Expression of the Ets family of transcription factors in early bovine and ovine embryo development Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/j3860977w

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  • Maternally-derived transcripts and proteins support early bovine and ovine embryo development until the 8- to 16-cell stage, at which time embryonic transcripts become essential for continued development. One purported mechanism for the switch from maternal to zygotic control of development (maternal to zygotic genome activation; MZGA) is the appearance of transcription factors that activate specific genes in the embryonic genome. Members of the E26 transformation specific (Ets) family are unique transcription factors involved in development, differentiation, and protease regulation. This study was undertaken to evaluate expression and function of the Ets transcription factors, Ets-1, Ets-2, and Elf-1, in early bovine and ovine embryos from the one-cell stage to Day 15 of pregnancy (Day 0 onset of estrus). In the first experiment, bovine embryos from the one- to 16-cell stages were derived by in vitro maturation, fertilization, and culture. Days 6, 8, 10, 12, and 14 embryos were collected nonsurgically from estrous synchronized and superovulated cows. RNA was extracted at the appropriate time interval and reverse transcribed. The resultant cDNA was amplified by PCR using primers designed for Ets-1, Elf-1, and Ets-2. Ets-1 transcripts were present in both primary and matured oocytes, cleavage stage embryos, and Days 10, 12, and 14 embryos, as well as in the positive control, bovine ovary. Elf-1 transcripts were detected in the matured oocyte, cleavage stage embryos, and Days 6, 10, and 14 embryos. Ets-2 transcripts were not observed in the embryonic stages investigated or the bovine ovary. Ovine embryos were surgically collected from synchronized and superovulated ewes and similarly analyzed for Ets-1 and Elf-1 expression using the same RNA extraction and RT-PCR technique. Embryos expressed both transcripts at Days 13 and 15, but did not show expression at any of the earlier stages evaluated. The second experiment was designed to determine if inhibition of ETS-1 translation would interfere with development and plasminogen activator (PA) production in bovine embryos. Plasminogen activator production was evaluated in Days 5 and 6 embryos nonsurgically collected from superovulated cows and cultured in 1, 2.5, 5, or 10 μM concentrations of sense or antisense Ets-1 oligonucleotides. In preliminary experiments, 1 μM antisense was ineffective in suppressing PA production, and 10 μM oligonucleotides were detrimental to development. Day 5 embryos treated with 2.5 μM oligonucleotides inhibited developmental effect and total PA production was (P<0.05) lower in antisense treatments when compared to either control or sense treatments. No difference (P>0.10) in PA production was observed between Day 6 embryos treated with 2.5 or 5 μM sense and antisense oligonucleotides. A significant time effect on PA production was observed in both Day 5 and Day 6 embryos cultured in either 2.5 or 5 μM concentrations of oligonucleotides. Based on these results, it is unlikely that Elf-1 and Ets-2 are involved in MZGA because the former is constituitively expressed throughout development, and the latter was not observed. There is some uncertainty regarding the expression of Ets-1 during MZGA. This factor may be expressed after MZGA for controlling PA production and other proteases involved in extracellular matrix turnover and early germ layer formation.
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