Freezing-thawing behaviour and cell membrane ultrastructure of mouse embryos pre-cultured in B2-Menezo medium before cryopreservation.
Mouse embryos were pre-cultured from the 2-cell to the 8-cell stage inserum-free B2-Menezo medium and in B2-Menezo medium with 15% fetal cord serum before cryopreservation with a slow freezing-thawing method (-0.3 degree C/min in 1.5 M DMSO). Viability after freezing-thawing was tested by continuing in-vitro culture and evaluating the percentage of embryos developing to the blastocyst stage. An extreme decrease in viability after freezing and thawing was found for the embryos pre-cultured in serum-free B2-Menezo medium compared with the embryos pre-cultured in B2-Menezo medium with 15% fetal cord serum (12 versus 81% blastocysts). Ultra-thin sections of the pre-cultured embryos and of freshly collected controls were prepared for transmission electron microscopy before and after the freezing-thawing procedure. A drastic decrease in the number of microvilli on the cell surface and a high degree of cell damage after freezing and thawing was observed in the embryos pre-cultured in serum-free B2-Menezo medium. In contrast a high number of microvilli and an intact cell structure after cryopreservation was observed in the embryos pre-cultured in B2-Menezo medium with 15% fetal cord serum and in the non-pre-cultured controls. It is suggested that the smaller cell surface and the resulting lower permeability of the cell membrane in the embryos with few microvilli are due to sub-optimal culture conditions and are the main reasons for the extremely decreased viability after freezing and thawing.
Department of Obstetrics and Gynaecology, University of Heidelberg, FRG.
W
W
Bernart
T
T
Rabe
E
E
Schleiermacher
I
I
Anton-Lamprecht
B
B
Runnebaum
D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure*; D003470:Culture Media; D004625:Embryo, Nonmammalian / Q000648:ultrastructure*; D005260:Female; D005615:Freezing; D051379:Mice; D008815:Mice, Inbred Strains; D008854:Microscopy, Electron; D010062:Ovulation Induction; D014021:Tissue Preservation / Q000379:methods*
D003470:Culture Media