Development of a predictive model for optimal zona pellucida binding using insemination volume and sperm concentration

Objective: To develop a predictive model under hemizona assay (HZA) conditions for human spermatozoa concentrations and insemination volume for optimum zona pellucida (ZP) binding. Design: Analysis of 20 different insemination volumes for zona binding and sperm morphology under HZA conditions. Setting: Reproductive biology unit, tertiary medical center. Patients: Four proven fertile sperm donors. Main Outcome Measures: 5-, 20-, 50-, 80-, and 100-μL droplets were analyzed with four different concentrations of 0.5 x 106, 1.0 x 106, 2.0 x 106, and 4.0 x 106 cells/mL to determine the number of sperm bound to each hemizona. Fifteen hemizonae were used for each insemination volume or microdroplet. Response surface regression model with volume and concentration as the regressor variables has been used. Results: The response surface of binding for the factors concentration and volume showed nonlinear association. A formula, indicating the optimal sperm insemination volume for maximum sperm binding to the ZP, V(max) = -(b1 + b5c)/2b6c, is described. The transformed data indicated 60 μL containing 4 x 106 sperm/mL to be optimal. Although morphology of zona spermatozoa is superior compared with seminal and postswim-up samples, no difference among the percentage of the normal morphology in different microdroplets could be demonstrated. Conclusion: Optimal volume for the obtained concentration of spermatozoa from a patient can be calculated and therapeutically used for cases of severe oligozoospermic patients by microvolume inseminations in IVF practice.Articl

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

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

Comparison of motility characteristics and normal sperm morphology of human semen samples separated by Percoll density gradient centrifugation

Tile aim of this study was twofold: to investigate the ability of Percoll gradient centrifugation (52, 68, 84%) to fractionate semen samples according to motility quality and percentage normal morphology and to determine whether there is an association between sperm motility quality and percentage normal morphology. Sperm motility was evaluated using a Hamilton Thorn analyzer and normal sperm morphology was manually assessed according to the strict criteria (≤4, 5-14, and >14%). The majority of motility parameters and the percentage normal morphology were found to be significantly improved in the 84% Percoll fraction. The greatest effect was on the 14% group, shifting the mean normal morphology percentage from 2.6 to 5.2%. Curvilinear velocity (VCL) and average path velocity (VAP) were the only two motility parameters that were significantly associated with the percentage normal morphology. Using a combined VCL,VAP vector the >14% group was found to have a significantly different value as compared to the 5-14 and <4% groups. Percoll (discontinuous) gradient centrifugation can therefore play a significant role in the improvement of semen samples for use in assisted reproduction procedures. The VCL,VAP vector identified may also serve as an additional tool in the prediction of the fertility potential of sperm samples.Articl

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

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

Defining bioassay conditions to evaluate sperm/zona interaction: Inhibition of zona binding mediated by solubilized human zona pellucida

Objective: Our goal was to study the influence of solubilized human zonae pellucidae on zona binding potential and acrosome reaction. Materials and Methods: Zona pellucida (ZP) solutions were prepared by dissolving zona in acidic buffer, NaH2PO4 (pH 2.5), to obtain 0.1 and 0.5 zona pellucida/μl. Zona binding capacity was evaluated by the addition of oocytes (10-fold) to sperm/zona pellucida solution droplets. The number of sperm bound to each oocyte was recorded. Zona pellucida mediated acrosome activity was evaluated after 60 min of coincubation of sperm and 0.5 ZP/μl. Results: The mean (±SE) number of sperm bound for control, 0.1 ZP/μl, and 0.5 ZP/μl was 181.2 ± 12, 79.6 ± 5, and 38.8 ± 3, respectively. Zona pellucida-exposed sperm populations showed significant more acrosome-reacted sperm compared to control sperm, namely, 78 versus 32%, respectively (P = 0.001). Conclusions: The observed zona binding inhibition might be ascribed to zona receptor blocking on the sperm surface or the inability of acrosome-reacted sperm to bind to the zona pellucida.Articl

The effect of pentoxifylline on sperm movement characteristics and zona pellucida binding potential of teratozoospermic men

This study investigates the effect of pentoxifylline on sperm movement and zona pellucida binding. Spermatozoa from nine teratozoospermic patients (10.2 ± 6% normal forms) were included in the study. Test samples were diluted with a 4 mM solution of pentoxifylline, and control samples with Ham's F-10 culture medium only. Zona pellucida binding potential was measured under hemizona assay conditions (HZA). Sperm motility was evaluated at the start (0 h post-swim-up) and end (4 h) of the HZA, under test-tube conditions and under HZA conditions (50 μl droplet under oil). Motility parameters tested included the curvilinear velocity (VCL), straight line velocity (VSL) and linearity (LIN). Compared to the controls, pentoxifylline-treated samples showed an immediate stimulation of sperm motility, under test-tube conditions, with a significant elevation of VCL at 0 h incubation (102.77 ± 14.4 versus control value 84.60 ± 10.6 μm/s; P = 0.005), which however was reversed after 4 h incubation (73.16 ± 4.6 versus control value 85.47 ± 12.8 μm/s; P < 0.005). A decline in sperm motility from 0 to 4 h incubation was noted for all the pentoxifylline-treated samples, under both test tube conditions (VCL: 102.77 ± 14.4 versus 73.16 ± 4.6 μm/s, P < 0.005; VSL: 27.2 ± 10 versus 10.66 ± 2.2 μm/s, P < 0.005; LIN: 23.65 ± 7.1 versus 11.86 ± 1.8%, P < 0.005) and HZA conditions (VCL: 100.04 ± 13.1 versus 76.00 ± 7 μm/s, P < 0.005; VSL: 26.40 ± 8.7 versus 9.14 ± 4.5 μm/s, P < 0.005; LIN: 26.2 ± 12 versus 11.05 ± 4.3%, P < 0.005). Pentoxifylline also showed no effect on the zona binding capacity of the sperm population, since no significant difference in zona binding capacity was seen between control and test samples. In conclusion, the effect of pentoxifylline on teratozoospermic spermatozoa is short-lived, and despite its stimulatory potential pentoxifylline shows no beneficial use in enhancing zona pellucida binding of teratozoospermic spermatozoa.Articl

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responsesThis work was funded by a Wellcome Trust Principal Research Fellowship (D.A.C.), Program Grant nos. 065975/Z/01/A and 097418/Z/11Z, and Tenovus Scotlan