Sperm acquire epididymis-derived proteins through epididymosomes.

STUDY QUESTION: Are epididymosomes implicated in protein transfer from the epididymis to spermatozoa? SUMMARY ANSWER: We characterized the contribution of epididymal secretions to the sperm proteome and demonstrated that sperm acquire epididymal proteins through epididymosomes. WHAT IS KNOWN ALREADY: Testicular sperm are immature cells unable to fertilize an oocyte. After leaving the testis, sperm transit along the epididymis to acquire motility and fertilizing abilities. It is well known that marked changes in the sperm proteome profile occur during epididymal maturation. Since the sperm is a transcriptional and translational inert cell, previous studies have shown that sperm incorporate proteins, RNA and lipids from extracellular vesicles (EVs), released by epithelial cells lining the male reproductive tract. STUDY DESIGN, SIZE, DURATION: We examined the contribution of the epididymis to the post-testicular maturation of spermatozoa, via the production of EVs named epididymosomes, released by epididymal epithelial cells. An integrative analysis using both human and mouse data was performed to identify sperm proteins with a potential epididymis-derived origin. Testes and epididymides from adult humans (n = 9) and adult mice (n = 3) were used to experimentally validate the tissue localization of four selected proteins using high-resolution confocal microscopy. Mouse epididymal sperm were co-incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled epididymosomes (n = 4 mice), and visualized using high-resolution confocal microscopy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult (12-week-old) C57BL/CBAF1 wild-type male mice and adult humans were used for validation purposes. Testes and epididymides from both mice and humans were obtained and processed for immunofluorescence. Mouse epididymal sperm and mouse epididymosomes were obtained from the epididymal cauda segment. Fluorescent epididymosomes were obtained after labeling the epididymal vesicles with CFSE dye followed by epididymosome isolation using a density cushion. Immunofluorescence was performed following co-incubation of sperm with epididymosomes in vitro. High-resolution confocal microscopy and 3D image reconstruction were used to visualize protein localization and sperm-epididymosomes interactions. MAIN RESULTS AND THE ROLE OF CHANCE: Through in silico analysis, we first identified 25 sperm proteins with a putative epididymal origin that were conserved in both human and mouse spermatozoa. From those, the epididymal origin of four sperm proteins (SLC27A2, EDDM3B, KRT19 and WFDC8) was validated by high-resolution confocal microscopy. SLC27A2, EDDM3B, KRT19 and WFDC8 were all detected in epithelial cells lining the human and mouse epididymis, and absent from human and mouse seminiferous tubules. We found region-specific expression patterns of these proteins throughout the mouse epididymides. In addition, while EDDM3B, KRT19 and WFDC8 were detected in both epididymal principal and clear cells (CCs), SLC27A2 was exclusively expressed in CCs. Finally, we showed that CFSE-fluorescently labeled epididymosomes interact with sperm in vitro and about 12-36% of the epididymosomes contain the targeted sperm proteins with an epididymal origin. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The human and mouse sample size was limited and our results were descriptive. The analyses of epididymal sperm and epididymosomes were solely performed in the mouse model due to the difficulties in obtaining epididymal luminal fluid human samples. Alternatively, human ejaculated sperm and seminal EVs could not be used because ejaculated sperm have already contacted with the fluids secreted by the male accessory sex glands, and seminal EVs contain other EVs in addition to epididymosomes, such as the abundant prostate-derived EVs. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that epididymosomes are capable of providing spermatozoa with a new set of epididymis-derived proteins that could modulate the sperm proteome and, subsequently, participate in the post-testicular maturation of sperm cells. Additionally, our data provide further evidence of the novel role of epididymal CCs in epididymosome production. Identifying mechanisms by which sperm mature to acquire their fertilization potential would, ultimately, lead to a better understanding of male reproductive health and may help to identify potential therapeutic strategies to improve male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competividad; fondos FEDER 'una manera de hacer Europa' PI13/00699 and PI16/00346 to R.O.; and Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109 to J.C.), by National Institutes of Health (grants HD040793 and HD069623 to S.B., grant HD104672-01 to M.A.B.), by the Spanish Ministry of Education, Culture and Sports (Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario, FPU15/02306 to F.B.), by a Lalor Foundation Fellowship (to F.B. and M.A.B.), by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337 to M.J.), by Fundació Universitària Agustí Pedro i Pons (to F.B.), and by the American Society for Biochemistry and Molecular Biology (PROLAB Award from ASBMB/IUBMB/PABMB to F.B.). Confocal microscopy and transmission electron microscopy was performed in the Microscopy Core facility of the Massachusetts General Hospital (MGH) Center for Systems Biology/Program in Membrane Biology which receives support from Boston Area Diabetes and Endocrinology Research Center (BADERC) award DK57521 and Center for the Study of Inflammatory Bowel Disease grant DK43351. The Zeiss LSM800 microscope was acquired using an NIH Shared Instrumentation Grant S10-OD-021577-01. The authors have no conflicts of interest to declare.

Accuracy of contrast-enhanced ultrasound in the detection of bladder cancer.

OBJECTIVE: To assess the accuracy contrast-enhanced ultrasound (CEUS) in bladder cancer detection using transurethral biopsy in conventional cystoscopy as the reference standard and to determine whether CEUS improves the bladder cancer detection rate of baseline ultrasound. METHODS: 43 patients with suspected bladder cancer underwent conventional cystoscopy with transurethral biopsy of the suspicious lesions. 64 bladder cancers were confirmed in 33 out of 43 patients. Baseline ultrasound and CEUS were performed the day before surgery and the accuracy of both techniques for bladder cancer detection and number of detected tumours were analysed and compared with the final diagnosis. RESULTS: CEUS was significantly more accurate than ultrasound in determining presence or absence of bladder cancer: 88.37% vs 72.09%. Seven of eight uncertain baseline ultrasound results were correctly diagnosed using CEUS. CEUS sensitivity was also better than that of baseline ultrasound per number of tumours: 65.62% vs 60.93%. CEUS sensitivity for bladder cancer detection was very high for tumours larger than 5 mm (94.7%) but very low for tumours <5 mm (20%) and also had a very low negative predictive value (28.57%) in tumours <5 mm. CONCLUSION: CEUS provided higher accuracy than baseline ultrasound for bladder cancer detection, being especially useful in non-conclusive baseline ultrasound studies.

Adenosquamous carcinoma of the head and neck: criteria for diagnosis in a study of 12 cases.

AIMS: Adenosquamous carcinoma (ASC) of the head and neck is an unusual neoplasm in which a general consensus with regard to diagnostic criteria has not yet been reached. In this study we report the clinicopathological results of 12 ASCs, with special attention to their histological and immunohistochemical characteristics in order to define this neoplasm more precisely. METHODS AND RESULTS: All the patients were male with a peak incidence in the sixth decade of life. The tumours were located most frequently in the larynx and oral cavity, followed by the nasal cavity and pharynx. ASCs had two distinct histological components. The most extensive one was an usual keratinizing squamous cell carcinoma, arising from the surface epithelium, where characteristically severe dysplasia or carcinoma in situ was found in all cases. The second component was an adenocarcinoma, usually displayed in the deepest areas of the tumour. Evidence of origin from salivary or seromucinous glands was not found. Immunohistochemical studies demonstrated in most cases positivity of glandular differentiated areas for carcinoembryonic antigen (CEA) (11/12), CK7 (9/12) and CAM5.2 (7/12), whereas the squamous cell component was unreactive or reacted only focally for these markers. High-molecular-weight cytokeratin 34BE12 was positive in both components and CK20 was always negative. All cases showed high expression of Ki67 antigen. Most of them had overexpression of p53 (8/12) and DNA aneuploidy (10/12). Fifty percent of patients with ASC died of disease after a mean period of 23 months (range 12-35 months). CONCLUSIONS: ASC of the head and neck is an aggressive neoplasm that originates in the surface epithelium of the upper respiratory tract. Severe dysplasia or carcinoma in situ is usually found and its recognition helps to make the diagnosis. In addition to mucin stains, positive immunoreactivity for CEA, CK7 and CAM5.2 helps to identify the glandular component.

Chromosomal high-polysomies predict tumour progression in T1 transitional cell carcinoma of the bladder.

UNLABELLED: The main prognostic factor generally accepted for tumour progression in T1 transitional cell carcinoma (TCC) of the bladder is histological grade. Despite this fact it is considered inaccurate to make clinical decisions on individuals. It appears that progression from minimally invasive to deeply invasive cancer is concurrent with the acquisition of genomic alterations that increase the malignant potential of cancer cells. The aim of this study is to determine if changes in chromosomes 7, 8, 9 and 17 copy number can be used to predict recurrence and progression in patients with T1 TCC of the urinary bladder. METHODS: Thirty-one T1 TCC samples were analyzed for chromosomal alterations by fluorescence in situ hybridization using centromeric probes for chromosomes 7, 8, 9 and 17. Clinical data were collected from the patients' clinical records and correlated with chromosomal studies. RESULTS: Histological grade was confirmed as a prognostic factor of tumour progression (p=0.01). None of the cytogenetic alterations demonstrated in the studied group could be related to tumour recurrence. The high-polysomies (five or more copies) of chromosomes 8, 9 and 17 showed predictive value (p=0.05, 0.05, 0.03 respectively) for tumour progression since it was observed that patients with high-polysomy of these chromosomes showed more risk of tumour progression towards muscle-invasive disease than those without high-polysomy alteration. CONCLUSION: Our findings suggest a possible prognostic significance of highly aneuploid cells (high-polysomies of chromosomes 8, 9 and 17) in tumour progression of T1 TCC bladder tumours. FISH analysis is a reproducible technique for evaluating cytogenetic alterations and could contribute to the assessment of the individual prognosis of T1 transitional cell carcinoma of the bladder.

Screening of selected genomic areas potentially involved in thyroid neoplasms.

Loss of heterozygosity (LOH) studies have been used to identify sites harbouring tumour suppressor genes (TSGs) involved in tumour initiation or progression. To further elucidate the genetic mechanisms for follicular and papillary thyroid tumours development, we studied the frequency of LOH in 36 thyroid tumours (21 follicular thyroid adenomas (FAs) and 15 papillary thyroid carcinomas (PTCs)) on 10 specific genomic areas: 3p22, 3p25, 7q21, 7q31, 10q23, 10q25-26, 11q13, 11q23, 13q13 and 17p13.3-13.2 using 20 polymorphic markers. We have selected these areas for two reasons: (a) Even though LOH in thyroid neoplasms has been described in some of these areas, results are controversial, and (b) we have also studied areas described as involved in other epithelial or endocrine tumour types, but not studied up to now in thyroid neoplasms. Two areas showed a high percentage of LOH: 7q31 and 11q23. A 62% LOH was found at 7q31 in the FAs, suggesting, as other authors have proposed, that at least one TSG must be present in the vicinity of the c-met locus. The second area in frequency was at the 11q23 locus, with a 45% LOH in the FAs. This area was studied because it has been described as being involved in the development of epithelial and endocrine cancers. This locus had not been studied before in thyroid neoplasms. This result is interesting because the LOH11CR2A gene is localised at this locus. We suggest that this gene and/or an other TSG nearby may be involved in the progression to FA. In our study, a low percentage of LOH was found in the PTC samples, indicating that TSGs present in the areas we have studied are not significantly involved in their progression. Our data also suggest that TSGs located in areas where no LOH was detected (PTEN, MEN1, Cyclin D1, BRCA2 and RFC3) are not involved or do not have an important role in tumour progression.

Leiomioma vesical / Leiomyoma of the bladder

Presentamos un nuevo caso de leiomioma vesical en una paciente con clínica inespecífica, y en la que las pruebas de imagen preoperatorias no lo orientaron. El diagnóstico definitivo lo dio el estudio anatomopatológico de la pieza quirúrgica (AU)

Differential expression of cdc25 cell-cycle-activating phosphatases in human colorectal carcinoma.

cdc25 is a family of cell-cycle phosphatases that activate the cyclin-dependent kinases. cdc25A and B, but not C, have oncogenic potential in vitro. In this study, we analyzed the possible implication of cdc25 genes in the progression of colorectal tumors. RNA and DNA were extracted from 34 paired tumor and normal colorectal tissues and examined by Northern blot, RT-PCR, and Southern blot, respectively. Protein expression was analyzed by Western blot in a subset of normal and tumor samples. The expression levels were correlated with the clinicopathologic characteristics and survival of the patients. cdc25B mRNA was overexpressed in 19 carcinomas (56%). A significant correlation was observed between high cdc25B mRNA levels and the relapse-free, overall, and cancer-related survival of the patients. The cdc25B2 splicing variant was detected in 27 carcinomas (79%) but only in 9 normal samples (26%) and was associated with the grade of the differentiation of the tumors. cdc25A mRNA was overexpressed in four tumors (12%) and cdc25C1 mRNA was overexpressed in nine tumors (26%). A new cdc25C2 splicing variant lacking exon 4 and 5 was identified in all of the tumors and in 56% of the normal samples. No amplifications or gene rearrangements of these genes were detected. In conclusion, these findings indicate that cdc25 isoforms and splicing variants are differentially regulated in colorectal carcinomas and may participate in the development of these tumors. Additionally, the correlation between cdc25B mRNA levels and the survival of the patients also suggest that the cdc25B isoform may be involved in the progression of the disease.

High-grade prostate intraepithelial neoplasia shares cytogenetic alterations with invasive prostate cancer.

BACKGROUND: High-grade prostate intraepithelial neoplasia (PIN) is the most likely precursor of prostate adenocarcinoma. However, the relationship between this lesion and prostate cancer has not yet been established. The detection of cytogenetic changes in the lesions prior to prostate adenocarcinoma would be useful in demonstrating such a pathogenic relationship. METHODS: Twenty eight high-grade PIN cases were found among 57 specimens of radical prostatectomy performed for clinically localized prostate cancer. Fluorescence in situ hybridization (FISH) analysis using centromeric probes to enumerate chromosomes 7, 8, 10, and 12 was performed to study the numerical chromosome alterations. FISH analysis was carried out over isolated nuclei obtained from high-grade PIN areas and prostate cancer foci in the same prostatectomy specimen. RESULTS: Of the 28 suitable cases it was possible to complete the study in 26 tumor and 20 PIN areas. The remaining cases were excluded because of insufficient tissue or poor preservation. Cytogenetic alterations (aneuploidy) were found in 16 of the 26 (62%) tumors studied. The most frequent chromosome alteration was trisomy 7, detected in 12 (75%) aneuploid tumors, followed by monosomy 8 present in 5 (31%) aneuploid tumors. Trisomy 7 was also the most frequent isolated chromosome alteration since it was detected in 7 (44%) tumors. Thirteen of 20 (65%) PIN cases were aneuploid when studied by FISH. Trisomy 7, trisomy 8, and monosomy 8 were the most common cytogenetic alterations in the 20 PIN areas studied, being observed in nine (45%), six (30%), and four (20%) cases, respectively. FISH analysis showed a high correlation (75% cases) in ploidy and pattern of cytogenetic alterations between high-grade PIN areas and the paired prostate cancer focus in the same specimen. CONCLUSIONS: The above results show a cytogenetic link between high-grade PIN and prostate cancer, suggesting that the former could be an early form of prostate cancer.

[Bladder leiomyoma]. / Leiomioma vesical.

A new case of leiomyoma of the bladder is presented in a patient with unspecific symptoms and the preoperatives patterns don't give to a certainty diagnosis. The conclusive diagnosis was obtained with pathoanatomical study of the quirurgic piece.

Rabdomioma de laringe: Un raro caso de disfonía. Revisión de la literatura / Laryngeal rhabdomyoma: a rare case of disphony. Review of the literature

Los rabdomiomas de laringe son tumores benignos muy raros. Sólo 25 casos han sido bien documentados hasta la actualidad. Se caracterizan por presentar un crecimiento lento. El diagnóstico se basa en los estudios inmunohistoquímicos y de microscopía elec trónica, distinguiéndose tres tipos histológicos: adulto, fetal y fe tal mixoide o genital. El tratamiento consiste en la extirpación quirúrgica. Se presenta un nuevo caso de rabdomioma tipo adulto y una revisión de la bibliografía (AU)

Rhabdomyomas of the larynx are extremely rare benign tumors. Only 25 have been well documented until now. These tumors display a low growing pattern. Diagnosis is based on immunocytochemical studies and electron microscopy. Three histological types are distinguished: adult, fetal and fetal myxoidal or genital. Surgery is the treatment of choice. A new case, adult type, is presented and the literature reviewed (AU)

Hipertensión arterial de larga evolución en un varón de 71 años diagnosticado de sarcoidosis / Long lasting hypertension in a 71 year-old man with sarcoidosis

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[Laryngeal rhabdomyoma: unusual case of dysphonia. Review of the literature]. / Rabdomioma de laringe: un raro caso de disfonía. Revisión de la literatura.

Rhabdomyomas of the larynx are extremely rare benign tumors. Only 25 have been well documented until now. These tumors display a low growing pattern. Diagnosis is based on immunocytochemical studies and electron microscopy. Three histological types are distinguished: adult, fetal and fetal myxoidal or genital. Surgery is the treatment of choice. A new case, adult type, is presented and the literature reviewed.

High-grade carcinoma component in epithelial-myoepithelial carcinoma of salivary glands clinicopathological, immunohistochemical and flow-cytometric study of three cases.

Three cases of epithelial-myoepithelial carcinoma (EMC) with coexisting areas of high grade carcinoma are reported. In two of the cases there was a previous recurrence, and in all three patients there had been a sudden increase in size before final surgery. The typical ductal and myoepithelial components of EMC showed the usual biphasic pattern and the expected immunophenotypes, with expression of wide spectrum cytokeratins, Cam 5.2 and EMA in the ductal part, and muscle-specific actin, smooth muscle actin, S-100 protein, vimentin and cytokeratins in the myoepithelial component. These areas also had a low mitotic count and low proliferation rate as measured by immunohistochemistry and by flow cytometry. Conversely, areas of high-grade tumour had the features of a large cell carcinoma, with focal mucin secretion in two cases. This high-grade component showed an epithelial immunophenotype in two cases, and was negative for all tested markers in the third one. The mitotic counts and the proliferation rates were much higher in these anaplastic areas. One of the patients died 3 months after treatment; another developed lymph node metastases 1 year later and was alive after 6 years of follow-up. The third patient was alive without evidence of disease 7 months after wide surgical resection of the tumour. The possibility of anaplastic transformation in EMC makes thorough sampling mandatory in this type of neoplasm.

Numeric alterations in chromosomes 7 and 8 detected by fluorescent in situ hybridization correlate with high-grade localized prostate cancer.

OBJECTIVE: To compare the ability of flow cytometry (FCM) and fluorescent in situ hybridization (FISH), using a small set of 4 enumeration chromosome probes to detect aneuploidy in prostate tumors, and to correlate it with histological grade and pathological stage. METHODS: Among 28 suitable cases, 21 could be analyzed by FISH and FCM techniques. DNA centromeric probes were used in FISH analysis to enumerate chromosomes 7, 8, 10 and 12. RESULTS: (a) Of the 21 cases studied by FISH, 5 were diploid, 14 aneuploid and 2 were tetraploid. When studied by FCM, these tumors were: 14 diploid, 6 aneuploid, and 1 tetraploid. FISH proved to have a higher ability for detecting DNA aneuploidy than FCM while been equally specific, since all tumors aneuploid by FCM were also found to be aneuploid by FISH. (b) Of the 14 aneuploid tumors, 12 were of high histological grade, while only 2 of the 7 nonaneuploid were of high grade. A statistically significant association was observed between high histological grade and FISH aneuploidy (p = 0.033). (c) All the aneuploid tumors showed chromosome 7 and/or 8 aneusomy. Trisomy 7 and monosomy 8 were the most frequent alterations present in 56 and 42% of the aneuploid tumors, respectively. CONCLUSION: FISH analysis of chromosome 7 and 8 alterations proved to be more sensitive than FCM in the detection of aneuploid prostate tumors. This aneuploidy was significantly associated with a poor pathological prognosis.

p21WAF1/Cip1 is associated with cyclin D1CCND1 expression and tubular differentiation but is independent of p53 overexpression in human breast carcinoma.

p21WAF1/Cip1 is an inhibitor of cdk/cyclin complexes, and thus regulates the cell cycle. p21 is also related to cell differentiation and is regulated by wild-type p53, although p53-independent regulatory pathways have been proposed. In order to analyse p21 expression as well as its relationship with p53 in human breast cancer, an immunohistochemical analysis was undertaken of 77 breast carcinomas, 16 of them with an in situ component; 30 adjacent normal tissue samples; and five non-neoplastic specimens. Forty-four infiltrating carcinomas (57 per cent) were p21-positive. Expression of p21 was also observed in pre-invasive lesions, whereas normal ducts were negative or focally and weakly positive. p21 expression was associated with high histological grade (II + III) (P = 0.017) and poor tubule formation (P = 0.002), and was significantly less frequent in lobular carcinomas (P = 0.0001). p21 positivity also correlated with increased proliferation, but this seemed to be dependent on the histological grade. Twenty carcinomas (26 per cent) showed p53 overexpression, but this was not associated with p21 negativity, suggesting the existence of p53-independent mechanisms for p21 regulation in vivo. Cyclin D1CCND1 expression was analysed in the same series and an association between p21 and cyclin D1 expression was found, since 23 of 26 cyclin D1-positive carcinomas were p21-positive (P < 0.001 ...). In conclusion, p21 is frequently overexpressed in breast carcinomas and this occurs in the early stages of neoplastic progression. This overexpression seems to be independent of p53 status and might be involved in cyclin D1 modulation.

cdc25 cell cycle-activating phosphatases and c-myc expression in human non-Hodgkin's lymphomas.

cdc25A, cdc25B, and cdc25C are a family of human phosphatases that activate the cyclin-dependent kinases at different points of the cell cycle. cdc25A and cdc25B have been shown to have oncogenic potential, and they have been identified as transcriptional targets of c-myc. To determine the role of cdc25 genes in the pathogenesis of human lymphomas and their possible correlation with c-myc deregulation, we have analyzed the expression of cdc25A, cdc25B, and cdc25C and c-myc genes in a series of 63 non-Hodgkin's lymphomas and 8 nonneoplastic lymphoid tissues. The mRNA levels of the three phosphatases in the nonneoplastic tissues were negative or negligible. cdc25B overexpression was detected in 35 tumors (56%). This overexpression was more frequently found in aggressive (81%) than in indolent lymphomas (36%; P < 0.01). cdc25B overexpression was also significantly associated with a higher proliferative activity of the tumors. No cdc25B gene amplification or rearrangements were detected by Southern blot analysis. A biallelic EcoRI polymorphism of cdc25B gene was identified with a similar distribution in patients with lymphoma and in a normal population. cdc25A was overexpressed in three aggressive lymphomas. No detectable cdc25C mRNA levels were seen in any of the tumors. c-myc was overexpressed in 43% of tumors, and it correlated significantly with the presence of cdc25B up-regulation. Twenty-six of 35 (74%) lymphomas with high levels of cdc25B mRNA also showed c-myc overexpression, whereas 27 of 28 (96%) tumors without detectable or with very low cdc25B expression also had undetectable c-myc levels (P < 0.0001). In addition, a significant linear correlation was found between the cdc25B and c-myc mRNA levels (r = 0.575, P < 0.001). These findings suggest that cdc25B overexpression in non-Hodkin's lymphoma may participate in the pathogenesis of aggressive variants, and it may cooperate with c-myc oncogene in the development of these tumors.