ZP Binding Research Articles

P–064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer: The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings: In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number Not applicable

P-064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number not applicable

Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals.

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies.

Ready for the journey: a comparative proteome profiling of porcine cauda epididymal fluid and spermatozoa

In the present study, we describe the proteome of porcine cauda epididymis fluid and spermatozoa by means of Multidimensional Protein Identification Technology (MudPIT). Ten sexually mature healthy boars were surgically castrated and epididymides were dissected to obtain the cauda epididymal content. Polled protein extracts of cauda epididymal fluid (CEF) and spermatozoa (CESperm) were loaded in an Agilent 1100 quaternary HPLC and peptides eluted from the microcapillary column were electro-sprayed directly into a LTQ Orbitrap XL mass spectrometer. Using bioinformatics, identified proteins were classified by their molecular functions, involvement in biological processes and participation in relevant metabolic pathways associated with spermatozoa physiology, fertility potential and protection. A total of 645 proteins were identified in the CEF, with epididymal-specific lipocalin-5, beta-hexosaminidase subunit beta precursor and phosphatidylethanolamine-binding protein 4 being the most abundant proteins found. A total of 2886 proteins were identified in the CESperm proteome with 81 proteins being considered more abundant (spectral counts > 100). CEF and CESperm data were compared and 345 proteins were present in both proteomes. Phosphatidylethanolamine-binding protein 4 precursor was the only protein found most abundant in both CEF and CESperm proteomes. Based on Gene Ontology analysis, we identified CEF and CESperm proteins associated with sperm protection against ROS and immune mediated response, glycosaminoglycan degradation, ubiquitin-proteasome system, metabolic process and maturation, modulation of acrosome reaction and ZP binding and oocyte penetration. These results provide a better comprehension about the molecular process and biological pathways involved in sperm epididymis maturation and establishment of the cauda epididymis sperm reservoir.

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.

BackgroundIn order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding.ResultsExperiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents (P < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10–17 replicates; n = 429).Experiment 2: Twelve ejaculates and 28 straws of semen were used (11–19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively (P < 0.05).Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5–6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) (P > 0.05).ConclusionsChemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm, but between sperm treatments no significant differences were obtained.

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 μm) and EGCG (10, 20 and 60 μm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 μm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.

Sperm-oocyte interaction and in vitro fertilization clinical outcomes in patients with unexplained infertility

To determine the sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction in patients with unexplained infertility, and to discuss the relationship between ZP-induced acrosome reaction and fertilization rate. We compared the fertilization rate and good embryo rate in patients with unexplained infertility after fertilization in 2 ways. Based on the causes of infertility, patients were divided into an unexplained infertility group (Group A) and a pure female tubal factor group (Group B). Oocytes which were obtained by super ovulation from 25 patients with unexplained infertility were randomly divided into 2 groups with conventional in vitro fertilization (IVF) (Group A1) and intracytoplasmic sperm injection (ICSI) fertilization (Group A2). The pure female tubal factor group (Group B) had conventional IVF. We conducted sperm-ZP binding and ZP-induced acrosome reaction experiments with 2 groups of men's sperms separately. We compared the number of sperm-egg binding and ZP-induced acrosome reaction rate and discussed the relationship between the ZP-induced acrosome reaction and fertilization rate, and also the fertilization rate, good embryo rate and pregnancy rate in patients with unexplained infertility after fertilization in 2 ways. The average number of sperm-egg binding (78.29 ± 16.31) and the ZP-induced acrosome reaction rate (55.87 ± 27.69) % in Group A were lower than those of Group B [94.63 ± 6.72, (82.53 ± 17.99)%]. The difference between the average number of sperm-egg binding and the ZP-induced acrosome reaction was significant (P <0.01). The fertilization rate of Group A1 was significantly lower than that of Group B and Group A2 (P <0.01). But there was no significant difference in the good embryo rate among the 3 groups. There was no significant difference between Group A2 and B in fertilization rate and good embryo rate (P <0.05). There was no significant difference in pregnancy rate between Group A and B (P <0.05). Fertilization rate and the rate of acrosome reaction had marked positive correlation with statistical significance (r =0.932, P <0.01). ZP binding and ZP-induced acrosome reaction are very important experiments in sperm function test for patients with unexplained infertility. It can not only effectively avoid no embryo transferring due to complete failure of fertilization but also get a desirable outcome of pregnancy using half-ICSI fertilization in patients with unexplained infertility.

180. CHARACTERIZATION AND FUNCTION OF THE BULL SPERM PROTEIN Spam1: TWO DISTINCT ISOFORMS WITH DISTINCT ROLES

We identified an 80 kDa bull sperm protein (p80) that possesses homology with the Sperm adhesion molecule 1 (Spam1), a GPI-anchored glycoprotein conserved amongst mammals that is required for fertilization. Since bovine Spam1 had not been identified, the aim of this project was to determine if p80 is the bovine Spam1 and to test the hypothesis that it plays a role in gamete interaction during bovine fertilization. Amino acid sequence deduced from 3`/5`Race confirmed that homology between p80 and Spam1 in various species ranged from 47 to 61%.It also revealed specific differences including the absence of a GPI-anchor, the presence of a transmembrane domain, and N- and O- glycosylation sites. By generating and characterising antibodies against p80 N- and C-terminal domains, the protein orientation in the sperm membrane was evaluated. We identified two populations of p80 on the sperm head: one internalised in the anterior region and the second localised on the post-acrosomal region with its hyaluronidase domain exposed to the extracellular environment. Proteomic and immunologic analyses revealed that the p80 post-acrosomal population is a shorter isoform originating from the epididymis while the full length p80 located on the anterior region originates from the testis. Finally, the potential function of p80 during the sperm/zp interaction was evaluated by sperm/zona pellucida (zp) binding assay. The C-terminal extremity of p80 was implicated in sperm binding to the zp by antibody and native protein competition. Furthermore, glycosylation was not required during this interaction since deglycosylated p80 in the incubation medium had the same inhibitory effect on zona binding as the native p80. Collectively, the results demonstrated that p80 is the bovine Spam1, and that two isoforms are present on bull sperm. The hyaluronidase activity of the post-acrosomal isoform is required for cumulus penetration, while the other one participates in sperm/zp binding during fertilization.

Effect of Protease Inhibitors on the Acrosome Reaction and Sperm‐Zona Pellucida Binding in Bovine Sperm

Acrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin-like, chymotrypsin-like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim-up method (4 x 10(6) cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N-alpha-p-tosyl-l-lysine-chloromethyl-ketone (trypsin inhibitor); Trypsin inhibitor (I-S Type from soybean); N-tosyl-l-phenylalanine-chloromethyl-ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm-zona binding capacity was evaluated using cumulus cell-free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome-reacted sperm was observed only in cells incubated with trypsin (10.2 +/- 1%) and chymotrypsin inhibitors (18.5 +/- 1%) in relation to the control (52.2 +/- 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 +/- 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.

Zona pellucida binding test: a method for functional sperm seletion for ICSI

OBJECTIVE: Sperm and egg interactions consist of a series of events involving species recognition, adhesion and fusion. In infertile men, a small proportion of sperm is capable of binding to the zona pellucida in vitro and there is a relationship between sperm morphology and the proportion of sperm bound to the ZP. In addition, sperm with single stranded or denatured DNA generally do not bind to the ZP. These evidences suggest that the ZP has the capacity to selectively bind normal functional sperm. We evaluate weather ZP binding test may be a toll to select more functionally competent spermatozoa for ICSI.DESIGN: Case-control study.MATERIALS AND METHODS: We evaluated 316 oocytes collected from 26 patients undergoing ICSI cycles. For sperm-ZP binding test, each patient, had a metaphase I-stage oocyte incubated with 5μL of ejaculated sperm sample (concentration: 2 x106/mL) for 1 h. The sperm bounded to the ZP were mechanically removed and used to inject the metaphase II (MII) oocytes. For each patient, MII oocytes were split in two groups: control-group (n=164), in which the routine selection of sperm was conducted, and ZP-group (n=152), in which ZP-bounded sperm was injected. Fertilization and high quality embryos rates were compared between the groups and also, to study the influence of the ZP-bounding sperm injection on the embryo quality, a logistic regression model was conducted. The results were presented as odds ratios and its 95% confidential interval.RESULTS: The fertilization rate was similar among the groups (control: 71.9% ZP: 80.2%, p=0.084). However, the high quality embryos rate was higher when a ZP-bounded sperm was selected for injection (control: 68.6% vs ZP: 85.9%, p=0.001). Furthermore, ZP-bound sperm injection positively influenced the embryo quality (OR = 2.79 IC 95% = 1.47 – 5.32; p = 0.001).CONCLUSIONS: The present study showed that the fertilization rate was not affected by the ZP-bound sperm injection; however, once fertilized, embryo development may be positively affected. Sperm capable of binding to the ZP represents a population that satisfies a necessary condition for fertilization. The in vivo sperm binding to the ZP initiate the chain reaction that culminates with fertilization; nonetheless, the molecular, biochemical, and physiological changes in the sperm while it binds to ZP, and whether these changes are associated with embryo development in vitro is unclear. Our findings suggest that ZP sperm binding may select more competent sperm, able to improve embryo development. OBJECTIVE: Sperm and egg interactions consist of a series of events involving species recognition, adhesion and fusion. In infertile men, a small proportion of sperm is capable of binding to the zona pellucida in vitro and there is a relationship between sperm morphology and the proportion of sperm bound to the ZP. In addition, sperm with single stranded or denatured DNA generally do not bind to the ZP. These evidences suggest that the ZP has the capacity to selectively bind normal functional sperm. We evaluate weather ZP binding test may be a toll to select more functionally competent spermatozoa for ICSI. DESIGN: Case-control study. MATERIALS AND METHODS: We evaluated 316 oocytes collected from 26 patients undergoing ICSI cycles. For sperm-ZP binding test, each patient, had a metaphase I-stage oocyte incubated with 5μL of ejaculated sperm sample (concentration: 2 x106/mL) for 1 h. The sperm bounded to the ZP were mechanically removed and used to inject the metaphase II (MII) oocytes. For each patient, MII oocytes were split in two groups: control-group (n=164), in which the routine selection of sperm was conducted, and ZP-group (n=152), in which ZP-bounded sperm was injected. Fertilization and high quality embryos rates were compared between the groups and also, to study the influence of the ZP-bounding sperm injection on the embryo quality, a logistic regression model was conducted. The results were presented as odds ratios and its 95% confidential interval. RESULTS: The fertilization rate was similar among the groups (control: 71.9% ZP: 80.2%, p=0.084). However, the high quality embryos rate was higher when a ZP-bounded sperm was selected for injection (control: 68.6% vs ZP: 85.9%, p=0.001). Furthermore, ZP-bound sperm injection positively influenced the embryo quality (OR = 2.79 IC 95% = 1.47 – 5.32; p = 0.001). CONCLUSIONS: The present study showed that the fertilization rate was not affected by the ZP-bound sperm injection; however, once fertilized, embryo development may be positively affected. Sperm capable of binding to the ZP represents a population that satisfies a necessary condition for fertilization. The in vivo sperm binding to the ZP initiate the chain reaction that culminates with fertilization; nonetheless, the molecular, biochemical, and physiological changes in the sperm while it binds to ZP, and whether these changes are associated with embryo development in vitro is unclear. Our findings suggest that ZP sperm binding may select more competent sperm, able to improve embryo development.

Recombinant Mouse Sperm ZP3-binding Protein (ZP3R/sp56) Forms a High Order Oligomer That Binds Eggs and Inhibits Mouse Fertilization in Vitro

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Dipeptidase-Inactivated tACE Action In Vivo: Selective Inhibition of Sperm-Zona Pellucida Binding in the Mouse1

The angiotensin-converting enzyme (ACE) plays a crucial role in male fertilization and is a key regulator of blood pressure. Testicular ACE (tACE), the germinal specific isozyme expressed on different promoters, exclusively carries out the role of ACE in fertility, although the site and mode of action are not well known. To investigate the contribution of tACE in fertilization, we produced transgenic mouse lines carrying a dipeptidase-inactivated mutant. Although the transgenic mice showed normal blood pressure, kidney morphology, and fertility, reduced fertilization was observed after in vitro fertilization (IVF). The sperm-zona pellucida (ZP) binding was exclusively impaired in these lines in a manner similar to that observed in an Ace knockout mouse. The dipeptidase activity was reduced in epididymal ingredients but not in the testis. Furthermore, direct application of mutant protein did not suppress sperm-ZP binding of intact sperm during IVF, implying that the dipeptidase-inactivated mutant affects sperm modification in the epididymis for ZP binding. Our results indicate that the dipeptidase-inactivated tACE acts in vivo, suggesting that tACE contributes to fertilization as a dipeptidase at least in the epididymis.

New insights into sperm-zona pellucida interaction: involvement of sperm lipid rafts

Sperm-zona pellucida (ZP) binding is the first step of gamete interaction. This binding occurs in two sequential steps, starting with the primary binding of acrosome-intact sperm to the ZP followed by the secondary ZP binding of acrosome reacting/reacted sperm. While there are only a few ZP sulfoglycoproteins involved in these binding events, a large number of sperm surface molecules have been shown to possess ZP affinity. In this review, we have given explanations to the existence of these many ZP binding molecules. We have also summarized their origin and the mechanisms of how they are targeted to the sperm surface and acrosome. Recently, we have shown that sperm lipid rafts have affinity for the ZP. A number of ZP binding molecules are also present in sperm lipid rafts. In this review, we have provided an argument that sperm lipid rafts may be the platforms on the sperm surface for ZP interaction.

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.

World Health Organization grade ‘a’ motility and zona-binding test accurately predict IVF outcome for mild male factor and unexplained infertilities

The aim of this study was to determine the pronostic value of a sperm-zona pellucida (ZP) binding assay, combined with World Health Organization (WHO) grade 'a' sperm motility on the day of the IVF attempt, to predict sperm fertilizing ability in unexplained and moderate male factor infertilities. In total, 84 couples (64 unexplained infertility; 20 male factor) underwent both a sperm-ZP binding assay and an IVF attempt, irrespective of the test's result. The test was negative when grade 'a' motility was #5% and/or the ZP binding index was <0.7. Fertilization and pregnancy rates were related to the test's results. Thirty-one patients had a negative test (group N) and 53 a positive test (group P). A difference was observed concerning the fertilization rate [median (range): 0 (0-75%) versus 50 (0-100%); P = 0.0001] and the number of cycles with fertilization rate <20% (65 versus 23%; P = 0.0002) between groups N and P respectively. In the group of unexplained and male factor infertilities, the test showed a sensitivity of 83 and 60%, specificity of 50 and 90%, positive predictive value of 76 and 86%, and negative predictive value of 61 and 69% respectively. Sperm-ZP binding test, combined with WHO grade 'a' motility assessment, is relevant to prevent IVF fertilization failures in unexplained infertility and, most particularly, in moderate male factor infertility.

HUMAN SYNTHETIC PEPTIDE VACCINE FOR CONTRACEPTION TARGETING SPERM

A vaccine targeting sperm is an interesting approach to contraception. The acceptability of a sperm antigen for contraceptive vaccine development is contingent upon its sperm-specificity, surface expression for antibody binding, and involvement in fertilization. Sperm-zona pellucida (ZP) binding is a pivotal step and constitutes an attractive site for immunointerception. Using phage display technology, we have identified a novel dodecamer peptide sequence, designated as YLP12, that is present on sperm, and is involved in ZP binding and sperm capacitation/acrosome reaction in man/mouse. Extensive search in the human and mouse genome sequence databases did not indicate a complete identity with the YLP12nucleotide sequence. It is peptide mimetic with sperm receptor carbohydrate mimicking action that is involved in oocyte binding. The sperm-specific YLP12sequence is also involved in involuntary immunoinfertility in men, indicating its autoantigenicity, sperm-specificity, and involvement in fertility/infertility in humans. Vaccination of female animals with synthetic YLP12peptide causes a long-term reversible contraception by raising a sperm-specific immune response. The contraceptive effect could also be completely reversed voluntarily at any time by immunoneutralization of antibodies using intravaginal administration of the peptide. Thus, the novel synthetic YLP12peptide is an attractive candidate for contraceptive vaccine development, and diagnosis and treatment of immunoinfertility in humans.

HUMAN SYNTHETIC PEPTIDE VACCINE FOR CONTRACEPTION TARGETING SPERM

A vaccine targeting sperm is an interesting approach to contraception. The acceptability of a sperm antigen for contraceptive vaccine development is contingent upon its sperm-specificity, surface expression for antibody binding, and involvement in fertilization. Sperm-zona pellucida (ZP) binding is a pivotal step and constitutes an attractive site for immunointerception. Using phage display technology, we have identified a novel dodecamer peptide sequence, designated as YLP(12), that is present on sperm, and is involved in ZP binding and sperm capacitation/acrosome reaction in man/mouse. Extensive search in the human and mouse genome sequence databases did not indicate a complete identity with the YLP(12) nucleotide sequence. It is peptide mimetic with sperm receptor carbohydrate mimicking action that is involved in oocyte binding. The sperm-specific YLP(12) sequence is also involved in involuntary immunoinfertility in men, indicating its autoantigenicity, sperm-specificity, and involvement in fertility/infertility in humans. Vaccination of female animals with synthetic YLP(12) peptide causes a long-term reversible contraception by raising a sperm-specific immune response. The contraceptive effect could also be completely reversed voluntarily at any time by immunoneutralization of antibodies using intravaginal administration of the peptide. Thus, the novel synthetic YLP(12) peptide is an attractive candidate for contraceptive vaccine development, and diagnosis and treatment of immunoinfertility in humans.

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

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

Sperm from the Calmegin-Deficient Mouse Have Normal Abilities for Binding and Fusion to the Egg Plasma Membrane

Calmegin is a putative testis-specific molecular chaperone required for the heterodimerization of fertilin α/β and the appearance of fertilin β on the sperm surface. Calmegin-deficient mice are almost completely sterile. The cause of the sterility initially was considered to be impaired abilities in sperm/zona pellucida (ZP) and sperm/egg plasma membrane (EPM) binding, and in the ascension of sperm to the oviduct, phenotypes similar to those seen in sperm from fertilin β-deficient animals. We have developed a new method in which eggs were prepared without any detectable ZP3 on their surfaces by using a piezo-driven micromanipulator. Using these eggs and sperm containing the green fluorescent protein in their acrosomes, which can distinguish acrosome-intact from acrosome-reacted sperm, the binding and fusing abilities of calmegin-deficient sperm were reexamined. Under these conditions, acrosome-reacted sperm retained their ability to bind to and fuse with the EPM. The reduction in EPM binding of sperm from the calmegin −/− animals was apparently due to the artifactual binding of large numbers of acrosome-intact sperm from calmegin +/− mice to ZP remnants remaining on the EPM prepared with acidic Tyrode's solution. Thus, the sperm defect in calmegin-null animals is not at the level of sperm–EPM binding but rather may involve either sperm–ZP binding and/or sperm transit to the oviduct. Because fertilin β is absent from calmegin-deficient mice, these results also suggest that the role of fertilin β in sperm–EPM interaction needs to be reevaluated.

Sperm from the Calmegin-Deficient Mouse Have Normal Abilities for Binding and Fusion to the Egg Plasma Membrane

Calmegin is a putative testis-specific molecular chaperone required for the heterodimerization of fertilin α/β and the appearance of fertilin β on the sperm surface. Calmegin-deficient mice are almost completely sterile. The cause of the sterility initially was considered to be impaired abilities in sperm/zona pellucida (ZP) and sperm/egg plasma membrane (EPM) binding, and in the ascension of sperm to the oviduct, phenotypes similar to those seen in sperm from fertilin β-deficient animals. We have developed a new method in which eggs were prepared without any detectable ZP3 on their surfaces by using a piezo-driven micromanipulator. Using these eggs and sperm containing the green fluorescent protein in their acrosomes, which can distinguish acrosome-intact from acrosome-reacted sperm, the binding and fusing abilities of calmegin-deficient sperm were reexamined. Under these conditions, acrosome-reacted sperm retained their ability to bind to and fuse with the EPM. The reduction in EPM binding of sperm from the calmegin −/− animals was apparently due to the artifactual binding of large numbers of acrosome-intact sperm from calmegin +/− mice to ZP remnants remaining on the EPM prepared with acidic Tyrode's solution. Thus, the sperm defect in calmegin-null animals is not at the level of sperm–EPM binding but rather may involve either sperm–ZP binding and/or sperm transit to the oviduct. Because fertilin β is absent from calmegin-deficient mice, these results also suggest that the role of fertilin β in sperm–EPM interaction needs to be reevaluated.