C1639

The fusion assay was performed as described previously (9 (link)). Specifically, cauda epididymal spermatozoa were incubated in TYH drops for 2 h to 3 h before the fusion assay. Unfertilized oocytes were collected from hormone-treated females 14 h after hCG injection. To remove the ZP, oocytes were treated with 1 mg/mL collagenase (C1639; Sigma Aldrich) for 10 min. After washing in TYH, oocytes were stained with Hoechst 33342 (1:10,000) for 10 min and washed repeatedly in clean TYH drops. For insemination, 1 × 10⁵ spermatozoa per mL were used. After 30 min of incubation, oocytes were fixed with 0.25% glutaraldehyde (Wako) or 0.2% paraformaldehyde (PFA).

ASCs were isolated from the adipose tissue of mice aged 6–8 weeks as described previously^{13 (link)}. Briefly, adipose tissue was excised from the inguinal area and digested by collagenase type I (C1639; Sigma). Digested tissue was centrifuged and resuspended in mixed medium of Dulbecco’s modified Eagle’s medium and Nutrient Mixture F-12 (DMEM/F12; 11330032; Gibco) supplemented with 10% fetal bovine serum (FBS; 16140071; Gibco) and 1% penicillin/streptomycin (GNM-15140; GENOM). The resuspended cells were incubated at 37 °C, 5% CO₂ until the cells were 80–90% confluence. Cells were passaged every 2–3 days by trypsinization when they reached 70–80% confluence and were used for the experiments at passages 4–6. ASCs were seeded onto 12-well plates at a density of (1–2) × 10⁵ cells per well, followed by incubation overnight.