Cytoplasmic maturation in human oocytes: an ultrastructural study †.

Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

Sub-chronic inhalation of lead oxide nanoparticles revealed their broad distribution and tissue-specific subcellular localization in target organs.

BACKGROUND: Lead is well known environmental pollutant, which can cause toxic effects in multiple organ systems. However, the influence of lead oxide nanoparticles, frequently emitted to the environment by high temperature technological processes, is still concealed. Therefore, we investigate lead oxide nanoparticle distribution through the body upon their entry into lungs and determine the microscopic and ultramicroscopic changes caused by the nanoparticles in primary and secondary target organs. METHODS: Adult female mice (ICR strain) were continuously exposed to lead oxide nanoparticles (PbO-NPs) with an average concentration approximately 106 particles/cm3 for 6 weeks (24 h/day, 7 days/week). At the end of the exposure period, lung, brain, liver, kidney, spleen, and blood were collected for chemical, histological, immunohistochemical and electron microscopic analyses. RESULTS: Lead content was found to be the highest in the kidney and lungs, followed by the liver and spleen; the smallest content of lead was found in brain. Nanoparticles were located in all analysed tissues and their highest number was found in the lung and liver. Kidney, spleen and brain contained lower number of nanoparticles, being about the same in all three organs. Lungs of animals exposed to lead oxide nanoparticles exhibited hyperaemia, small areas of atelectasis, alveolar emphysema, focal acute catarrhal bronchiolitis and also haemostasis with presence of siderophages in some animals. Nanoparticles were located in phagosomes or formed clusters within cytoplasmic vesicles. In the liver, lead oxide nanoparticle exposure caused hepatic remodeling with enlargement and hydropic degeneration of hepatocytes, centrilobular hypertrophy of hepatocytes with karyomegaly, areas of hepatic necrosis, occasional periportal inflammation, and extensive accumulation of lipid droplets. Nanoparticles were accumulated within mitochondria and peroxisomes forming aggregates enveloped by an electron-dense mitochondrial matrix. Only in some kidney samples, we observed areas of inflammatory infiltrates around renal corpuscles, tubules or vessels in the cortex. Lead oxide nanoparticles were dispersed in the cytoplasm, but not within cell organelles. There were no significant morphological changes in the spleen as a secondary target organ. Thus, pathological changes correlated with the amount of nanoparticles found in cells rather than with the concentration of lead in a given organ. CONCLUSIONS: Sub-chronic exposure to lead oxide nanoparticles has profound negative effects at both cellular and tissue levels. Notably, the fate and arrangement of lead oxide nanoparticles were dependent on the type of organs.

Recent approaches in tooth engineering research.

Tooth absence and defects caused by various reasons are frequent events in humans. They are not life threatening but may bring about social consequences. Recent dentistry provides solutions in the form of prosthetics or dental implants; however, several complications and distinct limitations favour bioengineering of dental and periodontal structures. At least two types of cells (epithelial and mesenchymal) have to be recombined to produce a new functional tooth. Moreover, the tooth must be vascularized, innervated and properly anchored in the bone. To study these issues, different approaches have been established in both basic and applied research. In this review, recent strategies and techniques of tooth engineering are comprehensively summarized and discussed, particularly regarding manipulation using stem cells.

Properties of neural crest-like cells differentiated from human embryonic stem cells.

Neural crest cells (NCCs) derive early in vertebrate ontogenesis from neural tube as a population of migratory cells with exquisite differentiation potential. Abnormalities in NCC behaviour are cause of debilitating diseases including cancers and a spectrum of neurocristopathies. Thanks to their multilineage differentiation capacity NCCs offer a cell source for regenerative medicine. Both these aspects make NCC biology an important issue to study, which can currently be addressed using methodologies based on pluripotent stem cells. Here we contributed to understanding the biology of human NCCs by refining the protocol for differentiation/propagation of NCClike cells from human embryonic stem cells and by characterizing the molecular and functional phenotype of such cells. Most importantly, we improved formulation of media for NCC culture, we found that poly-L-ornithine combined with fibronectin provide good support for NCC growth, we unravelled the tendency of cultured NCCs to maintain heterogeneity of CD271 expression, and we showed that NCCs derived here possess the capacity to react to BMP4 signals by dramatically up-regulating MSX1, which is linked to odontogenesis.

[Theoretic and practical content of the clinical embryology]. / Teoretická a praktická nápln oboru klinická embryologie.

Clinical embryology is in whole Europe respected health care specialization. The specialists in this field are certified by the European Society of Human Reproduction and Embryology (ESHRE). In the Czech Republic the specialization has been formally established by the Government Order No 31/ 2010 after 30 years unformal existence. Concurrently with the wide development of new techniques in assisted reproduction, there is necessary to define the content of clinical embryology as a health care, scientific and educational discipline. The definition authorized by the Committee of Association of Reproductive Embryology (ARE) is given in this article..

Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin.

BACKGROUND AIMS: Microvesicles (MV) shed from the plasma membrane of eukaryotic cells, including human embryonic stem cells (hESC), contain proteins, lipids and RNA and serve as mediators of cell-to-cell communication. However, they may also contain immunogenic membrane domains and infectious particles acquired from xenogenic components of the culture milieu. Therefore, MV represent a potential risk for clinical application of cell therapy. METHODS: We tested the ability of hESC and their most commonly used feeder cells, mouse embryonic fibroblasts (MEF), to produce MV. We found that hESC are potent producers of MV, whereas mitotically inactivated MEF do not produce any detectable MV. We therefore employed a combined proteomic approach to identify the molecules that constitute the major components of MV from hESC maintained in a standard culture setting with xenogenic feeder cells. RESULTS: In purified MV fractions, we identified a total of 22 proteins, including five unique protein species that are known to be highly expressed in invasive cancers and participate in cellular activation, metastasis and inhibition of apoptosis. Moreover, we found that hESC-derived MV contained the immunogenic agents apolipoprotein and transferrin, a source of Neu5Gc, as well as mouse retroviral Gag protein. CONCLUSIONS: These findings indicate that MV represent a mechanism by which hESC communicate; however, they also serve as potential carriers of immunogenic and pathogenic compounds acquired from environment. Our results highlight a potential danger regarding the use of hESC that have previously been exposed to animal proteins and cells.

In vitro labelling of mouse embryonic stem cells with SPIO nanoparticles.

Labelling of mammalian cells with superparamagnetic iron oxide (SPIO) nanoparticles enables to monitor their fate in vivo using magnetic resonance imaging (MRI). However, the question remains whether or not SPIO nanoparticles affect the phenotype of labelled cells. In the present study, the effects of SPIO nanoparticles from two producers on the growth and differentiation of mouse embryonic stem (ES) cells in vitro were investigated. Our observations have shown that SPIO nanoparticles have no effect on the self-renewal of ES cells. Subsequently, we studied the effect of SPIO on the formation of embryoid bodies and neural differentiation of ES cell in monolayer culture. The cavitation of embryoid bodies was partially inhibited and neural differentiation was supported regardless the type of SPIO nanoparticles used. Thus for the first time we documented the effects of SPIO nanoparticles on ES cells and their differentiation.

Impact of acute and subchronic inhalation exposure to PbO nanoparticles on mice.

Lead nanoparticles (NPs) are released into air from metal processing, road transport or combustion processes. Inhalation exposure is therefore very likely to occur. However, even though the effects of bulk lead are well known, there is limited knowledge regarding impact of Pb NPs inhalation. This study focused on acute and subchronic exposures to lead oxide nanoparticles (PbO NPs). Mice were exposed to PbO NPs in whole body inhalation chambers for 4-72 h in acute experiment (4.05 × 106 PbO NPs/cm3), and for 1-11 weeks in subchronic experiment (3.83 × 105 particles/cm3 in lower and 1.93 × 106 particles/cm3 in higher exposure group). Presence of NPs was confirmed in all studied organs, including brain, which is very important considering lead neurotoxicity. Lead concentration gradually increased in all tissues depending on the exposure concentration and duration. The most burdened organs were lung and kidney, however liver and brain also showed significant increase of lead concentration during exposure. Histological analysis documented numerous morphological alterations and tissue damage, mainly in lung, but also in liver. Mild pathological changes were observed also in kidney and brain. Levels of glutathione (reduced and oxidized) were modulated mainly in lung in both, acute and subchronic exposures. Increase of lipid peroxidation was observed in kidney after acute exposure. This study characterized impacts of short to longer-term inhalation exposure, proved transport of PbO NPs to secondary organs, documented time and concentration dependent gradual increase of Pb concentration and histopathological damage in tissues.

Hematological findings in non-treated and gamma-irradiated mice deficient for MIC-1/GDF15.

Several members of the TGF-beta family are known to effectively regulate the fate of hematopoietic progenitor cells in a complex and context-dependent manner. Growth differentiation factor-15 (GDF15) is a divergent member of the TGF-beta family. This stress-induced cytokine has been proposed to possess immunomodulatory functions and its high expression is often associated with progression of a variety of pathological conditions. GDF15 is also induced by chemotherapy and irradiation. Very few fundamental studies have been published regarding the effect of GDF15 in hematopoiesis. In this study, we analyzed the hematological status of untreated and gamma-irradiated mice deficient for GDF15 as a result of genetic knock-out (KO), in order to clarify the regulatory role of GDF15 in hematopoiesis. Significant differences between GDF15 KO mice and their pertinent WT controls were found in the parameters of blood monocyte numbers, blood platelet size, and distribution width, as well as in the values of bone marrow granulocyte/macrophage progenitor cells. Different tendencies of some hematological parameters in the GDF15 KO mice in normal conditions and those under exposure of the mice to ionizing radiation were registered. These findings are discussed in the context of the GDF15 gene function and its lack under conditions of radiation-induced damage.

Neural differentiation potentiated by the leukaemia inhibitory factor through STAT3 signalling in mouse embryonal carcinoma cells.

LIF is a cytokine playing a key role in the regulation of self-renewal and maintenance of undifferentiated state in mouse ES cells. The response of pluripotent cells to LIF is mediated mainly by the STAT3 and ERK signalling pathways. Recently, we have shown that LIF potentiated retinoic acid-induced neural differentiation of pluripotent mouse embryonal carcinoma P19 cells. Here we demonstrate that pro-neural effects of LIF and partially also of retinoic acid are abolished by inhibition of the JAK2->STAT3 signalling pathway. In contrast, inhibition of the MEK1->ERK signalling pathway does not exhibit any effect. These results suggest that in neurogenic regions, cooperative action of LIF and other neuro-differentiation-inducing factors, such as retinoic acid, may be mediated by the STAT3 signalling pathway.

Impact of acute and chronic inhalation exposure to CdO nanoparticles on mice.

Cadmium nanoparticles can represent a risk in both industrial and environmental settings, but there is little knowledge on the impacts of their inhalation, especially concerning longer-term exposures. In this study, mice were exposed to cadmium oxide (CdO) nanoparticles in whole body inhalation chambers for 4 to 72 h in acute and 1 to 13 weeks (24 h/day, 7 days/week) in chronic exposure to investigate the dynamics of nanoparticle uptake and effects. In the acute experiment, mice were exposed to 2.95 × 106 particles/cm3 (31.7 µg CdO/m3). The same concentration and a lower one (1.18 × 106 particles/cm3, 12.7 µg CdO/m3) were used for the chronic exposure. Transmission electron microscopy documented distribution of nanoparticles into all studied organs. Major portion of nanoparticles was retained in the lung, but longer exposure led to a greater relative redistribution into secondary organs, namely the kidney, and also the liver and spleen. Accumulation of Cd in the lung and liver occurred already after 24 h and in the brain, kidney, and spleen after 72 h of exposure, and a further increase of Cd levels was observed throughout the chronic exposure. There were significant differences in both Cd accumulation and effects between the two exposure doses. Lung weight in the higher exposure group increased up to 2-fold compared to the control. Histological analyses showed dose-dependent alterations in lung and liver morphology and damage to their tissue. Modulation of oxidative stress parameters including glutathione levels and increased lipid peroxidation occurred mainly after the greater chronic exposure. The results emphasize risk of longer-term inhalation of cadmium nanoparticles, since adverse effects occurring after shorter exposures gradually progressed with a longer exposure duration.

Increased expression of fibroblast growth factor receptor 3 in CD34+ BCR-ABL+ cells from patients with chronic myeloid leukemia.

Previously, we showed that expression of myeloma-associated (proto)oncogene fibroblast growth factor receptor 3 (FGFR-3) is increased in white blood cells from patients with chronic myeloid leukemia (CML). The abnormal expression was returned back to the normal levels as soon as these patients reconstituted their hematopoiesis following transplantation of allogeneic peripheral blood stem cells. The aims of this study were: (1) to define population(s) of cells overexpressing FGFR-3, and (2) to determine the expression of FGFR-3 during the clinical course of the disease. We show that the vast majority of FGFR-3 transcripts as well as FGFR-3 protein arise from CD34+ BCR-ABL+ cells. Although increased levels of FGFR-3 were found in majority of late chronic phase patients treated with interferon alpha or hydroxyurea, the expression of FGFR-3 was always lowered following treatment with BCR-ABL tyrosine kinase inhibitor STI571. Compared to unstimulated cells, high levels of FGFR-3 were also identified in CD34+ cells from granulocyte colony-stimulating factor-mobilized blood stem cell harvests from healthy donors, suggesting a potential growth factor-dependent basis for elevated expression of FGFR-3 in CML. These findings have implications for the involvement of FGFR-3 in malignant hematopoiesis and depict FGFR-3 tyrosine kinase in CD34+ leukemic cells as a possible target for tyrosine kinase inhibitors.

FGF-2 abnormalities in B cell chronic lymphocytic and chronic myeloid leukemias.

An elevated level of fibroblast growth factor-2 (FGF-2) in peripheral blood is considered to play a role in regulating the growth of leukemia cells. Here, we show that the level of plasma FGF-2 is increased in 54% of B cell chronic lymphocytic leukemias (B-CLL) and in 44% of chronic myeloid leukemias (CML). Notably, white blood cells (WBCs) from B-CLL patients contain 18, 22 and 24 kDa isoforms of FGF-2 whereas WBCs from CML patients contain only the 24 kDa isoform. Furthermore, as cultured B-CLL WBCs release 18 kDa FGF-2 into the medium, they constitute a potential source of FGF-2 in the blood. In a receptor binding assay, 125I-FGF-2 binds weakly to B-CLL WBCs, whereas the ligand binds more strongly to CML WBCs. Correspondingly, FGF-2 is unable to activate mitogen-activated protein kinase kinase (MEK) and its substrate, extracellular signal-regulated kinase (ERK), in B-CLL cells, whereas phosphorylation of both these cell growth-related kinases increases following treatment of CML WBCs. We conclude that B-CLL WBCs secrete FGF-2 with no apparent autocrine actions. In contrast, WBCs in CML bind FGF-2 provided by other FGF-2-hyperproducing cells and activate the MEK/ERK kinase cascade, possibly to modulate cell growth.

Retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells is potentiated by leukemia inhibitory factor.

Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferation, survival and differentiation in a wide range of cell types. Here we show that LIF potentiates retinoic acid-mediated neural induction in pluripotent P19 embryonal carcinoma cells. This activity of LIF was demonstrated by a profounded neural morphology followed by increased expression of neural-specific proteins (N-CAM, III beta-tubulin, and GAP-43), up-regulation of early neural lineage-specific gene Mash-1, and down-regulation of early endoderm-specific genes -fetoprotein and GATA-4. Moreover, LIF also slows growth and increases the level of apoptosis in differentiating cells.

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum.

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.

O-linked carbohydrates are required for FGF-2-mediated proliferation of mouse embryonic cells.

During development, fibroblast growth factors (FGFs) serve highly specific functions that are mediated through high-affinity transmembrane receptors and modulated by membrane-bound proteoglycans. Proteoglycans, in an embryonic environment called embryoglycans, contain numerous carbohydrate ectodomains, the structure of which undergoes rearrangement. Since they can be lost from the cell surface, they are sometimes found in extracellular space where they may also serve some regulatory function. Here we address the potential roles of three naturally occurring isoforms of Lewis X (LeX) in FGF-2-mediated proliferation of embryonic stem (ES) cells. We have found that the addition of sulfated LeX to ES cells at a concentration of 17 nM promotes FGF-2 mitogenic activity while a 10-fold higher concentration leads to a reduction of FGF-2-mediated proliferation. Notably, this dose-dependent modulation operated only for sulfated LeX. Other fucosylated motifs, basic LeX trisaccharide and sialylated LeX, also affected ES cell proliferation but the mechanism cannot be clearly correlated with the presence or absence of FGF-2. The suppression of biosynthesis of O-linked carbohydrates including LeX reduced basal proliferation of ES cells and interfered with the mitogenic effect of FGF-2. However, in inhibitor-treated cells, the stimulatory activity of FGF-2 can be reestablished to its original level by exogenous LeX oligosaccharides. Our results show that (A) O-linked LeX oligosaccharides can regulate mitogenic activity of FGF-2 in embryonic cells, (B) and this ability varies with subtle modifications in their structure. Importantly, our data represent the first insight into the mechanism of how growth factor activities might be modulated by shedded embryoglycan ectodomains.

Mouse Incisor Stem Cell Niche and Myb Transcription Factors.

Dental hard tissues are formed particularly by odontoblasts (dentin) and ameloblasts (enamel). Whereas the reparation of dentin is often observed, enamel does not regenerate in most species. However, in mouse incisor, a population of somatic stem cells in the cervical loop is responsible for the incisor regeneration. Understanding of the specificities of these cells is therefore of an interest in basic research as well as regenerative therapies. The Myb transcription factors are involved in essential cellular processes. B-Myb is often linked to the stem cell phenotype, and c-Myb expression marks undifferentiated and proliferating cells such as the stem cells. In the presented study, temporo-spatial expression of B-Myb and c-Myb proteins was correlated with localisation of putative somatic stem cells in the mouse incisor cervical loop by immunohistochemistry. B-Myb expression was localised mostly in the zone of transit-amplifying cells, and c-Myb was found in the inner enamel epithelium, the surrounding mesenchyme and in differentiated cells. Taken together, neither B-Myb nor c-Myb was exclusively present or abundant in the area of the incisor stem cell niche. Their distribution, however, supports recently reported novel functions of c-Myb in differentiation of hard tissue cells.

N-(2-hydroxypropyl) methacrylamide based cryogels--synthesis and biomimetic modification for stem cell applications.

The design of favorable mechanical properties and suitable surface modifications of hydrogels in order to stimulate specific cell response is a great challenge. N-(2-Hydroxypropyl) methacryl-amide (HPMA) was utilized to form macroporous cryogel scaffolds for stem cell applications. Furthermore, one group of scaffolds was enhanced by copolymerization of HPMA with methacryloyl-GGGRGDS-OH peptide in an effort to integrate biomimetic adhesion sites. The cryogels were characterized by stiffness and equilibrium swelling measurements as well as by scanning electron microscopy. Cell culture experiments were performed with human adipose-derived stem cells and substrates were found completely non-toxic. Moreover, RGDS-enriched cryogels supported cell attachment, spreading and proliferation, so they can be considered suitable for designed aims.

Three-dimensional Imaging Reveals New Compartments and Structural Adaptations in Odontoblasts.

In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.