When idiopathic male infertility is rooted in maternal malnutrition during the perinatal period in mice†.

Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.

Identification of the Role of TGR5 in the Regulation of Leydig Cell Homeostasis.

Understanding the regulation of the testicular endocrine function leading to testosterone production is a major objective as the alteration of endocrine function is associated with the development of many diseases such as infertility. In the last decades, it has been demonstrated that several endogenous molecules regulate the steroidogenic pathway. Among them, bile acids have recently emerged as local regulators of testicular physiology and particularly endocrine function. Bile acids act through the nuclear receptor FXRα (Farnesoid-X-receptor alpha; NR1H4) and the G-protein-coupled bile acid receptor (GPBAR-1; TGR5). While FXRα has been demonstrated to regulate testosterone synthesis within Leydig cells, no data are available regarding TGR5. Here, we investigated the potential role of TGR5 within Leydig cells using cell culture approaches combined with pharmacological exposure to the TGR5 agonist INT-777. The data show that activation of TGR5 results in a decrease in testosterone levels. TGR5 acts through the PKA pathway to regulate steroidogenesis. In addition, our data show that TGR5 activation leads to an increase in cholesterol ester levels. This suggests that altered lipid homeostasis may be a mechanism explaining the TGR5-induced decrease in testosterone levels. In conclusion, the present work highlights the impact of the TGR5 signaling pathway on testosterone production and reinforces the links between bile acid signaling pathways and the testicular endocrine function. The testicular bile acid pathways need to be further explored to increase our knowledge of pathologies associated with impaired testicular endocrine function, such as fertility disorders.

Farnesoid X receptor alpha (FXRα) is a critical actor of the development and pathologies of the male reproductive system.

The farnesoid-X-receptorα (FXRα; NR1H4) is one of the main bile acid (BA) receptors. During the last decades, through the use of pharmalogical approaches and transgenic mouse models, it has been demonstrated that the nuclear receptor FXRα controls numerous physiological functions such as glucose or energy metabolisms. It is also involved in the etiology or the development of several pathologies. Here, we will review the unexpected roles of FXRα on the male reproductive tract. FXRα has been demonstrated to play functions in the regulation of testicular and prostate homeostasis. Even though additional studies are needed to confirm these findings in humans, the reviewed reports open new field of research to better define the effects of bile acid-FXRα signaling pathways on fertility disorders and cancers.

Nuclear Receptor Metabolism of Bile Acids and Xenobiotics: A Coordinated Detoxification System with Impact on Health and Diseases.

Structural and functional studies have provided numerous insights over the past years on how members of the nuclear hormone receptor superfamily tightly regulate the expression of drug-metabolizing enzymes and transporters. Besides the role of the farnesoid X receptor (FXR) in the transcriptional control of bile acid transport and metabolism, this review provides an overview on how this metabolic sensor prevents the accumulation of toxic byproducts derived from endogenous metabolites, as well as of exogenous chemicals, in coordination with the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Decrypting this network should provide cues to better understand how these metabolic nuclear receptors participate in physiologic and pathologic processes with potential validation as therapeutic targets in human disabilities and cancers.

Nuclear receptors as pharmacological targets, where are we now?

Knowledge of integrative physiology is a major challenge for scientists, as even small deregulation could lead to diseases. Cells communicate with each other to control many processes such as growth, migration, survival, or differentiation. Such interaction could be achieved via several mechanisms either through cell-cell interactions and/or through the signaling of molecules that bind to receptors on the membrane or in the target cells. The produced molecules could have either autocrine, paracrine stimulations, or even act on distant organs (endocrine signaling).

Liver x receptors protect from development of prostatic intra-epithelial neoplasia in mice.

LXR (Liver X Receptors) act as "sensor" proteins that regulate cholesterol uptake, storage, and efflux. LXR signaling is known to influence proliferation of different cell types including human prostatic carcinoma (PCa) cell lines. This study shows that deletion of LXR in mouse fed a high-cholesterol diet recapitulates initial steps of PCa development. Elevation of circulating cholesterol in Lxrαß-/- double knockout mice results in aberrant cholesterol ester accumulation and prostatic intra-epithelial neoplasia. This phenotype is linked to increased expression of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2), which results in the down-regulation of the tumor suppressors Msmb and Nkx3.1 through increased methylation of lysine 27 of histone H3 (H3K27) on their promoter regions. Altogether, our data provide a novel link between LXR, cholesterol homeostasis, and epigenetic control of tumor suppressor gene expression.

Bile acids alter male fertility through G-protein-coupled bile acid receptor 1 signaling pathways in mice.

UNLABELLED: Bile acids (BAs) are signaling molecules that are involved in many physiological functions, such as glucose and energy metabolism. These effects are mediated through activation of the nuclear and membrane receptors, farnesoid X receptor (FXR-α) and TGR5 (G-protein-coupled bile acid receptor 1; GPBAR1). Although both receptors are expressed within the testes, the potential effect of BAs on testis physiology and male fertility has not been explored thus far. Here, we demonstrate that mice fed a diet supplemented with cholic acid have reduced fertility subsequent to testicular defects. Initially, germ cell sloughing and rupture of the blood-testis barrier occur and are correlated with decreased protein accumulation of connexin-43 (Cx43) and N-cadherin, whereas at later stages, apoptosis of spermatids is observed. These abnormalities are associated with increased intratesticular BA levels in general and deoxycholic acid, a TGR5 agonist, in particular. We demonstrate here that Tgr5 is expressed within the germ cell lineage, where it represses Cx43 expression through regulation of the transcriptional repressor, T-box transcription factor 2 gene. Consistent with this finding, mice deficient for Tgr5 are protected against the deleterious testicular effects of BA exposure. CONCLUSIONS: These data identify the testis as a new target of BAs and emphasize TGR5 as a critical element in testicular pathophysiology. This work may open new perspectives on the potential effect of BAs on testis physiology during liver dysfunction.

Liver X receptors interfere with the deleterious effect of diethylstilbestrol on testicular physiology.

Liver X receptors LXRα (NR1H3) and LXRß (NR1H2) are transcription factors belonging to the nuclear receptor superfamily, activated by specific oxysterols, oxidized derivatives of cholesterol. These receptors are involved in the regulation of testis physiology. Lxr-deficient mice pointed to the physiological roles of these nuclear receptors in steroid synthesis, lipid homeostasis and germ cell apoptosis and proliferation. Diethylstilbestrol (DES) is a synthetic estrogen considered as an endocrine disruptor that affects the functions of the testis. Various lines of evidences have made a clear link between estrogens, their nuclear receptors ERα (NR3A1) and ERß (NR3A2), and Lxrα/ß. As LXR activity could also be regulated by the nuclear receptor small heterodimer partner (SHP, NR0A2) and DES could act through SHP, we wondered whether LXR could be targeted by estrogen-like endocrine disruptors such as DES. For that purpose, wild-type and Lxr-deficient mice were daily treated with 0.75 µg DES from days 1 to 5 after birth. The effects of DES were investigated at 10 or 45 days of age. We demonstrated that DES induced a decrease of the body mass at 10 days only in the Lxr-deficient mice suggesting a protective effect of Lxr. We defined three categories of DES-target genes in testis: those whose accumulation is independent of Lxr; those whose accumulation is enhanced by the lack of both Lxrα/ß; those whose accumulation is repressed by the absence of Lxrα/ß. Lipid accumulation is also modified by neonatal DES injection. Lxr-deficient mice present different lipid profiles, demonstrating that DES could have its effects in part due to Lxrα/ß. Altogether, our study shows that both nuclear receptors Lxrα and Lxrß are not only basally important for testicular physiology but could also have a preventive effect against estrogen-like endocrine disruptors.

Farnesoid X receptor alpha: a molecular link between bile acids and steroid signaling?

Bile acids are cholesterol metabolites that have been extensively studied in recent decades. In addition to having ancestral roles in digestion and fat solubilization, bile acids have recently been described as signaling molecules involved in many physiological functions, such as glucose and energy metabolisms. These signaling pathways involve the activation of the nuclear receptor farnesoid X receptor (FXRα) or of the G protein-coupled receptor TGR5. In this review, we will focus on the emerging role of FXRα, suggesting important functions for the receptor in steroid metabolism. It has been described that FXRα is expressed in the adrenal glands and testes, where it seems to control steroid production. FXRα also participates in steroid catabolism in the liver and interferes with the steroid signaling pathways in target tissues via crosstalk with steroid receptors. In this review, we discuss the potential impacts of bile acid (BA), through its interactions with steroid metabolism, on glucose metabolism, sexual function, and prostate and breast cancers. Although several of the published reports rely on in vitro studies, they highlight the need to understand the interactions that may affect health. This effect is important because BA levels are increased in several pathophysiological conditions related to liver injuries. Additionally, BA receptors are targeted clinically using therapeutics to treat liver diseases, diabetes, and cancers.

Liver X receptors, lipids and their reproductive secrets in the male.

Liver X receptor (LXR) α and LXRß belong to the nuclear receptor superfamily. For many years, they have been called orphan receptors, as no natural ligand was identified. In the last decade, the LXR natural ligands have been shown to be oxysterols, molecules derived from cholesterol. While these nuclear receptors have been abundantly studied for their roles in the regulation of lipid metabolism, it appears that they also present crucial activities in reproductive organs such as testis and epididymis, as well as prostate. Phenotypic analyses of mice lacking LXRs (lxr-/-) pointed out their physiological activities in the various cells and organs regulating reproductive functions. This review summarizes the impact of LXR-deficiency in male reproduction, highlighting the novel information coming from the phenotypic analyses of lxrα-/-, lxrß-/- and lxrα;ß-/- mice. This article is part of a Special Issue entitled: Translating nuclear receptor from health to disease.

Metallome deregulation and health-related impacts due to long-term exposure to recent volcanic ash deposits: New chemical and isotopic insights.

Volcanic ash exposure can lead to significant health risks. Damage to the respiratory and pulmonary systems are the most evident toxic side effects although the causes of these symptoms remain unclear. Conversely, the effects on other organs remain largely under-explored, limiting our understanding of the long-term volcanic ash-related risk at the whole-body scale. The metallome i.e. metal concentrations and isotopic compositions within the body, is suspected to be affected by volcanic ash exposure, having thus the potential for capturing some specificities of ash toxicity. However, the means by and extent to which the metallome is affected at the entire body scale and how the consequent chemical and isotopic deregulations correlate with pathophysiological dysfunctions are currently poorly understood. Here, we adopt a transdisciplinary approach combining high precision chemical analyses (major and trace element concentrations) and CuZn isotope measurements in seven organs and two biological fluids of isogenic mice (C57BL/6) exposed to eruption products from La Soufrière de Guadeloupe (Eastern Carribean), in tandem with biological parameters including physiological and morphological data. Based on principal component analysis, we show that after one month of exposure to volcanic ash deposits, the mice metallome; originally organ-specific and isotopically-typified, is highly disrupted as shown for example by heavy metal accumulation in testis (e.g., Fe, Zn) and Cu, Zn isotopic divergence in liver, intestine and blood. These metallomic variations are correlated with early testicular defects and might reflect the warning signs of premature (entero)hepatic impairments that may seriously affect fertility and favor the emergence of liver diseases after prolonged exposure. Monitoring the temporal evolution of the Cu and Zn isotope compositions seems to be a promising technique to identify the main biological processes and vital functions that are vulnerable to environmental volcanogenic pollutants although this will require further validation on human subjects.

Identification of a Crosstalk among TGR5, GLIS2, and TP53 Signaling Pathways in the Control of Undifferentiated Germ Cell Homeostasis and Chemoresistance.

Spermatogonial stem cells regenerate and maintain spermatogenesis throughout life, making testis a good model for studying stem cell biology. The effects of chemotherapy on fertility have been well-documented previously. This study investigates how busulfan, an alkylating agent that is often used for chemotherapeutic purposes, affects male fertility. Specifically, the role of the TGR5 pathway is investigated on spermatogonia homeostasis using in vivo, in vitro, and pharmacological methods. In vivo studies are performed using wild-type and Tgr5-deficient mouse models. The results clearly show that Tgr5 deficiency can facilitate restoration of the spermatogonia homeostasis and allow faster resurgence of germ cell lineage after exposure to busulfan. TGR5 modulates the expression of key genes of undifferentiated spermatogonia such as Gfra1 and Fgfr2. At the molecular level, the present data highlight molecular mechanisms underlying the interactions among the TGR5, GLIS2, and TP53 pathways in spermatogonia associated with germ cell apoptosis following busulfan exposure. This study makes a significant contribution to the literature because it shows that TGR5 plays key role on undifferentiated germ cell homeostasis and that modulating the TGR5 signaling pathway could be used as a potential therapeutic tool for fertility disorders.

Analysis of the Reversible Impact of the Chemodrug Busulfan on Mouse Testes.

Spermatogenesis is a process within the testis that leads to the production of spermatozoa. It is based on a population of spermatogonial stem cells, which have the capacity to self-renew and to differentiate throughout life to ensure the functions of reproduction are maintained. Male fertility disorders are responsible for half of the cases of infertility in couples worldwide. It is well known that cancer treatments are associated with reversible or irreversible fertility disorders. Busulfan (Bu) is an alkylating agent that significantly inhibits spermatogenesis. The present study relied on a combination of in vivo and in vitro approaches as well as RNAseq analysis to characterize the effects of Bu, in which mouse testes were used as a model. An in silico analysis revealed that many of the Bu-modulated genes are potentially regulated by the SIN3 Transcription Regulator Family Member A (SIN3A) and E2F Transcription Factor (E2F) families of transcription factors. The results demonstrate that the deregulated genes function in processes related to the cell cycle, DNA repair, and cell death mechanisms, including the Tumor Protein 53 (TP53) pathway. This reinforces the role of the TP53 signaling pathway as a major player in Bu effects. In addition, Bu altered the patterns of mRNA accumulation for various genes in undifferentiated spermatogonia. This work provides significant insight into the kinetics and impacts of busulfan, which could pave the way for developing strategies to minimize the impact of chemodrugs and, thus, could lead to germ cell lineage regeneration following anticancer treatments.

FXRα modulates leydig cell endocrine function in mouse.

The hypothalamic-pituitary axis exert a major control over endocrine and exocrine testicular functions. The hypothalamic-pituitary axis corresponds to a cascade with the Gonadotropin Releasing Hormone secreted by the hypothalamus, which stimulates the synthesis and the release of Luteinizing Hormone (LH) and Follicle Stimulating Hormone by the gonadotropic cells of the anterior pituitary. The LH signaling pathway controls the steroidogenic activity of the Leydig cells via the activation of the luteinizing hormone/choriogonadotropin receptor. In order to avoid a runaway system, sex steroids exert a negative feedback within hypothalamus and pituitary. Testicular steroidogenesis is locally controlled within Leydig cells. The present work reviews some local regulations of steroidogenesis within the Leydig cells focusing mainly on the roles of the Farnesoid-X-Receptor-alpha and its interactions with several orphan members of the nuclear receptor superfamily. Further studies are required to reinforce our knowledge of the regulation of testicular endocrine function, which is necessary to ensure a better understanding of fertility disorders and then proposed an adequate treatment of the diseases.

Liver X receptors regulate adrenal cholesterol balance.

Cholesterol is the obligate precursor to adrenal steroids but is cytotoxic at high concentrations. Here, we show the role of the liver X receptors (LXRalpha and LXRbeta) in preventing accumulation of free cholesterol in mouse adrenal glands by controlling expression of genes involved in all aspects of cholesterol utilization, including the steroidogenic acute regulatory protein, StAR, a novel LXR target. Under chronic dietary stress, adrenal glands from Lxralphabeta-/- mice accumulated free cholesterol. In contrast, wild-type animals maintained cholesterol homeostasis through basal expression of genes involved in cholesterol efflux and storage (ABC transporter A1 [ABCA1], apoE, SREBP-1c) while preventing steroidogenic gene (StAR) expression. Upon treatment with an LXR agonist that mimics activation by oxysterols, expression of these target genes was increased. Basally, Lxralphabeta-/- mice exhibited a marked decrease in ABCA1 and a derepression of StAR expression, causing a net decrease in cholesterol efflux and an increase in steroidogenesis. These changes occurred under conditions that prevented the acute stress response and resulted in a phenotype more specific to the loss of LXRalpha, including hypercorticosteronemia, cholesterol ester accumulation, and adrenomegaly. These results imply LXRalpha provides a safety valve to limit free cholesterol levels as a basal protective mechanism in the adrenal gland, where cholesterol is under constant flux.

Fxralpha gene is a target gene of hCG signaling pathway and represses hCG induced steroidogenesis.

The bile acid receptor Farnesoid-X-Receptor alpha (FXRα), a member of the nuclear receptor superfamily, is well known for its roles in the enterohepatic tract. In addition, FXRα regulates testicular physiology through the control of both endocrine and exocrine functions. The endocrine function of the Leydig cells is mainly controlled by the hypothalamo-pituitary axis viaLH/chorionic gonadotropin (CG). If FXRα was demonstrated to control the expression of the Lhcgr gene, encoding the LH receptor; the impact of the LH/CG signaling on the Fxrα expression has not been defined so far. Here, we demonstrate that hCG increases the Fxrα gene expression through the protein kinase-A signaling pathway. Fxrα is then involved in a negative feedback of steroid synthesis. These data improve our knowledge of the local control of the testicular steroidogenesis with the identification of the link between the hypothalamo-pituitary axis and the FXRα signaling pathway.

In vivo imaging of farnesoid X receptor activity reveals the ileum as the primary bile acid signaling tissue.

We generated and characterized a firefly luciferase reporter mouse for the nuclear receptor farnesoid X receptor (FXR). This FXR reporter mouse has basal luciferase expression in the terminal ileum, an organ with well-characterized FXRalpha signaling. In vivo luciferase activity reflected the diurnal activity pattern of the mouse, and is regulated by both natural (bile acids, chenodeoxycholic acid) and synthetic (GW4064) FXRalpha ligands. Moreover, in vivo and in vitro analysis showed luciferase activity after GW4064 administration in the liver, kidney, and adrenal gland, indicating that FXRalpha signaling is functional in these tissues. Hepatic luciferase activity was robustly induced in cholestatic mice, showing that FXRalpha signaling pathways are activated in this disease. In conclusion, we have developed an FXR reporter mouse that is useful to monitor FXRalpha signaling in vivo in health and disease. The use of this animal could facilitate the development of new therapeutic compounds that target FXRalpha in a tissue-specific manner.

Multiple roles of the nuclear receptors for oxysterols liver X receptor to maintain male fertility.

Oxysterol nuclear receptors liver X receptor (LXR)alpha and LXRbeta are known to regulate lipid homeostasis in cells exposed to high amounts of cholesterol and/or fatty acids. In order to elucidate the specific and redundant roles of the LXRs in the testis, we explored the reproductive phenotypes of mice deficient of LXRalpha, LXRbeta, and both, of which only the lxralpha;beta-/- mice are infertile by 5 months of age. We demonstrate that LXRalpha-deficient mice had lower levels of testicular testosterone that correlated with a higher apoptotic rate of the germ cells. LXRbeta-deficient mice showed increased lipid accumulation in the Sertoli cells and a lower proliferation rate of the germ cells. In lxralpha;beta-/- mice, fatty acid metabolism was affected through a decrease of srebp1c and increase in scd1 mRNA expression. The retinoid acid signaling pathway was also altered in lxralpha;beta-/- mice, with a higher accumulation of all-trans retinoid receptor alpha, all-trans retinoid receptor beta, and retinoic aldehyde dehydrogenase-2 mRNA. Combination of these alterations might explain the deleterious phenotype of infertility observed only in lxralpha;beta-/- mice, even though lipid homeostasis seemed to be first altered. Wild-type mice treated with a specific LXR agonist showed an increase of testosterone production involving both LXR isoforms. Altogether, these data identify new roles of each LXR, collaborating to maintain both integrity and functions of the testis.

Cholesterol: A Gatekeeper of Male Fertility?

Cholesterol is essential for mammalian cell functions and integrity. It is an important structural component maintaining the permeability and fluidity of the cell membrane. The balance between synthesis and catabolism of cholesterol should be tightly regulated to ensure normal cellular processes. Male reproductive function has been demonstrated to be dependent on cholesterol homeostasis. Here we review data highlighting the impacts of cholesterol homeostasis on male fertility and the molecular mechanisms implicated through the signaling pathways of some nuclear receptors.