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Abstract
Zona pellucida (ZP)-induced acrosomal exocytosis in mammalian spermatozoa is thought to be mediated by signal transduction cascades similar to those found in hormonally responsive cells. In order to characterize this process further, we have examined the role of GTP-binding regulatory proteins (G proteins) in coupling sperm-ZP interaction to intracellular second messenger systems in mouse sperm. An in vitro signal transduction assay was developed to assess ZP-G protein dynamics in sperm membrane preparations. Guanosine 5'-3-O-(thio)triphosphate (GTP gamma S), a poorly hydrolyzable analogue of GTP, bound to these membranes in a specific and concentration-dependent fashion which reached saturation at 100 nM. Incubation of the membrane preparations with heat-solubilized ZP resulted in a significant increase in specific GTP gamma S binding in a concentration-dependent fashion with a half-maximal response at 1.25-2 ZP/microliters. Solubilized ZP also caused a significant increase in high affinity GTPase activity in the membranes over basal levels. Mastoparan increased specific GTP gamma S binding to the sperm membranes and stimulated high-affinity membrane GTPase activity to levels consistently greater than that seen with the solubilized ZP. Mastoparan, together with solubilized ZP, gave the same level of stimulation of GTP gamma S binding as mastoparan alone. Pertussis toxin completely inhibited the ZP-stimulated GTP gamma S binding, but only decreased mastoparan-stimulated GTP gamma S binding by 70-80%. Purified ZP3, the ZP component which possesses quantitatively all of the acrosomal exocytosis-inducing activity of the intact ZP, stimulated GTP gamma S binding to the same level as solubilized ZP; ZP1 and ZP2 did not stimulate GTP gamma S binding. ZP from fertilized eggs (ZPf), which does not possess acrosome reaction-inducing activity, also failed to stimulate GTP gamma S binding to sperm membranes. These data demonstrate the direct activation of a Gi protein in sperm membrane preparations in response to the ZP glycoprotein, ZP3, that induces the acrosome reaction. These data imply that Gi protein activation is an early event in the signal sequence leading to sperm acrosomal exocytosis.
Highlights
GTP-binding regulatory proteins (G proteins) in cou- acrosomal membrane, resulting in release of acrosomal conplingsperm-ZPinteraction tointracellular second tents [3]
Z P from fertilized eggs (ZPf), which In mouse sperm, the Gi proteins are localized to the plasma does not possess acrosome reaction-inducing activity, and/or outer acrosomal membrane region overlying the acroalso failed to stimulateGTP-ySbinding to spermmem- some [21]
Characteristics of GTPyS Binding to Sperm Membrane Preparations-In order to examineZP-stimulated signal transduction mediated by one or more membrane-bound G proteins associated with putative sperm plasma membrane Zona pellucida (ZP) receptors, membranes from capacitated sperm were prepared
Summary
Characteristics of GTPyS Binding to Sperm Membrane Preparations-In order to examineZP-stimulated signal transduction mediated by one or more membrane-bound G proteins associated with putative sperm plasma membrane ZP receptors, membranes from capacitated sperm were prepared. These membranes displayed high affinity and specific GTPyS binding which was concentration-dependent and reached saturation at 100 nM (Fig. la). The results shown in panel b are the mean f S.D. from triplicates within a representative experiment whichwas repeated three times with similar results
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