97 Raman microspectroscopy to characterize secondary structure of zona pellucida proteins of in vitro produced and in vivo derived bovine embryos

The zona pellucida (ZP) is a multilaminar, highly glycosylated glycoprotein structure composed of long filaments of ZP2 and ZP3 crosslinked by ZP1. When sperm fuses with the oocyte it triggers a biochemical change termed “zona hardening,” which is characterised by the release of cortical granules into the perivitelline space, resulting in the crosslinking of ZP glycoproteins to form a modified secondary structure with increased β-sheets and decreased α-helices. The crosslinking of zona proteins causes the ZP to become refractory to sperm binding and penetration and increases resistance to dissolution by a variety of agents. It has been hypothesised that in vitro-produced (IVF) embryos have zona proteins that are not as tightly crosslinked as in vivo-derived (IVO) embryos, making them a less efficient barrier to viruses and more susceptible to polyspermy. In this study Raman microspectroscopy (RMS) was used to assess the composition and structure of the ZP glycoproteins and compare the α-helices/β-sheet ratio on IVO and IVP embryos. Bovine embryos from various collection and cryopreservation methods were donated for analysis. For warming, embryos were thawed in straws and then washed in a warming media of 1 M sucrose and incubated for 1 min, transferred to dilution media of 0.5 M sucrose and incubated for 3 min, and then incubated in IVC media overnight. Measurements were then performed on the Renishaw InVia Raman Microscope; 785 nm excitation wavelength at 50 mW of power and a water-immersion 50× objective. Embryos were placed on a reflective metal slide and then fully immersed in 10 mL of phosphate-buffered saline within a culture plate. A cross section of the ZP was selected at random and focused underneath the laser for spectral acquisition, with 10 spectra taken from each embryo (n = 3 per treatment). Spectra from each embryo were then baseline-corrected, vector-normalized, and then divided into treatment groups where the average spectrum per group was calculated. The zona protein components were analysed by focusing on the amide III (1230–1300 cm⁻¹) spectral range, where the respective peak frequencies of the α-helix and β-sheet bands were found to be at 1267 cm⁻¹ and 1239 cm⁻¹. Spectral processing was done using the Origin software, and a best-fitting procedure was applied to quantify the α-helix/β-sheet ratio by dividing the area of the α-helix band by the area of the β-sheet band. The mean α-helix/β-sheet (1267/1239) ratio for the IVP group was 1.061 ± 0.929 and the mean ratio for the IVO group was 1.039 ± 0.946. A nested t-test was run on GraphPad Prism using the results of the spectral processing, which found that the crosslinking of zona proteins was not significantly different between IVO and IVP embryos. This study demonstrates the utility of Raman microspectroscopy as a noninvasive tool to study molecular structure on individual cells. Further studies would be useful to determine whether the rate of crosslinking is consistent between IVP and IVO systems, or if crosslinking occurs at a slower rate in IVP embryos and if cryopreservation affects crosslinking.