Ovaries from 4-week-old mice were sliced at a thickness of several hundred micrometers using a microtome blade (Leica Biosystems, Nussloch, Germany) under a stereoscopic microscope (Leica Biosystems). Ovarian tissue slices were
placed in a 30-mm cell culture insert (Merck Millipore, Darmstadt, Germany), which was subsequently placed in a 3.5-cm culture dish (AGC Techno Glass, Shizuoka, Japan). Ovarian slices were cultured in the minimum essential medium
alpha (MEM-alpha) GlutaMax (Gibco, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), 100 mIU/ml FSH from human pituitary gland (Sigma-Aldrich, St Louis, MO, USA), and 10 mIU/ml LH from equine pituitary
gland (Sigma-Aldrich) under conditions of 5% CO2 and 37°C. The cultured ovarian tissue slices were treated with 100 mIU/ml of LH for 12 h every 4 days to reproduce the physiological LH surge. Concentrations of FSH and
LH used were based on previous experiments [10 (link), 11 (link)]. The effect of P4 on follicle growth was evaluated after adding P4 (10 ng/ml,
100 ng/ml, and 1 μg/ml) (Sigma-Aldrich) to the culture medium, followed by culture for 18 days. In this experiment, one ovary was cut into four slices; two of these slices were treated with dimethyl sulfoxide (DMSO) alone and the
other two, with P4 dissolved in DMSO. Four or five ovaries were cultured with each concentration of P4.
placed in a 30-mm cell culture insert (Merck Millipore, Darmstadt, Germany), which was subsequently placed in a 3.5-cm culture dish (AGC Techno Glass, Shizuoka, Japan). Ovarian slices were cultured in the minimum essential medium
alpha (MEM-alpha) GlutaMax (Gibco, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), 100 mIU/ml FSH from human pituitary gland (Sigma-Aldrich, St Louis, MO, USA), and 10 mIU/ml LH from equine pituitary
gland (Sigma-Aldrich) under conditions of 5% CO2 and 37°C. The cultured ovarian tissue slices were treated with 100 mIU/ml of LH for 12 h every 4 days to reproduce the physiological LH surge. Concentrations of FSH and
LH used were based on previous experiments [10 (link), 11 (link)]. The effect of P4 on follicle growth was evaluated after adding P4 (10 ng/ml,
100 ng/ml, and 1 μg/ml) (Sigma-Aldrich) to the culture medium, followed by culture for 18 days. In this experiment, one ovary was cut into four slices; two of these slices were treated with dimethyl sulfoxide (DMSO) alone and the
other two, with P4 dissolved in DMSO. Four or five ovaries were cultured with each concentration of P4.
