Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation

In previous studies we identified an epididymal gene that exhibits homology to the cystatin family of cysteine protease inhibitors. The expression of this gene, termed CRES (cystatin-related epididymal and spermatogenic), was shown to be highly restricted to the proximal caput epididymal epithelium with less expression in the testis and no expression in the 24 other tissues examined. In this report, studies were carried out to examine CRES gene expression in the testis as well as to characterize the CRES protein in the testis and epididymis. In situ hybridization experiments revealed that within the testis CRES gene expression is stage-specific during spermatogenesis and is exclusively expressed by the round spermatids of Stages VII-VIII and the early elongating spermatids of Stages IX and X. Immunohistochemical studies demonstrated that CRES protein was transiently expressed in both the testis and epididymis. Within the testis the protein was localized to the elongating spermatids, whereas within the epididymis CRES protein was exclusively synthesized by the proximal caput epithelium and then secreted into the lumen. Surprisingly, the secreted CRES protein had completely disappeared from the epididymal lumen by the distal caput epididymidis. Western blot analysis of testicular and epididymal proteins showed that the CRES antibody specifically recognized a predominant 19 kDa CRES protein and a less abundant 14 kDa form. These observations suggest that the CRES protein performs a specialized role during sperm development and maturation. © 1995 Wiley-Liss, Inc.     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.     

Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice

Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.     

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.     

Intra-acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: A study using a monoclonal antibody MC101

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley-Liss, Inc.     

Isolation and proteomic characterization of the mouse sperm acrosomal matrix

A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Because of its important role during fertilization, efforts have been put toward isolating the AM for biochemical study and to date AM have been isolated from hamster, guinea pig, and bull spermatozoa. However, attempts to isolate AM from mouse spermatozoa, the species in which fertilization is well-studied, have been unsuccessful possibly because of the small size of the mouse sperm acrosome and/or its fusiform shape. Herein we describe a procedure for the isolation of the AM from caput and cauda mouse epididymal spermatozoa. We further carried out a proteomic analysis of the isolated AM from both sperm populations and identified 501 new proteins previously not detected by proteomics in mouse spermatozoa. A comparison of the AM proteome from caput and cauda spermatozoa showed that the AM undergoes maturational changes during epididymal transit similar to other sperm domains. Together, our studies suggest the AM to be a dynamic and functional structure carrying out a variety of biological processes as implied by the presence of a diverse group of proteins including proteases, chaperones, hydrolases, transporters, enzyme modulators, transferases, cytoskeletal proteins, and others.     

Induction of the acrosome reaction in dog sperm cells is dependent on epididymal maturation: the generation of a functional progesterone receptor is involved

In the current study we investigated the progesterone receptor exposure on the sperm from the testis and different parts of the epididymis, the relation to the sperm maturation stage, the functionality of the progesterone receptor and the capacity of sperm to undergo acrosome reaction. Exposed progesterone receptors on spermatozoa were detected using Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) or a monoclonal antibody against progesterone receptor, C-262. Either progesterone or calcium ionophore was used to induce acrosome reaction. A high percentage (69 +/- 8%; mean +/- SD) of spermatozoa from the cauda epididymis showed P-BSA-FITC labeling at the onset of incubation, whereas only 0.1 +/- 1 and 4 +/- 2%, of spermatozoa from the testes, caput, and corpus epididymis, respectively, were labeled. There was no significant increase in P-BSA-FITC binding during the course of a 6 hr incubation. Treatment with either 10 microM progesterone or 5 microM calcium ionophore induced acrosome reaction in cauda epididymal sperm but not in testicular sperm, caput or corpus epipidymal sperm. It is concluded that the matured sperm of the dog from cauda epididymis and freshly ejaculated sperm demonstrate a functional membrane-bound progesterone receptor while less matured spermatozoa from the testicle, caput, and corpus epididymis fail to demonstrate such a receptor. Acrosome reaction of dog sperm can be induced using either progesterone or calcium ionophore; however, the maturation stages of spermatozoa influence this occurrence.     

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida

Glioma pathogenesis‐related 1‐like protein1 (GliPr1L1) was identified by liquid chromatography‐tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine‐rich secretory proteins, Antigen 5 and pathogenesis‐related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N‐glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N‐glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti‐GliPr1L1 showed that this protein is involved in sperm–zona pellucida interaction. J. Cell. Physiol. 227: 3876–3886, 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of membrane-associated actin in boar spermatozoa

Biochemical, immunological, and electron microscopic methods have been used to provide semi-quantitative estimates and to localize actin in membranes of boar spermatozoa. Immunoblots, using a monoclonal antibody raised against actin from chicken gizzard, detected the protein in caput and cauda sperm plasma membranes. Immunoassay indicated that approximately 1% of the total plasma membrane protein was actin. Monomeric actin accounted for more than one-half of the membrane actin. Approximately 30-40% of plasma membrane actin was insoluble in Triton X-100, and approximately 10% of the total actin remained insoluble after treatment with guanidine hydrochloride. The presence of F-actin in sperm plasma membranes and in plasma membrane detergent-insoluble proteins was detected by fluorescence microscopy using the specific probe NBD phallacidin. When S1 myosin subfragments attached to colloidal gold were used to localize F-actin by electron microscopy, the label was restricted to the outer acrosomal membrane of intact epididymal and ejaculated sperm. Filaments appeared in short arrays along the anterior region of the membrane. S1/gold labeled detergent-insoluble plasma membrane fractions but did not label the plasma membrane in intact sperm. Filaments were least prominent in intact caput spermatozoa and most prominent in ejaculated spermatozoa. We conclude that most actin associated with sperm membranes is in monomeric form in boar spermatozoa, but that actin filaments or protofilaments are components of the outer acrosomal membrane. These filaments may also associate with the plasma membrane overlying the acrosome.     

Protein tyrosine phosphorylation of a heparin-binding sperm membrane mitogen (HBSM) is associated with capacitation and acrosome reaction

Protein tyrosine phosphorylation in spermatozoa is associated with epididymal maturation and though to be central for attainment of a capacitated state and expression of hyperactivated motility. Heparin, the most highly sulfated glycosaminoglycans, was also the most potent at stimulating the acrosomal reaction in bovine epididymal spermatozoa. Studies using radiolabeled inorganic phosphate showed 11-fold increase (32)Pi incorporation in heparin-binding sperm membrane protein (HBSM) during spermatozoal capacitation, and the phosphorylation occurs at the tyrosine residue. Epididymal spermatozoa were induced to undergo capacitation and acrosome reaction by 70% when the cells were incubated in BWW medium supplemented with heparin. The spermatozoa pre-treated with anti-HBSM antibody showed 46% reduction in the hyperactivated motility and lowers the acrosome reaction. This was confirms by measuring the hydrolysis of benzoyl-l-arginine ethyl ether (BAEE) by the acrosomal enzyme; acrosin. The preliminary finding suggests that HBSM may play an important role in the sperm capacitation and acrosome reaction.     

A 105- to 94-kilodalton protein in the epididymal fluids of domestic mammals is angiotensin I-converting enzyme (ACE); evidence that sperm are the source of this ACE

SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was purified from ram cauda epididymal fluid, and a rabbit polyclonal antiserum was obtained. This antiserum showed that membranes of testicular sperm and sperm from the initial caput were positive for the presence of an immunologically related antigen. The protein was immunolocalized mainly on the flagellar intermediate piece, whereas in some corpus and caudal sperm, only the apical ridge of the acrosomal vesicle was labeled. The purified protein was microsequenced: its N-terminal was not found in the sequence database, but its tryptic fragments matched the sequence of the angiotensin I-converting enzyme (ACE). Indeed, the purified 94-kDa protein exhibited a carboxypeptidase activity inhibited by specific blockers of ACE. All the soluble seminal plasma ACE activity in the ram was attributable to the 94-kDa epididymal fluid ACE. The polyclonal antiserum also showed that a soluble form of ACE appeared specifically in the caput epididymal fluid of the boar, stallion, and bull. This soluble form was responsible for all the ACE activity observed in the fluid from the distal caput to the cauda epididymidis in these species. Our results strongly suggest that the epididymal fluid ACE derives from the germinal form of ACE that is liberated from the testicular sperm in a specific epididymal area.     

Prostasome-like particles are involved in the transfer of P25b from the bovine epididymal fluid to the sperm surface

In bulls, P25b is a sperm protein associated with the plasma membrane covering the acrosome. The amount of P25b bound to a constant number of spermatozoa varies from one individual to the other, low levels being associated with bull subfertility. In this study, we describe the epididymal origin of P25b using Western blot analysis. Whereas P25b was undetectable on caput spermatozoa, the amount of P25b associated to a constant number of spermatozoa increases from the corpus to the cauda. Prostasome-like particles were prepared by ultracentrifugation of epididymal fluid. P25b appears to be also associated with those membranous vesicles in increasing amounts along the epididymis. P25b is anchored to the plasma membrane of spermatozoa through glycosylphosphatidyl-inositol as shown by the ability of phospholipase C. but not of high salt treatment, to release P25b. Coincubation experiments revealed that prostasome-like particles are able to transfer P25b to spermatozoa, this process being more efficient at slightly acidic pH. P25b thus appears to be a marker of sperm epididymal maturation in bulls.     

Identification of epididymal proteins associated with hamster sperm.

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.     

Binding and inactivation of the germ cell-specific protein phosphatase PP1gamma2 by sds22 during epididymal sperm maturation

Testis- and sperm-specific protein phosphatase, PP1gamma2, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP1gamma2 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP1gamma2 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1gamma2 in motile caudal spermatozoa (Huang Z, et al. Biol Reprod 2002; 67:1936-1942). The objectives of this study were to determine: 1) stoichiometry of PP1gamma2-sds22 binding and 2) whether PP1gamma2 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP1gamma2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP1gamma2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP1gamma2 coeluted in the gradient fraction. In contrast, caput sperm sds22 and PP1gamma2 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP1gamma2-sds22 complex from caudal sperm was 88 kDa in size. Caput sperm sds22 and PP1gamma2 eluted at 60 kDa and 39 kDa, respectively. SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP1gamma2 (39 kDa), suggesting a 1:1 complex between these two proteins. PP1gamma2 bound to sds22 in this complex was inactive. Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17). This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP1gamma2. Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP1gamma2 during epididymal sperm maturation.     

Selected proteins of "prostasome-like particles" from epididymal cauda fluid are transferred to epididymal caput spermatozoa in bull

During epididymal transit, spermatozoa acquire selected proteins secreted by epithelial cells. We recently showed that P25b, a protein with predictive properties for bull fertility, is transferred from prostasome-like particles present in the cauda epididymal fluid (PLPCd) to the sperm surface. To further characterize the interactions between PLPCd and epididymal spermatozoa, PLPCd were prepared by ultracentrifugation of bull epididymal fluid, then surface-exposed proteins were biotinylated and coincubated in different conditions with caput epididymal spermatozoa. Western blot analysis revealed that only selected proteins are transferred from PLPCd to spermatozoa. MALDI-TOF analysis revealed that these transferred proteins are closely related. The pattern of distribution of the PLPCd transferred varied from one sperm cell to the other, with a bias toward the acrosomal cap. This transfer appeared to be temperature sensitive, being more efficient at 32-37 degrees C than at 22 degrees C. Transfer of PLPCd proteins to spermatozoa was also pH dependant, the optimal pH for transfer being 6.0-6.5. The effect of divalent cations on PLPCd protein transfer to caput spermatozoa was investigated. Whereas Mg(2+) and Ca(2+) have no effect on the amount of proteins remaining associated with spermatozoa following coincubation, Zn(2+) had a beneficial effect. These results are discussed with regard to the function of PLPCd in epididymal sperm maturation.     

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.     

Isolation, immunolocalization, and sperm-association of three proteins of 18, 25, and 29 kilodaltons secreted by the mouse epididymis.

Three murine epididymal secretory proteins have been characterized by their site of synthesis, sperm association, and tissue localization by use of polyclonal antisera and immunochemistry. Mouse epididymal protein 7 (MEP 7) was localized initially within the supranuclear regions of some principal epithelial cells in the proximal corpus while other cells remained unstained. In the mid-proximal corpus, all principal cells and stereocilia were stained, and luminal staining increased from corpus to cauda. Some clear cells in the distal corpus and cauda also showed immunoperoxidase staining. Sequential extraction of caudal spermatozoa indicated that MEP 7 was predominantly loosely associated with spermatozoa and that only a small amount of MEP 7 required detergent to extract it from spermatozoa. Examination of other rodent caudal fluids revealed a related protein in rat caudal fluid of 32 kDa, and amino acid sequence analysis of MEP 7 showed a 68% sequence similarity with rat proteins AEG and D/E. MEP 9 immunolocalized within the cytoplasm of all principal cells of the distal caput. In a transition zone between the distal caput and the corpus, some principal cells were stained while others were not. Distal to the corpus, the principal cell staining gradually decreased. In the distal caput and proximal corpus, large heavily stained droplets associated with spermatozoa were seen in the lumen. The staining intensity of these droplets also decreased from corpus to cauda. The clear cells of the distal corpus and cauda did not stain with the antibody to MEP 9. Sequential extraction of caudal spermatozoa showed that some MEP 9 was extractable under low-salt conditions, whereas extraction with 0.1% Triton X-100 was required to remove all MEP 9, indicating it was firmly associated with spermatozoa. The antibody to MEP 9 cross-reacted with a 25-kDa protein present in rat caudal fluid. MEP 10 was localized within the cytoplasm of the principal cells, the stereocilia, and the lumen of the epididymis at the junction of the distal caput and corpus. In the distal corpus, a large number of clear cells were stained, but very few of these cells stained in the cauda. MEP 10 dissociated completely from caudal spermatozoa under low-salt conditions, indicating that it was not firmly bound to spermatozoa. The antiserum to MEP 10 cross-reacted with proteins present in rat and guinea pig caudal fluid. The related rat protein migrated at approximately 20 kDa. Amino acid sequence analysis of MEP 10 revealed an 86% sequence similarity with rat proteins B and C.(ABSTRACT TRUNCATED AT 400 WORDS)