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

Department of Physiology, National University of Ireland, Galway, University Road, Galway, Ireland

Correspondence should be addressed to A C Hynes; Email: ailish.hynes{at}nuigalway.ie

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 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 x 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.

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