NORMAL OXYGEN ATMOSPHERE IS ESSENTIAL FOR THE SOLITARY LONG-TERM CULTURE OF EARLY PREANTRAL MOUSE FOLLICLES

This study compares the effects of reduced (5%) or normal (5% CO2 in a ir; 20% O-2) oxygen tension on the in vitro maturation of early preant ral ovarian follicles isolated from 14-day-old (C57BI/6J x CBAca) F1 m ice. Intact follicles (100-130 mu m) are singly cultured in 20 mu l dr oplets alpha-MEM enriched with FCS and rFSH under mineral oil at 37 de grees C and 100% humidity. In this culture system the follicles are al lowed to attach to the bottom of the petri dishes. Follicle in vitro g rowth, hormone secretory capacity, and in vitro ovulation were studied under the two oxygen tensions. Spontaneous oocyte release from the fo llicle during a 16-day culture period was observed significantly more under 5% oxygen. Antrallike cavity formation was not observed under 5% O-2 The follicles in the 5% O-2 cultures reaching day 16 were strippe d of their granulosa cell layers, and 83% of the retrieved oocytes had already undergone spontaneous germinal-vesicle breakdown (GVBD). Unde r 20% O-2, the GV stage was maintained until day 16 in 77% of the oocy tes. Under 5% O-2, intact follicle survival up to day 12 was significa ntly reduced as compared to the 5% CO2 in air conditioning. The hCG st imulus on day 12 induced mucification in a significantly larger propor tion of follicles cultured under 20% O-2 (79% VS. 47%). Germinal-vesic le breakdown (20% O-2:95%, 5%, O-2:42%) and first polar body extrusion (20% O-2,:40%, 5% O-2:15%) were significantly more prevalent under no rmal oxygen tension. A reduced secretory capacity of E(2) and inhibin was demonstrated for follicles cultured under 5% O-2 The histological study of serially sectioned follicles showed increased areas of centra lly located granulosa cell necrosis and pyknosis in the cumulus cells. Gassing follicle cultures using 5% COP in air provided appropriate co nditions for normal growth, enhanced whole-follicle survival, differen tiation, and hormone production, and improved the yield of meiotic com petent oocytes. (C) 1996 Wiley-Liss, Inc.