Czech J. Anim. Sci., 2013, 58(5):217-226 | DOI: 10.17221/6750-CJAS

Transcript analysis of Heat shock protein 72 and protein 53 of 4-cell mouse embryos following Cryotop vitrificationOriginal Paper

A. Habibi¹, N. Farrokhi², F. Morreira da Silva³, A. Hosseini⁴, B.F. Bettencourt⁵, F. Amidi⁶

¹ Department of Anatomical Sciences, International Branch, Shiraz University of Medical Sciences, Kish, Iran

² Department of Agronomy and Plant Breeding, Faculty of Agriculture, Shahrood University of Technology, Shahrood, Iran

³ Animal Reproduction, Department of Agrarian Sciences, University of the Azores, Angra do Heroísmo, Portugal

⁴ Cellular and Molecular Biology Researcher Center, Medical School of Shahid Beheshti University of Medical Sciences and Health Services, Tehran, Iran

⁵ Hospital Santo Espírito de Angra do Heroísmo, SEEBMO, Angra do Heroísmo, Azores, Portugal

⁶ Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

The effects of two different concentrations of cryoprotectants on survival and developmental capacity of four-cell mouse embryos were compared by Cryotop vitrification to demonstrate that lower concentrations provide the same results as higher previously reported concentrations with lesser negative molecular impact on embryo cells. For this latest, embryos were compared via transcript analyses of Heat shock protein 72 (Hsp72) and protein 53 (p53). Four-cell embryos were obtained from superovulated female mice and randomly assignedto one of three following groups: (i) control (non-vitrified), (ii) vit₁ (15% v/v: 7.5% ethylene glycol(EG) and 7.5% dimethyl sulfoxide (DMSO), and (iii) vit₂ (30% v/v: 15% EG + 15% DMSO).The cells vitrified by Cryotop were thawed and side-by-side to the control group divided into two parts: one part was used to analyze the morphological traits, survival rate, and embryo cleavage ability to form blastocysts, and the other part was examined for changes in transcript levels of Hsp72 (Hspa1a + Hspa1b), p53, and Hprt1 (reference gene) by quantitative Real-Time polymerase chain reaction (qPCR). The results were analyzed by One Way Analysis of Variance and the mean values compared with LSD (P < 0.05). The relative expression of p53 in vit₂ (30% v/v) was significantly higher than in vit₁ (15% v/v) and in vit₁ it was higher than in the control. The relative expression of Hsp72 was the same in vit₁ and vit₂ and significantly higher than in the control.The survival, cleavage, and blastocyst rates were the same for both vitrification treatments and significantly lower than in the control group. The up-regulations of Hsp72 and p53 following vitrification were suggestive of imposed heat shock, cold stress, and DNA damage to the mouse 4-cell embryos.

Keywords: cryopreservation; in vitro culture; murine; Real-Time PCR

Published: May 31, 2013  Show citation