Angiogenesis in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Abstract

Impaired placental angiogenesis during early pregnancy may result in placental defects that adversely affect development of nuclear-transfer (NT) embryos later in pregnancy. These experiments were designed to quantify and compare development of placental microvasculature and expression of genes associated with angiogenesis, including members of the VEGF and angiopoietin (Ang) families, in maternal and embryonic placental tissues of day 30 bovine concepti derived from NT or in vitro fertilization (IVF) followed by in vivo development to the blastocyst stage in the sheep oviduct. Microvascular volume density (MVD) within the caruncular tissues, as determined using Periodic Acid-Schiff's staining as well as immunohistochemical staining for von Willebrand's factor, was not different between NT- and IVF- derived pregnancies. Expression of genes implicated in angiogenic mechanisms, including VEGF-A and -C, placental growth factor (PlGF), VEGF receptors (Flt-1, Flk-1, and Flt-4), angiopoietin-1 (Ang1), Ang2, Tie1, Tie2, and hypoxia-inducible factor (HIF), were determined. In chorio-allantoic membranes, levels of PlGF transcripts were significantly lower in NT- than IVF-derived tissues (p<0.05), whereas HIF-1alpha transcription in chorio-allantoic membranes of cloned concepti was higher at p<0.10. Caruncular expression of HIF-1alpha and Ang1 also was increased in NT-derived pregnancies at p <or= 0.10. Immunohistochemical staining of caruncular tissues for VEGF-A and the Flt-1 receptor revealed few differences in protein expression between NT- and IVF-derived pregnancies. These results indicate that expression of most angiogenic factors at day 30 of gestation is not altered as a result of the NT procedure; however, given reports of impaired placental vascular development in NT-derived bovine embryos, perturbations in angiogenesis may occur subsequently during early placental development and throughout gestation. Elevated expression of the HIF-1alpha gene in maternal and chorio-allantoic tissues of cloned concepti may suggest a generalized hypoxic condition in early placental tissues of NT-derived concepti, which could adversely affect subsequent development of the placenta.