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 microg/ml of ethidium bromide or 31.2 microg/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.

Mitochondrial DNA replication in sea urchin oocytes.

Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [(3)H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.

Complex mitochondrial DNA in animal thyroids. A comparative study.

1. The frequency of circular dimers and catenanes was determined in thyroid mitochondrial DNA (mtDNA) from rabbits, mice, pigs, sheep and cattle. 2. The mtDNA from freshly removed thyroids was isolated by buoyant density centrifugation in ethidium bromide/CsCl gradients after DNAase treatment of the mitochondrial pellet. Typically, more than 90% of the recovered mtDNA was found in the lower band, indicating a low rate of nicking during isolation. A sample of the total mtDNA (upper and lower bands) was examined by electron microscopy after preparation by the aqueous protein film technique. 3. The frequency of circular dimers generally ranged from 0.1 to 0.3%. However, in an mtDNA sample from cow thyroid, the frequency of circular dimers was 0.6% (0.9% if circular dimers occuring in catenanes are included(, differing significantly from the frequency of these forms in bull thyroid, 0.1%. A small but significant variability also occurred in the frequency of catenanes ranging from 2 to 8% in the different groups; this variation is within the limits usually observed in normal tissues. 4. These observations indicate that thyroids, like other normal tissues examined so far, have a low content of circular dimers. A high frequency of these forms seems to be the trademark of some genetically and physiologically abnormal cells such as certain established cell lines, virus-transformed cells and malignant or otherwise pathological tissues.

Sequence of a mouse embryo cDNA clone encoding proteolipid subunit 9 (P1) of the mitochondrial H(+)-ATP synthase.

A cDNA clone to an abundantly expressed mRNA in cleavage stage mouse embryos has been sequenced and identified as encoding subunit 9 (P1) of the mitochondrial H(+)-ATP synthase. The deduced amino acid sequence of the mature subunit 9 protein differs in a single residue from the corresponding rat, ovine, bovine and human subunits.

Structural and replicative forms of mitochondrial DNA from human leukocytes in relation to age.

The structure and replication of human leukocyte mitochondrial DNA (mtDNA) was investigated in healthy young adult males (23--37 years old), middle-aged males (42--52 years old) with secondary polycythemia, and elderly males (80--89 years old) who exhibited different degrees of age-related disease syndromes. The distribution of the various cell types within the white cell population was within normal limits in all samples. Total mtDNA was isolated in ethidium bromide--CsCl gradients and examined by electron microscopy after spreading by the aqueous and formamide techniques. The individual frequencies of catenated forms ranged from 2 to 6% but showed relatively little change (declining slightly) with age. The individual frequencies of circular dimers varied from 0 to 0.1% in the young adult and polycythemic groups and in 10 out of 12 elderly individuals. One elderly individual had a circular dimer frequency of 0.3% (including a circular molecular of tetramer size) and another had 4.5%. This finding suggest that agerelated cellular pathology may exist in the blood-forming system in some cases. The mode of replication of leukocyte mtDNA agrees well with that described for mouse L cells. There was no evidence of aberrant mtDNA replication as a result of aging.

Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: evidence for an increased frequency of deletions/additions with aging.

To obtain information on the extent of random nucleotide changes in mitochondrial DNA (mtDNA) from different organs of young adult and senescent Fischer 344 rats, the temperature of thermal denaturation (tm) was measured in (1) the native mtDNA cut at a single SstI site and (2) the reannealed duplexes formed after the initial melting of the mtDNA sample. No change was found between the two tm values in either young or senescent mtDNA, suggesting that the overall mismatch in nucleotide sequence in these samples was below the resolution of the method estimated at about 0.2%. In another experiment, mtDNA samples from young adult or senescent BALB/c mouse liver were digested with EcoRI, denatured and allowed to reanneal. The duplexes formed by the 14-kb EcoRI fragment were analyzed in randomly taken electron micrographs for the occurrence of mismatched segments. About 1.8% of reconstituted duplexes in adult mtDNA and 11% of those in senescent mtDNA contained small loops or knobs suggestive of deletions/additions of about 400 +/- 150 nucleotides. These data correspond to about 1% of the native mtDNA population in adult liver and about 5% in senescent liver having deleted/inserted segments. Although deletions/insertions may occur at variable sites, their distribution appears to be non-random. These findings suggest that small sequence rearrangements, which have been observed previously in unicircular dimers of mouse and human mtDNA, occur also in monomeric mtDNA from normal tissues and accumulate with aging.

Structural and replicative forms of mitochondrial DNA in tissues from adult and senescent BALB/c mice and Fischer 344 rats.

Age-related changes in the structure and replication of mitochondrial DNA (mtDNA) were investigated in different organs from young adult (9-10 months' old) and senescent (28-29 months' old) BALB/c mice and Fischer 344 rats. Total mtDNA from brain, heart, kidney and liver was isolated by centrifugation in ethidium bromide-CsCl gradients and examined for the occurrence of complex forms and replicative intermediates by electron microscopy. The frequency of catenated mtDNA (interlinked molecules containing two or more circular units) varied from about 2.5% to 5% in adult tissues and showed a small increase in the majority of senescent organs. The frequency of double-sized circular molecules, or circular dimers, was very low in adult tissues, with an average of about 0.04% in mice and 0.1% in rats. The frequency of circular dimers increased with aging to 1.9% in mouse brain and 1.5% in rat kidney, with smaller increases (0.4% and 0.7%) in heart mtDNA from both species; there was no significant increase in the other organs. It is suggested that the increase in the frequency of circular dimer mtDNA reflects an overall deterioration of tissue physiology rather than intrinsic senescent changes in the mitochondria. The frequencies and types of the various replicative forms of mtDNA varied significantly according to tissue but not according to species or donor age. The only exception was a significant increase in the frequency of larger replicative forms in senescent mouse liver, to about 20% compared with 12% in adult liver, suggesting an age-related change in the rate of mtDNA replication and/or turnover in this organ.

Variation in the frequency of complex forms of mitochondrial DNA in different brain regions of senescent mice.

It was shown previously that the frequency of an aberrant form of mitochondrial DNA (mtDNA), double-sized circular molecules or circular dimers, increased significantly in the brain of senescent mice, to about 2% versus less than 0.1% in the brain of adult mice. To follow up these observations, we isolated total mtDNA from 6 different brain regions of 29-month-old male BALB/c mice and examined it for the occurrence of circular dimers and other complex forms by electron microscopy. There was a statistically highly significant variability in the occurrence of circular dimer mtDNA among the 6 brain regions. The frequencies of circular dimers were: medulla, 3.3%; cortex, 1.7%; midbrain, 1.1%; cerebellum, 0.9%; hippocampus, 0.5%; and striatum, 0.2%. The frequency of catenated (topologically interlinked) molecules varied only slightly, from 4 to 6%. On the basis of the available literature, a correlation appears to exist between age-related tissue pathology of the mouse brain and the increased incidence of circular dimer mtDNA. Although no cause-effect relationships can be established, it is suggested that the frequency of circular dimer mtDNA may be a useful marker in assessing the general physiological condition of the aging brain.

Complex forms and replicative intermediates of mitochondrial DNA in tissues from adult and senescent mice.

The occurrence and types of complex forms and replicative intermediates of mitochondrial DNA (mtDNA) were investigated in tissues from C57BL/6J mice aged 10-11 months or 29-30 months. Total mtDNA from brain, heart, kidney and liver was isolated in ethidium bromide-CsCl gradients and examined by electron microscopy after aqueous or formamide spreading. Contour length measurements indicated no difference in the monomer size of mtDNA according to either tissue or donor age. The frequencies of catenated mtDNA, ranging from 4 to 8%, varied significantly according to tissue but changed relatively little as a result of donor age. The main age-related effect observed in this study was a significant increase in the frequency of circular dimers, from about 0.05% in adult tissues to 0.3% in kidney, 0.5% in liver, 0.6% in heart and 1.9% in brain of senescent mice. The frequency of D-loop DNA varied from 30 to 60% and that of larger replicative intermediates from 1 to 10%, suggesting differences in the rate of mtDNA replication according to tissue. The frequencies and types of the various replicative intermediates were unaffected by donor age.

Poly(A) length, cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos.

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B. Clegg, 1982, Dev. Biol. 89, 362-378). To shed light on the significance of these changes, we analyzed the size distribution of total poly(A) from embryos at different stages of development by gel electrophoresis and hybridization with [3H]poly(U). The number-average size of poly(A) tracts varies only slightly, from 61 to 77 nucleotides, indicating that the changes in poly(A) content are due primarily to changes in the number of poly(A) sequences, i.e., the number of poly(A)+ mRNA. From these data, the number of poly(A)+ mRNA can be estimated as follows: ovulated egg, 1.7 x 10(7); one-cell embryo, 2.4 x 10(7); late two-cell, 0.7 x 10(7); late eight-cell, 1.3 x 10(7); and early blastocyst, 3.4 x 10(7). These results suggest the elimination of the bulk of maternal poly(A)+ mRNA at the two-cell stage, to be replaced by newly synthesized mRNA derived from the embryonic genome. To study the synthesis of poly(A)+ mRNA, we cultured mouse embryos in vitro with [3H]adenosine and analyzed the labeled poly(A)+ RNA as to molecular size, length of the poly(A) tail, and relative distribution of label in poly(A) vs internal locations. We observed an active incorporation of label into large-molecular-weight (average size about 2 kb) poly(A)+ RNA at all stages from the one-cell to the blastocyst. However, in the one-cell embryo, about 70% of the label was localized in the poly(A) tail, suggesting cytoplasmic polyadenylation, and only about 30% was localized in the remainder of the molecule, suggesting the complete new synthesis of a small amount of poly(A)+ RNA. Differences in the size distribution of the labeled poly(A) as compared with the total poly(A) in the one-cell embryo indicate that the labeling is not due to a general turnover of poly(A) tails, but rather to the polyadenylation of previously nonpolyadenylated, stored RNA. Significant new synthesis of poly(A)+ RNA is evident from the two-cell stage onward and most likely accounts for the sharp rise in the number of poly(A)+ RNA molecules by the early blastocyst stage.

Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos.

Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for periods of up to 320 min during which the specific activity of the ATP pool was constant. The time course of the molar accumulation of adenosine was calculated for tRNA, high-molecular-weight poly(A)- RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of the adenosine incorporation into tRNA is due to turnover of the 3'-terminal AMP but some new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate of about 0.2 pg/embryo/h. In the poly(A)- RNA fraction, an unstable component which is assumed to be heterogeneous nuclear RNA is synthesized at a high rate and accumulates at a steady-state level of about 1.5 pg/embryo in the 1-cell embryo and about 3.0 pg/embryo in the 2-cell embryo. Both 1-cell and 2-cell embryos synthesize relatively stable heterogeneous poly(A)- RNA, assumed to be mRNA, at a rate of about 0.3 pg/embryo/h; 2-cell embryos also synthesize mature ribosomal RNA at a rate of about 0.4 pg/embryo/h. Internally labelled poly(A)+ RNA is synthesized at a low rate in the 1-cell embryo, about 0.045 pg/embryo/h, but the rate increases to about 0.2 pg/embryo/h by the 2-cell stage. A striking feature of the 1-cell embryo is the high rate of synthesis of poly(A) tails, about 2.5 X 10(6) tails/embryo/h of an average length of (A)43, due almost entirely to cytoplasmic polyadenylation. This and other evidence suggests a turnover of the poly(A)+ RNA population in 1-cell embryos as a result of polyadenylation of new RNA sequences and degradation of some of the pre-existing poly(A)+ RNA. In the 2-cell embryo, the rate of synthesis of poly(A) tails (average length (A)93) is estimated at about 0.8 X 10(6) tails/embryo/h and a significant fraction of poly(A) synthesis appears to be nuclear.

Mitochondrial biogenesis in early mouse embryos: expression of the mRNAs for subunits IV, Vb, and VIIc of cytochrome c oxidase and subunit 9 (P1) of H(+)-ATP synthase.

The mouse egg contains about 90,000 mitochondria which undergo a buildup of mitochondrial cristae and increase in respiratory activity during cleavage. The mitochondrial DNA does not replicate during preimplantation development but is transcribed actively from the two-cell stage onward (Pikó and Taylor, 1987: Dev Biol 123:364-374). To gain further insight into mitochondrial biogenesis, we have now determined the steady state amounts of the mRNAs for the cytochrome c oxidase (COX) subunits IV, Vb and VIIc and the H(+)-ATPase subunit 9 (P1) (all encoded by nuclear genes) in slot hybridization experiments of total RNA from oocytes and early embryos. All four mRNAs showed a similar developmental pattern of prevalence, characterized by a steady decline in mRNA copy numbers from the late growth-phase oocyte through the two-cell embryo, and an about 30-fold rise during cleavage through the blastocyst stage. However, the ATPase subunit 9 (P1) mRNA was about three times more prevalent in cleavage-stage embryos than the COX mRNAs. A similar pattern was obtained previously for the mitochondrial-encoded COX I and II mRNAs, but the latter accumulate at a 30-50-fold excess over the nuclear-encoded COX subunit mRNAs during the cleavage stages. The results suggest a coordinated activation and transcription of the mitochondrial and nuclear genes for the components of the respiratory apparatus beginning with the two-cell stage. It is estimated that new respiratory chains are produced at a rate of 50-100 chains hr-1/mitochondrion in the early blastocyst, accounting for 3.5-7% of the total protein synthetic activity at this stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos.

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.

Expression of the rig gene in mouse oocytes and early embryos.

A clone selected from a two-cell mouse embryo cDNA library has been sequenced and identified as rig cDNA. The rig gene codes for a highly conserved nuclear protein, which may have a general role in cell growth or replication (Shiga et al.: Proc Natl Acad Sci USA 87:3594, 1990). The quantitative changes in rig mRNA were studied in blot hybridization experiments with total RNA from oocytes and early embryos. The amount and relative abundance of rig mRNA change considerably during early development. There are about 1.6 x 10(4) rig mRNA molecules in a late growth-stage oocyte; this number is reduced to about one-tenth in the ovulated egg but increases about twenty-fold during cleavage through the blastocyst stage. In F9 embryonal carcinoma cells, the relative abundance of rig mRNA is similar to that in blastocysts (about 0.1% of the mRNA population), but it is about eight-fold higher in the mouse myeloma cell line MOPC-104E. The high level of rig mRNA in late growth-stage oocytes suggests that the rig gene product may be important for overall transcriptional activity rather than DNA replication and mitosis. Alternatively, the rig protein may be a storage product of oogenesis and have a role in the initiation of development.