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.     

Injection of mitochondria into oocytes and fertilized eggs]

The suspension of mitochondria isolated from the loach embryos or the frog heart were injected in the oocytes or fertilized eggs of the loach, newt, toad and frog in the amount roughly equivalent to the content of mitochondria in the egg. After the injection the oocytes did not differ during several days from the normal ones and the fertilized eggs of the loach, newt and South Afican clawed toad developed normally. The activity of cytochrome oxidase in the injected oocytes was kept at a somewhat higher level (1.4 to 1.9 vs 1.0 in the control) during several days. In the developing eggs the activity of cytochrome oxidase began to decrease from the blastula stage and attained rapidly the control level. The decrease of the enzyme activity is due to non-specific degradation of excessive mitochondria or to compensatory inactivation of the enzyme ensuring the maintenance of its normal activity during the development.     

Studies of heterogeneous mitochondrial populations in a mouse cell line: the effects of selection for or against mitochondrial genomes that confer chloramphenicol resistance

A single nucleotide substitution in a highly conserved region of the mitochondrial genome of a mouse cell line confers both chloramphenicol resistance and an alteration to the recognition site for the endonuclease Eco RV. This has enabled a detailed study on the effects of selection on a mitochondrial population comprising initially both chloramphenicol-resistant and chloramphenicol-sensitive mitochondrial genomes. The mutation confers advantage to cells grown in the presence of chloramphenicol, but is apparently deleterious in its absence. Selection at the cellular level is sufficient to explain the results observed. Fixation, which results in cells having mitochondria of only a single type, is slow. It is probable, therefore, that mammalian oocyte mitochondria are derived from only a small number of progenitors. This would allow fixation of new mutations and explain the observed uniformity in mitochondrial genomes of the individual in the presence of extensive variation between different members of the population.     

Transferring isolated mitochondria into tissue culture cells

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.     

mRNA translation during oocyte maturation plays a key role in development of primordial germ cells in<Emphasis Type="Italic">Xenopus</Emphasis> embryos

It is believed that cytoplasmic localization in the egg is necessary for development of primordial germ cells (PGCs) inXenopus embryos. In this study, we sought to determine if translation of maternal mRNA during oocyte maturation is involved in the development of PGCs. Donor oocytes were collected from both stimulated (those who receive gonadotropin) and unstimulated females, artificially matured and fertilized using a host transfer technique. Using chloramphenicol (50 μM and 500 μM RNA), RNA translation was inhibited during oocyte maturation. Our results showed that in unstimulated embryos treated with 50 μM chloramphenicol, there was a significant reduction in the number of PGCs reaching genital ridges. In stimulated embryos, however, the number of PGCs was unchanged unless a higher concentration (500 (μM) of chloramphenicol was used. From these results it is suggested that maternal mRNA translation during oocyte maturation plays a key role in development of PGCs.     

Retention of mitochondrial DNA species in somatic cell hybrids using antibiotic selection

Interspecific cell hybrids were constructed by fusion of an antimycin-resistant, thymidine kinase- (TK-) Chinese hamster cell line with a chloramphenicol-resistant, hypoxanthine-guanine phosphoribosyl transferase- (HPRT-) mouse cell line. Hybrids were selected in HAT medium alone, or HAT supplemented with chloramphenicol, antimycin, or both antibiotics. Analysis of the mitochondrial DNA (mtDNA) of these hybrids indicates that antibiotic selection directed at the mitochondrial populations results in retention of the resistant parental genome and loss of the sensitive parental genome.     

Distribution of foreign mitochondrial DNA during the first splitting of transmitochondrial mouse embryos

The distribution of human mitochondrial DNA (mtDNA) among single murine blastomeres was analyzed during the splitting of embryos injected with a suspension of human mitochondria at the one- or two-cell stage. Human mtDNA was detected by PCR with species-specific primers. The total amount of the- and four-cell murine embryos analyzed in the study was 315. In all embryos examined together with murine mtDNA copies of human mitochondrial genome were revealed indicating the phenomenon of an artificially modeled heteroplasmy. Foreign mtDNA was not ubiquitous in blastomeres of transmitochondrial embryos. Mathematical treatment of the results showed that, in the period between the injection of human mitochondria and the subsequent embryo cleavage, an uneven distribution of human mtDNA occurred in the cytoplasm. These results also indicate the presence of more than two to three segregation units of mtDNA in the entire pool of mitochondria (about 500) introduced into an embryo by microinjection.     

Statistical and frequency-amplitude characteristics of ultra-weak emissions of loach eggs and embryos under normal conditions and on their optic interactions. II. Changes in characteristics of ultra-weak emissions upon optic interaction of groups of embryos of different ages.

We compared the characteristics of ultraweak emissions from groups of loach embryos of different ages in the presence or absence of optic interaction. The percentage of zero values of emission gradually increased during the first hour of optic interaction. The number and height of rare big pulses estimated by the value of kurtosis increased in parallel. In addition, the correlation between the Fourier spectra of optically interacting samples decreased at a higher rate than in the absence of optical contact. Just after the 1-hour optic interaction was terminated, the number of high pulses decreased in a younger interacting group and increased in the older one and the farther away the partner groups were in developmental stages, the more pronounced these differences were. Measurements of the Fourier spectra after long-term (12-22-hour) optic interactions have shown that an "exchange" of autocorrelation characteristics of the spectra took place among the samples: the sums of autocorrelation coefficients were inverted in the vast majority of cases, often with an "overshoot" or, at least, were smoothed over with reference to the control samples. We conclude that the previously described effects of optic interactions between groups of loach embryos of different ages could be due to changes in the frequency spectra of their ultraweak emissions.     

Persistence and expression of circular DNAs encoding Drosophila amylase, bacterial chloramphenicol acetyltransferase, and others in Xenopus laevis embryos

We injected circular forms of several different DNAs into fertilized eggs of Xenopus laevis, and studied the persistence and expression of the injected DNAs during early embryonic development. When we injected plasmids which contained Drosophila amylase genes, the copy number of the injected DNA increased only slightly during cleavage, started to decrease at the blastula stage, then became very small at the tadpole stage. In such embryos, Drosophila amylase activity was detected at and after the gastrula stage. When we injected other kinds of circular DNAs (pX1r101A, cDm2055, pII25.1, pBR322, and pSP-64-L14), their copy number did not increase throughout the early stages. When circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes were injected, their copy number usually did not increase, but sometimes, for unknown reasons, it increased extensively throughout the blastula to gastrula stages. In both cases, CAT enzyme activity started to be expressed during the blastula to gastrula stages and disappeared at the 2 day-old tadpole stage. The level of CAT enzyme activity was roughly proportional to the amount of CAT mRNA formed, and also to the copy number of injected genes. From these results, we concluded that in Xenopus embryos, exogenously-injected circular DNAs are preserved for the most part as circular DNAs, and that the increase in their copy number within the embryos is not prerequisite for the expression of their genetic information.     

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.     

Developmental stages and germ cell lineage of the loach (Misgurnus anguillicaudatus)

The staging of embryonic and larval development, and the germ cell lineage of the loach, Misgurnus anguillicaudatus, are described. Fertilized eggs were obtained by artificial insemination. For the convenience of detailed observation and photography of the external appearance, we use dechorionated embryos. Through a series of operations, these embryos were cultured at 20 degrees C in an incubator. Embryonic and larval development of the loach was divided into five periods: cleavage, blastula, gastrula, segmentation, and hatching. Stages were assigned within each of these periods. Developmental stages were determined and named by morphological features and somite number. The staging series were photographed and tabulated. The germ cell lineage was then elucidated by whole mount in situ hybridization of mRNA expression of the germ-cell-specific marker vasa and histological analysis. Primordial germ cells (PGCs) of the loach derived from the cleavage furrows of 8-cell stage embryos began proliferation in the late blastula period and migrated to the gonadal anlagen through a migration pathway similar to that of the zebrafish. However, it is characteristic of the loach that PGCs migrate a long distance and stay in the posterior part of the yolk-extension region.     

ROLE OF THE MITOCHONDRIAL GENOME DURING EARLY DEVELOPMENT IN MICE : Effects of Ethidium Bromide and Chloramphenicol

The role of the mitochondrial genome in early development and differentiation was studied in mouse embryos cultured in vitro from the two to four cell stage to the blastocyst (about 100 cells). During this period the mitochondria undergo morphological differentiation: progressive enlargement followed by an increase in matrix density, in number of cristae, and in number of mitochondrial ribosomes. Mitochondrial ribosomal and transfer RNA synthesis occurs from the 8 to 16 cell stage on and contributes to the establishment of a mitochondrial protein-synthesizing system. Inhibition of mitochondrial RNA- and protein-synthesis by 0.1 µg/ml of ethidium bromide or 31.2 µg/ml of chloramphenicol permits essentially normal embryo development and cellular differentiation. Mitochondrial morphogenesis is also nearly normal except for the appearance of dilated and vesicular cristae in blastocyst mitochondria. Such blastocysts are capable of normal postimplantation development when transplanted into the uteri of foster mothers. Higher concentrations of these inhibitors have general toxic effects and arrest embryo development. It is concluded that mitochondrial differentiation in the early mouse embryo occurs through the progressive transformation of the preexisting mitochondria and is largely controlled by the nucleocytoplasmic system. Mitochondrial protein synthesis is required for the normal structural organization of the cristae in blastocyst mitochondria. Embryo development and cellular differentiation up to the blastocyst stage are not dependent on mitochondrial genetic activity.     

Cytoplasmic transfer of microtubule organizing centers in mouse tissue culture cells

A chloramphenicol-resistant, aminopterin-sensitive cell line (AMT) derived from a mouse mammary tumor MT-29240 was enucleated, and the cytoplasts were fused with nucleated chloramphenicol-sensitive, HAT-resistant SV40 3T3 mouse cells. The resulting cytoplasmic hybrids (cybrids) were selected for their resistance to chloramphenicol and the chromosome complement of the SV40 3T3 cells. In addition to transfer of chloramphenicol resistance, these cybrid clones, as studied in the electron microscope, contained the intracisternal A particle phenotype characteristic of only the AMT cells. The cytoplasmic microtubule complex (CMTC) in these cybrids was also studied and appears to resemble the elaborate CMTC of the AMT cells more closely than the more reduced CMTC of the SV40 3T3. When treated with a colcemid block and then a brief reverse, the microtubule organizing centers (MTOC) appear as bright fluorescent foci when tubulin antibody and indirect immunofluorescence techniques are used. When AMT or SV40 3T3 cells are treated in this manner, only one MTOC is present in interphase cells. One clone of these cybrids, however, contained two MTOCs in interphase cells. This CMTC and MTOC phenotype has persisted in this cybrid clone for over 3 months of continuous culture.     

Visualization of primordial germ cells in vivo using GFP-nos1 3'UTR mRNA

In some teleost fish, primordial germ cells (PGCs) inherit specific maternal cytoplasmic factors such as vasa and nanos 1 (nos1) mRNA. It has been shown that the 3'untranslated regions (UTRs) of vasa and nos1 have critical roles for stabilization of these RNAs in zebrafish PGCs. In this study, to determine whether this role of the nos 1 3'UTR is conserved between teleost species, we injected artificially synthesized mRNA, combining green fluorescent protein (GFP) and the zebrafish nos 1 3'UTR (GFP-nos 1 3'UTR mRNA), into the fertilized eggs of various fish species. The 3'UTR of the Oryzias latipes vasa homologue (olvas ) mRNA was assayed in the same manner. We demonstrate that the PGCs of seven teleost species could be visualized using GFP-nos 1 3'UTR mRNA. GFP-olvas 3'UTR mRNA did not identify PGCs in herring or loach embryos, but did enable visualization of the PGCs in medaka embryos. Our results indicate that the 3'UTR of the zebrafish nos1 mRNA can promote maintenance of RNAs in the PGCs of different fish species. Finally, we describe and compare the migration routes of PGCs in seven teleost species.     

Assignment of the chloramphenicol resistance gene to mitochondrial deoxyribonucleic acid and analysis of its expression in cultured human cells.

The mitochondrial deoxyribonucleic acids (mtDNA's) from human HeLa and HT1080 cells differed in their restriction endonuclease cleavage patterns for HaeII, HaeIII, and HhaI. HaeII digestion yielded a 9-kilobase fragment in HT1080, which was replaced by 4.5-, 2.4-, and 2.1-kilobase fragments in HeLa. HaeIII and HhaI yielded distinctive 1.35- and 0.68-kilobase HeLa fragments. These restriction endonuclease polymorphisms were used as mtDNA markers in HeLa-HT1080 cybrid and hybrid crosses involving the cytoplasmic chloramphenicol resistance mutation was used. mtDNA's were purified and digested with the restriction endonucleases, the fragments were separated on agarose gels, and the bands were detected by ethidium bromide staining and Southern transfer analysis. Three cybrids and four hybrids (four expressing HeLa and three expressing HT1080 chloramphenicol resistance) contained 2- to 10-fold excesses of the mtDNA of the chloramphenicol-resistant parent. One cybrid, which was permitted to segregate chloramphenicol resistance and was then rechallenged with chloramphenicol, had approximately equal proportions of the two mtDNA's. Only one hybrid was discordant. These results indicated that chloramphenicol resistance is encoded in mtDNA and that expression of chloramphenicol resistance is related to the ratio of chloramphenicol-resistant and -sensitive genomes within cells.     

Chloramphenicol effects on DNA replication in UV-damaged bacteria

Increasing UV-doses to cultures of Escherichia coli strain B/r decreased progressively the amount of DNA which was formed in the presence of chloramphenicol (160 μg/ml) from the amount formed in unirradiated control cultures in chloramphenicol-containing medium. This is attributed to the progressive inactivation of active sites of DNA replication by UV. In order to form DNA the bacteria must then replicate from the chromosomal fixed origin, an activity which requires protein synthesis and thus cannot occur in the presence of chloramphenicol. Such damage was shown to be subject to photoreactivation after lower UV-doses and thus is the pyrimidine dimer. After higher doses non-photoreversible lesions began to accumulate so that all such damage became non-photoreversible after 96 erg/mm². The rate of synthesis of DNA in the presence of chloramphenicol was shown to be very close to the rate shown by bacteria incubated in the absence of chloramphenicol, indicating that all active sites of replication remaining after UV-damage remain active in the presence of chloramphenicol, as expected if the limiting effect of chloramphenicol is on initiation at the chromosomal origin and not due to reduction in rate of DNA replication.A much lower concentration of chloramphenicol (2 μg/ml) blocking only the chloramphenicol-sensitive event in control of DNA replication described by Ward and Glaser¹⁵, imposed a limitation in DNA accumulation in the culture of somewhat less than a doubling, as would be expected if the antibiotic at this concentration does not block the chloramphenicol-resistant control event. DNA degradation occured with incubation of bacteria given a UV-dose sufficient to inactivate all active DNA replication sites on their chromosomes, when in medium containing chloramphenicol concentrations (above 20 μg/ml) sufficient to block the chloramphenicol-resistant control event. Such breakdown resulted in death. The damage responsible for such death and DNA breakdown was not photoreversible after this dose, supporting the hypothesis that breakdown results from non-photoreversible inactivation of active DNA replication sites. This was in contrast to increased death in UV-damaged bacteria promoted by nalidixic acid, a specific inhibitor of DNA replication, which could be prevented in part by light exposure after the same UV-dose.     

Features of the ultrastructure of loach embryos under the influence of norfloxacin

Results from a study of the ultrastructure of embryos of loach, Misgurnus fossilis L., on the stages of the first and tenth blastomere division under the control and in the presence of the fluoroquinolone series antibiotic norfloxacin (5 and 25 μg/ml) in the incubation medium are presented. The action of norfloxacin leads to ultrastructural changes in the cell organelles, such as hypertrophy of the rough and smooth endoplasmic reticulum and disorganization of the mitochondria and the plasma membranes of the embryos. It is established that fluoroquinolone inhibits biosynthesis processes that directly influence the biosynthesis structure of the blastomeres. Destructive changes in the organelles are a consequence of disturbances in assimilation processes that ultimately lead to death of the embryos. Thus, the results that have been obtained indicate that high embryotoxicity is characteristic of norfloxacin.     

In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos

Gene targeting technology is not available in the rat which is an animal model of major importance, e.g., in cardiovascular research. This is due to the fact that the rat embryonic stem cell (ESC)-like cells established by several groups do not form germ-line chimeras when injected into blastocysts. In the mouse, the aggregation of ESC with tetraploid embryos has allowed the generation of animals completely derived from these cells. However, aggregation of rat ESC-like cells with tetraploid rat embryos has not yet been attempted to evaluate their developmental capacity. Therefore, we established a method to produce tetraploid rat embryos by fusion at the two-cell stage. Chemical fusion by polyethylene glycol (PEG) was shown to be less efficient (56.3% fused embryos) than electrofusion (96.1% fused embryos). The rate of development of fused embryos to blastocysts was independent of the fusion method and similar to the rate of control embryos. However, this rate was lower when the embryos had been cultured from the zygote state before fusion (14-20%) compared to freshly isolated two-cell embryos (41-63%). Alike for the mouse, blastocysts derived from fused two-cell rat embryos contained about half the number of cells as control blastocysts and were homogeneously tetraploid with no evidence of mosaicism. This method may be useful for the establishment of gene-targeting technology in the rat.     

Fate of genetically marked mitochondrial DNA from spermatocytes microinjected into mouse zygotes.

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 x DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4-5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.     

Comparative study of superoxide dismutase and glutathione peroxidase activity in embryos and skeletal muscles of adult fish

The activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase in loach and sturgeon embryogenesis as well as in red and white skeletal muscles of loach was studied. The specific activity of cytoplasmic and mitochondrial forms of superoxide dismutase in developing sturgeon embryos was higher than in loach embryos, which may be due to oxygen conditions under which these species develop in nature. A similar dependence was also observed for the activity of glutathione peroxidase in embryos of these fish species. A comparative study of specific superoxide dismutase activity in loach and sturgeon embryos and in loach skeletal muscles showed that the activity of cytoplasmic superoxide dismutase is maximum in red and white muscles and minimum in loach embryos, whereas the activity of the mitochondrial form of this enzyme is maximum in red skeletal muscles.