Nonpathological Extracellular Amyloid Is Present during Normal Epididymal Sperm Maturation

Amyloids are aggregated proteins characterized by a specific cross-β-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

Maturation of the functional mouse CRES amyloid from globular form

The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming β-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid β-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms

Reduced Fertility In Vitro in Mice Lacking the Cystatin CRES (Cystatin-Related Epididymal Spermatogenic): Rescue by Exposure of Spermatozoa to Dibutyryl cAMP and Isobutylmethylxanthine1

The cystatin CRES (cystatin-related epididymal spermatogenic; Cst8) is the defining member of a reproductive subgroup of family 2 cystatins of cysteine protease inhibitors and is present in the epididymis and spermatozoa, suggesting roles in sperm maturation and fertilization. To elucidate the role of CRES in reproduction, mice lacking the Cst8 gene were generated and their fertility examined. Although both male and female Cst8−/− mice generated offspring in vivo, spermatozoa from Cst8−/− mice exhibited a profound fertility defect in vitro. Compared to spermatozoa from Cst8+/+ mice, spermatozoa from Cst8−/− mice were unable to undergo a progesterone-stimulated acrosome reaction and had decreased levels of protein tyrosine phosphorylation, suggesting a defect in the ability of Cst8−/− spermatozoa to capacitate. Incubation of Cst8−/− spermatozoa with dibutyryl cAMP and 3-isobutyl-1-methylxanthine rescued the fertility defect, including the capacity for sperm protein tyrosine phosphorylation. Both untreated Cst8+/+ and Cst8−/− spermatozoa, however, exhibited similar increased total levels of cAMP and protein kinase A (PKA) activity throughout the capacitation time course compared to spermatozoa incubated under noncapacitating conditions. Taken together, these results suggest that mice lacking CRES may have altered local levels of cAMP/PKA activity, perhaps because of improper partitioning or tethering of these signaling molecules, or that the CRES defect does not directly involve cAMP/PKA but other signaling pathways that regulate protein tyrosine phosphorylation and capacitation

Amyloid properties of the mouse egg zona pellucida.

The zona pellucida (ZP) surrounding the oocyte is an extracellular fibrillar matrix that plays critical roles during fertilization including species-specific gamete recognition and protection from polyspermy. The mouse ZP is composed of three proteins, ZP1, ZP2, and ZP3, all of which have a ZP polymerization domain that directs protein fibril formation and assembly into the three-dimensional ZP matrix. Egg coats surrounding oocytes in nonmammalian vertebrates and in invertebrates are also fibrillar matrices and are composed of ZP domain-containing proteins suggesting the basic structure and function of the ZP/egg coat is highly conserved. However, sequence similarity between ZP domains is low across species and thus the mechanism for the conservation of ZP/egg coat structure and its function is not known. Using approaches classically used to identify amyloid including conformation-dependent antibodies and dyes, X-ray diffraction, and negative stain electron microscopy, our studies suggest the mouse ZP is a functional amyloid. Amyloids are cross-β sheet fibrillar structures that, while typically associated with neurodegenerative and prion diseases in mammals, can also carry out functional roles in normal cells without resulting pathology. An analysis of the ZP domain from mouse ZP3 and ZP3 homologs from five additional taxa using the algorithm AmylPred 2 to identify amyloidogenic sites, revealed in all taxa a remarkable conservation of regions that were predicted to form amyloid. This included a conserved amyloidogenic region that localized to a stretch of hydrophobic amino acids previously shown in mouse ZP3 to be essential for fibril assembly. Similarly, a domain in the yeast protein α-agglutinin/Sag 1p, that possesses ZP domain-like features and which is essential for mating, also had sites that were predicted to be amyloidogenic including a hydrophobic stretch that appeared analogous to the critical site in mouse ZP3. Together, these studies suggest that amyloidogenesis may be a conserved mechanism for ZP structure and function across billions of years of evolution

Identification of amyloidogenic regions in mouse ZP proteins.

<p>A) Schematic diagram of mature ZP1, ZP2, and ZP3 with amyloidogenic regions predicted by AmylPred 2 indicated as red bars above the individual domains. Yellow box, trefoil domain. Numbers signify amino acid number. B) Structure based sequence alignment of the ZP polymerization domain of mouse ZP1 (aa 268–541), ZP2 (aa 361–630), and ZP3 (aa 42–305) showing amyloidogenic sites (blue highlighting), as predicted by the Amylpred2 algorithm. Cysteine residues are noted by black boxes. The internal hydrophobic patch (IHP) is indicated by a red box [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129907#pone.0129907.ref035" target="_blank">35</a>]. The β-strand secondary structure based on the crystal structure of chicken ZP3 is noted by orange bars (ZP-N subdomain) and green bars (ZP-C subdomain) above the amino acid sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129907#pone.0129907.ref008" target="_blank">8</a>]. C) Structure based alignment of ZP-N domains in mouse ZP1 (N1, N2), ZP2 (N1-N4), ZP3 (N), abalone VERL repeat 10 (R10) and yeast α-agglutinin/Sag 1p showing predicted amyloidogenic sites in blue highlighting as determined by the AmylPred2 algorithm. The β-strand secondary structure, based on structure of mZP-N is indicated by orange bars above the amino acid sequence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129907#pone.0129907.ref007" target="_blank">7</a>]. Cysteine residues are noted by black boxes. <b>*</b>, indicate sites essential or important for ZP2-sperm and α-agglutinin-a-agglutinin binding [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129907#pone.0129907.ref007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129907#pone.0129907.ref036" target="_blank">36</a>].</p

ZP exhibit structural characteristics of amyloid.

<p>Aa-Ae) Isolated ZP were digested with chymotrypsin followed by spotting on to grids and staining with 2% uranyl acetate. TEM micrographs show the various amyloid-like structures observed in the dispersed ZP. Af) Negative control in which buffer containing chymotrypsin but not ZP was spotted on to a grid. Ag) TEM micrograph of human Aβ amyloid fibrils. Scale Bar = 100 nm. B) X-ray diffraction of mouse ZP. Numbers represent Angstroms. Small arrows indicate the 4.65Å reflection.</p