Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos.
The effect of modified droplet vitrification was assessed on cellular actin filament organization, apoptosis related gene expression and development competence in mouse embryos cultured in vitro. Mouse zygotes, 2-cell embryos and morulae were vitrified in ethylene glycol (VS-1) and ethylene glycol plus DMSO (VS-2) and thawed by directly placing the vitrified drop into 0.3M sucrose solution at 37 degrees C. High recovery (93-99%) of morphologically normal embryos was evident following vitrification and thawing. No detectable actin filament disruption was observed in the embryos at any development stage following vitrification and thawing and/or in vitro culture. The expression pattern of Bax, Bcl2 and p53 genes was altered (P<0.05) in vitrified zygotes and 2-cell embryos, but not in morulae. Although a large proportion of the vitrified zygotes (59.5+/-4.4% in VS-1 and 57.9+/-4.5% in VS-2; mean+/-S.E.M.) and 2-cell embryos (63.1+/-4.4% in VS-1 and 59.2+/-4.3% in VS-2) developed into blastocysts, development of control embryos (70.2+/-5.0% of zygotes and 75.5+/-4.4% of 2-cell embryos) into blastocysts was higher (P<0.05). In contrast, development of the control and vitrified morulae into blastocysts (more than 85%) was similar. We concluded that the modified droplet vitrification procedure supported better survival of morula stage compared to zygotes and 2-cell mouse embryos.
Dhali
A
A
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0315, USA.
Anchamparuthy
V M
VM
Butler
S P
SP
Pearson
R E
RE
Mullarky
I K
IK
Gwazdauskas
F C
FC
D000199:Actins / Q000032:analysis; D000818:Animals; D001755:Blastocyst / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods*; D003599:Cytoskeleton / Q000648:ultrastructure; D046149:Embryo Culture Techniques; D047108:Embryonic Development; D005260:Female; D015870:Gene Expression; D016158:Genes, p53 / Q000235:genetics; D051379:Mice; D008813:Mice, Inbred ICR; D018613:Microscopy, Confocal; D009028:Morula / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D011247:Pregnancy; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D014021:Tissue Preservation / Q000379:methods*; D015053:Zygote / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D051028:bcl-2-Associated X Protein / Q000235:genetics
D000199:Actins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C052617:Bcl2 protein, mouse