I4011

To prepare luteinizing and non-luteinizing GCs, the culture medium was replaced with a fresh medium containing 0.1% of BSA, 5 ng/ml sodium selenite
(Sigma-Aldrich; S5261), 5 µg/ml transferrin (Sigma-Aldrich; T4132), and 0.5 mM ascorbic acid (Wako-Pure Chemical Industries Osaka, Japan; 031-12061), and the
cells were then incubated in a normal culture atmosphere (20% O₂, 5% CO₂, and 75% N₂) with or without insulin (2 µg/ml;
Sigma-Aldrich; I4011) in the medium in combination with forskolin (10 µM; Research Biochemicals International, Natick, MA, USA; 70-0501-05) for 24 h. Insulin
and insulin-like growth factor I (IGF-I) are known to stimulate proliferation of (and P4 production in) GCs [27 (link),28 (link),29 (link),30 (link),31 (link)]. In addition, forskolin increases intracellular cyclic AMP concentration via activation of adenylate cyclase [32 (link)]. Insulin in combination with forskolin mimics the effects of luteinizing hormone (LH) and activates adenylate cyclase via upregulation of P4
[33 (link)]. The concentration of insulin and forskolin was selected according to other reports [26 (link), 34 ].

The stromal vascular fraction in inguinal WATs was stained with fluorophore‐conjugated antibodies as described above, and the adipose‐derived stem cells (ADSCs, CD45⁻CD31⁻Sca1⁺ cells) were sorted by the FACSAria II and cultured in adipocyte maintenance medium (DMEM supplemented with 10% FBS, 1% Pen Strep (10378‐016, Life Technologies), 20 nM insulin (I4011, Sigma‐Aldrich)). For adipocyte differentiation, confluent cells were incubated in adipocyte differentiation medium (DMEM supplemented with 10% FBS, 1% Pen Strep, 20 nM insulin, 0.5 mM 3‐isobutyl‐1‐methylxanthine [I5879, Sigma‐Aldrich], 0.5 µM dexamethasone [D1756, Sigma‐Aldrich] and 125 µM indomethacin [I7378, Sigma‐Aldrich]). After 48 h of exposure to the adipocyte differentiation medium, cells were maintained in the adipocyte maintenance medium with changing the medium every 2 days until analysis. To delete Zfat gene ex vivo, cells were treated with 1 µM 4‐hydroxytamoxifen (H7904, Sigma‐Aldrich) in the adipocyte maintenance medium for 4 days.