42 ANGIOGENESIS IN CLONED AND IVF-DERIVED BOVINE PREGNANCIES AT DAY 30 OF GESTATION

Abstract

New and expanded microvascular networks in maternal and embryonic tissue are vital to development of a functional placenta. Typically a large proportion of bovine pregnancies derived from somatic cell nuclear transfer (NT) are lost between Days 25 and 45 of gestation. Several laboratories have noted aberrant placental development of NT embryos and fetuses; one factor that may contribute to these defects is faulty maternal or embryonic angiogenesis during placentation. The aim of this study was to determine if angiogenesis in maternal caruncular tissues differs in bovine NT- and IVF-derived pregnancies at the time of placentation. Cloned embryos were produced using cultured ear skin fibroblasts from a 15-year-old Hereford cow; in other experiments, these donor cells resulted in a term live calf and 67% of viable Day-30 embryos dying by Day 51 of gestation. For IVF-derived embryos, IVM oocytes were fertilized with semen from Hereford or Angus bulls. All embryos were cultured in vivo in ligated sheep oviducts, and at Day 8 blastocysts were transferred to synchronized recipient heifers. Whenever possible, one embryo was transferred to each uterine horn. A total of 41 NT and 41 IVF grade 1, 2 and 3 embryos were transferred to 48 recipients. Viable NT (n = 9; 22.0% of transferred) and IVF (n = 9; 22.0%) embryos were recovered at Day 30, and caruncular tissue adjacent to each embryo was sampled. Expression of genes implicated in angiogenesis was measured by real-time quantitative RT-PCR and normalized to histone 2A expression. In addition, paraformaldehyde-fixed sections were stained by the Periodic Acid-Schiffs method to identify blood vessels. Microvascular density was determined as percentage of total tissue area composed of blood vessels in the luminal caruncular stroma as measured by two observers. Data were analyzed by ANOVA using the GLM procedure of SAS (SAS Institute, Cary, NC, USA). Quantification of gene expression revealed no differences between NT and IVF pregnancies for angiogenesis-promoting growth factors and their receptors: vascular endothelial growth factor-A (VEGF-A), VEGF-C, placental growth factor, VEGF receptor-1 (VEGFR-1/flt-1), VEGFR-2 (flk-1/KDR), VEGFR-3 (flt-4), angiopoietin-1 (Ang-1), and Ang receptor-1 (tie-1). Likewise, NT and IVF pregnancies demonstrated similar mean microvascular densities in the caruncles; however, density differed between individual pregnancies across both groups (P < 0.05). For these experiments, the timing of NT and IVF, embryo transfer, and tissue collection on Day 30 was as consistent as possible. Despite these efforts, embryo morphology and developmental stage were highly variable even within the IVF group. It follows that the progress of placentation, and hence the signaling between each embryo and the maternal tissues, may have differed as well, accounting for the variation observed. Our results suggest that failure of maternal tissue to increase angiogenesis during placentation is not a primary cause of aberrant placental development in cloned cattle.