The spermicidal effect of ethylene dibromide in bulls and rams

The spermicidal effect of ethylene dibromide (EDB) in bulls and rams is reviewed. Following oral or parenteral administration EDB was found to cause, by its alkylating effect in the testes of treated bulls and rams, lysis of the chromatin of the elongating spermatids during the time of somatin histones replacement by the sperm protamines. However, while in the bulls the abnormal spermatozoa issued from the affected spermatids were also collected in the ejaculates, this was not the case with treated rams. In the latter animals the abnormal spermatids seem to be phagocytized in the epididymis before their arrival in the ejaculate. In addition, whereas the alkylating effect of EDB occurred also in the upper parts of the epididymis of the bulls, causing tail and acrosome defects to the spermatozoa, in the rams such an effect seems to occur all along the epididymal duct. These differences between bulls and rams in the sites of the genital tract where the drug takes effect, and in the mechanism of this effect, reveal probable differences in the physiology of the reproductive tract between these species.     

Changes in the morphology of spermatozoa during their passage through the genital tract in dairy bulls with normal and impaired spermat ogenesis

An investigation was conducted on changes in sperm morphology along the excurrent duct of bulls. The study comprised 20 bulls with proven fertility and normal semen picture, and 10 bulls with pathologic semen. The morphology of spermatozoa was evaluated on ejaculates and on postslaughter collected contents from the excurrent duct. The incidence of pathologic sperm heads decreased along the duct in both groups of bulls. The main decrease generally occurred in caput epididymidis. In bulls with pathologic semen, the decrease continued in lower regions of epididymis and was deferens. The rate and pattern of sperm removal seem to primarily depend on the quality of spermatozoa entering the excurrent duct. The removal was clearly selective and is assumed to be associated with phagocytosis of spermatozoa mainly in the efferent ductules and proximal part of caput epididymis. Proximal cytoplasmic droplets were present on almost all sperm in the efferent ductules. The incidence decreased during passage along the genital tract. Migration of cytoplasmic droplets from a proximal to a distal position took place between regions C and D of the caput epididymidis. The incidence of middle-piece abnormalities decreased during passage along the genital tract, while the incidence of sperm tail abnormalities increased in the corpus and cauda epididymidis in all bulls.     

Nature of the rabbit acrosome reaction-inducing activity of follicular fluid.

Follicular fluid samples from rabbits, cats, pigs, women and cows had acrosome reaction-inducing activity (ARIA) on rabbit spermatozoa as determined by differential staining after incubation with these fluids. Activity was retained after dialysis and at least 50% was found to be labile when heated to 56 degrees C for as little as 20 min. The induction of the acrosome reaction by bovine follicular fluid showed a dependence on the concentration of follicular fluid and spermatozoa and on calcium ions, and had a pH optimum of approximately 8. Enzyme treatments showed that proteases destroyed the ARIA and this activity was completely blocked by treatment of bovine follicular fluid with goat anti-bovine antiserum. Electron microscope observations indicated the similarity of the reactions observed to that occurring in vivo. It is concluded that the rabbit acrosome reaction inducing-activity of bovine follicular fluid is a serum component(s), probably a protein(s) of high molecular weight.     

Intracellular calcium in hamster spermatozoa in testis,epididymis and during the acrosome reaction

Hamster spermatozoa from the testis and cauda epididymis were examined for intracellular localization of calcium using a pyroantimonate technique. Calcium precipitates were found in spermatozoa from the testis but not in those from cauda epididymis. Spermatozoa from the epididymis which had been induced to undergo capacitation and the acrosome reaction in vitro, were also investigated. In these, the anterior region of the acrosome showed precipitates indicating calcium binding at specific sites, possibly prior to transport across the membranes. The additional presence of calcium precipitates in the postacrosomal region suggests that the appearance of free calcium in that region is an important step in the acrosome reaction.     

The localization of protein carboxyl-methylase in sperm tails

Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility.     

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.     

Phosphoinositide specific phospholipase C activity of goat spermatozoa in transit from the caput to the cauda epididymis.

Goat sperm lysate from cauda epididymis was incubated in the presence of [14C]phosphatidyl-choline, -ethanolamine, -inositol and diphosphatidyl-glycerol. The release of [14C]diacylglycerol from only phosphatidyl inositol confirmed the presence of phosphatidyl inositol specific phospholipase C. The enzyme activity was linear up to 1 hr of incubation at a sperm concentration of 1-10 x 10(9). It had pH optimum of 7.5 in a broad range of pH activity profile (pH 6-9). Maximum activity was observed at 8 mM calcium ion concentration. EDTA and EGTA (5 mM) did not inhibit the activity completely. A comparative study on spermatozoa and fluid from caput, corpus and cauda epididymis revealed 6.5-fold increase of activity in spermatozoa and a 4-fold decrease in case of fluid during the epididymal transit. However, the total protein content remained unchanged in fluid and decreased up to the extent of 2.4-fold in spermatozoa during this process. Thirty five percent of the caudal sperm activity was soluble and the rest was associated with head (44%), mid-piece (10%) and tail (10%).     

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14? fluorescent stain), mitochondrial activity (Mitotraker? fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer - CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.     

Movement characteristics and acrosomal status of rabbit spermatozoa recovered at the site and time of fertilization

Rabbit spermatozoa were recovered from the oviductal ampullae 11 h postcoitus by an oil microflush technique. Their movement was evaluated in the ampullar fluid, or in ampullar fluid diluted with in vitro fertilization medium, in slide preparations which were approximately 25 micron or 100 micron deep. The movement of these sperm was compared with the movement of ejaculated sperm in diluted semen. Movement parameters measured from videotapes recorded by a high-speed camera were coded according to treatment and entered into a microcomputer for statistical analysis. A total of 157 spermatozoa were recovered from the oviducts of 16 does: 152 were motile and 126 were free-swimming. Nearly all of the free-swimming sperm swam in trajectories whose average paths were circular. The flagellar beat pattern of the circular swimmers was asymmetric and nearly planar, and the sperm did not roll. Spermatozoa observed in 25-micron slide preparations produced smaller flagellar bends than sperm swimming in 100-micron preparations and tended to swim in larger circles which were oriented in the plane of the slide. Spermatozoa observed within the cumulus matrix moved in a slow, erratic, sinuous manner, but resumed rapid circling upon leaving the matrix. It was concluded that the ampullar sperm were hyperactivated, retaining this physiological condition as they entered the cumulus. The movement qualitatively resembled that of hyperactivated guinea pig and hamster spermatozoa because these species effectively swim in circles. In contrast, 80% of the ejaculated spermatozoa swam in linear trajectories, resulting from relatively symmetrical, flagellar beat patterns. The percentage of rolling spermatozoa and the rolling frequencies were less in the 25-micron than the 100-micron slide preparations. Thus, the movement parameters of both ampullar and ejaculated spermatozoa were affected by the geometry of their observation chambers. This influence should be taken into account when observing sperm motility in vitro. It could also be important in vivo, where changes in sperm movement in response to epithelial surfaces might provide an advantage for reaching the cumulus mass. Ninety-eight percent of the motile ampullar sperm were observed to have acrosomes, including all spermatozoa found within the cumulus matrix.     

Effect of insemination with doses of 2 or 15 million frozen-thawed spermatozoa and semen deposition site on pregnancy rate in dairy cows

The effects of low-dose artificial insemination (AI) on pregnancy rates have seldom been studied in lactating dairy cows. We evaluated the pregnancy results after AI with doses of 2 and 15 million frozen-thawed spermatozoa and the effect of semen deposition in lactating dairy cows. A total of 284 first inseminations with 2 million spermatozoa and 312 first inseminations with 15 million spermatozoa were performed on 480 dairy farms. Low-dose inseminations (2 million spermatozoa) under field conditions in commercial dairy herds, without estrus synchronization, generally resulted in significantly reduced pregnancy rates compared with normal doses (15 million spermatozoa). The bull x technician effect on fertility was statistically significant. This finding indicates that there is a high variability in fertility among bulls using 2 million spermatozoa per dose. The semen deposition site did not influence pregnancy rates. It is concluded that a dose of 2 million frozen-thawed spermatozoa is probably too low for most bulls to achieve acceptable pregnancy rates in dairy cows.     

An epididymal form of cauxin, a carboxylesterase-like enzyme, is present and active in mammalian male reproductive fluids

Mass spectrometric analysis of a prion protein (PrP)-containing complex isolated from ram cauda epididymal fluid revealed a protein that showed homology to a carboxylesterase-like protein previously identified in cat urine (cauxin). Using anti-cauxin antibodies, immunoreactive bands were detected in corpus and cauda epididymal fluid from all mammals tested (ram, boar, mouse, and cat). In the ram, the protein was also present in seminal fluid but not found to be associated with sperm. The bands reacting with the anti-cauxin antibody coincided with those having esterase activity in a zymographic assay and its levels paralleled the esterase activity of native epididymal fluids. A partial nucleotide sequence of 1143 bp, corresponding to 380 amino acids, was obtained by RT-PCR amplification from total RNA from the corpus epididymis (zone 6). The deduced protein sequence shows a high degree of homology (up to 90%) with the different cauxin proteins found in databases but only up to 60% with other known carboxylesterases. By PCR, strong mRNA expression was found in the corpus and cauda epididymis, while the testis, kidney, and caput epididymis had low expression. No mRNA was detected in the lung, heart, or liver. These data demonstrate that an epididymal form of the cauxin enzyme is secreted into mammalian epididymal fluid. In the ram, it is associated with a high molecular-weight PrP-associated complex and may be responsible for the majority of the esterase activity in the cauda epididymal fluid of this species.     

Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen

The purpose of the current study was to further investigate the role of the antioxidant selenium-dependent enzyme glutathione peroxidase (GPx) in reproductive organs and semen from bulls. To this end a fast and convenient combined method for immune detection and substrate localization was adapted, which allows the assessment of both molecular weight and peroxidase activity of proteins on one and the same SDS-PAGE gel plate. After routine semen analysis of ejaculates, a spectrophotometrical assay of GPx activity in bovine semen was performed. For the immunological analyses performed, a rabbit polyclonal monospecific antibody against GPx was raised. Substrate detection and immunolocalization of GPx in lysates from bovine testis, epididymis, spermatozoa, and seminal plasma was performed. In order to determine the localization of GPx in spermatozoa, immunofluorescence analysis was performed. A positive correlation was established between GPx activity in semen and the number of motile spermatozoa. A negative correlation was observed between GPx activity and the number of immotile spermatozoa. The combined method for immunodetection and substrate localization was tested and proved reliable. Both tetramer and monomer forms of GPx were detected in lysates from testis, epididymis, and spermatozoa. We found no GPx activity in seminal plasma. Immunofluorescence shows the presence of GPx chiefly in the mitochondrial and in the acrosome regions of spermatozoa. GPx activity remained stable under the extreme experimental conditions.     

Morphological features of the epididymal epithelium of gerbil, Meriones unguiculatus

Principal cells of the ducts epididymis of the Mongolian gerbil showed ultrastructural characteristics of lining epithelium cells close related to processes of protein secretion, and transcytosis occurring between adjacent principal cells which were mainly verified in the initial segment. Principal cells also presented roles of fluid phase and adsorptive endocytoses, as well as autophagic and heterophagic lysosomal activities mainly observed in the caput epididymis. Columnar (principal) cells of the corpus epididymidis presented great number of variable vesicles and vacuoles distributed in all the cytoplasmic levels occurring a progressive coalescence pattern among them, which help to guarantee formation of cytoplasmic channels for fluid phase transport between the tubular lumen and epididymal interstitium. Clear cells were presented in the initial segment and predominately in the cauda epididymis epithelium of the gerbil and showed marked ultrastructural characteristics of endocytosis activities occurrence, perhaps directly related to the turnover of fluid phase of spermatozoa stored into the lumen of the distal tail. Other epididymal epithelium cells were verified and described such as basal, halo, apical and dark cells, but they did not presented special ultrastructural features.     

Epididymal cysts in European bison

We describe gross and histopathology of cysts found in the epididymis of the European bison (Bison bonasus). The material was collected from 107, 3 mo to 20 yr old, free-ranging, culled males from the Bia?owieza Primeval Forest (Poland). Epididymal cysts were observed in 65 (61%) of bison of varying ages. They were primarily in the head of epididymis and less often in the corpus or tail. The cysts contained opalescent fluid devoid of spermatozoa. They were lined by epithelium resembling that of efferent ducts and appeared to be congenital abnormalities.     

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

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

A simplified fertility test in the dairy bull utilizing superovulated dairy cows

Dairy bull fertility level has received less attention than production transmitting ability. A simplified fertility test may be beneficial. A study was designed to test the use of tris-(1-aziridinyl)-phosphine oxide (TEPA) treated sperm, which arrests early cell division of the fertilized egg, in heterospermic insemination of superovulated cows. Semen samples were collected and pooled from University of Illinois dairy bulls. Semen samples were washed once, suspended in Illini Variable Temperature diluent (IVT) and incubated with or without TEPA (1.0 to 5.0 mg/ml) for 15 min. Samples were then washed again to remove excess TEPA. Additions of 1.0 to 5.0 mg/ml TEPA to sperm concentrations of 8 x 10(8) sperm/ml had no adverse effect on motility or morphology. The first part of the study utilized superovulated cows inseminated with treated (six cows) or untreated (six cows) sperm in different samples from the same bulls. Secondly, superovulated cows (eight cows) were artificially inseminated with treated and untreated split ejaculates from the same bulls. Lastly, superovulated cows (five cows) were heterospermically inseminated with treated (bull No. 1) and untreated (bull No. 2) spermatozoa. Out of 54 and 39 ova recovered in control and test cows, 40 blastocysts and 31 embryos arrested at the one- to five-cell stage resulted, respectively. Out of a predicted 123 ovulations, 78 fertilized ova were recovered; 40 of these were fertilized by control spermatozoa and 36 by TEPA-treated spermatozoa for parts one and two of the study respectively. These results indicated no significant difference in fertilizability of ova between control and TEPA-treated spermatozoa. Of 41 fertilized ova recovered (part 3), bull No. 1 fertilized significantly more ova (mean +/- standard deviation 5.0 +/- 2.3) than bull No. 2 (2.6 +/- 1.8). Results indicate a difference in fertility between bulls.     

Embryo quality and andrological study of two subfertile bulls versus five control bulls with normal fertility

Andrological studies and embryo morphology evaluation of superovulated cows were performed on 2 randomly selected subfertile dairy bulls whose semen was used for artificial insemination and on 5 control bulls with normal fertility. Neither sperm motility studies, nor sperm morphology or testicular measurements differed between the subfertile and the control bulls. Altogether 315 ova were recovered from 41 superovulated cows inseminated with semen collected from either the subfertile or the normal control bulls. The spermatozoa of one of the 2 subfertile bulls was shown to have a decreased ability to fertilize superovulated ova, while the other subfertile animal, the bull with the lowest noreturn rate, was found by chromosome analysis to have a reciprocal translocation (60, XY, rcp 20:24), causing embryonic death. We suggest that subfertile bulls should not be used in commercial embryo transfer programs nor in artificial insemination and that andrological studies on subfertile bulls with good sperm motility should include evaluation of 6- to 7-day-old ova from superovulated cows to determine if the fertilization rate is normal or impaired. A chromosome analysis should also be performed when a subjertile bull has a normal fertilization rate of ova.     

Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.     

Can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated super- and single-ovulating cattle?

The collective efficiency of barriers in the female tract against spermatozoa with abnormal heads was studied. In Experiment 1, Day 6 ova/embryos were recovered nonsurgically from superovulated (n = 24) and single-ovulating (n = 44) cows following artificial insemination with semen of bulls selected for normal spermatozoal motility (> or = 50%) and high content (> 30%) of spermatozoa with misshapen heads, random nuclear vacuoles or the diadem defect. To assess characteristics of spermatozoa capable of traversing barriers in the female tract, accessory spermatozoa were classified morphologically (x 1250) and compared with those of the inseminate. Superovulated cows proved inadequate for assessment of accessory spermatozoa due to evidence of poor sperm retention in the zona pellucida; thus, only single-ovulating cows were used. Accessory spermatozoa (n = 479) from 31 ova/embryos recovered from 44 cows were more normal in head shape than those in the inseminate (76 vs 62%; P < 0.05). Spermatozoa with normal head shape, but with nuclear vacuoles appeared as accessory spermatozoa at the same frequency as they were found in the inseminate (20 vs 17%, respectively). Only sperm cells with subtly misshapen heads appeared as accessory spermatozoa. In Experiment 2, semen pooled from 4 bulls having large numbers of spermatozoa exhibiting a gradation from severely asymmetrically misshapen heads to subtly misshapen heads was evaluated. Again, the accessory sperm population (960 sperm cells recovered from 64 ova/embryos) was enriched with spermatozoa of normal head shape relative to the inseminate (53 vs 26%, respectively; P < 0.05). Sperm cells with only nuclear vacuoles and those with subtly misshapen heads were not different between the accessory and inseminate populations (11 vs 8%, and 20 vs 25%, respectively). We conclude that morphologically abnormal spermatozoa are excluded from the accessory sperm population based upon severity of head shape distortion.     

Secreted epididymal glycoprotein 2D6 that binds to the sperm's plasma membrane is a member of the beta-defensin superfamily of pore-forming glycopeptides

The plasma membrane of spermatozoa undergoes substantial remodeling during passage through the epididymal duct, principally because of changes in phospholipid composition, exchange of glycoproteins with epididymal fluid, and processing of existing membrane proteins. Here, we describe the interaction of an epididymal glycoprotein recognized by monoclonal antibody 2D6 with the plasma membrane of rat spermatozoa. Our goals have been to understand more about the mechanism of secretion of epididymal glycoproteins, how they interact with the sperm's plasma membrane, and their disposition within it. Reactivity to 2D6 monoclonal antibody was first detectable in principal cells in the distal caput epididymidis and as a soluble high-molecular-weight complex in the secreted fluid. It was not associated with membranous vesicles in the duct lumen. On cauda spermatozoa 2D6 monoclonal antibody recognized a 24-kDa glycoprotein (the subunit of a disulfide cross-linked homodimer of 48 kDa) that was present on the plasma membrane overlying the sperm tail. Binding of 2D6 to immature spermatozoa in vitro was cell-type specific but not species specific, and the antigen could only be extracted from cauda spermatozoa with detergents. Sequencing studies revealed that the 24-kDa glycoprotein was a member of the beta-defensin superfamily of small pore-forming glycopeptides of which several others (ESP13.2, Bin1b, E-2, EP2, HE2) are found in the epididymis. This evidence suggests that some epididymal glycoproteins are secreted into the luminal fluid in a soluble form and bind to specific regions of the sperm's surface via hydrophobic interactions. Given the antimicrobial function of beta-defensins, they have a putative role in protecting spermatozoa and the epididymis from bacterial infections.