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Melatonin is a pineal hormone that regulates testicular activity (i.e., steroidogenesis and spermatogenesis) through two complementary mechanisms, indirect effects exerted via the hypothalamic-adenohypophyseal axis and direct actions that take place on the different cell populations of the male gonad. The effects of increased age on the testis and the general mechanisms involved in testicular pathology leading to infertility are still only poorly understood. However, there is growing evidence that link testicular aging and idiopathic male infertility to local inflammatory and oxidative stress events. Because literature data strongly indicate that melatonin exhibits anti-inflammatory and anti-oxidant properties, this review focuses on the potential benefits exerted by this indoleamine at testicular level in male reproductive fertility and aging. Taking into account that the effects of melatonin supplementation on testicular function are currently being investigated, the overview covers not only promising prospects but also many questions concerning the future therapeutic value of this indoleamine as an anti-aging drug as well as in the management of cases of male infertility for which there are no medical treatments currently available., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The implications of SARS-CoV-2 infection on male fertility remain largely unknown. Besides their well-known pro- and anti-inflammatory actions, prostaglandins and polyamines are present in semen, where they play key roles in sperm quality., Objectives: To analyze semen parameters, oxidative profile and the seminal fluid prostaglandin and polyamine systems in samples collected from individuals without coronavirus disease 2019 diagnosis and men who recovered from coronavirus disease 2019., Materials and Methods: This study compared semen collected from men without positive coronavirus disease 2019 diagnosis with samples obtained from individuals 1-6 months and 7-30 months post SARS-CoV-2 infection. Semen parameters, thiobarbituric acid reactive substances, cyclooxygenase 2 expression by fluorescence immunocytochemistry and immunoblotting, prostaglandin levels by enzyme immunoassay, ornithine decarboxylase activity by a radioactive assay, and polyamine and acetylated polyamine levels by thin-layer chromatography were assessed., Results: In both groups of semen samples from coronavirus disease 2019 recovered men, sperm vitality, total and progressive sperm motility, and putrescine levels were significantly decreased when compared with samples from the uninfected group. In contrast, lipid peroxidation, leukocyte-associated cyclooxygenase 2 expression, and prostaglandin D2 levels were higher in semen from coronavirus disease 2019 recovered men than in samples from uninfected individuals. While sperm concentration and morphology, ornithine decarboxylase activity, and N-acetylputrescine levels were statistically diminished in semen obtained up to 6 months after coronavirus disease 2019 recovery, these parameters remained unchanged when samples were collected 7-30 months after coronavirus disease 2019 recovery. Coronavirus disease 2019 vaccination did not show negative effects on any of the parameters evaluated., Discussion and Conclusion: Our work provides insights into the detrimental impact of coronavirus disease 2019 on several sperm parameters, in some cases, even more than a year after SARS-CoV-2 infection, which would be accompanied by alterations in the seminal fluid prostaglandin and polyamine profiles. Therefore, future treatments targeting the prostaglandin and polyamine pathways in coronavirus disease 2019 recovered men could lead to a successful reinstatement of semen parameters., (© 2023 American Society of Andrology and European Academy of Andrology.)

Antioxidant actions of melatonin and its impact on testicular function and fertility have already been described. Considering that Sertoli cells contribute to provide structural support and nutrition to germ cells, we evaluated the effect of melatonin on oxidative state and lactate metabolism in the immature murine TM4 cell line and in immature hamster Sertoli cells. A prooxidant stimulus applied to rodent Sertoli cells expressing MT1/MT2 receptors, increased lipid peroxidation whereas decreased antioxidant enzymes (superoxide dismutase 1, catalase, peroxiredoxin 1) expression and catalase activity. These changes were prevented by melatonin. Furthermore, melatonin stimulated lactate dehydrogenase (LDH) expression/activity via melatonin receptors, and increased intracellular lactate production in rodent Sertoli cells. Interestingly, oral melatonin supplementation in infertile men positively regulated LDHA testicular mRNA expression. Overall, our work provides insights into the potential benefits of melatonin on Sertoli cells contributing to testicular development and the future establishment of a sustainable spermatogenesis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Peritubular myoid cells are emerging as key regulators of testicular function in adulthood. However, little is known about the role of testicular peritubular myoid cells (TPMCs) in the development of the male gonad. We found that, compared to testes of young adult hamsters, gonads of 21 day-old animals show increased melatonin concentration, seminiferous tubular wall thickening and a heterogeneous packaging of its collagen fibers thus raising the question whether melatonin may be involved in the regulation of TPMCs., Methods: We established primary cultures of TPMCs from immature hamsters (ihaTPMCs), which we found express melatonergic receptors., Results: Exogeneous melatonin decreased the levels of inflammatory markers (NLRP3 inflammasome, IL1β) but increased the expression of cyclooxygenase 2 (COX2, key enzyme mediating prostaglandin synthesis) and of the glial cell line-derived neurotrophic factor (GDNF) in ihaTPMCs. Melatonin also stimulated ihaTPMCs proliferation and the expression of extracellular matrix proteins such as collagen type I and IV. Furthermore, collagen gel contraction assays revealed an enhanced ability of ihaTPMCs to contract in the presence of melatonin., Conclusion: Melatonin regulates immune and inflammatory functions as well as contractile phenotype of the peritubular wall in the hamster testis., General Significance: If transferable to the in vivo situation, melatonin-dependent induction of ihaTPMCs to produce factors known to exert paracrine effects in other somatic cell populations of the gonad suggests that the influence of melatonin may go beyond the peritubular wall and indicates its contribution to testicular development and the establishment of a normal and sustainable spermatogenesis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The evolutionary theory of aging supports a trade-off relationship between reproduction and aging. Aging of the male reproductive system primarily affects the testes, leading to a decrease in the levels of sexual hormones, alterations in sperm quality and production, and a decline in fertility that does not necessarily involve a complete cessation of spermatogenesis. Inflammation, oxidation, and apoptosis are events considered as predictors of pathogenesis and the development of age-related diseases that are frequently observed in aged testes. Although the molecular mechanisms are still poorly understood, accumulating evidence points toward pro-inflammatory molecules and reactive oxygen species as primary contributing factors for testicular aging. However, the real impact of aging-related testicular alterations on fertility, reproductive health, and life span is far from being fully revealed. This work discusses the current knowledge on the impact of aging in the testis, particularly of aging-related dysregulated inflammation and oxidative damage on the functioning of its different cell populations. More interestingly, this review covers the potential benefits of anti-aging interventions and therapies using either pharmacological compounds (such as non-selective non-steroidal anti-inflammatory medication) or more natural alternatives (such as various nutraceuticals or even probiotics) that exhibit anti-inflammatory, antioxidant, and anti-apoptotic properties. Some of these are currently being investigated or are already in clinical use to delay or prevent testicular aging.

Ageing is linked to changes in the hypothalamic-pituitary-gonadal axis and a progressive decline in gonadal function. While women become infertile when they enter menopause, fertility decline in ageing men does not necessarily involve a complete cessation of spermatogenesis. Gonadal dysfunction in elderly people is characterized by morphological, endocrine and metabolic alterations affecting the reproductive function and quality of life. With advancing age, sexuality turns into a critical emotional and physical factor actually defining the number of years that ageing people live a healthy life. Gonadal ageing correlates with comorbidities and an increased risk of age-related diseases including diabetes, kidney problems, cardiovascular failures and cancer. This article briefly summarizes the current state of knowledge on ovarian and testicular senescence, explores the experimental models used in the study of gonadal ageing, and describes the local pro-inflammatory, oxidative and apoptotic events and the associated signalling pathways that take place in the gonads while people get older. Overall, literature reports that ageing exacerbates a mutual crosstalk among oxidative stress, apoptosis and the inflammatory response in the gonads leading to detrimental effects on fertility. Data also highlight the clinical implications of novel therapeutic interventions using antioxidant, anti-apoptotic and anti-inflammatory drugs on health span and life span., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have previously shown an inverse correlation between testicular melatonin concentration and inflammation/oxidative stress-related markers levels in infertile men showing unexplained azoospermia. Here, we evaluated the impact of melatonin oral supplementation (daily 3 mg dose used to treat sleep disorders) in the incidence of local inflammation, oxidative stress, and tubular wall fibrosis development in young and middle-aged infertile adult men. Compared with testes without histological alterations, gonads with morphological abnormalities showed lower melatonin concentration along with increased macrophage numbers, TBARS generation, and expression levels of inflammation-related markers and antioxidant enzymes, as well as tubular wall collagen fibers disorganization and thickening. Melatonin oral supplementation not only increased its own testicular levels but also decreased inflammation- and oxidative stress-related markers levels, and improved the tubular wall aspect. Overall, our work provides insights into the potential benefits of melatonin on the inflammatory and oxidative status in testes of patients suffering from unexplained infertility., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Continuously elevated levels of growth hormone (GH) during life in mice are associated with hepatomegaly due to hepatocytes hypertrophy and hyperplasia, chronic liver inflammation, elevated levels of arachidonic acid (AA) at young ages and liver tumors development at old ages. In this work, the hepatic expression of enzymes involved in AA metabolism, cPLA2α, COX1 and COX2 enzymes, was evaluated in young and old GH-transgenic mice. Mice overexpressing GH exhibited higher hepatic expression of cPLA2α, COX1 and COX2 in comparison to controls at young and old ages and in both sexes. In old mice, when tumoral and non-tumoral tissue were compared, elevated expression of COX2 was observed in tumors. In contrast, exposure to continuous lower levels of hormone for a short period affected COX1 expression only in males. Considering the role of inflammation during liver tumorigenesis, these findings support a role of alterations in AA metabolism in GH-driven liver tumorigenesis., Competing Interests: Declaration of competing interest All the authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Metformin (MET) is the most widely prescribed hypoglycemic drug in type 2 diabetes and Polycystic Ovary Syndrome. Besides its effects on glucose metabolism, MET exerts beneficial effects on these patients' fertility. However, the exact mechanisms of action of MET on female fertility are still unclear. In this work, we analyzed a possible direct effect of MET on ovarian cells. We found expression of the organic cation transporters OCT1, OCT2 and OCT3, responsible for MET uptake into the cells, in rat granulosa cells and human cumulus cells. Furthermore, MET increased pAMPK and decreased VEGF levels both in vivo and in rat granulosa cells in culture. These last effects were reversed when OCTs were inhibited. Our results suggest that MET acts directly on ovarian cells regulating cell metabolism and VEGF expression. Our findings are relevant to optimize PCOS fertility treatment and to explore ovarian MET actions in other female pathologies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Catecholaminergic neuronal elements (CNE) and macrophages (MACs) are increased in testes of patients with idiopathic infertility. Now, we describe an anatomical proximity between CNE and MACs, expression of specific α- and β-adrenergic receptors (ADRs) subtypes in MACs, and a positive correlation between the number of MACs and cyclooxygenase (COX2) expression - key enzyme in prostaglandin (PG) synthesis and an inflammatory marker - in testes of infertile men. To examine a potential effect of adrenergic input on COX2 expression, we used two additional experimental models: non-testicular human MACs (THP1 cell line) and non-human testicular MACs purified from adult Syrian hamsters. We found that epinephrine and norepinephrine up-regulate COX2 expression and PGD2 production through β1-and β2-ADRs. Our results demonstrate the existence of a yet unknown link between CNE and MACs in the human testis that could trigger inflammation and tissue homeostatic dysregulation associated with pathogenesis or maintenance of infertility states., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Male meiotic germ cell including the spermatozoa represent a great challenge to the immune system, as they appear long after the establishment of normal immune tolerance mechanisms. The capacity of the testes to tolerate autoantigenic germ cells as well as survival of allogeneic organ engrafted in the testicular interstitium have led to consider the testis an immunologically privileged site. Disruption of this immune privilege following trauma, tumor, or autoimmune orchitis often results in male infertility. Strong evidence indicates that indoleamine 2,3-dioxygenase (IDO) has been implicated in fetal and allograft tolerance, tumor immune resistance, and regulation of autoimmune diseases. IDO and tryptophan 2,3-dioxygenase (TDO) catalyze the same rate-limiting step of tryptophan metabolism along a common pathway, which leads to tryptophan starvation and generation of catabolites collectively known as kynurenines. However, the relevance of tryptophan metabolism in testis pathophysiology has not yet been explored. Here we assessed the in vivo role of IDO/TDO in experimental autoimmune orchitis (EAO), a model of autoimmune testicular inflammation and immunologically impaired spermatogenesis. EAO was induced in adult Wistar rats with testicular homogenate and adjuvants. Control (C) rats injected with saline and adjuvants and normal untreated rats (N) were also studied. mRNA expression of IDO decreased in whole testes and in isolated Sertoli cells during EAO. TDO and IDO localization and level of expression in the testis were analyzed by immunostaining and Western blot. TDO is expressed in granulomas from EAO rats, and similar protein levels were observed in N, C, and EAO groups. IDO was detected in mononuclear and endothelial cells and reduced IDO expression was detected in EAO group compared to N and C rats. This phenomenon was concomitant with a significant reduction of IDO activity in EAO testis measured by tryptophan and kynurenine concentrations (HPLC). Finally, in vivo inhibition of IDO with 1-methyl-tryptophan increased severity of the disease, demonstrating down regulation of IDO-based tolerance when testicular immune regulation was disrupted. We present evidence that an IDO-based mechanism is involved in testicular immune privilege.

Testicular aging is linked to histological, morphological and functional alterations. In the present study, we investigated whether aging affects the inflammatory and oxidative status in the testis by comparing young adult, middle-aged adult and aged hamsters. The Syrian hamster, a thoroughly studied seasonal breeder, was chosen as the experimental model since it allows further investigations on the role of photoperiod and melatonin in testicular aging with a minimal impact of the experimental intervention on the animal well-being and the subsequent results achieved. In testes of aged hamsters, we found a decrease in melatonin concentration, a thickening of the wall of the seminiferous tubules as well as a significant increase in IL-1β, NLRP3 and cyclooxygenase 2 expression, PGD2 production, macrophages numbers, lipid peroxidation and anti-oxidant enzyme catalase levels. Interestingly, when aged hamsters were transferred from a long day (LD) to a short day (SD) photoperiod for 16 weeks, testicular melatonin concentration increased while local inflammatory processes and oxidative stress were clearly reduced. Overall, these results indicate that melatonin might display anti-inflammatory and anti-oxidant capacities in the aged testes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adolescence is a transitional period from childhood to adulthood characterized by puberty and brain maturation involving behavioral changes and environmental vulnerability. Diet is one of the factors affecting brain health, potentially leading to long-lasting effects. Hence, we studied the impact of early exposure (P21-60) to a high-fat diet (HFD) on mouse hippocampus, analyzing inflammation, adult neurogenesis, dendritic spine plasticity, and spatial memory. Glycemia and seric pro-inflammatory IL1β were higher in HFD mice without differences on body weight. In the HFD hippocampus, neuroinflammation was evidenced by Iba1+ cells reactivity together with a higher expression of TNFα and IL1β while the neurogenic capability in the dentate gyrus was strongly reduced. We found a predominance of immature Dil-labeled dendritic spines from CA1 neurons along with diminished levels of the scaffold protein Shank2, suggesting a defective connectivity. Moreover, the HFD group exhibited spatial memory alterations. To elucidate whether microglia could be mediating HFD-associated neuronal changes, the lipotoxic context was emulated by incubating primary microglia with palmitate, a saturated fatty acid present in HFD. Palmitate induced a pro-inflammatory profile as shown by secreted cytokine levels. The isolated exosome fraction from palmitate-stimulated microglia induced an immature dendritic spine phenotype in primary GFP+ hippocampal neurons, in line with the in vivo findings. These results provide novel data concerning microglia to neuron communication and highlight that fat excess during a short and early period of life could negatively impact on cognition and synaptic plasticity in a neuroinflammatory context, where microglia-derived exosomes could be implicated. Graphical Abstract ᅟ.

Ageing is usually characterised by a mild chronic proinflammatory state. Despite the tight association between both processes, the phenomenon has recently been termed inflammageing. Inflammation in the male reproductive tract is frequently linked with bacterial or virus infections but also with a broad range of noninfectious processes. Prostatitis, epididymitis and orchitis, among others, can lead to infertility. However, in spite of the inflammation theory of disease, chronic inflammation in male urogenital system does not always cause symptoms. With advancing age, inflammatory processes are commonly observed in the male reproductive tract. Nevertheless, the incidence of inflammation in reproductive organs and ducts varies greatly among elderly men. Inflammageing is considered a predictor of pathogenesis and the development of age-related diseases. This article briefly summarises the current state of knowledge on inflammageing in the male reproductive tract. Yet, the precise aetiology of inflammageing in the male urogenital system, and its potential contribution not only to infertility but most importantly to adverse health outcomes remains almost unknown. Thus, further investigations are required to elucidate the precise cross-links between inflammation and male reproductive senescence, and to establish the impact of anti-inflammatory drug treatments on elder men's general health status., (© 2018 Blackwell Verlag GmbH.)

Reactive oxygen species (ROS) regulate testicular function in health and disease. We previously described a prostaglandin D2 (PGD2) system in Sertoli cells. Now, we found that PGD2 increases ROS and hydrogen peroxide (H2O2) generation in murine TM4 Sertoli cells, and also induces antioxidant enzymes expression suggesting that defense systems are triggered as an adaptive stress mechanism that guarantees cell survival. ROS and specially H2O2 may act as second messengers regulating signal transduction pathways and gene expression. We describe a stimulatory effect of PGD2 on lactate dehydrogenase (LDH) expression via DP1/DP2 receptors, which is prevented by the antioxidant N-acetyl-L-cysteine and the PI3K/Akt pathway inhibitor LY 294002. PGD2 also enhances Akt and CREB/ATF-1 phosphorylation. Our results provide evidence for a role of PGD2 in the regulation of the oxidant/antioxidant status in Sertoli cells and, more importantly, in the modulation of LDH expression which takes place through ROS generation and the Akt-CREB/ATF-1 pathway., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Aged testes undergo profound histological and morphological alterations leading to a reduced functionality. Here, we investigated whether variations in longevity affect the development of local inflammatory processes, the oxidative state and the occurrence of apoptotic events in the testis. To this aim, well-established mouse models with delayed (growth hormone releasing hormone-knockout and Ames dwarf mice) or accelerated (growth hormone-transgenic mice) aging were used. We hereby show that the testes of short-lived mice show a significant increase in cyclooxygenase 2 expression, PGD2 production, lipid peroxidation, antioxidant enzymes expression, local macrophages and TUNEL-positive germ cells numbers, and the levels of both pro-caspase-3 and cleaved caspase-3. In contrast, although the expression of antioxidant enzymes remained unchanged in testes of long-lived mice, the remainder of the parameters assessed showed a significant reduction. This study provides novel evidence that longevity confers anti-inflammatory, anti-oxidant and anti-apoptotic capacities to the adult testis. Oppositely, short-lived mice suffer testicular inflammatory, oxidative and apoptotic processes.

Several organ systems can be affected by psychostimulant toxicity. However, there is not sufficient evidence about the impact of psychostimulant intake on testicular physiology and catecholaminergic systems. The aim of the present study was to further explore potential toxic consequences of chronic exposure to cocaine, caffeine, and their combination on testicular physiology. Mice were injected with a 13-day chronic binge regimen of caffeine (3x5mg/kg), cocaine (3×10mg/kg), or combined administration. Mice treated with cocaine alone or combined with caffeine showed reduced volume of the seminiferous tubule associated to a reduction in the number of spermatogonia. Cocaine-only and combined treatments induced increased lipid peroxidation evaluated by TBARS assay and decreased glutathione peroxidase mRNA expression. Importantly, caffeine-cocaine combination potentiated the cocaine-induced germ cell loss, and induced pro-apoptotic BAX protein expression and diminished adenosine receptor A1 mRNA levels. We analyzed markers of dopaminergic function in the testis and detected the presence of tyrosine hydroxylase (TH) in the cytoplasm of androgen-producing Leydig cells, but also in meiotic germs cells within seminiferous tubules. Moreover, using transgenic BAC-Drd1a-tdTomato and D2R-eGFP mice, we report for the first time the presence of dopamine receptors (DRs) D1 and D2 in testicular mouse Leydig cells. Interestingly, the presence of DRD1 was also detected in the spermatogonia nearest the basal lamina of the seminiferous tubules, which did not show TH staining. We observed that psychostimulants induced downregulation of DRs mRNA expression and upregulation of TH protein expression in the testis. These findings suggest a potential role of the local dopaminergic system in psychostimulant-induced testicular pathology.

Prostaglandins (PGs) are synthesized through the action of the rate-limiting enzyme cyclooxygenase (COX) and further specific enzymes. The development of Cox-deficient mice in the 1990s gave insights into the reproductive roles of PGs. Female Cox-knockout mice were subfertile or infertile. Interestingly, fertility was not affected in male mice deficient in Cox, suggesting that PGs may not be critical for the functioning of the testis. However, this conclusion has recently been challenged by observations of important roles for PGs in both physiological and pathological processes in the testis. The two key somatic cell types in the testis, Leydig and Sertoli cells, express the inducible isoenzyme COX2 and produce PGs. Testicular COX2 expression in these somatic cells is regulated by hormonal input (FSH, prolactin (PRL), and testosterone) as well as by IL1β. PGs modulate steroidogenesis in Leydig cells and glucose uptake in Sertoli cells. Hence, the COX2/PG system in Leydig and Sertoli cells acts as a local modulator of testicular activity, and consequently may regulate spermatogenic efficiency. In addition to its expression in Leydig and Sertoli cells, COX2 has been detected in the seminiferous tubule wall, and in testicular macrophages and mast cells of infertile patients. These observations highlight the possible relevance of PGs in testicular inflammation associated with idiopathic infertility. Collectively, these data indicate that the COX2/PG system plays crucial roles not only in testicular physiology (i.e., development, steroidogenesis, and spermatogenesis), but more importantly in the pathogenesis or maintenance of infertility status in the male gonad. Further studies of these actions could lead to new therapeutic approaches to idiopathic male infertility., (© 2015 Society for Reproduction and Fertility.)

Melatonin acting through the hypothalamus and pituitary regulates testicular function. In addition, direct actions of melatonin at the testicular level have been recently suggested. We have described that melatonin inhibits androgen production in hamster Leydig cells via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. The initial events of the melatonin/CRH signalling pathway have also been established. Melatonin and all components of the melatonergic/CRH system were also detected in Leydig cells of infertile men. This study attempted to search for additional targets of melatonin in the human testis, and to investigate the effects of melatonin on proliferation and the oxidative state in these novel target cells. To this aim, evaluation of human testicular biopsies of patients suffering from hypospermatogenesis or Sertoli cell only syndrome and cell culture studies were performed. Melatonergic receptors were found in macrophages (MACs) and mast cells (MCs) of the human testis. In biopsies of patients suffering idiopathic infertility, melatonin testicular concentrations were negatively correlated with MAC number per mm(2) and TNFα, IL1β and COX2 expression, but positively correlated with the expression of the anti-oxidant enzymes SOD1, peroxiredoxin 1 and catalase. Melatonin inhibited proliferation and the expression of pro-inflammatory cytokines and cyclooxygenase 2 (COX2) in both the human non-testicular THP-1 MAC cell line and primary cell cultures of hamster testicular MACs. In the human HMC-1 MC line, melatonin increased the expression of anti-oxidant enzymes and decreased reactive oxygen species (ROS) generation. The results reveal new testicular targets of melatonin and describe anti-proliferative and anti-inflammatory effects of this hormone on testicular MACs. Furthermore, melatonin might provide protective effects against oxidative stress in testicular MCs., (© 2014 American Society of Andrology and European Academy of Andrology.)

Adult mice with a Leydig cell specific deletion of MAPK kinase (MEK) 1 and 2 (Mek1(f)(/)(f);Mek2(-/-);Cre(+)) mice display Leydig cell hypoplasia and hypergonadotropic hypogonadism. We used radioimmunoassays and quantitative PCR to evaluate the function and expression of the Leydig cell genes involved in the conversion of cholesterol to testosterone (Star, Cyp11a1, Hsd3b6, Cyp17a1 and Hsd17b3), androgen metabolism (Srda1 and Dhrs9), and four transcription factors (Creb1, Nr5a1, Nr4a1 and Nr0b1) that regulate the expression of steroidogenic genes. We show that Star, Hsd3b6, Cyp17a1 and Hsd17b3 are downregulated in Ledyig cells of adult Mek1(f)(/)(f);Mek2(-/-);Cre(+) mice whereas Srda1 and Dhrs9 are upregulated and Creb1, Nr5a1, Nr4a1 and Nr0b1 are unchanged or upregulated. Functionally, all the downregulated genes but none of the upregulated genes contribute to the decrease in testosterone synthesis in Leydig cells of adult Mek1(f)(/)(f);Mek2(-/-);Cre(+) mice because they produce low testosterone and dihydrotestosterone when stimulated with hCG or when incubated with testosterone precursors such as progesterone or androstenedione., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

In Leydig cells, hormonal stimulation by LH/hCG entails increased intracellular Ca(2+) levels and steroid production, as well as hyperpolarization of the cell membrane. The large-conductance Ca(2+)-activated K(+)-channel (BK(Ca)) is activated by raised intracellular Ca(2+) and voltage and typically hyperpolarizes the cell membrane. Whether BK(Ca) is functionally involved in steroid production of Leydig cells is not known. In order to explore this point we first investigated the localization of BK(Ca) in human and hamster testes and then used a highly specific toxin, the BK(Ca) blocker iberiotoxin (IbTx), to experimentally dissect a role of BK(Ca). Immunohistochemistry and RT-PCR revealed that adult Leydig cells of both species are endowed with these channels. Ontogeny studies in hamsters indicated that BK(Ca) becomes strongly detectable in Leydig cells only after they acquire the ability to produce androgens. Using purified Leydig cells from adult hamsters, membrane potential changes in response to hCG were monitored. HCG hyperpolarized the cell membrane, which was prevented by the selective BK(Ca) blocker IbTx. Steroidogenic acute regulatory (StAR) mRNA expression and testosterone production were not affected by IbTx under basal conditions but markedly increased when hCG, in submaximal and maximal concentration or when db-cAMP was added to the incubation media. A blocker of K(V)4-channels, expressed by Leydig cells, namely phrixotoxin-2 (PhTx-2) was not effective. In summary, the data reveal BK(Ca) as a crucial part of the signaling cascade of LH/hCG in Leydig cells. The hyperpolarizing effect of BK(Ca) in the Leydig cell membrane appears to set in motion events limiting the production of testosterone evoked by stimulatory endocrine mechanisms., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Fibrosis, increased amounts of immune cells and expression of COX-2 in the testes of infertility patients provide circumstantial evidence for a specific testicular milieu, in which reactive oxygen species (ROS) could be increased. If ROS level increase and/or ROS scavengers decrease, the resulting testicular oxidative stress may contribute to human male infertility. Primary peritubular cells of the human testis, from men with normal spermatogenesis (HTPCs) and infertile patients (HTPC-Fs), previously allowed us to identify an end product of COX-2 action, a prostaglandin derivative (15dPGJ2), which acts via ROS to alter the phenotype of peritubular cells, at least in vitro. Using testicular biopsies we now found 15dPGJ2 in patients and hence we started exploring the ROS scavenger systems of the human testis. This system includes catalase, DJ-1, peroxiredoxin 1, SOD 1 and 2, glutathione-S-transferase and HMOX-1, which were identified by RT-PCR/sequencing in HTPCs and HTPC-Fs and whole testes. Catalase, DJ-1, peroxiredoxin 1 and SOD 2 were also detected by Western blots and in part by immunohistochemistry in testicular samples. Western blots of cultured cells further revealed that catalase levels, but not peroxiredoxin 1, SOD 2 or DJ-1 levels, are significantly higher in HTPC-Fs than in HTPCs. This particular difference is correlated with the improved ability of HTPC-Fs to handle ROS, which became evident when cells were exposed to 100 μm H(2)O(2). H(2)O(2) induced stronger responses in HTPCs than in HTPC-Fs, which correlates with the lower level of the H(2)O(2)-degrading defence enzyme catalase in HTPCs. The results provide evidence for an adaptation to elevated ROS levels, which must have occurred in vivo and which persist in vitro in HTPC-Fs. Thus, in infertile men with impaired spermatogenesis elevated ROS levels likely exist, at least in the tubular wall., (© 2012 The Authors. International Journal of Andrology © 2012 European Academy of Andrology.)

We have previously described a stimulatory effect of testosterone on cyclooxygenase 2 (COX2) expression and prostaglandin (PG) synthesis, and the involvement of PGs in the modulation of testosterone production in Leydig cells of the seasonal breeder Syrian hamster. In this study, we investigated the existence of a COX2/PGs system in hamster Sertoli cells, its regulation by testosterone and FSH, and its effect on glucose uptake. COX2 expression was observed in Sertoli cells of both reproductively active and inactive adult hamsters. Testosterone and the plasma membrane-impermeable testosterone-BSA significantly induced COX2 expression, mitogen activated protein kinases 1/2 (MAPK1/2) phosphorylation and 15d-Δ(12,14)PGJ(2) production in Sertoli cells purified from photoperiodically regressed hamsters. These actions were abolished by the antiandrogen bicalutamide and by the inhibitor of MAPK kinase (MEK1/2) U0126, suggesting that testosterone exerts its stimulatory effect on COX2/PGs through a non-classical mechanism that involves the presence of androgen receptors and MAPK1/2 activation. FSH also stimulated COX2/PGs via MAPK1/2 phosphorylation. FSH and testosterone stimulate, whereas 15d-Δ(12,14)PGJ(2) via PPARγ inhibits, [2,6-(3)H]-2-deoxy-d-glucose ([(3)H]-2-DOG) uptake. Meloxicam, a selective COX2 inhibitor, further increases [(3)H]-2-DOG uptake in the presence of FSH or testosterone. Thus, in addition to their positive effect, FSH and testosterone may also exert an indirect negative regulation on glucose uptake which involves the COX2/15d-Δ(12,14)PGJ(2)/PPARγ system. Overall, these results demonstrate the presence of a COX2/PG system in hamster Sertoli cells which might act as a local modulator of FSH and testosterone actions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have previously described that melatonin inhibits androgen production in hamster testes via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. This study attempted to determine the initial events of the melatonin/CRH signaling pathway. In Leydig cells from reproductively active Syrian hamsters, Western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and a colorimetric assay demonstrated that melatonin and CRH activate tyrosine phosphatases and subsequently reduce the phosphorylation levels of extracellular signal-regulated kinase (erk) and c-jun N-terminal kinase (jnk), down-regulate the expression of c-jun, c-fos and steroidogenic acute regulatory (StAR), and inhibit the production of testosterone. These effects were prevented by a highly selective CRH antagonist, thus indicating that melatonin does not exert a direct role. Specific mitogen-activated protein kinase kinase (MEK) and jnk blockers inhibited expression of c-jun, c-fos, StAR and the production of testosterone, confirming that these are events triggered downstream of erk and jnk. In Leydig cells from photoperiodically regressed adult hamsters, CRH inhibited the production of androstane-3α,17β-diol (3α-diol), the main androgen produced, through the same signaling pathway. Testicular melatonin concentration was 3-4-fold higher in reproductively inactive hamsters than that detected in active animals. Since melatonin, CRH, and their receptors are present not only in hamster testes but also in testicular biopsies of infertile men, we can conjecture about the relevance of this previously uncharacterized pathway in human fertility disorders. In summary, our study identifies crucial intracellular events triggered by melatonin/CRH in the testis that lead to a down-regulation of the steroidogenic process., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Serum prolactin (PRL) variations play a crucial role in the photoperiodic-induced testicular regression-recrudescence transition in hamsters. We have previously shown that cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins (PGs), is expressed mostly in Leydig cells of reproductively active hamsters with considerable circulating and pituitary levels of PRL. In this study, we describe a stimulatory effect of PRL on COX2/PGs in hamster Leydig cells, which is mediated by IL-1β and prevented by P38-MAPK and JAK2 inhibitors. Furthermore, by preparative isoelectric focusing (IEF), we isolated PRL charge analogues from pituitaries of active [isoelectric points (pI): 5.16, 4.61, and 4.34] and regressed (pI: 5.44) hamsters. More acidic PRL charge analogues strongly induced COX2 expression, while less acidic ones had no effect. Our studies suggest that PRL induces COX2/PGs in hamster Leydig cells through IL-1β and activation of P38-MAPK and JAK2. PRL microheterogeneity detected in active/inactive hamsters may be responsible for the photoperiodic variations of COX2 expression in Leydig cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: In non-obstructive azoospermia, histological patterns of Sertoli cell-only Syndrome (SCO) and hypospermatogenesis (H) are commonly found. In these pathologies, Leydig cell hyperplasia (LCH) is detected in some patients. Since TGF-β1 is involved in cellular proliferation/development, the aim of this work was to analyze the expression of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), and the co-receptor endoglin in human biopsies from patients with idiopathic infertility., Methods: Specific immunostaining of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), co-receptor endoglin and Smads proteins, were carried out in testicular biopsies from normal and infertile men with SCO or H. Gene expression of TGF-β1 system were made in biopsies from infertile patients with semi-quantitative and quantitative PCR., Results: Immunohistochemical studies revealed that TGF-β1 and its specific receptors are present in Leydig cells in biopsies from normal tissue or patients with SCO or H with or without LCH. Smad proteins, which are involved in TGF-β1 signaling, are also detected in both their phosphorylated (activated) and dephosphorylated form in all samples TGF-β1, ALK-1 and endoglin gene expression are stronger in human biopsies with LCH than in those with SCO or H. Neither TGFBRII nor ALK-5 gene expression showed significant differences between pathologies. A significant correlation between ALK-1 and endoglin expression was observed., Conclusions: In conclusion, the high levels of mRNA and protein expression of the TGF-β1 system in patients with LCH, particularly ALK1 and its correlation with endoglin, suggest that these proteins acting in concert might be, at least in part, committed actors in the Leydig cell hyperplasia.

As we previously reported, testes of men suffering from hypospermatogenesis and germ cell arrest or Sertoli cell-only syndrome show a major increase in the number of macrophages expressing interleukin-1β (IL-1β) and abundant expression of cyclooxygenase-2 (COX-2), the inducible isoform of the key enzyme in the biosynthesis of prostaglandins (PGs), in Leydig cells. In the present study we report [1] a positive correlation between IL-1β levels and COX-2 expression in testes of infertile patients, [2] the induction of COX-2 by IL-1β in mouse Leydig cells (TM3) and human macrophages (THP-1), and therefore [3] evidence for an IL-1β-dependent induction of testicular inflammatory states., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

We have previously observed expression of prostaglandin-endoperoxide synthase 2 (PTGS2), the key enzyme in the biosynthesis of prostaglandins (PGs), in reproductively active Syrian hamster Leydig cells, and reported an inhibitory role of PGF(2alpha) on hamster testicular steroidogenesis. In this study, we further investigated PTGS2 expression in hamster Leydig cells during sexual development and photoperiodic gonadal regression. Since PTGS2 is mostly expressed in pubertal and reproductively active adult hamsters with high circulating levels of LH and androgens, we studied the role of these hormones in the regulation/maintenance of testicular PTGS2/PGF(2alpha). In active hamster Leydig cells, LH/hCG and testosterone induced PTGS2 and PGF(2alpha) production, and their actions were abolished by the antiandrogen bicalutamide (Bi). These results indicate that LH does not exert a direct effect on PG synthesis. Testosterone also stimulated phosphorylation of the mitogen-activated protein kinase isoforms 3/1 (MAPK3/1) within minutes and hours, but the testosterone metabolite dihydrotestosterone had no effect on PTGS2 and MAPK3/1. Because Bi and U0126, an inhibitor of the MAP kinase kinases 1 and 2 (MAP2K1/2), abolished testosterone actions on MAPK3/1 and PTGS2, our studies suggest that testosterone directly induces PTGS2/PGF(2alpha) in hamster Leydig cells via androgen receptors and a non-classical mechanism that involves MAPK3/1 activation. Since PGF(2alpha) inhibits testosterone production, it might imply the existence of a regulatory loop that is setting a brake on steroidogenesis. Thus, the androgen environment might be crucial for the regulation of testicular PG production at least during sexual development and photoperiodic variations in hamsters.