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International audience; Macrophages comprise a heterogeneous immune cell population and display niche-specific phenotypes and functions in almost all organs. Testicular macrophages (TMs) perform essential immune and non-immune functions in the mammalian male gonads. Here, we discuss the most recent findings on TM ontogeny, heterogeneity, and function under steady state and inflammatory conditions. We also highlight new discoveries regarding the functions of macrophages during bacterial and viral infections of the testes and how macrophages may indirectly help the establishment of a reservoir through virus seeding. Understanding TM function and macrophage-related mechanisms of disease might assist in developing new opportunities for intervention in male infertility. © 2021 Elsevier Ltd

Regionalised interaction of the activins, follistatin and inhibin was investigated in the male reproductive tract of mice lacking the inhibin α-subunit (Inha-/-). Serum and intratesticular activin B, although not activin A and follistatin, were increased in Inha-/- mice at 25 days of age, but all three proteins were elevated at 56 days. None of these proteins were altered within the epididymis and vas deferens at either age. At 25 days, histology of the epididymis and vas deferens was similar to wild-type. At 56 days, the testis contained extensive somatic cell tumours, leading to Leydig cell regression and testosterone deficiency. The epididymis and vas deferens showed epithelial regression and increased prominence of the interstitial stroma. Immunoregulatory and fibrotic gene expression in the epididymis and vas deferens were unchanged. Thus, absence of the inhibin α-subunit has marginal effects on activins in the epididymis and vas deferens, and regression of these tissues is associated with androgen deficiency.

Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis.

The fetus in the gravid uterus and the developing spermatogenic cells in the adult testis both comprise special challenges for the host immune system. Protection of the neoantigens of the fetus and male germ cells from immune attack, defined as immune privilege, is fundamental for the propagation of species. Immune privilege is not simply the absence of leukocytes, but involves immune and non-immune cells acting synergistically together at multiple levels to create a unique tolerogenic environment. A number of the pathways are shared by the testis and gravid uterus. Amongst them steroid hormones, namely testosterone in the male and progesterone in the female, seem to function as key molecules that govern the local production of immunoregulatory factors which finally control the overall immune environment.

Protection of the spermatogenic cells from the host immune response is fundamental to male fertility. Significantly, this protection extends to the tolerance of foreign tissue grafts placed within the testicular environment, a phenomenon that is called 'immune privilege'. This privilege of the testis appears to involve several levels of immune control, encompassing the normal mechanisms of immune tolerance, antigen sequestration behind the blood-testis barrier, reduced immune activation, localised immunosuppression and antigen-specific immunoregulation. Central to these regulatory processes are the somatic cells of the testis, particularly the Sertoli cells, and testicular secretions, including androgens, cytokines, peptides and bioactive lipids. Failure of these protective mechanisms, which may be precipitated by trauma, inflammation or infection, or as the consequence of genetic factors, can lead to androgen deficiency, infertility and autoimmunity.

Spermatogonial stem cells (SSCs) provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and differentiation into sperm. SSC-derived cell lines called multipotent adult germline stem cells (maGSCs) were recently shown to be pluripotent and to have the same potential as embryonic stem cells (ESCs). In a differentiation protocol using retinoic acid (RA) and based on a double selection strategy, we have shown that ESCs are able to undergo meiosis and produce haploid male germ cells in vitro. Using this differentiation protocol we have now succeeded to generate haploid male germ cells from maGSCs in vitro. maGSCs derived from a Stra8-EGFP transgenic mouse line were differentiated into stable spermatogonial stages and further cultured. These cells were transfected with a postmeiotic specific promoter construct Prm1-DsRed to monitor retinoic acid (RA) induced differentiation into haploid male gametes. Our protocol is another approach for the production of pluripotent stem cell derived gametes (PSCDGs) and is an alternative for the investigation of mammalian spermatogenesis, germ line gene modification and epigenetic reprogramming. If reproducible with pluripotent cell lines derived from human SSCs, it could also be used as a therapeutic approach for the treatment of male infertility.

PubMed: 19826004 Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/ VCP seems to control the ubiquitination status of recruited substrates. TheCOP9signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

SummaryMale gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed to adult mice. This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.

Glucocorticoid hormones are important anti-inflammatory agents because of their anti-inflammatory and proapoptotic action within the immune system. Their clinical usefulness remains limited however by side effects that result in part from their growth inhibitory action on sensitive target tissues. The protein mediator, macrophage migration inhibitory factor (MIF), is an important regulator of the host immune response and exhibits both glucocorticoid-antagonistic and growth-regulatory properties. MIF has been shown to contribute significantly to the development of immunopathology in several models of inflammatory disease. Although there is emerging evidence for a functional interaction between MIF and glucocorticoids in vitro, little is known about their reciprocal influence in vivo. We investigated the expression of MIF in rat tissues after ablation of the hypothalamic-pituitary-adrenal axis and after high-dose glucocorticoid administration. MIF expression is constitutive and independent of the influence of adrenal hormones. Hypophysectomy and the attendent loss of pituitary hormones, by contrast, decreased MIF protein content in the adrenal gland. Administration of dexamethasone was found to increase MIF protein expression in those organs that are considered to be sensitive to the growth inhibitory effects of glucocorticoids (immune and endocrine tissues, skin, and muscle). This increase was most likely because of a posttranscriptional regulatory effect because tissue MIF mRNA levels were not influenced by dexamethasone treatment. Finally, MIF immunoneutralization enhanced lymphocyte egress from blood during stress-induced lymphocyte redistribution, consistent with a functional interaction between MIF and glucocorticoids on immune cell trafficking in vivo. These findings suggest a role for MIF in both the homeostatic and physiological action of glucocorticoids in vivo.

Development of germ cells during spermatogenesis is characterized by a complex series of differentiation events finally leading to the production of spermatozoa. Beside the main hormonal regulators, paracrine interactions are thought to play a major role in this process. Mitochondria in germ cells pass through unique alterations ranging from the ‘typical’ cristae-rich mitochondria found in spermatogonia to the ‘condensed’ form in pachytene spermatocytes. This study provides further support that paracrine factors produced by Sertoli cells, most likely activin A, are involved in germ cell differentiation as monitored by the maintenance of the physiological ‘condensed’ mitochondrial phenotype in primary spennatocytes. Culture of primary spermatocytes in Sertoli cell conditioned medium (SCCM) for 18 h resulted in the maintenance of a high percentage of ‘condensed-type’ mitochondria in comparison to cells cultured in Dulbecco's minimum essential medium (DMEM). Activin A, a product of Sertoli cells, showed at subnanogram concentrations a similar ability to SCCM to maintain a high percentage of spematocyte mitochondria in the ‘condensed’ state, while inhibin had no effect. The addition of an antiserum specific for activin A resulted in a neutralization of the effect caused by activin A or SCCM. This strongly suggested that the active substance in SCCM was activin A. Taken together these data indicate that activin A is the first Sertoli cell product that has been identified to influence differentiation of male meiotic germ cells.

Inhibin A and B are dimeric proteins capable of suppressing FSH both in vitro and in vivo. The principal form in the male is inhibin B which is produced in the testis and circulates to inhibit pituitary FSH secretion. Activin A, B and AB are dimeric proteins that share the same beta subunits with the inhibins but, in contrast, stimulate FSH secretion. Although activin A circulates, castration does not lead to a decrease in serum concentrations, indicating that the testis is not the major source of activin A. In the circulation, the activins are bound to a structurally unrelated binding protein, follistatin, that neutralizes the biological actions of these proteins. The subunits of the inhibins/activins as well as follistatin are also produced locally within the pituitary and their levels can be modulated by testosterone and gonadotrophin releasing hormone as well as by autocrine mechanisms. Consequently, the output of FSH is dependent of the balance between local processes and the circulating feedback exerted by testosterone and inhibin. There is increasing data to support the local gonadal production of not only inhibin but also activin and follistatin by both germ cells and somatic cells such as the Sertoli cells. Evidence is accumulating to support the concept that these proteins exert local regulatory mechanisms in the testis.

cycles were excluded as they did not have embryos available for transfer. The remaining 256 cycles were included in the analysis (Table 1). Patients who did not adhere were younger, had higher number of oocytes retrieved and fertilized, and higher number of cleavage stage embryos, as compared to patients who adhered. The remaining clinical characteristics were similar between the two groups. Analysis of outcome data showed a higher LBR and twinning rate in non-adherent patients. Questionnaire analysis revealed financial burden as the major contributor to non-adherence (38%).

High mobility group box protein 1 (HMGB1), the non-histone chromosomal protein, plays an important role in onset and chronification of autoimmune diseases once released from the nuclei. In this study, we analyzed how HMGB1 can regulate inflammatory reactions in vivo in a rat model of experimental autoimmune orchitis (EAO) and in vitro in primary testicular cells. HMGB1 was translocated from the nuclei in EAO testis and in the testis of infertile men with leukocytic infiltrates. HMGB1 levels in EAO testis were elevated at late chronic phase of disease as compared to early proinflammatory cytokines such as IL-6 and TNF- α . We found that testicular somatic cells show a cell-specific expression profile of HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE). The highly sensitive and specific proximity ligation assay was used to analyze HMGB1 receptor binding in testicular cells. HMGB1-TLR4 binding was dominant in testicular macrophages. However, Sertoli and peritubular cells showed higher levels of HMGB1-RAGE interaction. In support, HMGB1 triggered RAGE-dependent ERK1/2 MAPK and CREB activation in Sertoli and peritubular cells, whilst in testicular macrophages HMGB1 induced TLR4-signaling as evidenced by p38 MAPK and p65 NF-?B phosphorylation which stimulated an increase in mRNA levels of TNF- α and IL-6. Recent data shows that RAGE induced ERK activation leads to enhanced autophagy levels. In line with these data, extracellular HMGB1 triggered formation of autophagosomes in Sertoli cells. Increased autophagy levels in Sertoli cells might explain how these cells survive the inflammatory environment in EAO testis. Considering HMGB1’s late phase of action, inhibition of HMGB1 may be a putative target for therapeutic intervention in treatment of chronic testicular inflammation.

Introduction Infection and inflammation of the urinary tract are aetiological factors contributing to 10–15% of infertility cases in men. Epididymitis is often the consequence of ascending bacterial infection into the urinary tract. Specific strains of Uropathogenic E. coli (UPEC) produce the toxin alpha-haemolysin (hlyA), which has been shown to manipulate host immune responses by degrading components of the NF- κ B signalling cascade following infection of bladder epithelial cells. The aim of this study was to elucidate the consequences of hlyA production on innate immune responses in the UPEC induced murine experimental epididymitis model. Methods To evaluate the consequences of hlyA on cytokine production in vitro, RAW 267.4 cells or single epididymides dissected from C57BL/6 N mice were left untreated, treated with LPS (100 ng/mL) or infected with the clinically relevant pyelonephritic?-haemolytic (UPEC CFT073) or non-haemolytic E. coli strain NPEC 470. Cell supernatants were collected following treatment and cytokine production analysed using the BD ™ Cytometric Bead Array. The establishment of the murine experimental epididymitis model was achieved through injection of 40,000 UPEC CFT073 or NPEC 470 in total, into both left and right vas deferens of C57BL/6 N mice. PBS injections were used as a sham control for in vivo experiments. Following 3 days infection, epididymides were dissected and snap frozen. qPCR analyses was performed to quantify pro-inflammatory gene expression. Immuno-fluorescent staining was used for the detection of macrophages (F4/80) and T-cells (CD3). Statistical analysis was performed by One-way ANOVA followed by the Bonferroni’s Multiple Comparison Test where appropriate. P ⩽ 0.05 is considered statistically significant. Results In this study we show that the?-haemolytic UPEC strain CFT073 significantly reduced secretion of pro-inflammatory cytokines TNF-?, IL-6, IL-1 and IFN-? in epididymal organ cultures and RAW 267.4 cells following infection. Conversely, in vivo qPCR analyses revealed expression of early response genes TNF-? and IL-6 were significantly increased by day 3 following UPEC CFT073 infection. Interestingly, RANTES/CCL5 and IFN-? gene expression were differentially modulated dependent on the hylA status of the E. coli strain. Our findings illustrate that hlyA production significantly enhances RANTES expression following UPEC CFT073 infection in contrast to PBS and NPEC 470 infected mice. Conversely, IFN-? gene expression in UPEC CFT073 infected mice was comparable to PBS infected mice, whereas NPEC 470 significantly increased IFN-? expression. In addition, a decrease in the number of F4/80 and CD3+ cells within the epididymal interstitium of UPEC CFT073 infected mice was observed in comparison to NPEC 470 infected mice. Conclusion Overall these finding suggest that hlyA produced by UPEC attenuates host innate immune responses in the initial phases of infection for successful colonisation within the epididymis. Furthermore, the down-regulation of IFN-? attenuates maturation of adaptive immune responses thereby evading elimination and contributing to persistence.

Introduction High mobility group box protein 1 (HMGB1) is a non-histone nucleosomal protein. Under inflammatory conditions HMGB1 is secreted from activated immune cells or released from necrotic cells and acts as an endogenous danger signal. Despite its immune privileged status, the testis is prone to inflammatory infertility. In this study we analyzed the role of HMGB1 in mediating autoimmune responses in experimental autoimmune orchitis (EAO) – a rodent model of chronic testicular inflammation. Methods EAO was induced in WKY rats by immunization with testicular homogenate in complete Freund’s adjuvant. HMGB1 localization was analyzed in EAO and adjuvant control rat testes by immunofluorescence staining. Levels of HMGB1, IL-6 and TNF- α were measured in testes and serum using ELISA. Isolated testicular macrophages (TM), Sertoli cells (SC) and peritubular cells (PTC) were stimulated with recombinant HMGB1. Cytokine mRNA levels were measured by qRT-PCR. Testosterone production was evaluated using RIA assay in HMGB1 treated Leydig cells (LC). Expression of RAGE as well as activation of NF-?B (p65), MAPK (p38, ERK1/2) and Akt pathway were investigated in TM, SC and pPTC by Western blot. Possible binding of HMGB1 to TLR4 was investigated in isolated testicular cells using Duolink PLA assay. Results In control testes, HMGB1 was expressed mainly in the nucleus of testicular somatic cells. In contrast, EAO testes showed translocation of HMGB1 from the nucleus into the cytoplasm. Concentrations of IL-6 and TNF- α were increased 50 days after first immunization in EAO testes, whilst HMGB1 levels increased 80 days post-immunization. RAGE was highly expressed in SC as well as PTC and to a lower level in TM. In contrast, TLR4 levels were higher in TM. Our preliminary results show that HMGB1 binding to TLR4 is significantly higher in TM than in PTC. In vitro stimulation of isolated TM using recombinant HMGB1 activates phosphorylation of p38 and p65. However, HMGB1 treated PTC and SC activated ERK1/2. HMGB1 induced a significant increase in mRNA expression of TNF- α and IL-6 in TM. In HMGB1 treated SC the levels of IL-6 and TNF- α mRNA did not show a statistically significant difference. HMGB1 induced a significant increase in testosterone production by LC. Conclusion In control testis, HMGB1 was expressed mainly in the nucleus of testicular somatic cells. In contrast, EAO testes showed translocation of HMGB1 from the nucleus into the cytoplasm. Concentrations of IL-6 and TNF- α were increased 50 days after first immunization in EAO testis, whilst HMGB1 levels increased 80 days post-immunization. RAGE was highly expressed in SC as well as PTC and to a lower level in TM. In contrast, TLR4 levels were higher in TM. Our preliminary results show that HMGB1 binding to TLR4 is significantly higher in TM than in PTC. In vitro stimulation of isolated TM using recombinant HMGB1 activates phosphorylation of p38 and p65. In contrast, HMGB1 treated PTC and SC activated ERK1/2. HMGB1 induced a significant increase in mRNA expression of TNF- α and IL-6 in TM. In HMGB1 treated SC the levels of IL-6 and TNF- α mRNA did not show a statistically significant difference. HMGB1 induced a significant increase in testosterone production by LC.