<p>Crypreservation of oocytes is recently being considered to be a valid choice in infertility treatments.Low survival and fertilization rates due to inefficient slow freeze protocols have been the outcome ofmany previous studies done in the field. However, introduction of the vitrification technique and itsapplication in reproductive medicine and to some extent new improved slow freeze protocols haveshown that oocytes can be cryopreserved with successful outcome.</p><p>In this project the survival rate of oocytes after vitrification with MediCult Vitrification andWarming Media has been studied. Also, a comparison of the carriers Cryoloop (an open system) andCryopette (a closed system) has been performed.</p><p>A total of 43 oocytes were vitrified and warmed according to MediCult's protocol, of which 21oocytes with Cryoloop and 22 with Cryopette. The cells were post-thaw incubated in a physiologicalenvironment for 24h. During that time the morphology and viability were observed and noted after 2h,over night and after 24h. No significant difference in survival rates of the oocytes' was observed usingthe two carriers, and the total survival rate of the oocytes was 83.7%. This indicates thatcryopreservation of oocytes is a valid choice in fertility treatments and that the new product Cryopettecan be used clinically with just as good results as the well established Cryoloop technique. This merits further studies on the applicability of vitrification on oocytes and the Cryopette technique.</p>

Crypreservation of oocytes is recently being considered to be a valid choice in infertility treatments.Low survival and fertilization rates due to inefficient slow freeze protocols have been the outcome ofmany previous studies done in the field. However, introduction of the vitrification technique and itsapplication in reproductive medicine and to some extent new improved slow freeze protocols haveshown that oocytes can be cryopreserved with successful outcome. In this project the survival rate of oocytes after vitrification with MediCult Vitrification andWarming Media has been studied. Also, a comparison of the carriers Cryoloop (an open system) andCryopette (a closed system) has been performed. A total of 43 oocytes were vitrified and warmed according to MediCult's protocol, of which 21oocytes with Cryoloop and 22 with Cryopette. The cells were post-thaw incubated in a physiologicalenvironment for 24h. During that time the morphology and viability were observed and noted after 2h,over night and after 24h. No significant difference in survival rates of the oocytes' was observed usingthe two carriers, and the total survival rate of the oocytes was 83.7%. This indicates thatcryopreservation of oocytes is a valid choice in fertility treatments and that the new product Cryopettecan be used clinically with just as good results as the well established Cryoloop technique. This merits further studies on the applicability of vitrification on oocytes and the Cryopette technique.

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Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-04Bitstream added on 2014-06-13T20:59:35Z : No. of bitstreams: 1 blanco_idp_me_botfmvz.pdf: 957805 bytes, checksum: d8fe74b65ba19ca53f1ec69c651d4a0a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Em éguas, o aprimoramento e a aplicação de técnicas como a ICSI, TO e transferência nuclear é restrita devido à dificuldade de obtenção de oócitos in vivo, atribuída a particularidades dos folículos ovarianos da espécie. Em folículos pré-ovulatórios, a expansão do cumulus que ocorre com a progressão da maturação folicular e deposição de ácido hialurônico aumenta os índices de recuperação de oócitos por folículo, enquanto o tratamento superovulatório, embora não seja eficiente em aumentar os índices de recuperação por folículo, melhora os índices de recuperação por égua. Este trabalho teve como objetivo avaliar o efeito da administração de hCG na expansão das células da granulosa e do cumulus e no índice de recuperação de oócitos a partir de folículos imaturos de éguas superovuladas. Para isso 10 éguas foram submetidas à OPU após três diferentes tratamentos hormonais (G1: EPE, G2: EPE/hCG e G3:controle). Cada égua foi submetida aos três tratamentos, tendo sido alternados um ciclo onde era permitido que houvesse ovulação, sem interferência, e um ciclo com OPU. No ciclo onde devia ocorrer aspiração, as éguas recebiam aplicações de prostaglandina no sétimo e oitavo dia após a ovulação. Para os grupos 1 e 2, iniciava-se o tratamento com EPE no D7. Quando a maioria dos folículos atingia entre 22 e 27 mm, somente o grupo 1 recebia hCG, a EPE era suspensa e efetuava-se OPU 24 horas depois para ambos os grupos. Para o grupo controle, após a aplicação de prostaglandina a OPU era efetuada 24 horas após o folículo dominante atingir 27-30 mm. Em todos os grupos foram aspirados folículos entre 10 e 35 mm. Não houve diferença entre os 3 grupos quanto aos índices de recuperação de oócitos por folículo, com médias de 15% a 16,7%. Entretanto, a hCG foi capaz de induzir uma expansão e luteinização precoce das células foliculares. / In mares, the use of artificial reproductive techniques (ART) such as intracitoplasmatic sperm injection (ICSI), oocyte transfer (OT), and cloning is limited by the low availability of oocytes obtained either in vitro or in vivo. The low recovery rate of oocytes from immature follicles seems to be linked with anatomical particularities of the follicular wall. In pre-ovulatory follicles (>35mm in diameter), final oocyte maturation is accompanied by the expansion of the cumulus, which releases the oocyte from the follicular wall resulting in increased recovery rates after follicular aspiration. The number of oocytes recovered in each OPU (Ovum Pick Up) section should be higher with superovulation. However, although the total number of oocytes recovered by each individual mare is increased, the recovery rate per follicle is still low. Treatment with hCG modifies the intra-follicular environment by inducing precocious expansion of the granulosa cells, leading to a higher recovery rate during OPU of pre-ovulatory follicles. Therefore, this study aimed to evaluate the effect of hCG treatment on the recovery rate of oocytes obtained from immature follicles in mares superovulated with Equine Pituitary Extract (EPE). Ten mares were rotated in three different treatments: G1 – superovulated with EPE; G2 – Superovulated with EPE and treated with hCG, and G3 – Control. For all treatments, when the biggest follicle reached 27 – 30 mm, all follicles bigger than 10 mm were aspirated. Each OPU cycle was alternated with a rest cycle when a spontaneous ovulation occurred. Although treatment with hCG increased the number of follicles presenting expanded granulosa and high intra-follicular progesterone concentration, no statistically significant differences were observed between groups regarding oocyte recovery.

It is now more than 35 years since the first world test-tube baby, Louise Brown, was born (1978) in England and it is estimated that since then more than 4 000 000 of children were produced by in vitro fertilization (IVF) worldwide. The initial success of IVF was less than 20% in best clinics, but now it reaches about 40%. This is a consequence of introduction of new methods, standardization and exploitation of new manipulation and culture media, as well as the incorporation of research results. Nevertheless, the most important still remains the skill and experience of IVF clinics and IVF laboratories staff, especially their ability to critically evaluate the quality of biological material and to decide which cure and treatment are the best one. At least, some biological material (immature and low quality oocytes) can be used for training and also for some experiments aiming to explain some questions, which are not yet fully understood (for example aneuploidies in human oocytes and embryos). In addition, this training can facilitate the introduction of new progressive approaches and may also improve indirectly the quality of infertility treatments. The first part of thesis is focused on the quality evaluation of oocytes collected by aspiration from follicles of stimulated patients. For labeling...

Errors in chromosome segregation during the first division of female meiosis are very common in humans and result in aneuploidy leading to reproduction problems. Chromosome segregation depends on the formation and function of the meiotic spindle as well as the structure of chromosomes, which need to condense to be able to orient and segregate properly. It is important to understand the mechanisms underlying the female meiotic spindle function and chromosome condensation to gain insight into female fertility problems. The female meiotic spindle assembles without centrosomes, so the mechanisms ensuring microtubule nucleation, spindle assembly and establishment of bipolarity act differently from those of mitosis or male meiosis. I identified a set of genes that are required for microtubule nucleation, spindle maintenance and centromere orientation in Drosophila female meiosis. This was accomplished by mapping previously uncharacterized Drosophila mutants and depleting already known genes by RNAi. I discovered that several proteins have a different role in female meiosis as compared to mitosis, which provides insight into the major differences between these systems. Little is known about the molecular mechanisms of chromosome condensation. The roles of only a few factors, such as condensin complexes, have been studied previously, and the evidence suggests that there are more molecular players required for chromosome condensation. To discover molecular mechanisms critical to this process, I depleted various chromosomal proteins by RNAi and screened for abnormalities of metaphase chromosome morphology in Drosophila oocytes by immunostaining and live imaging. I found that the conserved kinase NHK-1 plays a role in chromosome condensation in female meiosis. BAF is a critical NHK-1 substrate in this process and its phosphorylation is required for detachment of the chromosomes from the nuclear envelope to allow proper condensation. Also, I discovered that the nucleosome remodelling complex NuRD is crucial for chromosome condensation, especially for the chromosome arms. As a result of my PhD project I identified multiple factors required for meiotic spindle function. I also discovered two novel pathways of chromosome condensation that require the NuRD complex and NHK-1 activity.

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Mammalian odorant receptors (ORs) are Class I G-protein coupled receptors (GPCRs) located within the nasal epithelium. Odorant receptors interact with Galpha olfactory, a Galpha S type G-protein. Activated Galpha olfactory stimulates adenylate cyclase and the resulting increase in cAMP concentration opens cyclic nucleotide gated channels allowing Ca2+ to enter the cell. The increased Ca2+ then activates a Ca2+ activated Cl- channel which further depolarizes the cell. This depolarization initiates an action potential that reaches the axon of the olfactory sensory neuron located in the main olfactory bulb. Information from the main olfactory bulb is then transmitted to higher regions of the brain. Olfactory information is initially coded through the interaction of odorant molecules with hundreds of distinct ORs, but difficulty in exogenous expression of odorant receptors has delayed the identification of ligands for individual ORs. However, expression of mouse odorant receptors in Xenopus laevis oocytes allows for a systematic screening for potential ligands, as well as for efficient study of the structure-function relationship of the receptors and their ligands. My screening of odorant receptors using Xenopus oocytes included the coexpression of a signal transduction system and the use of robotic two-electrode voltage clamp electrophysiology. In this study, I investigated the structural basis for ligand recognition in mouse odorant receptors. First, I expanded the molecular receptor ranges of seven Class I odorant receptors. By use of a high throughput assay, I was able to expand upon current knowledge in the field for the mouse odorant receptors 23-1, 31-4, 32-11, 40-4, 42-1, 42-2 and 42-3. I then examined one receptor (MOR23-1) in more detail. I used the substituted cysteine accessibility method to identify residues within transmembrane domain five of this receptor that are accessible from the extracellular space. These residues may line the ligand binding site or the ligand access pathway. Conventional mutations of A205 caused little alteration in the molecular receptive range of the receptor, suggesting that this residue may not play a significant role in ligand interaction within the binding pocket. Mutagenesis of G111, a residue within transmembrane domain three caused significant shifts in the molecular receptive range of the receptor, but the location of this residue within the binding pocket could not be confirmed by the substituted cysteine method. Previous reports had suggested significant similarity between the molecular receptive ranges of the seven mouse odorant receptors that I used in my research. By expanding upon the known aliphatic ligands for each receptor identified new ligands for each receptor, I was able to show that the molecular receptive ranges of these receptors are in fact distinct. The experimental identification of residues located within the binding pocket on transmembrane five of mouse odorant receptor 23-1 provides an improved understanding of ligand recognition by this receptor class and will aid in better computer modeling of these receptors. This increased accuracy of the computer models of these basic Class I GPCRs may aid in future drug discoveries. Since GPCRs constitute a significant fraction of current drug targets, understanding the mechanism of ligand interactions with mouse odorant receptors may aid in the development of more efficacious compounds in the treatment of many common ailments.

Cryopreservation of gametes and embryos is crucial in fertility treatment and fertility preservation. Preservation of oocytes is a more complicated process than preservation of sperm and embryos. According to recent studies, a new preservation technique called vitrification is found to have higher rates of oocytes-survival after warming    The aim of this study was to evaluate VitriBlast™ Kit, for vitrification of oocytes. Vitrification is an ultra-rapid freezing method in the presence with of high concentrations of cryoprotectants which avoids intracellular ice-crystal formation during the freezing and warming process.     In this study, a total number of 117 immature oocytes were used and 62 of these oocytes were vitrified with VitroBlast™ Kit. During this process two different vitrification devices were used, VitroLoop™ and Cryopette®. After warming, the average survival rate for vitrified oocytes was found to be 61% for VitroLoop™ and 15% for Cryopette® (p&lt;0.001). The remaining 55 oocytes were used as a non-frozen control group and the same incubation method as for vitrified oocytes was used. The survival rate for the control groups was higher than for the vitrified groups (93% versus 35%, p&lt;0.001). The results of this study indicate that VitriBlast™ Kit is not suitable for vitrification of oocytes.

Acquisition of full developmental competence of oocytes not only occurs during growth stage, and the final preparation during oocyte maturation is also critical. Previous studies have shown that nuclear maturation can occur spontaneously following culture in vitro; however, there may be some insufficiency in cytoplasmic maturation of the in vitro matured oocytes. But till now, the differences of the events of cytoplasmic maturation between in vitro and in vivo matured oocytes are still not clear. Ovarian stimulation by gonadotrophins is used to permits the growth and development of follicles, to time the initiation of pre-ovulatory oocyte maturation, and to increase the numbers of oocytes ovulated. It is one of the foundations of current treatments of human infertility. The success of clinical IVF has been depending on generation of matured oocytes at high frequency. However, ovarian stimulation with gonadotropins is associated with side effects and complications. / In order to illuminate mechanisms which affect the developmental competence of oocytes produced in vitro, in the present study, we have compared the difference of the quality of oocytes produced in vitro with that of the oocytes produced in vivo using mouse model. In order to understand the relationship between oocyte competence and ovarian responses to stimulation in the mouse, we also have compared difference of the quality of oocytes produced in vitro or in vivo from gonadotrophns stimulated ovaries with that of from natural cycling ovaries. / In-vitro matured oocytes were collected from (1) naturally ovulated mice and (2) superovulated (PMSG + hCG) mice. Immature oocytes were retrieved from (3) naturally cycling mice, and (4) from mice primed with PMSG. The results indicate that the percentages of cleavage and blastocyst formation are significantly different (P&lt;0.05) between in-vivo and in-vitro matured oocytes. Blastocyst formation rate is significantly higher (P&lt;0.05) in immature oocytes derived from PMSG primed mice compared to immature oocytes derived from naturally cycling mice. The percentages of oocytes with comet tails and the length of comet tails are significantly higher and longer respectively in in-vitro matured oocytes compared to in-vivo matured oocytes. Total cell numbers of blastocyst are also significantly different (P&lt;0.05) between in-vivo and in-vitro matured oocytes. However, there are no differences in ratio of trophectoderm (TE)/inner cell mass (ICM) between in-vivo and in-vitro matured oocytes. In conclusion, in-vivo matured mouse oocytes are more competent than those of matured in-vitro, suggesting that it may be due to its less damage of DNA. Embryonic development capacity of in-vivo matured oocytes is not promoted by ovarian stimulation. Gonadotropin priming prior to immature mouse oocyte retrieval is beneficial to subsequent embryonic development. / Keywords. mouse oocyte, IVM, IVF, gonadotropin, development