Combined Application of Methyl-Beta-Cyclodextrin and Reduced Glutathione to the In Vitro Fertilization of Cryopreserved C57BL/6 Mouse Sperm.

Abstract

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. The benefits of sperm cryopreservation include the saving of space and costs relating to the upkeep of each mouse lineage, the potential avoidance of a genetic drift during the storage term, and the ease of transporting the mice between research facilities. However, embryos need to be obtained using in vitro fertilization (IVF) in order to reproduce live animals from frozen/thawed sperm. In general, the fertility of frozen/thawed sperm taken from C57BL/6 mice, which is mainly used as a base for genetically engineered mice, is severely lower than that of fresh sperm. Previously, we reported that methyl-β-cyclodextrin (MBCD) dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. MBCD effectively induces sperm capacitation by facilitating a cholesterol efflux from the cells. Recently, an interesting report was published on the topic of the supportive effects of reduced glutathione (GSH) on improving the fertility of mouse sperm. However, the action mechanism of GSH during the fertilization process is not fully understood. In this study, we examined the most efficient timing of GSH treatment and the combined effects of MBCD and GSH on the rate of fertilization for IVF using frozen/thawed C57BL/6 mouse sperm. As a result, we found that the presence of GSH in the fertilization medium increased the fertilization rate whereas GSH had no effects in the medium of sperm preincubation, and that the elevation of fertilization rates started at 0.5 mM GSH and reached a peak at 1.0 mM GSH (0 mM: 66%, 0.5 mM: 79% and 1.0 mM: 92%). We also discovered that the MBCD and GSH independently increased the rate of fertilization, while a combination of MBCD and GSH exhibited the highest fertilization rate in each group (control: 14%, MBCD: 65%, GSH: 36% and MBCD + GSH: 87%). In summary, we have demonstrated a novel IVF method using the combined application of MBCD for sperm preincubation and GSH for IVF, in order to achieve a high and stable fertilization rate in frozen/thawed C57BL/6 mouse sperm. (poster)