Sperm cryostorage in a dry tank: An accurate alternative.

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at -194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.

Bile acid homeostasis controls CAR signaling pathways in mouse testis through FXRalpha.

Bile acids (BAs) are molecules with endocrine activities controlling several physiological functions such as immunity, glucose homeostasis, testicular physiology and male fertility. The role of the nuclear BA receptor FXRα in the control of BA homeostasis has been well characterized. The present study shows that testis synthetize BAs. We demonstrate that mice invalidated for the gene encoding FXRα have altered BA homeostasis in both liver and testis. In the absence of FXRα, BA exposure differently alters hepatic and testicular expression of genes involved in BA synthesis. Interestingly, Fxrα-/- males fed a diet supplemented with BAs show alterations of testicular physiology and sperm production. This phenotype was correlated with the altered testicular BA homeostasis and the production of intermediate metabolites of BAs which led to the modulation of CAR signaling pathways within the testis. The role of the CAR signaling pathways within testis was validated using specific CAR agonist (TCPOBOP) and inverse agonist (androstanol) that respectively inhibited or reproduced the phenotype observed in Fxrα-/- males fed BA-diet. These data open interesting perspectives to better define how BA homeostasis contributes to physiological or pathophysiological conditions via the modulation of CAR activity.

Bile acid-FXRα pathways regulate male sexual maturation in mice.

The bile acid receptor Farnesol-X-Receptor alpha (FRXα) is a member of the nuclear receptor superfamily. FRXα is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. In adult, short term treatment (12 hours) with FRXα agonist inhibits the expression of steroidogenic genes via the induction of the Small heterodimer partner (SHP). However the consequences of FRXα activation on testicular pathophysiology have never been evaluated. We demonstrate here that mice fed a diet supplemented with bile acid during pubertal age show increased incidence of infertility. This is associated with altered differentiation and increase apoptosis of germ cells due to lower testosterone levels. At the molecular level, next to the repression of basal steroidogenesis via the induction expression of Shp and Dax-1, two repressors of steroidogenesis, the main action of the BA-FRXα signaling is through lowering the Leydig cell sensitivity to the hypothalamo-pituitary axis, the main regulator of testicular endocrine function. In conclusion, BA-FRXα signaling is a critical actor during sexual maturation.

Bile Acid Alters Male Mouse Fertility in Metabolic Syndrome Context.

Bile acids have recently been demonstrated as molecules with endocrine activities controlling several physiological functions such as immunity and glucose homeostases. They act mainly through two receptors, the nuclear receptor Farnesol-X-Receptor alpha (FXRα) and the G-protein coupled receptor (TGR5). These recent studies have led to the idea that molecules derived from bile acids (BAs) and targeting their receptors must be good targets for treatment of metabolic diseases such as obesity or diabetes. Thus it might be important to decipher the potential long term impact of such treatment on different physiological functions. Indeed, BAs have recently been demonstrated to alter male fertility. Here we demonstrate that in mice with overweight induced by high fat diet, BA exposure leads to increased rate of male infertility. This is associated with the altered germ cell proliferation, default of testicular endocrine function and abnormalities in cell-cell interaction within the seminiferous epithelium. Even if the identification of the exact molecular mechanisms will need more studies, the present results suggest that both FXRα and TGR5 might be involved. We believed that this work is of particular interest regarding the potential consequences on future approaches for the treatment of metabolic diseases.

Identification of the link between the hypothalamo-pituitary axis and the testicular orphan nuclear receptor NR0B2 in adult male mice.

The small heterodimer partner (SHP, nuclear receptor subfamily 0, group B, member 2; NR0B2) is an atypical nuclear receptor known mainly for its role in bile acid homeostasis in the enterohepatic tract. We previously showed that NR0B2 controls testicular functions such as testosterone synthesis. Moreover, NR0B2 mediates the deleterious testicular effects of estrogenic endocrine disruptors leading to infertility. The endocrine homeostasis is essential for health, because it controls many physiological functions. This is supported by a large number of studies demonstrating that alterations of steroid activity lead to several kinds of diseases such as obesity and infertility. Within the testis, the functions of the Leydig cells are mainly controlled by the hypothalamo-pituitary axis via LH/chorionic gonadotropin (CG). Here, we show that LH/CG represses Nr0b2 expression through the protein kinase A-AMP protein kinase pathway. Moreover, using a transgenic mouse model invalidated for Nr0b2, we point out that NR0B2 mediates the repression of testosterone synthesis and subsequent germ cell apoptosis induced by exposure to anti-GnRH compound. Together, our data demonstrate a new link between hypothalamo-pituitary axis and NR0B2 in testicular androgen metabolism, making NR0B2 a major actor of testicular physiology in case of alteration of LH/CG levels.

Hepatotoxicity induced by neonatal exposure to diethylstilbestrol is maintained throughout adulthood via the nuclear receptor SHP.

BACKGROUND: Liver physiology is sensitive to estrogens, which suggests that the liver might be a target of estrogenic endocrine disrupters (EED). However, the long-term consequences of neonatal exposure to EED on liver physiology have rarely been studied. The nuclear receptor small heterodimer partner (SHP) mediates the deleterious effects of neonatal exposure to diethylstilbestrol (DES) on male fertility. OBJECTIVES: As SHP is involved in liver homeostasis, we aimed to determine whether neonatal estrogenic exposure also affected adult liver physiology through SHP. Male mouse pups were exposed to DES in the first 5 days of life. RESULTS: DES exposure leads to alterations in the postnatal bile acid (BA) synthesis pathway. Neonatal DES-exposure affected adult liver BA metabolism and subsequently triglyceride (TG) homeostasis. The wild-type males neonatally exposed to DES exhibited increased liver weight and altered liver histology in the adult age. The use of deficient male mice revealed that SHP mediates the deleterious effects of DES treatment. These long-term effects of DES were associated with differently timed alterations in the expression of epigenetic factors. CONCLUSIONS: However, the molecular mechanisms by which neonatal exposure persist to affect the adult liver physiology remain to be defined. In conclusion, we demonstrate that neonatal DES exposure alters adult hepatic physiology in an SHP-dependent manner.

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.

Male fertility: Is spermiogenesis the critical step for answering biomedical issues?

Regarding male fertility, biomedical issues have opposite goals to treat infertility or develop contraceptive drugs. Recently, the identification of the molecular mechanisms involved in germ cell differentiation suggest that spermiogenesis has to be put at the crossroad to reach these goals. Concerning fertility issues, citizens in our modern world are schizophrenic. On one side, couples have the possibility to control conception; and on the other side, more and more couples suffer from the misfortune of being infertile. These two societal problems lead to intensive research and conflicting government policies. However, these opposing goals rely on a better understanding of germ cell differentiation.

Oxysterol receptors and their therapeutic applications in cancer conditions.

INTRODUCTION: Oxysterols are implicated in various cellular processes. Among their target proteins, liver X receptors (LXRs) α and ß modulate the cell cycle in a large range of cancer cell lines. Besides their role as cholesterol sensors, LXRs are also involved in the proliferation/apoptosis balance regulation in various types of cancers. AREAS COVERED: This review covers oxysterols and derivatives of cholesterol as well as synthetic or natural ligands (agonist/antagonist) of LXRs. Most tumor cell lines are sensitive to LXR activation. Indeed various cancers are concerned such as prostate, breast, glioblastoma, colorectal, and ovary tumors, and leukemia. EXPERT OPINION: Developing the use of LXR ligands in human health, especially in the field of cancer, represents a novel and promising strategy. Despite a wide spectrum of applications, numerous adverse effects of LXR activation need to be solved before genuine clinical trials in humans. Future directions will be based on the engineering of selective LXRs modulators (SLiMs) as already done for nuclear steroid receptors.

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.

Neonatal exposure to zearalenone induces long term modulation of ABC transporter expression in testis.

Mycotoxin zearalenone (ZEN) is a cereal contaminant produced by various species of Fusarium fungi. When interacting with estrogen receptors, ZEN leads to animal fertility disturbances and other reproductive pathologies. Few data are available on the effects of perinatal exposure to ZEN, particularly in the blood-testis barrier. The aim of this study was to assess the impact of ZEN in adult rats exposed neonatally. We focused on the expression and cellular localization of major ABC transporters expressed in adult rat testis, comparing ZEN effects with those of Estradiol Benzoate (EB) neonatal exposure. Dose-dependent and long term modulations of mRNA and protein levels of Abcb1, Abcc1, Abcg2, Abcc4 and Abcc5 were observed, along with Abcc4 protein cellular delocalization. ZEN exposure of SerW3 Sertoli cells showed modulation of Abcb1, Abcc4 and Abcc5. Comparison with EB exposure showed similar modulation profiles for Abcg2 but differential modulations for Abcb1, Abcc1, Abcc4 and Abcc5 in vivo, and a similar profile for Abcb1 modulation by ZEN and EB, but differential modulation for Abcc4 and Abcc5 in vitro. ZEN and EB effects were inhibited by in vitro addition of the pure anti-estrogen ICI 182.780, suggesting the at least partial implication of ZEN estrogenic activity in these modulations. These results suggested that ZEN neonatal exposure could affect the exposure of testis to ABC transporter substrates, and negatively influence spermatogenesis and male fertility.

Bile acids: from digestion to cancers.

Bile acids (BAs) are cholesterol metabolites that have been extensively studied these last decades. BAs have been classified in two groups. Primary BAs are synthesized in liver, when secondary BAs are produced by intestinal bacteria. Recently, next to their ancestral roles in digestion and fat solubilization, BAs have 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 FXRα or of the G-protein-coupled receptor TGR5. These two receptors have selective affinity to different types of BAs and show different expression patterns, leading to different described roles of BAs. It has been suggested for long that BAs could be molecules linked to tumor processes. Indeed, as many other molecules, regarding analyzed tissues, BAs could have either protective or pro-carcinogen activities. However, the molecular mechanisms responsible for these effects have not been characterized yet. It involves either chemical properties or their capacities to activate their specific receptors FXRα or TGR5. This review highlights and discusses the potential links between BAs and cancer diseases and the perspectives of using BAs as potential therapeutic targets in several pathologies.

Cholesterol and male fertility: what about orphans and adopted?

The link between cholesterol homeostasis and male fertility has been clearly suggested in patients who suffer from hyperlipidemia and metabolic syndrome. This has been confirmed by the generation of several transgenic mouse models or in animals fed with high cholesterol diet. Next to the alteration of the endocrine signaling pathways through steroid receptors (androgen and estrogen receptors); "orphan" and "adopted" nuclear receptors, such as the Liver X Receptors (LXRs), the Proliferating Peroxisomal Activated Receptors (PPARs) or the Liver Receptor Homolog-1 (LRH-1), have been involved in this cross-talk. These transcription factors show distinct expression patterns in the male genital tract, explaining the large panel of phenotypes observed in transgenic male mice and highlighting the importance of lipid homesostasis and the complexity of the molecular pathways involved. Increasing our knowledge of the roles of these nuclear receptors in male germ cell differentiation could help in proposing new approaches to either treat infertile men or define new strategies for contraception.

Epigenetic: a molecular link between testicular cancer and environmental exposures.

In the last decades, studies in rodents have highlighted links between in utero and/or neonatal exposures to molecules that alter endocrine functions and the development of genital tract abnormalities, such as cryptorchidism, hypospadias, and impaired spermatogenesis. Most of these molecules, called endocrine disrupters exert estrogenic and/or antiandrogenic activities. These data led to the hypothesis of the testicular dysgenesis syndrome which postulates that these disorders are one clinical entity and are linked by epidemiological and pathophysiological relations. Furthermore, infertility has been stated as a risk factor for testicular cancer (TC). The incidence of TC has been increasing over the past decade. Most of testicular germ cell cancers develop through a pre-invasive carcinoma in situ from fetal germ cells (primordial germ cell or gonocyte). During their development, fetal germ cells undergo epigenetic modifications. Interestingly, several lines of evidence have shown that gene regulation through epigenetic mechanisms (DNA and histone modifications) plays an important role in normal development as well as in various diseases, including TC. Here we will review chromatin modifications which can affect testicular physiology leading to the development of TC; and highlight potential molecular pathways involved in these alterations in the context of environmental exposures.

GPR30, the non-classical membrane G protein related estrogen receptor, is overexpressed in human seminoma and promotes seminoma cell proliferation.

BACKGROUND: Testicular germ cell tumours are the most frequent cancer of young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown but epidemiological and clinical data have suggested that fetal exposure to environmental endocrine disruptors (EEDs) with estrogenic effects, could participate to testicular germ cell carcinogenesis. However, these EEDs (like bisphenol A) are often weak ligands for classical nuclear estrogen receptors. Several research groups recently showed that the non classical membrane G-protein coupled estrogen receptor (GPER/GPR30) mediates the effects of estrogens and several xenoestrogens through rapid non genomic activation of signal transduction pathways in various human estrogen dependent cancer cells (breast, ovary, endometrium). The aim of this study was to demonstrate that GPER was overexpressed in testicular tumours and was able to trigger JKT-1 seminoma cell proliferation. RESULTS: We report here for the first time a complete morphological and functional characterization of GPER in normal and malignant human testicular germ cells. In normal adult human testes, GPER was expressed by somatic (Sertoli cells) and germ cells (spermatogonia and spermatocytes). GPER was exclusively overexpressed in seminomas, the most frequent testicular germ cell cancer, localized at the cell membrane and triggered a proliferative effect on JKT-1 cells in vitro, which was completely abolished by G15 (a GPER selective antagonist) and by siRNA invalidation. CONCLUSION: These results demonstrate that GPER is expressed by human normal adult testicular germ cells, specifically overexpressed in seminoma tumours and able to trigger seminoma cell proliferation in vitro. It should therefore be considered rather than classical ERs when xeno-estrogens or other endocrine disruptors are assessed in testicular germ cell cancers. It may also represent a prognosis marker and/or a therapeutic target for seminomas.

Perinatal programming of adult rat germ cell death after exposure to xenoestrogens: role of microRNA miR-29 family in the down-regulation of DNA methyltransferases and Mcl-1.

Different studies have pointed out that developmental exposure to environmental endocrine disruptors can induce long-term testicular germ cell death probably through epigenetic mechanisms. By using a model of early neonatal post-natal day (PND) 1 to 5 exposure of male rats to a xenoestrogen, estradiol benzoate (EB), we investigated the role of microRNA and DNA methyltransferases (DNMT) on the developmental effects of EB on the adult germ cell death process. Neonatal exposure to EB induced adult germ cell apoptosis together with a dose-dependent increase in miR-29a, miR-29b, and miR-29c expression. Increased miR-29 expression resulted in a decrease in DNMT1, DNMT3a, and DNMT3b and antiapoptotic myeloid cell leukemia sequence 1 (Mcl-1) protein levels as shown in 1) germ cells of adult rats exposed neonatally to EB and 2) in spermatogonial GC-1 transfected with miR-29. The DNMT decrease was associated with a concomitant increase in transcript levels of DNA methylation target genes, such as L1td1-1 ORF1 and ORF2, Cdkn2a, and Gstp1, in correlation with their pattern of methylation. Finally, GC-1 cell lines transfection with miR-29a, miR-29b, or miR-29c undergo apoptosis evidenced by Annexin-V expression. Together, the increased miR-29 with a subsequent reduction in DNMT and Mcl-1 protein levels may represent a basis of explanation for the adult expression of the germ cell apoptosis phenotype. These observations suggest that the increased expression of the "apoptomir" miR-29 family represents the upstream mechanism identified until now that is involved in adult germ cell apoptosis induced by a neonatal hormonal disruption.

Expression of embryonic stem cell markers in cultured JKT-1, a cell line derived from a human seminoma.

Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (alphaFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2gamma and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results.