Alterations in calcium oscillatory activity in vitrified mouse eggs impact on egg quality and subsequent embryonic development

Cryopreservation of mature eggs is a useful technique that can be applied in assisted reproductive technology. However, the method has some limitations, such as cryodamage induced by biophysical modifications during the cryopreservation process. To assess these biophysical damage, we analyzed the relationship between intracellular calcium ([Ca2+](i)) oscillatory activity via type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) distribution after vitrification and efficiency of cryopreservation according to cryoprotectant (CPA) composition. In immunostaining, results of IP(3)R1with eggs after the vitrification performed using ethylene glycol (EG) alone or EG + dimethylsulfoxide (DMSO) as CPAs, CPA-treated, and fresh eggs displayed a homogeneous IP(3)R1 distribution which is spread uniformly throughout cytoplasm with clusters on the cortex. However, after vitrification and warming process, more than 60% of eggs displayed a heterogeneous distribution which is non-uniform distribution with patches and disconnection of IP(3)R1. In 90-min incubation for recovery from cryodamage, eggs from the EG + DMSO group recovered from with a heterogeneous IP(3)R1 distribution to the homogeneous distribution, but not in EG alone group. In ICSI experiments, vitrified eggs in the EG-alone group presented significantly low blastocyst formation compared to those of the fresh and EG + DMSO groups. These results suggest that the vitrification process influences IP(3)R1 distribution, and subsequently, [Ca2+](i) oscillatory activity and embryonic development. Accordingly, we propose that IP(3)R1 distribution and [Ca2+](i) oscillatory activity are correlated with egg quality and developmental potential after vitrification, and may thus be applied as an effective indicator to evaluate the efficiency of oocyte cryopreservation methods.