Artificial blastocyst collapse prior to vitrification significantly improves Na⁺/K⁺-ATPase-dependent post-warming...

Blastocoel expansion during embryo development is known to be reliant on the Na+/K+-ATPase pump, but little is known about the relative contribution of active (Na+/K+-ATPase pump) and facilitated diffusion (aquaporins) water transport during blastocoel re-expansion after vitrification. The aims of this study were to examine potential effects of artificial blastocoel collapse (ABC) on markers of embryo stress and the contribution of active and facilitated diffusion water transport mechanisms to blastocoel re-expansion. Day 5 mouse embryos were vitrified using either a standard protocol, laser pulse ABC, a hyperosmotic sucrose ABC protocol or both laser pulse and sucrose. Using real-time polymerase chain reaction, no differences were found in the gene expression of the endoplasmic reticulum (ER) stress markers activating transcription factor 4 (Atf4) or heat shock protein 90-alpha (Hsp90α) 2h after warming. Similarly, expression of the Na+/K+-ATPase pump gene, ATPase, Na+/K+ transporting, beta 1 polypeptide (Atp1b1) and protein did not differ between groups. Aquaporin 8 (Aqp8) gene expression was significantly lower in the laser+sucrose ABC group than in fresh controls, and aquaporin 3 (Aqp3) expression significantly higher in standard vitrified embryos compared with all other groups. Ouabain, a potent and specific Na+/K+-ATPase pump inhibitor, inhibited blastocoel re-expansion in both standard protocol- and laser ABC-vitrified embryos, reducing both groups to the same rate of re-expansion 3h after warming. These results demonstrate that ABC before vitrification does not alter mRNA or protein expression of Na+/K+-ATPase, or mRNA levels of ER stress genes Atf4 and Hsp90α. Activity of the pump may be increased in ABC embryos, with potential compensation by AQP3 when it is compromised.