Naslov
De novo DNA methylation dynamics and cell cycle regulators Rb and p53 expression in testicular development of human, mouse and rat.

Autori
Himelreich Perić, Marta

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

Izvornik
Knjiga sažetaka - Dan doktorata / - , 2019, 125-125

Skup
Dan doktorata

Mjesto i datum
Zagreb, Hrvatska, 24.05.2019

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
testis, development, de novo DNA methylation, quiescence, human, rat, mouse

Sažetak
Introduction Disturbances of the complex prenatal testicular development are a possible cause of male reproductive system disorders, diagnosed however later in adult life, such as infertility and testicular tumors. It is known that critical epigenetic events like DNA de novo methylation (sex-specific imprinting and retrotransposonal sequences) in male germ cells take place in the fetal period, suggesting its importance for germ cell homeostasis in adult testis. Importantly, de novo DNA methylation takes place in quiescent germ cells, arrested in G1 phase of the cell cycle, which is thought to be caused by p53 and/or Rb signalling pathways. Hypothesis De novo DNA methylation in the fetal testes is concomitant with the expression of negative cell cycle regulators pRb and p53 during late gestation in human, mouse and rat. Aims The aim of this study is to systematically analyse and compare the cell cycle, global methylation and imprinting dynamics of male germ cells in developmental stages of human, mouse and rat testis. For the first time, time correlation of an expression of Rb and p53 to DNA methylation will be analysed in human samples. 1. Determine global DNA, H19 and Rasgrf1 gene methylation level in different developmental stages of fetal testicular tissue samples in human and complementary mouse and rat developmental stages. 2. Determine gene expression dynamics and epigenetic modifications of cell cycle regulators pRb and p53, with their phosphorylated forms on protein and mRNA level in the same samples. 3. Investigate whether silencing of Rb and p53 gene via siRNA affects germ cell quiescence period that starts in the late pregnancy of mice and rat in in vitro culture. Materials and methods The study will be conducted on formalin-fixed, paraffin-embedded archive human fetal samples, 14.-35. week of pregnancy, a prepubertal sample ; mouse and rat fetal testes samples isolated from pregnant animals in a timeline of the second trimester to the juvenile postnatal period. An in vitro testis organoid culture will be established to investigate siRNA-Rb effect on the germ cell fate analysing the proliferation , apoptosis, de novo DNA methylation). Formalin-fixed, paraffin-embedded (FFPE) samples will be accessed by histological, immunohistochemical/immunofluorescence and sterological methods. Frozen samples from in vitro culture will be stored on -80 °C and together with FFPE, used for molecular diagnostics – DNA methylation, pyrosequencing, DNA isolation and gene expression by ddPCR method. Expected scientific contribution The results of the study will contribute in validating the animal model in investigation of factors that affect the development and function of testis and possibly in clarifying causes of male infertility and/or germ cell neoplasms.

Izvorni jezik
Engleski

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