IN-VITRO MATURATION, FERTILIZATION AND EMBRYO DEVELOPMENT OF IMMATUREOOCYTES 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 culture d singly in 20 mu l droplets under oil in medium supplemented with rec ombinant follicle stimulating hormone (r-FSA) at 37 degrees C and 5% C O2 in air. The follicles grew and became attached to the bottom of the dish, progressively lost their spherical structure by outgrowth of th e granulosa cells through the basal membrane and developed follicles w ith antral-like cavities. The normal three-dimensional follicular stru cture was lost but all components, i.e. theca, granulosa and oocyte, r emained functional, as was proven by the oestradiol, inhibin and proge sterone secretion patterns. Follicle survival exceeded 80% and histolo gical analysis proved the absence of atresia and cell death in granulo sa 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 metaphas e II oocytes were obtained on days 12 and 14 (39 and 41%). The fertili zing potential of the in-vitro matured oocytes was comparable to in-vi vo matured controls. A 50% hatched-blastocyst development rate was obs erved.