Sperm-zona Interaction Research Articles

Identification and in silico Characterization of Deleterious Single Nucleotide Variations in Human ZP2 Gene.

ZP2, an important component of the zona matrix, surrounds mammalian oocytes and facilitates fertilization. Recently, some studies have documented the association of mutations in genes encoding the zona matrix with the infertile status of human females. Single nucleotide polymorphisms are the most common type of genetic variations observed in a population and as per the dbSNP database, around 5,152 SNPs are reported to exist in the human ZP2 (hZP2) gene. Although a wide range of computational tools are publicly available, yet no computational studies have been done to date to identify and analyze structural and functional effects of deleterious SNPs on hZP2. In this study, we conducted a comprehensive in silico analysis of all the SNPs found in hZP2. Six different computational tools including SIFT and PolyPhen-2 predicted 18 common nsSNPs as deleterious of which 12 were predicted to most likely affect the structure/functional properties. These were either present in the N-term region crucial for sperm-zona interaction or in the zona domain. 31 additional SNPs in both coding and non-coding regions were also identified. Interestingly, some of these SNPs have been found to be present in infertile females in some recent studies.

Transcriptome and Proteome Analysis Revealed Key Pathways Regulating Final Stage of Oocyte Maturation of the Turkey (Meleagris gallopavo).

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte—called the inner perivitelline layer—is involved in sperm–zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1–4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte–granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation.

LYPD4, mouse homolog of a human acrosome protein, is essential for sperm fertilizing ability and male fertility†

Fertilization is one of the fundamental biological processes, but so far, we still do not have a full understanding of the underlying molecular mechanism. We have identified a human acrosome protein, LY6/PLAUR domain containing 4 (LYPD4), expressed specifically in human testes and sperm, and conserved within mammals. Mouse Lypd4, also specific to the testis and sperm, is essential for male fertility. LYPD4 protein first appeared in round spermatids during acrosome biogenesis and became part of acrosomes during spermatogenesis and in mature sperm. Lypd4 knockout mice are infertile with normal sperm number and motility. Mutant sperm, however, failed to reach oviduct during sperm migration inside the female reproductive tract, leading to fertilization failure and infertility. In addition, Lypd4 mutant sperms were unable to fertilize denuded egg via IVF (in vitro fertilization) but could fertilize eggs within intact Cumulus-Oocyte Complex, supporting an additional role in sperm-zona interaction. Out of more than five thousand spermatozoa proteins identified by mass spectrometry analysis, only a small subset of proteins (26 proteins) was changed in the absence of LYPD4, revealing a whole proteome picture of mutant sperm defective in sperm migration and sperm-zona binding. ADAM3, a key component of fertilization complex, as well as other sperm ADAM proteins are significantly reduced. We hence propose that LYPD4 plays an essential role in mammalian fertilization, and further investigation of its function and its interaction with other sperm membrane complexes may yield insights into human fertilization and novel strategy to improve IVF success.

The bioactivities of the central segment of Zp2 polypeptide.

In order to understand the role of the protein zona pellucida 2 in fertilization, an antibody against a central segment of the zona pellucida 2 peptide, segment 190-505 (Z2eH), was prepared. The influence of the antibody on sperm-zona interaction was tested using the sperm-egg binding assay. The effect of the antibody on fertility was evaluated by passive immunization with anti-Z2eH antibody. Immunohistochemical assay showed that an antibody from rabbit reacted specifically with the natural zona pellucida on mouse ovarian sections. Immunofluorescence assay showed that the antibody bound specifically to the zonae pellucidae of the ovulated oocytes and 2-cell embryos after passive immunization. The antibody-treated oocytes bound capacitated sperm as control oocytes, passive immunization did not impede the action of sperm to fertilize the oocyte in vivo. These findings suggest that the central peptide of ZP2 (190-505) is immunogenic and contains zona pellucida-specific epitopes, however the central polypeptide might not be the crucial part from which to construct a functional domain to bind sperm.

Co‐evolution of gametes of the Greater Bandicoot Rat, Bandicota indica – a murine rodent from South‐East Asia

AbstractMost Old World mice and rats, subfamily Murinae, have a spermatozoon with an apical hook, a long tail and, as seen typically in eutherian mammals, a bilaterally flattened head. Dramatically different from this are the sperm of the Greater Bandicoot Rat, Bandicota indica. Here, we ask the question has the structure of the sperm head co‐evolved with that of the egg coat, the zona pellucida? For this, we first summarise the morphological features of the spermatozoon of B. indica that may relate to zona pellucida penetration at the time of fertilisation, and we confirm that the sperm head is generally round, not bilaterally flattened, in profile and has a huge acrosome. We then show that the zona pellucida around oocytes in tertiary follicles also differs from that of the other murine rodents in being only about 4 μm thick and, as demonstrated by lectin staining, has an unusual abundance of alpha‐L‐fucose. These findings indicate that both the male and female gametes of this South‐East Asian murine rodent are highly divergent in their structural organisation. One of the functional implications of this probably relates to sperm–zona interactions and the release of acrosomal enzymes that probably facilitate penetration by digestion of the zona matrix at the time of fertilisation.

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm-oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm-zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.

002. PIG SPERM EGG INTERACTION AND FORMATION OF A ZONA PELLUCIDA BINDING COMPLEX

In order to achieve fertilization sperm cells first need to successfully interact with the zona pellucida. Before reaching the zona pellucida the sperm cell undergoes extensive remodeling both in the male and female genital tract. These changes serve to mediate optimal recognition of the zona pellucida in the oviduct (primary zona pellucida binding). Optimal sperm-zona interactions are crucial for porcine oocyte fertilization: The zona pellucida- attached sperm cell is triggered to undergo the acrosome reaction and will also become hypermotile. Together these two responses allow the sperm cell to drill through the zona pellucida (secondary zona pellucida binding) this coincides with local sperm zona drilling so that a few sperm can reach the perivitellin space. This delaying strategy allows only one sperm cell in a given time-point to bind and fuse with the oocyte (fertilization) and thus minimizes the risk of polyspermy. The polyspermy block is essentially executed by the fertilized oocyte that immediately secretes its cortical granule content into the perivitellin space. This content blocks sperm-oocyte fusion either by sticking to the oolemma or by the induction of a biochemical reaction of the zona pellucida (zona pellucida hardening). The cortical reaction thus blocks sperm-zona pellucida binding and/or sperm-zona pellucida drilling. Note that zona pellcuida interactions under pig IVF conditions relatively frequently result in polyspermy. It is not clear whether this is also the case in vivo after natural mating. More importantly we do not know how other artificial reproductive technologies affect polyspermy rates. This is especially relevant for new sperm treatments and insemination technologies in which sperm are activated by capacitation media essentially mimicking the IVF media. Therefore, better understanding of sperm activation and of the arrangement of proteins involved in zona pellucida interactions are relevant for designing strategies to further improve pig reproduction.

Functional consequences of cleavage, dissociation and exocytotic release of ZP3R, a C4BP-related protein, from the mouse sperm acrosomal matrix

The acrosome is an exocytotic vesicle located on the apical tip of the sperm head. In addition to having different morphological regions, two biochemically distinct compartments can be defined within the acrosome: a particulate acrosomal matrix and a soluble partition. The domains within the acrosome participate in the release of acrosomal proteins from the sperm during exocytosis, depending on whether the proteins partition into either the soluble or matrix compartments of the acrosome. We have examined the mechanism of differential release by evaluating the solubilization of acrosomal matrix protein ZP3R (sp56) from mouse sperm during the course of spontaneous acrosomal exocytosis. Using indirect immunofluorescence and immunoblotting, we found that the ZP3R monomer is processed from 67,000 M(r) to 43,000 M(r) by proteases coincident with release from the acrosome. Sperm require a maturational step, termed capacitation, before they are competent for acrosomal exocytosis and the processing of ZP3R is dramatically reduced under non-capacitating conditions. The cleavage probably takes place in complement control protein domain (CCP) 6 or the bridge region between CCP6 and CCP7, which is not present in the guinea pig orthologue AM67. The cleaved form of ZP3R does not bind to unfertilized eggs. We have incorporated these structural considerations into a model to explain the functional consequences of acrosomal exocytosis on sperm-zona interactions.

Developmental sperm contributions: fertilization and beyond

The objective of this review was to examine the role of the various spermatozoal components suspected of actively participating in early human development. The contributions of the fertilizing spermatozoon to the oocyte include, as a minimum, the delivery of the DNA/chromatin, a putative oocyte-activating factor (OAF), and a centriole. Recent data indicate that spermatozoa may also provide the zygote with a unique suite of paternal mRNAs; some transcripts might be crucial for early and late embryonic development and deficient delivery, or aberrant transcription might contribute to abnormal development and arrest. Clinical evidence from assisted reproduction suggests that failure to complete the fertilization process, syngamy, or early cleavage might be the result of an early paternal effect. It is speculated that an abnormal release of a putative OAF and/or dysfunctions of the centrosome and cytoskeletal apparatus may mediate these effects. On the other hand, a later paternal effect resulting in embryonic failure to achieve implantation, pregnancy loss, and/or developmental abnormalities resulting from "carried over" sublethal effects may be associated with sperm nuclear/chromatin defects, including the presence of aneuploidy, genetic anomalies, DNA damage, and possibly other causes. These findings highlight the need for continuous monitoring of clinical results.

Recombinant Mouse Sperm ZP3-binding Protein (ZP3R/sp56) Forms a High Order Oligomer That Binds Eggs and Inhibits Mouse Fertilization in Vitro

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Analysis of chaperone proteins associated with human spermatozoa during capacitation

Mammalian spermatozoa must undergo a post-ejaculatory period of maturation, known as capacitation, before they can engage in the process of fertilization. Studies in the mouse have established that capacitation facilitates sperm-zona recognition via mechanisms that involve the appearance of tyrosine phosphorylated chaperone proteins on the sperm surface overlying the acrosome, the site of sperm-zona recognition. In this study, we examined whether a similar relationship existed between the tyrosine phosphorylation events associated with capacitation and sperm-zona interaction in human spermatozoa. These studies confirmed that capacitation is associated with an increase in both sperm-zona binding and an increase in tyrosine phosphorylation over the sperm tail. However, we could not detect the surface expression of phosphotyrosine residues over the sperm head, as observed with murine spermatozoa. Moreover, although we could clearly detect a number of chaperone proteins in human spermatozoa including HSPE1, DNAJB1, HSPD1, HSPA1A, HSPCA, HSPH1, HSPA5 and TRA1, none of these molecules were expressed on the sperm surface. On the basis of these results, it is unlikely that these proteins play an active role in the remodeling of the sperm surface during capacitation. We conclude that strong species-specific differences exist in the molecular mechanisms that drive sperm-egg recognition and that alternative, chaperone-independent, mechanisms must underpin sperm-zona interaction in the human.

Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte

An important step in fertilization is the recognition and primary binding of the sperm cell to the zona pellucida (ZP). Primary ZP binding proteins are located at the apical plasma membrane of the sperm head. In order to exclusively study primary zona binding proteins, plasma membranes of sperm heads were isolated, highly purified and subsequently solubilized with a mild or a strong solubilization procedure. Native, highly purified ZP ghosts were used as the binding substrate for solubilized sperm plasma membrane proteins, and a proteomic approach was employed to identify ZP binding proteins. Two-dimensional gel electrophoresis of ZP fragments with bound sperm proteins showed very reproducibly 24 sperm protein spots to be associated to the zona ghosts after mild plasma membrane solubilization whereas only three protein spots were detected after strong plasma membrane solubilization. This indicates the involvement of multiple sperm proteins in ZP binding. The three persistently bound proteins were identified by a tandem mass spectrometry as isoforms of AQN-3 and probably represent the main sperm protein involved in ZP binding. P47, fertilin beta and peroxiredoxin 5 were also conclusively identified. None of the identified proteins has a known acrosomal origin, which further indicated that there was no sample contamination with secondary ZP binding proteins from the acrosomal matrix. In this study, we showed and identified multiple zona binding proteins involved in primary sperm-zona binding. Although we were not able to identify all of the proteins involved, this is a first step in understanding the event of primary sperm-zona interactions and the relevance of this for fertilization is discussed.

Study on the in vitro effect of zearalenone and α‐zearalenol on boar sperm–zona pellucida interaction by hemizona assay application

The mycotoxin zearalenone (zen) impairs fertility in farm animals. The aim of the present study was to investigate the effect of zearalenone and its major metabolite (alpha-zearalenol) on boar semen binding capacity, under in vitro conditions. Extended boar semen was exposed to three different concentrations of zen and alpha-zen (40, 60 and 80 microg ml(-1) of semen) for 1 h. Afterwards, the semen was washed and incubated with homologous oocyte hemizona for 4 h. A significant decrease (P < 0.001) in the number of tightly attached spermatozoa on the hemizona was obtained at concentrations of 60 microg ml(-1) and 80 microg ml(-1) of zen and alpha-zen. In conclusion, zen and alpha-zen affected the sperm-zona interaction by reducing the ability of boar spermatozoa to bind to the zona pellucida.

Antihyaluronidase Action of Ellagic Acid Effectively Prevents Polyspermy As a Result of Suppression of the Acrosome Reaction Induced by Sperm-Zona Interaction During In Vitro Fertilization of Porcine Oocytes

The present study was conducted to examine the effects of three tannin relatives (tannic acid, TA; gallic acid, GA; and ellagic acid, EA) on antihyaluronidase and reactive oxygen species (ROS) scavenging activity, in vitro fertilization (IVF) parameters, and the acrosome reaction (AR) induced by sperm-zona interaction. Among the three tannin relatives, TA and EA showed the strongest potency for blocking the hyaluronidase activity of boar sperm, with concentration-dependent inhibition over the range of 2-10 microg/ml. In contrast, ROSs were effectively scavenged by TA and GA, but not EA. When cumulus-free oocytes were inseminated in IVF medium containing 5 microg/ml of the tannin relatives, polyspermy was significantly reduced by TA and EA (32 and 29%, respectively) compared with oocytes treated with or without GA (51 and 69%, respectively) under conditions that maintained a high sperm penetration rate (P<0.05). Interestingly, induction of the AR by treatment of preincubated sperm with progesterone was blocked by TA and GA as a result of their higher levels of ROS scavenging activity, while EA, which possessed weak ROS scavenging activity, did not disturb induction of the AR with progesterone. However, the incidence of AR induced by sperm-zona interaction was significantly decreased by the strong antihyaluronidase actions of TA and EA compared with that in the absence of these compounds. Treatment with the compounds caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization nor a reduction in acrosomal proteolytic activity or the number of zona-bound sperm. These findings suggest that the antihyaluronidase action of EA effectively prevents polyspermy by suppression of AR functionality induced by sperm-zona interaction and that hyaluronidase intervention is therefore required during porcine IVF.

Characterization of galactose-binding proteins in equine testis and spermatozoa

Carbohydrate-binding proteins are thought to be involved in a myriad of sperm functions including sperm–oviductal and sperm–zona interactions. Recent studies in our laboratory have characterized galactose-binding proteins on equine spermatozoa as possible candidate molecules for sperm adhesion to oviduct epithelial cells. In the current study, equine sperm membrane proteins were subjected to galactose-affinity chromatography, and bound proteins were eluted with excess galactose in a calcium-free buffer. The eluted fraction recovered after galactose-affinity chromatography was used for generation of a polyclonal antibody which was immobilized on an affinity column to recover a purified protein from equine sperm extracts. Several protein bands of approximately 70, 25, and 20–18 kDa were detected with a major band at 25 kDa on immunoblots which was subjected to N-terminal amino acid sequencing. These galactose binding proteins (GBP) were specific to sperm and testis and were absent in all the somatic tissues tested. Based upon immunocytochemistry, GBP were localized over the sperm head. In noncapacitated sperm, fluorescent labeling was observed over the rostral sperm head as well as the postacrosomal area; whereas in capacitated sperm, the labeling was localized primarily in the equatorial segment. Immunohistochemistry of equine testis demonstrated abundant staining in the adluminal region of the seminiferous tubules corresponding to round spermatids. In summary, this study demonstrates the presence of testis- and sperm-specific galactose binding proteins in the horse. The function of these proteins remains to be determined.

The extracellular protein coat of the inner acrosomal membrane is involved in zona pellucida binding and penetration during fertilization: Characterization of its most prominent polypeptide (IAM38)

A consequence of the acrosome reaction is to expose the inner acrosomal membrane (IAM), which is a requirement for the sperm's ability to secondarily bind to and then penetrate the zona pellucida (ZP) of the mammalian oocyte. However, the proteins on the IAM responsible for binding and presumably penetrating the zona have not been identified. This issue can be resolved if direct information is made available on the composition of the IAM. For this purpose, we devised a methodology in order to obtain a sperm head fraction consisting solely of the IAM bound to the detergent-resistant perinuclear theca. On the exposed IAM surface of this fraction, we defined an electron dense protein layer that we termed the IAM extracellular coat (IAMC), which was visible on sonicated and acrosome-reacted sperm of several mammalian species. High salt extraction removed the IAMC coincident with the removal of a prominent 38 kDa polypeptide, which we termed IAM38. Antibodies raised against this polypeptide confirmed its presence in the IAMC of intact, sonicated and acrosome-reacted sperm. By immunoscreening of a bovine testicular cDNA library and sequencing the resulting clones, we identified IAM38 as the equivalent of porcine Sp38 [Mori, E., Kashiwabara, S., Baba, T., Inagaki, Y., Mori, T., 1995. Amino acid sequences of porcine Sp38 and proacrosin required for binding to the zona pellucida. Dev. Biol., 168, 575–583], an intra-acrosomal protein with ZP-binding ability, whose precise localization in sperm was unknown. The blockage of IVF at the level of the zona with anti-IAM38 antibodies and the retention of IAM38 after sperm passage through the zona support its involvement in secondary sperm–zona binding. This study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM for identifying other candidates for sperm–zona interactions.

Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF

The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccessful IVF trials. In theory, this may result in gradual reduction of sperm-zona interaction and eventually in total fertilization failure (TFF). Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1-87 T --> G) and one in exon 6 [c. 894 G --> A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.

Serine protease activity, bovine sperm protease, 66 kDa (BSp66), is present in hamster sperm and is involved in sperm–zona interaction

Bovine sperm protease, 66 kDa (BSp66) is a serine protease previously characterized in bovine spermatozoa. Like other proteases, it may be present in sperm from other mammalian species different from bovine, playing a role in the fertilization process. In this study, we looked for BSp66 in hamster spermatozoa using heterologous antibodies against bovine BSp66. An immunoreactive protein was detected by Western blotting in mature and immature sperm. The detected protein had two isoforms similar to the ones reported in bovine sperm. Furthermore, indirect immune detection by fluorescence and electron microscopy assays, showed BSp66 signal at the acrosomal region similar to bovine sperm. As it was determined in bovine sperm, the acrosomal reaction displays the antigen within the acrosomal content. When live hamster sperm was incubated with polyclonal antibody against bovine BSp66 a decrease in the number of sperm bound to zona pellucida in homologous IVF and an impairment of head-head agglutination, were observed. These results suggest that a protease homologous to bovine BSp66 is present in golden hamster spermatozoa, with a conserved molecular mass and cellular location. Moreover, hamster BSp66 is probably involved in zona pellucida recognition.

Macaque sperm release ESP13.2 and PSP94 during capacitation: the absence of ESP13.2 is linked to sperm-zona recognition and binding.

ESP13.2 coats the entire surface of macaque sperm and remains until sperm become capacitated (Yudin et al., 2003: Biol Reprod 69: 1118-1128). Capacitation of macaque sperm is synchronized by treatment with dibutyrl cAMP (dbcAMP) and caffeine. ESP13.2 and PSP94 constituted approximately 95% of the proteins released from the sperm surface following treatment with caffeine + dbcAMP. Caffeine and dbcAMP alone induce different patterns of ESP13.2 release. As determined by ELISAs of supernatants and immuno-fluorescent labeling of sperm heads, caffeine alone and caffeine + dbcAMP induced comparable release of ESP13.2, while dbcAMP-treated sperm did not differ from controls. Sperm treated with caffeine + dbcAMP showed a reduction of ESP13.2 from the entire surface, while caffeine treatment alone induced removal of ESP13.2 from the sperm head and midpiece. As confirmed with immunofluorescence, ESP13.2 could be added back to the surfaces of sperm that had been previously exposed to caffeine. Treatment with caffeine significantly increased the number of sperm that bound tightly to the zona pellucida as compared with controls (42 +/- 9 and 13 +/- 3 sperm/zona, respectively; P < or = 0.01). This increase in binding was inhibited by "adding back" ESP13.2 to the sperm surface (12.8 +/- 3; P < or = 0.01). Alexa-conjugated anti-ESP13.2 Ig labeling of live sperm showed that only sperm lacking ESP13.2 over the head were capable of tight binding to the zona. Our results suggest that ESP13.2 masks zona pellucida ligands on the sperm surface and its release, as part of capacitation, is required for sperm-zona interaction.

Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition.

Mammalian spermatozoa undergo a series of molecular and biochemical changes collectively termed capacitation prior to acquiring the ability to fertilise the oocyte. Although phosphorylation of sperm proteins on tyrosine residues has been recognised as an important component of this process, the precise relationship between the phosphorylation status of mammalian spermatozoa and their capacity for fertilisation has remained unclear. In this study we demonstrate a causal relationship between tyrosine phosphorylation in spermatozoa and sperm-zona interaction. The phosphotyrosine expression associated with sperm capacitation localised to internal flagellar structures in permeabilised cells but could also be detected on the exterior surface of the sperm head in live cells. Importantly, almost all spermatozoa bound to the zona pellucida demonstrated this pattern of phosphoprotein localisation, compared to fewer than 15% of the free-swimming population. These data suggest that tyrosine phosphorylation plays a significant role in remodelling the sperm surface, so that these cells are able to recognise the zona pellucida. Phosphoproteome analysis yielded the first evidence of molecular chaperones, endoplasmin (erp99) and heat shock protein 60 (hsp60), as targets for phosphorylation on the surface of mouse spermatozoa, whereas immunofluorescence localised these proteins to the precise region of the sperm head that participates in zona recognition. Based on these results, we propose a novel mechanism for mammalian gamete interaction whereby the activation of sperm-surface chaperones by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of mammalian spermatozoa.