Reduced HSP70 expression under mild hypothermia is involved in bovine embryo growth suppression

Abstract

Bovine blastocysts cultured under mild hypothermia (MH) can be maintained with non-hatching viable embryos compared to normothermic controls (38.5°C). However, the mechanism by which mildly hypothermic culture delays embryonic growth has not yet been elucidated. This study evaluated the number of cells in embryos cultured under MH conditions and the expression of genes involved in embryonic differentiation. Bovine blastocysts cultured under MH conditions exhibited reduced cell numbers and interferon-tau mRNA expression. Both forkhead box O3 (FOXO3) mRNA expression and FOXO3 protein level in blastocysts cultured under MH conditions were higher than those in normothermic controls (P < 0.05). On the phosphorylated FOXO3 protein level, there was no significant difference between blastocysts cultured under MH and normothermic conditions. In contrast, no significant difference was observed in the ATP content of blastocysts between the MH and normothermic groups. In blastocysts cultured under MH conditions, cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) mRNA expression increased, and heat shock protein 70 (HSP70) mRNA expression decreased compared to that in normothermic controls (P < 0.05). Considering that HSP70 is involved in preventing apoptosis, these results suggest that MH retards embryonic development via apoptosis induced by HSP70 downregulation during the culture period.

Keywords: Apoptosis; Cattle; Embryo; HSP70; Mild hypothermia.

Fig. 1.

Total cell number in bovine blastocysts cultured in vitro from days 7 to 10 at different temperatures (33 and 38.5°C). Morphological categories: blastocyst (Bl) and hatched blastocyst (Hatch-Bl). Error bar means the standard error of the mean. * P < 0.05.

Fig. 2.

mRNA levels of differentiation-associated genes in bovine blastocysts cultured at different temperatures (33 and 38.5 °C) from days 7 to 10. Relative mRNA levels of IFNT (A), POU5F1(B), and CDX2 (C). The mRNA level of each gene was normalized to that of β-actin. Morphological categories: blastocyst (Bl) and hatched blastocyst (Hatch-Bl). The experiments were repeated thrice. Error bar means the standard error of the mean. * P < 0.05.

Fig. 3.

mRNA levels of apoptosis-related genes in bovine blastocysts cultured at different temperatures (33 and 38.5°C) on day 8. Relative mRNA levels of BAX, BCL2, FOXO3, P53, and FAS. The mRNA level of each gene was normalized to that of β-actin. The experiments were repeated thrice. Error bar means the standard error of the mean. * P < 0.05.

Fig. 4.

Protein levels of FOXO3 and phosphorylated FOXO3 in bovine blastocysts cultured at different temperatures (33 and 38.5°C) on day 8. (A) Representative images of bovine blastocysts cultured at 38.5°C (left) and 33°C (right) groups with anti-FOXO3 antibody staining. (B) Mean pixel intensities of FOXO3. Individual mean pixel intensity per embryo was quantified using ImageJ software. Error bar means the standard error of the mean. * P < 0.05. (C) Representative images of bovine blastocysts cultured at 38.5°C (left) and 33°C (right) groups with anti-phosphorylated FOXO3 antibody staining. (D) Mean pixel intensities of phosphorylated FOXO3. Individual mean pixel intensity per embryo was quantified using ImageJ software. Error bar means the standard error of the mean.

Fig. 5.

Effect of mild hypothermia on embryonic metabolic functions on day 8. ATP concentrations in bovine blastocysts cultured at different temperatures (33 and 38.5°C). Error bar means the standard error of the mean. * P < 0.05.

Fig. 6.

mRNA levels of heat shock protein and cold shock protein-related genes in bovine blastocysts cultured at different temperatures (33 and 38.5°C) on day 8. Relative mRNA levels of the heat shock proteins HSP60, HSP70, and HSP90 (A) and cold shock proteins CIRP and RBM3 (B). The expression level of each gene was normalized to that of β-actin. The experiments were repeated thrice. Error bar means the standard error of the mean. * P < 0.05.