Glycosidic residues involved in human sperm-zona pellucida binding in vitro

Glycosidic residues of the mammalian zona pellucida (ZP) are known to be involved in sperm binding, suggesting the presence of complementary carbohydrate binding sites on spermatozoa. However, in previous studies, in which sperm suspensions were incubated with monosaccharides, no inhibitory effect was observed. Results of studies in which sperm were treated shortly after swim-up suggest that the use of non-capacitated cells may explain the apparently conflicting results. In the present report, we studied the effect of preincubation of capacitated spermatozoa with different monosaccharides on their ability to bind to ZP. After 5 h under capacitating conditions, spermatozoa were incubated in medium with or without a monosaccharide, resuspended in fresh medium and used for hemizona (HZ) binding assay. When ZH were incubated with spermatozoa treated with N-acetyl-D- glucosamine, D-mannose, D-fucose, L-fucose or D-galactose, a significant decrease in the number of spermatozoa bound was observed (level of inhibition: 62, 58, 82, 68 and 48% respectively) while treatment of spermatozoa with D-glucose produced no inhibition. Sugar treatment neither altered sperm motility nor the rate of acrosome reaction. These results suggest that N-acetylglucosamine, mannose, fucose and galactose residues are involved in human sperm-zona pellucida binding in vitro.      

The effect of seminal plasma on human sperm-zona pellucida binding

The aim of this study was to determine the effect of differentconcentrations of seminal plasma (SP) in insemination mediumon human sperm-zona pellucida (ZP) binding.The effects of freshSP [50%(n = 10) and 1% (n = 9)],frozen-thawed versus fresh SP[5% (n = 10)] and frozenthawed SP [5% (n = 10) and 1% (n = 9)]on sperm-ZP binding were examined using hemizona assays. Thevalidity of the hemizona assay as performed in this study wasalso examined. Relative sperm binding percentages were determinedfor respective hemizonae, and the hemizona index was calculatedfor each experimental group and compared statistically. The50% native SP inhibited sperm-ZP binding by -70% (P = 0.0051),while 1% native SP enhanced sperm-ZP binding by –350%(P =0.0051). Significantly more spermatozoa (mean ± SD,172.00± 54.12) bound to zonae in the presence of 5%frozen-thawedSP than with 5% SP that had not been frozen (mean ±SD,127.00 ± 69.18; P = 0.0431). The 1 and 5% frozen-thawedSP stimulated sperm-ZP binding by -400 and -250% respectively(P = 0.0077 and 0.0051 respectively). It is concluded that SPconcentrations found in insemination media during assisted reproductivetechniques do not inhibit but in fact enhance sperm-ZP binding.     

重组人卵透明带蛋白及其多克隆抗体对精子-透明带结合的影响

为了探讨毕赤酵母表达的重组人卵透明带ZP3蛋白(recombinant human zona pellucida-3 protein,rhZP3)及其多克隆抗体对小鼠和人精卵结合的影响,采用不同浓度的rhZP3以及空白培养液分别处理小鼠精子,然后再与小鼠卵子进行结合实验,观察经过不同处理的精子对透明带黏附及体外受精率的影响;用rhZP3以及空白培养液分别处理人精子,然后再与人卵子进行结合实验,观察经过不同处理的精子对透明带黏附的影响;用抗rhZP3抗体与阴性血清分别处理小鼠和人卵子,再与精子进行结合实验,观察多克隆抗体对精子粘附以及小鼠体外受精率的影响。实验结果表明,rhZP3和抗rhZP3多克隆抗体既能抑制人的体外精卵结合,也能抑制小鼠体外精卵结合,提示rhZP3具有天然透明带的特性,有发展成避孕疫苗和作为检测透明带抗体检测试剂的可能。     

Molecular basis of human sperm-zona pellucida interaction

The recognition of carbohydrate sequences by complimentary receptors has been shown to be a critical factor in gamete interaction in many different animal species. We have proposed the hypothesis that, in the human, sperm binding to the zona pellucida requires a 'selectin-like' interaction. We have used the hemizona assay (a unique internally controlled bioassay that evaluates tight binding of human sperm to the homologous zona pellucida) and advanced methods of carbohydrate analysis to test this hypothesis. We provide compelling evidence that demonstrates that oligosaccharide recognition is also required for specific, tight human gamete binding. Hapten inhibition tests, zona pellucida lectin-binding studies and removal/modification of functional carbohydrates by chemical and enzymatic methods provide evidence for the involvement of defined carbohydrate moieties in initial binding. Our studies suggest the existence of distinct zona-binding proteins on human sperm that can bind to selectin ligands. Additionally, results suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Full characterization of the glycoconjugates that manifest selectin-ligand activity on the human zona pellucida will allow for a better understanding of human gamete interaction in physiological and pathological situations.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in- vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.      

Evidence for the participation of beta-hexosaminidase in human sperm-zona pellucida interaction in vitro

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.     

The influence of sera, follicular fluids and seminal plasma on human sperm-zona pellucida binding

The purpose of this study was to assess the influence of male, female and fetal cord sera, follicular fluid, and seminal plasma on human sperm-zona pellucida binding, using the hemizona assay. Steroids, gonadotrophins, growth hormone and prolactin concentrations in follicular fluid and sera were also analysed. The influence of follicular fluid (10 or 50%, v/v) and sera (10%) on sperm-zona pellucida binding was investigated by supplementing the sperm processing medium as well as the sperm-hemizona incubation medium. Different seminal plasma concentrations (1 or 10%) were added to the sperm-hemizona incubation medium. Supplementation with 10% day 3 donor serum was used as a control throughout experimentation. Although supplementation with male sera and fetal cord serum exerted a stimulatory effect (36 and 90% respectively; P < 0.029) on sperm-zona pellucida binding, hemizona indices obtained with addition of male sera, fetal cord serum and sera obtained from sub-fertile in-vitro fertilization (IVF) patients on day 12 of their menstrual cycle did not differ significantly (P > 0.05). Final progesterone concentrations in sperm-zona pellucida incubation media (10% follicular fluid supplementation), which ranged from 0.788 to 3.85 microg/ml, enhanced sperm binding to the zonae by >100% (P < 0.02). The utilization of follicular fluid (10%) as a natural physiological stimulus to enhance sperm-zona pellucida binding in an IVF setting is recommended. The presence of seminal plasma in the spermzona pellucida incubation media showed no beneficial effect on the binding ability of sperm, and can be viewed as an unfavourable substance in the proximity of the oocyte.      

Role of acrosomal matrix proteases in sperm-zona pellucida interactions

The roles of acrosomal matrix proteases in sperm-zona pellucida interactions have long been one of the most puzzling questions in mammalian fertilization. Of the acrosomal proteases identified, acrosin exhibits two distinct activities: (i) an enzymatic activity as a trypsin-like serine protease; and (ii) a lectin-like carbohydrate-binding activity. Thus, acrosin has been postulated to function both in the limited proteolysis of the zona pellucida, and in the maintenance of binding of acrosome-reacted sperm to the zona. However, analysis of acrosin-deficient mouse sperm demonstrates that this protein is not essential for sperm-zona pellucida interactions, including the sperm penetration of the zona pellucida. Acrosin is presumably involved in the limited proteolysis and/or processing of other proteins in the acrosome and on the membranes during the acrosome reaction at least in the mouse, although the serine protease system in mouse sperm is very different from those in sperm of other rodent species. It is concluded that membranous proteases rather than acrosomal proteases may play an important role(s) in the sperm-zona pellucida interactions.     

Effect of antisperm antibodies present in human follicular fluid upon the acrosome reaction and sperm-zona pellucida interaction

PROBLEM: To determine the ability of IgGs isolated from follicular fluids (hFFIgGs) to induce the acrosome reaction (AR) in human spermatozoa and to inhibit sperm-zona pellucida (ZP) interaction. METHOD OF STUDY: Incubation of capacitated spermatozoa with hFFIgGs (n = 40) and assessment of their effect on the AR or hemizona (HZ) assay in a condition that allows sperm-ZP interaction, avoiding acrosomal exocytosis. RESULTS: hFFIgGs from different women varied in their ability of inducing the AR. Those hFFIgGs with the highest AR-inducing capacity evoked the exocytotic response in most of the different sperm donors tested [high Induction Frequency (IF)]. Some of these antibodies were also able of inhibiting sperm binding to ZP [low HZ Index (HZI)]. A significant correlation was found between the IF and the HZI for each hFFIgG. CONCLUSIONS: Human follicular fluid contains antibodies capable of inducing the AR and inhibiting sperm-ZP binding, suggesting that they could be directed towards ZP receptors. hFFIgGs would constitute a tool for the identification of sperm entities involved in fertilization.     

High frequency of defective sperm-zona pellucida interaction in oligozoospermic infertile men

BACKGROUND: The ability of sperm to interact with the zona pellucida (ZP) plays a critical role during the process of human fertilization. The aim of this study is to determine frequency of defective sperm-ZP interaction in oligozoospermic infertile men. METHODS: Sperm-ZP binding assays and the ZP-induced acrosome reaction (AR) were performed in 72 infertile men with a sperm concentration <20 x 10(6)/ml. Oocytes that had previously failed to fertilize in a clinical IVF programme were used for the tests. Motile sperm (2 x 10(6)/ml) selected by swim-up from each semen sample were incubated with four oocytes for 2 h. The number of sperm bound per ZP and the ZP-induced AR were assessed. Under these conditions, an average of < or =40 sperm bound/ZP was defined as low sperm-ZP binding and a ZP-induced AR < or =16% was defined as low ZP-induced AR. RESULTS: In the 72 oligozoospermic men, 28% (20/72) had low sperm-ZP binding. Of those with normal sperm-ZP binding, 69% (36/52) had low ZP-induced AR. Overall, 78% (56/72) had either low ZP-binding or normal ZP binding but low ZP-induced AR. This means that only 22% (16/72) had both normal sperm-ZP binding and normal ZP-induced AR. CONCLUSION: Oligozoospermic men have a very high frequency of defective sperm-ZP interaction, consistent with their low natural fertility or low fertilization rate in conventional IVF. Infertile couples with oligozoospermic semen should be treated by ICSI rather than by conventional IVF.     

Frequency of defective sperm-zona pellucida interaction in severely teratozoospermic infertile men

BACKGROUND: The frequency of defective sperm-zona pellucida (ZP) interaction in teratozoospermic infertile men was investigated. METHODS: Sperm-ZP binding and the ZP-induced acrosome reaction (ZPIAR) were performed in 125 infertile men with <5% of their sperm with normal morphology (strict criteria), but with a sperm count > or =20x10(6)/ml and total motility >30% in semen. Oocytes that failed to fertilize in clinical IVF were used for the tests. Four oocytes were incubated for 2 h with 2x10(6)/ml motile sperm selected by swim-up. The number of sperm bound per ZP and ZPIAR were assessed. Under these conditions, an average < or sperm bound per ZP was defined as poor sperm-ZP binding, and a ZPIAR < or = was defined as low ZPIAR. RESULTS: Among 125 teratozoospermic men, 31% (39/125) had poor sperm-ZP binding. Of those without poor ZP binding, 48% (41/86) had low ZPIAR. Some 64% (28/44) with sperm counts between 20 and 60x10(6)/ml had low ZPIAR. Only 36% (45/125) had normal sperm-ZP binding and ZPIAR. CONCLUSIONS: Defective sperm-ZP interaction was present in 64% of teratozoospermic infertile men: 31% had defective sperm-ZP binding, and 33% low ZPIAR. The frequency of low ZPIAR was higher in men with sperm counts between 20-60x10(6)/ml.     

Analysis of the participation of N-acetylglucosamine in the different steps of sperm-zona pellucida interaction in hamster

Glycoproteins and lectin-like proteins mediate sperm-zona pellucida interaction. The present study analysed the participation of carbohydrates in the different stages of sperm interaction with the zona pellucida in hamster, by determining the effects of different monosaccharides. N-acetylglucosamine (GlcNAc, 1 mM) reduced sperm ability to bind to the zona pellucida. Surprisingly, spontaneous acrosome reaction (AR) was also inhibited by this sugar. In order to analyse the effect of GlcNAc on sperm-zona pellucida binding, independent of its effect on the AR, strontium (Sr) was used as a calcium (Ca) replacement in the sperm capacitation and co-incubation medium. Sr seemed to be able to replace Ca for sperm capacitation, at least when measured as the ability to bind to the zona pellucida, and undergo AR when Ca is provided. Moreover, sperm-zona pellucida binding could also take place in a Sr-modified medium. When binding assays were carried out in the Sr medium, GlcNAc also produced an inhibitory effect. This could be reproduced when sperm, but not oocytes, were pre-incubated with the monosaccharide. IVF assays were also carried out to analyse the participation of GlcNAc in the different steps of sperm-oocyte interaction. Taken together, the results support the involvement of the GlcNAc residues of the zona pellucida in the early steps of the interaction with sperm.     

Heterogeneity of the zona pellucida carbohydrate distribution in human oocytes failing to fertilize in vitro

The mammalian zona pellucida contains several glycoproteins whose oligosaccharide moieties are known to play a key role in the interaction with spermatozoa. Since zona pellucida defects may represent one of the most likely causes of failed fertilization in human in-vitro reproduction, we have studied the carbohydrate composition and distribution over the human zona pellucida by means of lectins. Donated, not inseminated cumulus-oocyte complexes, from cohorts with high fertilization rates, and fertilization-failed oocytes from cohorts inseminated with proven fertile donor semen, were analysed using 11 fluorescein-labelled lectins, on deplasticized semi-thin epoxy sections. Results showed that wheat germ agglutinin (WGA), Maclura pomifera (MPA) and Pisum sativum (PSA) bound to the extracellular matrix bordering the zona pellucida-corona radiata interface of cumulus- oocytes complexes, while the zona pellucida was labelled by WGA, Concanavalin A (ConA) and PSA. WGA labelling and correlative electron microscopy on the cumulus-oocyte complexes demonstrated that this lectin is a useful tool to trace the cortical granule distribution in the human oocyte. Surprisingly, in the failed-fertilized oocytes the zona pellucida was also labelled by MPA and showed three different patterns: (i) labelling of the zona pellucida outer surface; (ii) uniform labelling; (iii) labelling of an outer zona pellucida layer with variable thickness. Comparative analysis of WGA and MPA labelling on single failed-fertilized oocytes demonstrated that MPA zona pellucida patterns are not related to the cortical reaction. The nature and meaning of the MPA pattern of failed-fertilized oocytes were discussed in the light of zona pellucida defects impairing sperm receptivity.      

Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm-zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction

BACKGROUND: Protein tyrosine phosphorylation (TP) of human sperm is related to sperm capacitation and zona pellucida (ZP) binding. The aim of this study was to determine whether the TP of capacitated sperm is a useful marker for the ability of sperm to bind to the ZP and undergo the ZP-induced acrosome reaction (AR). METHODS: Semen samples were obtained from 115 subfertile men with sperm count > or =20 x 10(6)/ml, motility > or =25% and variable morphology. Motile sperm (2 x 10(6)/ml) selected by swim-up were incubated with four oocytes for 2 h, and the number of sperm bound to the ZP and the ZP-induced AR was examined. TP of sperm tail was assessed by immunofluorescence (IF) with anti-phosphotyrosine monoclonal antibody. The time course and effects of dibutyryl cyclic adenosine monophosphate (dbcAMP) and phorbol myristate acetate (PMA) on TP were also studied. RESULTS: TP was stimulated more by dbcAMP (P < 0.001) and less by PMA (P < 0.05). TP increased significantly with time of incubation of sperm. TP was not detectable on the surface of unfixed live sperm by either Dynabeads or IF. Sperm TP at 2, 4 and 20 h incubation was all significantly correlated with sperm-ZP binding but not with the ZP-induced AR. CONCLUSION: Sperm TP detected by IF correlates strongly with sperm-ZP binding capacity but not with the ZP-induced AR. This simple IF assay of TP may be a clinically useful test of sperm function that is predictive of normal sperm ZP-binding capacity.     

Enhancement of sperm-zona pellucida (ZP) binding capacity by activation of protein kinase A and C pathways in certain infertile men with defective sperm-ZP binding

BACKGROUND: Defective sperm–zona pellucida (ZP) binding (DSZPB) isa common cause of failure of fertilization in vitro. This studywas to determine if DSZPB is caused by defective pathways upstreamof protein kinase A (PKA) and C (PKC), or reduced protein tyrosinephosphorylation (TP). METHODS: Infertile men with DSZPB and either normal sperm morphology(NSM) 14% (n = 15) or 5% (n = 15) were studied. Sperm–ZPbinding test was performed by incubation of motile sperm withoocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP,PKA activator) or phorbol myristate acetate (PMA, PKC activator).TP of capacitated sperm in medium was assessed by immunofluorescencewith an anti-phosphotyrosine monoclonal antibody. RESULTS: For normal sperm with normal sperm–ZP binding, both PMAand dbcAMP significantly enhanced sperm–ZP binding ina dose–response manner. Only dbcAMP, but not PMA, significantlyincreased TP of capacitated sperm. In DSZPB men with severeteratozoospermia (NSM 5%), neither PMA nor dbcAMP enhancedsperm–ZP binding, despite dbcAMP significantly increasingthe TP of capacitated sperm for all samples. In contrast, forDSZPB with NSM 14%, PMA caused significantly increased spermbinding up to normal levels (40 sperm bound/ZP) in five men,and dbcAMP had a similar result in two men. Again TP was significantlyenhanced only by dbcAMP, but not by PMA. CONCLUSIONS: There is defective signalling in pathways upstream of PKC andPKA in some men with DSZPB and normal semen analysis. Stimulationof TP by dbcAMP does not enhance sperm–ZP binding capacityin DSZPB men with low TP, regardless of sperm morphology.     

Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization

Sperm-zona pellucida binding and penetration were assessed on the oocytes that failed to fertilize from couples with >/=3 oocytes treated by standard in-vitro fertilization (IVF). There were four groups: fertilization rate 0% (n = 369), 1-25% (n = 194), 26-50% (n = 81) and 51-95% (n = 100). Of the couples with zero fertilization rate 70% had 相似文献   

Direct evaluation of antigen binding to human T lymphocyte clones: involvement of major histocompatibility complex products in antigen binding

Cloned human helper T lymphocytes reactive with a defined peptide (p20; residues 306-329) of the HA-1 molecule of influenza virus hemagglutinin were analyzed for their capacity to specifically bind peptide antigen. Three different methods of analyzing antigen binding to T cell receptors were compared. One method involved the binding of radiolabeled T cells to antigen-pulsed populations of sheep erythrocyte rosette-negative (E-) cells (B cells and monocytes). The binding was antigen specific, in that only E- cells pulsed with the appropriate antigen bound the treated T cells, and was inhibitable by free peptide. Furthermore, antigen binding was major histocompatibility complex-restricted in that only E- cells histocompatible at the HLA-D region locus bound the T cells, and monoclonal antibody of the relevant specificity was able to inhibit the binding. Secondly, it was demonstrated that tritiated T cells could bind to insolubilized antigen (p20) in the absence of E- cells. The binding was inhibited by anti-class II antibody suggesting that the interaction of antigen with the T cells involves recognition of T cell major histocompatibility complex class II determinants. Finally, radiolabeled peptides were also used to detect binding to the appropriate clones in the absence of presenting cells. This binding was specific, inhibitable by the appropriate unlabeled peptide and temperature dependent. These studies demonstrate that the process of antigen binding to receptors is analyzable and should in turn facilitate the analysis of the mechanism of T cell activation.     

Comparison of the frequency of defective sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen

BACKGROUND: The aim of this study was to compare the frequency of defective sperm-zona pellucida (ZP) binding (DSZPB) and defective ZP-induced acrosome reaction (DZPIAR) in subfertile men (i.e. male partners of infertile couples) with normal and abnormal semen analyses. METHODS: A total of 1030 subfertile men with normal semen analysis (n=255), oligozoospermia (count<20x10(6)/ml, n=136), severe teratozoospermia (strict normal morphology相似文献   

Gonadotrophin-releasing hormone antagonists inhibit sperm binding to the human zona pellucida.

Previous work from our laboratory indicated that gonadotrophin-releasing hormone (GnRH) increases human sperm-zona pellucida binding. Here we present evidence that GnRH antagonists inhibit sperm-zona pellucida binding in humans. Motile spermatozoa (10(7) cells/ml) were incubated in modified Tyrode's medium at 37 degrees C, in 5% CO(2) in air. After 4.5 h, aliquots of spermatozoa were treated with saline (control) or with different concentrations of GnRH antagonists (test). Each sperm aliquot was then tested in the hemizona binding assay. In this assay, the control aliquot was incubated with half a human zona pellucida (hemizona) and the test aliquot was incubated with the matching half. After 20 min, the hemizonae were withdrawn and the number of zona-bound spermatozoa counted using phase-contrast microscopy. In addition, the effect of GnRH antagonists upon the pattern of sperm movement, frequency of sperm-zona pellucida collisions, and percentage of living and acrosome-reacted spermatozoa was determined. The results indicated that treatment with GnRH antagonists decreased the number of zona-bound spermatozoa and did not change the pattern of sperm movement, frequency of sperm-zona collisions, and percentage of acrosome-reacted spermatozoa. We suggest that this action of GnRH antagonists may be due to an effect on zona receptors on the sperm plasma membrane.