Meiosis requires cells to undergo two successive rounds of division without an intermediate synthesis or S-phase. This results in a gamete with half the number of chromosomes that can then combine with those of another gamete to begin the development of a new offspring. Once an oocyte enters meiosis it is arrested in late prophase I and remains in that state until the animal reaches sexual maturity and undergoes ovulation. Upon hormonal stimuli, the oocyte is released from its first meiotic arrest, undergoes oocyte maturation, arrests in metaphase II and is ovulated to await fertilisation. The process of oocyte maturation is governed by the interaction of many different protein kinases, phosphatases and proteases. How these proteins co-localise with each other during oocyte maturation is a central aim of this thesis. One central protein is maturation promoting factor or MPF. MPF is composed of a catalytic subunit, cdc2 and a regulatory subunit, cyclin B1. Throughout oocyte maturation MPF activity is dynamic and changes in MPF activity coordinate the progression of oocyte maturation. The ubiquitin-proteasome pathway (UPP) plays a role in regulating the activity of MPF at specific times during oocyte maturation by targeting and degrading the cyclin B1 subunit. In this thesis the 20S proteasome ‘core’ of the 26S proteasome was localised in bovine and murine oocytes undergoing maturation in vitro. The 20S proteasome was found in the germinal vesicle prior to maturation in both species. Once germinal vesicle breakdown occurred, the proteasome consistently localised around the developing spindle in both species. Cyclin B1, an important target of the UPP was also localised in murine oocytes to see how the UPP interacted with its substrates during oocyte maturation. Cyclin B1 was found to have similar localisation patterns throughout oocyte maturation to the 20S proteasome. This demonstrates that the 26S proteasome moves to the location of its substrates in order to degrade them. To confirm the role of the UPP in cyclin B1 degradation, we inhibited proteasome function using MG132, a known inhibitor of proteasome function. Polar body 1 (PB1) extrusion declined significantly (P<0.05) in a dose dependent manner, confirming that the UPP is essential for the continuation of meiosis. Our localisation data indicated that cyclin B1 interacted with the UPP during the GV stage and around GVBD. Inhibition of the proteasome, again using MG132, prevented oocytes from undergoing GVBD and arrested them in the GV stage. This demonstrated that the UPP was essential for oocyte maturation as well as indicating a potential switch in substrate during this transition. The deubiquitinylating enzyme, Fat Facets in Mouse (FAM also know as USP9x), an antagonist of the UPP, was also localised in murine oocytes undergoing maturation in vitro. FAM was found primarily in the cytoplasm prior to germinal vesicle breakdown however it relocalised to the developing spindle upon germinal vesicle breakdown. FAM is known to rescue a number of specific substrates from ubiquitinylation and degradation, one being I- catenin, a multifunctional protein, not previously implicated in oocyte maturation. Localisation of I-catenin during oocyte maturation revealed distinct staining at specific stages of meiosis. Staining was observed on the plasma membrane and in the GV prior to maturation. During MI and MII it appeared to co-localise with FAM. When homologous chromosomes divided at AI/TI I-catenin distinctly localised to the cleavage furrow indicating its involvement during cytokinesis. To determine if FAM activity was essential for oocyte maturation we inhibited FAM function by microinjecting anti-FAM serum into eggs post GVBD. Inhibition of FAM prevented PB1 extrusion. Some oocytes appeared to attempt PB1 but failed to complete (Hoechst staining of the chromatin revealed these cells remained in MI). This finding strongly indicates that FAM maybe rescuing I-catenin from degradation during MI until it is needed at AI/TI. However further examination of this interaction are needed to clarify the data. This research demonstrates that regulation of turnover of key proteins is essential for progression through oocyte maturation. It also demonstrates that multiple pathways may regulate the same processes. This may add to the complexity of studying the cell, but it also demonstrates the elegance of a highly regulated and sophisticated system.
| Identifer | oai:union.ndltd.org:ADTP/252403 |
| Creators | Edgecumbe, Paul Christopher |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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