Naslov
Impact of 5-azacytidine on placentas in pre- and postgastrulating stages of development

Autori
Šerman, Ljiljana ; Sinčić, Nino ; Vlahović, Maja ; Bulić-Jakuš, Floriana ; Jurić-Lekić, Gordana ; Katušić, Ana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Zbornik radova - 2.hrvatski mikroskopijski kongres / Gajović, Srećko - Zagreb : Hrvatsko društvo za elektronsku mikroskopiju, 2006, 155-156

Skup
2nd Croatian Congress on Microscopy with International Participation

Mjesto i datum
Topusko, Hrvatska, 18.05.2006. - 21.05.2006

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
PCNA; placenta; 5-azacytidine

Sažetak
DNA methylation represents one of the major epigenetic mechanisms of gene expression regulation. Methylation of mammalian genome correlates with gene expression in a way that tissue-specific genes are mostly methylated in every tissue where they are not expressed, while unmethylated in their tissue of expression. Moreover, the process of cytosine methylation at CpG sequences appears to be essential for normal mammalian development (1). Specific methylation pattern of differential gene activation has to be established very early during the period of embryonic development, and it is stably inherited from one cell generation to the next (2). 5-azacytidine (5azaC) a known demethylating agent is able to inhibit postreplication methylation by its incorporation into DNA which causes subsequent inhibition of DNA cytosine methyltransferase and loss of methylation followed by a change in gene expression. Compared to somatic tissue both oocyte and sperm genomes are undermethylated, with much heavier methylation of sperm DNA. After fertilization, the extent of methylation remains constant during early cleavages. In an 8-cell embryo the extent of DNA methylation corresponds to a mixture of undermethylated maternal and methylated paternal DNA. Towards the blastocyst stage there is a global decrease in methylation. Following implantation, there is a huge increase in methylation due to global de novo DNA methylation, affecting the whole embryo (3). As well as embryos, placentas are subjected to methylation pattern changes during their development (4), as shown by our previous results (5). During development of rat placenta the formation of ectoplacental cone derived from polar trophoblast cells at day 8 of gestation, and onset of early placental function which starts at day 11 are considered to be crucial for normal fetal development. Single dose of 5azaC (5mg/kg body weight) was administered to pregnant Fischer rats on days 8 (before onset of gastrulation) or 11 (after gastrulation) of gestation. Animals were sacrificed on day 20 of pregnancy and placentas were measured and analyzed by classical histological methods (HE) and by indirect immunohystochemistry. Monoclonal Mouse Anti-PCNA, Clone PC 10, (M 0879, DAKO), was diluted to 1:50. Negative control was a standard negative reagent (V 1617, DAKO). Samples were treated with primary antibodies for 15 minutes and washed. DAKO Animal Research Kit, (Peroxidase) was used for detection of monoclonal mouse anti-PCNA antibody. In all treated animals significantly smaller placentas were found. The 5azaC treatment at day 8 of gestation completely upset the placental structure. There was no clearly visible border between labyrinth and basal layer. On the other hand application of 5azaC at day 11 demonstrated recurrent establishment of natural border between those two layers, although compared to controls labyrinth wass significantly reduced with predominant basal layer (Fig.1). At day 8 of gestation positive intranuclear PCNA signal was detected in only few cells of the basal plate. In contrast to this finding, in day 11 placentas PCNA was expressed in a much higher number of cells but not in as many as in controls.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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