The present human study was approved by the Ethics Committee of Shenzhen Ethics Review Committee on Biomedical Research (Shenzhen, China) [(2023) approval no. 001]. All participants provided written informed consent to participate. The subjects were patients who were treated in Peking University Shenzhen Hospital from July 2023 to January 2024. The present study included 25 male patients aged between 22 and 45 years with asthenozoospermia (sperm concentration, ≥15 million cells/ml; progressive motility, <32%; total motility, <40%; morphologically normal forms, ≥4%) and 25 participants with normal semen parameters. Semen samples were collected by masturbation following 3–7 days of abstinence. Samples were evaluated by computer-assisted semen analysis (version SSA-II, Suijia Software Co., Ltd) system. Inclusion criteria were as follows: i) Patients aged between 20 and 45 years with complete clinical data and ii) no syphilis, hepatitis, acquired immune deficiency syndrome or other infectious disease. Exclusion criteria were as follows: i) Patients with genitourinary tract infection; ii) patients with a history of recent use of drugs that interfere with sperm or semen quality and iii) malignant tumor or abnormal liver and kidney function.

To compare the expression of SIRT5 among asthenozoospermic, asthenoteratozoospermic and normal sperm from fertile individuals, mRNA expression data was retrieved from the GEO database (accession no. GSE160749) (ncbi.nlm.nih.gov/geo/geo2r/?acc=GSE160749). In addition, the protein expression of SIRT5 in testicular cells was queried using the Human Protein Atlas (HPA) database (proteinatlas.org/ENSG00000124523-SIRT5/single+cell+type).

A single male C57BL/6 J wildtype mouse (age, 2 months; weight, 22 g) was obtained from the Nanjing University Experimental Animal Institute (Nanjing, China). The mouse was housed in a specific-pathogen-free animal facility (22°C, 55% humidity) with a 12/12-h light/dark cycle and free access to standard food and water. Health and behavior were monitored every day. The duration of the experiment was 20 weeks. Humane endpoints were as follows: Complete anorexia or signs of depression accompanied by hypothermia (body temperature <37°C) without anesthesia or sedation. The mouse was euthanized by intraperitoneal injection of an overdose of 1% pentobarbital sodium (150 mg/kg). Animal death was confirmed by respiratory and cardiac arrest and pupil dilation was observed for ≥10 min. All animals were treated according to the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources for the National Research Council. The present study was approved by the Ethics Committee of Shenzhen Peking University-Hong Kong University of Science and Technology Medical Center (approval no. 2021-007).

After the mouse was euthanized, the testicles were dissected and removed. Testes were fixed with 4% paraformaldehyde for 15–20 h at room temperature and dehydrated using a fully automatic tissue dehydrator (Excelsior AS; Thermo Fisher Scientific, Inc.). The samples were embedded in paraffin using an embedding machine (HistoStar; Thermo Fisher Scientific, Inc.), and sectioned to 3–5 µm thickness. Sections were deparaffinized in xylene at room temperature, rehydrated using descending ethanol concentrations, immersed in 0.01 M citrate buffer (Wuhan Servicebio Technology Co., Ltd.) at 100°C for 10 min to facilitate antigen retrieval, and cooled for 2–3 h at room temperature. Sections were washed three times for 5 min each at room temperature with PBS (Gibco; Thermo Fisher Scientific, Inc.). Sections were blocked with 5% bovine serum albumin (BSA; Merck KGaA) at room temperature for 1h. Sections were incubated with SIRT5 (1:100; Cat. No. 15122-1-AP, Proteintech Group, Inc.) and γH2AX (1:100; ab26350, Abcam) in a humid environment at 4°C overnight. Sections were washed in PBS and incubated with Alexa Fluor secondary antibody (1:100; Invitrogen; cat. # A-11008 and Cat. # A32742, Thermo Fisher Scientific, Inc.) for 1 h at room temperature and treated with VECTASHIELD^® antifading mounting agent containing DAPI (Vector Laboratories, Inc.) for 10 min at room temperature. Sections were observed under a confocal microscope (STELLARIS 5; Leica GmbH).

To observe the mitochondrial staining, the GC-2 spd cells were cultivated in glass coverslips for 24 h and stained with Mito-Tracker red (Thermo Fisher Scientific, Inc.) for 30 min, all at 37°C. Cells were blocked in 5% BSA for 1 h and incubated with SIRT5 (1:100) for 1 h at room temperature and sealed with DAPI, then observed under the confocal laser scanning microscope.

GC-2 spd cell line was procured from the American Type Culture Collection. Cells were maintained in a 25-cm² petri dish with DMEM containing 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin. Cells were maintained at 37°C with 5% CO₂.

GC-2 spd cells at 60–70%. GC-2 spd cells were transfected with 3 µg/ml pCDNA3.1-SIRT5 plasmid (Sangon Biotech Co. Ltd.) using Lipofectamine^TM 3000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated in 5% CO₂ at 37°C for 8 h to overexpress (OE) SIRT5. Cells transfected with 3 µg/ml pCDNA3.1 plasmid (Sangon Biotech Co. Ltd.) were used as a control. Cells were treated in a 5% CO₂ incubator at 37°C for 48 h before subsequent experiments.

Small interfering (si)RNA transfection. SIRT5 and negative control siRNAs were synthesized by Suzhou GenePharma. The antisense sequences (5′→3′) were as follows: Negative control, ACGUGACACGUUCGGAGAATT; SIRT5 siRNA-1, CCAGUUGUGUUGUAGACGATT and SIRT5 siRNA-2, GGCUCGUCCAAGUUCAAAUTT. When the cell density reached ~30% confluency, 1 µg/ml SIRT5 and control siRNAs were transfected into GC-2 spd cells. The cells were incubated at 37°C in 5% CO₂ for 48 h before subsequent experiments.

Total RNA was extracted from sperm samples and 1×10⁶ GC-2 spd cells using SteadyPure RNA extraction kit (Hunan Accurate Bio-Medical Technology Co., Ltd.). According to the manufacturer's protocol, the extracted RNA was reverse-transcribed into cDNA using Evo M-MLV RT Mix Tracking kit with gDNA Clean for qPCR (Hunan Accurate Bio-Medical Technology Co., Ltd.). qPCR was carried out using SYBR Green Premix Pro Taq HS qPCR kit (Hunan Accurate Bio-Medical Technology Co., Ltd.). The following thermocycling conditions were used: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The housekeeping gene GAPDH was used as an internal control for normalization. RT-qPCR was performed out using a two-step process. The 2^−ΔΔCq method was employed to evaluate the relative expression of the target genes (25). The primers are listed in Table I.

Western blotting was performed as previously described (17). The antibodies were as follows: Anti-β-actin (1:10,000; cat. No. 66009-1-IG, Proteintech Group, Inc.), anti-SIRT5 (1:5,000; Cat. No. 15122-1-AP, Proteintech Group, Inc.), anti-PI3K (1:1,000; Cat. # 13666, Cell Signaling Technology, Inc.), anti-AKT (1:1,000; Cat. no. 9272, Cell Signaling Technology, Inc.,), anti-phosphorylated (p-)AKT (1:1,000; Cat. # 4060, Cell Signaling Technology, Inc.), anti-Bax (1:1,000; Santa Cruz Biotechnology, Inc.) and anti-Bcl-2 (1:1,000; Santa Cruz Biotechnology, Inc.). The secondary antibodies were anti-rabbit (1:2,000; Cat. #7074, Cell Signaling Technology, Inc.). and anti-mouse IgG (1:2,000; Cat. #7076, Cell Signaling Technology, Inc.).

CCK-8 assay (Dojindo Molecular Technologies Inc.) was performed to evaluate cellular viability. Transfected GC-2 spd cells were seeded into a 96-well plate at a density of 3,000 cells/well. A volume of 10 µl CCK-8 (Beyotime Institute of Biotechnology) was added to the DMEM and incubated at 37°C for 2 h. Subsequently, the optical density at 450 nm was measured every 24 h using a Multiskan Go plate reader (Beckman Coulter, Inc.).

An EdU incorporation assay was carried out with the EdU cell proliferation kit (cat. no. C0078L, Beyotime Institute of Biotechnology). Sterilized slides were placed in a 24-well plate and transfected cells were seeded into each well at a density of 30%. After culturing with 50 µmol/l EdU reagent for 2 h at room temperature, slides were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature (Beyotime Institute of Biotechnology). After staining the nucleus with DAPI, the cells were imaged and photographed with a confocal laser scanning microscope.

Cell migration was detected using a wound healing assay. 5×10⁵ transfected cells were plated in a 6-well plate and cultured to 90% confluency. Subsequently, monolayers were scratched with a 200 µl pipette tip and washed with PBS. The medium was replaced with a serum-free DMEM and images were captured 0 and 24 h by a Lecia DMi8 fluorescence microscope. All cells were grown at 37°C in a 5% CO₂ incubator.

An apoptosis assay was performed using Annexin V, 633 Apoptosis Detection kit (Dojindo Laboratories, Inc.). In brief, after digestion with trypsin, the cells were centrifuged at 700 × g for 2 min at 4°C, washed twice with PBS and resuspended in binding buffer at a final density of 1×10⁶ cells/ml. Annexin V-633 and PI (5 µl each) were added to 100 µl cell suspension. The cell suspension was mixed and incubated for 15 min at room temperature in the dark. A total of 200 µl binding buffer was added and cells were measured by flow cytometry using Calibur (BD Accuri C6 Plus; BD Biosciences). Data were analyzed using BD Accuri C6 Plus software (version 1.0.264.21; BD Biosciences) and the apoptotic rate was calculated as the percentage of early (Annexin V-FITC) + late apoptotic (Annexin V-FITC and PI) cells.

A total of 1×10⁷ GC-2 cells were lysed in 1 ml NP-40 buffer (Beyotime Institute of Biotechnology) containing protease inhibitor cocktail, centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was removed for use. 1 ml lysates were incubated with PI3K (1:100; Cat. # 13666, Cell Signaling Technology, Inc.), FLAG (1:200; Cat. No. 20543-1-AP, Wuhan Sanying Biotechnology) or mouse IgG (1:100; sc-515946, Santa Cruz Biotechnology, Inc.) antibody for 12 h at 4°C and incubated with 20 µl Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The beads were washed with PBST (0.1% Tween-20) and incubated at 95°C for 10 min in 1× loading buffer (Beyotime Institute of Biotechnology). The beads were separated using a magnetic rack and the supernatant was used for Western blotting, as aforementioned.

Data analysis was performed using GraphPad Prism 8 (Dotmatics). The results from three experiments are reported as mean ± standard deviation. Student's unpaired t-test was employed for comparisons between two independent groups, while one-way ANOVA followed by the Tukey's post hoc test was used to assess significance between >2 groups. P<0.05 was considered to indicate a statistically significant difference.

In our previous study, proteomic analysis revealed that SIRT5 was downregulated in asthenozoospermia compared with normal sperm samples (Fig. 1A) (26). In addition, the expression levels of SIRT5 were assessed in asthenozoospermia, asthenoteratozoospermia and normal sperm from fertile men by downloading the data from the GEO database (GSE160749). SIRT5 expression was significantly downregulated in asthenozoospermia and asthenoteratozoospermia spermatozoa compared with normal (Fig. 1B). We obtained samples from clinical patients and the basic semen parameters in normal and asthenozoospermic patients are described in Table II. Furthermore, expression of SIRT5 was assessed in normal and asthenozoospermia sperm samples, which revealed that its mRNA and protein expression was significantly downregulated in asthenozoospermia sperm samples (Fig. 1C and D).

To determine the SIRT5 expression in the testicular tissue, immunofluorescence analysis was conducted using testicular tissue from a 2-month-old wild-type mouse. Analysis revealed widespread expression of SIRT5 in testicular tissue, including its presence in spermatocytes (Fig. 1E). This was congruent with the expression profile of SIRT5 in the HPA database (Fig. 1G). In the mouse spermatocyte cell line GC-2 spd, SIRT5 was mainly localized to mitochondria (Fig. 1F).

Both gain- and loss-of-function assays were performed to further elucidate the biological function of SIRT5 in GC-2 spd cells. GC-2 spd cells were infected with lentivirus harboring pLV-SIRT5 or siRNAs for SIRT5 knockdown. The efficacy of SIRT5 modulation in GC-2 spd cells was evaluated by RT-qPCR and western blot analysis (Fig. 2A-D). CCK-8 assays revealed the viability of OE-SIRT5 GC-2 spd cells increased compared with the vector control group (Fig. 2E). Conversely, knockdown of SIRT5 in GC-2 spd cells decreased viability (Fig. 2E). EdU assay revealed that SIRT5 knockdown inhibited cell proliferation while OE-SIRT5 increased cell proliferation (Fig. 2F). These results suggested that SIRT5 promoted the proliferation of GC-2 spd cells.

Wound healing assay was performed to investigate the effects of SIRT5 on the motility of GC-2 spd cells. Knockdown of SIRT5 significantly impaired the migratory capacities of GC-2 spd cells (Fig. 3A), whereas OE increased migration (Fig. 3B).

To determine the effects of SIRT5 on apoptosis in GC-2 spd cells, flow cytometry was used. Notably, the proportion of apoptotic cells was significantly elevated in the SIRT5 knockdown compared with the control group (Fig. 4A). Conversely, the proportion of apoptotic cells with OE-SIRT5 significantly decreased (Fig. 4B).

To clarify the molecular mechanism underlying regulation of proliferation and apoptosis by SIRT5 in GC-2 spd cells, western blotting was performed. Knockdown of SIRT5 suppressed the expression of Bcl-2 and p-AKT, but promoted the expression of Bax (Fig. 5A and B). Conversely, OE-SIRT5 markedly enhanced the expression of Bcl-2 and p-AKT, while decreasing expression of Bax (Fig. 5A and B). To explore how SIRT5 regulates the PI3K/AKT pathway, the binding of SIRT5 to PI3K was assayed using co-immunoprecipitation (Fig. 5C). SIRT5 could bind with PI3K, which might inhibit the phosphorylation of PI3K/AKT signaling members.