Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Maturation-dependent glycoproteins containing both N- and O-linked oligosaccharides in epididymal sperm plasma membrane of rhesus monkeys (Macaca mulatta)

Glycosylation is one of the important post-translational modifications of sperm plasma membrane proteins during the maturation of epididymal spermatozoa that results in the development of motility and fertilizing capability. The aim of the present study was to identify and characterize the maturation-dependent asparagine-linked (N-linked) and serine- and threonine-linked (O-linked) glycoproteins of the epididymal spermatozoa of rhesus monkeys. The presence of N- and O-linked glycoproteins was confirmed by treatment of sperm membranes with N-glycosidase F and O-glycosidase. The major maturation-dependent sperm membrane glycoproteins identified on blots of SDS-PAGE-fractionated proteins of purified sperm plasma membranes from five segments of epididymis, probed with biotinylated lectins and Vectastain-ABC reagent included O-linked 170, 150, 86 and 60/58 kDa glycoproteins; N-linked 68, 56, 48 and 38 kDa glycoproteins and N- and O-linked 116 kDa glycoprotein, all of which exhibited marked differences in the degree of glycosylation between immature and mature sperm surfaces. These glycoproteins can be used as markers of sperm maturation in the epididymis of rhesus monkeys, during the screening of antifertility agents acting at the epididymis, or may be developed as potential sperm antigens. The 100% inhibition of fertility in female rats and rabbits immunized with major maturation-dependent 116 kDa glycoprotein showed the significance of glycosylation changes in the maturation status of epididymal spermatozoa. This 116 kDa protein can be used as a marker parameter of sperm maturation in the rhesus monkey, which is often the preferred animal model for preclinical studies. These results will contribute to the identification of an appropriate animal model for the development of male contraceptives in humans.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Lectin activity in pollen from plants representative of thirty orders of spermatophyta

Summary Pollen extracts from a variety of species representative of thirty orders of spermatophyta, including gymnosperms, dicotyledons and monocotyledons, were examined for the presence of lectin activity by means of a hemagglutination assay. Hemagglutinating activity (HA) was detected in the pollen extracts of all the species examined, indicating that lectins are generally present in the pollen of spermatophyta. The response of this pollen hemagglutinating activity to the sugars and glycoproteins tested as potential inhibitors was identical in all species examined. Moreover, the hemagglutinating activity of pollen extracts from eight species which had been selected as representative of the gymnosperms and both subclasses of angiosperms exhibited similar properties (e.g. distribution by differential centrifugation, stability to heat, response to bivalent ions). The bulk of the hemagglutinating activity was always recovered in the pellet after centrifugation at 1000 g for 5 min. Although sequential treatments with 1% Triton X-100 and 1 M KCl were ineffective, subsequent incubation of the pellet with saline phosphate buffer released hemagglutinating activity. The solubilized hemagglutinating activity was destroyed by protease treatment, indicating that the substance(s) responsible for the activity is (are) protein in nature and, consequently, might be considered to be a lectin. The sugar specifity of the pollen lectin activity from wheat, potato and bean was compared with that of wheat germ agglutinin (WGA), potato agglutinin and bean agglutinin — the lectins present in sporophytic tissues of these plants. For all three plants, the response of the pollen lectin activity to sugars and glycoproteins was different from that shown by the lectin from sporophytic tissues.Abbreviations HA Hemagglutinating activity - PBS 150 mM Na-phosphate buffer (pH 7.2) containing 0.9% NaCl - PHA Phaseolus vulgaris agglutinin - STA Solanum tuberosum agglutinin - WGA wheat germ agglutinin     

Binding of epididymal proteins to rat spermatozoa in vivo.

The secretion of epididymal proteins and their binding to spermatozoa in rats were examined after retrograde perfusion of the superior and inferior epididymal arteries with [35S]methionine. PAGE revealed that the pattern of radioactive proteins in the luminal fluid was markedly different from the well-characterized pattern of secretory proteins obtained by in vitro incubation of epididymal minces with labeled methionine. Of the proteins secreted into the lumen, about 1% were associated with Percoll-purified spermatozoa. More proteins were associated with the spermatozoa in the corpus epididymidis than in the caput. Sequential extraction of spermatozoa with an isotonic buffer, a high-salt buffer, Triton X-100, and SDS revealed that almost half of the radiolabeled proteins could be extracted with the isotonic buffer. The firmly bound radioactive proteins remaining, which were extracted with Triton X-100 or SDS, consisted of one major band of 25 kDa and two minor bands of 30 kDa and 32 kDa. Analysis of the sperm-associated proteins at various times after the isotope was administered indicated that tight binding of proteins to spermatozoa occurs within 3 h after isotope injection.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.     

Identification of UEA I-binding surface glycoproteins of cultured human endothelial cells

Ulex europaeus agglutinin (UEAI) binds mainly to endothelial cells in human tissues. In cultured human umbilical vein endothelial cells TRITC-UEAI gave an even surface staining but no binding to pericellular material. After permeabilization of the cells UEAI decorated the Golgi apparatus as a juxtanuclear structure. Electrophoresis of Triton X-100 lysates of 35S-methionine labeled cells bound to lectin agarose beads showed that a similar set of polypeptides was recognized by UEA-I and WGA while distinctly different polypeptides were bound to LcA-agarose. Surface labelling revealed major glycoproteins with Mr 220 kD, 160 kD, 140 kD, 120 kD, 80 kD and 50 kD, most of which could be extracted with Triton X-100. However, only the 140 kD gp, 120 kD gp and 80 kD gp showed binding to UEA 140 kD gp, 120 kD gp and 80 kD gp showed binding to UEA I-lectin. The results show that among a distinct set of surface glycoproteins in cultured human endothelial cells only a few have alpha-l-fucosyl moieties capable of binding to UEAI lectin.     

Immunochemical localization of secretory antigens in the human epididymis and their association with spermatozoa

Ejaculated spermatozoa were washed and extracted with 0.6 M NaCl (2 h at 0 degree C) and the extract used to immunize rabbits. The crude antibody reacted with epididymal fluid and cytosol and with prostatic cytosol but did not recognize blood serum and testicular cytosol. After adsorption with prostatic proteins, the serum was specific for epididymis. Using immunoelectrophoresis and affinity chromatography, it was found that the antibody reacted with antigens which co-electrophoresed with androgen-dependent proteins (mobility relative to albumin, Ra) 0.3, 0.43 and 1.0, previously identified in human epididymis. Weak immunofluorescence in the epithelium of proximal caput tubules was detected on tissue sections. In contrast, distal caput and corpus tubules displayed a strong fluorescence in the cytoplasm of basal and principal cells as well as in spermatozoa present in lumen. Intense fluorescence was limited to the luminal content and the apical border and sterociliae of principal cells in caudal tubules. When applied to isolated spermatozoa, the reaction was negative for testicular sperm, while 49%, 82% and 100% of spermatozoa from caput, corpus and cauda, respectively, had a fluorescent acrosomal cap. An apparent gradient of increasing fluorescent intensities was also observed in this sequence. The reaction was strongest over the acrosomal cap, apparently absent in the postacrosomal region and weaker over the midpiece and principal piece. These results are interpreted as suggestive of the progressive coating of human spermatozoa with androgen-dependent epididymal proteins during epididymal transit.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

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)     

Glycoproteins of ram sperm plasma membrane. Relationship of protein having affinity for Con A to epididymal maturation

Con A Receptors from the sperm plasma membrane were quantitated (using ³H acetyl-Con A) along the epididymal duct; they diminished in the second part of the epididymis as compared to the epididymal head. Glycoproteins having affinity for Con A were partially characterized: washed spermatozoa from rete testis (= testicular spermatozoa), middle corpus and distal cauda epididymis were labelled (¹²⁵I Na). Proteins of their plasma membrane were extracted (Triton ×100, 0.1% and chromatography affinity): differences appeared in ACA44 profiles from ¹²⁵I Con A Glycoprotein extractions between testicular spermatozoa (2 major peaks Kav= 0.41 and 0.52) and epididymal spermatozoa (3 major peaks Kav= 0.33–0.34, 0.41 and 0.52 and additional minor peaks between 0.66 and 1.00). The peak Kav= 0.41 diminished considerably on epididymal spermatozoa as compared to testicular spermatozoa.     

Tyrosine phosphorylation as evidence of epididymal cauda participation in the sperm maturation process of Corynorhinus mexicanus bat

The bat Corynorhinus mexicanus provides an interesting experimental model for the study of epididymal sperm maturation because after spermatogenesis and the regression of the testes, this bat stores sperm in the epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this species must be completed in the caudal region of the epididymis. One of the major signal transduction events during sperm maturation is the tyrosine phosphorylation of sperm proteins. The aim of the present study was to comparatively evaluate tyrosine phosphorylation in spermatozoa obtained from the caput, corpus and cauda of the epididymis during the sperm storage period. The maturation status of the sperm was determined by the percentage of capacitation and tyrosine phosphorylation in sperm obtained from the epididymis. The highest proportion of tyrosine phosphorylation was registered after the sperm had reached the cauda epididymis during the middle of the storage period. In conclusion, in Corynorhinus mexicanus and most likely in other chiropteran species with an asynchronous male reproductive pattern, epididymal sperm maturation ends in the caudal region of the epididymis and is related to the time that the sperm remains in the epididymis before mating activity.     

Immunolocalization of a guinea pig sperm surface antigen recognized by a monoclonal antibody E74

E74 is a mouse monoclonal antibody raised against the acrosome-reacted guinea pig spermatozoa. This study describes immunolocalization of the E74 antigen in guinea pig spermatozoa. Immunoelectron microscopy of guinea pig spermatozoa shows that the E74 antigen is localized on the equatorial segment plasma membrane following the acrosome reaction but not associated with the surface of the acrosome-intact spermatozoa. Immunoblot analysis of Triton X-100 extract of cauda epididymal guinea pig spermatozoa following one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that E74 antibody recognizes a protein with an apparent molecular weight of 45,000 dalton. Immunoblot of sperm extracts separated by two dimensional gel electrophoresis indicates a broad spot of 45,000 dalton in the 5 to 7.5 isoelectric focusing range.     

Distribution of Pz-peptidase in bovine epididymal and ejaculated semen

Bovine epididymal or ejaculated semen was fractionated by density gradient centrifugation in Percoll, and seminal components recovered from the gradients were subjected to additional separation and washing steps. This procedure resulted in isolation of four major seminal constituents: particle-free extracellular fluid, washed light particulates, washed cytoplasmic droplets, and washed spermatozoa. When assayed using the Pz-peptide substrate, all the isolated seminal fractions contained substantial Pz-peptidase activity. The extracellular fluid Pz-peptidase was present in soluble form, but Triton X-100 was required for complete extraction of the Pz-peptidase activity from the spermatozoa, cytoplasmic droplets, and light particulates. The greatest Pz-peptidase activities were observed in the cytoplasmic droplet and epididymal sperm extracts, whereas the activities in extracellular fluid, extracts of light particulates, and extracts of ejaculated spermatozoa were relatively low. Most of the Pz-peptidase activity in extracts of epididymal spermatozoa was attributable to cytoplasmic droplets. The specific Pz-peptidase activities found by regression analysis were 6.1 mU/billion attached cytoplasmic droplets and 1.1 mU/billion spermatozoa. These results established that in the bovine, cytoplasmic droplets were the major source of Pz-peptidase activity in semen and that Pz-peptidase was not primarily a spermatozoal enzyme.     

Characterization of fibronectin as a marker for human epididymal sperm maturation.

Fertilization involves adhesive interactions between gametes similar to those mediated by fibronectin (FN) in other cellular systems. Fibronectin has been found on the equatorial segment of ejaculated human serum. As sperm capacity to interact with the oocyte is acquired during epididymal transit, the possible participation of FN in human sperm maturation was studied. The presence of FN in both epididymal sperm and fluid was demonstrated by the detection of a major component of 220 kD in immunoblot studies using anti-FN antisera. The concentration of FN in soluble tissue extracts of epididymis was determined by enzyme-linked immunosorbent assay (ELISA). A gradual increase along the length of the organ, averaging 12-fold from proximal caput to distal corpus, was detected. Immunocytochemistry assays indicated that the number of spermatozoa with immunoreactive FN over the equatorial segment increased from 18% in caput to 64% in distal corpus epididymis. Immunoprecipitation of medium from epididymal explants culture with anti-FN antiserum demonstrated the de novo synthesis of FN in vitro. The greater number of FN-positive sperm coincident with FN accumulation in distal regions of the epididymis supports the role of FN in sperm maturation.