In vitro glycation assays

To establish the effect glycating agents have on sperm vitality and motility in an in vitro system, sperm were incubated with various glycating compounds over long-term (6 days) and a short-term incubation periods (0, 2 and 4 hours). 3 donors were used for the long-term experiments, and samples from another set of 3 donors were used for the short-term experiments. Long-term experiments were carried out to show the glycating effects of glucose, which has low reactivity relative to glucose-6-phosphate⁵⁷ or other reducing sugars such as D-ribose⁵⁸. A period of 6 days was chosen as glucose has previously shown no glycating effects on cultured cells over 2 days⁵⁸. Mannitol - a non-reducing sugar - was included in initial experiments to control for osmotic effects of glucose⁵⁹. The highly reactive AGE intermediates, MG and GO, were used as an accelerated model for the glycation of sperm. Cellular levels of these compounds are predicted to be approximately 1–5 µM and 0.1–1 µM, respectively⁶⁰. However, physiological levels of MG have been reported to be in the nM⁶¹, µM⁶² and mM⁶³ range. The concentrations implemented in this study were higher in order to simulate a disease environment and were in line with those used in other studies³⁹.

For the long-term in vitro glycation experiments, motile sperm separated by density gradient centrifugation were pelleted and resuspended at a concentration of 20 × 10⁶/ml in solutions of SPM containing D-glucose (30 mM or 50 mM), Mannitol (30 mM). These concentrations were used to mimic diabetic hyperglycemia and to asses sperm response to massive doses of glucose⁴⁰. Sperm were also exposed to methylglyoxal (MG) (50 μM), glyoxal (GO) (50 μM) or SPM alone. Sperm were incubated at 34 °C on a rotator for 6 days before analysis of CML formation, ROS levels and oxidative DNA damage. Incubations were carried out at a temperature of 34 °C in order to best represent the scrotal environment of normospermic men⁶⁴. Sperm vitality was assessed after the long-term incubation experiments using the eosin-nigrosin stain as outlined above and motility was measured using the CASA⁶⁵.

For the short-term in vitro glycation experiments, sperm at a concentration of 20 × 10⁶/ml were treated with solutions of SPM containing MG (50 μM) and GO (50 μM); cells were removed at 0, 2 and 4 hour time points for analysis of motility, CML formation and hyaluronan binding capacity.

To measure sperm motility for both short-term and long-term glycation experiments,, 5 μl sperm from each sample was applied to a cell counting slide in duplicate (CellVision®, Mitrone) and analysed by a Computer Assisted Sperm Analyser (CASA – Sperminator®, Procreative). Automated CASA technology allowed standardization of measurements, as used elsewhere^65,66. Three CASA measurements were recorded for each sample and 200 sperm were analyzed per read⁶⁷. For the long-term glycation experiments - progressive sperm motility was assessed at three different time points (24, 48 and 72 hours) as a period of 3 days was chosen as the longest period that could be used without any major loss of sperm motility in the control sample⁴⁷.

The concentration of MG and GO to use in the glycation experiments was determined using a toxicity assay in which sperm (20 × 10⁶/ml) were incubated with 0, 50, 100, 250 and 500 μM MG in SPM for 48 hours at 34 °C. Due to the limited availability of donor samples, the toxicity assay was carried out on a single sample. Vitality was measured using the one-step eosin-nigrosin stain. The stain was prepared as described in WHO 5^th edition⁵⁵ Briefly, eosin Y (0.67%) (Sigma, UK) and sodium chloride (NaCl) (0.9%) (Sigma, UK) were dissolved in water with gentle heating, before nigrosin (10%) (Sigma, UK) was added, and the solution was boiled, then allowed to cool and filtered. Equal volumes of eosin-nigrosin stain and resuspended sperm sample were mixed and smeared onto a microscope slide using the feathering technique. Duplicate smears were made for each concentration of MG using a fresh aliquot of sperm. Using a light microscope (100 × oil objective), 200 sperm were counted on each slide and the average number of live sperm was recorded. The percentage of live sperm were calculated as [# live cells/# total cell count] × 100.

The chosen working concentration of MG was 50 µM as the toxicity assay showed only 8% cell death over the course of the experiment, in comparison to 28% and 77% cell death at 100 µM and 500 µM MG, respectively.

The level of CML on sperm was measured by flow cytometry after both long-term and short-term in vitro glycation, as has been demonstrated elsewhere⁶⁸.

Sperm were removed from glycating solutions after long-term (n = 3) and short-term periods (n = 3) and pelleted by centrifugation at 300 g to remove glycation media. Sperm cells were resuspended in paraformaldehyde (4%) (Sigma, UK) and incubated at room temperature on a tube rotator (Eppendorf, UK) for 20 minutes. Cells were pelleted again and permeabilized by resuspension in 0.1% PBS-tween for 15 minutes. Sperm were then blocked in 10% normal goat serum (Vector laboratories, UK) in 1xPBS for 1 hour at room temperature. Cells were incubated with a mouse anti-CML primary antibody (1:50) (Abcam, UK, ab125145) for one hour at room temperature, followed by a goat anti-mouse Alexa Fluor 488 secondary antibody (1:2000) (Life Technologies, UK) for 45 minutes at room temperature in the dark. Cells were then washed once and resuspended in 1xPBS before acquisition using a BD FACSCalibur™ (BD Biosciences, UK) and analysed with BD CellQuest™ Pro software (BD Biosciences, UK). Cells were gated and debris was excluded in the forward scatter (FSC) and side scatter (SSC) plot. Fluorescein isothiocyanate (FITC) fluorescence was detected in the FL-1 channel (488 nm). Each test sample was measured in triplicate and 10,000 events were counted each time. A FITC-negative cell population was gated using an unstained control sample in the FL-1 channel. Relative CML levels were recorded as the mean fluorescence intensity (MFI) of cells.

Spontaneous glycation of biomolecules and later stages of the Maillard reaction are associated with the generation of ROS and oxidative stress. Flow cytometric methods have been established for numerous evaluation parameters in sperm⁶⁹, including for ROS measurement. Intracellular ROS in sperm was measured after long-term glycation incubation (n = 3) with either glucose, MG or GO using the CM-H2DCFDA General Oxidative Stress Indicator (Invitrogen, UK). CM-H2DCFDA diffuses into cells where it is oxidized to yield a fluorescent adduct, measured in the fluorescein spectrum^70,71. The assay was carried out as instructed by the manufacturer. Briefly, treated sperm were washed in pre-warmed PBS and resuspended in PBS containing the ROS probe. The cells were incubated at 34 °C for 30 minutes. The dye was then removed by centrifugation and the cells resuspended in PBS and FITC-positive cell fluorescence was detected by flow cytometry under the same conditions as for CML measurement, in the FL-1 channel. A negative control of sperm unexposed to the dye was made and a positive control of sperm exposed to H₂O₂ for 15 minutes prior to the assay to induce oxidative damage. These controls were used to gate the fluorescence-negative and -positive cells prior to ROS measurement. 10,000 cells were counted in triplicate for each experimental sample (n = 3) and relative ROS levels were expressed as MFI values.

The level of oxidative DNA damage in spermatozoa incubated with glycating agents over 6 days (n = 3) was determined using the fluorometric OxyDNA Assay Kit (Calbiochem®, EMD Millipore, US). The assay is based on a FITC-conjugate that binds to the 8-oxoguanine moiety of oxidized DNA, as performed on sperm cells elsewhere⁶⁹. Sperm cells were centrifuged at 300 g for 7 minutes to pellet the cells and remove media. Cells were fixed in 4% PFA and permeabilised with 70% ethanol. Cells were washed with Wash Solution (1×) provided with the kit and resuspended in 100 μl FITC-conjugate (1×), before incubation in the dark for 1 hour at room temperature. Cells were washed again in Wash Solution (1×), resuspended in 1xPBS and analyzed by flow cytometry as for CML. Three flow cytometry measurements were made for each experimental sample (n = 3).

To identify the localization of AGE formation on sperm, samples from the long-term incubation experiment (n = 3) were stained for AGEs using a general anti-AGE antibody⁷² (Abcam, UK, ab23722) and specifically for CML using an anti-CML antibody⁷³ (Abcam, UK, ab125145). Sperm that had been incubated with SPM, MG and GO were stained for AGEs and CML. 10 μl of sperm was placed on one end of a glass microscope slide and smeared across the slide using the feathering technique and allowed to air dry. Slides were then fixed by submersion in ice-cold methanol (100%) for 15 minutes. Once dry, a water-resistant pen (Life technologies, UK) was used to seal the area around the cells. Slides were rehydrated in PBS-tween (PBS-T) for 3 minutes before blocking in 10% normal goat serum in PBS-T (0.05%) for 1 hour at room temperature. Slides were washed in 3× fresh changes of PBS-T for 1 minute each. Primary antibodies were diluted in PBS-T (1:200), added to the slides and incubated in a humidified chamber overnight at 4 °C. Negative controls were included in which the primary antibody was omitted. The following day, slides were washed in 3× changes of PBS-T and FITC-conjugated goat anti-mouse (1:2000) or goat anti- rabbit (1:2000) secondary antibodies were added to the slides staining for CML and AGEs, respectively. Slides were incubated with secondary antibodies for 1 hour at room temperature in a darkened humidified chamber. Slides were washed a final 3 times in PBS-T before excess reagent was drained off and sperm nuclei were stained for using Vectashield Mounting Medium with DAPI (Vectashield, UK) and finally coverslipped. Fluorescent images were taken using an Axio Imager Z1 (Zeiss, HBO 100 mercury lamp) with AxioVision 4 software. Sperm head fluorescence was quantified using ImageJ software and the Corrected Total Cell Fluorescence (CCF)⁷⁴ was obtained using the equation CCF = initial cell density − (cell area × background intensity).

Functional activity of plasma membrane hyaluronidase in glycated and non- glycated sperm was measured using Hyaluronan Binding Assay (HBA) slides⁷⁵ (Origio, Denmark). At 0, 2 and 4 hours of incubation with MG, GO or SPM, 8 μl of sperm suspension was removed and placed onto a hyaluronan-coated slide and a coverslip was applied. After a 10-minute incubation period in which sperm were allowed to bind to hyaluronan, the number of bound motile sperm and the number of unbound motile sperm on a 10 × 10 square grid were counted at 40 × magnification. Immotile sperm were not counted. Bound sperm are differentiated from unbound sperm by their beating tails with heads that make no progressive movement. Sperm motility was also assessed at these stages using the CASA recording grades A (fast progressive motility), B (slow progressive motility), C (non-progressive motility) and D (immotile).

Statistical analysis was carried out using GraphPad Prism 6 Software (Version 6.01). Descriptive statistics were analyzed for each variable, observing means and standard deviation (SD). Ordinary one-way ANOVA and Tukey’s multiple comparisons tests were used to identify significant differences across different treatment groups in both short-term and long-term glycation experiments. A repeated measures ANOVA and Tukey’s multiple comparisons tests were used to test statistical significance across treatment groups and over multiple time points. Statistical significance was established when the above tests returned a p-value < 0.05.