ACTIVATION OF A G(I) PROTEIN IN DIGITONIN CHOLATE-SOLUBILIZED MEMBRANE PREPARATIONS OF MOUSE SPERM BY THE ZONA-PELLUCIDA, AN EGG-SPECIFIC EXTRACELLULAR-MATRIX

Citation
Xp. Ning et al., ACTIVATION OF A G(I) PROTEIN IN DIGITONIN CHOLATE-SOLUBILIZED MEMBRANE PREPARATIONS OF MOUSE SPERM BY THE ZONA-PELLUCIDA, AN EGG-SPECIFIC EXTRACELLULAR-MATRIX, Molecular reproduction and development, 40(3), 1995, pp. 355-363
Citations number
57
Categorie Soggetti
Reproductive Biology","Developmental Biology",Biology
ISSN journal
1040452X
Volume
40
Issue
3
Year of publication
1995
Pages
355 - 363
Database
ISI
SICI code
1040-452X(1995)40:3<355:AOAGPI>2.0.ZU;2-S
Abstract
Mammalian sperm possess guanine nucleotide-binding regulatory proteins (G proteins) that are involved in signal transduction pathways leadin g to zona pellucida (ZP)-mediated acrosomal exocytosis. We have previo usly examined ZP-G protein dynamics in mouse sperm homogenates, as wel l as cell-free membrane preparations, and our data support the existen ce of ZP receptor-G protein complexes in sperm membranes. However, the composition of this complex has not been identified due to experiment al limitations of the membrane preparations. In the present study, a d etergent-solubilized preparation from mouse sperm membranes that retai ned the signaling properties of cell homogenates and cell-free membran e preparations was developed using buffers containing digitonin and ch olate. GTP gamma S, a poorly hydrolyzable analogue of GTP, bound to th ese solubilized preparations in a specific and concentration-dependent fashion that reached saturation at 100 nM. Incubation of this solubil ized membrane preparation with heat-solubilized ZP resulted in an incr ease in specific GTP gamma S binding in a concentration-dependent mann er, with a maximal response at 4-6 ZP/mu l. Mastoparan (50 mu M) incre ased GTP gamma S binding to levels similar to that seen with solubiliz ed ZP. Mastoparan plus ZP stimulated GTP gamma S binding to the same e xtent as mastoparan or ZP alone. Pertussis toxin completely inhibited ZP-stimulated GTP gamma S binding and decreased mastoparan-stimulated GTP gamma S binding by 50-60%. Purified ZP3, the ZP component that pos sesses quantitatively all of the sperm binding and acrosomal exocytosi s-inducing activities of the intact ZP, stimulated GTP gamma S binding to an extent similar to that of solubilized ZP. The properties of thi s solubilized membrane preparation are similar to those found in the c ell homogenates and cell-free membrane preparations, suggesting that t he components involved in ZP3-mediated signal transduction are effecti vely solubilized and are responsive to the ZP3 ligand. (C) 1995 Wiley- Liss, Inc.