ANALISI DELLE MODIFICAZIONI INDOTTE DALLA CRIOCONSERVAZIONE SULLA ZONA PELLUCIDA DELL’OOCITA OVINO MEDIANTE MICROSPETTROSCOPIA RAMAN

"Morphological and structural damage is the most likely adverse event associated with. oocyte cryopreservation affecting oocyte quality and fertilization, embryonic. developmental and offspring generation rates1. Current cell biology techniques. employed to assess oocyte status after cryopreservation are invasive and lack the ability. to completely identify and quantify changes induced by chilling treatment. This. evidence contributes to the need to develop new evaluation methods to completely. assess oocyte cryoinjuries. Raman microspectroscopy (RMS) is a powerful noninvasive. technique for studying the molecular composition of cells, which provides a. unique fingerprint of the macromolecular components based on their chemical. composition and spatial distribution. An increasing number of RMS studies on single. cells describe numerous applications, while little research has been carried out on the. oocyte2.In the present study, we investigated molecular changes induced by. cryopreservation on the zona pellucida (ZP) of vitrified\/warmed in vitro matured sheep. oocytes by means of Raman microspectroscopy (RMS).Cumulus-oocyte complexes. were recovered from ovaries of slaughtered adult sheep, matured in vitro and vitrified. following the Minimum Essential Volume method using cryotops. ZP from fresh (n=20). and vitrified\/warmed (n=20) in vitro matured oocytes were analyzed by a Bruker. Senterra confocal Raman microscope. For measurements, entire oocytes were immersed. in a 50 ml drop of PSB on BaF2 windows. Three 4x4-point square grids were defined. on the ZP of each oocyte and on each grid point a spectrum was acquired averaging 15. acquisitions each lasting 2 seconds. RMS spectra were divided into two groups,. corresponding to fresh and vitrified ZP, cut in the 900-1800 cm-1. range, vector. normalized and baseline corrected. For each group, the average spectra were calculated.. To analyze the secondary structure of ZP proteins, we focused on the Amide III (1220-. 1320 cm-1. spectral range) and Amide I (1655 cm-1. ) protein bands. To analyze the. carbohydrate component we considered a 1020-1140 cm-1. spectral range.Following. oocyte vitrification\/warming, the results highlighted changes in the secondary structure. of the ZP proteins which involved a significant increase in β-sheet content while the α-. helix content decreased. Carbohydrate components were also modified after oocyte. vitrification. In conclusion, our study demonstrated, by means of RMS, that vitrification. of ovine oocytes induced biochemical changes to the ZP, mainly related to the. secondary structure of proteins and carbohydrate residues. The functional significance. of changes to the secondary structure of ZP proteins after vitrification may be the result. of the biochemical modifications that occur during the zona hardening. Raman. microspectroscopy may offer a powerful, non-invasive tool to assess molecular. alterations induced by cryopreservation in the oocyte. "