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Title: DUAL LABELING OF THE CYTOSKELETON AND DNA STRAND BREAKS IN PORCINE EMBRYOS PRODUCED IN VIVO AND IN VITRO

Author
item Long, Charles
item Dobrinsky, John
item Johnson, Lawrence

Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/23/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: In vitro production of porcine embryos exhibit decreased developmental capacity. Evaluation of embryos by standard light microscopy indicates that cleavage and cavitation of embryos occurs with reduced cell number compared to in vivo produced embryos. It is therefore imperative to develop procedures for evaluating the subcellular components of embryos to better understand their developmental ability. In this project, techniques were developed to label DNA fragmentation and either microtubules or microfilaments of the cytoskeleton. Both in vitro and in vivo embryos were evaluated. These procedures successfully identified blastomeres of embryos possessing DNA fragmentation. Cytoskeletal labeling was informative in identifying irregular shaped and fragmenting blastomeres. The developed procedures are the first to evaluate DNA strand breaks and cytoskeletal components concurrently. These techniques will allow researchers to investigate changes to culture conditions, improvements to the Beltsville Sperm Sexing Technology and also provide greater insight to the sensitivity of porcine embryos to cryopreservation.

Technical Abstract: Porcine embryos which undergo in vitro fertilization and culture 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 procedures to minimize deleterious effects. Whole mount techniques were developed to utilize the 3' end labeling of broken DNA with terminal transferase (TDT) incorporation of biotin-16-UTP (bUTP) to identify nuclei that contain broken DNA. Subsequent labeling of either tubulin or actin filaments provides further evidence of cytological damage of cellular structure. Porcine embryos produced in vitro or in vivo were evaluated throughout the cleavage and pre-implantation stages of development. Early cleavage stages up to the 8 cell stage never contained TDT labeled nuclei. However, labeling of in vitro produced morula, detected a few blastomeres with broken DNA. Nearly all in vitro produced blastocysts displayed some cells labeled with TDT, whereas, in vivo collected porcine embryos at a similar stage displayed few, if any, TDT labeled nuclei. Microtubule and microfilament labeling distinguished blastomeres of unequal size and shape, perhaps losing cellular integrity. These data suggest that the combination of these labeling techniques will be useful in evaluating cellular damage due to in vitro conditions and manipulations such as cryopreservation.