The datasets GSE149084 and GSE25518 related to cryptorchidism were obtained from the GEO database. DEGs in cryptorchidism compared to normal testes were identified with a significance level of P < 0.01. Autophagy-related differential expression genes were sourced from the HAMdb database. Protein interaction analysis was conducted using STRING to identify core targets. The hTFtarget system was used to predict upstream transcription factors of EIF4EBP1. The Jvenn system was utilized to generate Venn diagrams showing the intersections among different datasets.

Reagents: Male BALB/c mice (4 weeks old, 12–16 g) were purchased from Hunan Slyke Jinda Experimental Animal Co., Ltd., with production license No.: SCXK (Xiang)2024–0,009; The short hairpin RNA targeting EIF4EBP1 (sh-EIF4EBP1), the EIF4EBP1 overexpression plasmid (oe-EIF4EBP1), and the negative controls for lentiviral infection (including sh-NC and oe-NC) were purchased from Genomeditech, Ltd. (Shanghai, China); GC-1 spg (CL-0600, Procell Life, Wuhan, China); Fetal Bovine Serum (FBS, A5670701, Thermo Fisher Scientific, MA, United States); Penicillin-Streptomycin (10,000 U/mL, 15140122, Thermo Fisher Scientific, MA, United States); Dulbecco’s Modified Eagle Medium (DMEM, 11965092, Thermo Fisher Scientific, MA, United States); Lipofectamine™ 2000 Transfection Reagent (11668027, Thermo Fisher Scientific, MA, United States); HLM006474 (324461, Merck, Darmstadt, Germany); Dimethyl Sulfoxide (DMSO, D2650, Sigma-Aldrich, St. Louis, MO, United States); MTT Solution (HY-15924, Medchemexpress, New Jersey, United States); PBS (AM9624, Thermo Fisher Scientific, MA, United States); Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Apoptosis Detection Kit (C1090, Beyotime, Shanghai, China); DCFH-DA (HY-D0940, Medchemexpress, New Jersey, United States); Anti-Fade Mounting Medium (F4680, Sigma-Aldrich, MO, United States); Dual-Luciferase Reporter Assay Kit (RG027, Beyotime, Shanghai, China); Simple ChIP Enzymatic Chromatin IP Kit (9002S, Cell Signaling, Danvers, MA, United States); IgG Antibody (SAB5600195, Sigma-Aldrich, St. Louis, Missouri, United States); Isoflurane (R510-22-16, RWD, Shenzhen, China); Metronidazole (HY-B0318, Medchemexpress, New Jersey, United States); Hematoxylin and Eosin (HE) Staining Kit (C0105S, Beyotime, Shanghai, China); Masson’s Trichrome Staining Kit (C0189S, Beyotime, Shanghai, China); RIPA Lysis Buffer (P0013B, Beyotime, Shanghai, China); BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China); SDS-PAGE (P0531S, Beyotime, Shanghai, China); PVDF Membranes (ab133411, Abcam, Cambridge, UK); Goat Serum (C0265, Beyotime, Shanghai, China); Primary Antibodies: Cleaved Caspase-3 (9661, 1:1000, Cell Signaling Technology, Beverly, MA, United States), LC3 (A5618, 1:500, ABclonal, Wuhan, China), Beclin-1 (ab207612, 1:2000, Abcam, Cambridge, England), P62 (ab314504, 1:1000, Abcam, Cambridge, England), EIF4EBP1 (A24691, 1:500, ABclonal, Wuhan, China), E2F1 (ab314311, 1:1000, Abcam, Cambridge, England), GAPDH (A19056, 1:50000, ABclonal, Wuhan, China); HRP-Conjugated Secondary Antibody (AS014, 1:2000, ABclonal, Wuhan, China); Trizol Reagent (R0016, Beyotime, Shanghai, China); SOD Assay Kit (19160, Sigma-Aldrich, St. Louis, MO, United States); MDA Assay Kit (ab238537, Abcam, Cambridge, UK); GSH/GSSG Ratio Detection Assay Kit (ab138881, Abcam, Cambridge, UK); Testosterone ELISA Kit (PT872, Beyotime, Shanghai, China); RT-qPCR Kit (D7268S, Beyotime, Shanghai, China); Non-fat Milk Powder (P0216, Beyotime, Shanghai, China).

Instruments: ELISA Reader (1410101, Thermo Fisher Scientific, MA, United States); Fluorescence Microscope (DM2500, Leica, Wetzlar, Germany); BeyoECL Star (A38554, Thermo Fisher, Massachusetts, United States); Luciferase Detection System (E1500, Promega, Madison, Wisconsin, United States); ImageJ Software (V1.8.0.112, NIH, Madison, WI, United States); FlowJo (v10.8.2, BD, Ashland, Oregon, United States); GraphPad Prism 9.0 (Dotmatics, Boston, MA, United States).

Before the experiment, the mice were acclimated for 3 days in a suitable environment (temperature 18°C–26°C, humidity 40%–70% RH, 12-h light/dark cycle, with free access to water and food). This study strictly adhered to the 3 R principles and was approved by the Animal Committee of Hunan Evidence-based Biotechnology Co., Ltd. (Ethics Number: XZ23032).

The mice were randomly divided into 6 groups, with 6 mice in each group: Sham, Surgery, Saline, HLM, HLM + oe-EIF4EBP1, and HLM + oe-NC. The Surgery group mice were anesthetized with 3% isoflurane gas delivered via a mask, and 1% isoflurane gas was maintained during the surgery. The mice were placed on the operating table in a supine position, and a 2 cm incision was made in the midline of the lower abdomen. The abdominal cavity was opened, and the bilateral testes were gently exteriorized and pulled into the abdominal cavity. The testicular pedicles were cut, and the testes were sutured and fixed within the abdominal cavity. In the Sham group, the abdominal cavity was opened, and the bilateral testes were exposed without being cut or fixed, allowing them to remain in their original position (Zheng et al., 2019).

For the lentivirus injection, oe-EIF4EBP1 and oe-NC expressing lentiviruses were injected into the bilateral testes of the mice before suturing (Ikawa et al., 2002). After suturing the abdominal cavity, all mice were administered metronidazole to prevent infection. The E2F1-inhibitor, HLM006474 (12.5 mg/kg) was administered via intraperitoneal injection to the mice three times a week for 6 weeks (Yi et al., 2023). The HLM group received only HLM006474, while the Saline group was injected with an equivalent volume of saline as a negative control. All mice were kept under identical suitable conditions.

At week 6, the mice were euthanized via an intraperitoneal injection of 1% sodium pentobarbital (150 mg/kg). Peripheral blood, testicular tissue, and epididymides were collected. Testicular volume and weight were measured using a precision caliper and electronic balance. The epididymides were dissected to collect sperm, which were then observed under a microscope, and the sperm count and motility ratio (motility ratio = motile sperm count/total sperm count) were recorded using a hemocytometer.

Mouse testicular tissues were fixed in 10% neutral buffered formalin, then dehydrated in a graded ethanol series and cleared with xylene. The tissues were embedded in paraffin and sectioned. The paraffin sections were dewaxed in xylene and rehydrated through an alcohol-to-water gradient. The sections were stained with an HE staining kit, cleared again in xylene, and dehydrated through graded ethanol. Finally, the sections were observed and photographed using a standard optical microscope.

Deparaffinize mouse testicular tissue sections to water. Stain with hematoxylin for 5 min, followed by staining with Biebrich Scarlet-Acid Fuchsin solution for 10 min. Then, treat with phosphomolybdic acid solution for 2 min, and finally stain with light green solution for 1 min. After rinsing with 1% acetic acid solution, dehydrate the sections using a graded series of ethanol, clear with xylene, and observe under a standard light microscope for photography.

The GC-1 spg mouse spermatogonial cells were cultured in DMEM medium with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO2. To create a cryptorchidism model, cells were exposed to 42°C for 2 h (Shahat et al., 2020). For HLM006474 treatment, cells were incubated with 40 μM HLM006474 dissolved in DMSO for 24 h (Ma et al., 2008), with an equal volume of DMSO added to the medium as a negative control.

The mouse spermatogonial cell line GC-1 spg was seeded into culture plates, and lentiviral infection was performed when the cells reached 60% confluence. After 48 h of infection, stable infected cells were selected by adding the appropriate antibiotics, and the successful alteration of gene expression was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) analysis.

GC-1 spg cells in the logarithmic growth phase were plated into 96-well plates at a density of 5,000 cells per well, with five replicates per group. Then, 10 μL of MTT solution was added to each well. The plates were incubated at 37°C for 4 h. Absorbance at 562 nm was measured using a microplate reader.

Cell culture supernatants, mouse serum, and testicular tissue homogenates were collected, and the levels of SOD, MDA, GSH/GSSG, and testosterone in the samples were measured using commercial assay kits. The procedures were performed according to the instructions provided with each kit. Absorbance measurements were taken using a microplate reader.

Add 10 μM DCFH-DA to each group of GC-1 spg cells and incubate in the dark for 30 min in a cell culture incubator. Remove the unreacted probe, and then mount the slides using an anti-fade mounting medium. Observe and capture images using a fluorescence microscope. Fluorescence intensity was evaluated using ImageJ software.

Fix each group of GC-1 spg cells with 4% paraformaldehyde for 30 min, followed by incubation with PBS containing 0.1% Triton X-100 at room temperature for 5 min. Prepare the TUNEL detection solution according to the instructions provided with the TUNEL assay kit, and add 50 μL of the solution to each sample. Incubate in the dark at 37°C for 60 min. Add DAPI working solution (1 μg/mL) and incubate in the dark at room temperature for 30 min. After mounting the slides with an anti-fade mounting medium, observe the samples under a fluorescence microscope. The apoptosis rate was calculated using ImageJ software.

The mouse EIF4EBP1 promoter region DNA sequence was retrieved from the UCSC database (http://genome.ucsc.edu/), followed by prediction of E2F1 binding sites in the EIF4EBP1 promoter region using JASPAR (https://jaspar.elixir.no/). Mutations at the top three binding sites were performed. Subsequently, the amplified wild-type (WT) and mutated (MUT) EIF4EBP1 promoter sequences were inserted into the pGL3-Basic luciferase reporter vectors, which were transfected with sh-NC and sh-E2F1 plasmids into GC-1 spg cells using Lipofectamine 2000 and incubated at 37°C with 5% CO₂ for 48 h. The luciferase activity was measured using a luciferase reporter assay kit.

According to the Simple ChIP Enzymatic Chromatin IP Kit instructions, treat GC-1 spg cells with formaldehyde for 15 min. Quench the protein-DNA crosslinking with glycine. Digest the chromatin with micrococcal nuclease and resuspend in ChIP buffer. Perform the immunoprecipitation (IP) reaction by adding E2F1 antibody (1:30) or IgG and incubate overnight at 4°C. Elute the chromatin and reverse the crosslinks, then purify the DNA for qPCR analysis.

Obtain total protein lysates from cell and testicular tissue homogenates using RIPA lysis buffer. Measure protein concentration using a BCA protein assay kit, then load the protein samples onto an SDS-PAGE gel for electrophoresis. Transfer the samples to PVDF membranes, block with 5% non-fat milk, and incubate overnight at 4°C with primary antibodies against Cleaved Caspase-3, LC3, Beclin-1, P62, E2F1, EIF4EBP1, and GAPDH. Subsequently, incubate with HRP-conjugated secondary antibodies at room temperature for 2 h. Develop the blots using BeyoECL Star and analyze band density with ImageJ software, using GAPDH as an internal control for quantification.

According to the manufacturer’s instructions for the Trizol reagent kit, extract total RNA from cell and testicular tissue homogenates. Use RT-qPCR kits and specific primers (see Table 1) to set up reactions for E2F1 and EIF4EBP1. Calculate relative expression levels using the 2^−ΔΔCt formula, with GAPDH as the internal control.

Statistical analyses were conducted using Prism 9 software, with data presented as mean ± SD. T-tests were applied for comparisons between two groups, while one-way or two-way ANOVA followed by Tukey’s post hoc test was used for comparisons among three or more groups. A P-value of <0.05 was considered statistically significant.

A cryptorchidism mouse model was established surgically to investigate the pathological mechanisms of the condition. Results demonstrated a significant reduction in testicular volume and weight in the Surgery group compared to the Sham group (Figure 1A). HE staining revealed pronounced testicular injury in the Surgery group (Figure 1B). Additionally, testosterone levels in both serum and testis were markedly lower in the Surgery group, along with reduced sperm count and motility (Figures 1C,D), which confirmed the successful establishment of the cryptorchidism model.

The results of WB analysis showed that the Surgery group exhibited significantly increased levels of Cleaved Caspase-3, LC3II/LC3I, and Beclin-1, while P62 were downregulated (Figures 1E,F). These findings indicated enhanced autophagy and apoptosis in the cryptorchidism model. To explore the mechanism underlying excessive autophagy in germ cells, we used jvenn (https://jvenn.toulouse.inrae.fr/app/example.html) to perform a cross-analysis of the differentially expressed genes (DEGs) from GSE149084 and GSE25518 (Figure 1G) alongside autophagy-related genes. This analysis identified nine overlapping factors: SDHB, TMEM59, E2F1, EIF4EBP1, ATG15L1, SMPD1, RRAGA, CHMP4B, and COPZ1 (Figure 1H). Subsequently, we conducted a protein-protein interaction (PPI) analysis of these nine overlapping factors using STRING (https://cn.string-db.org/cgi/input?sessionId=bhaAMvIiNIFZ&input_page_active_form=multiple_identifiers) (Szklarczyk et al., 2023). The results revealed that EIF4EBP1 was a key gene among the DEGs related to cryptorchidism and autophagy (Figure 1I) (Bardou et al., 2014). Subsequent RT-qPCR and WB analyses revealed that EIF4EBP1 expression was significantly upregulated in the testicular tissues of cryptorchidism model mice (Figures 1J,K).

The expression of EIF4EBP1 in cryptorchidism was further investigated using in vitro experiments. A cryptorchid spermatogonial cell model was established under heat stress conditions. Results from the MTT assay revealed significantly reduced cell viability in the Heat group compared to the Control group (Figure 2A). Biochemical analyses and fluorescence staining showed that oxidative stress markers, such as SOD and GSH/GSSG, were significantly reduced, while MDA and ROS levels were markedly elevated in the Heat group (Figures 2B,C), confirming successful model establishment.

TUNEL staining and WB analyses results further demonstrated that the percentage of apoptotic cells was significantly higher in the Heat group (Figure 2D). Consistently, the expression of apoptosis-related protein Cleaved Caspase-3 and autophagy-related proteins LC3II/LC3I and Beclin-1 were upregulated, while autophagy-related protein P62 were significantly downregulated (Figures 2E,F). Furthermore, RT-qPCR and WB demonstrated a significant increase in EIF4EBP1 expression in the Heat group compared to the Control group (Figures 2G,H). These findings suggest that the upregulation of EIF4EBP1 was associated with enhanced signs of apoptosis and autophagy in germ cells, providing insight into its potential role in cryptorchidism pathology.

GC-1 spg cells were transduced with a recombinant lentivirus expressing sh-EIF4EBP1. This established an EIF4EBP1 knockdown model within the cryptorchid spermatogonial cell model. RT-qPCR and WB analyses confirmed a significant reduction in EIF4EBP1 expression in the sh-EIF4EBP1 group compared to the sh-NC group (Figures 3A,B). Following EIF4EBP1 knockdown, cell viability in the cryptorchid spermatogonial cell model significantly improved (Figure 3C), accompanied by reductions in ROS and MDA (Figures 3D,E).

Assessment of apoptosis and autophagy revealed that EIF4EBP1 knockdown significantly reduced the percentage of apoptotic cells (Figure 3F). Notably, the expression levels of apoptosis-related proteins Cleaved Caspase-3 and autophagy-related proteins LC3II/LC3I and Beclin-1 were decreased, whereas autophagy-related protein P62 was significantly increased in the sh-EIF4EBP1 group (Figures 3G,H). Taken above, EIF4EBP1 knockdown reduces signs of autophagy and apoptosis in germ cells exposed to cryptorchid conditions. The results highlight EIF4EBP1 as a potential therapeutic target for managing cryptorchidism-associated germ cell damage.

To further investigate the mechanisms underlying excessive autophagy in germ cells during cryptorchidism, bioinformatics analysis identified E2F1 as an upstream transcription factor of EIF4EBP1, intersecting with differentially expressed genes associated with both cryptorchidism and autophagy (Figure 4A). WB analysis of E2F1 expression in cryptorchidism spermatogonial cells revealed significant upregulation in the Heat group compared to the Control group (Figure 4B). Moreover, treatment with an E2F1-inhibitor significantly decreased EIF4EBP1 expression in the cryptorchid spermatogonial cell model (Figure 4C).

To elucidate the regulatory relationship between E2F1 and EIF4EBP1 at the transcriptional level, we conducted dual-luciferase reporter assays and ChIP experiments. The dual-luciferase reporter assay demonstrated that E2F1 knockdown reduced the activity of the wild-type EIF4EBP1 promoter, but had no effect on the mutated version (Figure 4D). Furthermore, ChIP analysis revealed significantly greater enrichment of E2F1 at the EIF4EBP1 promoter region compared to the IgG control group (Figure 4E). These findings confirm that E2F1 functions as an upstream transcription factor for EIF4EBP1.

To further confirm whether E2F1 influences apoptosis and autophagy in cryptorchidism germ cells via EIF4EBP1, we generated GC-1 spg cells overexpressing EIF4EBP1 and established a cryptorchid spermatogonial cell model. These cells were treated with the E2F1-inhibitor, HLM006474. RT-qPCR and WB analyses revealed that EIF4EBP1 expression was significantly lower in the HLM006474-treated group compared to the DMSO control group (Figures 5A,B). Moreover, the overexpression of EIF4EBP1 restored its levels, confirming successful infection.

E2F1 inhibition significantly improved cell viability and reduced oxidative stress in spermatogonial cells compared to the DMSO group. However, when EIF4EBP1 was overexpressed, cell viability decreased again, and oxidative stress levels increased (Figures 5C–E).

Next, we assessed apoptosis and autophagy. The results indicated that inhibition of E2F1 significantly reduced the proportion of apoptotic cells (Figure 5F) and increased the levels of the apoptosis-related protein Cleaved Caspase-3 and autophagy-related proteins LC3II/LC3I and Beclin-1, while decreasing the levels of the autophagy-related protein P62 (Figures 5G,H). Conversely, EIF4EBP1 overexpression exacerbated both signs of apoptosis and autophagy.

To investigate whether E2F1 affects testicular damage and fertility in the cryptorchidism mouse model through EIF4EBP1, we injected oe-EIF4EBP1-expressing recombinant lentivirus into the testes of cryptorchidism mice and treated them with the E2F1-inhibitor HLM006474. Successful lentiviral infection was confirmed by fluorescence microscopy (Supplementary Materials). RT-qPCR and WB analyses confirmed that EIF4EBP1 expression was significantly lower in the HLM006474-treated group compared to the saline control group. Overexpression of EIF4EBP1 reversed this decrease, validating successful infection (Figures 6A,B).

Measurements of testicular volume and weight indicated that E2F1 inhibition significantly increased both parameters, whereas EIF4EBP1 overexpression reduced them again (Figure 6C). HE staining revealed that E2F1 inhibition improved testicular damage, while EIF4EBP1 overexpression exacerbated the damage further (Figure 6D).

Biochemical analyses showed that E2F1 inhibition significantly increased testosterone levels in both the serum and testes, but overexpression of EIF4EBP1 reversed this effect (Figure 6E). Additionally, E2F1 inhibition resulted in significant improvements in sperm count and motility, which were diminished upon EIF4EBP1 overexpression (Figure 6F). WB results confirmed that E2F1 inhibition significantly reduced apoptosis and autophagy in the testes, yet EIF4EBP1 overexpression promoted apoptosis and autophagy (Figures 6G,H).

In conclusion, E2F1 inhibition protects the testes from damage in a cryptorchidism mouse model by reducing EIF4EBP1 transcription.