Ethics statement

Eight-week-old male ICR mice were obtained from DAEHAN Biolink (Eumsung, Korea). The mice were housed in the pathogen-free authorized facility at Hanyang University, where the temperature was 24 ± 2 ℃, the humidity was 50 ± 10%, and the dark/light cycle was 12 h. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University (IACUC No. 2021-0134A).

Sperm preparation and incubation

Cauda epididymides were isolated and decapsulated in phosphate-buffered saline (PBS) to eliminate blood. Spermatozoa were isolated from epididymides in Tyrode's basal medium (without Ca2+, bovine serum albumin [BSA], or bicarbonate) and centrifuged at 600 × g for 20 min in 35% Percoll. The pellets were washed in PBS and centrifuged at 800 × g for 10 min at 4℃. The sperm concentration was adjusted to 1 × 106 cells/mL through the addition of Tyrode’s complete medium (TCM) containing with Ca2+, BSA, and bicarbonate. This medium condition spermatozoa to initiate capacitation. Sperm suspensions were incubated with GSK3 inhibitor 6-bromo-indirubin-3'-oxime (BIO), proteasome inhibitor Z-Leu-Leu-Norvalinal (MG115), and cyclosporin A (CsA) obtained from Sigma for 1 h in a CO2 incubator at 37 °C. After incubation, spermatozoa were subjected to motility analysis, protein isolation, and dry smear.

Western blot analysis

Spermatozoa were homogenized in RIPA lysis buffer (WSE-7420; ATTO, Tokyo, Japan) containing 1% (v/v) protease and phosphatase inhibitor cocktail. After five rounds of sonication for 5 s at 4 °C, protein samples were obtained by centrifugation at 18,000× g for 20 min, and supernatants were subjected to western blot. Antibodies specific for rabbit anti GSK3α/β (#5676; Cell Signaling, Beverly, MA, USA), rabbit anti p-GSK3α/β (Ser21/9) (#9327; Cell Signaling), mouse anti cyclophilin D (ab110324; Abcam, Cambridge, UK), rabbit anti ATP synthase F1 subunit alpha (ATP5A) (ab176569; Abcam), rabbit anti β-tubulin (ab108342; Abcam), and rabbit anti GAPDH (sc-25778; SantaCruz Biotechnology, Dallas, TX, USA) were mixed in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with the membranes overnight at 4 °C. After being rinsed three times with TBST for 20 min, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG (ab6721; Abcam) and anti-mouse IgG (ab6728; Abcam) mixed with skim milk in TBST for 1 h. After washing three times with TBST for 20 min, the signals were detected using ECL Reagent (RPN2232; Amersham Bioscience) and Fusion SL (Vilber Lourmat, Marne-la-Vallée, France). The relative band intensities were analyzed using ImageJ (Ver.1.51j8; National Institutes of Health) and are expressed in arbitrary units (AU).

Immunocytochemistry

Dry spermatozoa smeared on poly-L-lysine-coated slides were fixed in acetone:methanol (1:1) solution at 4 °C for 10 min. Slides then were blocked in 5% donkey serum in 0.1% Triton X-100 PBS for 1 h and incubated with GSK3α/β, ATP5A, and cyclophilin D antibodies mixed in 1.5% donkey serum in a humidified chamber overnight at 4˚C. Rabbit IgG (ab172730; Abcam) and mouse IgG (sc-2023; Santa Cruz) were used as negative controls. After washing three times in PBS, slides were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (ab150061; Abcam) and Alexa Fluor 568-conjugated anti-mouse IgG (ab175472; Abcam) mixed in 1.5% donkey serum PBS for 1 h at room temperature. After washing three times in PBS, slides were mounted with DAPI (P36931; Invitrogen). Fluorescence images were captured by a microscope system with a cooled CCD (DP71; Olympus, Tokyo, Japan).

Live mitochondria isolation

For isolation of live mitochondria in spermatozoa, the Mitochondria Isolation Kit (C3601; Beyotime Biotechnology, Shanghai, China) was used. Briefly, spermatozoa were suspended in cold mitochondrial isolation buffer containing 1 mM phenylmethanesulfonyl fluoride, incubated at 4℃ for 1 h, and punched 15 times with a cold glass homogenizer. Punched samples were centrifuged at 600 × g for 10 min and supernatants were collected. Acquired supernatants were centrifuged at 11,000 × g for 10 min at 4℃. The isolated mitochondria pellet was suspended in mitochondria digestion solution and subjected to western blot analysis together with cytosolic protein-containing supernatant.

Co-immunoprecipitation assay

Spermatozoa were lysed in 0.1% Triton X-100 PBS and 1% protease and phosphatase inhibitor cocktails and subjected to co-immunoprecipitation (co-IP). Protein samples were precleared by protein A/G bead (sc-2003; Santa Cruz) at 4℃ for 2 h and obtained by centrifugation at 600 × g for 5 min. The supernatants were incubated with CypD antibodies and mouse IgG in a rolling machine at 4℃ overnight. The next day, protein A/G beads were added and incubated at 4℃ for 2 h. After washing five times in cold PBS by centrifugation at 600 × g for 5 min at 4℃, pellets were suspended in SDS-PAGE loading buffer and subjected to western blot analysis.

Computer-assisted sperm analysis

Sperm motility was analyzed using video recording and computer-assisted sperm analysis (CASA). Briefly, 20-μL samples were released onto a MAKLER® counting chamber (Irvine Scientific, Santa Ana, CA, USA) at 37 °C. Videos were recorded by a Nikon Diaphot microscope system with a CoolSnap EZ CCD camera (Photometrics, Tucson, AZ, USA) using iSPERM software (CNC Biotech, Suwon, Korea). Sperm motility was measured at least 200 spermatozoa.

Mitochondrial membrane potential assay

Mitochondrial membrane potential (MMP) of spermatozoa was measured using a tetramethylrhodamine, ethyl ester (TMRE) assay kit (ab113852; Abcam). Briefly, sperm were treated with TMRE and incubated for 30 min, and mesoxalonitrile 4-trifluoromethoxyphenylhydrazone was used as a negative control 10 min before TMRE treatment. Spermatozoa were washed in pre-warmed 0.2% BSA PBS and obtained by centrifugation at 500 g for 5 min. After centrifugation, the supernatant was discarded, and the pellet was suspended in PBS. TMRE fluorescence in spermatozoa was measured with a microplate reader (Varioskan; Thermo Scientific, Waltham, MA, USA) with an excitation wavelength of 549 nm and an emission wavelength of 575 nm. For TMRE fluorescence examination in spermatozoa, images were captured with a fluorescence microscope system with cooled CCD (DP71; Olympus).

Measurement of mPTP opening

To measure mPTP opening in spermatozoa cobalt quenching of calcein fluorescence method was conducted [26]. Briefly, spermatozoa were incubated in media containing 1 μM calcein AM (Sigma) and 1 mM CoCl2 (Sigma) for 30 min at 36℃ and washed in PBS by centrifugation at 500 g for 5 min. The spermatozoa were resuspended in PBS, and calcein fluorescence was measured using a microplate reader (Varioskan; Thermo Scientific) with an excitation wavelength of 496 nm and an emission wavelength of 516 nm. For calcein fluorescence examination in spermatozoa, images were captured with a fluorescence microscope system with cooled CCD (DP71; Olympus).

ATP assay

Sperm ATP level was analyzed using the CellTiter-Glo ® kit (G7570; Promega, Madison, WI, USA), which uses the enzymatic activity of the luciferase. Briefly, an equal volume of reagent was added to medium containing spermatozoa. The mixtures were shaking-incubated for 2 min to induce cell lysis and incubated at room temperature for 10 min to stabilize the luminescent signal. The luminescence of oxyluciferin was measured with a microplate reader (Varioskan) under an emission wavelength of 570 nm. Luminescence values are presented as relative light units (RLU).

Statistical analysis

Student’s t-test and one-way analysis of variance (ANOVA) followed by Tukey’s test were used to evaluate statistical significance with SPSS (version 17.0; SPSS Inc, Chicago, IL, USA). Significant difference was determined at p < 0.05.