New aspects of sperm-zona pellucida binding

Sperm-zona pellucida binding tests have become a widely used diagnostic application for clinicians to obtain guidance in so far as the therapeutic approach of the subfertile couple is concerned. Expanding the oocyte sources is imperative to ensure the consistent use of sperm-zona binding assays. Sources include oocytes derived from post-mortem ovaries, inseminated non-fertilized IVF oocytes and recycled hemizonae. Identification of specific gamete dysfunction is one of the most formidable tasks and fertilization disorders due to defective sperm-zona pellucida interaction are relatively common in the clinical practice, thereby emphasizing the importance of sperm-zona binding tests as diagnostic/predictive tests. Independent publications from Norfolk (USA), Melbourne (Australia), Tygerberg (South Africa) and Israel of highly comparable results confirm that sperm-zona binding tests are good predictors of fertilization. Studies using solubilized human pellucida recently evaluated the influence of solubilized human pellucida on spermatozoa during the capacitational process and subsequent sperm-zona binding. Involvement of G protein and carbohydrate moieties in sperm-zona pellucida binding emphasized the biological and biochemical properties of lectin and have afforded much weight to their employment as membrane probes to evaluate cell surface components. Attention has been focused on the alterations of sperm surface receptors (oligosaccharides) during the differential pathway, epididymal transit and capacitation.     

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.     

The paradoxical effects of pentoxifylline on the binding of spermatozoa to the human zona pellucida

In the presence of pentoxifylline, human spermatozoa are induced to increase certain motion characteristics; however, the role of this drug in fertilization remains equivocal. In this study, the influence of pentoxifylline on one aspect of fertilization, that is sperm-zona binding, has been examined. Results from a fluorescence label competitive zona binding (CZB) test showed that spermatozoa exposed to a pentoxifylline challenge of between 0.1 and 5 mM, which was curtailed after 1 h by washing, had a decreased (P < 0.01) ability to bind to intact zona compared with control spermatozoa. The washing procedures also removed (decrease P < 0.01 compared with peak values) some of the enhanced motion characteristics induced by pentoxifylline. These results were in contrast with those obtained using experimental conditions that maintained an increased curvilinear velocity (VCL) and lateral head displacement (ALH) (increase P < 0.001 above baseline controls) in the continued presence of pentoxifylline. Using a hemizona binding (HZB) assay, 3 mM pentoxifylline increased (P < 0.001) sperm-zona binding almost 20% above zona binding with unexposed control spermatozoa. It was concluded that, in the presence of pentoxifylline, there is increased sperm binding to the zona pellucida; however, if the drug is removed by washing, the sperm binding to the zona is decreased in concert with the removal of the enhanced motion characteristics. The application of zona solubilization by acidic conditions in a microchamber enabled the precise determination of sperm numbers in both of the sperm-zona binding assays, and the results demonstrated that a wide variation in sperm numbers was observed in each test, with 63-580 spermatozoa bound in the CZB assay and 56-1340 spermatozoa bound on a hemizona.     

Expression of mannose-ligand receptors on human spermatozoa: effect of lecithin and association with sperm binding to the zona pellucida

OBJECTIVE: To evaluate the change in the expression of mannose-ligand receptors and sperm binding capacity after the incubation of sperm cells with lecithin liposomes. DESIGN: A randomized, blinded-controlled experiment. SETTING: Andrology laboratory at the Lis Maternity Hospital. PATIENT(S): Fifteen fertile sperm donors and 10 subfertile men. INTERVENTION(S): Incubation of sperm samples with either control medium or 1 mg/mL of liposomal lecithin for 2 hours. MAIN OUTCOME MEASURE(S): Expression of mannose-ligand receptors as evaluated by mannosylated bovine serum albumin-fluorescein isothiocyanate and sperm binding to the zona pellucida as evaluated by the hemizona assay. RESULT(S): The mean +/- SE percentages of spermatozoa with patterns I, II, and III were 86% +/- 4.8%, 11% +/- 3.4%, and 3% +/- 1.6%, respectively, after treatment with control medium and 71% +/- 5.7%, 22% +/- 3.5%, and 7% +/- 2.5%, respectively, after treatment with lecithin. The same effect of lecithin was observed in the 10 sperm samples from subfertile men. The mean +/- SE numbers of sperm that bound to hemizonae after treatment with control medium or lecithin were 116 +/- 32.4 and 176 +/- 29.6, respectively. Statistically significant correlations were observed between the shift in patterns II and III and the enhancement of sperm binding after lecithin treatment (r = 0.44 and 0.6, respectively). CONCLUSION(S): Lecithin shifts the expression of mannose-ligand receptors to the capacitated and acrosoine-reacted patterns and enhances the binding capacity of the sperm cells.     

Pentoxifylline improves sperm binding to the zona pellucida in the hemizona assay

OBJECTIVE: To evaluate the effect of pentoxifylline on sperm binding capacity to zona pellucida (ZP) using the hemizona assay (HZA). DESIGN: The fertility potential of 82 men was evaluated by routine semen analysis. Each ejaculate was incubated with or without pentoxifylline (3 mM) in Ham's F-10 medium (Flow Laboratories, Irvine, Scotland) before the HZA. The effect of the pentoxifylline treatment on sperm-binding capacity to ZP was assessed by the hemizona index. RESULTS: The mean hemizona indexes with medium or pentoxifylline treatment were 23% +/- 2.1% (mean +/- SE) and 41% +/- 3.4%, respectively. Taking into consideration a significant change of the hemizona index on rising above the intra-assay coefficient of variation (+/- 8%) after pentoxifylline treatment, 73.1% of specimens improved, 19.5% deteriorated, and 7.4% remained unchanged. Using a threshold hemizona index of 23% as a discriminator between fertile and infertile specimens, 43.5% of the "pentoxifylline-improved" samples ascended to the fertile zone (> 23%). No correlations were found between sperm variables in the raw semen and the effect of pentoxifylline on sperm binding capacity. CONCLUSION: Pentoxifylline may improve the binding capacity of human spermatozoa. However, this effect is confined to a selected group of patients and cannot be predicted by the basic sperm variables. Thus, to avoid uncertain or damaging effects of pentoxifylline while preparing sperm suspension for assisted reproductive techniques, it is recommended that its effect be tested by the HZA system before its use.     

Boar spermadhesins AQN-1 and AQN-3: oligosaccharide and zona pellucida binding characteristics

AQN-1 and AQN-3 form part of the complement of surface-associated lectins which coat the plasma membrane overlying the acrosomal cap of in vitro capacitated boar spermatozoa. They belong to the spermadhesin protein family and have binding affinity for glycoconjugates of the zona pellucida, the extracellular investment surrounding mammalian eggs. The oligosaccharide and zona pellucida binding characteristics of spermadhesins AQN-1 and AQN-3 were investigated using a solid-phase assay and biotinylated glycoprotein ligands. Both sperm proteins bind glycoproteins containing Gal beta (1-4)-GlcNAc and Gal beta-(1-3)-GalNAc oligosaccharide sequences with dissociation constants (Kd) of 0.08 to 0.8 microM, and to zona pellucida glycoproteins with Kd = 0.15-0.25 microM. However, 5-N-acetyl neuraminic acid alpha (2-3/6)-linked to the galactose residue decreases the affinity of glycosylated ligands to AQN-1 three-fold, although it did not affect oligosaccharide binding to AQN-3. In addition, AQN-3 binds preferentially to glycoproteins with either a linear or tri- and tetraantennary carbohydrates than to those containing diantennary N-acetyllactosamine structures. The similar but distinct oligosaccharide recognition capabilities of spermadhesins AQN-1 and AQN-3 (this work) and AWN-1 (Dostálová, Z, Calvete, J.J., Sanz, L., and T?pfer-Petersen, E. (1995) Eur. J. Biochem. 230, 329-336) suggest that, in the pig, sperm-zona pellucida binding might be mediated by lectins displaying similar although distinct carbohydrate-recognition abilities.     

Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA I-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction).     

Species-specific binding of sperm proteins to the extracellular matrix (zona pellucida) of the egg

The zona pellucida is an extracellular matrix surrounding the mammalian egg where species-specific gamete recognition and signaling occur. Pig zona pellucida were isolated in large amounts and used as an affinity matrix for detergent-solubilized, biotinylated membrane proteins of pig spermatozoa. On non-reducing SDS-polyacrylamide gel electrophoresis, specifically bound sperm proteins migrated with M(r) 170,000, 150,000, 130,000, 56,000, and 50,000 (p50). Disulfide bond reduction separated each of the M(r) 130,000-170,000 proteins into M(r) 105,000 (p105) and M(r) 45,000 (p45) subunits, indicating that these high M(r) proteins are related. Based on two-dimensional electrophoresis, the M(r) 56,000 band was composed of three to four proteins that migrated with M(r) 56,000-62,000 (p56-62) in the second (reducing) dimension. p50 bound to heterologous zona pellucida (murine, bovine) and to Xenopus laevis oocyte envelopes, demonstrating a lack of species specificity to its binding and was identified as proacrosin/acrosin based on amino acid sequences of two tryptic peptides and its interaction with monospecific antibodies to proacrosin. In contrast, p105/p45 and one or more of the p56-62 proteins bound to pig zona pellucida but not to the egg extracellular matrices of the other species; these proteins therefore exhibited the species-specific binding to the zona pellucida expected for molecules involved in specific gamete adhesion. Amino acid sequences of nine tryptic peptides derived from p105/p45 did not match peptide sequences in existing databases, establishing it as a unique protein. These (p105/p45 and at least one p56-62 protein) are the first sperm membrane proteins to be identified that bind in a species-specific manner to the egg extracellular matrix.     

Clinical significance.

This article initiates the special section on clinical significance. Within a brief précis and overview, the 4 methodological articles and the integrative commentary of the special section are introduced. A call for the inclusion of the assessment of clinical significance in treatment evaluations is extended. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Clinical significance of snoring

Habitual snoring, nocturnal apnea, and excessive daytime sleepiness are leading symptoms of the obstructive sleep apnea syndrome. However, simple snoring without apnea is a more common and normal phenomenon. In certain habitual snorers increased upper airway resistance during sleep may lead to sleep fragmentation and hypersomnolence even in the absence of frank apnea; this condition is termed upper airway resistance syndrome. There is no convincing evidence that snoring in the absence of sleep apnea is an independent risk factor for cardiovascular disease. The evaluation of symptomatic snorers includes a specific history and physical exam, followed by a sleep study if treatment is considered necessary. The choice of treatment modality for snoring is guided by the individual needs and symptoms of the patient. Weight loss, nocturnal application of continuous positive airway pressure, or intraoral appliances which hold the mandible in protrusion during sleep are non-surgical treatment options. According to the patients' subjective assessment conventional or laser-assisted uvulo-palato-pharyngoplasty (UPPP) has a high cure rate for snoring. However, objective documentation of the effect of these interventions on measured snoring noise is scant.     

Clinical significance of virus diseases

After introductory explanations the following topics are treated: acute respiratory diseases, virus infections of the central nervous system and virus infections of the intestine. In the last paragraph the present state of virus-diagnostic possibilities is described. From this is derived that the routine use of modern virus diagnostics brings great advantages for the clinician. The intensive construction of virological laboratories for the treatment of patients is emphasized.     

Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes

The ZP2 protein is a zona pellucida glycoprotein that plays a major role in fertilization. It mediates secondary binding of spermatozoa and is one of the proteins that are involved in zona 'hardening'. ZP2 proteins were identified in various mammalian zonae pellucidae. Their primary structures are highly conserved as revealed by cDNA cloning. Antisera were used against synthetic peptides generated either against a ZP2 amino acid that is homologous in human and mouse ZP2 amino acid sequences (AS ZP2-20) or antibodies against a synthetic human ZP2 peptide (AS ZP2-26). Immunoblots showed that antiserum AS ZP2-20 and AS ZP2-26 strongly recognized human ZP2 protein with an apparent molecular mass of about 72 kDa; both antisera reacted with a minor immunoreactive polypeptide at 96 kDa. In human ovary sections, both antisera revealed immunoreactivity to human zonae pellucidae. Immuno-electron microscopy demonstrated an equal distribution of ZP2 throughout the human zona pellucida. Considerable amounts of immunoreactive material were observed in the ooplasm; some ramification-like extensions of zona pellucida antigen were found close to cells surrounding the oocyte. Our results indicate that antisera against synthetic ZP2 peptides can be used as specific markers for the identification of ZP2 protein in human oocytes.     

Clinical significance of insulin resistance

Insulin resistance has emerged in the last 20 years as a concept familiar to clinicians in many specialties. No easily performed laboratory test is available to assess insulin action in routine clinical practice. Several rare syndromes of severe insulin resistance are recognized but most clinicians will encounter insulin resistance as a manifestation of common diseases. Physiological and treatment induced changes in insulin sensitivity influence insulin doses required in insulin-dependent diabetes mellitus. In non-insulin-dependent diabetes mellitus insulin resistance is fundamental to pathogenesis but it is also influenced by standard dietary and pharmacological treatment, and probably mediated by changes in prevailing glycaemia. Antihypertensive agents appear to have diverse effects on the insulin resistance of essential hypertension though the long term significance of these remains unclear. The importance of insulin as a risk factor for coronary heart disease and the concept of an insulin resistance syndrome as a common precursor of conditions with increased vascular risk remains controversial.     

Sperm characteristics and zona pellucida binding in relation to field fertility of frozen-thawed semen from dairy AI bulls

There is still debate in the literature on whether or not endurance athletes tend to have low iron stores. In this article, we propose that endurance athletes really are at risk of becoming iron deficient due to an imbalance between absorption of dietary iron and exercise-induced iron loss. The purpose of this article is to present a critical review of the literature on iron supplementation in sport. The effect of iron deficiency on performance, its diagnosis and suggestions for treatment are also discussed. Studies of the nutritional status of athletes in various disciplines have shown that male, but not female, athletes clearly achieve the recommended dietary intake of iron (10 to 15 mg/day). This reflects the situation in the general population, with menstruating women being the main risk group for mild iron deficiency, even in developed countries. Whereas the benefit of iron supplementation in athletes with iron deficiency anaemia is well established, this is apparently not true for non-anaemic athletes who have exhausted iron stores alone (prelatent iron deficiency); most of the studies in the literature show no significant changes due to supplementation in the physical capacity of athletes with prelatent iron deficiency. However, the treatment protocols used in some of these studies do not meet the general recommendations for the optimal clinical management of iron deficiency, that is, with respect to adequate daily dosage, mode of administration and treatment period. For future studies, we recommend a prolonged treatment period (> or = 3 months) with standardised conditions of administration (use of a pharmaceutical iron preparation with known high bioavailability and a dosage of ferrous (Fe++) iron 100 mg/day, taken on an empty stomach). Currently, decisions regarding iron supplementation are best made on the basis of taking care of individual athletes. We believe that there are sufficient arguments to support controlled iron supplementation in all athletes with low serum ferritin levels. Firstly, the development of iron deficiency is prevented. Secondly, the nonspecific upregulation of intestinal metal ion absorption is reverted to normal, thus limiting the hyperabsorption of potentially toxic lead and cadmium even in individuals with mild iron deficiency.     

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.     

Clinical significance of L-arginine--nitric oxide system

The ELISA was standardized to detect monoclonal antibodies of dengue virus proteins E and NS1. One indirect ELISA was applied, using C6-36 cells inoculated with the A-15 strain, isolated during the dengue 2 epidemic in 1981 as an antigen source. These cells were fixed in ELISA plates at a 200,000 cell/well concentration. A cell control in similar conditions was used. Specific monoclonal antibodies to both proteins were used to standardize the system. Studies at different incubation periods, to determine the highest expression moment of these proteins in the cell membrane, were carried out. The results show a full response at 72 hours postinoculation for both proteins; a 14.7 ng/mL sensitivity was obtained for the detection of NS1, and of 1.43 ng/mL for E protein. This system allows the monoclonal antibodies primary screening to dengue 2 virus E and NS1 proteins.