ASSESSMENT OF VITRIFICATION-INDUCED STRUCTURAL DAMAGES IN MATURED BOVINE OOCYTES BY RAMAN MICROSPECTROSCOPY

Vitrification induces ultrastructural and structural damages in different oocyte structures such as membrane, zona pellucida (ZP) and cytoplasm.1 Recently Raman Microspectroscopy (RMS) has been used to assess the changes caused by vitrification/ warming of in vitro matured ovine oocytes.2 RMS is a noninvasive technique for studying the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. Aim of the present study was to investigate the structural modifications of ZP and cytoplasm of vitrified/warmed in vitro matured bovine oocytes by RMS. Abattoir derived in vitro matured oocytes (n=171) were denuded and divided in three experimental groups: control untreated oocytes (CTR), oocytes only exposed to vitrification/warming solutions (CPA) and vitrified/warmed oocytes (VITRI). Oocytes were exposed/vitrified in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3 M). RMS analysis was carried out on CTR matured oocytes (after 22h) and on CPA and VITRI oocytes at different incubation times (0,1,2,3 and 4h) after exposure/warming. The analysis of the large spectral date set, acquired by RMS were performed by Principal Component Analysis (PCA). Our experimental outcomes suggest that vitrification induce a transformation of the protein secondary structure from the α-helices to the b-sheet form in the ZP, while lipids tend to assume a more ordered configuration. Both effects induce a mechanical stiffening of ZP, which could explain the reduced fertility of vitrified oocytes with respect to the untreated ones. Intriguingly, these transformations present a certain degree of reversibility, which renders vitrified oocytes more similar to untreated cells after 2h of warming at room temperature.