The annulus is a higher order septin cytoskeletal structure located between the midpiece and principal piece regions of the sperm tail. The annulus has been hypothesized to generate the diffusion barrier that exists between these two membrane domains. We tested this premise directly on septin 4 knockout mice, whose sperm are viable but lack an annulus, by following the diffusing membrane protein basigin. Basigin is normally confined to the principal piece domain on testicular and caput sperm, but undergoes relocation into the midpiece during sperm epididymal transit. On Sept4(-/-) sperm, domain confinement was lost, and basigin localized over the entire plasma membrane. Both immunofluorescence and immunoblotting further revealed reduced levels of basigin expression on sperm from the knockout. Testicular immunohistochemistry showed similar basigin expression and tail targeting in wild-type (WT) and Sept4(-/-) tubules until step 15 of spermatid development, at which point basigin was redistributed throughout the plasma membrane of Sept4(-/-) spermatids. The basigin outside of the tail was subsequently lost around the time of sperm release into the lumen. The redistribution in the knockout coincides with the time in WT sperm when the annulus completes its migration from the neck down to the midpiece-principal piece junction. We posit that basigin may not diffuse freely until after the annulus arrives at the midpiece-principal piece junction to restrict lateral movement. These results are the strongest evidence to date of a mammalian septin structure establishing a membrane diffusion barrier.

Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggests that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation.

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