This study reports a novel, simple method for culture of mouse follicles which results in follicles with cell numbers similar to in vivo fully grown follicles. Using this method, follicles (180 -240 mm in diameter) were cultured in a 100 ml inverted drop of medium without oil and compared with culture in upright drops with and without a mineral oil overlay. Follicles, isolated from C57BL/6 3 CBA/ca crossbred and MF1 inbred mice, were cultured individually at 37 8C in 96-well round-bottomed suspension cell tissue culture plates for 6 days. Follicles grown in the inverted drop culture system reached a markedly higher final diameter (means6S.E.M.; 471 6 6.0 mm) as compared with the upright with oil (363 6 2.7 mm) and without oil (358 6 4.0) systems. There was no significant effect of mouse strain on follicle diameter. Follicular secretion of oestradiol and lactate into the medium was measured on days 2, 4 and 6 of culture. Secretion of oestradiol per follicle on day 6 was 2.49 6 0.45 ng in the inverted and 0.90 6 0.17 ng in the upright without oil system (P < 0.001). Follicular secretion of lactate on a per unit of follicle volume basis remained constant in the inverted system over days 2, 4 and 6 and was less (P < 0.001) than secretion in both the upright with and without oil systems. Follicle cell proliferation was markedly increased in the inverted as compared with the upright with oil system; the increases in cell numbers were significant on day 3 (P < 0.01) and on all subsequent days (P < 0.001). These results are discussed in relation to the supply of oxygen to the follicle in culture.

Aerobic metabolism and an adequate oxygen supply are essential for normal follicular development.