Raw RNA-seq data collection and processing for meta-analysis

The raw public datasets were mined from the gene expression omnibus (GEO) database by searching for the terms “boar testis” and “mouse testis”. These datasets were identified and chosen under the following criteria: (1) the datasets were derived from untreated testis samples of boars and mice; (2) boars were from postnatal 0 to 180 days (P0-P180) and mice were from postnatal 0 to 35 days (P0-P35); (3) the dataset was unprocessed; (4) the dataset was based on high-throughput RNA-seq strategy. Any data that did not meet the above criteria were excluded. Additionally, two boar testis RNA-seq datasets were obtained from our previous research [2]. The datasets in each layout were divided into sexually immature groups (IMM, the ages of mouse: P0-P20; the ages of pig: P0-P60) and maturing groups (MAT, the ages of mouse: P21-P35; the ages of pig: P61-P180), based on the occurrence of round spermatids which marks the beginning of testis maturation [2, 27]. A total of 185 datasets including 70 datasets from pigs and 115 datasets from mice were collected in this meta-analysis. To pre-process the raw datasets, the index of genome was first constructed by Hisat2 software. Then, the paired-end or single-end RNA reads were aligned to the Ensembl Sscrofa 11.1 genome or Ensembl GRCm36 genome using Hisat2 software. The aligned reads were counted by featureCounts software in subreads. At last, The DEseq2 software was used to normalize the gene count data from samples of the same layout. These normalized values were used for subsequent meta-analysis as previously described [28].

Meta-analysis

In each comparison, fold-change values (Cohen’s d value) of each gene were calculated as log2 fold-enrichment (IMM vs. MAT) divided by pooled standard deviation. Then, the J factor was used to calculate Hedge’s g values of each gene and the variances of each g value. The g values and variances of two layout groups were integrated to calculate combined effect sizes. The calculation of the combined effect sizes (M*) was based on the random effect model using the inverse variance method, considering various effects on the true effect sizes. Finally, Z-scores were computed and used for p-value and false discovery rate adjusted p values (q values) calculation. The differentially expressed genes (DEGs) were filtered by setting cutoffs based on M* values and q values. The relevance between DEGs and spermatogenesis was predicted by SpermatogenesisOnline1.0 database [29].

Germ cell-conditional knockout Dnajc5b mice

Dnajc5b-floxed mice and Stra8‐Cre mice were generated by Cyagen Biosciences with CRISPR–Cas9. The Flox elements were inserted at both ends of the third exon of Dnajc5b to create a large deletion of Dnajc5b gene. Dnajc5b germ cell–conditional knockout (Stra8Cre/+; Dnajc5bFlox/−, CKO) mice were developed by crossing Dnajc5bFlox/Flox mice with Dnajc5bCre/+; Flox/+ mice. The animal procedures were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University (HZAUMO-2024-0186), and the mice were housed in the specific pathogen-free facility of Huazhong Agricultural University. All experiments with mice were conducted ethically according to the Guide for the Care and Use of Laboratory Animal guidelines. Total genomic DNAs of murine toes were utilized for genotyping using the primers in Supplementary Table S2. Wild-type and knockout male mice were housed with wild-type female mice at two months of age, and the total number of offspring was counted over a period of seven months. At least 3 cages were set up for each genotype, and the number of newborn pups was counted from each litter.

Cell culture

The swine testicular (ST) cells (RRID: CVCL_2204) that have been identified as immature Sertoli cells [30], and the Chinese hamster ovarian (CHO) cells (RRID: CVCL_0213) were purchased from the Cell Bank of Wuhan University (Wuhan, China). ST and CHO cells were cultured in MEM/EBSS medium (SH30024.01, Cytiva) and F12 medium (Gibco, C11330500BT) at 37 °C with 5% CO2, respectively. Both culture mediums are supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin-Amphotericin B (Solarbio, P7630).

Cell treatment

The coding sequences of murine Dnajc5b (ENSMUSG00000027606) and porcine DNAJC5B (ENSSSCG00000006217) were amplified and cloned into pCMV-T7-MCS-3×FLAG-WPRE-Neo (Bena Culture Collection) individually. The DNAJ-domain and Cys-domain deleted vectors were generated based on full-length DNAJC5B vector by overlap-extension PCR using the primers listed in Supplementary Table S2. The plasmids were transfected into ST cells or CHO cells using Exfect Transfection Reagent (Vazyme, T101) or Lipomaster 2000 Transfection Reagent (Vazyme, TL201).

For inducing autophagy, the ST cells or CHO cells were starved at serum-free EBSS (Beyotime, C0213) for 8 h, or treated with FCCP (10 µM, MedChemExpress, HY-100410) or CCCP (10 µM, Sparkjade, SJ-MX4008) in culture medium for 6 h. For the oxidative stress assay, the ST cells were exposed to hydrogen peroxide (100 µM) for 2 h. For inhibition of lysosomal acidification, chloroquine (Sparkjade, SJ-MA0048A) was added to the cell culture medium at a final concentration of 50 µM at the last 2 h of starvation, FCCP or CCCP treatment. For inhibition of 26 S proteasome, MG-132 (MedChemExpress, HY-13259) was added to the cell culture medium for 8 h.

Tissues and spermatozoa morphological staining

Testes and epididymis were fixed in a modified formaldehyde solution containing ethanol and acetic acid (Servicebio, G1121), dehydrated with increasing concentrations of ethanol (70-80%-90-100%-100%), embedded in paraffin, and cut into 5 μm thick sections. Then, the sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (Servicebio, G1076) or PAS staining solution B, solution A, and solution C (Servicebio, G1008). The sections were dehydrated and sealed with neutral gum. The murine cauda epididymis was removed and placed in pre-warmed PBS at 37 ℃. Cauda epididymis was then gently cut into a few pieces with scissors to allow the spermatozoa to swim out freely. The spermatozoa were smeared on the slide, and fixed with cold methanol for 10 min. Then fixed spermatozoa were stained with Wright-Giemsa solution and washed with water to remove unstained dyes. The images were observed and acquired from the Olympus microscope.

Tandem mass tag (TMT) proteomics

The TMT proteomic analysis was supported by Jingjie PTM BioLabs. In brief, the experimental procedures for TMT proteomic analysis on testes and sperms included protein preparation, trypsin digestion, TMT labeling, HPLC fractionation, and affinity enrichment. Then the peptides were separated by Liquid Chromatography and detected by Mass Spectrometry/ Mass Spectrometry. Tandem mass spectra were searched against Mus_musculus_10090_SP_20230103.fasta (17132 entries) concatenated with reverse decoy and contaminants database. The false discovery rate (FDR) of protein, peptide and peptide-spectrum matches was adjusted to < 1%. The differentially expressed proteins (DEPs) were filtered by setting the cutoffs based on the foldchange and p values (FC > 1.5 or FC < 0.66, and p < 0.05).

Quantitative real-time PCR (qRT-PCR) analysis

Total RNAs of testes and cells were extracted using the TRIZOL extraction method and tissue/cell total RNA extraction kit (Sparkjade, AC0202) and subsequently reversely transcribed into cDNA using Prime Script Fast RT reagent kit (TaKaRa, RR092A). The quantitative real-time PCR (qRT-PCR) mixture was prepared using TB Green Premix (TaKaRa, RR820A) and the primers listed in Supplementary Table S2. The PCR was performed with an initial denaturation at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing/extension at 60 °C for 30 s. The melt curve was established to range from 58 ℃ to 95 ℃. Each gene was detected in at least 3 individual samples. And each gene of each sample was replicated triply. Relative mRNA expression was calculated using the 2−ΔΔCt method with beta-actin as control.

Western blot

Total protein lysates of tissues and cells were extracted with RIPA lysis buffer. Then, the protein samples were loaded and separated in an SDS-PAGE gel and transferred onto the PVDF membranes (BioRad, 1620177). The membranes were blocked with 5% nonfat milk for 2 h and then incubated with primary antibodies overnight at 4 ℃. The primary antibodies included DNAJC5B (1:1000, Proteintech, 17364-1-AP), P62 (1:1000, Zen-Bio, 380612), LC3B (1:2000, Abclonal, A7198), FBXL4 (1:1000, Zen-Bio, 863894), LONP1 (1:1000, Abclonal, A4293), ATP1A1 (1:5000, Abmart, T55159), ATF5 (1:1000, Abclonal, A3563), ACTB (1:10000, Abclonal, AC026), GRP75 (1:1000, Abclonal, A11256), α- TUBULIN (1:5000, Abclonal, AC012), GAPDH (1:10000, Abclonal, AC033), ODF1 (1:2000, Abcam, ab197029), and FLAG (1:3500, Abmart, M20008). On the next day, the membranes were washed four times with TBST and incubated with secondary antibodies (1:3500, Bio-Rad, 170–6516/ 172–1034) at room temperature for 2 h. Proteins on the blot were visualized using ECL reagent.

Immunofluorescence

The testis sections were deparaffinized, rehydrated, and boiled in sodium citrate buffer for 10 min to retrieve antigen. The cells were spread on slides and fixed with 4% polyformaldehyde or cold methanol for 10 min, respectively. The samples were blocked with 5% BSA in PBST (0.1% Tween-20 in PBS) and then incubated with primary antibody overnight at 4 ℃. The primary antibodies used here targeted DNAJC5B (1:100, Proteintech, 17364-1-AP), LC3B (1:100, Abclonal, A7198), and FLAG (1:100, Abmart, M20008). The next day, the samples were incubated with the secondary antibody and DAPI at room temperature for 2 h. Finally, the samples were mounted in an anti-fade mounting Medium (Servicebio, G1401).

Sperm protein fractionation

The separation of sperm fractions was carried out with triton and SDS solution. Briefly, murine and porcine sperms were washed three times with PBS. Subsequently, the sperms were first lysed for 2 h at room temperature by 1% triton X-100 buffer (20 mM Tris-HCl, pH 7.4; 50 mM NaCl; 1% Triton X-100; 1% protease inhibitor cocktail). The triton-soluble supernatant was removed, and unlysed parts were then treated with 1% SDS buffer (24 mM EDTA-2Na, pH 6; 75 mM NaCl; 1% SDS; 1% protease inhibitor cocktail) for 2 h at room temperature to extract SDS-soluble phase. Finally, the residual pellet was resuspended in 1×SDS sample buffer and boiled for 10 min to extract the SDS-resistant phase. The Triton-soluble, SDS-soluble, and SDS-resistant fractions were analyzed by western blot. The sperm membrane proteins were extracted by Membrane and Cytosol Protein Extraction Kit (Beyotime, P0033).

Mitochondria extraction and mitochondrial functional assays

The mitochondria of the testes and cells were isolated using a mitochondria extraction kit (Solarbio, SM0020). The mitochondria were stained with specific reagents to detect the mitochondrial functions of cells under different treatments. For mitochondria staining, the cells were incubated with Mito-Tracker Red CMXRos (200 nM, Beyotime, C1035) at 37 ℃ for 30 min. For cell oxidant detection, the cells were incubated with DCFH-DA (10 µM, Beyotime, S0033S) in serum-free medium at 37 ℃ for 30 min, and washed with serum-free medium three times. For mitochondrial membrane potential detection, the cells were incubated with 1×JC-1 staining solution (Solarbio, M8650) in an equal volume of serum-free medium at 37 ℃ for 20 min, and washed twice with pre-cold wash buffer. For mitochondrial permeability transition pore assay (Beyotime, C2009S), all cells were incubated with Calcein AM probes and CoCl2 for 30 min; and the negative cells were additionally supplemented with ionomycin in an incubation medium; after incubation, the medium was replaced with culture medium for an additional 30 min. The above assays were detected by fluorescence microscopes.

Transmission electron microscopy and scanning electron microscopy

For transmission electron microscopy, testes were sectioned into less than 2 mm thick slices. Testis and sperm samples were fixed in freshly prepared electron microscopy fixation solution (2.5% glutaraldehyde in PBS) at 4 °C for preservation. Subsequently, the samples were post-fixed with 1% OsO4 in PB for 2 h at room temperature. Fixed samples were dehydrated at room temperature through a graded ethanol series, followed by washes in acetone. The samples were then embedded in a mixture of acetone and EMBed 812 kept overnight at 37 ℃ and polymerized at 60 ℃ for more than 48 h. Ultrathin sections with 60–80 nm in thickness were cut from the resin blocks and stained with uranium acetate-saturated alcohol solution and lead citrate, and subsequently examined using a transmission electron microscope (Hitachi, HT7800).

For scanning electron microscopy, murine spermatozoa were collected from the cauda epididymis. The spermatozoa were fixed immediately in electron microscopy fixative for 2 h at room temperature, then stored at 4 ℃. The samples were incubated with 1% OsO4 in PB for 1–2 h at room temperature. Then the samples were dehydrated through a series of ethanol solutions. Finally, the samples were soaked in isoamyl acetate for 15 min and dried using a Critical Point Dryer. Specimens were mounted on metallic stubs with carbon stickers, sputter-coated with gold for 30 s. Images were observed and obtained with a scanning electron microscope (Hitachi, SU8100).

Acyl–biotin exchange assay

The acyl-biotin exchange assay was employed to detect protein palmitoylation and oxidation using a modified method [31, 32]. In brief, CHO or ST cells transiently expressing 3*Flag-tagged DNAJC5B were harvested 36 h after transfection and washed with cold PBS. Immediately before cell lysis, N-ethylmaleimide (NEM, Sigma-Aldrich, E3876) was added to the lysis buffer of pH 7.4 (50 mM Tris-HCl pH 7.4; 1% NP-40; 150 mM NaCl; 10% Glycerol) to a final concentration of 50 mM. The cells were then suspended in NEM-containing lysis buffer for 1 h at 4 °C and the supernatants were incubated with anti-Flag nanobody magarose beads (KTSM, KTSM1338) at 4 °C overnight. After incubation, the beads were incubated with hydroxylamine (1 M, HAM, Macklin, H828371) to remove the palmitoyl, or with TCEP buffer (10 mM, Beyotime, ST046) for reducing the oxidized group at room temperature for 1 h. Then, the beads were washed three times with lysis buffer of pH 7.2, and three times with lysis buffer of pH 6.2. Subsequently, beads were treated with Biotin-maleimide (0.5 µM, Sigma-Aldrich, B1267) in a lysis buffer of pH 6.2 at 4 ℃ for 1 h. The immunoprecipitated samples were eluted by 1×SDS sample buffer and analyzed by western blot using anti-Flag antibody (1:3500, Abmart, M20008) and streptavidin-HRP (1:10000, Aladdin, np156148).

Cell viability

The cell viability was detected by cell counting kit-8 (CCK8) assays. Briefly, the ST cells were cultured in 96-well plates and transfected with DNAJC5B or empty vectors for 24 h, followed by FCCP or CCCP treatments for 1 h, 2 h, or 6 h. Then, 10 µL CCK8 reagent was added into a well and incubated with cells for 1 h. Cell viability was calculated as the absorbance at 450 nm of treated cells compared with an untreated control group.

Bioinformatic analysis

Gene ontology (GO) analysis of DEGs and DEPs was performed by the online GO tool (https://geneontology.org/). The transmembrane domain of DNAJC5B was predicted by the DeepTMHMM tool (https://dtu.biolib.com/DeepTMHMM). 3D structure of DNAJC5B protein was constructed by Alphafold and displayed by PyMOL. Protein-protein interactions were analyzed by STRING and visualized by Cytoscape.

Statistical analysis

All data were analyzed using GraphPad Prism and expressed as mean ± standard error of the mean (SEM). The experiments were repeated at least three times. Each independent experiment was set up with at least three replicates. The data were normalized by the controls. The two-tailed student’s t-test was applied to calculate p values.