In humans and other mammals, 1-10% of fertilized eggs become triploid (and die) most often due to fertilization by more than one sperm. This is normally prevented by the release of egg cortical granules (CGs) whose contents modify the zona pellucida (ZP).
The Aims will determine the biochemical basis for the failure of CG release in pre-ovulatory oocytes, and the roles of intracellular calcium (Ca2+) oscillations in CG release and cell cycle resumption in mature mouse eggs, as follows. 1) In mature eggs, the hypothesis will be tested that CG exocytosis and cell cycle resumption have different Ca2+ requirements encoded in Ca2+ oscillation parameters. Although Ca2+ oscillations are known to cause CG release, ZP modifications, and cell cycle resumption (via decreases in H1 and MAP kinases) upon fertilization, it is not known whether these events require different numbers or types of oscillations for their activation and/or timing. Three hypotheses will be tested by experimentally changing the amplitude, frequency, or number of oscillations, and quantifying changes in the aforementioned events of egg activation. 2) In pre ovulatory oocytes, the blocked step in the CG secretory pathways will be identified. Three hypothesis will be investigated: (A) Failure of CG translocation from the cortex to the plasma membrane, (B) normal translocation followed by a failure in CG docking, and (C) failure of membrane fusion after translocation/docking. For the blocked step (A, B, or C), relevant structural and regulatory proteins will be compared in secretion-competent mature eggs and incompetent oocytes for protein amount, localization, and phosphorylation status to begin to identify the biochemical deficiency responsible for the failure of exocytosis (including secretory-machinery proteins, calmodulin-depth kinase II, synapsin, & relevant cytoskeletal proteins). Significance (of long-range objectives):
Aim 1 : The precise Ca2+ requirements (this study) coupled with the identification of the Ca2+ effectors (Aim 2 & other studies) will provide fundamental information about the molecular mechanism by which Ca2+ causes egg activation events responsible for the onset of mammalian development. Using the methods in the proposal, in vitro matured human oocytes could be tested for activation competence without sperm (not applied for herein).
Aim 2 : Identification of the proteins involved in secretion and the (pre-ovulatory) biochemical change(s) responsible for CG release will provide valuable markers of oocyte maturation to evaluate emerging protocols to mature animal and human oocytes in vitro or in vivo.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD024191-12
Application #
6387543
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Tasca, Richard J
Project Start
1988-07-01
Project End
2001-07-31
Budget Start
2001-04-01
Budget End
2001-07-31
Support Year
12
Fiscal Year
2001
Total Cost
$6,120
Indirect Cost
Name
Tufts University
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
Ducibella, Tom; Fissore, Rafael (2008) The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol 315:257-79
Ducibella, Tom; Schultz, Richard M; Ozil, Jean-Pierre (2006) Role of calcium signals in early development. Semin Cell Dev Biol 17:324-32
Matson, Sara; Markoulaki, Styliani; Ducibella, Tom (2006) Antagonists of myosin light chain kinase and of myosin II inhibit specific events of egg activation in fertilized mouse eggs. Biol Reprod 74:169-76
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
Abbott, A L; Ducibella, T (2001) Calcium and the control of mammalian cortical granule exocytosis. Front Biosci 6:D792-806
Abbott, A L; Fissore, R A; Ducibella, T (2001) Identification of a translocation deficiency in cortical granule secretion in preovulatory mouse oocytes. Biol Reprod 65:1640-7
Gross, V S; Wessel, G; Florman, H M et al. (2000) A monoclonal antibody that recognizes mammalian cortical granules and a 32-kilodalton protein in mouse eggs. Biol Reprod 63:575-81
Abbott, A L; Fissore, R A; Ducibella, T (1999) Incompetence of preovulatory mouse oocytes to undergo cortical granule exocytosis following induced calcium oscillations. Dev Biol 207:38-48
Ducibella, T (1998) Biochemical and cellular insights into the temporal window of normal fertilization. Theriogenology 49:53-65
Abbott, A L; Xu, Z; Kopf, G S et al. (1998) In vitro culture retards spontaneous activation of cell cycle progression and cortical granule exocytosis that normally occur in in vivo unfertilized mouse eggs. Biol Reprod 59:1515-21

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