The long-term objectives of this research are to understand how sperm initiate a set of events that are collectively called egg activation and how these events of egg activation lead to successful embryonic development. The first clearly observed event following fertilization is a transient increase in the concentration of intracellular free Ca2+ that is then followed by a series of Ca2+ oscillations; these changes in Ca2+ are mediated about IP3. While evidence suggests that these Ca2+ oscillations are critical for successful development, little is known regarding the underlying molecular mechanisms that link these oscillations to successful development. This grant renewal will focus on (1) the molecular mechanisms underlying the acquisition of a Ca2+ oscillatory behavior observed following fertilization that is acquired during oocyte maturation, (2) the linkage between these Ca2+ oscillations in the 1-cell embryo and changes in gene expression that occur during preimplantation development, and (3) the linkage between these changes in gene expression and post-implantation development. The overarching hypothesis tested in this renewal application is that the fertilization-induced Ca2+ oscillations entrain a hierarchy of changes in gene expression that in turn are responsible for the ability of a preimplantation embryo to develop to the blastocyst stage, implant, and develop to term.
Specific Aim 1 will use RNA interference (RNAi) to prevent the maturation-associated increase in the type 1 IP3 receptor to test the hypothesis that this increase confers increased in IP3 sensitivity in the metaphase II-arrested egg, and that this increase is required for the Ca2+ oscillatory behavior of fertilized eggs, and later developmental events.
Specific Aim 2 will use RNAi to prevent the maturation-associated increase in CaMKII to test the hypothesis that this increase is critical for CaMKII-mediated signaling events in the fertilized egg, and is required for the Ca2+ oscillatory behavior of fertilized eggs. A positive correlation exists between the number of Ca2+ oscillations that occur following fertilization and the incidence of development to the blastocyst stage. Although it is assumed that these Ca2+ oscillations encode information that ultimately dictates preimplantation development, the molecular events that constitute the readout of this information are unknown.
In Specific Aim 3 the Ca2+ oscillations will be experimentally manipulated in eggs injected with a sperm nucleus that lacks sperm activating factor activity (thus bypassing the intrinsic problem using parthenogenotes) to test the hypothesis that Ca2+ oscillations are linked to the initial reprogramming of gene expression that in turn confers the capacity to develop to term. Results of these studies will provide new information regarding the mechanistic linkage between fertilization-induced Ca2+ oscillations and developmental potential.
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