All chemical reagents were obtained from Sigma Aldrich (St. Louis, MO, USA) unless otherwise noted. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.

C57BL/6J mice were purchased from the Animal Resources Centre or produced by the animal breeding facilities of the Institute of Biotechnology. The mice were housed in the IMG animal facilities, Institute of Molecular Genetics of Czech Academy of Science, Prague, and food and water were supplied ad libitum. The mice were healthy 10–12 weeks old animals with no sign of stress or discomfort. Transgenic reporter C57BL/6J^Acr3-EGFP mice expressing green fluorescent protein in the acrosome of developing spermatids and mature spermatozoa³⁶ were used for testicular histology sections. These reporter transgenic male mice were generated in the Transgenic Unit of the Czech Center for Phenogenomics at the Institute of Molecular Genetics CAS and they are property of the Laboratory of Reproductive Biology, IBT CAS, Vestec, Czech Republic. The mice were housed in animal facilities of the Institute of Molecular Genetics of Czech Academy of Science, Prague, and food and water were supplied ad libitum. All the animal procedures and all the experimental protocols were approved by the Animal Welfare Committee of the Czech Academy of Sciences (Animal Ethics Number 66866/2015-MZE-17214, 18 December 2015).

Freshly ejaculated or frozen-thawed bovine sperm were obtained from bulls (Bos taurus) of Slovak Breeding Services, Inc., Luzianky, Slovak Republic. The bull epididymides were obtained at a local slaughterhouse (Mala Maca, Slovakia). The study was carried out according to the Council Directive 98/58/EC, Council Regulation (EC) No. 1099/2009, Regulation (EU) 2016/1012, Slovak National Council No. 39/2007 and guidelines of the Slovak legislation (directive 432/2012 Z. z.).

Elutriation protocol was performed according⁶¹. The elutriation was done in PBS at 4 °C on a Centrifuge J26XP with elutriation rotor JE-5.0 (Beckman Coulter, Indianapolis, IN). The precise conditions are described in Table S1. The cells were collected into 50 ml tubes on ice. Cells in each tube were pelleted by centrifugation (400 × g, 15 min, 4 °C) and resuspended in Tri-reagent (Sigma-Aldrich). The total RNA was isolated according to manufacturer’s instructions and stored at 70 °C. The protein fractions were precipitated by acetone, dissolved in reducing sample solution (2% SDS in Tris-HCl, pH 6.8; 5% mercaptoethanol) with 0.5% Protease Inhibitor Cocktail, incubated for 30 min at 4 °C and boiled for 5 min.

The bull tissues segments (testes and epididymides) were preserved by TissueTek (Sakura Finetek, Alphen aan den Rijn, NL) and frozen in liquid nitrogen. The frozen sections (5-µm) were cut using a Leica Cryocut 1800 cryostat (Leica Microsystems, Wetzlar, Germany), fixed for 5 min in a cold acetone-ethanol mixture (1:1), air-dried and washed in PBS.

Total RNA was isolated from testicular fractions prepared by elutriation and testes samples using TRI Reagent (Sigma-Aldrich). Firstly, RNA extracts (2 μg) were treated with DNase I (1 U/μL, Fermentas, Hanover, MA) in presence of DNase I buffer 10× (Thermo Scientific) with MgCl₂ for 30 min at 37 °C and EDTA (Fermentas) was added for 10 min at 65 °C. The reverse transcription reaction contained 5x reaction Buffer (Fermantas), Riboblock Inhibitor (20 U/μL, Sigma-Aldrich), Universal RNA Spike II (0.005 ng/μl, TATAA biocenter, Sweden), 10 mM dNTP Mix (Thermo Scientific), oligo(dt)18 (Thermo Scientific) mixed 1:1 with Random primers (Thermo Scientific) and M-MuLV RevertAid transcriptase (200 U/μL, Fermentas), and run for 60 min at 42 °C followed by 10 min at 70 °C to generate cDNA.

For q-RT-PCR cDNA (10 ng/µl), two times Maxima SYBR Green qPCR Master Mix (Thermo Scientific), reverse and forward primer (1 μM, Generi Biotech, Hradec Kralove, Czech Republic) and nuclease free water were used.

The Ribosomal protein S2 (Rps2) gene was used as the reference gene. Specific gene markers for germinal cells and somatic cells were selected to determine elutriation fractions (the enrichment of individual elutriation fractions is listed in the Supplementary Table 1).

Mouse spermatozoa were obtained from cauda epididymis, released into M2 medium, incubated for 15 min at 37 °C in 5% CO₂, washed in PBS and centrifuged at 300 × g for 10 min twice at room temperature (RT).

Bull spermatozoa were obtained from epididymides divided into three segments: the caput, corpus and cauda. The segments of caput and corpus were cut into small pieces, incubated in 10 ml of PBS for 15 min at 37 °C. Spermatozoa from ducts of cauda were blown with syringe. Spermatozoa were washed with PBS and centrifuged at 200 × g for 10 min at RT.

Freshly ejaculated spermatozoa were separated from seminal plasma by centrifugation at 200 × g for 10 min at RT and washed with PBS. The pellets of frozen-thawed spermatozoa were washed in PBS at 200 × g for 10 min at RT.

Human ejaculates were obtained from Centrum for assisted reproduction Gennet (Prague, Czech Republic) with the informed consent of healthy donors and in accordance with the Institutes’ Human Ethics Committee guidelines, from men after 3-4 days of sexual abstinence. After liquefaction, the spermatozoa was separated from seminal plasma by centrifugation gradient (55%/80%) of SupraSperm System (Origio, Måløv, Denmark) and centrifuged at 300 × g for 20 min at 37 °C. Biological materials and experimental protocols were approved by Ethics committee of the General University Hospital, Prague, number 617/17 S-IV.

Mouse sperm from the distal regions of the cauda epididymis were released into a 200 μL droplet of M2-fertilizing medium (M7167) under paraffin oil and pre-tempered at 37 °C in 5% CO₂. Released sperm were assessed for motility and viability under a light inverted microscope with a thermostatically controlled stage at 37 °C. Sperm (5 × 10⁶/mL) were left freely to capacitate for 90 min in 100 μL M2 medium under paraffin oil. The acrosomal reaction was induced by Calcium Ionophore (CaI, A 23187) at a final concentration of 5 μM for 90 min at 37 °C in 5% CO₂.

Bull sperm were re-suspended in a commercially supplied TL medium for bovine sperm cell capacitation (Minitube, Celadice, Slovak Republic), supplemented with 6 mg/mL BSA, 0.02 mol/L Na pyruvate and 0.5 mg/mL gentamycin, to a final concentration of 10⁷ cells/mL and capacitated for 4 h at 39 °C in 5% CO₂. Acrosome reaction was induced by 10 µMol/L CaI A23 187 for 1 h under the same conditions.

Human sperm was capacitated in 0.5 ml of Sperm Preparation Medium (Origio, Måløv, Denmark) for 2 h at 37 °C under 5% CO₂. The acrosomal reaction was inducted by CaI A 23187 in a final concentration of 10 µM for 1 h at 37 °C in 5% CO₂.

For super-resolution microscopy spermatozoa were fixed at a high precision cover glasses with cold acetone-ethanol for 5–8 min, blocked with Super Block® Blocking Buffer (Thermo Scientific, Rockford, IL, USA) for 30 min at RT and treated with primary antibody or combination of two antibodies in case of double staining, overnight at 4 °C. Secondary antibody for 1 h at RT were applied. The intactness of spermatozoa acrosomes was assessed by Peanut agglutinin (PNA)-Alexa568 (1:500). The nuclear DNA of cells was stained by Hoechst (1:200). After washing, 90% glycerol with 5% anti-fade-N-propyl gallate as a mounting medium was used. For SIM visualisation, following antibodies were used: primary antibodies rabbit polyclonal anti- CD151 antibody (ab125363, Abcam, antibodies, Cambridge, UK) diluted 1:100 in PBS, rabbit polyclonal anti-β1 integrin antibody (sc- 8978, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) 1:10, rabbit polyclonal anti-α3 integrin antibody (H-43, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) 1:10, mouse monoclonal anti-α6 integrin antibody (F-6, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) 1:10, rabbit polyclonal anti-β4 integrin antibody (bs-4115R, Bioss antibodies, Woburn, MA, USA) 1:10; secondary antibodies Alexa fluor 488 goat anti-rabbit IgG and Alexa fluor 488 goat anti- mouse IgG or Alexa fluor 568 donkey anti- mouse IgG (Molecular Probes, Eugene, OR, USA) diluted 1:300 in PBS. SIM super-resolution images were obtained by Zeiss Elyra PS.1 inverted microscope at Laboratory of confocal and fluorescent microscopy of Faculty of Science (Charles University, Prague, Czech Republic). Representative results are shown.

Cryo-sections of testicular tissue of Transgenic C57BL/6J^Acr3-EGFP mice expressing green fluorescent protein in the acrosome of developing spermatids and mature spermatozoa were used for immunolabeling. Tissue was fixed for 10 min with cold acetone-methanol (1:1) and dried. After 30 min of blocking with Super Block® Blocking Buffer (Thermo Scientific, Rockford, IL, USA), primary anti-CD151 antibody (ab125363, Abcam, antibodies) in concentration 1:50 in PBS was applied for 2 hours, followed by secondary antibody Alexa fluoro 568 goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) (1:300) for 1 h at room temperature. In the end, the slides were mounted into a Vectashield mounting medium with DAPI (Vector Lab., Burlingame, CA, USA). Images were obtained with high-end confocal microscope Carl Zeiss LSM 880 NLO (Imaging Methods Core Facility at BIOCEV, Vestec, Czech Republic). An open source software Fiji⁶² was used for further image processing.

Bull tissue sections and sperm smears were fixed for 5 min by cold acetone-methanol (1:1) and dried. After blocking, anti-CD151 antibody or rabbit IgG isotype control (1–2 µg/mL) was applied. Goat anti-rabbit IgG-fluorescein (FITC) conjugated secondary antibody (1:300) (Vector laboratories, Burlingame, CA, USA) was applied, followed by PNA-TRITC staining. The nuclear DNA of cells was stained by Vectashield mounting medium with DAPI (Vector laboratories). Immunostaining was evaluated under a Leica DM5500 B epifluorescence microscope at 400× and 1000× magnifications. The fluorescence images were recorded with a Leica DFC340 FX digital camera and processed using Leica Advanced Fluorescence software. Representative results are shown.

Fractionation was made according to Somanath and Gandhi (2004). Fresh bull ejaculate (10 ml) was diluted 1:2 with Krebs Ringer Bicarbonate (KRB) medium: 0.7 mM Na₂HPO₄ + 0.49 mM MgCl₂ + 4.56 mM KCl + 0.1198 M NaCl + 0.0013 M NaH₂PO₄ + 2.37 mM fructose + 0.0149 M NaHCO₃ (pH 7–7.6)). Human and mouse sperm were prepared as above and diluted 1:2 with KRB medium (devoid of BSA, containing 2.37 mM/l glucose). Diluted semen of bull, mouse and human were layered on solution of 1.3 M sucrose with 0.9% NaCl and centrifuged for 30 min at 2000 × g at 4 °C. Pellet of sperm was resuspended in 0.15 M NaCl with 5 mM HEPES (pH 7.0), layered on solution of 1.3 M sucrose with 0.9% NaCl and centrifuged for 20 min at 34000 × g at 4 °C. The pellets were resuspended in 0.15 M NaCl with 5 mM HEPES (pH7.0) supplemented with protease inhibitors and sonicated 1 × 5 s and 3 × 2 s for mouse and 20 × 10 s intervals for human (Ultrasonic, 80 Amplitude microns power), and 10 × 10 s intervals (Soniprep 150, 30 Amplitude microns power) for bull. Membranes from homogenate were separated by discontinuous sucrose gradient consisting of 1.75 M sucrose and 1.3 M sucrose (1:1) and centrifugation for 3 h at 95000 × g at 4 °C. The plasma membrane fraction was at the interface between the sample and 1.3 M sucrose; acrosomal membrane fraction was the interface between 1.3 M/1.75 M sucrose. Pellet contained IAM and remaining equatorial segments closely associated with the sperm heads. The fractions of plasma membrane and acrosomal membrane fraction were diluted with PBS and pelleted by centrifugation for 30 min at 120000 × g at 4 °C. The fraction of IAM was washed by PBS and centrifuged at 3500 × g for 10 min at 4 °C. Subsequently the separate membrane fractions were solubilized with 1% (v/v) Triton X-100 for 1 h at 4 °C. Proteins from human and mouse fractions were precipitated by acetone, all protein samples were then incubated at 100 °C for 5 min in reducing sample solution.

Pellets of human and bull ejaculated and mouse epididymal spermatozoa were dissolved in reducing sample solutions for 30 min at 4 °C and subsequently boiled for 5 min at 100 °C.

The protein extracts from elutriation fractions of mice testes, membrane fractions and spermatozoa of mouse, bull and human were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane (Advantec Toyo Kaisha Ltd., Tokyo, Japan). The molecular weights of the separated proteins were estimated using Dual Color Prestained Protein Standards (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were incubated with anti-CD151 antibody (ab125363, Abcam) overnight at 4 °C, followed by incubation with secondary antibody: goat anti-rabbit IgG conjugated to horseradish peroxidase, or goat anti-rabbit IgG conjugated to alkaline phosphatase for 1 h at RT. Antibody reaction was visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) for HRP-conjugated antibody (mouse and human) or with NBT (4-nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolyl-phosphate) solution for antibody conjugated with alkaline phosphatase (bull).

Mouse sperm suspension (5 × 10⁷) was lysed in 100 µl of Tris buffer (20 mM Tris-HCl, 137 mM NaCl, pH 8) with 1% CHAPS at 4 °C for 1 h. Lysates were centrifuged at 10,000 g for 5 min at 4 °C. Then supernatant was incubated with polyclonal antibody anti-CD151 (ab125363, Abcam) in final amount 2 µg per sample overnight at 4 °C in rotator. As a control, rabbit IgG (Abcam) in the same concentration as antibody was used. Twenty µl of washed Agarose Protein A/G (Thermo Scientific) were added and incubated for 2 h at RT. Precipitates bound to Protein A/G were washed twice with lysis buffer for 5 min at 4 °C in rotator. This procedure was repeated for three times. Co-immunoprecipitated complexes were eluted from Protein A/G agarose by incubation in reducing sample buffer for 5 min at 100 °C. After electrophoretic separation in 10% polyacrylamide gel and transfer into PVDF membrane, the CD151 immunoprecipate was incubated with mouse antibody against α6 integrin (F-6, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and rabbit antibody α3 integrin (H-43, Santa Cruz Biotechnology, Inc.) diluted 1:100 in PBS overnight at 4 °C. After washing and incubation with secondary anti-mouse or anti-rabbit antibody conjugated with HRP (Bio-Rad) diluted 1:3000, antibody reaction was visualised by SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific).