Matching Hemizonae Research Articles

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.

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.

P-402: Fertility loss in the sperm of tobacco smokers may be reversed after washing with a cannabinoid agonist

Human sperm carry a nicotinic acetylcholine receptor and respond to nicotine from tobacco. They also have cannabinoid (CB) receptors and respond to marijuana and endocannabinoids or synthetic CB chemicals. We previously reported that sperm from 2/3 of tobacco smokers showed a significant drop in fertilizing capacity, using the Hemizona Assay (HZA). Their sperm showed reduced binding to the zona pellucida (cover) of the human egg. In Part II, we hypothesized that treatment of the smoker’s sperm with a CB agonist would improve sperm binding capacity. We have studied the effects of a CB agonist (AM-1346; analog of the endocannabinoid anandamide). After obtaining a positive response when donor sperm were treated with nicotine plus a CB agonist, we studied the sperm from 8 chronic tobacco smokers in 24 in vitro HZA tests. Each smoker was previously tested in the HZA, where his sperm fertilizing function was compared to a non-smoking donor. Four of 8 smokers (Group I) had shown normal sperm fertilizing function (HZI > 65). The other 4 smokers (Group II) had failed the zona assay. In Part II, each new test used the sperm from one tobacco smoker. His sperm were washed with and without a CB ligand (Control had no ligand present). We tested the effects of a potent CB1 agonist, AM-1346 (n = 17; 100 pM to 100 nM). After a “swim-up,” the motile sperm fraction was used for the HZA. Here, a nonviable human oocyte was cut in half. One half was incubated with smoker’s sperm that had been treated with a CB ligand. The matching Hemizona (HZ) was placed with that smoker’s Control sperm. Post-incubation, each HZ was rinsed and counted for the number of sperm tightly bound to the outside. The ratio of [# bound for the treated sperm / # bound for the Control sperm] was calculated as the Hemizona Index (HZI). HZI = 100 indicates that the treated sperm bound to the zona as well as the Control sperm. We hypothesized that the HZI would improve with in vitro CB treatment. After exposing the sperm to AM-1346, at any concentration, we found that Group II (low fertility) responded with markedly improved zona binding, compared to the Control sperm (4 out of 8 experiments). Specifically, when semen quality was poor, Group II showed a mean HZI of 177% (77% improvement compared to the untreated sperm). However, the sperm from smokers with normal fertility (Group I) behaved differently. Here, only 1 of 9 tests showed a zona binding enhancement (p < 0.05). In Groups I & II, there were 7 experiments where the smoker was tested with a low concentration of AM-1346 (one assay) and also tested at a higher 1346 level (second assay). In 6 of 7 cases, the higher 1346 dose produced a better HZI score (improved binding). We conjecture that most smoking men would show a stimulation of zona binding if the dose of AM-1346 is high enough. Based on our previous data, and published literature, it is clear that a majority of tobacco smokers will exhibit a small or significant decline in fertility potential. Infertility clinics worldwide are trying to meet the challenge of male and female tobacco smokers who have only borderline fertility. Smoking cessation is the best solution. However, the in vitro use of synthetic cannabinoid analogs may significantly improve the fertilizing capacity of tobacco smokers who cannot quit.

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Application of a 1.48-microm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay.

Laser systems are very promising new technical tools in assisted reproduction. It was investigated if laser radiation can replace the mechanical cutting procedure via micromanipulator in the hemizona assay (HZA), a commonly used bioassay to determine the sperm-zona pellucida binding capacity. An oocyte was bisected precisely into two identical hemizonae with approximately 20 laser pulses (pulse length 30 msec) using a 1.48-microm diode laser. Compared with the conventional method using microscalpels for zona bisection, laser treated hemizonae showed equivalent sperm-binding and within the two groups there was no detectable difference between matching hemizonae in their capacity for tight sperm-binding. To evaluate whether laser radiation affects the outcome of the HZA when effects of certain substances are investigated, the spermatozoa were preincubated with human follicular fluid (hFF), which inhibits the binding of spermatozoa to zona pellucida in vitro. Supplementation with follicular fluid exerted an inhibitory effect in both groups. The hemizona index (HZI) showed no statistical differences between the two methods. Therefore, the 1.48-microm diode laser is a suitable new instrument for generating equally sized hemizonae. There is no use for holding pipettes and microscalpels, on the contrary, for performing the HZA the laser is a precise, very quick and easy to use new working tool.

Macrophage secretory products and sperm zona pellucida binding

OBJECTIVE: To determine if exposure of human gametes to macrophage secretory products reduces sperm binding to the zona pellucida, and to determine which cytokine(s) may be responsible for this effect. METHODS: A human macrophage cell line was cultured and either activated with lipopolysaccharide for 2 hours and then washed or left unactivated. Culture-conditioned media from activated or unactivated cells was used in hemizona assay. Hemizonae were incubated with sperm suspended in culture medium from either unactivated macrophages or activated macrophages, with the matching hemizona incubated with sperm suspended in control medium. Matching hemizonae were incubated with sperm suspended in unactivated macrophage medium paired with sperm suspended in activated macrophage culture medium. Conditioned medium from activated macrophages was found to have elevated levels of tumor necrosis factor-α (TNF-α), interleukin-1β, and transforming growth factor-β, therefore, gametes were also exposed to these cytokines followed by the hemizona assay. After each incubation, the number of sperm tightly bound to the outer surface of each hemizona was determined. RESULTS: Exposure of gametes to activated and unactivated macrophage culture-conditioned media significantly decreases sperm binding to the zona pellucida, with medium from activated macrophages inducing the greatest effect ( P < .05). Exposure of sperm to TNF-α significantly impaired sperm binding ( P < .05), whereas other cytokines tested had no effect. CONCLUSION: These results suggest that macrophage secretory products in the basal and activated state may be a factor in endometriosis-associated infertility through the interference of sperm binding to the zona pellucida, and that TNF-α is a key cytokine responsible for this effect.

Relationship between sperm-zona pellucida binding assays and the 56-day nonreturn rate of cattle inseminated with frozen-thawed bull semen

Assays based on sperm-zona pellucida binding have been developed as diagnostic tests to predict the fertilizing potential of mammalian spermatozoa. Recently, we reported on the development of a sperm-zona pellucida binding assay (SZBA) for bull spermatozoa. The aim of the present study was to develop a hemi-zona assay (HZA) for bull spermatozoa and to investigate the relationship between SZBA and HZA outcomes and in vivo fertility. Frozenthawed semen samples from 8 fertile Swedish Red and White bulls (one ejaculate per bull) designated as the test semen samples and a single ejaculate from a fertile Holstein-Friesian bull designated as the control semen sample were used in this study. In the SZBA, 2 groups of 20 oocytes per semen test sample and in the HZA a minimum of 6 matching pairs of hemizonae were used for comparison of sperm binding with control semen. Sperm binding to matching hemi-zonae of individual semen samples was equal, and clearly demonstrated the feasibility of the HZA for cattle. A significant correlation was found between the SZBA and the HZA indices obtained from the different semen test samples (r = 0.42, P < 0.001; n = 67). There was no significant relation between the SZBA indices and the 56-d nonreturn rate of the test samples. However, the HZA indices of the semen test samples and the 56-d nonreturn rate were significantly correlated (r = 0.46, P < 0.0001; n = 67). It is concluded that HZA can be regarded as a potential assay for predicting the fertilizing ability of bovine semen samples. However, further studies using more semen samples are necessary to confirm this view.

Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function

To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. Prospectively designed, blinded, internally controlled study. Tertiary care academic center. Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3-6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3-7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-antiperoxidase method and diaminobenzidine as color reagent. A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3-6-treated hemizonae than patients with sperm factor. These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting.

Relation between stallion sperm binding to homologous hemizonae and fertility

The hemizona assay (HZA) has been developed as a diagnostic test to predict the fertilisation potential of human spermatozoa. The aim of this study was to develop an HZA for stallion spermatozoa and to investigate a possible relationship between fertility and the outcome of the HZA in this species. Equine oocytes were obtained from ovaries collected at a slaughterhouse and by transvaginal, ultrasound-guided follicle aspiration. They were then denuded from cumulus cells and stored in salt solution at 4 °C until use. On the day of the experiments the oocytes were bisected, thus providing 2 equal matching hemizonae from each oocyte. Semen samples from Dutch Warmblood stallions with known fertility data were used to assess the number of spermatozoa bound to the outer side of the hemizona after incubation in vitro. Sperm binding to matching hemizonae of a particular stallion was similar and confirmed the feasibility of using the HZA for the horse. Sperm hemizona binding capacity of 10 pairs of stallions was compared by incubating 1 hemizona with the semen of a stallion and the matching hemizona with the semen of another stallion from the same stud farm. Five matching pairs of hemizonae were used for each pair of stallions. There was a significant relationship between the mean number of spermatozoa bound to matching hemizonae and the fertility indices of stallions from each stud farm (P < 0.0001). It is concluded that HZA can be used as a valuable parameter in stallion semen analysis.

Development of a sperm Hemizona binding assay for boar semen

The Hemizona assay (HZA) was developed as a diagnostic test for predicting the fertilization potential of human spermatozoa. The aim of this study was to develop an HZA for porcine species to allow for boar semen analysis and the study of gamete interaction in this species. Porcine oocytes were obtained from slaughterhouse ovaries, denuded, and stored in buffered ammonium sulphate solution at 4 °C. On the day of the experiments the oocytes were bisected, thus providing 2 equal matching hemizonae from each oocyte. Semen samples from fertile boars were used to assess the number of spermatozoa bound to the outer side of the hemizona during incubation in vitro. The HZA index was defined as the ratio of the number of test spermatozoa bound to the hemizona divided by that of the control spermatozoa. Sperm binding to matching hemizonae of a particular boar was equal and clearly demonstrated the feasibility of using HZA for the pig. The number of spermatozoa bound to hemizona was dependent on sperm concentration in the incubation droplets. Hemizonae obtained from oocytes stored in buffered ammonium sulphate and in ammonium sulphate showed binding which was similar to that of hemizonae from fresh oocytes. However, there was a significant difference between HZA indices when fresh zonae binding was compared with hemizonae from buffered magnesium chloride-stored oocytes. A combination of monoclonal antibody 18.6 and Hoechst dye 33342 was used to assess boar sperm acrosomal status. More than 80% of the spermatozoa bound to hemizonae were partially acrosome-reacted or had lost their acrosome completely. In contrast, spermatozoa at the onset of incubation or after 4 h of incubation irrespective of exposure to hemizona showed a low incidence of acrosome reactions (14 to 25%). In conclusion, this paper presents the development and validation of an HZA for boar semen. The HZA in porcine species has several potential areas of use in which the functional status of sperm-oocyte interaction is vital.

Use of sperm binding to homologous hemizona pellucida to predict stallion fertility

SummaryEquine oocytes were obtained post mortem from slaughterhouse ovaries and by transvaginal ultrasound‐guided follicle aspiration. The oocytes were denuded and stored in salt solution at 4°C. On the day of the experiment the oocytes were bisected by micromanipulation surgery, thus providing two equal matching hemizonae from each oocyte. Three pairs of stallions were formed, each consisting of a fertile and subfertile Dutch Warmblood stallion. Matching hemizonae from 5 oocytes were used for each pair of fertile and subfertile stallions and the number of spermatozoa bound to the outer‐side of each hemizona during incubation in vitro was assessed. The numbers of bound spermatozoa from fertile stallions were significantly higher than those from subfertile stallions (P&lt;0.05). The results of this investigation encourage further use of the hemizona assay for evaluation of stallion fertility.

The specificity of human spermatozoa/zona pellucida interaction under hemizona assay conditions.

The objective of this prospective study was to evaluate the specificity of human sperm/zona pellucida interaction under hemizona assay (HZA) conditions in experiments with gametes from the same and different species. Human, cynomolgus monkey and hamster oocytes were used after salt-storage. Oocytes were bisected into matching hemizonae by micromanipulation and used in the HZA. Semen was obtained from healthy men (donors) and male cynomolgus monkeys and prepared by wash and swim-up. Sperm binding to matching hemizonae was assessed (tight binding) after 4-h coincubation in the HZA in homologous and interspecies experiments. Acrosome reaction was evaluated in the sperm droplets using FITC-PSA and on the hemizonae using the T-6 monoclonal antibody. On human hemizonae, the number of tightly bound sperm for human and monkey were 93.2 +/- 15.8 and 3.9 +/- 1.3, respectively (P < 0.001). On monkey hemizonae, the number of tightly bound sperm for monkey and human were 126.0 +/- 34.8 and 2.8 +/- 1.6, (P = 0.02) respectively. On hamster hemizonae, there was negligible binding of human and monkey sperm. There was a significantly higher incidence of acrosome reacted sperm on the zona pellucida in homologous compared to heterologous experiments. These results demonstrate a high species-specificity of human gamete functions under HZA conditions, providing further support for the use of this bioassay in infertility and contraception testing.

Hemizona assay and teratozoospermia: increasing sperm insemination concentrations to enhance zona pellucida binding

This study aimed: (1) to evaluate the zona-binding capacity of patients with abnormal sperm morphology, using standard hemizona assay (HZA) conditions and increasing sperm insemination concentration during the assay and (2) to determine the insemination concentration needed to obtain equality in the number of tightly bound sperm to matching hemizonae, using sperm from teratozoospermic patients versus proven fertile controls. The minimum concentration of motile sperm from fertile controls necessary to validate HZA results was 250,000/mL (35.4 +/- 5.6 tightly bound sperm; mean +/- SE). The "effective number of sperm" (morphologically normal with high motility) was 60,750/mL. Each teratozoospermic patient had a unique, (higher) sperm insemination concentration (range: 0.5 X 10(6) to 2.0 X 10(6) motile sperm/mL) necessary to equal the number of tightly bound sperm representing the lower 95% confidence interval for the control sample (at 0.5 X 10(6) motile sperm/mL) with the matching hemizona. These results suggest that the HZA may be used as an indicator of the sperm insemination concentration during in vitro fertilization in patients with teratozoospermia.

The hemizona assay (HZA): a predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment.

The hemizona assay (HZA) was developed to assess human sperm fertilizing potential. This blinded study investigated the relationship between sperm binding to the hemizona and in vitro fertilization (IVF) success (36 patients). Nonliving human oocytes were recovered from excised ovaries and stored. Each zona pellucida was cut into equal hemispheres by micromanipulation. For the HZA, one droplet exposed a hemizona to abnormal spermatozoa, while the control droplet contained the matching hemizona and spermatozoa from normal semen. After 4 hr, the number of tightly bound spermatozoa was counted. Binding to the hemizona was significantly higher for those having IVF success (mean of 36.1 +/- 7, versus 10.4 +/- 4 from the failure group; P less than 0.05). Fewer sperm from the failure group had a strictly normal morphology (3.2 versus 12.7%; P less than 0.05, Kruger method). Tight zona binding was significantly correlated with the percentage motile sperm, percentage normal morphology, and seminal sperm concentration. These results enhanced our confidence that the HZA is diagnostic for identification of patients at high risk of failing to achieve fertilization in vitro.

Hemizona assay using salt‐stored human oocytes: Evaluation of zona pellucida capacity for binding human spermatozoa

Human oocytes were stored (25 degrees C) in 1.5 M MgCl2 for 6-30 days, then utilized in the new hemizona assay (HZA) for tight binding of human spermatozoa [Burkman et al.: Fertil Steril 49:688-697, 1988]. We have compared 1) the ability of matching salt-treated hemizonae or dimethylsulfoxide (DMSO)-treated hemizonae to distinguish between sperm from semen having normal versus subnormal characteristics and, 2) the kinetics of fertile sperm binding to salt-treated or DMSO-treated hemizonae. After sperm preparation one salt-treated hemizona was incubated with normal spermatozoa and the matching hemizona was placed with sperm from the subnormal group. As a control, DMSO-treated hemizonae were incubated in additional sperm droplets. After 4 hours, the number of sperm tightly bound to each hemizona was counted. Within the normal semen group, there was equivalent binding to salt- or DMSO-treated hemizonae (54.0 +/- 12 and 49 +/- 14, respectively, mean +/- SEM). Similarly, tight binding of sperm from the subnormal group was not affected by the zona storage method (21 +/- 8 and 17 +/- 5, respectively). For either storage approach, binding of subnormal sperm was significantly less (P less than 0.01) compared with the number of normal sperm attached to the matching hemizona. For the kinetics study, the hemizona binding of proven fertile spermatozoa was followed throughout 8.5 hours. The shape of the binding curve was the same for zonae stored by either method and was consistent with our published kinetics data. Salt storage offers a simple and inexpensive means for accumulating and transporting human zonae pellucida; the resulting hemizonae function effectively in the HZA for estimating sperm binding potential.