Cuo np

The copper nanoproducts (NPs) of Cu NPs and CuO NPs of similar primary particle size of around 50 nm and ionic form CuSO₄ were purchased from Sigma Aldrich. The basic suspension of compounds 1 g Cu·L⁻¹ was prepared in artificial seminal plasma (ASP: 125 mM NaCl, 40 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 50 mM Tris, pH 8.5, 363 mOsm·kg⁻¹, composition by Morisawa and Morisawa [55 (link)] modified by Dziewulska) and sonicated in an ultrasonic bath for 30 min. Following stock suspension: 0, 1, 5, 10, 25, 50 mg Cu·L⁻¹ of testing compounds were prepared by mixing in artificial seminal plasma. Four milt samples were mixed individually with artificial seminal plasma containing an adequate nanoproduct concentration in the ratio 1:10 and stored in thin layers (5 mm), at 6 °C, for 96 h. The samples were mixed every 12 h. At defined incubation times: 0, 2, 12, 24, 48, 72, and 96 h activation of spermatozoa motility was determined in CASA following the method used in Kowalska-Góralska et al. [23 (link)].

The materials and reagents used in this study are as follows: CuONPs (Cat#544868, Sigma, St. Louis, MO, USA), MG132 (Cat#S1748, Beyotime, Shanghai, China), arsenite (Cat#H4525, Xiya Reagent Co. Ltd., Shandong, China). NAC (N-acetyl-L-cysteine, Cat#S0077, Beyotime), DHE (dihydroethidium, Cat#S0063, Beyotime), 7-AAD (7-aminoactinomycin D, Cat#AP104, MultiSciences, Hangzhou, China), MitoSOX (Cat#M36008, Thermo Fisher Scientific, Waltham, MA, USA), Calcein-AM (Cat#sc-203865, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CHX (cycloheximide, Cat#AC466, Genview, Houston, TX, USA), tBHQ (tert-butylhydroquinone, Cat#HY-100489, MedChemExpress, Shanghai, China), 3-MA (3-methyladenine, Cat#HY-19312, MedChemExpress), CQ (chloroquine diphosphate salt, Cat# C6628, Sigma), Wort (wortmannin, Cat#S2758, Selleck Chemicals, Houston, TX, USA), DMEM (Dulbecco's Modified Eagle Medium, Cat#C11995500BT, Gibco, Grand Island, NY, USA), FBS (fetal bovine serum, Cat#S711-001S, Lonsera, Uruguay), penicillin–streptomycin (Cat#15140122, Thermo Fisher Scientific) and puromycin (Cat#P8230, Solarbio, Beijing, China).

CuO NPs (primary particle size <50 nm) and CNTs were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Nanjing XF Nanotech Port Co. Ltd. (China), respectively. Before experiments, CNTs were treated according to the literature21 (link). Firstly, CNTs were heated at 350 °C for 3 h. After being cooled down, nanotubes were placed in 12 M HCl for 8 h, bath sonicated under ambient conditions for 1 h, and then washed by copious amounts of Milli-Q water until neutral pH. Finally the powder CNTs were gotten after being dried in an oven (60 °C) overnight. The stock nanomaterial suspension was prepared by ultrasonicating the powder CuO NPs or CNTs in Milli-Q water for 1 h.

CuONPs (#544868), mitochondrial division inhibitor 1 (Mdivi-1, #M0199) and chloroquine (CQ, # C6628) were purchased from Sigma Aldrich (St. Louis, MO, USA). Wortmannin (Wort, #S2758) was purchased from Selleck Chemicals (Shanghai, China). Urolithin A (UA, #HY-100599) were purchased from MedChemExpress (Shanghai, China). MitoTracker™ Green FM(#M7514), MitoSOX™ Red (#M36008) and Dulbecco’s modified Eagle’s medium (DMEM, #11965-092) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS, #04-001-1ACS) was purchased from Biological Industries (Kibbutz Beit Haemek, Israel).

CuO NPs (particle size 50 nm), DHEA and quercetin were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). All other chemicals were of the highest analytical grade available.

CuO NPs (particle size < 50 nm) were purchased from Sigma-Aldrich (St. Louis, MO). X-ray diffraction (XRD) analysis was conducted on a Bruker D8 Advance diffractometer with a Cu Kα radiation source and the result is shown in Figure S8 (Supplementary information). The powder CuO NPs were ultrasonicated in alcohol and then dropped into Ni grids for TEM analysis (Figure S9, Supplementary information). Also, the 50 mg/L NPs stock suspension was prepared by dispersing 0.05 g NPs powder in 1 L Milli-Q water, which was followed by 1 h ultrasonication (25 °C, 500 W, 40 kHz). The size distribution of particles in stock suspension was determined by laser diffraction technique via a Mastersizer 3000 (Malvern UK).