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.

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.

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.

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.

Acquired renal cystic disease in HD: a study of 82 nephrectomies in young patients.

In order to study the development of acquired renal cystic disease (ARCD) and its potential complications, we studied, macro- and microscopically, 82 surgical specimens of nephrectomy carried out on young patients with chronic renal failure previous to renal transplantation. Statistical correlation of pathological findings with age, sex and time on hemodialysis (HD) have been done. There were 72 cases of ARCD (87.8%). It was statistically correlated with male sex (p less than 0.02) and prolonged time on HD (p less than 0.001) as has been previously reported. Hyperplasia of the cystic epithelium was found in 42 cases (52%), with 18 (22%) showing marked papillary proliferation. Also, there were 22 cases (27%) with renal adenomas. This incidence of hyperplastic and neoplastic proliferations, more than would be common in such a young population (males: 33.5 +/- 9.3 years; females: 35.4 +/- 11.7 years), suggests the potential of patients affected by ARCD to develop neoplasms. Thus, we consider that these patients must be checked periodically to detect possible malignant neoplasms.

Expression of cytokeratins in squamous cell carcinomas of the larynx: immunohistochemical analysis and correlation with prognostic factors.

The expression of distinct cytokeratin subtypes in squamous cell carcinomas (SQCC) of the larynx was examined by immunohistochemistry with a panel of monoclonal antibodies (MABs) and different immunophenotypes were correlated with known prognostic factors, such as tumor site, local extension, degree of morphologic differentiation and lymph node metastasis. Among 50 cases of laryngeal SQCC tested, all 22 low grade tumors (Broders I and II) reacted strongly with MABs AE1, AE3 and KB.12, which corresponds to the phenotype of non-neoplastic squamous epithelium of the larynx. MABs K8.13 and K8.60 were negative only in 1 and 4 cases respectively. In these tumors CAM5.2 was either negative (18 cases) or weakly positive (4 cases). In contrast, we found that of 28 high grade SQCC (Broders III and IV) tested, strong reactivity with MABs AE1, AE3, K8.12 and K8.13 was restricted to smaller subsets, whereas CAM5.2 immunoreactivity was seen in 16 cases (57%). By morphological criteria 31 out of 50 cases in our series of SQCC showed keratinization and 30 of these 31 showed coexpression of cytokeratins identified by MABs AE3, K8.12, K8.13 and K8.60 and 29 cases by MAB AE1. The remaining non-keratinizing SQCC showed heterogeneous immunoreactivity for AE1, AE3 and K8.12 in 13 cases, for K8.13 in 12 cases and for K8.60 in 5 cases. Thirty of the 50 SQCC tested had no known lymph node metastasis and of these, 29 reacted with MABs AE1, AE3, K8.12 and K8.13 respectively, the remaining cases being either unreactive or only weakly reactive. Statistical analysis showed no significant differences between cytokeratins expression in SQCC and either anatomical location or local extension (pT).(ABSTRACT TRUNCATED AT 250 WORDS)

Myoepithelial tumors of salivary glands: a clinicopathologic, immunohistochemical, ultrastructural, and flow-cytometric study.

Myoepitheliomas of the salivary glands remain a controversial entity. To contribute to the knowledge of this entity, 16 myoepithelial tumors of the salivary glands were studied: 12 benign myoepitheliomas (BME) and 4 malignant myoepitheliomas (MME). The clinical and the histologic findings of each case were studied Immunohistochemistry and flow-cytometry analysis were performed from the paraffin-embedded material in 15 cases. An electron-microscopy study was performed in 8 cases. The myoepithelial tumors affected patients of both sexes equally. The mean age of the patients with BME was 54 years, and the mean age of patients with MME was 62 years. Eight cases of BME originated in the parotid gland and 4 cases originated in the minor salivary glands. All the MME developed from a benign preexistent tumor: two developed from a pleomorphic adenoma in the parotid gland, and the other two MME developed in the minor salivary gland from a BME. The myoepithelial tumors were composed of epithelioid, plasmacytoid, spindle, or clear cell types, and they showed a solid or a myxoid pattern of growth. Immunohistochemical studies revealed marked and diffuse positivity to cytokeratins, vimentin, and S-100 protein in all cases. Glial fibrillary acidic protein was positive in 8 cases (53%), and muscle-specific actin and smooth-muscle actin were positive in only 3 cases (20%); they were all cases of BME. Desmin was negative in all tumors. Ultrastructural studies showed the presence of basal membrane, tight junctions, intermediate filaments, and microvilli as well as actin-like filaments lacking focal densities in all cases. But actin-like filaments with focal densities were not identified. Flow cytometry determined that all BME were diploid with a mean proliferative index of 7.73%. Two of the MME were diploid and the other two MME were aneuploid. The mean proliferative index of MME was 11.93%. In conclusion, BME and MME originated in major and minor salivary glands can display different histologic patterns and cellular features. Some immunohistochemical and ultrastructural characteristics have been found in all these neoplasms, which supports the idea that myoepitheliomas are composed by neoplastic modified myoepithelial cells, not fully differentiated. These techniques can be useful for the diagnosis of these tumors.

Primary adenocarcinoma of the endometrium associated with genital tuberculosis. A case report.

BACKGROUND: Adenocarcinoma of the endometrium is a common tumor of the female reproductive tract, and tuberculosis accounts for only about 1% of postmenopausal metrorrhagia. Tuberculosis of the fallopian tubes is the most frequent location; that of the cervix is the most uncommon. CASE: A case of primary adenocarcinoma of the endometrium was diagnosed after curettage in a 58-year-old, postmenopausal woman complaining of a bloody vaginal discharge. A laparotomy was performed, and histologic examination of the uterus disclosed an endometrial carcinoma, grade 2/3, with extension to both tubes. The tumor was associated with an extensive granulomatous reaction, with numerous tubercles composed of epithelioid cells, multinucleate giant cells of Langhans and central necrosis type. These granulomas were seen close to the carcinoma in the endometrium and tubes and far from the neoplasm in sections of the cervix, myometrium and lymph nodes. CONCLUSION: The association of endometrial adenocarcinoma with genital tuberculosis is extremely rare. Moreover, similar granulomas may appear in the surrounding stroma of the tumor. For these reasons a diagnosis of genital tuberculosis associated with this neoplasm should be made with caution.

[Carcinoma of the kidney as a complication of acquired cystic disease of the kidney in patients subjected to treatment with periodical hemodialysis]. / Carcinoma renal como complicación de la enfermedad renal quística adquirida en pacientes sometidos a tratamiento con hemodiálisis periódica.

Three cases of acquired cystic renal disease (ACRE) are reported in patients treated with periodical hemodialysis (HD) who developed neoplastic degeneration. In the first patient, diagnosis was a chance finding of nephrectomy during renal transplantation. The second patient was discovered following hematuria, and in the third patient disseminated metastases were the presenting feature of the disease. The finding of renal tumours associated with ACRE is a well known fact; however, metastatic disease has been infrequently reported. Generally, the denomination "adenoma" is adopted for tumours with a maximal diameter shorter than 3 cm, as they seldom develop metastases. However, the third patient had an adenocarcinoma measuring 2 cm in diameter. One of the parameters that best correlates the occurrence of ACRE and tumour development is the duration of HD; therefore, it is suggested that those patients who have received substitution therapy for longer than 3 years should undergo radiological studies for the early identification of these complications.

[A cellular proliferation study in patients with colorectal cancer, solitary, synchronous and with polyps]. / Estudio de la proliferación celular en pacientes con cáncer colorrectal solitario, sincrónico y con pólipos.

Colonic carcinogenesis is probably related to a disturbance in cell proliferation of the colonic mucosa. The present study was designed to determine whether patients with adenomatous polyps of the colon, which have a tendency to develop synchronous malignancies of the colon, have any disturbance in mucosal cell proliferation. Using flow cytometry the proliferative index, and the S and G2M phases of normal mucosa and tumoral tissue of patients with colorectal cancer (synchronous, alone, or associated with adenomatous polyps) were studied. No differences were found between the there groups of study at the level of proliferation pattern of normal mucosa. Our findings do not support the development of synchronous or metachronous colon cancer in patients with polyps on the basis of different patterns of cell proliferation.

[DNA as a prognostic factor in colorectal cancer. A retrospective analysis of 34 patients]. / Estudio del DNA como factor pronóstico en cáncer colo-rectal. Análisis retrospectivo de 34 pacientes.

OBJECTIVE: We study the tumoral DNA as a prognostic factor in patients operated for colo-rectal cancer. DESIGN: Retrospective. We analyze by flow cytometry the DNA content of tumoral tissue: Ploidy, DNA Index, Proliferative Index, S Phase. PATIENTS: 34 operated patients, for colo-rectal cancer, with a minimum 3 years of follow-up. RESULTS: Ploidy doesn't show any relation with other prognostic factors. Only Dukes C aneuploid patients had more positive nodes than diploid. Ploidy was not related to better evolution or survival. Neither DNA Index, Proliferative Index and S Phase were related to the evolution of the disease. CONCLUSIONS: The DNA study by flow cytometry has no better prognostic value then other classical factors.

[Changes in the cellular proliferation of the healthy mucosa in patients with colorectal cancer]. / Alteraciones de la proliferación celular de la mucosa sana en pacientes con cáncer colorrectal.

AIM: To analyze differences in the proliferative pattern of normal mucosa between patients with colorectal cancer and healthy subjects. PATIENTS AND METHODS: By using flow cytometry we examined the proliferative pattern (Proliferative Index and S-Phase) in samples of normal mucosa from 34 patients operated on for colorectal cancer at our hospital and from 14 healthy subjects as a control group. RESULTS: All examined samples were diploid. The Proliferative Index and S-Phase in the Cancer Group were significantly higher than in the Control Group (16.7 +/- 5.9 vs 11.4 +/- 4, p < 0.003, and 11.9 +/- 3.9 us 6.6 +/- 3 p < 0.0004). CONCLUSIONS: These findings provide evidence for an altered proliferative pattern in the healthy colonic mucosa of patients with colorectal 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.

[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.

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.