RESUMEN
STUDY QUESTION: Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function? SUMMARY ANSWER: While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action. WHAT IS KNOWN ALREADY: Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). LIMITATIONS, REASONS FOR CAUTION: The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. WIDER IMPLICATIONS OF THE FINDINGS: We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. STUDY FUNDING/COMPETING INTEREST(S): D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.
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Acetaminofén , Motilidad Espermática , Acetaminofén/farmacología , Adulto , Ácidos Araquidónicos , Calcio/metabolismo , Canales de Calcio/metabolismo , Humanos , Masculino , Preparaciones Farmacéuticas/metabolismo , Progesterona/metabolismo , Espermatozoides/metabolismoRESUMEN
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.
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Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Ganglios Linfáticos/patología , Neoplasias de Células Germinales y Embrionarias/patología , Células Madre Pluripotentes/patología , Neoplasias Testiculares/patología , Animales , Proliferación Celular , Humanos , Ganglios Linfáticos/efectos de los fármacos , Masculino , Ratones , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Células Madre Pluripotentes/efectos de los fármacos , Transducción de Señal , Neoplasias Testiculares/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
STUDY QUESTION: Does experimental manipulation of fibroblast growth factor 9 (FGF9)-signalling in human fetal gonads alter sex-specific gonadal differentiation? SUMMARY ANSWER: Inhibition of FGFR signalling following SU5402 treatment impaired germ cell survival in both sexes and severely altered the developing somatic niche in testes, while stimulation of FGF9 signalling promoted Sertoli cell proliferation in testes and inhibited meiotic entry of germ cells in ovaries. WHAT IS KNOWN ALREADY: Sex-specific differentiation of bipotential gonads involves a complex signalling cascade that includes a combination of factors promoting either testicular or ovarian differentiation and inhibition of the opposing pathway. In mice, FGF9/FGFR2 signalling has been shown to promote testicular differentiation and antagonize the female developmental pathway through inhibition of WNT4. STUDY DESIGN, SIZE, DURATION: FGF signalling was manipulated in human fetal gonads in an established ex vivo culture model by treatments with recombinant FGF9 (25 ng/ml) and the tyrosine kinase inhibitor SU5402 (10 µM) that was used to inhibit FGFR signalling. Human fetal testis and ovary tissues were cultured for 14 days and effects on gonadal development and expression of cell lineage markers were determined. PARTICIPANTS/MATERIALS, SETTING, METHODS: Gonadal tissues from 44 male and 33 female embryos/fetuses from first trimester were used for ex vivo culture experiments. Tissues were analyzed by evaluation of histology and immunohistochemical analysis of markers for germ cells, somatic cells, proliferation and apoptosis. Culture media were collected throughout the experimental period and production of steroid hormone metabolites was analyzed in media from fetal testis cultures by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with SU5402 resulted in near complete loss of gonocytes (224 vs. 14 OCT4+ cells per mm2, P < 0.05) and oogonia (1456 vs. 28 OCT4+ cells per mm2, P < 0.001) in human fetal testes and ovaries, respectively. This was a result of both increased apoptosis and reduced proliferation in the germ cells. Addition of exogenous FGF9 to the culture media resulted in a reduced number of germ cells entering meiosis in fetal ovaries (102 vs. 60 γH2AX+ germ cells per mm2, P < 0.05), while in fetal testes FGF9 stimulation resulted in an increased number of Sertoli cells (2503 vs. 3872 SOX9+ cells per mm2, P < 0.05). In fetal testes, inhibition of FGFR signalling by SU5402 treatment altered seminiferous cord morphology and reduced the AMH expression as well as the number of SOX9-positive Sertoli cells (2503 vs. 1561 SOX9+ cells per mm2, P < 0.05). In interstitial cells, reduced expression of COUP-TFII and increased expression of CYP11A1 and CYP17A1 in fetal Leydig cells was observed, although there were no subsequent changes in steroidogenesis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ex vivo culture may not replicate all aspects of fetal gonadal development and function in vivo. Although the effects of FGF9 were studied in ex vivo culture experiments, there is no direct evidence that FGF9 acts in vivo during human fetal gonadogenesis. The FGFR inhibitor (SU5402) used in this study is not specific to FGFR2 but inhibits all FGF receptors and off-target effects on unrelated tyrosine kinases should be considered. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study suggest that dysregulation of FGFR-mediated signalling may affect both testicular and ovarian development, in particular impacting the fetal germ cell populations in both sexes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.JØ. Additional funding was obtained from the Erichsen Family Fund (A.JØ.), the Aase and Ejnar Danielsens Fund (A.JØ.), the Danish Government's support for the EDMaRC programme (A.JU.) and a Wellcome Trust Intermediate Clinical Fellowship (R.T.M., Grant no. 098522). The Medical Research Council (MRC) Centre for Reproductive Health (R.T.M.) is supported by an MRC Centre Grant (MR/N022556/1). The authors have no conflict of interest to disclose.
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Regulación del Desarrollo de la Expresión Génica , Células Germinativas/efectos de los fármacos , Ovario/embriología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Testículo/embriología , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Femenino , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Humanos , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Embarazo , Primer Trimestre del Embarazo , Pirroles/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Células de Sertoli/efectos de los fármacos , Transducción de Señal , Proteína Wnt4/metabolismoRESUMEN
BACKGROUND: Cholesterol is essential for cell membrane stability, permeability, and fluidity. Cholesterol is present in seminal plasma, but whether a relationship between the level of cholesterol in seminal plasma and semen quality exists remains to be elucidated. OBJECTIVES: To explore the association between cholesterol levels in seminal plasma and serum cholesterols, semen quality, and serum reproductive hormones. Secondly, to explore whether the associations are biologically plausible. MATERIALS AND METHODS: An association study between cholesterol levels in seminal plasma and semen quality in 403 men, median age 19 years, from the general population. Additionally, an immunohistochemical evaluation of proteins involved in cholesterol metabolism and transport in tissues from the male reproductive tract (testis, epididymis, prostate, and seminal vesicle). Tissue specimens were investigated by immunohistochemistry for markers of cholesterol metabolism and transport (ABCA1, ABCG1, CYP11A1, CYP51A1, HMGCR, LAL, LCAT, LDLR, and SOAT1). RESULTS: Trend analyses showed that total amount of total cholesterol in seminal plasma was positively associated with sperm concentration, total sperm count, sperm motility, and morphology (all p < 0.008, adjusted). Cholesterol concentrations in seminal plasma were neither associated with serum cholesterol and lipid levels nor serum reproductive hormone (FSH, LH, testosterone, estradiol, sex-hormone-binding globulin, inhibin b) levels. All investigated markers of cholesterol metabolism and transport were expressed in the investigated tissue specimens to varying degrees. DISCUSSION: Seminal plasma level of cholesterol was positively associated with semen parameters. The presence of proteins and enzymes involved in cholesterol metabolism in Leydig cells, Sertoli cells, and maturing germ cells in the seminiferous tubules supports the view that cholesterol may be important for spermatogenesis. CONCLUSION: Cholesterol level in seminal plasma may be an indicator of semen quality. Investigations are needed to corroborate or refute our findings and to clarify the exact role of cholesterols for semen quality.
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Colesterol/análisis , Genitales Masculinos/química , Inmunohistoquímica , Semen/química , Recuento de Espermatozoides , Espermatogénesis , Adolescente , Transporte Biológico , Biomarcadores/análisis , Estudios Transversales , Dinamarca , Hormonas/análisis , Humanos , Masculino , Motilidad Espermática , Adulto JovenRESUMEN
Perceived stress has been associated with decreased semen quality but the mechanisms have not been elucidated. It is not known whether cortisol, the major stress hormone in humans, can act directly via receptors in the testis, and whether variants in the gene encoding the glucocorticoid receptor (NR3C1) can possibly modulate the effect. To address these questions, we investigated the expression of the glucocorticoid receptor in human testicular tissue, including adult and fetal samples (n = 20) by immunohistochemical staining, and in silico analysis of publicly available datasets. In the adult testis NR3C1 protein was detected in peritubular cells, a subset of Leydig cells, Sertoli cells (weak), and spermatogonia, but not in spermatids. The NR3C1 expression pattern in fetal testis samples differed by a notably stronger reaction in Sertoli cells, lack of staining in gonocytes but the presence in a subset of pro-spermatogonia, and the almost absent reaction in nascent peritubular cells. In parallel, we explored the association between adult testicular function and three single nucleotide NR3C1 polymorphisms (BcII [rs41423247], 9ß [rs6198], and Tth111I [rs10052957]) affecting glucocorticoid sensitivity. Testicular function was determined by semen analysis and reproductive hormone profiling in 893 men from the general population. The NR3C1 SNP BclI was associated with semen quality in an over-dominant manner with heterozygotes having better semen parameters compared to both homozygote constellations, and with sperm motility showing the strongest association. This association was supported by a higher inhibin B and inhibin B/FSH ratio, as well as a lower FSH in BclI heterozygotes. The SNPs 9ß and Tth111I were not associated with semen parameters. Although the clinical impact of the findings is limited, the results substantiate a suggested link between stress and testicular function. Hence this investigation should be regarded as a discovery study generating hypotheses for future studies.
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Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Análisis de Semen , Testículo/metabolismo , Adolescente , Adulto , Feto , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder characterized by motor, psychiatric, and cognitive manifestations. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene but the exact pathogenesis remains unknown. Dopamine imbalance has previously been shown in HD, and furthermore dopamine is thought to be implicated in cognition, behavioral and motor disturbances. A substantiated inverse correlation between motor onset and the elongated CAG repeat in the HTT has been established. This relation does not account for the full variability of the motor onset, and efforts have been put into finding genetic modifiers of motor onset, however, mostly with unsuccessful outcome. In this study, we took an alternative approach focusing on symptom complexes and searched for modifiers of cognitive impairment and psychiatric symptoms in a well-described cohort of Danish HD gene-expansion carriers. We show that cognitive impairment and psychiatric symptoms in HD are modified by polymorphisms in the monoamine oxidase A (MAOA) and catechol-O-methyltransferase (COMT) genes and by the 4p16.3 B haplotype. These results support the theory of dopamine imbalance in HD, and point toward more personalized treatment modalities of HD in the future.
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Catecol O-Metiltransferasa/genética , Cognición , Enfermedad de Huntington/psicología , Monoaminooxidasa/genética , Polimorfismo Genético , Adulto , Anciano , Conducta , Catecol O-Metiltransferasa/metabolismo , Catecolaminas/metabolismo , Femenino , Haplotipos , Humanos , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/genética , Masculino , Persona de Mediana Edad , Monoaminooxidasa/metabolismo , Adulto JovenRESUMEN
STUDY QUESTION: What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? SUMMARY ANSWER: RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures. WHAT IS KNOWN ALREADY: One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo. STUDY DESIGN, SIZE, DURATION: This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE: A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology. LIMITATIONS, REASONS FOR CAUTION: The number of samples included in this study was relatively small due to the limited availability of human fetal tissue. WIDER IMPLICATIONS OF THE FINDINGS: The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest.
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Técnicas de Cultivo de Embriones , Meiosis/efectos de los fármacos , Técnicas de Cultivo de Órganos/métodos , Testículo/efectos de los fármacos , Testículo/embriología , Tretinoina/química , Hormona Antimülleriana/metabolismo , Apoptosis , Factor de Transcripción COUP II/metabolismo , Proliferación Celular , Femenino , Feto/patología , Células Germinativas/citología , Células de la Granulosa/citología , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Células Intersticiales del Testículo/metabolismo , Masculino , Oocitos/citología , Oogonios/patología , Ovario/efectos de los fármacos , Ovario/embriología , Fenotipo , Diferenciación Sexual , Transducción de Señal , Testículo/patología , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Finding early and dynamic biomarkers in Huntington's disease is a key to understanding the early pathology of Huntington's disease and potentially to tracking disease progression. This would benefit the future evaluation of potential neuroprotective and disease-modifying therapies, as well as aid in identifying an optimal time point for initiating a potential therapeutic intervention. METHODS: This explorative proteomics study evaluated cerebrospinal fluid from 94 Huntington's disease gene-expansion carriers (39 premanifest and 55 manifest) and 27 Huntington's disease gene-expansion negative individuals using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry. Differences in peak intensity from SELDI-TOF spectra were evaluated. RESULTS: Levels of 10 peaks were statistically significantly different between manifest gene-expansion carriers and controls. One of them identified as ubiquitin was shown to be dependent on the Unified Huntington Disease Rating Scale Total Functional Capacity, a pseudo-measure of disease severity (P = 0.001), and the Symbol Digit Modalities Test (0.04) in manifest and CAG-age product score (P = 0.019) in all gene-expansion carriers. CONCLUSIONS AND RELEVANCE: Multiple studies have shown that the ubiquitin-proteasome system is involved in Huntington's disease pathogenesis and understanding of this involvement may have therapeutic potential in humans. This is the first study on cerebrospinal fluid to confirm the involvement of the ubiquitin-proteasome system in Huntington's disease. Furthermore it is shown that ubiquitin increases with disease progression and CAG-age product score and therefore may have the potential as a Huntington's disease progression marker, also prior to motor onset.
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Progresión de la Enfermedad , Enfermedad de Huntington/líquido cefalorraquídeo , Ubiquitina/líquido cefalorraquídeo , Adulto , Anciano , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteómica , Adulto JovenRESUMEN
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.
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Biglicano/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Espermatogonias/metabolismo , Testículo/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Masculino , Unión Proteica , Isoformas de Proteínas/metabolismo , Espermatogonias/citología , Testículo/citologíaRESUMEN
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.
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Diferenciación Celular , Insulina/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Células Intersticiales del Testículo/citología , Proteínas de la Membrana/genética , Proteínas/genética , Enfermedades Testiculares/patología , Actinas/genética , Actinas/metabolismo , Adolescente , Adulto , Factor de Transcripción COUP II/genética , Factor de Transcripción COUP II/metabolismo , Proteínas de Unión al Calcio , Niño , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Regulación de la Expresión Génica , Humanos , Lactante , Recién Nacido , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Síndrome de Klinefelter/genética , Síndrome de Klinefelter/metabolismo , Síndrome de Klinefelter/patología , Células Intersticiales del Testículo/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Estudios Retrospectivos , Enfermedades Testiculares/genética , Enfermedades Testiculares/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologíaRESUMEN
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.
Asunto(s)
Activinas/farmacología , Técnicas de Cultivo de Célula , Folistatina/farmacología , Seminoma/patología , Neoplasias Testiculares/patología , Testículo/citología , Adulto , Antígenos de Neoplasias/análisis , Apoptosis/efectos de los fármacos , Carcinoma in Situ/patología , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Antígeno Ki-67/análisis , Masculino , Morfogénesis/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Cultivo Primario de Células/métodos , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Proteínas Proto-Oncogénicas c-kit/genética , Espermatogénesis/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Factor de Transcripción AP-2/biosíntesis , Factor de Transcripción AP-2/genética , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Carcinoma in Situ/genética , Metilación de ADN/genética , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias Testiculares/genética , Carcinoma in Situ/patología , Diferenciación Celular , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Feto/metabolismo , Feto/patología , Genoma Humano , Humanos , Inmunohistoquímica , Masculino , Neoplasias de Células Germinales y Embrionarias/patología , Neoplasias Testiculares/patología , Testículo/metabolismo , Testículo/patologíaRESUMEN
OBJECTIVES: Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurodegenerative disorders characterized by a progressive gait disorder, lower limb spasticity, hyper-reflexia, weakness and extensor plantar responses. Recently, large intronic hexanucleotide repeat expansions (GGGGCC) in C9ORF72 have been found to cause frontotemporal dementia (FTD), amyotrophic lateral sclerosis and FTD with motor neuron disease. Owing to the overlapping phenotypes among HSP, amyotrophic lateral sclerosis and FTD with motor neuron disease along with shared pathological findings, we hypothesized that C9ORF72 expansions might be a genetic risk factor or modifier of HSP. METHODS: Clinically characterized HSP patients were investigated for elongations in the hexanucleotide repeat of C9ORF72. RESULTS: Upon analyses of the repeat lengths in the C9ORF72 gene in a Danish cohort of HSP patients, we found no expansions. CONCLUSION: We conclude that HSP is most likely not associated with repeat expansions in C9ORF72.
Asunto(s)
Expansión de las Repeticiones de ADN/genética , Proteínas/genética , Paraplejía Espástica Hereditaria/genética , Secuencia de Bases , Proteína C9orf72 , Dinamarca , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
UNLABELLED: The SPG5A subtype of Hereditary Spastic Paraplegia (HSP) is a rare autosomal recessive neurodegenerative disorder caused by mutations in the CYP7B1 gene, which encodes a steroid cytochrome P450 7α-hydroxylase. This enzyme provides the primary metabolic route for neurosteroids. Clinically, SPG5A has been characterized as a pure form of HSP with a variable age of onset, but recently a broader spectrum of phenotypes has been described. OBJECTIVE: This study characterizes four unrelated SPG5A patients through clinical evaluation. METHODS: The investigations included blood biochemistry, electrophysiology, brain MRI and MR spectroscopy. RESULTS: One patient had saccadic pursuit eye movements in addition to a pure HSP phenotype. Motor evoked potential (MEP) examinations revealed prolonged central conduction time. MRI of the brain showed white matter hyperintensities (WMH) in one patient. MRS showed elevated mI/Cr ratio in white matter in two patients; in the one patient with WMH and in one patient with normal MRI. Four novel mutations were identified; one frameshift (c.509 delT p.L170fs), one premature stop codon (c.334 C>T p.R112X), one amino acid changing (c.440 G>A p.G147D) and one duplication (c.945_947 dupGGC p.A316AA). CONCLUSION: SPG5A could be characterized as a predominantly pure HSP. MRS showing elevated mI/Cr ratio in the white matter may be indicative of SPG5A.
Asunto(s)
Encéfalo/metabolismo , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/fisiopatología , Esteroide Hidroxilasas/genética , Adolescente , Adulto , Encéfalo/patología , Estudios de Cohortes , Familia 7 del Citocromo P450 , Análisis Mutacional de ADN , Potenciales Evocados Motores , Potenciales Evocados Somatosensoriales , Femenino , Humanos , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Persona de Mediana Edad , Mutación , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Conducción Nerviosa/fisiología , Paraplejía Espástica Hereditaria/patología , Adulto JovenRESUMEN
Recently, a hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 was reported as the cause of chromosome 9p21-linked frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS). We here report the prevalence of the expansion in a hospital-based cohort and associated clinical features indicating a wider clinical spectrum of C9ORF72 disease than previously described. We studied 280 patients previously screened for mutations in genes involved in early onset autosomal dominant inherited dementia disorders. A repeat-primed polymerase chain reaction amplification assay was used to identify pathogenic GGGGCC expansions. As a potential modifier, confirmed cases were further investigated for abnormal CAG expansions in ATXN2. A pathogenic GGGGCC expansion was identified in a total of 14 probands. Three of these presented with atypical clinical features and were previously diagnosed with clinical olivopontocerebellar degeneration (OPCD), atypical Parkinsonian syndrome (APS) and a corticobasal syndrome (CBS). Further, the pathogenic expansion was identified in six FTD patients, four patients with FTD-ALS and one ALS patient. All confirmed cases had normal ATXN2 repeat sizes. Our study widens the clinical spectrum of C9ORF72 related disease and confirms the hexanucleotide expansion as a prevalent cause of FTD-ALS disorders. There was no indication of a modifying effect of the ATXN2 gene.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Ataxia/genética , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , Proteínas/genética , Adulto , Anciano , Esclerosis Amiotrófica Lateral/diagnóstico , Ataxia/diagnóstico , Secuencia de Bases , Proteína C9orf72 , Estudios de Cohortes , Salud de la Familia , Femenino , Demencia Frontotemporal/diagnóstico , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas , Humanos , Masculino , Persona de Mediana Edad , SíndromeRESUMEN
Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human spermatozoa. The VD-inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression at the sperm annulus. Here, we investigated if CYP24A1 expression serves as a marker for VD metabolism in spermatozoa, and whether CYP24A1 expression was associated with semen quality. We included 130 men (53 healthy young volunteers and 77 subfertile men) for semen analysis and immunocytochemical (ICC) detection of CYP24A1. Another 40 men (22 young, 18 subfertile) were tested for in vitro effects of 1,25(OH)(2)D(3) on intracellular calcium concentration ([Ca(2+)](i)) and sperm motility. Double ICC staining showed that CYP24A1 and VDR were either concomitantly expressed or absent in 80% of the spermatozoa from young men. The median number of CYP24A1-expressing spermatozoa was 1% in subfertile men and thus significantly (p < 0.0005) lower than 25% in spermatozoa from young men. Moreover, CYP24A1 expression correlated positively with total sperm count, -concentration, -motility and -morphology (all p < 0.004), and the percentage of CYP24A1-positive spermatozoa increased (15 vs. 41%, p < 0.0005) after percoll-gradient-centrifugation. We noticed that the presence of >3% CYP24A1-positive spermatozoa distinguished young men from subfertile men with a sensitivity of 66.0%, a specificity of 77.9% and a positive predictive value of 98.3%. Functional studies revealed that 1,25(OH)(2)D(3) increased [Ca(2+)](i) and sperm motility in young healthy men, while 1,25(OH)(2)D(3) was unable to increase motility in subfertile patients. In conclusion, we suggest that CYP24A1 expression at the annulus may serve as a novel marker of semen quality and an objective proxy for sperm function.
Asunto(s)
Infertilidad Masculina/diagnóstico , Análisis de Semen/métodos , Espermatozoides/enzimología , Esteroide Hidroxilasas/biosíntesis , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/biosíntesis , Adulto , Biomarcadores , Calcio , Colestanotriol 26-Monooxigenasa/biosíntesis , Familia 2 del Citocromo P450 , Humanos , Masculino , Receptores de Calcitriol/metabolismo , Recuento de Espermatozoides , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Vitamina D/metabolismo , Vitamina D3 24-Hidroxilasa , Adulto JovenRESUMEN
Kufor-Rakeb syndrome (KRS) is a rare autosomal recessive inherited juvenile parkinsonian syndrome caused by mutations in ATP13A2. We describe six patients from a consanguineous Greenlandic Inuit family, homozygous for a novel frame-shift mutation in exon 22 of ATP13A2 (c.2473C>AA, p.Leu825AsnfsX32). Disease onset varied from 10 to 29 years of age, the latest reported, and the clinical features were highly variable within a wide spectrum of an extrapyramidal-pyramidal syndrome with cognitive/psychiatric features. Ataxia was seen in two patients and axonal neuropathy in one, features not previously related to KRS. Dopamine transporter scans showed symmetrical, severely reduced uptake in striatum in two patients. Magnetic resonance imaging was without atrophy in one patient despite disease duration of 17 years, and cerebral and cerebellar atrophy was seen in another patient after 4 years of disease duration. The molecular pathogenic mechanisms of ATP13A2 mutations are discussed. The observation that the mutant transcript is not degraded by nonsense-mediated RNA decay and the fact that none of the eight heterozygous carriers from the family have KRS symptoms suggest that the mutant protein does not interfere and destroy the function of the wild-type ATP13A2 protein.
Asunto(s)
Mutación , Trastornos Parkinsonianos/genética , ATPasas de Translocación de Protón/genética , Adulto , Encéfalo/patología , Genotipo , Groenlandia , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Degradación de ARNm Mediada por Codón sin Sentido , Trastornos Parkinsonianos/enzimología , Fenotipo , ATPasas de Translocación de Protón/metabolismoRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma in Situ/diagnóstico , Proteínas de Homeodominio/análisis , Neoplasias de Células Germinales y Embrionarias/diagnóstico , Semen/química , Coloración y Etiquetado/métodos , Neoplasias Testiculares/diagnóstico , Factor de Transcripción AP-2/análisis , Fosfatasa Alcalina/análisis , Humanos , Inmunohistoquímica , Isoenzimas/análisis , Masculino , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/análisis , Semen/citología , Testículo/enzimologíaRESUMEN
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.