Maternal-fetal interactions and angiogenesis in early bovine pregnancies derived from nuclear transfer

The high incidences of embryonic mortality and placental abnormalities reported by researchers studying nuclear transfer (NT) appear to be related. Early NT-derived bovine concepti exhibit reduced placental vascularity and poor structural organization of placentomes. These experiments were designed to identify alterations in maternal-fetal interactions during establishment of the placentas of NT-derived embryos at Day 30 of gestation. Bovine NT embryos were produced using cultured fibroblast cells from a Hereford donor cow, and control embryos were derived from in vitro fertilization (IVF). Following in vivo culture, Day-8 blastocysts were transferred to synchronized recipient heifers. Tissues recovered from viable Day-30 pregnancies were analyzed by real-time RT-PCR, immunohistochemistry, and quantitative histological techniques. Immunoperoxidase staining of caruncular tissues from NT- and IVF-derived pregnancies revealed no significant differences in expression of extracellular matrix proteins (collagen type IV and laminin) or integrin subunits alpha 1 and alpha3, suggesting that altered expression of these proteins at Day 30 does not cause abnormal placentome structure in cloned concepti. Percentage of binucleate cells (BNC) within the trophoblast also was similar; however, expression of the BNC-specific placental lactogen (PL) transcript was elevated in NT-derived concepti (P < 0.05), indicating that regulation of PL transcription was altered in cloned placentas. Determination of microvascular density in caruncular endometrium revealed no difference in the vascular morphology of NT- and IVF-derived pregnancies. Likewise, expression of many genes implicated in the regulation of angiogenesis, including vascular endothelial growth factor (VEGF)-A and -C, VEGF receptors Flt-1, Flk-l, and Flt-4, and angiopoietin-2 and its receptors Tie1 and Tie2, was not different in the extraembryonic membranes or caruncles of cloned and IVF-derived concepti. In contrast, expression of the placental growth factor gene was elevated (P < 0.05) in cloned extraembryonic membranes, and expression of angiopoietin-1 tended (P < 0.10) to be higher in caruncular tissues from cloned pregnancies. Transcripts of hypoxia-inducible factor-1alpha tended (P < 0.10) to be higher in both extraembryonic membranes and caruncles of NT-derived pregnancies. These results suggest the presence of relatively hypoxic conditions in the placental tissues of Day-30 cloned concepti, which subsequently could alter regulation of placental vascular development, undermining continued development of NT-derived concepti.