MTT is a commonly used cell vitality probe which forms insoluble formazan deposits at cellular locations of intense oxidoreductase activity. While the majority of this activity occurs within the mitochondria, extra-mitochondrial sites of MTT reduction have been recognized in the spermatozoa of several mammalian species. Therefore, the aim of this study was to determine the major sites and causative mechanisms of MTT reduction in stallion spermatozoa.
Spermatozoa were collected from miniature pony stallions, and ejaculates were separated into high- and low-density fractions using discontinuous Percoll gradients. Cells were incubated with 0.5 mg/mL MTT for 1 h at 37 oC, before being scored for formazan deposition via microscopy. A number of reagents were also assessed for their impact on MTT reduction by incubation with spermatozoa for 20 min before MTT addition. These reagents included the NADPH oxidase (NOX5) inhibitors VAS2870 (10 µM) and zinc (0.5 mM). Additionally, spermatozoa were incubated under capacitating conditions (0.72 mg/mL pentoxifylline, 0.6 mg/mL methyl-beta-cyclodextrin and 2.45 mg/mL dibutyryl-cyclic AMP), before the addition of MTT.
Our results show that both mitochondrial and extra-mitochondrial MTT reduction (formazan deposition) was greater in high-density compared to low-density spermatozoa (mitochondrial: 83.7±1.6 vs 30±5.2%; P≤0.001; extra-mitochondrial: 20.8±2.5 vs 12.2±3.2; P≤0.05). NOX5 activity was implicated in extra-mitochondrial MTT reduction, with NOX5 inhibitors suppressing this activity (VAS2870: 6.6±1.9 vs 19.3±2.9%; P≤0.01; zinc: 11.7±4.4 vs 22.6±3.7; P≤0.05; for treated vs control respectively), without impacting sperm vitality. Furthermore, extra-mitochondrial formazan deposition was greater in capacitated compared to non-capacitated spermatozoa (20.4±1.7 vs 34.6±3.4; P≤0.001), suggesting that NOX5 generated ROS play a role in mediating the redox regulation of equine sperm capacitation.
In conclusion, MTT reduction patterns by stallion spermatozoa are reflective of a species dependent on OXPHOS and one exhibiting NOX5 activity, with extra-mitochondrial redox activity being reflective of sperm quality.