In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system

A simplified culture system was developed for the in-vitro maturation of early preantral mouse ovarian follicles, The follicles were cultured singly in 20 mu l droplets under oil in medium supplemented with recombinant follicle stimulating hormone (r-FSA) at 37 degrees C and 5% CO2 in air. The follicles grew and became attached to the bottom of the dish, progressively lost their spherical structure by outgrowth of the granulosa cells through the basal membrane and developed follicles with antral-like cavities. The normal three-dimensional follicular structure was lost but all components, i.e. theca, granulosa and oocyte, remained functional, as was proven by the oestradiol, inhibin and progesterone secretion patterns. Follicle survival exceeded 80% and histological analysis proved the absence of atresia and cell death in granulosa cells up to day 16. Oocytes of 55 (+/- 4) mu m diameter on the day of isolation reached 74 (+/- 3) mu m by day 16 of culture. The optimal moment for inducing the final meiotic maturation with human chorionic gonadotrophin was investigated: the highest absolute numbers metaphase II oocytes were obtained on days 12 and 14 (39 and 41%). The fertilizing potential of the in-vitro matured oocytes was comparable to in-vivo matured controls. A 50% hatched-blastocyst development rate was observed.