Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development.
In vitro culture of mouse embryos results in loss of imprinting. The aim of the present study was to examine how two of the techniques commonly used during assisted reproduction, namely embryo culture and embryo transfer, affect genomic imprinting after implantation in the mouse. F1 hybrid mouse embryos were subjected to three experimental conditions: control (unmanipulated), embryo transfer and in-vitro-culture followed by embryo transfer. Concepti were collected on d9.5 of development and allelic expression determination of ten imprinted genes (H19, Snrpn, Igf2, Kcnq1ot1, Cdkn1c, Kcnq1, Mknr3, Ascl2, Zim1, Peg3) was performed. Although control concepti had monoallelic imprinted gene expression in all tissues, both manipulated groups had aberrant expression of one or more imprinted genes in the yolk sac and placenta. Culture further exacerbated the effects of transfer by increasing the number of genes with aberrant allelic expression in extraembryonic, as well as embryonic tissues. Additionally, placentae of both groups of manipulated concepti exhibited reduced levels of Igf2 mRNA and increased levels of Ascl2 mRNA when compared with their unmanipulated counterparts. Furthermore, we show that biallelic expression of Kcnq1ot1 coincided with loss of methylation on the maternal allele of the KvDMR1 locus, a phenotype often associated with the human syndrome Beckwith-Wiedemann. In conclusion, our results show that even the most basic manipulation used during human-assisted reproduction, namely, embryo transfer, can lead to misexpression of several imprinted genes during post-implantation development. Additionally, our results serve as a cautionary tale for gene expression studies in which embryo transfer is used.
Rivera
Rocío M
RM
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Stein
Paula
P
Weaver
Jamie R
JR
Mager
Jesse
J
Schultz
Richard M
RM
Bartolomei
Marisa S
MS
D000483:Alleles; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000235:genetics; D019175:DNA Methylation; D046149:Embryo Culture Techniques; D004624:Embryo Transfer / Q000009:adverse effects*; D047108:Embryonic Development / Q000235:genetics*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D018392:Genomic Imprinting; D005865:Gestational Age; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010920:Placenta / Q000196:embryology / Q000378:metabolism; D011247:Pregnancy; D062085:RNA, Long Noncoding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics; D027724:Reproductive Techniques, Assisted / Q000009:adverse effects; D015017:Yolk Sac / Q000196:embryology / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D050761:Cyclin-Dependent Kinase Inhibitor p57; D007335:Insulin-Like Growth Factor II; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D022661:RNA, Untranslated; C489311:Ascl2 protein, mouse; C493965:Cdkn1c protein, mouse; C094335:H19 long non-coding RNA; C513121:IGF2 protein, mouse