A novel follicle culture system markedly increases follicle volume, cell number and oestradiol secretion.
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 microm in diameter) were cultured in a 100 microl 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 degrees 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 microm) as compared with the upright with oil (363 +/- 2.7 microm) 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.
Wycherley
G
G
Department of Physiology, National University of Ireland, Galway, University Road, Galway, Ireland.
Downey
D
D
Kane
M T
MT
Hynes
A C
AC
D000818:Animals; D002452:Cell Count; D002455:Cell Division; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D006080:Ovarian Follicle / Q000166:cytology / Q000378:metabolism / Q000502:physiology*
D003470:Culture Media; D004958:Estradiol