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

Abstract

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 assigned to one of three following groups: (i) control (non-vitrified), (ii) vit 1 (15% v/v: 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO), and (iii) vit 2 (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 vit 2 (30% v/v) was significantly higher than in vit 1 (15% v/v) and in vit 1 it was higher than in the control. The relative expression of Hsp72 was the same in vit 1 and vit 2 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.

The Effect of Vitrification Device and Embryonic Stage on Survival and Developmental Competence of Gabali Rabbit Embryos

“…Vitrification was carried out in two steps: in the 1 st step, embryos were pipetted into vitrification solution 1 (VS1) containing BM + 12.5% (v:v) ethylene glycol (EG) + 12.5% (v:v) dimethyl-sulfoxide (DMSO) + 0.5 M sucrose) in disposable sterile Petri dish for 2 min. In the 2 nd step, the embryos were pipette into VS2 (BM + 20% (v:v) EG+20% (v:v) DMSO + 0.5 M sucrose) for 30 s. Then, three embryos were loaded into cryotop with <0.1 μl VS2, and immediately submerged in liquid nitrogen (Habibi et al, 2013). Also, eight embryos were suspended in VS2, loaded in 0.25 ml plastic straws (IMV, L'Aigle, France), sealed and plunged directly into liquid nitrogen for at least 15 days.…”

“…Vitrification is routinely being used in the artificial reproductive technologies and has become increasingly used than slow freezing method with no or little damage to provide the chance to maintain scientifically important stocks, strains, and lines. Also, vitrification methods have been made possible to store embryos for extended periods and allowed the import and export of embryos and gametes (Habibi et al, 2013).…”