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 t echniques were developed to utilize terminal deoxynucleotidyl-transfer ase 3' nick end labeling (TUNEL) to detect broken DNA. Subsequent labe ling 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 devel opment. Early cleavage stages up to the 8-cell stage never contained T UNEL-labeled nuclei. However, TUNEL labeling of in vitro-produced moru la revealed some blastomeres with broken DNA. Nearly all in vitro-prod uced blastocysts displayed some TUNEL positive cells, whereas in vivo- collected embryos at a similar stage displayed few, if any, TUNEL-labe led nuclei. The ratio of TUNEL-labeled DNA to total DNA area of in vit ro-derived blastocysts was significantly greater than their in vivo co unterparts (P < 0.05). Microtubule and microfilament labeling identifi ed blastomeres of unequal size and shape that were losing cellular int egrity. These data suggest that the combination of these labeling tech niques may be useful in evaluating cellular damage in embryos produced under in vitro conditions. Mel. Reprod. Dev. 51:59-65, 1998. (C) 1998 Wiiey-Liss, Inc.