A novel trans-complementation assay suggests full mammalian oocyte activation is coordinately initiated by multiple, submembrane sperm components

To initiate normal embryonic development, an egg must receive a signal to become activated at fertilization. We here report that the ability of demembranated sperm heads to activate is abolished after incubation over the range 20-44 degrees C and is sensitive to reducing agents. On the basis of this observation, we have developed a microinjection-based, trans-complementation assay in order to dissect the heat-inactivated sperm-borne oocyte-activating factor(s) (SOAF). We demonstrate that the failure of heat-inactivated sperm heads to activate an egg is rescued by coinjection with dithiothreitol-solubilized SOAF from demembranated sperm heads. The solubilized SOAF (SOAF(s)) is trypsin sensitive and is liberated from demembranated heads in a temperature-dependent manner that inversely correlates with the ability of sperm heads to activate. This argues that SOAF(s) is a proteinaceous molecular species required to initiate activation. Injection of oocytes with mouse or hamster sperm cytosolic factors, but not SOAF(s) alone, induced resumption of meiosis, further suggesting that these cytosolic factors and SOAF are dis tinct. Collectively, these data strongly suggest that full mammalian oocyte activation is initiated by the coordinated action of one or more heat-sensitive protein constituents of the perinuclear matrix and at least one heat-stable submembrane component.