Program freezing of diapausing embryos in the mouse

Embryonal diapause is a characteristic feature of about 130 mammalian species. However, very few studies have addressed cryopreservation of diapausing embryos. This work is aimed to apply program freezing to blastocysts obtained from CD1 mice, which were at diapause state after ovariectomy and the subsequent hormonal therapy. Blastocysts collected from non-operated mice of the same strain served as controls. Some diapausing as well as non-diapausing frozen-thawed blastocysts demonstrated blastocoel re-expansion after 24 h of in vitro culture (IVC) indicating their viability after cryopreservation. Raman spectroscopy assessment of phenylalanine accumulation revealed that the fraction of new synthesized proteins was lower for non-frozen as well as for frozen-thawed diapausing blastocysts compared to non-diapausing ones. Although protein metabolism was reduced in diapausing embryos, most of the protein synthesis remained active. Cell number increased after 24 h of IVC in non-frozen as well as in the frozen-thawed blastocysts of the control but not of the diapause group. However, cell numbers were increased in frozen-thawed diapausing blastocysts after 47 h of IVC in a medium supplemented with putrescine. This indicates viability of frozen-thawed diapausing embryos after cryopreservation. Besides, protein metabolism was not affected by cryopreservation in diapausing and non-diapausing murine embryos indicating their viability. Our results demonstrated the possibility of successful cryopreservation of diapausing murine embryos.