Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.     

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer’s disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.     

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB⁺¹, to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Regulatory roles of ubiquitin-proteasome pathway in pig oocyte meiotic maturation and fertilization

The ubiquitin-proteasome pathway is involved in the degradation of proteins related to cell cycle progression including cyclins. The present study, using two specific proteasome inhibitors, for the first time investigated the roles of ubiquitin-proteasome in cell cycle progression during pig oocyte meiotic maturation and after fertilization. In contrast to its effect in rodent oocytes, proteasome inhibition strongly prevented germinal vesicle breakdown (GVBD). After GVBD, proteasome inhibition disrupted meiotic apparatus organization, cell cycle progression, and first polar body (PB1) extrusion. Sperm penetration into the oocytes was completely inhibited when proteasome inhibitors were added at the beginning of insemination. However, sperm chromatin decondensation and metaphase-interphase transition were not affected when inhibitors were added once sperm penetrated. The results suggest that ubiquin-proteasome complex is one of the critical regulators of meiotic cell cycle, but proteasome inhibitors do not affect major fertilization events when added after sperm penetration into the oocytes in the pig.     

Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling

ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132

Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.     

Phosphoinositide-dependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction

Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCzeta. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.     

Distribution of α-D-mannose residues on zona pellucida and their role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cumulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost completely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4,spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice

In this study the role of two intra-acrosomal molecules, acrin 1 (MN7) and acrin 2 (MC41), during in vitro fertilization (IVF) was examined. The pertinent monoclonal antibodies mMN7 and mMC41 specifically recognize a 90 kDa protein (acrin 1) localized to the entire acrosome and a 200 kDa protein (acrin 2) localized to the cortex region of the anterior acrosome, respectively. Experiments were designed to assess the effects of mMN7 and mMC41 on fertilization in mice using TYH medium containing mMN7 or mMC41 at 0.0, 0.025, 0.05 and 0.1 mg ml-1. Under these conditions, capacitated spermatozoa inseminated the cumulus-invested oocytes. Acrosome-reacted spermatozoa inseminated the zona pellucida-free oocytes. The antibodies had no effect on sperm motility and primary binding to the zona pellucida, but significantly inhibited the rate of fertilization of zona pellucida-intact oocytes in a dose-dependent manner. A significantly small number of spermatozoa remained attached to the zona pellucida at 5 h after insemination in the presence of mMC41. mMC41 and mMN7 antibodies did not affect the fertilization rate of zona pellucida-free oocytes. Confocal laser scanning microscopy with indirect immunofluorescence traced the effect of the monoclonal antibodies on the zona pellucida-induced acrosome reaction, and revealed that mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the zona pellucida after the acrosome reaction but before penetration of the zona pellucida. Thus, the intra-acrosomal antigenic molecules acrin 1 and acrin 2 are essential for distinct events before sperm penetration of the zona pellucida in mice.     

Zona pellucida characteristics and sperm-binding patterns of in vivo and in vitro produced porcine oocytes inseminated with differently prepared spermatozoa

IVF of porcine oocytes has been carried out in many laboratories. However, polyspermic fertilization is still a major issue to be solved. It is well known that besides the nucleus, oocyte organelles and the cytoplasm have to undergo a final maturation process before they become fully competent for fertilization. Until now, it is still uncertain whether the zona pellucida (ZP) must also undergo a maturation process and what impact the maturation status may have on sperm recognition and monospermic fertilization. Our data show that the ZP undergoes biochemical changes in the final maturation phase of the oocyte prior to fertilization. During zona maturation, the induction of the acrosome reaction in spermatozoa bound to the zona pellucida shows a different time pattern. Additionally, it was shown by 2D gel electrophoresis that after maturation, ZPA moved 0.8 pI units and ZPB/ZPC 1.3 pI units in the direction of the anode, indicating increased acidity. These preliminary studies indicate that the maturation processes of the oocyte involves biochemical and functional alterations in the zona pellucida. In addition, the morphology of the porcine ZP was investigated before and after maturation at the GVI and metaphase II stage as well as 1h after onset of IVF. No significant consistent structural changes were seen between immature oocytes and those matured in vitro for 48 h. However, at 24 h, the zona structures were more similar to those in in vivo matured oocytes. This phenomenon needs to be elucidated. So far, the only way to avoid polyspermic penetration is to reduce the number of spermatozoa per oocyte used for IVF. The amount depends on the treatment of the sperm and has to be set for each individual boar.     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.     

Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.     

Zona pellucida-induced acrosome reaction in boar sperm

Induction of the acrosome reaction in boar sperm by the zona pellucida (ZP) was investigated. A modified cytochemical staining method for measuring the acrosome reaction in boar sperm gave equivalent results to those obtained with transmission electron microscopy. Isolated heat-solubilized ZP effectively induced the acrosome reaction in boar sperm at a concentration of 25 micrograms/ml. Electrophoretically purified ZP components were also tested for acrosome reaction-inducing activity; both the 55,000 and 90,000 components of the ZP were effective. The carbohydrate moiety of the 55,000 component was necessary for activity because the polypeptides derived by chemical deglycosylation of the two glycoproteins did not induce the acrosome reaction.     

Distribution of α-D-mannose residue on zona pellucida and its role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cu- mulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost com- pletely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4, spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.     

ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization

The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization.     

Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility.