Morphological and functional characterization of placenta during gestation in bovine clones derived by somatic nuclear transfer

Although somatic cell nuclear transfer (SCNT) has been shown to be successful, there are still problems with this technique that is inhibiting its use in industry.  Altered placental formation and development results in the high incidence of pregnancy loss and prenatal morbidity and mortality found in SCNT pregnancies.   The objective of the initial study was to characterize morphological changes in fetal membranes of a group of bovine recipients carrying cloned fetuses and comparing them with control AI fetuses at various stages of gestation. Several morphological anomalies of placentomes such as edematous chorioallantoic membrane, hyper-echodense spikes or irregularities in the amniotic membrane as well as the pathological presence of degenerated inflammatory cells accompanied by disappearance of the placental epithelium were observed. It was concluded that these anomalies compromise fetal development. The results of this work also showed that ultrasonography may be a reliable technique to monitor and to characterize the placental changes in bovine pregnancies that can be used to assess fetal well-being.  The next study compared the functionality of the placenta of cloned fetuses with controls. The concentration of three steroids (progesterone (P4), estrone sulphate (E1S), and estradiol (E2)) and pregnancy-specific protein B (PSPB) in maternal peripheral circulation were assessed and their associations with gestational anomalies were determined. The hormones profiles in the SCNT recipients deviated from the control group at certain stages of pregnancy.  We observed higher concentrations of E2 throughout the study period, lower levels of P4 at day 80 as well as elevated PSPB concentrations at day 150 in SCNT recipients which coincided with high rate of abortion in these animals shortly after this stage. So, it is proposed that these hormonal changes together with the morphological anomalies of the placenta result in compromised fetal development.  Finally, the molecular mechanism that could be responsible for the abnormal disappearance of the epithelial layer observed in SCNT placenta was investigated. To do so, we measured the expression of two major epithelial adherens junction proteins (E-cadherin and β-catenin) and determined if their expression is altered in relation to the aberrant placentation in SCNT embryos. Cotyledonary tissues from SCNT gestations and control pregnancies were analyzed by Western blot, quantitative RT-PCR and immunohistochemical analysis. Both candidates were significantly (P < 0.05) under-expressed in SCNT trophoblast cells at the protein level. Also, qRT-PCR confirmed that the Wnt/β-catenin signaling pathway target genes CCND1, CLDN1 and MSX1 were significantly down-regulated in SCNT placentas. So, we inferred that impaired E-cadherin and β-catenin protein expression, along with defective β-catenin signaling during embryo attachment, specifically in the window of placentation, results in loose attachment and contributes to insufficient placentation in bovine SCNT-derived embryos.  Overall, we concluded that during the high-risk pregnancy of cloned fetuses, characterization of the morphological and functional changes of the placenta is critical to enable us to predict normal fetal development and wellbeing through a standardized procedure during clone gestations and to intervene medically in emergency cases to improve the overall efficiency of cloning in cattle.

Keyphrases: Bovin, Clone, Bovine, Pregnancy monitoring, Cloned, Hormone profile, Adhesion molecules