Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mu l suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1 % HSA at 37 C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5',6,6'-tetracllloro-1-1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1 +/- 3.2%, P<0.05) when compared with controls (19.4 +/- 1.9%). The combination of HSA and sucrose (65.2 +/- 2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6 +/- 4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.