This proposal will test the hypothesis that maternal nutritional effects in developing oocytes and procedures used in assisted reproduction (ART) can program fetal development and adult health via heritable epigenetic changes in DNA methylation at specific gene loci in the oocyte and pre-implantation embryo. It will utilize two well established animal models of pre-and postnatal development to determine the effects of methionine-deficient (hypomethylation) diets offered during the period of oocyte growth and maturation, and pre-implantation embryo development on: (i) pre- and postnatal development, (ii) genome wide alterations in DNA methylation, and methylation of specific loci on imprinted genes in fetal and placental tissues, and (iii) methylation of imprinted and non-imprinted loci in oocytes and pre-implantation embryos. In addition, the effects of dietary treatment on oocyte DNA methylation with and without gonadotrophin stimulation (super ovulation) will be examined. Changes in oocyte methylation status will be correlated to indices of methylation cycle activity (e.g., S-adenosyl methionine/S-adenosyl homocysteine ratio) in granulosa cells and follicular fluid; and related to genetic allelic variations in methyl donor group enzymes. Finally, additional effects of culturing zygotes from FSH-stimulated donor animals from different dietary backgrounds in the presence or absence of serum on DNA methylation in pre-implantation embryos will be complemented by assessing effects on fetal and postnatal development. Zygotes and embryos will be collected from sheep offered either a methionine deficient or supplemented diet and transferred to surrogate dams. Rats offered either control or hypomethylating diets will revert to control diets at a similar stage of pregnancy (Day 4 post fertilization). Fetal tissues will be harvested for analysis at specific points of gestation. Some fetuses will be taken to term, and post-natal measurements of growth and development determined (by X-ray CT in the case of sheep). Insulin resistance, blood pressure and immune function will be determined in adult rats. Defining the effects of diet and ART on DNA methylation within the oocyte, as well as genotype variations and DNA methyltransferase gene expression, will provide novel markers for defining oocyte programming of specific future phenotypes in ART, and form the basis for establishing appropriate nutritional intervention in the periconception period.
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