RESUMEN

Heat waves, defined as periods with daily temperatures surpassing the historical average for a specific region, have become more frequent worldwide in recent years. Previous studies have reported a negative association between temperature and semen quality, but the focus has mainly been on Asian and European populations. The study included 54,926 men (18-60 years) undergoing routine semen analysis between 2005 and 2023 at CEUSA-LAEH andrology unit, in Buenos Aires, Argentina. Hourly temperature readings were provided by the Servicio Meteorológico Nacional. R programming (R Studio v2022.07.2) was used to define heat waves, calculate key characteristics, visualize results, and perform statistical tests at the IBYME laboratory. During the period studied, a total of 124 days had heat waves (defined after at least 3 consecutive days with 32.3 °C and 22 °C). Men exposed to heat waves during spermatogenesis exhibited lower sperm number (concentration and count; P < 0.0001) and decreased normal morphology (percentage of normal sperm and normal motile count; P < 0.05) compared to those not exposed. These differences were most pronounced between semen samples from years with several heat waves (2013, 2023) and none (2005, 2007, 2016), displaying 4-5 times higher fold changes (P < 0.05). Further analysis employing multiple regression revealed a significantly negative association between semen quality and heat wave length, suggesting that a prolonged exposure may be more detrimental than an acute exposure. Subsequent analysis focusing on prolonged exposure (≥6-days heat wave) during spermatogenesis revealed a negative (P < 0.05) association between early exposure (spermatocytogenesis: 64-90 days prior semen collection) and semen quality. This study underscores the negative association between early exposure to heat waves during sperm development and semen quality, raising concerns about its possible association with the worldwide declining male fertility. A comprehensive collaborative approach is crucial, involving global governmental policies, sustainable practices, and coordinated efforts across scientific, healthcare, and policy domains.

Asunto(s)

Análisis de Semen , Masculino , Humanos , Argentina , Adulto , Estudios Retrospectivos , Adulto Joven , Calor , Adolescente , Persona de Mediana Edad , Recuento de Espermatozoides , Semen/fisiología

RESUMEN

OBJECTIVES: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters. STUDY DESIGN: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318). RESULTS: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients. CONCLUSIONS: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.

Asunto(s)

Fragmentación del ADN , Etiquetado Corte-Fin in Situ , Infertilidad Masculina , Análisis de Semen , Espermatozoides , Humanos , Masculino , Adulto , Análisis de Semen/métodos , Infertilidad Masculina/diagnóstico , Persona de Mediana Edad , Motilidad Espermática

RESUMEN

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Asunto(s)

Bison , Preservación de Semen , Masculino , Animales , Caballos , Porcinos , Humanos , Perros , Semen , Criopreservación/veterinaria , Criopreservación/métodos , Espermatozoides , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Coloides , Centrifugación/veterinaria , Centrifugación/métodos , Búfalos , Motilidad Espermática

RESUMEN

[This corrects the article DOI: 10.3389/fonc.2019.01306.].

Asunto(s)

Células del Cúmulo , Oocitos , Células del Cúmulo/metabolismo , ADN Mitocondrial/metabolismo , Femenino , Humanos , Mitocondrias , Oocitos/metabolismo

RESUMEN

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Asunto(s)

Cadherinas/metabolismo , Fertilización/fisiología , Mamíferos/fisiología , Actinas/metabolismo , Animales , Anticuerpos/farmacología , Células del Cúmulo/efectos de los fármacos , Células del Cúmulo/metabolismo , Epidídimo/metabolismo , Femenino , Fertilización/efectos de los fármacos , Fertilización In Vitro , Humanos , Masculino , Ratones , Modelos Animales , Modelos Moleculares , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Péptidos/farmacología , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Testículo/metabolismo , beta Catenina/metabolismo

RESUMEN

Endometrial cancer (EC) is the sixth most common cancer in women worldwide. Early diagnosis is critical in recurrent EC management. The present study aimed to identify biomarkers of EC early recurrence using a workflow that combined text and data mining databases (DisGeNET, Gene Expression Omnibus), a prioritization algorithm to select a set of putative candidates (ToppGene), protein­protein interaction network analyses (Search Tool for the Retrieval of Interacting Genes, cytoHubba), association analysis of selected genes with clinicopathological parameters, and survival analysis (Kaplan­Meier and Cox proportional hazard ratio analyses) using a The Cancer Genome Atlas cohort. A total of 10 genes were identified, among which the targeting protein for Xklp2 (TPX2) was the most promising independent prognostic biomarker in stage I EC. TPX2 expression (mRNA and protein) was higher (P<0.0001 and P<0.001, respectively) in ETS variant transcription factor 5­overexpressing Hec1a and Ishikawa cells, a previously reported cell model of aggressive stage I EC. In EC biopsies, TPX2 mRNA expression levels were higher (P<0.05) in high grade tumors (grade 3) compared with grade 1­2 tumors (P<0.05), in tumors with deep myometrial invasion (>50% compared with <50%; P<0.01), and in intermediate­high recurrence risk tumors compared with low­risk tumors (P<0.05). Further validation studies in larger and independent EC cohorts will contribute to confirm the prognostic value of TPX2.

Asunto(s)

Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Neoplasias Endometriales/mortalidad , Endometrio/patología , Proteínas Asociadas a Microtúbulos/genética , Recurrencia Local de Neoplasia/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Estudios de Casos y Controles , Línea Celular Tumoral , Quimioterapia Adyuvante , Biología Computacional , Conjuntos de Datos como Asunto , Supervivencia sin Enfermedad , Neoplasias Endometriales/diagnóstico , Neoplasias Endometriales/patología , Neoplasias Endometriales/terapia , Endometrio/cirugía , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Clasificación del Tumor , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Proyectos Piloto , Pronóstico , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas/genética , Procesamiento Proteico-Postraduccional , Curva ROC , Medición de Riesgo/métodos

RESUMEN

Bladder cancer (BC) is the ninth most common cancer worldwide, but molecular changes are still under study. During tumor progression, Epithelial cadherin (E-cadherin) expression is altered and ß-catenin may be translocated to the nucleus, where it acts as co-transcription factor of tumor invasion associated genes. This investigation further characterizes E-cadherin and ß-catenin associated changes in BC, by combining bioinformatics, an experimental murine cell model (MB49/MB49-I) and human BC samples. In in silico studies, a DisGeNET (gene-disease associations database) analysis identified CDH1 (E-cadherin gene) as one with highest score among 130 BC related-genes. COSMIC mutation analysis revealed CDH1 low mutations rates. Compared to MB49 control BC cells, MB49-I invasive cells showed decreased E-cadherin expression, E- to P-cadherin switch, higher ß-catenin nuclear signal and lower cytoplasmic p-Ser33-ß-catenin signal, higher Ephrin-B1 ligand and EphB2 receptor expression, higher Phospho-Stat3 and Urokinase-type Plasminogen Activator (UPA), and UPA receptor expression. MB49-I cells transfected with Ephrin-B1 siRNA showed lower migratory and invasive capacity than control cells (scramble siRNA). By immunohistochemistry, orthotopic MB49-I tumors had lower E-cadherin, increased nuclear ß-catenin, lower pSer33-ß-catenin cytoplasmic signal, and higher Ephrin-B1 expression than MB49 tumors. Similar changes were found in human BC tumors, and 83% of infiltrating tumors depicted a high Ephrin-B1 stain. An association between higher Ephrin-B1 expression and higher stage and tumor grade was found. No association was found between abnormal E-cadherin signal, Ephrin-B1 expression or clinical-pathological parameter. This study thoroughly analyzed E-cadherin and associated changes in BC, and reports Ephrin-B1 as a new marker of tumor aggressiveness.

RESUMEN

Objective: Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models. Methods: FXYD5/Dys messenger RNA (mRNA) levels were determined in EC tissue and UA biopsies. FXYD5/Dys expression was evaluated in EC RNAseq data from The Cancer Genome Atlas (TCGA) and GENEVESTIGATOR tools. FXYD5/Dys impact on E-cadherin expression and cell behavior was assessed in EC Hec1a cells treated with transforming growth factor (TGF)-ß1, stably transfected with ETV5, and transiently transfected with FXYD5/Dys small interfering RNA (siRNA) or pcDNA3-FXYD5/Dys plasmid. Results: FXYD5/Dys was associated with EC aggressiveness, finding high mRNA levels in tumors depicting MI > 50%, Grade 3, and intermediate/high risk of recurrence. FXYD5/Dys was highly expressed at the tumor invasive front compared to the superficial area. Most results were recapitulated in UA biopsies. FXYD5/Dys modulation in Hec1a cells altered cell migration/adhesion and E-cadherin expression. TGF-ß1 treatment of Hec1a cells induced FXYD5/Dys expression. TCGA-UCEC RNAseq analysis revealed a positive correlation between FXYD5/Dys, TGF-ß1, and plasminogen activator inhibitor (PAI)-1 mRNA levels. FXYD5/Dys induced nuclear factor (NF)-κB pathway activation in Hec1a cells. FXYD5/Dys mRNA levels positively correlated with transcriptional activation of NF-κB p65-regulated genes. Survival analysis revealed patient segregation into low- and high-risk groups, the latter depicting the highest FXYD5/Dys, PAI-1, tumor necrosis factor (TNF)-α, and TGF-ß1 mRNA levels and shorter survival rates. Conclusion: FXYD5/Dys is a novel biomarker of EC progression related to TGF-ß1 and NF-κB pathways that collectively promote tumor dissemination and result in poor patient prognosis.

Asunto(s)

Humanos , Masculino , Paternidad , Tabaquismo , Alcoholismo , Fertilidad , Obesidad

RESUMEN

BACKGROUND: Breast cancer (BC) is the most common female cancer and the leading cause of cancer death in women worldwide. Alterations in epithelial cadherin (E-cadherin) expression and functions are associated to BC, but the underlying molecular mechanisms have not been fully elucidated. We have previously reported a novel human E-cadherin splice variant (E-cadherin variant) mRNA. Stable transfectants in MCF-7 human BC cells (MCF7Ecadvar) depicted fibroblast-like cell morphology, E-cadherin wild-type downregulation, and other molecular changes characteristic of the epithelial-to-mesenchymal transition process, reduced cell-cell adhesion, and increased cell migration and invasion. In this study, a two-dimensional differential gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS) protein identification and bioinformatics analyses were done to characterize biological processes and canonical pathways affected by E-cadherin variant expression. RESULTS: By 2D-DIGE and MS analysis, 50 proteins were found differentially expressed (≥ Δ1.5) in MCF7Ecadvar compared to control cells. Validation of transcript expression was done in the ten most overexpressed and underexpressed proteins. Bioinformatics analyses revealed that 39 of the 50 proteins identified had been previously associated to BC. Moreover, metabolic processes were the most affected, and glycolysis the canonical pathway most altered. The lactate dehydrogenase B (LDHB) was the highest overexpressed protein, and transcript levels were higher in MCF7Ecadvar than in control cells. In agreement with these findings, MCF7Ecadvar conditioned media had lower glucose and higher lactate levels than control cells. MCF7Ecadvar cell treatment with 5 mM of the glycolytic inhibitor 2-deoxy-glucose led to decreased cell viability, and modulation of LDHB expression in MCF7Ecadvar cells with a specific small interfering RNA resulted in decreased cell proliferation. Finally, a positive association between expression levels of the E-cadherin variant and LDHB transcripts was demonstrated in 21 human breast tumor tissues, and breast tumor samples with higher Ki67 expression showed higher LDHB mRNA levels. CONCLUSIONS: Results from this investigation contributed to further characterize molecular changes associated to the novel E-cadherin splice variant expression in BC cells. They also revealed an association between expression of the novel variant and changes related to BC progression and aggressiveness, in particular those associated to cell metabolism.

RESUMEN

In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/deficiencia , Espermatogénesis , Espermatozoides/fisiología , Animales , Peso Corporal , Epidídimo/metabolismo , Femenino , Fertilidad , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Espermatozoides/ultraestructura , Testículo/metabolismo

RESUMEN

OBJECTIVE: To assess the impact of aging on routine semen and computer-assisted sperm analysis (CASA) motility parameters according to the current World Health Organization guidelines; and to evaluate the effect of obesity and lifestyle (alcohol consumption, cigarette smoking) in older men's semen. DESIGN: Blind cross-sectional study. SETTING: Research laboratory and andrology and reproduction laboratory. PATIENT(S): A population of 11,706 men. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen analysis: routine (semen volume, sperm concentration and count, motility, vitality, morphology, hypo-osmotic swelling test, round and peroxidase-positive cell concentration) and CASA (straight-line velocity, curvilinear velocity, average path velocity, linearity, straightness, beat cross frequency, wobble, amplitude of lateral head displacement, and mean angular displacement) parameters; and body mass index. RESULT(S): A negative correlation was found between age and routine semen parameters: volume, sperm count, motility, vitality, total motile spermatozoa and normal-motile spermatozoa, round cell concentration, and hypo-osmotic swelling test values. Several CASA variables (straight-line velocity, curvilinear velocity, average path velocity, beat cross frequency, amplitude of lateral head displacement, and mean angular displacement) were also negatively affected. Using 40 years as a cut-off value, significant differences in most parameters correlated to age. In a selected subpopulation of men unexposed to known fertility-compromising factors, the same evaluations were performed, finding some parameters still decreased. Although obesity exerted a significant deleterious effect on older patients' semen quality, alcohol consumption and cigarette smoking mildly affected it. CONCLUSION(S): Male aging, with the contribution of unhealthy conditions, are paramount effectors of sperm quality deterioration.

Asunto(s)

Envejecimiento/fisiología , Infertilidad Masculina/etiología , Estilo de Vida , Espermatozoides/citología , Espermatozoides/fisiología , Adolescente , Adulto , Factores de Edad , Anciano , Consumo de Bebidas Alcohólicas/epidemiología , Consumo de Bebidas Alcohólicas/fisiopatología , Fumar Cigarrillos/efectos adversos , Fumar Cigarrillos/epidemiología , Estudios Transversales , Humanos , Infertilidad Masculina/epidemiología , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Obesidad/epidemiología , Obesidad/fisiopatología , Análisis de Semen , Recuento de Espermatozoides , Motilidad Espermática , Adulto Joven

RESUMEN

Fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) have been described in several tissues, where they regulate cellular proliferation, differentiation, motility and apoptosis. Although FGF2/FGFRs expression in the male reproductive tract has been reported, there is scarce evidence on their presence in the female reproductive tract and their involvement in the modulation of sperm function. Therefore, the objective of this study was to determine the expression of FGF2 in the female reproductive tract and to assess the role of the FGF2/FGFRs system in the regulation of sperm physiology using the murine model. FGF2 was detected in uterus and oviduct protein extracts, and it was immunolocalized in epithelial cells of the uterus, isthmus and ampulla, as well as in the cumulus oophorus-oocyte complex. The receptors FGFR1, FGFR2, FGFR3 and FGFR4 were immunodetected in the flagellum and acrosomal region of sperm recovered from the cauda epididymis. Analysis of testis sections showed the expression of FGFRs in germ cells at different stages of the spermatogenesis, suggesting the testicular origin of the sperm FGFRs. Sperm incubation with recombinant FGF2 (rFGF2) led to increased sperm motility and velocity and to enhanced intracellular Ca2+ levels and acrosomal loss compared to the control. In conclusion, this study shows that FGF2 is expressed in tissues of the female reproductive tract. Also, the fact that functional FGFRs are present in mouse sperm and that rFGF2 affects sperm motility and acrosomal exocytosis, suggests the involvement of this system in the in vivo regulation of sperm function.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/metabolismo , Genitales Femeninos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Espermatozoides/fisiología , Animales , Femenino , Masculino , Ratones , Testículo/metabolismo

Asunto(s)

Oocitos , Vitrificación , Criopreservación , Transferencia de Embrión , Humanos

RESUMEN

Ovarian cancer (OC) is the fifth cancer death cause in women worldwide. The malignant nature of this disease stems from its unique dissemination pattern. Epithelial-to-mesenchymal transition (EMT) has been reported in OC and downregulation of Epithelial cadherin (E-cadherin) is a hallmark of this process. However, findings on the relationship between E-cadherin levels and OC progression, dissemination and aggressiveness are controversial. In this study, the evaluation of E-cadherin expression in an OC tissue microarray revealed its prognostic value to discriminate between advanced- and early-stage tumors, as well as serous tumors from other histologies. Moreover, E-cadherin, Neural cadherin (N-cadherin), cytokeratins and vimentin expression was assessed in TOV-112, SKOV-3, OAW-42 and OV-90 OC cell lines grown in monolayers and under anchorage-independent conditions to mimic ovarian tumor cell dissemination, and results were associated with cell aggressiveness. According to these EMT-related markers, cell lines were classified as mesenchymal (M; TOV-112), intermediate mesenchymal (IM; SKOV-3), intermediate epithelial (IE; OAW-42) and epithelial (E; OV-90). M- and IM-cells depicted the highest migration capacity when grown in monolayers, and aggregates derived from M- and IM-cell lines showed lower cell death, higher adhesion to extracellular matrices and higher invasion capacity than E- and IE-aggregates. The analysis of E-cadherin, N-cadherin, cytokeratin 19 and vimentin mRNA levels in 20 advanced-stage high-grade serous human OC ascites showed an IM phenotype in all cases, characterized by higher proportions of N- to E-cadherin and vimentin to cytokeratin 19. In particular, higher E-cadherin mRNA levels were associated with cancer antigen 125 levels more than 500 U/mL and platinum-free intervals less than 6 months. Altogether, E-cadherin expression levels were found relevant for the assessment of OC progression and aggressiveness.

Asunto(s)

Cadherinas/metabolismo , Neoplasias Ováricas/metabolismo , Antígenos CD , Ascitis/metabolismo , Ascitis/patología , Biomarcadores de Tumor/metabolismo , Antígeno Ca-125/sangre , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/sangre , Invasividad Neoplásica/patología , Invasividad Neoplásica/fisiopatología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Neoplasias Ováricas/cirugía , Pronóstico , ARN Mensajero/metabolismo

RESUMEN

Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them acquisition of a fibroblastic-like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 232: 1368-1386, 2017. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Empalme Alternativo/genética , Cadherinas/genética , Transición Epitelial-Mesenquimal/genética , Adulto , Empalme Alternativo/efectos de los fármacos , Secuencia de Aminoácidos , Antígenos CD , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cadherinas/química , Cadherinas/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Medios de Cultivo Condicionados/farmacología , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Humanos , Masculino , Modelos Biológicos , Invasividad Neoplásica , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección

RESUMEN

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-ß1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.

Asunto(s)

Acrosina/inmunología , Precursores Enzimáticos/inmunología , Infertilidad Masculina/metabolismo , Vacunas de ADN/inmunología , Acrosina/genética , Animales , Tasa de Natalidad , Microambiente Celular , Citocinas/metabolismo , Precursores Enzimáticos/genética , Femenino , Inmunización Secundaria , Inmunoglobulina G/sangre , Masculino , Ratones , Ratones Endogámicos , Embarazo , Vesículas Seminales/metabolismo

RESUMEN

PROBLEM: Antisperm antibodies (ASA) are associated with male subfertility. However, results on sperm surface autoantibodies are controversial, the relationship between ASA and semen parameters (WHO, 2010) is unknown, and data on ASA and sperm kinematics are scarce. METHOD OF STUDY: A retrospective study carried out in men undergoing routine semen analysis (WHO 2010), ASA evaluation (direct SpermMAR(™) (IgG) test), and computer-assisted sperm analysis (CASA). RESULTS: A 2.6% and a 5.9% incidence of ASA-positive cases were found (cut-off 50% and 10%, respectively; n = 7492). ASA-positive samples had lower (P < 0.0001) sperm concentration, count, motility, and hypo-osmotic swelling (HOS) test score. HOS results did not correlate with sperm vitality in normozoospermic samples with high ASA levels. In unselected samples, ASA-positive samples (cut-off 50%) showed decreased sperm kinematics (VSL, VAP, LIN, ALH, STR, BCF, WOB), but in normozoospermic samples, ASA-positive and ASA-negative subgroups had similar CASA results. CONCLUSIONS: ASA evaluation is highly relevant in full semen assessment.

Asunto(s)

Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Motilidad Espermática/inmunología , Espermatozoides/inmunología , Adulto , Humanos , Masculino