Molecular demarcation of surface domains as established by label-fracture cytochemistry of boar spermatozoa.

Abstract

We used "label-fracture" (J Cell Biol 99:1156, 1984) to establish high-resolution maps of wheat germ agglutinin (WGA) and concanavalin A (ConA) receptor sites on the cell surface of boar spermatozoa and to investigate the possible association of these receptors to integral membrane components. Label-fracture reveals intense WGA labeling over the region of the plasma membrane that overlies the acrosome, including the equatorial segment. The density of WGA receptors decreases from the post-acrosomal area to the posterior ring. The WGA receptor domain changes abruptly into a microdomain with an unusually high density of WGA receptors over a sharply delimited, particle-free zone at the base of the head. Over the tail, the density of WGA receptors in the tail is high and uniform over the midpiece, annulus, and principal piece, but narrow patches of rectilinear arrays of pits close to the annulus are not labeled. Labeling of the entire sperm head by ConA is both intense and uniform, except for the particle-free zone at the base of the head, which is barren of receptors. Over the tail, ConA labeling is strong over the midpiece, absent over the annulus, and sparse over the principal piece and the end piece. In contrast to WGA, ConA receptors co-distribute with the intramembrane particles. Our results confirm the potential of label-fracture for high-resolution mapping of the distribution of cell surface receptors. They show that in this specialized cell membrane domains can be sharply defined, i.e., apparent without free lateral diffusion of components.

Topographical distribution of phospholipids in boar sperm plasma and intracellular membranes as revealed by freeze-fracture cytochemistry.

“…The mean density of PLA2-CG labeling over the E face is relatively low (25.8 k 1.8 particles/m2; see Figure 18). We then used the label-fracture technique to examine the distribution of PLAz-CG labeling over the sperm head, since label-fracture is a high-resolution method for labeling cell surfaces (Kan and Pinto da Silva, 1987). As expected, label-fracture preparation of boar spermatozoa also showed a uniform distribution of PLA2-CG labeling over the E face of the sperm head ( Figure 2), confirming the results obtained by fracture-label shown in Figure 1.…”

“…It has been suggested that the regional specificity of sperm membranes is decided mainly by differences in the organization of plasma membrane constituents in individual membrane domains (Tesarik and Flechon, 1986). When mammalian spermatozoa are in different physiological states, the flow of molecules in the regionalization of the membrane is differentially modulated ( Cowan et al, 1987;Kan and Pinto da Silva, 1987;Myles et al, 1987;Wolf, 1987;Wolf et al, 1986;O'Rand. 1982).…”

The Annulus of the Mouse Sperm Tail Is Required to Establish a Membrane Diffusion Barrier That Is Engaged During the Late Steps of Spermiogenesis1

“…Such proteins could then act as a ''fence'' to keep basigin, and presumably other diffusing proteins, corralled within either the principal piece or midpiece membrane domain. This idea is supported by freeze-fracture analysis of the membrane directly over the annulus, which shows circumferential rows of tightly aligned particles (presumably transmembrane proteins) visible on the E-face of the membrane [19,20,[46][47][48]. One transmembrane protein, Tat1, an anion transporter, has also been localized to the membrane directly over the annulus, and it has been suggested that it binds this structure and thus could participate in such a fence [49].…”

Freeze‐fracture cytochemistry: Review of methods

“…C: Label-fracture permits the high resolution mapping of surface domains of different composition. This example, from the study of Kan and Pinto da Silva (1987), shows the distribution of wheat germ agglutinin receptors in boar sperm. A high labeling density occurs in a margin, free of IMP, a t the base of the head.…”

“…Label that does not correspond to any structural component visible on the E face is, of course, easily visualized as well. Some of the most impressive demonstrations of the potential of label-fracture for the high-resolution mapping of membrane domains of different composition have been provided by recent studies on the sperm cell (Kan and Pinto da Silva, 1987;Fig 7C). The technique is also capable, at low magnification, of providing extensive survey views of cell surface receptor distribution.…”