Vecteshield mounting medium with dapi

Sperm were collected from the cauda of the epididymis with a 27-G syringe in HTF medium. Additionally, the sperm cells localized in seminiferous tubule were collected from testis using 0.1% collagenase contained HTF medium. After incubation for 30 min and centrifuging, the pellet was washed using 5% (w/v) BSA/PBS. The sperm or sperm cells were fixed with 100% methanol for 10 min at room temperature and then permeabilized by 0.1% (v/v) TritonX-100/PBS for 10 min. Sperm were incubated in 5% (w/v) BSA/PBS for 30 and then probed with the primary antibody (anti-TLR7 antibody conjugated Alexa fluor 647 [bs-6601R-A647,Bioss], anti-TLR8 antibody, or anti-acetylated tubulin antibody [#T7451; Sigma] were used at 1:100). After washing by 5% (w/v) BSA/PBS, the antigens were visualized with Alexa fluor 488 anti-rabbit IgG (1:200; # 4030–30, Southern Biotech, Birmingham, AL, USA), Alexa fluor 647 anti-mouse IgG (1:200; #ab150115, Abcam), or DAPI (VECTESHIELD Mounting Medium with DAPI, Vector Laboratories, Burlingame, CA, USA). The intensity was analyzed by flow cytometry (Attune NxT Acoustic Focusing Cytometer, Invitrogen).

Sperm were mounted on glass slides and air-dried. The sperm were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.3% (v/v) Triton X-100/PBS. The sperm were probed with anti-SR-BI antibody (Cat No. NB-400-113; Novus Biologicals, Littleton, CO, USA) at a 1:100 dilution. The specificity of this antibody was confirmed by western blotting (Supplementary Fig. S2A). After washing with 0.3% (v/v) Triton X-100 in PBS (−), the antigens were visualized with Cy3-conjugated goat antirabbit IgG (1:200, Sigma-Aldrich) and DAPI (VECTESHIELD Mounting Medium with DAPI, Vector Laboratories, Newark, CA, USA). The negative control stained with only the secondary antibody is shown in Supplementary Fig. S2B. Digital images were captured using a confocal microscope (Nikon, Tokyo, Japan).

Sperm was thawed and washed with BSA‐free BO medium, mounted on glass slides, and air‐dried. Sperm was fixed with 100% methanol for 10 min at room temperature. Then, it was probed with a 1:100 diluted primary antibodies (Anti‐CD36 antibody (NB 110‐59724, Novus Biologicals) and anti‐GOT2 antibody (NBP2‐16708, Novus Biologicals). After washing with PBS, the antigens were visualized using Cy3‐conjugated goat anti‐mouse IgG (1:100, Sigma), FITC‐conjugated goat anti‐rabbit IgG (1:100, Sigma), and DAPI (VECTESHIELD Mounting Medium with DAPI, Vector Laboratories). Digital images were captured using a Keyence BZ‐9000 microscope (Keyence Co.).