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.     

Phagocytosis of sperm cytoplasmic droplets by a specialized region in the epididymis of the brushtailed possum, Trichosurus vulpecula

During sperm maturation in the brushtailed possum, Trichosurus vulpecula, cytoplasmic droplets are shed from maturing spermatozoa in the distal regions of the head of the epididymis. Examination of luminal contents from various regions of the epididymis showed that the proportion of detached droplets in the luminal contents was reduced from about 45% in the proximal corpus epididymidis to less than 10% in the distal corpus and cauda epididymides. In contrast, the proportion of droplet-free spermatozoa increased from about 45% to more than 90% in the luminal contents. Disappearance of detached cytoplasmic droplets from the lumen was found to be associated with a region of specialized principal cells lining Regions 6 and 7 of the epididymis which selectively sequester and phagocytose free droplets from the luminal milieu. The luminal surfaces of these cells are characterized by a complex system of interdigitating processes which appear as waves of microfolds . These processes contrast with the stereocilia which cover the luminal surfaces of principal cells in adjacent, nonphagocytic regions of the duct. Cytoplasmic droplets are phagocytosed with their limiting membrane intact and gradually become condensed as they are transported deeper into the cell. Membrane lamellae are gradually compacted, transformed into concentrically arranged membrane stacks and then condensed into small electron-dense vesicles, which are probably degraded by the epithelial cells. The presence of a specific recognition factor on cytoplasmic droplets is suggested by the observation that phagocytic principal cells are able to selectively remove detached cytoplasmic droplets from the lumen in the presence of sperm-associated droplets and spermatozoa.     

Characteristics of donkey spermatozoa along the length of the epididymis

In mammals, the epididymis has numerous interrelated functions including absorptive and secretory activity that affect luminal environment and cell membrane, and the maturation and storage of sperm. Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymis and the motility of epididymal spermatozoa should be a balance between the maturation of flagellum and the inhibition of the flagellar machinery. In this study maturational change in sperm characteristics were evaluated in the epididymis of donkey. Spermatozoa collected from four portions of the epididymis (head, cranial corpus, caudal corpus, tail) were compared before and after ejaculation for viability, mitochondrial activity, kinetic parameters, and morphology. A significant increase in the mitochondrial activity along the epididymis was reported, suggesting a possible involvement in the motion mechanism. This should be corroborated by the significant correlation between mitochondrial activity and the total and progressive motility and the increase in velocities of spermatozoa recorded by computer-assisted sperm analysis. The percentage of most of the abnormal spermatozoa were similar in all tracts, with a great variability between jackasses. Only the bent midpiece percentage decreased significantly along epididymis. A significant increase in the percentage of distal cytoplasmic droplets (DCD), and a simultaneous decrease in the proximal cytoplasmic droplets (PCD), was found. The DCD fell down after ejaculation suggesting the late loss of the cytoplasmic residual (DCD) in the donkey, as hypothesized in the stallion. Because the prevalence of PCD were similar in both tail epididymal and ejaculated spermatozoa, a defect of the maturative process in the PCD sperm should be speculated.     

Reproduction in the male honey possum (Tarsipes rostratus: Marsupialia): the epididymis

The epididymis of the adult honey possum, Tarsipes rostratus, is enclosed by a heavily pigmented tunica vaginalis and lies with the testis in a prominent prepenile scrotum. It is connected to the testis by a single ductus efferentis and is lined by approximately equal numbers of cuboidal ciliated and principal cells. It is unusual for marsupials in having no well-defined compartments or fibrous septae and in having extensive convolutions of the duct only at the caudal flexure. Three principal functional zones (initial, middle, and terminal segments) were identified in the epididymis, based on epithelial type and ultrastructural evidence of sperm maturation. Luminal diameter increases progressively throughout the tract, and epithelial height variations (from about 2 to 20 microns) are greatest in the terminal segment. The epithelium itself is remarkably low (maximum of 21.6 microns) compared with that seen in the epididymis of other mammals. The thickness of the peritubular smooth muscle coat increases close to the junction of the epididymis and ductus deferens. Sperm concentrations were estimated from counts of sperm nuclei and thus can be no more than approximations. The figures are consistent, however, with a rapid increase in concentration in the initial segment, indicating extensive fluid resorption. Sperm concentrations appear to peak in the distal zone of the terminal segment, although sampling problems and wide variations in count make such a conclusion only tentative. Principal and basal cells are the predominant cell types in the epididymal epithelium. Basal cells are most abundant in the initial and distal middle segment. Principal cells show structural evidence of active exchange with the luminal contents and have abundant apical stereocilia, the structure of which depends on the epididymal zone. Other cell types occur less commonly in the epithelium. Lipid-rich and phagocytic principal cells are restricted to the middle and distal zones of the middle segment, respectively. Clear cells, restricted to the terminal segment, and halo cells were found in very low numbers. As in some other marsupials, principal cells (possibly specialized for this function) selectively remove cytoplasmic droplets and probably other cellular debris from the luminal contents. In Tarsipes, however, this process is not very efficient, and many discarded droplets pass through to the terminal segment where they form large masses of debris associated with aggregates of degenerating spermatozoa.     

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.     

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)     

Comparison of lectin-staining pattern in testis and epididymis of gerbil, guinea pig, mouse, and nutria

The testis and epididymis of gerbil, guinea pig, nutria, and mouse were studied after staining with seven rhodamine-conjugated lectins to disclose the distribution of glycoproteins with different sugar residues. In the testis, the lectins showed a variable affinity for Leydig cells, tubular basement membrane, cytoplasm, acrosome, and plasma membrane of maturing spermatids as well as for Sertoli cell extensions. During acrosomal development, the staining pattern showed characteristic changes with different lectins indicating a gradual processing of the glycoprotein components. The staining in the Sertoli cell extensions displayed a cyclic change linked with the release of spermatozoa. A nuclear staining was prominent in zygotene and pachytene spermatocytes in the mouse, weak in the nutria, but absent in gerbil and guinea pig. The principal cells of epididymis showed a lectin-stained Golgi region as well as a similar staining in the apical surface, microvilli, and tubular contents. This staining was most prominent in the caput/corpus regions with some interspecies differences indicating the epididymal areas active in secretion. Narrow cells active in absorption of testis-derived material were lectin-positive in the initial segment of mouse, gerbil, and nutria epididymis. Large light cells with a strong affinity for some lectins were found in the proximal cauda of gerbil and guinea-pig epididymis. In the nutria, corresponding cells were arranged as islands within the low epithelium. The distal cauda of mouse, gerbil, and nutria was the site for lectin-stained light cells interspersed among the low principal cells. It is concluded that the high and low light cells may be active in the absorption and phagocytosis of residual bodies/cytoplasmic droplets and surplus epididymal secretory material, respectively. Thus, labeled lectins formed a useful tool in the analysis of glycoprotein distribution, processing, secretion, absorption, and degradation in the male reproductive tissues.     

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.     

Effects of breeding season and mating on total number and distribution of spermatozoa in the epididymis of the brown marsupial mouse, Antechinus stuartii

Changes in the number and distribution of spermatozoa in the epididymis of the adult brown marsupial mouse were examined during July/August in mated and unmated males. The effects of mating on epididymal sperm populations were studied in 2 groups of males each mated 3 times and compared with the number and distribution of spermatozoa in the epididymides of 4 unmated control groups. One testis and epididymis were removed from each animal (hemicastration) either before or early in the mating season to provide information on initial sperm content and distribution. The contralateral side was removed later in the mating season to examine the effects of mating or sexual abstinence on epididymal sperm distribution. Epididymal sperm number peaked in both the distal caput and distal corpus/proximal cauda epididymidis in late July. The total number of spermatozoa, including those remaining in the testis, available to each male at the beginning of the mating season in early August was approximately 4.4 x 10(6)/side. Although recruitment of spermatozoa into the epididymis from the testis continued until mid-August, sperm content of the epididymis reached a peak of about 3.5 x 10(6)/epididymis in early August. At this time approximately 0.9 x 10(6) spermatozoa remained in the testis which had ceased spermatogenic activity. Throughout the mating season, epididymal spermatozoa were concentrated in the distal corpus/proximal cauda regions of the epididymis and were replenished by spermatozoa from upper regions of the duct. Relatively few spermatozoa were found in the distal cauda epididymidis, confirming a low sperm storage capacity in this region. A constant loss of spermatozoa from the epididymis, probably via spermatorrhoea, occurred throughout the mating season and very few spermatozoa remained in unmated males in late August before the annual male die-off. Mating studies showed that an average of 0.23 x 10(6) spermatozoa/epididymis were delivered per mating in this species, but the number of spermatozoa released at each ejaculation may be as few as 0.04 x 10(6)/epididymis when sperm loss via spermatorrhoea is taken into account. We suggest that the unusual structure of the cauda epididymidis, which has a very restricted sperm storage capacity, may function to limit the numbers of spermatozoa available at each ejaculation and thus conserve the dwindling epididymal sperm reserves in order to maximize the number of successful matings which are possible during the mating season.     

Characterization of a 23 kDa sperm-binding polypeptide of the golden hamster epididymis

A 23 kDa polypeptide has been identified on the flagellum of sperm obtained from the cauda epididymis of the golden hamster. A monospecific antiserum to the 23 kDa hamster polypeptide was prepared and used to study its distribution on sperm, in the epididymis, and in epididymal fluid. In the cauda, the polypeptide is found on the midpiece and endpiece of the sperm tail, in detergent extracts of sperm, and in epididymal luminal fluid-enriched fractions. It is not present on sperm or in luminal fluid-enriched fractions from the caput epididymis. Immunocytochemical staining of epididymal tissue has demonstrated the 23 kDa polypeptide in the Golgi region of the principal cells of the proximal cauda and on sperm in the tubules of this segment and in tubules distal to it. Antiserum to the 23 kDa golden hamster polypeptide cross-reacts with sperm from rats and Chinese hamsters, but not with sperm from rabbits, cattle, mice, and guinea pigs. The antigen is localized to the tail of sperm obtained from the cauda of the rat and from the distal caput of the Chinese hamster. Immunoblots of detergent extracts of sperm and luminal fluid-enriched fractions from these two species reveal a 26 dKa polypeptide that is immunologically related to the golden hamster polypeptide.     

Localization of angiotensin converting enzyme (dipeptidyl carboxypeptidase) in swine sperm by immufluorescence

Testis and epididymis are known to have high amounts of angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). We investigated the localization of the enzyme in these tissues by an immunofluorescent technique and found that the enzyme was localized in the spermatids and residual bodies in the Sertoli cells of the testis. Furthermore, the enzyme was shown to be present in the cytoplasmic droplet of epididymal sperm and also in detached cytoplasmic droplets in semen. The enzyme was not detected in the interstitium of testis and epididymis except for the endothelial cells of the vessel.     

Structural features of the epididymis in a dasyurid marsupial (Antechinus stuartii)

Summary The ductus epididymidis of the marsupial mouse Antechinus stuartii was divided into caput, corpus, and caudal regions using several constant morphological landmarks. Tubule diameter and epithelial height increased gradually from caput to cauda. In contrast, the surface area of the lumen of the ductus epididymidis increased to a maximum in the distal caput region, but decreased markedly in the distal cauda in association with characteristic changes in lumen shape (from circular to slit-shaped) and epithelial height. Epithelial cells of the ductus epididymidis were generally similar in structure to those described in other mammalian species. Principal and basal cells were common throughout the epithelium. Clear and mitochondria-rich cells were also identified, but occurred less frequently. Regional variations in cell ultrastructure were observed only in principal cells. Numerous vesicular inclusions occurred in the apical cytoplasm of cells in caput segments, membrane-bounded, electron-dense bodies were common in distal corpus regions, and a brush border of microvilli characterized the luminal surface of principal cells in caudal segments. Sperm index increased in the proximal caput, declined to basal levels in the distal caput and proximal corpus, and then increased to a maximum in segment 9 of the distal corpus and remained at about this level throughout the cauda epididymidis. Nuclear rotation, loss of cytoplasmic droplets, and other sperm maturational changes were observed along the epididymis. Discarded cytoplasmic droplets collected in large masses interspersed between aggregates of spermatozoa throughout the distal regions of the duct. There was no evidence of phagocytosis by principal cells of cytoplasmic droplets. The epididymis of A. stuartii differs from that of other mammals. The unusual caudal region, which has little storage capacity for sperm, is an unusual adaptation in a species in which the male is known to be polygamous.     

Identification of bovine CD52-like molecule by monoclonal antibody IVA-543: distribution of CD52-like molecule in the bull genital tract

The bovine maturation-associated sperm membrane antigen CD52-like molecule has been analysed using a mouse anti-sperm monoclonal antibody developed against bull spermatozoa. The antigen recognised by monoclonal antibody IVA-543 was detected on blood mononuclear cells (including lymphocytes and monocytes) and on a minor population of polymorphonuclear leukocytes. The bovine CD52-like molecule is secreted by the epididymal epithelium and then it is inserted into the sperm membrane during the epididymal transport in the distal part of epididymis. The CD52-like molecule was absent from spermatozoa derived from testes, and the highest proportion of IVA-543-reactive sperm was observed in the cauda epididymis (91.6%).This study has shown that the new molecule identified on bovine cells has properties analogous to those previously described for CD52 molecules in man, mouse, rat, monkey, and dog.     

Carbonic Anhydrase in Mouse Testis and Epididymis; Transfer of Isozyme IV to Spermatozoa During Passage

Spermatozoa are subjected to major changes as they pass through the epididymal duct. The aim of the present study was to describe the distribution of carbonic anhydrase (CA) in the mouse testis and epididymis using a histochemical technique showing total catalytic activity, in combination with immunohistochemistry for the two important isoforms CAs II and IV. By comparing normal mice with CA II-deficient mice, we were able to study membrane-bound CA without influence from the ubiquitous cytoplasmic CA II. Spermatozoa, when studied in both the scanning electron and light microscope, were found to pickup membrane-bound CA IV during their passage through the epididymal duct. The transfer appeared to take place in the proximal part of the corpus, where the apical membrane and vesicles of principal cells were richly supplied with CA IV. In addition to CA IV, another membrane-bound isozyme was located in basolateral membranes of principal cells. Cytoplasmic CA II was found in varying amounts in apical/narrow cells and principal cells of the corpus in control animals. The significance of CA for pH-regulating processes vital for sperm storage and motility is discussed. A function in HCO3- transport during sperm capacitation at fertilization is suggested for the CA IV found in spermatozoa.     

Sperm morphological abnormalities appearing in the male rabbit reproductive tract

The role of the excurrent duct system in producing and/or eliminating morphologically abnormal spermatozoa may modify the semen parameters and interfere with sperm fertilizing capacity. To study this process, changes in the morphology of spermatozoa during their transit through the reproductive tract in sexually mature rabbits were investigated. The incidence of head, midpiece and tail abnormalities as well as of multiple defects in a single spermatozoon, and the position of the cytoplasmic droplet along the sperm midpiece were evaluated in samples from the testis, 6 regions of the epididymis and the vas deferens. Spermatozoa were characterized by rapid migration of the cytoplasmic droplet when passing from the proximal to the distal caput of the epididymis, and spermatozoa with no droplet predominated in the distal epididymis and vas deferens. In passing from the testis to the proximal caput of the epididymis, the incidence of spermatozoa with an abnormal midpiece and those with multiple defects decreased significantly. The proportion of spermatozoa with abnormal heads was also lower in the testis, but no statistically significant differences were found, whereas there was no change in the proportion of those with abnormal tails. These results indicate that there must be a mechanism for the disposal of defective spermatozoa. No evidence of spermiophagy by luminal macrophages was observed in the extracts, although a few spermatozoa exhibited signs of degeneration, suggesting, that although intraepithelial phagocytosis has not been clearly demonstrated in the nonexperimental rabbit, sperm cells may undergo a form of autolysis within the lumen of the duct.     

Osteopontin expression and regulation in the testis, efferent ducts, and epididymis of rats during postnatal development through to adulthood

Osteopontin (OPN), a multifunctional phosphoprotein found in both hard and soft tissues, was examined in the male reproductive tract. The expression and regulation of OPN in the rat testis, efferent ducts, and epididymis was examined during postnatal development through to adulthood using immunocytochemistry at the light- and electron-microscopic level. Immunoblot analysis revealed a major 30-kDa band for epididymal tissue and a major 60-kDa band for the testis. In the testis, immunostaining of OPN was noted in early germ cells from spermatogonia to early pachytene spermatocytes, suggesting a role for OPN as an adhesive protein binding these cells to the basement membrane and adjacent Sertoli cells. Nonciliated cells of the efferent ducts expressed OPN, whereas a cell- and region-specific distribution of OPN was observed in the epididymis. Reactivity of OPN in the apical region of the cell corresponded to labeling of microvilli, small endocytic vesicles, and endosomes, where OPN may serve to remove calcium from the epididymal lumen and, thus, prevent mineral accumulation and subsequent decrease in sperm fertility. Regulation and postnatal studies revealed that circulating androgens regulate OPN expression in principal cells of the epididymis only. Taken together, the data reveal cell- and region-specific expression and regulation of OPN in the epididymis.     

Identification of a hamster epididymal region-specific secretory glycoprotein that binds nonviable spermatozoa

Even though the epididymis produces an environment promoting sperm maturation and viability, some sperm do not survive transit through the epididymal tubule. Mechanisms that segregate the epididymal epithelium and/or the viable sperm population from degenerating spermatozoa are poorly understood. We report here the identification and characterization of HEP64, a 64-kDa glycoprotein secreted by principal cells of the corpus and proximal cauda epididymidis of the hamster that specifically binds to and coats dead/dying spermatozoa. The HEP64 monomer contains approximately 12 kDa carbohydrate and, following chemical deglycosylation, migrates as a approximately 52-kDa polypeptide. Both soluble (luminal fluid) and sperm-associated HEP64 are assembled into disulfide-linked high molecular weight oligomers that migrate as a doublet band of 260/280 kDa by nonreducing SDS-PAGE. In the epididymal lumen, HEP64 is concentrated into focal accumulations containing aggregates of structurally abnormal or degenerating spermatozoa, and examination of sperm suspensions reveals that HEP64 forms a shroudlike coating surrounding abnormal spermatozoa. The HEP64 glycoprotein firmly binds degenerating spermatozoa and is not released by either nonionic detergent or high salt extraction. Electron microscopic immunocytochemistry demonstrates that HEP64 localized to an amorphous coating surrounding the abnormal spermatozoa. The potential mechanisms by which this epididymal secretory protein binds dead spermatozoa as well as its possible functions in the sperm storage function of the cauda epididymidis are discussed.