Somatic cell nuclear transfer in the sheep induces placental defects that likely precede fetal demise

Abstract

The efficiency of cloning by somatic cell nuclear transfer (SCNT) is poor in livestock with w5% of transferred cloned embryos developing to term. SCNT is associated with gross placental structural abnormalities. We aimed to identify defects in placental histology and gene expression in failing ovine cloned pregnancies to better understand why so many clones generated by SCNT die in utero. Placentomes from SCNT pregnancies (nZ9) and age matched, naturally mated controls (nZ20) were collected at two gestational age ranges (105-134 days and 135-154 days; termZ147 days). There was no effect of cloning on total placental weight. However, cloning reduced the number of placentomes at both gestational ages (105-134 days: control 55.0G4.2, clone 44.7G8.0 and 135-154 days: control 72.2G5.1, clone 36.6G5.1; P!0.001) and increased the mean individual placentome weight (105-134 days: control 10.6G1.3 g, clone 18.6G2.8 g and 135-154 days: control 6.6G0.6 g, clone 7.0G2.0 g; P!0.02). Placentomes from cloned pregnancies had a significant volume of shed trophoblast and fetal villous hemorrhage, absent in controls, at both gestational age ranges (P!0.001) that was shown to be apoptotic by activated caspase-3 immunoreactivity. Consequently, the volume of intact trophoblast was reduced and the arithmetic mean barrier thickness of trophoblast through which exchange occurs was altered (P!0.001) at both gestational age ranges in clones. In addition, cloning reduced placental expression of key genes in placental differentiation and function. Thus, cloning by SCNT results in both gross and microscopic placental abnormalities. We speculate that trophoblast apoptosis, shedding, and hemorrhage may be causal in fetal death in ovine clones. Reproduction (2007) 133 243-255

Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium

“…Thus, multiple lines of experimental evidence have led to the conclusion that EET is the origin of the developmental pathologies in placentation of SCNTderived clones (17,(22)(23)(24)(25)(26). Therefore, study of the communication between cloned conceptuses and the endometrium should yield important insights into the mechanisms underlying average losses of ∼60% of cloned embryos before implantation and 80% of fetuses within the first trimester of gestation (27,28).…”

Review Paper: A Review of the Pathology of Abnormal Placentae of Somatic Cell Nuclear Transfer Clone Pregnancies in Cattle, Sheep, and Mice

“…56 Placental vasculogenesis was significantly reduced as indicated by lack of CD34 expression by individual foetal blood vessels, 79 similar to the abnormal placental vasculogenesis observed with spontaneous abortions in humans. 101 In both ovine and bovine SCNT pregnancies, some investigators report that reduction in villous vascularization is accompanied by hypoplasia of trophoblastic epithelial cells, apoptosis and shedding of trophoblastic epithelial cells from foetal villi, 28 and/or reduced numbers of the trophoblastderived binucleate cells. 28,39,64,79 In these studies, caruncular and cotyledonary tissues were collected at 60 days of gestation from slaughtered cattle carrying SCNT motile and immotile embryos 39 and in sheep pregnancies at term with alive and healthy conceptus 28,64,79 or within 2 to 24 hours of fetal death.…”

“…gestation and characterized by an increase in fetal, maternal, and binucleate cell numbers. 85 In sheep, reduced amounts of IGF2 mRNA were observed from 105 to 154 days of gestation in SCNT placentae, 28 which could promote trophoblast cell apoptosis, inasmuch as IGF2 has high antiapoptotic activity in the pancreatic islet cells of neonatal rats. 83 Transforming growth factor-betas (TGF-Bs) are highly conserved growth factors controlling cell growth and differentiation and tissue remodeling during menstruation, proliferation, decidualization, and establishment of pregnancy through opposite promotory and inhibitory effects.…”

“…Placental gene expression, which is reduced in ovine SCNT placentae at 105 to 154 days of gestation, includes the glucose transporters GLUT1, GLUT3, and GLUT8, with subsequent reduced fetal plasma glucose concentration after 135 days of pregnancy. 28 The underlying cases of SCNT placental pathology are uncertain but usually appear to involve inefficient epigenetic reprogramming of the somatic cell genome by the oocyte cytoplasm. 53,73 Such reprogramming is necessary for a somatic cell nucleus to reacquire totipotency and become functionally equivalent to that of a zygote, which requires reestablishment of the coordinated pattern of gene expression necessary for embryonic and fetal development.…”

Somatic cell reprogrammed by oocyte: process and barricade

“…Moreover, it should be noted that nuclear envelope was broken in MII oocyte, and MFs should disperse into ooplasm, so how enucleation can specifically remove nuclear Dnmt1o. Abnormal placentation has been described in several cloned species (Hill et al 2000;Tanaka et al 2001;Fletcher et al 2007;Wei et al 2010), and this defect can be partly attributed to the abnormalities in the expression and epigenetic modification of imprinted genes. The first evidence is that some genes are only imprinted in placenta, not in fetus, such as Slc22a2, Slc22a3, and Ascl2 in mouse genome (these imprinted genes were named placentalspecific imprinted gene in this review), so placenta has more imprinted genes than fetus (Hudson et al 2010).…”