# Effect of Hydroxyapatite Nanoparticles on the Ultrastructure, Developmental Competence, and Expression of ZP3, MFN1, and NPM2 in Vitrified Bovine GV Oocytes

## TL;DR

Using hydroxyapatite nanoparticles with lower cryoprotectant levels improves the survival and development of frozen bovine egg cells.

## Contribution

This study shows that hydroxyapatite nanoparticles combined with reduced cryoprotectants enhance bovine oocyte cryopreservation.

## Key findings

- Combining HA nanoparticles with lower CPA concentrations increased mitochondrial membrane potential and developmental competence in vitrified oocytes.
- VS1-HA group showed higher maturation, cleavage, and blastocyst rates compared to controls.
- ZP3, MFN1, and NPM2 gene expression was upregulated in oocytes treated with HA nanoparticles.

## Abstract

The cryopreservation efficiency of bovine germinal vesicle (GV) oocytes remains relatively low. Although hydroxyapatite (HA) nanoparticles have been investigated for use in cryopreservation, the effects of combining HA nanoparticles with reduced concentrations of permeable cryoprotective agents (CPAs) on bovine GV oocytes remain unclear. This study therefore examined the synergistic effects of HA nanoparticles and permeable CPAs on the ultrastructure, developmental competence, and gene expression of bovine GV oocytes following vitrification. The results demonstrated that the combination of HA nanoparticles with lower concentrations of permeable CPAs increased mitochondrial membrane potential (MMP) level, enhanced developmental competence, alleviated vitrification-induced ultrastructural damage, and upregulated the expression of the related genes, ZP3, MFN1, and NPM2, in vitrified bovine GV oocytes. These findings provide experimental evidence supporting the development of nanomaterial-based cryopreservation strategies for bovine oocytes and may contribute to improvements in reproductive technologies and germplasm conservation.

To improve the vitrification efficiency of bovine germinal vesicle (GV) oocytes, the use of hydroxyapatite (HA) nanoparticles as a novel cryopreservation additive represents a promising approach. This study aimed to investigate the effects of HA nanoparticles and permeable cryoprotective agents (CPAs) on the ultrastructure, developmental competence, and gene expression of bovine GV oocytes following vitrification. Oocytes were vitrified in vitrification solutions containing HA nanoparticles of different sizes (20, 40, or 60 nm) and concentrations (0.01%, 0.05%, or 0.1%) to determine the optimal conditions based on survival rate, mitochondrial membrane potential (MMP) level, and developmental competence. Subsequently, the synergistic effects of HA nanoparticles and permeable CPAs (VS: 20% EG + 20% DMSO; VS1: 17.5% EG + 17.5% DMSO) were further evaluated. The optimal treatment (40 nm 0.05% HA nanoparticles) significantly increased MMP level, and improved developmental competence compared with the vitrified control group (p < 0.05). Among the vitrified groups, vitrified oocytes in the VS1-HA group (combining HA nanoparticles with reduced concentrations of permeable CPAs) exhibited the highest MMP level (1.89), maturation rate (50.39%), cleavage rate (27.07%), and blastocyst rate (10.53%) (p < 0.05). Ultrastructural analysis further revealed that the VS1-HA group maintained more intact zona pellucida structures and showed reduced mitochondrial swelling compared with the vitrified control group. Moreover, the expression levels of genes associated with zona pellucida formation (ZP3), mitochondrial fusion (MFN1), and chromatin remodeling (NPM2) were significantly upregulated in the VS1-HA group relative to the vitrified control group. Overall, these findings indicate that the combination of HA nanoparticles with lower concentrations of permeable CPAs enhances MMP level, alleviates vitrification-induced ultrastructural damage, and upregulates the expression of key developmental genes, thereby improving the developmental competence of vitrified bovine GV oocytes.