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
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/31845 |
| Date | 27 November 2002 |
| Creators | CoreyAyne, Singleton |
| Contributors | Menino, Alfred R. Jr |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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