Activation of a Gi protein in digitonin/cholate-solubilized membrane preparations of mouse sperm by the Zona pellucida, an egg-specific extracellular matrix
✍ Scribed by Xiaoping Ning; Cynthia R. Ward; Gregory S. Kopef
- Publisher
- John Wiley and Sons
- Year
- 1995
- Tongue
- English
- Weight
- 1020 KB
- Volume
- 40
- Category
- Article
- ISSN
- 1040-452X
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✦ Synopsis
Abstract
Mammalian sperm possess guanine nucleotide‐binding regulatory proteins (G proteins) that are involved in signal transduction pathways leading to zona pellucida (ZP)‐mediated acrosomal exocytosis. We have previously examined ZP‐G protein dynamics in mouse sperm homogenates, as well as cell‐free membrane preparations, and our data support the existence of ZP receptor‐G protein complexes in sperm membranes. However, the composition of this complex has not been identified due to experimental limitations of the membrane preparations. In the present study, a detergent‐solubilized preparation from mouse sperm membranes that retained the signaling properties of cell homogenates and cell‐free membrane preparations was developed using buffers containing digitonin and cholate. GTPγS, a poorly hydrolyzable analogue of GTP, bound to these solubilized preparations in a specific and concentration‐dependent fashion that reached saturation at 100 nM. Incubation of this solubilized membrane preparation with heat‐solubilized ZP resulted in an increase in specific GTPγS binding in a concentration‐dependent manner, with a maximal response at 4‐6 ZP/μl. Mastoparan (50 μM) increased GTPγS binding to levels similar to that seen with solubilized ZP. Mastoparan plus ZP stimulated GTPγS binding to the same extent as mastoparan or ZP alone. Pertussis toxin completely inhibited ZP‐stimulated GTPγS binding and decreased mastoparan‐stimulated GTPγS binding by 50–60%. Purified ZP3, the ZP component that possesses quantitatively all of the sperm binding and acrosomal exocytosis‐inducing activities of the intact ZP, stimulated GTPγS binding to an extent similar to that of solubilized ZP. The properties of this solubilized membrane preparation are similar to those found in the cell homogenates and cell‐free membrane preparations, suggesting that the components involved in ZP3‐mediated signal transduction are effectively solubilized and are responsive to the ZP3 ligand. © 1995 Wiley‐Liss, Inc.