Development and characterization of an in vitro ovulation model using mouse ovarian follicles

To investigate ovulation, an in vitro model with cultured mouse follicles w as developed and compared with an in vivo ovulation model. In this model, s econdary follicles were grown in vitro with immature mouse serum (5%) and r ecombinant human FSH. Addition of ascorbic acid and selenium to the medium increased follicular survival (from 29% to 86%) and resulted in the develop ment of healthy preovulatory follicles (> 400 mu m) producing estradiol. De pending on the starting size of the follicles, the preovulatory stage was r eached after 4-6 days. The ovulatory response to hCG was maximal in follicl es exceeding a diameter of 400 pm. The in vitro-ovulated oocytes could be f ertilized and were able to develop to the blastocyst stage. Ovulation induc ed by hCG was dose dependent, reaching a maximum of 80% at 1 IU/ml. Concomi tantly, progesterone production increased from 3.6 +/- 0.5 to 29 +/- 2 ng/m l. Both in vivo and in vitro, hCG induced expression of the progesterone re ceptor and the prostaglandin endoperoxide synthase-2 (PGS-2) gene within 3 h. Ovulation could be completely blocked with the anti-progestogen Org-3171 0 and partially (50%) with the PGS inhibitor indomethacin in vitro and in v ivo. Org-31710 and indomethacin did not affect progesterone production. In summary, a physiologically relevant in vitro ovulation model of cultured mouse follicles that can be used to study the process of follicular ruptur e has been developed.