The annulus of the mouse sperm tail is required to establish a membrane diffusion barrier that is engaged during the late steps of spermiogenesis

Abstract

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.

FIG. 1.

A cartoon of a mature sperm showing the location of the cytoskeletal annulus with relationship to the various domains of the sperm tail.

FIG. 2.

Representative micrograph overlays of mouse caput (A) and cauda (B and C) epididymal sperm double labeled for basigin (green) and the annular protein septin 4 (red). Basigin labels the principal piece of caput sperm (A) and the midpiece of cauda sperm (B), stopping discretely at the annulus in both cases. Cauda sperm initially frozen and thawed before fixation (C) show coincident labeling for both proteins at the annulus (yellow). Bar = 20 μm.

FIG. 3.

Cold-field emission scanning electron micrographs of demembranated sperm tails focusing on the principal piece-midpiece junction. A) shows the annulus (arrow) of a WT sperm, while B shows the gap (arrow) created by the missing annulus of a Sept4^−/− sperm. In both pictures, the mitochondrial sheath of the midpiece is to the left of the arrow, and the fibrous sheath of the principal piece is to the right of the arrow. Bar = 1.0 μm.

FIG. 4.

Indirect immunofluorescent labeling of basigin on WT and Sept4^−/− sperm. WT caput (A) and cauda (E) sperm show basigin localization within the principal piece and midpiece domains, respectively. Basigin is hardly detectable in Sept4^−/− caput (B) and cauda (F) sperm at identical photographic exposures. Contrast enhancement of these images (C and G) reveals a weak and patchy basigin distribution over the whole membrane at both stages; D and H show similar enhancements of Sept4^−/− sperm that served as serum controls. Micrographs are representative of findings from at least three experiments. Bar = 10 μm.

FIG. 5.

Indirect immunofluorescent labeling of basigin on a caput sperm with a prominent cytoplasmic droplet (CD) from a Sept4^−/− mouse. The image was taken with a different exposure time from those in Figure 4; thus, fluorescence intensity cannot be directly compared between the images. The image was contrast enhanced. Bar = 20 μm.

FIG. 6.

Immunoblot showing reduced basigin levels in sperm from Sept4^−/−mice at various stages. Lanes consist of cell equivalent loads of sperm isolated from the testes (Test), as well as caput (Cap), corpus (Corp), and cauda regions of the epididymides of WT and Sept4^−/− mice and visualized for basigin. Basigin undergoes proteolytic cleavage on caput sperm, leading to increased signal detection of the lower molecular weight form with the polyclonal antibody. The blot was stripped and reprobed for β-tubulin to confirm the loads. The figure is representative of results obtained from three separate experiments.

FIG. 7.

Indirect immunofluorescent labeling of basigin on testicular tissue sections from WT (A) and Sept4^−/− (B) mice, along with serum controls (C and D, respectively). Bar = 200 μm.

FIG. 8.

Testicular cross sections labeled for basigin using indirect immunofluorescence. Micrographs depict tubules from WT (A–C) and Sept4^−/− (D–F) mice, along with magnifications (a–f) of regions within the tubules (white rectangles) showing developing spermatids. Tubule stages are indicated by roman numerals. Black arrowheads point to sperm heads, and white arrowheads point to basigin on corresponding sperm tails. Bars = 50 μm (A–F) and 10 μm (a–f).

FIG. 9.

Indirect immunofluorescent labeling of the annular protein septin 4 on testicular tissue sections from WT mice showing annulus migration during spermiogenesis. The micrographs depict seminiferous tubules at stages II–III (A), IV–V (B), and VII–VIII (C) of the cycle and a region within each tubule (the white boxes) that has been magnified to show detail on the elongating spermatids. On step 14 spermatids (D), the annuli appear at the base of the condensed nuclei. During step 15 of spermatid development (E), the annuli migrate down the length of the midpiece. At step 16 (F), the annuli are seen at the midpiece-principal piece junction. White arrowheads point to the labeled annulus and black arrowheads point to the corresponding sperm heads. Bars = 50 μm (A–C) and 10 μm (D–F).