ABSTRACT
Mono (2-ethylhexyl) phthalate (MEHP), an environmental contaminant, is known to cause many serious diseases, especially in reproductive system. However, little is known about the effect of MEHP on preimplantation embryo development. In this study, we found that the development of mouse 2-cell embryo was blocked by 10(-3) M MEHP. A significant increase in the level of reactive oxygen species (ROS) was observed in arrested 2-cell embryo following 10(-3) M MEHP treatment for 24 h. However, antioxidants, catalase (CAT), and superoxide dismutase (SOD), reduced intracellular ROS and protected MEHP-exposed embryos from death but failed to return the arrested embryos. Further experiments demonstrated that the level of apoptosis was not altered in live arrested 2-cell embryo and increased in dead arrested 2-cell embryo after MEHP treatment, which implied that ROS and apoptosis were not related with 2-cell block. During analysis of the indicators of embryonic genome activation (EGA) initiation (Hsc70, MuERV-L, Hsp70.1, eIF-1A, and Zscan4) and maternal-effect genes (OCT4 and SOX2), we found that MEHP treatment could significantly decline Hsc70, MuERV-L mRNA level and SOX2 protein level, and markedly enhance Hsp70.1, eIF-1A, Zscan4 mRNA level, and OCT4 protein level at 2-cell to 4-cell stage. Supplementation of CAT and SOD did not reverse the expression tendency of EGA related genes. Collectively, this study demonstrates for the first time that MEHP-induced 2-cell block is mediated by the failure of EGA onset and maternal-effect genes, not oxidative stress and apoptosis.
Subject(s)
Diethylhexyl Phthalate/analogs & derivatives , Embryonic Development/drug effects , Animals , Antioxidants/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Catalase/genetics , Catalase/metabolism , Diethylhexyl Phthalate/pharmacology , Embryonic Development/genetics , Eukaryotic Initiation Factor-1/genetics , Eukaryotic Initiation Factor-1/metabolism , Female , Gene Expression Regulation, Developmental/drug effects , HSC70 Heat-Shock Proteins/genetics , HSC70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Mice , Mice, Inbred ICR , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Oxidation-Reduction/drug effects , Proteins/genetics , Proteins/metabolism , RNA, Messenger/genetics , Reactive Oxygen Species/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
The expression of the zygotic genome starts at the late one-cell stage in mouse embryos, and its regulation changes dynamically until the late two-cell stage. To understand this process, it is important to accumulate the profiles of the genes transcribed at any given instant at each stage of development. However, because large amounts of maternal mRNA accumulate in embryos to sustain early development, it is difficult to determine the profile of newly synthesized mRNA just after gene activation. To overcome this difficulty, we established a novel method of isolating nascent mRNA from the large pool of preexisting mRNA. Briefly, the procedure was as follows. Embryos were electrically permeabilized and loaded with 5-bromouridine-5'-triphosphate (BrUTP). Nascent mRNA with incorporated BrU was isolated by immunoprecipitation with an antibody recognizing BrU. The cDNA was synthesized from the isolated mRNA, and its abundance was evaluated using semiquantitative real-time PCR. Using this method, we examined the amounts of newly synthesized eIF-1A, MuERV-L, and cyclin-A2 transcripts in two-cell mouse embryos and compared them with the quantities of these transcripts present in the total mRNA pool. The amount of each transcript in the nascent mRNA fraction and in the total mRNA pool changed differently over time, demonstrating that this method can be used to obtain profiles of genes transcribed during development.
Subject(s)
Blastocyst/metabolism , Genetic Techniques , RNA, Messenger/biosynthesis , RNA, Messenger/isolation & purification , Uridine Triphosphate/analogs & derivatives , Animals , Cells, Cultured , Computer Systems , Cyclin A/genetics , DNA, Complementary/metabolism , Embryo Culture Techniques , Eukaryotic Initiation Factor-1/genetics , Genetic Techniques/standards , Immunoprecipitation , Mice , Proteins/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Uridine Triphosphate/metabolismABSTRACT
Protein synthesis is very sensitive to NaCl. However, the molecular targets responsible for this sensitivity have not been described. A cDNA library of the halotolerant plant sugar beet was functionally screened in a sodium-sensitive yeast strain. We obtained a cDNA clone (BveIF1A) encoding the eukaryotic translation initiation factor eIF1A. BveIF1A was able to partially complement the yeast eIF1A-deficient strain. Overexpression of the sugar beet eIF1A specifically increased the sodium and lithium salt tolerance of yeast. This phenotype was not accompanied by changes in sodium or potassium homeostasis. Under salt stress conditions, yeast cells expressing BveIF1A presented a higher rate of amino acid incorporation into proteins than control cells. In an in vitro protein synthesis system from wheat germ, the BveIF1A recombinant protein improved translation in the presence of NaCl. Finally, transgenic Arabidopsis plants expressing BveIF1A exhibited increased tolerance to NaCl. These results suggest that the translation initiation factor eIF1A is an important determinant of sodium tolerance in yeast and plants.