A novel follicle culture system markedly increases follicle volume, cell number and oestradiol secretion

Abstract

This study reports a novel, simple method for culture of mouse follicles which results in follicles with cell numbers similar toin vivofully grown follicles. Using this method, follicles (180–240 μm in diameter) were cultured in a 100 μl 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 × CBA/ca crossbred and MF1 inbred mice, were cultured individually at 37 °C 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 (means±s.e.m.; 471 ± 6.0 μm) as compared with the upright with oil (363 ± 2.7 μm) and without oil (358 ± 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 ± 0.45 ng in the inverted and 0.90 ± 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.