Bilirubin helps to overcome the two-cell block in mouse oocyte cultures.

PURPOSE: In vitro fertilization and culture of mouse oocytes, under normal atmospheric oxygen tension, subjects them to severe oxidative stress. Oocytes from some strains of mice lack the natural protective mechanism that guards them against this oxidative stress and fail to develop beyond the two-cell stage.

METHODS

We could overcome the toxic effects of oxygen metabolites by adding 0.2-0.4 mg/dl bilirubin in a lactate-pyruvate culture medium defined by Whitten (1971). Six- to 8-week-old ICR (Institute of Cancer Research) female mice were super ovulated by intra peritoneal injection of 5 IU PMSG (pregnant mare serum gonadotropin) followed by 10 IU hCG 48 h later. The oocytes were collected from the distended fallopian tubes and inseminated with 1-2 million sperm from 3-4-month-old ICR male mice. The eggs were scored at 24, 48, and 72 h after the hCG injection.

CONCLUSIONS

With 0.4 mg/dl bilirubin supplement, by the end of 72 h, 82% of the eggs progressed from the two-cell stage to the four-cell stage. Routine inclusion of bilirubin can improve embryo development in vitro.