39 PLACENTA ABNORMALITIES IN SHEEP SOMATIC CELL CLONES AT 20 DAYS OF GESTATION

Abstract

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, ranging from therapeutic cloning, production of transgenic animals, drug production, regenerative medicine and even for the rescue of endangered species. It is already more than 16 years since Dolly, first cloned mammal, was born, and many improvements in SCNT have been made by using different epigenetic and technical approaches but the efficiency is still disappointingly low. Only ~0.1 to 3% of reconstructed embryos develop to term. SCNT is associated with high rate of fetal, perinatal, and neonatal losses and production of abnormal offspring. Many factors have been implied in the pathogenesis of the NT fetal losses, but it seems that the highest incidence is placental abnormalities, confirmed by our first reports studied on full-term sheep placentas. Reports strongly suggest that post-mortality in cloned lambs is mainly caused by placental abnormalities such as placentomegaly, hypoplasia of trophoblastic epithelium, altered basement membrane, and reduced vascularisation. The pathogenesis of these changes is poorly understood, so here we analysed early sheep placenta (20 days) for better understanding of mechanisms involved. Blastocysts obtained by nuclear transfer of somatic cells (adult sheep fibroblasts) were transferred into recipient ewes. Naturally mated ewes were analysed as controls (CTR). Conceptuses were recovered at Day 20 of gestation. Then, part of their placentas were fixed for histological and transmission electron microscope (TEM) analysis; other parts of the tissues were snap frozen in liquid nitrogen for subsequent mRNA analysis. Expression of genes regulating vasculo- and angiogenesis (ANG1, ANG2, FGF-2, FGF-2R, VEGF, and Tie-2) as well as imprinted genes (IGF-2, H19, PHLDA-2) were analysed. Statistical analysis was performed using GraphPad software (GraphPad Software Inc., San Diego, CA, USA). Gene expression was analysed using the nonparametric Mann-Whitney test; P < 0.05 was considered significant. Expression of all analysed imprinted genes as well as vasculo- and angiogenesis factors in NT placentas at Day 20 of development were down-regulated (v. CTR). Histological analysis showed reduced vascularization and increased apoptosis in placental tissue. The observation of ultrathin sections confirmed placental abnormalities. Cells presented decreased density of mature mitochondria, high number of cytoplasmic vacuolization, numerous cytoplasmic vesicles and autophagosomes, less developed cells to cell junctions, and disruption of chorionic villi, being irregularly and sparsely arranged. The results of the present study indicate that the placental abnormalities affecting particularly the vascular compartment start from early stage of placenta development. Moreover, imprinted genes, which have been already shown to determine the transport capacity of the placenta by regulating its growth, morphology and nutrient abundance, were deregulated in NT early placentas. All those factor can lead to the developmental failure of cloned fetuses.