During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix (ECM), located on the blastocoelic side of the trophectoderm, to form a continuous layer of extraembryonic endoderm. Cell migration events depend on a family of cell surface proteins known as integrins that bind specific ECM proteins. In an effort to understand the mechanisms involved in bovine endodermal cell migration, two experiments were conducted. In the first experiment, expression of the ECM proteins fibronectin, laminin and vitronectin was evaluated by immunofluorescent staining in in vivo and in vitro developing embryos during Day 6-10 and Day 7-10, respectively (Day 0=onset of estrus). Fibronectin was detected in all stages of in vivo and in vitro embryos, however no difference (P>0.10) was observed due to day or developmental stage. Laminin staining was moderately expressed in all stages of in vivo embryos, with an increase (P<0.05) in Day 10 in vivo embryos. Laminin staining in Day 9 in vitro embryos was less intense (P<0.05) than Day 7 and 8 in vitro embryos. Higher (P<0.05) expression of laminin was observed in Day l0 in vivo embryos as compared to Day 10 in vitro. Vitronectin staining was expressed throughout all stages of development. Day 6 in vivo embryos exhibited more intense (P<0.05) staining compared to Day 8 in vivo embryos. Day 10 in vivo embryos expressed more (P<0.05) vitronectin than Day 10 in vitro embryos. In the second experiment, the effects of ECM-type and inhibitors of integrin binding on bovine endodermal cell outgrowth from the ICM were evaluated. Day 7 embryos were nonsurgically collected and cultured for 96 h on either fibronectin-layered microdrops containing 0 (control), 0.5 or 1.0 mg/ml RGD and/or EILDV peptides or vitronectin-layered microdrops containing 0, 0.5 or 1.0 mg/ml RGD peptides. At 24-h intervals, ICM were photographed and the numbers of cells leaving the ICM were counted. Areas of cellular outgrowth were calculated from the photomicrographs. Compared to the control, addition of 0.5 or 1.0 mg/ml RGD, EILDV or RGD and EILDV did not (P>0.10) reduce the areas of cellular outgrowth from the ICM on matrices of fibronectin. Numbers of cells in outgrowths were greater (P<0.05) in control ICM compared to 0.5 mg/ml RGD, but this effect was eliminated (P>0.10) when the inhibitor concentration was increased to 1.0 mg/ml. Addition of 0.5 or 1.0 mg/ml RGD did not reduce (P>0.10) the area of cellular outgrowth from the ICM on vitronectin and had no effect (P>0.10) on numbers of cells in the outgrowths. Detection of fibronectin, laminin and vitronectin by immunofluorescence suggests these proteins are present in the developing bovine embryo to support endodermal cell migration and stabilization in extraembryonic endoderm formation. Because cell migration over fibronectin and vitronectin was not inhibited by the RGD and EILDV peptides, endodermal cells must use either an integrin that recognizes alternative binding sites in fibronectin and vitronectin or an alternative cell adhesion system. / Graduation date: 2003

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

Graduation date: 1996