Mature mammalian oocytes arrest meiosis in metaphase II (MII). If the oocyte is not fertilized, it can spontaneously break the MII arrest. Spontaneous activation and postovulatory aging hinder precisely timed and regulated embryonic development. To elucidate the role of Src family protein tyrosine kinases (SFKs) in porcine oocyte MII arrest, activation, and aging, we used a specific SFK inhibitor and immunolocalization. The 24h-prolonged oocyte culture in the presence of SFK inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) increased (P < 0.05) the proportion of spontaneously activated porcine oocytes compared to controls. Further culture with PP2 inhibitor led to an increase (P < 0.05) in the parthenogenetic embryos and a decrease (P < 0.05) in lytic oocytes. SFK inhibition did not affect (P > 0.05) the proportion of ionophore A23187-activated oocytes. SFKs were localized in the perichromosomal region, in the pronuclei, in the cytoplasm, and on the plasma membrane of oocytes and parthenogenetic embryos after 24, 48, and 72 h of prolonged in vitro culture. The greatest SFKs fluorescence was detected after a 24h-prolonged culture on the plasma membrane of MII oocytes. In embryos and fragmented oocytes, intense fluorescence was detected in the cleavage furrow region and on the membrane of apoptotic vesicles, respectively. Our results reveal the involvement of SFKs in MII arrest maintenance, though they don't appear to modulate the early processes of ionophore-stimulated parthenogenetic activation. Changes in the distribution of SFKs during prolonged culture suggest their role in signaling cascades associated with actin filament cytoskeleton organization.
- Klíčová slova
- Meiotic arrest, Pig, Postovulatory aging, Tyrosine kinases,
- MeSH
- meióza * fyziologie MeSH
- metafáze * fyziologie MeSH
- oocyty * fyziologie enzymologie účinky léků MeSH
- partenogeneze MeSH
- prasata fyziologie MeSH
- pyrimidiny farmakologie MeSH
- skupina kinas odvozených od src-genu * metabolismus antagonisté a inhibitory fyziologie MeSH
- stárnutí buněk fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- pyrimidiny MeSH
- skupina kinas odvozených od src-genu * MeSH
Angiogenesis is the process by which new blood vessels are formed through the migration and proliferation of endothelial cells from existing vessels. A balance between pro- and anti-angiogenic factors is essential for the process to occur. In adults, the most common causes are inflammation, wound healing, and neoplastic processes. The female reproductive system is the only organ in the body that exhibits cyclical and repetitive angiogenesis, irrespective of the aforementioned factors, which occurs as a consequence of dynamic and continuous tissue remodelling associated with folliculogenesis, decidualization, implantation, and embryo development. The process of ovarian angiogenesis is contingent upon the participation of a multitude of angiogenic and anti-angiogenic factors. The principal angiogenic factors are vascular endothelial growth factor (VEGF), which plays a pivotal role in the regulation of angiogenesis, as well as fibroblast growth factor (FGF), insulin-like growth factor (IGF), and angiopoietin. This article provides an overview of the molecular aspects of ovarian angiogenesis as well as presents studies on the potential clinical applications of molecular factors in the diagnosis of reproductive system diseases. The objective of research on ovarian angiogenesis is to gain insight into the mechanisms regulating this process and to develop new diagnostic and therapeutic methods for reproductive disorders. A significant proportion of ovarian diseases and infertility problems can be attributed to vascular dysfunction and angiogenesis. Therefore, it is of great importance to perform a detailed analysis of the ovarian vasculature to provide a basis for further studies on potential vascular dysfunctions.
- Klíčová slova
- Angiogenesis, Ovarian follicle, Proangiogenic factors,
- MeSH
- fyziologická neovaskularizace * genetika MeSH
- genetické markery MeSH
- lidé MeSH
- ovarium * krevní zásobení fyziologie MeSH
- ovum růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- genetické markery MeSH
Translational regulation plays a pivotal role during pre-implantation development. However, the mechanisms by which messenger RNAs (mRNAs) are selectively regulated over time, along with their dynamic utilization and fate during this period, remain largely unknown. Here, we performed fraction-resolved polysome profiling and characterized translational dynamics across oocytes and early embryo development. This approach allowed us to examine the changes in translation during pre-implantation development in high resolution and uncover previously unrecognized modes of translational selectivity. We observed a stage-specific delay in translation, characterized by the postponed recruitment of stored mRNAs-either unbound or associated with light ribosomal fractions-into actively translating polysomes (heavy fraction). Comparative analysis of translatome with proteomics, RNA N6-methyladenosine modifications, and mRNA features further revealed both coordinated and distinct regulatory mechanisms during pre-implantation development. Furthermore, we identified a eukaryotic initiation factor 1A domain containing 3, Eif1ad3, which is exclusively translated at the two-cell stage and is essential for embryonic development by regulating ribosome biogenesis and protein synthesis. Collectively, our study provides a valuable resource of spatiotemporal translational regulation in mammalian pre-implantation development and highlights a previously uncharacterized translation initiation factor critical for early embryos.
- MeSH
- adenosin analogy a deriváty metabolismus MeSH
- blastocysta metabolismus MeSH
- embryonální vývoj * genetika MeSH
- eukaryotický iniciační faktor 1 metabolismus genetika MeSH
- messenger RNA * metabolismus genetika MeSH
- myši MeSH
- oocyty metabolismus růst a vývoj MeSH
- polyribozomy metabolismus genetika MeSH
- proteosyntéza * MeSH
- ribozomy metabolismus MeSH
- vývojová regulace genové exprese * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adenosin MeSH
- eukaryotický iniciační faktor 1 MeSH
- messenger RNA * MeSH
- N-methyladenosine MeSH Prohlížeč
Mammalian oocytes are notoriously prone to chromosome segregation errors leading to aneuploidy. The spindle provides the machinery for accurate chromosome segregation during cell division. Mammalian oocytes lack centrioles and, therefore, mouse meiotic spindle relies on the organization of numerous acentriolar microtubule organizing centers into two poles (polar microtubule organizing centers, pMTOCs). The traditional view is that, in mammalian oocytes, microtubules are the sole cytoskeletal component responsible for regulating pMTOC organization and spindle assembly. We identify a previously unrecognized F-actin pool that surrounds pMTOCs, forming F-actin cage-like structure. We demonstrate that F-actin localization on the spindle depends on unconventional myosins X and VIIb. Selective disruption of spindle-localized F-actin, using myosin X/VIIb knockdown oocytes or photoswitchable Optojasp-1, perturbs pMTOC organization, leading to unfocused spindle poles and chromosome missegregation. Here, we unveil an important function of spindle-localized F-actin in regulating pMTOC organization, a critical process for ensuring the fidelity of meiotic spindle formation and proper chromosome segregation.
- MeSH
- aktiny * metabolismus MeSH
- aparát dělícího vřeténka * metabolismus MeSH
- meióza * fyziologie MeSH
- mikrotubuly metabolismus MeSH
- myosiny metabolismus genetika MeSH
- myši MeSH
- oocyty * metabolismus cytologie MeSH
- organizační centrum mikrotubulů * metabolismus MeSH
- segregace chromozomů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aktiny * MeSH
- myosiny MeSH
Mitochondria in the egg are suggested to be crucial for the onset of new life. However, there is ambiguous knowledge about the necessity for fertilization and early embryonic development. Therefore, we created a conditional Tfam knockout (TfamloxP/loxP; Zp3-Cre) to produce Tfamnull oocytes for investigation of the mitochondrial abundance in oocytes and early embryos. This created mtDNA-depleted eggs, although the abundance of mitochondria did not change. Despite decreased mitochondrial membrane potential, Tfamnull oocytes matured and were fertilized, which led to embryo formation. These Tfamnull eggs were developed into mtDNA-deficient blastocysts. Both TFAM and mtDNA appear to be dispensable for the success of embryo implantation. Tfam expression and mtDNA replication rescue the mtDNA-deficient embryo after implantation, enabling passage through a post-implantation bottleneck, and allowing survivor embryos to develop into healthy individuals. Our findings highlight the uncoupled relationship between mtDNA replication and mitochondrial abundance in the growing oocyte and show the importance of the oocyte bulk mtDNA for successful mitochondrial activation in post-implantation embryos.
- Klíčová slova
- embryo, fertilization, mitochondrial, mitochondrion, oocyte, transcription factor A,
- MeSH
- blastocysta metabolismus MeSH
- embryo savčí metabolismus MeSH
- embryonální vývoj * genetika MeSH
- genom mitochondriální * genetika MeSH
- implantace embrya * genetika MeSH
- mitochondriální DNA * genetika MeSH
- mitochondrie * genetika metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- replikace DNA MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- mitochondriální DNA * MeSH
Our study investigates the interaction of two bis-quinolinium ligands, Phen-DC3 and 360A, with the quadruplex-duplex hybrid (QDH) derived from the promoter region of the PIM1 oncogene. While the QDH is polymorphic in vitro, with a hybrid and antiparallel conformation, we demonstrate that it predominantly adopts the antiparallel conformation within the intracellular environment of Xenopus laevis oocytes (eukaryotic model system). Notably, both ligands selectively bind to the hybrid QDH conformation in vitro and in a cellular context. High-resolution nuclear magnetic resonance (NMR) structures of the complexes between the hybrid QDH and the ligands reveal distinct binding modes at the quadruplex-duplex (Q-D) junction. Specifically, Phen-DC3 binds rigidly, while 360A dynamically reorients between two positions. Our findings provide a crucial paradigm highlighting the differences in structural equilibria involving QDH in vitro compared to its behavior in the intracellular space. They also underscore the potential to modulate these equilibria under native-like conditions through ligand interactions. The observed differences in the binding of Phen-DC3 and 360A lay the groundwork for designing next-generation bis-quinolinium compounds with enhanced selectivity for the Q-D junction. Methodologically, our study illustrates the potential of 19F-detected in-cell NMR methodology for screening interactions between DNA targets and drug-like molecules under physiological conditions.
- MeSH
- chinolinové sloučeniny * chemie metabolismus MeSH
- DNA chemie MeSH
- G-kvadruplexy * MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely MeSH
- oocyty metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- protoonkogenní proteiny c-pim-1 * genetika chemie MeSH
- Xenopus laevis MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chinolinové sloučeniny * MeSH
- DNA MeSH
- ligandy MeSH
- PIM1 protein, human MeSH Prohlížeč
- protoonkogenní proteiny c-pim-1 * MeSH
Mammalian oocytes and embryos are known to exhibit a markedly low frequency of de novo mutations compared to somatic cells. We still lack efficient tools to carry out functional studies of the intergenerational mechanism of genome protection, and our view of this phenomenon is constantly being modified in light of the new results. Although oocytes were originally considered a cell type lacking DNA repair, new results indicate that mammalian oocytes might possess a set of unique properties that make them and their descendants resistant to accumulation of DNA damage. Here, we review various factors that can influence oocyte and embryo genome stability and discuss the functional evidence for the uniquely efficient response to DNA damage, particularly in the presence of minor DNA lesions and single-strand breaks. We discuss whether high levels of DNA repair proteins might be the basis for the observed low mutation rate. Finally, we present the idea that the unique characteristics of the chromatin landscape, as well as the limited replication, rather than the abundance of repair factors alone, may be responsible for the intergenerational protection of the genome.
- MeSH
- blastocysta * metabolismus MeSH
- chromatin genetika metabolismus MeSH
- lidé MeSH
- mutace MeSH
- mutační rychlost * MeSH
- nestabilita genomu MeSH
- oocyty * metabolismus MeSH
- oprava DNA MeSH
- poškození DNA MeSH
- savci genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- chromatin MeSH
Congression of all chromosomes on the equatorial plane is in mitosis required for metaphase-anaphase transition, and for successful segregation of sister chromatids during anaphase. In contrast to somatic cells, mouse oocytes, which are undergoing the first meiotic division, are capable to undergo metaphase-anaphase transition without congression of all chromosomes. Consequently, oocytes exhibit an increased frequency of chromosome segregation errors, which might lead to aneuploidy. In various experimental conditions, for example, during gene function studies, or during evaluation of the effects of pharmaceutical inhibitors, it might be beneficial to assess chromosome congression in live oocytes. In this chapter, we describe all steps necessary for performing such experiments, including isolation of mouse oocytes, microinjection-free labeling of chromosomes and spindle, the time-lapse recording of chromosome division during meiosis I, using confocal live cell microscopy, and also steps necessary for data analysis.
- Klíčová slova
- Chromosome congression, Chromosomes, Live imaging, Metaphase, Oocyte, Spindle,
- MeSH
- aparát dělícího vřeténka metabolismus MeSH
- časosběrné zobrazování MeSH
- konfokální mikroskopie MeSH
- meióza MeSH
- myši MeSH
- oocyty * cytologie metabolismus MeSH
- segregace chromozomů * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Electron microscopy represents a powerful visualizing technique, capable of a million times magnification. Prior to imaging, biological samples must undergo complex preparation to withstand the exposition to electrons in the vacuum inside the electron microscope. Here, we describe a preparation technique allowing preservation of scarce and delicate human oocytes for ultrastructural investigation.
- Klíčová slova
- Electron microscopy, Focused ion beam scanning electron microscopy, Human oocytes, Transmission electron microscopy, Ultrastructure,
- MeSH
- elektronová mikroskopie * metody MeSH
- lidé MeSH
- oocyty * ultrastruktura MeSH
- transmisní elektronová mikroskopie metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
RNA metabolism plays an essential role in the development of both oocytes and embryos. Their dependence on stored maternal transcripts is due to a long period of silenced transcription in which the oocyte undergoes meiotic maturation and fertilization. Although maternal RNAs are unusually stable, they should be replaced by "zygotic" transcripts during the transition from oocyte to zygote. Analysis of ncRNA and mRNA distribution in the oocyte can provide clues to the fate of RNA in the single-cell environment.Our work focuses on the visualization of the subcellular distribution of specific RNAs in mammalian oocytes and early embryos. The localization of many RNAs in the oocyte and embryo is still not fully understood. In this chapter, we describe an optimized protocol for RNAscope, a type of RNA fluorescence in situ hybridization (RNA FISH), which is a valuable technique for exploring mammalian oocytes and embryos. The method is based on a specific probe design strategy that enables signal amplification together with simultaneous background suppression. Additionally, it is suitable for quantitative spatio-temporal analysis of specific RNA transcripts. RNAscope, a simple and a reliable protocol, contributes to advancing our understanding of RNA biology in the context of germ cell development.
- Klíčová slova
- Embryo, Oocyte, RNA FISH, Single-molecule imaging, mRNA,
- MeSH
- embryo savčí * metabolismus MeSH
- hybridizace in situ fluorescenční * metody MeSH
- messenger RNA genetika MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- RNA * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- messenger RNA MeSH
- RNA * MeSH
