Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen

Abstract

STUDY QUESTION: Do the CRES (cystatin-related epididymal spermatogenic) subgroup members, including CRES2, CRES3 and cystatin E2, contribute to the formation of a nonpathological, functional amyloid matrix in the mouse epididymal lumen?SUMMARY ANSWER: CRES2, CRES3 and cystatin E2 self-assemble with different aggregation properties into amyloids in vitro, are part of a common amyloid matrix in the mouse epididymal lumen and are present in extracellular vesicles.WHAT IS KNOWN ALREADY: Although previously thought only to be pathological, accumulating evidence has established that amyloids, which are highly ordered protein aggregates, can also carry out functional roles in the absence of pathology. We previously demonstrated that nonpathological amyloids are present in the epididymis; specifically, that the reproductive cystatin CRES forms amyloid and is present in the mouse epididymal lumen in a film-like amyloid matrix that is intimately associated with spermatozoa. Because the related proteins CRES2, CRES3 and cystatin E2 are also expressed in the epididymis, the present studies were carried out to determine if these proteins are also amyloidogenic in vitro and in vivo and thus may coordinately function with CRES as an amyloid structure. STUDY DESIGN, SAMPLES/MATERIALS, METHODS:The epididymides from CD1 and Cst8 (CRES)129SvEv/B6 gene knockout (KO) and wild-type mice and antibodies that specifically recognize each CRES subgroup member were used for immunohistochemical and biochemical analyzes of CRES subgroup proteins. Methods classically used to identify amyloid, including the conformation-dependent dyes thioflavin S (ThS) and thioflavin T (ThT), conformation-dependent antibodies, protein aggregation disease ligand (which binds any amyloid independent of sequence) and negative stain electron microscopy (EM) were carried out to examine the amyloidogenic properties of CRES subgroup members. Immunofluorescence analysis and confocal microscopy were used for colocalization studies. MAIN RESULTS AND THE ROLE OF CHANCE:Immunoblot and immunofluorescence analyzes showed that CRES2, CRES3 and cystatin E2 were primarily found in the initial segment and intermediate zone of the epididymis and were profoundly downregulated in epididymides from CRES KO mice, suggesting integrated functions. Except for CRES3, which was only detected in a particulate form, proteins were present in the epididymal lumen in both soluble and particulate forms including in a film-like matrix and in extracellular vesicles. The use of amyloid-specific reagents determined that all CRES subgroup members were present as amyloids and colocalized to a common amyloid matrix present in the epididymal lumen. Negative stain EM, dot blot analysis and ThT plate assays showed that recombinant CRES2, CRES3 and cystatin E2 formed amyloid in vitro, albeit with different aggregation properties. Together, our studies demonstrate that a unique amyloid matrix composed of the CRES family of reproductive-specific cystatins and cystatin C is a normal component of the mouse ...

The epididymal amyloid matrix: structure and putative functions

“…Within the epididymis, CRES, CRES2, CRES3, and cystatin E2 are synthesized and secreted by the initial segment region suggesting interrelated functions (Cornwall & Hann, ; Li et al ., , , ; Hsia & Cornwall, ). In support of this, using antibodies that specifically recognize each subgroup member, we established that CRES, CRES2, CRES3, and cystatin E2 colocalize with each other and with the prototypical cystatin, cystatin C, in amyloid matrix isolated from the mouse epididymal initial segment lumen, and within the luminal amyloid matrix in situ, as represented by the colocalization of CRES and CRES3 in immunofluorescence studies (Whelly et al ., ; Fig. ).…”

“…). Additional biochemical approaches including pulldown of epididymal amyloid using the protein aggregation disease (PAD) ligand followed by immunoblot with anti‐CRES antibodies further indicated the four epididymal CRES subgroup members were indeed present as amyloids in the epididymal lumen (Whelly et al ., ).…”

“…All four epididymal CRES subgroup members are also highly amyloidogenic in vitro and exhibit distinct kinetics of amyloidogenesis and form unique amyloid structures including matrices, films, and polygons (Fig. B) (Whelly et al ., ). Similarly, cystatin C is an established amyloid in vitro and in vivo (Wahlbom et al ., ).…”

“…Extracellular vesicles (EVs) of various sizes, including epididymal exosomes (epididymosomes), are also found within this matrix, and their delivery of cargo to spermatozoa is an essential part of the maturation process (Fig. A, arrows) (Caballero et al ., ; Sullivan, ; Whelly et al ., ). Various other aggregate structures, including electron‐dense bodies that contain heat‐shock protein 1 (HSPD1, HSP60) and tumor rejection antigen (TRA1), a member of the heat‐shock protein 90 family, are also in the epididymal lumen; however, their functions have not been determined (Asquith et al ., ).…”

“…Amyloids exhibit SDS and/or protease resistance and often require harsh denaturants, such as formic acid, guanidine, or DMSO for solubilization (Loksztejn & Dzwolak, ; Whelly et al ., , ). The extreme stability of amyloid can be problematic in pathological states but functionally beneficial for its biological roles as scaffolds and structures that provide protection and mediate cell–cell interactions including the chorion surrounding oocytes in fish and its equivalent, the zona pellucida in mammals (Iconomidou et al ., ; Podrabsky et al ., ; Louros et al ., ; Egge et al ., ).…”