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1
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.
2
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.
3
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.)
4
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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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.
6
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.
7
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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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.
9
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.
10
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
11
Zhu, Jie. "Pig oocyte activation and developmental competence of parthenogenetically activated oocytes : in vitro and in vivo studies". Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/27741.
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In somatic cell nuclear transfer in mammals, to clone a piglet is still a big challenge. Although many factors could contribute to the low success rate, such as quality of donor and recipient cells, types of donor cell including sources of animal breeds and tissues, number of passages and culture conditions, timing of cell cycle, procedures of nuclear transfer, techniques and embryos transfer, one of the factors is believed to be poor oocyte activation, especially in pig nuclear transfer. Therefore studies presented in this thesis aimed at the establishment of an in vitro culture system for pig oocyte maturation and embryo culture, based on this system an electrical activation protocol for pig oocytes was optimized and also tested by monitoring in vivo development of activated pig oocytes. Finally, the protocol was used for activating pig embryos reconstructed by transfer of somatic cells into enucleated ovulated oocytes and for production of pig parthenotes to maintain pregnancies of cloned pig embryos, which resulted in the birth of a cloned male piglet. The thesis comprises a total of 6 chapters. In addition to the review of literature (Chapter 1), general materials and methods (Chapter 2) and general discussion (Chapter 6), in Chapter 3 and 4, the studies focused on optimizing electrical parameters on pig oocyte activation and investigating the effects of activation conditions including temperature, activation medium, and concentrations of Ca2+ and Mg2+ in activation medium and diploidization of activated oocytes. These experiments were carried out in vitro, whereas experiments in Chapter 5 were conducted in vivo to assess the in vivo developmental competence of in vitro matured (IVM) pig oocytes activated by the improved activation protocol.
12
Fineschi, Benedetta. "Selection of competent oocytes by morphological features. Can an artificial intelligence - based model predict oocyte quality?" Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1211555.
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Infertility is a significant problem of humanity.
Despite the many advances in the field of assisted reproductive techniques (ART), accurately
predicting the outcome of an in vitro fertilization cycle (IVF) has yet to be achieved.
The focus of a great deal of research Is to improve on the current 30% success rate of IVF.
Assessment of oocyte quality is probably the most important and difficult task in the ART.
Oocyte quality has a direct impact on the fertilization and oocyte competence; the identification of
oocyte quality markers is particularly important to select embryos with higher developmental
potential and thus increase the success rates of IVF cycles.
Nevertheless, the assessment of the oocyte morphology is still performed more arbitrarily.
Over the past years, the ARTs have been accompanied by constant innovations; the use of artificial
intelligence (AI) techniques has become increasingly popular in the medical field and is being
leveraged in the embryology laboratory to help improve IVF outcomes.
The aims of this study are to evaluate the influence of specific morphological characteristics of
oocytes on the outcome of intracytoplasmic sperm injection (ICSI) and to develop an AI-based model
that predicts oocyte quality and fertilization outcome.
13
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.
14
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.
15
Januschke, Jens. "MRNA localization in the Drosophila oocyte". Paris 7, 2005. http://www.theses.fr/2005PA077101.
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Plachynta, Maksym. "Studies on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling". Thesis, University of Bedfordshire, 2007. http://hdl.handle.net/10547/622016.
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Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.
17
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.
18
GARCIA, BARROS RODRIGO. "DEVELOPMENT OF NEW OOCYTE IN VITRO CULTURE STRATEGIES TO ENHANCE THE OUTCOME OF ASSISTED REPRODUCTIVE TECHNOLOGIES". Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/809746.
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Fertility preservation has received unprecedented attention nowadays. In addition to cryopreservation and re-implantation of embryos, oocytes, and ovarian tissue pieces, in vitro culture system for follicles/oocytes has been considered an alternative strategy for fertility preservation. Reproduction strategies based on the recovery of oocytes' population from antral follicles are unsatisfactory, and the success of this approach has not exceeded 35% of embryos produced in vitro for over 30 years. The possibility of accessing the reserve of smaller follicles (primordial, secondary, and up to the preantral stage) would amplify the number of gametes available for increasing reproductive potential. Furthermore, this would open enormous prospects for the rescue of fertility in various conditions in the human clinic and genetic rescue in animal breeding and biodiversity preservation programs. However, this would require developing protocols capable of growing immature oocytes to the stage in which they can be matured and fertilized in vitro.
Culture systems to achieve in vitro growth (IVG) of immature oocytes to maturity and subsequent fertilization in vitro (IVF) have been the subject of research for almost 40 years. Several systems that support the growth of later stages of follicle development from rodents have been developed, with some reporting the production of live young, but they are still at an experimental stage, and further research is required before the protocols could be clinically applied.
One of the significant limitations is identifying growth factors, hormones, and nutrients necessary for each specific follicle development stage. This evidence has led to hypothesize the development of culture systems consisting of a step-by-step approach, although no reliable protocols have been developed so far. The oocyte culture at the early stages of development represents an alternative to maximize the potential source of gamete used for fertility preservation. Several attempts have been made to recreate these conditions in vitro, but no reliable protocols have been developed to date. The lack of knowledge in the mechanisms involved in the early development of the oocyte and this passage from growing to fully grown stage be one of the most critical steps during oocyte development, these still represent the significant limiting factor for this technology.
The studies conducted during the doctorate program led to defining a physiological culture system that successfully differentiated growing bovine oocytes. This study used parameters predictive of oocyte differentiation to evaluate the current technique's efficiency and efficacy.
Based on previous observations from our laboratory, we initially hypothesized that zinc plays a role during the latest stages of oocyte growth and differentiation, particularly in controlling transcription during the final stage of oocyte growth. This first study demonstrated that zinc supplementation improves the meiotic competence of growing oocytes, affects the global transcription activity and the global DNA methylation. This information was used in the next part to better define a culture system for growing oocytes.
The subsequent study provided a 5-days protocol named L-IVCO (long in vitro culture of oocytes) to promote growing oocyte differentiation until the acquisition of meiotic and embryonic developmental competencies in a significantly higher proportion of the published protocols. This study demonstrated that a physiological medium could support a gradual transition of the oocyte from immature to mature stage, thus generating suitably quality blastocysts after fertilization.
In conclusion, our studies provide an improved protocol that can increase the source of fertilizable gametes in preservation programs and gives a prospective approach in human clinics, animal breeding programs, and salvage intervention of threatened species. Moreover, our studies defined a model to perform in-depth studies of the cellular and molecular processes that regulate the acquisition of meiotic and developmental competence during oocyte differentiation.
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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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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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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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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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Sleep, Darrell. "Molecular analysis of Xenopus oocyte maternal RNA". Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/11917.
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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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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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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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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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Zapater, Cardona Cinta. "Molecular physiology of a teleost oocyte aquaporin: evolution, regulation and role during oocyte hydration / Fisiología molecular de una acuaporina ovocitaria de teleósteos: Evolución, regulación y papel durante la hidratación del oocito". Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/117374.
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In marine teleosts that spawn pelagic eggs (pelagophils), the process of oocyte hydration that occurs during meiosis resumption is a key physiological process for the survival of the eggs in the ocean. Previous studies have discovered the role of a teleost-specific aquaporin water channel (Aqp1b) during fish oocyte hydration, but direct experimental evidence for the function of Aqp1b in oocytes is still lacking. In addition, the molecular regulation of the Aqp1b-mediated mechanism remains poorly understood. In this context, the main objectives of the present thesis were to investigate the evolutionary origin of aqp1ab in teleosts, to provide functional evidence of the role of Aqp1b during oocyte hydration, and to begin to dissect the molecular mechanisms involved in the transcriptional regulation of aqp1b in the oocyte of marine teleosts.
By integrating the molecular phylogeny with synteny and structural analyses we show that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication, and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut (Hippoglossus hippoglossus), a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. Using immunological inhibition of Aqp1ab in halibut oocytes and artificial expression of the halibut paralog Aqp1aa, we demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation.
To investigate the aqp1ab transcriptional regulation in oocytes, we isolated the 5’-flanking region of the gilthead seabream (Sparus aurata) aqp1ab gene which contains regulatory cis-elements for the nuclear progestin receptor (Pgr) and SOX transcription factors. The Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oo-gonia, whereas in primary growth oocytes, when aqp1ab mRNA and protein are synthesized, the Pgr is translocated into the nucleus. In contrast, sox9b is highly expressed in more advanced oocytes showing the depletion of aqp1ab transcripts. In the seabream, four different pgr transcript variants are expressed in primary growth ovaries which are generated by alternative pre-mRNA splicing. Seabream wild-type Pgr shows the highest transactivation response to progestins such as 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), whereas two of the N-terminally truncated Pgr isoforms regulate novel nuclear and cytosolic mechanisms of dominant-negative repression of Pgr-mediated transcription. Transactivation assays on the aqp1ab promoter demonstrated that aqp1ab transcription is dependent on wild-type Pgr, with Sox3 and -8b acting synergistically, while Sox9b acts as a repressor. Incubation of primary ovarian explants in vitro with 17,20β-P, followed by chromatin immunoprecipitation, confirmed that 17,20β-P-activated Pgr enhanced aqp1ab promoter activation.
The production of 17,20β-P in seabream primary growth ovaries in vivo was consistent with the expression of P450c17-II (Cyp17a2) and 20β-hydroxysteroid dehydrogenase (Cbr1), enzymes needed for progestins synthesis, in granulosa cells associated with primary growth oocytes, and with a high concentration of 17,20β-P. Incubation of primary ovarian explants with recombinant piscine follicle-stimulating hormone (rFsh) in vitro stimulated 17,20β-P synthesis, which was reduced in the presence of Cbr1 inhibitors. The rFsh-mediated production of 17,20β-P correlated with the up-regulation of cyp17a2 and cbr1 transcription, as well as of wild-type pgr mRNA and protein levels. Altogether, these data suggest that aqp1ab transcription in seabream primary growth oocytes is under Fsh regulation through the synthesis of progestins.
The results of this thesis show that the Aqp1ab mediated mechanism for oocyte hy-dration is likely conserved in marine teleosts. In addition, the tight transcriptional reg-ulation of Aqp1ab during oogenesis highlights the essential physiological role of this water channel and opens new research avenues for understanding the molecular basis of egg formation in marine fish.La hidratación de los oocitos de teleósteos marinos que producen huevos pelágicos es clave para la supervivencia de los embriones en el océano. Estudios previos han descu-bierto el papel de una acuaporina específica de teleósteos (Aqp1b) durante este proceso, pero se carece todavía de evidencias experimentales directas, así como información sobre la regulación molecular de la Aqp1ab. En la presente tesis, estudios iniciales con el fletan Atlántico (Hippoglossus hippoglossus) confirman que los parálogos aqp1aa y -1ab de teleósteos han surgido probablemente por duplicación génica en tándem, y han demostrado el papel esencial de la Aqp1ab durante la hidratación del oocito. Para investigar el control transcripcional del gen aqp1ab en los oocitos de la dorada (Sparus aurata) se ha aislado su región promotora, la cual contiene elementos cis-reguladores de unión al receptor nuclear de progestinas (Pgr), como la 17α,20β-dihidroxi-4-pregnen-3-ona (17,20β-P), y factores de transcripción Sox. Estudios de localización subcelular indican que el Pgr aparece en el citoplasma de las oogonias, así como en el núcleo de oocitos en crecimiento primario (pre-vitelogénicos) coincidiendo con la activación de la expresión de aqp1ab. En este estadio también se expresan cuatro isoformas diferentes del Pgr, dos de las cuales pueden inhibir la transcripción mediada por el Pgr de forma dominante negativa. Las oogonias también expresan sox3 y -8b, mientras que el sox9b aparece en el estadio de alveolo cortical, cuando se reduce la expresión de aqp1ab. Ex-perimentos de transactivación indican que el Pgr activa la transcripción de aqp1ab, con Sox3 y -8b actuando de forma sinérgica, mientras que el Sox9b reprime este mecanismo. El papel del Pgr se ha investigado sobre explantes ováricos pre-vitelogénicos incubados in vitro, lo cual ha demostrado que la 17,20β-P, producida en las células de la granulosa en respuesta a la hormona folículo estimulante, activa el promotor de aqp1ab en el oocito induciendo la síntesis de Aqp1ab. Los resultados de esta tesis revelan por primera vez una estricta regulación transcripcional del gen aqp1ab durante la oogénesis de teleósteos marinos, lo cual remarca la función fisiológica esencial de este canal de agua durante la formación de los huevos pelágicos.
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Yokoi, Hayato y 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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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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Ralph, John Hunter. "Factors affecting follicle and oocyte development in cattle". Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/11889.
Texto completoResumen
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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Xie, Sancai. "Oocyte maturation and early embryo development in swine /". The Ohio State University, 1989. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487676261011605.
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Sartini, Becky Lynn. "Characterization of boar sperm plasma membrane candidate oocyte membrane fusion proteins and investigation of oocyte membrane binding sites in boar sperm populations /". For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.
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Roura, Llerda Montserrat. "Fatty acids goat follicular fluid: effect on oocyte competence". Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/399336.
Texto completoResumen
Els àcids grassos (AAGG) són una important font d’energia pels oòcits en fase de creixement. Per tant, l’estudi del fluid fol·licular (FF) on aquests oòcits maduren és d’especial interès. En estudis previs, Romaguera i col. (2011) va concloure que els oòcits de fol·licles grans (≥ 3mm) eren més competents que els de fol·licles petits. A més, Catalá i col. (2015) van demostrar que hi havia una producció diferencial d’embrions entre les estacions de l’any, sent significativament més baixes a la tardor que a l’hivern.
La nostra hipòtesi va ser que el perfil d’AAGG de cabres adultes i prepúbers, així com de diferents mides de fol·licle i de diferents estacions de l’any, ens podria donar informació per poder-ho relacionar amb la competència oocitària. El nostre objectiu va ser determinar si es podria fer servir aquest perfil com a marcador molecular per la qualitat oocitària.
En l’experiment 1, vàrem analitzar el perfil d’AAGG en FF de cabres adultes i prepúbers, segons edat, mida del fol·licle i època de l’any. Entre d’altres, vàrem trobar diferències en el total d’AAGG poliinsaturats (PUFA), la ràtio n6:n3 segons edat de la femella i mida de fol·licle i per estacions de l’any.
En l’experiment 2, vàrem avaluar la competència de l’oòcit provinent de femelles prepúbers afegint diferents ràtios dels àcids linoleic (LA: n6 PUFA) i α-linolenic (ALA: n3 PUFA) en el medi de MIV. Vàrem observar que la ràtio LA:ALA 200:50 µM tenia un efecte perjudicial sobre el desenvolupament embrionari dels oòcits quan es comparava amb els grups control i la resta dels tractaments (100:50 i 50:50 µM), quan els oòcits eren fecundats in vitro (FIV) (2.63 % vs ≈ 13 %) però no quan eren activats partenogenèticament.
L’experiment 3 consistia en l’estudi de l’efecte de les diferents ràtios de LA:ALA en l’oòcit de cabra prepúber avaluant l’activitat i distribució mitocondrial, la concentració d’ATP i l’expressió gènica relativa. Vàrem observar que hi havia un canvi si es comparava els oòcits en el moment de recol·lecció i després de 24h de MIV en l’activitat mitocondrial, en la distribució d’aquests orgànuls i en la producció d’ATP. A més, en el grup de 200:50 µM l’activitat mitocondrial era més alta que en la resta de grups. Es van analitzar l’expressió relativa de 9 gens que es troben alterats si la cèl·lula pateix estrès, comprometent la seva viabilitat. D’entre aquests gens, es varen trobar diferències d’expressió entre les 0 i 24h de maduració pels gens: GPX1, RPL19 i SOD1, però no entre els diferents tractaments.
En conclusió, es va trobar que el perfil d’AAGG de FF de cabra és similar a la del fluid fol·licular de vaques, ovelles i dona. Les principals diferències trobades en les cabres es devien principalment a l'edat de la femella. Es va trobar una relació directa entre la ràtio n6:n3 present en el FF segons mida del fol·licle i estació de l’any amb resultats previs en el nostre laboratori en la producció in vitro d’embrions, suggerint que aquesta ràtio podria ser un bon biomarcador de la competència dels oòcits. L'efecte negatiu de la més alta ràtio de LA:ALA en la competència dels oòcits no estava relacionada, tal com s’havia observat en altres estudis, amb una funció mitocondrial alterada, ni amb un augment de la producció d’espècies reactives d’oxigen o d’estrès del reticle endoplasmàtic com a conseqüència dels resultats obtinguts en l'expressió relativa dels gens estudiats. Per tant, hem hipotetitzat que l'addició d'altes concentracions de LA: ALA es pot relacionar amb una alteració en l'estructura de la membrana plasmàtica causada per una incorporació d'aquests àcids grassos en els fosfolípids de la membrana acompanyats amb el consum d'ATP.Fatty acids (AAGG) are an important source of energy for oocyte growth phase. Therefore, the study of follicular fluid (FF) where they mature, is of special interest. In previous studies, Romaguera et al. (2011) concluded that oocytes from large follicles (≥ 3mm) were more competent than the small follicles. Moreover, Catalá et al. (2015) showed that there was a differential production of embryos between seasons, being significantly lower in autumn than in winter. Our hypothesis was that the FA profile of FF from prepubertal and adult goats, as well as from different follicle sizes and different seasons, could give us information of the oocyte competence. Our objective was to determine whether this profile could be used as a molecular marker for the quality oocytes. In the first experiment, we analysed the profile of FA in FF. Among others, we found differences in total AAGG acids (PUFA), and n6: n3 ratio according to age, size of the follicle and seasons. In the second experiment, we evaluate the competence of oocytes from prepubertal females adding different ratios of linoleic acid (LA: n6 PUFA) and α-linolenic acid (ALA: n3 PUFA) in IVM. We observed ratio LA: ALA 200: 50 µM had a detrimental effect on embryo development of oocytes when compared with control groups and other treatments (100: 50 and 50:50 µM) when oocytes were in vitro fertilized (IVF) (≈ 2.63% vs 13%) but not when they were activated partenogenèticament. According to the results of the experiment 2, the aim of the experiment 3 was to study the effect of LA:ALA ratios on prepubertal goat oocyte quality by assessing mitochondrial distribution and activity, ATP concentration and relative gene expression. Assessing mitochondrial activity, active mitochondria distribution and ATP concentration in the oocyte, we found that there was a change in this parameters when they were analysed on immature oocytes (collection point) compared to IVM oocytes (after 24 h of maturation). Moreover, the addition of 200:50 µM at IVM modified the mitochondrial activity of these oocytes, being higher compared with the other treatment groups, but no changes were observed in the active mitochondria distribution or ATP concentration. Concerning mRNA relative expression, we analysed 9 genes that are shown to be altered if the cell is under stress, and which development could be compromised: ATF4, DNMT1, GAPDH, GCLC, GPX1/GSH-Px, HSPA5/GPR78, RPL19, SLC2A1/GLUT1, SOD1/CuZnSOD. Among these genes, GPX1, RPL19 and SOD1 showed significant differences when comparing immature and IVM oocytes, but not among groups of treatment. In conclusion, we found that FA profile of goat FF is similar to the follicular fluid found in cows, sheep and woman. The main differences that we found in goats were mainly due to the age of the female. However, we found a direct relationship between n6:n3 PUFA composition in follicular fluid regarding follicular size and season of the year, with previous results in our lab suggesting that this ratio could be a biomarker of oocyte competence. Moreover, we found that adding 200:50 µM LA:ALA had a detrimental effect on blastocyst production of prepubertal goat oocytes produced by IVF but not by parthenogenetic activation. Contrarily to what was previously concluded in another studies found in the literature, the negative effect of the highest LA:ALA ratio on oocyte competence was not related to impaired mitochondrial function, ROS production or ER stress according to the relative expression of the studied genes. Thus, we hypothesized that the effect of the addition of high concentrations of LA:ALA was related to an alteration on the structure of the plasma membrane caused for an incorporation of these fatty acids in the membrane phospholipids accompanied with ATP consumption.
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Cornet, Bartolomé David. "Molecular determinants of human oocyte quality in assisted reproduction". Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/667566.
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Infertility, defined by the World Health Organization (WHO) as the inability to conceive after a year of unprotected sex, is a disease that affects 1 in 9 couples of reproductive age worldwide. Most of these couples will only achieve pregnancy through assisted reproduction techniques. In recent decades, many improvements have been made in this field, but no method has been able to achieve 100% success. There are many variables that could affect the outcome of assisted reproduction cycles, one of the most important is the quality of the woman's oocytes. Maternal age is the most important factor affecting women’s ability to conceive and give birth, since female reproductive aging is associated with reduced oocyte quality; however, the underlying molecular mechanisms remain poorly understood. It is also well established that as woman age increases, her ovarian reserve diminishes. However, the role of ovarian reserve in the decline of oocyte quality with age is currently unknown.
The developmental competence of an oocyte is its ability to sustain embryonic development until embryonic genome activation. It is determined by the transcripts (coding and non-coding RNAs) accumulated during oocyte maturation. The lack of transcription during final oocyte maturation suggests that regulation of the genes involved in this process occurs at the post-transcriptional level. Among the possible mechanisms, alternative splicing (AS) of the messenger RNA could be involved. Studying oocyte gene expression and the spliced mRNA isoforms might provide novel information on the molecular mechanisms driving early development, and might be a source of potential biomarkers of oocyte quality.
The main objective of this thesis is to explore new possibilities for the identification of oocyte quality biomarkers at the molecular level in order to better understand the oocyte and how its developmental competence can be improved. For this, the results of oocytes from women with different age and ovarian reserve have been compared. In addition, to identify non-invasive biomarkers for oocyte developmental competence, the evaluation of the association between the expression analysis of different aging markers in human cumulus cells (CCs), the age and ovarian reserve and the oocyte maturation rates was conducted. Finally, because many of the oocytes used were vitrified, the effect of vitrification on oocyte developmental competence was analysed by comparing the reproductive outcomes of fresh and vitrified oocytes from the same stimulation cycle.
The results suggest an important role for ncRNAs and alternative splicing in human oocyte biology. Age and ovarian reserve have been shown to independently affect the ncRNAs transcriptome of in vivo matured oocytes. These results might provide valuable information for the search of oocyte quality markers, and for the (re)interpretation of existing dataset. On the other hand, differences in transcribed splicing variants can also provide biomarkers of oocyte quality, since the profile of confirmed AS events could determine the specific transcriptome of the mature oocyte.
The expression of common somatic aging markers in CCs didn’t show a clear correlation between the analysed genes and age, suggesting that CCs of reproductively old women do not present the typical transcriptome of aged tissues. In addition, when looking at future clinical applications, these markers have not been found useful for the development of non-invasive markers for oocyte developmental competence, since no correlation was observed either with the ovarian reserve or the oocyte maturation rates.
Finally, this study showed that oocyte vitrification per se maintained the developmental potential of human oocytes within a reasonable biological range, clinically comparable to fresh oocytes. As a consequence, we established that the main reason for the reported lower clinical results in vitrified cycles has to be attributed to the loss of oocytes during the warming step. This has important repercussions in the clinical practice, as measures can be easily put in place to offset oocyte loss.La competencia de desarrollo ovocitaria se define como la capacidad del ovocito para mantener el desarrollo embrionario hasta que el embrión activa su propio genoma. Está determinada por los transcritos (ARN codificantes y no codificantes) acumulados durante la maduración de los ovocitos. La falta de transcripción durante la maduración final de los ovocitos sugiere que la regulación de los genes involucrados en este proceso ocurre a nivel postranscripcional. Entre los posibles mecanismos, el “splicing alternativo” del ARN mensajero, podría estar involucrado. El objetivo principal de esta tesis es explorar nuevas posibilidades para la identificación de biomarcadores de calidad de ovocitos a nivel molecular con el fin de comprender mejor el ovocito y cómo se puede mejorar su competencia de desarrollo. Además, para identificar biomarcadores no invasivos de la competencia de desarrollo de ovocitos, se ha evaluado la expresión de diferentes marcadores de envejecimiento somático en células de cúmulos humanos que rodean los ovocitos, y se ha correlacionado con la edad y la reserva ovárica de las mujeres y con las tasas de maduración de los ovocitos. Finalmente, debido a que muchos de los ovocitos utilizados estaban vitrificados, se ha analizado el efecto de la vitrificación sobre la competencia de desarrollo de los ovocitos comparando los resultados reproductivos de ovocitos frescos y vitrificados del mismo ciclo de estimulación. Los resultados sugieren que los ARNs no codificantes y el splicing alternativo representan un papel importante en el proceso de adquisición de la competencia de desarrollo en ovocitos humanos. Estos resultados pueden proporcionar información valiosa para la búsqueda de marcadores de calidad de ovocitos y para la (re)-interpretación de conjuntos de datos existentes. El estudio de la expresión de marcadores de envejecimiento somático en células de cúmulos humanos no ha mostrado una clara correlación entre los genes analizados y la edad, lo que sugiere que las células del cúmulo de mujeres de edad reproductiva avanzada no presentan el transcriptoma típico de los tejidos envejecidos. Finalmente, este estudio ha demostrado que la vitrificación de ovocitos mantiene per se el potencial de desarrollo de los ovocitos humanos dentro de un rango biológico razonable, clínicamente comparable a los ovocitos frescos.
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Booth, Ronald A. "Signalling pathways controlling the initiation of Xenopus oocyte maturation". Thesis, University of Ottawa (Canada), 2002. http://hdl.handle.net/10393/6440.
Texto completoResumen
Xenopus laevis oocytes are physiologically arrested at the first meiotic prophase. Re-initiation of meiosis, or oocyte maturation, is triggered in vivo by progesterone, but can also be triggered by insulin and insulin-like growth factor-1 (IGF-1) in vitro. The mechanism by which these two very different hormones regulate the same physiological process is poorly understood. Chapter 2 describes my research that contributed to the characterization of the progesterone receptor responsible for inducing oocyte maturation. We demonstrated that the Xenopus progesterone receptor (xPR) is a dual functional protein. When expressed in the heterologous COS-7 cells, OR is imported into the nucleus and functions as a progesterone-regulated transcription factor. In contrast, the endogenous OR in Xenopus oocytes is restricted in the cytoplasm and appears to mediate cytoplasmic signalling. Chapter 3 describes a functional link between the IGF-1 receptor and G-protein signalling in the control of oocyte maturation. The Xenopus homologue of GIPC, a PDZ-domain-containing protein, was identified as a binding partner for the cytoplasmic domain of the IGF-1 receptor. GIPC is known to interact with the C-terminus of a Galphai-specific GAP, RGS-GAIP. Expression of two dominant negative forms of xGIPC blocked insulin-induced MAPK activation and oocyte maturation, while full-length xGIPC synergized with human RGS-GRIP to enhance insulin signalling. This is the first demonstration that the GIPC/RGS-GAIP complex acts positively in IGF-1 receptor signalling.
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Wang, Jing. "Function of protein kinase A in Xenopus oocyte maturation". Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/29377.
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Like most other vertebrate oocytes, oocytes from Xenopus laevis are physiologically arrested at the first meiotic prophase. Re-initiation of meiosis, or oocyte maturation, is triggered by progesterone. Progesterone-induced oocyte maturation is thought to involve inhibition of cAMP-dependent protein kinase (protein kinase A or PKA). However, changes in PKA activity in live oocytes have never been demonstrated. I have developed a novel approach to monitor endogenous PKA activity in live oocytes, by expressing a PKA-specific substrate in oocytes followed by monitoring its PKA phosphorylation status during progesterone-induced oocyte maturation. I demonstrate that PKA is fully activated in prophase oocytes and that progesterone causes a rapid and permanent inhibition of PKA (Chapter 3). Utilizing this PKA activity indicator in live oocytes, I further demonstrate that progesterone-induced oocyte maturation requires persistent inhibition of PKA such that critical maturation-specific proteins can be synthesized and accumulated to threshold levels necessary for activation of maturation promoting factor (MPF) (chapter 4). In addition to analyzing PKA activity dynamics during oocyte maturation, I have also provided the first biochemical evidence supporting the notion that in prophase oocytes, a constitutively activated G protein coupled receptor system is responsible for elevated cAMP and the high levels of PKA activity (chapter 2).
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Iatropoulou, Aikaterini. "Genesis of chromosomal abnormalities during oocyte growth and maturation". Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408762.
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Davies, Mark Richard. "Nuclear receptors in Xenopus oocyte maturation and early development". Thesis, University of Warwick, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396851.
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Dingwall, C. "The accumulation of proteins in the Xenopus oocyte nucleus". Thesis, Open University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354967.
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The ability of proteins to accumulate in the nucleus has been studied by injecting nucleoplasmin and calf thymus histone H1 into the cytoplasm of Xenopus oocytes. Nucleoplasmin, the most abundant protein in the Xenopus oocyte nucleus is pentameric and proteolysis of the nuceloplasmin pentamer produces a relatively protease resistant 'core' molecule that cannot enter the nucleus after microinjection into the cytoplasm. The polypeptide domain ('lq tail') of each subunit removed by proteolysis was obtained as a discrete fragment and has the ability to accumulate in the nucleus. Partially cleaved pentameric molecules with a single intact sub unit can still accumulate in the nucleus. Therefore a polypeptide domain of nucleoplasmin has been found that is both necessary and sufficient for accumulation in the nucleus. When the `core' molecule was injected directly into the oocyte nucleus it remained there, indicating that the 'tail' region confers selective entry rather than selective retention. In the case of histone H1 a proteolytic fragment encompassing the carboxyterminal domain can accumulate in the nucleus. The amino acids lysine, proline and alanine comprise 75 of the 89 amino acids in this fragment. Since the remaining 14 amino acids are scattered throughout the fragment and not clustered any primary sequence specifying entry into the nucleus would seem necessarily to involve the amino acids lysine, proline and alanine. Positive charge alone cannot explain the accumulation of this gragment since poly L-lysine does not accumulate after microinjection into the cytoplasm. Fragments encompassing other domains of the molecule are so unstable in the oocyte that their ability to accumulate in the oocyte nucleus cannot be assayed. The gene for nucleoplasmin has been cloned and sequences have been found in the 'tail' region of nuceloplasmin that show homology to sequences identified in other nuclear proteins that appear to constitute a signal specifying nuclear localisation.
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Ilozue, Tagbo. "Cytoplasmic dynamics in the mouse oocyte and preimplantation embryo". Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708499.
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Zhao, Tongtong. "Mechanisms of oocyte polarisation and axis formation in Drosophila". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610207.
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Ford, Nicola. "Interactions between translation initiation factors in the Xenopus oocyte". Thesis, University of Sussex, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413319.
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BUCHHOLZ, JANDA LEIGH. "DONOR FERTILITY AFTER PARTICIPATION IN AN OOCYTE DONATION PROGRAM". University of Cincinnati / OhioLINK, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1025724050.
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