The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.     

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution.

The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.     

Functional aspects of human sperm binding to the zona pellucida using the hemizona assay

The hemizona assay (HZA) has facilitated investigations of sperm function in relation to zona pellucida binding. In this study, the authors examined: 1) the association between hyperactivated sperm motility and HZA binding; 2) the binding kinetics and efficiency of sperm from subfertile men; and 3) the influence of sperm freezing and thawing on binding capacity. For each HZA, a nonviable human oocyte was cut into equal zona hemispheres. The mean number of bound sperm and the incidence of hyperactivation were significantly greater for samples of sperm from fertile men compared with sperm from subfertile men (P less than 0.05). Subfertile sperm had a binding curve that paralleled the curve for fertile sperm, although the magnitude of binding was markedly reduced. Freezing and thawing of sperm from fertile samples impaired their capacity to bind to the zona pellucida. The HZA binding efficiency was reduced by 30%, although the binding curves for fresh versus frozen samples remained parallel.     

In vitro fertilization failure: identification of gamete defects by investigation of sperm-zona pellucida binding capacity of unfertilized oocytes

Summary. The objective of the study was to determine whether fertilization failure was due to spermatozoal or oocyte factors. Twenty-five unfertilized oocytes from 12 IVF/GIFT couples showing total or partial fertilization failure were evaluated for sperm zona binding potential under hemizona assay (HZA) conditions. Hemizonae were separately incubated with a sperm sample from the husband and that of a fertile control. Tight sperm binding to hemizonae was assessed. First, among the 12 patients, results showed a possible zona defect thought to be the cause of fertilization failure in five cases. Second, in two cases, fertilization failure was possibly caused by poor sperm binding potential of spermatozoa. Third, in two cases, fertilization failure was possibly caused by an oocyte defect, and fourth, three cases showed a mixture of possible causes. The results stress the need to develop a sequential analytic programme for those couples with repeated total or partial fertilization failure.     

人精子膜结合腺苷脱氨酶单克隆抗体的制备和初步鉴定

本研究以正常人精子，用ｐｅｒｃｏｌｌ分离，经脾直接免疫、细胞融合和克隆化后，以标准ＡＤＡ为抗原筛选，得到２８株单克隆抗体（ＭｃＡｂ）细胞株。其腹水效价均在１：６４００以上（ＥＬＩＳＡ）。免疫球蛋白分类显示，１株为ＩｇＭ，其余各株皆为ＩｇＧ。ＭｃＡｂ对ＡＤＡ活性的抑制实验结果显示，有２２株ＭｃＡｂ对ＡＤＡ有抑制作用而其余６株对ＡＤＡ无抑制作用。初步研究结果显示我们得到的ＭｃＡｂ具有特异性，可用于对精子中ＡＤＡ的性质研究。     

Hemizona assay and its impact on the identification and treatment of human sperm dysfunctions

Summary. The HZA, a functional test for human gamete interaction, has become a useful and valuable experimental tool for physiological and cellular analysis of the early events leading to fertilization. The analysis of the conventional semen parameters with emphasis on sperm morphology (as judged by strict criteria) and motion characteristics (evaluated by computer assisted analysis) constitutes the first obligatory step for a critical evaluation of male-factor patients. Patients in whom fertilization disorders are suspected should be evaluated through bioassays of sperm function of established accuracy. The HZA, a bioassay of sperm-zona binding capacity is here proven to be highly predictive of IVF outcome. Ultimately, our increasing knowledge of sperm biology and dysfunction will provide a basis for a better diagnosis (membrane receptor defects and metabolic/biochemical abnormalities?) as well as better therapeutic interventions in patients with sperm disorders. It seems likely that the HZA may be eventually replaced by a standardized test kit in which recombinant human DNA-derived zona receptors mimic the natural function of the hemizonae currently used. This ZP3 reagent may also be a useful antigen for contraceptive development. The HZA therefore constitutes a useful adjuvant in the armentarium for the diagnosis and therapy of male-factor patients.     

The influence of sperm/zona pellucida collision rates on zona binding

Summary. The satisfying success rates reported with intra-cytoplasmic sperm injection (ICSI) urged clinicians and scientists to re-address the emphasis in the management of the male factor patient towards gamete manipulation in order to circumvent the underlying problem causing fertilization failure. We have designed a study to (i) calculate the collision rate of a sperm population with the human zona pellucida, using a mathematical hypothesis and (ii) use the calculated collision rate to evaluate subsequent zona binding results obtained under hemizona assay conditions. Microdroplets were used to co-incubate sperm and human oocytes in order to evaluate zona binding. Using microvolumes, the track followed by sperm, as well as the maximum distance travelled were employed to calculate the collision rate of sperm and zona pellucida. The sperm concentrations of swim up samples were adjusted to 4 × 10⁶ and 0.8 × 10⁶ sperm ml⁻¹. Five separate droplets each of 20 μl containing 4 × 10⁶ sperm ml⁻¹ (80000 motile sperm) and 200 μl containing 0.8 × 10⁶ sperm ml⁻¹ (160000 motile sperm), respectively, were prepared. Both volumes were incubated for 18 h at 37°C. The mean (±SD) number of spermatozoa tightly bound to hemizona, incubated in 20 μl and 200 μl sperm droplets, was 2444±612 and 548±315, respectively ( P = 0.0001). The results can be used as a guideline to calculate the optimum insemination concentration needed for a specific sperm population to ensure the maximum collision rates with the oocyte.     

Modulation of human sperm function by follicular fluid

Human follicular fluid (hFF), present in the ampullary environment, can reduce the number of sperm bound to the zona pellucida. The aim of the present study was to investigate the effects of follicular fluid on sperm function. The presence of 50% v/v follicular fluid resulted in a significant reduction in the number of bound spermatozoa with respect to control medium (12.7 +/- 5.5 sp HZ(-1) versus 24.6 +/- 5.7 sp HZ(-1), P = 0.03) as measured by the hemizona binding assay. This reduction in zona binding capacity was not associated with a loss of sperm viability, motility or a premature acrosomal reaction. When capacitated spermatozoa were previously exposed 1 h to follicular fluid, a significant reduction in the number of alpha-d-mannose binding sites on sperm head was detected (23.7 +/- 3.1% versus 15.5 +/- 2.4%, P < 0.05). In addition, sperm fertilizing capacity (assessed as the acrosome reaction to ionophore challenge score) in the presence of follicular fluid was also diminished (38.0 +/- 4.8% versus 22.6 +/- 4.9%, P < 0.01). No modification in the pattern of protein tyrosine phosphorylation which occurs during capacitation was observed in the presence of the fluid. Taken together, the results indicate that the decrease in sperm zona-binding capacity observed in the presence of hFF was related to a lower number of sperm containing alpha-d-mannose receptors.     

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.     

Utilisation of sperm‐binding assay combined with computer‐assisted sperm analysis to evaluate frozen‐thawed bull semen

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm² of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r² = 0.91 and CASA: r² = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.     

人精子制动单抗的制备、鉴定及其腹水型单抗的纯化

用杂交瘤技术建立的一株分泌抗人精浆蛋白的使人精子制动单抗的细胞株，Ｉｇ亚类分析表明，此单抗为ＩｇＭ。间接ＥＬＩＳＡ试验、精子制动试验、精子凝集试验和间接免疫荧光等试验分别证明所得单抗能与人精浆和无精子症患者的精浆产生特异免疫反应，有制动人精子的作用，并能特异结合到人精子的颈部，从而证实此单抗是精子制动单抗，其相应的抗原系精浆和精子共有的精子膜抗原，用ＦＰＬＣ－羟基磷灰石柱层析成功地从小鼠腹水纯化了此单抗。     

Sperm antigens and reactivity of antisperm monoclonal antibodies in elisa

Summary. Several types of sperm antigenic suspensions as well as the whole sperm, either methanol-fixed or air-dried, were checked for intensity of binding to monoclonal antisperm antibodies with known characteristics of reactivity to sperm. The activity of sperm antigen—antibody binding was measured by elisa (enzyme-linked immunosorbent assay) and compared in several variations (parallely run) of the assay where different types of sperm antigen preparations were applied. The obtained results were then evaluated for statistical significance in Wilcox test. It was shown that antibody reactivity was markedly higher in experiments where the whole sperm was coated in a solid-phase in comparison to results obtained with adhered different sperm antigenic suspensions. However, one exception was noted, where the results from elisa, run with sperm organic extract, were (statistically) insignificantly lower than those obtained with the whole sperm. Therefore, organic sperm extracts (containing mostly glycolipids) can be a valuable alternative to screening for antisperm antibody activity and/or infertility background.     

Localization of binding sites of naturally occurring antisperm antibodies on human spermatozoa by immunofluorescence

Antisperm antibodies (ASA) may affect sperm motility, acrosome reaction, sperm penetration of cervical mucus, binding to the zona pellucida, and sperm-egg fusion. We investigated the localization of ASA of infertile men or men after vasectomy bound on the sperm surface using an immunofluorescence method. Binding occurred in the acrosomal region, midpiece, and tail. Most of the ASA in both groups of patients bound to the midpiece alone or in combination with other regions of spermatozoa. Only few ASA samples showed binding to all the three sperm regions. A combination of binding to the acrosomal region and to the midpiece was never observed. In infertile patients with ASA, the binding site was compared with sperm parameters. ASA binding to the sperm head influenced the acrosome reaction. Binding of ASA on tail and/or midpiece was not associated with a significant alteration of viability and motility. Immunofluorescence appears to be a valuable tool in the diagnosis of immune infertility, in particular when impairment of the acrosome activity is suggested.     

The influence of a mineral oil overlay on the zona pellucida binding potential of human spermatozoa

Summary.  The objective of the study was to evaluate the effect of mineral oil on zona pellucida binding potential of human spermatozoa. The study compared zona binding using micro volume droplets under mineral oil as apposed to micro droplets in cryopreservation straws. Spermatozoa from eight proven fertile sperm donors were used. One hundred and fifty five matched hemizonae in 50 μl, 100 μl and 200 μl insemination sperm droplets were co-incubated; (i) under mineral oil and (ii) 0.5 ml plastic cryopreservation straws. The results were analysed to determine the number of the zona bound spermatozoa during each experiment. Microvolumes with an oil overlay had a decrease in sperm bound per hemizona of 38% (mean±SD; 563±415 vs. 921±597), 51% (mean±SD; 392±359 vs. 800±566 sperm) and 18% (mean±SD; 502±369 vs. 618±445) in 200 μl, 100 μl and 50 μl respectively, compared to microvolumes in cryopreservation straws. It was concluded that mineral oil may have some detrimental factors which interfere with zona binding of spermatozoa.     

The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein (tNASP) could result in reproductive failure, we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP (mtNASP). Using mouse as a model, recombinant mtNASP (rmtNASP) and a synthetic peptide, human tNASP_393-408 (htNASP_393–408), were investigated for their antifertility effect. Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice. Sperm-egg binding and fusion assay were carried out in 8–10-week-old BALB/c mice. Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP. There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide. The effect on fertility in the mice immunized with the synthesized peptide was reversible. Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.     

Comparison of two techniques to evaluate the acrosomal status of zona pellucida bound sperm in humans

In this work, we have compared two procedures that evaluate the acrosomal status of human sperm bound to the human zona pellucida. Motile sperm, selected by a Percoll gradient, were capacitated by incubation at 37 degrees C, 5% CO2, for 4.5 h, at 20 x 10(6) cells ml(-1). Then, the sperm were incubated with nonviable human oocytes for 10 min at 37 degrees C, 5% CO2. The oocytes with bound sperm were transferred to 500 microl phosphate-buffered saline (PBS) and washed to remove loosely bound sperm. The oocytes were then processed according to the procedures of Cross et al. (1986) or Liu & Baker (1996). In the Cross's procedure, the sperm were labelled while they were bound to the zona. In the Liu's procedure, the sperm were first dislodged from the zona into a droplet of PBS and labelled in there. Both procedures gave equivalent percentages of acrosome-reacted sperm. However, the total number of zona-bound sperm available for assessment with the procedure of Liu & Baker was greater than that of Cross et al. We suggest to use the former procedure to evaluate the acrosomal status of zona-bound sperm in humans. Moreover, this procedure also provided information about sperm ability to bind to the zona pellucida.     

Flow cytometric determination of spermatozoa binding monoclonal antibodies does not improve the prediction of fertility potential

Summary. In a previous publication the determination of binding of several monoclonal antibodies to human spermatozoa by flow cytometry was described in 223 patients. In normozoo-spermic samples the percentage of spermatozoa binding antibodies was higher than in samples with oligozoospermia. However, only weak correlations were found between the percentage of antibody binding spermatozoa and sperm count, motility and morphology. Thus the antibody binding may represent a property different from the classical parameters and might improve the prediction of fertility in the patients. A questionnaire was sent to all the patients inquiring about a conception in their female partners. One hundred and twenty one out of the 223 patients replied, 103 of whom we were able to evaluate. Forty five of them had induced a pregnancy in their partners. The mean values of sperm count, motility and morphological normal cells, as well as the percentages of cells that bound antibodies, were significantly higher in the group of men whose partners became pregnant, than in those who had not achieved conception. The differences, however, were only marginal. We conclude from our results that the determination of the percentage of spermatozoa binding the monoclonal antibodies used in our study is not likely to improve the prediction of conception probability.     

Significance of sperm antibodies detected by the mixed antiglobulin reaction and the tray agglutination test

Summary. The most widely used tests to detect seminal and serum sperm antibodies are the mixed antiglobulin reaction (MAR as recommended by WHO) and the tray agglutination test (TAT). It has been suggested that the prognostic significance of sperm antibody tests might be influenced by a concomitant reduction of sperm numbers and/or sperm motility. Furthermore, the relative sensitivity of these sperm antibody tests to detect sperm antibodies is not known. We therefore compared TAT, performed with serum and MAR results retrospectively for 565 infertile patients and MAR IgA and MAR IgG results for 1189 infertile patients. The association of TAT and MAR results with changes in sperm number, morphology and motility was assessed for 565 and 1185 patients, respectively. The influence of MAR and TAT results on sperm cervical mucus penetration test (SCMPT) results was investigated for 349 and 434 patients, respectively. Whereas only 23% of all MAR IgG positive patients were also MAR IgA positive, 82% of all MAR IgA positive patients were also MAR IgG positive. There was a significant ( P < 0.0001) correlation between serum TAT, and MAR results. Positive MAR and TAT results were not associated with reductions in sperm number, motility and morphology. There was a significant correlation between MAR IgG and MAR IgA results and the sperm cervical mucus penetration test (SCMPT) results. According to these results, the MAR IgG would be sufficient as an initial screening for seminal sperm antibodies. MAR IgG negative patients with strong indication for immunologic infertility should also be investigated with the MAR IgA and the serum TAT. Serum and seminal sperm antibodies do not seem to influence male fertility via reductions of sperm number, motility and morphology. However because of their significant influence on SCMPT results, MAR IgG and MAR IgA results seem to be of prognostic significance.     

Sperm binding capacity of human zona pellucida derived from oocytes obtained from different sources

Summary. The important contributions of sperm-oocyte interaction to infertility diagnostics is well established. Scientists are urged to search for methods to improve the assessment of gamete interaction. Sperm binding and penetration assays have frequented the literature, reporting on various aspects of sperm-oocyte interaction using either microbisected or whole human oocytes during the assay procedure. The objective of the study was to evaluate additional zona pellucida sources which can be used during zona binding studies. Hemizonae were obtained from the following oocytes: 1) experiment 1, prophase I oocytes from post-mortem ovarian tissue from different age groups namely, 7 months, 5 years, 7 years, 12 years and 30 years; 2) experiment 2 used donated immature Prophase I oocytes from the IVF treatment program and 3) experiment 3 evaluated zona binding for hemizonae which were previously used in hemizona assays. Results indicated that, in experiment 1, ovarian age does not have any influence on the zona pellucida's capacity to bind spermatozoa. The mean number of bound sperm among the different age groups did not differ significantly, namely 38.9±17 (7 months), 31.0±27 (5 years), 49.3±21 (7 years), 32.8 ± 18 (12 years) and 39.5 ± 17 (30 years). The pooled mean ±SD binding for all the age groups in experiment 1 was 37.7 ± 7. Likewise, the mean number of sperm bound (experiment 2) to zonae collected from oocytes using different ovulation induction regimes were 31.1±20 (unstimulated), 54.4±12 (HMG/HCG) and 15.3 ± 9 (HMG alone). The pooled binding data for experiment 2 were 33.0 ± 20. Results of experiment 3 indicated metaphase II oocytes with previous exposure to sperm retained its binding capacity indicating that hemizonae can be recycled for at least a second binding experiment. Zonae that had been exposed to sperm and that were subsequently stripped from bound sperm, revealed a mean number of bound sperm after re-insemination that were significantly higher than the prophase I oocytes namely, 115.0 ± 2.8 versus 35.6±12 (P = 0.0001). In conclusion, the data highlights (i) new sources of human oocytes needed for sperm-oocyte interaction studies; (ii) the capability of the human zona pellucida to bind sperm after previous exposure and (iii) the importance of nuclear competence to obtain increased zona pellucida binding.