Human Zona Pellucida Research Articles

168. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

Upon leaving the testis mammalian spermatozoa are functionally incompetent and are thus unable to fertilize an oocyte. As the spermatozoa ascend the female reproductive tract, functional maturity is achieved through a complex cascade of biophysical and biochemical changes known as capacitation. An important aspect of this final maturation phase is the remodelling of the sperm surface architecture to enable it to interact with the zona pellucida, a glycoprotein matrix that surrounds the oocyte, and initiate fertilisation. While originally thought to be underpinned by a simple lock and key mechanism, emerging evidence has suggested that this interaction may instead be mediated by a multimeric recognition complex that is formed on the sperm surface during capacitation. However, to date the presence and composition of such a complex has yet to be described. Through the application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have provided evidence that human spermatozoa express a number of high molecular weight protein complexes on their surface. Furthermore, the affinity of these surface expressed complexes for the zona pellucida was assessed utilising solubilised human zona pellucida and the technique of Far Western Blotting. Among the complexes that showed affinity for the zona pellucida we identified one comprising 14 subunits of the 20S proteasome. Interestingly, the 20S proteasome has previously been implicated in various aspects of mammalian fertilisation, including zona pellucida penetration and the acrosome reaction, although its precise role in these events has yet to be elucidated. Collectively, these results demonstrate the presence of multimeric protein complexes on the surface of human spermatozoa, and support their putative role in the initial interaction between the sperm and the zona pellucida. Our current research is focused on elucidation of the role of the 20S proteasome in human sperm-zona binding and further investigation of surface expressed protein complexes.

DNA vaccine encoding chimeric protein encompassing epitopes of human ZP3 and ZP4: Immunogenicity and characterization of antibodies

Immunization with zona pellucida (ZP) glycoproteins leads to curtailment of fertility often associated with ovarian dysfunction. To avoid ovarian dysfunction, synthetic peptides corresponding to ZP glycoproteins have been proposed as candidate immunogens. In the present study, plasmid DNA encoding a human ZP glycoprotein-3 (ZP3) epitope corresponding to amino acid (aa) residues 334–343 and a human ZP glycoprotein-4 (ZP4) epitope corresponding to aa residues 251–273 separated by a triglycine spacer was constructed using the mammalian expression vector, VR1020. The plasmid DNA construct expressed both human ZP3 and ZP4 epitopes, as revealed by transient transfection of COS-1 (African green monkey, kidney) mammalian cells. Active immunization of female BALB/cJ mice with the DNA vaccine led to generation of antibodies reactive with baculovirus-expressed recombinant human ZP3, ZP4 and ZP3 (334–343aa)-GGG-ZP4 (251–273aa) synthetic peptide in an ELISA as well as T cell responses. Antibodies generated by the DNA vaccine also recognized native ZP. The immune sera significantly inhibited ( p < 0.005) the binding of FITC-labeled ZP3 to capacitated human sperm, whereas no inhibition in the binding of FITC-labeled ZP4 was observed. However, a significant decrease in acrosomal exocytosis mediated by both recombinant human ZP3 ( p < 0.005) and ZP4 ( p < 0.005) was observed in presence of the immune sera. These studies demonstrate that a DNA vaccine can be designed to elicit antibodies against small epitopes of ZP glycoproteins.

The zona pellucida birefringence in immature and mature oocytes collected after controlled ovarian stimulation: preliminary results

The zona pellucida birefringence in immature and mature oocytes collected after controlled ovarian stimulation: preliminary results

Effects of Native Human Zona Pellucida Glycoproteins 3 and 4 on Acrosome Reaction and Zona Pellucida Binding of Human Spermatozoa

Acrosome reaction is crucial to the penetration of spermatozoa through the zona pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinase-C, protein tyrosine kinase, T-type Ca2+ channels, and extracellular Ca2+ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca2+ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding.

Purification of Native Human Zona Pellucida Glycoproteins from Eggs: Binding Characteristics to Human Spermatozoa, Hyper-activation, Acrosomal Exocytosis, and Sperm-Oocyte Interaction.

Purification of Native Human Zona Pellucida Glycoproteins from Eggs: Binding Characteristics to Human Spermatozoa, Hyper-activation, Acrosomal Exocytosis, and Sperm-Oocyte Interaction.

Acrosin antibodies and infertility. I. Detection of antibodies towards proacrosin/acrosin in women consulting for infertility and evaluation of their effects upon the sperm protease activities

To detect the presence of antibodies to the proacrosin/acrosin system and to evaluate their effect on the sperm acrosomal protein activities in women consulting for infertility. Retrospective study. Basic research laboratory. Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10) and recombinant human zona pellucida (ZP) glycoprotein A( *) (rec-hZPA). Development of an ELISA-Acro to test for antiacrosin antibodies using Rec-40 and truncated acrosin proteins as antigens. Evaluation of: 1) the presence of antiacrosin antibodies; 2) the protein regions recognized by the antibodies; 3) the relationship between antiacrosin antibodies and surface antisperm antibodies (ASA) identified by the immunobead binding test (IBT); and 4) the effect of antiacrosin antibodies upon proacrosin/acrosin binding activity to ZPA and acrosin amidase activity. Antiacrosin antibodies were detected in sera from 34 of 179 women (19%). Detection of ASA by the IBT resulted in a similar incidence (36 of 179, 20%), although only six of them showed correspondence between both assays; five of these six sera were IBT-positive IgGs to the sperm head. Antiacrosin antibodies directed toward different protein regions inhibited proacrosin binding activity to rec-hZPA as well as its activation and acrosin amidase activity in protein sperm extracts. Antiacrosin antibodies are present in sera of women consulting for infertility in both IBT-positive and IBT-negative samples, and they affect proacrosin/acrosin activities.

Native human zona pellucida glycoproteins: purification and binding properties

Fertilization starts with the binding of the spermatozoa to the zona pellucida (ZP) of the oocyte. Such binding is a carbohydrate-mediated event and consists of a series of tightly regulated events. Molecular interactions between spermatozoon and ZP in human are not well characterized due to limited availability of oocytes for research. Our current technology cannot generate recombinant human ZP (hZP) glycoproteins with native glycosylation. In this study, hZP glycoproteins, hZP2 (approximately 120 kDa), hZP3 (approximately 58 kDa) and hZP4 (approximately 65 kDa) were purified from ZP (purity >88%) by immunoaffinity columns. The binding sites of the purified native hZP3 and hZP4 were localized to the acrosome region of the capacitated human spermatozoa, and were lost after acrosome reaction. Purified human hZP2 bound to this region only in acrosome-reacted spermatozoa. Differential binding of the three glycoproteins to the post-acrosomal region and the midpiece of the spermatozoa was observed. In addition, hZP3, but not hZP2 and hZP4, induced hyperactivation. The stimulatory activity was dependent partly on N-linked glycosylation of hZP3. This manuscript describes the biological activities of purified hZP glycoproteins from the native source for the first time.

Stability of Human Zonae Pellucidae During Extended Culture In Vitro

Background: Literature data on the fate of the zona pellucida or its remnants, including intact zonae pellucidae containing cellular debris, empty zonae, and/or zona fragments and unfertilized oocytes from previous nonfertile reproductive cycles observed in the oviducts, uterus, and peritoneal cavity, is available for at least two mammalian species: mice (A. McLaren, 1970) and the little bulldog bat (John J. Rasweiler IV, 1977, 1979) however, no similar data were found for humans.

Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles

The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.

Detection of zona pellucida proteins during human folliculogenesis

The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.

Recombinant human zona pellucida protein C produced in Chinese hamster ovary cells binds to human spermatozoa and inhibits sperm–zona pellucida interaction

Recombinant human zona pellucida protein C expressed in Chinese hamster ovary cells associates to the acrosomal region of human spermatozoa and inhibits sperm-zona pellucida interaction in the hemizona assay. Recombinant human zona pellucida protein C may be a useful tool toward the development of diagnostic methods for male factor infertility and the elucidation of the molecular basis of fertilization.

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.

Human sperm bound to the zona pellucida have normal nuclear chromatin as assessed by acridine orange fluorescence

The aim of this study was to determine if only sperm with double stranded DNA could bind to the human zona pellucida (ZP). Sperm samples from 124 infertile men with a range of semen abnormalities were studied. Oocytes that had failed to fertilize in IVF or ICSI were used for the sperm-ZP binding test. A group of four oocytes were incubated for 2 h with 2 x 10(6)/ml motile sperm selected by colloidal silica gradient centrifugation (CSGC). After assessing the number of sperm bound per ZP, all sperm bound to the surface of the ZP of four oocytes were dislodged and placed on a glass slide. The double (green fluorescence) or single stranded (denatured, red fluorescence) DNA of sperm in semen, motile sperm selected by CSGC and ZP-bound sperm, was assessed by acridine orange (AO) fluorescence. The percentage of sperm with green fluorescence was significantly correlated with normal sperm morphology in semen and after CSGC preparation. The proportion of sperm with green fluorescence was significantly higher in motile sperm selected by CSGC than ejaculated sperm. There were very few sperm (average <8%) with red fluorescence bound to the ZP, even in men who had very high (>70%) proportions of sperm with red fluorescence in their semen. Sperm binding to human ZP is highly selective for double stranded DNA. Sperm with single stranded or denatured DNA bind less or do not bind at all to the ZP, probably because of defects of motility and, more especially, morphology.

The influence of homogenous zona pellucida on human spermatozoa hyperactivation, acrosome reaction and zona binding

The study aimed to evaluate the changes in sperm motion characteristics and the occurrence of hyperactivation among sperm populations after exposure to human zona pellucida. Motile spermatozoa samples were used to evaluate the sperm-zona binding capacity, zona-induced acrosome reaction and changes in sperm motion characteristics. Sperm motion characteristic changes studied included straight line velocity, curvilinear velocity, amplitude of lateral head displacement, straightness and beat cross frequency. Recordings were performed on semen immediately after liquefaction, 3 h capacitation and after exposure to solubilised human zona pellucida. The semen samples were divided into morphology categories, namely six (16 +/- 1.4% normal forms, normal patterns), 31 (8 +/- 1.7% normal forms, G-pattern) and 27 (3 +/- 1.3% normal forms, P-pattern). The Hemizona Indices for the three morphology groups namely normal, G-patterns and P-patterns, were 77 +/- 6%, 61 +/- 5% and 41 +/- 5% respectively (P <or= 0.05). The zona-induced acrosome reaction for the three groups were 25.0 +/- 2% (normal group), 18.0 +/- 2% (G-patterns) and 14.1 +/- 0.6% (P-pattern) (P <or= 0.05). The number of hyperactivated sperm showed an increase in the ejaculates (1%), after capacitation (2%) and exposure to zona pellucida (5%) (P <or= 0.05). When divided into the three morphology groups, the number of spermatozoa with hyperactivated motility was more profound in men with normal morphology compared with G- and P-pattern men. Human zona pellucida is a strong inducer of hyperactivated motility among spermatozoa and the effect is more profound among sperm populations with normal morphology.

Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.

Citation classics in Fertility and Sterility, 1975–2004

Citation classics in Fertility and Sterility, 1975–2004

P-628: Sperm bound to hyaluronic acid or to human zona pellucida exhibit similar levels of improvement in tygerberg strict morphology scores

During spermiogenesis, a plasma membrane remodeling step facilitates formation of zona pellucida and hyaluronic acid(HA) binding sites in sperm. Thus, diminished maturity sperm, which contain surplus cytoplasm and show consequential abnormal morphology, are unable to bind to HA. We have tested the hypothesis that there will be an improvement in the proportion of sperm with normal Kruger strict morphology in HA-bound sperm fractions of semen samples, similar to the 4.2x increase reported by the Tygerberg, South Africa (TSA) group for zona pellucida bound sperm fractions of teratozoospermic specimens (Mol. Repr. Dev., 1991). Strict sperm morphology was determined in semen and in their respective HA-bound fractions in 30 men (Sperm conc. range: 7.4-115x106/mL, motility: 45.6±3.1%) by three independent investigators. Two readers were from the USA (US1 and US2) and the third, TSA, who was blinded to the A and B surface of slides, was from South Africa. METHODS: For sperm evaluation, a two-chamber version of the HBA slide (MidAtlantic Diagnostics) was used. An aliquot of semen was smeared on the uncoated A side. In another aliquot, the sperm were allowed to bind to the HA-coated surface of B side, to determine HA-binding score. After Diff-Quik staining, 200 sperm were evaluated for strict morphology by each investigator on the A and B sides. The sperm maturity markers, CK-activity (cytoplasmic retention) and HspA2 chaperone were assessed, as published previously. Statistical analysis by paired T-test, Wilcoxon ranked sum test, and Spearman correlations were by Sigma Stat (Jandel). All values are mean±SEM. Comparing the three readers, US1 and US2 used stricter criteria than TSA. The % normal scores by the three investigators for A semen and the respective B HA-bound sides were: US1: 6.6±0.7% (range:14.7) and 13.5±1.4% (range: 33.2): US2: 8.9±0.9 (range:20.7) and 14.5±1.4 (range:29.8); TSA: 11.0±1.1 (range: 23) and 26.5±2.1 (range: 50), A vs. B p<0.003 in all readers. There were similar variations in Kruger strict morphology score improvements from the A to B sides: US1: 2.2x, US2:1.8x and TSA: 3.1x. However, despite the differences in “strictness,” the evaluations were internally consistent among USA1, USA2, and TSA between r=0.38 and 0.66 (p=0.039 - <0.001). The rationale for the morphology improvements is supported by the relationships between HA-binding scores and sperm maturity markers: HA-binding vs. CK activity: r= -0.56, p=0.004; HA-binding vs. HspA2 levels: r=0.55, p=0.005. Most revealing are the % increase in sperm with normal morphology bound to HA from the 19 teratozoospermic (<14% normal) samples: 226% and 194% for the stricter US1 and US2. However, TSA found a 376% improvement, a value nearly identical to the 420% improvement found in zona pellucida-bound sperm by the TSA group in 1991. CONCLUSIONS: Consistent with our hypothesis, there was a significant improvement in normal strict morphology scores in the HA-bound vs. semen sperm fractions. The improvement was comparable to those reported earlier for zona pellucida-bound sperm. The relationships between sperm maturity, HA-binding and normal morphology are further supported by the HA-binding scores and sperm maturity markers. This selection/separation method holds promise for semen evaluation and ICSI programs, as mature normal forms are highly selected.

Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen

Six donor semen samples were evaluated after 28 years cryopreservation in liquid nitrogen. The results showed that the samples retained good postthaw motility recovery and normal levels of binding to the human zona pellucida and that four of the five samples tested also gave normal levels of zona-induced acrosome reaction. In conclusion, human sperm can survive very long-term storage, which is pertinent information for clinicians referring boys and young men for sperm banking before chemotherapy.

Sperm-zona pellucida interaction and immunological infertility.

Immune reactions against gametes appear to be physiologically important for the maintenance of homeostasis in reproduction. In contrast, aberration of the immune homeostasis might give rise to 'immunological infertility'. Antisperm antibodies cause infertility by blocking fertilization. The mechanism can be explained as inhibiting the acrosome reaction of sperm by their blocking effect on capacitation through inhibiting an increase of fluidity of the sperm membrane. Autoantibodies against zona pellucida also cause infertility by blocking sperm-zona pellucida interaction, though the definitive mechanism has not been elucidated. Pretreatment of spermatozoa with D-mannnose completely inhibited sperm penetration through, but not binding to, the zona pellucida. Furthermore, very rapid kinetics between sperm extracts and D-mannnose by a BIAcore apparatus suggest that a D-mannose ligand of the sperm surface is easy to bind to and dissociate from a D-mannose residue in the sperm receptor site on the zona pellucida. Thus, D-mannnose on the human zona pellucida might be an essential molecule acting as a second sperm receptor, through which sperm penetrate into the zona pellucida. Because these antibodies appear to not cause any deleterious clinical symptoms, sperm and zona pellucida antigens are promising candidates in the development of an immunocontraceptive. (Reprod Med Biol 2006; 5: 95-104).

Sperm–zona pellucida interaction and immunological infertility

Immune reactions against gametes appear to be physiologically important for the maintenance of homeostasis in reproduction. In contrast, aberration of the immune homeostasis might give rise to ‘immunological infertility’. Antisperm antibodies cause infertility by blocking fertilization. The mechanism can be explained as inhibiting the acrosome reaction of sperm by their blocking effect on capacitation through inhibiting an increase of fluidity of the sperm membrane. Autoantibodies against zona pellucida also cause infertility by blocking sperm–zona pellucida interaction, though the definitive mechanism has not been elucidated.Pretreatment of spermatozoa with D‐mannnose completely inhibited sperm penetration through, but not binding to, the zona pellucida. Furthermore, very rapid kinetics between sperm extracts and D‐mannnose by a BIAcore apparatus suggest that a D‐mannose ligand of the sperm surface is easy to bind to and dissociate from a D‐mannose residue in the sperm receptor site on the zona pellucida. Thus, D‐mannnose on the human zona pellucida might be an essential molecule acting as a second sperm receptor, through which sperm penetrate into the zona pellucida.Because these antibodies appear to not cause any deleterious clinical symptoms, sperm and zona pellucida antigens are promising candidates in the development of an immunocontraceptive. (Reprod Med Biol 2006; 5: 95–104)