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SummaryIn contrast with most cancers, adrenocortical carcinomas (ACC) are more frequent in women than men, but the underlying mechanisms of this sexual dimorphism remain elusive. Homozygous deletion of the negative WNT pathway regulator ZNRF3 is the most frequent alteration in ACC patients. Here, we show that Cre-mediated inactivation of Znrf3 in steroidogenic cells of the mouse adrenal cortex is associated with sexually dimorphic tumour progression. Indeed, although most knockout female mice develop metastatic carcinomas over an 18 month-time course, adrenal hyperplasia gradually regresses in male knockout mice. This male-specific regression is associated with induction of senescence and recruitment of macrophages, which differentiate as active phagocytes that clear-out senescent preneoplastic cells. Macrophage recruitment is also observed in female mice. However, it is delayed and dampened compared to males, which allows for tumour progression. Interestingly, testosterone treatment of female knockouts is sufficient to induce senescence, recruitment of phagocytic macrophages and regression of hyperplasia. We further show that although macrophages are present within adrenal tumours at 18 months, MERTKhigh active phagocytes are mostly found in indolent lesions in males but not in aggressive tumours in females. Consistent with our observations in mice, analysis of RNA sequencing data from the TCGA cohort of ACC shows that phagocytic macrophages are more prominent in men than women and associated with better prognosis. Altogether, these data establish that phagocytic macrophages prevent aggressive ACC development in male mice and suggest that they may play a key role in the unusual sexual dimorphism of ACC in patients.

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide for the first time a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development.Graphical abstractSummary of the expansion of the conventional and hybrid lymphatic vasculature during postnatal development of the murine epididymis.

Objective Adrenocortical carcinoma (ACC) is an aggressive cancer that originates from steroidogenic cells within the adrenal cortex. The most common alteration in ACC patients is inactivation of the transmembrane E3 ubiquitin-ligase Zinc and Ring Finger 3 (ZNRF3), which is responsible for inhibiting the canonical WNT/Beta-catenin pathway. We show that inactivation of Znrf3 (ZKO) in the adrenal cortex results in an initial hyperplasia by 6-weeks, after which sexually dimorphic phenotypes arise. Materials & methods Adrenal cortex specific ablation of Znrf3 & Trp53 was achieved by Cre/LoxP recombination technology. Results In males, but not females, Znrf3 inactivation induces senescence and release of senescence associated secretory phenotype (SASP) factors. This is associated with recruitment of macrophages (Mφ) that phagocytose neoplastic cells, which is associated with regression of hyperplasia. In ACC patients, ZNRF3 inactivation is frequently associated with mutations of TP53, a key inducer of senescence, suggesting that inhibition of senescence may be required for tumorigenesis to proceed. Preliminary data suggests that combined inactivation of Trp53 and Znrf3 (DKO) results in blunted senescence, reduced Mφ recruitment and acceleration of tumorigenesis, ultimately culminating with metastatic ACC development at 6 months. Discussion Our current aim is to use this metastatic ACC mouse model to highlight the role of Mφ in immunosurveillance and tumor inhibition within the adrenal cortex.

Testes produce spermatozoa that transit through and are stored in the epididymis where they acquire their fertilising capacities. Spermatozoa appear in the genital tract at puberty, long after the immune system was trained to self-antigens. As a consequence, this organ has to set strategies to tolerate sperm antigens to avoid autoimmune responses that would specifically target and destroy them. A recent study pointed the Transforming Growth Factor-beta (TGF-β) signalling in the dendritic cells as a crucial mechanism for epididymal tolerance to spermatozoa. In the mouse, TGF-β exists under three isoforms, and three distinct receptors have been described. Using RT-qPCR, immunohistochemistry and ELISA techniques, we investigated the expression and spatial distribution of the epididymal TGF-β isoforms and of their receptors in young and adult mice. We showed that both ligands and receptors were produced by immune and non-immune cells in the epididymis, whatever the age mice have. These data bring new clues as to the mechanisms of peripheral tolerance to sperm cells in the murine epididymis and raise potential other implications of the cytokine isoforms.

Despite increasing evidence that epididymal immune disorders can lead to infertility, the cells and mechanisms underlying epididymal immunity remain poorly understood. In this study, we propose a rather exhaustive overview of innate and adaptive immune cells present in the murine caput and cauda epididymis. Using flow cytometry and a wide set of markers, we screened the broadest panel of immune cells ever, in this organ. For the first time, we unequivocally quantified the innate populations of monocytes, macrophages, and dendritic cells subtypes. We also revealed the presence of B cells, gamma delta T cells, and double negative T cells in the murine epididymis. They were localized by immunofluorescence stainings, and appeared to be all present in the interstitium and epithelium along the organ, but with respective preferential regional distribution. Altogether, these findings provide new insights on the actors and potential mechanisms involved in the immune responses against genital tract ascending pathogens and in the setting and maintenance of tolerance toward the sperm cells.

International audience; Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endo-crine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phospho-diesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state. adrenal | differentiation | progenitors | PKA signaling | EZH2

Recent studies have revealed a well-defined higher order of chromosome architecture, named chromosome territories, in the human sperm nuclei. The purpose of this work was, first, to investigate the topology of a selected number of chromosomes in murine sperm, second, to evaluate whether sperm DNA damage has any consequence on chromosome architecture. Using fluorescence in situ hybridization, confocal microscopy, and 3D-reconstruction approaches we demonstrate that chromosome positioning in the mouse sperm nucleus is not random. Some chromosomes tend to occupy preferentially discrete positions, while others, such as chromosome 2 in the mouse sperm nucleus are less defined. Using a mouse transgenic model (Gpx5&minus, /&minus, ) of sperm nuclear oxidation, we show that oxidative DNA damage does not disrupt chromosome organization. However, when looking at specific nuclear 3D-parameters, we observed that they were significantly affected in the transgenic sperm, compared to the wild-type. Mild reductive DNA challenge confirmed the fragility of the organization of the oxidized sperm nucleus, which may have unforeseen consequences during post-fertilization events. These data suggest that in addition to the sperm DNA fragmentation, which is already known to modify sperm nucleus organization, the more frequent and, to date, the less highly-regarded phenomenon of sperm DNA oxidation also affects sperm chromatin packaging.

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Pan-genomic analyses identified p53/Rb and WNT/β-catenin signaling pathways as main contributors to the disease. However, isolated β-catenin constitutive activation failed to induce malignant progression in mouse adrenocortical tumors. Therefore, there still was a need for a relevant animal model to study ACC pathogenesis and to test new therapeutic approaches. Here, we have developed a transgenic mice model with adrenocortical specific expression of SV40 large T-antigen (AdTAg mice), to test the oncogenic potential of p53/Rb inhibition in the adrenal gland. All AdTAg mice develop large adrenal carcinomas that eventually metastasize to the liver and lungs, resulting in decreased overall survival. Consistent with ACC in patients, adrenal tumors in AdTAg mice autonomously produce large amounts of glucocorticoids and spontaneously activate WNT/β-catenin signaling pathway during malignant progression. We show that this activation is associated with downregulation of secreted frizzled related proteins (Sfrp) and Znrf3 that act as inhibitors of the WNT signaling. We also show that mTORC1 pathway activation is an early event during neoplasia expansion and further demonstrate that mTORC1 pathway is activated in ACC patients. Preclinical inhibition of mTORC1 activity induces a marked reduction in tumor size, associated with induction of apoptosis and inhibition of proliferation that results in normalization of corticosterone plasma levels in AdTAg mice. Altogether, these data establish AdTAg mice as the first preclinical model for metastatic ACC.

Summary Spermatogenesis is the process by which spermatozoa are generated from spermatogonia. This cell population is heterogeneous, with self-renewing spermatogonial stem cells (SSCs) and progenitor spermatogonia that will continue on a path of differentiation. Only SSCs have the ability to regenerate and sustain spermatogenesis. This makes the testis a good model to investigate stem cell biology. The Farnesoid X Receptor alpha (FXRα) was recently shown to be expressed in the testis. However, its global impact on germ cell homeostasis has not yet been studied. Here, using a phenotyping approach in Fxrα−/− mice, we describe unexpected roles of FXRα on germ cell physiology independent of its effects on somatic cells. FXRα helps establish and maintain an undifferentiated germ cell pool and in turn influences male fertility. FXRα regulates the expression of several pluripotency factors. Among these, in vitro approaches show that FXRα controls the expression of the pluripotency marker Lin28 in the germ cells., Graphical Abstract, Highlights • FXRα regulated germ cell apoptotis independently of androgen homeostasis • FXRα controls germ cell differentiation • FXRα regulates the establishment and maintenance of undifferentiated germ cells • In germ cells, FXRα controls the expression of pluripotency markers such as Lin28, Martinot et al. report that FXRα controls the fate of undifferentiated spermatogonia.

Gamete DNA integrity is one key parameter conditioning reproductive success as well as the quality of life for the offspring. In particular, damage to the male nucleus can have profound negative effects on the outcome of fertilization. Because of the absence of repair activity of the quiescent mature spermatozoa it is easily subjected to nuclear damage, of which oxidative damage is by far the most prominent. In relation to the organization of the mammalian sperm nucleus we show here that one can correlate the nuclear regions of lower compaction with areas preferentially showing oxidative damage. More precisely, we show that oxidative DNA damage targets primarily histone-rich and nuclear matrix-attached domains located in the peripheral and basal regions of the mouse sperm nucleus. These particular sperm DNA domains were recently shown to be enriched in genes of paramount importance in postfertilization DNA replication events and in the onset of the embryonic developmental program. We propose that monitoring of sperm DNA oxidation using the type of assay presented here should be considered in clinical practice when one wants to estimate the integrity of the paternal nucleus along with more classical assays that essentially analyze DNA fragmentation and nucleus compaction.

After its production in the testis, a spermatozoon has to undergo posttesticular maturation steps to become fully motile and fertile. The first step is epididymal maturation, during which immature spermatozoa are transformed into biochemically mature cells ready to proceed to the next step, capacitation, a physiological process occurring in the female genital tract. The biochemical transformations include modification of sperm lipid composition during epididymal transit, with significant changes in fatty acids, phospholipids, and sterols between the caput and the cauda epididymal spermatozoa. Although quantitative aspects of these changes are well documented for several mammalian species, molecular mechanisms governing these steps are poorly understood. Transgenic male mice invalidated for the two liver X receptors (LXRalpha and LXRbeta, nuclear oxysterol receptors regulating cholesterol and lipid metabolism) become sterile when aging, showing an epididymal phenotype. We used single-knockout-model mice to characterize the role of each LXR isoform during sperm maturation in the epididymis. We show here that although a certain redundancy exists in the functions of the two LXR isoforms, some physiological processes are more under the influence of only one of them. In both cases, aging males showed slight subfertility, associated with dyslipidemia, emphasizing the importance of lipid metabolism in relation with male fertility.

The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1(-/-) model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1(-/-) animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed.

International audience; Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1(-/-)-deficient mice. In the caput epididymis of Ido1(-/-) animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.

Adrenocortical carcinoma is a rare but aggressive cancer with unknown aetiology. Constitutive activation of beta-catenin is the most frequent alteration in benign and malignant adrenocortical tumours in patients. Here, we show that constitutive activation of beta-catenin in the adrenal cortex of transgenic mice resulted in progressive steroidogenic and undifferentiated spindle-shaped cells hyperplasia as well as dysplasia of the cortex and medulla. Over a 17 months time course, transgenic adrenals developed malignant characteristics such as uncontrolled neovascularization and loco-regional metastatic invasion. These oncogenic events were accompanied by ectopic differentiation of glomerulosa at the expense of fasciculata cells, which caused primary hyperaldosteronism. Altogether these observations demonstrate that constitutively active beta-catenin is an adrenal oncogene which triggers benign aldosterone-secreting tumour development and promotes malignancy.

This work shows that an overload of dietary cholesterol causes complete infertility in dyslipidemic male mice (the Liver X Receptor-deficient mouse model). Infertility resulted from post-testicular defects affecting the fertilizing potential of spermatozoa. Spermatozoa of cholesterol-fed lxr-/- animals were found to be dramatically less viable and motile, and highly susceptible to undergo a premature acrosome reaction. We also provide evidence, that this lipid-induced infertility is associated with the accelerated appearance of a highly regionalized epididymal phenotype in segments 1 and 2 of the caput epididymidis that was otherwise only observed in aged LXR-deficient males. The epididymal epithelial phenotype is characterized by peritubular accumulation of cholesteryl ester lipid droplets in smooth muscle cells lining the epididymal duct, leading to their transdifferentiation into foam cells that eventually migrate through the duct wall, a situation that resembles the inflammatory atherosclerotic process. These findings establish the high level of susceptibility of epididymal sperm maturation to dietary cholesterol overload and could partly explain reproductive failures encountered by young dyslipidemic men as well as ageing males wishing to reproduce.

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.