Ti3C2 nanosheets disrupt spermatogenesis in male mice via alterations in proliferation and apoptosis of spermatogonia mediated by the ATM/p53 signaling pathway

doi:10.21203/rs.3.rs-2491632/v1

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Ti3C2 nanosheets disrupt spermatogenesis in male mice via alterations in proliferation and apoptosis of spermatogonia mediated by the ATM/p53 signaling pathway

https://doi.org/10.21203/rs.3.rs-2491632/v1

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published 13 Jun, 2023

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Background: Two-dimensional ultrathin Ti₃C₂ nanosheets are increasingly being used in biomedical applications owing to their special physicochemical properties. Meanwhile, the biological effects of its exposure, especially on the reproductive system, deserve attention. However, this effect on the reproductive system has not been studied yet.

Results: In the present study, we established an in vivo Ti₃C₂ nanosheet exposure model in mice and an in vitro Ti₃C₂ nanosheet exposure model in GC-1 cells. In vivo, Ti₃C₂ nanosheets accumulated in the testes of mice, leading to male reproductive dysfunction, including diminished sperm quantity and quality. Furthermore, they also induced elevated reactive oxygen species (ROS) and DNA damage in testis tissues and activated the DNA damage response signaling pathway (ATM/p53). In vitro, they entered the GC-1 cytoplasm and reduced cell viability. Ti₃C₂ nanosheets also induced elevated ROS levels and DNA double-strand breaks, activated the ATM/p53 signaling pathway, caused cell cycle arrest in the G1/G0 phase, inhibited proliferation, and increased apoptosis in GC-1 cells.

Conclusions: Ti₃C₂ nanosheet-induced suppression of spermatogonia proliferation disrupted normal spermatogenic function, which was mediated by the ATM/p53 signaling pathway. Thus, our findings shed more lights on the mechanisms of male reproductive toxicity induced by Ti₃C₂ nanosheets.

Spermatogenesis

Ti3C2 nanosheets

Spermatogonia

DNA damage

With an increasing decline in global fertility, reproductive health has gained the spotlight in medical research. It is estimated that 20–30% of infertility cases are caused by men [1]. Epidemiological studies have shown that sperm quality and quantity have shown a downward trend [2], with male toxicity caused by environmental pollutants being an important factor. Exposure to environmental pollutants results in changes in gene expression, hormonal imbalances, reproductive disorders, and sperm abnormalities [3]. Many studies have focused on the effects of endocrine-disrupting chemicals (EDCs), pesticides, and heavy metals on the reproductive system. However, an increasing relevance of nanomaterials to human beings (cosmetics, paints, medical products, textiles, personal care, and other products) warrants the verification of their biosafety as they pose potentially high risks to human health [4]. For instance, exposure to ZnO reduces sperm quality in mice by inducing lipid metabolism disorders [5], and La₂O₃ nanoparticles (NPs) induce abnormal spermatogenesis in mice via Nrf-2/ARE signaling [6].

Recently, MXenes have gained grounds as a new class of 2D transition metal carbides or nitrides [7]. The general formula of MXene is M_n+1X_nT_x (n = 1 − 3), where M represents an early transition metal (such as Ti, Zr, Hf, or Ta), X refers to carbon and/or nitrogen, and T represents surface terminal groups (such as oxygen, hydroxyl, chlorine, or fluorine). It was derived from the corresponding MAX phase by the selective etching of the interlayer Al atoms of the transition metal carbide, Ti₃AlC₂ [8]. Owing to their physicochemical properties, they have increasingly gained attention for biomedical applications such as photothermal therapy [9, 10], diagnostic imaging [11], antimicrobial formulations [12, 13], biosensing [14, 15], and drug delivery [16, 17]. Recently, it has been shown that exposure to Ti₃C₂ nanosheets leads to their accumulation in the lungs, thereby interfering with respiratory function through the downregulation of SP-B levels in alveolar epithelial cells [18]. Abnormal placental development and altered placental lipid metabolism following exposure to Ti₃C₂ nanosheets cause neurodevelopmental disorders in offspring mice [19]. Previously, we found that Ti₃C₂ nanosheet exposure inhibited the growth of villus explants and impaired the function of HTR-8/SVneo cells [20]. However, experimental studies on the biosafety of Ti₃C₂ nanosheets are limited, and the effects of Ti₃C₂ nanosheets on the male reproductive system have not been studied yet.

Previous studies have shown that the biological effects of NPs are closely associated with DNA damage. Human DNA is damaged by endogenous and exogenous factors tens of thousands of times per day [21]. Recent investigations have indicated that exposure to cobalt nanoparticles causes DNA damage in A549 cells, and exposure to nickel nanoparticles causes DNA damage in BEAS-2B cells [22, 23]. Nanoparticles inhibit spermatogenesis by inducing cell cycle arrest and inhibiting proliferation in the testes [24]. Studies have equally demonstrated that oxidative stress can lead to testicular damage and apoptosis in rats, resulting in reduced sperm quality [25]. Oxidative stress is a key factor in many diseases causing cell damage, and the generation of reactive oxygen species (ROS) is associated with DNA damage, membrane lipid peroxidation, and apoptosis. Studies have shown that environmental toxicant-induced male reproductive toxicity is associated with increased oxidative stress and reduced antioxidant capacity in the testes [26, 27].

In this study, we investigated the effects of Ti₃C₂ nanosheet exposure on spermatogenesis and its potential mechanisms. Kunming male mice and GC-1spd cell lines were exposed to different doses of Ti₃C₂ nanosheets, and the underlying mechanisms were investigated. Our findings complement those from the study of the biosafety of Ti₃C₂ nanosheet exposure as well as provide clinical applications of this nanomaterial.

2.1 Synthesis and characterization of Ti₃C₂ nanosheets

Following earlier techniques [28]. Ti₃C₂ nanosheets were created by etching Ti₃AlC₂ in an HCl/LiF solution. In a nutshell, 4 g of LiF and 2 g of Ti₃AlC₂ powder were slowly added to 80 mL of HCl (9 M) and stirred for 24 hours at 35 °C. The compound was washed with distilled water numerous times until the pH of the supernatant reached 6. After being sonicated for 1 hour under an Ar flow, the MXene powder was recovered using centrifugation at 3500 rpm for 1 hour. Transmission electron microscopy (TEM, JEM-2100FEG, JEOL, Japan), scanning electron microscopy (SEM, JSM 6701 F, JEOL, Japan), atomic force microscopy (AFM, Multimode 8, Bruker, USA), and X-ray photoelectron spectroscopy were all used to analyze the ultrastructure and morphology of the nanosheets.

2.2 Animal model

In this study, male mice aged 8 weeks (KM-strain, 28-30g) were acquired from the lab animal center of Chongqing Medical University (Certificate: SICXK (YU) 2007-0001). The Institutional Animal Care and Use Committee of Chongqing Medical University (Chongqing, China) gave its approval to all animal experiments. They were given unlimited access to food and water, kept in a stable environment with a relative humidity of 55% ± 10%, and kept at 22 °C ± 2 °C with a 12-hour light/dark cycle.

Based on research on Ti₃C₂ nanosheets as a new photothermal agent for cancer therapy, we selected the lowest known dose as the highest dose per week in the present study [29]. After being adapted to the environment for 1 week, 60 male mice were randomly assigned to four groups of 15 mice each, three experimental groups were exposed to Ti₃C₂ nanosheets (1.25, 2.5, and 5 mg/kg body weight) through intravenous injections once weekly for 4 weeks, and the control group were injected with saline. Ti₃C₂ nanosheets were dispersed in saline. The volume of the Ti₃C₂ nanosheet suspension used for exposure was 0.05 mL/10 g body weight. All mice were sacrificed 7 days after exposure to Ti₃C₂ nanosheets, and the testes and epididymis were sampled for analyses.

2.3 ICP-MS analysis

To track the Ti₃C₂ nanosheets' biodistribution, testicular tissue was gathered. Ti₃C₂ nanosheet accumulation in the testes was carried out by Beijing Zhongkebaice Technology Co., Ltd. Using inductively coupled plasma mass spectrometry, the (Ti) content was quantified (ICP-MS, 7800, Agilent Technologies Inc., USA).

2.4 Sperm quality evaluation

The left epididymis was utilized to calculate the rate of sperm malformation, while the right epididymis was removed for the sperm count test. In order to fully release the sperm, the epididymis was divided into pieces and placed in 1 mL of normal saline that had been preheated to 37°C. 10µL of the sperm suspension was placed on a hemocytometer, and the number of sperm was counted under a microscope. The suspension was placed onto a slide and gently pushed in order to test for sperm deformity. Slides were dried, methanol-fixed, and eosin-stained. Finally, under a microscope, abnormal sperms were counted. During morphological analyses, at least 500 sperm cells per animal were counted to assure precision.

2.5 Testicular histopathology

Fresh mouse testes were excised, preserved for 4-6 hours at 25–37 °C in 4% paraformaldehyde, and then dehydrated with a gradient series of alcohol before being embedded in paraffin (melting point, 60 °C). Hematoxylin and eosin (H&E) staining was done on the testicular sections (5-μm thick), and the results were seen under a microscope (BX40, Olympus, Tokyo, Japan).

2.6 Immunohistochemistry

Immunohistochemistry was used to identify the corresponding antigens. After being deparaffinized in xylene and heated at 60 °C for one hour, the tissue sections were rehydrated using an alcohol gradient series. The endogenous peroxidase activation was suppressed for 10 minutes after being incubated with 1% H₂O₂ following the 15-minute boiling of sodium citrate buffer to extract the antigens. The next step was blocking with 10% goat serum for 15 minutes at 25 °C. The sections were then treated with secondary antibodies (goat anti-rabbit IgG, 1:1000 dilution, Santa Cruz Biotechnology Inc., CA, USA) for 30 min. at 37 °C after being exposed to primary antibodies, such as Ki67 (1:200, Abcam Inc., Cambridge, MA, USA), at 4 °C overnight. The sections were then stained with 3,3-diaminobenzidine for 3 minutes and hematoxylin for 2 minutes after three PBS rinses. They were then observed and photographed under an Olympus microscope.

2.7 Immunofluorescence (IF)

Mouse testicular tissue sections were dewaxed and hydrated, and the repair antigens were prepared. After three PBS washes, testicular tissue and GC-1 cell slices were blocked with 10% goat serum in 5% bovine serum albumin. They were then treated with primary antibodies against Ki67 and -H2AX (1:200, Cell Signaling Technology, Danvers, MA, USA) for an overnight period at 4 °C. The sections were incubated with Dylight 488-conjugated affinity purified goat anti-rabbit IgG (1:200, Abcam Inc., Cambridge, MA, USA) for 1 h in the dark at 37 °C the next day after being washed three times with PBS. The nuclei were then stained with DAPI, and the pictures were captured on camera and examined under a microscope.

2.8 TUNEL staining

To detect apoptotic germ cells in the testes of the mice, a Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, China) was utilized. Tissue sections were deparaffinized in xylene, heated for 1 hour at 60 °C, and rehydrated using an alcohol gradient series. Proteinase K solution (20 μg/mL) was applied to the sections for 15-30 minutes at 20-37 °C. The tissue sections were rinsed three times with PBS before being incubated in 3% hydrogen peroxide and cleaned with PBS. Biotin labeling solution (50μL) was applied to the sections and incubated for 60 minutes at 37 °C, light shielded. The slices were incubated at room temperature for 30 minutes after being washed three times in PBS with 50 mL streptavidin-HRP working fluid. They were then stained with 3,3-diaminobenzidine and hematoxylin, observed and photographed under a microscope.

2.9 Cell culture and treatment

Mouse spermatogonia GC-1 cell line was obtained from the Concorde Cell Bank. GC-1 cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell lines were incubated at a constant temperature 37 °C and 5% CO₂ in an incubator. GC-1 cells were exposed to Ti₃C₂ nanosheets (0, 6.25, 12, 25, and 50 μg/mL) for 24 h. Control cells were treated with saline solution.

2.10 Transmission electron microscopy (TEM)

After being exposed to various doses of Ti₃C₂ nanosheets for 24 hours, GC-1 cells were washed in PBS and digested with trypsin solution. The supernatant from the centrifugation of the cell suspensions at 1200 rpm for 10 min was discarded. In order to evaluate the samples, the acquired cells were pre-fixed with 2.5% glutaraldehyde at the College of Life Sciences of Chongqing Medical University.

2.11 Assessment of ROS, malondialdehyde (MDA), superoxide dismutase (SOD) levels

Testicular tissues of the mice were collected 28 days after Ti₃C₂ nanosheets treatment. Mouse testicular tissue was homogenized (tissue: normal saline = 1:9), and the supernatant was collected. Levels of oxidative stress-related factors, including ROS, MDA, and SOD, were quantified using enzyme-linked immunosorbent assay (ELISA) kits.

2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), a fluorescent probe, was used to measure intracellular ROS levels (Beyotime, China). Ti₃C₂ nanosheets at varying concentrations were applied to GC-1 cells for 24 hours. 200 mL of 10 μmol/L DCFH-DA (DCFH-DA: DMEM = 1:1,000) were added after the medium was aspirated, and the mixture was then incubated at 37 °C for 30 minutes. Using a microplate reader with excitation and emission settings at 485 nm and 530 nm, respectively, the DCFH fluorescence of the cells was detected. With the aid of the appropriate kits, the MDA and SOD levels were determined (Beyotime, China). Additionally, cells were taken out of the culture dishes and lysed for five minutes. The lysate was then centrifuged for 5 minutes at 4 °C at a 12,000rpm speed. According to the manufacturer's instructions, the supernatant was collected and the intracellular levels of MDA and SOD were found.

2.12 Comet assay

Following a 24-hour exposure to Ti₃C₂ nanosheets, GC-1 cells were collected. Then, 10 μL of the cell suspension was added to a low melting point agarose (LMPA) solution, transferred to glass slides that were covered in a normal melting point agarose (NMPA), and a third layer of LMPA agarose was added to plug any remaining holes in the second agarose layer. Slides were soaked in lysis buffer for two hours at 4 °C after the agaroses had solidified. At 25 V, electrophoresis was carried out for 20 minutes. The slides were exposed to neutralization buffer for 10 minutes after electrophoresis before being stained with ethidium bromide. Finally, photos were captured on camera and viewed under a microscope.

2.13 Flow cytometry analysis

An Annexin V-FITC Apoptosis Detection Kit was used for flow cytometry analysis (E-CK-A212, Elabscience, Wuhan, China). The cells were digested with trypsin before being transferred to 1.5 mL tubes for further investigations. The cells were placed in 1 mL of PBS buffer and centrifuged at 1000 rpm for 5 minutes, yielding approximately 1×10⁶ cells. Following two PBS washes, 100μL of binding buffer was added and mixed. Flow cytometry was used to detect apoptosis using Annexin V-FITC (5 μL) and PI (5 μL).

GC-1 cells were collected and washed twice with PBS before being resuspended in 0.1 mL of PBS. To avoid aggregation, 0.9 mL of ice-cold 75% ethanol was injected dropwise on a vortex. After all samples were stored in the dark at room temperature for 30 minutes, the prepared working fluid (RNase A: PI = 1:9) was added for cell cycle analysis by flow cytometry.

2.14 Cell viability assay

Cell viability was determined using the manufacturer's cell fluorescence counting kit (CCK-F) (C2013S, Beyotime, Jiangsu, China). GC-1 cells were grown in BeyogoldTM all-black 96-well plates at a density of 5×10³ cells/well for 24 hours. The cells were subsequently exposed to various concentrations of Ti₃C₂ nanosheets and saltwater. After adding 100 μL of calcein AM detection solution to each well and incubating the mixture at 37 °C for 30 minutes, the fluorescence intensity was measured using fluorescence microscopy. Finally, fluorescence intensity was used to calculate cell viability.

2.15 Alignment of RNA-seq data and differential expression analysis

The testicular tissues were delivered on dry ice to Nanjing Personal gene technology CO., LTD (Nanjing, China) for RNA sequencing. The DESeq2 software was used to identify differentially expressed genes (DEGs), and the GO stats tool was used to perform functional enrichment for KEGG.

2.16 Western blotting analysis

Testicular tissues and GC-1 cells were lysed in RIPA buffer containing protease inhibitor from a protein extraction kit (Beyotime, China), and the BCA protein assay (Beyotime, China) was used to measure the protein concentration. Proteins were separated using 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 1 h at 37 °C with 5% dried fat-free milk in PBS-Tween 0.1% (PBST), and were incubated with specific primary antibodies against γ-H2AX (1:5,000), Cleaved Caspase3, p21, p-p53, p-CHK2, ATM (1:1,000, Cell Signaling Technology, Danvers, MA, USA), Bax, Bcl-2, CyclinD1 and CDK4 (1:500, Wanleibio, Shenyang, China), PCNA (1:5,000, Abcam Inc., Cambridge, MA, USA), PUMA, p-ATM, CHK2 (1:1000, BOSTER, Wuhan, China), p53, and β-actin (1:1,000, ZSGB-BIO, Beijing, China) overnight at 4 °C. The next day, the secondary antibodies (1:1,000, BOSTER, Wuhan, China) were incubated for 1 h at 37 °C with anti-mouse or anti-rabbit HRP-conjugated antibodies. Protein bands were detected using enhanced chemiluminescence (NCM Biotech, Suzhou, China), and the densities of those bands were quantified by ImageJ software.

2.17 Statistical analysis

GraphPad Prism software (version 8.0) (Graph-Pad Software Inc., La Jolla, CA, USA) was used for the data analysis. Student's t-test was used to analyze two-group comparisons, and ANOVA was used to compare more than two groups. P < 0.05 was considered significant. All variables were expressed as mean ± standard deviation (SD).

3.1 Ti₃C₂ nanosheets can enter testicular tissue and GC-1 cells

The morphology of the Ti₃C₂ nanosheets was evaluated using TEM and SEM, the lateral size of the ultrathin nanosheet structure was 0.05-1 μm (Fig. 1a-b). The thickness of the well-dispersed nanosheets, determined through AFM analysis, was 1-4 nm (Fig. 1c). In addition, the chemical state and elemental composition were analyzed using XPS (Fig. 1d-e), showing characteristic peaks in the region of the Ti and C core layers. The Raman spectra of the Ti₃C₂ nanosheets exhibited two peaks at approximately 1342.2 and 1581.9 cm⁻¹, implying that the Ti₃AlC₂ phase was completely converted into Ti₃C₂ nanosheets (Fig. 1f). ICP-MS analysis was performed to measure the biodistribution of Ti in testes. The Ti content was significantly increased (p < 0.05) in the testes compared to that in the control group after intravenous injection of Ti₃C₂ nanosheets in the 2.5 and 5 mg/kg group (Fig. 1g). TEM was used to observe the distribution of the nanosheets and the ultrastructural variation in GC-1 cells treated with Ti₃C₂ nanosheets. Images of treated GC-1 cells clearly demonstrate the cellular uptake of the nanosheets. All Ti₃C₂ nanosheets were distributed mainly within the cytoplasm of GC-1 cells (Fig. 1h).

3.2 Ti₃C₂ nanosheets exposure reduces sperm count in mouse

The effect of Ti₃C₂nanosheet exposure on sperm quality was also evaluated. There was no significant change in body weight and organ coefficient of the testis between Ti₃C₂nanosheet-exposed mice and controls (Fig. 2a-c). The epididymal sperm number was considerably lower in Ti₃C₂nanosheet-exposed mice than in controls (Fig. 2d). The epididymal sperm malformation rate was remarkably higher in the 2.5 and 5 mg/kg groups than in the controls (Fig. 2e). We examined pathological changes in the testes using hematoxylin and eosin (H&E) staining after Ti₃C₂ nanosheets exposure. Mice in the control group showed normal seminiferous tubules and germinal cell arrangement. In the 2.5 and 5 mg/kg groups, H&E staining showed vacuoles and disorganization of the germ cell layers in the seminiferous tubules (Fig. 2f).

3.3 Ti₃C₂ nanosheets exposure induces oxidative stress in mouse testes and GC-1 cells

The oxidative stress levels were measured using ELISA kits and oxidation-antioxidation assay kits to explore the effect of Ti₃C₂ nanosheets on oxidative stress in mouse testes and GC-1 cells. The ROS levels and MDA content in Ti₃C₂ nanosheets-treated mice groups were significantly higher than those in the control group. The SOD activity in Ti₃C₂ nanosheets-treated mice groups were significantly lower than those in the control group (Fig. 3a-c). In vitro, the intracellular ROS levels of GC-1 cells in the Ti₃C₂ nanosheet-treated groups (12.5, 25, and 50 μg/mL groups) were remarkably higher than those in the control group. The MDA content in the Ti₃C₂ nanosheet-treated groups (25 and 50 μg/mL groups) was remarkably higher than that in the control group. The SOD activity in the Ti₃C₂ nanosheet-treated groups (the 25 and 50 μg/mL groups) was significantly lower than that in the control group (Fig. 3d-f). These findings indicate that Ti₃C₂ nanosheets induced oxidative stress in mice testes and GC-1 cells.

3.4 Ti₃C₂ nanosheets exposure causes DNA damage in mouse testes and GC-1 cells

To determine the effect of Ti₃C₂nanosheets on DNA damage in testicular tissues and GC-1 cells, γ-H2AX (a sensitive marker of DNA damage) was assessed through IF staining of testicular sections. As shown in Fig. 4a, many spermatogonia stained positively in the spermatogenic tubules of the testes after treatment with Ti₃C₂ nanosheets. The protein expression level of γ-H2AX was significantly elevated in Ti₃C₂ nanosheet-exposed mouse testes and GC-1 cells (Fig. 4b-e). In addition, DNA damage was also observed in Ti₃C₂ nanosheet-exposed GC-1 cells, as assessed by the comet assay (Fig. 4f). As shown in Fig. 4g, γ-H2AX-positive cells were significantly enhanced in the Ti₃C₂ nanosheet-exposed GC-1 cells. These findings strongly indicate that Ti₃C₂ nanosheet exposure induces DNA damage in the mouse testes and GC-1 cells.

3.5 Ti₃C₂ nanosheets exposure inhibits proliferation in mouse testes and GC-1 cells

Ki67 immunohistochemical staining was used to measure the proliferation of mouse testicular germ cells to explore the effect of the nanosheets on spermatogonia proliferation. The proportion of positive Ki67 stained nuclei in the Ti₃C₂ nanosheet-exposed mouse testes was significantly than that in the control group (Fig. 5a-b). The expression level of PCNA, an additional marker of cell proliferation, was dramatically decreased in the testes of Ti₃C₂ nanosheet-exposed mice (Fig. 5c-d). Next, we studied the effect of the Ti₃C₂ nanosheets on the proliferation of GC-1 cells, as well as the protein expression levels of PCNA (Fig. 5e-f). Cell viability decreased in Ti₃C₂ nanosheet-exposed GC-1 cells in a dose-dependent manner (Fig. 5g). Ki67-positive cells significantly reduced in Ti₃C₂ nanosheet-exposed GC-1 cells (Fig. 5h). These results strongly indicate that Ti₃C₂ nanosheet exposure inhibits the proliferation of mice testicular tissues and GC-1 cells.

3.6 Ti₃C₂ nanosheets exposure induces cell cycle arrest in mouse testes and GC-1 cells

G1/G0-related proteins were measured in Ti₃C₂ nanosheet-exposed mice testes to determine the effects of the exposure to these nanosheets on the cell cycle. CyclinD1 protein expression levels significantly decreased, while CDK4 and p21 protein expression levels significantly increased in the testes of Ti₃C₂ nanosheet-exposed mice (Fig. 6a-d). Compared with the control group, the results revealed that the protein expression levels of CDK4 and CyclinD1 were significantly lower, and p21 protein expression was increased in the GC-1 cells after Ti₃C₂ nanosheet exposure (Fig. 6e-h). Flow cytometry was used to detect the cell cycle of GC-1 cells after exposure to different concentrations of Ti₃C₂ nanosheets (0, 6, 12.5, 25, and 50 μg/mL) for 24 h. Compared to the control, the proportion of cells in the G2/M phase was reduced and that of cells in the G1/G0 phase was increased, whereas that of cells in the S phase did significantly change in the Ti₃C₂ nanosheet-exposed group (Fig. 6i-j). These results strongly indicate that Ti₃C₂ nanosheet exposure induced cell cycle arrest in GC-1 cells and mouse testes.

3.7 Ti₃C₂ nanosheets exposure induces apoptosis in mouse testes and GC-1 cells

To further evaluate the effect of Ti₃C₂ nanosheets on spermatogenesis, we conducted TUNEL staining of testicular sections. As shown in Fig. 7a, the number of TUNEL-positive cells in the Ti₃C₂ nanosheet-exposed group significantly increased. Moreover, the expression levels of PUMA, Cleaved-caspase3 and Bax/Bcl-2 were remarkably higher in the Ti₃C₂ nanosheet-treated groups than in the control group (Fig. 7b-g). In addition, flow cytometry showed that the apoptotic rate of GC-1 cells was significantly higher in Ti₃C₂ nanosheet-exposed cells than in the control group (Fig. 7h-i). The expression levels of PUMA, Cleaved-caspase3, and Bax/Bcl-2 were remarkably higher in Ti₃C₂ nanosheet-treated GC-1 cells than in the control group (Fig. 7j-o). These findings reveal that Ti₃C₂ nanosheet exposure induced apoptosis in mouse testes and GC-1 cells.

3.8 ATM/p53 signaling pathway is involved in Ti₃C₂ nanosheets exposure-induced spermatogenesis disorder

To identify possible upstream regulators of reduced spermatogonia proliferation and increased apoptosis due to Ti₃C₂ nanosheet exposure, we analyzed DEGs between Ti₃C₂ nanosheet-exposed and control tissues. The volcano map (Fig. 8a) and heat map (Fig. 8b) showed 106 differential genes between the Ti₃C₂ nanosheet-exposed and control groups (adjusted p-value < 0.1), with 51 and 55 upregulated and downregulated genes in testicular tissue, respectively. KEGG pathway enrichment analysis was performed using these DEGs in mice testicular tissue. We found that the enriched KEGG pathways included the p53 signaling pathway in mouse testes (Fig. 8c). These findings imply that exposure to Ti₃C₂ nanosheets altered the expression of the p53 signaling pathway in the testes. We explored the role of the ATM/p53 signaling pathway in Ti₃C₂ nanosheet-induced DNA damage. Western blotting was used to evaluate the expression of proteins related to the ATM/P53 signaling pathway. In mice testicular tissues and GC-1 cells, the phosphorylation levels of ATM, CHK2, and p53 were significantly elevated in Ti₃C₂ nanosheet-treated groups compared to the control group (Fig. 8d-k). These results indicate that the ATM/p53 signaling pathway was upregulated after Ti₃C₂ nanosheet exposure and induced DNA damage.

Ti₃C₂ nanosheets are relatively new nanomaterials that have increasingly gained scientific attention for various applications. Owing to their unique photothermal properties, they are used in biomedical applications [30, 31]. Thus, humans are usually exposed to these nanomaterials, and their effects on the reproductive system are of interest. Its effects on placental and ovarian functions have been previously reported by our group, and the present study was concerned with its effect on spermatogenesis.

The decrease in sperm number and increase in sperm malformation rate reflect the decline in male fertility. Research has shown a clear correlation between infertile men and low sperm count [32]. In the present study, we found that Ti₃C₂ nanosheets accumulated in the testes, and in vitro studies indicated that Ti₃C₂ nanosheets infiltrate GC-1 cells in a dose-dependent manner. These findings reveal that Ti₃C₂ nanosheets traverse the BTB, as with other nanoparticles [33, 34]. In addition, Ti₃C₂ nanosheets reduced the sperm count and increased sperm malformation in mice. Ti₃C₂ nanosheet exposure possibly triggers a decline in sperm quality. Sperm formation is the main function of the testes and is dependent on their normal structure. It is known that exposure to environmental pollutants can induce histopathological changes in testes [35, 36]. The present study demonstrates that Ti₃C₂ nanosheets exposure can cause testicular damage, such as vacuoles in seminiferous tubules and disorganization of the germ cell layer. However, there were no significant changes in body weight gain or relative testicular weight in the mice. These results are in accordance with the findings of Adebayo et al. [27], who found that cerium oxide nanoparticles did not change the relative weight of the testis. This reveals that Ti₃C₂ nanosheets cause abnormalities in the structure of testicular tissue, an important reason for spermatogenesis disorders.

Normally, oxidative and antioxidant systems in the body are in a dynamic balance. However, a disruption of the balance produces oxidative stress, which mainly manifests as an imbalance between ROS and antioxidants [37]. MDA is a typical indicator of membrane lipid peroxidation. SOD functions as an antioxidant and protects the cells from oxidative damage. Excessive ROS causes excessive production of lipid radicals such as MDA [38]. Oxidative stress often induces cellular DNA damage and is an important reason exposure to many environmental pollutants impairs homeostasis. In this study, we found that Ti₃C₂ nanosheet exposure induced oxidative stress and DNA damage in tissues and cells in vitro, suggesting that oxidative stress and its induced DNA damage are important links for Ti₃C₂ nanosheet toxicity.

Spermatogenesis is a unique male germ cell differentiation process and a unique cell cycle process, mainly through the proliferation of spermatogonia, meiosis of spermatocytes, and spermatocyte metamorphosis. Maintaining normal spermatogonia proliferation is important for sperm production [39]. Several studies have shown that nanoparticles or environmental pollutants can impair spermatogenesis by affecting germ cell proliferation and inducing apoptosis [40, 41]. To further reveal the cause of reduced spermatogenesis, we observed cell proliferation and apoptosis and found that Ti₃C₂ nanosheets inhibited proliferation and increased apoptosis in testicular tissues and GC-1 cells. This explains the previously observed decrease in sperm count. Cell cycle regulation is essential for maintaining genomic integrity against DNA damage and can provide more time for DNA repair to avoid replication and mutation. When the integrity of the cellular genome is challenged by extrinsic and intrinsic stresses, cells initiate the DNA damage response (DDR), in order to deliver intact genetic information to offspring [42]. When DNA damage is too severe, excessive DSBs trigger cell cycle arrest, leading to cell proliferation inhibition and irreversible apoptosis [43]. Cell cycle arrest explains why Ti₃C₂ nanosheets inhibit proliferation and promote apoptosis.

Transcriptome sequencing suggested that Ti₃C₂ nanosheets could lead to significant changes in the ATM/P53 signaling pathway. ATM kinase is the most upstream DDR kinase in mammalian cells [44]. Once DSBs are present, ATM is immediately recruited to the DNA damage site and activated. and can also activate downstream CHK2, which in turn activates downstream P53 [45]. A recent study showed that decabromodiphenyl ether (BDE-209) activates the ATM/p53 signaling pathway in mouse testes through oxidative stress-mediated DNA damage [40]. In the present study, the levels of phosphorylated ATM, CHK2, and p53 were remarkably elevated in mouse testes and GC-1 cells after Ti₃C₂ nanosheet exposure. As a protein located at a key point in many regulatory networks, p53 plays a critical role in maintaining cellular genomic integrity and various cellular stress responses through cell cycle control. P53 participates in cell cycle regulation by controlling the checkpoint at which proliferating cells migrate from G1 to S phase of the cell cycle. This forces cells with DNA damage to stop in the G1 phase and perform DNA repair [46]. p53 is a major transcriptional regulator of p21, and DNA damage and oxidative stress upregulate p53 activity, leading to p21 expression [47]. Previous studies have confirmed that p21 binds to the cell cycle protein, Cyclin D/Cdk4 in a complex, resulting in cell cycle arrest in the G1 phase [48]. In this study, our results showed that the Ti₃C₂ nanosheet-exposed GC-1 cells were blocked in the G1/G0 phase. P53 plays a key role in cell fate determination and prevents expansion of damaged and mutated cells. When DNA damage exceeds a certain threshold or is too severe to be repaired, p53 promotes apoptosis by activating the mitochondrial apoptotic pathway. p53 plays an important role in inducing apoptosis. p53 specifically activates its downstream targets and induces apoptosis [49]. Several studies have shown that Bcl-2 family and mitochondrial proteins are associated with p53-dependent apoptosis [50]. Following DNA damage, PUMA is triggered in a p53-dependent manner [51]. PUMA plays a core role in mitochondria-mediated cell death by reacting with all known members of the anti-apoptotic Bcl-2 family [52]. In the mitochondrial apoptotic pathway, an increase in the ratio of Bax to Bcl-2 activates caspase9. Upon activation, caspase9 activates caspase3, as a key initiator of apoptosis [26]. In the present study, Ti₃C₂ nanosheets promoted apoptosis in testicular tissue and GC-1 cells via the activation of the mitochondrial apoptotic pathway. Thus, the ATM/p53 signaling pathway is a potential molecular mechanism by which spermatogenesis is impaired by Ti₃C₂ nanosheets.

To the best of our knowledge, this is the first study that explores the effects of Ti₃C₂ nanosheet exposure on male reproductive toxicity. In vivo experiments indicated that Ti₃C₂ nanosheet exposure inhibited the proliferation of mice spermatogonia, resulting in reduced sperm quality. In vitro experiments showed that Ti₃C₂ nanosheet exposure induced cell cycle arrest and inhibited the proliferation of GC-1 cells. In addition, Ti₃C₂ nanosheet exposure induced excessive ROS production, DNA damage, genotoxic stress, and apoptosis in GC-1 cells and mouse testes. These findings reveal that ROS-induced DNA damage mediates Ti₃C₂ nanosheet exposure-induced inhibition of spermatogonia proliferation and decreases sperm quality in mice. This provides new experimental evidence on the mechanism of toxicity of Ti₃C₂ nanosheets on the male reproductive system.

EDCs	Endocrine-disrupting chemicals
ROS	Reactive oxygen species
TEM	Transmission electron microscopy
SEM	Scanning electron microscopy
AFM	Atomic force microscopy
IHC	Immunohistochemistry
IF	Immunofluorescence
MDA	Malondialdehyde
SOD	Superoxide dismutase
ELISA	Enzyme-linked immunosorbent assay
DCFH-DA	2,7-Dichlorodihydrofluorescein diacetate
LMPA NMPA	Low melting point agarose Normal melting point agarose
CCK-F	Cell fluorescence counting kit
DEGs WB	Differentially expressed genes Western Blot
PVDF	Polyvinylidene fluoride
SD	Standard deviation
H&E	Hematoxylin and eosin

Authors’ contributions

Yanqing Geng and Xueqing Liu were responsible for experimental design and data interpretation. Yang Wei and Ruilin Bao performed experiments. Le Hu synthesized the nanomaterials. Xuemei Chen, Yixian Wen, Yingxiong Wang and Mao Qin provided technical support. Yang Wei, Yue Zhang wrote the manuscripts and Xueqing Liu, Yue Zhang acquired the funding.

Funding

This work was supported by the Postdoctoral Program of Chongqing Natural Science Foundation (No. CSTB2022NSCQ-BHX0660) and the Natural Science Foundation of Gansu (No. 21JR1RA108).

Availability of data and materials

All the original data are available upon reasonable request for correspondence authors.

Ethics approval and consent to participate

This work was approved by the ethics committee of Chongqing Medical University.

Consent for publication

All authors read and agree this version be published.

Competing interests

All authors declared no conflict of interests.

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Ti3C2 nanosheets disrupt spermatogenesis in male mice via alterations in proliferation and apoptosis of spermatogonia mediated by the ATM/p53 signaling pathway

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