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1
Pugh, Hedley James. "Deposition and adhesion of cryptosporidium oocysts on surfaces." Thesis, University College London (University of London), 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300120.
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Kazem, Rahnuma. "Oocyte cryopreservation." Thesis, University of Aberdeen, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282706.
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A questionnaire based survey was done to assess the views of fertile individuals, infertile individuals, egg donors and recipients towards gamete donation. The survey showed that fertile individuals were significantly less inclined towards the use of donated eggs in research and treatment, compared to infertile individuals. Acceptability of gamete donation was found to be very high in all groups regardless of their fertility, but the majority of individuals, whether fertile or infertile, were opposed to the use of fetal and cadaveric sources of obtaining eggs. The effect of modifications of the freeze-thaw process was investigated in the mouse model. It was seen that slight modifications of the slow freeze protocol affected survival rates and that ultrarapid freezing achieved better survival rates than slow freezing. Human oocyte cryopreservation was performed using a slow freeze-rapid thaw protocol. In total, 34.4% of oocytes survived cryopreservation and these were randomly allocated for fertilisation by conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Resulting embryos were spread for chromosomal analysis. ICSI significantly improved the rates of normal fertilisation (43.2% versus 2.7%) compared to IVF (P<0.001). A normal diploid karyotype was achieved by ICSI. These studies show that oocyte donation is acceptable to the majority of both fertile and infertile individuals. Further research is required to improve the methods of oocyte cryopreservation. Once the techniques of cryopreservation have been established, ICSI may successfully be applied to enhance subsequent fertilisation rates.
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Korich, Dick Gary. "Cryptosporidium oocyst viability: Assessment and correlation with infectivity." Diss., The University of Arizona, 1993. http://hdl.handle.net/10150/186125.
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Outbreaks of cryptosporidiosis have been traced to Cryptosporidium oocysts in finished drinking water. Indeed, water contaminated with oocysts may be judged perfectly safe by conventional coliform tests. Although oocysts can be specifically identified using immunofluorescence, it is not yet possible to determine their viability. The lack of a viability test means that each oocyst detected in finished water must be regarded as potentially infective even though water treatment may have killed them. The goal of this research was to develop a test for oocyst viability. In vitro excystation, oocyst morphology, vital dyes, and a monoclonal antibody were tested. In vitro excystation expressed as percent of theoretical sporozoite yield correlated best with neonatal mouse infectivity. Although not directly applicable to testing water samples, excystation provided a basis for screening other testing methods. None of the eight vital dyes tested showed any relationship between oocyst staining and viability. This was presumably due to inability of the dyes to penetrate the oocyst wall. Pretreatment strategies designed to increase oocyst wall permeability were either ineffective or damaged the oocysts in ways that rendered them nonviable. Initially, microscopic appearance appeared to be related to oocyst infectivity. However, regression analysis showed that phase contrast microscopic appearance had marginal utility for use as a viability test. Indeed, microscopic identification of internal structures of intact oocysts is not a reliable viability indicator because DAPI staining showed intact sporozoite nuclei within obviously dead oocysts that would not excyst. A monoclonal antibody (MAb OW64) was found which binds to internal sites along the oocyst suture. There was positive correlation between binding of this MAb and decreasing oocyst infectivity indicating that MAb OW64 bound preferentially to nonviable oocysts. Regression analysis showed that OW64 binding overestimated oocyst viability because many nonviable oocysts did not bind the MAb. Nevertheless, MAb OW64 is a candidate for producing an immunofluorescence based test in which oocysts that bind OW64 are nonviable whereas those that do not bind are not necessarily viable. Before such a test can be recommended, however, the nonviability of oocysts that bind OW64 must be demonstrated by neonatal mouse infectivity using oocysts sorted by a fluorescence activated cell sorter.
4
Hardy, Scott Andrew. "Effectiveness of static mixers for disinfection of cryptosporidium oocysts." Thesis, Georgia Institute of Technology, 1999. http://hdl.handle.net/1853/20925.
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Liyanage, Lalith R. J. "Chlorine dioxide inactivation of Cryptosporidium parvum oocysts in water." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0007/NQ29064.pdf.
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Marsh, Adam. "Oocyte-follicle interactions." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12684/.
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The ovarian follicle is an individual functional unit that provides the optimal environment for the oocyte within to develop. This thesis outlines the research in the field of ovarian follicular dynamics that has already been established, and further develops these findings to explore in greater detail the relationship between the oocyte and its environment, both in an in vitro and in vivo setting, using a variety of species. The first major research area involved studying the role of oocyte-secreted factors, which was examined using a series of dose response experiments. These were performed using an ovine granulosa cell culture model, and elucidated a possible role for a collaborative action of BMP15 and GDF9 in the promotion of oestradiol synthesis, while inhibiting production of progesterone in this species. This finding was then further investigated using an ovine in vivo immune-neutralisation study, the endocrine and histological results of which confirmed these findings in a proportion of these animals, although this study was limited by the animals appearing to have been in seasonal anoestrus. The second major topic that was investigated was based around the ovarian microenvironment, in terms of angiogenesis and hypoxia. Again, ovine granulosa cell cultures were used, in this instance to examine the effect of hypoxic conditions on steroid hormone production. These experiments indicated that somatic cell steroid hormone production is likely to be compromised by a hypoxic environment, and therefore that the provision of oxygen through a local blood supply may be a vital requirement for these cells. To investigate the relevance of studying ovarian blood supply and physiology in a clinical setting, perfusion studies were carried out based on a series of bovine phantom experiments, which were used to study the effect of varying flow rate on the parameters routinely measured using this technology. The routine clinical ultrasonographic methods of ovarian assessment such as 4D ViewTM, SonoAVCTM and VOCAL were also examined, based on bovine phantom experiments, revealing possible weaknesses in the data provided by ultrasound that are increasingly relied upon in the clinical setting. Finally, a clinical trial was carried out to try and encompass all of the findings of the in vitro and in vivo work, in order to place these theories into context in a human IVF setting. This work was unfortunately limited severely by a lack of patient numbers, but some interesting results were observed with regard to oocyte developmental potential relationships with follicular fluid and somatic cell factors, as well as ultrasound measures of peri-follicular blood supply.
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Pfender, Sybille Helen. "Studies of asymmetric oocyte division and new genes controlling oocyte maturation." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648232.
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Wang, Ling. "Mouse oocyte maturation: How similar is it to frog oocyte maturation?" Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/27075.
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In this study, I have attempted to address the similarities/differences between amphibian and mouse oocytes by focusing on two aspects of mouse oocyte maturation. In the first project, I investigated the ability of several antagonists of serotonin receptors to initiate follicle-enclosed mouse oocytes to undergo maturation. I demonstrated that ritanserin, but not any others, was capable of inducing oocyte maturation in the intact mouse follicles. Significantly, ritanserin is also capable of inducing frog oocyte maturation, as demonstrated by others in our lab. These results therefore suggested that a similar cAMP-elevating G protein coupled receptor, the target of ritanserin, is responsible for maintaining prophase arrest in both frog and mouse oocytes. In the second project, I have investigated the ability brefeldin A (BFA), a specific inhibitor of a small G protein ARF1, to initiate mouse oocyte maturation, as it has been suggested that BFA is capable of inducing frog oocyte maturation. I demonstrated that BFA indeed was as potent as human chorionic gonadotropin (HCG) to initiate follicle-enclosed oocytes to undergo germinal vesicle breakdown. However, BFA-treated oocytes failed to complete maturation and, instead, were arrested at metaphase I with apparently normal bipolar spindles. We further demonstrated a dominant negative mutant of ARF1 (ARF1-T31N-HA) similarly arrested the maturing oocytes at metaphase I.
These studies helped reinforce the idea that oocyte maturation is fundamentally the same in mammals as it is in amphibians. The experimentally observed differences may not be very significant biologically. This concept will be discussed in conjunction with recently published literature. (Abstract shortened by UMI.)
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Lewin, Nicola. "Effects of sequential exposure of Cryptosporidium oocysts to chemical disinfectants." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0010/MQ60147.pdf.
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Yang, Min. "Evaluation of Oocyte Developmental Competence and Potential Strategies to Improve Oocyte Quality." DigitalCommons@USU, 2018. https://digitalcommons.usu.edu/etd/6914.
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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.
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Davies, S. "Oocyte maturation in mice." Thesis, University of Essex, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377928.
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Abdelsalam, Selima Mohamed. "Impact of oocyte vitrification." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/impact-of-oocyte-vitrification(d112e86b-faac-4b95-abff-06934e923ebd).html.
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Safe and effective oocyte cryopreservation will have a considerable impact on clinical practice in assisted reproduction. Great improvements have been made in recent years to oocyte vitrification procedures, although further controlled trials are necessary to ensure safety, and it is necessary to know more about pregnancy and live birth outcomes. This study aims to validate various methods of oocyte vitrification as assessed by comparative target gene analysis, hence contributing to information available to clinicians advising women about fertility preservation options before cancer treatment. Target genes investigated were: the maternal effect genes Deleted in Azoospermia Like (DAZL), Maternal Antigen That Embryos Require (MATER/NLRP5) and Zygote Arrest 1 (ZAR1); three genes involved in cell cycle progression and cell death, tumour suppressor protein 53 (p53), B-cell lymphoma 2 (BCL2), BCL2-Associated X Protein (BAX); three genes known to affect spindle and chromatin structure, oocyte-specific histone 1 (H1FOO), kinesin family member 11 (KIF11) and mitotic arrest deficient 2 (MAD2); together with Factor In the GermLine, Alpha (FIGLα) which regulates zona pellucida proteins, octamer-binding transcription factor 4 (OCT4/POU5F1) which is associated with pluripotency and oocyte developmental competence, and superoxide dismutase 2, mitochondrial (SOD2) which responds to oxidative stress in the mitochondria. These genes may be useful indicators of oocyte quality following vitrification. Lysis, complementary DNA (cDNA) amplification, polyadenylic acid polymerase chain reaction (polyA PCR) and quantitative polymerase chain reaction (QPCR) were used to investigate gene expression patterns in failed-to-fertilize non-vitrified, vitrified and slow frozen human MII oocytes. Comparative gene analyses included oocytes vitrified using closed and open carriers, and two different media. Results indicate that the impact of vitrification varies by gene and oocyte variability, highlighting the importance of studies based on single oocytes and the need for caution in interpretation of generalised findings. OCT4 and also β-actin were significantly affected by all methods investigated, while FIGLα, MAD2, ZAR1 and DAZL were affected by some methods. Oocyte survival rate after thawing and the number of genes expressed by individual oocytes were higher with media incorporating dimethyl sulfoxide (DMSO) and Dextran Serum Supplement (DSS) and first-step warming in a larger volume. All methods led to altered expression of target genes, most noticeably when the second media was used. Further quantitative studies of the impact of OCT4, FIGLα and β-actin should be conducted, together with clinical comparisons between media and a longitudinal multi-centre study regarding outcomes arising from different vitrification methods.
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Tomiak, Robert B., Jason L. Rennecker, Benito J. Marinas, Richard J. Miltner, and James H. Owens. "Modeling Cryptosporidium spp. Oocyst inactivation in bubble-diffuser ozone contactors." Thesis, Urbana, Illinois, Univeristy of Illinois at Urbana-Champaign, 1998. http://hdl.handle.net/10945/37776.
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CIVINSThe CT concept (product of disinfectant concentration and characteristic contact time) is currently used to demonstrate compliance with disinfection requirements for Giarda lamblia (G. lamblia) and viruses under the Surface Water Treatment Rule (SWTR). Minimum CT requirements include large safety factors to account for possible deviations from actual disinfection efficiencies achieved in full-scale contactors. The application of this conservative regulatory approach for Cryptosporidium parvum (C. parvum) might result in unrealistic disinfection requirements under the Enhanced SWTR due to the much stronger resistance of this protozoan parasite to inactivation by all chemical disinfectants used in drinking water applications. There is a need for the development of approaches that could provide a more accurate assessmant of actual inactivation efficiency achieved in disinfection contactors. The main objective of this study is to develop and apply a mathematical model predicting the inactivation of Cryptosporidium app. (C. parvum and C. muris) oocysts in ozone bubble-diffusers contactors. The model is calibrated with semi-batch kinetic data, verified with pilot-scale inactivation experiments, and used for predicting and optimizing full-scale disinfection efficiency.
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Akinosoglou, Karolina-Anthoula. "Anopheles/Plasmodium interactions at the ookinete-to-oocyst developmental transition." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/14690.
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The ookinete to oocyst developmental transition of the Plasmodium parasite represents amajor population bottleneck in the malaria life cycle. This suggests that it could be a target forintervention strategies, such as transmission blocking vaccines, provided essential parasite targetmolecules can be identified. A recent microarray analysis has identified a large number of transcriptsdifferentially expressed during the parasite?s developmental transitions. Genes differentiallyregulated during the ookinete-to-oocyst transition may determine the development of the parasitewithin the mosquito host, as well as, participating directly in parasite/mosquito interactions. Yet, thefunction of the majority of such molecules is largely unknown.This PhD thesis aims to identify and functionally characterise genes putatively involved inookinete development and/or the interactions between the parasite and the mosquito host in the modelsystem Plasmodium berghei. Thirty three proteins likely to be implicated in the parasite?s interactionwith the mosquito immune system and local epithelial response were identified based on theirexpression pattern and predicted structural features. Generation of knock-out mutants throughtargeted gene disruption by homologous recombination was the first step towards functionalcharacterization of these candidates. Successful mutants were assessed for their ability to completetheir sexual sporogonic development, as well as, their impact on mosquito immunity followinginfection of Anopheline mosquitoes of various immune backgrounds. Interestingly, two of thesuccessful mutants were hampered in their ability to undergo normal differentiation during ookinetedevelopment while the third one?s ability to invade the mosquito midgut epithelium was impaired.The inability to invade implies a potential interaction of this gene product with mosquito midgutligands. Eventually malaria transmission through Anopheline mosquitoes was affected in all threemutants. Moreover, challenging of a mosquito protein LRIM1, a major parasite antagonist, alsorevealed potential involvement of the three mutants in mosquito/parasite immune response pathways.Genetic crosses with parasite lines deficient in the production of either male or female fertile gametesdemonstrated in the case of two mutants that, this defect in ookinete development is sex dependent,thus underlining the critical importance of maternal and/or paternal control during the first few hoursof parasite development in the mosquito.
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Nazir, Mozamel. "Novel methods for the separation of Cryptosporidium parvum oocysts from water." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252194.
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Crockett, Christopher Scott Haas Charles N. "The significance of streambed sediments as a reservoir of Cryptosporidium oocysts /." Philadelphia, Pa. : Drexel University, 2004. http://dspace.library.drexel.edu/handle/1860/290.
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Cox, Lindsay. "Oocyte Quality: Molecular Constituents Altered in the Oocyte Due to Various Environmental Factors." DigitalCommons@USU, 2016. https://digitalcommons.usu.edu/etd/5042.
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An estimated 1.6 million American couples struggle with infertility. Some causes for poor fertility can be clearly defined but in many instances, subfertility is unexplained. Poor oocyte quality is now considered to be a main contributing factor for many causes of infertility. Good oocyte quality is crucial for many processes including embryo development and maintaining pregnancy. There is a possibility that any alterations to the oocyte can have long lasting effects on embryo development and the health of the offspring. The oocyte is very sensitive to any perturbations to its surrounding environment. Transcripts for apoptosis inhibitors and epigenetic modifiers were found to be significantly more abundant in in vivo-matured oocytes compared to oocytes that were matured in vitro. RNA degradation and chromatin remodeling pathways may also be perturbed in in vitro-matured oocytes. While examining the effects of maternal age on the oocyte, there are age-related differences in gene expression in equine cumulus-oocyte complexes. Differences in gene expression may lead to a decrease in oocyte developmental competence. Age related alterations to gene expression in the equine cumulus-oocyte complexes might be caused by increased rates of oxidative stress and subsequent DNA damage. These alterations could directly impact many processes within the oocyte. Higher incidences of apoptosis may be possible in the cumulus cells from aged mares, which would directly impact the developmental competence of the oocyte. Lastly, oocyte quality may be impacted by western dietary consumption patterns, which could lead to many genes being differentially expressed in oocytes. Alterations to the abundance of these genes have been shown to lead to effects that are commonly seen with metabolic syndrome, such as glucose intolerance, insulin resistance, obesity and diabetes. The results of this work will ultimately provide insight into the effects environmental influences have on the oocyte at the molecular level.
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Weingarten, Lisa Suzanne. "The oocyte-to-embryo transition : regulation of oocyte maturation and egg activation in Drosophila." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/79191.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 33-39).
In oogenesis, meiosis must be highly regulated to ensure that growth of the oocyte and chromosomal segregation are coordinated properly. To do this, meiosis arrests at two points to permit oocyte differentiation and coordination with fertilization. In Drosophila, the first arrest in prophase I is released by oocyte maturation, and the second arrest in metaphase I is released by egg activation. This thesis explores mechanisms controlling these two processes. First, the putative role of the Deadhead (DHD) thioredoxin in Drosophila female meiosis is examined. Possible roles that DHD may play in DNA replication, ROS/RNS redox pathways, and vitelline membrane crosslinking are explored. Furthermore, current research into the role of Ca²+ as a regulator of Drosophila egg activation is summarized. Recent studies have suggested that Sarah (Sra), a regulator of Calcineurin (CN), is required for egg activation and meiotic completion. A model for Sra/CN signaling is presented, highlighting the role of Ca²+ in Drosophila activation, and emphasizing aspects of meiotic activation conserved across species. Finally, proteins recovered from a large-scale proteomic screen undertaken by our lab are discussed. This screen identified proteins that increase or decrease significantly during the processes of maturation and activation through quantitative mass spectrometry. Pairwise comparison of protein levels between pre- and post- maturation oocytes (stage 10 vs. stage 14 oocytes) or pre- and post-activation eggs (stage 14 vs. unfertilized eggs) identified candidate proteins up- and downregulated during one or both of these processes. These candidates include proteins involved in calcium binding and transport, the ubiquitination pathway, steroid biosynthesis and metabolism, and a gap junction protein. Additional characterization of these proteins may provide further insight into the regulation of Drosophila maturation and activation.
by Lisa Suzanne Weingarten.
S.M.
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Duffié, Rachel. "Epigenetic inheritance from the oocyte." Paris 6, 2013. http://www.theses.fr/2013PA066077.
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La méthylation de l’ADN est essentielle pour le développement des mammifères. Cette marque épigénétique peut être héritée des gamètes parentaux et influencer les phénotypes dans la descendance, un phénomène reconnu sous le nom d’empreinte parentale lorsque la transmission se fait de manière asymétrique depuis l’ovocyte et le spermatozoïde. Mon travail de thèse a concerné la méthylation spécifiquement héritée de l’ovocyte chez le modèle murin. J’ai tout d’abord montré que la méthylation ovocytaire est globalement dépendante du cofacteur d’ADN méthyltransférase DNMT3L. De manière importante, j’ai mis en évidence de nouvelles formes d’empreinte héritées de l’ovocyte, maintenues de manière tissu-spécifique ou très transitoirement au cours du développement. Le gène Cdh15 est soumis à une empreinte tissu-spécifique : la méthylation maternelle est maintenue et conduit à l’expression paternelle d’un transcrit alternatif uniquement dans l’hypothalamus. J’ai également identifié un ARN régulateur au locus Gpr1/Zdbf2 comme soumis à une empreinte maternelle transitoire : son expression mono-allélique très brève dans l’embryon péri-implantatoire induit en cis des marques de méthylation permanentes associées à l’expression paternelle de Zdbf2 tout au long de la vie. Ces découvertes ouvrent des perspectives nouvelles quant à la régulation spatio-temporelle de l’empreinte parentaleDNA methylation is essential for mammalian development. Genomic imprinting regulates parent-of-origin phenotypes through differential gametic inheritance of DNA methylation at imprinting control regions, ICRs, which is maintained in the progeny ubiquitously and lifelong. During my PhD, I focused on DNA methylation inherited from the oocyte using the mouse model. I found that DNMT3L is required for global oocyte methylation. I also provide evidence for new forms of imprinting, demonstrating that maternal imprints can be maintained in a tissue-specific manner or very transiently during development. The Cdh15 gene is subject to tissue-specific imprinting: maternal-specific methylation is maintained in the hypothalamus where it leads to paternal expression of an alternative transcript. I also identified a regulatory RNA at the Gpr1/Zdbf2 locus under the control of a transient maternal imprint. Its brief monoallelic expression in the periimplantation embryo is associated with establishment of permanent methylation marks in cis and subsequent lifelong paternal expression of neighboring Zdbf2. These findings provide new perspectives about the permanency and regulation of genomic imprinting in mammals
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Amburgey, James E. "Improving filtration for removal of cryptosporidium oocysts and particles from drinking water." Diss., Georgia Institute of Technology, 2002. http://hdl.handle.net/1853/20723.
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Bukhari, Zia. "Cryptosporidium infections in livestock and the significance of environmental contamination with oocysts." Thesis, University of Strathclyde, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309045.
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Bushell, Ellen S. C. "Plasmodium genes responsible for oocyst development and interaction with its Anopheline vector." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/5542.
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The transmission of the malaria parasite Plasmodium is governed by a complex developmental cycle. This PhD thesis describes the transcriptional profiling of the rodent malaria parasite Plasmodium berghei developmental migration through its A. gambiae vector. The study was conducted in vivo, using a near complete P. berghei genome microarray platform. Emphasis was placed on the oocyst stage, as little is known about the genes implicated in the ookinete to oocyst transition, and oocyst maturation. The data presented here provide novel transcriptional information about Plasmodium transmission. The analysis revealed a large shift in gene utilisation as the parasite makes its transition from the motile ookinete to the sessile oocyst. Furthermore, this work has shown that different sets of co-regulated genes are important for early and late oocyst development. In addition, this PhD thesis outlines the characterisation of a novel Plasmodium formin-like protein essential for rodent malaria transmission named the male inherited sporulation factor important for transmission (misfit). MISFIT is expressed in the early mosquito stages, where the protein localises to the parasite nucleus. Misfit exhibits an absolute requirement for paternal inheritance, which is in accordance with an observed male-biased expression pattern. pbmisfitΔ ookinetes display significant ultrastructural and gene expression defects and fail to complete zygotic meiosis. However, pbmisfitΔ ookinetes retain functionality and can successfully cross the midgut epithelial barrier. In contrast, mosquito infections with pbmisfitΔ resulted in an arrest immediately upon ookinete-oocyst transformation, where defective oocysts fail to sporulate. An essential role in chromosome segregation during mitosis / meiosis is postulated for MISFIT. In conclusion, the work presented in this thesis has established the ookinete-oocyst transition as a major cell cycle check point during malaria transmission and identified misfit as the first male inherited Plasmodium gene known to affect development post-fertilisation.
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Hurtubise, Patricia. "Intracellular signalling during murine oocyte growth." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=31239.
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During the growth phase of oogenesis, mammalian oocytes increase several hundred-fold in volume. Although it is known that ovarian granulosa cells send growth promoting signals, neither these external signals nor the transduction pathways that become activated in the oocyte are known. Therefore, the presence and the activity of candidate signaling pathways in growing murine oocytes were investigated. By immunoblotting, the MAP kinases, ERK1 and ERK2, as well as their activating kinase MEK, were detected in oocytes at all stages of growth. However, using a phospho-specific anti-ERK antibody, no immunoreactive species were detectable in isolated granulosa cells or oocytes at any stage of growth, except metaphase II. Phosphorylated ERK was also present, although in smaller quantities, in oocyte-granulosa cell complexes at the later stages of growth. Furthermore, when ovarian sections were stained with an anti-ERK antibody, the protein was found to be highly concentrated in the cytoplasm of oocytes at all stages of growth, with lower levels in the nucleus. Another member of the MAP kinase family, Jun kinase (JNK), was investigated. By immunoblotting, JNK was detected in growing oocytes. Experiments using an anti-JNK antibody on ovary sections revealed the protein to be uniformly distributed in non-growing and growing oocytes with no evidence of preferential nuclear localization. These results imply that an interaction between the oocyte and the granulosa cells may be required to generate phosphorylated ERK. They also imply that growth signals probably are not relayed through ERK, but do not exclude a role for Jun kinase in mediating oocyte growth.
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Dalton, C. M. "Mitochondrial dynamics during mouse oocyte maturation." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1335718/.
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Mitochondria provide the primary source of ATP for the oocyte and pre-implantation embryo and undergo a number of redistributions during oocyte maturation which may be related to developmental competence. The experiments presented in this thesis aim to examine the changes in distribution and function of mitochondria during the transition from the germinal vesicle stage to the mature metaphase II arrested egg. Mitochondrial distribution was monitored throughout oocyte maturation and accumulation of mitochondria around the first meiotic spindle was observed. This was dependent on the activities of microtubules and their associated motor proteins dynein and kinesin. Migration of the spindle to the oocyte cortex was accompanied by mitochondria but at polar body extrusion a dramatic reorganisation of mitochondria away from the cortical domain occurred, suggesting that a mechanism exists for retention of these important organelles in the oocyte during this asymmetric cell division. The role of the mitochondrial adapter proteins Trak and Miro in establishing redistribution of mitochondria was also addressed. Finally, a novel recombinant FRET probe for measuring ATP was validated for use in oocytes. Use of this probe revealed alterations to both ATP levels and ATP consumption at different stages of oocyte maturation. Furthermore, whilst the first meiotic spindle was found to be dependent on mitochondrial activity to retain its structure and function, attempts to identify subcellular heterogeneity in ATP supply and demand related to the distribution of mitochondria around the spindle did not reveal any differences. However, the presence of cumulus cells surrounding the oocyte as part of a cumulus-oocyte complex was found to influence ATP levels in the oocyte; oocytes matured as part of a cumulus-oocyte complex had higher ATP levels than those observed in oocytes which had been denuded of cumulus cells. This was found to be dependent on the presence of gap junctional communication between the somatic and germ cell compartments, since inhibition of gap junctions abolished the higher ATP levels observed in cumulus enclosed oocytes.
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Januschke, Jens. "MRNA localization in the Drosophila oocyte." Paris 7, 2005. http://www.theses.fr/2005PA077101.
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Amdani, Siti Nornadhirah. "The oocyte-activation factor, phospholipase C zeta (PLCζ) : clinical prognosis, diagnosis, and treatment of oocyte activation deficiency." Thesis, University of Oxford, 2018. https://ora.ox.ac.uk/objects/uuid:af4c4f98-497a-4666-9eec-a46bb579dd59.
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Oocyte activation deficiency (OAD) is an infertile condition observed in patients who have experienced recurrent total fertilisation failure (TFF) following intracytoplasmic sperm injection treatment. This condition was considered to be an idiopathic factor for a long time but strong clinical evidence now suggests that dysfunctional forms of phospholipase C zeta (PLCζ) may be predominant causative factors for OAD. Genetic contribution has played a role in patients suspected of having OAD, as four PLCζ exonic mutations have been discovered and characterised as being the cause of infertility. In this study, a novel nonsense mutation, PLCζK322Stop, was identified in the PLCζ XY-linker region of Patient LR. This variant results in the truncation of approximately half of PLCζ, therefore was non-functional when activity was tested. Patient LR, which also exhibited a previously reported mutation, PLCζH233L, may suggest that the patient is sub-fertile, as opposed to being infertile, as initially expected. Although research has purely focused upon the coding regions of PLCζ, it was obvious that our knowledge of PLCζ regulatory elements remain very limited. Next generation sequencing (NGS) was therefore employed to detect variants in the non-coding regions of PLCζ, promoter and introns, which may have resulted in the observed phenotypic diversity of PLCζ expression in fertile and infertile patients. As a result of mapping failure, an alternative approach was considered to identify variants within human PLCζ, and this involved using the single nucleotide polymorphism (SNP) database. Over 2500 SNPs were localised in the intronic regions of PLCζ and thus, it could be speculated that these variants may help elucidate the wide variation of PLCζ expression reported. Additionally, two particular patients with TFF (79 and 107) were investigated in this study to identify an association with PLCζ and their infertile state. For Patient 79, multiple PLCζ immunofluorescence analysis was performed and a significant improvement in PLCζ expression was observed one year after his first investigation. This may have been the result of an external factor, which influenced protein expression. As for Patient 107, a novel substitution mutation, PLCζV193E, was identified and was predicted to affect PLCζ stability and folding. There is global interest to create a safer and alternative OAD therapy, namely a human recombinant PLCζ protein (hrPLCζ). The first method, using a bacterial cell line resulted in successful purification and identification but the product proved to be inactive following mouse oocyte microinjection. The second method involved production of a mammalian-expressed hrPLCζ, which was successfully purified and identified but due to time restrictions, could not be tested for functionality. Concurrently, the findings in this thesis have reinforced the association between PLCζ and OAD, and provided improved options for the diagnosis and treatment of OAD.
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Chen, Hong, Stefanie Wiedmer, Sacha Hanig, Rolf Entzeroth, and Michael Kurth. "Development of Eimeria nieschulzi (Coccidia, Apicomplexa) Gamonts and Oocysts in Primary Fetal Rat Cells." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-127190.
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The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.
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Basak, Shibesh Chandra. "The identification of oocysts of chicken Eimeria species : biochemical, immunological and molecular biological approaches." Thesis, University of Liverpool, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356983.
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Jellison, Kristen L. (Kristen Leigh) 1975. "Molecular and genetic analysis of Cryptosporidium spp. oocysts : sources and genotypes in the environment." Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/29359.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 2003.Includes bibliographical references.
Cryptosporidium parvum is responsible for an acute gastrointestinal disease that is self-limiting in immunocompetent people but potentially life-threatening for the immunocompromised. Until recently, C. parvum was the only species of Cryptosporidium known to cause disease in people, however, reports of C. muris, C. felis, and C. meleagridis in immunocompetent adults have raised questions about the extent to which Cryptosporidium spp. are infectious for humans. Until more is known, presence of any Cryptosporidium oocysts in the environment should be considered a potential public health risk. Cryptosporidium spp. can infect a wide range of animal hosts, and environmental sources may include wildlife, agricultural animals, or human sewage. Transmission of Cryptosporidium spp. via fecally-contaminated food and water has been well-documented, and outbreaks of cryptosporidiosis have occurred around the world. The exogenous stage of the organism, the oocyst, is difficult to remove from drinking water supplies because it is resistant to chlorine disinfection and inefficiently filtered. Therefore, a better understanding of the sources, fate, and transport of oocysts in the environment is critical to protect source waters from oocyst contamination. In this work, a sensitive and specific molecular detection assay for Cryptosporidium spp. in environmental samples was developed and applied to surface water and fecal samples from the Wachusett Reservoir watershed, the drinking water source for metropolitan Boston, to establish links between oocyst sources and surface water contamination. Multiple species of Cryptosporidium were detected, and previously uncharacterized genetic diversity at the 18S rRNA locus was observed.
(cont.) Each surface water site had a hypothesized oocyst source, but results showed that the sources detected were often very different from those hypothesized to be most important. Cryptosporidium spp. from wildlife was detected in surface waters hypothesized to be contaminated by human sewage, and surface waters susceptible to agricultural runoff were observed to be more impacted by birds. In addition, Cryptosporidium spp. contamination occurred seasonally, with the seasonal pattern of detection distinct for surface waters with different oocyst sources. Results of this work contribute to a growing characterization of Cryptosporidium in the environment that will ultimately help minimize public exposure to this waterborne parasite.
by Kristen L. Jellison.
Ph.D.
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Chen, Hong, Stefanie Wiedmer, Sacha Hanig, Rolf Entzeroth, and Michael Kurth. "Development of Eimeria nieschulzi (Coccidia, Apicomplexa) Gamonts and Oocysts in Primary Fetal Rat Cells." Hindawi, 2013. https://tud.qucosa.de/id/qucosa%3A27286.
Full textAbstract:
The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.
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McClellan, Kelly Anne. "Murine oocyte loss occurs during fetal development." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79047.
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Recently, the timing of oocyte loss during murine development has been brought into question as authors using mouse vasa homologue (MVH) as a germ cell marker did not observe a loss of oocytes during fetal life. Instead the major loss was observed in the days following birth, after chromosome pairing has occurred.In this study the controversy was addressed by establishing a new and reliable method to quantify murine oocytes in meiotic prophase, as well as to determine the gestation age and meiotic prophase stage of oocyte loss. Earlier limitations were overcome through the use of Germ Cell Nuclear Antigen-1 (GCNA-1) antibody as a germ cell specific marker, and the novel addition of a cytospin centrifugation step to the method. Progress through meiotic prophase was examined in chromosome spread preparations where meiotic stages were assessed using an antiserum against synaptonemal complex (SC) proteins. Quantification was accomplished by counting the number of GCNA-1 immunoreactive cells in chromosome spread preparations and estimated in histological sections using the ratio estimation model. (Abstract shortened by UMI.)
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Martínez, Marchal Ana. "Regulation of the oocyte pool in mammals." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667797.
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Durant la oogènesi dels mamífers, les oogònies proliferen forman els anomenats cists. Les oogònies entren en meiosis progressant en la profase I i els cists es trenquen al mateix temps que es produeix una mort massiva perinatal dels oòcits. En la profase I, s’indueixen trencaments de doble cadena (DSBs) per tot el genoma, que son reparats per recombinació homòloga per a promoure la sinapsi dels cromosomes homòlegs. Existeixen diferents mecanismes que s’activen en resposta a errors en aquests processos i que aturen el cicle cel·lular i produeixen l’apoptosi de les cèl·lules danyades. La resposta al dany al DNA (DDR) es activada en presència d’oòcits i d’espermatòcits amb errors de recombinació en l’anomenat checkpoint de recombinació. Per l’altre banda, errors en la sinapsi activen el checkpoint de sinapsi. El nostre objectiu era caracteritzar les funcions de la DDR i del checkpoint de sinapsi durant l’oogènesi en mamífers. Contràriament al que succeeix en espermatòcits, els oòcits presenten un alt número de DSBs no reparats a l’estadi de paquitè en el moment en que es produeix la mort oocitària massiva i el trencament del cists. Per tal d’esbrinar si el checkpoint de recombinació participa en la regulació del número d’oòcits en mamífers, hem analitzat el número de DSBs, el número d’oòcits en femelles perinatals i adultes, el trencament dels cists, la formació de fol·licles i la vida reproductiva de femelles de ratolí control i mutants per a la quinasa efectora de la via de la DDR, la proteïna CHK2. Les nostres dades han revelat la implicació de CHK2 en la regulació del número d’oòcits, però només en ovaris fetals, obrint la possibilitat de l’existència d’una via alternativa regulant el número d’oòcits després del naixement. Els nostres estudis utilitzant ovaris cultivats in vitro en presència d’inhibidors, suggereixen que CHK1 podria compensar l’absència de CHK2 in vivo. Per tant, la via de la DDR controlaria el número d’oòcits en mamífers. A més, hem trobat un augment del número d’oòcits en adultes velles mutants per CHK2 suggerint que la DDR controla la llargada de la vida reproductiva en mamífers. Finalment, hem estudiat el possible paper de TRIP13 en el checkpoint de sinapsi. La proteïna TRIP13 es necessària per a la recombinació, però també per a la sinapsi dels cromosomes sexuals i per a la formació de la vesícula sexual, suggerint un possible rol al checkpoint de sinapsi. Hem analitzat el número d’oòcits en ovaris Spo11-/- Trip13mod/mod i Dmc1-/- Chk2-/- Trip13mod/mod per a esbrinar si TRIP13 es necessària per a activar el checkpoint de sinapsi en femelles. Les nostres dades han revelat un rescat en el número d’oòcits en el triple mutant, però no en el doble. Aquest resultats obren la possibilitat de que TRIP13 participi en el checkpoint de sinapsis, però com a alternativa, proposem que aquesta participació podria ser compatible amb una possible regulació per part de TRIP13 de la elecció de la via de reparació dels DSBs.During mammalian oogenesis, oogonia proliferate forming the so-called cysts. The oogonia enter meiosis progressing through prophase I and the cysts break down concomitantly to massive perinatal oocyte death. During meiotic prophase I, double strand breaks (DSBs) are induced throughout the genome and repaired by homologous recombination to promote the synapsis of the homologous chromosomes. In response to errors in these processes, different response pathways are activated triggering cell cycle arrest or even apoptosis. The DNA damage response (DDR) is activated in response of meiocytes with recombination failure in the recombination checkpoint; while errors in synapsis trigger the synapsis checkpoint. We aimed to characterize the roles of the DDR and synapsis checkpoint in mammalian oogenesis. Contrary to what occurs in spermatocytes, oocytes present high numbers of unrepaired DSBs at pachynema, at the time of the massive oocyte death and cyst breakdown. In order to know if the recombination checkpoint participates in the regulation of the oocyte number in mammals, we analyzed the presence of DSBs, the oocyte number in both perinatal and adult females, the cyst breakdown, the formation of follicles and the reproductive lifespan using control and mutant mice for the effector kinase of the DNA damage response pathway, CHK2. Our data revealed the involvement of CHK2 in the regulation of the oocyte number but only in fetal ovaries prior to birth, raising the question of a possible alternative regulator acting just after birth. Our studies using in vitro ovarian cultures using inhibitors, suggest that CHK1 may compensate the loss of CHK2 perinatally in vivo. Thus, revealing that the DDR pathway controls the oocyte number in mammals. Furthermore, we found an increased number of oocytes in elder Chk2 mutant females suggesting that the DDR controls the reproductive lifespan extension in mammals. Finally, we studied the possible involvement of TRIP13 in the synapsis checkpoint. The protein TRIP13 is required for recombination, but it is also needed for the synapsis of sex chromosomes and the sex body formation. Thus, suggesting a possible role in the synapsis checkpoint. We analyzed the oocyte number in females from Spo11-/- Trip13mod/mod and Dmc1-/- Chk2-/- Trip13mod/mod ovaries in order to infer if TRIP13 is required to implement the synapsis checkpoint in females. Our data revealed a rescue in the number of oocytes in the triple mutant, but not in the double mutant. These results leave open the possibility of a participation of TRIP13 in the synapsis checkpoint, but as an alternative, they could be compatible with a possible role of TRIP13 regulating the DSB repair pathway choice.
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Carroll, John. "Cryopreservation and development of the mammalian oocyte /." Title page, contents and abstract only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phc319.pdf.
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Siordia, Jimena Carolina. "Analysis of Toxic Chemicals Affecting the Oocyte." Thesis, The University of Arizona, 2009. http://hdl.handle.net/10150/192989.
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Davidson, Bryony Kathryn. "Raman spectroscopic investigation of the murine oocyte." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4641.
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Over recent years, the application of assisted reproductive techniques in the treatment of infertility has increased exponentially, yet these methodologies still remain inherently inefficient. It has long been established that the single greatest obstacle to improving the success of these treatments is determining the quality of the oocytes used. However, currently the methods available for oocyte assessment are mainly qualitative, and suffer due to a lack of standardisation. As such, the efficiency of fertility treatments could benefit from the introduction of a rigorous quantitative measure of oocyte quality and maturation. The principal aim of this thesis was to determine the potential of Raman spectroscopy when applied to the field of oocyte biology. Consequently, this thesis addressed three main areas of investigation: I. the intra-oocyte biochemical variation; II. the biochemistry of oocyte maturation; and finally, III. the effect of environment on the mature oocyte in vivo and in vitro. I. To investigate the presence of intra-oocyte biochemical variation, oocytes from various stages of development were analysed using high resolution Raman mapping, in combination with univariate and multivariate analysis. Images revealed variation between the germinal vesicle and ooplasm in immature oocytes, as well as intra-ooplasmic variation in all oocytes. II. The spectral analysis of oocytes derived from pre-antral and in vitro cultured follicles revealed significant variation: It was found that Raman spectroscopy could successfully discriminate between immature and mature oocytes. III. Finally, the spectral analysis of oocytes derived from unstimulated and stimulated ovulation cycles, as well as those derived from in vitro follicle cultures, revealed that although biochemically similar, in vitro matured oocytes demonstrated reduced protein content. Furthermore, greater spectral variation was observed in superovulated oocytes, which was found to describe the corresponding morphological quality. In conclusion, this thesis has demonstrated the effective application of Raman spectroscopy to the study of fixed murine oocytes. Raman mapping experiments have demonstrated this technique for the visualisation of biochemical variation which exists within the oocyte. Furthermore, using Raman spectroscopy, the identification of the biochemical variation resulting from different maturation mechanisms has been achieved, as has the discrimination of immature and mature oocytes. These results indicate that Raman spectroscopy holds promise as a quantitative analysis method in the field of fertility treatment.
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Huynh, J. R. "Oocyte determination and polarisation during Drosophila oogenesis." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604905.
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The antero-posterior and dorso-ventral axes of the Drosophila embryo are set up during oogenesis. These axes of polarity are the results of several symmetry-breaking steps that characterise the development of the Drosophila oocyte. During my PhD, I have studied two of these steps. Oocyte determination. During early oogenesis, one cell is selected to become the oocyte within a cyst of 16 germ cells. These 16 germ cells are all siblings and share the same cytoplasm. By using several oocyte-specific markers, I have found that there at least three different pathways to restrict the oocyte fate to one cell. The restriction of cytoplasmic proteins depends on the microtubules and on the activity of the Egl/BicD complex. The restriction of meiosis also depends on the same complex but is independent of the microtubules. Finally, the restriction of the centrosomes is independent of both the microtubules and the Egl/BicD complex. From this analysis, I also concluded that the selection of the oocyte does not depend on the microtubules and that the oocyte is probably selected while the cyst is still dividing, as shown by the asymmetric inheritance of the fusome. By homology with the polarisation of the C. elegans one cell embryo, I have shown that the Drosophila homologues of par-1, par-3 and par-6 are required for the first polarisation of the oocyte. This polarisation is then required to maintain the oocyte fate. Oocyte polarisation. During mid-oogenesis, the A/P and D/V axes of the oocyte are defined by the asymmetric localisation of several mRNAs. I have conducted a germline clone screen to find lethal genes involved in this polarisation. In particular, I report here that one of the major Drosophila hnRNP, hrp48, is required for the localisation of oskar mRNA within the oocyte.
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McKenzie, Andy. "Modelling spiral waves in Xenopus laevis oocyte." Thesis, University of Canterbury. Mathematics and Statistics, 1997. http://hdl.handle.net/10092/6954.
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An investigation was made into the spiral waves solutions for the Atri et al model, a partial differential equation model for Ca²⁺ dynamics in the Xenopus laevis oocyte. Spiral wave solutions, both stable and unstable, were found to exist in the oscillatory regime for this model. The spiral wave solutions were found to have a period that decreased as the initial IP₃ bolus increased. Increasing the initial IP₃ bolus also lead to destabilisation of the spiral waves solutions. After the break up of spiral wave solutions complex spatio-temporal patterns occurred. In some cases spirals reformed after breaking up.
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Sleep, Darrell. "Molecular analysis of Xenopus oocyte maternal RNA." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/11917.
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Assidi, Mourad. "Oocyte competence and cumulus cells gene expression." Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/27679/27679.pdf.
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Hemmings, Karen Emily. "Cellular and molecular markers of oocyte quality." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445945.
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Mola, Choulia. "Identification of oocyte reprogramming factors and mechanisms." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/42492/.
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The ability of the oocyte to reprogram paternal chromatin is widely accepted. The oocyte cytoplasm has been reported to contain activities that alter the histone composition, demethylate and unwind highly methylated paternal DNA upon fertilization and lead to zygote formation. The same events are mimicked during the course of cell reprogramming, where somatic cells exposed to the oocyte environment acquire a pluripotent character. Amphibian oocytes have been widely used to study the reprogramming attributes of the female germ cells for over half a century. The size of amphibian oocytes renders them good candidates for technical manipulations, as well as protein biochemistry studies. Oocytes harvested from the amphibian axolotl were utilised in the present study to delineate the oocyte reprogramming machinery. Previous studies have reported the presence of key pluripotency factor orthologs, such as Nanog, in the axolotl oocyte, thus rendering it an interesting tool for the study of the chromatin remodelling and pluripotency networks as well as their interplay. To study these networks, identification of the physical interactors of the Nanog protein or the Nanog promoter was attempted. One of the key events taking place during cell and paternal chromatin reprogramming is demethylation. The removal of the methyl mark that is deposited over genetic loci during cell differentiation is linked with the transcriptional activation of the respective loci. Demethylation is therefore fundamental for the activation of pluripotency-associated factors both in fertilisation and cell reprogramming. The leading theory behind demethylation is the successive oxidation of the methyl mark to formyl and carboxyl and its subsequent removal from Thymine DNA Glycosylase (TDG). Ten Eleven Translocation (TET) family proteins catalyse methyl oxidation and therefore utilise demethylation. Additional demethylation pathways have also been supported by findings, specifically in the context of the oocyte. Base Excision Repair (BER) and Nucleotide Excision Repair (NER) factors have been associated with DNA demethylation occurring immediately after fertilisation. It is therefore fundamental to delineate the demethylation mechanism facilitated by the oocyte in the early stages of development. The identification of oocyte demethylation factors and mechanisms will greatly improve our understanding of demethylation as well as improve current cell reprogramming methodologies by using the more efficient oocyte demethylation mechanism. To delineate the demethylation mechanism employed by the oocyte and contribute to the debate of TET oxidation versus NER or BER demethylation via DNA repair in the context of the oocyte, oocyte factors preferentially binding to different cytosine modifications were analysed. The murine Nanog promoter, a genetic locus activated during cell reprogramming, harboured the different cytosine modifications in an attempt to see how each one would affect its transcriptional status. As a result, TET factors were not discovered to bind any of the cytosine modifications while both NER and BER pathway proteins were found to be significantly enriched in the case of methylcytosine. The results obtained therefore support previous findings and advocate for a TET independent DNA demethylation occurring through DNA repair. While teams researching demethylation through DNA repair have supported either NER or BER, the findings unveiled in this thesis advocate for the two pathways acting in concert in the recognition and potential removal of DNA methylation. It also has to be noted that it is the first time an experiment of this kind is carried out in the context of the oocyte and the findings offer a unique insight into oocyte demethylation dynamics. Taking into account the data obtained and previous research, a new DNA demethylation model is proposed. The model advocates for NER taking the lead in DNA demethylation, creating patchy demethylated sites that are subsequently recognised and demethylated via the BER pathway.
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Connolly, Amy. "Oocyte Meiotic Spindle Assembly in Caenorhabditis Elegans." Thesis, University of Oregon, 2014. http://hdl.handle.net/1794/18492.
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As in many organisms, Caenorhabditis elegans oocytes assemble bipolar meiotic spindles in the absence of centrosomes. While the assembly of the mitotic spindle in C. elegans has been studied in some detail, how the poles assemble in the absence of centrosomes remains poorly understood. In an ongoing screen for temperature-sensitive (TS), embryonic-lethal mutants, we have identified TS mutations in multiple genes required for oocyte meiotic spindle pole assembly. We have so far identified mutations in four genes: or1178ts in mei-1, which encodes the catalytic domain of the microtubule severing complex katanin; or447ts in klp-18, which encodes a kinesin 12 family member; or645ts in aspm-1, which encodes a microtubule scaffolding protein; and or1092ts and or1292ts in klp-7, which encode a kinesin 13/MCAK family member. By using live cell imaging of oocytes from transgenic strains expressing GFP and mCherry fusion to proteins associated with the spindle, we have found and confirmed other findings that klp-18 promotes spindle bipolarity and that MEI-1 promotes pole assembly both by severing microtubules and by recruiting ASPM-1. More recently, we have found that klp-7 is required for maintaining bipolarity in the meiotic spindle by preventing the number of poles that can form. In klp-7(-) mutants, we observed in addition to extraneous poles an excess accumulation of microtubules during Meiosis I. Futhermore, reducing klp-7 function can restore bipolarity in a klp-18(-) monopolar spindle mutant background. We also observed that disruption of the kinetochore factor KNL-1 in klp-7(-) mutants exacerbates the extra spindle pole phenotype. We suggest that in oocyte meiosis, klp-7 is required to limit microtubule accumulation and pole assembly and that it may carry out these functions in a kinetochore-dependent manner.
This dissertation includes previously published co-authored material.
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Lee, Young Shin. "MATERNAL DETERMINANTS OF OOCYTE AND EMBRYO QUALITY." Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/111983.
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Molecular Biology and GeneticsPh.D.
Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oocyte quality, I have compared oocytes and cumulus cells of different qualities at a molecular level. I present in this thesis the pathways and molecules that may determine the developmental competence of oocyte as well as candidate molecular markers of oocyte and embryo quality. A cDNA microarray analysis was performed, comparing the transcriptomes of rhesus monkey MII oocytes of different qualities, high quality VVM oocytes and poor quality IVM oocytes. A small set of 59 Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oo was identified as differentially expressed between the two types of oocytes. These mRNAs are involved in steroid metabolism, cell-cell interactions, cellular homeostasis, cell adhesion, mRNA stability and translation. In addition, the overexpression of several imprinted genes in IVM oocytes were detected, indicating a possible loss of correct epigenetic programming during IVM. These results indicate that normal oocyte-somatic cell interactions may be disrupted during IVM and the interruptions of these interactions during the final phase of oocyte maturation may be the prime cause of reduced developmental competence of IVM oocytes. To elucidate oocyte quality factors linked to the cumulus cell phenotype, the transcriptomes of two types of rhesus monkey cumulus cells, IVM and VVM, were compared using a cDNA microarray analysis. In contrast to a relatively small difference between IVM and VVM oocytes, a large number of genes were differentially expressed between IVM and VVM rhesus cumulus cells. Moreover, a much larger number of differential mRNA expressions were observed comparing the transitions from pre-maturation cumulus cells to the IVM and VVM cumulus cells. The results from these array comparisons indicated that the cumulus cells may fail to achieve successfully normal gene regulation during IVM and thus make a remarkable amount of changes in gene expression to compensate for the loss. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not suppressed during IVM. In addition, a panel of 24 cumulus cell markers of oocyte quality was identified. Genetic effects on oocyte quality were explored by comparing transcriptomes of oocytes obtained from two different inbred mouse strains, B6 and D2, and F1 hybrid. A clustering analysis and statistical tests showed that the transcriptome of F1 oocytes is more similar to the B6 transcriptome than to the D2 at both GV and MII stages. Also, comparison analyses of GV stage oocyte transcriptomes with MII oocyte transcriptomes from three different mouse strains indicated that the number of overdominance genes at the MII stage is bigger than the number of overdominance genes at the GV stage. In order to investigate how the genes gain the overdominance during the GV to MII transition, overdominance genes were categorized according to their mRNA expression patterns at GV and MII stages. The results showed that more than 80% of overdominance genes belong to one of the four major transition groups. The further evaluation of changes in array intensities from GV to MII stage transition revealed that F1 oocytes and inbred strain oocytes differentially regulate the mRNA abundance during oocyte maturation and that the differential regulation of mRNA abundance by the F1 genotype is responsible for the increase of the number of overdominance genes during maturation from GV stage to MII stage. A mRNA sequence analysis indicated that the gain of overdominant low in F1 mRNA expression pattern during maturation may be regulated by 3'UTR motif elements. The number of dominance genes also increase during GV to MII transition. At both GV and MII stages, there are more genes with B6 dominant mRNA expression pattern than those with D2 dominance pattern. Lipid metabolism, small molecule biochemistry and cell death are biofunctions overrepresented in both dominance and overdominance genes. In addition, `blebbing' was identified as a biofunction significantly downregulated in the F1 and B6 MII eggs, indicating that the cellular function may be involved in oocyte maturation.
Temple University--Theses
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Heisz, Marianne. "Studies on the survival and viability testing of Cryptosporidium parvum oocysts in the water environment." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/mq21991.pdf.
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Kayed, Dima 1960. "METHODS FOR THE ISOLATION OF OOCYSTS OF CRYPTOSPORIDIUM FROM SLUDGE AND GIARDIA CYSTS FROM STOOL." Thesis, The University of Arizona, 1986. http://hdl.handle.net/10150/276355.
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Yokoi, Hayato, and Kenjiro Ozato. "Injection of DNA into the medaka oocyte nucleus." Laboratory of Freshwater Fish Stocks Bioscience Center Nagoya University, 1995. http://hdl.handle.net/2237/13805.
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Alton, Michelle. "Control of the oocyte population in mouse ovaries." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81585.
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Oocyte loss and meiotic prophase progression was studied in XY sex-reversed and XO female mice, two mouse models that lack pairing between their sex chromosomes. An arrest at the pachytene stage of meiosis was not observed, nor was a significant loss of oocytes at this stage compared to normal XX control mice. Thus, it was concluded that a pairing checkpoint either does not exist in oocytes or is not as stringent as the one observed in males.The effect of mutating the pro-apoptotic Bax molecule was studied at three distinct ages corresponding to the time when female germ cells are premeiotic, in meiotic prophase, and arrested in dictyotene. Although it appeared that more germ cells were retained in the Bax homozygous mutant compared to the wild-type and heterozygous mice at 18.5 dpc, by 24.5 dpc all of the mice possessed similar numbers of germ cells. These results indicate a role for Bax in germ cell death but also support the idea that an alternative pathway can compensate for the elimination of this molecule.
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Allsopp, Janet. "'Oocyte maturation in the Manila clam, Tapes philippinarum'." Thesis, Bangor University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357212.
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Ralph, John Hunter. "Factors affecting follicle and oocyte development in cattle." Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/11889.
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The mechanisms governing development of mammalian oocytes are not well understood. Isolation and in vitro growth of immature cattle follicles will enable determination of the factors affecting bovine follicular development, have potential applications in assisted reproduction and provide a suitable model for studying human infertility. Intercommunication of the oocyte and somatic cells is necessary for normal oocyte and follicle development. Studies using systems where oocyte-somatic cell communication is preserved allows an accurate assessment of the factors affecting follicular development. The aims of this project were to examine early follicle and oocyte development in cattle and determine whether the bovine oocyte plays a role in follicular development. A non-enzymatic isolation procedure was developed which allowed intact bovine follicles to be isolated. On the basis of follicle size, these could be divided into 3 distinct stages: large preantral, large preantral/early antral and antral follicles. A culture technique was devised which supported in vitro follicle and oocyte development, the key elements of which were: volume of medium (0.25 ml/follicle), serum and insulin minimal number of medium changes and a substrate of collagen. The effect of FSH on preantral to early antral follicles in culture was examined. Initial experiments on large preantral/early antral follicle growth found that all FSH doses stimulated an increase in follicle diameter. The dose of FSH was important as low levels did not stimulate proliferation or affect oocyte size whilst high levels reduced proliferation, inhibited oocyte growth and reduced oocyte quality. Oocyte localised granulosa cell proliferation was observed in some follicles only when a healthy oocyte was present, demonstrating the importance of oocyte-somatic cell communication in granulosa cell proliferation and differentiation. The intensity of oocyte localised proliferation was reduced at high FSH doses, confirming its dose dependent inhibitory effect on follicular development. FSH stimulated the growth of large preantral/early antral and antral follicles but not oocyte growth in any of the stages. The increase in size was due to an increase in intercellular spacing and, as antral cavities were neither maintained or formed during culture, this may be analogous to antrum development. FSH maintained granulosa cell proliferation in all follicle size classes. No detectable effect of FSH on preantral follicles were found, therefore the effect of FSH depends on the stage of follicle examined.
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Ghafari, Fataneh. "Oocyte progression and death during first meiotic prophase." Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409952.
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