RESUMO
PURPOSE: Germ cell neoplasia in situ (GCNis), the precursor of adult testicular germ cell tumours (GCTs), is found in 5-6% of contralateral testicles in patients with testicular GCT and in the tumour-surrounding tissue of >â¯90% of testes undergoing testis-sparing surgery (TSS) for GCT. Local radiotherapy to the testis with 18-20â¯Gy eradicates GCNis while preserving Leydig cells. The frequency of treatment failures is so far unknown. METHODS: A 22-year-old patient with right-sided seminoma clinical stage I and contralateral GCNis received radiotherapy with 18â¯Gy to his left testicle. Fifteen years later he underwent orchiectomy of the irradiated testis for seminoma with adjacent GCNis. The patient is well 1 year postoperatively while on testosterone-replacement therapy. The literature was searched for further cases with GCTs arising despite local radiotherapy. RESULTS: Six failures of radiotherapy have been reported previously. An estimated total number of 200 and 100 radiotherapeutic regimens with 18-20â¯Gy applied to cases with contralateral GCNis and with TSS, respectively, are documented in the literature. CONCLUSION: Cumulative experience suggests that radiotherapy with 18-20â¯Gy to the testis may fail with an estimated frequency of around 1%. Reasons for failure are elusive. A primary radioresistant subfraction of GCNis is hypothesized as well as technical failures regarding application of the radiotherapeutic dose volume in small and mobile testes. Caregivers of patients with TSS and contralateral GCNis should be aware of local relapses occurring after intervals of >â¯10 years.
Assuntos
Neoplasias Embrionárias de Células Germinativas , Seminoma , Neoplasias Testiculares , Adulto , Masculino , Humanos , Adulto Jovem , Seminoma/radioterapia , Seminoma/cirurgia , Recidiva Local de Neoplasia , Neoplasias Testiculares/radioterapia , Neoplasias Testiculares/cirurgia , Neoplasias Testiculares/patologia , Neoplasias Embrionárias de Células Germinativas/radioterapia , Neoplasias Embrionárias de Células Germinativas/cirurgiaRESUMO
STUDY QUESTION: Is it possible to induce in vitro reorganization of primary human testis cells from testicular sperm extraction (TESE) biopsies, maintain their long-term cultivation in a 2D system and identify cellular compositions? SUMMARY ANSWER: In vitro reorganization of primary human testis cells from TESE biopsies and their long-term cultivation on uncoated cell culture dishes is feasible and the cellular compositions can be uncovered through gene expression and microscopic analyses. WHAT IS KNOWN ALREADY: It has been shown in the rodent model that mixtures of testicular cell types are able to reassemble into clusters when cultivated on different kinds of surfaces or three-dimensional matrices. Two recent publications demonstrated the ability of primary human testicular cells to assemble into testicular organoids and their cultivation for a period of 3-4 weeks. STUDY DESIGN SIZE, DURATION: Primary human testis cells from TESE biopsies from 16 patients were reorganized in vitro and the clusters were cultivated long term on uncoated cell culture dishes, providing a solid ground for in vitro spermatogenesis. Gene expression analysis as well as fluorescence/transmission electron microscopy (TEM) were employed to uncover the cellular composition of the clusters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsies from adult, normogonadotropic patients displaying full spermatogenesis (n = 11), hypospermatogenesis (n = 2), predominantly full spermatogenesis with some hypospermatogenic tubules (n = 1), meiotic arrest (n = 1) or mixed atrophy (n = 1) were enzymatically digested and dispersed cells were cultivated on 96-well plates or chamber dishes as aggregate-free cell suspensions. Time-lapse imaging of cluster formation was performed over a period of 48 h. For receptor tyrosine kinase inhibition of cluster formation, cells were treated twice with K252a within 2-3 days. Immunofluorescence staining and confocal microscopy was carried out on clusters after 1-3 weeks of cultivation to identify the presence of Sertoli cells (SC) (SOX9), peritubular myoid cells (SMA), Leydig cells (LC) (STAR), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Single clusters from four patients and a pool of eight larger clusters from another patient were manually picked and subjected to quantitative real-time PCR to evaluate the presence of SC (SOX9, AR), LC (INSL3, STAR, HSD3B1), peritubular myoid cells (ACTA2), fibroblasts (FSP1), endothelial cells (CD34), macrophages (CD68), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Finally, an ultrastructural investigation was conducted based on TEM of clusters from six different patients, among them 3-month cultivated large clusters from two patients. MAIN RESULTS AND THE ROLE OF CHANCE: Quantitative PCR-based analysis of single-picked testicular cell clusters identified SC, peritubular myoid cells, endothelial cells, fibroblasts, macrophages, spermatids and LC after 1, 2 or 3 weeks or 3 months of cultivation. Immunofluorescence positivity for SC and peritubular myoid cells corroborated the presence of these two kinds of testis niche cells. In addition, round as well as elongated spermatids were frequently encountered in 1 and 2 weeks old clusters. Transmission electron microscopical classification confirmed all these cell types together with a few spermatogonia. Macrophages were found to be of the proinflammatory M1 subtype, as revealed by CD68+/CD163-/IL6+ expression. Time-lapse imaging uncovered the specific dynamics of cluster fusion and enlargement, which could be prevented by addition of protein kinase inhibitor K252a. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Cell composition of the clusters varied based on the spermatogenic state of the TESE patient. Although spermatids could be observed with all applied methods, spermatogonia were only detected by TEM in single cases. Hence, a direct maintenance of these germ cell types by our system in its current state cannot be postulated. Moreover, putative dedifferentiation and malignant degeneration of cells in long-term cluster cultivation needs to be investigated in the future. WIDER IMPLICATIONS OF THE FINDINGS: This work demonstrates that the reorganization of testicular cells can be achieved with TESE biopsies obtained from men enroled in a standard clinical assisted reproduction program. The formed clusters can be cultivated for at least 3 months and are composed, to a large extent, of the most important somatic cell types that are essential to support spermatogenesis. These findings may provide the cellular basis for advances in human in vitro spermatogenesis and/or the possibility for propagation of spermatogonia within a natural stem cell niche-like environment. STUDY FUNDING AND COMPETING INTERESTS: The project was funded by a DFG grant to K.v.K. (KO 4769/2-1). The authors declare they have no conflicts of interest.
Assuntos
Espermatogônias/metabolismo , Testículo/metabolismo , Biópsia , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Humanos , Masculino , Reação em Cadeia da Polimerase , Protaminas/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Espermatogônias/citologia , Espermatozoides/citologia , Espermatozoides/fisiologia , Testículo/citologiaRESUMO
OBJECTIVE: Mutations in the cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), serine protease inhibitor Kazal type 1 (SPINK1), and chymotrypsin C (CTRC) genes are associated with an elevated risk for chronic pancreatitis, which is a known risk factor for pancreatic cancer (PC). Therefore, we analyzed whether PRSS1, CFTR, SPINK1, and/or CTRC mutations are associated with pancreatic adenocarcinoma. METHODS: The study cohort was composed of 121 PC patients, of whom 74 were classified as having chronic pancreatitis, 102 patients with idiopathic chronic pancreatitis, and 130 as healthy controls. Mutation analyses for the CFTR, SPINK1, PRSS1, and CTRC genes were performed for the presence of the most common mutations. RESULTS: The frequency of CFTR mutations in patients with PC was not significantly different in comparison with healthy controls and controls with pancreatitis. The SPINK1 mutation frequency was significantly decreased in patients with PC in comparison with patients with idiopathic pancreatitis but varied not significantly in comparison with healthy controls. None of the selected 121 PC samples showed a pancreatitis-predisposing mutation in the PRSS1 or CTRC gene. CONCLUSIONS: Mutations in the genes CFTR, SPINK1, PRSS1, and CTRC do not seem to significantly increase the risk for pancreatic adenocarcinoma.
Assuntos
Carcinoma Ductal Pancreático/genética , Proteínas de Transporte/genética , Quimotripsina/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Tripsina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Carcinoma Ductal Pancreático/epidemiologia , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Predisposição Genética para Doença , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/epidemiologia , Pancreatite Crônica/epidemiologia , Pancreatite Crônica/genética , Lesões Pré-Cancerosas/epidemiologia , Lesões Pré-Cancerosas/genética , Estudos Retrospectivos , Inibidor da Tripsina Pancreática de KazalRESUMO
Human spermatogonia (Spg) and their fetal precursors express fibroblast growth factor receptor 3 (FGFR3). To further elucidate the role of FGFR3 in the control of Spg self-renewal, proliferation, and/or differentiation, and to narrow down the FGFR3-positive cell type(s) in the normal adult human testis, tissue sections and whole mount preparations of seminiferous tubules were analyzed combining immunofluorescence and confocal fluorescence microscopy. FGFR3 protein was chiefly observed in cellular membranes and cytoplasmic vesicles of a subpopulation of type A Spg, which comprised the chromatin rarefaction zone-containing type A(dark). Cytoplasmic expression of FGFR3 and nuclear expression of proliferation-associated antigen KI-67 were mutually exclusive. Similarly, FGFR3-positive Spg were negative for Doublesex and Mab-3 related transcription factor 1 (DMRT1). By contrast, undifferentiated embryonic cell transcription factor 1 (UTF1) and survival time-associated PHD finger in ovarian cancer 1 protein (SPOC1) were co-expressed in the nuclei of FGFR3-positive Spg. Whole mounted seminiferous tubules illustrated the clonogenic arrangement of the FGFR3/UTF1 double-positive Spg, which mainly occurred as pairs or quadruplets and, different from the KIT-positive Spg, showed no overlap with KI-67 labeled clusters. Taken together, in the adult human testis, FGFR3 expression is a feature of small clones of rarely dividing type A Spg which resemble "undifferentiated" Spg, including the spermatogonial stem cells.
Assuntos
Divisão Celular , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/biossíntese , Espermatogônias/metabolismo , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Imunofluorescência , Humanos , Antígeno Ki-67/biossíntese , Masculino , Microscopia Confocal , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas c-kit/análise , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/análise , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Espermatogênese/fisiologia , Testículo/citologia , Testículo/metabolismo , Transativadores/biossíntese , Fatores de Transcrição/análise , Fatores de Transcrição/biossínteseRESUMO
It is unclear whether the distinct nuclear morphologies of human A(dark) (Ad) and A(pale) (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear 'vacuolar spaces' representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex- and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing.
Assuntos
Espermatogônias/citologia , Espermatogônias/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Espermatogênese , Espermatogônias/classificação , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Y-chromosomal microdeletions (YCMD) are the major genetic cause of male infertility. To date, it is not known which global changes are induced by the presence of AZFc or AZFb+c deletions in the human testicular transcriptome. We investigated this question by microarray analysis in which we had to eliminate the 'germ cell effect', i.e., the dominating effect of germ cell transcripts due to the quantitative difference in germ cell composition in samples with/without YCMD. This problem was tackled by selecting 26 samples from an initial cohort of 34 samples by their homogeneity in respect to cellular composition as obtained from gene expression clustering. This way, the 'germ cell effect' was minimized, and a distinct 'deletion effect' became more apparent. Several hundred genes are influenced by YCMD as shown on the three different phenotypes hypospermatogenesis, meiotic arrest, and Sertoli-cell only syndrome. We validated on an independent cohort of samples five genes by quantitative real-time PCR that are expressed in germ cells or the somatic compartment and which are exclusively altered by the presence of YCMD. We conclude that the deletion of Y-chromosomal genes has a significant effect on spermatogenesis by modulating the transcriptional network of the germ cell and somatic compartment.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Y/genética , Perfilação da Expressão Gênica , Testículo/metabolismo , Biópsia , Análise por Conglomerados , Estudos de Coortes , Loci Gênicos , Células Germinativas/metabolismo , Células Germinativas/patologia , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Meiose/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plasma Seminal/genética , Síndrome de Células de Sertoli/genética , Espermatogênese/genética , Testículo/patologiaRESUMO
BACKGROUND: A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type. METHODS: The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets. RESULTS: Highly significant differences in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast growth factor receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-1 (CTAG1A/B), undifferentiated embryonic cell transcription factor 1 (UTF1) and synaptosomal-associated protein, 91 kDa homolog (SNAP91) were shown to represent specific biomarkers of human spermatogonia. CONCLUSIONS: These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation.