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Effects of vitrification on the expression of pluripotency, apoptotic and stress genes in in vitro-produced porcine blastocysts

Miriam Castillo-Martín A D , Marc Yeste B , Eva Pericuesta C , Roser Morató A , Alfonso Gutiérrez-Adán C and Sergi Bonet A

A Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Campus Montilivi, E-17071 Girona, Spain.
B Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain.
C Department of Animal Reproduction, INIA, Ctra de la Coruña Km 5.9, E-28040 Madrid, Spain.
D Corresponding author. Email: miriam.castillo@udg.edu

Reproduction, Fertility and Development - http://dx.doi.org/10.1071/RD13405
Submitted: 27 November 2013  Accepted: 12 March 2014   Published online: 3 April 2014


 
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Abstract

The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX : BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9 ± 0.4% vs 11.9 ± 2.0%) and peroxide levels (80.4 ± 2.6 vs 97.2 ± 3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r = –0.561; HSPA1A, r = 0.604) and peroxide levels (POU5F1, r = –0.590; HSPA1A, r = 0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression.

Additional keywords: Cryotop, DNA fragmentation, peroxide levels, real-time reverse transcription–polymerase chain reaction.


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