The epididymal amyloid matrix: structure and putative functions

Abstract

Background: We previously demonstrated the normal mouse epididymal lumen contains a non-pathological amyloid matrix that surrounds spermatozoa and plays important roles in sperm maturation and protection.

Objective: The objective herein was to present a review of this work, including studies showing the amyloid structures of four members of the CRES (cystatin-related epididymal spermatogenic) subgroup are integral and essential components of the amyloid matrix.

Methods: We used conformation-dependent reagents that recognize the cross-β-sheet structure characteristic of amyloid, including thioflavin S (ThS), thioflavin T (ThT), anti-amyloid antibodies, and X-ray diffraction, as well as negative-stain transmission electron microscopy (TEM) to visualize amyloid structures in the epididymal lumen. Antibodies that specifically detect each CRES subgroup family member were also used in indirect immunofluorescence analysis.

Results and discussion: The epididymal lumen contains an amyloid matrix that surrounds maturing spermatozoa and represents a functional amyloid. Alterations in the structure of the amyloid matrix by the loss of the CRES subgroup members or the overexpression of cystatin C result in epididymal pathologies, including infertility. Preliminary data suggest the epididymal amyloid matrix is structurally and functionally similar to bacterial biofilms.

Conclusion: Together, these results suggest the amyloid matrix serves important roles in epididymal function including sperm maturation and protection.

Keywords: amyloid; antimicrobial; bacterial biofilm; epididymis; mouse; sperm maturation.

Figure 1.. An amyloid matrix is present in the mouse epididymal lumen.

A) Transmission electron micrograph of the initial segment region of the mouse epididymis showing particulate material within the lumen. SP, spermatozoon. Arrows, extracellular vesicles. Micrograph kindly provided by A. Parent and L. Hermo, McGill University, Montreal, Quebec Canada. Reprinted from (Cornwall et al., 2011). Bar, 0.5 μm. B) Indirect immunofluorescence analysis of the initial segment epididymal lumen using the anti-oligomeric amyloid antibody (A11) (green fluorescence). DAPI, blue fluorescence, indicates staining of epithelial cell nuclei. Bar, 20 μm. C) Thioflavin S staining of amyloid matrix isolated from the mouse initial segment; and D) cauda epididymis. Bar, 5 μm. E) X-ray diffraction of epididymal amyloid matrix isolated from the initial segment region of the epididymis. Reprinted from (Whelly et al., 2012).

Figure 2.. Colocalization of CRES and CRES3 in amyloid matrix in the initial segment epididymal lumen.

Indirect immunofluorescence analysis was performed using an affinity-purified rabbit anti-mouse CRES antibody followed by an Alexa Fluor 594 conjugated secondary antibody (red fluorescence) and an Alexa Fluor 488 conjugated CRES3 antibody (green fluorescence) on paraformaldehyde-fixed paraffin embedded mouse epididymal tissue sections. Images were captured with a Nikon Ti-E confocal microscope. Bar, 20 μm.

Figure 3.. CRES subgroup members form amyloid in vitro.

A) Recombinant proteins in 6 M guanidine-Cl were diluted to 10 µM in buffer, ThT added to 20 µM and fluorescence immediately determined using a Synergy HT plate reader with excitation at 450 nm and emission at 485 nm. Values represent the mean relative fluorescence units (RFU) + SEM of four replicates. B) Negative stain EM of recombinant proteins following dilution into buffer. Scale bar, CRES left, CRES2, 366 nm; cystatin E2 left, 616 nm; CRES right, CRES3, cystatin E2 right, 100 nm. C) Dot blot analysis of immature (A11 positive) and mature (OC positive) forms of amyloid in CRES subgroup proteins. Five micrograms of each recombinant CRES subgroup protein were diluted into buffer and immediately spotted onto nitrocellulose membrane in a dot blot apparatus. The membranes were incubated with the conformation-dependent anti-oligomer A11 and anti-fibrillar OC antibodies. (Modified and reprinted from Whelly et al., 2016).

Figure 4.. Epididymal amyloid matrix structure is altered in CRES KO mice.

The epididymal amyloid matrix was isolated from the initial segment region from age-matched CRES wildtype (WT) and CRES knockout (KO) mice and incubated with the protein aggregation disease (PAD) reagent (Microsens Biotechnologies, London, UK) to pulldown amyloid structures (Whelly et al., 2012). Proteins were eluted from the PAD beads by incubation in Laemmli buffer at 65°C for 15 min, spotted on to formvar/carbon coated 200 mesh nickel grids (Ted Pella, Redding, CA, USA) and stained with 2% uranyl acetate. Images were captured with a Hitachi H-8100 transmission electron microscope. Bar, top panels, 500 nm; Bar, bottom panels, 100 nm.