Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes.
We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.
Succu
S
S
Department of Animal Biology, University of Sassari, Sassari, Italy. succus@uniss.it
Leoni
G G
GG
Bebbere
D
D
Berlinguer
F
F
Mossa
F
F
Bogliolo
L
L
Madeddu
M
M
Ledda
S
S
Naitana
S
S
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D015925:Cryopreservation / Q000295:instrumentation*; D004864:Equipment and Supplies / Q000009:adverse effects*; D005260:Female; D005307:Fertilization in Vitro; D016200:Maturation-Promoting Factor / Q000378:metabolism; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism*; D009866:Oogenesis / Q000502:physiology; D008941:Spindle Apparatus / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D002843:Chromatin; D014404:Tubulin; D016200:Maturation-Promoting Factor