Developmental fate of mitochondria microinjected into murine zygotes

A greater understanding of the fate of mitochondria injected into early preimplantation embryos would provide insights into mitochondrial biology and dynamics associated with development and disease. The ability to introduce foreign mitochondria into mouse embryos provides a means of tracking or following mitochondrial populations in vivo. Previously, injection of foreign mitochondria into the cytoplasm of the zygote was used to produce heteroplasmic mice. However, populations of introduced mitochondria decreased rapidly during development beyond the blastocyst stage. Therefore, the fate of exogenous mitochondria introduced into mouse ova was examined to determine viability and localization in comparison to endogenous mitochondria. Microinjection of murine mitochondria labeled with mitochondria-specific MitoTracker fluorophores allowed evaluation of subsequent viability and functionality of exogenous mitochondria populations in vivo. Characterization of mitochondrial survival and migration following microinjection illustrated toxic effects of MitoTracker Red upon exposure to laser confocal examination. In contrast, mitochondrial-specific fluorophores effectively detected foreign mitochondrial migration post-microinjection. The subsequent viability of the introduced mitochondria was observed through the blastocyst stage. Through the use of mitochondria-specific fluorophores, newly introduced mitochondria were further characterized and tracked post-transfer.     

Dichloroacetate-induced developmental toxicity and production of reactive oxygen species in zebrafish embryos

Dichloroacetate (DCA) is one of the toxic by products that are formed during the chlorine disinfection process of drinking water. In this study, the developmental toxicity of DCA has been determined in zebrafish (Danio rerio) embryos. Embryos were exposed to different concentrations (4, 8, 16, and 32 mM) of the compound at the 4 h postfertilization (hpf) stage of development, and were observed for different developmental toxic effects at 8, 24, 32, 55, 80, and 144 hpf. Exposure of embryos to 8-32 mM of DCA resulted in significant increases in the heart rate and blood flow of the 55 and 80 hpf embryos that turned into significant decreases at the 144 hpf time point. At 144 hpf, malformations of mouth structure, notochord bending, yolk sac edema and behavioral effects including perturbed swimming and feeding behaviors were also observed. DCA was also found to produce time- and concentration-dependent increases in embryonic levels of superoxide anion (O2*-) and nitric oxide (NO), at various stages of development. The results of the study suggest that DCA-induced developmental toxic effects in zebrafish embryos are associated with production of reactive oxygen species in those embryos.     

Similar nature of inhibition of mitochondrial respiration of heart tissue and malignant cells by methylglyoxal. A vital clue to understand the biochemical basis of malignancy

The effect of methylglyoxal on the oxygen consumption of mitochondria of heart and of several other organs of normal animals of different species has been tested. The results indicate that methylglyoxal (3.5 mM) strongly inhibits ADP-stimulated -oxoglutarate and malate plus pyruvate-dependent respiration of exclusively heart mitochondria of normal animals of different species. Whereas, with the same substrates, but at a higher concentration of methylglyoxal (7.5 mM), the respiration of mitochondria of other organs of normal animals is not inhibited. Methylglyoxal also inhibits the respiration of slices of rat and toad hearts. But this inhibition is less pronounced. However, methylglyoxal (15 mM) fails to have any effect on perfused toad heart. Using rat heart mitochondria as a model, the effect of methylglyoxal on the oxygen consumption was also tested with different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-spe inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal. The results strongly suggest that methylglyoxal inhibits the electron flow through complex I of rat heart mitochondrial respiratory chain. Moreover, lactaldehyde (0.6 mM), a catabolite of methylglyoxal, can exert a protective effect on the inhibition of rat heart mitochondrial respiration by methylglyoxal (2.5 mM). The effect of methylglyoxal on heart mitochondria as described in the present paper is strikingly similar to the results of our previous work with mitochondria of Ehrlich ascites carcinoma cells and leukemic leukocytes. We have recently proposed a new hypothesis on cancer which suggests that excessive ATP formation in cells may lead to malignancy. The above mentioned similarity apparently provides a solid experimental foundation for the proposed hypothesis which has been discussed.     

Regulation of the number and function of mitochondria during artificial increase of their mass in fish embryos

The mechanisms of mitochondrial mass reduction were investigated by microinjection of mitochondria in developing loach embryos. This reduction can be due to the degradation of the injected mitochondria or to the triggering of regulatory mechanisms. In the latter case the decrease of mitochondrial excess should be caused by exogenous and endogenous mitochondria of the embryos. When the protein-labelled mitochondria were injected into unlabelled eggs or the unlabelled mitochondria were injected into the eggs containing labelled mitochondria, the label content in the mitochondrial protein was decreased 2-fold within 12 hours and then remained unchanged at later stages of embryogenesis. After injection of 3H-labelled mitochondria into the 14C-labelled eggs the 3H/14C ratio in the mitochondrial protein during embryogenesis remained unchanged. These data suggest that the restoration of the normal amount of the mitochondrial mass is caused by the triggering of regulatory mechanisms. Oxygen uptake in the embryos with the artificially increased amount of mitochondria is maintained at a control level or even below control, i. e. undergoes regulation. In the homogenates of these embryos the regulatory control is absent and oxygen uptake is proportional to the amount of mitochondria.     

Changes in mitochondrial respiration during the development of Xenopus laevis

Mitochondria isolated from Xenopus laevis embryos at various developmental stages show a good oxidative capacity and an acceptable respiratory control provided that certain requirements are fulfilled. The rates of respiration with pyruvate and Krebs' cycle intermediates, especially with citrate and isocitrate, are very low during cleavage stages and increase after gastrulation. Glutamate in the presence of malate is the only substrate to be readily oxidized during early development and its rate of oxidation decreases after gastrulation. These results, together with the altered sensitivity of embryonic mitochondria towards azide, support the view that the oxidative metabolism undergoes important changes around gastrulation and is associated with mitochondrial differentiation.     

Mitochondria and metazoan epigenesis

In eukaryotes, mitochondrial activity controls ATP production, calcium dynamics, and redox state, thereby establishing physiological parameters governing the transduction of biochemical signals that regulate nuclear gene expression. However, these activities are commonly assumed to fulfill a ‘housekeeping’ function: necessary for life, but an epiphenomenon devoid of causal agency in the developmental flow of genetic information. Moreover, it is difficult to perturb mitochondrial function without generally affecting cell viability. For these reasons little is known about the extent of mitochondrial influence on gene activity in early development. Recent discoveries pertaining to the redox regulation of key developmental signaling systems together with the fact that mitochondria are often asymmetrically distributed in animal embryos suggests that they may contribute spatial information underlying differential specification of cell fate. In many cases such asymmetries correlate with localization of genetic determinants (i.e., mRNAs or proteins), particularly in embryos that rely heavily on cell-autonomous means of cell fate specification. In such embryos the localized genetic determinants play a dominant role, and any developmental information contributed by the mitochondria themselves is likely to be less obvious and more difficult to isolate experimentally. Hence, ‘regulative’ embryos that make more extensive use of conditional cell fate specification are better suited to experimental investigation of mitochondrial impacts on developmental gene regulation. Recent studies of the sea urchin embryo, which is a paradigmatic example of such a system, suggest that anisotropic distribution of mitochondria provides a source gradient of spatial information that directs epigenetic specification of the secondary axis via Nodal–Lefty signaling.     

Threatened abortion, hormone therapy and malformed embryos

Causal relations between maternal genital bleeding, supportive hormone therapy and external malformations of the embryos were investigated with special reference to the critical period of organogenesis. This was done using morphological and obstetrical data obtained by Nishimura and his associates from 667 undamaged embryos derived from induced abortions whose mothers had genital bleeding in early pregnancy. In addition, data from 90 embryos with polydactyly and 38 with limb reductions in the Nishimura collection were used for case history studies. Evidence was presented to demonstrate that, for major malformations such as CNS anomalies, cleft lip, polydactyly and limb reductions, maternal genital bleeding was not a cause but a consequence of the conception of an abnormal embryo. No indication was revealed that exogenous female hormones currently used in Japan for preventing miscarraiges could produce major malformations recognizable at the embryonic stage, including limb reductions, nor salvage the severely malformed embryos. This does not however mean to exclude the possible relationship of progestogens/estrogens intake during early pregnancy with an increased incidence at birth of certain internal and/or external malformations. It was suggested that most, if not all, of the minor anomalies observed at certain embryonic stages are kinds of normal variants without any functional impairment of embryonic development.     

Mitochondrial degeneration and not apoptosis is the primary cause of embryonic lethality in ceramide transfer protein mutant mice

Ceramide transfer protein (CERT) functions in the transfer of ceramide from the endoplasmic reticulum (ER) to the Golgi. In this study, we show that CERT is an essential gene for mouse development and embryonic survival and, quite strikingly, is critical for mitochondrial integrity. CERT mutant embryos accumulate ceramide in the ER but also mislocalize ceramide to the mitochondria, compromising their function. Cells in mutant embryos show abnormal dilation of the ER and degenerating mitochondria. These subcellular changes manifest as heart defects and cause severely compromised cardiac function and embryonic death around embryonic day 11.5. In spite of ceramide accumulation, CERT mutant mice do not die as a result of enhanced apoptosis. Instead, cell proliferation is impaired, and expression levels of cell cycle–associated proteins are altered. Individual cells survive, perhaps because cell survival mechanisms are activated. Thus, global compromise of ER and mitochondrial integrity caused by ceramide accumulation in CERT mutant mice primarily affects organogenesis rather than causing cell death via apoptotic pathways.     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.     

Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2‐PN) embryos

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Δψ) and microtubule distribution in mouse 2‐PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2‐PN embryos was significantly lower than in fresh ones (67.3 ± 3.0% vs. 84.9 ± 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2‐PN embryos without mitochondrial rings (61.7 ± 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 ± 2.8%). (3) Following staining by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbo‐cyanine iodide (JC‐1), most red‐colored mitochondria (high Δψ) were distributed peripherally around pronuclei and along cell membranes of fresh 2‐PN embryos. Conversely, red‐colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Δψ) evenly distributed throughout the cytoplasm. The proportion of fresh 2‐PN embryos with obvious aggregation of high Δψ mitochondria (84.2 ± 2.2%) was significantly higher than that of vitrified embryos (26.7 ± 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 ± 3.4%) was similar to that of vitrified embryos (74.7 ± 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2‐PN embryos, events which may affect subsequent developmental viability of such embryos. Mol. Reprod. Dev. 76: 1056–1063, 2009. © 2009 Wiley‐Liss, Inc.     

Sirtuin inhibitor Ex‐527 causes neural tube defects,ventral edema formations,and gastrointestinal malformations in Xenopus laevis embryos

Chemical reagent Ex‐527 is widely used as a major inhibitor of Sirtuin enzymes, which are a family of highly conserved protein deacetylases and have been linked with caloric restriction and aging by modulating energy metabolism, genomic stability, and stress resistance. However, the extent to which Ex‐527 controls early developmental events of vertebrate embryos remains to be understood. Here, we report an examination of Ex‐527 effects during Xenopus early development, followed by a confirmation of expressions of xSirt1 and xSirt2 in embryonic stages and enhancement of acetylation by Ex‐527. First, we found that reductions in size of neural plate at neurula stages were induced by Ex‐527 treatment. Second, tadpoles with short body length and large edematous swellings in the ventral side were frequently observed. Moreover, Ex‐527‐treated embryos showed severe gastrointestinal malformations in late tadpole stages. Taken together with these results, we conclude that the Sirtuin family start functioning at early embryonic stages and is required for various developmental events.     

The role of arabinogalactan proteins binding to Yariv reagents in the initiation, cell developmental fate, and maintenance of microspore embryogenesis in Brassica napus L. cv. Topas

Arabinogalactan proteins (AGPs) are extracellular proteoglycans involved in plant growth and development. The addition of beta-D-glucosyl Yariv reagent (betaGlcY), a synthetic phenylglycoside that specifically reacts with AGPs, to the culture medium notably disturbed microspore embryogenesis in a concentration-dependent manner. The initiation of microspore embryogenesis was clearly inhibited by 30 microM betaGlcY and completely inhibited by 50 microM betaGlcY. The transfer of microspore-derived embryos at different developmental stages into NLN6 medium containing 50 microM betaGlcY prohibited their normal development, as approximately 21.24, 43.99, and 59.73%, respectively, of the treated globular-, heart-, and torpedo-stage embryos exhibited numerous root hair-like structures. Both heart-stage and torpedo-stage embryos showed a rapid growth of roots with a large number of clustered root hairs. Some root hair-like structures were also observed on the apical portions of embryos. Microscopy of the treated embryos revealed that the basic patterns of cells at both the radial and apical-basal axes were greatly altered, such that the cells lost their ability to carry out programmed embryogenesis. These results show that the betaGlcY-AGP interaction modulates the developmental fate of embryonic cells, especially epidermal cells, and thereby strongly affects root generation and development. Immunofluorescence microscopy revealed that both JIM8 and JIM13 binding to AGP co-localize with betaGlcY-binding sites. Thus, AGPs binding to betaGlcY, co-localized with Jim8- and Jim13-binding protein, appear to play a crucial role in the initiation of Brassica microspore embryogenesis and the maintenance of cell differentiation during embryonic development. In addition, these proteins may also be involved in the regulation of root generation.     

Miro proteins coordinate microtubule‐ and actin‐dependent mitochondrial transport and distribution

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho‐GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double‐knockout mouse embryos and single‐ and double‐knockout embryonic fibroblasts, we demonstrate the essential and non‐redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double‐knockout cells. In contrast, we show that TRAK2‐mediated retrograde mitochondrial transport is Miro1‐dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin‐dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double‐knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine‐tune actin‐ and tubulin‐dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.     

Vinyl Chloride Monomer (VCM) Induces High Occurrence of Neural Tube Defects in Embryonic Mouse Brain During Neurulation

The aim of this study was to explore the direct embryonic teratogenicity of vinyl chloride monomer (VCM), especially the toxic effects on the early development of the nervous system and its underlying mechanisms. Pregnant mice at embryonic day 6.5 (E6.5) were injected with different doses of VCM (200, 400 and 600 mg/kg) and embryos were harvested at E10.5. Our results showed that doses higher than 400 mg/kg of VCM increased the incidence of malformed embryos, especially the neural tube defects (NTDs). In addition, high-dose of VCM decreased mitotic figure counts in the neuroepithelium and enhanced the percentage of cells in G0/G1 phase, while they were reduced in S phase. The more VCM was injected into mice, the fewer positive PCNA cells were seen and the more positive TUNEL cells were observed in the neuroepithelium. Moreover, significant increases in the levels of caspase-3 protein were observed in NTD embryos. Our results demonstrate that during early pregnancy, exposure to doses higher than 400 mg/kg of VCM increases the incidence of malformations and particularly the rate of NTDs. High-dose of VCM inhibits the proliferation of neural cells and induces cell apoptosis, leading to an imbalance in the ratio of proliferation and apoptosis. Meanwhile, the apoptosis of neuroepithelial cells might be accelerated by the activation of the caspase-3 pathway, and it might be a reason for NTDs.     

Nicotine-induced embryonic malformations mediated by apoptosis from increasing intracellular calcium and oxidative stress

BACKGROUND: Tobacco smoking by women during pregnancy increases the risk of congenital birth defects in the infants. Among the smoke products, nicotine is believed to be the major teratogenic factor that perturbs embryonic development. However, the role of nicotine in embryonic malformations has not been addressed, and the mechanisms by which nicotine affects embryonic development remain to be delineated. METHODS: To investigate the effects of nicotine on early embryogenesis, murine embryos at embryonic day (E) 8.5 were dissected out of the uteri, cultured in a roller bottle system, and treated with nicotine (0.6-6 microM) or vehicle. Embryonic morphogenesis and growth were examined in terms of structural morphology and crown/rump length, respectively. Programmed cell death (apoptosis) was assessed using LysoTracker Red staining of whole mount embryos and TUNEL assay of tissue sections. Changes in intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) production were assessed using fluorescent dyes (Flu-4, AM; H2DCFDA, respectively) under a confocal microscope. To further investigate the role of intracellular calcium and ROS in nicotine-induced embryopathy, embryos were treated with BAPTA-AM (2 microM) to inhibit [Ca2+]i elevation and ascorbic acid (vitamin C; 100 microg/ml) to scavenge ROS in presence of nicotine (6 microM). RESULTS: The embryos treated with nicotine in 3-6 microM were smaller than those treated with vehicle. Most of the embryos had open neural tube in the anterior (brain) regions. The embryos treated with 6 microM nicotine also exhibited severe defects in the posterior trunk, resembling caudal dysplasia. Excessive apoptosis was observed in the deformed structures. Significant increases in [Ca2+]i and ROS were seen in the tissues that had higher levels of apoptosis. Furthermore, inhibition of [Ca2+]i and scavenging of ROS significantly reduced embryonic malformation and apoptotic rates in the embryos. CONCLUSIONS: Nicotine affects embryonic development in a concentration-dependent manner. The nicotine-induced embryonic malformations are, in part, a result of excessive cell death. Nicotine increases [Ca2+]i and ROS level, which play a role in nicotine-induced embryonic apoptosis and malformations. These studies identify the molecular pathway of nicotine action in embryonic apoptosis and malformations, and provide a promising approach for ameliorating the teratogenic effects of nicotine.     

核移植与线粒体

线粒体是哺乳动物的产能、供能细胞器,与生长、发育、衰老和凋亡等多种细胞事件及疾病有关。哺乳动物核移植可能导致克隆胚胎及后代中线粒体的杂合性,从而影响到个体的表型甚至导致线粒体疾病。文章阐明了哺乳动物中线粒体的生物学功能及遗传特性,并分析了核移植中供体细胞和受体卵胞质两种来源的线粒体在同种胚胎细胞核移植、同种及异种体细胞核移植重构胚发育进程中的变化以及可能影响线粒体杂合性的一些因素,对其可能导致的线粒体疾病及解决方法进行了简单的阐述。

     

Influence of oxidative processes in mitochondria on contractility of the frog Rana temporaria heart muscle. Effects of cadmium

The inotropic Cd2+ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd2+ at concentrations of 1, 10, and 20 microM is established to decrease dosedependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Ca2(+)-channels of the L-type (Ca 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd2+ at concentrations of 15 and 25 microM produces swelling of non-energized and energized mitochondria in isotonic (with KNO2 and NH4NO3) and hypoosmotic (with 25 mM CH3COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 microM Cd2+ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd2+ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP-uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd2+ is due not only to its direct blocking action on Ca2(+)-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K+ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

The absence of mitochondrial thioredoxin 2 causes massive apoptosis,exencephaly, and early embryonic lethality in homozygous mice

Thioredoxin 2 (Trx-2) is a small redox protein containing the thioredoxin active site Trp-Cys-Gly-Pro-Cys that is localized to the mitochondria by a mitochondrial leader sequence and encoded by a nuclear gene (Trx-2). Trx-2 plays an important role in cell viability and the regulation of apoptosis in vitro. To investigate the role of Trx-2 in mouse development, we studied the phenotype of mice that have the Trx-2 gene silenced by mutational insertion. Homozygous mutant embryos do not survive to birth and die after implantation at Theiler stage 15/16. The homozygous mutant embryos display an open anterior neural tube and show massively increased apoptosis at 10.5 days postcoitus and are not present by 12.5 days postcoitus. The timing of the embryonic lethality coincides with the maturation of the mitochondria, since they begin oxidative phosphorylation during this stage of embryogenesis. In addition, embryonic fibroblasts cultured from homozygous Trx-2-null embryos were not viable. Heterozygous mice are fertile and have no discernible phenotype visible by external observation, despite having decreased Trx-2 mRNA and protein. These results show that the mitochondrial redox protein Trx-2 is required for normal development of the mouse embryo and for actively respiring cells.