RESUMO
Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes. The volume of both the female and the male pronucleus was found to correlate with the volume of the oocyte cytoplasm. The NC ratio of the male pronucleus was greater than that of the female pronucleus. The NC ratio of the average volume of the female and male pronuclei was greater than the NC ratio of the mononucleate oocytes. The occurrence of 1PN oocytes was significantly higher when the volume of cytoplasm was lower than the cut-off value. These results indicated that the NC ratio is retained during pronuclear formation. A higher NC ratio in male compared with the female pronucleus indicated structural and/or molecular difference between the two pronuclei. 1PN formation may occur when sperm enters close to the MII spindle.
Assuntos
Fertilização in vitro , Zigoto , Animais , Núcleo Celular , Citoplasma , Feminino , Masculino , Camundongos , OócitosRESUMO
Ovarian cancers include several disease subtypes and patients often present with advanced metastatic disease and a poor prognosis. New biomarkers for early diagnosis and targeted therapy are, therefore, urgently required. This study uses antibodies produced locally in tumor-draining lymph nodes (ASC probes) of individual ovarian cancer patients to screen two separate protein microarray platforms and identify cognate tumor antigens. The resulting antigen profiles were unique for each individual cancer patient and were used to generate a 50-antigen custom microarray. Serum from a separate cohort of ovarian cancer patients encompassing four disease subtypes was screened on the custom array and we identified 28.8% of all ovarian cancers, with a higher sensitivity for mucinous (50.0%) and serous (40.0%) subtypes. Combining local and circulating antibodies with high-density protein microarrays can identify novel, patient-specific tumor-associated antigens that may have diagnostic, prognostic or therapeutic uses in ovarian cancer.
Assuntos
Adenocarcinoma de Células Claras/diagnóstico , Adenocarcinoma Mucinoso/diagnóstico , Antígenos de Neoplasias/imunologia , Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Cistadenocarcinoma Seroso/diagnóstico , Neoplasias Ovarianas/diagnóstico , Adenocarcinoma de Células Claras/sangue , Adenocarcinoma de Células Claras/imunologia , Adenocarcinoma Mucinoso/sangue , Adenocarcinoma Mucinoso/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/sangue , Autoanticorpos/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Cistadenocarcinoma Seroso/sangue , Cistadenocarcinoma Seroso/imunologia , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/imunologia , Prognóstico , Análise Serial de Proteínas , Adulto JovemRESUMO
Tumor antigens are responsible for initiating an immune response in cancer patients, and their identification may provide new biomarkers for cancer diagnosis and targets for immunotherapy. The general use of serum antibodies to identify tumor antigens has several drawbacks, including dilution, complex formation, and background reactivity. In this study, antibodies were generated from antibody-secreting cells (ASC) present in tumor-draining lymph nodes of 20 breast cancer patients (ASC-probes) and were used to screen breast cancer cell lines and protein microarrays. Half of the ASC-probes reacted strongly against extracts of the MCF-7 breast cancer cell line, but each with a distinct antigen recognition profile. Three of the positive ASC-probes reacted differentially with recombinant antigens on a microarray containing cancer-related proteins. The results of this study show that lymph node-derived ASC-probes provide a highly specific source of tumor-specific antibodies. Each breast cancer patient reacts with a different antibody profile which indicates that targeted immunotherapies may need to be personalized for individual patients. Focused microarrays in combination with ASC-probes may be useful in providing immune profiles and identifying tumor antigens of individual cancer patients.
RESUMO
OBJECTIVE: Cryopreservation of embryos and oocytes has become an essential service for infertility treatment. The clinical application of this technology should ensure optimal survival of the embryos and oocytes that are stored and subsequently thawed for transfer. The aim of this review is to compare the widely employed slow cooling procedures with vitrification to evaluate and recommend the more effective and safer procedure. DESIGN: The review is mainly based on a comparison of the principles, procedures, and results reported in the literature. A historical description of vitrification and personal experiences with this technology are also included. SETTING: University-based hospitals and private clinics that treat infertility and have published information on cryopreservation. PATIENT(S): Women being treated for infertility and reproductive technology clinics. INTERVENTION(S): The application of slow cooling involving a range of cooling rates is compared with vitrification using rapid and ultrarapid cooling in simple containers. The purpose of both techniques is the induction of a glasslike state in cells to protect them from damage by ice crystals. The early development of vitrification involved the use of long pre-equilibration procedures. Improved methods resulted from the use of mixtures of penetrating and nonpenetrating solutes that are not toxic and a range of cooling rates. MAIN OUTCOME MEASURE(S): Reported number of pregnancies established after transfer of embryos that were cryopreserved by vitrification, or transfer of embryos derived from vitrified oocytes. RESULT(S): Both slow cooling and vitrification procedures have resulted in the successful cryopreservation of human embryos and oocytes. Both procedures have resulted in healthy births, although the slow cooling of oocytes gives very low success rates. Vitrification is a promising novel technique in assisted reproductive technology, but comparative success rates are yet to be established. CONCLUSION(S): Vitrification is a simple procedure that requires less time and is likely to become safer and more cost effective than slow cooling.
Assuntos
Criopreservação/métodos , Embrião de Mamíferos , Oócitos , Animais , Fertilização in vitro/métodos , HumanosRESUMO
OBJECTIVE: To determine whether leiomyoma, adenomyosis and endometrial polyps are associated with changes in uterine cavity matrix metalloproteinases (MMP-2 and MMP-9) and cytokines. STUDY DESIGN: Uterine cavity irrigation was performed in women with leiomyoma, adenomyosis and endometrial polyps, and in women with a normal uterus. MMP-2 and MMP-9 were assayed in the uterine washings by gelatin zymography. For individual subjects, the total MMP level was obtained by adding the semi-quantitative scores of band densities related to gelatinases in the zymograms. Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1) were measured using enzyme-linked immunosorbant assay (ELISA) kits. RESULTS: The uterine cavity of patients with leiomyoma, adenomyosis and endometrial polyps had significantly higher MMP scores than controls. Although the mean IL-1beta levels were elevated in uteri harboring a pathology compared with the normal uteri, the cytokine was significantly elevated only in the adenomyotic group. Significantly elevated levels of IFN-gamma were found in uteri with leiomyoma and endometrial polyps. Uterine washings from leiomyoma and adenomyosis contained significantly elevated mean levels of TGF-beta1 compared with controls, while TNF-alpha was significantly higher only in leiomyoma. When uterine cytokine levels were compared in relation to individual MMP levels a significant relationship was found between TGF-beta1 and elevated levels of MMP-9 and total MMPs in leiomyoma. A significant relationship was also found between IL-1beta and elevated levels of MMP-2, MMP-9 and total MMPs in the endometrial polyp group. CONCLUSION: The uterine cavity in leiomyoma, adenomyosis and endometrial polyps contains elevated levels of MMPs and cytokines compared with the normal uterus. In some pathologies elevated cytokines are associated with elevated MMPs.
Assuntos
Citocinas/análise , Metaloproteinases da Matriz/análise , Doenças Uterinas/metabolismo , Útero/química , Útero/enzimologia , Neoplasias do Endométrio/metabolismo , Endometriose/metabolismo , Feminino , Humanos , Interferon gama/análise , Interleucina-1/análise , Leiomioma/metabolismo , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Pólipos/metabolismo , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/análise , Neoplasias Uterinas/metabolismoRESUMO
OBJECTIVE: The known overexpression of matrix metalloproteinases (MMPs) by various tumors prompted a study to determine whether endometrial cancer could be detected by measuring MMPs in uterine cavity washings. METHODS: The study populations comprised 95 women being treated for endometrial cancer and 98 women with other gynecological conditions. A simple method was developed for performing uterine lavage and preparing cell-free uterine supernatants for MMP analysis. Gelatin zymography revealed elevated levels of latent and active forms of MMP-2 and MMP-9 in patients with endometrial cancer. For each patient individual bands of gelatinase activity were scored from 0 to 5 and summed to provide a total MMP score for analysis. RESULTS: The mean MMP score in uterine washings of patients with endometrial cancer was 10.0 (range 1 to 22) compared with 0.8 (range 0 to 15) in the group without this cancer (P < 0.001). Receiver operating characteristic analysis showed that an MMP cutoff score of 3 gave a sensitivity of 98% and specificity of 91% for detecting endometrial cancer. With this MMP cutoff, a positive result was 11 times as likely in endometrial cancer compared to other conditions. The mean MMP score in the group with nodal metastases (14.1) was significantly higher than that without nodal involvement (9.4, P = 0.005). MMP-9 but not MMP-2 was significantly associated with nodal metastasis (P = 0.01). There was no significant association between MMP score and histological grade of tumor, vascular invasion, or depth of myometrial invasion. CONCLUSION: Gelatinase measurement in uterine washings was reliable for confirming the presence of endometrial cancer in the population studied.
Assuntos
Carcinoma Endometrioide/enzimologia , Neoplasias do Endométrio/enzimologia , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Adulto , Carcinoma Endometrioide/patologia , Neoplasias do Endométrio/patologia , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Irrigação TerapêuticaRESUMO
BACKGROUND: The actions of the extracellular-matrix degrading enzymes, matrix metalloproteinases (MMPs), are implicated in tumorigenesis. The cellular localization of MMP-2, MMP-9, membrane type 1 (MT1)-MMP, tissue inhibitors of metalloproteinases (TIMPs) 1-3, and the presence of active gelatinases were investigated in endometrial carcinoma. METHODS: Endometrial carcinomas were grouped according to histologic grade (Grades 1-3), depth of myometrial invasion (0, < 50%, > 50%) and the presence of vascular/lymphatic invasion. Twenty-nine endometrial carcinoma biopsies were investigated immunohistochemically to determine the tissue localization of MMP-2 (gelatinase A), MMP-9 (gelatinase B), MT1-MMP, and TIMPs 1-3. In situ hybridization was performed to localize MMP-2 and MMP-9 mRNA. The presence of active gelatinases was assessed using in situ zymography. RESULTS: Epithelial tumor cells were the main site of MMP-2, MMP-9, and MT1-MMP protein. Variable stromal cell localization was also observed, particularly in areas adjacent to tumor nests. Semiquantitative analysis revealed increases in MMP-9 and MMP-2 but not MT1-MMP staining scores in tumor epithelial cells in the transition from histologic Grade 1 to Grades 2 and 3. Matrix metalloproteinase-9 and MT1-MMP staining scores in tumor cells were significantly associated with the presence of myometrial invasion and vascular/lymphatic invasion, while MMP-2 did not correlate with these factors. In addition, MT1-MMP was co-localized with MMP-2, supporting its role in the activation of proMMP-2. Tumor cells from all histologic grades stained intensely for TIMP-2 and TIMP-3 proteins, while variable stromal staining was observed. In Grade 1 carcinomas TIMP-1 was predominantly immunolocalized to the stromal compartment with variable tumor cell localization being observed in Grades 2 and 3 carcinomas. Matrix metalloproteinase-9 and MMP-2 mRNAs were predominantly observed in tumor epithelial cells as well as in the stroma to varying degrees. In situ zymography revealed active forms of gelatinases at the cellular surface and in association with tumor epithelial cells within endometrial carcinoma tissues. CONCLUSIONS: These data suggest that increasing expression of MMPs and endometrial carcinoma progression are closely related. Active gelatinases are present in endometrial carcinoma, resulting in alterations to the microenvironment that promote tumor invasion and metastasis.