Vascularization and VEGF expression altered in bovine yolk sacs from IVF and NT technologies.

ABSTRACT

Reproductive technologies are widely used in cattle, although many are associated with high-embryonic mortality, especially during early gestation, when the yolk sac undergoes macroscopic changes in structure. We hypothesized that vasculogenesis and angiogenesis are affected, thereby affecting embryonic and placental differentiation. To test this, we studied yolk sac development and gene expression of the vascular endothelial growth factor system (VEGF-A, VEGFR-1/Flt-1, VEGFR-2/KDR). Samples from Days 25 to 40/41 of pregnancy from control cattle (n = 8) and from pregnancies established with IVF, (n = 7) or somatic cell nuclear transfer/clones (n = 5) were examined by histology, immunohistochemistry, and quantitative reverse transcriptase PCR. Yolk sacs in IVF- and nuclear transfer-derived pregnancies were immature. Development of villi was sparse in IVF yolk sacs, whereas vascularization was barely formed in clones and was associated, in part, with thin or interrupted endothelium. Transcript levels of the genes characterized exceed minimum detection limits for all groups, except in the mentioned clone with interrupted endothelium. Levels of mRNA for VEGF-A and VEGFR-2 were significantly higher in IVF yolk sacs. Clones had substantial individual variation in gene expression (both upregulation and downregulation). Our data confirmed the broad range in expression of VEGF genes. Furthermore, overexpression in IVF yolk sacs may compensate for an immature yolk sac structure, whereas in clones, patchy expression may cause structural alterations of blood vessels. In conclusion, we inferred that disturbances of yolk sac vasculature contributed to increased early embryonic mortality of bovine pregnancies established with reproductive technologies.