Regulatory mechanisms in cumulus expansion, oocyte maturation and block to polyspermy in mammals

The main purpose of this dissertation was to determine the effect of gonadotropins and the interaction between different second messengers on regulation of cumulus expansion and cytoplasmic maturation. The first objective was to verify the effect of gonadotropins on mucification of cumulus cells and on cytoplasmic maturation of mouse oocytes using a serum-free medium. Percentages of fertilized oocytes were greater (p $<$ 0.05) when LH was present. In contrast, expansion of cumulus cells was only observed when COC were matured in the presence of FSH. The percentage of male and female pronuclei formation after 5 h of incubation with sperm was significantly reduced in penetrated oocytes (p $<$ 0.02) suggesting that FSH delays cytoplasmic maturation. Development to two-cell embryos was not affected by the presence of gonadotropins in the oocyte maturation medium.The second objective of this study was to investigate the effect of fetuin (a proteinase inhibitor) in the block to polyspermy. The inhibition of proteinases during fertilization induced polyspermy in 52.4% of the eggs. A significant reduction in the percentage of polyspermic eggs over time following penetration (F = 7.34; p = 0.0190) occurred. Structures similar to cytoplasmic blebs were observed in some eggs cultured for 6 and 24 hours. The results demonstrate that fetuin inhibits the block to polyspermy when present in the fertilization medium. More relevant was the demonstration of a mechanism for eliminating supernumerary sperm from within the egg.Finally, an investigation was undertaken on the role of cAMP, Ca$\sp{\rm 2+}$-calmodulin (CaM), diacylglycerol (DG) and the interaction between these intracellular messengers on the mucification of the cumulus cells. Activators of protein kinase A stimulated cumulus expansion while activators of protein kinase C induced mucification of cumulus cells only under conditions that increased levels of cAMP. Effect of activators of protein kinase A to stimulate cumulus expansion was totally suppressed by an inhibitor of the Ca$\sp{\rm 2+}$CaM complex (W7). The compounds were not toxic since their effects were reversible. These results suggest that not only cAMP but other intracellular messengers such as Ca$\sp{\rm 2+}$-CaM complex and DG are involved and interact in the regulation of cumulus expansion.U of I OnlyETDs are only available to UIUC Users without author permissio