, significantly increased androgen and progesterone production by Leydig cells isolated from rats exposed to repeated IMO. In contrast, when other steroid substrates (22(R)-OH-cholesterol, pregnenolone, progesterone, Delta4-androstenedione) were present in the culture media, Leydig cell steroidogenesis was still reduced by IMO. Thus, PKA-mediated phosphorylation of StAR protein is an important mechanism in the adaptive response of Leydig cells to repeated IMO.

Steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives have been prepared using 3beta,17beta-dihydroxy-17alpha-picolyl-androst-5-ene (1), 3beta-acetoxy-17-picolinylidene-androst-5-ene (2), and 3beta-hydroxy-17-picolinylidene-androst-5-ene (3) as synthetic precursors. The compounds 2 and/or 3 were reacted with m-chloroperoxybenzoic acid (MCPBA). The compounds synthesized from 2 were 17-picolinylidene-N-oxide 4, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 5 and 6, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 7 and 8. Starting from compound 3, a mixture of 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene 9 and 10, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 11 and 12, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 13 and 14 were obtained. From compounds 15 and 18, obtained from 1 and 3 by the Oppenauer oxidation, the 4alpha,5alpha-epoxy and 4beta,5beta-epoxy derivatives 16, 17 and 20, 21 were prepared by oxidation with 30% H(2)O(2). Oxidation of 18 with MCPBA yielded only the N-oxide 19. The structures of compounds 15 and 18 were proved by the X-ray analysis. Compounds 1-6, 9, 15, 17, 18, and 21 were tested on activity against the enzyme aromatase. Antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Three tested compounds (1, 4, and 19) showed strong activity against PC3, the IC(50) values being in the range 0.55-10microM, whereas compound 17 showed strong activity against MDA-MB-231 (IC(50) 10.4microM).

The G protein-coupled receptors in excitable cells have prominent roles in controlling Ca2+-triggered secretion by modulating voltage-gated Ca2+ influx. In pituitary lactotrophs, spontaneous voltage-gated Ca2+ influx is sufficient to maintain prolactin release high. Here we show that endothelin in picomolar concentrations can interrupt such release for several hours downstream of spontaneous and high K+-stimulated voltage-gated Ca2+ influx. This action occurred through the Gz signaling pathway; the adenylyl cyclase-signaling cascade could mediate sustained inhibition of secretion, whereas rapid inhibition also occurred at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. In a nanomolar concentration range, endothelin also inhibited voltage-gated Ca2+ influx through the G i/o signaling pathway. Thus, the coupling of seven-transmembrane domain endothelin receptors to Gz proteins provided a pathway that effectively blocked hormone secretion distal to Ca2+ entry, whereas the cross-coupling to G i/o proteins reinforced such inhibition by simultaneously reducing the pacemaking activity.

Starting from 3-(benzyloxy)-16-(hydroxyimino)-estra-1,3,5(10)-trien-17-one (1) several 17-methyl-16,17-secoestratriene derivatives were synthesized. In the first step of synthesis, hydroxyimino ketone 1 was transformed into 3-(benzyloxy)-16-(hydroxyimino)-17α-methylestra-1,3,5(10)-trien-17β-ol (2), the Beckmann fragmentation of which gave 3-(benzyloxy)-17-methyl-17-oxo-16,17-secoestra-1,3,5(10)-triene-16-nitrile (3a). Reduction of 3a with sodium borohydride yielded (17S)-3-(benzyloxy)-17-hydroxy-17-methyl-16,17-secoestra-1,3,5(10)-triene-16-nitrile (4a), whose configuration at the newly formed chiral center was established by X-ray structural analysis. Catalytic hydrogenation of compound 3a under different reaction conditions yielded 3-hydroxy-17-methyl-17-oxo-16,17-secoestra-1,3,5(10)-triene-16-nitrile (3b) and 16-amino-17-methyl-16,17-secoestra-1,3,5(10)-triene-3,17-diol (6b). Biological tests in vivo of compounds 3b and 6b showed their moderate antiestrogenic activity.

The starting compound for synthesis of new 16,17-seco-estratriene derivatives was 3-benzyloxy-17-hydroxy-16,17-secoestra-1,3,5(10)-triene-16-nitrile (1b), obtained from estrone in several synthetic steps. 17-Tosyl, -chloro-, bromo-, and -iodo- derivatives 2b, 4b, 5b, and 6b were prepared directly from secocyanoalcohol 1b, while the 17-fluoro-derivative 3b was obtained from tosylate 2b in the reaction with tetrabutyl ammonium fluoride. The corresponding 3-hydroxy derivatives of these compounds were produced by action of hydrogen in presence of Pd/C, except the 3-hydroxy-17-iodo derivative 6a, which was obtained from 3-hydroxy-17-tosyloxy derivative 2a. All the newly synthesized compounds in biological tests on experimental animals exhibited an almost total loss of estrogenic activity, while most of them even prevented the action of endogenous estrogens. On the other hand, most of them, except compounds 3a and 6b, partially hindered the action of estradiol benzoate, behaving as moderate antagonists.

Two Ca(2+)-mobilizing receptors expressed in lactotrophs, endothelin-A (ET(A)) and thyrotropin-releasing hormone (TRH), induce a rapid Ca(2+) release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX) treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca(2+) signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs(+), a relatively specific blocker of inward rectifier K(+) channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca(2+) signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs(+) was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca(2+)-activated SKand BK-type K(+) channels, respectively; E-4031, a blocker of ether a-go-go K(+) channel; and linopirdine, a blocker of M-type K(+) channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs(+)-sensitive channels, presumably the Gi/o-controlled inward rectifier K(+) channels, and that this agonist also inhibits PRL release, but downstream of Ca(2+) influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

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.

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.