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

Abstract

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