title: Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen.
abstract: 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?
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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 epididymal lumen and may play a functional role in sperm maturation by coordinating interactions between the luminal fluid and spermatozoa.
  
  
  
  The structures examined in our studies were isolated from luminal fluid obtained by puncture of the epididymis and therefore we cannot rule out some contamination by epithelial cells. Although our studies show CRES family members are associated with extracellular vesicles, we have yet to determine if proteins are present on the surface or are within the vesicles. We also have not established if narrow/apical cells are the source of the CRES family extracellular vesicles. CRES and CRES2 have been previously found in the human epididymis and associated with spermatozoa; however, we have yet to determine if the human CRES subgroup proteins are amyloidogenic and if an amyloid matrix is present in the human epididymal lumen.
  
  
  
  Understanding the regulation and biological roles of amyloids, such as the CRES subgroup amyloid matrix that functions without causing pathology, could have broad implications for understanding pathological amyloids including those associated with neurodegenerative diseases and prionopathies.
  
  
  
  None.
  
  
  
  This work was supported by NIH grants RO1HD033903 and RO1HD056182 to G.A.C. The authors declare there are no conflicts of interest.
authors.affiliation: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
authors.forename: Sandra
authors.initials: S
authors.lastname: Whelly
authors.affiliation: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
authors.forename: Archana
authors.initials: A
authors.lastname: Muthusubramanian
authors.affiliation: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Powell
authors.affiliation: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
authors.forename: Seethal
authors.initials: S
authors.lastname: Johnson
authors.affiliation: College of Arts and Sciences Microscopy, Texas Tech University, Canton and Main, Lubbock, TX 79409, USA.
authors.forename: Mary Catherine
authors.initials: MC
authors.lastname: Hastert
authors.affiliation: Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA gail.cornwall@ttuhsc.edu.
authors.forename: Gail A
authors.initials: GA
authors.lastname: Cornwall
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D055332:Cystatin M / Q000235:genetics / Q000378:metabolism; D015891:Cystatins / Q000235:genetics / Q000378:metabolism; D004822:Epididymis / Q000378:metabolism*; D000067128:Extracellular Vesicles / Q000378:metabolism*; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013079:Sperm Maturation / Q000235:genetics / Q000502:physiology*; D013091:Spermatogenesis / Q000235:genetics / Q000502:physiology*
chemical_list: D000682:Amyloid; C472453:Cst11 protein, mouse; C472454:Cst12 protein, mouse; D055332:Cystatin M; D015891:Cystatins; D011994:Recombinant Proteins; C078310:Cst8 protein, mouse
keywords: amyloid; cystatin; epididymal protein; extracellular vesicles; sperm maturation