The fact that optical force is very significant in the microscopic world and can be used to manipulate microparticles has triggered an evolution in micromanipulation, in particular, the manipulation of biological species and colloidal particles. The induced optical force can easily be more than 103 times of the particle's weight. The particle size that are accessible to optical forces ranges from tens of nanometers to hundreds of micrometers. One of the most well-known tools in optical manipulation is called optical tweezers, which is, in essence, performing optical trapping by a strongly focused light beam. The optical force induced by the incident light wave can be generally decomposed into two mathematically and physically distinct components, namely the conservative (gradient force) and non-conservative (scattering and absorption force) forces. Such a split helps in the study of optical forces and elucidates the underlying physics (e.g., the optical trapping). For example, in optical trapping, the conservative gradient force drives the particles toward the intensity maxima and traps the particles there, whereas the non-conservative scattering and absorption force tends to push the particles away and thus has some destabilizing effects. However, while a significant portion of paper dealing with optical trapping explicitly mentioned gradient and scattering forces, the true and exact force profiles of the decomposed optical forces have been mysteries for decades. Researchers still use these concepts, and to certain extent, they imagine the force profile according to their own convenience. This thesis is mainly devoted to the analytical and numerical studies of the decomposition of optical forces. The intrinsic nature of the decomposed optical forces will be discussed, and the approaches of generating a purely conservative force field are presented.. First, the analytical approaches for decomposing the optical force into the gradient force and the scattering and absorption force are described. These approaches can be applied to different particle sizes (smaller than 40% of the wavelength if the multipoles are only considered up to the electric octopole or much larger than the wavelength under the geometrical optics limit), but they still cannot describe the experimentally accessible particle size, which is on the order of micrometer. Second, within the dipole limit, the origin of scattering force is shown to be resulted from the radiation reaction, the polarizations, and the topological charges. In addition, it is found that the conservativeness of the force is closely related to the force constant matrix (the linear term in the Taylor expansion of the optical force) at every point, and certain symmetries in these force constant matrix can guarantee the force to be conservative.. A numerical method that utilizes the fast Fourier transform (FFT) was developed to decompose the conservative and non-conservative forces. This approach is valid when the total force field is spatially localized and decayed sufficiently fast as we move away from the beam center (e.g., optical tweezers or alike) or is spatially periodic (e.g. plane incident waves). We also considered spherical aberration due to the mismatch of the refractive indices between the oil and water media in a typical optical tweezers setup within the FFT method. Various particle sizes, materials, and numerical apertures were also considered. For the periodic force field generated by a collection of plane waves, it is demonstrated that an incident 2-dimensional standing wave could generate a purely conservative force field. The accuracy of this fast Fourier transform approach is analyzed in details and shown to be quite accurate. Moreover, an incident 3-dimensional standing wave could also induce a conservative force field for intermediately sized particles.. Finally, three counter-intuitive examples obtained with the fast Fourier transform approach are presented. These examples clearly demonstrated the need to calculate the gradient and scattering forces accurately, as not doing so would lead to qualitatively wrong results.

Eight functionally identified group Ia muscle afferents from triceps surae or plantaris muscles were labeled intraaxonally with horseradish peroxidase (HRP) in seven adult cats. Subsequently, HRP was injected into two to six homonymous or heteronymous α-motoneurons per animal (total = 22), each identified by motor unit type and located near the site of afferent injection. The complete trajectories of labeled afferents were reconstructed, and putative synaptic contacts on HRP-labeled motoneurons were identified at high magnification. Dendritic paths from each contact were also mapped and measured. A total of 24 contact systems (the combination of a group Ia afferent and a postsynaptic motoneuron) were reconstructed, of which 17 were homonymous, and seven were heteronymous. Overall, homonymous contact systems had an average of 9.6 boutons, whereas heteronymous contact systems had an average of 5.9 boutons. The average number of boutons found on type S motoneurons in homonymous contact systems was smaller (6.4, range 3-17) than in systems involving types FF or FR motoneurons (FF: 10.4, range 4-18; FR: 11.3, range 4-32). Neither of these differences were statistically significant. In contrast to earlier reports, a majority (15/24) of contact systems included more than one collateral from the same Ia afferent. The complexity (number of branch points) in the arborization pathway leading to each contact (overall mean 8.4 ± 3.3) was virtually identical in all contact systems, irrespective of the type of the postsynaptic motoneuron. The three- dimensional distribution of group Ia contacts was not coextensive with the radially organized dendrites of motoneurons: Dendrites oriented in the ventromedial to dorsolateral axis had the fewest (8%) contacts, whereas rostrocaudal dendrites had the most (63%) contacts. Nevertheless, contacts were widely distributed on the motoneuron surface, with few on and near the soma (≤200 μm radial distance from the soma) or on the most distal parts of the tree (≤1,000 μm). The boutons in individual contact systems also showed wide spatial and estimated electrotonic distributions; only 3/24 systems had all contact located within a restricted spatial/electrotonic region. The relations between these anatomical results and existing electrophysiological data on group Ia synaptic potentials are discussed.

No description available.

Different types of alumina containing coatings were made on lithiumand copper in the purpose to mechanically hinder the growth ofdendrites. Lithium, coated with polymer-alumina composites wereplaced in symmetric cells for in situ studies by a light microscope.The coatings did not block the dendrites, but they did change thegrowth rate and morphology of them, probably throw both chemicalinteractions and changes in ion transportation. Also the stability ofcapacity were tested for the same coatings, the result showed abigger capacity drop for cells containing coated lithium versus cellswithout coatings.Attempted alumina coatings were also made by a solgel technique, bydirect reaction with the compound trimethylaluminium and with analumina containing acetonitrile solution.The theses also includes a study of the stability of lithium inadiponitrile. A higher amount of LiTFSI salt in adiponitrile could bythis study be reported to inhibit the dissolution of lithium that wasseen for lower salt concentrations. The dissolution appeared when thesolution was used as an electrolyte in a symmetric lithium cell. Somedifferences could be seen when the lithium surface were studied byXPS after interaction with high, low and zero concentration LiTFSI.Both the XPS studies and the absences of lithium dissolutionindicates that a more or less stable SEI had been formed.

Multicellular development in fungi is fundamentally different from that of animals or plants. In filamentous fungi, multicellular structures are formed by aggregation and adhesion of hyphae, followed by septation and specialisation of hyphal compartments within the aggregate. The perithecium, a flask-shaped sexual fruitbody produced by both Neurospora crassa and Sordaria macrospora, provides a model system in which to study fungal multicellular development. This study presents a detailed description of the morphological stages of perithecial morphogenesis in N. crassa and S. macrospora and its early stages, the ascogonial and protoperithecial stages, using a range of microscopical techniques. Details of the development of several mutants impaired in perithecial development are described, including: gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; and three mutants pro22, pro40 and pro41 of S. macrospora, and their corresponding gene-deletions in N. crassa. The results confirm that all three MAP kinase cascades are required for sexual development. However, only the pheromone response and cell-wall integrity MAP kinase pathways, but not the osmoregulatory MAP kinase pathway, are essential for hyphal cell fusion. Evidence of cell fusion-related processes, regulated through MAP kinase signalling, have been identified as novel features important for the construction of fertilisable protoperithecia. These cell-fusion related processes include extracellular matrix deposition, hyphal attachment and envelopment. A novel phenotype of S. macrospora with defective ascogonial septation is presented. This pro22 mutant also has impaired hyphal cell fusion and produces only small, defective protoperithecia. The pro22 gene encodes a protein that is highly conserved throughout eukaryotes. Live-cell imaging revealed that this PRO22 protein is localised in the dynamic tubular and vesicular vacuolar-network of the colony periphery and in ascogonia. PRO22 is absent from the large spherical vacuoles in the vegetative hyphae of the sub-peripheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar-network. Furthermore, the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.

No description available.

Canola (Brassica napus) contributes $19.3 billion dollars to the Canadian economy each year as a result of its oil- and protein-rich seeds. These economically important seed products are produced in highest concentration in the embryo. Embryo development is supported nutritionally and structurally by the maternal subregions, which include the inner (ISC) and outer distal seed coat (OSC), the chalazal seed coat (CZSC), and the chalazal proliferating tissue (CPT). Research on the maternal seed subregions is limited to the SC as a result of its accessibility; the embedded CZSC and CPT subregions have yet to be characterized in canola. Using light and transmission electron microscopy, I found the CZSC and CPT to be anatomically distinct and experience profound changes throughout seed development. To understand these changes at the RNA level, laser microdissection and RNA sequencing were used to profile these subregions spatially and temporally from the ovule to mature green stage of seed development. Employing vigorous bioinformatics analyses, I found that the maternal subregions are transcriptomically distinct and possess unique RNA populations. From here I began to elucidate the biological processes operating within the maternal subregions. As a whole, the maternal subregions appear to have a critical role in transporting nutrients to the filial subregions as well as in coping with oxidative stress produced during these energy-rich processes. Additionally, using CanEnrich, I was able to generate predictive transcriptional circuits regulating the biological processes occurring within the maternal seed. This research has produced the most comprehensive dataset on the canola seed to date and will provide a valuable resource for research on seed development as well as seed improvement. / October 2016

Introdução: O fuso meiótico dos oócitos de mamíferos pode se despolimerizar quando exposto a pequenas variações de temperatura. Este fato já está bem estabelecido e estudado em oócitos maduros em metáfase II (MII). No entanto, pouco se sabe a respeito da influência da diminuição da temperatura sobre o fuso meiótico dos oócitos imaturos. Desse modo, este estudo tem como objetivos: 1) avaliar a influência da diminuição da temperatura sobre o fuso meiótico de oócitos de camundongas maturados in vitro e 2) avaliar o fuso meiótico em oócitos humanos maturados in vitro submetidos à criopreservação pela técnica de congelação lenta ou por vitrificação quando em estágio de vesícula germinativa. Métodos: Realizaram-se dois experimentos, denominados 1 e 2, sendo o primeiro em oócitos de camundongas e o segundo em oócitos humanos. No experimento 1 oócitos imaturos de camundongas nos estágios de metáfase I (MI), telófase I(TI) e MII foram cultivados nas seguintes temperaturas: 37º C (controle), temperatura ambiente (22oC) e 4º C por 0, 10, 30 e 60 minutos. Após este período de tempo o fuso meiótico oocitário foi avaliado por meio de microscopia de luz polarizada (MLP) (LC-Polscope-Oosight image software) e imunocitoquímica (IC). No experimento 2 oócitos em estágio de vesícula germinativa (GV) coletados de pacientes submetidas à indução da ovulação e fertilização in vitro, foram divididos de forma randômica em três grupos: oócitos a fresco (A), oócitos congelados pela técnica de congelação lenta (B) e oócitos congelados pela técnica de vitrificação (C). Os oócitos a fresco, os descongelados e os aquecidos foram maturados in vitro até estágio de (MII). A análise do fuso meiótico foi realizada por microscópio invertido equipado com uma câmera de vídeo analógica e um sistema de imagens que combina luz polarizada em cristal líquido (ICSI Guard Octax). Resultados: Experimento 1: No tempo 0 e à 37º C, todos os oócitos apresentavam o fuso meiótico visível tanto pela MLP quanto pela IC. À 4º C, o número de oócitos em MI com fuso meiótico visível por meio da MLP foi menor do que com a IC, e descresceu com o tempo, fato que também ocorreu, em menor proporção, com os oócitos em TI. No entanto, a 4º C, o reconhecimento do fuso meiótico dos oócitos em TI foi semelhante tanto para MLP como para IC. Quando os oócitos MII foram expostos à 4º C, a detecção do fuso meiótico teve descréscimo diretamente proporcional ao tempo de cultura quando foi utilizada a MLP, sendo que o mesmo ocorreu para a IC, porém de forma menos pronunciada. À temperatura ambiente houve um pequeno descéscimo na visualização do fuso meiótico tanto por MLP quanto por IC, porém este não foi estatisticamente significativo para os oócitos em TI. Experimento 2: A taxa de sobrevivência imediatamente após o descongelamento/ aquecimento foi de 44,6% para o grupo B e de 79% para o grupo C. Após 24 horas em cultura , estas taxas passaram para 29,2% e 69%, respectivamente. A mediana de tempo para maturação foi de 26 horas para os grupos A e C, e de 27 horas para o grupo B. Ao final da maturação in vitro a porcentagem de oócitos em MII foi menor no grupo B e semelhante nos grupos A e C. Assim como para a detecção do fuso meiótico que foi menor no grupo B e similar nos grupos A e C. Conclusões: Houve diferença na porcentagem de despolimerização do fuso meiótico em resposta à baixa temperatura entre os oócitos de camundongas nos diferentes estágios da divisão meiótica, sendo menor nos oócitos em TI. A porcentagem de despolimerização do fuso meiótico foi diretamente proporcional ao tempo de cultivo, à exceção dos oócitos em TI à temperatura ambiente. Os oócitos hmanos em GV vitrificados apresentaram melhores taxas de sobrevivência quando comparados com oócitos humanos em GV criopreservados pelo congelamento lento. Os oócitos humanos em GV vitrificados apresentaram taxas semelhantes de maturação in vitro e detecção do fuso meiótico polimerizado quando comparados a oócitos a fresco / Introduction: The meiotic spindle of most mammals is sensitive to cooling and depolymerizes even after a slight reduction in temperature. This is well described and studied on matured oocytes at metaphase II (MII). However, little is known about the influence of low temperatures under meiotic spindle of imature oocytes. In this way, we sougth to evaluate: 1) the influence of low temperatures on mice oocyte meiotic spindle matured in vitro e 2) the oocyte meiotic spindle from human oocytes matured in vitro and cryopreserved by slow-rate freezing or vitrification at GV stage. Methods: Two experiments were done: the first one on mice and the second one on women.At experiment 1, immature mice oocytes at metaphase I (MI), telophase I (TI) and MII were cultured at 37º C (control), room temperature (22oC) and 4º C for 0, 10, 30 and 60 minutes and then spindle analysis was made with polarized light microscopy (PLM) (LC-Polscope-Oosight image software) or immunocytochemistry (ICC). At experiment 2, GV oocytes retrieved from women submitted to ovulation induction and in vitro fertilization were randomly divided in three groups: fresh oocytes (A), cryopreserved by slow-freezing (B) and cryopreserved by vitrification (C). Fresh, thawed and warmed oocytes were matured in vitro to metaphase II oocytes (MII). A meiotic spindle analysis was done by polarized light microscopy (ICSI Guard Octax). Results: Experiment 1: At time 0 min and 37º C, all oocytes had polymerized spindles both at PLM or ICC. At 4º C, the number of MI oocytes with detectable spindles at PLM was smaller than those analysed by ICC, and it decreased with time, which had also occured with TI oocytes at a smaller proportion. However, at 4º C, TI meiotic spindle recognition with polarized light microscopy and ICC was comparable. When MII oocytes were cultured at 4º C, the spindle visualization decreased proportionally in correlation with culture time at PLM, and the same happened with ICC in a less pronounced manner. At room temperature there was a little descrease regarding visualization of meiotic spindle, both at PLM and ICC, altought it was not significant for TI oocytes. Experiment 2: Oocyte survival immediately after thawing/warming were 44.6% for group B and 79% for group C. After 24 hours of culture, oocyte survival was 29.2% and 69%, respectively. The median time for maturation was 26 hours for groups A and C, and 27 hours for group B. The percentage of MII after maturation in vitro were smaller in group B and similar between groups A and C. The same oocured for spindle visualization which were lower in group B and similar between groups A and C. Conclusions: There was a difference on the percentages of meiotic spindle depolymerization in response to cooling in mice oocytes at different stages of meiotic division. Spindle depolymerization was lower in TI. Also, meiotic spindle depolimerization was proportional to culture time, except for TI oocytes at room temperature.Vitrified GV oocytes had a better survival when warmed, compared to slow-rate frozen oocytes. Vitrified GV oocytes had similar maturation in vitro rates and polymerized spindles detection when compared to fresh oocytes

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-10-23T18:30:38Z No. of bitstreams: 1 2013 - Enely Maris da Silveira Firmiano.pdf: 6302423 bytes, checksum: d767e55220a0e40f0b8ee86826ca1a3e (MD5) / Made available in DSpace on 2018-10-23T18:30:38Z (GMT). No. of bitstreams: 1 2013 - Enely Maris da Silveira Firmiano.pdf: 6302423 bytes, checksum: d767e55220a0e40f0b8ee86826ca1a3e (MD5) Previous issue date: 2013-02-18 / CAPES / Phrynops geoffroanus is a freshwater turtle popularly known as Geoffroy's toadhead turtle or Geoffroy's side-necked turtle. Like other species of the Testudines order, it is oviparous, meaning the females lay their eggs in the environment. The objectives of this article are to describe the morphology of the oviduct of P. geoffroanus as observed through light microscopy after performing standard histochemical techniques (AB pH 2.5, PAS, Mallory?s trichrome, xylidine Ponceau), besides identifying the endocrine cells in the various regions of the oviduct that produce motilin, serotonin and somatostatin through immunohistochemistry. The oviduct of this turtle is composed of five regions: the infundibulum, which receives the oocyte released at the moment of oocytation; the tube uterine (magnum), the spiraled region that produces the albumen; the isthmus, a transition region; the uterus, responsible for producing the egg shell; and the vagina, the final portion of the oviduct, which leads to the cloaca. The structure of the oviduct of P. geoffroanus is similar to that of other oviparous reptile species. Its description can be used for phylogenetic morphological comparisons. The immunohistochemistry study revealed the absence of neuroendocrine cells that produce motilin, serotonin and somatostatin in all regions of the oviduct. However, the existence of these cells in the gut of this turtle was verified, enabling the suggestion that the regulatory peptides produced by these cells are carried by the bloodstream and reach specific receptors on target cells located along the oviduct, to regulate the peristaltic movements of this organ. / Phrynops geoffronus ? uma esp?cie de r?ptil representante da ordem dos Testudines, popularmente conhecida como c?gado-de-barbichas ou c?gado-de-barbelas. Como os demais representantes desta ordem, ? uma esp?cie ov?para, ou seja, as f?meas colocam ovos no ambiente durante a reprodu??o. Os objetivos deste trabalho foram a descri??o histol?gica do oviduto das f?meas de P. geoffroanus com aux?lio da microscopia de luz, ap?s ser submetido ?s t?cnicas histol?gicas e histoqu?micas (AB pH 2,5, PAS, Tricr?mico de Mallory, Xylidine Ponceau), al?m de procurar identificar c?lulas end?crinas produtoras de motilina, serotonina e somatostatina atrav?s da imuno-histoqu?mica, ao longo dos diversos segmentos do oviduto. O oviduto deste c?gado ? composto por cinco diferentes regi?es: infund?bulo, que recebe o ov?cito liberado no momento da ovocita??o; a tuba uterina (magno), regi?o espiralada produtora do alb?men; o istmo, uma regi?o de transi??o; o ?tero, respons?vel pela produ??o da casca do ovo, e a vagina, por??o final do oviduto que leva ? cloaca. A estrutura do oviduto de P. geoffroanus ? semelhante ? de outras esp?cies de r?pteis ov?paros e pode ser utilizada para compara??es morfol?gicas filogen?ticas. O estudo imuno-histoqu?mico revelou aus?ncia de c?lulas neuroend?crinas produtoras de motilina, serotonina e somatostatina em todas as regi?es do oviduto da esp?cie estudada. Entretanto, a exist?ncia dessas c?lulas foi verificada no intestino deste c?gado (teste-controle), tornando poss?vel sugerir que provavelmente os pept?deos regulat?rios produzidos por tais c?lulas, sejam transportados pela corrente sangu?nea e atinjam receptores espec?ficos em c?lulas-alvo localizadas ao longo do oviduto para atuarem na regula??o dos movimentos perist?lticos deste ?rg?o.

Introdução: O fuso meiótico dos oócitos de mamíferos pode se despolimerizar quando exposto a pequenas variações de temperatura. Este fato já está bem estabelecido e estudado em oócitos maduros em metáfase II (MII). No entanto, pouco se sabe a respeito da influência da diminuição da temperatura sobre o fuso meiótico dos oócitos imaturos. Desse modo, este estudo tem como objetivos: 1) avaliar a influência da diminuição da temperatura sobre o fuso meiótico de oócitos de camundongas maturados in vitro e 2) avaliar o fuso meiótico em oócitos humanos maturados in vitro submetidos à criopreservação pela técnica de congelação lenta ou por vitrificação quando em estágio de vesícula germinativa. Métodos: Realizaram-se dois experimentos, denominados 1 e 2, sendo o primeiro em oócitos de camundongas e o segundo em oócitos humanos. No experimento 1 oócitos imaturos de camundongas nos estágios de metáfase I (MI), telófase I(TI) e MII foram cultivados nas seguintes temperaturas: 37º C (controle), temperatura ambiente (22oC) e 4º C por 0, 10, 30 e 60 minutos. Após este período de tempo o fuso meiótico oocitário foi avaliado por meio de microscopia de luz polarizada (MLP) (LC-Polscope-Oosight image software) e imunocitoquímica (IC). No experimento 2 oócitos em estágio de vesícula germinativa (GV) coletados de pacientes submetidas à indução da ovulação e fertilização in vitro, foram divididos de forma randômica em três grupos: oócitos a fresco (A), oócitos congelados pela técnica de congelação lenta (B) e oócitos congelados pela técnica de vitrificação (C). Os oócitos a fresco, os descongelados e os aquecidos foram maturados in vitro até estágio de (MII). A análise do fuso meiótico foi realizada por microscópio invertido equipado com uma câmera de vídeo analógica e um sistema de imagens que combina luz polarizada em cristal líquido (ICSI Guard Octax). Resultados: Experimento 1: No tempo 0 e à 37º C, todos os oócitos apresentavam o fuso meiótico visível tanto pela MLP quanto pela IC. À 4º C, o número de oócitos em MI com fuso meiótico visível por meio da MLP foi menor do que com a IC, e descresceu com o tempo, fato que também ocorreu, em menor proporção, com os oócitos em TI. No entanto, a 4º C, o reconhecimento do fuso meiótico dos oócitos em TI foi semelhante tanto para MLP como para IC. Quando os oócitos MII foram expostos à 4º C, a detecção do fuso meiótico teve descréscimo diretamente proporcional ao tempo de cultura quando foi utilizada a MLP, sendo que o mesmo ocorreu para a IC, porém de forma menos pronunciada. À temperatura ambiente houve um pequeno descéscimo na visualização do fuso meiótico tanto por MLP quanto por IC, porém este não foi estatisticamente significativo para os oócitos em TI. Experimento 2: A taxa de sobrevivência imediatamente após o descongelamento/ aquecimento foi de 44,6% para o grupo B e de 79% para o grupo C. Após 24 horas em cultura , estas taxas passaram para 29,2% e 69%, respectivamente. A mediana de tempo para maturação foi de 26 horas para os grupos A e C, e de 27 horas para o grupo B. Ao final da maturação in vitro a porcentagem de oócitos em MII foi menor no grupo B e semelhante nos grupos A e C. Assim como para a detecção do fuso meiótico que foi menor no grupo B e similar nos grupos A e C. Conclusões: Houve diferença na porcentagem de despolimerização do fuso meiótico em resposta à baixa temperatura entre os oócitos de camundongas nos diferentes estágios da divisão meiótica, sendo menor nos oócitos em TI. A porcentagem de despolimerização do fuso meiótico foi diretamente proporcional ao tempo de cultivo, à exceção dos oócitos em TI à temperatura ambiente. Os oócitos hmanos em GV vitrificados apresentaram melhores taxas de sobrevivência quando comparados com oócitos humanos em GV criopreservados pelo congelamento lento. Os oócitos humanos em GV vitrificados apresentaram taxas semelhantes de maturação in vitro e detecção do fuso meiótico polimerizado quando comparados a oócitos a fresco / Introduction: The meiotic spindle of most mammals is sensitive to cooling and depolymerizes even after a slight reduction in temperature. This is well described and studied on matured oocytes at metaphase II (MII). However, little is known about the influence of low temperatures under meiotic spindle of imature oocytes. In this way, we sougth to evaluate: 1) the influence of low temperatures on mice oocyte meiotic spindle matured in vitro e 2) the oocyte meiotic spindle from human oocytes matured in vitro and cryopreserved by slow-rate freezing or vitrification at GV stage. Methods: Two experiments were done: the first one on mice and the second one on women.At experiment 1, immature mice oocytes at metaphase I (MI), telophase I (TI) and MII were cultured at 37º C (control), room temperature (22oC) and 4º C for 0, 10, 30 and 60 minutes and then spindle analysis was made with polarized light microscopy (PLM) (LC-Polscope-Oosight image software) or immunocytochemistry (ICC). At experiment 2, GV oocytes retrieved from women submitted to ovulation induction and in vitro fertilization were randomly divided in three groups: fresh oocytes (A), cryopreserved by slow-freezing (B) and cryopreserved by vitrification (C). Fresh, thawed and warmed oocytes were matured in vitro to metaphase II oocytes (MII). A meiotic spindle analysis was done by polarized light microscopy (ICSI Guard Octax). Results: Experiment 1: At time 0 min and 37º C, all oocytes had polymerized spindles both at PLM or ICC. At 4º C, the number of MI oocytes with detectable spindles at PLM was smaller than those analysed by ICC, and it decreased with time, which had also occured with TI oocytes at a smaller proportion. However, at 4º C, TI meiotic spindle recognition with polarized light microscopy and ICC was comparable. When MII oocytes were cultured at 4º C, the spindle visualization decreased proportionally in correlation with culture time at PLM, and the same happened with ICC in a less pronounced manner. At room temperature there was a little descrease regarding visualization of meiotic spindle, both at PLM and ICC, altought it was not significant for TI oocytes. Experiment 2: Oocyte survival immediately after thawing/warming were 44.6% for group B and 79% for group C. After 24 hours of culture, oocyte survival was 29.2% and 69%, respectively. The median time for maturation was 26 hours for groups A and C, and 27 hours for group B. The percentage of MII after maturation in vitro were smaller in group B and similar between groups A and C. The same oocured for spindle visualization which were lower in group B and similar between groups A and C. Conclusions: There was a difference on the percentages of meiotic spindle depolymerization in response to cooling in mice oocytes at different stages of meiotic division. Spindle depolymerization was lower in TI. Also, meiotic spindle depolimerization was proportional to culture time, except for TI oocytes at room temperature.Vitrified GV oocytes had a better survival when warmed, compared to slow-rate frozen oocytes. Vitrified GV oocytes had similar maturation in vitro rates and polymerized spindles detection when compared to fresh oocytes