Characterization of the acrosome reaction-inducing substance in Xenopus (ARISX) secreted from the oviductal pars recta onto the vitelline envelope

We previously demonstrated that Xenopus sperm undergo an acrosome reaction on the vitelline envelope (VE) in response to the materials secreted from the oviductal pars recta [Dev. Biol. 243 (2002), 55]. A monoclonal antibody against the acrosome reaction-inducing substance in Xenopus (ARISX) was obtained by immunizing mice with pars recta extract (PRE). The acrosome reaction by PRE or on the VE was effectively inhibited by the intact anti-ARISX antibody as well as its Fab fragment, indicating that the antibody recognizes the epitopes localized on the acrosome reaction-inducing substance. On Western blots, the anti-ARISX antibody recognized a molecule with an apparent molecular mass of 300 kDa in PRE and in the VE, but this molecule was not detected in the coelomic envelope. The amount of ARISX in PRE was increased by the treatment of females with pregnant mare serum gonadotropin. Periodate oxidation of PRE completely abolished the acrosome reaction-inducing activity, indicating the involvement of the carbohydrate moieties of ARISX in inducing the acrosome reaction. On immunofluorescence observation, ARISX was localized in the epithelial cells in the posterior region of the pars recta and on the VE as fibrous structures.     

Biochemical characterization of inner sugar chains of acrosome reaction-inducing substance in jelly coat of starfish eggs

The inception of the acrosome reaction (AR) in the starfish Asterias amurensis is perceived to be strongly associated with sulfated polysaccharide chains derived from an extremely large proteoglycan-like molecule called AR-inducing substance (ARIS), in which one of the sugar fragments, named fragment 1 (Fr. 1), was composed of the repeating units of [-->4]-beta-D-Xylp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Fucp-4 (SO3-)-(1-->3)- alpha-L-Fucp-4(SO3-)-(1-->4)-alpha-L-Fucp-(1-->)n. In the current study, this sugar chain is inferred to link to the peptide part by O-glycosidic linkage through a sugar chain with different structural features from Fr. 1. This inner sugar portion of ARIS was isolated as Fr. 2 from the sonicated products of pronase digest of ARIS. Fr. 2, which retains AR-inducing activity to an admirable extent and has an apparent molecular size of 400 kDa, is composed of Gal, Xyl, Fuc, GalNAc, and GlcNAc in a molar ratio of 5:1:5:4:2 with O-sulfate substitutions at Gal-4, Gal-2, Gal-2,3 and Gal-2,4 (disulfated), Fuc-4, and GlcNAc-6. The study of Fr. 2 revealed that the major portion of the inner sugar chain of ARIS is composed of the heptasaccharide units of -->3)-Galp-(1-->3)-Fucp-(1-->3)-Galp-(1-->4)-GalNAcp-(1-->4)-GlcNAcp-6(SO3-)-(1-->6)-Galp-4(SO3-)-(1-->4)-GalNAcp-(1-->. This new structure of inner sugar chains of ARIS is elucidated by using electrospray ionization MS along with tandem mass analysis, sugar composition analysis, and methylation analysis of the sugar fragments obtained by acid-catalyzed resin-based partial hydrolysis of Fr. 2. Furthermore, this study corroborates that the sulfate groups are solely liable to the anionic character of ARIS, which ought to be present in the sugar chains of ARIS for its biological activity.     

Novel conserved structural domains of acrosome reaction-inducing substance are widespread in invertebrates

In the starfish Asterias amurensis, acrosome reaction inducing substance (ARIS) is the main factor responsible for allowing sperm to recognize the egg jelly and begin the acrosome reaction (AR). ARIS is a large proteoglycan-like molecule, and its pentasaccharide repeat, Fragment 1 (Fr. 1), is responsible for inducing AR. Here, we investigated the primary structure of ARIS for the first time in order to improve our understanding of its functionality. Electrophoretic analysis revealed that ARIS is a complex of three proteins, all of which are modified by the Fr. 1 sugar chain. Sequencing indicated that there are two novel, conserved domains in all three ARIS proteins: ARIS N-terminus (AR-N) and ARIS C-terminus (AR-C) domains. We also found that other echinoderms possess ARIS proteins that are capable of inducing the AR for homologous sperm, indicating that ARIS proteins may be a ubiquitous component for echinoderm fertilization. Moreover, we identified ARIS-like genes from Ctenophora to Protochordata.     

Egg and sperm recognition systems during fertilization

Fertilization is a programmed process that has many molecules and sequential events amenable to study. The biochemistry of fertilization has identified cellular and acellular components fundamental to the interactions between sperm and egg. Recent studies highlight the molecular details of the species-specificity of fertilization that involve protein–protein and protein–carbohydrate interactions. Although the diversity of structure and mechanism may imply rapid evolution of fertilization proteins, understanding the structure–function relationships has become important. Here, we introduce the molecules controlling the sperm AR, sperm attachment to, and penetration through, the egg investments.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Primary structure of a 120 kDa protein associated with the fucose sulfate glycoconjugate constituting the acrosome reaction-inducing substance of the sea urchin, Hemicentrotus pulcherrimus

A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction-inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus . The FSG is composed primarily of four constituents: a 120kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide-containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGE-PELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bp) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40% is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.     

Sperm-egg ratios and the site of the acrosome reaction during in vivo fertilization in the hamster

Little is known about the timing of the mammalian sperm acrosome reaction during fertilization in vivo. To study this problem, female hamsters were inseminated at about the time of ovulation, and the contents of the ampullary regions of their oviducts were subsequently examined at various intervals. No living spermatozoa were recovered from ampullae earlier than 4 hr after insemination. The first appearance of living spermatozoa coincided closely with the first appearance of fertilized eggs in the same oviduct. The total numbers of living spermatozoa did not start to exceed the number of eggs in the same ampulla, until after 50% or more of the eggs had been fertilized. Hamster spermatozoa are highly efficient at making contact with eggs, and the fertilizing spermatozoon probably spends no more than 2½ –5½ min in penetrating the cumulus oophorus. Spermatozoa that enter the ampulla appear to be ready to undergo the acrosome reaction, and complete it while they are passing through the cumulus or shortly before, or after, contacting the surface of the zona pellucida.     

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

Possible involvement of CD81 in acrosome reaction of sperm in mice

Tetraspanin CD81 is closely homologous in amino acid sequence with CD9. CD9 is well known to be involved in sperm-egg fusion, and CD81 has also been reported to be involved in membrane fusion events. However, the function of CD81 as well as that of CD9 in membrane fusion remains unclear. Here, we report that disruption of the mouse CD81 gene led to a reduction in the fecundity of female mice, and CD81-/- eggs had impaired ability to fuse with sperm. Furthermore, we demonstrated that when CD81-/- eggs were incubated with sperm, some of the sperm that penetrated into the perivitelline space of CD81-/- eggs had not yet undergone the acrosome reaction, indicating that the impaired fusibility of CD81-/- eggs may be in part caused by failure of the acrosome reaction of sperm. In addition, we showed that CD81 was highly expressed in granulosa cells, somatic cells that surround oocytes. Our observations suggest that there is an interaction between sperm and CD81 on somatic cells surrounding eggs before the direct interaction of sperm and eggs. Our results may provide new clues for clarifying the cellular mechanism of the acrosome reaction, which is required for sperm-egg fusion.     

Effects of surfactants on the fertilizing capacity and acrosome reaction of sea urchin spermatozoa

The effects of seven surfactants on spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, were studied. All these surfactants induced the acrosome reaction and inhibited the fertilizing capacity of spermatozoa. There was a statistically significant correlation between the concentrations that induce the acrosome reaction and inhibit fertilization. The critical micelle concentrations (CMC) of surfactants in sea water were almost even and these values, which are inherent physical properties of surfactants, did not provide a direct measure of their inhibitory effect of fertilization. Among seven surfactants, p-menthanyl-phenol polyoxyethylene (8.8) ether (TS-88) with a characteristic hydrophobes was the most potent both in the induction of acrosome reaction and in the inhibition of fertilization. Various ethylene oxide adducts to p-menthanyl-phenol were also tested for the purpose of comparison. It is suggested that the effects of surfactants on sea urchin spermatozoa at low concentrations reflect their activity associated with the hydrophobic group inherent in each surfactant.     

Sperm-egg interaction in the painted frog (Discoglossus pictus): An ultrastructural study

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration. Gamete-binding and fusion take place between D1 (the egg-specific site for sperm interaction) and the perpendicularly oriented sperm. Spermatozoa visualized at their initial interaction (15 sec postinsemination) with the oolemma are undergoing vesiculation. The first interaction is likely to occur between the D1 glycocalyx and the plasma membrane of the hybrid vesicles surrounding the apical rod. As fusion is observed between the internal acrosome membrane and the oolemma, it can be postulated that gametic interaction might be followed by fusion of the latter with the apical rod internal membrane that extends posteriorly into the inner acrosome membrane. Insemination of the outermost jelly layer (J3) dissected out of the egg, and observations of the ultrastructural changes of spermatozoa in this coat, indicate that J3 rather than the vitelline coat (VC) induces the AR. Interestingly, at the late postinsemination stage, VC fibrils are seen crosslinking the inner acrosome membrane. The role of this binding is here discussed. Mol. Reprod. Dev. 47:323–333, 1997. © 1997 Wiley-Liss, Inc.     

Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction

The effects of lipids on the survival, acrosome reaction, and fertilizing capacity of guinea pig spermatozoa were studied by incubating the spermatozoa in media containing various concentrations of the lipids. Lipids tested were: phosphatidyl-choline (PC), -ethanolamine (PE), -inositol (PI), -serine (PS), sphingomyelin (S), cholesterol (C), lysophosphatidyl-choline (LC), -ethanolamine (LE), -inositol (LI), -serine (LS), and glyceryl monooleate (M). When spermatozoa were incubated in a regular medium (containing 2 mM Ca²⁺) with M, the majority underwent the acrosome reaction within 1 hour. None of the other lipids were as effective as M, and some were totally ineffective under the same conditions. However, when spermatozoa were preincubated in Ca²⁺-free medium containing LC, LE, or LI, they gained the ability to undergo the acrosome reaction. One hour of preincubation in Ca²⁺-free medium with LC, LE, or LI was enough to render the vast majority of spermatozoa capable of undergoing the acrosome reaction in response to Ca²⁺. The optimum concentrations for LC, LE, and LI were approximately 85 μg/ml, 210 μg/ml, and 140 μg/ml, respectively. Spermatozoa that had undergone the acrosome reaction by pretreatment with LC, LE, or LI remained actively motile and were capable of fertilizing eggs. LS was totally ineffective in rendering the spermatozoa capable of undergoing the acrosome reaction, and in fact it inhibited the acrosome reaction by itself and also inhibited the LC-, LE-, or LI-mediated acrosome reaction. LS did not prevent acrosome-reacted spermatozoa from penetrating the zona pellucida, but did prevent sperm-egg fusion. Based on these findings, it is suggested that lysophospholipids are intricately involved in the sperm acrosome reaction and perhaps in sperm-egg fusion.     

Possible causal relation between the acrosome reaction and cross-fertilization in the sea urchins hemicentrotus pulcherrimus and pseudocentrotus depressus

Sperm of Hemicentrotus pulcherrimus undergo the acrosome reaction in the jelly coat or on the surface of the vitelline layer of Pseudocentrotus depressus eggs and can fertilize the latter, whereas those of P depressus do not undergo the acrosome reaction even after they strike the vitelline layer of H pulcherrimus eggs and cannot cross-fertilize. In the latter combination, however, if cross-insemination is performed in the presence of homologous egg water, cross-fertilization becomes less difficult than in normal seawater, and percentage cross-fertilization rises as does percentage acrosome reaction. It is suggested that the cross-fertilizability of this combination depends highly on the inductivity of the acrosome reaction. The reason why such a causal relation is observed is discussed.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

NMDA‐type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm

The N‐methyl d ‐aspartate type glutamate receptor (NMDAR) is a ligand‐gated cation channel that causes Ca²⁺ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA‐seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg‐jelly substances along with acrosome reaction‐inducing substance (ARIS) and sperm motility‐initiating substance (SMIS). In the egg‐jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg²⁺, which blocks Ca²⁺ influx through gated NMDARs, was removed from the ST. In addition, the ARIS‐induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg²⁺ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

Rabbit spermatozoa undergo an acrosome reaction in the presence of glycosaminoglycans

Rabbit-ejaculated spermatozoa were incubated in a chemically defined medium containing comercially available glycosaminoglycans (GAGs) at concentrations ranging from 0.1 to 100 μg/ml. Sperm were stained and examined for the degree of acrosome reaction and viability after 9 h of incubation. There were significant dose and treatment effects of the induction of the acrosome reaction. Viability did not differ significantly betweendoses or treatment. Heparin enhanced the acrosome reaction between concentrations of 0.1 to 1.0 μg/ml, whereas higher levels depressed the percentage of sperm undergoing the acrosome reaction. Seminal plasma added to sperm cultures depressed the stimulatory effect of GAGs. Treatment of chondroitin-4-sulfate with chondro-4-sulfatase prohibited the stimulatory effect. It is concluded that GAGs, components of the female reproductive tract, may promote the acrosome reaction so that successful fertilization can occur.     

Sperm entry into zona-free oocytes in the hamster oviduct: Implications for the mechanisms of acrosome reaction induction

The possible importance of the zona pellucida for induction of the acrosome reaction (AR) and establishment of sperm/egg associations in the fallopian tube was investigated by instilling zona-free eggs into the oviductal ampulla of hamsters that had been inseminated with epididymal spermatozoa 6–7 hours previously. The eggs were recovered only 60–90 minutes later because of increasing difficulty with time of collecting zona-free eggs from the oviduct. In the zona-free group, 41 (4%) of 1,101 transferred eggs were recovered, of which 20% contained spermatozoa with decondensing nuclei (mean 4.4/egg). A similar (22%) fertilization rate (mean 3.2 spermatozoa/egg) was found among intact (control) eggs recovered after instillation into the contralateral oviduct. Mammalian spermatozoa are not incorporated even into zona-free eggs before AR occurs. These results thus demonstrate that an AR in functional hamster spermatozoa in vivo and establishment of sperm/egg associations in vivo require no interaction with the zona pellucida nor with other products of ovulation.