Mouse preimplantation embryo development in vitro: effect of sodium concentration in culture media on RNA synthesis and accumulation and gene expression.
Results of previous studies indicate that culture of preimplantation mouse embryos in SOM medium containing 85 mM NaCl promotes better development in vitro, as well as supporting higher rates of protein synthesis, when compared to culture in SOM containing 125 mM NaCl (Anbari and Schultz, 1993, Mol Reprod Dev 35:24-28; Biggers et al., 1993, Mol Reprod Dev 34:380-390). In the present study we compare the effect of culturing embryos in these 2 media on several aspects of RNA synthesis and gene expression in order to determine whether the reduced development in SOM containing 125 mM NaCl and lower rates of protein synthesis are correlated with decreases in RNA synthesis and stability and changes in gene expression. Although no apparent differences in the metabolism of [3H]uridine to UMP, UDP, and UTP and its incorporation into total RNA are observed when 2-cell embryos are cultured to the morula stage in either medium, a 20% decrease in the rate of mRNA synthesis is found when embryos are cultured in SOM containing 125 mM NaCl. In addition, pulse-chase experiments reveal that total mRNA is less stable when the embryos are cultured in SOM containing 125 mM NaCl. Using a reverse transcription-polymerase chain reaction to assay for changes in the relative amounts of specific mRNAs, the relative amounts of mRNAs for IGF-I and IGF-II and their cognate receptors are dramatically reduced in embryos cultured in SOM containing 125 mM NaCl, whereas only a mild reduction is observed in the relative amount of actin mRNA. In contrast, when freshly isolated morulae are cultured to the blastocyst stage in either of these 2 media, similar amounts of these mRNAs are observed. Last, high-resolution, 2-dimensional gel electrophoresis reveals significant changes in the pattern of protein synthesis when the embryos are cultured in SOM containing 125 mM NaCl. Results of these experiments suggest that culture of embryos in medium containing lower concentrations of NaCl that are normally present in various culture media results in higher rates of mRNA synthesis and greater mRNA stability. These changes in RNA synthesis may underlie, at least in part, the improved development in vitro that is fostered by SOM containing 85 mM NaCl.
Ho
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Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
Doherty
A S
AS
Schultz
R M
RM
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism; D011247:Pregnancy; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012965:Sodium Chloride / Q000008:administration & dosage; D014529:Uridine / Q000378:metabolism
D000199:Actins; D003470:Culture Media; D017931:DNA Primers; D012333:RNA, Messenger; D011061:Poly A; D012965:Sodium Chloride; D012313:RNA; D014529:Uridine