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United States Department of Agriculture

Agricultural Research Service

Research Project: REDUCING COST OF EFFICIENT BEEF PRODUCTION

Location: Livestock and Range Research Laboratory (LARRL)

Title: Establishment of a phenotypical model of adverse outcomes associated with assisted reproductive technologies

Authors
item Robbins, K -
item Wells, K -
item Geary, Thomas
item O'Gorman, C -
item Macneil, Michael
item Smith, M -
item Pohler, K -
item Jinks, E -
item Rivera, R -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2010
Publication Date: July 30, 2010
Citation: Robbins, K.M., Wells, K.D., Geary, T.W., O'Gorman, C., Macneil, M.D., Smith, M.F., Pohler, K., Jinks, E., Rivera, R.M. 2010. Establishment of a phenotypical model of adverse outcomes associated with assisted reproductive technologies. Society for the Study of Reproduction Abstract #316.

Technical Abstract: Genomic imprinting is an epigenetic modification that directs parent-specific gene expression. Imprinted genes are involved in regulating growth and development of the conceptus (fetus and placenta). Beckwith-Wiedemann Syndrome (BWS) is an overgrowth condition that is associated with loss-of-imprinting in humans. BWS has been shown to have an increased incidence in children conceived by the use of assisted reproductive technologies (ART). Currently, there are no animal models that recapitulate the ART-induced human overgrowth phenotype. As in humans, ART can induce an overgrowth syndrome in ruminants which is phenotypically similar to BWS. In ruminants this syndrome is called large offspring syndrome (LOS). We hypothesize that BWS and LOS share epigenetic origins. The goal of our research is to determine the baseline methylation and allele-specific expression in bovids of imprinted loci known to be misregulated in BWS as a result of minimal ART procedures. First, DNA was isolated from Bos taurus taurus (Angus, Hereford, Holstein breeds) and Bos taurus indicus (Nelore breed) tissues to determine DNA sequence polymorphisms between the two breeds at differentially methylated regions (DMRs) as well as within the transcriptional unit (i.e. exons) of BWS-associate loci. For DNA methylation analyses, bisulfite-specific PCR primers were designed to amplify over the polymorphic regions at putative DMRs. For gene expression analyses, PCR primers which encompassed the polymorphic regions were developed and were used to determine parent-specific gene expression. We produced three day 65 Bos taurus taurus x Bos taurus indicus F1 concepti by artificially inseminating three Bos taurus taurus heifers with Nelore semen to establish baseline DMR methylation and imprinted gene expression. The following tissues were collected from the day 65 concepti; chorioallantois, amnion, brain, tongue, lung, heart, liver, kidney, intestines, and reproductive tract. Results show that, as expected, the BWS-associated genes KCNQ1OT1 and CDKN1C are monoallelicaly expressed in the chorioallantois of the F1 concepti. Methylation analyses for the DMRs that regulate these genes is underway. In order to determine if ART causes misregulation of imprinted genes in bovine embryos, cumulus-oocytes complexes from Bos taurus taurus cows were matured in vitro and inseminated with Nelore semen. Putative zygotes were stripped of their cumulus cells after fertilization and allowed to develop for 8 days. Half of the embryos received 10% fetal calf serum supplementation on day 5. The addition of serum is known to cause LOS in ruminants and therefore the blastocysts exposed to serum are expected to have biallelic expression of KCNQ1OT1 and loss of expression in CDKN1C. Blastocysts were collected and RNA and DNA isolated from individual embryos for expression and methylation analyses, respectively. Preliminary data suggest that serum supplementation results in silencing of the maternally-expressed cell cycle regulator CDKN1C. Studies in mice demonstrate that silencing of Cdkn1c can result in placental overgrowth. In vivo-produced F1 blastocyst will be generated by inseminating Bos taurus taurus heifers with Nelore semen. Embryos will be flushed from the uterus on day 8 gestation to use as controls for the in vitro-produced F1 blastocysts. These data will serve to determine the appropriateness of using LOS as a epigenetic model for BWS.

Last Modified: 8/30/2014
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