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