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. 2026 Feb 27;12(9):eaea3591. doi: 10.1126/sciadv.aea3591

Epididymal macrophage senescence contributes to sperm motility decrease upon environmental stress

Xin-Run Wang 1,2,†, Hao Li 3,1,2,†, Yong-Wei Xiong 1,2,†, Zhi Yuan 1,2, Xin-Yi Cheng 1,2, Yi-Fan Hu 1,2, Xiao-Jin He 4, Long-Long Fan 1,2, Ye-Xin Luo 1,2, Jian Sun 1,2, Cheng-Fang Sun 1,2, Xin-Xin Zhang 1,2, Yan Wang 5, Qi Lu 1,2, Kai-Wen Wang 1,2, Lu-Lu Tan 1,2, Xiao-Han Jiang 5, Hua-Long Zhu 1,2, De-Xiang Xu 1,2, Hua Wang 1,2,6,*
PMCID: PMC12947854  PMID: 41758944

Abstract

Environmental stressors–induced male infertility has become a major public health issue. Sperm motility is the key to conception. However, the immunological mechanism for environmental stress–induced sperm motility reduction remains unknown. Our experiments find that three classical environmental stressors, including lead, cadmium, and mercury, are the key heavy metals reducing sperm motility. We reveal that environmental stress induces epididymal macrophage senescence, whereas the clearance of above cells significantly alleviates environmental stress–reduced sperm motility. In vivo and in vitro experiments further demonstrate that environmental stress induces macrophage senescence via down-regulating Foxo3 expression. Mechanistically, environmental stress increases the epididymal Foxo3 N6-methyladenosine level and inhibits the FTO. Overexpression of Fto in vitro and S-adenosylhomocysteine supplementation in vivo alleviates environmental stress–induced macrophage senescence. Together, these results suggest that epididymal macrophage senescence contributes to sperm motility decrease upon environmental stress. Clearing senescent epididymal macrophages will provide a previously unknown strategy for preventing male infertility.

Eliminating senescent epididymal macrophages can significantly alleviate the decline in sperm motility upon environmental stress.

INTRODUCTION

Infertility affects about one in six people globally, with male factors accounting for about 30% (1, 2). One study from China showed that even more than 50% of male infertility is caused by asthenospermia (3). Study has found that the disease burden of asthenospermia-associated male infertility continues to rise in recent years (3). Environmental stress is one of the main factors causing sperm motility decline (4, 5). Heavy metals are typical environmental stressors. With the rapid development of the economy and society, environmental heavy metals pollution has become a global problem (6). Heavy metal pollutants from electronic waste and other sources are nonbiodegradable and can accumulate in the environment for a long time, thereby disrupting the ecological balance of aquatic and terrestrial environments (6–9). It is estimated that the global annual emissions of heavy metals such as cadmium (Cd) amount to as much as 1 million tons, and the annual emissions of mercury (Hg) are ~1500 tons (6). Humans commonly face combined heavy metal exposure in the environment (10, 11). Previous animal studies have initially found that the combined heavy metals exposure such as Cd and lead (Pb), Cd, and copper (Cu) can reduce sperm motility in recent years (12–14), but the key heavy metals that impair sperm motility remain controversial. The mechanism of combined heavy metal exposure on sperm motility is still unclear.

There is a unique immune microenvironment in the epididymis to ensure the normal maturation of sperm (15–18). The delicate balance between sperm tolerance and immune defense protects sperm from the influence of stressors, and the disruption of the epididymal immune microenvironment is an important cause of male infertility (15–18). Characteristics of epididymal immune microenvironment disorders include immune cell subsets imbalance and proinflammatory cytokines release (19). It is worth noting that inflammation and disorders in the immune microenvironment are also important characteristics of immunosenescence (20). Otherwise, senescent immune cells produce proinflammatory senescence-associated secretory phenotypes (SASPs), thereby exacerbating inflammation (20, 21). Environmental stress has been confirmed as a common trigger of immunosenescence (22, 23). A previous study has found that environmental stress activates immune imbalance to induce epididymal inflammation and damage reproductive ability (24). Mononuclear phagocytes (MPs) are the most crucial immune cell subsets in epididymis (17, 18). Among them, macrophages that release proinflammatory cytokines during inflammation are the main culprits promoting the disorder of the epididymal microenvironment (17, 18). Above all, we infer that environmental stress causes sperm motility decline possibly by inducing epididymal macrophage senescence to release proinflammatory cytokines.

Epigenetic alterations are one of the important causes of senescence (25), N6-methyladenosine (m6A) modification is the most common mRNA modification in eukaryotes, which is a vital epigenetic regulatory mechanism (26). The m6A modification is conferred by demethylases (ALKBH5 and FTO) and methylases (METTL3 and METTL4), which regulates transcription stability, degradation, and translation (27). m6A has been confirmed to regulate cellular senescence, such as a previous study has found that demethylase inhibition promotes cell senescence (28). Previous studies from our group found that environmental stress increases m6A levels in testis (29–31). We hypothesize that environmental stress may induce epididymal macrophage senescence through m6A modification.

In this study, we screened the key heavy metals that can damage sperm motility through our cross-sectional study. Subsequently, we constructed a sperm motility decline mouse model with the key heavy metal mixture and cleared senescent epididymal macrophages to clarify the role of senescent macrophages in environmental stress–induced sperm motility decrease. Furthermore, we used S-adenosylhomocysteine (SAH) supplementation in vivo and Fto overexpression in vitro experiments to confirm the role of m6A modification mechanism in environmental stress–induced macrophage senescence.

RESULTS

Urinary Pb, Hg, and Cd are negatively correlated with sperm motility

To explore the association between environmental heavy metal exposure and sperm motility, a cross-sectional study was designed on the basis of participants from the First Affiliated Hospital of Anhui Medical University (fig. S1). The characteristics of participants in the cross-sectional study are shown in Table 1. The levels of heavy metals in the urine samples were detected by inductively coupled plasma mass spectrometry (ICP-MS), which benefits from its unique advantages such as the ability to simultaneously detect multiple heavy metals and high sensitivity (Fig. 1A and tables S1 and S2). The results of quantile-based g-computation model showed that Pb, Hg, and Cd were the top three metals negatively correlated with sperm motility (Fig. 1B). Restricted cubic spline analysis indicated that the levels of Pb, Hg, and Cd were all nonlinear related to sperm motility (Fig. 1, C to E). Linear regression analysis was further performed to evaluate the effect of Pb, Hg, and Cd exposure on sperm motility (Fig. 1, F to H). The results showed that urinary Pb (β = −6.77, P = 0.007) and Cd (β = −5.07, P = 0.028) had a significant negative effect on sperm motility, while Hg could not be excluded (β = −8.36, P = 0.051). It is worth noting that multiple linear regression analysis indicated that the negative association between Pb, Hg, and Cd and sperm motility had a combined effect (β = −2.06, P = 0.001) (Fig. 1I). The analysis of Bayesian kernel machine regression (BKMR) model also yielded similar results (Fig. 1J). Together, these results confirmed that urinary Pb, Hg, and Cd are negatively correlated with sperm motility and have a combined effect.

Table 1. Characteristics of participants in the cross-sectional study on male reproductive health.

BMI, body mass index; PR, progressive motility.

Characteristics Overall (n = 170) Qualified sperm motility (n = 122) Unqualified sperm motility (n = 48) P value
Age (years) 30.58 ± 4.01 30.40 ± 4.31 31.02 ± 3.12 0.367
BMI (kg/m2) 25.51 ± 4.08 25.17 ± 4.05 26.35 ± 4.09 0.089
Height (cm) 174.45 ± 5.16 174.62 ± 5.13 174.00 ± 5.26 0.480
Weight (kg) 77.62 ± 13.01 76.76 ± 13.08 79.81 ± 13.00 0.172
Sperm motility, PR (%) 40.79 ± 17.16 48.81 ± 12.56 20.40 ± 7.79 <0.001
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Fig. 1. Urinary Pb, Hg, and Cd are negatively correlated with sperm motility.

Fig. 1.

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(A to J) Two hundred sixty-two male urine samples were collected from the First Affiliated Hospital of Anhui Medical University, and 170 urine samples and sperm report data were lastly included for the cross-sectional study. Informed consent was obtained from the participants for use of all data and biological samples. (A) Workflow for the cross-sectional study. Created in BioRender. Wang, S. (2025) https://BioRender.com/87n1yql. (B) The result of quantile-based g-computation (QGCOMP) model between urinary heavy metals and sperm motility. (C to E) Restricted cubic splines (RCSs) of Cd, Pb, and Hg with sperm motility. (F to I) Results of unadjusted and adjusted regression of Cd, Pb, and Hg with sperm motility, with body mass index (BMI) and age as covariates. (J) Associations between heavy metal mixture and sperm motility calculated by Bayesian kernel machine regression (BKMR) model. CI, confidence interval; ppb, parts per billion; PR, progressive motility.

Environmental stress decreases sperm motility in mice

To further verify that the combined effect of Pb, Hg, and Cd reduced sperm motility, the mouse model of single and combined exposure to Pb, Hg, and Cd was established (Fig. 2A). As shown in Fig. 2 (B and C), combined heavy metal exposure significantly reduced sperm motility and progressive motility in mice. Subsequently, the mouse experiment with different exposure doses and time was performed (Fig. 2D). The result on hematoxylin and eosin (H&E) staining indicated that the combined heavy metal exposure impaired the lumen of each segment of the epididymis (Fig. 2E and fig. S2, K and L). Figure 2 (F to K) confirmed that the decrease of sperm motility was more obvious with the increase of heavy metal exposure time or dose. Collectively, the above results indicate that environmental stress decreases sperm motility in mice.

Fig. 2. Environmental stress declines sperm motility in mice.

Fig. 2.

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(A to C) The male mice were exposed to Cd (CdCl2, 15 mg/liter), Pb [Pb(C2H3O2)2·3H2O, 100 mg/liter], Hg (HgCl2, 1 mg/liter), and high-dose metal mixture [HM; CdCl2, 15 mg/liter; Pb(C2H3O2)2·3H2O, 100 mg/liter; and HgCl2, 1 mg/liter] for 20 weeks through drinking water, respectively. (A) Experimental designs of the single heavy metal exposure in male mice. (B) Sperm motility. (C) Progressive motility. (D to K) The male mice were exposed to low-dose metal mixture group [LM; CdCl2, 1.5 mg/liter; Pb(C2H3O2)2·3H2O, 10 mg/liter; and HgCl2 0.1 mg/liter], medium-dose metal mixture group [MM; CdCl2, 5 mg/liter, Pb(C2H3O2)2·3H2O, 30 mg/liter; and HgCl2, 0.3 mg/liter], and high-dose metal mixture group [HM; CdCl2, 15 mg/liter; Pb(C2H3O2)2·3H2O, 100 mg/liter; and HgCl2, 1 mg/liter] or vehicle for 10 weeks through drinking water, respectively. (D) Experimental designs of combined heavy metal exposure in male mice. (E) Representative hematoxylin and eosin (H&E) staining images of the mouse epididymal caput. Black arrowhead denotes lumen destruction. (F) Representative images of sperm motility tests. Green curves mark motile sperm, and red dots mark stationary sperm. (G) Sperm motility. (H) Progressive motility. (I) Straight-line velocity. (J) Amplitude of lateral head displacement. (K) Straightness. All data were expressed as the means ± SEM; n = 10 to 15 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the NC group. VSL, straight-line velocity; ALH, amplitude of lateral head displacement; STR, straightness.

Environmental stress induces epididymal macrophage senescence

To investigate the mechanism of the reduction in sperm motility induced by environmental stress, markers of senescence in epididymis were evaluated. As shown in Fig. 3 (A and B), the SPiDER-β-Gal–positive areas of epididymis caput in the high-dose metal mixture (HM) group were markedly increased compared to that in the negative control (NC) group. Moreover, environmental stress significantly up-regulated the SASP gene Il-6 expression (Fig. 3C). The expression of senescence-related proteins p16 and p21 was also up-regulated in HM-exposed epididymis (Fig. 3, D to F). The effect of environmental stress on epididymal macrophages was then analyzed by flow cytometry. Results indicated that environmental stress caused a significant reduction in the number of macrophages in the epididymis (Fig. 3, G and H) but no significant effects on dendritic cells and monocytes (fig. S3, D and E). Further multiplex immunohistochemistry (mIHC) results showed that p16+ F4/80+ cells in mouse epididymis were significantly increased after HM exposure (Fig. 3I and fig. S4C). RAW264.7 cells (a commonly used macrophage cell line) were selected as an in vitro model to expose the mixture of heavy metals. As shown in Fig. 3J and fig. S4 (A and B), the expression of senescence-related proteins p16 and p21 was significantly up-regulated in medium-dose metal mixture (MM)–exposed RAW264.7 cells. Combined heavy metal exposure significantly increased the cellular senescence-associated β-Galactosidase (SA-β-Gal)–positive areas (Fig. 3K and fig. S4D). In addition, environmental stress increased the protein level of interleukin-6 (IL-6) in both the epididymis and RAW26.7 cells (fig. S4, E and F). In a word, our results show that environmental stress induces epididymal macrophage senescence.

Fig. 3. Environmental stress induces epididymal macrophage senescence.

Fig. 3.

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(A to I) The male mice were exposed to low-dose metal mixture group [LM; CdCl2, 1.5 mg/liter; Pb(C2H3O2)2·3H2O, 10 mg/liter; and HgCl2, 0.1 mg/liter], medium-dose metal mixture group [MM; CdCl2, 5 mg/liter; Pb(C2H3O2)2·3H2O, 30 mg/liter; and HgCl2, 0.3 mg/liter], and high-dose metal mixture group [HM; CdCl2, 15 mg/liter; Pb(C2H3O2)2·3H2O, 100 mg/liter; and HgCl2, 1 mg/liter] or vehicle for 10 weeks through drinking water, respectively. (A and B) Representative images and analysis of SPiDER-β-Gal fluorescence staining for the detection of senescent cells in mouse epididymis. Orange represents SPiDER-β-Gal, and blue represents 4′,6-diamidino-2-phenylindole (DAPI; nucleus). (C) The mRNA levels of Il-1α, Il-1β, Il-6, Il-18, Mmp1, Mmp3, and Mmp9 in mouse epididymis were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). (D to F) The protein expressions of p16 and p21 in mouse epididymis were measured using immunoblotting. (G and H) Representative images and analysis of F4/80+ CD11b+ cells in the mouse epididymis using flow cytometry. (I) Representative images showing p16+ F4/80+ cells in the mouse epididymis, as detected by multiplex immunohistochemistry (mIHC). (J and K) The RAW264.7 cells were exposed to metal mixture for 0, 6, 12, and 24 hours, respectively. (J) The protein expressions of p16 and p21 were measured using immunoblotting. (K) Representative SA-β-gal staining images and analysis. All data were expressed as the means ± SEM; n = 3 to 9 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the NC or 0-hour group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FITC, fluorescein isothiocyanate; h, hours.

Depletion of senescent macrophages protects environmental stress–decreased sperm motility

To explore the effect of senescent macrophages on HM-decreased sperm motility, we depleted macrophages in mice with clodronate liposomes and adoptively transferred bone marrow–derived macrophages (BMDMs) from young mice (Fig. 4A). As shown in Fig. 4 (C to E) and fig. S5 (E to G), the sperm motility in the HM and macrophage (MФ) group was markedly restored compared to that in the HM group. The expression of p16, p21 and IL-6 was down-regulated in the HM and MФ group compared to that in the HM group (Fig. 4, F to H, and fig. S5I). As expected, mIHC results confirmed that depletion of senescent macrophages significantly decreased p16+ F4/80+ cells in mouse epididymis (Fig. 4I and fig. S5H). As above, depletion of senescent macrophages protects environmental stress–decreased sperm motility.

Fig. 4. Depletion of senescent macrophages protects environmental stress–declined sperm motility.

Fig. 4.

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(A to I) The male mice were exposed to HM or NC through drinking water for 10 weeks. After 5 weeks of HM exposure, mice were injected with clodronate liposomes twice on 3 days. Twenty-four hours later, bone marrow–derived macrophages (BMDMs) were injected into mice via the tail vein and named the NC, MФ, HM, and HM and MФ groups, respectively. (A) Experimental design of the macrophage depletion and adoptive transfer in male mice. Created in BioRender. Wang, S. (2025) https://BioRender.com/rzppeoh. (B) Representative images of F4/80+ CD11b+ cells in mouse epididymis were detected by flow cytometry. (C) Representative images of sperm motility tests. Green curves mark motile sperm, and red dots mark stationary sperm. (D) Sperm motility. (E) Progressive motility. (F to H) The protein expressions of p16 and p21 in mouse epididymis were measured using immunoblotting. (I) Representative images of p16+ F4/80+ cells in mouse epididymis. All data were expressed as the means ± SEM; n = 3 to 8 per group. *P < 0.05 and **P < 0.01 compared to the NC group. #P < 0.05 and ##P < 0.01 compared to the HM group. DMEM, Dulbecco’s modified Eagle’s medium; M-CSF, macrophage colony-stimulating factor.

Environmental stress induces epididymal macrophage senescence by inhibiting Foxo3 expression

To investigate the mechanism of macrophage senescence induced by environmental stress, the key aging-related genes in epididymis were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Result indicated that environmental stress significantly inhibited the expression of the antiaging gene Foxo3 (Fig. 5, A and B). Environmental stress significantly down-regulated the protein expression of FOXO3 in mouse epididymis and RAW264.7 cells (Fig. 5, C to F). To further explore the role of Foxo3 in environmental stress–induced macrophage senescence, overexpressed plasmid transfection of Foxo3 in vitro was performed. Results confirmed that overexpressed Foxo3 significantly restored the environmental stress–up-regulated expression of p16 and p21 in RAW264.7 cells (Fig. 5, G to I). In addition, overexpressed Foxo3 significantly alleviated the environmental stress–increased cellular SA-β-gal–positive area (Fig. 5, J and K). In addition, environmental stress increased the protein level of IL-6 in RAW26.7 cells (Fig. 5L). Above all, our results show that environmental stress induces epididymal macrophage senescence by inhibiting Foxo3 expression.

Fig. 5. Environmental stress induces epididymal macrophage senescence by inhibiting Foxo3 expression.

Fig. 5.

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(A to D) The male mice were exposed to low-dose metal mixture group [LM; CdCl2, 1.5 mg/liter; Pb(C2H3O2)2·3H2O, 10 mg/liter; and HgCl2, 0.1 mg/liter], medium-dose metal mixture group [MM; CdCl2, 5 mg/liter; Pb(C2H3O2)2·3H2O, 30 mg/liter; and HgCl2, 0.3 mg/liter], and high-dose metal mixture group [HM; CdCl2, 15 mg/liter; Pb(C2H3O2)2·3H2O, 100 mg/liter; and HgCl2, 1 mg/liter] or vehicle for 10 weeks through drinking water, respectively. (A and B) The mRNA levels of Sirt3, Apoe, Ins, Klotho, Ppagc1a, Igf1, Nqo1, Stat1, Becn1, Tert, Prkaa2, Igf1r, Stat3, Tgfb2, Tgfb3, Prkaa1, Tfam, Terc, Gpx1, Sirt1, Foxo3, Stat6, and Tgfb1 were assessed by RT-qPCR. (C and D) The FOXO3 protein expressions in epididymis were measured using immunoblotting. (E and F) The RAW264.7 cells were exposed to metal mixture for 0, 6, 12, and 24 hours, respectively. The FOXO3 protein expressions were measured using immunoblotting. (G to L) The RAW264.7 cells were exposed to metal mixture for 24 hours after pcDNA-Foxo3 pretreatment. (G to I) The protein expressions p16 and p21 were measured using immunoblotting. (J and K) Representative SA-β-gal staining images and analysis. (L) The level of IL-6 in the cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). All data were expressed as the means ± SEM; n = 3 to 6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the NC or control (Ctrl) (cell) group. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the HM or MM (cell) group. h, hours.

Environmental stress inhibits Foxo3 expression through FTO-mediated m6A modification

Given that m6A modification plays a crucial role in regulating transcription, stability, degradation, and translation of transcripts, the total RNA m6A modification level was detected in mouse epididymis. As seen in Fig. 6A, environmental stress significantly increased the total m6A level in the epididymis. Methylated RNA immunoprecipitation (MeRIP)–qPCR assay confirmed that the m6A level at the predicted site 3 of Foxo3 mRNA in the HM group was enhanced compared to that in the NC group (Fig. 6, B and C). Meanwhile, environmental stress significantly increased the level of METTL14 and reduced the level of FTO but did not change the level of ALKBH5 and METTL3 in mouse epididymis. Further, results confirmed that environmental stress significantly reduced the level of FTO but did not change the level of METTL14 in RAW264.7 cells (Fig. 6, D to H). To further explore the role of the FTO-mediated m6A level in the reduced FOXO3 expression, overexpressed plasmid transfection of Fto was performed in RAW264.7 cells. Enzyme-linked immunosorbent assay (ELISA) and SA-β-gal staining showed that overexpressed Fto significantly alleviated the environmental stress–elevated cellular SA-β-gal–positive area and the cellular IL-6 level (Fig. 6I and fig. S6, A and B). Overexpressed Fto significantly alleviated the environmental stress–up-regulated expression of p16 and p21 and restored the expression of FOXO3 protein in RAW264.7 cells (Fig. 6, J to M). Equally important, we treated RAW264.7 cells with actinomycin and subjected them to combined heavy metal exposure. The results indicated that, compared to that in the MM group, the degradation rate of Foxo3 mRNA in the overexpressed MM and pcDNA-Fto group was significantly reduced (fig. S6C). In summary, our results show that environmental stress inhibits Foxo3 expression through FTO-mediated m6A modification.

Fig. 6. Environmental stress inhibits Foxo3 expression through FTO-mediated m6A modification.

Fig. 6.

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(A to E) The male mice were exposed to low-dose metal mixture group [LM; CdCl2, 1.5 mg/liter; Pb(C2H3O2)2·3H2O, 10 mg/liter; and HgCl2, 0.1 mg/liter], medium-dose metal mixture group [MM; CdCl2, 5 mg/liter; Pb(C2H3O2)2·3H2O, 30 mg/liter; and HgCl2, 0.3 mg/liter], and high-dose metal mixture group [HM; CdCl2, 15 mg/liter; Pb(C2H3O2)2·3H2O, 100 mg/liter; and HgCl2, 1 mg/liter] or vehicle for 10 weeks through drinking water, respectively. (A) The total RNA m6A levels in epididymis. (B) The three m6A modification sites in Foxo3 mRNA were obtained using sequence-based N6-methyladenosine (m6A) modification site predictor (SRAMP). (C) The m6A level of epididymal Foxo3 mRNA was assessed by MeRIP-qPCR. (D and E) The METTL14, METTL3, FTO, and ALKBH5 protein expressions in mouse epididymis were measured using immunoblotting. (F to H) The RAW264.7 cells were exposed to metal mixture for 0, 6, 12, and 24 hours, respectively. The METTL14 and FTO protein expressions were measured using immunoblotting. (I to M) The RAW264.7 cells were exposed to metal mixture for 24 hours after pcDNA-Fto pretreatment. (I) The level of IL-6 in the cell culture supernatant was detected by ELISA. (J to M) The FOXO3, p16, and p21 protein expressions were measured using immunoblotting. All data were expressed as the means ± SEM; n = 3 to 5 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the NC or control (Ctrl) (cell) group. #P < 0.05 and ##P < 0.01 compared to the HM or MM (cell) group. h, hours.

SAH supplementation alleviates environmental stress–induced epididymal macrophage senescence

To further investigate the effect of m6A modification on Foxo3 expression, SAH was injected into mice by intraperitoneal injection (Fig. 7A). As shown in Fig. 7 (B to D) and fig. S7 (D to F), the sperm motility in the HM and SAH group was markedly restored compared to that in the HM group. Correspondingly, SAH supplementation significantly alleviated the environmental stress–up-regulated expression of p16, p21, IL-6, and the level of total m6A in the mouse epididymis (Fig. 7, E to J, and fig. S7, G and J). It also restored the expression of FOXO3, FTO protein in the mouse epididymis, and Foxo3 mRNA in F4/80+ cells (Fig. 7, E, H, and I, and fig. S7, H and I). As expected, the mIHC results showed that p16+ F4/80+ cells in the mouse epididymis were significantly decreased in the HM and SAH group compared to that in the HM group (Fig. 7, J and K). As above, SAH supplementation alleviates epididymal macrophage senescence and sperm motility decrease.

Fig. 7. SAH supplementation alleviates environmental stress–induced epididymal macrophage senescence.

Fig. 7.

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(A to K) The male mice were exposed to HM or NC through drinking water. Among them, half of the mice were intraperitoneally injected with SAH (10 mg/kg) every Monday, Wednesday, and Friday for 10 weeks and named the NC, SAH, HM, and HM and SAH groups, respectively. (A) Experimental design of the SAH supplementation in male mice. (B) Sperm motility. (C) Progressive motility. (D) Representative images of sperm motility tests. Green curves mark motile sperm, and red dots mark stationary sperm. (E to I) The protein of p16, p21, FOXO3, and FTO in mouse epididymis expressions were measured using immunoblotting. (J and K) Representative images and analysis of p16+ F4/80+ cells in mouse epididymis using mIHC. All data were expressed as the means ± SEM; n = 3 to 11 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the NC group. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the HM group.

DISCUSSION

The immunological mechanism for environmental stress–induced sperm motility reduction remains unknown. In this study, we explored this issue through a series of experiments and analyses on humans, animals, and cells. We firstly report that epididymal macrophage senescence plays a key role in environmental stress–evoked sperm motility reduction. Our data support that FTO-mediated Foxo3 m6A modification regulates senescence phenotype of epididymal macrophages. We found that clearance of epididymal senescent macrophages is necessary to prevent the decline in sperm motility induced by environmental stress, which represents a previously unknown potential therapeutic target in the male reproduction field.

Heavy metals, as typical environmental stressors, have been confirmed to reduce sperm motility when exposed in combination (12–14). However, the key heavy metals that damage sperm motility remain controversial. In this research, results showed that Pb, Cd, and Hg were the key heavy metals that correlated negatively with sperm motility according to the BKMR model. In line, these three heavy metals were listed in the top 10 in the list of 275 toxic substances issued by the United States Agency for Toxic Substances and Disease Registry in 2022 (32). Our human investigation found that Pb, Cd, and Hg have a negative combined effect on sperm motility. Animal experiment further demonstrated that the combined exposure to Pb, Cd, and Hg reduced sperm motility in mice. Previous studies have also found that single exposure to heavy metals can reduce sperm motility (33, 34). In brief, environmental stress decreases sperm motility.

The maintenance of the epididymal immune microenvironment homeostasis is crucial for the normal capacitation and maturation of sperm (16, 19). MPs are the guardian of the epididymal immune microenvironment (17, 35). Our results showed that environmental stress reduced the number of macrophages in the epididymis, while the number of dendritic cells and monocytes did not change. The change in the number of immune cells is one of the vital manifestations of immune senescence (20, 36). We found that environmental stress significantly increased the number of senescent macrophages in the epididymis. The accumulation of senescent immune cells is an important feature of immunosenescence, and senescent immune cells also secrete large amounts of SASP to further damage tissues (20, 36, 37). We found that environmental stress significantly increased the levels of IL-6 in the epididymis and RAW264.7. Previous studies have found that IL-6 is significantly negatively correlated with sperm motility (38, 39). Further, we found that the clearance of senescent epididymal macrophages significantly alleviated the reduction in sperm motility caused by environmental stress. Analogously, clearance of senescent macrophages has been found to ameliorate tumorigenesis in lung cancer (40). Above all, environmental stress decreases sperm motility by inducing epididymal macrophage senescence.

Multiple biological mechanisms have been confirmed to be involved in regulating the process of cellular senescence (41–45), involving 12 hallmarks (42). We focused on the main key genes of the abovementioned 12 hallmarks. RT-qPCR screened that environmental stress significantly down-regulates the antisenescence gene. FOXO3 is a transcription factor that regulates various cellular functions in a cell-type–dependent manner, including aging, longevity, and cell cycle progression (46, 47). It is worth noting that FOXO3 also shows antiaging potential in the regulation of inflammation and that inflammation is one of the important characteristics of cellular senescence (42, 48, 49). FOXO3 can directly bind to the promoter region of the IL-6 gene and inhibit its transcription (50). Previous studies have shown that the release of FOXO3 inhibition can significantly down-regulate IL-6 expression (48, 50, 51). For instance, in the lipopolysaccharide-induced inflammation model, the activation of FOXO3 can alleviate liver IL-6 (51). Overexpression of Foxo3 significantly alleviated macrophage senescence and IL-6 activation caused by environmental stress. In line with our findings, a previous study found that the reduction of Foxo3 levels leads to the proinflammatory senescence phenotype in gut macrophages (52). The increase in proinflammatory cytokine levels has been confirmed to induce a decrease in sperm motility (53–55). In short, environmental stress induces epididymal macrophage senescence by suppressing the expression of Foxo3, which releases IL-6 and reduces sperm motility.

m6A is the most abundant mRNA modification in eukaryotes and regulates transcription stability, degradation, and translation (56). It has been reported that m6A modification is associated with cellular senescence (27), and our data suggested that environmental stress increases the Foxo3 m6A level in the epididymis. Meanwhile, the expression of demethylase FTO was significantly down-regulated after environmental stress. FTO is known to specifically remove m6A modification, affecting the stability and translation efficiency of mRNA (57). Further, we found that overexpression of Fto significantly alleviates Foxo3 inhibition and macrophage senescence phenotypes induced by environmental stress. A previous study has also found that FTO inhibition promotes granulosa cells senescence (28). SAH is a classic m6A inhibitor (58, 59). Our data showed that SAH supplementation alleviated environmental stress–induced epididymal macrophage senescence. Above all, environmental stress inhibits Foxo3 expression through FTO downregulation–mediated m6A modification.

In summary, environmental stress induces epididymal macrophage senescence through FTO-mediated Foxo3 m6A modification and ultimately reduces sperm motility by releasing the inflammatory cytokine IL-6 (Fig. 8). Clearance of senescent epididymal macrophages will provide a previously unknown strategy for targeted prevention of male infertility.

Fig. 8. Epididymal macrophage senescence contributes to sperm motility decrease upon environmental stress.

Fig. 8.

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Graphical abstract of epididymal macrophage senescence contributes to sperm motility decrease upon environmental stress. Created in BioRender. Wang, S. (2025) https://BioRender.com/p8x4loa.

MATERIALS AND METHODS

Cross-sectional study

The cross-sectional study was designed on the basis of participants from the First Affiliated Hospital of Anhui Medical University. Two hundred sixty-two participants were enrolled in the cross-sectional study between June and September 2023 after informed consent was provided. We excluded the participants with idiopathic infertility and other specific diseases (n = 22). Participants with missing data were also excluded (n = 50). Other exclusion criteria included age older than 45 or younger than 18 (n = 20). Ultimately, 170 individuals were included in our analysis (Fig. 1). Participant information is provided in Table 1. Urinary metals (Pb, Hg, Cd, Mo, Ca, Fe, AI, As, Cu, and V) were all measured by ICP-MS. The characteristics of urinary heavy metal were shown in table S1. The data of sperm motility were obtained from the laboratory reports of the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University, and reports were based on WHO fifth edition laboratory standards (approval no. PJ2023-04-12).

Measurements of metals

The frozen urine samples were allowed to thaw slowly at normal atmospheric temperature. Following centrifugation, the supernatants were extracted and diluted with a mixed reagent consisting of Triton X-100 and HNO3. The epididymis tissues were stored at −80°C until further analysis. Before digestion, the frozen tissues were thawed on ice, and each sample was accurately weighed into precleaned digestion vessels. The tissue samples were digested using a closed-vessel microwave digestion system (ETHOS UP, Milestone Inc., Italy). A reagent blank, containing only the acids without any tissue, was processed simultaneously through the entire preparation procedure to account for any potential background contamination. Then, the urinary and epididymal levels of 12 selected metals, namely, Cd, Fe, Ca, Cu, Mo, V, Pb, Hg, AI, Mg, Ba, and As, were measured using ICP-MS (Nexion 350X, PerkinElmer, USA). To ensure accuracy, standard reference materials were analyzed as quality control samples every 20 to 25 specimens. The spiked recovery rates of the 12 metals fell within the range of 93.18 to 114.82%. Limits of detection, within-day precision, and between-day precision were shown in (table S2). Urine metal concentrations were corrected for specific gravity to normalize the urinary essential metals levels.

Animal treatments

Male C57BL/6J mice were obtained from Beijing Vital River. All animals were acclimated to standard conditions (ambient temperature of 20° to 25°C, air humidity of 50 to 60%, and a 12-hour light/dark cycle) for 1 week. During this period, all mice were fed a standard diet and provided with reverse osmosis water. All experimental procedures for mouse feeding and testing complied with the standard operating protocols established by the Experimental Animal Ethics Committee of Anhui Medical University (ethical approval no. LLSC20220640).

Experiment 1: To investigate the impact of combined heavy metal exposure on the sperm motility of mice, referring to the heavy metal administration design in previous studies (60, 61), we established the heavy metal exposure model in male C57BL/6J mice. Five-week-old male C57BL/6J mice were exposed to CdCl2 (15 mg/liter), Pb(C2H3O2)2·3H2O (100 mg/liter), HgCl2 (1 mg/liter), and HM for 20 weeks through drinking water, respectively. Meanwhile, other 5-week-old male C57BL/6J mice were exposed to low-dose metal mixture group, MM group, and HM group or vehicle for 10 weeks through drinking water, respectively. Dose information is shown in table S5.

Experiment 2: To explore the effect of senescent macrophages on HM-decreased sperm motility, five-week-old C57BL/6J male mice were exposed to HM or NC through drinking water for 10 weeks. All the mice were divided into four groups: NC, MФ, HM, and HM and MФ. After 4 weeks of HM exposure, 7-week-old C57BL/6J male mice were sourced from Beijing Vital River. Following a week of acclimatization, 8-week-old male mice were euthanized, and BMDMs removed from the tibia were cultured in a cell incubator for 4 days. Then, MФ and HM and MФ group mice were injected with clodronate liposomes twice on 3 days. In addition, 24 hours later, BMDMs collected from cell culture dishes were injected into mice through the tail vein (62, 63).

Experiment 3: To investigate the effect of m6A modification on macrophage senescence, five-week-old C57BL/6J male mice were exposed to HM or NC through drinking water. All the mice were divided into four groups: NC, SAH, HM, and HM and SAH. Among them, mice in the SAH and HM and SAH groups were intraperitoneally injected with SAH (10 mg/kg) every Monday, Wednesday, and Friday, and the remaining mice were injected with normal saline for 10 weeks (59, 64, 65).

Cell culture

RAW264.7 cells, a mouse macrophage line, were obtained from the Chinese Academy of Science. RAW264.7 cells were cultured with Dulbecco’s modified Eagle’s medium high-glucose medium and 10% fetal bovine serum. All cell cultures were performed at 37°C and containing 5% CO2. RAW264.7 cells were incubated with various concentrations of the metal mixture. The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to measure cell viability at different time points, aiming to determine the impact of the metal mixture on the growth of RAW264.7 cells. To explore the effect of combined heavy metal exposure on RAW264.7 cell senescence, RAW264.7 cells were treated with metal mixture for 0, 6, 12, and 24 hours. To explore the effect of Foxo3 on RAW264.7 cell senescence induced by combined heavy metal exposure, metal mixture was used to stimulate the cells after pcDNA-Foxo3 pretreatment. To explore the effect of Fto on RAW264.7 cell senescence induced by combined heavy metal exposure, metal mixture was used to stimulate the cells after pcDNA-Fto pretreatment. Dose information is shown in table S6.

Western blotting

The target tissues or cells were completely homogenized with lysate, and its concentration was adjusted to the specified level. Subsequently, an equal volume of sample and marker were loaded into the prefabricated SDS–polyacrylamide gel electrophoresis wells. Electrophoresis is initially performed at a constant voltage of 55 V until the marker bands are clearly separated. Then, the electrophoresis continued at a constant voltage of 110 V until completion. Afterward, the proteins were transferred onto the polyvinylidene difluoride membrane at a constant current of 200 mA for a duration of 2 to 3.5 hours. Next, the membrane was blocked using 5% skim milk prepared with tris-buffered saline with tween 20 (TBST) for 1.5 hours. Following this, it was incubated with the primary antibody for 1.5 hours and then with the secondary antibody for 1.5 hours. Last, the enhanced chemiluminescence (ECL) reagent was added for incubation, and the resulting image was visualized using a chemiluminescence imager. All results used glyceraldehyde-3-phosphate dehydrogenase as the internal reference.

Flow cytometric

After the cells were counted and their density was adjusted, the epididymal single-cell suspension was divided into different tubes, with ~5 × 106 cells in each tube. Then, it was stained with APC-F4/80, AF488-CD11b, SB600-CD11c, and PECy7-Ly6c for 30 min at 4°C. Flow cytometric detection was carried out using a CytoFlex flow cytometry system (Beckman Coulter, USA), and the analysis was conducted with FlowJo V10.8.1 software.

Multiplex immunohistochemistry

Following fixation in 4% paraformaldehyde, the epididymides were cut into 15-mm paraffin sections. Subsequently, the sections underwent dewaxing and rehydration procedures. Endogenous peroxidase was quenched using 3% H2O2. Antigen retrieval of the sections was then carried out with tris-EDTA buffer (pH 9.0). Next, the sections were incubated with F4/80 antibody (diluted 1:1000) overnight at 4°C. On the following day, after incubation with the horseradish peroxidase (HRP)–labeled secondary antibody, the sections were treated with fluorescein isothiocyanate–labeled tyramide working solution for conjugation.

Afterward, another round of antigen retrieval was performed on the sections using tris-EDTA buffer (pH 9.0). Then, the sections were incubated with p16 antibody (at a dilution of 1:1000) overnight at 4°C. The next day, after incubation with the HRP-labeled secondary antibody, Cy3-labeled tyramide working solution was used to combine with the sections. The cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). Confocal microscopy (Thunder, Leica) was used to take photographs. The number of p16+F4/80+ cells in the epididymides was calculated using ImageJ software.

Histological examination

The epididymides were first fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Sections with a thickness of 5 μm were stained using H&E. All images were obtained through an optical microscope (Leica DM4B, Germany).

Assessment of sperm motility

Mouse sperm motility was assessed using an automatic sperm analyzer (Hamilton Thorne, USA). The left epididymis of the mouse was cut into slices and put into a prewarmed medium. Subsequently, the medium was incubated at 37°C for 5 min until the sperm was fully released. In the last step, the sperm suspension was diluted with the prewarmed medium, and 50 μl of the suspension was dropped onto a slide for parameter evaluation.

Isolation of total RNA and real-time RT-qPCR

The epididymis was lysed on ice with TRIzol. Subsequently, RNA was extracted using the chloroform method. Then, the RNA was reverse transcribed into cDNA by means of the Roche reverse transcription kit. The mRNA level of the target gene was measured via LightCycler 480 real-time fluorescent qPCR (Roche), with 18S as a control. The primer sequences used are shown in table S7.

Assessment of m6A levels

In every well of the test plate, 80 μl of the binding solution along with 2 μl of the diluted positive control was added. After that, 200 ng of RNA was put into each well. The substances in the well were gently blended and then incubated at 37°C for 90 min. Following this, the wells were washed three times using 150 μl of the cleaning solution. Next, 50 μl of the diluted antibody capture solution was added to each well and incubated at room temperature for 60 min. After the incubation, each well was washed three times with 150 μl of the cleaning solution. Then, each well was incubated with 50 μl of the diluted antibody solution at room temperature for 30 min. Afterward, the wells were washed four times with 150 μl of the cleaning solution. Subsequently, 50 μl of the diluted antibody enhanced solution was added, and the wells were incubated at room temperature for 30 min. Last, the wells were rinsed five times. To measure the absorbance of the sample at 450 nm, 100 μl of the developer was added to each well for 10 min. This made it possible to calculate the m6A level in the RNA.

MeRIP–polymerase chain reaction

The Arraystar Seq-Star poly(A) mRNA Isolation Kit (Maryland, USA) was used to isolate RNA from total epididymal RNA. As a control, 10% of the purified mRNA was reserved. Next, 2 μg of an anti-m6A polyclonal antibody (Synaptic System, 202003) was added to the reaction system, and the mixture was incubated at 4°C for 2 hours. Then, Dynabeads M-280 Sheep anti-Rabbit immunoglobulin G (Thermo Fisher Scientific, 11204D) magnetic beads were introduced, and the mixture was allowed to react at 4°C for another 2 hours. After being washed with buffer and wash buffer, elution buffer was added to the mixture, which was then incubated at 50°C for 30 min. Eventually, chloroform was used to extract the m6A-modified mRNA.

Enzyme-linked immunosorbent assay

The quantity of IL-6 in mouse epididymis and cells was measured using the mouse IL-6 ELISA kit, respectively. Assays were implemented according to the manufacturer’s specifications. To detect epididymal IL-6, the epididymis was accurately weighed and homogenized in 1 ml ice phosphate-buffered saline (PBS) with protease inhibitor. After grinding and centrifugation at 6000g for 15 min, the supernatant was collected for IL-6 detection. To detect IL-6 in the cell culture medium supernatant, the medium was collected and centrifuged before the RAW264.7 cells were collected and then stored at −80°C.

Macrophage depletion and adoptive transfer

Ten-week-old C57BL/6J male mice in the MФ and HM and MФ groups were injected with 200 μl of clodronate liposomes twice on 3 days first. Twenty-four hours later, BMDMs from 8-week-old male C57BL/6J mice were differentiated and matured in the cell culture incubator for 4 days using macrophage colony-stimulating factor, and, then, 1 × 106 BMDMs were injected into the MФ and HM and MФ group mice via the tail vein.

SA-β-gal staining

The β-gal kit (Solarbio, China) was used to perform the SA-β-gal staining. Specifically, after fixed for 15 min, RAW264.7 cells were rinsed three times with PBS, followed by staining with the working staining solution at 37°C for 24 hours. The percentage of SA-β–positive area in glass coverslips was quantitatively analyzed.

SPiDER-β-Gal staining

Optimal cutting temperature compound (OCT)–embedded tissue sections were first left to thaw at room temperature. Subsequently, they were incubated at 37°C for 30 min to improve the adhesion of the tissue to the glass slide. The sections were then rinsed with 1× PBS and incubated with the SPiDER-β-Gal staining solution (SG04, Dojindo) at 4°C throughout the night. Images of the stained tissues were obtained by using a ZEISS LSM8800 confocal microscope and were quantified with the help of ImageJ software. When conducting costaining, the immunofluorescent antibody staining procedures were implemented after the SPiDER-β-Gal staining, following the methods described in previously published protocols.

RNA stability assay

Actinomycin D (0.5 μg/ml) inhibited transcription in RAW264.7 cells. The mRNA expression of Foxo3 was detected by RT-qPCR after 0, 3, and 6 hours of heavy metal treatment.

Fluorescence in situ hybridization

The simultaneous detection of Foxo3 mRNA and F4/80 protein was performed using the RNA Fluorescence In Situ Hybridization Kit (SA-Biotin System, GenePharma) combined with standard immunofluorescence protocols. All steps were carried out under ribonuclease-free conditions. Images were acquired using a confocal laser scanning microscope (Thunder, Leica) equipped with appropriate lasers and filters for DAPI, Alexa Fluor 488, and cyanine 3. Sequential scanning was applied to avoid cross-talk between channels. Multiple fields of view from at least three independent biological replicates were captured using an oil immersion objective. Image processing and analysis, including background subtraction, channel merge, and cell counting, were performed using ImageJ software. Colocalization analysis was conducted to identify F4/80-positive macrophages expressing Foxo3 mRNA.

Statistical analysis

Quantified data were expressed as the means ± SEM. The data were statistically analyzed using SPSS 23.0 software. The sample size (n) represents the number of biologically independent experiments. To determine the statistical significance of the differences, parametric tests [Student’s t test or analysis of variance (ANOVA)] and nonparametric tests (Mann-Whitney U test or Kruskal-Wallis H test) were carried out. In the case of ANOVA, after the variance homogeneity test, the Bonferroni or Tamhane’s T2 method was further used. P < 0.05 was regarded as statistically significant.

Acknowledgments

We thank the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University for the valuable assistance provided for the cross-sectional study and thank the Anhui Provincial Center for Disease Control and Prevention for the valuable assistance offered for the animal experiments. We thank the Center for Scientific Research of Anhui Medical University for valuable help in our experiment. The main flowcharts were created with BioRender.com.

Funding:

This work was supported by National Key Research and Development Program of China (2022YFC2702904), Graduate Research and Practice Innovation Program of Anhui Medical University (YJS20240050), Anhui Provincial University Scientific Research Project (2024AH050731), Research Funds of Center for Big Data and Population Health of IHM (JKS2022016), Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0542300), and Doctoral Research Start-up Project of Wannan Medical College (WYRCQD2025019).

Author contributions:

Conceptualization: X.-R.W., X.-J.H., and H.W. Methodology: Y.-F.H., X.-H.J., X.-J.H., Y.W., Investigation: Y.-F.H., L.-L.F., Y.-X.L., J.S., X.-X.Z., Q.L., K.-W.W., L.-L.T., D.-X.X., and H.-L.Z. Visualization: X.-R.W., H.L., and Z.Y. Supervision: H.W. and C.-F.S. Writing—original draft: X.-R.W., H.L., Y.-W.X., and Z.Y. Funding acquisition: Y.-W.X., H.L., and H.W. Software: X.-Y.C. Validation: X.-Y.C. Writing—review and editing: X.-Y.C., X.-J.H., and H.W. Resources: X.-J.H. and C.-F.S. Formal analysis: X.-J.H.

Competing interests:

The authors declare that they have no competing interests.

Data, code, and materials availability:

All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. The cross-sectional study data are saved in data S1. Code for the data analyzes used for this study is available on Zenodo (https://doi.org/10.5281/zenodo.17605431). This study did not generate new reagents or physical materials.

Supplementary Materials

The PDF file includes:

Figs. S1 to S7

Tables S1 to S8

Legend for data S1

sciadv.aea3591_sm.pdf (1.9MB, pdf)

Other Supplementary Material for this manuscript includes the following:

Data S1

sciadv.aea3591_data_s1.zip (45KB, zip)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S7

Tables S1 to S8

Legend for data S1

sciadv.aea3591_sm.pdf (1.9MB, pdf)

Data S1

sciadv.aea3591_data_s1.zip (45KB, zip)

Data Availability Statement

All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. The cross-sectional study data are saved in data S1. Code for the data analyzes used for this study is available on Zenodo (https://doi.org/10.5281/zenodo.17605431). This study did not generate new reagents or physical materials.

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