Plin4 exacerbates cadmium-decreased testosterone level via inducing ferroptosis in testicular Leydig cells

Abstract

Strong evidence indicates that environmental stressors are the risk factors for male testosterone deficiency (TD). However, the mechanisms of environmental stress-induced TD remain unclear. Based on our all-cause male reproductive cohort, we found that serum ferrous iron (Fe²⁺) levels were elevated in TD donors. Then, we explored the role and mechanism of ferroptosis in environmental stress-reduced testosterone levels through in vivo and in vitro models. Data demonstrated that ferroptosis and lipid droplet deposition were observed in environmental stress-exposed testicular Leydig cells. Pretreatment with ferrostatin-1 (Fer-1), a specific ferroptosis inhibitor, markedly mitigated environmental stress-reduced testosterone levels. Through screening of core genes involved in lipid droplets formation, it was found that environmental stress significantly increased the levels of perilipins 4 (PLIN4) protein and mRNA in testicular Leydig cells. Further experiments showed that Plin4 siRNA reversed environmental stress-induced lipid droplet deposition and ferroptosis in Leydig cells. Additionally, environmental stress increased the levels of METTL3, METTL14, and total RNA m6A in testicular Leydig cells. Mechanistically, S-adenosylhomocysteine, an inhibitor of METTL3 and METTL14 heterodimer activity, restored the abnormal levels of Plin4, Fe²⁺ and testosterone in environmental stress-treated Leydig cells. Collectively, these results suggest that Plin4 exacerbates environmental stress-decreased testosterone level via inducing ferroptosis in testicular Leydig cells.

Keywords: Environment stress; Ferroptosis; Lipid droplet; Perilipins; Testosterone deficiency; m6A modification.

Graphical abstract

Fig. 1

The association between elevated serum ferrous iron (Fe²⁺) and all-cause testosterone deficiency. (A–I) Serum was collected from 310 men aged 20–40 years. (A) Flowchart of the study population. (B) Visual representation of Spearman correlations among various indices and correlation strength indicated by the color depth and rectangle size. A blank grid denotes a P-value of more than 0.05. (C) Male age. (D) Adult males were divided into two groups based on serum testosterone (T) levels: normal testosterone group (T > 10.4 nmol/L) and testosterone deficiency group (T < 10.4 nmol/L). (E) Ferrous iron (Fe²⁺). t = 4.942, P < 0.0001. (F) Triglycerides (TG). t = 4.371, P < 0.0001. (G) HDL cholesterol (HDL-C). t = 3.260, P = 0.0012. (G and H) LDL cholesterol (LDL-C) and total cholesterol (TC) levels (P > 0.05). (J and K) Urine was collected from 200 men aged 20–40 years. (J) urine Fe levels. (P > 0.05). (K) Urine Cd content. t = 2.262, P = 0.0248. N = 317 for human serum samples and N = 200 for human urine samples. *P＜0.05, **P < 0.01 and ns P > 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Environmental stress suppresses testosterone synthesis in mouse testicular Leydig cells. (A–I) Male mice received an intraperitoneal injection of 1 mg/kg CdCl₂ daily for three days. Mouse sera and testes were collected. (A) Diagram illustrating the experimental design for the animal experiment. (B) Mouse body weight. (C) Testicular coefficient. (D) Testosterone level in serum. (E–G) The protein expression levels of 3β-HSD and StAR were assessed using immunoblotting in mouse testes. (H and I) Representative images and quantitative results of immunofluorescence staining for StAR, with DAPI highlights the nuclei. Scale bar: 100 μm. (J–M) Mouse testicular Leydig cells (TM3) were exposed to CdCl₂ (20 μM) for 0–24 h. (J) Flowchart of TM3 cell culture and CdCl₂ treatment. (K) Testosterone levels in cell media. (L and M) The protein expression levels of 3β-HSD and StAR were assessed using immunoblotting in TM3 cells. Data are presented as mean ± S.E.M. (n = 3–8 per group). *P < 0.05, **P < 0.01.

Fig. 3

Environmental stress induces ferroptosis in mouse testicular Leydig cells. (A–M) Male mice received an intraperitoneal injection of 1 mg/kg CdCl₂ daily for three days. Mouse sera and testes were collected. (A) Representative H&E staining images and their magnified versions of testes. (B) Quantitative analysis of nuclei in mouse testicular Leydig cells. (C) Representative transmission electron microscopy (TEM) images and their magnified versions in mouse testicular Leydig cells. (D) Mitochondrial area for TEM images. (E) Total iron content in sera. (F) Ferrous iron content in sera. (G) Malondialdehyde (MDA) content in testes. (H) Glutathione (GSH) content in testes. (I–K) The protein expression levels of GPX4 and 4-HNE were evaluated using immunoblotting in mouse testes. (L and M) Representative images and quantitative results of immunofluorescence staining for GPX4, with DAPI marking the nuclei. Scale bar: 20 μm. (N–R) Mouse testicular Leydig cells (TM3) were exposed to CdCl₂ (20 μM) for 0–24 h. (N and O) Exemplary images and quantitative results of C11-BODIPY staining for the detection of lipid peroxidation. Scale bar: 200 μm. (P–R) The protein expression levels of GPX4 and 4-HNE were assessed using immunoblotting in TM3 cells. Data are shown as mean ± S.E.M. (n = 3–4 per group). *P < 0.05, **P < 0.01.

Fig. 4

Environmental stress reduces testosterone levels by inducing ferroptosis in mouse testicular Leydig cells. All male mice were intraperitoneally injected with 2 mg/kg ferrostatin-1, and 30 min later, 1 mg/kg CdCl₂ was injected once daily for three days. Mouse sera and testes were collected. (A) Representative transmission electron microscopy (TEM) images and their magnified images of mouse testicular Leydig cells. (B) Mitochondrial area for TEM images. (C) Malondialdehyde (MDA) content in testes. (D) Glutathione content in testes. (E–G) The protein expression levels of GPX4 and 4-HNE were assessed using immunoblotting in mouse testes. (H and I) Immunohistochemical detection of 4-HNE positive areas in the testes. (J) Serum testosterone levels. (K–M) The protein expression levels of 3β-HSD and StAR were evaluated using immunoblotting in mouse testes. Data are shown as mean ± S.E.M. (n = 3–15 per group). *P < 0.05, **P < 0.01.

Fig. 5

Environmental stress upregulates Plin4 expression and induces lipid droplets deposition in mouse testicular Leydig cells. (A–K) Male mice received an intraperitoneal injection of 1 mg/kg CdCl₂ daily for three days. Mouse sera and testes were collected. (A) Representative transmission electron microscopy (TEM) images and their magnified images of mouse testicular Leydig cells. (B) Quantitative results of lipid droplets (LDs) in TEM images. (C) Total cholesterol (TC) in serum. (D) Triglycerides (TG) in serum. (E) HDL cholesterol (HDL-C) in serum. (F) LDL cholesterol (LDL-C) in serum. (G and H) Quantitative results of oil red O staining and LDs area ratio in testes. Scale bar: 100 μm. (I) Heatmap of RT-qPCR detection of Plin1, Plin2, Plin3, and Plin4 mRNA levels in the testes. (J and K) The protein expression levels of PLIN4 were assessed using immunoblotting in mouse testes. (L–P) Mouse testicular Leydig cells (TM3) were exposed to CdCl₂ (20 μM) for 0–24 h. (L and M) Representative images and quantitative results of BODIPY staining for the number of LDs in TM3 cells. Scale bar: 150 μm. (N) Heatmap of RT-qPCR analysis of Plin1, Plin2, Plin3, Plin4 and Plin5 mRNA levels in TM3 cells. (O and P) The protein expression levels of PLIN4 were evaluated using immunoblotting in TM3 cells. Data are shown as mean ± S.E.M. (n = 3–10 per group). *P < 0.05, **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Environmental stress induces ferroptosis via enhancing Plin4-mediated lipid droplet deposition in mouse testicular Leydig cells. TM3 cells were exposed with CdCl₂ (20 μM) for 0–24 h, with or without Plin4 siR. (A–D) In TM3 cells, BODIPY fluorescence staining was used to detect the number of lipid droplets, while C11-BODIPY fluorescence staining was employed for lipid peroxidation detection. (A) Exemplary images of BODIPY fluorescence staining. Scale bar: 150 μm. (B) Exemplary images of C11-BODIPY fluorescence staining. Scale bar: 150 μm. (C) Quantitative results of lipid droplets (LDs). (D) Quantification for lipid peroxidation. (E–G) The protein expression levels of GPX4 and 4-HNE were assessed using immunoblotting. (H and I) The protein expression levels of PLIN4 were evaluated using immunoblotting. Data are shown as mean ± S.E.M. (n = 3–4 per group). *P < 0.05, **P < 0.01.

Fig. 7

S-adenosylhomocysteine (SAH) supplementation alleviates environmental stress-increased Plin4 and Fe²⁺ levels in mouse testicular Leydig cells. (A–D) Mouse testicular Leydig cells (TM3) were exposed to CdCl₂ (20 μM) for 0–24 h. (A) The total RNA m6A levels. (B–D) The protein expression levels of METTL3 and METTL14 were assessed using immunoblotting. (E–K) TM3 cells were treated with CdCl₂ (20 μM) for 24 h, with or without 10 μM S-adenosylhomocysteine (SAH). (E) Measurement of total RNA m6A levels. (F and G) The protein expression levels of PLIN4 were assessed using immunoblotting. (H–J) Exemplary immunofluorescence staining images and quantitative results of PLIN4 and lipid droplets, with nuclei marked by DAPI. Scale bar: 150 μm. (K and L) Exemplary images of FerroOrange fluorescence staining and quantitative results of ferrous iron (Fe²⁺), with nuclei marked by Hoechst 33342. Scale bar: 150 μm. (M) Testosterone levels in cell media. Data are shown as mean ± S.E.M. (n = 3–4 per group). *P < 0.05, **P < 0.01.