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 ��M concentrations of sense or antisense Ets-1
oligonucleotides. In preliminary experiments, 1 ��M antisense was ineffective in
suppressing PA production, and 10 ��M oligonucleotides were detrimental to
development. Day 5 embryos treated with 2.5 ��M 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 ��M 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 ��M 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. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/32048 |
| Date | 10 June 2002 |
| Creators | Collins, Jonna Erin |
| Contributors | Menino, Alfred R. Jr |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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