Characterization of the zona pellucida of bovine oocytes during in vitro maturation using Raman spectroscopy

Abstract

The plasma membrane of mammalian oocytes is surrounded by an extracellular matrix composed mainly of glycoproteins, called the zona pellucida (ZP). This matrix is composed of glycoproteins and it has previously been shown that there is a correlation between its morphology and the degree of maturation of the oocytes. ZP plays an important role in fertilization, since it is the primary receptor for sperm, more precisely the sialic acid present in glycoproteins. The aim of this study was to determine by Raman spectroscopy the chronological order of the main structural changes experienced by ZP glycoproteins during in vitro maturation (IVM).ZP spectra were taken from immature bovine oocytes (ZP-0H) and oocytes that were in vitro matured for 6, 18 and 22h (ZP-6H, ZP-18H and ZP-22H). The spectra of the ZP-6H group showed that the intensities of the sugar bands decreased while the intensities of the protein signals increased considerably, with respect to the ZP-0H spectrum. When comparing the spectra of ZP-18H with those of the ZP-6H group, a marked increase in the intensity of the specific signal of sialic acid was observed; this modification suggests a greater exposure of this molecule (relevant in the fertilization of bovine oocytes) in the last stage of IVM. Finally, the ZP-22H spectra showed weak sugar bands compared to the other 3 groups while the sialic acid signal continued to stand out. A principal component analysis (PCA), a multivariate statistical calculation, was implemented for the signals that presented the greatest spectral difference. The dispersion graph obtained by PCA revealed that the oocytes of the ZP-18H and ZP-22H groups are grouped in the sector of the graph that represents the presence of an intense sialic acid signal. These results suggest that during the IVM process there was a structural reorganization of the ZP glycoproteins and that sialic acid could be useful to discriminate between the different states of oocyte maturation.