Análise invasiva e não invasiva do fuso meiótico de oócitos humanos obtidos de ciclos estimulados: dados preliminares

OBJETIVOS: Avaliar a concordância entre as técnicas de microscopia de polarização e microscopia confocal na avaliação do fuso meiótico de oócitos humanos maturados in vivo. MÉTODOS: Estudo prospectivo que avaliou oócitos com o primeiro corpúsculo polar extruído obtidos de mulheres inférteis submetidas à estimulação ovariana para realização de injeção intracitoplasmática de espermatozoide. Os oócitos com o primeiro corpúsculo polar extruído foram avaliados por meio da microscopia de polarização e, imediatamente após, foram fixados e corados para avaliação dos microtúbulos e cromatina pela microscopia confocal de alto desempenho. Foram comparadas as técnicas de microscopia de polarização e confocal, de acordo com a visualização ou não do fuso meiótico pela microscopia de polarização e a presença ou não de anomalias meióticas à análise pela microscopia confocal. Foram calculados os intervalos de confiança, o índice de Kappa e a concordância entre as metodologias, considerando a análise da microscopia de imunofluorescência como padrão-ouro para avaliação de normalidade do fuso e distribuição cromossômica oocitária. RESULTADOS: Observou-se que 72,7% dos oócitos em metáfase II com fuso celular não visível à polarização apresentaram anormalidades meióticas à análise confocal e que 55,6% dos oócitos em metáfase II com fuso celular visível à polarização apresentaram-se como oócitos anormais à análise confocal. Somente 44,4% dos oócitos com fuso celular visível à polarização apresentaram-se como normais à análise confocal. A concordância entre os métodos foi de 51,1% (Kappa: 0,11; IC95% -0,0958 - 0,319). CONCLUSÕES: A baixa concordância entre a microscopia de polarização e a confocal na avaliação do fuso meiótico oocitário sugere que a visualização do fuso meiótico de oócitos humanos em metáfase II pela microscopia de polarização tem limitado o valor preditivo de normalidade meiótica oocitária.

Oocyte meiotic-stage-specific differences in spindle depolymerization in response to temperature changes monitored with polarized field microscopy and immunocytochemistry

Objective: To compare the polymerization status of mouse oocyte spindles exposed to various temperatures at various stages of meiosis. Design: Experimental animal study. Setting: University animal laboratory. Animal(s): CF1 mice. Intervention(s): Immature oocytes matured to metaphase I (MI), telophase I (TI), and metaphase II (MII) were incubated at 37 degrees C (control), room temperature (RT), or 4 degrees C for 0, 10, 30, and 60 minutes. Spindle analysis subsequently was performed using polarized field microscopy and immunocytochemistry. Spindles of TI and MII oocytes that underwent vitrification and warming were analyzed also by immunocytochemistry. Main Outcome Measure(s): Detection of polymerized meiotic spindles. Result(s): At RT, and after 60 minutes at 4 degrees C, a significant time-dependent decrease in the percentage of polymerized meiotic spindles was observed in MI and MII oocytes, but not in TI oocytes. The polymerization of TI spindles at 4 degrees C was similar to that of TI spindles at 4 degrees C that underwent vitrification and warming. Conclusion(s): Significant differences in the microtubule dynamics of MI, TI, and MII oocytes incubated at different temperatures were observed. In particular, meiotic spindles in TI oocytes exhibited less depolymerization than did metaphase spindles. (Fertil Steril (R) 2012; 97: 714-9. (C) 2012 by American Society for Reproductive Medicine.)

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

Meiotic segregation and interchromosomal effect in the sperm of a double translocation carrier: a case report

Abstract Background Infertility is a natural mechanism of selection intended to prevent the delivery of a child with malformations or mental retardation. Male infertility is closely related to chromosomal abnormalities. This study was focused on the analysis of meiotic segregation involving a Robertsonian translocation, 45,XY,der(13;13) [56]/45,XY,der(13;14) [44] and the evaluation of possible interchromosomal effects. Results Hybridisation with LSI 13q14 and subtelomere 14q probes and WCP13 SpectrumGreen and WCP14 SpectrumOrange probes showed a high proportion of unbalanced gametes, corresponding to 71.2% of the spermatozoa. The disomic frequencies of the sexual chromosomes and chromosome 18 of the patient were higher (5.28% and 2.55%, respectively) than those of the control (0.6% and 0.59%, respectively). Conclusion Meiotic segregation studies in sperm are an important tool for genetic counselling of chromosomal aberrations, allowing for a prediction of the risks and consequent implications for the reproductive life. The patient with this rare translocation exhibited meiotic segregation fidelity, and a high rate of unbalanced gametes with disomic spermatozoa.

The imaging and pathological features of a mucinous tubular and spindle cell carcinoma of the kidney: a case report

A mucinous tubular and spindle cell carcinoma (MTSCC) is a rare and recently described kidney neoplasm with distal nephron differentiation. It can affect patients of all ages and is more prevalent among women. In this case report, we present a 50-year-old woman who had a renal mass, which was accidently discovered during an investigation for chronic anemia. The final diagnosis of MTSCC was made after the lesion was removed and a pathology work-up was performed. The clinical, pathological and imaging findings of this rare neoplasm are described in this report.

Spermatogenesis in goats with and without scrotum bipartition

The objective of the present research was to quantify the seminiferous epithelium cells, spermatogenesis efficiency and characterize the ultrastrucure of Sertoli cells in goats. Eighteen goats were used and divided into three groups: Group I - goats without bipartition of the scrotum; Group II - animals with bipartition of the scrotum in up to 50% of the testicular length; Group III - goats with bipartition of the scrotum in more than 50% of the testicular length. The goat testes in Group III had a greater number of primary spermatocytes (25.37 +/- 4.55 cells per cross sections), spermatids (112 +/- 15.12 cells per cross sections), and Sertoli cells (9.46 +/- 1.74 cells per cross sections) than the animals in Groups land (P <0.05). The spermatogenic mitotic, meiotic, and general efficiency were greater in animals in Group III (1.25 +/- 0.28; 5.12 +/- 1.63; 6.44 +/- 1.96) when compared to those in Groups I and II. Sheet-like processes originated from the Sertoli cell body as simple and smooth structures which involved almost all the surface of germ cells. Slender cord-like processes originated from Sertoli cells and also from the sheet-like processes. The relative frequency of the cycle stages showed differences among the groups of goats studied, and the highest frequency was in Stage 3 (20.68% for goats in Group I, 21.15% for those in Group II, and 16.89% for the animals in Group III). In conclusion, goats with bipartition of the scrotum have a greater number of germ and Sertoli cells per cross section of seminiferous tubule, that indicated a greater sperm production when compared to the other groups, and the ultrastructure of the Sertoli cell process did not present any relationship with bipartition of the scrotum. (C) 2012 Elsevier B.V. All rights reserved.

Predictive models for mutations in mismatch repair genes: implication for genetic counseling in developing countries

Background: Lynch syndrome (LS) is the most common form of inherited predisposition to colorectal cancer (CRC), accounting for 2-5% of all CRC. LS is an autosomal dominant disease characterized by mutations in the mismatch repair genes mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), postmeiotic segregation increased 1 (PMS1), post-meiotic segregation increased 2 (PMS2) and mutS homolog 6 (MSH6). Mutation risk prediction models can be incorporated into clinical practice, facilitating the decision-making process and identifying individuals for molecular investigation. This is extremely important in countries with limited economic resources. This study aims to evaluate sensitivity and specificity of five predictive models for germline mutations in repair genes in a sample of individuals with suspected Lynch syndrome. Methods: Blood samples from 88 patients were analyzed through sequencing MLH1, MSH2 and MSH6 genes. The probability of detecting a mutation was calculated using the PREMM, Barnetson, MMRpro, Wijnen and Myriad models. To evaluate the sensitivity and specificity of the models, receiver operating characteristic curves were constructed. Results: Of the 88 patients included in this analysis, 31 mutations were identified: 16 were found in the MSH2 gene, 15 in the MLH1 gene and no pathogenic mutations were identified in the MSH6 gene. It was observed that the AUC for the PREMM (0.846), Barnetson (0.850), MMRpro (0.821) and Wijnen (0.807) models did not present significant statistical difference. The Myriad model presented lower AUC (0.704) than the four other models evaluated. Considering thresholds of >= 5%, the models sensitivity varied between 1 (Myriad) and 0.87 (Wijnen) and specificity ranged from 0 (Myriad) to 0.38 (Barnetson). Conclusions: The Barnetson, PREMM, MMRpro and Wijnen models present similar AUC. The AUC of the Myriad model is statistically inferior to the four other models.

Oral Lesions in Renal Transplant

To prevent rejection of kidney transplants, patients must be kept in immunosuppressive therapy for a long time, which includes the use of drugs such as cyclosporine, azathioprine, cyclophosphamide, and prednisone. The action of these drugs reduces the general immune response of transplant patients and thus increases their susceptibility to infections. Moreover, these drugs increase the potential of developing lesions. Therefore, oral hygiene in kidney transplant recipients contributes to maintenance of the transplanted organ and its function. Thus, an investigation of oral lesions could be counted as a notable work. The aim of this study was to investigate oral lesions in a group of 21 kidney transplant patients under immunosuppressive therapy attended during a 1-year period in the Nephrology Department of the Federal University of Sergipe, Brazil. Data related to sex, age, etiology of renal disease, types of renal transplant, time elapsed after transplantation, immunosuppressive treatment, use of concomitant agents, and presence of oral lesions were obtained. All patients received a kidney transplant from a living donor, and the mean posttransplantation follow-up time was 31.6 months; 71.5% used triple immunosuppressive therapy with cyclosporine A, azathioprine, and prednisone. Ten patients were also treated with calcium-channel blockers. Of the 21 transplant patients, 17 (81%) presented oral lesions. Gingival overgrowth was the most common alteration, followed by candidiasis and superficial ulcers. One case of spindle cell carcinoma of the lower lip was observed. Oral cavity can harbor a variety of manifestations related to renal transplantation under immunosuppressive therapy.

Clinicopathologic and immunohistochemical features of oral neurofibroma

Objective. The purposes of this study were to assess clinical, histopathological and immunohistochemical features of 22 oral neurofibromas (NFs) and discuss with previously described literature, addressing the main aspects regarding the differential diagnosis. Materials and methods. Immunohistochemical reactions included S-100, CD34, GLUT-1, EMA, Ki-67, p53 and Collagen IV and histochemical reactions for Alcian blue. Results. Clinically, the preferential location was the maxillary bones, tongue and buccal mucosa. Microscopically, widely spread spindle-shaped cells with scant cytoplasm and elongated nuclei were observed. Immunostaining revealed that the tumor cells weakly expressed GLUT-1, Collagen IV, Ki-67 and p53. They were variably positive for CD34, S-100 protein and membrane epithelial antigen (EMA). Conclusions. The different types of nerve sheath cells observed in the present series reinforce the presence of heterogeneous population in NFs. The strong positivity for S-100 suggests that the lesions were more composed by S-100-positive Schwann cells than other cells. Besides, the high number of CD34-positive cells suggests that this marker can be useful for the differential diagnosis of NFs against PEN, traumatic neuromas and Schwannomas. Finally, the low immunostaining for p53 and Ki-67 may indicate that NFs massively composed by S-100-positive Schwann cells present low potential of aggressiveness and malignant transformation.

Crossing over does occur in males of Drosophila ananassae from natural populations

Spontaneous crossing over in males of Drosophila ananassae has been well demonstrated using F-1 individuals from crosses between marker stocks and wild type strains. However, the question of its occurrence in males from natural populations remained open. Here we present the cytological evidence that crossing over does occur in males of D. ananassae from two Brazilian populations, sampled nearly 21 years apart, and in two recently sampled populations, one from Indonesia and one from Okinawa, Japan. Cytological analysis of meiosis in males collected from nature and in sons of females from the same population inseminated in nature revealed the presence of chiasmata, inversion chiasmata, and isosite chromosome breakages in the diplotene cells in all sampled populations. These data demonstrate that reciprocal and nonreciprocal exchanges and chromosome breakages, previously reported as related events of male crossing over, do occur at variable frequencies among males from natural populations.

Cutaneous Fibroma in a Captive Common Snapping Turtle (Chelydra serpentina)

An adult female common snapping turtle (Chelydra serpentina) had a mass on the plantar surface of the right forelimb that was removed surgically. Microscopical examination revealed many spindle cells with mild anisocytosis and anisokaryosis and a surrounding collagenous stroma. There were no mitoses. Immunohistochemistry showed that the spindle cells expressed vimentin, but not desmin. A diagnosis of cutaneous fibroma was made. Tumours are reported uncommonly in chelonian species. Cutaneous fibroma has been diagnosed in an alligator snapping turtle (Macrochelys temminckii), but not previously in a common snapping turtle. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Infantile Encephaloneuromyopathy and Defective Mitochondrial Translation Are Due to a Homozygous RMND1 Mutation

Defects of mitochondrial protein synthesis are clinically and genetically heterogeneous. We previously described a male infant who was born to consanguineous parents and who presented with severe congenital encephalopathy, peripheral neuropathy, myopathy, and lactic acidosis associated with deficiencies of multiple mitochondrial respiratory-chain enzymes and defective mitochondrial translation. In this work, we have characterized four additional affected family members, performed homozygosity mapping, and identified a homozygous splicing mutation in the splice donor site of exon 2 (c.504+1G>A) of RMND1 (required for meiotic nuclear division-1) in the affected individuals. Fibroblasts from affected individuals expressed two aberrant transcripts and had decreased wild-type mRNA and deficiencies of mitochondrial respiratory-chain enzymes. The RMND1 mutation caused haploinsufficiency that was rescued by overexpression of the wild-type transcript in mutant fibroblasts; this overexpression increased the levels and activities of mitochondrial respiratory-chain proteins. Knockdown of RMND1 via shRNA recapitulated the biochemical defect of the mutant fibroblasts, further supporting a loss-of-function pathomechanism in this disease. RMND1 belongs to the sif2 family, an evolutionary conserved group of proteins that share the DUF155 domain, have unknown function, and have never been associated with human disease. We documented that the protein localizes to mitochondria in mammalian and yeast cells. Further studies are necessary for understanding the function of this protein in mitochondrial protein translation.

Cancer cell proliferation is inhibited by specific modulation frequencies

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology. British Journal of Cancer (2012) 106, 307-313. doi:10.1038/bjc.2011.523 www.bjcancer.com Published online 1 December 2011 (C) 2012 Cancer Research UK

Phospholipase D2:A Pivotal Player Modulating RBL-2H3 Mast Cell Structure

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells. (J Histochem Cytochem 60:386-396, 2012)