Dual labeling of the cytoskeleton and DNA strand breaks in porcine embryos produced in vivo and in vitro.
In vitro-produced embryos exhibit decreased cell numbers, small inner cell masses and reduced pregnancy rates after transfer. Evaluation of intracellular components of in vitro-produced or -manipulated embryos will lead to improved methodology for embryo production. Whole mount techniques were developed to utilize terminal deoxynucleotidyl-transferase 3' nick end labeling (TUNEL) to detect broken DNA. Subsequent labeling of either tubulin or actin filaments provides further evidence of cytological damage. Porcine embryos produced in vitro or in vivo were evaluated throughout the cleavage and preimplantation stages of development. Early cleavage stages up to the 8-cell stage never contained TUNEL-labeled nuclei. However, TUNEL labeling of in vitro-produced morula revealed some blastomeres with broken DNA. Nearly all in vitro-produced blastocysts displayed some TUNEL positive cells, whereas in vivo-collected embryos at a similar stage displayed few, if any, TUNEL-labeled nuclei. The ratio of TUNEL-labeled DNA to total DNA area of in vitro-derived blastocysts was significantly greater than their in vivo counterparts (P < 0.05). Microtubule and microfilament labeling identified blastomeres of unequal size and shape that were losing cellular integrity. These data suggest that the combination of these labeling techniques may be useful in evaluating cellular damage in embryos produced under in vitro conditions.
Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, USA.
C R
CR
Long
J R
JR
Dobrinsky
W M
WM
Garrett
L A
LA
Johnson
D000818:Animals; D001757:Blastomeres; D046508:Culture Techniques; D003599:Cytoskeleton; D004249:DNA Damage; D005260:Female; D020287:In Situ Nick-End Labeling; D009028:Morula; D011247:Pregnancy; D013552:Swine