MEIOTIC PROGRESSION OF MOUSE OOCYTES THROUGHOUT FOLLICLE GROWTH AND OVULATION IN-VITRO

Intact ovarian follicles can be grown in culture but the normality of their oocyte development has not yet been established. This work examined meiotic progression in oocytes from mouse ovarian follicles grown in vitro and in vivo. Follicles of initial diameter 112-279 pm were isolated from ovaries of B6 CBA F-1 mice and cultured individually in 25 mu l minimal essential medium-alpha containing 5% mouse serum, 100 mIU/ml follicle stimulating hormone and various supplements. Oocytes were isolated from follicles of various sizes after culture for less than or equal to 5 days and oocyte chromatin was examined by fluorescence microscopy with Hoechst 33258. The stage of meiosis was classed as prophase I [germinal vesicle (GV) stages I-IV], metaphase I, anaphase I or metaphase II and the results were compared with oocytes from fresh follicles of unprimed or gonadotrophin-primed immature mice, and mature mice in oestrus. Small pre-antral follicles grew in vitro to intact pre-ovulatory follicles and some ovulated spontaneously despite negligible luteinizing hormone (LH). Progression through GV stages I-IV in vitro was related to initial and final follicle diameter, follicle growth rate and antrum formation. All the normal stages of GV development were observed in vitro; however, the cultured follicles were significantly larger than the freshly dissected follicles for stages II and III of GV development. GV breakdown had occurred in 7/11 analysable ovulated oocytes and 3/69 intra-follicular oocytes. We conclude that, under the conditions employed, the majority of oocytes cultured in intact immature follicles retain the GV. Meiotic prophase progresses apparently normally but in vitro follicles are larger and maturation of oocytes after spontaneous ovulation is variable.