Analysis of a protein family mediating membrane-microfilament interactions in cortical dynamics and effective tension during mammalian oocyte maturation and early embryonic development

The mammalian egg cortex undergoes dramatic remodeling during oocyte maturation and fertilization. The integrity of the cortical cytoskeleton is integral to these events and the plasma membrane block to polyspermy, the prevention of fertilization by multiple sperm. Although these cortical remodeling events and the importance of monospermy in zygotic development are well described, the molecular modulators underlying these cortical changes are not fully understood. This dissertation research undertook a variety of studies to characterize cortical dynamics during oocyte maturation and fertilization in mouse eggs.;Chapter 2 examined the roles of actin and myosin in post-fertilization cortical and membrane changes. We show that the extent of cortical granule (CG) exocytosis was similar in control eggs and eggs treated with the actin microfilament inhibitor cytochalasin D, indicating that increased polyspermy observed in cytochalasin D-treated eggs is not due to a failure of CG exocytosis. Treatment of eggs with the myosin light chain kinase inhibitor ML-7 increased the incidence of polyspermy in fertilized eggs and also caused arrest in anaphase II, indicating that myosin has multiple functions in the egg cortex.;Chapter 3 investigated the ERM (Ezrin/Radixin/Moesin) family of proteins in oocytes and eggs. Phosphorylated, activated ERM proteins modulate interactions between the actin cytoskeleton and plasma membrane in many cell types, and thus are candidates of interest to mediate cortical dynamics in eggs. We show that all three ERMs are expressed in eggs, and levels of ERM phosphorylation decrease precipitously during oocyte maturation and rise significantly upon fertilization. Disruption of phosphoERM function resulted in abnormal actin localization in metaphase II eggs and failed spindle rotation upon fertilization.;Work in Chapter 4 characterized effective cortical tension (Teff ) in oocytes and eggs. We show that prophase I oocytes have high T eff values that decrease significantly during oocyte maturation and increase upon fertilization. This pattern correlates with that of phosphoERM proteins, and reduction of phosphoERM levels significantly reduces T eff in both oocytes and metaphase II eggs. These results indicate T eff varies dynamically as the oocyte prepares for fertilization and early embryonic cell divisions and that ERM proteins mediate cortical mechanics of mouse oocytes and eggs.