Effect of Culture Stress on Expression of RNA Binding Proteins and Target mRNAs in Mouse Embryos.

RNA binding proteins (RBP) are expressed in mammalian cells and they affect storage, splicing, localization, adenylation, half-life and translation of mRNAs. Previously, we detected expression of ELAVL1, KHSRP and ZFP36 RBPs in mouse embryos by real-time PCR and immunofluorescence and showed abundance of two RBPs and one target mRNA were affected by culture. Generally, ELAVL1 increases expression of target mRNAs while KHSRP and ZFP36 decrease mRNA expression. Furthermore, all are known to be regulated by MAPK, especially p38. The roles of environmental stress and MAPKs in affecting RBP and target mRNA expression were investigated. Three potential ARE-containing targets were chosen based on the likelihood that their expression would be culture-responsive, GCLC (glutathione synthesis), SLC2A1 (GLUT-1, glucose transport) and CAT-1 (amino acid transport). We investigated two methods of inducing environmental stress: 1) hyperoxia (H, 20% O2) versus low oxygen (L, 5% O2); and 2) culturing embryos in a good culture medium, KSOMaa (K) versus a poor medium without amino acids, Whitten's (W), ± the p38 MAPK inhibitor, SB220025 (10 ìM) or 3) culture in K or W ± the ERK inhibitor U0126 (10 ìM). In expt 1, embryos were cultured from the 2 cell stage in KSOMaa under a 5% or 20% O2 atmosphere to approximately 96h post hCG, and only blastocysts were collected. In expts 2 and 3, embryos were cultured from 2-cell to blastocyst in KSOMaa in 5% O2, then embryos were washed twice and cultured in a) K; b) K + inhibitor, c) W; d) W + inhibitor for approximately 18h. Embryos were frozen, reverse transcribed with oligodT and used for real-time PCR. GCLC (P < 0.001) and CAT-1 mRNAs (P< 0.01) were expressed at higher levels in H vs. L medium, and there was a tendency towards higher expression of SLC2A1 in H than L (P < 0.065). In expt. 2, there were no significant differences due to media, but p38 MAPK inhibition caused expression of CAT-1 mRNA to be markedly reduced (P < .001). In experiment 3, GCLC mRNA was more highly expressed in W than K (P < 0.003), and there was a tendency towards higher expression of CAT-1 mRNA in W than K (P < 0.06) but no differences in the presence of the ERK inhibitor. Further experiments will examine the role of the JUNK/SAPK pathway and examine whether stress affects mRNA half-life. We have therefore identified a culture sensitive stress response pathway that mediates affects on transcript levels likely by affecting mRNA stability rather than transcription. Culture stress acting through the p38 MAPK pathway may influence embryonic mRNA transcript levels via RBP mediated mechanisms controlling target mRNA abundance. Variations in mRNAs levels observed in cultured embryos may therefore occur due to effects to mRNA stability and half-life in addition to transcriptional control mechanisms. Research support by the Natural Science and Engineering Council of Canada (NSERC). (poster)

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.