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Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C

A ROLE FOR AMYLOID IN MALE INFERTILITY*
Open AccessPublished:February 05, 2014DOI:https://doi.org/10.1074/jbc.M113.515759
      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.

      Introduction

      Cystatin C (Cst3) belongs to the family 2 of the cystatin superfamily of reversible inhibitors of cysteine proteases of the papain and legumain families (
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      The cystatins. Protein inhibitors of cysteine proteinases.
      ). Cystatin C is a 13-kDa secreted protein that is broadly expressed and is present in all human biological fluids and tissues examined with very high concentrations in seminal plasma and cerebrospinal fluid (
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      Cystatin C–properties and use as diagnostic marker. Cystatin C. Properties and use as diagnostic marker.
      ). As a potent inhibitor of C1 family of cysteine proteases including the lysosomal cysteine proteases cathepsins B, H, L, and S, cystatin C is thought to play a protective role against tissue destruction, whereas other roles in bone resorption, tumor metastasis, protein processing, and parasite infections have also been proposed (
      • Wang P.H.
      • Do Y.S.
      • Macaulay L.
      • Shinagawa T.
      • Anderson P.W.
      • Baxter J.D.
      • Hsueh W.A.
      Identification of renal cathepsin B as a human prorenin-processing enzyme.
      ,
      • Calkins C.C.
      • Sloane B.F.
      Mammalian cysteine protease inhibitors. Biochemical properties and possible roles in tumor progression.
      ,
      • Huh C.G.
      • Håkansson K.
      • Nathanson C.M.
      • Thorgeirsson U.P.
      • Jonsson N.
      • Grubb A.
      • Abrahamson M.
      • Karlsson S.
      Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene.
      ,
      • Brage M.
      • Abrahamson M.
      • Lindström V.
      • Grubb A.
      • Lerner U.H.
      Different cysteine proteinases involved in bone resorption and osteoclast formation.
      ,
      • Siricoon S.
      • Grams S.V.
      • Grams R.
      Efficient inhibition of cathepsin B by a secreted type 1 cystatin of Fasciola gigantica.
      ). Cst3 resides on human chromosome 20 and mouse chromosome 2, where it clusters with several other cystatin family 2 members including members of a reproductive subgroup comprising Cst8 (cystatin-related epididymal spermatogenic (CRES)
      The abbreviations used are: CRES
      cystatin-related epididymal spermatogenic
      AD
      Alzheimer disease
      HCCAA
      human cystatin C amyloid angiopathy
      KSOM
      K+-modified simplex optimized medium
      MTT
      thiazolyl blue tetrazolium bromide
      CASA
      computer-assisted sperm analysis
      RT
      room temperature
      PAD
      protein aggregation disease.
      ), Cst9 (testatin), Cst11 (CRES2), Cst12 (CRES3), cystatin E2, and others (
      • Schnittger S.
      • Rao V.V.
      • Abrahamson M.
      • Hansmann I.
      Cystatin C (CST3), the candidate gene for hereditary cystatin C amyloid angiopathy (HCCAA), and other members of the cystatin gene family are clustered on chromosome 20p11.2.
      ,
      • Abrahamson M.
      • Islam M.Q.
      • Szpirer J.
      • Szpirer C.
      • Levan G.
      The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20.
      ,
      • Cornwall G.A.
      • Hsia N.
      • Sutton H.G.
      Structure, alternative splicing, and chromosomal localization of the cystatin-related epididymal spermatogenic gene.
      ,
      • Cornwall G.A.
      • Hsia N.
      A new subgroup of the family 2 cystatins.
      ).
      Human cystatin C has been implicated in neurodegenerative diseases and in particular Alzheimer disease, as suggested by the genetic linkage of a cystatin C polymorphism with late-onset AD (
      • Finckh U.
      • von der Kammer H.
      • Velden J.
      • Michel T.
      • Andresen B.
      • Deng A.
      • Zhang J.
      • Müller-Thomsen T.
      • Zuchowski K.
      • Menzer G.
      • Mann U.
      • Papassotiropoulos A.
      • Heun R.
      • Zurdel J.
      • Holst F.
      • Benussi L.
      • Stoppe G.
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      • Staehelin H.B.
      • Rebeck G.W.
      • Hyman B.T.
      • Binetti G.
      • Hock C.
      • Growdon J.H.
      • Nitsch R.M.
      Genetic association of a cystatin C gene polymorphism with late-onset Alzheimer disease.
      ,
      • Crawford F.C.
      • Freeman M.J.
      • Schinka J.A.
      • Abdullah L.I.
      • Gold M.
      • Hartman R.
      • Krivian K.
      • Morris M.D.
      • Richards D.
      • Duara R.
      • Anand R.
      • Mullan M.J.
      A polymorphism in the cystatin C gene is a novel risk factor for late-onset Alzheimer's disease.
      ) and that cystatin C colocalizes with amyloid-β in plaques associated with Alzheimer disease (
      • Levy E.
      • Sastre M.
      • Kumar A.
      • Gallo G.
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      • Ghetti B.
      • Tagliavini F.
      Codeposition of cystatin C with amyloid-β protein in the brain of Alzheimer disease patients.
      ). However, cystatin C seems to play a protective role as in vitro studies showed that cystatin C association can inhibit amyloid-β fibril formation (
      • Tizon B.
      • Ribe E.M.
      • Mi W.
      • Troy C.M.
      • Levy E.
      Cystatin C protects neuronal cells from amyloid-β-induced toxicity.
      ,
      • Sastre M.
      • Calero M.
      • Pawlik M.
      • Mathews P.M.
      • Kumar A.
      • Danilov V.
      • Schmidt S.D.
      • Nixon R.A.
      • Frangione B.
      • Levy E.
      Binding of cystatin C to Alzheimer's amyloid β inhibits in vitro amyloid fibril formation.
      ,
      • Selenica M. L
      • Wang X.
      • Ostergaard-Pedersen L.
      • Westlind-Danielsson A.
      • Grubb A.
      Cystatin C reduces the in vitro formation of soluble Aβ1–42 oligomers and protofibrils.
      ) and in vivo cystatin C inhibited the deposition of amyloid-β in several amyloid precursor protein mouse models (
      • Mi W.
      • Pawlik M.
      • Sastre M.
      • Jung S.S.
      • Radvinsky D.S.
      • Klein A.M.
      • Sommer J.
      • Schmidt S.D.
      • Nixon R.A.
      • Mathews P.M.
      • Levy E.
      Cystatin C inhibits amyloid-β deposition in Alzheimer's disease mouse models.
      ,
      • Sun B.
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      • Halabisky B.
      • Lo I.
      • Cho S.H.
      • Mueller-Steiner S.
      • Devidze N.
      • Wang X.
      • Grubb A.
      • Gan L.
      Cystatin C-cathepsin B axis regulates amyloid β levels and associated neuronal deficits in an animal model of Alzheimer's disease.
      ,
      • Kaeser S.A.
      • Herzig M.C.
      • Coomaraswamy J.
      • Kilger E.
      • Selenica M.L.
      • Winkler D.T.
      • Staufenbiel M.
      • Levy E.
      • Grubb A.
      • Jucker M.
      Cystatin C modulates cerebral β-amyloidosis.
      ). Interestingly, cystatin C itself has also been shown to self-aggregate and form amyloid fibrils (
      • Wahlbom M.
      • Wang X.
      • Lindström V.
      • Carlemalm E.
      • Jaskolski M.
      • Grubb A.
      Fibrillogenic oligomers of human cystatin C are formed by propagated domain swapping.
      ). Cystatin C crystallized as a domain-swapped dimer in which the tertiary structure elements of the monomeric-fold were exchanged between two participating monomers (
      • Janowski R.
      • Kozak M.
      • Jankowska E.
      • Grzonka Z.
      • Grubb A.
      • Abrahamson M.
      • Jaskolski M.
      Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping.
      ,
      • Janowski R.
      • Kozak M.
      • Abrahamson M.
      • Grubb A.
      • Jaskolski M.
      3D domain-swapped human cystatin C with amyloid-like intermolecular β-sheets.
      ). Domain swapping has been observed in several amyloidogenic proteins including prion protein and β-2-microglobulin and, therefore, has been proposed as a mechanism for the formation of amyloid fibrils (
      • Knaus K.J.
      • Morillas M.
      • Swietnicki W.
      • Malone M.
      • Surewicz W.K.
      • Yee V.C.
      Crystal structure of the human prion protein reveals a mechanism for oligomerization.
      ,
      • Liu C.
      • Sawaya M.R.
      • Eisenberg D.
      β2-Microglobulin forms three-dimensional domain-swapped amyloid fibrils with disulfide linkages.
      ,
      • Jaskólski M.
      3D domain swapping, protein oligomerization, and amyloid formation.
      ,
      • Bennett M.J.
      • Sawaya M.R.
      • Eisenberg D.
      Deposition diseases and 3D domain swapping.
      ). Indeed, prevention of domain swapping by the generation of stabilized disulfide bonds or a hinge loop mutation inhibited cystatin C dimerization and formation of amyloid fibrils (
      • Nilsson M.
      • Wang X.
      • Rodziewicz-Motowidlo S.
      • Janowski R.
      • Lindström V.
      • Onnerfjord P.
      • Westermark G.
      • Grzonka Z.
      • Jaskolski M.
      • Grubb A.
      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.
      ,
      • Kolodziejczyk R.
      • Michalska K.
      • Hernandez-Santoyo A.
      • Wahlbom M.
      • Grubb A.
      • Jaskolski M.
      Crystal structure of human cystatin C stabilized against amyloid formation.
      ,
      • Orlikowska M.
      • Jankowska E.
      • Kołodziejczyk R.
      • Jaskólski M.
      • Szymańska A.
      Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C.
      ).
      In addition to amyloid formation in wild type cystatin C, a single point mutation (leucine 68 to glutamine, L68Q) in human cystatin C results in a highly unstable and highly amyloidogenic protein that readily forms an amyloid at 37 °C (
      • Calero M.
      • Pawlik M.
      • Soto C.
      • Castaño E.M.
      • Sigurdsson E.M.
      • Kumar A.
      • Gallo G.
      • Frangione B.
      • Levy E.
      Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.
      ,
      • Gerhartz B.
      • Abrahamson M.
      Physico-chemical properties of the N-terminally truncated L68Q cystatin C found in amyloid deposits of brain haemorrhage patients.
      ,
      • Wei L.
      • Berman Y.
      • Castaño E.M.
      • Cadene M.
      • Beavis R.C.
      • Devi L.
      • Levy E.
      Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.
      ). Patients with this hereditary form of cystatin C amyloid angiopathy (HCCAA), also known as hereditary cerebral amyloid with amyloidosis, Icelandic type, die in their 30–40s as a result of cerebral hemorrhage due to L68Q cystatin C deposits in the cerebral arteries (
      • Olafsson I.
      • Thorsteinsson L.
      • Jensson O.
      The molecular pathology of hereditary cystatin C amyloid angiopathy causing brain hemorrhage.
      ,
      • Ghiso J.
      • Jensson O.
      • Frangione B.
      Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of γ-trace basic protein (cystatin C).
      ). In HCCAA patients cystatin C deposits have also been found outside the central nervous system including the testis (
      • Palsdottir A.
      • Snorradottir A.O.
      • Thorsteinsson L.
      Hereditary cystatin C amyloid angiopathy. Genetic, clinical, and pathological aspects.
      ,
      • Löfberg H.
      • Grubb A.O.
      • Nilsson E.K.
      • Jensson O.
      • Gudmundsson G.
      • Blöndal H.
      • Arnason A.
      • Thorsteinsson L.
      Immunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis.
      ), and anecdotal evidence suggests that affected males have fertility problems. The HCCAA disease is autosomal dominant with patients expressing both the wild type and the L68Q variant form of human cystatin C (
      • Palsdottir A.
      • Abrahamson M.
      • Thorsteinsson L.
      • Arnason A.
      • Olafsson I.
      • Grubb A.
      • Jensson O.
      Mutation in the cystatin C gene causes hereditary brain hemorrhage.
      ).
      To develop a mouse model for HCCAA, transgenic mice expressing the human L68Q cystatin C in addition to mouse cystatin C proteins were generated. Heterozygous L68Q male mice were unable to generate offspring, suggesting that L68Q cystatin C amyloid may be detrimental to reproductive function. The present studies were carried out to identify the fertility defect in L68Q male mice and to examine whether human L68Q cystatin C amyloid present in epididymal fluid may be detrimental for sperm function and thus play a causative role in male infertility.

      DISCUSSION

      A single point mutation (L68Q) in the human cystatin C gene results in a highly unstable and highly amyloidogenic cystatin C variant. The L68Q cystatin C readily forms amyloid at body temperature as compared with the more stable wild type cystatin C protein that can also form amyloid but to a much lesser degree under normal biological conditions. Because of its highly amyloidogenic properties, patients with this mutation (HCCAA, human cystatin C amyloid angiopathy) rapidly develop L68Q cystatin C amyloid deposits in the cerebral arteries such that detrimental effects including stroke, dementia, and death can often occur as early as in their 30s (
      • Palsdottir A.
      • Snorradottir A.O.
      • Thorsteinsson L.
      Hereditary cystatin C amyloid angiopathy. Genetic, clinical, and pathological aspects.
      ). Although L68Q cystatin C deposits are also present in organs outside of the brain including the testis, less is known regarding the pathophysiology of these inclusions since the primary cause of death in affected individuals is due to cerebral hemorrhage (
      • Palsdottir A.
      • Snorradottir A.O.
      • Thorsteinsson L.
      Hereditary cystatin C amyloid angiopathy. Genetic, clinical, and pathological aspects.
      ,
      • Löfberg H.
      • Grubb A.O.
      • Nilsson E.K.
      • Jensson O.
      • Gudmundsson G.
      • Blöndal H.
      • Arnason A.
      • Thorsteinsson L.
      Immunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis.
      ).
      Using a mouse model for the study of HCCAA, our studies presented herein demonstrate that L68Q amyloids develop within the reproductive tract and can have detrimental effects on fertility. Specifically, our studies show that heterozygous mice expressing human L68Q cystatin C are unable to generate offspring. Examination of L68Q sperm function in in vitro fertilization experiments showed that the spermatozoa were unable to fertilize unless normal barriers to fertilization, including the cumulus cells and zona pellucida that surround the oocyte, were removed. The L68Q spermatozoa also showed impaired motility such that few cells exhibited progressive movement. Because penetration of the spermatozoa through the cumulus cells and zona pellucida requires a vigorously motile cell, these data suggest that the impaired fertility may be a consequence of the poor motility of the spermatozoa. This was supported by our observations that when the cumulus cells and zona pellucida were removed, L68Q spermatozoa were able to fertilize and generate blastocysts as efficiently as WT spermatozoa. Although there were no obvious structural defects present in the L68Q spermatozoa, at this time we cannot rule out that other abnormalities in the spermatozoa also contribute to the fertility and motility defects.
      Further examination of the L68Q spermatozoa showed that when diluted into aqueous buffers, the viability was reduced compared with that of WT spermatozoa and that large agglutinated clumps of spermatozoa were present. Taken together these studies suggested that in the L68Q mice components in the epididymal luminal fluid surrounding the spermatozoa may bind to the sperm surface causing agglutination and damaging cell membranes such that viability was reduced that ultimately affected sperm motility and fertility. Indeed, when WT spermatozoa were incubated in epididymal fluid from L68Q mice they exhibited a time-dependent reduction in cell viability and motility. The recapitulation of the phenotype of L68Q spermatozoa in WT spermatozoa by the addition of luminal fluid from L68Q mice suggests a causative role for the luminal fluid proteins in the generation of the phenotype.
      Our studies further suggest that the presence of the highly amyloidogenic L68Q cystatin C in the epididymal fluid contributed to the infertility phenotype of the L68Q spermatozoa. Increased amounts of cystatin C amyloid were detected in the epididymal fluid surrounding the spermatozoa, and increased amounts of cystatin C amyloid were associated with the sperm surface and membranes. Also, after centrifugation to deplete insoluble material from the luminal fluid, the depleted luminal fluid from L68Q mice was no longer damaging to spermatozoa, and motility was similar to spermatozoa incubated in WT fluid. PAD pulldown assays showed that the depleted luminal fluid no longer contained cystatin C amyloids, suggesting a role for the amyloids in affecting sperm function. Our studies also showed that the L68Q mice contained different types of amyloid structures in the epididymal fluid compared with that in the WT mice. Although we were unable to determine that in particular human L68Q cystatin C amyloid rather than increased amounts of mouse cystatin C amyloid or a combination of these two was the causative factor, our studies do show that the presence of the human L68Q cystatin C amyloid changed the epididymal luminal environment such that it was incompatible with normal sperm function.
      Other transgenic mouse models overexpressing human L68Q cystatin C have been generated; however, none showed fertility defects. This may reflect the different promoters that were used to direct cystatin C expression as the neuron-specific Thy1 promoter used by Kaeser et al. (
      • Kaeser S.A.
      • Herzig M.C.
      • Coomaraswamy J.
      • Kilger E.
      • Selenica M.L.
      • Winkler D.T.
      • Staufenbiel M.
      • Levy E.
      • Grubb A.
      • Jucker M.
      Cystatin C modulates cerebral β-amyloidosis.
      ) would likely not result in human cystatin C expression in the mouse epididymis. Similarly, the use of the human promoter (
      • Mi W.
      • Pawlik M.
      • Sastre M.
      • Jung S.S.
      • Radvinsky D.S.
      • Klein A.M.
      • Sommer J.
      • Schmidt S.D.
      • Nixon R.A.
      • Mathews P.M.
      • Levy E.
      Cystatin C inhibits amyloid-β deposition in Alzheimer's disease mouse models.
      ,
      • Pawlik M.
      • Sastre M.
      • Calero M.
      • Mathews P.M.
      • Schmidt S.D.
      • Nixon R.A.
      • Levy E.
      Overexpression of human cystatin C in transgenic mice does not affect levels of endogenous brain amyloid β peptide.
      ) may result in different levels or patterns of expression of human cystatin C in the epididymis as compared with the mouse cystatin C promoter used in our studies. L68Q human cystatin C expression was not examined in the epididymis in any of these other L68Q mouse models.
      Although amyloids play a causative role in a number of incurable neurodegenerative diseases including Alzheimer disease, amyloid structures also have been shown to carry out biological roles in the absence of pathology (
      • Fowler D.M.
      • Koulov A.V.
      • Alory-Jost C.
      • Marks M.S.
      • Balch W.E.
      • Kelly J.W.
      Functional amyloid formation within mammalian tissue.
      ,
      • Maji S.K.
      • Perrin M.H.
      • Sawaya M.R.
      • Jessberger S.
      • Vadodaria K.
      • Rissman R.A.
      • Singru P.S.
      • Nilsson K.P.
      • Simon R.
      • Schubert D.
      • Eisenberg D.
      • Rivier J.
      • Sawchenko P.
      • Vale W.
      • Riek R.
      Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.
      ). We have previously established that cystatin (CRES) amyloids are a normal component of the mouse epididymal lumen (
      • Whelly S.
      • Johnson S.
      • Powell J.
      • Borchardt C.
      • Hastert M.C.
      • Cornwall G.A.
      Nonpathological extracellular amyloid is present during normal epididymal sperm maturation.
      ). Although studies are in progress to determine their roles in the epididymis, these functional amyloids are likely formed under controlled conditions such that pathologies do not result as generally occurs during amyloidogenesis. Our studies suggest that environments containing functional amyloids, however, can become pathological if the delicate balance between monomer and amyloid is disturbed possibly due to quality control mechanisms that become overwhelmed or perhaps as a result of an alteration of the functional amyloid structure itself by association/integration of additional amyloids. In our studies the epididymal fluid from the L68Q mice contained more cystatin C protein in the film-like amyloid that is a component of the normal luminal environment as well as additional and distinct stable DMSO resistant cystatin C amyloid structures, suggesting that both mechanisms may have contributed to the fertility defect in the L68Q mouse model.
      Amyloids are present in human semen and have been implicated in facilitating HIV infection (
      • Münch J.
      • Rücker E.
      • Ständker L.
      • Adermann K.
      • Goffinet C.
      • Schindler M.
      • Wildum S.
      • Chinnadurai R.
      • Rajan D.
      • Specht A.
      • Giménez-Gallego G.
      • Sánchez P.C.
      • Fowler D.M.
      • Koulov A.
      • Kelly J.W.
      • Mothes W.
      • Grivel J.C.
      • Margolis L.
      • Keppler O.T.
      • Forssmann W.G.
      • Kirchhoff F.
      Semen-derived amyloid fibrils drastically enhance HIV infection.
      ,
      • Roan N.R.
      • Müller J.A.
      • Liu H.
      • Chu S.
      • Arnold F.
      • Stürzel C.M.
      • Walther P.
      • Dong M.
      • Witkowska H.E.
      • Kirchhoff F.
      • Münch J.
      • Greene W.C.
      Peptides released by physiological cleavage of semen coagulum proteins form amyloids that enhance HIV infection.
      ); however, whether amyloids can affect human sperm function and contribute to male factor infertility has not been established. It is of interest, however, that ALS patients show reduced fecundity (
      • Johnson W.G.
      • Lucek P.R.
      • Chatkupt S.
      • Furman Y.
      • Lustenberger A.
      • Lazzarini A.
      Reduced fecundity in male ALS gene-carriers.
      ), and individuals with hereditary apolipoprotein A-I amyloidosis exhibit infertility and hypergonadotropic hypogonadism (
      • Scalvini T.
      • Martini P.R.
      • Obici L.
      • Tardanico R.
      • Biasi L.
      • Gregorini G.
      • Scolari F.
      • Merlini G.
      Infertility and hypergonadotropic hypogonadism as first evidence of hereditary apolipoprotein A-I amyloidosis.
      ). Our studies examining the L68Q mouse model for the human disease HCCAA suggest that pathological amyloids can indeed play a role in male infertility by affecting sperm function. Together these studies suggest that the presence of amyloids be considered as a potential causative factor in cases of idiopathic male infertility.

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