Physico-chemical properties of the N-terminally truncated L68Q cystatin C found in amyloid deposits of brain haemorrhage patients

Cystatin C, a major extracellular cysteine proteinase inhibitor, is deposited as amyloid in brain haemorrhage patients with hereditary cystatin C amyloid angiopathy (HCCAA). A disease-causing mutation on the genetic level results in the substitution Leu68-->Gln (L68Q) in cystatin C, which causes protein instability. Besides carrying the L68Q substitution, cystatin C in amyloid deposits isolated from patients is N-terminally truncated by 10 amino acids. To elucidate the role of the N-terminal truncation for protein stability and aggregation properties, (delta1-10,L68Q)-cystatin C was produced in an Escherichia coli expression system and characterised. Unlike wild-type cystatin C, this variant rapidly dimerised under physiological conditions. Two unfolding intermediates of (delta1-10,L68Q)-cystatin C were identified, under the same pH and ionic strength conditions as required to form intermediates of full-length L68Q cystatin C. No evidence was found that the N-terminal truncation per se alters protein stability and leads to higher forms of aggregation. Monomeric as well as dimeric L68Q cystatin C incubated with neutrophil elastase was truncated as in HCCAA patients' amyloid. A protein variant with a thrombin cleavage site placed in front of residue Gly11 in L68Q cystatin C was constructed and used to confirm that the N-terminal segment is similarly accessible to proteinases in the monomeric and dimeric states of L68Q cystatin C. Thus, the N-terminal segment of L68Q cystatin C is exposed to proteolytic attack and does not seem to be involved in intramolecular contacts leading to dimerisation or higher-order aggregation. We conclude that the N-terminal truncation likely is an event secondary to amyloid formation, and of no relevance for the development of HCCAA.     

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: why is the L68Q variant amyloidogenic?

Human L68Q cystatin C is one of the known human amyloidogenic proteins. In its native state it is a monomer with alpha/beta structure. Experimental evidence suggests that L68Q variant associates into dimeric intermediates and that the dimers subsequently self-assemble to form amyloid deposits and insoluble fibrils. Details of the pathway of L68Q mutant amyloid formation are unclear; however, different experimental approaches with resolutions at molecular level have provided some clues. Probably, the stability and flexibility of monomeric L68Q variant play essential roles in the early steps of amyloid formation; thus, it is necessary to characterize early conformational changes of L68Q cystatin C monomers. In this paper, we demonstrate the possibility that the differences between the monomeric forms of wild-type (wt) cystatin C and its L68Q variant are responsible for higher tendency of the L68Q cystatin C amyloidogenesis. We started our studies with the simulations of wt and L68Q cystatin C monomers. Nanosecond time scale molecular dynamics simulations at 308K were performed using AMBER7.0 program. The results show that the structure of the L68Q monomer was changed, relative to the wt cystatin C structure. The results support earlier speculation that the L68Q point mutation would easily lead to dimer formation.     

Oligomerization and transglutaminase cross-linking of the cystatin CRES in the mouse epididymal lumen: potential mechanism of extracellular quality control

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.     

Molecular dynamics simulations of human cystatin C and its L68Q varient to investigate the domain swapping mechanism

Human cystatin C variant (L68Q), one of the amyloidgenic proteins, has been shown to form dimeric structure spontaneously via domain swapping and easily cause amyloid deposits in the brains of patients suffering from Alzheimer's disease or hereditary cystatin C amyloid angiopathy. The monomeric L68Q and wild-type (wt) HCCs share similar structural feature consisting of a core with a five-stranded anti-parallel beta-sheet (beta-region) wrapped around a central helix. In this study, various molecular dynamics simulations were conducted to investigate the conformational fluctuations of the monomeric L68Q and wt HCCs at various combinations of temperature (300 and 500K) and pH (2 and 7) to gain insights into the domain swapping mechanism. The results show that elevated temperature accelerates the disruption of the hydrophobic core and acidic condition promotes the destruction of three salt bridges between beta2 and beta3 in both HCCs. The results also indicate that the interior hydrophobic core of the L68Q variant is relatively unstable, leading to domain swapping more readily comparing to wt HCC under conditions favoring this process. However, these two monomeric HCCs adopt the same mechanism of domain swapping as follows: (i) first, the interior hydrophobic core is disrupted; (ii) subsequently, the central helix departs from the beta-region; (iii) then, the beta2-L1-beta3 hairpin structure unfolds following the so-called "zip-up" mechanism; and (iv) finally, the open form HCC is generated.     

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.     

Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C: use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C

Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.     

Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Cystatins – Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the elevated cellular cystatin levels seen in hereditary cystatin C amyloid angiopathy.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

Molecular Diagnosis of Hereditary Cystatin C Amyloid Angiopathy

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant disorder characterized by the deposition of amyloid in most investigated tissues. The main component of the amyloid deposits is a variant of the cysteine proteinase inhibitor cystatin C, and the most serious consequence of the disease is that amyloid deposition in the cerebral arteries leads to a massive brain hemorrhage and death before 40 years of age. HCCAA has been shown to be caused by a T → A point mutation in the codon for leucine at position 68 in exon 2 of the cystatin C gene, which results in a leucine → glutamine amino acid substitution in the cystatin C molecule. Since the HCCAA-causing mutation abolishes an AluI restriction site in the cystatin C gene, analysis of this AluI restriction fragment-length polymorphism (RFLP) enables simple and accurate molecular diagnosis of HCCAA. One hundred ninety-one individuals have now been screened for the HCCAA causing mutation, including a fetus for prenatal diagnosis. Thirty-six individuals belonging to nine Icelandic families have been found to have the mutation and it is highly probable that these families descend from a common ancestor.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.     

Induction and enhancement of progressive motility in hamster caput epididymal spermatozoa

Progressive motility was induced in hamster caput epididymal spermatozoa incubated in Tyrodes medium containing 50 mM theophylline, 1.0% Fraction V bovine serum albumin, and 15% (v/v) heat-treated human seminal plasma. Under these induction conditions, however, the maximum percent of caput spermatozoa exhibiting progressive motility (21%) and the time during which motility was sustained (120 min) were significantly less (p less than 0.05) than that of controls from the cauda epididymidis. Moreover, in contrast to caudal spermatozoa, the majority of the induced caput spermatozoa exhibited some degree of flagellar bending at the neck or midpiece. In subsequent experiments the procedure for motility induction was modified to achieve levels of motility in caput spermatozoa equivalent to those observed for caudal spermatozoa. The addition of 5 microM diamide, a sulfhydryl oxidant, to the induction medium prevented the flagellar angularity observed in induced caput sperm preparations. The percentage of caput spermatozoa induced to progressive motility was increased to levels characteristic of caudal spermatozoa (48%) by the addition of hamster caudal epididymal fluid (CEF) to the induction medium. Finally, the viability of the induced caput spermatozoa was significantly enhanced (p less than 0.05) by the removal of Fraction V albumin from the induction medium. In the presence of CEF and in the absence of albumin, 50% of the caput spermatozoa acquired progressive motility and sustained this motility for 4 h. Moreover, when fatty acid-free, charcoal-extracted albumin instead of Fraction V albumin was utilized in the induction procedure, a maximum of 43% of the caput spermatozoa acquired progressive motility and maintained this motility for 4 h, suggesting that the decreased sperm viability observed in the presence of Fraction V albumin was due to a contaminant of albumin, possibly fatty acids. The studies described herein demonstrate for the first time that immature quiescent caput epididymal spermatozoa can be induced to acquire progressive and sustained motility equivalent to that observed in mature caudal epididymal spermatozoa.     

Role of Cystatin C in Amyloid Precursor Protein-induced Proliferation of Neural Stem/Progenitor Cells

The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion.     

Recovery and cryopreservation of sika deer (Cervus nippon) spermatozoa from epididymides stored at 4 degrees C

The aim of this study is to clarify influence of cold storage of deer epididymides on sperm quality and suitability for cryopreservation. The epididymides were obtained postmortem from sika deer during the breeding season. When epididymides were removed 8-12h postmortem and stored at 4 degrees C for 1-4 days, the collected spermatozoa showed low motility (6.4%). When spermatozoa were collected from epididymides removed within 4h postmortem, sperm motility and viability were 71.8 and 82.4%, respectively. Sperm motility decreased as prolongation of the storage period of the epididymides continued up to 7 days, but sperm viability was not affected. Pyknosis of the epithelial cells and their release into the lumen were observed in the stored epididymides. Epididymal spermatozoa frozen on Day 0 showed 58.1% motility and 83.2% viability. Motility of the frozen-thawed spermatozoa from epididymides stored at 4 degrees C for 1 day (41.9%) was similar to that of nonfrozen spermatozoa from epididymides stored for 4 days (41.8%). These results suggest that refrigeration of deer epididymides or cryopreservation of spermatozoa from refrigerated epididymides can be used for assisted reproductive techniques when epididymal spermatozoa cannot be collected immediately.     

Altered molecular dynamics and antioxidant status in the spermatozoa in testosterone-induced oligospermia in mouse

Though supraphysiological doses testosterone (T) and its derivatives are known to suppress spermatogenesis in mammals by interfering with the hypothalamus-pituitary axis leading to oligozoospermia, no study has been performed to evaluate the integrity of the sperm cells produced by such individuals. In T-induced oligozoospermia in the mouse, the spermatozoa showed suppressed zona-binding ability though the motility and viability remained unchanged. In order to assess whether this decreased zona-binding ability is due to perturbations in the mechanical properties of the sperm membranes, we attempted to examine the molecular dynamics employing a lipophilic spin label (16-doxyl stearate) and a protein-binding label (Mal-Net) in two sets of independent experiments. The results showed that the rotational freedom of lipophilic molecules reduced significantly within the first week of T-treatment. During weeks 1 through 4, the protein rotation was found to be retarded significantly. We observed a sharp increase in the ascorbyl radical associated with the cauda epididymal spermatozoa and epididymal fluid of testosterone-treated mice. Moreover, the glutathione (GSH) content in the spermatozoa and the epididymal fluid increased significantly after testosterone-treatment. Further, there was a elevation in the superoxide dismutase (SOD) activity and suppression in the superoxide anion radical generated by the cauda epididymal spermatozoa of testosterone-treated animals. A change in the mechanical properties of a bilayer could modify both the mechanical properties and the function of incorporated proteins. In many instances, a liquid-crystalline bilayer is necessary for protein function. It is likely that the change in the physical properties of sperm membranes might cause the inhibition of enzymes associated with spermatozoa after T-treatment. The alterations in the sperm membrane structure and the antioxidant potentials of both the spermatozoa and the cauda epididymal fluid could also account for the decrease in the zona-binding index of the spermatozoa in T-treated animals. Thus, this study demonstrates for the first time that supraphysiological doses of testosterone could modify the mechano-dynamic properties of sperm membranes and could perturb the redox status of both spermatozoa and the epididymal fluid.     

Synthesis and physicochemical studies of amyloidogenic hexapeptides derived from human cystatin C

Human cystatin C (hCC) is a low molecular mass protein that belongs to the cystatin superfamily. It is an inhibitor of extracellular cysteine proteinases, present in all human body fluids. At physiological conditions, hCC is a monomer, but it has a tendency to dimerization. Naturally occurring hCC mutant, with leucine in position 68 substituted by glutamine (L68Q), is directly involved in the formation of amyloid deposits, independently of other proteins. This process is the primary cause of hereditary cerebral amyloid angiopathy, observed mainly in the Icelandic population. Oligomerization and fibrillization processes of hCC are not explained equally well, but it is proposed that domain swapping is involved in both of them. Research carried out on the fibrillization process led to new hypothesis about the existence of a steric zipper motif in amyloidogenic proteins. In the hCC sequence, there are 2 fragments which may play the role of a steric zipper: the loop L1 region and the C‐terminal fragment. In this work, we focused on the first of these. Nine hexapeptides covering studied hCC fragment were synthesized, and their fibrillogenic potential was assessed using an array of biophysical methods. The obtained results showed that the studied hCC fragment has strong profibrillogenic propensities because it contains 2 fragments fulfilling the requirements for an effective steric zipper located next to each other, forming 1 super‐steric zipper motif. This hCC fragment might therefore be responsible for the enhanced amyloidogenic properties of dimeric or partially unfolded hCC.     

Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process

Experiments were conducted to investigate the effect of seminal plasma on sperm motility during the cryopreservation process. Ejaculated and epididymal spermatozoa from the ram and the bull were washed by centrifugation and resuspended in either seminal plasma or a modified Tyrode's medium (TALP) prior to dilution in medium suitable for cryopreservation. Resuspension of washed ejaculated ram spermatozoa in seminal plasma resulted in higher percentages of motile spermatozoa than resuspension in TALP after the spermatozoa were cooled to 5 degrees C (52 vs 35%), and after thawing (14 vs 9%), respectively. Resuspension of epididymal ram spermatozoa in seminal plasma had no beneficial effect in maintaining sperm motility after cooling (78 vs 73%); however, seminal plasma was beneficial to epididymal ram spermatozoa after thawing (34 vs 3%), respectively. Resuspension of washed ejaculated bull spermatozoa in either seminal plasma or TALP had no effect on the percentage of motile spermatozoa after cooling to 5 degrees C (73 vs 75%) or after thawing (60 vs 60%), respectively. In addition, seminal plasma had no beneficial effect on the percentage of motile epididymal bull spermatozoa when compared with that of TALP-treated spermatozoa after cooling (75 vs 72%) or after thawing (66 vs 63%), respectively. Seminal plasma from different sires (ram and bull) affected epididymal sperm motility. The ability of sperm cells to withstand damage during cryopreservation, however, appears to reside in the sperm cells themselves, probably due to sperm cell composition.     

The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma.

Epididymal spermatozoa from bull, rabbit and ram were incubated in homologous epididymal plasma or seminal plasma in a buffered saline-based medium with or without serum albumin. The spermatozoa were either diluted directly into the medium or were washed first. No effect of washing was observed on the subsequent reaction of the cells to the different media. A considerable proportion of the populations of epididymal spermatozoa survived (i.e. continued to exhibit motility) for up to 22 h at 30 degrees C in the simple saline-based medium. Initially epididymal plasma had a slight stimulatory effect on sperm motility in ram and bull but it had no effect on sperm survival in any of the 3 species. Seminal plasma stimulated motility markedly in ram initially, but in all 3 species seminal plasma was detrimental to survival: in ram even a 15-min exposure to the fluid reduced survival. Serum albumin also stimulated motility; it delayed, but did not prevent, the detrimental effect of seminal plasma, although it had no effect itself on survival. The effects of epididymal plasma, seminal plasma and serum albumin on surface properties of epididymal spermatozoa, i.e. agglutination, sticking-to-glass and eosinophilia, were also noted. These varied between species and there was no correlation between these effects and the effects on motility and survival.