Egg activation events (e.g., cortical granule exocytosis, cell cycle resumption, and recruitment and degradation of maternal mRNA) are initiated by changes in egg Ca2+ homeostasis following sperm-egg fusion. In mammals, these changes consist of repetitive oscillations in intracellular Ca2+ that terminate at the time of pronucleus formation. The long-term goals of this application are to determine how Ca2+ oscillations in the egg are generated in response to the fertilizing sperm and how these Ca2+ oscillations entrain a hierarchy of changes in gene expression that lead to successful development. In mammals, the Ca2+ oscillations are initiated by a sperm-derived phospholipase C, PLC-?.
Specific Aim 1 will test the hypothesis that each Ca2+ oscillation is the result of a two-component system in which PLC-? triggers an initial rise in intracellular Ca2+ whereas an egg-derived PLC generates the exponential phase.
This aim will be done by reducing the amount of specific PLCs in the egg and then assaying the effect of altering egg PLC levels on the sperm-induced Ca2+ oscillatory pattern. Events of egg activation are governed by the total amount of Ca2+ released during the oscillatory period. Prematurely terminating the Ca2+ oscillations or subjecting eggs to hyperstimulation of Ca2+ signaling does not affect development to the blastocyst stage, but differentially affects both gene expression in the blastocysts and implantation/post-implantation development. Thus, the Ca2+ oscillations that occur prior to pronucleus formation have long-term consequences for development.
Specific Aim 2 will test the hypothesis that these long-term consequences are due to inappropriate reprogramming of gene expression that occurs during the maternal-to-zygotic transition. We will employ a microarray approach to determine the effects of altering Ca2+ oscillations on gene expression in the 2-cell stage embryo.
This aim will be conducted in collaboration with J.P. Ozil (INRA, France), who over the past decade has developed unique technologies to modulate precisely the frequency and amplitude of changes in Ca2+ in embryos. Maternal mRNA recruitment by cytoplasmic polyadenylation is essential for development and requires almost the full complement of Ca2+ oscillations. We have used our published microarray data to identify candidate maternally recruited transcripts.
Specific Aim 3 will assess the function of these mRNAs by RNAi. In addition, an unbiased proteomic approach will be taken to identify maternally recruited mRNAs whose function will also be assessed by RNAi. Results of these studies will provide new information regarding how fertilization-induced Ca2+ oscillations are generated and the mechanistic linkage between these Ca2+ oscillations and developmental potential.

Public Health Relevance

The proposed studies will provide new information regarding how fertilization-induced Ca2+ oscillations are generated and the mechanistic linkage between these Ca2+ oscillations and developmental potential. The results of these studies will likely impact on the treatment of human infertility and assisted reproduction technologies.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Special Emphasis Panel (ZRG1-EMNR-G (07))
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Ravindranath, Neelakanta
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University of Pennsylvania
Schools of Arts and Sciences
United States
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Igarashi, Hideki; Knott, Jason G; Schultz, Richard M et al. (2007) Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization. Dev Biol 312:321-30
Gardner, Allison J; Knott, Jason G; Jones, Keith T et al. (2007) CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs. J Cell Physiol 212:275-80
Deng, Manqi; Suraneni, Praveen; Schultz, Richard M et al. (2007) The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes. Dev Cell 12:301-8
Knott, Jason G; Gardner, Allison J; Madgwick, Suzanne et al. (2006) Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations. Dev Biol 296:388-95
Toth, Szabolcs; Huneau, Daniel; Banrezes, Bernadette et al. (2006) Egg activation is the result of calcium signal summation in the mouse. Reproduction 131:27-34
Ducibella, Tom; Schultz, Richard M; Ozil, Jean-Pierre (2006) Role of calcium signals in early development. Semin Cell Dev Biol 17:324-32
Ozil, Jean-Pierre; Banrezes, Bernadette; Toth, Szabolcs et al. (2006) Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term. Dev Biol 300:534-44
Williams, Carmen J; Schultz, Richard M (2006) Transgenic RNAi: a tool to study testis-specific genes. Mol Cell Endocrinol 247:1-3
Schultz, Richard M (2005) From egg to embryo: a peripatetic journey. Reproduction 130:825-8
Ozil, Jean-Pierre; Markoulaki, Styliani; Toth, Szabolcs et al. (2005) Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse. Dev Biol 282:39-54

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