Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells.
The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.
Bogliolo
L
L
Department of Pathology and Veterinary Clinic, University of Sassari, Via Vienna 2, 07100 Sassari, Italy. luis@uniss.it
Ariu
F
F
Fois
S
S
Rosati
I
I
Zedda
M T
MT
Leoni
G
G
Succu
S
S
Pau
S
S
Ledda
S
S
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002843:Chromatin / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology; D054885:Cumulus Cells / Q000502:physiology*; D003571:Cytochalasin B / Q000494:pharmacology; D005260:Female; D016200:Maturation-Promoting Factor / Q000032:analysis; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D012756:Sheep / Q000502:physiology*; D016019:Survival Analysis; D013997:Time Factors
D002843:Chromatin; D003451:Cryoprotective Agents; D003571:Cytochalasin B; D016200:Maturation-Promoting Factor; D020928:Mitogen-Activated Protein Kinases