Reagents and animals

The PKA inhibitor H89 dihydrochloride (H89, ab120341) and 1,2-bis-(2-aminophenoxy) ethane-N, N, N’, N’-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM, ab120503) were procured from Abcam (Cambridge, UK). PKC inhibitor chelerythrine chloride (CC, A10196) was sourced from AdooQ BioScience (Irvine, CA, USA). N-Ethylmaleimide (NEM, HY-D0843) and BMCC-biotin (21900) were purchased from Thermo Fisher Scientific Inc. (NC, USA). BeyoMag™ streptavidin magnetic beads (P2151), loading buffer (5×), HRP-streptavidin (A0303), bovine serum albumin (BSA, ST023), and all other reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were acquired from Beyotime Institute of Biotechnology (BIB, Shanghai, China). Human tubal fluid (HTF, MR-070-D) and ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid tetrasodium salt (EGTA, E8145) were sourced from Merck (Darmstadt, Germany). Hydroxylamine hydrochloride (NH2OH, 379921), dimethyl sulfoxide (DMSO, C10265), and 2-bromopalmitic acid (2BP, 2151) were procured from Sigma-Aldrich (MO, USA).

The animal study procedures were approved by the Animal Ethics Committee of the Zhejiang Academy of Medical Sciences (merged into Hangzhou Medical College). All experiments involving animals adhered to the guidelines set by the Animal Care and Use Committee of Hangzhou Medical College. Sexually mature male ICR mice, with a license for production (SCXK(Zhe) 2019–0011) and application (SYXK(Zhe) 2019-0002), aged over eight weeks, were obtained from the Experimental Animal Center of Zhejiang Province (Hangzhou, China). The animals were housed in plastic cages under specific pathogen-free conditions in a temperature-controlled room (23 ± 2 °C) with 60% ± 10% relative humidity and a 12 h light-dark cycle, as previously described [27]. Animal reporting in this study complied with the ARRIVE guidelines.

Sperm preparation

Sperm were prepared as previously described [6]. According to experimental requirements, one–12 mice were euthanized using an overdose of CO2 in a manual chamber in each experiment, following the American Veterinary Medical Association Guidelines for the Euthanasia of Animals (2020). Caudal epididymal tissues were collected, minced, and incubated in pre-warmed HTF (1 mL/mouse) at 37 °C for 10 min to release the sperm. The sperm suspension was separated from the tissue and collected via gravity settling or centrifugation at 500 × g for 10 s. Sperm were harvested after centrifugation at 500 × g for 20 min, ensuring no contamination with somatic or immature spermatogenic cells. The sperm concentration was adjusted to approximately 10 × 106 cells/mL using HTF, divided into aliquots, and treated with 2BP, H89, CC, BAPTA-AM, or EGTA, according to the experimental design. These aliquots were then incubated in a 5% CO2 incubator at 37 °C for 90 min, or the required time for the design.

Different inhibitors were used for the experimental design. 2BP, an inhibitor of protein palmitoylation [28], was used to explore whether protein palmitoylation was inhibited in mouse sperm and the effects of protein palmitoylation on sperm motility, protein tyrosine phosphorylation, and ROS. The PKA inhibitor H89 [26] and PKC inhibitor CC [29] were used to examine the effect of PKA/PKC-mediated protein phosphorylation on protein palmitoylation. Moreover, BAPTA-AM, an intracellular calcium chelator [30], and EGTA, an extracellular calcium chelator [31], were used to detect the effect of intracellular and extracellular Ca2+ on protein palmitoylation, respectively.

Sperm motility analysis

Sperm motility was assessed using a computer-assisted sperm analysis (CASA) system (IVOS, Hamilton-Thorne Bio-Sciences, MA, USA) at 0 and 90 min after treatment with 25, 50, or 100 µM 2BP. The CASA parameters were set as follows [16]: frame rate, 60 Hz; acquisition frame, 45; minimum contrast, 50; minimum cell size, 5 pixels; cell intensity, 90; path velocity, 10.0 μm/s; straightness threshold, 0; slow cell, average path velocity (VAP) and straight line velocity (VSL) of less than 5.0 μm/s and 0 μm/s; temperature, 37 °C; and chamber depth, 20 μm. For analysis, 5 µL sperm samples were loaded into a pre-warmed 20 μm-depth Goldcyto chamber (SCA20-04-01; Microptic, Barcelona, Spain). Motion parameters, including VAP, VSL, straightness (STR), curvilinear velocity (VCL), linearity (LIN), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and the percentage of motile and progressive sperm were assessed (with at least 200 sperm). Data from four replicates were collected for statistical analyses.

Click chemistry

Click chemistry was conducted using a copper-catalyzed azide/alkyne reaction to label in situ palmitoylated proteins in sperm, following the manufacturer’s instructions. Sperm aliquots (1 mL) were cultured with or without 50 µM Click® IT palmitate azide (C10265; Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 environment for 0, 60, 90, 120, and 180 min. Sperm were washed twice with PBS and fixed with 4% paraformaldehyde at 25°C for at least 15 min. Fixed samples were smeared onto Silane-Prep Slides (S4651-72EA; Sigma Aldrich, MO, USA) and air-dried. Permeabilization was achieved using 0.25% Triton X-100 for 15 min, followed by three washes with 3% BSA in PBS. Endogenous biotin was blocked using a biotin-blocking reagent (P0101; BIB, Shanghai, China) at 25°C for 30 min, followed by three washes with 3% BSA in PBS. The samples were then incubated with the Click-IT® Cell Reaction Buffer Kit (C10269; Invitrogen, Carlsbad, CA, USA) and 5 µM biotin alkyne (B10185; Invitrogen) at 25°C for 30 min, followed by three washes with 3% BSA in PBS. The samples were treated with 1 µg/mL Alexa Fluor® 488 conjugated streptavidin (S11223; Invitrogen). for one hour at 25°C, followed by five washes with PBS. Nuclear staining was performed with 0.5 µg/mL 4’,6-diamidino-2-phenylindole (DAPI, D9542; Roche, Mannheim, Germany) for 5 min, followed by three washes with PBS. Observations were made under a fluorescence microscope, and images were captured using the NIS-Elements software. The mean fluorescence intensity was determined, processed by subtraction of the background, and analyzed using the ImageJ software.

Protein extraction

The sperm samples were washed twice with PBS prior to resuspension in lysis buffer (P0013, BIB, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride, cOmplete™ EDTA-free Protease Inhibitor Cocktail (11836170001; Roche, Mannheim, Germany), and phosphatase inhibitor PHOSTOP cocktail (11836170001; Roche, Mannheim, Germany). The sperm were then lysed on ice via ultrasonication at a frequency of 20 kHz, employing a cycle of 5 s, followed by 10 s off for a total duration of 30 s. Subsequently, the lysate was centrifuged at 14,000 × g at 4 °C for 30 min, and the supernatant was collected. Protein concentration in the supernatant was determined using an enhanced BCA Protein Assay Kit (P0010; BIB, Shanghai, China).

Acyl-biotin exchange (ABE) method and streptavidin precipitation

The ABE method, as previously described [6], was employed to quantify protein palmitoylation levels. The protein lysate supernatant was precipitated using ice-cold acetone. The resulting pellets were harvested by centrifugation at 14,000 × g at 4 °C for 15 min and subsequently solubilized in cell lysis buffer (P0013; BIB, Shanghai, China) containing 50 mM NEM at 4 °C for two hours. This mixture underwent precipitation with pre-cooled acetone at − 20 °C and was split into two portions. The first portion was mixed with 150 µL of 1.5 M NH2OH buffer, and the second portion was mixed with 150 µL of 0.1 M Tris-HCl buffer as a control. Further precipitation was achieved using pre-cooled acetone at − 20 °C for 90 min. The pellets were treated with 0.8 µM BMCC-Biotin in Tris-HCl buffer and maintained at 4 °C for one hour. The resulting mixtures were precipitated with pre-cooled acetone at − 20 °C and re-dissolved in lysis buffer (P0013). An enhanced BCA Protein Assay Kit (P0010; BIB, Shanghai, China) was used to determine the protein concentration. The quantified protein was used for ABE experiments and streptavidin precipitation.

For the ABE experiment, the quantified protein buffer was heated in 5× loading buffer at 100 °C for 5 min. The samples were separated by SDS-PAGE and incubated with streptavidin-conjugated horseradish peroxidase (A0303; BIB, Shanghai, China). Protein bands were probed using an Enhanced Chemiluminescence Kit (Pierce Biotechnology, Rockford, IL, USA) and visualized using an Imager 600 system (Amersham™, GE Healthcare, IL, USA). When a loading control was needed, the membranes were probed using Western blotting with a β-tubulin antibody. The molecular weights of the detected proteins were compared with those of a pre-stained protein ladder (26616; Fermentas, Thermo Fisher Scientific Inc.). Gray intensity was quantified using ImageJ software.

For streptavidin precipitation, the quantified protein was incubated overnight at 4 °C with magnetic beads conjugated with streptavidin (P2151; BIB, Shanghai, China) and washed three times with PBS. The centrifuged beads, which served as protein aliquots, were subjected to Western blot analysis to detect palmitoylated HSP90.

Western blotting analysis

This procedure was performed as previously described [26, 27]. Extracted protein aliquots were boiled in 5× loading buffer at 100 °C for 5 min. Protein aliquots were resolved using SDS-PAGE in a gel containing 12% acrylamide and transferred to polyvinylidene difluoride membranes (FFP28, BIB, Shanghai, China). The membranes were blocked with Tris-buffered saline Tween-20 (TBST; pH 7.4, 20 mM Tris-HCl, 50 mM NaCl, 0.1% Tween 20) containing 3% BSA for 2 h. After blocking, the membranes were incubated with primary antibodies against HSP90 (ab203126; Abcam, Cambridge, UK; RRID, AB_2800428; Lot, GR3359427-3; 1:1000), tyrosine phosphorylation (61-5800; Invitrogen; RRID, AB_2533927; Lot, 801118A2; 1:1000;), β-tubulin (ab6046; Abcam; RRID, AB_2210370; Lot, GR126811-1; 1:1000), and peroxidase-conjugated secondary antibodies (656120; Invitrogen; RRID, AB_2533967; Lot, VL313544; 1:10000) at 4 °C overnight, followed by three washes (5 min each) with TBST containing 0.01% (v/v) Tween-20 at 25 °C. Protein bands were detected by the SuperSignal™ West Pico PLUS chemiluminescent substrate kit (34580; Thermo Fisher Scientific Inc.), and the gray intensity was quantified using ImageJ software.

Measurement of [Ca2+]i level

As previously described [16], intracellular calcium ([Ca2+]i) levels in spermatozoa were measured using a fluorescent probe Fluo4-AM (S1060; BIB, Shanghai, China). Briefly, spermatozoa were loaded with 5 µL Fluo-4 AM in the dark at 37 °C in a 5% CO2 incubator for 30 min and then washed three times with HTF. The loaded spermatozoa aliquots were treated with 2BP or DMSO. The fluorescence intensity at 488/20 nm excitation and 516/20 nm emission wavelengths was monitored using a Cytation 3™ multifunctional microplate reader (Biotek, USA). The [Ca2+]i levels were standardized by setting the initial recorded fluorescence intensity values as 1.00 in the blank group (the Fluo4-AM-loaded spermatozoa aliquots in HTF without any treatment) at 0 min.

Determination of ROS levels

ROS levels were measured using the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) in ROS assay kits (50202ES01; Yeasen, Shanghai, China) [32]. Similar to the Ca2+ level detection method, the prepared sperm in HTF were loaded with 10 µL DCFH-DA for 30 min in the dark and then washed three times with HTF. Equal aliquots were treated with 2BP, DMSO, or Rosup (positive control). The fluorescence intensity at 488/20 nm excitation and 525/20 nm emission wavelengths was monitored using the same microplate reader. ROS levels were standardized as described for [Ca2+]i measurement.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were conducted using the IBM SPSS Statistics version 23.0 software (IBM, USA). Initially, the normality of distribution was assessed using the Shapiro-Wilk test, and homogeneity of variance was assessed using the Levene test. If the data followed a normal distribution, one-way ANOVA, paired Student’s t-test, or Dunnett’s T3 test was employed. Alternatively, the Mann–Whitney or Wilcoxon tests were used. The threshold for statistical significance was set at P < 0.05.