The goal of this project is to understand the molecular and cellular mechanisms that control sperm CA2+ channel activity. Sperm T-type CA2+ channels are activated by adhesive contact with the egg's zona pellucida and mediate the acrosome reaction, a secretory event that is required for fertilization. This channel may also be the site of action of the reported human male contraceptive effect of 1,4-dihydropyridines. Conductance through T channels is regulated by membrane potential and is further modulated by voltage-dependent mechanisms and tyrosine phosphorylation state. Studies during the next proposed funding period focus on the control of sperm T-type CA2+ channels during capacitation and interaction with the egg's zona pellucida. The essential question being asked by the applicant is the following: how do sperm develop the ability to undergo ZP3-dependent acrosome reactions and yet simultaneously prevent excessive premature acrosome reactions? To this end, Dr. Florman believes that the fine control of calcium flux through low voltage activated T-type channels plays a key role in this modulation of acrosome activity. A multi-step concerted hypothesis is stated: (1) channels must be in a high conductance open state for acrosome reactions to occur; (2) spontaneous acrosome reactions are minimized in uncapacitated sperm by low membrane potential that prevents channel opening; (3) spontaneous acrosome reactions are also minimized as sperm capacitate by a tyrosine phosphorylation dependent process that allows the T channel to open only into a low conductance state; and (4) only when T channels are dephosphorylated, which occurs during ZP3 stimulations, can they open to a high conductance state and thus trigger the acrosome reaction. Thre are four specific aims designed to test each aspect of this hypothesis.
In Aim 1, Dr. Florman will determine the conductance state of T channels during capacitation using electrophysiological and fluorescent probe methods.
In Aim 2 the modulation state of sperm T channels will be assayed during capacitation and interaction with the zona pellucida.
For Aim 3, the regulation of sperm tyrosine phosphatase activity by ZP3 will be determine in reversibly permeabilized sperm. Finally, the mechanism of T channel modulation by tyrosine phosphorylation will be tested in Specific Aim 4. The excised-patch configuration of the patch clamp technique will be used to examine the single T channel characteristics while tyrosine phosphorylation state is altered. Health problems are associated with both unbounded and impeded fertilization. An understanding of sperm Ca2 channel regulation during capacitation and egg contact is essential to our models of mammalian fertilization. Moreover, an understanding of the regulation of this channel is a necessary step in attempts to design channel-based contraceptive agents.
|Galantino-Homer, Hannah L; Florman, Harvey M; Storey, Bayard T et al. (2004) Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation. Mol Reprod Dev 67:487-500|
|Lopez-Gonzalez, I; De La Vega-Beltran, J L; Santi, C M et al. (2001) Calmodulin antagonists inhibit T-type Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction. Dev Biol 236:210-9|
|Gonzalez-Martinez, M T; Galindo, B E; de De La Torre, L et al. (2001) A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm. Dev Biol 236:220-9|
|O'Toole, C M; Arnoult, C; Darszon, A et al. (2000) Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction. Mol Biol Cell 11:1571-84|
|Arnoult, C; Kazam, I G; Visconti, P E et al. (1999) Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3 and by membrane hyperpolarization during capacitation. Proc Natl Acad Sci U S A 96:6757-62|
|Arnoult, C; Villaz, M; Florman, H M (1998) Pharmacological properties of the T-type Ca2+ current of mouse spermatogenic cells. Mol Pharmacol 53:1104-11|
|Florman, H M; Arnoult, C; Kazam, I G et al. (1998) A perspective on the control of mammalian fertilization by egg-activated ion channels in sperm: a tale of two channels. Biol Reprod 59:12-6|
|Arnoult, C; Cardullo, R A; Lemos, J R et al. (1996) Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida. Proc Natl Acad Sci U S A 93:13004-9|
|Arnoult, C; Zeng, Y; Florman, H M (1996) ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization. J Cell Biol 134:637-45|
|Zeng, Y; Clark, E N; Florman, H M (1995) Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion. Dev Biol 171:554-63|
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