Examining the Relationship Between Polystyrene Microplastics and Male Fertility: Insights From an In Vivo Study and In Vitro Sertoli Cell Culture

Abstract

Background: While polystyrene microplastics (PS-MPs) are emerging as potentially significant health threats, linked to cancer and reproductive dysfunction, their precise effects on human health remain largely unknown. We aimed to investigate the underlying mechanisms promoting microplastic-induced damage in the reproductive system.

Methods: Thirty C57BL/6 male mice were randomly allocated into six equal-sized groups. Mice were exposed to fluorescent PS-MPs (5 µm, < 18%, green) at a dose of 1 and 3 mg/dL via oral gavage for 28 and 56 days, respectively (control, 0 mg/dL). The presence of antibodies and inflammatory and oxidative stress markers were evaluated using western blotting. Sperm analysis was also performed. Mouse testis Sertoli TM4 cells were divided into two groups: control (medium only) and PS-MPs (medium containing, 1,000 μg/mL) groups and cultured in vitro for 1, 24, 48, or 72 hours. The cells were cultured in a Ham's F12: Dulbecco's Modified Eagle Medium medium with 0.25% fetal bovine serum at 37°C with humidified atmosphere of 5% carbon dioxide in the air. Protein analyses for interleukin (IL)-6, IL-10, NADPH-oxidase (NOX)-2, NOX-4, hypoxia-inducible transcription factor (HIF)-2α, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β were performed using western blotting.

Results: The testes were evaluated after 28 and 56 days of exposure. Varying sizes of PS-MPs were detected in the testes (ranging from 5.870 to 7.768 µm). Significant differences in sperm concentration, motility, and the proportion of normal sperm were observed between the two groups. An increase in TGF-β, HIF-2α, and NOX-4 levels was observed using western blot analysis. However, no dose-dependent correlations were observed between the two groups. In vitro evaluation of the PS-MPs group displayed PS-MP penetration of the lumen of Sertoli cells after 1 hour. Further PS-MP aggregation within Sertoli cells was observed at 24, 48, and 72 hours. A significant increase in inflammatory protein expressions (IL-10, TGF-β, MCP-1, IL-6, TNF-α, and HIF-2α) was observed through western blotting, although oxidative agents did not show a significant increase.

Conclusion: PS-MPs induced reproductive dysfunction in male mice provide new insights into PS-MPs-associated toxicity in mammals.

Keywords: Infertility; Male; Mice; Microplastics; Sertoli Cells.

Fig. 1. Morphology and characterization of primary cell cultures and colony formation of Sertoli cells exposed to PS-MPs.

PS-MPs = polystyrene microplastics.

Fig. 2. Western blot analysis of antibody expression in Sertoli cells. The protein level of (A) NOX-2, (B) NOX-4, (C) IL-10, (D) IL-6, (E) TGF-β, (F) MCP-1, (G) TNF-α, and (H) HIF-2α.

NOX = NADPH-oxidase, PS-MPs = polystyrene microplastics, IL = interleukin, TGF-β = transforming growth factor-β, MCP-1 = monocyte chemoattractant protein-1, TNF-α = tumor necrosis factor-α, HIF-2α = hypoxia-inducible transcription factor-2α.

Fig. 3. Fluorescent PS-MPs in the testes of mice after exposure for 28 days.

PS-MPs = polystyrene microplastics, 1M = 1 month.

Fig. 4. Western blot analysis of antibody expression in mice testes (TGF-β, MCP-1, TNF-α, NOX-2, NOX-4, NOX-5, and HIF-2α).

PS-MPs = polystyrene microplastics, 1M = 1 month, 2M = 2 months, TGF-β = transforming growth factor-β, MCP-1 = monocyte chemoattractant protein-1, TNF-α = tumor necrosis factor-α, NOX = NADPH-oxidase, HIF-2α = hypoxia-inducible transcription factor-2α.