Improvement of cumulus-free oocyte maturation in vitro and its application to microinsemination with primary spermatocytes in mice

In micromanipulation experiments using immature oocytes, final ooplasmic maturation is often compromised because the oocytes are usually first freed from their nurturing cumulus cells. This study was undertaken to determine whether cumulus-free in vitro maturation (IVM) in mice could be improved by modifying IVM medium having defined components. Cumulus-free germinal vesicle (GV) stage oocytes were subjected to IVM in either alpha MEM medium, TYH medium, or a 1:1 mixture of the two (termed TaM). TYH medium produced a better maturation rate (181/196; 92.3%) than alpha MEM (184/257; 71.6%). However, alpha MEM supported better embryo development to the morula/blastocyst stage than TYH following in vitro fertilization (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse following IVM in alpha MEM, but was aggregated with TYH. The maturation promoting factor (MPF) activity in MII oocytes was significantly higher in TYH than in alpha MEM (P < 0.05). Oocytes cultured in TaM had intermediate characteristics and essentially resembled in vivo matured oocytes, with the mitochondrial distribution pattern being most typical of that condition. The highest rate of development from GV oocytes to full-term fetuses following in vitro fertilization and embryo transfer to foster mothers (23.8%) was obtained using TaM. When this IVM system was applied to MI oocytes injected with spermatocytes, offspring were first obtained without cytoplasmic replacement at MII. Thus, optimization of the culture medium can considerably improve the quality of cumulus-free oocyte IVM in mice.