The conditions of oxidative stress that can be generated during physiological events, such as post-ovulatory aging and reproductive aging, as well as by the PMA procedures, can seriously degrade the oocyte developmental competence. The ability to identify predictive markers of oxidative stress using noninvasive techniques may provide a useful diagnostic tool for the assessment of oocyte quality. The aim of the present work is to evaluate the potential of Raman spectroscopy (RMN) as a tool to detect molecular modifications induced by aging-related oxidative stress in mouse oocytes. The research was carried out using CD-1 mice; at the age of 4 to 8 weeks (young mice) and 48 to52 weeks (old mice), females were superovulated and oocytes at metaphase II stage were recovered from oviducts. The MII oocytes from young animals were divided into 3 experimental groups: (A) young oocytes, processed immediately after collection; (B) in vitro aged oocytes, cultured in vitro for 10 h before processing; (C) oxidative-stressed oocytes, exposed to 10 mM hydrogen peroxide for 2 min before processing (oocytes with a fully oxidized status). Oocytes from reproductively old mice were referred to as old oocytes (D). After fixation in 3.7% paraformaldehyde, oocytes (n = 10 for each group) were immersed in a 50-µL drop of PBS on quartz windows and analyzed using a Bruker Senterra confocal Raman microscope. Measurements were performed by recording 3 line scans across the oocyte with 5-µm step size, totalling 32 point spectra for each oocyte. The spectra were statistically analyzed using principal component analysis. Principal component analysis showed a clear discrimination between the spectra of young oocytes (A), in vitro aged oocytes (B), oxidative-stressed oocytes (C), and old oocytes (D). Compared with the control group (A), B, C, and D groups revealed significant differences in the bands attributable to lipid components; specifically, a reduction in the intensity of the peaks at 1653 and 1602 cm–1 (stretching of the C = C bond) and of the peaks at 1485, 1462, 1437, 1396 cm–1 (CH3-CH2 vibration) was recorded. With regard to the protein component, spectra of B, C, and D groups showed modifications in the intensities of peaks 1297 and 850 cm–1, which refer respectively to amide III and to CNC symmetric stretching compared with group A. Principal component analysis also revealed an overlap between the spectra of in vitro aged oocytes, old oocytes, and oxidative-stressed oocytes, suggesting that the molecular damage caused by ageing has similar characteristics to chemically induced oxidative damage. In conclusion, the results of our study show that Raman spectroscopy is a valuable tool for the identification of molecular biochemical markers of oxidative stress. This technique could represent a highly informative method of investigation to evaluate the oocyte quality in response to various stress conditions (in vitro maturation, aging, cryopreservation, and so on) that may negatively affect its potential development.
Visualization of in vitro deep blood vessels using principal component analysis based laser speckle imaging