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Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy

Bogliolo, Luisa and Murrone, Ombretta and Piccinini, Massimo and Ariu, Federica and Ledda, Sergio and Tilocca, Sara and Albertini, David F. (2015) Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy. Journal of Assisted Reproduction and Genetics, Vol. 32 (2), p. 185-193. ISSN 1058-0468. eISSN 1573-7330. Article.

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DOI: 10.1007/s10815-014-0389-7

Abstract

Purpose: 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.

Item Type:Article
ID Code:10996
Status:Published
Refereed:Yes
Uncontrolled Keywords:Oocyte, cryopreservation, raman microspectroscopy, actin, sheep
Subjects:Area 07 - Scienze agrarie e veterinarie > VET/10 Clinica ostetrica e ginecologia veterinaria
Divisions:001 Università di Sassari > 01-a Nuovi Dipartimenti dal 2012 > Medicina Veterinaria
Publisher:Springer US
ISSN:1058-0468
eISSN:1573-7330
Deposited On:10 Jun 2015 12:18

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