Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos
Razif Dasiman, Nor-Shahida Abdul Rahman, Salina Othman, Mohd-Fazirul Mustafa, Norhazlin Jusoh, Wan-Hafizah W. Jusof, Mohd Hamim Rajikin, Gabriele Ruth Anisah Froemming, Nor-Ashikin Mohamed Noor Khan
Med Sci Monit Basic Res 2013; 19:258-266
This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM).
Material and Methods: Fifty female mice, aged 4–6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3.
Results: The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos.
Conclusions: Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.
Keywords: Blastocyst - metabolism, Animals, Actins - metabolism, Cell Nucleus - metabolism, Cryopreservation, Cytoskeleton - metabolism, Embryonic Development, Fluorescence, Mice, Mice, Inbred ICR, Microscopy, Confocal, Tubulin - metabolism