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.     

Zona pellucida mediated acrosome reaction and sperm morphology

Summary. Sperm samples from 29 men randomly selected from the andrology laboratory, were used to evaluate acrosome reaction response to solubilized human zona pellucida. Capacitated sperm samples were exposed to a solution containing 2 zona pellucidae (ZP) per μl for 60 min, after which acrosomal status were recorded using a PSA-FITC technique. Controls included samples supplied by fertile sperm donors. After completion of acrosome reaction studies, patient samples were divided according to the percentage of morphologically normal spermatozoa. Three basic groups were identified, namely, fertile donors, teratozoo-spermic (normal sperm morphology 5–14%; n = 25) and severely teratozoospermic (normal sperm morphology <4%; n = 4) groups. The mean percent normal sperm were 15.8 ± 0.9, 10.4 ± 0.7 and 2.7 ± 0.7, respectively, for normozoospermic donors, teratozoospermic and severely teratozoospermic men. The mean percentage (± SE) ZP mediated acrosome reacted sperm among teratozoospermic and severely teratozoospermic cases was 25.8% ± 0.9 and 19.0% ± 0.9 (P = 0.001), compared to 36.8% ± 0.9 for the donor controls. Results were analysed and expressed as correlations between sperm morphology and acrosomal response to human solubilized zona pellucida, spontaneous and calcium ionophore induced acrosome reaction. Predictive values for acrosome responsiveness were depicted with ROC curve analyses. Sperm morphology evaluated by strict criteria correlated positively and highly significantly with the responsiveness of the acrosome reaction (r = 0.91, P = 0.0001). At a morphology cut-off value of 4%, the ROC curve analysis showed sperm morphology to be highly predictive of zona pellucida induced acrosome responsiveness with a sensitivity of 100% and negative predictive value of 100%. Spontaneous and calcium ionophore induced acrosome reactions revealed no correlation with sperm morphology. It was concluded that (i) morphological features of human spermatozoa are indicative of specific functional characteristics; (ii) zona pellucida induction of the acrosome reaction is superior, as a predictor of sperm morphology, compared to calcium ionophore induced and spontaneous acrosome reactions.     

Acrosome reaction in the head-attached human sperm

Summary: The physiological acrosome reaction of the sperm occurs on the surface of the oocyte. Although its occurrence on the zona pellucida is considered to be a specific response to zona compounds, whether the simple attachment itself could influence the reaction of motile spermatozoa to a fixed surface and promote onset of the reaction is questioned. A motile human sperm head fixation method was developed recently, through which a group of head-attached sperm can be established easily. These head-attached sperm were investigated for their acrosome reactions. After staining by fluorescein iso-thiocyanate conjugated peanut lectin (FITC-PNA), many head-attached spermatozoa (75.7%) were found to have undergone an acrosome reaction.     

Hemizona assay (HZA) demonstrates effects of characterized mouse antihuman sperm antibodies on sperm zona binding.

Characterized antihuman sperm monoclonal antibodies from mice were evaluated using the hemizona assay (HZA) to determine whether sperm:zona binding was effected. The seven monoclonal antibodies were characterized using human sperm in agglutination, immobilization, and penetration assays. Semen was provided by four fertile men and used in the HZA to determine if the presence of a monoclonal antibody would affect tight binding of the sperm to the zona pellucida. Pre-incubation of MA-14 for 1 h with the sperm induced a 33-54% reduction of the number of tightly bound sperm. This antibody reacts to an antigen located on the acrosome and midpiece. Experiments in which there was no pre-incubation of the antibody with sperm, resulted in no significant reduction in the number of sperm bound in the HZA. These findings suggest that an anti-human sperm antibody produced in mice can modulate sperm:zona binding. Reduction in zona binding could indicate a cause of immune-related infertility and this test may be useful in selecting an antigen for contraceptive vaccine development.     

Early penetration of human sperm through the vestments of human eggs in vitro

Penetration of human sperm through the vestments of human oocytes during the first 3 h after insemination was investigated to determine the time taken for sperm capacitation, which precedes the acrosome reaction and fertilization. Twelve oocytes from women who became pregnant by IVF, recovered at laparoscopy after appropriate stimulation, were examined by electron microscopy. Follicular development was controlled by administration of clomiphene citrate, hMG, and hCG. Oocytes were cultured in Whittingham's T6 medium for 7-14 h before insemination and were fixed 1-3 h after insemination with preincubated sperm obtained from fertile men. All oocytes had matured and eight were normally fertilized 2-3 h after insemination. The acrosome reaction had already begun 1 h after insemination when 10-30% of sperm had reacted. Sperm-oocyte membrane fusion occurred 2 h after insemination, and sperm decondensation and pronuclear formation were in progress 3 h after insemination. Variable numbers of sperm (30-60%) had reacted acrosomes after 2-3 h of insemination. Many sperm penetrating the cumulus were intact or had partially reacted acrosomes. Intact, partially and fully reacted sperm were found at the surface of the zona. Intact sperm were bound to the zona by their plasma membranes. Sperm penetrating the zona had reacting or reacted acrosomes exposing their inner acrosome membranes. Those approaching the perivitelline space had a persistent equatorial vestige of the acrosome with intact plasma membrane. The acrosome reaction involved multiple fusions of the sperm plasma and outer acrosome membranes, resulting in vesiculation. This study shows that human sperm could complete capacitation and initiate the acrosome reaction within 1 h of insemination in vitro.     

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.     

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.     

Extracellular signal-regulated kinase activation involved in human sperm-zona pellucida binding

In a previous study involving the inhibition the mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK), we found that the very specific MAPK kinase (MEK) inhibitor, PD098059, inhibited the zona pellucida (ZP) induced acrosome reaction. As an intact acrosome on the spermatozoa is a prerequisite in ensuring tight binding to the ZP, we investigated the zona binding potential of spermatozoa after PD098059 treatment of sperm, followed by exposure to solubilised human ZP and calcium ionophore (A23187). PD098059 treated spermatozoa, exposed to solubilised ZP, bound significantly more to the ZP, as compared to control spermatozoa also exposed to solubilised ZP (26.5 +/- 3.7 vs. 13.8 +/- 2.8, P < 0.05). No significant differences in binding to the ZP were observed between PD098059 treated and untreated sperm populations after A23187 exposure. These results can be interpreted to support the idea that the ZP-induced AR is the physiologically relevant exocytotic event, as it is the ZP-induced AR, and not the spontaneous (culture medium) or A23187 induced AR, that appears to be mediated through an ERK-mediated signal transduction process.     

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.     

Sperm—oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10⁷ cells ml⁻¹ after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.     

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 相似文献   

Inhibition of sperm-zona pellucida binding by a 55 kDa pig sperm protein in vitro

This study was conducted to evaluate the role of a 55 kDa pig sperm protein on the oocytesperm binding process, and its location in situ. For this purpose, in vitro matured oocytes were incubated with isolated and purified protein, and incubated with capacitated spermatozoa. In addition, capacitated sperm were incubated with anti-55 kDa antiserum and later with mature oocytes. Immunolocalization assays were performed using non-capacitated, capacitated and acrosome reacted sperm, which were incubated independently with anti-55 kDa protein antibodies and analyzed under fluorescence light microscopy. The 55 kDa protein concentrations correlated negatively with the amounts of sperm bound to the zona pellucida (ZP); the presence of the anti-55 kDa protein totally inhibited this binding. The immunolocalization assays revealed that fluorescence was located preferentially at the apical edge of the head in capacitated sperm, but not in acrosome reacted sperm. It would appear that the 55 kDa protein binds specifically to the oocyte ZP, and that it may be responsible for primary gamete binding during fertilization.     

Correlation between sperm penetration into the human zona pellucida and in vitro fertilization rates

Summary.  Sperm penetration into the zona pellucida of unfertilized oocytes, and its correlation with in vitro fertilization rates of the sibling oocytes, were assessed. This was performed in order to evaluate the prediction rate of the sperm penetration test into the zona pellucida. Unfertilized oocytes ( n =1872) from 371 cycles were pipetted through a microcapillary, and the remaining sperm cells penetrating the zona pellucida were counted. The mean (±SD) number of spermatozoa that penetrated the zona pellucida of unfertilized oocytes was 12.9±16.37. A significant correlation was found between the fertilization rate and the mean number of spermatozoa that penetrated into the zona pellucida of the unfertilized sibling oocytes (r = 0.48; P < 0.001), or the percent of unpenetrated zonae pellucidae in a cohort (r= —0.43; P < 0.001). However, a distinct variation in the number of spermatozoa that penetrated into the zona pellucida was detected. A step-wise regression analysis proved the number of spermatozoa penetrating the zona pellucida to be more predictive for fertilization rates than the variable of percent of unpenetrated zonae pellucidae. The results imply that although there is interdependence between penetration into the zona pellucida and fertilization rate, the predictive value of sperm penetration test for prognosis and future management after the first in vitro fertilization attempt, is limited.     

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.     

In vitro tests for essential sperm functions using the phyto-oestrogen genistein as a test substance

Sperm motility, binding of spermatozoa to the zona pellucida and induction of the acrosome reaction are prerequisites for successful oocyte fertilization. Examination of the physiological and nonphysiological effects of particular compounds on sperm functions requires high-quality in vitro test systems. In this short methodological overview, a reliable combined in vitro test system with bovine gametes is described. The purpose of the study was to evaluate whether aliquots of pooled post-thaw spermatozoa are suitable for examination of environmental substances that affect essential sperm functions. The combined test system includes a number of known methods for the assessment of sperm vitality and motion parameters, acrosomal status, inducibility of acrosome reaction and sperm zona pellucida binding. First observations indicate that genistein inhibits the induction of acrosomal exocytosis and binding of spermatozoa to the zona pellucida. Motility parameters and the viability of bovine spermatozoa were not affected by this substance. It is concluded that genistein, a phyto-oestrogen which is abundant in several plants, can be used as a test substance for the evaluation of effects upon essential bovine sperm functions in vitro.     

Laser-assisted cryopreservation of single human spermatozoa in cell-free zona pellucida

Improved procedure for efficient cryopreservation of single human spermatozoa in cell-free human zona pellucida is reported. We used a diode laser system for efficient and precise creation of a single hole into the zona pellucida of a degenerated or immature human oocyte. This allowed the extraction of the cytoplasm using a micropipette with a diameter of 10–15  μm. Through the same opening, human spermatozoa were inserted into the empty zona. We used motile and laser immobilized spermatozoa. Immobilized sperm were obtained by a single laser irradiation delivered in the vicinity of the sperm tail prior to insertion. This new immobilization procedure was shown to have no deleterious effect on membrane integrity and sperm viability. Following sperm transfer into the zona, the laser-drilled hole was closed with an oil droplet which was expelled from the micropipette during withdrawal to avoid loss of spermatozoa. This facilitated detection of the otherwise translucent zona during the cryopreservation procedure. After thawing, all cryopreserved zonae (20/20) could be successfully retrieved. Spermatozoa were recovered from the zona pellucida through the hole used for insertion. The rate of sperm recovery for initially motile spermatozoa was 80% vs. 92% for laser immobilized spermatozoa. Sperm viability was 81% and 84%, respectively, detected by a Hoechst stain. This technique makes cryopreservation of single human spermatozoa easy and feasible and appears beneficial for couples with severe male infertility and for those facing repeated surgical sperm extraction.     

Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility

Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.     

Effect of genistein on acrosome reaction and zona pellucida binding independent of protein tyrosine kinase inhibition in bull

Aim： To investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa. Methods： The effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin （FITC-PSA）/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline （CTC） assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa. Results： Immunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively. Conclusion： Our results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.     

Can a cumulus cell complex be used to select spermatozoa for assisted reproduction?

Since the onset of intracytoplasmic sperm injection, researchers have intensified the search for the ideal spermatozoa to be used for injection. The aim of this study was to record the functional role of cumulus cell interaction with human spermatozoa as far as capacitation, acrosome reaction, morphology, zona binding and chromatin packaging quality are concerned. Using a previously described cumulus oophorus model, we recorded specific sperm functional aspects of sperm populations that transverse a cumulus cells mass. Control spermatozoa were kept under similar experimental conditions in the culture media only. Results indicated cumulus cells to be beneficial to spermatozoa as far as functional and capacitational events are concerned. The mean percentage of morphologically normal spermatozoa in the control sample was 6.9%, while the spermatozoa that traversed the cumulus oophorus (test) had a significantly higher percentage of normal forms (mean 9.5%; P  ≤   0.01). We observed a decline in the percentage of CMA3-positive spermatozoa when we compared the control population (49.1%) to the test, i.e. 38.4%, ( P  = <0.05), thus implying that the spermatozoa with good chromatin condensation increased during cumulus penetration. Significantly more ( P  ≤   0.01) acrosome-reacted spermatozoa were found in the penetrated spermatozoa (mean 23%) than in the control spermatozoa (mean 11%). The test spermatozoa had a higher zona binding capacity with significantly more ( P  ≤   0.01) tightly bound spermatozoa on the hemizona (61 ± 15) than the control spermatozoa (47 ± 18). In the absence of sophisticated and expensive sperm selection products, the use of a cumulus model to select spermatozoa for intracellular sperm injection seems to be an alternative method.     

A new method to produce equally sized hemizonae pellucidae for the hemizona assay

The hemizona assay (HZA) is a valuable tool to study the binding potential and interaction of spermatozoa with the zona pellucida. Its accuracy strongly depends on the use of equally sized hemizonae. Usually, manipulator-guided microblades are used for cutting the zona pellucida. Recently, lasers were introduced for precise local thermolysis of the zona. The use of a 1.48 microm diode laser for the generation of hemizonae from human oocytes was investigated. This laser allowed drilling of equally sized hemizonae which were used for hemizona binding assays. It is concluded that the 1.48 microm diode laser system can be applied for the production of hemizonae. The method is easy to perform and offers a fast and efficient access to hemizonae of identical size.