The aim of the present study is to provide an ultrastructural description of the major placentalabnormalities occurred in ovine somatic ce11 clones deriving fiom the injection of granulosa cellsinto enucleated oocytes.At the gross examination, an extensive placental degeneration was found in the three clonesdelivered stillborn at term.By means of histological and irnmunohistochemical analysis (vWF, calponin, smooth muscleactin, desmin, CD34), these features were noted in cloned samples: reduced villousI vascularization, hypoplasia of trophoblastic epithelium, lack of binucleate cells, irnmaturity of Iplacenta1 vessels (reduced expression of von Willebrand Factor and calponin) and reduced lvasculogenesis (scarce CD34 immunolabelling).For the ultrastructural studies, semithin and ultrathin sections'were obtained fiom control andlcloned samples fixed in 2.5% gluteraldehyde, postfixed in 1% Oso4 in cacodylate buffer andembedded in epoxy resin. On semithin sections, the effect of fixation on cellular shrinkage andithe villous vascularization were evaluated using a digital image system. The differenceconceming the vessel number/mm2 (control vs. clone = 726,9 vs. 193,66), the capillariesnumber/mm2 (541,36 vs. 140,61), the vessels total arealvillous stroma area (0,31 vs. 0,041) and Ithe capillaries total area/villous stroma area (0,345 vs. 0,0088) was very high. TEM analysisconftrrned the morphological abnormalities of trophoblastic epithelium and villous capillarieslIrevealed at light microscopy. Moreover, a diffuse thickening and a larnellated appearance ofsubtrophoblastic basement membrane (SBM) were noted in cloned placentae and the former wasquantified on digitized images at final magnification of 12,000~b y means of two methodsl Ii(simplified and detailed). The two methods were equally eficient for SBM thickness estimation(p > 0.05) and the difference of SBM thickness was statistically significant (p < 0.05), with a imean value of 0,7 pm for the control placentae and 0,19 pm for cloned ones. Anotherultrastnictural feature in cloned placentae was the thickening of capillaries basa1 lamina.These results led us to hypothesize, through an autoamplification model, that the abnormal 1 !vascularization and the thickening of basement membranes, probably related to genetic factors,were the main reasons of the reduced rate of fetal oxygen and nutrient uptake. This couldexplaine the increase in fetal losses occurring after somatic cells nuclear transfer.[...]

Qualitative and quantitative ultrastructural examination of ovine somatic cell clones placentae

PALMIERI, CHIARA;LOI, Pasqualino;PTAK, Grazyna;DELLA SALDA, Leonardo
2005-01-01

Abstract

The aim of the present study is to provide an ultrastructural description of the major placentalabnormalities occurred in ovine somatic ce11 clones deriving fiom the injection of granulosa cellsinto enucleated oocytes.At the gross examination, an extensive placental degeneration was found in the three clonesdelivered stillborn at term.By means of histological and irnmunohistochemical analysis (vWF, calponin, smooth muscleactin, desmin, CD34), these features were noted in cloned samples: reduced villousI vascularization, hypoplasia of trophoblastic epithelium, lack of binucleate cells, irnmaturity of Iplacenta1 vessels (reduced expression of von Willebrand Factor and calponin) and reduced lvasculogenesis (scarce CD34 immunolabelling).For the ultrastructural studies, semithin and ultrathin sections'were obtained fiom control andlcloned samples fixed in 2.5% gluteraldehyde, postfixed in 1% Oso4 in cacodylate buffer andembedded in epoxy resin. On semithin sections, the effect of fixation on cellular shrinkage andithe villous vascularization were evaluated using a digital image system. The differenceconceming the vessel number/mm2 (control vs. clone = 726,9 vs. 193,66), the capillariesnumber/mm2 (541,36 vs. 140,61), the vessels total arealvillous stroma area (0,31 vs. 0,041) and Ithe capillaries total area/villous stroma area (0,345 vs. 0,0088) was very high. TEM analysisconftrrned the morphological abnormalities of trophoblastic epithelium and villous capillarieslIrevealed at light microscopy. Moreover, a diffuse thickening and a larnellated appearance ofsubtrophoblastic basement membrane (SBM) were noted in cloned placentae and the former wasquantified on digitized images at final magnification of 12,000~b y means of two methodsl Ii(simplified and detailed). The two methods were equally eficient for SBM thickness estimation(p > 0.05) and the difference of SBM thickness was statistically significant (p < 0.05), with a imean value of 0,7 pm for the control placentae and 0,19 pm for cloned ones. Anotherultrastnictural feature in cloned placentae was the thickening of capillaries basa1 lamina.These results led us to hypothesize, through an autoamplification model, that the abnormal 1 !vascularization and the thickening of basement membranes, probably related to genetic factors,were the main reasons of the reduced rate of fetal oxygen and nutrient uptake. This couldexplaine the increase in fetal losses occurring after somatic cells nuclear transfer.[...]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11575/1840
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