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Methylation Status of the Male Pronucleus in the Horse

B. Gadsden, Y.H. Choi, and K. Hinrichs 

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466, USA


After fertilization in mice, humans, pigs and cattle, the paternal chromatin is  demethylated by the cytoplasm of the oocyte before the two pronuclei combine.  However, in other species such as rabbits and sheep, the paternal pronucleus is not demethylated.  No information is available on the methylation status of horse embryos.  In this study, we developed a staining protocol that allowed the visualization of the methylation status of equine cumulus cell, oocyte and embryonic nuclei. Equine oocytes with attached cumulus cells were used to develop the protocol.  Once developed, the protocol was used to evaluate the methylation status of the male pronucleus in the horse at 8 and 16 hours after fertilization via intracytoplasmic sperm injection.  The results show that no pronucleus had formed 8 hour after injection; however, by 16 hours, zygotes had developed pronuclei and showed methylation of both  the paternal and maternal pronuclei.