Bodies Of Embryos Research Articles

Seed oil storage in three contrasted legume species: implications for oil improvement

Cellular comparisons on three legume species indicate oil body size is factor in mediating oil content. Key transcription factors and oleosins determining seed oil content were identified in somatic embryos of Medicago. Legume seeds provide a significant oil alternative to meat fat needs. Increasing demand for oil nutrition in the context of sustainable crop production has stimulated the exploration of legume seed oil storage regulation. This study investigated the cellular characteristics of seed oil storage using three legume species i.e. Medicago truncatula, Glycine max and Pongamia pinnata representing different oil/protein ratios, and then examined in vitro approaches for assessing strategies in enhancing seed oil storage. A greater range of oil body sizes was in higher oil/protein content species, with highest species having the largest oil bodies; and the smallest oil body size being relatively similar across species, suggesting that the arrangement of oil body size may be factor in mediating oil content. The expression of four key transcription factors i.e. LEC1, L1L, FUS3 and ABI3, and four oleosin genes in determining seed oil content was compared in vivo and in vitro using somatic embryos in Medicago, along with cellular evidence of oil bodies in somatic embryos, indicating that somatic embryos may be suitable models for rapid assessment of seed oil enhancement. This study revealed the cellular characteristics for legume seed oil storage with different nutritional compositions, and identified the associated molecular basis for boosting seed oil storage via regulating oil body size. In addition, somatic embryogenesis may be an effective system for examining oil production by modifying the expression of candidate genes prior to in vivo testing.

Heterochromatin Morphodynamics in Late Oogenesis and Early Embryogenesis of Mammals.

During the period of oocyte growth, chromatin undergoes global rearrangements at both morphological and molecular levels. An intriguing feature of oogenesis in some mammalian species is the formation of a heterochromatin ring-shaped structure, called the karyosphere or surrounded “nucleolus”, which is associated with the periphery of the nucleolus-like bodies (NLBs). Morphologically similar heterochromatin structures also form around the nucleolus-precursor bodies (NPBs) in zygotes and persist for several first cleavage divisions in blastomeres. Despite recent progress in our understanding the regulation of gene silencing/expression during early mammalian development, as well as the molecular mechanisms that underlie chromatin condensation and heterochromatin structure, the biological significance of the karyosphere and its counterparts in early embryos is still elusive. We pay attention to both the changes of heterochromatin morphology and to the molecular mechanisms that can affect the configuration and functional activity of chromatin. We briefly discuss how DNA methylation, post-translational histone modifications, alternative histone variants, and some chromatin-associated non-histone proteins may be involved in the formation of peculiar heterochromatin structures intimately associated with NLBs and NPBs, the unique nuclear bodies of oocytes and early embryos.

The nucleolus-like and precursor bodies of mammalian oocytes and embryos and their possible role in post-fertilization centromere remodelling.

In nearly all somatic cells, the ribosome biosynthesis is a key activity. The same is true also for mammalian oocytes and early embryos. This activity is intimately linked to the most prominent nuclear organelles - the nucleoli. Interestingly, during a short period around fertilization, the nucleoli in oocytes and embryos transform into ribosome-biosynthesis-inactive structures termed nucleolus-like or nucleolus precursor bodies (NPBs). For decades, researchers considered these structures to be passive repositories of nucleolar proteins used by the developing embryo to rebuild fully functional, ribosome-synthesis competent nucleoli when required. Recent evidence, however, indicates that while these structures are unquestionably essential for development, the material is largely dispensable for the formation of active embryonic nucleoli. In this mini-review, we will describe some unique features of oocytes and embryos with respect to ribosome biogenesis and the changes in the structure of oocyte and embryonic nucleoli that reflect this. We will also describe some of the different approaches that can be used to study nucleoli and NPBs in embryos and discuss the different results that might be expected. Finally, we ask whether the main function of nucleolar precursor bodies might lie in the genome organization and remodelling and what the involved components might be.

Haloxyfop-P-methyl induces developmental defects in zebrafish embryos through oxidative stress and anti-vasculogenesis

Haloxyfop-P-methyl, an aryloxyphenoxypropionate herbicide, is widely used to eliminate unwanted plants by inhibiting lipid synthesis and inducing oxidative stress. Since haloxyfop-P-methyl targets are limited within plants, few negative side effects on non-target crops have been reported. However, dissolved haloxyfop-P-methyl in rain or groundwater contaminates aquatic environments and affects marine ecosystems. In the present study, treatment with haloxyfop-P-methyl for 48 h induced developmental deficiencies in the eyes and bodies of the zebrafish embryos as a whole and was also linked to increases in the incidence of pericardial edema. Additionally, haloxyfop-P-methyl treatment decreased hatching ratio, embryo viability, and heart rate, while simultaneously increasing the expression levels of apoptotic and inflammatory genes. Moreover, haloxyfop-P-methyl hampered vasculogenesis in the embryos through down-regulation of functional genes, and disruption of vessel formation caused neurodegeneration in the olig2-positive notochord. Collectively, this study newly discovered the oxidative stress-related toxic mechanism of haloxyfop-P-methyl during embryonic development through anti-vasculogenesis, which suppresses neurogenesis of the notochord. This toxicity assessment of haloxyfop-P-methyl on embryogenesis may contribute to establishment of safety profiling of herbicide and to support hazard control in aquatic environment.

Characterization of trace metal content in the developing zebrafish embryo.

Trace metals are essential for health but toxic when present in excess. The maintenance of trace metals at physiologic levels reflects both import and export by cells and absorption and excretion by organs. The mechanism by which this maintenance is achieved in vertebrate organisms is incompletely understood. To explore this, we chose zebrafish as our model organism, as they are amenable to both pharmacologic and genetic manipulation and comprise an ideal system for genetic screens and toxicological studies. To characterize trace metal content in developing zebrafish, we measured levels of three trace elements, copper, zinc, and manganese, from the oocyte stage to 30 days post-fertilization using inductively coupled plasma mass spectrometry. Our results indicate that metal levels are stable until zebrafish can acquire metals from the environment and imply that the early embryo relies on maternal contribution of metals to the oocyte. We also measured metal levels in bodies and yolks of embryos reared in presence and absence of the copper chelator neocuproine. All three metals exhibited different relative abundances between yolks and bodies of embryos. While neocuproine treatment led to an expected phenotype of copper deficiency, total copper levels were unaffected, indicating that measurement of total metal levels does not equate with measurement of biologically active metal levels. Overall, our data not only can be used in the design and execution of genetic, physiologic, and toxicologic studies but also has implications for the understanding of vertebrate metal homeostasis.

Ultrastructure of Cell Organelles in Pre-implantation Embryos from Cows with Different Body Condition Score.

Morphology of important cell organelles (mitochondria, lipid droplets, vacuoles, inclusion bodies and apoptotic bodies) in embryos derived from cows with different body condition score (BCS) was analysed by transmission electron microscopy (TEM). Embryos were recovered on 7th day after the insemination by a standard non-surgical flushing of the uterine horns from superovulated Holstein Friesian cows with BCS 2, 3, 4 and 5. Thereafter, the good quality blastocysts were processed for TEM. The electronograms were evaluated by stereological analysis. The relative volume of lipid droplets in BCS4 and BCS5 embryos increased significantly (18.53 and 22.40%) when compared to BCS3 embryos (5.46%). In the embryos from the BCS4 or BCS5 cows, we observed different morphological patterns of mitochondria, as well as the mitochondria containing vacuoles. BCS4 and BCS5 embryo cell nuclei showed the structure typical for low transcription activity (none or very few reticular nucleoli); also dilated inter-cellular spaces were often observed in these embryos. In conclusion, differences in the ultrastructural morphology of embryos from over-conditioned cows (BCS4 and BCS5), particularly the higher lipid content in the cytoplasm, can be a marker of their low quality, and this fact can be a contributing factor to subfertility in over-conditioned cows.

Pannexin-1 expression in developing mouse nervous system: new evidence for expression in sensory ganglia.

Pannexin1 (Panx1) is one of three members of the pannexin protein family. The expression of Panx1 mRNA has been extensively investigated from late embryonic to adult stages. In contrast, expression during early embryonic development is largely unknown. Our aim is to examine the temporal and spatial expression of Panx1 in mouse embryonic development by focusing on embryonic days (E) 9.5 to 12.5. Whole embryos are investigated in order to provide a comprehensive survey. Analyses were performed at the mRNA level by using reverse transcription plus the polymerase chain reaction and whole-mount in situ hybridization. Panx1 mRNA was detected in the heads and bodies of embryos at all developmental stages investigated (E9.5, E10.5, E11.5, E12.5). In particular, the nervous system expressed Panx1 at an early time point. Interestingly, Panx1 expression was found in afferent ganglia of the cranial nerves and spinal cord. This finding is of particular interest in the context of neuropathic pain and other Panx1-related neurological disorders. Our study shows, for the first time, that Panx1 is expressed in the central and peripheral nervous system during early developmental stages. The consequences of Panx1 deficiency or inhibition in a number of experimental paradigms might therefore be predicated on changes during early development.

Localisation of RNAs and proteins in nucleolar precursor bodies of early mouse embryos

Early embryos of all mammalian species contain morphologically distinct but transcriptionally silent nucleoli called the nucleolar precursor bodies (NPBs), which, unlike normal nucleoli, have been poorly studied at the biochemical level. To bridge this gap, here we examined the occurrence of RNA and proteins in early mouse embryos with two fluorochromes - an RNA-binding dye pyronin Y (PY) and the protein-binding dye fluorescein-5'-isothiocyanate (FITC). The staining patterns of zygotic NPBs were then compared with those of nucleolus-like bodies (NLBs) in fully grown surrounded nucleolus (SN)-type oocytes, which are morphologically similar to NPBs. We show that both entities contain proteins, but unlike NLBs, NPBs are significantly impoverished for RNA. Detectable amounts of RNA appear on the NPB surface only after resumption of rDNA transcription and includes pre-rRNAs and 28S rRNA as evidenced by fluorescence in situ hybridisation with specific oligonucleotide probes. Immunocytochemical assays demonstrate that zygotic NPBs contain rRNA processing factors fibrillarin, nucleophosmin and nucleolin, while UBF (the RNA polymerase I transcription factor) and ribosomal proteins RPL26 and RPS10 are not detectable. Based on the results obtained and data in the contemporary literature, we suggest a scheme of NPB assembly and maturation to normal nucleoli that assumes utilisation of maternally derived nucleolar proteins but of nascent rRNAs.

Histochemical evaluation of induction of somatic embryogenesis in Passiflora edulis Sims (Passifloraceae)

The aim of this study was to describe the accumulation of reserves during the somatic embryogenesis process in Passiflora edulis Sims FB-300, obtained from mature zygotic embryos, using histochemical methods. Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 31.06 μM of picloram, 2.22 μM of benzyladenine, and 2.27 μM of thidiazuron. The zygotic embryo explants, at different developmental stages, were collected and fixed in Karnovsky solution and subsequently subjected to dehydration in an ethanol series and embedded in acrylic resin. Transverse and longitudinal sections (5-μm thick) were stained with toluidine blue for anatomical characterization, using Xylidine Ponceau for the detection of total protein, periodic acid-Schiff reagent for neutral polysaccharides, Sudan black B to detect lipids, and Lugol’s reagent for the starch detection. Histological sections revealed the formation of protuberances and globular stage somatic embryos in the cotyledonary region of the zygotic embryo. Histochemical tests revealed the presence of large quantities of protein bodies in zygotic embryos of P. edulis, which were gradually metabolized during somatic embryo development. Lipid bodies and starch grains were identified only after 20 d of culture, suggesting the use of these compounds as energy source for de novo synthesis. The present study describes the anatomical changes and the pattern of reserve accumulation during the somatic embryogenesis process in P. edulis.

Immunocytochemical study of YB-1 nuclear distribution in different cell types.

In the present work we studied the distribution of YB-1 in the nuclei of mouse hepatocytes, early embryos and human skin fibroblasts with the use of light and electron microscopy. To reveal YB-1, we applied rat polyclonal antibody against the C-terminal fragment of YB-1 molecule and rabbit polyclonal antibody against full-length YB-1 molecule. YB-1 distribution patterns varied significantly in different cell types. YB-1 was found to be colocalized with RNA polymerase I in mouse hepatocytes and embryos. Besides, YB-1 was revealed in a population of Cajal bodies in 2-cell mouse embryos but not in other cells studied.

Knockdown of regulator of G-protein signalling 2 (Rgs2) leads to abnormal early mouse embryo development in vitro

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

Chromosomal stability of second polar bodies in mouse embryos

Incorporation of a second polar body (PB2) into one of the blastomeres has been considered as a causal mechanism underlying diploid/triploid mixoploidy in humans. Using a mouse model, we examined whether PB2s can participate in the formation of mixoploidy. Uptake of BrdU was examined to determine DNA synthesis in PB2s up to 28 h after fertilization. PB2s from embryos at 4-6 (1-cell), 24 (2-cell), 48 (4-cell), and 72 h (morula) were fused with MII oocytes to induce premature chromosome condensation. Caspase and TUNEL assays were used to detect apoptotic PB2s at 24, 48, and 72 h. PB2s were fused with one of the blastomeres of the 2-cell embryos to produce mixoploid embryos. DNA synthesis in the PB2s continued until 22 h after fertilization. At 4-6 h, nearly all of the PB2s showed G1-type chromosomes and there was no significant increase in chromosome damage. At 24, 48, and 72 h, S-type chromatin predominated. Few PB2s showed apoptotic response until 72 h. Regardless of the fusion with the PB2, more than 90 % of the embryos developed to 4-cell stage, and over 80 % of the resultant 4-cell embryos had daughter blastomeres with a morphologically normal nucleus. Some of the daughter blastomeres displayed triploidy. The PB2 is viable for at least 72 h after fertilization, with slow progression through the cell cycle. Once the PB2 has been incorporated into a blastomere, the cell cycle of the PB2 might be synchronized with that of the host resulting in diploid/triploid mixoploidy.

Drosophila patterning is established by differential association of mRNAs with P bodies

The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization. In Drosophila melanogaster, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA in the oocyte and embryo. Both transcripts are translationally silent while being localized within the oocyte along microtubules by cytoplasmic dynein. Once localized, grk is translated at the dorsoanterior of the oocyte to send a TGF-α signal to the overlying somatic cells. In contrast, bcd is translationally repressed in the oocyte until its activation in early embryos when it forms an anteroposterior morphogenetic gradient. How this differential translational regulation is achieved is not fully understood. Here, we address this question using ultrastructural analysis, super-resolution microscopy and live-cell imaging. We show that grk and bcd ribonucleoprotein (RNP) complexes associate with electron-dense bodies that lack ribosomes and contain translational repressors. These properties are characteristic of processing bodies (P bodies), which are considered to be regions of cytoplasm where decisions are made on the translation and degradation of mRNA. Endogenous grk mRNA forms dynamic RNP particles that become docked and translated at the periphery of P bodies, where we show that the translational activator Oo18 RNA-binding protein (Orb, a homologue of CEPB) and the anchoring factor Squid (Sqd) are also enriched. In contrast, an excess of grk mRNA becomes localized inside the P bodies, where endogenous bcd mRNA is localized and translationally repressed. Interestingly, bcd mRNA dissociates from P bodies in embryos following egg activation, when it is known to become translationally active. We propose a general principle of translational regulation during axis specification involving remodelling of transport RNPs and dynamic partitioning of different transcripts between the translationally active edge of P bodies and their silent core.

Do embryonic polar bodies commit suicide?

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

Functional and heterologous expression of human protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 in zebrafish

Although membrane-associated proteins are related to many diseases and are important targets for drug discovery, their expression is often difficult in bacterial hosts such as Escherichia coli. To overcome this limitation, here, we focused on a novel host-vector system in zebrafish for the expression of human protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 (hPOMGnT1) which is related to muscle-eye-brain disease. For the expression of hPOMGnT1, the vector pZex-EGFP-pXI-hPOMGnT1 was constructed and injected into fertilized eggs. Using this system, we demonstrated that recombinant hPOMGnT1 was successfully expressed in the whole bodies of zebrafish embryos.

Acute Toxicity of the Antifouling Compound Butenolide in Non-Target Organisms

Butenolide [5-octylfuran-2(5H)-one] is a recently discovered and very promising anti-marine-fouling compound. In this study, the acute toxicity of butenolide was assessed in several non-target organisms, including micro algae, crustaceans, and fish. Results were compared with previously reported results on the effective concentrations used on fouling (target) organisms. According to OECD's guideline, the predicted no effect concentration (PNEC) was 0.168 µg l−1, which was among one of the highest in representative new biocides. Mechanistically, the phenotype of butenolide-treated Danio rerio (zebrafish) embryos was similar to the phenotype of the pro-caspase-3 over-expression mutant with pericardial edema, small eyes, small brains, and increased numbers of apoptotic cells in the bodies of zebrafish embryos. Butenolide also induced apoptosis in HeLa cells, with the activation of c-Jun N-terminal kinases (JNK), Bcl-2 family proteins, and caspases and proteasomes/lysosomes involved in this process. This is the first detailed toxicity and toxicology study on this antifouling compound.

Perinuclear P granules are the principal sites of mRNA export in adult C. elegans germ cells

Germline-specific granules of unknown function are found in a wide variety of organisms, including C. elegans, where they are called P granules. P granules are cytoplasmic bodies in oocytes and early embryos. Throughout most of the C. elegans life cycle, however, P granules are associated with clusters of nuclear pore complexes (NPCs) on germ cell nuclei. We show that perinuclear P granules differ from cytoplasmic P granules in many respects, including structure, stability and response to metabolic changes. Our results suggest that nuclear-associated P granules provide a perinuclear compartment where newly exported mRNAs are collected prior to their release to the general cytoplasm. First, we show that mRNA export factors are highly enriched at the NPCs associated with P granules. Second, we discovered that the expression of high-copy transgenes could be induced in a subset of germ cells, and used this system to demonstrate that nascent mRNA traffics directly to P granules. P granules appear to sequester large amounts of mRNA in quiescent germ cells, presumably preventing translation of that mRNA. However, we did not find evidence that P granules normally sequester aberrant mRNAs, or mRNAs targeted for destruction by the RNAi pathway.

Perinuclear P granules are the principal sites of mRNA export in adult C. elegans germ cells.

Germline-specific granules of unknown function are found in a wide variety of organisms, including C. elegans, where they are called P granules. P granules are cytoplasmic bodies in oocytes and early embryos. Throughout most of the C. elegans life cycle, however, P granules are associated with clusters of nuclear pore complexes (NPCs) on germ cell nuclei. We show that perinuclear P granules differ from cytoplasmic P granules in many respects, including structure, stability and response to metabolic changes. Our results suggest that nuclear-associated P granules provide a perinuclear compartment where newly exported mRNAs are collected prior to their release to the general cytoplasm. First, we show that mRNA export factors are highly enriched at the NPCs associated with P granules. Second, we discovered that the expression of high-copy transgenes could be induced in a subset of germ cells, and used this system to demonstrate that nascent mRNA traffics directly to P granules. P granules appear to sequester large amounts of mRNA in quiescent germ cells, presumably preventing translation of that mRNA. However, we did not find evidence that P granules normally sequester aberrant mRNAs, or mRNAs targeted for destruction by the RNAi pathway.

A quantification model for apoptosis in mouse embryos in the early stage of fetation

Apoptosis is the most important inducement and modulator for embryos in the early stage of fetation, i.e. after the 8-cell stage, mostly the morula and blastula stage, to proceed to the stage of nonlinear development. Using a two-photon laser scanning microscopy (TPLSM) system, we obtained 3-dimensional (3D) fluorescent images of preimplantation mouse embryos. A model for quantification was established. The statistical results for the spatial location of apoptosis bodies in embryos was obtained following image processing, as well as investigation of the kinetics of apoptosis. It was found that most (70%) apoptosis occurred in the trophectoderm, and the departure between the centroid and geometric center of embryos had a step transition when embryos developed into the 32-cell stage, which was consistent with the theoretical prediction that the blastocele would induce a symmetry break of the distribution of cells in embryos.

Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction

The cell volume alteration participates in a wide variety of cellular functions that may interfere with intra-cellular homeostasis. The most adequate approach of estimation of the volume changes induced by osmotic misbalance, alteration in shape and size due to the action shape forming substances, etc., is the direct measurement of volumetric parameters of embryos. In the given research, the volume magnitude and kinetics of changes in volume and surface area of blastomere and polar bodies of early mouse embryos were determined using three-dimensional reconstruction of the optical section stack obtained with laser scanning microscope (LSM). The size and surface area were determined for isotonic and anisosmotic conditions. The physiological significance of the findings is discussed.