Proximal Corpus Epididymidis Research Articles

Molecular cloning and characterization of EPI-1, the major protein in chimpanzee (Pan troglodytes) cauda epididymal fluid.

A 27-kDa glycoprotein comprises approximately 20% of the total protein in chimpanzee (Pan troglodytes) cauda epididymal fluid. Polyclonal antibodies generated against this glycoprotein react with 27- and 25-kDa components in chimpanzee cauda epididymal fluid and in human, gorilla, chimpanzee, and monkey seminal fluid. According to microsequencing, the 27- and 25-kDa components (chimpanzee EPI-1) are identical to the cloned putative human epididymal protein HE1. Screening of a chimpanzee epididymal cDNA library enabled isolation of a cDNA clone of chimpanzee EPI-1. On the cDNA level, chimpanzee EPI-1 and human HE1 are 99% identical. Northern analysis localized chimpanzee EPI-1 mRNA to the distal caput epididymidis. With EPI-1 primers, polymerase chain reaction of reverse-transcribed rhesus monkey (Macaca mulatta) epididymal RNA enabled isolation of a rhesus monkey EPI-1 cDNA clone. The derived amino acid sequence of rhesus monkey EPI-1 is identical to chimpanzee EPI-1 and to human HE1. Northern analysis localized rhesus monkey EPI-1 mRNA to the distal caput and the proximal corpus epididymidis. Northern analysis also showed that chimpanzee EPI-1 and rhesus monkey EPI-1 gene products are expressed specifically in the epididymis and not in any other tissue examined.

Detection of 25-kDa Anti-Agglutinin in Epididymal Plasma and Spermatozoa Collected from Various Regions of Boar Epididymis.

Distribution of the 25-kDa epididymal anti-agglutinin for spermatozoa was examined by the Western blotting technique in fluid collected from boar rete testes and epididymides. Fluid was collected from the rete testes and various regions of the epididymis, and then plasma and sperm extracts were prepared. Antiserum was raised against the anti-agglutinin, and specific antibody was purified from it. In the plasma, a reaction between the anti-agglutinin and purified antibody was first observed in the proximal corpus epididymidis and became stronger in the more distal regions. In extracts from washed spermatozoa using the original sample buffer, anti-agglutinin was detected between the proximal corpus and the distal cauda and more strongly in the distal corpus. A thorough washing of spermatozoa on a discontinuous Percoll gradient brought about only minor changes in the detection pattern of anti-agglutinin. However, in samples extracted from washed spermatozoa using a modified sample buffer containing twofold detergent and twofold 2-mercaptoethanol, a stronger reaction was observed between the anti-agglutinin and purified antibody in the distal cauda than in the distal corpus. These results indicate that free or loosely sperm-bound anti-agglutinin increases in the corpus and cauda epididymides. It is also suggested that solubility of the anti-agglutinin associated with spermatozoa in the original sample buffer decreases in the cauda epididymidis.

Characterization of the motility of maturing rat spermatozoa by computer-aided objective measurement.

A computer-aided sperm analysis system was optimized for objective assessment of the movement characteristics of mature and immature rat spermatozoa by testing different settings. Measurements of straight line velocity of individual motile cells were validated by manual tracking with a digitizer. Better agreement between the two methods and better performance in distinguishing between mature and immature spermatozoa was obtained by reducing the tracking rate to increase the time of analysis. However, numbers of motile and immotile cells could not be determined accurately. Manual counting of videotaped images revealed no significant differences in percentage motility of spermatozoa from five epididymal regions. Caput spermatozoa were characterized by low straight-line (VSL) and averaged-path (VAP) velocities and low path straightness (STR), whereas mature cells displayed high VSL, VAP and STR. An increase in curvilinear velocity on maturation was less obvious. Spermatozoa in the proximal corpus epididymidis were heterogeneous in their acquisition of motility maturation and the uniformity of movement pattern achieved in the distal corpus and proximal cauda regions tended to decrease again in the distal cauda epididymidis. Such objective measurements of motility patterns will facilitate studies on the regulation of motility development upon sperm maturation.

Loss of Sperm Surface Sialic Acid Induces Phagocytosis: An Assay with a Monoclonal Antibody T21, Which Recognizes a 54K Sialoglycoprotein

Using a monoclonal antibody T21, we reported that a mouse sperm maturation-associated antigen sialoglycoprotein of 54000 daltons (54K sialoglycoprotein) was secreted at the distal caput to proximal corpus epididymidis and that the 54K sialoglycoprotein had a hidden determinant (cryptodeterminant), which could be eliminated by sialidase treatment (Toshimori et al. (1988): Histochemistry 90:195-200; (1990a): Biol Reprod 42:151-160; (1990b): Arch Histol Cytol 53:339-349). This study evaluated the mouse sperm susceptibility to phagocytosis by macrophage in vitro. Comparisons were made between sperm from the caput epididymidis (caput sperm) incubated in modified Krebs Ringer's solution (MKR) and caput sperm incubated in MKR containing cauda fluid, and between sialylated (sialidase-untreated) sperm from the corpus and cauda epididymidis (corpus/cauda sperm) and desialylated (sialidase-treated) corpus/cauda sperm. The results showed that macrophages were least actively engaged in phagocytosis for caput sperm incubated in MKR containing cauda fluid, and most active for desialylated corpus/cauda sperm. Incubation of caput sperm in MKR containing cauda fluid revealed that the 54K sialoglycoprotein in cauda fluid could be bound to the flagellar surface of caput sperm. These results together with previous findings strongly suggest that the 54K sialoglycoprotein bound to immature sperm during maturation in the epididymis is implicated in the protection of sperm from phagocytosis with the aid of sialic acid residues.

Proteins in luminal fluid of the ram excurrent ducts: changes in composition and evidence for differential endocytosis.

Rete testis fluid (RTF) and luminal fluid collected by micropuncture at selected epididymal sites were analyzed to characterize the spectrum of proteins and to quantify the net gain or loss of total/bulk protein and androgen-binding protein (ABP) between successive regions within the ductus epididymidis. Based on one-dimensional SDS gel electrophoresis, the spectra of proteins in RTF and fluids from the proximal, central, and distal caput through proximal corpus epididymidis differed from each other. Concentrations of sperm, bulk protein, and ABP increased from the rete testis through the central caput epididymidis. Electron microscopic studies following intraluminal microinjections of RTF proteins conjugated to colloidal gold at specific sites in the excurrent ducts revealed that 145 times more protein-gold was endocytosed in the ductuli efferentes than in any of the four regions of the caput epididymidis. Thus, ductuli efferentes were the major extra-testicular site of endocytosis of bulk protein present in RTF; at least a portion of the uptake was specific. On a per sperm basis, the amount of protein present in the central caput epididymidis was less than 15% of that leaving the testis. Although most of the protein present in RTF (greater than or equal to 86 mg/d) must be absorbed in the ductuli efferentes and the initial segment of the epididymis and replaced by newly secreted proteins (greater than or equal to 34 mg/d), there was negligible loss of ABP in these regions. Net loss of ABP occurred primarily in the distal caput and proximal corpus epididymidis. These studies demonstrate that ABP is spared from endocytosis along with the bulk protein in RTF and conserved for functions in epididymal regions far distal to the site of bulk protein loss.

Immunohistochemical localization of 54,000 dalton sialoglycoprotein in the epididymal duct of the germ cell-free W/WV mouse.

A monoclonal antibody T21 specifically recognizes the mouse epididymal sialoglycoprotein of 54,000 dalton (SGP54). The localization of SGP54 was studied in the epididymal duct of germ cell-free WBB6F1W/Wv mutant mice (W/Wv mice) by avidin biotin complex (ABC) immunohistochemistry using T21. None of the testis cells showed immunoreaction. No spermatozoa were present in the epididymal duct lumen. The duct luminal fluid was stained weakly in the proximal corpus epididymidis, and strongly in the cauda epididymidis. Degenerated cells appeared in the duct lumen. The degenerated cells located at the corpus epididymidis showed strong immunostaining in the cytoplasmic region, while the degenerated cells located at the cauda epididymidis showed weak immunostaining. Immunoreaction was also detected between and on microvilli along the epididymis, the intensity being very strong at the distal caput and proximal corpus epididymidis. Invaginations and coated vesicles at the luminal surface of the principal cells were frequently immunostained at the corpus epididymidis. Giant inclusions frequently occurred in the principal cells of the distal caput and corpus epididymidis, with these being very intensely immunostained. These inclusions are ultrastructurally confirmed to be giant multivesicular bodies reported by ABE et al. (1984) in the mouse with the efferent duct cutting. These results suggest that the majority of excess SGP54 are absorbed by the principal cells at the distal caput to corpus epididymidis and catalyzed in the giant multivesicular bodies.

Morphological Features of Principal Cells in the Ovine Epididymis: A Quantitative and Qualitative Study1

Functions of the epididymis differ by region, and this may be reflected in epithelial structure. Therefore, tissues from the initial segment (IS), proximal and central caput (PCap, CCap), and proximal and central corpus (PCor, CCor) epididymidis were examined by light and transmission electron microscopy. The proportion of principal cells in the epithelium was highest (p less than 0.05) in the CCap (74%) and lowest in the CCor (68%), whereas proportions of basal cells (25%), apical cells (1.4%), and white blood cells (2%) were similar in all regions. Volume density (VD) of the nucleus was lower (p less than 0.05) in principal cells in the IS (7%) than in other regions (10%). There was no regional difference in VD of the Golgi complex (14%) or endoplasmic reticulum (19%) in principal cells. The VD of mitochondria averaged 4% in the IS through CCap, but only 2.5% in PCor or CCor (p less than 0.05). The VD of clear vesicles + multivesicular bodies (8%) and dense vesicles (6%) were higher (p less than 0.05) in the CCap than in other regions (1% each), while there were more lipid droplets (12%) in the PCor than in other regions (less than or equal to 2%). Most quantitative differences in VD of organelles within principal cells were small even though significant. However, there were profound differences in the morphological features of the Golgi complex, endoplasmic reticulum, and mitochondria among regions.

Regional and Seasonal Differences in Concentrations of Androgen and Estrogen Receptors in Ram Epididymal Tissue1

Dihydrotestosterone (DHT) is essential for sperm maturation within the epididymis, but the roles of estradiol-17 beta (E2) and progesterone (P) in epididymal function are unknown. To identify sites of potential action of these hormones, and any effect of season on their concentrations, specific binding of steroids to receptors in extracts of ram epididymal tissue was quantified in two studies. Tissue was taken from three broad regions of the epididymis (caput, corpus, and cauda; Study 1) or from seven discrete regions of the epididymis (Study 2) in February to May (nonbreeding season; NBS) or late August to October (breeding season; BS). Specific binding of P was not detected. Saturable high-affinity binding sites specific for DHT (Ka = 2.6 x 10(8).M-1) and E2 (Ka = 5.4 x 10(8).M-1) were detected. Binding was not to androgen-binding protein, testosterone-estradiol-binding globulin, or sperm nuclei. There was no regional or seasonal difference in affinity of DHT or E2 binding. In both studies, concentration of DHT-binding sites (fmol/mg protein for low- plus high-salt extracts) was higher (p less than 0.05) in the BS than NBS. In Study 1, mean concentration of DHT-binding sites was higher (p less than 0.05) in the caput than in the corpus and cauda. The more definitive localization possible in Study 2 revealed that concentration of DHT-binding sites was highest in the distal caput, lowest in the proximal cauda (p less than 0.05), and intermediate in other regions. For E2, however, concentration of binding sites was higher (p less than 0.05) in the BS than NBS only in Study 1, and was higher (p less than 0.05) in the cauda or corpus than in the caput epididymidis. In Study 2, the season by region interaction was significant (p less than 0.05); concentration of E2-binding sites was higher in the distal cauda during the NBS. These data support the concept that the central caput through proximal corpus epididymidis are most dependent on androgenic stimulation, whereas distal regions may respond to estrogenic stimulation.

Secretion of proteins and glycoproteins by perifused rabbit corpus epididymal tubules: effect of castration.

Protein synthesis in epididymal tissue of intact and castrated rabbits was studied after incubation of epididymal minces with [35S]-cysteine or [35S]-methionine and protein separation by two-dimensional gel electrophoresis. Regional differences in the pattern of protein synthesized were observed. Castration did not change overall protein synthesis, but it reduced these regional differences. The presence of 5 alpha-DHT in the culture medium of the proximal corpus epididymidis perfused for 24 hr did not increase overall protein synthesis in tubules from intact or castrated rabbits and did not reinitiate synthesis of the proteins that had disappeared after castration. The kinetics of glycoprotein synthesis and secretion were studied by light and electron microscopy autoradiography at 0.5, 2, 6, and 24 hr after exposure to [3H]-mannose, [3H]-fucose, and [3H]-glucosamine. Changes in the distribution of mannose- and glucosamine-labeled material indicated that the decline in grain density over the epithelium from 30 min to 24 hr coincided with an increasing reaction over the stereocilia border from 30 min to 2 hr and in the lumen from 2 to 24 hr. The distribution of fucose-labeled material indicated that the grain reaction over the epithelium declined more rapidly than with the mannose label. When the glucosamine-labeled sperm mass was released from the tubules, the labeled material was lost after the first washing, indicating that the glucosamine-labeled glycoproteins did not bind firmly to corpus spermatozoa within 24 hr. After castration, both mannose- and fucose-labeled materials migrated to the cell apex more rapidly than in the intact animal, but they were not released as readily into the lumen. The culture of epididymal tubules from castrated males with 5 alpha-DHT for 24 hr did not promote the release of either mannose- or fucose-labeled material into the lumen. However, testosterone given in vivo for 2 weeks restored secretion of mannose-labeled material into the lumen.

Steroidogenesis and testosterone metabolism in cultured principal cells from the ram epididymis.

To determine if ram principal cells can synthesize or metabolize testosterone, or metabolize other steroids present in rete testis fluid, principal cells from the initial segment, central caput, and proximal corpus epididymidis were isolated and cultured in a floating collagen matrix with medium containing 20% dialyzed rete testis fluid. In the first experiment, each matrix was washed twice in testosterone-free medium on day 2.8, transferred into culture medium containing 100 nM of a tritiated steroid and incubated for 4 hours at 34 C. The tritiated steroids were pregnenolone, 5-androstene-3 beta,17 beta-diol, progesterone, 4-androstene-3,17-dione, testosterone, and dihydrotestosterone. Since testosterone was not formed from 5-androstene-3 beta,17 beta-diol or 4-androstene-3,17-dione, testosterone synthesis by ram principal cells is unlikely Pregnenolone and 5-androstene-3 beta,17 beta-diol were not metabolized and only slight metabolism of dihydrotestosterone occurred. Progesterone, 4-androstene-3,17-dione, and testosterone were metabolized to 5 alpha-reduced products tentatively identified as 5 alpha-pregnane-3,20-dione and 5 alpha-pregnan-3 beta-ol-20-one and/or 5 alpha-pregnan-20 alpha-ol-3-one; 5 alpha-androstane-3,17-dione and 5 alpha-androstan-3 alpha-ol-17-one, and dihydrotestosterone, respectively. The second experiment evaluated testosterone metabolism by both cultured principal cells and minced epididymal tissue. On day 1 of culture, during 12 hours the accumulation of dihydrotestosterone in medium from cells of the central caput was 48 X and 1.1 X that in medium from cells of the initial segment and proximal corpus epididymidis.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis.

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.

A determinant of Mr 34 000 expressed by hamster epididymal epithelium binds specifically to spermatozoa in co-culture

A murine monoclonal antibody raised against hamster cauda epididymal spermatozoa was shown to recognize an Mr 34,000 component of epididymal epithelium. Antigen was localized by immunocytochemistry on the surface and in the apical cytoplasm of principal cells in the proximal corpus epididymidis but not in the caput or initial segment regions. Spermatozoa from the corpus epididymidis expressed antigen on their post-acrosomal plasma membrane and annulus. Epididymal principal cells from the proximal corpus region when cultured in vitro bound antibody on their apical surface for at least 5 days. Spermatozoa from the caput epididymidis co-cultured with epithelium expressed antigen after incubation for 8 and 24 h. These results suggest that a surface change to epididymal spermatozoa during maturation in vivo may also be elicited during in-vitro culture.

Development of a maturation antigen on the plasma membrane of rat spermatozoa in the epididymis and its fate during fertilization

The ontogeny of a surface membrane antigen on rat spermatozoa has been investigated using the monoclonal antibody, 2D6. Using indirect immunofluorescence microscopy the 2D6 antigen was first detected on spermatozoa from the proximal corpus epididymidis; no reaction was present on testicular cells. The 2D6 antibody also bound to spermatozoa flushed from the uterus of mated rats and to a sperm-derived antigen on the surface of newly fertilized eggs. When frozen sections of epididymal tissues were stained with 2D6 monoclonal antibody immunofluorescence was confined to the epithelium lining the duct in the proximal and distal corpus epididymidis. Fluorescence in the tissue was androgen-dependent. Immunoblots of proteins in luminal secretions collected by micropuncture from different sites along the epididymal duct showed that in the proximal corpus epididymidis the 2D6 monoclonal antibody recognized a 32 kD antigen, but in secretions from the distal corpus and cauda epididymidis the monoclonal antibody also recognized antigens with molecular weights of 28, 23 and 20 kD. Immunoblots of proteins from spermatozoa collected from the corpus epididymidis revealed a reaction over a 32 kD antigen, while on spermatozoa from the cauda epididymidis the 2D6 monoclonal antibody recognized only a 23 kD antigen. Two hypotheses are proposed to account for the varied reactivity of the monoclonal antibody and their relative merits are discussed.

Culture of principal cells from the ram epididymis. A comparison of the morphology of principal cells in culture and in situ.

A procedure was developed to isolate and culture principal cells from the initial segment, central caput, distal caput, and proximal corpus epididymidis. The morphology of cultured cells and of cells in situ was compared. Over four to ten days, principal cells cultured in a floating collagen matrix with serum-free medium formed clusters that developed into either large sheets of columnar cells or tubular structures of cuboidal cells. Structural polarity was evident and junctional complexes reformed. The distribution and relative abundance of organelles in principal cells in situ differed depending on the region examined, and most of these regional characteristics were retained by principal cells in culture. Microvilli and membrane-bound vesicles were less conspicuous in cultured cells. Cultured principal cells were shorter than principal cells in situ, but were of similar volume. The high purity (90%) and viability (70%) of principal cells after seven to ten days in culture, their retention of morphologic characteristics unique to the region of origin, and the formation of function units are evidence that such cultures should be valuable for studying regional differences in the function of principal cells.

Epididymal proteins mimic the androgenic effect on zona pellucida recognition by immature hamster spermatozoa.

The increase in zona pellucida binding caused by the exposure of cultured proximal corpus epididymidis to 2 microM-5 alpha-DHT (0.87 and 4.29 spermatozoa/egg for control and 5 alpha-DHT group respectively) was lost when 20 microM-cycloheximide was also added to the medium (0.72 spermatozoa/egg). These results were interpreted as meaning that de-novo protein synthesis was required to obtain the effect of androgens. When a fraction enriched in epididymal glycoproteins EP2-EP6 (18% total protein in epididymal cytosol and 30% in enriched fraction) and depleted of androgens (less than 120 pg testosterone + DHT/ml) was added to the cultured epididymal tubules, the zona pellucida-binding ability of the contained spermatozoa increased from 0.55 in controls to 2.73 spermatozoa/egg in the extract-treated group (P less than 0.02). When the enriched fraction was prepared from epididymides of 30-day castrates, the stimulatory effect was lost (1.04 spermatozoa/egg). We suggest that proteins synthesized in the epididymis are required to obtain the effect of androgens and that the glycoproteins EP2-EP6 may be involved.

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.

Epididymal origin of a coiled-tail sperm defect in a boar.

The development of a tail-coiling defect in spermatozoa from Welsh boar was studied by light and electron microscopy. The onset of tail-coiling was coincident with the passage of spermatozoa through the epididymis, since only cells isolated from regions of the duct distal to the caput epididymidis exhibited this characteristic. An intermediate form of this abnormality was detected in the proximal corpus epididymidis, where migration of the cytoplasmic droplets mainly occurred. It is suggested that defective migration of the cytoplasmic droplet might be associated with the pathogenesis of the coiled-tail defect in this animal.

An assessment of the fertilizing ability of spermatozoa in the epididymis of the marmoset monkey (Callithrix jacchus).

The fertilizing ability of spermatozoa from differ regions of the epididymis of the marmoset monkey, Callithrix jacchus, was assessed by determining their rate of fusion with zonaless hamster ova in vitro. This technique tests for functional competence and was validated using epididymal spermatozoa of the hamster whose fertility have been measured previously by in vivo fertilization experiments. The results suggest that some marmoset spermatozoa first acquire the ability to fertilize in the distal caput and proximal corpus epididymidis although the majority become fertile on reaching the proximal cauda region. Acquisition of fertilizing capacity was associated with a change in the degree and character of sperm motility. However, it is considered that modifications to the sperm plasmalemma which allow capacitation and the acrosome reaction to occur are of primary importance for sperm fertility. The development of sperm fertilizing ability in the marmoset is discussed in relation to that in other primates.

Concentrations of sperm, protein and a sperm membrane glycoprotein within boar epididymal luminal fluids.

Boar rete testis fluid and luminal contents from nine epididymal segments were collected for analysis. From proximal to distal, segments were designated A, B, C (caput), Da, Db, Ea (corpus) and Eb, Fa, Fb (cauda). Concentrations of sperm, protein and soluble sperm membrane glycoprotein S2P3 were lowest in the caput epididymidis (region A) and highest in the proximal corpus epididymidis (region Da). At region Da, approximately 94% of the fluid that had entered the epididymis had been reabsorbed. More total protein was found in the luminal fluid of region Da than could be accounted for on the basis of concentration via fluid removal. The proximal cauda epididymidis was the major luminal protein-removing region. However, large amounts of glycoprotein S2P3 were removed from the luminal fluids during passage through the caput epididymidis, and smaller amounts were removed during passage through the cauda epididymidis.

Localization of specific glycoproteins secreted by the rabbit and hamster epididymis.

To investigate the origin and fate of a number of specific epididymal proteins, the luminal contents from the cauda epididymides of sexually mature New Zealand White rabbits and Syrian hamsters were collected by retrograde perfusion. Epididymal fluid, separated from spermatozoa by centrifugation over a 10% Ficoll solution, was then subjected to isoelectric focussing on polyacrylamide flat bed gels. Three epididymal-specific proteins from the rabbit, pI 3.45, 4.15 and 4.65 and two from the hamster, pI 3.40 and 4.15 were identified as glycoproteins by their low isoelectric point and their affinity for concanavalin A. These fractions were isolated and implanted s.c. in guinea pigs and rabbits, respectively, to produce monospecific antisera as verified by immunodiffusion and immunoelectrophoresis. Localization of glycoprotein antigen within the epididymis was performed on longitudinal sections of snap-frozen epididymis using the unlabeled antibody enzyme method. Both rabbit and hamster glycoproteins were secreted mainly by epididymal epithelial cells in the caput and proximal corpus region. However, the initial segments and testis were devoid of antigen. Differences were also apparent in the staining pattern of individual glycoproteins. In the rabbit, glycoproteins were first associated with spermatozoa in the distal caput and proximal corpus epididymidis while glycoproteins in the hamster epididymis were first associated with spermatozoa in the proximal caput region. Reaction product to glycoproteins in the cauda epididymidis of both species was present on spermatozoa and at the adluminal border of epithelial cells. The significance of these results with regard to changes in the surface composition of the sperm plasmalemma during maturation is discussed.