Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration.

STUDY QUESTION: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests.

T cells in testicular germ cell tumors: new evidence of fundamental contributions by rare subsets.

BACKGROUND: Immune cell infiltration is heterogeneous but common in testicular germ cell tumors (TGCT) and pre-invasive germ cell neoplasia in situ (GCNIS). Tumor-infiltrating T cells including regulatory T (Treg) and follicular helper T (Tfh) cells are found in other cancer entities, but their contributions to TGCT are unknown. METHODS: Human testis specimens from independent patient cohorts were analyzed using immunohistochemistry, flow cytometry and single-cell RNA sequencing (scRNA-seq) with special emphasis on delineating T cell subtypes. RESULTS: Profound changes in immune cell composition within TGCT, shifting from macrophages in normal testes to T cells plus B and dendritic cells in TGCT, were documented. In most samples (96%), the CD4+ T cell frequency exceeded that of CD8+ cells, with decreasing numbers from central to peripheral tumor areas, and to tumor-free, contralateral testes. T cells including Treg and Tfh were most abundant in seminoma compared to mixed tumors and embryonal carcinoma. CONCLUSION: Despite considerable heterogeneity between patients, T cell subtypes form a key part of the TGCT microenvironment. The novel finding of rare Treg and Tfh cells in human testis suggests their involvement in TGCT pathobiology, with implications for understanding tumor progression, to assess patients' prognosis, and as putative targets for personalized immunotherapy.

New aspects of activin biology in epididymal function and immunopathology.

The activins (A and B) and their binding protein, follistatin, play crucial roles in development, immunoregulation and inflammation throughout the body. In the male reproductive tract of the mouse, activin A and B production is largely confined to the initial segment and proximal caput of the epididymis and the efferent ducts, under normal conditions, with very low expression in the corpus, cauda and vas deferens. However, activin A protein is present throughout the epididymis and vas deferens and is largely associated with the epithelium and interstitial macrophages. Conversely, the activin-binding protein follistatin is produced in the distal epididymis, with very high expression in the vas deferens. Activin activity in the distal tract is inhibited by follistatin, and the activin-follistatin balance is important for regulating coiling of the duct during epididymal development. In further experiments, as described in this report, in situ hybridisation was used to localise activin A mRNA principally to cells in the periductal zone and interstitium in the efferent ducts and proximal caput. Activin B mRNA, on the other hand, was localised to periductal cells in the efferent ducts and proximal epididymis and, most notably, to epithelial cells in the initial segment. Activin A is implicated in the regulation of mononuclear phagocyte function and immune responses in the caput and stimulates the expression of the key immunoregulatory protein, indoleamine 2,3-dioxygenase in this region. Activin A production in the corpus and cauda increases dramatically during bacterial epididymitis in mice, promoting inflammation and fibrosis and causing damage to the epithelium and obstruction of the epididymal duct. Consequently, it appears that the activin-follistatin axis is crucial for maintaining normal epididymal structure and function, but disruption of this balance during inflammation has deleterious effects on male fertility. Follistatin has therapeutic potential in ameliorating the proinflammatory and profibrotic effects of activins.

The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis.

The epididymis functions as transition zone for post-testicular sperm maturation and storage and faces contrasting immunological challenges, i.e. tolerance towards spermatozoa vs. reactivity against pathogens. Thus, normal organ function and integrity relies heavily on a tightly controlled immune balance. Previous studies described inflammation-associated tissue damage solely in the distal regions (corpus, cauda), but not in the proximal regions (initial segment, caput). To understand the observed region-specific immunity along the epididymal duct, we have used an acute bacterial epididymitis mouse model and analyzed the disease progression. Whole transcriptome analysis using RNAseq 10 days post infection showed a pro-inflammatory environment within the cauda, while the caput exhibited only minor transcriptional changes. High-dimensional flow cytometry analyses revealed drastic changes in the immune cell composition upon infection with uropathogenic Escherichia coli. A massive influx of neutrophils and monocytes was observed exclusively in distal regions and was associated with bacterial appearance and tissue alterations. In order to clarify the reasons for the region-specific differences in the intensity of immune responses, we investigated the heterogeneity of resident immune cell populations under physiological conditions by scRNASeq analysis of extravascular CD45+ cells. Twelve distinct immune cell subsets were identified, displaying substantial differences in distribution along the epididymis as further assessed by flow cytometry and immunofluorescence staining. Macrophages constituted the majority of resident immune cells and were further separated in distinct subgroups based on their transcriptional profile, tissue location and monocyte-dependence. Crucially, the proximal and distal regions showed striking differences in their immunological landscapes. These findings indicate that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for addressing the contrasting immunological challenges and thus, preserving tissue integrity and organ function.

Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis.

Experimental autoimmune-orchitis (EAO), a rodent model of chronic testicular inflammation and fibrosis, replicates pathogenic changes seen in some cases of human spermatogenic disturbances. During EAO, increased levels of pro-inflammatory and pro-fibrotic mediators such as TNF, CCL2, and activin A are accompanied by infiltration of leukocytes into the testicular parenchyma. Activin A levels correlate with EAO severity, while elevated CCL2 acting through its receptor CCR2 mediates leukocyte trafficking and recruits macrophages. CCR2 + CXCR4 + macrophages producing extracellular matrix proteins contribute widely to fibrogenesis. Furthermore, testicular macrophages (TMs) play a critical role in organ homeostasis. Therefore, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. Following EAO induction, we observed lower levels of organ damage, collagen deposition, and leukocyte infiltration (including fibronectin+, collagen I+ and CXCR4+ TMs) in Ccr2-/- mice than in WT mice. Furthermore, levels of Il-10, Ccl2, and the activin A subunit Inhba mRNAs were lower in Ccr2-/- EAO testes. Notably, fibronectin+ TMs were also present in biopsies from patients with impaired spermatogenesis and fibrotic alterations. Overexpression of the activin A antagonist follistatin reduced tissue damage and collagen I+ TM accumulation in WT EAO testes, while treating macrophages with activin A in vitro increased the expression of Ccr2, Fn1, Cxcr4, and Mmp2 and enhanced migration along a CCL2 gradient; these effects were abolished by follistatin. Taken together, our data indicate that CCR2 and activin A promote fibrosis during testicular inflammation by regulating macrophage function. Inhibition of CCR2 or activin A protects against damage progression, offering a promising avenue for therapeutic intervention.

The changing landscape of immune cells in the fetal mouse testis.

Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0; PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80+ cells were the most abundant, comprising 90% of CD45+ cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80+ cells have different origins during embryonic life. CD3+ cells and F4/80-/MHCII+ were absent to rare until PND0. Ly6G+ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45+ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80+ and Ly6G+ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80+ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80Hi/CD206+) were different from macrophages at PND0 (F4/80Dim/CD206-), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.

Regulation of macrophage number and gene transcript levels by activin A and its binding protein, follistatin, in the testes of adult mice.

The cytokine activin A is expressed throughout testicular development and is a critical regulator of macrophage function, but its effects on the testicular macrophages are not well-defined. Macrophage distribution and gene transcript levels were examined in testes of adult mice with reduced levels of either activin A (Inhba+/-), or its binding protein, follistatin (TghFST315). Macrophages were identified using F4/80 immunohistochemistry and enumerated by morphometry. Transcript levels were measured in testis extracts by qRT-PCR and Fluidigm ™ analyses. Interstitial macrophages were twice as numerous as peritubular macrophages in Inhba+/- and TghFST315 mice and their littermate controls. Macrophage numbers were significantly reduced in all regions of the Inhba+/- testis, and the volume density of peritubular and subcapsular macrophages was significantly reduced compared to littermate controls (by 52.9% and 36.3% respectively). Transcripts encoding macrophage chemokines, Csf1 and Ccl2, and receptor Csf1r, were elevated (by 35%, 44% and 27% respectively) in Inhba+/- testes, but Cx3cl1 and their receptors, Cx3cr1 and Ccr2, were not altered. Transcripts encoding MHC class II antigens and the co-stimulatory molecule Cd86, also increased (by 32% and 60% respectively), but other co-stimulatory molecules Cd80 and Cd274, and the scavenger receptor Mrc1 (CD206), were unaffected. In the follistatin-deficient testes, macrophage numbers and most macrophage-specific transcripts were not significantly affected, but Mrc1 expression was reduced by 35%. These data indicate that activin A maintains macrophage numbers, but selectively inhibits the levels of key transcripts associated with macrophage antigen-presentation, recruitment and differentiation in the adult mouse testis.

Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput.

BACKGROUND: Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. OBJECTIVES: This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis. MATERIALS AND METHODS: The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. RESULTS: Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. CONCLUSION: These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

Experimental Cryptorchidism Causes Chronic Inflammation and a Progressive Decline in Sertoli Cell and Leydig Cell Function in the Adult Rat Testis.

Cryptorchidism causes spermatogenic failure and reduced serum androgen levels, as well as testicular oedema and fibrosis, which are hallmarks of inflammation. However, the role of inflammation and the effects of cryptorchidism on Sertoli cell and Leydig cell function at the molecular level remain ill-defined. Bilateral cryptorchidism was surgically induced in adult rats for 7 and 14 weeks. Testis weights decreased to 40% of normal within 7 weeks, due to loss of all developing spermatogenic cells except spermatogonia, but did not decrease further at 14 weeks. Serum FSH and LH were increased at both time points, consistent with a loss of feedback by inhibin and testosterone. This damage was accompanied by progressive accumulation of interstitial fluid and peritubular fibrosis, and a progressive decline of several critical Sertoli cell genes (Sox9, Inha (inhbin α-subunit), Cldn11 (claudin 11), Gja1 (connexin 43), and Il1a (interleukin-1α)) and the Leydig cell steroidogenic enzymes, Cyp11a1, Hsd3b1, and Hs17b3. Activin B and the activin-binding protein, follistatin, also declined, but the intratesticular concentration of activin A, which is a regulator of inflammatory responses, was largely unaffected at either time point. Expression of genes involved in inflammation (Tnf, Il10, Il1b, Mcp1) and fibrosis (Acta2, Col1a1) were considerably elevated at both time points. These data indicate that induction of experimental cryptorchidism, which causes complete failure of spermatogenesis in the adult rat, also induces chronic testicular inflammation, manifesting in oedema and fibrosis, and a progressive decline of Sertoli and Leydig cell gene expression and function.

Region-specific immune responses to autoimmune epididymitis in the murine reproductive tract.

Epididymitis is a common pathology of the male reproductive tract, potentially leading to infertility. Studies on bacterial epididymitis indicate that the cauda epididymis is more susceptible to inflammatory damage than the caput. These regional differences in immunoregulation are further investigated using an experimental autoimmune epididymo-orchitis model. Adult mice were immunized against testicular antigens and tissues were collected at 30 and 50 days following the first immunization. Epididymitis developed progressively; 70% of the mice developed disease at 30 days after the initial immunization and 93% at 50 days. Epididymitis was characterized by epithelial damage, immune cell infiltrates and fibrosis in the cauda, with minimal changes in the corpus, while the caput was unaffected. The incidence of epididymitis was greater than that of orchitis but similar to vasitis. The severity of epididymitis was positively correlated with the orchitis severity. Expression of key genes implicated in epididymal immunoregulation, inflammation and fibrosis, such as Ido1, Tnf, Tgfb1, Ccl2, Il1b, Il10, Cx3cl1 and Col1a1, was unchanged in the caput but increased in proportion to damage severity in the cauda at 50 days. Activin receptor mRNA expression in the cauda was negatively correlated with disease severity. These data suggest that the cauda is highly susceptible to inflammatory damage following an autoimmune challenge but the caput is minimally affected. This may be because the cauda is required to combat ascending infections through a robust inflammatory response, while the caput provides a more tolerogenic environment in order to protect the auto-antigenic sperm released from the testis.

Intravascular Follistatin gene delivery improves glycemic control in a mouse model of type 2 diabetes.

Type 2 diabetes (T2D) manifests from inadequate glucose control due to insulin resistance, hypoinsulinemia, and deteriorating pancreatic ß-cell function. The pro-inflammatory factor Activin has been implicated as a positive correlate of severity in T2D patients, and as a negative regulator of glucose uptake by skeletal muscle, and of pancreatic ß-cell phenotype in mice. Accordingly, we sought to determine whether intervention with the Activin antagonist Follistatin can ameliorate the diabetic pathology. Here, we report that an intravenous Follistatin gene delivery intervention with tropism for striated muscle reduced the serum concentrations of Activin B and improved glycemic control in the db/db mouse model of T2D. Treatment reversed the hyperglycemic progression with a corresponding reduction in the percentage of glycated-hemoglobin to levels similar to lean, healthy mice. Follistatin gene delivery promoted insulinemia and abundance of insulin-positive pancreatic ß-cells, even when treatment was administered to mice with advanced diabetes, supporting a mechanism for improved glycemic control associated with maintenance of functional ß-cells. Our data demonstrate that single-dose intravascular Follistatin gene delivery can ameliorate the diabetic progression and improve prognostic markers of disease. These findings are consistent with other observations of Activin-mediated mechanisms exerting deleterious effects in models of obesity and diabetes, and suggest that interventions that attenuate Activin signaling could help further understanding of T2D and the development of novel T2D therapeutics.

Differential tissue-specific damage caused by bacterial epididymo-orchitis in the mouse.

Ascending bacterial urinary tract infections can cause epididymo-orchitis. In the cauda epididymidis, this frequently leads to persistent tissue damage. Less coherent data is available concerning the functional consequences of epididymo-orchitis on testis and caput epididymidis. This in vivo study addresses the functional and spatial differences in responsiveness of murine epididymis and testis to infection with uropathogenic Escherichia coli (UPEC). Whole transcriptome analysis (WTA) was performed on testis, caput, corpus and cauda epididymidis of adult C57BL/6 J wildtype mice. Following UPEC-induced epididymo-orchitis in these mice, epididymal and testicular tissue damage was evaluated histologically and semi-quantitatively at 10 days and 31 days post-inoculation. Expression of inflammatory markers and candidate antimicrobial genes were analysed by RT-qPCR. WTA revealed distinct differences in gene signatures between caput and cauda epididymidis, particularly amonst immunity-related genes. Cellular and molecular signs of testicular inflammation and disruption of spermatogenesis were noticed at day 10, but recovery was observed by day 31. In contrast to the cauda, the caput epididymidis did not reveal any signs of gross morphological damage or presence of pro-inflammatory processes despite confirmed infection. In contrast to beta-defensins, known UPEC-associated antimicrobial peptides (AMP), like Lcn2, Camp and Lypd8, were inherently highly expressed or upregulated in the caput following infection, potentially allowing an early luminal protection from UPEC. At the time points investigated, the caput epididymidis was protected from any obvious infection/inflammation-derived tissue damage. Studies addressing earlier time-points will conclude whether in the caput epididymidis a pro-inflammatory response is indeed not essential for effective protection from UPEC.

Pre-pubertal growth, muscle and fat accumulation in male and female sheep-Relationships with metabolic hormone concentrations, timing of puberty and reproductive outcomes.

Metabolic homeostasis is aligned with changes in growth and body composition, through processes mediated by circulating metabolites and metabolic hormones, and is eventually linked to reproductive success. In the present study with sheep, we determined the relationships among phenotypic and genotypic rates of growth, muscle and fat accumulation, and the circulating concentrations of metabolic and tested for relationships with the timing of puberty and subsequent reproductive outcomes. We used 64 females and 62 males with known phenotypic values for depth of eye muscle (EMD) and fat (FAT) and known Australian Sheep Breeding Values at post-weaning age for live weight (PWT), depth of eye muscle (PEMD) and depth of fat (PFAT). Blood plasma sampled every 20 min for 8 hr via was assayed for growth hormone (GH), insulin-like growth factor I (IGF-I), insulin, leptin, ghrelin, follistatin, glucose and non-esterified fatty acids (NEFA). In males, PWT was positively related to the concentrations of GH, follistatin and glucose, whereas FAT and PFAT were positively related to IGF-I concentrations (p < .01). Testosterone concentration was negatively related to muscle variables (p < .001) and to PFAT (p < .05). In females, the only significant relationship detected was the positive link between EMD and insulin concentrations (p < .05). Reproductive variables were only measured in females. Live weight at first oestrus was related positively to insulin concentration and negatively to GH concentration (p < .05). No other relationships with reproductive variables were significant. The relationships that were detected suggest subtle differences between the sexes in the way their metabolic homeostasis responds to changes in the rates of growth, and muscle and fat accumulation, perhaps due to interference by testosterone in the males.

Acute heat-treatment disrupts inhibin-related protein production and gene expression in the adult rat testis.

Heat reversibly disrupts spermatogenesis, but the effects on Sertoli cell (SC) function and inhibin/activin-related proteins are less well-defined. Adult rat testis weights decreased by 40% within 2 weeks after heat-treatment (43 °C, 15 min), due to loss of pachytene spermatocytes and round spermatids. Coincident effects were reduced SC nuclear volume at one week and >50% reduction in expression of several critical SC genes (Inha, Cld11, Gja1, Tjp1, Cldn3) by 2 weeks. Leydig cell steroidogenic enzymes, Cyp11a1, Hsd3b1, were also reduced. Activin gene expression was unaffected at this time, but expression of the activin-binding protein, follistatin (Fst), increased >2-fold. At 4-8 weeks, coincident with the recovery of spermatocytes and early spermatids, but progressive loss of elongated spermatids, most SC genes had recovered; however, testicular activin A was reduced and activin B increased. At 8 weeks, serum inhibin was decreased and, consequently, serum FSH increased. Crucially, germ cell damage was not associated with a significant inflammatory response. At 14 weeks, most testicular parameters had returned to normal, but testis weights remained slightly reduced. These data indicate that, following acute heat-treatment, expression of several key Sertoli and Leydig cell genes declined in parallel with the initial loss of meiotic germ cells, whereas activins were responsive to the subsequent loss of mature spermatids, leading to an increase in testicular activin B production relative to activin A.

Rethinking ME/CFS Diagnostic Reference Intervals via Machine Learning, and the Utility of Activin B for Defining Symptom Severity.

Biomarker discovery applied to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a disabling disease of inconclusive aetiology, has identified several cytokines to potentially fulfil a role as a quantitative blood/serum marker for laboratory diagnosis, with activin B a recent addition. We explored further the potential of serum activin B as a ME/CFS biomarker, alone and in combination with a range of routine test results obtained from pathology laboratories. Previous pilot study results showed that activin B was significantly elevated for the ME/CFS participants compared to healthy (control) participants. All the participants were recruited via CFS Discovery and assessed via the Canadian/International Consensus Criteria. A significant difference for serum activin B was also detected for ME/CFS and control cohorts recruited for this study, but median levels were significantly lower for the ME/CFS cohort. Random Forest (RF) modelling identified five routine pathology blood test markers that collectively predicted ME/CFS at ≥62% when compared via weighted standing time (WST) severity classes. A closer analysis revealed that the inclusion of activin B to the panel of pathology markers improved the prediction of mild to moderate ME/CFS cases. Applying correct WST class prediction from RFA modelling, new reference intervals were calculated for activin B and associated pathology markers, where 24-h urinary creatinine clearance, serum urea and serum activin B showed the best potential as diagnostic markers. While the serum activin B results remained statistically significant for the new participant cohorts, activin B was found to also have utility in enhancing the prediction of symptom severity, as represented by WST class.

Investigation of activin A in inflammatory responses of the testis and its role in the development of testicular fibrosis.

STUDY QUESTION: Does activin A contribute to testicular fibrosis under inflammatory conditions? SUMMARY ANSWER: Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. WHAT IS KNOWN ALREADY: Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n = 6), 50 (n = 6) and 80 (n = 5) days after first immunization. Age-matched mice injected with adjuvant alone (n = 14) and untreated mice (n = 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n = 3) or vector with an empty cassette (empty vector controls; n = 2) 30 days prior to the first immunization, as well as appropriate adjuvant (n = 2) and untreated (n = 2) controls, were also examined.Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n = 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n = 7).Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Production of activin A by SC was stimulated by 25 and 50 ng/ml TNF (P < 0.01, P < 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P < 0.05), accompanied by increased total collagen (P < 0.01) and fibronectin deposition. Total testicular collagen (P < 0.0001) and fibronectin protein expression (P < 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r = 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P < 0.05) and the production of collagen I (P < 0.001; P < 0.01) and fibronectin (P < 0.05). Moreover, activin A also increased collagen IV mRNA (P < 0.05) in PTC, while αSMA mRNA (P < 0.01) and protein (P < 0.0001) were significantly increased by activin A in NIH 3T3 cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. WIDER IMPLICATIONS OF THE FINDINGS: Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government's Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1-2) on `Molecular pathogenesis on male reproductive disorders' funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

Dexamethasone improves therapeutic outcomes in a preclinical bacterial epididymitis mouse model.

STUDY QUESTION: Can dexamethasone improve infertility-related cauda epididymidal tissue damage caused by bacterial epididymitis? SUMMARY ANSWER: Dexamethasone in addition to anti-microbial treatment effectively reduces long-term deleterious epididymal tissue damage by dampening the host's adaptive immune response. WHAT IS KNOWN ALREADY: Despite effective anti-microbial treatment, ~40% of patients with epididymitis experience subsequent sub- or infertility. An epididymitis mouse model has shown that the host immune response is mainly responsible for the magnitude of epididymal tissue damage that is fundamentally causative of the subsequent fertility issues. STUDY DESIGN, SIZE, DURATION: Bacterial epididymitis was induced in male mice by using uropathogenic Escherichia coli (UPEC). From Day 3 after infection onwards, mice were treated with daily doses of levofloxacin (20 mg/kg, total n = 12 mice), dexamethasone (0.5 mg/kg, total n = 9) or both in combination (total n = 11) for seven consecutive days. Control animals were left untreated, i.e. given no interventional treatment following UPEC infection (total n = 11). Half of the animals from each group were killed either at 10 or 31 days post-infection. PARTICIPANTS/MATERIALS, SETTING, METHODS: A mouse model of induced bacterial epididymitis was applied to adult male C57BL/6J mice. At the respective endpoints (10 or 31 days post-infection), epididymides were collected. Effectiveness of antibiotic treatment was assessed by plating of epididymal homogenates onto lysogeny broth agar plates. Overall tissue morphology and the degree and nature of tissue damage were assessed histologically. Quantitative RT-PCR was used to assess local cytokine transcript levels. Blood was drawn and serum analysed for systemic IgG and IgM levels by ELISA. In addition, correlation analyses of clinical data and serum-analyses of IgG and IgM levels in patients with epididymitis were performed. MAIN RESULTS AND THE ROLE OF CHANCE: The addition of dexamethasone to the standard anti-microbial treatment did not further worsen epididymal tissue integrity. In fact, an obviously dampened immune response and reduced tissue reaction/damage was observed at both 10 and 31 days post-infection following combined treatment. More specifically, epididymal duct continuity was preserved, enabling sperm transit. In contrast, in untreated or antibiotic-treated animals, damage of the epididymal duct and duct constrictions were observed, associated with a lack of cauda spermatozoa. In line with the bacteriostatic/bactericidal effect of levofloxacin (alone as well as in combination), local cytokine transcript levels were significantly and similarly reduced in animals treated with levofloxacin alone (P < 0.01) or in combination with dexamethasone (P < 0.05) compared to UPEC-infected untreated animals. Interestingly, the addition of dexamethasone to the anti-microbial treatment induced a unique dampening effect on adaptive immunity, since systemic IgG and IgM levels as well as the pan-T cell marker CD3 were reduced at both 10 and 31 days post-infection. LIMITATIONS, REASONS FOR CAUTION: Breeding studies to address the fertility-protecting effect of the combined treatment were not possible in the experimental animals because the vas deferens was ligated (model specific). WIDER IMPLICATIONS OF THE FINDINGS: Whereas innate immunity is necessary and involved in acute bacterial clearance, adaptive immunity seems to be responsible for long-term, subclinical immunological activities that may negatively affect the pathogenesis of bacterial epididymitis even after effective bacterial eradication. These effects can be reduced in mice by the additional treatment with dexamethasone. This immunological characteristic of bacterial epididymitis shows similarities to the Jarisch-Herxheimer reaction known from other types of bacterial infection. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants from the Deutsche Forschungsgemeinschaft, Monash University and the Medical Faculty of Justus-Liebig University to the International Research Training Group on 'Molecular pathogenesis of male reproductive disorders' (GRK 1871). R.W., K.L.L. and M.P.H. were supported by grants from the National Health and Medical Research Council of Australia (ID1079646, ID1081987, ID1020269 and ID1063843) and by the Victorian Government's Operational Infrastructure Support Program. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: No clinical trial involved.

Interplay between Follistatin, Activin A, and BMP4 Signaling Regulates Postnatal Thymic Epithelial Progenitor Cell Differentiation during Aging.

A key feature of immune functional impairment with age is the progressive involution of thymic tissue responsible for naive T cell production. In this study, we identify two major phases of thymic epithelial cell (TEC) loss during aging: a block in mature TEC differentiation from the pool of immature precursors, occurring at the onset of puberty, followed by impaired bipotent TEC progenitor differentiation and depletion of Sca-1lo cTEC and mTEC lineage-specific precursors. We reveal that an increase in follistatin production by aging TECs contributes to their own demise. TEC loss occurs primarily through the antagonism of activin A signaling, which we show is required for TEC maturation and acts in dissonance to BMP4, which promotes the maintenance of TEC progenitors. These results support a model in which an imbalance of activin A and BMP4 signaling underpins the degeneration of postnatal TEC maintenance during aging, and its reversal enables the transient replenishment of mature TECs.

Cumulin and FSH Cooperate to Regulate Inhibin B and Activin B Production by Human Granulosa-Lutein Cells In Vitro.

The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally, and it is hypothesized that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine whether cumulin regulates granulosa cell inhibin and activin production and whether this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from patients undergoing in vitro fertilization were cultured with or without FSH with various forms of recombinant cumulin (native and cysteine mutants, with or without the prodomains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature forms and proforms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin with or without FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced regulation of inhibin B and activin B. The prodomains of cumulin may have a minimal role in its actions on granulosa cells.