Localization of Low-Density Detergent-Resistant Membrane Proteins in Intact and Acrosome-Reacted Mouse Sperm1

Abstract

Mammalian sperm become fertile after completing capacitation, a process associated with cholesterol loss and changes in the biophysical properties of the sperm membranes that prepares the sperm to undergo the acrosome reaction. Different laboratories have hypothesized that cholesterol efflux can influence the extent and/or movement of lipid raft microdomains. In a previous study, our laboratory investigated the identity of sperm proteins putatively associated with rafts. After extraction with Triton X-100 and ultracentrifugation in sucrose gradients, proteins distributing to the light buoyant-density fractions were cored from polyacrylamide gels and microsequenced. In this study, a subset of these proteins (TEX101, basigin, hexokinase 1, facilitated glucose transporter 3, IZUMO, and SPAM1) and other molecules known to be enriched in membrane rafts (caveolin 2, flotillin 1, flotillin 2, and the ganglioside GM3) were selected to investigate their localization in the sperm and their behavior during capacitation and the acrosome reaction. These molecules localize to multiple sperm domains, including the acrosomal cap (IZUMO, caveolin 2, and flotillin 2), equatorial segment (GM3), cytoplasmic droplet (TEX101), midpiece (basigin, facilitated glucose transporter 3, and flotillin 2), and principal piece (facilitated glucose transporter 3). Some of these markers modified their immunofluorescence pattern after sperm incubation under capacitating conditions, and these changes correlated with the occurrence of the acrosome reaction. While GM3 and caveolin 2 were not detected after the acrosome reaction, flotillin 2 was found in the equatorial segment of acrosome-reacted sperm, and IZUMO distributed along the sperm head, reaching the post- and para-acrosomal areas. Taking into consideration the requirement of the acrosome reaction for sperm to become fusogenic, these results suggest that membrane raft dynamics may have a role in sperm-egg membrane interaction.

The association between CDC42 and caveolin-1 is involved in the regulation of capacitation and acrosome reaction of guinea pig and mouse sperm

“…We can assume that the lack of CAV1 is not critical for sperm fertility, and it does not seem to be upregulated by other caveolin isoforms, although CAV2 is present in mouse sperm (Miranda et al 2009) but is unstable in the absence of CAV1 (Razani et al 2001). CAV3 is exclusively expressed in striated muscle cells and cardiac and skeletal muscles (Song et al 1996).…”

Tssk6 is required for Izumo relocalization and gamete fusion in the mouse

“…Deficient actin polymerization in Tssk6-null sperm Changes in the immunofluorescence pattern of sperm surface proteins during capacitation and acrosome reaction have been described previously (Miranda et al, 2009;Saxena et al, 1986;Primakoff and Myles, 2007;Selvaraj et al, 2007). It has been shown that changes in protein localization are at least in part due to actincytoskeleton dynamics (Saxena et al, 1986;Hernandez-Gonzalez et al, 2000).…”

Calcium Channels in the Development, Maturation, and Function of Spermatozoa

“…Recently it was reported that Izumo relocalizes from the anterior acrosome where it is found in intact mouse spermatozoa, to other regions in the head engaged in fusion with the egg plasma membrane. Tssk6, a member of the testis-specific serine kinase family, is necessary for the movement of Izumo, and its role is mediated by the actin cytoskeleton (362,476).…”