title: Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida.
abstract: Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP. Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230-1300 cm(-1) amide III region and the 1020-1140 cm(-1) spectral range in RMS spectra, respectively. The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in β-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.
authors.affiliation: Department of Pathology and Veterinary Clinics, University of Sassari, Via Vienna 2, 07100 Sassari, Italy. luis@uniss.it
authors.forename: Luisa
authors.initials: L
authors.lastname: Bogliolo
authors.forename: Sergio
authors.initials: S
authors.lastname: Ledda
authors.forename: Plinio
authors.initials: P
authors.lastname: Innocenzi
authors.forename: Federica
authors.initials: F
authors.lastname: Ariu
authors.forename: Daniela
authors.initials: D
authors.lastname: Bebbere
authors.forename: Irma
authors.initials: I
authors.lastname: Rosati
authors.forename: Giovanni Giuseppe
authors.initials: GG
authors.lastname: Leoni
authors.forename: Massimo
authors.initials: M
authors.lastname: Piccinini
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D012756:Sheep; D034561:Sheep, Domestic; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013395:Sucrose / Q000494:pharmacology; D058989:Vitrification; D015044:Zona Pellucida / Q000187:drug effects
chemical_list: D003451:Cryoprotective Agents; D011506:Proteins; D013395:Sucrose; D019855:Ethylene Glycol; D000116:Acetylgalactosamine; D004121:Dimethyl Sulfoxide