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1.
Mol Hum Reprod ; 22(11): 729-744, 2016 11.
Article in English | MEDLINE | ID: mdl-27445316

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 epididymal lumen and may play a functional role in sperm maturation by coordinating interactions between the luminal fluid and spermatozoa. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by NIH grants RO1HD033903 and RO1HD056182 to G.A.C. The authors declare there are no conflicts of interest.


Subject(s)
Amyloid/metabolism , Epididymis/metabolism , Extracellular Vesicles/metabolism , Sperm Maturation/physiology , Spermatogenesis/physiology , Animals , Blotting, Northern , Cystatin M/genetics , Cystatin M/metabolism , Cystatins/genetics , Cystatins/metabolism , Immunohistochemistry , Male , Mice , Mice, Knockout , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sperm Maturation/genetics , Spermatogenesis/genetics
2.
J Biol Chem ; 289(11): 7718-29, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24500719

ABSTRACT

Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility.


Subject(s)
Amyloid/physiology , Cystatins/chemistry , Cystatins/genetics , Infertility, Male/genetics , Amyloid/chemistry , Animals , Enzyme-Linked Immunosorbent Assay , Epididymis/metabolism , Female , Fertilization in Vitro , Humans , Male , Mice , Mice, Transgenic , Microscopy, Electron , Microscopy, Fluorescence , Point Mutation , Sperm Motility , Spermatozoa/metabolism
3.
PLoS One ; 7(5): e36394, 2012.
Article in English | MEDLINE | ID: mdl-22570708

ABSTRACT

Amyloids are aggregated proteins characterized by a specific cross-ß-sheet structure and are typically associated with neurodegenerative diseases including Alzheimer's disease. Recently, however, several nonpathological amyloids have been found in intracellular organelles of normal mammalian tissues suggesting that amyloid may also carry out biological functions. We previously have shown that the epididymal cystatin CRES (cystatin-related epididymal spermatogenic), cst8, a reproductive-specific member of the cystatin superfamily of cysteine protease inhibitors, forms amyloid in vitro suggesting that CRES amyloid may also form in vivo within the epididymal lumen. Here we show that amyloid structures containing CRES are a component of the normal mouse epididymal lumen without any apparent cytotoxic effects on spermatozoa and that these structures change along the length of the tubule. These studies suggest the presence of a functional amyloid structure that may carry out roles in sperm maturation or maintenance of the luminal milieu and which itself may undergo maturational changes along the epididymis. In contrast to previous examples of functional amyloid which were intracellular, our studies now show that nonpathological/functional amyloid can also be extracellular. The presence of an extracellular and nonpathological amyloid in the epididymis suggests that similar amyloid structures may be present in other organ systems and may carry out distinctive tissue-specific functions.


Subject(s)
Amyloid/metabolism , Epididymis/metabolism , Extracellular Space/metabolism , Sperm Maturation/physiology , Amyloid/ultrastructure , Animals , Male , Mice , Mice, 129 Strain , Mice, Knockout
4.
J Biol Chem ; 282(45): 32912-23, 2007 Nov 09.
Article in English | MEDLINE | ID: mdl-17855342

ABSTRACT

CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDS-resistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway.


Subject(s)
Cystatins/chemistry , Cystatins/metabolism , Epididymis/metabolism , Extracellular Space/metabolism , Transglutaminases/metabolism , Amino Acid Sequence , Amyloid/metabolism , Amyloid/ultrastructure , Animals , Catalysis , Electrophoresis, Polyacrylamide Gel , Guinea Pigs , Male , Mice , Microscopy, Electron , Molecular Sequence Data , Molecular Weight , Protein Binding , Substrate Specificity , Tandem Mass Spectrometry
5.
Asian J Androl ; 9(4): 500-7, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17589787

ABSTRACT

The epididymal lumen represents a unique extracellular environment because of the active sperm maturation process that takes place within its confines. Although much focus has been placed on the interaction of epididymal secretory proteins with spermatozoa in the lumen, very little is known regarding how the complex epididymal milieu as a whole is maintained, including mechanisms to prevent or control proteins that may not stay in their native folded state following secretion. Because some misfolded proteins can form cytotoxic aggregate structures known as amyloid, it is likely that control/surveillance mechanisms exist within the epididymis to protect against this process and allow sperm maturation to occur. To study protein aggregation and to identify extracellular quality control mechanisms in the epididymis, we used the cystatin family of cysteine protease inhibitors, including cystatin-related epididymal spermatogenic and cystatin C as molecular models because both proteins have inherent properties to aggregate and form amyloid. In this chapter, we present a brief summary of protein aggregation by the amyloid pathway based on what is known from other organ systems and describe quality control mechanisms that exist intracellularly to control protein misfolding and aggregation. We then present a summary of our studies of cystatin-related epididymal spermatogenic (CRES) oligomerization within the epididymal lumen, including studies suggesting that transglutaminase cross-linking may be one mechanism of extracellular quality control within the epididymis.


Subject(s)
Epididymis/physiology , Sperm Maturation/physiology , Amino Acid Substitution , Amyloid/physiology , Amyloid/standards , Cystatin C , Cystatins/genetics , Dimerization , Humans , Male , Mutation , Protein Folding , Proteins/standards , Quality Control , Transglutaminases/physiology
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