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1.
BMC Cancer ; 20(1): 349, 2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32326899

RESUMO

BACKGROUND: Testicular germ cell tumours (TGCTs) are characterised by an overall high cisplatin-sensitivity which has been linked to their continued expression of pluripotency factors. Recently, the Nodal signalling pathway has been implicated in the regulation of pluripotency factor expression in fetal germ cells, and the pathway could therefore also be involved in regulating expression of pluripotency factors in malignant germ cells, and hence cisplatin-sensitivity in TGCTs. METHODS: We used in vitro culture of the TGCT-derived cell line NTera2, ex vivo tissue culture of primary TGCT specimens and xenografting of NTera2 cells into nude mice in order to investigate the consequences of manipulating Nodal and Activin signalling on pluripotency factor expression, apoptosis, proliferation and cisplatin-sensitivity. RESULTS: The Nodal signalling factors were markedly expressed concomitantly with the pluripotency factor OCT4 in GCNIS cells, seminomas and embryonal carcinomas. Despite this, inhibition of Nodal and Activin signalling either alone or simultaneously did not affect proliferation or apoptosis in malignant germ cells in vitro or ex vivo. Interestingly, inhibition of Nodal signalling in vitro reduced the expression of pluripotency factors and Nodal pathway genes, while stimulation of the pathway increased their expression. However, cisplatin-sensitivity was not affected following pharmacological inhibition of Nodal/Activin signalling or siRNA-mediated knockdown of the obligate co-receptor CRIPTO in NTera2 cells in vitro or in a xenograft model. CONCLUSION: Our findings suggest that the Nodal signalling pathway may be involved in regulating pluripotency factor expression in malignant germ cells, but manipulation of the pathway does not appear to affect cisplatin-sensitivity or tumour cell proliferation.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Linfonodos/patologia , Neoplasias Embrionárias de Células Germinativas/patologia , Células-Tronco Pluripotentes/patologia , Neoplasias Testiculares/patologia , Animais , Proliferação de Células , Humanos , Linfonodos/efeitos dos fármacos , Masculino , Camundongos , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais , Neoplasias Testiculares/tratamento farmacológico , Células Tumorais Cultivadas
3.
Andrology ; 6(5): 748-755, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29981219

RESUMO

A simple histological method to evaluate the Leydig cell compartment is lacking. We aimed to establish such a method and to investigate if Leydig cell hyperplasia of the biopsy contralateral to the tumour-bearing testicle in patients with testicular germ cell cancer is associated with biochemical signs of Leydig cell dysfunction after long-term follow-up. A case group of 50 long-term testicular germ cell cancer survivors without human chorionic gonadotropin elevation, 10 testicular germ cell cancer patients with elevated human chorionic gonadotropin and 10 controls without testicular malignancy were included. For each subject, 2-4 representative sections from their testicular biopsies were selected for analysis. Using the image processing program ImageJ (V.1.48, NIH), an area with a minimum of 50 tubules was selected and delineated (total selected area) and the total Leydig cell area was calculated by adding up every delineated Leydig cell group within the total selected area. Four different methods were tested for the ability to quantify the Leydig cell compartment. In the 50 testicular germ cell cancer survivors, associations between the area of the Leydig cell compartment and serum levels of testosterone and luteinising hormone were investigated using linear regression analysis. The Leydig cell compartment was best quantified by the total Leydig cell area/total selected area index, which was significantly larger in the human chorionic gonadotropin-positive patients than in controls (P = 0.00001). In the 50 human chorionic gonadotropin-negative testicular germ cell cancer survivors, increasing total Leydig cell area/total selected area was significantly associated with decreased levels of total testosterone and decreased total testosterone/luteinising hormone ratio after a median of 9-year follow-up. In conclusion, a new simple method, total Leydig cell area/total selected area, was established to estimate the Leydig cell compartment in testicular biopsies. The index identified Leydig cell hyperplasia in the contralateral biopsy in patients with testicular germ cell cancer, and it was associated with long-term biochemical Leydig cell dysfunction. Although in testicular germ cell cancer survivors, the clinical value is limited because the contralateral biopsies are not commonly available, we propose a closer andrological follow-up in any patient with an increased total Leydig cell area/total selected area index.


Assuntos
Biópsia/métodos , Sobreviventes de Câncer , Células Intersticiais do Testículo/patologia , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/patologia , Adulto , Humanos , Hiperplasia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
4.
Andrology ; 6(1): 176-183, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29179257

RESUMO

Testicular germ cell cancer (TGCC) is derived from germ cell neoplasia in situ (GCNIS), which arises due to niche disturbances affecting the Sertoli cells. It is believed that exogenous endocrine factors have a crucial role in governing neoplastic transformation but on a strong hereditary background. Follicle-stimulating hormone (FSH) is the major regulatory hormone of the Sertoli cells. FSH signalling-related single-nucleotide polymorphisms (SNPs) have previously been shown to affect FSH action in men at different levels. We aimed to investigate whether three FSH-related SNPs (FSHR 2039A>G, FSHR -29G>A and FSHB -211G>T) are associated with development of TGCC. A total of 752 Danish and German patients with TGCC from two tertiary andrological referral centres were included. Three control groups comprising 2020 men from the general population, 679 fertile men and 417 infertile men, were also included. Chi-squared test was performed to compare genotype- and allele frequencies. Kruskal-Wallis test was performed to compare age at diagnosis. Patients with TGCC had a higher frequency of the A-allele of FSHR 2039A>G compared to the group of fertile men with an AA-genotype frequency of 30.2% vs. 22.0%, respectively, p = 0.002. This variant is associated with higher FSH receptor activity. The distribution of the FSHR 2039A>G did not differ significantly between the patients with TGCC and the infertile or the general population. The frequency of the two other SNPs did not differ between patient with TGCC and any of the control groups. No differences were detected between genotypes and age distribution or histological subtype of the tumours. In conclusion, we observed that a genetic variant associated with FSHR activity may modulate the susceptibility to TGCC.


Assuntos
Predisposição Genética para Doença/genética , Neoplasias Embrionárias de Células Germinativas/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores do FSH/genética , Neoplasias Testiculares/genética , Adolescente , Adulto , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
5.
Hum Reprod ; 32(11): 2332-2339, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28927238

RESUMO

STUDY QUESTION: Is the thrombophilia mutation factor V Leiden (FVL) associated with an increased total sperm count? SUMMARY ANSWER: Carriers of FVL have a higher total sperm count than non-FVL-carriers, which could not be explained by genetic linkage or by observations in a FVL-mouse model. WHAT IS KNOWN ALREADY: FVL has a high prevalence in Caucasians despite detrimental health effects. Carriers have been shown to have higher fecundity, which might partly explain this evolutionary paradox. STUDY DESIGN, SIZE, DURATION: We determined FVL status in two cohorts (Dutch, n = 627; Danish, n = 854) of consecutively included men without known causes for spermatogenic failure, and performed an individual patient data meta-analysis of these two cohorts together with one previously published (Dutch, n = 908) cohort. We explored possible biological underpinnings for the relation between sperm count and FVL, by use of a FVL-mouse model and investigations of genetic linkage. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were male partners of subfertile couples (two Dutch cohorts) and young men from the general population (Danish cohort): FVL carrier rate was 4.0%, 4.6% and 7.3%, respectively. There were differences in smoking, abstinence time and age between the cohorts. We corrected for these in the primary analysis, which consisted of a mixed linear effects model, also incorporating unobjectified population differences. In public haplotype data from subjects of European descent, we explored linkage disequilibrium of FVL with all known single nucleotide polymorphisms in a 1.5 MB region around the F5 gene with an R2 cutoff of 0.8. We sequenced exons of four candidate genes hypothesized to be linked to FVL in a subgroup of FVL carriers with extreme sperm count values. The animal studies consisted of never mated 15-18-week-old C57BL/J6 mice heterozygous and homozygous for FVL and wild-type mice. We compared spermatogenesis parameters (normalized internal genitalia weights, epididymis sperm content and sperm motility) between FVL and wild-type mice. MAIN RESULTS AND THE ROLE OF CHANCE: Human FVL carriers have a higher total sperm count than non-carriers, with an adjusted mean difference of 31 × 106 (95%CI 0.2-61.7; P = 0.048). Mice with the FVL mutation do not have increased spermatogenesis as compared to wildtype mice. None of the studied polymorphisms was in linkage disequilibrium, either in the public databases or in a subgroup of FVL carriers with extremely high sperm counts. LIMITATIONS, REASONS FOR CAUTION: The difference in total sperm count would benefit from confirmation in other cohorts. The finding of higher count in carriers was consistent however, with no heterogeneity between the cohorts. The lack of effect of murine FVL might suggest there is no direct causality. The exploratory efforts on genetic linkage do not rule out that the association is a reflection of FVL co-inheritance with a non-studied causative polymorphism. WIDER IMPLICATIONS OF THE FINDINGS: A high sperm count in FVL-carrying males contributes to understanding the high prevalence of this otherwise disadvantageous mutation. The findings might provide directions for future research on male fertility. STUDY FUNDING/COMPETING INTEREST(S): No conflicts of interest. Research was conducted with funding from the Netherlands Organisation for Scientific Research (NWO, VIDI innovative research grant 016.126.364 awarded to S. Middeldorp). The Danish cohort was supported by the Innovation Fund Denmark (InnovationsFonden, grant no. 14-2013-4), The Danish Ministry of Health and the Danish Environmental Protection Agency. TRIAL REGISTRATION NUMBER: Not applicable.


Assuntos
Fator V/genética , Infertilidade Masculina/genética , Contagem de Espermatozoides , Motilidade dos Espermatozoides/genética , Adolescente , Adulto , Animais , Humanos , Masculino , Camundongos Endogâmicos C57BL , Análise do Sêmen , Adulto Jovem
8.
Ann Oncol ; 26(4): 737-742, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25542924

RESUMO

BACKGROUND: Screening programmes for contralateral carcinoma in situ (CIS) testis in patients with unilateral germ-cell cancer (GCC) have never been evaluated. We investigated the effect of screening for contralateral CIS in a large nation-wide, population-based study. PATIENTS AND METHODS: A contralateral single-site biopsy was offered to 4130 patients in whom GCC had been diagnosed in 1984-2007 (screened cohort); 462 patients in whom GCC was diagnosed in 1984-1988 comprised the unscreened cohort. Cases with CIS were offered radiotherapy. Initially CIS-negative biopsies in patients with metachronous GCC were revised according to today's standards. Risk for metachronous GCC was estimated using cumulative incidence and the Cox proportional hazards model. RESULTS: In the screened cohort, contralateral CIS was found in 181 (4.4%) patients. The cumulative incidence of metachronous GCC after 20 years was 1.9% in the screened cohort and 3.1% in the unscreened cohort (P = 0.097), hazard ratio (HR) for the unscreened cohort: 1.59 (P = 0.144). Expert revision with contemporary methodology of CIS-negative biopsy samples from patients with metachronous cancer revealed CIS in 17 out of 45 (38%) cases. Decreased risks for metachronous GCC were related to older age at diagnosis (HR 0.52 per 10 years, P < 0.001) and chemotherapy (HR 0.35, P = 0.002). Limitations include the small number of patients in the unscreened cohort and the retrospective study design. CONCLUSIONS: Our evaluation of a national population-based screening programme for contralateral CIS in patients with testicular cancer showed no significant difference in the risk for metachronous GCC between a screened and an unscreened cohort. Single-site biopsy including modern immunohistochemistry does not identify all cases of CIS.


Assuntos
Carcinoma in Situ/diagnóstico , Carcinoma in Situ/epidemiologia , Detecção Precoce de Câncer , Neoplasias Embrionárias de Células Germinativas/epidemiologia , Neoplasias Primárias Múltiplas/diagnóstico , Neoplasias Primárias Múltiplas/epidemiologia , Neoplasias Testiculares/epidemiologia , Adulto , Carcinoma in Situ/terapia , Estudos de Coortes , Terapia Combinada , Dinamarca/epidemiologia , Feminino , Seguimentos , Humanos , Incidência , Masculino , Estadiamento de Neoplasias , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Embrionárias de Células Germinativas/terapia , Neoplasias Primárias Múltiplas/terapia , Prognóstico , Medição de Risco , Neoplasias Testiculares/patologia , Neoplasias Testiculares/terapia
9.
Andrology ; 3(1): 19-26, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25538016

RESUMO

Basic research results can provide new ideas and hypotheses to be examined in epidemiological studies. We conducted a survey among testicular cancer researchers on hypotheses concerning the etiology of this malignancy. All researchers on the mailing list of Copenhagen Testis Cancer Workshops and corresponding authors of PubMed-indexed articles identified by the search term 'testicular cancer' and published within 10 years (in total 2750 recipients) were invited to respond to an e-mail-based survey. Participants of the 8th Copenhagen Testis Cancer Workshop in May 2014 were subsequently asked to rate the plausibility of the suggested etiologic hypotheses on a scale of 1 (very implausible) to 10 (very plausible). This report describes the methodology of the survey, the score distributions by individual hypotheses, hypothesis group, and the participants' major research fields, and discuss the hypotheses that scored as most plausible. We also present plans for improving the survey that may be repeated at a next international meeting of experts in testicular cancer. Overall 52 of 99 (53%) registered participants of the 8th Copenhagen Testis Cancer Workshop submitted the plausibility rating form. Fourteen of 27 hypotheses were related to exposures during pregnancy. Hypotheses with the highest mean plausibility ratings were either related to pre-natal exposures or exposures that might have an effect during pregnancy and in post-natal life. The results of the survey may be helpful for triggering more specific etiologic hypotheses that include factors related to endocrine disruption, DNA damage, inflammation, and nutrition during pregnancy. The survey results may stimulate a multidisciplinary discussion about new etiologic hypotheses of testicular cancer.


Assuntos
Pesquisadores/psicologia , Neoplasias Testiculares/etiologia , Consenso , Feminino , Humanos , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Medição de Risco , Fatores de Risco , Inquéritos e Questionários
10.
Mol Cell Endocrinol ; 399: 235-43, 2015 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-25260943

RESUMO

Regulation of spermatogonial maintenance in the human testis is currently not well understood. One pathway suggested to be involved is activated by fibroblast growth factor receptor 3 (FGFR3), which is expressed in a subset of spermatogonia. FGFR3-activating mutations have been identified in spermatocytic seminoma, thought to originate from clonal expansion of spermatogonia. In this study we aimed to characterize potential binding partners of FGFR3, and specifically its mesenchymal "c" splice isoform, in human spermatogonia. Based on expression patterns and homology to the binding site, we identified FGF1, FGF2, and FGF9 as the best candidates for natural ligands of FGFR3c in the testis. In addition, we screened non-FGF proteins and found that a proteoglycan biglycan (BGN) contains a sequence homologous to the FGFR3c binding site on FGF1, and is expressed in peritubular cells adjacent to FGFR3-expressing spermatogonia. Experiments in a cell-free system confirmed that BGN binds to FGFR3c and FGF1. In conclusion, our findings further clarify the complex regulation of FGFR3c in the human testis. We postulate that BGN is a factor secreted by peritubular cells to modulate FGFR3c signaling and thus contributes to the regulation of spermatogonial maintenance.


Assuntos
Biglicano/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Espermatogônias/metabolismo , Testículo/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Ligação Proteica , Isoformas de Proteínas/metabolismo , Espermatogônias/citologia , Testículo/citologia
11.
Hum Reprod ; 29(8): 1637-50, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24908673

RESUMO

STUDY QUESTION: What is the differentiation stage of human testicular interstitial cells, in particular Leydig cells (LC), within micronodules found in patients with infertility, testicular cancer and Klinefelter syndrome? SUMMARY ANSWER: The Leydig- and peritubular-cell populations in testes with dysgenesis contain an increased proportion of undifferentiated cells when compared with control samples, as demonstrated by increased delta-like homolog 1 (DLK1) and decreased insulin-like peptide 3 (INSL3) expression. WHAT IS KNOWN ALREADY: Normal LC function is essential for male development and reproduction. Signs of LC failure, including LC micronodules, are often observed in patients with reproductive disorders. STUDY DESIGN, SIZE, PARTICIPANTS: In this retrospective study, a panel of markers and factors linked to the differentiation of LCs was investigated in 33 fetal and prepubertal human specimens and in 58 adult testis samples from patients with testicular germ cell tumours, including precursor carcinoma in situ (CIS), infertility or Klinefelter syndrome. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression patterns of DLK1, INSL3, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and smooth muscle actin (SMA) were investigated by immunohistochemistry and quantitative RT-PCR. The percentage of positive LCs was estimated and correlated to total LC numbers and serum levels of reproductive hormones. MAIN RESULTS AND THE ROLE OF CHANCE: DLK1, INSL3 and COUP-TFII expression changed during normal development and was linked to different stages of LC differentiation: DLK1 was expressed in all fetal LCs, but only in spindle-shaped progenitor cells and in a small subset of polygonal LCs in the normal adult testis; INSL3 was expressed in a subset of fetal LCs, but in the majority of adult LCs; and COUP-TFII was expressed in peritubular and mesenchymal stroma cells at all ages, in fetal LCs early in gestation and in a subset of adult LCs. CYP11A1 was expressed in the majority of LCs regardless of age and pathology and was the best general LC marker examined here. SMA was weakly expressed in peritubular cells in the fetal and infantile testis, but strongly expressed in the adult testis. In pathological testes, the numbers of DLK1-positive interstitial cells were increased. The proportion of DLK1-positive LCs correlated with total LC numbers (R = 0.53; P < 0.001) and was higher in testis with enlargement of the peritubular layers (P < 0.01), which was also highly associated with DLK1 expression in the peritubular compartment (P < 0.001). INSL3 expression was absent in some, but not all LC micronodules, and in the majority of LCs, it was mutually exclusive of DLK1. LIMITATIONS, REASONS FOR CAUTION: The number of samples was relatively small and no true normal adult controls were available. True stereology was not used for LC counting, instead LCs were counted in three fields of 0.5 µm(2) surface for each sample. WIDER IMPLICATIONS OF THE FINDINGS: The population of LCs, especially those clustered in large nodules, are heterogeneous and comprise cells at different stages of differentiation. The study demonstrated that the differentiation and function of LCs, and possibly also peritubular cells, are impaired in adult men with testicular pathologies including testis cancer and Klinefelter syndrome. STUDY FUNDING/COMPETING INTERESTS: This work was funded by Rigshospitalet's research funds, the Danish Cancer Society and Kirsten and Freddy Johansen's foundation. The authors have no conflicts of interest.


Assuntos
Diferenciação Celular , Insulina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Intersticiais do Testículo/citologia , Proteínas de Membrana/genética , Proteínas/genética , Doenças Testiculares/patologia , Actinas/genética , Actinas/metabolismo , Adolescente , Adulto , Fator II de Transcrição COUP/genética , Fator II de Transcrição COUP/metabolismo , Proteínas de Ligação ao Cálcio , Criança , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Regulação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Síndrome de Klinefelter/genética , Síndrome de Klinefelter/metabolismo , Síndrome de Klinefelter/patologia , Células Intersticiais do Testículo/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Estudos Retrospectivos , Doenças Testiculares/genética , Doenças Testiculares/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia
12.
Br J Cancer ; 110(10): 2604-14, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24781282

RESUMO

BACKGROUND: Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. METHODS: Human testis and testis cancer specimens from orchidectomies were cultured in 'hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. RESULTS: Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. CONCLUSIONS: Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche.


Assuntos
Ativinas/farmacologia , Técnicas de Cultura de Células , Folistatina/farmacologia , Seminoma/patologia , Neoplasias Testiculares/patologia , Testículo/citologia , Adulto , Antígenos de Neoplasias/análise , Apoptose/efeitos dos fármacos , Carcinoma in Situ/patologia , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Antígeno Ki-67/análise , Masculino , Morfogênese/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Cultura Primária de Células/métodos , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/genética , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Fator de Transcrição AP-2/biossíntese , Fator de Transcrição AP-2/genética , Células Tumorais Cultivadas
13.
Mol Hum Reprod ; 20(8): 709-18, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24743772

RESUMO

The measurement of gene expression levels in cells and tissues typically depends on a suitable point of reference for inferring biological relevance. For quantitative (or real-time) RT-PCR assays, the method of choice is often to normalize gene expression data to an endogenous gene that is stably expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further recommend that such studies should be accompanied by additional assessment of histology and cellularity of each sample.


Assuntos
Neoplasias Embrionárias de Células Germinativas/metabolismo , Testículo/metabolismo , Adulto , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Neoplasias Embrionárias de Células Germinativas/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Br J Cancer ; 110(3): 668-78, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24292451

RESUMO

BACKGROUND: Developmental arrest of fetal germ cells may lead to neoplastic transformation and formation of germ cell tumours via carcinoma in situ (CIS) cells. Normal fetal germ cell development requires complete erasure and re-establishment of DNA methylation. In contrast to normal spermatogonia, the genome of CIS cells remains unmethylated in the adult testis. We here investigated the possible active and passive pathways that can sustain the CIS genome hypomethylated in the adult testis. METHODS: The levels of 5-methyl-cytosine (5mC) and 5-hydroxy-methyl-cytosine (5hmC) in DNA from micro-dissected CIS cells were assessed by quantitative measurements. The expression of TET1, TET2, APOBEC1, MBD4, APEX1, PARP1, DNMT1, DNMT3A, DNMT3B and DNMT3L in adult testis specimens with CIS and in human fetal testis was investigated by immunohistochemistry and immunofluorescence. RESULTS: DNA from micro-dissected CIS cells contained very low levels of 5hmC produced by ten eleven translocation (TET) enzymes. CIS cells and fetal germ cells expressed the suggested initiator of active demethylation, APOBEC1, and the base excision repair proteins MBD4, APEX1 and PARP1, whereas TETs - the alternative initiators were absent. Both maintenance and de novo methyltransferases were detected in CIS cells. CONCLUSION: The data are consistent with the presence of an active DNA de-methylation pathway in CIS cells. The hypomethylated genome of CIS cells may contribute to phenotypic plasticity and invasive capabilities of this testicular cancer precursor.


Assuntos
Carcinoma in Situ/genética , Metilação de DNA/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Carcinoma in Situ/patologia , Diferenciação Celular , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Feto/metabolismo , Feto/patologia , Genoma Humano , Humanos , Imuno-Histoquímica , Masculino , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/patologia , Testículo/metabolismo , Testículo/patologia
15.
Ann Oncol ; 24(4): 878-88, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23152360

RESUMO

In November 2011, the Third European Consensus Conference on Diagnosis and Treatment of Germ-Cell Cancer (GCC) was held in Berlin, Germany. This third conference followed similar meetings in 2003 (Essen, Germany) and 2006 (Amsterdam, The Netherlands) [Schmoll H-J, Souchon R, Krege S et al. European consensus on diagnosis and treatment of germ-cell cancer: a report of the European Germ-Cell Cancer Consensus Group (EGCCCG). Ann Oncol 2004; 15: 1377-1399; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part I. Eur Urol 2008; 53: 478-496; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part II. Eur Urol 2008; 53: 497-513]. A panel of 56 of 60 invited GCC experts from all across Europe discussed all aspects on diagnosis and treatment of GCC, with a particular focus on acute and late toxic effects as well as on survivorship issues. The panel consisted of oncologists, urologic surgeons, radiooncologists, pathologists and basic scientists, who are all actively involved in care of GCC patients. Panelists were chosen based on the publication activity in recent years. Before the meeting, panelists were asked to review the literature published since 2006 in 20 major areas concerning all aspects of diagnosis, treatment and follow-up of GCC patients, and to prepare an updated version of the previous recommendations to be discussed at the conference. In addition, ∼50 E-vote questions were drafted and presented at the conference to address the most controversial areas for a poll of expert opinions. Here, we present the main recommendations and controversies of this meeting. The votes of the panelists are added as online supplements.


Assuntos
Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Embrionárias de Células Germinativas/terapia , Europa (Continente) , Seguimentos , Humanos , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Embrionárias de Células Germinativas/classificação , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Taxa de Sobrevida
16.
Hum Reprod ; 27(6): 1547-55, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22466863

RESUMO

BACKGROUND: DDX3Y (DBY), located within AZoospermia Factor a (AZFa) region of the human Y chromosome (Yq11), encodes a conserved DEAD-box RNA helicase expressed only in germ cells and with a putative function at G1-S phase of the cell cycle. Deletion of AZFa results most often in germ cell aplasia, i.e. Sertoli-cell-only syndrome. To investigate the function of DDX3Y during human spermatogenesis, we examined its expression during development and maturation of the testis and in several types of testicular germ cell tumours (TGCTs), including the pre-invasive carcinoma in situ (CIS) precursor cells which are believed to originate from fetal gonocytes. METHODS: DDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry. RESULTS: Germ cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function. CONCLUSIONS: The fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y in CIS cells, but not in gonocytes, indicates phenotypic plasticity of CIS cells and suggests partial maturation to spermatogonia, likely due to their postpubertal microenvironment.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/fisiologia , Expressão Gênica , Espermatozoides/metabolismo , Neoplasias Testiculares/genética , Testículo/crescimento & desenvolvimento , Azoospermia/genética , Western Blotting , Carcinoma in Situ/genética , Cromossomos Humanos Y , RNA Helicases DEAD-box/análise , Deleção de Genes , Idade Gestacional , Humanos , Masculino , Antígenos de Histocompatibilidade Menor , Neoplasias Embrionárias de Células Germinativas/genética , Fenótipo , Puberdade , Seminoma/genética , Espermatogênese , Espermatogônias/citologia , Espermatogônias/metabolismo , Teratoma/genética , Testículo/química , Testículo/embriologia
17.
Andrologia ; 44(2): 78-85, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21486421

RESUMO

Prompted by the recently reported expression of POU5F1 (OCT3/4) in epididymis, a panel of markers for carcinoma in situ (CIS) testis and testicular germ cell tumours (TGCT), including AP-2γ(TFAP2C), NANOG, OCT3/4, KIT, placental-like alkaline phosphatase (PLAP), M2A/PDPN and MAGE-A4 were examined by immunohistochemistry or in situ hybridisation in urogenital epithelia, which may interfere with detection of CIS cells in semen. In addition to OCT3/4, the expression of AP-2γ and NANOG or their variants was detected in urogenital epithelia, while other CIS markers, including PLAP/alkaline phosphatase were absent. A combination of immunocytological staining for AP-2γ or OCT3/4 and rapid cytochemical alkaline phosphatase reaction was subsequently developed. This approach was tested in 22 patients with TGCT. In 14 patients (63.6%), double stained cells were found and thus the method was proven suitable for the detection of CIS cells in semen. In conclusion, transcription factors related to pluripotency and undifferentiated state of cells, which most likely have several variants or modifications, are unexpectedly detected using currently available antibodies in urogenital epithelial cells which may be shed into semen. Combining the immunohistochemical nuclear markers with a rapid cytochemical alkaline phosphatase reaction for detection of CIS cells in ejaculates may provide a more reliable diagnostic method.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma in Situ/diagnóstico , Proteínas de Homeodomínio/análise , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Sêmen/química , Coloração e Rotulagem/métodos , Neoplasias Testiculares/diagnóstico , Fator de Transcrição AP-2/análise , Fosfatase Alcalina/análise , Humanos , Imuno-Histoquímica , Isoenzimas/análise , Masculino , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/análise , Sêmen/citologia , Testículo/enzimologia
18.
Int J Androl ; 34(4 Pt 2): e122-32, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21696394

RESUMO

To search for disease-related copy number variations (CNVs) in families with a high frequency of germ cell tumours (GCT), we analysed 16 individuals from four families by array comparative genomic hybridization (aCGH) and applied an integrative systems biology algorithm that prioritizes risk-associated genes among loci targeted by CNVs. The top-ranked candidate, RLN1, encoding a Relaxin-H1 peptide, although only detected in one of the families, was selected for further investigations. Validation of the CNV at the RLN1 locus was performed as an association study using qPCR with 106 sporadic testicular GCT patients and 200 healthy controls. Observed CNV frequencies of 1.9% among cases and 1.5% amongst controls were not significantly different and this was further confirmed by CNV data extracted from a genome-wide analysis of 189 cases and 380 controls, where similar frequencies of 2.2% were observed in both groups (p=1). Immunohistochemistry for Relaxin-H1 (RLN1), Relaxin-H2 (RLN2) and their cognate receptor, RXFP1, detected one, and in some cases both, of the relaxins in Leydig cells, Sertoli cells and a subset of neoplastic germ cells, whereas the receptor was present in Leydig cells and spermatids. Collectively, the findings show that a heterozygous loss at the RLN1 locus is not a genetic factor mediating high population-wide risk for testicular germ cell tumour, but do not exclude a contribution of this aberration in some cases of cancer. The preliminary expression data suggest a possible role of the relaxin peptides in spermatogenesis and warrant further studies.


Assuntos
Variações do Número de Cópias de DNA , Neoplasias Embrionárias de Células Germinativas/genética , Relaxina/genética , Deleção de Sequência , Neoplasias Testiculares/genética , Adolescente , Adulto , Sequência de Bases , Hibridização Genômica Comparativa , Família , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética
19.
Int J Androl ; 34(4 Pt 2): e21-30; discussion e30-1, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21696398

RESUMO

Testicular cancer (TC) is usually diagnosed after manifestation of an overt tumour. Tumour formation is preceded by a pre-invasive and asymptomatic stage, carcinoma in situ (CIS) testis, except for very rare subtypes. The CIS cells are located within seminiferous tubules but can be exfoliated and detected in ejaculates with specific CIS markers. We have built a high throughput framework involving automated immunocytochemical staining, scanning microscopy and in silico image analysis allowing automated detection and grading of CIS-like stained objects in semen samples. In this study, 1175 ejaculates from 765 subfertile men were tested using this framework. In 5/765 (0.65%) cases, CIS-like cells were identified in the ejaculate. Three of these had bilateral testicular biopsies performed and CIS was histologically confirmed in two. In total, 63 bilateral testicular biopsy were performed in conjunction with analysis of the ejaculates because of infertility work-up. Histological analysis of the biopsies for the presence of CIS yielded a test sensitivity of 0.67 and a specificity of 0.98. In addition, ejaculates from 45 patients with clinical signs of an overt TC were investigated and yielded a slightly lower sensitivity (0.51), possibly because of obstruction. We conclude that this novel non-invasive test combining automated immunocytochemistry and advanced image analysis allows identification of TC at the CIS stage with a high specificity, but a negative test does not completely exclude CIS. On the basis of the results, we propose that the assay could be offered to subfertile men and other patients who are at increased risk of TC.


Assuntos
Carcinoma in Situ/diagnóstico , Diagnóstico por Imagem/métodos , Infertilidade Masculina/patologia , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Análise do Sêmen/métodos , Neoplasias Testiculares/diagnóstico , Adulto , Fosfatase Alcalina/análise , Biópsia , Carcinoma in Situ/patologia , Células Cultivadas , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Microscopia , Neoplasias Embrionárias de Células Germinativas/patologia , Sêmen/citologia , Coloração e Rotulagem/métodos , Neoplasias Testiculares/patologia
20.
Int J Androl ; 34(4 Pt 2): e175-87; discussion e187-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21651578

RESUMO

The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state', yet it appears that this categorization may be an oversimplification, because a number of sub-states appear to exist within this state. To increase the resolution of the undifferentiated state, we have generated eight novel monoclonal antibodies, all capable of recognizing undifferentiated human ES and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Carcinoma Embrionário/imunologia , Células-Tronco de Carcinoma Embrionário/imunologia , Células-Tronco Embrionárias/imunologia , Neoplasias Embrionárias de Células Germinativas/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Biomarcadores , Diferenciação Celular , Linhagem Celular/imunologia , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Testiculares/imunologia
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