RESUMO
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.
Assuntos
Neoplasias Embrionárias de Células Germinativas , Linfócitos T Reguladores , Neoplasias Testiculares , Microambiente Tumoral , Humanos , Neoplasias Testiculares/imunologia , Neoplasias Testiculares/patologia , Masculino , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Embrionárias de Células Germinativas/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Linfócitos T CD8-Positivos/imunologia , Análise de Célula Única , Testículo/patologia , Testículo/imunologia , AdultoRESUMO
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.
RESUMO
Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.
Assuntos
Infertilidade Masculina/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Epididimo/imunologia , Epididimo/metabolismo , Infertilidade Masculina/metabolismo , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Testículo/imunologia , Testículo/metabolismoRESUMO
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.
Assuntos
Orquite , Masculino , Humanos , Camundongos , Animais , Folistatina , Fibronectinas , Macrófagos , Fibrose , Inflamação , Receptores CCR2/genéticaRESUMO
BACKGROUND: Region-specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll-like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region-specific inflammatory responses to bacterial-derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR-associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. OBJECTIVES: To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. MATERIAL AND METHODS: Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC-treated animals and 6 and 24 h for LPS-/LTA-treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT-qPCR and Western blot assays were conducted. RESULTS: UPEC infection up-regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS- and LTA-induced up-regulation of TLR-associated signaling transcripts in the cauda epididymidis and initial segment, respectively. CONCLUSION: The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial-derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR-associated signaling pathways may contribute to fine-tuning inflammatory responses along the epididymis.
Assuntos
Epididimite , Lipopolissacarídeos , Transdução de Sinais , Ácidos Teicoicos , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Animais , Masculino , Epididimite/genética , Epididimite/metabolismo , Epididimite/microbiologia , Camundongos , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Ácidos Teicoicos/farmacologia , Escherichia coli Uropatogênica , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/genética , Receptor 6 Toll-Like/genética , Receptor 6 Toll-Like/metabolismo , Epididimo/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Camundongos Endogâmicos C57BL , Doença AgudaRESUMO
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.
Assuntos
Folistatina , Testículo , Ativinas , Animais , Proteínas de Transporte/metabolismo , Folistatina/genética , Folistatina/metabolismo , Humanos , Macrófagos/metabolismo , Masculino , CamundongosRESUMO
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.
Assuntos
Epididimo , Sêmen , Camundongos , Masculino , Animais , Maturação do Esperma , Espermatozoides , TestículoRESUMO
The epididymis is a tubular structure connecting the vas deferens to the testis. This organ consists of three main regions-caput, corpus, and cauda-that face opposing immunological tasks. A means of combating invading pathogens is required in the distally located cauda, where there is a risk of ascending bacterial infections originating from the urethra. Meanwhile, immune tolerance is necessary at the caput, where spermatozoa with immunogenic neo-antigens originate from the testis. Consistently, when challenged with live bacteria or inflammatory stimuli, the cauda elicits a much stronger immune response and inflammatory-inflicted damage than the caput. At the cellular level, a role for diverse and strategically positioned mononuclear phagocytes is emerging. At the mechanistic level, differential expression of immunoprotective and immunomodulatory mediators has been detected between the three main regions of the epididymis. In this review, we summarize the current state of knowledge about region-specific immunological characteristics and unveil possible underlying mechanisms on cellular and molecular levels. Improved understanding of the different immunological microenvironments is the basis for an improved therapy and counseling of patients with epididymal infections.
Assuntos
Infecções Bacterianas , Epididimite , Doença Aguda , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Infecções Bacterianas/terapia , Epididimo/imunologia , Epididimo/microbiologia , Epididimo/patologia , Epididimite/imunologia , Epididimite/microbiologia , Epididimite/patologia , Epididimite/terapia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Inflamação/terapia , MasculinoRESUMO
BACKGROUND: The precise movement of proteins and vesicles is an essential ability for all eukaryotic cells. Nowhere is this more evident than during the remarkable transformation that occurs in spermiogenesis-the transformation of haploid round spermatids into sperm. These transformations are critically dependent upon both the microtubule and the actin cytoskeleton, and defects in these processes are thought to underpin a significant percentage of human male infertility. OBJECTIVE AND RATIONALE: This review is aimed at summarising and synthesising the current state of knowledge around protein/vesicle transport during haploid male germ cell development and identifying knowledge gaps and challenges for future research. To achieve this, we summarise the key discoveries related to protein transport using the mouse as a model system. Where relevant, we anchored these insights to knowledge in the field of human spermiogenesis and the causality of human male infertility. SEARCH METHODS: Relevant studies published in English were identified using PubMed using a range of search terms related to the core focus of the review-protein/vesicle transport, intra-flagellar transport, intra-manchette transport, Golgi, acrosome, manchette, axoneme, outer dense fibres and fibrous sheath. Searches were not restricted to a particular time frame or species although the emphasis within the review is on mammalian spermiogenesis. OUTCOMES: Spermiogenesis is the final phase of sperm development. It results in the transformation of a round cell into a highly polarised sperm with the capacity for fertility. It is critically dependent on the cytoskeleton and its ability to transport protein complexes and vesicles over long distances and often between distinct cytoplasmic compartments. The development of the acrosome covering the sperm head, the sperm tail within the ciliary lobe, the manchette and its role in sperm head shaping and protein transport into the tail, and the assembly of mitochondria into the mid-piece of sperm, may all be viewed as a series of overlapping and interconnected train tracks. Defects in this redistribution network lead to male infertility characterised by abnormal sperm morphology (teratozoospermia) and/or abnormal sperm motility (asthenozoospermia) and are likely to be causal of, or contribute to, a significant percentage of human male infertility. WIDER IMPLICATIONS: A greater understanding of the mechanisms of protein transport in spermiogenesis offers the potential to precisely diagnose cases of male infertility and to forecast implications for children conceived using gametes containing these mutations. The manipulation of these processes will offer opportunities for male-based contraceptive development. Further, as increasingly evidenced in the literature, we believe that the continuous and spatiotemporally restrained nature of spermiogenesis provides an outstanding model system to identify, and de-code, cytoskeletal elements and transport mechanisms of relevance to multiple tissues.
Assuntos
Haploidia , Transporte Proteico/fisiologia , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Animais , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Microtúbulos/metabolismo , Espermátides/metabolismo , Espermatogênese/genética , Espermatozoides/fisiologiaRESUMO
Infection and inflammation of the male reproductive tract are relevant causes of infertility. Inflammatory damage occurs in the special immunosuppressive microenvironment of the testis, a hallmark termed testicular immune privilege, which allows tolerance to neo-antigens from developing germ cells appearing at puberty, long after the establishment of systemic immune tolerance. Experimental autoimmune orchitis (EAO) is a well-established rodent model of chronic testicular inflammation and organ specific autoimmunity that offers a valuable in vivo tool to investigate the pathological and molecular mechanisms leading to the breakdown of the testicular immune privilege. The disease is characterized by the infiltration of the interstitium by immune cells (mainly macrophages, dendritic cells, and T cells), formation of autoantibodies against testicular antigens, production of pro-inflammatory mediators such as NO, MCP1, TNFα, IL6, or activins and dysregulation of steroidogenesis with reduced levels of serum testosterone. EAO leads to sloughing of germ cells, atrophic seminiferous tubules and fibrotic remodeling, parameters all found similarly to changes in human biopsies from infertile patients with inflammatory infiltrates. Interestingly, testosterone supplementation during the course of EAO leads to expansion of the regulatory T cell population and inhibition of disease development. Knowledge of EAO pathogenesis aims to contribute to a better understanding of human testicular autoimmune disease as an essential prerequisite for improved diagnosis and treatment.
Assuntos
Doenças Autoimunes/imunologia , Orquite/imunologia , Animais , Humanos , MasculinoRESUMO
Ciliated bronchial epithelium 1 (CBE1) is a microtubule-associated protein localized to the manchette and developing flagellum during spermiogenesis and is associated with sperm maturation arrest in humans. It was hypothesized that CBE1 functions in microtubule-mediated transport mechanisms and sperm tail formation. To test this hypothesis, we analyzed Cbe1 expression and localization during spermiogenesis, and in mouse inner medullary collecting duct-3 (IMCD3) cells as a model of ciliogenesis. Furthermore, we generated and analyzed the fertility of a Cbe1 mutant mouse line. Mice containing a homozygous deletion in the long forms of Cbe1 were born at a lower frequency than predicted by Mendelian inheritance; however, adult male mice were fertile. An in-depth analysis of the Cbe1 gene revealed alternative transcript variants, which were not affected by the exon 2 mutation. To assess whether short variants compensate for the loss of long variants, exons 2 and 4 (which affect all variants) were individually mutated in IMCD3 cells and the effects on cell proliferation and ciliogenesis were analyzed. In wild-type IMCD3 cells, both variants were upregulated during cilia assembly. CBE1 protein was not a structural component of cilia; rather, CBE1 localized to the mitochondria and the contractile ring of dividing IMCD3 cells. Although IMCD3 cells carrying the mutation in long variants showed no phenotypic alterations, the mutation in exon 4 resulted in a significantly decreased proliferation rate. This study reveals that long isoforms of CBE1 are not essential for male fertility. Data, however, suggest that CBE1 is associated with intramanchette transport and midpiece formation of the sperm tail.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Espermátides/metabolismo , Espermatogênese , Animais , Divisão Celular , Linhagem Celular , Proteínas do Citoesqueleto/genética , Fertilidade , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
OBJECTIVE: To define the precise cellular localization of ciliated bronchial epithelium 1 (CBE1) in the human testis and test its relationship to impaired spermatogenesis. DESIGN: Gene expression analysis, and histologic and immunohistochemical evaluation. SETTING: University research laboratories and andrologic outpatient clinic. PATIENT(S): Forty-three human testicular biopsies: 12 biopsies showing normal spermatogenesis (NSP), 8 with maturation arrest at level of spermatocytes (STA), 8 with maturation arrest at level of spermatids (SDA), 4 with scattered elongating spermatids, and 12 with Sertoli cell-only syndrome, with an additional 5 semen samples from healthy donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Evaluation of CBE1 expression in normal as well as impaired spermatogenesis on mRNA (quantitative reverse-transcription polymerase chain reaction and in situ hybridization) and protein level (immunohistochemistry, Western blot analysis). RESULT(S): In normal spermatogenesis, CBE1 mRNA was expressed in late pachytene spermatocytes, and the protein was localized within the flagellum of elongating spermatids from stage V up to the spermiation in stage II. Immunoelectron microscopy showed CBE1 clearly associated with microtubules at the manchette, the head-tail coupling apparatus, and the flagellum, but the protein was absent in spermatozoa. Compared with normal spermatogenesis, CBE1 mRNA was statistically significantly reduced in samples with a maturation arrest at the level of round spermatids and primary spermatocytes, and was absent in samples showing Sertoli cell-only syndrome. CBE1 protein was completely missing in SDA samples showing few elongating spermatids. CONCLUSION(S): Our data strongly suggest an influence of CBE1 in ciliogenesis in spermatids due to the localization at the microtubules of the elongating spermatids, indicating a role in the intramanchette and/or intraflagellar transport mechanism. The absence of CBE1 in spermatozoa suggests that CBE1 is important for the spermatid development but not for the maintenance of mature spermatozoa as a component of the flagellum.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Infertilidade Masculina/metabolismo , Proteínas Nucleares/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Testículo/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Infertilidade Masculina/patologia , Masculino , Especificidade de Órgãos/fisiologia , Espermatozoides/patologia , Testículo/patologia , Distribuição TecidualRESUMO
OBJECTIVE: To define the stage-by-stage expression of KATNB1 during human spermatogenesis. DESIGN: Gene expression analysis, histologic and immunohistochemical evaluation. SETTING: University research laboratories and andrological clinic. PATIENT(S): Eighty human testicular biopsy samples: 43 showing normal spermatogenesis, 9 with maturation arrest at level of spermatocytes, 8 with maturation arrest at level of spermatogonia, and 20 with a Sertoli cell only syndrome. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Evaluation of katanin p80 expression in normal as well as impaired spermatogenesis on mRNA (RT-PCR, RT-qPCR, and in situ hybridization) and protein level (immunohistochemistry/immunofluorescence). RESULT(S): KATNB1 messenger RNA is exclusively expressed in germ cells, and quantitatively reduced in maturation arrests at the level of spermatogonia. The KATNB1 protein was detected in type B spermatogonia entering meiosis and in the Golgi complex of pachytene spermatocytes. Immediately before the first meiotic division, it is colocalized with the cleaving centriole. It was also detected in early round spermatids in the dictyosome. CONCLUSION(S): The expression and localization of KATNB1 support a role in spindle formation. The localization of KATNB1 in early round spermatids suggests an involvement in the formation of microtubule-based structures during spermiogenesis (manchette and flagellum). These data are consistent with the demonstrated role of KATNB1 in mouse meiosis, nuclear shaping, and flagellum formation of sperm and suggest the strong conservation of function even between distantly related species.