Inhibition of In Vitro Fertilizing Capacity of Cryopreserved Mouse Sperm by Factors Released by Damaged Sperm, and Stimulation by Glutathione

In vitro fertilization (IVF) of eggs by frozen and thawed C57BL/6J mouse sperm is inhibited by dead sperm and enhanced by preincubation of the sperm in calcium-free medium. In other species, the presence of sperm killed by freezing and thawing has been associated with the generation of hydrogen peroxide., reduced glutathione was added to the fertilization medium. This enhanced fertilization rate significantly (76.6%±2.0% versus 21.2%±1.9%), while addition of oxidized glutathione did not (82.7%±6.5% with reduced glutathione; 44.5±8.8% with oxidized glutathione; 47.8%±12.1% with no glutathione). Positive effects of reduced glutathione on IVF were also seen with frozen 129S1, FVB, and C3H sperm, and sperm from two lines of genetically modified C57BL/6J mice.IVF in cell culture inserts and addition of glutathione to fertilization medium significantly increased the proportion of eggs fertilized by cryopreserved mouse sperm from four inbred strains, suggesting that reactive oxygen species generated during fertilization inhibit fertilization. The modified IVF techniques developed here enhance the feasibility and efficiency of using cryopreserved sperm from genetically modified lines of inbred mice