Growth rates and antrum formation of mouse ovarian follicles in vitro in response to follicle-stimulating hormone, relaxin, cyclic AMP and hypoxanthine.
Hartshorne GM., Sargent IL., Barlow DH.
The culture of individual intact follicles in vitro, from small pre-antral to pre-ovulatory stages, will improve our ability to perform controlled experiments studying follicle growth and female gamete development. This study was undertaken to characterize further the conditions required for physiological follicle growth in vitro. We cultured a total of 398 pre-antral follicles of immature mice (aged 26-28 days) with an initial diameter of 140-340 microns for 4 days in vitro, using individual micro-cultures under paraffin oil. Summarizing the results of all groups, 50 follicles were damaged (12.6%) and, of those remaining intact (n = 348), 60 (17.2%) became atretic, 195 (56.0%) became antral and 26 (7.5%) ovulated. The most advanced follicles grew to 400-500 microns diameter. The presence of follicle-stimulating hormone (FSH) in the medium significantly stimulated follicle growth in vitro (P < 0.03), in a manner proportional to the initial diameter over the range of 140-250 microns initial diameter, with larger follicles being refractory. FSH also significantly increased the proportion of follicles forming antra (P < 0.001) and their likelihood of ovulating in vitro (P < 0.01), and reduced the frequency of atresia (P < 0.01). Dibutyryl-cyclic AMP mimicked FSH, significantly stimulating growth of large follicles (P < 0.05) and antrum formation (P < 0.01). Hypoxanthine also stimulated antrum formation (P < 0.01) but did not significantly affect follicle growth. Porcine relaxin had no significant effect on mouse follicle growth or antrum formation. The optimal conditions for mouse follicle growth in vitro have not yet been defined, but selection of follicles of < 250 microns diameter and inclusion of FSH or dibutyryl-cyclic AMP in the culture medium are recommended.