Classification,inhibition, and specificity studies of the vitelline coat lysin from toad sperm

The sonicated supernatant of the sperm of the toad, Bufo japonicus, can digest easily the vitelline coat (VC) of uterine eggs, and to a lesser extent the VC of coelomic eggs, but not that of activated eggs. The VC lysis and fertilization were competitively inhibited in the presence of t-butyloxycarbonyl-L-Gln-L-Arg-L-Arg-4-methylcoumaryl-7-amide (Boc-Gln-Arg-Arg-MCA), suggesting the involvement of proteases in the fertilization process. Starting from a sonicated supernatant, a potent VC lysin, possessing hydrolytic activity on Boc-Gln-Arg-Arg-MCA, was obtained by anion-exchange chromatography and gel filtration. The activity of the partially purified lysin was inhibited by diisopropyl fluorophosphate (DFP) and by such trypsin inhibitors as soybean trypsin inhibitor, leupeptin, and (p-amidinophenyl) methanesulfonyl fluoride hydrochloride, but not by chymostatin, E-64, and ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. The molecular weight of the lysin was estimated to be 32K, based on the fluorographic image of ³H-DFP binding to the lysin on sodium dodecyl sulfate gel electrophoresis. The VC lysin was most active at pH 7.0–7.6 and under low ionic strength equivalent to fresh water. The release of the VC lysin was induced upon incubation of sperm with the contents of oviducal pars recta granules (PRG), which are known to induce the acrosome reaction. We conclude that the protease studied here represents the VC lysin of toad sperm that is involved in fertilization by digesting the VC of uterine eggs, probably released as a result of the acrosome reaction induced by PRG.     

Effect of cholesterol and other sterols on human sperm acrosomal responsiveness

Human sperm become responsive to inducers of the acrosome reaction when they are washed free of seminal plasma and incubated in an appropriate medium. Previous work has shown that cholesterol-enriched medium prevents sperm from becoming responsive to the inducer, progesterone. Sperm that were incubated 24 hr in cholesterol-enriched medium and then treated with progesterone showed no evidence of membrane fusion, indicating that cholesterol acts at a stage before the earliest morphological change. Inhibition of acrosomal responsiveness by cholesterol was reversible. Among other sterols reported in mammalian sperm, desmosterol and cholesterol sulfate also inhibited sperm from becoming responsive, but cholesterol palmitate did not. Our results support a model in which sperm unesterified cholesterol, or a molecule in equilibrium with it, suppresses acrosomal responsiveness. Cholesterol-enriched medium also prevented sperm from becoming responsive to the calcium/proton exchanging ionophore, ionomycin, suggesting that cholesterol's effect may be, at least in part, at a point in the signal transduction pathway subsequent to the rise in intracellular-free calcium. © 1996 Wiley-Liss, Inc.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Production of motile acrosome-reacted mouse sperm with nanomolar concentration of calcium ionophore A23187

A method to generate a population of motile, acrosome-reacted mouse sperm is described. Sperm retrieved from the cauda epididymis and vas deferens were first capacited in a 3% bovine serum albumin (BSA) containing medium. Sperm were then resuspended in medium with low BSA content (0.01%) and treated with 30 nM of the calcium ionophore, A23187, which was added as a singel dose of 30 nM for 15 min at 37°C; or three sequential 10 nM doses over three 5 min intervals. Approximately 55–60% of the treated sperm population became acrosome reacted. The motility of the treated sperm sample was 40–65%, slightly lower than that of the control sperm, following addition of medium containing 3% BSA, This is in contrast to the <10% motility observed for capacitated mouse sperm treated with 10 μM A23187, a concentration that had been used by other investigators to induce the acrosome reaction. The ultrastructure of the 30 nM A23187-induced acrosome-reacted sperm ws similar to that of the acrosome-reacted sperm induced by solubilized zonae pellucidae. These motile, acrosome-reacted sperm were able to penetrate zone-free mouse eggs at a higher rate than the control sperm. Thus this method of treatment will be useful for further physiological experimentation with acrosome-reacted sperm. © 1994 Wiley-Liss, Inc.     

Changes in lectin binding to bovine sperm during heparin-induced capacitation

Changes in the plasma membrane of bovine sperm during heparin-induced capacitation were detected by the binding of fluorescent labeled lectins to unfixed sperm. Of the seven lectins evaluated, only binding of wheat germ agglutinin (WGA) changed with capacitation. Sperm were classified into one of 5 patterns (p1–p5) based on staining with WGA, presence or absence of propidium iodide (PI) staining (dead or alive), and acrosomal integrity (acrosome intact or reacted). The major changes associated with capacitation occurred in p1 and p2. Sperm in p1 exhibited diffuse WGA binding over the anterior sperm head, were alive, and had intact acrosomes. In p2, sperm were also acrosome intact and alive, but lacked WGA binding. When sperm were incubated under capacitating conditions with heparin, there was a decrease over time in the percentage of sperm classified in p1 (p < 0.05) and an increase in the percentage of sperm in p2 (p < 0.05). When capacitation by heparin was delayed by the inclusion of glucose in the culture medium, the same heparin-dependent changes in the percentage of sperm in p1 and p2 were delayed (p < 0.05). When capacitation by heparin was inhibited by including protamine in the culture medium, the percentage of sperm in p1 or p2 was not different from sperm incubated without heparin. Heparin-induced capacitation was associated with a loss of WGA binding to the bovine sperm head. © 1996 Wiley-Liss, Inc.     

Acrosome reaction in sperm of the toad,bufo bufo japonicus

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane-limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE-induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome-reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.     

Crypticity and functional distribution of the membrane associated alpha-L-fucosidase of human sperm

Two distinctive isoforms of the enzyme alpha-L-fucosidase are found within human semen in substantial amounts, suggesting specialized functions during reproduction. The membrane-associated isozyme of human sperm cells was previously characterized biochemically, and here we report on its subcellular localization. Intact, detergent permeabilized, capacitated, and acrosome-reacted sperm were investigated using antifucosidase immunofluorescence, binding of the fluorescent fucosylated glycoconjugate RITC-BSA-fucose (RBF), and enzyme activity in the presence and absence of selected inhibitors. Both immunolocalization and RBF binding show that fucosidase is broadly distributed over the membrane systems of human sperm, but is relatively enriched within the equatorial segment. Upon detergent treatment or induction of acrosome reaction (AR), a portion of enzyme activity is recoverable in the supernatant, presumably associated with released remnants of the outer acrosomal membrane. Surprisingly, cell-bound enzyme activity increases sharply following permeabilization of intact sperm, representing cryptic fucosidase that is relatively stable and corresponds with strong fluorescence in the equatorial segment and other sperm membranes. These observations support the notion that the fucosidase has a role in the intimate species signature interactions between sperm and oocyte.     

Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions

The kinetics of spontaneous and induced acrosomal loss have been studied in human sperm incubated in capacitating and noncapacitating media. Acrosomal status was quantitated using indirect immunofluorescence with a monoclonal antibody. The response of sperm to induction by calcium ionophores was time dependent reaching a maximum after 6 hours of incubation under capacitating conditions. The inducible population slowly decreased in size through the balance of a 24-hour incubation. The time-dependent development of ionophore responsiveness by sperm exposed to capacitating conditions corroborates the idea that only capacitated cells can respond to undergo acrosomal loss in response to ionophore. In contrast, only a small, constant percentage of sperm incubated under noncapacitating conditions responded to ionophore. Substitution experiments involving the addition or deletion of human serum albumin suggest that albumin is not absolutely required for capacitation but is essential for the maintenance of motility. Polyvinyl alcohol can be substituted for serum albumin, but it does not support capacitation or motility as well as HSA. These studies may provide a basis for optimizing capacitating conditions for human sperm in vitro as well as for diagnosing fertility or fertility potential based on measurements of spontaneous and ionophore induced acrosomal loss under defined culture conditions.     

Determining acrosomal status of the cynomolgus monkey (Macaca fascicularis) sperm by fluorescence microscopy

The acrosome of Macaca fascicularis sperm cannot be distinguished by conventional light microscopy, so determining whether sperm are acrosome-intact or-reacted is difficult. We describe methods for labeling the acrosomal region of sperm with two different probes: fluoresceinated Pisum sativum agglutinin and anti-sperm antiserum. Acrosome-intact sperm are much more heavily labeled in the acrosomal region than are acrosome-reacted sperm, providing a simple means of differentiating the two types of sperm. The two probes detect similar numbers of acrosome-reacted sperm following treatment with the divalent cation ionophore, A23187.     

Use of atomic force microscopy for morphological and morphometric analyses of acrosome intact and acrosome-reacted human sperm

The objective of this study was to use atomic force microscopy (AFM), with submicron resolution, for morphophologic and morphometric analyses of acrosome intact and acrosome-reacted human sperm heads. A mixed population of acrosome intact and reacted sperm was produced by treating capacitated sperm with A23187, which induced the acrosome reaction in approximately 50% of total sperm population. This A23187-treated sperm suspension was then plated onto a coverslip and acrosome reacted sperm were preidentified by their specific staining with rhodamine-conjugated Concanavalin A. The sperm coverslip was then air-dried and scanned by a Nanoscope IIIa atomic force microscope, using the contact mode. Top and side view images processed through the illuminate mode revealed three dimensional sperm head contour, with the highest point situated in the head posterior in both acrosome intact and acrosome reacted sperm. Maximum height, length, and width measured in 50 acrosome intact and 50 acrosome-reacted sperm were the same in both populations. However, head length at half maximum height was significantly decreased in acrosome reacted sperm (2.99 +/- 0.24 microm vs. 3.56 +/- 0.32 microm of acrosome intact sperm), due to the sudden change of the height contour from the maximum peak to the anterior tip of acrosome-reacted sperm. Our results described here can therefore be used to differentiate acrosome intact and reacted sperm from each other. This would allow future studies on subcellular changes, related to the acrosome reaction, at the submicron resolution level under more physiological conditions, since AFM does not require fixing or staining of the samples.     

Transitional states of acrosomal exocytosis and proteolytic processing of the acrosomal matrix in guinea pig sperm

In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.     

Induction of cross-fertilization between sea urchin eggs and starfish sperm by polyethylene glycol treatment

Cross-fertilization between sea urchin eggs (Strongylocentrotus nudus) and starfish sperm (Asterina pectinifera) was induced by treatment with polyethylene glycol (PEG). Without treatment with PEG, the denuded egg surface (jelly coat- and vitelline coat-free) engulfed the head of acrosome-reacted sperm; however, sperm penetration did not occur [Kyozuka and Osanai, 1988]. When these eggs were exposed briefly to PEG (molecular weight 3,000) in seawater, the sperm entered the egg by membrane fusion. Cortical granules were discharged, and embryogenesis began following sperm penetration. PEG did not induce parthenogenesis in Strongylocentrotus eggs. Egg activation is thus closely linked with gamete membrane fusion.     

Control of high affinity lectin binding to an integral membrane glycoprotein in lipid bilayers

High affinity binding of wheat germ agglutinin to glycophorin is demonstrated to be potently affected by non-specific interaction of the receptor with other protein and oligosaccharide structures present at the membrane surface. It is suggested that this may represent a significant general mechanism of receptor control.     

Characterization and biological properties of L-HGP, a glycoprotein from the amphibian oviduct with acrosome-stabilizing effects

BACKGROUND INFORMATION: The role of the jelly coat that surrounds the amphibian oocytes has been widely discussed, but is poorly understood. The presence of the jelly coat is essential for fertilization. However, the structure and function of the molecules that comprise the jelly coat have not been thoroughly documented. L-HGP (low-molecular-mass highly glycosylated protein) is a highly glycosylated protein that is present in the jelly coat of the toad, Bufo arenarum, oocytes and diffuses to the surrounding media. L-HGP, when purified from egg water, protects the sperm acrosome from breakdown induced by hypotonic solutions. RESULTS: L-HGP is an acidic glycoprotein, formed by two different subunits, linked by disulphide bonds. We raised polyclonal antibodies in rabbits against the deglycosylated protein. We determined that L-HGP is secreted along the oviduct, being hence present in all the jelly layers. The molecular mass of L-HGP is higher in the most cephalic region of the oviduct. The lower-M(r) L-HGP isoform, produced in the caudal regions of the oviduct, presents an acrosome-protecting property. L-HGP is produced by secretory cells in the oviduct and is deposited on the cilia at the oviduct lumen. CONCLUSIONS: Biochemical characterization of L-HGP has been carried out. It is synthesized by secretory cells in the oviduct and, when secreted, is deposited over the ciliated surface of the cells. The lower-M(r) isoform, secreted by the caudal region of the oviduct, protects acrosome integrity. This isoform diffuses into the medium. The role of the higher-M(r) L-HGP isoform in fertilization remains unknown.     

Assessment of sperm functional competence and sperm-egg interaction

A precise understanding in the functional competence of mammalian sperm is essential to generate clinical advances for the treatment of infertility and novel contraceptive strategies. The fundamental knowledge on the controlling parameters for spermatozoal activation process will help in the identifying the causes in fertilization failure due to male factor as well as in developing male contraceptive methodologies. The defects in the sperm-egg interaction seem to be one of the controlling mechanisms, however, none of the presently available methods for the evaluation of the fertilizing ability of sperm precisely indicates the reason for the failure or the success of sperm entry into egg. Adequate number of motile spermatozoa with normal morphology and timely occurrence of acrosome reaction are presumably the major prerequisites for the penetration through the egg investments. The present communication briefly reviews some of the main features of mammalian sperm which control the success or the failure of fertilization and existing clinical methods indicating the lack of fundamental knowledge on the sub-cellular and molecular aspects of this unique and species-specific cell-cell interaction.     

The composition, protein genesis and significance of the inner acrosomal membrane of eutherian sperm

As a consequence of the acrosomal reaction during fertilization, the inner acrosomal membrane (IAM) becomes exposed and forms the leading edge of the sperm for adhesive binding to and subsequent penetration of the zona-pellucida (ZP) of the metaphase-II-arrested oocyte. A premise of this review is that the IAM of spermatozoa anchors receptors and enzymes (on its extracellular side) that are required for sperm attachment to and penetration of the ZP. We propose a sperm cell fractionation strategy that allows for direct access to proteins bound to the extracellular side of the IAM. We review the types of integral and peripheral IAM proteins that have been found by this approach and that have been implicated in ZP recognition and lysis. We also propose a scheme for the origin and assembly of these proteins within the developing acrosome during spermiogenesis. During development, the extravesicular side of the membrane of the acrosomic vesicle is coated by peripheral proteins that transport and bind this secretory vesicle to the spermatid nucleus. The part of the membrane that binds to the nucleus becomes the IAM, while its extravesicular protein coat, which is retained between the IAM and the nuclear envelope of spermatozoa becomes the subacrosomal layer of the perinuclear theca (SAL-PT). Another premise of this review is that the IAM of spermatozoa is bound with proteins (on its intracellular side), namely the SAL-PT proteins, which hold the clue to the mechanism of acrosomal-nuclear docking. We propose a sperm cell fractionation strategy that allows for direct access to SAL-PT proteins. We then review the types of SAL-PT proteins that have been found by this approach and that have been implicated in transporting and binding the acrosome to the sperm nucleus.