Abstract
In the mammalian ovary, oocytes are contained within follicles, specialized structures that facilitate oocyte growth and development. During the reproductive cycle, several follicles are recruited into growth, and through a process of selection, one (human, cow) or several (mouse, pig) of these follicles eventually become dominant and obtain the ability
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to ovulate. The oocytes inside growing and developing follicles are arrested at prophase of meiosis I, until the follicle reaches the preovulatory stage and the oocyte resumes meiosis in response to the luteinizing hormone surge. The subsequent oocyte maturation process is characterized by progression of meiosis from prophase I to metaphase II and completion of this process is required for the oocyte to be fertilized and for the development of the resulting embryo. Oocyte maturation comprises intricately interweaved molecular events and structural changes, but it has become clear that current knowledge on this process constitutes merely the tip of the iceberg. The research presented in this thesis provides a substantial contribution to a better understanding of mammalian oocyte maturation using pig oocytes as a model system. The potential benefits of this research include improvements in fertility treatments, in vitro fertilization, prevention of genetic disorders, and production of embryonic stem cells. The protein kinase CDC2 plays a pivotal role in several key maturation events, in part through controlled changes in CDC2 localization. Research presented in this thesis shows that CDC2 transiently associates with a single domain that is composed of a cluster of endoplasmic reticulum exit sites in the cortex of maturing porcine oocytes. The results of this study further suggest that this specialized domain is involved in regulating membrane traffic, including redistribution of Golgi components, during the highly asymmetrical meiotic divisions. Unraveling the intricacies of intracellular events that govern oocyte maturation can be facilitated by manipulating the underlying processes at the molecular level. In order to achieve such delicate molecular interference, a novel method using pressure-based microinjection of fluorescent molecules and analysis of injected oocytes by epifluorescence microscopy was developed. Validation of this method is described in a second study presented in this thesis, and shows that it is an efficient means of manipulating oocytes using consistent concentrations of injected substances, without resulting in adverse effects on oocyte maturation. In a final experimental chapter, this method was applied to manipulate the functions of a ubiquitously expressed cytosolic protein, clathrin, which has originally been defined for its role in intracellular membrane traffic, but was recently shown to have a second independent function, involving the stabilization of kinetochore fibers in mitotic spindles of dividing somatic cells. To study whether clathrin also functions at meiotic spindles in oocytes we microinjected green fluorescent protein-tagged C-terminal and N-terminal clathrin protein constructs into isolated porcine oocytes prior to in vitro maturation. In oocytes, both constructs associated with meiotic spindles similar to endogenous clathrin, but induced severe meiotic defects, demonstrating a crucial role for clathrin at the first and second meiotic spindle in maturing oocytes.
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