Cacl2 2h2o

NaCl, KCl, KH₂PO₄, MgCl₂·6H₂O, CaCl₂·2H₂O, and gentamicin sulfate were purchased from Nacalai
Tesque (Kyoto, Japan). MgSO₄·7H₂O was purchased from Ishizu Pharmaceutical (Osaka, Japan). Furthermore, eCG (the trade name; Serotropin)
and hCG (the trade name; Gonatropin) were purchased from ASKA Pharmaceutical (Tokyo, Japan). Unless otherwise specified, other chemicals were purchased from
Sigma Aldrich (St. Louis, MO, USA).
Modified TL-HEPES-PVA medium composed of 114 mM NaCl, 3.2 mM KCl, 2 mM NaHCO₃, 0.34 mM NaH₂PO₄, 10 mM Na-lactate, 0.5 mM
MgCl₂·6H₂O, 2 mM CaCl₂·2H₂O, 12 mM sorbitol, 10 mM HEPES, 0.2 mM Na-pyruvate, 0.1% (w/v) polyvinyl alcohol (PVA),
25 µg/ml gentamicin sulfate, and 65 µg/ml potassium penicillin G was used for collecting and washing COCs. The basic IVM medium was a BSA-free,
chemically-defined, porcine oocyte medium (POM, Research Institute for the Functional Peptides, Yamagata, Japan) supplemented with 50 µM beta-mercaptoethanol
(mPOM) [20 (link)]. This IVM medium was equilibrated at 39°C in an atmosphere of 5% CO₂ overnight prior to use.

Human tubal fluid (HTF) was 101.6 mM NaCl, 4.96 mM KCl, 0.37 mM KH₂PO₄, 0.37 mM MgSO₄·7H₂O (Nacalai Tesque), 25 mM
NaHCO₃ (Nacalai Tesque), 2.04 mM CaCl₂·2H₂O (Nacalai Tesque), 2.78 mM glucose, 0.33 mM Na pyruvate (Nacalai Tesque), 21.4 mM
Na lactate 60% syrup (Sigma-Aldrich), 0.4% BSA (Sigma-Aldrich), Phenol red and 1% Penicillin streptomycin in ultrapure water. Sheep oviductal monolayer with a
higher K⁺ concentration (KSOM) medium was purchased from ARK Resource (Ark resource, Kumamoto, Japan).
HTF and KSOM medium were made two and one drop each and put at 37ºC and 5% CO₂ by the day before for gas equilibrium. Sperm was collected from cauda
epididymis of 3-month-old male mice and was incubated in the HTF for 90 min. After incubation, the number of sperm was counted by hemacytometer (Erma optical
works, Tokyo, Japan) under the upright microscope (Olympus, CH) and was diluted to final concentration at 2 × 10⁵ sperm/ml. Fifty COCs collected from
6-week-old ND and LFD female mice after 16 h later of hCG injection were cocultured with sperm in HTF for 6 h. Sperm and cumulus cells were removed from
co-cultured oocytes. The embryos were transferred to KSOM drop and were cultured at 37ºC and 5% CO₂ for 5 days. Embryos with both an embryoblast and
blastocoel were defined as blastocyst-stage embryos at 3 independent experiments (n = 3).

Chemical reagents such as Tris, glycine, CuCl₂·2H₂O, FeCl₃·6H₂O, Co(NO₃)₂·6H₂O, ZnSO₄·7H₂O, KCl, Na₂HPO₄·12H₂O, NaCl, KH₂PO₄, EDTA·2Na and IPTG were purchased from Wako Pure Chemical Industries Ltd, Osaka, Japan (Guaranteed Reagent). CaCl₂·2H₂O was purchased from Nacalai Tesque (Kyoto, Japan). The synthetic substrate peptidyl-pNA, Arg-pNA, was purchased from Peptides Institute Inc., Osaka, Japan. Tryptone and yeast extract were purchased from Becton-Dickinson and Company, NJ, USA. A commercially available recombinant PD-1 molecule was used (ENZO Life Sciences Inc., product number ENZ-PRT190; PD-1 (aa 25–167) containing a 5′-His-tag, V5 epitope tag spacer, and FLAG-tag; a doublet at 40 kDa and 50 kDa: Farmingdale, NY). In our SDS-PAGE analysis, the doublet was observed at smaller positions (∼36 kDa and ∼42 kDa) than those stated in the description manual. This might be caused by the reducing conditions we employed.