Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression.
Simplex optimization has generated several media that have improved the development of mouse preimplantation embryos in vitro. One objective of this study was to compare the development of preimplantation mouse embryos in one of these computer-optimized media, KSOM, with embryos that developed in vivo, in terms of the relative abundances of specific mRNAs involved in metabolism, transcription, and cell proliferation. First, however, since studies have indicated an improvement of other simple embryo culture media by addition of amino acids, the effects of the addition of amino acids to KSOM (KSOM/AA) on preimplantation development were assessed. We find that addition of both essential and nonessential amino acids to KSOM augments development in vitro, as compared to development supported by KSOM without amino acids. This augmentation is observed starting at the blastocyst stage, and is associated with increased rate of development to the blastocyst stage, increased frequency of hatching, and increased number of cells in the blastocysts. Reverse-transcription PCR was then used to assess the relative abundance of mRNAs for actin, glyceraldehyde-3-phosphate dehydrogenase, Na+, K(+)-ATPase, Sp1, TATA box-binding protein TBP, IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor in embryos that developed in vivo and in vitro using KSOM/AA. Eight out of 9 of these mRNAs were present in the 8-cell embryos and blastocysts raised in KSOM/AA in amounts that were indistinguishable from those in embryos that developed in vivo. It is concluded that KSOM/AA provides an environment in which preimplantation mouse embryos can undergo development that is quantitatively similar to that occurring in vivo.
Ho
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Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA.
Wigglesworth
K
K
Eppig
J J
JJ
Schultz
R M
RM
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects; D003470:Culture Media; D005314:Embryonic and Fetal Development / Q000235:genetics*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011247:Pregnancy
D000596:Amino Acids; D003470:Culture Media