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

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.