# In vitro exposure to polystyrene microplastics exerts oocyte toxicity through cumulus cells damage in the sheep model

## TL;DR

Exposure to polystyrene microplastics harms sheep oocytes by damaging surrounding cumulus cells, reducing fertility potential.

## Contribution

Demonstrates that microplastics impair sheep oocyte maturation via cumulus cell toxicity, revealing a novel reproductive risk.

## Key findings

- Polystyrene microplastics at 50-100 μg/mL reduced sheep oocyte maturation and developmental potential.
- Cumulus cells showed increased apoptosis and reduced antioxidant gene expression with microplastic exposure.
- Oocyte meiotic spindle and actin abnormalities increased regardless of microplastic concentration.

## Abstract

In recent years, the widespread environmental presence of microplastics (MPs) has raised major concerns regarding animal and human health, including potential risks to reproductive function and offspring.

This study aimed to evaluate the effects of increasing concentrations of polystyrene MPs (PS-MPs; 0, 5, 50, or 100 μg/mL) on ovine cumulus–oocyte complexes (COCs) during in vitro maturation (IVM). Fluorescent microspheres were used for uptake assessment into COCs and cumulus cells (CCs) monolayers, whereas non-fluorescent PS-MPs were employed to evaluate potential toxic effects induced on CCs and oocytes.

As regards CCs, increased PS-MPs uptake was highlighted at the highest exposure concentration (100 μg/mL), whereas no significant differences were observed in oocyte intracellular fluorescence intensity, compared to the control. The bioaccumulation increment in CCs monolayers was already visible after 6 h, both at 5 and 100 μg/mL, and confirmed at 24 h. The real-time PCR analysis in CCs revealed significant reductions in the expression levels of genes involved in antioxidant defense and alterations in those implicated in apoptosis. Finally, the TUNEL assay revealed a dose-dependent increase in CCs apoptotic index. Consequently, PS-MPs exposure impaired oocyte meiosis resumption by significantly reducing the maturation rates, particularly at 50 and 100 μg/mL, whereas no effects were observed at 5 μg/mL. Oocyte intracellular reactive oxygen species levels were significantly increased at all concentrations, whereas no differences in mitochondrial membrane potential were detected. The percentages of oocytes with abnormal configurations of meiotic spindle and cortical F-actin were found to be significantly increased, regardless of concentration. Finally, the cleavage rate was significantly reduced in oocytes exposed to 50 μg/mL, whereas no differences were found in the blastocyst rate at both 50 and 5 μg/mL.

In conclusion, in vitro exposure of sheep COCs to PS-MPs during IVM reduced oocyte quality and developmental potential through alterations induced in the CCs, which turned out to be the main target of these environmental contaminants.