Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy.
OBJECTIVE
Investigation of the changes induced by vitrification on the cortical F-actin of in vitro matured ovine oocytes by Raman microspectroscopy (RMS).


METHODS
Cumulus-oocyte complexes, recovered from the ovaries of slaughtered sheep, were matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. The cortical region of metaphase II (MII) oocytes (1) exposed to vitrification solutions but not cryopreserved (CPA-exp), (2) vitrified/warmed (VITRI), and (3) untreated (CTR) was analyzed by RMS. A chemical map of one quadrant of single CPA-exp, VITRI and CTR oocytes was, also, performed. In order to identify the region of Raman spectra representative of the cortical F-actin modification, a group of in vitro matured oocytes were incubated with latrunculin-A (LATA), a specific F-actin destabilizing drug, and processed for RMS analysis. Thereafter, all the oocytes were stained with rhodamine phalloidin and evaluated by fluorescence confocal microscopy. Raman spectra of the oocytes were, statistically, analyzed using Principal Component Analysis (PCA).


RESULTS
The PCA score plots showed a marked discrimination between CTR oocytes and CPA-exp/ VITRI groups. The main differences, highlighted by PCA loadings, were referable to proteins (1657, 1440 and 1300 cm(-1)) and, as indicated by LATA experiments, also included the changes of the F-actin. Analysis by confocal microscopy revealed a clear alteration of the cortical F-actin of CPA-exp and VITRI oocytes confirming RMS results.


CONCLUSIONS
Raman microspectroscopy may represent an alternative analytical tool for investigating the biochemical modification of the oocyte cortex, including the F-actin cytoskeleton, during vitrification of in vitro matured ovine oocytes.
Department of Veterinary Medicine, of Sassari, Via Vienna 2, 07100, Sassari, Sardegna, Italy, luis@uniss.it.
Luisa
L
Bogliolo
Ombretta
O
Murrone
Massimo
M
Piccinini
Federica
F
Ariu
Sergio
S
Ledda
Sara
S
Tilocca
David F
DF
Albertini
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000032:analysis; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D059471:In Vitro Oocyte Maturation Techniques; D018613:Microscopy, Confocal; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects; D034561:Sheep, Domestic; D013059:Spectrum Analysis, Raman / Q000379:methods*; D053778:Thiazolidines / Q000494:pharmacology; D058989:Vitrification
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003451:Cryoprotective Agents; D053778:Thiazolidines; C037067:latrunculin A