Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.     

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-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.     

羊精子表面的凝集素标记特征

用辣根过氧化物酶标记的蓖麻凝集素和伴刀豆素Ａ,对绵羊精子表面的凝集素标记特征进行了观察。蓖麻凝集素在睾丸内精子的顶体区有中等强度标记,尾部有弱标记,在附睾内成熟时,顶体区标记逐渐增强,尾部的标记消失,获能后标记强度则明显减弱。伴刀豆素Ａ的标记在睾丸内的精子仅限于顶体区,随着在附睾内成熟,顶体区的标记增强,尾部也出现弱的标记,获能后有部分精子的标记强度有所增加。实验结果表明,羊精子在成熟过程和获能过程表面糖复合物发生明显修饰。     

Selective binding of mouse and human spermatozoa to beads coated with extracellular matrix components

The ability of mouse epididymal and human ejaculated spermatozoa to bind to beads coated with various extracellular matrix components was examined. Mouse spermatozoa preferentially bound to beads coated with heparin (average values ranging between 6.2 and 8.8 sperm per bead were obtained in different experiments) and with chondroitinsulfate (6.2-7.0), and also, although with significant differences across replicate experiments, to beads coated with laminin (7.9-15.6 sperm per bead) and with collagen type I (6.1-18.5). Human spermatozoa bound to collagen-coated beads (15.4-22.6 sperm per bead) and, to a much lower extent, to chondroitin-sulfate-coated beads (3.2-4.7); they were also able to bind heparin-coated beads, although with ample differences between individual sperm donors (ranging between 0.8 and 18.7 sperm per bead). Very few human and mouse sperm bound fibronectin-coated beads; beads coated with albumin, hyaluronic acid, and chondronectin were always totally free of adhering sperm. The possible physiological role of the interactions between spermatozoa and extracellular matrix components are discussed.     

An investigation using lectins of glycocomponents of mouse spermatozoa during capacitation and sperm-zona binding

Ten different lectins conjugated to fluorescein isothiocyanate (FITC) were used to study the distribution of surface carbohydrates on mouse spermatozoa, and to monitor the possible changes of their distribution during capacitation in vitro and sperm-egg interaction. Most of the lectins gave a restricted pattern of binding to fixed or unfixed epididymal spermatozoa. Binding was highly specific because no staining occurred in the presence of appropriate monosaccharides. Binding of UEA I, DBA and Con A was unaffected by the type of fixative used, but it was influenced by mild centrifugation. While unwashed spermatozoa showed binding mainly over the acrosomal cap and equatorial or postacrosomal regions, spermatozoa washed by mild centrifugation showed a change in the staining of the equatorial segment. Binding of 5 different lectins to spermatozoa did not change during capacitation in vitro. In contrast, capacitated spermatozoa bound to the zona pellucida exhibited a UEA I binding pattern which was strikingly different from that of the capacitated but unbound spermatozoa. We conclude that glycocomponents of specific regions of mouse spermatozoa do not change dramatically during capacitation, but do alter significantly during binding to the zona pellucida.     

Capacitated acrosome-intact mouse spermatozoa bind to Sepharose beads coated with functional neoglycoproteins

Capacitated acrosome-intact mouse spermatozoa bind to the egg's extracellular coat, the zona pellucida (ZP), in a carbohydrate-mediated receptor-ligand manner. The tight irreversible binding of the opposite gametes triggers a signal transduction pathway resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction [AR]). Previously, we demonstrated that a hexose (mannose) and two amino sugars (N-acetylglucosamine and N-acetylgalactosamine), when covalently conjugated to bovine serum albumin (BSA) (functional neoglycoproteins, ngps), mimicked mZP3 and induced the AR [Biol. Reprod. 60 (1999) 94-101]. To further elucidate the specificity of sperm-ngp interaction and the mZP3 mimicking role of the functional ngps, we have examined binding of the mouse spermatozoa to Sepharose 4B beads coated with the functional and non-functional ngps as well as BSA, ovalbumin (OVA), or asialofetuin (ASF). A significantly greater number of capacitated acrosome-intact spermatozoa bound to the beads coated with functional ngps than the beads coated with non-functional ngps, BSA, OVA, or ASF. The binding was temperature-sensitive and was highest when the sperm-bead assay was carried out at 37 degrees C. Blocking of in vitro capacitation, by including calmodulin antagonists in the incubation medium, prevented sperm from binding to the beads. Furthermore, inclusion of free sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) in the binding assay, either individually or as a mixture, inhibited sperm-bead binding in a concentration-dependent manner. Taken together, our data provide evidence strongly suggesting that binding of capacitated spermatozoa to the ngp-coated Sepharose beads is specific. The beads that mimic zona-intact eggs provide an excellent tool for examining pharmacological effects of reagents that alter the sperm function. In addition, the immobilized ngp(s) will be useful as an affinity medium to isolate the sperm surface receptor(s) that recognize and bind to the sugar residues.     

Surface alterations during the capacitation of mammalian spermatozoa

Capacitation is the process by which mammalian sperm acquire the ability to undergo the acrosome reaction which, in turn, is a prerequisite for sperm-egg fusion and penetration. Until recently, it was thought that capacitation involved subtle physiological and chemical changes which had no morphological counterparts even at the electron microscopic level. However, it has now been shown by a number of investigators that material associated with the plasma membrane surface is either lost or extensively redistributed during in vitro or in vivo capacitation. We have made use of lectins and antibodies as probes of the sperm surface during capacitation and the acrosome reaction. Concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) have been used in conjunction with fluorescent tags (FITC) and ultrastructural markers (ferritin, hemocyanin) to study the surface of golden hamster, guinea pig, mouse and human spermatozoa. Con A and WGA label the plasma membrane overlying the acrosomal region quite uniformly on these species. After capacitation there is a specific loss (or masking) of lectin binding sites over the acrosomal region of the sperm head in all species examined. Antibodies prepared against sperm and specific antibodies to a cell surface protein (fibronectin) were also tagged with fluorescent or ultrastructural markers and used to label the surfaces of sperm before and after capacitation. These probes also indicate a specific loss of surface associated material over the acrosomal surface after capacitation. These results are consistent with the notion that there is a general removal of surface components during capacitation and that this denuding of the surface is a prerequisite for the following membrane fusion events involved in the acrosome reaction and sperm-egg fusion.     

Changes in lectin-binding features of ram sperm surfaces associated with epididymal maturation and ejaculation

Ram sperm, isolated from the caput, corpus, and cauda epididymidis, plus ejaculated cells were washed free of loosely bound components and tested for their ability to bind fluorescein-conjugated lectins (Con A, SBA, RCA, PNA, ECA and WGA) as assessed by epiluminescent-fluorescence light microscopy and flow cytometry. Detailed preliminary studies established an appropriate lectin-to-sperm ratio and incubation conditions for quantitative comparisons of sperm cell types and permitted a detailed analysis of both the amount of lectin bound as well as its distribution on the various aspects of the cell surface. Con A (mannose positive) bound weakly over the entire surface, with little change associated with maturation in the male tract. SBA (N-acetylgalactosamine positive) bound moderately strongly to caput sperm, with an emphasis on the apical ridge portion of the cell; during epididymal transit this binding was greatly diminished and was regained upon ejaculation. RCA, PNA, and ECA (galactose positive) gave generally equivalent results, where initially strong binding to the entire sperm surface decreased (over all parts of the surface except the anterior head) during epididymal maturation, with no change associated with ejaculation. WGA (sialic acid positive) binding initially was weak, but increased with epididymal transit and ejaculation. In vitro incubations with beta-galactosidase and neuraminidase confirmed the assignments given above. These data, when coupled with previous reports describing the heterogeneous distribution of proteins and lipids and changes in their distribution associated with epididymal maturation, serve to quantitatively describe changes in those aspects of the cell surface that are probably responsible for the acquisition of the capacity of the sperm to bind successfully to the oocyte.     

Epididymal maturation and ejaculation are key events for further in vitro capacitation of boar spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation, and the ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to assess the effects of epididymal maturation, ejaculation and in vitro capacitation on sperm viability, acrosome integrity, mitochondrial activity, membrane fluidity, and calcium influx, both as indicators of capacitation status and sperm motility. Results indicated that boar spermatozoa acquired the ability to move in the epididymal corpus; however, their motility was not linear until the ejaculation. Epididymal spermatozoa showed low membrane fluidity and intracellular calcium content; ejaculation led to an increased calcium content, while membrane fluidity showed no changes. Acrosome integrity remained constant throughout the epididymal duct and after ejaculation and in vitro capacitation. The frequency of viable spermatozoa with intact mitochondrial sheath was higher in caput and ejaculated samples than in corpus and cauda samples, whereas the frequency of spermatozoa with high membrane potential was significantly lower in cauda samples. In vitro capacitation resulted in a decreased frequency of viable spermatozoa with intact mitochondrial sheath and an increased frequency of spermatozoa with high membrane potential in ejaculated samples. These results indicated that both epididymal maturation and ejaculation are key events for further capacitation, because only ejaculated spermatozoa are capable of undergoing the set of changes leading to capacitation.     

SERPINE2, a serine protease inhibitor extensively expressed in adult male mouse reproductive tissues, may serve as a murine sperm decapacitation factor

SERPINE2, one of the potent serine protease inhibitors that modulates the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the present study, we purified SERPINE2 from mouse seminal vesicle secretion (SVS), using liquid chromatography and identified it by liquid chromatography/tandem mass spectrometry, and it showed potent inhibitory activity against the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was immunolocalized to the SVS and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testes, SERPINE2 was immunostained in spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa. SERPINE2 was also detected on the acrosomal cap of testicular and epididymal sperm and was suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind to epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was detected predominantly on uncapacitated sperm, indicating that SERPINE2 is lost before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.     

Electron microscopic study of the in vivo zona pellucida binding ability of epididymal ram spermatozoa

In the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.     

Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation.

Rat epididymal glycoprotein DE (37 kDa) associates with the sperm surface during maturation and is localized over the dorsal region of the acrosome. In the present study we examine, by indirect immunofluorescence, the localization of DE after in vitro and in vivo capacitation. While 49% of sperm capacitated in vitro for 5 hr still presented fluorescence over the dorsal region, 51% showed labeling distributed over a domain that corresponds to the equatorial segment of the sperm head. This change in the localization of fluorescence was not associated with sperm deterioration or death and increased gradually as a function of capacitation time, reaching the maximum at 5 hr. The presence of labeling over the equatorial segment results from protein migration and cannot be induced by permeabilization, proteinase, or high ionic strength treatments. The omission of Ca2+ from the standard capacitation medium inhibited the relocalization of DE, and incubation with Ca2+ ionophore A23187 for induction of the acrosome reaction (AR) significantly raised the percentage of cells with DE localized over the equatorial region. Finally, while free and cumulus-associated spermatozoa recovered from the oviducts of in vivo inseminated females presented 15% and 21% of cells with redistribution respectively, all perivitelline (acrosome reacted) spermatozoa showed DE over the equatorial segment. These results indicate that epididymal protein DE migrates to the equatorial segment under in vitro and in vivo capacitating conditions and suggest a possible association between the redistribution of DE and the occurrence of the AR.     

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

Changes in lipid diffusibility in the hamster sperm head plasma membrane during capacitation in vivo and in vitro

The technique of fluorescence recovery after photobleaching (FRAP) was employed on spermatozoa labeled with the fluorescent lipid analogue C₁₄diI to provide two measures of lateral diffusion in the plane of the sperm plasma membrane during capacitation in vivo and in vitro: the diffusion coefficient (D) for C₁₄diI and the fraction of C₁₄diI that is free to diffuse (%R) within the domain. To evaluate changes in lipid diffusibility during capacitation in vivo, spermatozoa were recovered from the uterus within 30 min after ejaculation or from the oviduct at 2, 4, 6 and 8 hr after mating. To compare the changes which occur in vivo with those which occur during capacitation in vitro, caudal epididymal spermatozoa were incubated under capacitating or non-capacitating (control) conditions for 4 hr. Although transient changes in D occurred during the course of capacitation, there was no net change in D for either anterior (AH) or posterior head (PH) domains following capacitation in vitro or in vivo. Significant differences in the lipid diffusion coefficient between the two head domains were observed during the course of capacitation. A transient decrease in %R was observed for the AH domain during capacitation in vitro and incubation under control conditions, but no significant change in %R was observed in the AH domain during capacitation in vivo. A significant decline in %R of the PH domain was observed for spermatozoa during capacitation in vivo, in vitro and following incubation under non-capacitating conditions. These data indicate that the changes in the lipid diffusibility of the AH and PH domains which occur during capacitation in vivo exhibit both similarities and differences to those which occur during capacitation in vitro. Mol. Reprod. Dev. 50:86–92, 1998. © 1998 Wiley-Liss, Inc.     

小鼠精子表面Con A结合糖复合物的形成与变化

用辣根过氧化物酶标记的ＣｏｎＡ（伴刀豆素Ａ）对小鼠睾丸与附睾切片,以及对取自附睾和子宫（交配后）内的精子涂片进行了标记,旨在认识精子在发生、成熟和获能过程中表面糖复合物的形成与变化。本研究表明,睾丸内的生精细胞和支持细胞均呈ＣｏｎＡ标记阳性。附睾的输出小管和附睾管上皮细胞,ＣｏｎＡ标记呈中度至强阳性,有部位的差别。附睾头和附睾尾内精子表面的标记无明显差别,标记位置均主要在顶体区和尾部。精子在子宫内存留１．５小时后,顶体后区出现中度阳性标记,但存留３小时和６小时后,顶体和顶体后区的标记均减弱或消失。这些结果提示,（１）精子发生期即可合成ＣｏｎＡ结合糖复合物,（２）精子在附睾成熟过程中表面的ＣｏｎＡ结合糖复合物无明显变化,（３）精子获能后顶体后区出现的ＣｏｎＡ结合糖复合物可能与受精能力有关。     

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.     

Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody

A monoclonal antibody generated against hamster epididymal spermatozoa and recognizing an antigen within the acrosome was used in conjunction with FITC-antimouse immunoglobulin as a marker of the human acrosome during sperm development, capacitation, and the acrosome reaction. The specificity of binding of the monoclonal antibody was assessed using immunolocalization by epi-fluorescence and electron microscopy. Immunofluorescence revealed that antibody bound over the entire anterior acrosome in hamster and human spermatozoa. Ultrastructural localization indicated that antigen was predominantly present on the inner face of the outer acrosomal membrane and within the acrosomal content. Qualitative specificity was studied using a highly purified preparation of hamster acrosomes in an enzyme-linked immunosorbent assay. Since the antibody rapidly visualized human acrosomes, it was used to detect abnormal acrosome morphology of mature spermatozoa and to mark spermatids present in the ejaculate. During incubation in capacitating medium, changes in the immunofluorescence of live or methanol fixed spermatozoa were correlated with incubation interval and the ability of spermatozoa to fuse with zona-free hamster oocytes. Spermatozoa bound to zona-free hamster oocytes displayed no fluorescence, confirming that acrosome loss occurred before spermatozoa attached to the vitellus.     

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.