Germ cell quality is a key prerequisite for successful fertilization and early embryo development. The quality is determined by the fine regulation of transcriptomic and proteomic profiles, which are prone to alteration by assisted reproduction technology (ART)-introduced in vitro methods. Gaining evidence shows the ART can influence preset epigenetic modifications within cultured oocytes or early embryos and affect their developmental competency. The aim of this review is to describe ART-determined epigenetic changes related to the oogenesis, early embryogenesis, and further in utero development. We confront the latest epigenetic, related epitranscriptomic, and translational regulation findings with the processes of meiotic maturation, fertilization, and early embryogenesis that impact the developmental competency and embryo quality. Post-ART embryo transfer, in utero implantation, and development (placentation, fetal development) are influenced by environmental and lifestyle factors. The review is emphasizing their epigenetic and ART contribution to fetal development. An epigenetic parallel among mouse, porcine, and bovine animal models and human ART is drawn to illustrate possible future mechanisms of infertility management as well as increase the awareness of the underlying mechanisms governing oocyte and embryo developmental complexity under ART conditions.

• Klíčová slova
• embryo development, epigenetics, oocyte maturation, protein translation,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

DNA double-strand breaks (DSBs), marked by ionizing radiation-induced (repair) foci (IRIFs), are the most serious DNA lesions and are dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold-standard method in radiation biodosimetry and allows research on DSB induction and repair at the molecular and single-cell levels. In this study, we introduce DeepFoci - a deep learning-based fully automatic method for IRIF counting and morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation. The proposed method was trained and tested on challenging datasets consisting of mixtures of nonirradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDFs, U-87) and primary cell cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 0.5-8 Gy γ-rays and fixed at multiple (0-24 h) postirradiation times. Under all circumstances, DeepFoci quantified the number of IRIFs with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software maintained its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This approach allowed multiparameter IRIF categorization of single- or multichannel data, thereby refining the analysis of DSB repair processes and classification of patient tumors, with the potential to identify specific cell subclones. The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to advanced DSB focus analysis and, in turn, a better understanding of (radiation-induced) DNA damage and repair.

• Klíčová slova
• 53BP1, P53-binding protein 1, Biodosimetry, CNN, convolutional neural network, Confocal Microscopy, Convolutional Neural Network, DNA Damage and Repair, DSB, DNA double-strand break, Deep Learning, FOV, field of view, GUI, graphical user interface, IRIF, ionizing radiation-induced (repair) foci, Image Analysis, Ionizing Radiation-Induced Foci (IRIFs), MSER, maximally stable extremal region (algorithm), Morphometry, NHDFs, normal human dermal fibroblasts, RAD51, DNA repair protein RAD51 homolog 1, U-87, U-87 glioblastoma cell line, γH2AX, histone H2AX phosphorylated at serine 139,
• Publikační typ
• časopisecké články MeSH

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

• MeSH
• chromatin účinky léků   genetika   MeSH
• dimethylsulfoxid farmakologie   MeSH
• dvouřetězcové zlomy DNA účinky léků   MeSH
• fibroblasty MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• kůže cytologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• S fáze účinky léků   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• zmrazování škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• chromatin MeSH
• dimethylsulfoxid MeSH
• kryoprotektivní látky MeSH