Leoni, Giovanni Giuseppe and Berlinguer, Fiammetta and Rosati, Irma and Bogliolo, Luisa and Ledda, Sergio and Naitana, Salvatore (2003) Resumption of metabolic activity of vitrified/warmed ovine embryos. Molecular Reproduction and Development, Vol. 64 (2), p. 207-213. eISSN 1098-2795. Article.
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To evaluate resumption of metabolic activity of vitrified ovine embryos during a short time immediately after warming, blastocysts collected from superovulated Sarda ewes were incubated with 35S-methionine. In vitrified/warmed embryo groups the protein secretion significantly (P < 0.05) increased from 0 to 24 hr of culture, reaching significantly (P < 0.01) higher activity at 18–24 hr and dropping to values similar to the control nonvitrified embryo group at 29–35 hr. Within the control group at 29–35 hr there was a significantly (P < 0.01) higher level of protein secretion compared to the other interval times. The electrophoretic pattern showed a 48–50 kDa secreted protein identified as urokinase-type plasminogen activator (PA). The caseinolytic assay of PA activity showed a course similar to protein secretion in both vitrified and control groups. During 29–35 hr of culture, we did not observe any improvement in PA activity as seen for secreted proteins. At this time, we observed the secretion of a new 20 kDa protein that was not present in vitrified/warmed embryos. Analysis of BrDU incorporation in newly synthesised DNA showed a significant (P < 0.01) improvement in positive cell number from 3 to 9 hr after warming, reaching a value similar to that of the control group at 12 hr of culture. Our results suggest that vitrified/warmed embryos require 9–12 hr of culture to complete resumption of DNA synthesis and 29–35 hr to re-acquire the full capacity of protein secretion but not the qualitative secretion pattern.
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