Compartmentalisation of the sperm plasma membrane: a FRAP, FLIP and SPFI analysis of putative diffusion barriers on the sperm head

Spermatozoa are highly polarised cells with a compartmentalised distribution of lipids and proteins in their plasma membrane. It is not known how these compartments are stably maintained in what is essentially a fluid environment. In this investigation we have examined the hypothesis that intramembranous diffusion barriers selectively retain some components within compartments, while allowing free passage of others. A fluorescence loss in photobleaching analysis of the behaviour of the lipid reporter probe 1,1'-dihexadecyl-3,3,3'3'-tetramethyindocarbocyanine (DiIC16) on the head of boar spermatozoa revealed that it was freely diffusing between all three compartments (anterior acrosome, equatorial segment and postacrosome). Spermatozoa also contained rapidly diffusing particles of DiIC16 over the anterior acrosome and equatorial segment. These particles, approximately 200 nm in diameter, were tracked in real time and their trajectories analysed by mean square displacement. Particle diffusion was essentially random over the anterior acrosome and equatorial segment but showed a periodicity in jump sizes and diffusion coefficients suggestive of microheterogeneities. Particles did not exchange between the equatorial segment and postacrosome, indicating a barrier at the junction between these two compartments. No barrier was detected between the equatorial segment and anterior acrosome. A model is proposed in which a molecular 'filter' is present at the equatorial segment-postacrosomal boundary that allows free passage of single molecules but not molecular complexes. Passage of heterogeneous complexes, such as lipid rafts, requires disassembly and reassembly on either side of the filter.