Female 7- to 8-week-old BDF1 mice (Orient Co., Seoul, Korea) were maintained under 12-hour light and a 12-hour dark conditions at 23℃ and fed ad libitum. All experiments were conducted ethically with the approval of the Institute of Animal Care and Use Committee of the Seoul National University Bundang Hospital (IACUC No. BA-1903-267-014-04).

After 1 week of adaptation, mice were divided into 4 groups and then treated with an intraperitoneal injection of each agent. In group A, which served as a control group, 0.1 mL of saline was injected once. In group B, Cp (25 mg/kg body weight) (Cp monohydrate; Cat. no. 29875, Sigma-Aldrich, St. Louis, MO, USA) was injected once. In group C, Cp (25 mg/kg) and imatinib (7.5 mg/kg; Enzo Life Sciences, Farmingdale, NY, USA) was injected once. In group D, Cp (25 mg/kg) and dasatinib (7.5 mg/kg; Enzo Life Sciences) was injected once. An imatinib or dasatinib stock solution was obtained by dissolving imatinib or dasatinib powder in phosphate-buffered saline (PBS). Three days later, the mice were killed by cervical dislocation, and the bilateral ovaries were collected in 1 mL of L-15 medium (WelGENE, Daegu, Korea) supplemented with 0.4% bovine serum albumin (BSA; Sigma-Aldrich).

Intact preantral follicles were mechanically isolated and cultured in 96-well plates (BD BioCoat; BD Falcon, Franklin Lakes, NJ, USA) at 37℃ in 5% CO₂ for 10 days in a growth medium. The growth medium was composed of alpha-minimum essential medium (WelGENE), 5% fetal bovine serum (Gibco, Paisley, UK), 10 mIU/mL recombinant follicle-stimulating hormone (Merck-Serono, Geneva, Switzerland), 1% insulin-transferrin-selenium mixture (Sigma-Aldrich), and 1% penicillin-streptomycin mixture (Sigma-Aldrich). Every 3-4 days, the medium was changed, and follicle survival and formation of the antrum were assessed. Oocytes were considered to be dead if they were not surrounded by granulosa cells or if the granulosa cells appeared to be dark and fragmented, and the follicle decreased in size.

After 10 days of culture in the growth medium, the follicles were transferred to maturation medium and cultured for 16 hours at 37℃ in 5% CO₂. The maturation medium was prepared by adding 1.5 IU/mL human chorionic gonadotropin (Merck-Serono), and 5 ng/mL recombinant mouse epidermal growth factor (Sigma-Aldrich) to the growth medium. The oocytes were then harvested either from spontaneously ruptured or non-ruptured follicles. The surrounding cumulus cells were removed by treating them with 0.3% hyaluronidase (Sigma-Aldrich) and gentle pipetting. Oocytes were classified as metaphase II (MII), metaphase I (MI), germinal vesicle, dead, or degenerated. If a polar body was present in the perivitelline space, the oocytes were regarded as MII oocytes. Fragmented or shrunken oocytes were classified as degenerated oocytes. Figure 1 shows mouse preantral follicles and various developmental stages of follicles during in vitro culture, as well as in the finally obtained oocytes.

The spindle integrity of the MII oocytes was assessed using previously described methods [14]. The MII oocytes were washed three times with 1% BSA in PBS for 5 minutes and then fixed with 4% paraformaldehyde for 1 hour at room temperature (RT). After washing twice with 1% BSA in PBS, permeabilization was performed with 0.25% Triton X-100 in PBS for 10 minutes at RT. After washing twice with 1% BSA in PBS, blocking was performed with 3% BSA in PBS for 1 hour at RT and then washing twice with 1% BSA in PBS. A primary antibody for α-tubulin (Cell Signaling, Danvers, MA, USA) diluted in 1% BSA (1:100) was added and incubated overnight at 4℃. After washing three times with 1% BSA in PBS, a secondary antibody (Alexa Fluor 488 goat anti-rabbit immunoglobulin G; Invitrogen, Carlsbad, CA, USA) diluted in 1% BSA (1:100) was added for 1 hour at RT in the dark. After washing three times with 1% BSA in PBS, the oocytes were air-dried on a silane-coated slide (DAKO, Glostrup, Denmark). The slide was counterstained with 4’,6’-diamidino-2-phenylindole (DAPI), and examined using a confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany). A typical barrel-shaped microtubule structure between both poles with centrally aligned chromosomes was considered normal (Figure 2).

In each experimental group, the final spent media, in which five ruptured follicles were present, were pooled and then frozen at –80℃. After thawing, the levels of 17β-estradiol and anti-Müllerian hormone (AMH) were measured by commercially available enzyme-linked immunosorbent assay kits. The limits of sensitivity for 17β-estradiol (Enzo Life Sciences) and AMH (Ansh Labs, Webster, TX, USA) were 10 pg/mL and 0.01 ng/mL, respectively.

The final follicular cells from 20 ruptured follicles were pooled (after isolation of oocytes) and then frozen at –80℃. After thawing, total RNA was extracted using the Dynabeads method (Dynabeads mRNA DIRECT kit; Ambion, Oslo, Norway), and cDNAs were synthesized using a PrimeScript first strand cDNA Synthesis Kit (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (PCR) was performed using a StepOne-Plus real-time PCR system with TaqMan probes (Applied Biosystems, Foster City, CA, USA). Real-time quantitative PCR was conducted in a 20-μL reaction volume containing 10 μL of Applied Biosystems TaqMan Universal PCR Master Mix I (Cat. no. 4427788), 2 μL of cDNA, and 6 μL of RNase-free water. Specific primers were purchased from Integrated DNA (Coraville, IA, USA). Primer sequences and their product sizes, accession number, annealing temperature, and total PCR cycles of each gene are listed in Table 1.

In this study, the mRNA level of steroidogenic acute regulatory protein (Star; a marker of luteinization), superoxide dismutase 1 (Sod1; a marker of oxidative stress), mitogen-activated protein kinase 3 (Mapk3; a marker of cell growth), and caspase 3 (Casp3; a marker of apoptosis) were quantitatively measured. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control. Star transports cholesterol from the outer mitochondrial membrane to the inner membrane and converts it into pregnenolone, which regulates steroid hormone synthesis, especially during the luteal phase [15]. Sod1 is present in the cytoplasm and catalyzes the disproportionation of superoxide into hydrogen peroxide and dioxygen by binding to copper or zinc ions [16]. Mapk3 serves as a signaling molecule and involves cell growth. In the granulosa cells of preovulatory follicles, it is activated by a luteinizing hormone (LH) surge and plays a role in the LH-induced oocyte resumption of meiosis, ovulation, and luteinization [17]. Casp3 is the last enzyme in the cell death process and is a marker of apoptosis [10,18].

In each experimental group, at least five repeats were conducted. Real-time quantitative PCR was repeated five times for each repeat, and the values were averaged. For each experiment, five different readouts were obtained for each gene of interest. The measured values were obtained as the cycle threshold (Ct) at a constant fluorescence intensity. The level of each transcript was inversely related to the observed Ct value. The relative expression (R) levels of the genes were normalized to that of Gapdh as an internal control. The ΔCt value was calculated as follows: the Ct of the target gene minus the Ct of Gapdh. The ΔΔCt value was calculated as ΔCt minus the mean value of each group. To determine the fold change for each gene, the relative gene expression of the four treatment groups was calculated using the 2^-ΔΔCt method as previously described [19].

Statistical analyses were performed using IBM SPSS ver. 22.0 (IBM Corp., Armonk, NY, USA), and p-values <0.05 were considered to indicate statistical significance. The Fisher exact test was used to compare the proportions among the groups. Numerical data were compared using the Kruskal-Wallis test. If the value was significant, the Mann-Whitney U test with the Bonferroni correction was used for further analysis.