Abnormal expression of the imprinted gene Phlda2 in cloned bovine placenta

Cloning in mammals suffers from high rates of pregnancy losses associated with abnormal placentation, mainly placentomegaly, leading to fetal death. Placental growth is dependent on the regulated expression of many genes of which imprinted genes play a fundamental role. Among them, the Phlda2 gene is expressed from the maternal allele and acts to limit placental growth in mouse and human. Here we used Northern blots, quantitative RT-PCR and in situ hybridization to analyze the expression patterns of bovine PHLDA2 and to compare its expression levels in normal and somatic cell nuclear transfer (SCNT) placentas over a range of gestational stages. PHLDA2 is not expressed in extra-embryonic tissues before d32 of gestation but the level of expression increases throughout pregnancy until term in the placental villi collected from pregnancy obtained by artificial insemination (AI). At all stages of pregnancy, PHLDA2 mRNA are specifically localized in the trophoblast mononucleated cells contrasting with lack of expression in the binucleated cells and uterine tissues. In SCNT placentas, a similar pattern of expression was observed during early pregnancy. In contrast the level of expression is significantly reduced around d200 of gestation in the placental villi from pathological clones. The reduced expression of PHLDA2 was obvious particularly in the placental villi anchored within the uterine crypts with expression confined to the trophoblast of the chorionic plate. Altogether, these results highlight a similarity in expression patterns for PHLDA2 bovine and human where expression is localized to the trophoblast throughout pregnancy and parallels the continuous growth of the placenta. Moreover, the lack of expression in the fetal villi from oversized bovine cloned placenta is consistent with the function of PHLDA2 in restraining placental growth and underlines an aberrant expression of this gene after somatic cloning.