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Methylation Status of Horse Embryos Produced by Intracytoplasmic Sperm Injection 

Patsy Morris, Y.H. Choi , K. Hinrichs 1
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA

AbstractPatsy Picture

Objective – We determined the methylation status of the nuclei of horse embryos derived by intracytoplasmic sperm injection (ICSI)  at 16, 18, 20, 22 and 24 hours, at  2, 4, and 6 days, and at the blastocyst stage.

Animals - Equine ICSI embryos, equine oocytes and mouse zygotes were used.

Procedure- A 5-methylcytosine (5-MeC) immunostaining protocol was performed on ICSI horse embryos.  In order to establish that our primary antibody (mouse monoclonal antibody to 5-MeC) was staining specifically for methylated cytosine residues in the DNA as opposed to all cytosine residues, a dilution protocol was developed in which the antibody was exposed to increasing  concentrations of 5-MeC or cytosine, before use in  immunocytochemistry.   The validity of our staining protocol was also tested using mouse zygotes whose methylation status has previously been established.

Results - All the ICSI horse embryos exhibited methylation of nuclear DNA without evidence of demethylation of the male pronucleus or of the nucleus during development to the blastocyst.  Validity of our staining protocol was verified: Staining of cumulus cell nuclei was quenched by exposure of the primary antibody to high concentrations of 5-MeC but not cytosine.  Staining of mouse zygotes demonstrated demethylation of the presumed male pronucleus.   

Conclusions and Clinical Relevance - The methylation status of the horse pronuclei demonstrates that pronuclear demethylation does not occur in the horse.  Our results can be used in testing the hypothesis that equine embryos produced by nuclear transfer show perturbations in DNA methylation in comparison to embryos produced by ICSI.