Investigating the Glycating Effects of Glucose, Glyoxal and Methylglyoxal on Human Sperm

Abstract

Glycation is the non-enzymatic reaction between reducing sugars, such as glucose, and proteins, lipids or nucleic acids, producing Advanced Glycation End (AGE) products. AGEs, produced during natural senescence as well as through lifestyle factors such as diet and smoking, are key pathogenic compounds in the initiation and progression of diabetes. Importantly, many of these factors and conditions also have influence on male fertility, affecting sperm count and semen quality, contributing to the decreasing trend in male fertility. This study investigated the impact of AGEs on sperm damage. In vitro sperm glycation assays were used to determine the levels and localization of the potent AGE compound, carboxymethyl-lysine (CML) in response to treatment with the glycating compounds glucose, glyoxal and methylglyoxal. Sperm function assays were then used to assess the effects of glycation on motility and hyaluronan binding, and levels of oxidative DNA damage were analyzed through measurement of the marker, 8-oxoguanine. Results showed that glyoxal, but not glucose or methylglyoxal, induced significant increases in CML levels on sperm and this correlated with an increase in 8-oxoguanine. Immunocytochemistry revealed that AGEs were located on all parts of the sperm cell and most prominently on the head region. Sperm motility and hyaluronidase activity were not adversely affected by glycation. Together, the observed detrimental effects of the increased levels of AGE on DNA integrity, without an effect on motility and hyaluronidase activity, suggest that sperm may retain some fertilizing capacity under these adverse conditions.

Figure 1

Glycation through the Maillard reaction. AGEs are formed through the Maillard reaction in which the carbonyl group of a reducing sugar, such as glucose, reacts with the amine group of a protein side chain residue, such as lysine. Initially a reversible Schiff base adduct is formed, which then undergoes a series of rearrangements to form an Amadori product. Further glycation and oxidation reactions lead to the formation of a primary AGE Nε-(carboxymethyl)lysine (CML). Fragmentation of the Schiff base generates the AGE intermediates methylglyoxal (MG) and glyoxal (GO). These reactive carbonyl compounds also react with biomolecules to produce AGEs.

Figure 2

Effect of glycating compounds on sperm vitality and motility. (A) The percentage of live sperm following incubation with glucose (Glc) (n = 3) (30 mM and 50 mM), (B) methylglyoxal, MG (n = 3) (50 µM) and glyoxal, GO (n = 3) (50 µM) for 6 days, measured using the eosin-nigrosin stain. Sperm vitality decreased in all treatment groups at the end of the incubation period, but the presence of glucose maintained vitality at a significantly higher level than Sperm Preparation Media, SPM (***p < 0.001). The presence of MG and GO also showed significantly higher vitality than SPM (*p < 0.05). (C) Sperm progressive motility had significantly decreased after 72 hours for all treatment groups (n = 3) (**p < 0.01) but did not significantly differ between the groups. Error bars represent mean +/− SD.

Figure 3

Carboxymethyl lysine formation in response to glycating agents. (A) Incubation of sperm with GO (50 µM) (n = 5) for 6 days resulted in a significant increase in the level (mean MFI +/− SD) of CML in comparison to SPM (n = 5) (p < 0.01) and MG (n = 5) (p < 0.05). (B) Incubation of sperm with 30 mM glucose (Glc) (n = 3) or 50 mM glucose (n = 3) had no effect on CML levels in comparison to SPM (n = 3). Error bars represent mean +/− SD.

Figure 4

Immunolocalisation of CML and AGE on sperm. (A) Immunocytochemical staining of CML on SPM (i), MG (ii) and GO (iii) sperm revealed strong positive staining (green) on the tail of MG and GO sperm, and uniquely on the head region of sperm incubated with GO (arrow) (n = 3). Negative control showed no CML staining (iv). (B) Mean (+/− SD) corrected total cell fluorescence (CTCF) was calculated from 10 cells of different experimental replicates and showed CML to be significantly higher in the head region of GO sperm (p < 0.05). (C and D) General AGE staining (green) indicated the presence of AGEs on all treatment groups, with both GO (iii) and SPM (i) showing significantly higher head fluorescence than MG (ii) (arrow) (p < 0.01, p < 0.05, respectively) (n = 3). Magnification was at 100× oil immersion. Nuclear staining is shown in blue (DAPI).

Figure 5

Intracellular ROS production and oxidative DNA damage. The effects of sugars and intermediate compounds on intracellular ROS levels in sperm. (A) Relative ROS levels in sperm treated with SPM (n = 3), glucose (Glc, 30 mM) (n = 3), MG (50 μM) (n = 3) or GO (50 μM) (n = 3) for 6 days. ROS is shown as mean fluorescence intensity (MFI) +/− SD of flow cytometry detection. One-way ANOVA analysis revealed no difference in ROS levels between any of the treatment groups. (B) 8-oxoguanine levels were measured in sperm treated with SPM (n = 3), glucose (Glc, 30 mM) (n = 3), MG (50 μM) (n = 3) and GO (50 μM) (n = 3). 8-oxoguanine levels are shown as MFI of flow cytometry detection. GO treatment caused significantly more 8-oxoguanine generation in comparison to MG (*p < 0.05), glucose (*p < 0.01) and SPM (*p < 0.01). Error bars represent mean +/− SD.

Figure 6

Short term glycation of sperm by GO. (A) CML levels (MFI +/− SD) in sperm treated with SPM, MG and GO (n = 3). Sperm incubated with GO showed an increase in CML at 2 hours (BA) and 4 hours (BB), both significantly higher than MG and GO at these time points (AA and AB). ****p < 0.0001, **p < 0.01. Key applies to (A), (B) and (C). (B) Sperm progressive motility (mean +/− SD) during glycation with SPM, MG and GO (n = 3). Two-way ANOVA showed no significant difference in progressive motility over 4 hours and between any treatment group. (C) HA binding of sperm incubated with MG and GO (n = 3). No difference was seen in hyaluronan binding capacity at 2 or 4 hours. Sperm maintained a high level of binding throughout. Error bars represent mean +/− SD.