Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-04-10T18:18:36Z No. of bitstreams: 1 2012 - Marcus Andr? Ferreira S?.pdf: 393876 bytes, checksum: 9d184a085854acf9828da371a7e2b732 (MD5) / Made available in DSpace on 2017-04-10T18:18:36Z (GMT). No. of bitstreams: 1 2012 - Marcus Andr? Ferreira S?.pdf: 393876 bytes, checksum: 9d184a085854acf9828da371a7e2b732 (MD5) Previous issue date: 2012-07-17 / CAPES / The present experiment aimed to verify if different vacuum pump negative pressures (150, 280 e 400 mmHg) can influence the oocyte recovery rate per preovulatory follicle aspirated. Hence, 21 estrous cycles from regularly cycling mares were subject to OPU. The estrous cycles were sorted in three groups (G150=150 mmHg; G280=280 mmHg; G400=400 mmHg) in the following sequence: G150= 150mmHg (n=6); G280= 280mmHg (n=7); G400= 400mmHg (n=6). During the estrus, the ovarian activity of the mares was daily monitored using the transrectal ultrasound technique until the largest follicle reached at least a diameter of 35mm and endometrial edema secore 2.5 was identified on ultrasonographic evaluation, when 1000UI of hCG administered intravenuous. Approximately 24 hours after the hCG administered, the mares were monitored by rectal palpation and ultrasonography every six hours to follicle evaluation. In the case of imminent indication of ovulation or formation of hemorrhagic follicle, the follicle would be immediately aspirate. The aspirations occurred in 32,45?1,92h after the hCG administration. The transvaginal aspirations were performed with ultrasound apparatus equipped with a convex transducer of 5,0mHz with polyethylene guide containing a double lumen needle of 12G. The follicular fluid collected from each follicle was frozen and the aspirated content was transferred to a Petri Dishes and thoroughly examined on the stereomicroscope to identify the oocytes presence. In order to statistically evaluate the influence of different pressures on the oocyte recovery, were used Chi-Squared test (a 5% significance) and Fisher Exact Test, when recommended. The recovery rate was 31,57% (6/19), being 16,66 % (1/6) in G150, 42,85 % (3/7) in G280 and 33,33 % (2/6) in G400. There was no difference among groups (p>0,05). From the results of the current study, it is possible to conclude that the negative pressure of the vacuum pump is not a determining factor to increase the oocyte recovery and other aspects would possibly have more significant influence . / O presente experimento visou investigar se diferentes press?es negativas da bomba de v?cuo (150, 280 e 400mmHg) podem influenciar a taxa de recupera??o oocit?ria por fol?culo preovulat?rio aspirado. Para tanto, foram submetidos a ovum pick up 21 ciclos estrais de ?guas ciclando regularmente, distribu?dos em tr?s grupos (G150= 150 mmHg; G280= 280 mmHg; G400= 400 mmHg), na seguinte ordem: G150= 150 mmHg (n=6); G280= 280 mmHg (n=7); G400= 400 mmHg (n=6), definida por meio de sorteio. Durante estro, a atividade ovariana das ?guas foi monitorada diariamente atrav?s da t?cnica ultrassonogr?fica transretal at? que o maior fol?culos atingisse pelo menos 35mm de di?metro e edema endometrial grau 2,5 durante a avalia??o ultrassonogr?fica, quando ent?o administrou-se 1000UI de hCG, por via endovenosa. Aproximadamente 24 horas ap?s a administra??o de hCG as ?guas foram submetidas a exame ultrassonogr?fico a cada seis horas para avalia??o folicular. Caso houvesse indica??o iminente de ovula??o ou forma??o de fol?culo hemorr?gico, o mesmo seria imediatamente aspirado. As aspira??es ocorreram em 32,45?1,92h ap?s a aplica??o do hCG fazendo uso de ultrassom equipado com um transdutor convexo de 5,0mHz com guia de polietileno contendo uma agulha de duplo l?men de 12G. O fluido folicular coletado de cada fol?culo foi congelado e o conte?do aspirado transferido para uma Placa de Petri e examinado minuciosamente ao estereomicrosc?pio para localiza??o dos o?citos. Para avaliar estatisticamente o efeito das diferentes press?es sobre a recupera??o oocit?ria, foi utilizado o teste Qui-Quadrado (a 5% de signific?ncia) e Fisher Exato, quando recomendado. A taxa de recupera??o foi de 31,57% (6/19), sendo 16,66 % (1/6) no G150, 42,85 % (3/7) no G280 e 33,33 % (2/6) no G400. N?o houve diferen?a entre os grupos (p>0,05). Atrav?s dos resultados obtidos no presente estudo ? poss?vel concluir que a press?o negativa da bomba de v?cuo utilizada n?o ? o determinante para elevar a recupera??o oocit?ria, possivelmente havendo outros fatores atuando de modo mais importante.

Este estudo foi realizado com o objetivo de: 1) determinar a influência da tensão de oxigênio na viabilidade das células do cumulus oriundas de oócitos caninos maturados em meio com alta concentração de glicose (11,0 mM); 2) determinar a taxa de maturação in vitro de oócitos caninos mantidos em meio contendo altas concentrações de glicose (11,0 mM), bem como avaliar o efeito da tensão de oxigênio sobre a taxa de maturação in vitro de oócitos caninos; 3) determinar a influência da adição de água de coco em pó (ACP-318®) ao meio de maturação com alta concentração de glicose (11,0 mM) na taxa de maturação in vitro de oócitos caninos. O meio de maturação usado nos experimentos foi TCM 199 suplementado com 26,19 mM de bicarbonato de sódio, 50 μg/ml de gentamicina, 0,20 mM de ácido pirúvico, 20 μg/ml de estradiol, 0,5 μg/ml de FSH e 0,03 UI/ml hCG. O meio de maturação era modificado de acordo com a proposta experimental apresentada. Os resultados do primeiro experimento mostraram diferença estatística nas taxas de apoptose nas células do cumulus entre os três grupos avaliados. Foi concluído que células do cumulus oriundas de COCs caninos cultivados em meio contendo altas concentrações de glicose apresentaram significativamente menos apoptose do que as cultivadas em meio com soro fetal bovino, e que a baixa tensão de oxigênio foi eficiente em reduzir a ocorrência de apoptose nas células do cumulus. No segundo experimento deste estudo, os índices mais elevados (p < 0,001) de oócitos degenerados foram obtidos, quando TCM 199 era suplementado com 10 % de FCS. Uma influência positiva sobre a retomada de meiose e aquisição de metáfase I (MI) foi observada quando os oócitos caninos foram maturados em meio livre de soro e com altas concentrações de glicose. Foi concluído que a adição de FCS ao meio de maturação de oócitos caninos resulta em altas taxas de degeneração dos oócitos e que a redução da tensão de oxigênio não resultou em melhora da taxa de maturação nuclear dos oócitos caninos. Os resultados obtidos no terceiro experimento mostraram que nos grupos experimentais cujo meio foi suplementado com ACP-318®, houve melhora significativa (p < 0,05) na taxa de maturação nuclear dos oócitos caninos. Oócitos maturados em meio suplementado com 5% de ACP-318® mostraram-se menos susceptíveis a alterações na configuração típica da cromatina que os maturados com 10% de ACP-318® no meio de maturação. Os resultados sugerem que tanto a integridade da morfologia nuclear, como a progressão da meiose dos oócitos caninos são positivamente influenciadas quando estes são expostos ao TCM 199 com alta concentração de glicose e suplementado com 5% de ACP-318®. / This study was designed: 1) determine the influence of oxygen tension on cumulus cell (CC) viability from canine oocytes in vitro matured in high glucose medium (11.0 mM); 2) determine the influence of two oxygen tensions on the nuclear maturation of canine oocytes in vitro matured in high glucose medium (11.0 mM); 3) determine the influence of powdered coconut water (ACP-318®) diluted in high glucose (11.0 mM) TCM199 in the achievement of nuclear in vitro maturation (IVM) of canine oocytes. Basic medium used in the experiments was TCM 199 suplemented with 26.19 mM sodium bicarbonate, 50 μg/ml gentamicin, 0.20 mM piruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml FSH and 0.03 IU/ml hCG. Maturation medium was modified following the beyond described experimental proposals. The results of the first experiment showed that there was statistical difference in the rate of CCs apoptosis among the three groups. It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O2 tension was efficient in reducing apoptosis in canine CCs. In the second experiment of this study, the highest rates (p < 0.001) of degenerated oocytes were achieved when TCM 199 was added with 10% FCS. A positive influence on the meiosis resumption and on the MI acquisition rate was observed when canine oocytes were matured in defined high-glucose medium. It was concluded that the addition of FCS in the maturation medium of canine oocytes result in a high level of degenerated oocytes, and that the low level of oxygen tension did not improve the nuclear maturation of canine oocytes. The results achieved on the third experiment showed that in the experimental groups with the medium supplemented with ACP-318® (groups 2 and 3) enhanced the rates of oocytes’ nuclear maturation (p < 0.05). Ooocytes matured in medium added with 5% powdered coconut water (ACP-318®) were less prone to deviation of typical chromatin configuration than those matured in medium added with 10% ACP-318®. The results suggest that oocytes’ nuclear morphology integrity and meiosis achievement were positively influenced when exposed to high glucose TCM199 supplemented with 5% powdered coconut water.

Poor egg quality is the leading cause of pregnancy loss and Down's syndrome. While even eggs in young women frequently contain an incorrect number of chromosomes and are therefore unlikely to give rise to a viable pregnancy, the incidence of chromosomally abnormal eggs increases strikingly with advancing maternal age. Why egg quality declines dramatically as women approach their forties remains one of the outstanding questions in developmental biology. This PhD thesis demonstrates how unforeseen features of kinetochore organization that are unique to meiosis render this cell division process in mammals particularly prone to errors. Firstly, my results uncovered an unexpected multi-subunit organization of the meiotic kinetochore, which is widely conserved across mammals and biases eggs towards errors. Secondly, I identified two independent mechanisms that predispose eggs from older women to aneuploidy. The first mechanism affects the fidelity of meiosis I. My analysis revealed that human oocytes challenge the paradigm that sister kinetochores are fully fused. Instead, I demonstrated that sister kinetochores disjoin as women get older, which promoted erroneous kinetochore-microtubule attachments. This in turn allowed chromosomes to rotate on the spindle and provided a mechanistic explanation for reverse segregation - a recently discovered meiotic error that is unique to humans. Secondly, I pioneered the use of super-resolution microscopy to study chromosome architecture in human eggs and discovered that individual kinetochores during meiosis II in mammals are composed of previously unidentified subdomains. In young females, these subdomains are joined together by cohesin complexes. With age, kinetochores fragment into two pieces. Fragmented kinetochores frequently attach merotelically to spindle microtubules, which predisposes aged eggs to errors. What severely hinders our progress in identifying causes of human infertility is that numerous features of human meiosis are not represented in mice. To overcome this challenge, I developed an experimental platform to mimic the age-related changes that occur in humans in oocytes from young mice. I achieved this by extending the applications of Trim-Away, a novel method to degrade endogenous proteins even in primary cells, to partially deplete proteins. Furthermore, I established a new experimental model system to study human-like aspects of meiosis in live non-rodent cells in real time: pig oocytes. Together, these results set foundations for new therapeutic approaches to extend reproductive lifespan by counteracting the age-related loss in kinetochore integrity that this study identified. Furthermore, partial Trim-Away and studying meiosis in pigs opens new directions for meiotic research.

Biotecnologias como a Ovum Pick-Up e a Produção In vitro de embriões (OPU-PIVE) tem sido uma importante ferramenta para alcançar o melhoramento genético rápido nos rebanhos, diminuindo o intervalo entre gerações. No Brasil, a PIVE está em fase de crescimento e representa 70,7% de toda a produção in vitro mundial. Contudo a OPU-PIVE é ainda ineficiente em vacas de leite, especialmente devido à sua reduzida população folicular. Muitos estudos têm mostrado um efeito positivo do FSH em gado de leite e corte. Recentemente, a pré-estimulação com FSH mostrou ser capaz de aumentar o diâmetro dos folículos aspirados e a porcentagem de embriões transferíveis. O FSH estimula o recrutamento dos folículos na fase antral, fazendo com que eles possam se desenvolver até o momento em que ocorre a divergência e um ou mais folículos se torne dominante. A hipótese do presente estudo é que o uso de FSH (200 mg), fracionado em 4 ou 6 aplicações, em vacas holandesas não lactantes, com emergência de onda folicular sincronizada, aumenta o número de folículos ovarianos, o número de oócitos viáveis e a quantidade de embriões na produção in vitro. Trinta e seis vacas Holandesas não lactantes foram utilizadas como doadoras de oócitos e distribuídas em três tratamentos: Controle (C), 4 aplicações de FSH (F4) e 6 aplicações de FSH (F6). Todas as vacas foram submetidas ao mesmo protocolo de sincronização de emergência de onda folicular, diferindo apenas pela administração e número de 4 ou 6 doses de FSH, conforme descrito acima. Em dia aleatório do ciclo estral (D0), todas as vacas receberam um dispositivo de P4 (Primer&reg;, Tecnopec-Agener União, São Paulo, SP, Brasil) e 2 mg de benzoato de estradiol (Ric-BE&reg;, Tecnopec-Agener União, São Paulo, SP, Brasil). Três dias após (D3), foi administrado 0,530 mg de Cloprostenol Sódico (Cioprostinn&reg;, Innovare Biotecnologia e Saúde Animal Ltda, Monte Aprazível, SP, Brasil), induzindo a luteólise com a intenção de liberar espaço no estroma ovariano para o crescimento folicular e facilitar a visualização de folículos na OPU. Vacas do grupo C não receberam tratamentos adicionais. Vacas do grupo F4 receberam 200 mg de FSH (Folltropin - Bioniche Anim,al Health, Belleville, ON, Canadá) fracionados em 4 aplicações de equivalentes concentrações em intervalos de 12 h, iniciando no D4 pela manhã. Vacas do grupo F6 receberam 200 mg de FSH fracionados em 6 aplicações de equivalentes concentrações em intervalos aproximados de 12 h, tendo início no D3 pela manhã. No D7, o dispositivo foi removido e a OPU realizada concomitantemente à contagem dos folículos existentes nos ovários. Os oócitos considerados viáveis foram fertilizados in vitro com sêmen sexado de touros da raça Holandesa. Os dados foram analisados pelo PROC GLIMIX do SAS 9.3, utilizando contrastes ortogonais C1 (C x FSH) e C2 (F4 x F6). Não houve efeito de tratamento no número de folículos (C = 53,3 &plusmn; 4,9 vs FSH = 51,36 &plusmn; 3,1;P = 0,89), número total de oócitos (C = 19,46 &plusmn; 1,64 vs FSH = 18,47 &plusmn; 1,27; P = 0,55), número de oócitos viáveis (C = 12,57 &plusmn; 1,26 vs FSH = 12,70 &plusmn; 1,03; P= 0,606), taxa de recuperação de oócitos (C = 36,5% vs FSH = 36,0%; P = 0,48) e produção de embriões in vitro (C = 4,11 &plusmn; 0,52 vs FSH = 4,32 &plusmn; 0,46; P = 0,79). Apesar de não ter havido efeito no número de folículos, o tratamento com FSH alterou a distribuição dos mesmos, proporcionando o aumento no número de folículos médios (6 a 10 mm). No entanto, não houve efeito do tratamento com FSH no número de oócitos totais e viáveis recuperados, nem na produção de embriões. / Reproductive biotechnologies such as Ovum Pick-Up and in vitro Embryo production (OPU-IVEP) have been widely used as important tools to achieve faster genetic improvement in herds, diminishing the intervals between generations. In Brazil, in vitro Embryo Production (IVEP) is growing in popularity and accounts for 70.7% of all in vitro embryo production worldwide. However, the OPU-IVEP is still poorly efficient in high-producing dairy cattle, especially because of their reduced follicular population. Several studies have shown a positive effect of FSH on OPU-IVEP yeld. Recently, FSH pre-stimulation has shown to be able to increase the diameter of aspirated follicles and the percentage of transferable embryos. The hormone FSH stimulates follicle recruitment in the antral phase, in the way that they develop until the moment of divergence and one or more follicles becomes dominant. The hypothesis of this study is that the use of 200mg FSH split into 6 doses in non-lactating Holstein cows with a synchronized follicular wave emergence increases the number of follicles, the recovery rate and the number of embryos produced in vitro. Thirty six Holstein cows used as oocyte donors were homogenously allocated to one of three treatment groups in a 3x3 Latin square design: Control (C); 4 doses of FHS (FSH4); 6 doses of FSH (F6). All cows were synchronized using the same protocol for synchronization of follicular wave emergence, except for the administration and number of doses of FSH as previously described. At random days of the estrous cycle known as D0, all cows received an intravaginal P4 device (Primer&reg;, Tecnopec-Agener União, São Paulo, Brazil) and 2mg estradiol benzoate (Ric-BE&reg;, Tecnopec-Agener União). Three days after (D3), all cows received 0.530 mg D-Cloprostenol (Cioprostinn&reg;, Innovare Biotecnologia e Saúde Animal Ltda, Monte Aprazível, SP, Brasil). Cows from the Control group received no additional treatment. Cows from group FSH4 were treated with 200 mg of FSH split in 4 doses of similar concentration given approximately 12 h apart, starting on D4 AM. Cows form group FSH6 were treated with 200 mg of FSH split in 6 doses of similar concentration given approximately 12 h apart, starting on D3 AM. On D7, the device was removed and OPU was performed concomitant with antral follicle count in each ovary. The oocytes considered as viable were sent to IVEP. Data was analyzed using the Glimmix of SAS 9.3, with orthogonal contrasts C1 (C x Treatment with FSH) and C2 (FSH4 x F6). There was no effect on the number of antral follicle (C = 53.3 &plusmn; 4.9 vs FSH = 51.36 &plusmn; 3.1;P = 0.89), number of total oocytes (C = 19.46 &plusmn; 1.64 vs FSH = 18.47 &plusmn; 1.27; P = 0.55), number of viable oocytes (C = 12.57 &plusmn; 1.26 vs FSH = 12.70 &plusmn; 1.03; P= 0.61), oocyte recovery rate (C = 36.5% vs FSH = 36.0%; P = 0.48) and number of embryos produced in vitro (C = 4.11 &plusmn; 0.52 vs FSH = 4.32 &plusmn; 0.46; P = 0.79). Although FSH treatment did not affect the number of follicles, it affected the distribution of them, increasing the number of follicles from 6 to 10 mm. However, FSH treatment did not alter the total number of oocytes and number of viable oocytes or embryo production.

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

O interesse em criopreservar tecido ovariano, embriões e oócitos, principalmente quando se trata de pacientes oncológicas que irão ser submetidas a tratamentos potencialmente esterilizantes, vem crescendo nas últimas duas décadas. Uma das técnicas propostas para se preservar a fertilidade destas pacientes é o congelamento de oócitos, podendo estes ser obtidos já maturados in vivo após a hiperestimulação ovariana controlada ou na forma de oócitos imaturos na ausência de estimulação, nestes casos procede-se a maturação in vitro (MIV) de oócitos pré congelamento. No entanto sabe-se que a criopreservação causa danos de viabilidade e perda do potencial reprodutivo destes oócitos. Alguns autores têm demonstrado que esses danos podem ser reduzidos por meio de cultivos que modulam o perfil lipídico tanto de oócitos como embriões, fazendo com que estes sejam menos susceptíveis ao congelamento. Uma das técnicas que permite a verificação da composição lipídica de células e outras estruturas é a espectrometria de massas. Os objetivos deste estudo foram comparar o perfil lipídico de oócitos maturados in vitro na presença ou ausência de PL e correlacionar com o perfil lipídico e desenvolvimento embrionário dos embriões produzidos in vitro. Além disso, avaliamos o perfil lipídico dos meios de maturação usando oócitos bovinos como modelo experimental. CCOs foram maturados em meio TCM ou TCM + PL, contendo 10% de soro fetal bovino, 0,5 µg/ml de FSH, 5 ng/ml de LH e 1 mg/mL de 17?-estradiol em atmosfera úmida, com 5% de CO2 durante 24 horas. Após a MIV, os oócitos foram desnudados mecanicamente, lavados em PBS e armazenados a -80 ° C, até a análise de perfil lipídico. Oócitos, meios de maturação e blastocistos foram submetidos à técnica de MALDI-MS (ionização e dessorção a laser assistida por matriz /espectrometria de massas). Diferenças no perfil lipídico foram identificadas por PCA (análise de componentes principais). O perfil lipídico dos meios de maturação determinado por MALDI-MS permitiu a diferenciação entre TCM e TCM + PL. No entanto, a análise dos oócitos maturados in vitro demonstrou que o perfil lipídico dos grupos controle ou suplementado com PL não foram diferentes. Da mesma forma, não foram observadas diferenças no perfil lipídico e na embriogênese dos embriões resultantes. No entanto, diferenças no perfil lipídico entre COC e oócitos desnudos (ODs) maturados in vitro foram detectadas. Oócitos maturados com as células do cumulus contêm íons PC com maiores graus de insaturação dos resíduos de ácidos graxos, enquanto ODs contêm espécies de PC com ácidos graxos insaturados (18:0) ou monoinsaturados (18:1). O MALDI-MS permite a obtenção de perfis lipídicos informativos para meios de cultura e oócitos maturados in vitro. A identificação de mudanças no metabolismo lipídico de oócitos durante a MIV pode contribuir para determinar a suplementação lipídica adequada dos meios de MIV e soluções de vitrificação, contribuindo para otimizar os protocolos de criopreservação de oócitos humanos. / The interest in ovarian tissue, embryos and oocytes cryopreservation has been growing in the last two decades, especially in patients who are faced with potentially sterilizing treatments. One of the techniques proposed to preserve the fertility of these patients is the oocyte cryopreservation. The oocytes can be obtained matured in vivo after controlled ovarian hyperstimulation or as immature oocytes, in the absence of stimulation. In these cases, an option is to proceed the in vitro maturation (IVM) of oocytes before cryopreservation. However, it is known that damage induced by cryopreservation is associated with loss of viability and reproductive potential of oocytes. Some authors have demonstrated that such damage can be reduced by culture media that modulate lipid profile in both oocytes and embryos, making them less susceptible to freezing. One technique that enables the determination of the lipid composition of cells and other structures is mass spectrometry. The objectives of this study were to compare lipid profiles of oocytes matured in vitro in the presence or absence of PL and relate this information to lipid profile and preimplantational development of IVM-derived embryos. Also, we evaluated the lipid profiles of culture media using bovine oocytes as an experimental model. COCs were matured in TCM or TCM + PL, both containing 10% fetal calf serum, 0,5µg/ml FSH, 5 µg/mL LH, and 1 µg/mL 17?-estradiol, with 5% CO2 for 24 h. After IVM, oocytes were mechanically denuded, washed in PBS and stored at -80°C, until lipid analysis. Oocytes, maturation media and blastocysts were submitted to the MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry). Differences in lipid profile were addressed by principal component analysis. Maturation media lipid fingerprint by MALDI-MS allows differentiation among TCM and TCM+PL. However, the MALDI-MS of the in vitro matured oocytes demonstrated that lipid profile of control or PL-supplemented groups were not different. Similarly, no differences were observed in the lipid profile and embryogenesis of resulting embryos. Nevertheless, differences in lipid profiles between COCs and denuded oocytes (DOs) matured in vitro were indicated to occur. The former contain PC ions with higher degrees of unsaturation in the fatty acid residues, while DOs contain PC ions with unsaturated (18:0) or monoenoic (18:1) fatty acids. The MALDI-MS has allowed obtaining informative lipid profiles for culture media and IVM-oocytes. Identification of lipid changes during IVM may contribute to determine appropriate lipid supplementation of IVM/vitrification media and to improve cryopreservation of human oocytes.

Un contrôle précis de la maturation ovocytaire et de la ponte sont essentiels au succès de la reproduction sexuée au sein le règne animal. Ces processus sont coordonnés précisément par des signaux endocriniens et/ou environnementaux, selon les espèces, mais beaucoup reste à apprendre sur leurs régulations. Chez les cnidaires, de nombreuses méduses du groupe des hydrozoaires sont connues pour produire des gamètes en réponse à la transition nuit/jour. Pour caractériser les machineries cellulaires et moléculaires liant la réception de la lumière à l'initiation de la maturation ovocytaire, j'ai étudié la méduse hydrozoaire Clytia hemisphaerica. Mon travail de thèse s’est découpé en trois parties, chacune impliquant l'identification d'un composant moléculaire clé de ce processus.Mon étude initiale faisait partie d'une collaboration avec N. Takeda (Asamushi) et R. Deguchi (Sendai), chercheurs qui avaient, avant le début de ma thèse, identifié chez Clytia les Hormones d'Incitation de Maturation ovocytaire endogènes (MIH) comme étant des tétrapeptides de type WPRPamide, produit par clivage de deux précurseurs à neuropeptides. J'ai montré par hybridation in situ et immunofluorescence que les deux gènes précurseurs du MIH sont exprimés par un type de cellules neurosécrétrices localisées au niveau de l’ectoderme de la gonade, et que les peptides MIH sont sécrétés par ces mêmes cellules suite à une stimulation lumineuse. Cette étude a posé les bases permettant l'identification des régulateurs agissant en amont et en aval du MIH, et plus spécifiquement ceux impliqués dans la photoréception de l’ectoderme de la gonade et la réception du MIH par les ovocytes.Pour identifier le récepteur du MIH de Clytia (CheMIHR) dans les ovocytes, j'ai compilé à partir de données transcriptomiques issues de tissus de gonades, une liste de 16 protéines candidates de la famille des Récepteurs Couplés aux Protéines G (GPCR). J'ai cloné les 16 cDNAs et, utilisant une méthode de « deorphelinisation » de GPCR basée sur de la culture cellulaire (collaboration avec P. Bauknecht et G. Jékély; MPI, Tübingen), j’ai pu identifier un GPCR activée par des peptides MIH synthétiques. Sa fonction in vivo comme récepteur essentiel du MIH a été confirmée par la méthode d'édition génétique CRISPR/CAS9. La délétion ainsi produite, entraînant un déplacement du cadre de lecture au sein du gène CheMIHR, a détérioré la croissance des colonies de polypes et le comportement de ponte des méduses matures. Confirmant la fonction de CheMIHR, la maturation ovocytaire chez des mutants CheMIHR ne pouvait pas être déclenchée par la lumière ou par addition de MIH synthétiques, mais pouvait être rétablie en utilisant des analogues au cAMP, molécule connue pour agir en aval de la réception du MIH dans les ovocytes d’hydrozoaires. Des analyses phylogénétiques ont montré que Clytia MIHR est affilié à un sous-ensemble de familles de neuropeptides de bilaterians impliqués dans divers processus physiologiques, notamment la régulation de la reproduction. Des hybridations in situ sur les méduses Clytia, ont en outre montré l'expression des précurseurs de CheMIH et de CheMIHR dans des cellules neurales hors de la gonade, suggérant un rôle plus large du couple CheMIH-MIHR que la seule initiation de la maturation ovocytaire.Pour mieux comprendre la photoréception des gonades chez Clyita, j'ai montré que la ponte est sélectivement incitée par la lumière bleu-cyan, et mis en évidence, grâce à l’analyse de données de transcriptome de gonade, qu’un photopigment de la famille des Opsin (Opsin9) est hautement exprimé dans l'ectoderme. De façon saisissante, les hybridations in situ ont montré que le gène Opsin9 est exprimé dans les mêmes cellules sécrétant le MIH. L'introduction d'une mutation de changement de cadre de lecture dans le gène Opsin9 via la technologie CRISPR/Cas9 a empêché la maturation ovocytaire et la ponte des méduses mutantes en réponse à la lumière... / Tight control of oocyte maturation and of gamete release is essential for successful sexual reproduction in the animal kingdom. These processes are precisely coordinated by endocrine and/or environmental cues, depending on the species, but much remains to be learned about their regulation. Within the Cnidaria, many hydrozoan jellyfish are known to spawn mature gametes following dark/light transitions. To characterise the cellular and molecular machinery linking light reception and oocyte maturation initiation, I have studied the hydrozoan jellyfish Clytia hemisphaerica. My thesis work had three parts, each involving the identification of a key molecular component of this process.My initial study was part of a collaboration with N. Takeda (Asamushi) and R. Deguchi (Sendai), who identified the endogenous oocyte Maturation-Inducing Hormones (MIH) in Clytia as WPRPamide-related tetrapeptides, generated by cleavage of two neuropeptide precursors. I showed by in situ hybridization and immunofluorescence that Clytia MIH is produced by neurosecretory cells of the gonad ectoderm that co-express the two precursor genes, and that it is secreted upon light stimulation. This study paved the way for identification of regulators acting upstream and downstream of MIH release in the gonads, specifically the ones involved in photoreception in the gonad ectoderm, and in MIH reception by the oocytes. To identify the Clytia MIH receptor (CheMIHR) in the oocytes, I compiled a shortlist of 16 candidate G protein-coupled receptors (GPCRs) from gonad transcriptome data. I cloned all 16 cDNAs and, using a cell culture-based "GPCR deorphanization" assay (collaboration with P. Bauknecht and G. Jékély; MPI, Tübingen), identified one GPCR that was activated by synthetic MIH peptides. Its in vivo function as the essential MIH receptor was confirmed by CRISPR/Cas9 gene editing. Introduction of a frame-shift mutation in the CheMIHR gene impaired growth of Clytia polyp colonies and also the spawning behaviour of mature medusae. Confirming the function of CheMIHR, oocyte maturation in CheMIHR mutants could not be triggered by light or by synthetic MIH, but could be restored using cell-permeable analogues of cAMP, known to act downstream of MIH reception in hydrozoan oocytes. Phylogenetic analyses showed that Clytia MIHR is related to a subset of bilaterian neuropeptide hormone receptor families involved in diverse physiological processes, including regulation of reproduction. Accordingly, in situ hybridization showed the expression of Clytia MIH precursors and MIHR in non-gonadal neural cells, suggesting a wider role of Clytia MIH-MIHR besides oocyte maturation initiation.To address gonad photoreception, I showed that Clytia spawning is selectively induced by blue-cyan light, and then identified using gonad transcriptome data an opsin photopigment (Opsin9) highly expressed in the ectoderm. Strikingly, in situ hybridization showed that Opsin9 is expressed in the MIH-secreting cells. Introduction of a frame-shift mutation into the Opsin9 gene via CRISPR/Cas9 prevented oocyte maturation and spawning of mutant jellyfish in response to light. Anti-MIH immunofluorescence and rescue experiments with synthetic MIH showed that the essential function of Opsin9 is upstream of MIH release. Spawning in Clytia thus appears to be regulated by a dual function photosensory-neurosecretory cell type, perhaps retained from a distant metazoan ancestor...

Orientador: Fernanda da Cruz Landim / Banca: João Carlos Pinheiro Ferreira / Banca: Maria Denise Lopes / Banca: Alício martins Júnior / Banca: Edson Guimarães Lo Turco / Resumo: Este trabalho teve como objetivo geral averiguar o efeito da BL-I e da ROS em bloquear temporariamente a retomada da meiose, atuando sobre os fatores que controlam o ciclo celular meiótico de oócitos bovinos e a expansão das células do cumulus oophoros. No Experimento I, os oócitos permaneceram em meio MIV durante 6, 12 e 24 h na presença de duas concentrações de ROS (12,5 μM e 25 μM) ou na presença de BL-I (50 μ e 100 μM) ou na presença da associação de ROS (6,25 μM) + BL-I (25 μM) e em seguida foram cultivados em meio MIV livre de fármacos por 18 h, 12 h ou 24 h. Após esse tempo, os oócitos inibidos por 6 ou 12 h foram fertilizados e cultivados in vitro. O tratamento com ROS e BL-I não resultou em atraso na quebra da vesícula germinativa em relação ao controle. Entretanto, quando utilizados por 24 h, elevadas taxas de degeneração oocitária foram encontradas. Quando estes fármacos foram utilizados por 12 h com reversão por mais 12 h, verificamos uma menor taxa produção de blastocistos em relação ao grupo controle. No entanto, após a retirada do bloqueio por 6 h e maturação por 18 h um maior número de oócitos tratados tanto com ROS, como BL-I encontraram-se em MII. Da mesma forma, ao reduzirmos a dose e o tempo de inibição, os grupos tratados por 6 h de inibição meiótica e reversão por 18 h apresentaram taxas de produção embrionária similares ao grupo controle. No Experimento II, os oócitos permaneceram em meio MIV durante 6h na presença de 12,5 μM de ROS ou na presença de 50 μM de BL-I ou com a associação de ROS (6,25 μM) + BL-I (25 μM) sendo em seguida cultivados em meio MIV livre de fármacos por 18 h. Após esse tempo, os oócitos inibidos foram fertilizados, cultivados in vitro e os embriões produzidos foram submetidos a vitrificação. Os grupos BI-I e ROS + BL-I apresentaram superioridade de produção embrionária, tanto em relação ao grupo ROS como o controle. O grupo ... / Abstract: This work had as main objective to investigate the effect of BL-I and ROS in temporarily blocking the resumption of meiosis, acting on the factors that control the meiotic cell cycle of bovine oocytes and expansion of cumulus oophoros cells. In Experiment I, the oocytes remained in IVM mediafor 6, 12 and 24h in the presence of two concentrations of ROS (12.5 μM and 25 μM) or in the presence of BL-I (50 μ to 100 mM) or the association of ROS (6.25 μM) + BL-I (25 μM) and then cultured in a drug-free IVM media for 18, 12 or 24 h. After this time, the inhibited oocytes by 6 or 12 h were fertilized and cultured in vitro. Treatment with ROS and BL-I did not resulted in delay in germinal vesicle breakdown in relation to control. However, when used for 24 h, higher rates of oocyte degeneration were found. When these drugs were used for 12 h with reversion for more than 12 h, a lower blastocyst production rate compared to the control group was verified. However, after removal of the blockage for 6 h and maturation for 18 h, a higher number of oocytes treated with both ROS and BL-I were found in MII. Similarly, when the dose and duration of inhibition were reduced, the groups treated for 6 h of meiotic inhibition and reversion for 18 h showed similar embryo production rates to the control group. In Experiment II, oocytes remained in IVM mediafor 6 h in the presence of 12.5 μM of ROS or the presence of 50 μM of BL-I or the association of ROS (6.25 μM) + BL-I (25 μM) and then cultured in drug-free IVM media for 18 h. After this time, the inhibited oocytes were fertilized in vitro and the produced embryos were submitted to vitrification. The BI-I and ROS + BL-I groups showed superiority in embryo production, both in relation to the control as ROS group. The ROS + BL-I group showed higher rate of re-expansion after vitrification. Additionally, embryos from BL-I and BL-I + ROS showed lower number of apoptotic cells when compared to ... / Doutor

In addition to contributing genes at fertilization, the sperm cell induces the oocyte to leave its arrested state and resume metabolism in the process of activation. A hallmark of oocyte activation is a release of intracellular calcium (Ca2+i) from the endoplasmic reticulum. The mediators of oocyte activation have been studied in many animal models, while little is known in the bovine model. Both Src Family Kinase (SFK) and Phospholipase C (PLC) enzymes have been reported to be involved in oocyte activation in other animal models. In this dissertation are described experiments that define the role of SFK and PLC enzymes in the pathway leading to Ca2+i and calcium induced calcium release in bovine oocyte activation. Western blotting was used to discover that SFKs Src, Hck, and Lck are present in matured bovine oocytes, and Src, Blk, and Yes are present in acrosome reacted bovine spermatozoa. The PLC δ1 and δ3 are present in both matured bovine oocytes and spermatozoa. PLC δ4, γ2, and η2 are present in matured bovine oocytes. Microinjecting a known general SFK inhibitor, PP2, significantly decreases both Ca2+i and cleavage rates. Microinjecting a 13 amino acid peptide that mimics the phosphorylated carboxyl terminal region of pp60c-src decreases both Ca2+i and cleavage rates. Microinjecting a downstream substrate of pp60c-src sequestered any signal produced by Src and decreased Ca2+i and cleavage rates. Microinjecting primary antibodies raised against PLC isotypes blocked both Ca2+i and cleavage rates, giving insight to the mechanism of calcium induced calcium release in the bovine model. The PLC isotypes δ3, δ4, and γ2 decreased Ca2+i oscillations and cleavage rates, indicating they are involved in both IP3R and RyR activation. PLC δ4 and η2 did not impact Ca2+i but did significantly decrease cleavage rates. The data presented in this dissertation increase the understanding of the pathway leading to bovine oocyte activation and further confirm that the detailed pathway differs among animal models.

Assisted reproductive technologies (ARTs) have now been extensively used to promote reproductive efficiency as a fertility treatment not only in human medicine but also animal reproduction. ARTs serve as an important tool to advance the fundamental knowledge of reproductive processes. The quality of female’s eggs defines its ability to undergo maturation, fertilization, and development. This quality is determined by various factors and is crucial for the success of ARTs. Any alternations happening during the egg growth and maturation process can result in the decreased quality, which could have long-lasting effects on development. Improving the developmental efficiency of the egg is quite challenging due to the limited knowledge on the underlying mechanism of how the egg regulates biological processes during the growth and maturation phase. We compared good-quality and poor-quality eggs to detect the key players in determining the egg quality at the molecular level. Our finding also provides information that benefits the understanding of how the nutrients in culture medium facilitate oocyte maturation, which will eventually help optimize the condition for oocyte culture. Based on the results from these comparative studies, we proposed a potential strategy for improving egg quality. The knowledge obtained from our research offers promise for many applications in the treatment of infertility and improvement of ART efficiency.