Hemizona Assay Research Articles

Symposium 1-4 - Immunological infertility caused by gamete immunity

1. Infertility caused by anti-sperm antibodies (ASA) 1) Female infertility caused by sperm-immobilizing antibodies in the sera In some women, ASA are produced as allogenic immunity, which causes infertility. Various methods for the antibody detection have been developed, but sperm-immobilization antibodies (SI-Ab) detected by the sperm immobilization test (SIT) have the greatest influence on the development of infertility. Sperm-immobilizing antibodies are present in the female genital tract, and also in the follicle fluid.SI-Ab cause a post-coital test (PCT) negative or impaired sperm transport from the uterine cavity to the Fallopian tubes, and fertilization disorders. There is a method to quantitatively measure SI-Ab titer, SI50 unit, and treatment methods are decided based on this SI 50 value. In cases with a SI50 value of 10 or more, it is difficult to conceive with AIH, and IVF is effective after preventing antibody contamination into the culture medium. As a mechanism for producing sperm-immobilizing antibodies, it has been speculated that women with disease-specific genes may be exposed to enough amount of sperm through sexual intercourse and develop symptoms triggered by such as chlamydia infection. CD52 is considered to be a promising antigen against sperm-immobilization antibodies, and is expected to be applied for the development of contraceptive vaccines. 2) Male infertility caused by ASA that bind to ejaculated sperm In some men, ASA are produced as autoantibodies, causing infertility. In men, it is not useful for measuring serum antibodies, therefore antibodies that bind to ejaculated sperm (sperm-binding antibodies) should be detected with ImmunoSpheres○R. However, since there are antibodies that are not related to the development of infertility, the treatment strategy can be determined by confirming whether the antibody has a biological effect. Inhibitory effect on cervical sperm penetration is confirmed by PCT and fertilization disorder is examined by the hemizona assay (HZA). If HZA cannot be performed, ICSI is recommended because it is likely to be accompanied by fertilization disorder in cases where the ratio of ASA-bound sperm to total motile sperm is 80% or more. 2. Female infertility caused by anti-zona pellucida (ZP) antibodies The ZP plays an important role in terms of egg formation, fertilization, protection of the embryo. These include the recognition of sperm species of animals, the induction of acrosome reaction, and the prevention of polysperm fertilization. On the other hand, since there is a strong antibody production ability as an antigen, if an anti-ZP antibodies are produced, the function of the ZP is impaired by the antibody, and there is a possibility of developing infertility. It was reported that anti-ZP antibodies were detected at a high rate in the sera of infertile women of unknown cause by the anti-ZP antibody detection method using porcine ZP, followed by controversial reports by another investigators. Therefore, we developed a detection method using human ZP. Anti-ZP antibodies disturb the follicular development, inhibit sperm-ZP binding and hatching. For the purpose of appropriate treatments, it was shown that ICSI is effective for fertilization disorder and assisted hatching (AHA) is effective for hatching disorder, respectively.

Development of homologous hemizona assay as a potential predictor of fertility in buffalo bulls: A preliminary approach

Although, myriads of tests are routinely used, no single test can accurately predict fertilization potential of semen. The hemizona assay (HZA) has advantages in two ways: (a) it determines multitude traits of sperm and (b) it is a controlled sperm function test. In the present study, we developed homologous HZA in buffaloes to predict bull fertility. In this experiment, bulls with fertility rate 53.3% and 48.5% were used as control, whereas bulls with fertility rate 32.6% and 32.2% were used as test semen samples. For HZA, matching buffalo hemizonae were co-incubated with processed buffalo sperm for 4 h. The number of sperms bound to the outer surface of hemizona was determined. No significant difference was observed in sperm binding for co-incubation of same bull sperm with matching hemizona (p < .05). Significant difference in sperm binding to matching hemizona was seen, while two halves were incubated with control and test semen, respectively (p < .05). Hemizona assay index (HZAI) of test bull semen has been determined from percentage of test-sperm bound to the matching hemizona in comparison to control-sperm. For finding relation, HZAI was correlated with respective fertility rates of semen samples, and it was found that a significant positive correlation was present with r=0.83, p < .1. A regression equation of Y=1.39X - 55.8 (where Y=pregnancy rate of test semen sample and X=HZAI of test semen sample) was presented to predict fertility rates of unknown semen samples. Thus, HZA can be used as a potential predictor of buffalo bull fertility.

An Insight into Novel Sperm Cell Proteins as Bio-markers for Male Infertility: A Review.

Male infertility is rising nowadays and accounts for a major part of infertility cases worldwide. Novel tests are being developed for better detection and management of male infertility. Though there are many tests available for diagnosing male infertility like acrosome reaction rate, hemizona assay, in vivo or in vitro sperm penetration assay, sperm DNA damage tests, however, a semen analysis is the most commonly used initial test for male infertility. It is usually associated with failure to detect the cause in many cases, as seminal composition gets affected by a number of factors and can give false reports. Furthermore, it does not give any information about defects in capacitation, sperm-zona pellucida interaction, and sperm's ability to fertilize oocytes. This results in failure of detection and delayed management of male infertility. Hence, the present review was conducted to identify various sperm proteins that play a significant role in spermatogenesis, sperm motility, sperm-zona pellucida interaction, and fertilization. These proteins can be used in the future as markers of male infertility and will aid in better detection and management of male infertility. Methodology: Search for literature was made from 1970 to 2020 from various databases like PUBMED, SCOPUS, Google Scholar on sperm proteins and their role in male fertility using keywords: "sperm protein as bio-markers", "novel sperm proteins as markers of infertility", "Sperm proteins essential for capacitation, sperm motility and oocyte fertilization". Inclusion criteria: All full-length research articles, systematic reviews, meta-analyses, or abstracts on sperm proteins and male infertility published in the English language in peer-reviewed journals were considered.

Sperm function tests in clinical practice.

Conventional semen analysis solely is not completely adequate to predict pregnancy outcomes. Therefore, advanced sperm function tests have been developed and introduced to clinical practice. These tests use different methods and techniques to evaluate different stages of fertilization steps. In this review, we reported some commonly used sperm function tests: sperm penetration assay, sperm-zona pellucida binding test (hemizona assay), acrosomal reaction test, hyaluronan binding test, hypo-osmotic swelling test, magnetic-activated cell sorting and zeta sperm selection. We discussed the literature concerning these tests, the utilization techniques and also purpose and mechanism of each test. We emphasized the importance of sperm function tests in predicting in vitro fertilization and pregnancy outcomes and in the management of infertile couples and also the limitations of these tests. Along with improvements in molecular biology techniques, we believe that more applicative and beneficial tests will be developed in the near future.

Suitability of the hemi-zona assay for the evaluation of the effect of the length of the equilibration period before cryopreservation

The aim of this study was to test the suitability of the interspecific hemizona assay (HZA) to predict the fertilizing capacity of bovine sperm after modifying the length of the equilibration period before freezing and thawing. Ejaculates from 10 proven fertile bulls were split after dilution, equilibrated at 4 °C for either 24 h (control sperm = CS) or 6, 48, 72 or 96 h (test sperm = TS) and cryopreserved. Hemizona (HZ) pairs from in vitro matured pig oocytes were used for the heterologous HZA: After thawing and swim-up (1 h) CS and TS were co-incubated with matching HZ (125,000 S/HZ in 25 μL Fert-TALP) for 4 h. Spermatological analyses (progressive motile sperm (PMS), plasma membrane- and acrosome-intact sperm (PMAI), sperm showing a high degree of DNA fragmentation (%DFI)) were performed after 0 and 3 h of incubation after thawing. After an equilibration time of 48 h and 72 h values for PMAI0h were higher (P < 0.05) compared to PMAI0h values of sperm equilibrated for 6 h, and %DFI3h values were higher after 96 h (P < 0.05) compared to 6 h equilibration. Between 12 and 90 TS and 13–97 CS were tightly bound to each HZ, respectively. The mean Hemizona Index (HZI) after a sperm equilibration for 48 h (HZI = 92.3 ± 12.7) or 72 h (HZI = 98.9 ± 16.23) was higher (P < 0.01) than after an equilibration for 6 h (HZI = 73.3 ± 13.93) or 96 h (HZI = 81.3 ± 11.41). The HZI for 96 h equilibration was moderately negatively related to PMS0h and PMS3h (r < −0.35, P < 0.05). Furthermore the HZI for 6 h equilibration was highly negatively correlated with DFI0h (r = −0,46, P < 0.01). On the basis of these results it can be concluded that the hemi-zona assay is a suitable test to detect alterations in the fertilizing capacity of bovine sperm after modifying the equilibration period.

Human sperm degradation of zona pellucida proteins contributes to fertilization

BackgroundThe mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process.MethodsPurified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay.ResultsThe results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP.ConclusionsThis study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

How accurate is sperm morphology as an indicator of sperm function?

Sperm morphology has been consistently correlated with fertilisation success or failure. The clinical relevance of the percentage normal spermatozoa has been a widely discussed topic amongst infertility specialists and scientists. This study aimed to evaluate the role of sperm morphology as an indicator of additional sperm functions among 114 andrology referrals. The sperm functions that were investigated included chromatin packaging quality (CMA3 test (n = 109), zona-induced acrosome reaction (ZIAR test; n = 36), hemizona assay (HZI; n = 36) and progressive motility (n = 47). Chromatin packaging quality had a negative and significant (P = 0.0001, r = -0.74) correlation with the percentage normal spermatozoa, while progressive motility had a significant and positive correlation (P = 0.0001, 0.59). Accurate sperm morphology scoring as described by the WHO 2010 manual can therefore be used as an indicator of specific sperm functions.

The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo quality

Oviductin is known to be a key player providing a convenient environment for the process of fertilization affecting this by direct interaction with oocytes and sperm. As in vitro embryo production in the context of assisted reproduction for endangered felids is still in the process of optimization, oviductin might be used to improve IVF results. Recombinant His-tagged feline oviductin was expressed by transformed Escherichia coli BL21DE3 cells. The protein was purified by immobilized metal ion affinity chromatography. The effect of the recombinant protein was characterized in three experiments: a hemizona assay for sperm binding analysis, the IVF outcome, and the relative mRNA expression levels in blastocysts after IVF. A significant higher number of bound sperm cells were found after incubation in oviductin. No significant effect on cleavage, morula, and blastocyst rates with or without oviductin incubation during IVF could be observed. However, the relative mRNA abundance of GJA1, a gene, whose expression level is known to be a marker of embryo quality, was significantly increased (P value less than 0.05) in blastocysts after oviductin treatment. In contrast to this, expression of OCT4, HSP70, DNMT1, DNMT3A, BAX, IGF1R, and GAPDH was not significantly affected. We assume that our recombinant oviductin in its current nonglycosylated form is able to enhance sperm binding. Despite of a missing significant effect on IVF outcome, embryo quality in terms of relative GJA1 expression is influenced positively. These promising results demonstrate the value of recombinant oviductin for the IVF in cats.

The role of carbohydrate recognition during human sperm-egg binding

What is the role of carbohydrates in the binding of human sperm to the zona pellucida (ZP) and what are potential implications for pathogenesis? Both lectin-like and protein-protein interactions play an essential role in human gamete interactions. Studies in the mouse and human indicate a role for both lectin-like and protein-protein interactions during sperm binding to the ZP. Non-systematic literature review. Ultrasensitive analysis by mass spectrometry of glycans linked to the human ZP has confirmed that this matrix is coated with a high density of complex type N-glycans terminated with the sialyl-Lewis(x) (sLe(x)) sequence, the universal selectin ligand. Selectins are essential for lymphocyte homing, and they participate in the initial binding of circulating leukocytes to activated endothelium at the sites of infection and tissue injury. Subsequent inhibition studies confirmed that either the sLe(x) tetrasaccharide or neoglycoproteins terminated with this sequence inhibited human sperm-ZP binding by 70% in the hemizona assay. These results support the hypothesis that both lectin-like and protein-protein interactions play an essential role in human gamete interactions. The sLe(x) sequence is also a ligand for siglec-9, a lectin-bearing immunoreceptor tyrosine-based inhibitory motif that transmits inhibitory signals. This siglec is expressed on a wide variety of different types of human leukocytes and lymphocytes. This result is consistent with the hypothesis that human ZP glycans are also being employed for immune recognition of the egg and the histoincompatible embryo prior to blastocyst hatching. This field of study is complex and more experimental work is needed to reveal fully the mechanism of sperm-ZP binding and how it varies between species. Knowledge about the glycans involved in sperm-egg binding may be relevant to infertility due to fertilization failure and also to the mother's immune tolerance of the preimplantation embryo. Studies focused on human sperm-egg interactions carried out by the author and coworkers have been supported by the Life Sciences Mission Enhancement Reproductive Biology Program funded by the State of Missouri, a Research Board Grant (CB000500) supported by the University of Missouri System and a grant from the Jeffress Memorial Trust of Virginia. Support from the Breeden-Adams Foundation has also been obtained to investigate potential linkages to tumor evasion. The author has no conflict of interest to declare.

Treatment of human sperm with serine protease during density gradient centrifugation

Seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective, whereby vertical or horizontal transmission of the infection could occur. Serine proteases have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could negatively impact on sperm parameters. This study investigated the effect of the addition of a recombinant, human-sequence protease (rhProtease) on sperm parameters during density gradient centrifugation. (i) Pooled semen samples (n = 9) were split and processed by density gradient centrifugation, with the top density layers supplemented, or non-supplemented with rhProtease at three different concentrations (diluted 2, 10 and 20 times). Sperm parameters were then analysed by flow cytometry and computer-assisted semen analyses. (ii) Semen samples (n = 5) were split and similarly processed using PureSperm® Pro, with rhProtease in the 40 % density gradient layer, or standard PureSperm® not supplemented with rhProtease (Nidacon, International) respectively. The Hemizona assay was then utilized to compare sperm-zona binding post processing. Evaluation of sperm parameters indicated that rhProtease did not, at any of the tested concentrations, have an impact on (i) mitochondrial membrane potential, vitality, motility, or (ii) zona binding potential. We report that the addition of rhProtease to density gradients is a non-detrimental approach that could improve the effectiveness of semen processing for the elimination of seminal pathogens, and benefit assisted reproduction outcome.

Genetic and physiological study of morphologically abnormal human zona pellucida

To investigate genetic, molecular and functional aspects of human zona pellucida (ZP) in oocytes with an abnormal appearance. The study included three women with unexplained infertility whose oocytes had an abnormal ZP appearance and the mother and fertile sister of one of them. The coding exons and their flanking intron regions of the four ZP genes and the regulatory element for the ZP3 gene were sequenced. Immunofluorescence staining of discarded oocytes using monoclonal antibodies against recombinant human ZP glycoproteins and a hemizona assay were performed. No new mutations were observed in the ZP1 (12 exons), ZP2 (19 exons), ZP3 (9 exons), ZP4 (12 exons) genes or in the ZP3 regulatory element of the three studied women. Sequencing of the genes revealed eight synonymous and non-synonymous reported polymorphisms only in ZP1, ZP2 and ZP3. Immunofluorescence staining of the discarded oocytes of two women showed clear and strong staining of the ZP1, ZP2 and ZP4 proteins, but weak staining of the ZP3 protein, although their ZP displayed normal sperm binding ability in the hemizona assay. Intracytoplasmic sperm injection yielded good pregnancy outcomes, even though few injected oocytes developed normally up to day 3. The abnormal oocyte ZP appearance in the three study women may not have been due to the genetic changes in the ZP genes. Moreover, sperm binding was normal despite low ZP3 staining observed, suggesting that ZP3 profile may play a subordinate role in the reported cases. Our findings support previous studies which claim that abnormal oocyte morphology is not associated with a decrease in fertilization rates or birth outcomes in couples undergoing intracytoplasmic sperm injection.

Involvement of the prostate and testis expression (PATE)-like proteins in sperm-oocyte interaction

The prostate and testis expression (PATE)-like family of proteins are expressed mainly in the male genital tract. They are localized in the sperm head and are homologous to SP-10, the acrosomal vesicle protein also named ACRV1. Our aim was to characterize the expression and functional role of three PATE-like proteins in the testis and ejaculated sperm. The expression and localization of PATE-like proteins in human testis biopsies (n= 95) and sperm cells were assessed by RT-PCR, immunohistochemistry and immunofluorescence staining (at least 600 sperm cells per specimen). The function of the PATE protein was tested by the hemizona assay and hamster egg penetration test (HEPT). PATE and PATE-M genes and proteins were present almost exclusively in germ cells in the testis: immunoflourescence showed that the percentage of germ cells positive for PATE, PATE-M and PATE-B was 85, 50 and 2%, respectively. PATE and PATE-M proteins were localized in the equatorial segment of the sperm head, while PATE-B protein was localized in the post-acrosomal region. A polyclonal antibody (Ab, at 1:50 and 1:200 dilutions) against the PATE protein did not inhibit sperm-zona binding in the hemizona assay (hemizona index of 89.6 ± 10 and 87 ± 36%, respectively). However, there was inhibition of sperm-oolemma fusion and penetration in the HEPT (penetration index: without Ab 7 ± 3.9; Ab dilution of 1:100, 4 ± 3.5; Ab dilution of 1:20, 0.6 ± 1.2, P < 0.001). Our data suggest that PATE protein is involved in sperm-oolemma fusion and penetration but not sperm-zona binding.

Selectivity of porcine zona pellucida to bind spermatozoa with normal chromatin structure

This study aimed to investigate the selective ability of swine zona pellucida (ZP) to bind spermatozoa with normal nuclear chromatin. Ten ejaculates of four boars were used, while hemizona assay was applied for evaluation of binding capability. The results of this study showed that swine ZP has the ability to select spermatozoa with normal chromatin structure for sperm-zona binding process.

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 μm² vs. 18.8 ± 1.4 μm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility

Couples with unexplained infertility (UI) tend to have low fertilization rates with current IVF procedures. Here, we attempted to identify spermatozoa with apoptotic markers in couples with UI and unsuccessful intrauterine insemination (IUI) and we investigated the efficiency and benefit of magnetic-activated cell sorting (MACS) for sperm preparation in such patients. Sixty couples with UI and two IUI failures were recruited. The sperm were prepared by conventional density gradient centrifugation (DGC) and divided into two aliquots. One aliquot was used as a control and the other was further processed by MACS (D + M). Apoptotic markers were identified using fluorescence-labeled dye and flow cytometry, including externalization of phosphatidylserine (EPS), disrupted mitochondrial membrane potential (MMP) and DNA fragmentation. The fertilization potential of prepared spermatozoa was analyzed by basic semen analysis, computer-aided sperm analysis and the induced acrosome reaction test (IART). After DGC, spermatozoa showed 18.6% EPS, 28.3% disrupted MMP and 13.5% DNA fragmentation. Numbers of spermatozoa with apoptotic markers were significantly reduced by D + M, versus DGC alone (P < 0.001). Although the motility of spermatozoa was slightly decreased after MACS, most sperm motion characteristics were not impaired. Interestingly, the IART significantly improved after D + M, versus DGC alone, especially for the couples with a normal hemizona assay (P < 0.001). The spermatozoa prepared by D + M showed a reduced level of apoptotic markers. Improvement in the IART suggests a high fertilization potential of the processed spermatozoa. The identification of apoptotic markers and use of MACS may be helpful in directing the management plan for patients with UI and multiple IUI failures.

Neural cadherin is expressed in human gametes and participates in sperm–oocyte interaction events

Neural cadherin (N-cadherin) is a transmembrane glycoprotein involved in calcium-dependent cell-cell adhesion and signalling events. The previous evidence shows N-cadherin expression in the human gonads and gametes; however, N-cadherin subcellular localization in human spermatozoa and oocytes, and its involvement in fertilization remain to be characterized. In this study, expression of N-cadherin in human spermatozoa and testis was confirmed by RT-PCR and protein forms were identified using Western immunoblotting. N-cadherin localization in testicular and ejaculated spermatozoa, in cells that had undergone capacitation and acrosomal exocytosis, as well as in oocytes was assessed using immunocytochemistry. Participation of the adhesion protein in fertilization was evaluated using the HemiZona Assay (HZA) and the zona pellucida (ZP)-free hamster oocyte sperm penetration assay (SPA). Both the N-cadherin transcript and the mature protein form (135 kDa) were found in spermatozoa and testis. The protein was mainly immunolocalized in the acrosomal region of testicular, non-capacitated and capacitated spermatozoa, and was found in the equatorial segment after acrosomal exocytosis. N-cadherin was also detected in oocytes, in conjunction with beta-catenin, a member of the adhesion complex. Sperm incubation with anti N-cadherin antibodies did not affect their ability to interact with homologous ZP in the HZA; by contrast, presence of the antibodies in the SPA led to a significant (p < 0.01) reduction in the percentage of penetrated oocytes. In conjunction, results indicate that N-cadherin is a sperm protein of testicular origin localized in cellular regions involved in gamete interaction. N-cadherin would not participate in sperm-ZP interaction, but it would have a role in sperm-oolemma adhesion/fusion events.

Identification of human sperm proteins that interact with human zona pellucida3 (ZP3) using yeast two-hybrid system.

Sperm proteins that interact with zona pellucida 3 (ZP3) have not been clearly identified in humans. In the present study, the yeast two-hybrid (Y2H) system was used to identify human sperm proteins that interact with human ZP3. Human ZP3 cDNA was cloned into pAS2-1 yeast vector and used as bait to find reactive proteins in the human testis cDNA library. Six specific clones were obtained that were further confirmed for interaction using the mammalian two-hybrid system. These six clones showed homologies with several proteins in the GenBank database. Of these, the strongest ZP3-interacting protein, that shows 97% homology with ubiquitin associated protein-2 like (UBAP2L), was tested in the hemizona assay. UBAP2L antibodies significantly (p<0.001) inhibited human sperm-zona binding in this assay. We conclude that the Y2H system is a useful strategy for identifying novel genes encoding proteins that interact with ZP proteins. To our knowledge, this is the first study using the Y2H system to identify sperm proteins that interact with human oocyte ZP3. Novel proteins identified using this system may find applications in elucidating the fertilization cascade, development of a new generation of non-steroidal contraceptives, and specific diagnosis and treatment of human infertility.

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

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 x 10(6) and 0.8 x 10(6) sperm ml-1. Five separate droplets each of 20 microliters containing 4 x 10(6) sperm ml-1 (80,000 motile sperm) and 200 microliters containing 0.8 x 10(6) sperm ml-1 (160,000 motile sperm), respectively, were prepared. Both volumes were incubated for 18 h at 37 degrees C. The mean (+/-SD) number of spermatozoa tightly bound to hemizona, incubated in 20 microliters and 200 microliters 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.

Effect of sperm preparation with TEST yolk buffer on sperm-binding capacity under hemizona assay conditions.

The study was conducted to evaluate the diverse effect and clinical significance of TEST yolk buffer treatment on sperm samples of 128 infertile men. Sperm samples were incubated with TEST yolk buffer and control medium (Ham's F-10) at room temperature for 2 h. The hemizona indices (mean +/- SE) of the TEST yolk buffer and medium-treated sperm samples were 29 +/- 2.3% and 22 +/- 1.6%, respectively. Inspection of the individual response of each sperm sample to TEST yolk buffer revealed that 63 samples (49%) improved (double the interassay variation = 28%) their binding to zona pellucida, 36 (28%) remained unchanged, whereas the binding capacity of 29 samples (23%) decreased. Furthermore, TEST yolk buffer treatment of 24 samples (19%) resulted in an increased binding beyond the hemizona index threshold set up at 23%. This level was previously shown to be the cut-off point between fertile and infertile sperm samples. It was concluded that when applied to an unselected group of infertile men, TEST yolk buffer significantly increased sperm binding capacity to the zona pellucida. However, only 19% of the sperm samples showed improvement with clinical significance. The other sperm samples may have improved, remained unchanged or even deteriorated independently on basic sperm variables. Thus, the effect of TEST yolk buffer treatment on sperm binding should be tested prior to its clinical use to avoid possible damage to certain sperm samples.

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.