The objective of this proposal is to continue our study of the acrosome reaction (AR) in mouse sperm induced by the zona pellucida (ZP) of the mouse egg. This is the physiological form of the AR in this species and in most mammalian species, including human. The AR is obligate for fertilization of the egg. Its study is undertaken in pursuit of the overall goal of providing a biochemical analysis of mammalian fertilization. the ZP-induced AR was first demonstrated in this laboratory by use of the potent and specific inhibitor of the reaction, 3- quinuclidinyl benzilate (QNB), that blocks the reaction at its point of initiation. The induced AR strongly resembles agonist ligand-mediated exocytosis in secretory somatic cells, and so is postulated to proceed by an intracellular signal transduction pathway similar to those operating in these cells. Investigation of this pathway is the major focus of this proposal, which has two specific aims.
The first aim i s to isolate and characterize the sperm plasma membrane protein that binds QNB. The hypothesis is that sperm QNB binding protein (SQBP) is one of a class of signal initiation components acting to transduce signals into cells from extracellular matrices. In this case, the ZP is the extracellular matrix. SQBP will be isolated, purified, and sequenced to establish its relationship to muscarinic receptors, long known to bind QNB as antagonist, and to other signal transduction components activated by glycoprotein hormones. Two approaches will be used. One will be covalent labelling with QNB analogs, purification by chromatographic and electrophoretic methods, followed by direct sequencing. The other will utilize oligonucleotide probes to screen a mouse germ cell library. Included in this aim are studies of possible linkage of GTP binding regulatory proteins (G proteins) to SQBP.
The second aim i s to determine the sequence and to quantify the kinetics of the mouse sperm intracellular reactions constituting the signal transduction chain that leads from initiation by the ZP to fusion and vesiculation of the plasma and outer acrosomal membranes that completes the AR. The hypothesis is that the acrosome reaction proceeds through a series of reactions with characteristic time constants, regulated primarily by a ZP-induced increase in intracellular Ca2+ concentration. Inhibitors and activators of the individual reactions will be used to alter the time course and outcome of the overall reaction in such a way as to order the reaction sequence. The kinetics will be measured by determinations of the rate of change of intracellular Ca2+ and of three other key intermediates affecting the activity of intracellular protein kinases. The results of this study should clarify the mechanism of sperm signal transduction and so provide a model for extracellular matrix signalling systems.
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