Rat Epididymal Protein Research Articles

Analysis of oxidative modification of rat epididymal mitochondrial proteins under normobaric chronic hypoxia and modulation of nitric oxide (II) synthesis

Background. The molecular mechanism of NO effects in adaptation to hypoxia is of interest as a potential point of application in the therapy of fertility disorders.
 Aim. To assess the degree of oxidative modification of epididymal mitochondrial proteins during hypoxia under conditions of experimentally modified NO synthesis.
 Material and methods. Sexually mature rats were divided into four groups of 8 individuals: (1) control (animals without hypoxia); (2) chronic normobaric hypoxia, the animals were kept in a sealed chamber with oxygen reduced to 10% once a day for 14 days; (3) animals with hypoxia were injected with an inhibitor of the synthesis of nitric oxide (II) L-nitroarginine methyl ester at a dose of 25 mg/kg intraperitoneally once a day for 7 days; (4) animals with hypoxia were injected with the substrate for NO synthesis L-arginine at a dose of 500 mg/kg intraperitoneally once a day for 10 days. In the mitochondrial fraction of the head and tail of the epididymis, the activity of superoxide dismutase, the amount of NO metabolites, and the degree of oxidative modification of proteins were evaluated. Statistical analysis was performed using the ShapiroWilk, MannWhitney, and Spearman tests; differences were considered significant at p 0.05.
 Results. Hypoxia led to an increase in the oxidative modification of proteins in the mitochondria of the head of the epididymis 319.12 [240.98; 363.63] c.u./mg protein relative to control 17.89 [15.31; 27.62] c.u./mg protein, p=0.0009. In the mitochondria of the tail of the epididymis, the oxidation of proteins under the studied conditions was less pronounced. The use of L-nitroarginine methyl ester, as well as L-arginine, led to a decrease in the level of oxidative modification of proteins in the head of the epididymis relative to the hypoxia model 39.89 [29.25; 43.17] and 37.25 [34.91; 40.96] c.u./mg of protein, respectively, p=0.0009.
 Conclusion. Mitochondrial proteins in the head of the epididymis are more susceptible to oxidative damage during hypoxia; a decrease in the level of NO metabolites under conditions of oxygen deficiency is associated with a decrease in the oxidative modification of mitochondrial proteins.

Evidence for the Involvement of Zinc in the Association of CRISP1 with Rat Sperm During Epididymal Maturation

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.

Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization.

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.

Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization

Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization

Evidence that human epididymal protein ARP plays a role in gamete fusion through complementary sites on the surface of the human egg.

Human epididymal sperm protein ARP, a member of the cysteine-rich secretory proteins (CRISP) family, exhibits significant homology with rat epididymal protein DE, a candidate molecule for mediating sperm-egg fusion in rodents. The aim of this study was to investigate the involvement of ARP in human gamete fusion. Sequential extraction of proteins from ejaculated human sperm revealed the existence of a population of ARP that is tightly associated with the sperm surface and thus, potentially capable of participating in gamete interaction. Exposure of capacitated human sperm to a polyclonal antibody against recombinant ARP (anti-ARP) produced a significant and concentration-dependent inhibition in the ability of human sperm to penetrate zona-free hamster eggs. This inhibition was not due to a deleterious effect on the gametes because anti-ARP affected neither sperm viability or motility, nor egg penetrability. The antibody did not inhibit the occurrence of spontaneous or Ca(2+) ionophore-induced acrosome reaction, nor did it inhibit the ability of sperm to bind to the oolema, supporting a specific inhibition of the antibody at the sperm-egg fusion level. As a relevant evidence for a role of ARP in gamete fusion, the existence of complementary sites for this protein on the surface of human eggs was investigated. Experiments in which zona-free human oocytes discarded from in vitro fertilization programs were exposed to ARP, fixed, and subjected to indirect immunofluorescence revealed the presence of specific ARP-binding sites on the entire surface of the human egg, in agreement with the fusogenic properties of the human oolema. Together, these results strongly support the participation of ARP in the sperm-egg fusion process, suggesting that this protein would be the functional homologue of DE in humans.

Mammalian sperm-egg fusion: evidence that epididymal protein DE plays a role in mouse gamete fusion.

Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.

Relationship between the association of rat epididymal protein “DE” with spermatozoa and the behavior and function of the protein

Relationship between the association of rat epididymal protein “DE” with spermatozoa and the behavior and function of the protein

Relationship between the association of rat epididymal protein "DE" with spermatozoa and the behavior and function of the protein.

Rat epididymal glycoprotein DE associates with the dorsal region of the sperm head during sperm maturation, migrates to the equatorial segment (ES) with the acrosome reaction (AR), and is involved in gamete membrane fusion. In the present study we examined the association of DE with the sperm surface and the relationship of this interaction with the behavior and function of the protein. Cloning and sequencing of DE revealed a lack of hydrophobic domains and the presence of 16 cysteine residues in the molecule. Experiments in which cauda epididymal sperm were subjected to different extraction procedures indicated that while most of the protein is removable from sperm by mild ionic strength, a low amount of DE, resistant to even 2 M NaCl, can be completely extracted by agents that remove integral proteins. However, the lack of hydrophobic domains in the molecule and the failure of DE to interact with liposomes, does not support a direct insertion of the protein into the lipid bilayer. These results, and the complete extraction of the tightly bound protein by dithiothreitol, suggest that this population would correspond to a peripheral protein bound to a membrane component by strong noncovalent interactions that involve disulfide bonds. While ELISA experiments showed that no protein could be extracted by NaCl from capacitated sperm, indirect immunofluorescence studies revealed the ability of the NaCl-resistant protein to migrate to the ES. Together, these results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly bound population that remains after capacitation, migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion.

Potential contraceptive use of epididymal proteins: immunization of male rats with epididymal protein DE inhibits sperm fusion ability.

Rat epididymal protein DE associates with the sperm surface during maturation and participates in sperm-egg fusion. Immunization of male rats with DE raised specific antibodies and produced a significant reduction in the animals' fertility. The present study focused on determining the in vivo mechanism involved in fertility inhibition. Wistar males were injected with DE, and antibody levels and animal fertility were evaluated. Results revealed an association between the two parameters, since animals with absorbance values lower than 0.5 in ELISA presented high fertility rates (66%, 100%) while those with absorbance values higher than 0.5 exhibited the lowest fertility rates (0%, 33%). Histological studies showed no evidence of orchitis, epididymitis, or vasitis in DE-immunized animals. ELISA results revealed the presence of anti-DE antibodies in epididymal and vas deferential fluids. Indirect immunofluorescence and ELISA experiments indicated that these antibodies would not interfere with the synthesis or secretion of DE or with its association with the sperm surface. Finally, while epididymal sperm recovered from DE-immunized animals presented no changes in motility, viability, or ability to undergo capacitation and acrosome reaction, they exhibited a significant decrease in their ability to fuse with zona-free eggs, with no effect on their ability to bind to the oolemma. Together these results indicate that immunization of male rats with epididymal protein DE specifically interferes with the sperm fertilizing ability, supporting the use of epididymal proteins for contraceptive vaccine development.

Genomic organization and chromosomal localization of the murine epididymal retinoic acid-binding protein (mE-RABP) gene.

The murine epididymal retinoic acid-binding protein (mE-RABP) is specifically synthesized in the mouse mid/distal caput epididymidis and secreted in the lumen. In this report, we have demonstrated by Southern blot analysis of genomic DNA that mE-RABP is encoded by a single-copy gene. A mouse 129/SvJ genomic bacterial artificial chromosome (BAC) library was screened using a cDNA encoding the minor form of mE-RABP. One positive BAC clone was characterized and sequenced to determine the nucleotide sequence of the entire mE-RABP gene. The molecular cloning of the mE-RABP gene completes the characterization of the 20.5-kDa-predicted preprotein leading to the minor and major forms of mE-RABP. Comparison of the DNA sequence of the promoter and coding regions with that of the rat epididymal secretory protein I (ESP I) gene showed that the mE-RABP gene is the orthologue of the ESP I gene that encodes a rat epididymal retinoic acid-binding protein. Several regulatory elements, including a putative androgen receptor binding site, "CACCC-boxes," NF-1, Oct-1, and SP-1 recognition sites, are conserved in the proximal promoter. Analysis of the nucleotide sequence of the mE-RABP gene revealed the presence of seven exons and showed that the genomic organization is highly related to other genes encoding lipocalins. The mE-RABP gene was mapped by fluorescent in situ hybridization to the [A3-B] region of the murine chromosome 2. Our data, combined with that of others, suggest that the proximal segment of the mouse chromosome 2 may be a rich region for genes encoding lipocalins with a genomic organization highly related to the mE-RABP gene.

Shedding of a rat epididymal sperm protein associated with infertility induced by ornidazole and alpha-chlorohydrin.

The protein composition of epididymal fluid and sperm extracts of rats treated with the nitroimidazole compound ornidazole was investigated by two-dimensional gel electrophoresis. Epididymal luminal fluid from the corpus and cauda regions of male animals rendered infertile by ornidazole treatment contained a prominent protein (contraception-associated protein 1, CAP1) with a molecular mass of approximately 25 kDa and an isoelectric point (pI) of 5.8; it was not found in fluids, but was present in sperm, from fertile vehicle-fed rats. Infrared matrix-assisted laser desorption/ionization mass spectrometry indicated that the molecular mass of CAP1 was 20420+/-120 daltons. Analysis of 17 amino acids demonstrated 49% homology to a diuretic hormone from an insect (Acheta domesticus). Densitometric quantitation of CAP1 on silver-stained gels indicated its presence in greater amounts in cauda than in corpus fluid from treated animals, whereas fluid from the rete testis lacked CAP1. In vitro incubations of tissue from the caput, corpus, and cauda epididymidal regions with [35S]methionine gave no hint that CAP1 was a secretion product of the epididymal epithelium. The absence of CAP1 from luminal fluid obtained from the sperm-depleted corpus epididymidis of efferent duct-ligated ornidazole-fed rats suggested a spermatozoal origin. CAP1 was present in spermatozoa from the caput epididymidis but not from the rete testis in control animals. Less CAP1 was present in detergent extracts of cauda sperm from ornidazole-treated rats than in sperm from control animals, suggesting a contraceptive-related displacement of protein from sperm to fluid. The association of ornidazole- and alpha-chlorohydrin-induced infertility with the presence of CAP1 in epididymal fluid, probably originating from spermatozoa, suggests a critical role for this protein in fertilization.

Mammalian sperm-egg fusion: the development of rat oolemma fusibility during oogenesis involves the appearance of binding sites for sperm protein "DE".

Rat epididymal protein DE mediates gamete fusion through complementary sites localized on the egg surface. To investigate whether these egg components are involved in the development of rat oolemma fusibility, both the presence of DE-binding components and the ability of the oolemma to fuse with sperm during oogenesis were examined. Localization of DE-complementary sites by indirect immunofluorescence revealed the absence of fluorescent labeling on growing oocytes with a diameter < 50 microns, and the presence of a uniform staining over the entire surface of germinal vesicle oocytes with a diameter > 50 microns. This localization of oolemma components changed progressively to a patchy distribution during maturation. Whereas sperm incorporation was observed only in maturing oocytes, the development of the Hoechst transfer technique to evaluate membrane fusion revealed that germinal vesicle oocytes with a diameter > 50 microns were already competent to fuse with sperm. The involvement of the DE-complementary sites in the oolemma fusibility of these oocytes was confirmed by the fact that the presence of DE during gamete coincubation significantly (p < 0.001) reduced the percentage of oocytes with fused sperm. Together, these observations indicate that the acquisition of fusibility by the rat oolemma occurs during the growth period and involves the appearance of DE-binding components on the oocyte surface. This study provides novel information on the molecular mechanism by which the mammalian egg plasma membrane becomes competent to fuse with sperm during oogenesis.

Potential contraceptive use of epididymal proteins: evidence for the participation of specific antibodies against rat epididymal protein DE in male and female fertility inhibition

Previous studies in our laboratory indicated that immunization of male and female Wistar and Lewis rats with epididymal protein DE, resulted in the development of anti-DE antibodies in over 90% of the animals, with a significant and reversible reduction of fertility. In the present study, ELISA assays performed to analyze the evolution of the immune response indicated that antibody levels in the sera of immunized animals reached a maximum at 8 weeks after the initial injection and then gradually decreased, returning to control values by the end of the sixth month. Western blot experiments demonstrated that the immune sera specifically recognized DE in epididymal sperm extracts and epididymal cytosol, while no reaction was observed with different reproductive and essential organs. The immune sera were also capable of recognizing DE on the surface of both fresh and capacitated sperm as indicated by indirect immunofluorescence experiments. Finally, the exposure of sperm to immune sera prior to uterine insemination resulted in a significant ( P < 0.05) reduction in the percentage of fertilized eggs compared to controls, with no effect on sperm motility and viability, nor on their ability to undergo capacitation. Together, these results support the participation of the raised antibodies as mediators of the antifertility effect and suggest a specific interference at the sperm-egg interaction level.

Identification of osteopontin (OPN) mRNA and protein in the rat testis and epididymis, and on sperm.

We have identified a bone cell adhesion molecule, osteopontin, in the rat testis and epididymis by Northern analysis, RT-PCR, Western immunoblot analysis and immunocytochemistry. A polyclonal antibody raised against rat epididymal fluid proteins was used to detect fusion proteins produced by a testis lambda gt11 cDNA library. Sequence analysis of one of four positive cDNA clones, designated as pREP5, revealed identity with the rat osteopontin (OPN) cDNA. The partial cDNA clone pREP5 encompasses 64% of the 1,457 residues reported by Oldberg et al. (1986; Proc Natl Acad Sci USA 83:8819-8823). Immunoblot analysis with a monoclonal antibody against OPN detects the presence of immunoreactive polypeptides in rat testis homogenates as well as in epididymal fluid and sperm extracts. Immunocytochemical localization to the basal and adluminal region of the seminiferous tubule suggests that OPN could be a Sertoli cell product. Indeed, Northern blot analysis of testicular cell preparations demonstrated positive hybridization to Sertoli cell-enriched RNA, but not to RNA isolated from interstitial cell preparations or to isolated germ cell RNA preparations. OPN is also detected in the rat epididymis and on epididymal spermatozoa. This is the first report on the presence of OPN mRNA and protein in rat testis and epididymis and on the presence of OPN on the surface of epididymal spermatozoa. The characterization of this protein in other tissue suggests that OPN could play a role in testicular cell adhesion during spermatogenesis and/or epididymal maturation, although other potential functions in the male reproductive tract are discussed.

In vivo regulation of rat epididymal proteins by retinoids: analysis by two-dimensional electrophoresis.

The role of retinoids in the regulation of epididymal fluid protein expression was investigated. We compared the patterns of two-dimensional electrophoretic gels of proteins from luminal fluids, cytosols and spermatozoa (from control rats only) of control, retinoid-depleted, retinoid-depleted retinoic acid-complemented and retinoid-depleted testosterone-supplemented rats. This study compared the luminal fluid patterns from the 4 diets and observed 13 proteins whose expression was dependent on nutritional status. Eight were either absent or very weakly expressed in retinoid-depleted animals only, while their presence was obvious in control rats and in the retinoid-deficient retinoic acid- and testosterone-complemented groups. The expression of 8 proteins was greatly enhanced in retinoid-depleted testosterone-supplemented fluids as compared to control fluids. Five of the regulated proteins seemed to be captured by spermatozoa as they were observed in sperm protein patterns of control rats. These results clearly show that the synthesis of several epididymal proteins is influenced by retinoids. Since testosterone-supplemented animals on retinoid-free diet elicited the same response as retinol and retinoic acid ones, testosterone is likely to be the mediator of retinoid action on epididymal protein synthesis. Nevertheless, the observation of one protein whose expression is stimulated by retinoic acid only and is totally independent of testosterone also favors the direct influence of this retinoid.

Mammalian sperm-egg fusion: The rat egg has complementary sites for a sperm protein that mediates gamete fusion

Rat epididymal protein DE is localized on the fusogenic region of the acrosome-reacted spermatozoa and has a potential role in sperm-egg fusion. We investigated the presence of DE binding sites on the egg surface by co-incubating zona-free eggs and capacitated sperm in different concentrations of pure DE. Results indicate that DE produced a concentration-dependent decrease in egg penetration by sperm (fusion), with almost complete inhibition at 200 μg/ml. This inhibition was not due to an effect of DE on initial sperm binding to the egg membrane, since the presence of this protein did not affect the percentage of oocytes with bound sperm nor the number of bound sperm per egg. Those sperm that failed to penetrate the egg in the presence of DE became able to do so after transfer of the eggs to protein- and sperm-free medium, indicating a role for DE in an event subsequent to binding and leading to fusion. Indirect immunofluorescence using a polyclonal antibody against DE revealed a patchy labeling over the entire egg surface, with the exception of the area overlying the second metaphase spindle. This conclusion was supported by the disappearance of the DE-negative area on the fertilized egg. Zona-free eggs, incubated with DE at 4°C or fixed before exposure to DE displayed a uniform staining, suggesting that the patchy labeling resulted from aggregation of DE binding sites by the purified protein. The aggregation of these egg components may represent a necessary step of the fusion process. To our knowledge, this is the first study reporting the existence and localization of complementary sites to a specific sperm protein on the plasma membrane of the mammalian egg.

Structure and expression of the rat epididymal secretory protein I gene. An androgen-regulated member of the lipocalin superfamily with a rare splice donor site

The complete rat epididymal secretory protein I (ESP I) gene was isolated from a genomic library constructed in bacteriophage lambda Charon 4A. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined. Interesting features include the presence of a rare, but functional, splice donor site (...GC) and the presence of a putative androgen-receptor-binding element. A detailed analysis of ESP I regulation was carried out after castration and subsequent testosterone treatment, demonstrating the requirement for androgens. Efferent-duct ligation and cryptorchism, on the other hand, had no effect on the steady-state concentrations of ESP I transcripts. Comparison of the exon/intron organization of the ESP I gene with those of members of the lipocalin superfamily provides strong support for a common ancestral origin.

A ram epididymal secretory protein shares common antigenic determinants with rat epididymal proteins and human seminal plasma proteins

A ram epididymal secretory protein shares common antigenic determinants with rat epididymal proteins and human seminal plasma proteins

Studies on the mechanism of the antiandrogenic effect of a putative 5α -reductase inhibitor

The mechanism of the antiandrogenic effect of 5,10-seco-19-norpregnane-4,5-diene-3,10,20-trione (secosteroid), reputedly an irreversible inhibitor of 5α -reductase, was investigated. Its addition (10 μM) to culture media effectively suppressed the synthesis of rat epididymal proteins specifically induced by 0.1 μM testosterone (T) or dihydrotestosterone (DHT). Under the same conditions, secosteroid did not change the rate at which labeled T was metabolized to 5α -reduced compounds. In a comparative study, secosteroid inhibited 5α -reductase in an isolated microsomal fraction while not affecting the enzyme activity in minced tissue. Secosteroid was shown to be a competitor of the binding of [ 3H]T and [ 3H]DHT (both at 4nM) to the epididymal cytosol androgen receptor, with ID 50 of 1 μM for the former and 4μM for the latter, thus explaining the mechanism involved in its antiandrogenic properties.

Immunochemical localization of epididymal protein DE on rat spermatozoa: Its fate after induced acrosome reaction

AbstractThe localization of rat epididymal protein DE on cauda epididymis spermatozoa was studied with a specific antibody and the peroxidase antiperoxidase (PAP) immunocyto‐chemical reaction. At the light microscopic level, all spermatozoa appeared to be labeled over the dorsal portion of the head, whereas tails were negative. This observation was confirmed using scanning electron microscopy. A large number of particles were seen on the external surface of the plasma membrane covering the acrosomal region and a smaller number on the postacrosomal portion. Flagella appeared free of particles. Sperm suspensions were incubated in conditions that induce capacitation and the acrosome reaction, and, in this instance, the permanence of protein DE on the vesicles and the postacrosomal region of the membrane were observed. The localization of this epididymal protein on the sperm surface is compatible with a role in the gamete interaction process.