Resumption of metabolic activity of vitrified/warmed ovine embryos.
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 (35)S-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.
Leoni
Giovanni
G
Department of Animal Biology, University of Sassari, Sassari, Italy. vetfis@ssmain.uniss.it
Berlinguer
Fiammetta
F
Rosati
Irma
I
Bogliolo
Luisa
L
Ledda
Sergio
S
Naitana
Salvatore
S
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D066298:In Vitro Techniques; D011247:Pregnancy; D012756:Sheep