Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.