The Impact of 5-Azacytidine on Placental Weight, Glycoprotein Pattern and Proliferating Cell Nuclear Antigen Expression in Rat Placenta

Abstract

During the placentation process, the expression of various glycoproteins plays an important role in embryonal development. Alterations in DNA methylation caused by 5-azacytidine (5azaC) can disturb normal glycoprotein expression as well as the proliferative ability of trophoblast cells. In order to assess this, a single dose of 5azaC was injected intraperitoneally into pregnant rats during days 1–19 of gestation. Animals were euthanised on day 20 and placental weight, as well as glycoprotein composition, was analysed together with immunohistological assessment of the degree of proliferation of the trophoblast cells. The placental weight was found to be significantly smaller in animals treated by 5azaC during days 4 to 14 of gestation (p < 0.01, Student's t-test). The treatment on days 4, 5, and 6 resulted in a lack of labyrinth with the strong proliferative activity of the cells in the basal layer. Expression of glycoproteins with molecular mass smaller than 60 kDa was reduced with treatment on day 6. The 5azaC administered from days 7 to 10 completely disturbed the placental structure and the proliferation of trophoblast cells was poor. During these days GP70 exhibited stronger expression in treated animals, contrary to GP40, which was stronger in controls. A natural border between the labyrinth and the basal layer was established on days 11 and 12. The basal layer was dominant with a lower proliferation of trophoblast cells compared with the controls. With the establishment of the labyrinth on day 13, the expression of GP40 was restored. Proliferation of the trophoblast cells from days 13 to 15 was higher compared with the controls. The changes in placental mass and the proliferative ability of trophoblast cells in rat placenta exposed to 5azaC represent more proof of the importance of epigenetics in the regulation of placental development.

Introduction

Normal embryonic human development is considered as one of contemporary medicine's hottest topics. It is impossible to imagine such development without the synchronised cooperation of the embryo and placenta [1].

The rat placenta represents one of the most convenient models for study of the molecules and their interactions in the processes of human implantation and placentation. Rat placenta resembles the human one in many respects [2]. While the human placenta is villous, the rodent placenta is of the labyrinth type, but both of them are haemochorial [3].

Any deviation in gene expression can bring about significant changes in the placenta, which are potentially important for ongoing pregnancy [4]. Considering mammals, gene expression in most cases is epigenetically regulated [5]. The modification of the mammalian DNA molecule may consequently change the gene expression at the level of transcription and is called DNA methylation [6]. Presently, numerous genes whose methylation appears to be crucial to the normal development of mammalian placenta are known. Mash2 is one of them. It is responsible for coding the transcription factor and it is indispensable for the maintenance of trophoblast stem cells [7]. The methylation process is of significant importance for the development of the rat placenta. The placental basal layer displays different patterns of methylation from the labyrinth layer, suggesting that the normal differentiation of placenta is regulated by precise DNA methylation mechanisms [8].

In order to investigate the influence of hypomethylation on the development of rat placenta we used demethylating agent 5-azacytidine (5azaC). 5azaC inhibits post-replication methylation by its incorporation into DNA causing subsequent inhibition of DNA methyltransferase and loss of methylation followed by the change in gene expression. In this study we analysed possible changes in placental weight, the proliferative ability of trophoblast cells and glycoprotein expression in the rat. All of this was performed while bearing in mind that the glycoprotein expression pattern in human placenta is consistent with a potential role in implantation and placentation [9].