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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 [3H]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.

1 Abbreviations used in this paper: Cairns' form, replicative intermediate in which both daughter segments are double stranded; D-loop DNA, mitochondrial DNA with a single-stranded displacement loop of approximately 3% genome size; Exp-D DNA, replicative intermediate with an expanded D loop which may be totally or partially single stranded; KTE, 0.55 M KC1, 0.01 M cyte and egg mtDNA has been well characterized as to amount, location, circularity, and physicalchemical properties in several animal groups: amphibians (2,3,5), echinoderms (13,14), and echiurids (4). However, there is relatively little information available on mitochondrial biogenesis and mtDNA replication in growing oocytes. In Tris-HCl, 0.001 M EDTA, pH 7.6; mtDNA, mitochondrial DNA; TE, 0.01 M Tris-HCl, 0.001 M EDTA, pH 7.6. The terminology adopted in this paper follows that of Robberson et al. (16). 146 THE JOURNAL OF CELL BIOLOGY . VOLUME 63, 1974 . pages 146-159 vivo long-term labeling experiments in the sea urchin have demonstrated incorporation of [3H]thymidine into oocyte mtDNA (11,14). Fine structural studies of oogenesis in sea urchins (20) as well as determinations of cytochrome c oxidase activity in developing oocytes of Urechis caupo (12) indicate that mitochondria are synthesized continuously during oogenesis. The formation of new mitochondria in growing sea urchin oocytes occurs in the absence of nuclear DNA replication (7). This paper presents a study of mtDNA replication in developing oocytes of the sea urchin Strongylocentrotus purpuratus. The mtDNA was isolated by buoyant density centrifugation in ethidium bromide-CsCl gradients and analyzed by electron microscopy after formamide spreading. The replicative intermediates of oocyte mtDNA can be arranged in a pattern similar to that found in mouse L cells (8,9,16) and various normal and tumor tissues (22,23). The following variations were observed: the oocyte mtDNA contains Dloop DNA at a low frequency; duplex synthesis on the displaced strand occurs early and is initiated at multiple sites; and the frequency of Cairns' forms is the highest reported so far for mtDNA. Replicative intermediates with expanded D loops were recovered primarily in the closed circular form, a result previously found for L-cell mtDNA (15)and for simian virus 40 (SV40) DNA (17). These findings demonstrate that a nicking-closing cycle is active during the course of replication.
The mtDNA in mature unfertilized eggs from two species of sea urchins was exclusively in the form of clean circular duplexes. This form is therefore considered to be the stable physiological storage form of mtDNA in these eggs.
We also describe some novel structural variants of superhelical mtDNA that have been observed on specimen grids prepared with the formamide technique.

Isolation of Mature Eggs
Mature unfertilized eggs of the sea urchins S. purpuratus and Lytechinus pictus were collected by KCI injection of the animals (19). The eggs were washed three times in sea water and three additional times in 0.55 M KCl to remove the jelly coats.

Isolation of Oocytes
Oocytes were isolated by a procedure similar to that described by Giudice et al. (6). Ovaries from six to ten S. purpuratus females were removed after KC1 shedding of the mature eggs. After three washes in Millipore-filtered Instant Ocean sea water (Aquarium Systems, Inc., Eastlake, Ohio) and three additional washes in 0.55 M KC1, 0.01 M Tris-HC1, 0.001 M EDTA, pH 7.6 (KTE), the ovaries (10 12 g wet weight) were teased and dissected into small pieces and digested for about 15 min under constant stirring in about 40 ml of a Pronase solution (1 mg/ml, B grade, Calbiochem, San Diego, Calif.) in KTE at room temperature. The suspension (excluding large debris) was washed three times with 200-ml ice-cold KTE by centrifugation at about 100g for 10-15 s in a Sorvall HB-4 rotor (DuPont Co., Instrument Products Div., Newtown, Conn.). The final pellet was resuspended in 20 ml of KTE which was divided into four batches of 5 ml each, and each batch was layered on 40 ml of an isotonic linear sucrose gradient (0.15-0.85 M) constructed in the following manner: 1.5 parts I M sucrose in 0.01 M Tris-HC1, 0.001 M EDTA, pH 7.6 (TE buffer) was diluted with 8.5 parts KTE to obtain an isotonic 0.15 M sucrose solution, and 8.5 parts 1 M sucrose in TE was diluted with 1.5 parts KTE to give an isotonic 0.85 M sucrose solution; these two solutions were then used to form an isotonic linear sucrose gradient. After centrifugation for 1 1.5 min at about 100 g in a Sorvall HB-4 rotor, the suspension was separated into the following main components: ovarian tissue cells in the upper portion of the gradient; mature eggs, large oocytes, and debris near the bottom of the tube; and an enriched band of small to medium oocytes in the middle of the gradient. The oocytes were collected by pipette, washed twice with KTE, resuspended in 10 ml KTE, and recentrifuged on the isotonic 0.15-0.85 M sucrose gradient as above. The oocytes were washed twice in KTE (for the isolation of mitochondria) or in sea water (for incubation with [3H]thymidine). The yield of oocytes was about 2 6% of the wet weight of the ovaries used. The yield was higher early in the reproductive season.

Isolation of Mitochondria
Mature unfertilized eggs or oocytes (either isolated freshly or after incubation with [3H]thymidine) were suspended in eightfold volume of ice-cold 0.4 M sucrose in TE and were homogenized with 10 strokes in a Potter-Elvehjem homogenizer (Arthur H. Thomas Co., Philadelphia, Pa.). The molarity of the homogenate was adjusted to 1 M sucrose with 2 M sucrose in TE. After dilution with 1 M sucrose in TE as needed, the homoge-MATSUMOTO, KASAMATSU, PIK6, AND VINOGRAD Mitochondrial DNA Replication 147 hate was layered on 3 ml of 1.5 M sucrose and centrifuged in an SW25.1 rotor at 23 krpm for 30 min at 5°C in a Beckman L2-65B ultracentrifuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.). Dense cellular constituents pelleted at the bottom of the tube, mitochondria migrated to the 1.0 1.5 M sucrose interface, and yolk formed a cap at the top of the gradient. The mitochondria were collected by pipette and washed once with KTE.

Isolation of mtDNA
The final mitochondrial pellet was suspended in 1 ml of 0.5 M NaCI, 0.1 M Tris-HCl, 0.01 M EDTA, pH 8.0, and lysed with an equal volume of prewarmed (37°C) 4% dodecyl sodium sulfate (M C & B Manufacturing Chemists, Cincinnati, Ohio) in distilled water. A sample of 14C-labeled SV40 marker DNA containing components I and II was added at this point. The lysate was adjusted to a final density of 1.55 g/ml with CsCI (optical grade, Harshaw Chemical Co., Solon, Ohio) and to about 400 #g/ml ethidium bromide (Boots Pure Drug Co., Ltd., Nottingham, England) in a total volume of 3.5 ml in a 5-ml polyallomer tube. After centrifugation for 18 h at 40 krpm, 20°C, in the Beckman SW50.1 rotor the contents of the tube (except for the pellet and top layer) were transferred to a second tube and centrifuged for 44 h at 38 krpm. In the experiments with labeled oocytes, the gradients were fractionated in 40-#1 fractions and an aliquot of each fraction was assayed for radioactivity in a Beckman LS-200 scintillation counter. The Whatman 3 MM discs were air dried, washed in three changes of cold 5% TCA, and dehydrated in ethanol and ether for counting. The remainder of the fractions was used for analyses by electron microscopy. In the experiments with unlabeled oocytes, all of the mtDNA from the region spanning the upper and lower bands in the gradient was collected and prepared for electron microscopy. In the study of mtDNA from mature unfertilized eggs, the lower band DNA (containing the major portion of the total DNA) was collected from the EthBr-CsCl gradient; this DNA was prepared for electron microscopy either directly or after a further purification step by band velocity sedimentation in EthBr-CsCl (9).

Electron Microscopy
Fractions collected from the EthBr-CsCl gradient were dialyzed in the dark at 4°C against several changes of 0.1 M NaCI, 0.1 M Tris, 0.01 M EDTA, pH 8.5, containing Dowex 50WX8 resin (Bio-Rad Laboratories, Richmond, Calif.). The DNA was concentrated in the dialysis tubing with dry Sephadex 200 (Pharmacia, Uppsala, Sweden) and dialyzed again. Formamide spreading of the DNA was carried out as described (9) except that the formamide concentration in the hypophase was 17% instead of 10% and the final cytochrome c concentration was 40 ~g/ml. Films were picked up on parlodion-coated 300-mesh copper grids, rotary shad-owed with Pt-Pd, and examined in a Philips EM200 or 300 at an accelerating voltage of 40 or 60 kV. Photographs were taken at a magnification of about × 5,000. The lengths of the single-and double-stranded regions in the molecules were measured from tracings of negatives enlarged on a Nikon 6F projection comparator. For an analysis of duplex synthesis on the displaced strand (Figs. 3 and 4), randomly selected squares on the grid were scanned and all Exp-D molecules (except for those having extensive entangled regions) were photographed; molecules containing D loops (less than 5% of genome size) were generally excluded from this sample. In these preparations the contour lengths of single-stranded 4~X DNA and duplex RF ~bX DNA, which were used as internal standards, were equal.

Isolated Oocytes
The isolated oocytes and the purity of the preparations used in these studies are illustrated in Fig. 1. The mean oocyte diameter of 186 randomly measured oocytes was 41 ± 9 #m SD, with a size range of 20 76 ~tm. The bulk of the oocytes was of medium size, 30 60 #m, and was in the vitellogenesis stage (20). Mature S. purpuratus oocytes are about 80 ~m in diameter (18).

Replicative Forms in Total Oocyte MtDNA
The types and frequencies of the various replicatire forms in the total mtDNA from S. purpuratus oocytes are shown in Table I. Contamination with linear DNA amounted to about 10% in these samples. In two such preparations, the replicative forms accounted for 21 and 14% of the circles. Molecules with D loops were relatively infrequent, 7 and 4%, and constituted about 30% of replicative forms; the average size of the D loop in 17 molecules was 3.2 ± 0.7% of the genome size. Gapped circular molecules ( Fig. 2 e) were scarce. Molecules with expanded D loops represented by far the most frequent class and were used for an analysis of the occurrence and the initiation of duplex synthesis.

Duplex Synthesis in Exp-D Molecules
Electron microscope examination revealed that duplex synthesis had occurred in 90% of Exp-D molecules. Cairns' forms, i.e., totally doublestranded replicating intermediates, were frequent and accounted for 25% of the Exp-D molecules, and molecules with partial duplex synthesis accounted for 65%. Fig. 2 illustrates the various types of replicating intermediates and Fig. 3 148 THE JOURNAL OF CELL BIOLOGY . VOLUME 63, 1974  presents the data from which the above percentages were derived. Fig. 3 shows the relative size of the duplex region(s), in the expanded segment of 79 Exp-D molecules and nine D-loop molecules from preparation a of Table I Fig. 4 were examined, but no clearcut pattern emerged to indicate that initiation of the duplex segments occurred at unique sites on the displaced strand.  presumably associated with sea urchin egg polysaccharide (13); electron microscope examination failed to reveal DNA in this band. The frequencies of various forms of mtDNA in different regions of the gradient (Fig. 5, fractions B-G) were determined by electron microscopy and are shown in Table II. Samples from the intermediate region of the gradient (Fig. 5, fractions D, E) were greatly enriched in mtDNA molecules with expanded D loops, a high proportion of which (about 80%) was totally duplex or Cairns' forms. A substantial portion of these molecules exhibited double-branch migration (Fig. 9) and several Exp-D molecules contained single-stranded denaturation loop(s) (Fig. 7 b); these forms are discussed below. Gapped circular molecules were present only in the upper band region (Fig. 5, fractions F, G); most of the gaps were on the order of 1-3% genome size. D-loop molecules were observed infrequently in the lower and intermediate regions of the gradient. The reason for the scarcity of D-loop DNA is not clear but it could have been due to the occurrence of D-loop expansion in the absence of D-loop initiation in the isolated oocytes. Nicking in the system appeared to be low and the bulk of the mtDNA sedimented in the lower band as observed by fluorescence in the gradient when viewed with ultraviolet light. Table III shows the results of an electron microscope study of mtDNA from mature, unfertilized eggs of two sea urchin species. Virtually all the DNA in these eggs was found as clean circular duplexes (Fig. 6).

MtDNA of Mature Eggs
We measured the size of the mtDNA from S.   4.45 4.85 urn, on the basis of electron microscope calibration methods (13,14).

MOLECULES WITH DENATURED REGIONS:
In the course of this work several unreported DNA structures were observed. Simple closed circular mitochondrial and SV40 DNA molecules with single-stranded denaturation loops (Fig. 7 a) were seen at frequencies of about 2 and 5%, respectively, in the DNA from the lower band of an EthBr-CsCl gradient (Fig. 5, fractions B D). These DNAs are regarded as closed molecules because of their buoyant positions. They were absent in the open circular DNA sample from the upper band (Fig. 5, fractions F, G). The denatured region accounted for 2 and 4% of the contour length in the mitochondrial and SV40 DNA. A study of the occurrence of such molecules in preparations of SV40, PM2, and mitochondrial DNA, to be reported elsewhere, has indicated that these denaturation loops arise as a result of the "early melting" (21) of superhelical DNAs having negative superhelicai turns. The removal of the superhelical turns results in a relaxed circular conformation and the formation of a denatured (unwound) duplex region localized in a presumed A-T-rich region. These molecules have previously escaped detection because, in part, they were unexpected and also possibly because of minor variations in the technique of specimen preparation. For example, these structures are not observed if the salt concentration in the DNA-cytochrome solution is elevated over that used in this work as described in Materials and Methods. Denaturation loop(s) were also observed in closed replicative intermediates isolated from an intermediate region between the lower and upper bands in an EthBr-CsC1 gradient (Fig. 5, fraction E). As in the example shown in Fig. 7 b, the denaturation loop always occurs in the nonreplicated region which we assume was originally negatively supercoiled and melted as described. The duplex in the replicated region contains a 152 THE JOURNAL OF CELL BIOLOGY • VOLUME 63, 1974 minimum of two swivels, one at each fork, and, therefore, is not subject to the thermodynamic instability referred to above as causing the early melting of negatively superhelical DNA.

REPLICATIVE INTERMEDIATES WITH LIN-
EAR DUPLEX SEGMENT(S) EMERGING FROM THE FORK(S); Several complex structures were observed on specimen grids prepared with fractions C-F, Fig. 5 (see Table II). Among the larger replicative intermediates in these fractions, 20% were interpretable as double branch-migrated (1, 10) closed Exp-D molecules illustrated diagrammatically in Fig. 8 B. These molecules all contained denaturation loops in the nonreplicated region. Electron micrographs of three such molecules are shown in Fig. 9. In the first two ( Fig. 9 a  and b), the junction of the four duplexes at each of the replicating forks is clearly visible as a small,   Table 11). Fractions from the gradient shown in Fig. 5.
MATSUMOTO, KASAMATSU, Pro6, AND VINOGRAD Mitochondrial DNA Replication almost rectangular, single-stranded loop. In the third example ( Fig. 9 c), no junction loops are visible. The size of the junction loop is variable but on the average corresponds to about 100 nucleotides and could have resulted from the symmetric melting of 12 base pairs in each of the four duplexes forming the junction region as illustrated diagrammatically in Fig. 8 C. The linear duplex segments with free ends that emerge from the replicating forks (Fig. 8 B) were usually of unequal length. In one molecule out of 17, one of the emerging linears was entirely single stranded; in the others the emerging linear segments were entirely duplex. We propose that the emerging duplex segments have arisen by double-strand branch migration as shown diagrammatically in Fig. 8 A and B. The complementary progeny strands form an emerging linear duplex segment at each fork while the corresponding parental strands base pair to form a duplex within the closed circle. This process requires the introduction of negative superhelical turns or denaturation loops to maintain the topological winding number constant. Alternatively, if a denaturation loop is formed in the structure shown in Fig. 8 A, branch migration would occur (in the absence of supercoiling) to conserve the topological winding number (21). The observed molecules ( Fig. 9) contain some crossovers and two to four denatured single-stranded looped regions. The loops are always in the nonreplicated region and are not seen in the nonconstrained replicated regions. If the total length of the emerging linear duplex segments is taken to be a

154
THE JOURNAL OF CELL BIOLOGY • VOLUME 63, 1974 FIGURE 7 Electron micrographs of oocyte mtDNA molecules with denaturation loops. Fig. 7 a, a mtDNA circle with a denaturation loop; inset, a SV40 DNA circle with a denaturation loop. Fig. 7 b, a Cairns' form with a denaturation loop and a denatured region at one of the forks, x 60,000. In the corresponding line drawings the thin line represents single strands, the medium-heavy line represents double-stranded portions of the parent molecule, and the extra-heavy line represents double-stranded daughter segments.
measure of the extent of duplex formation within the closed circle, we would expect that the total length would be equal to one-half the sum of the contour lengths of the denaturation loops in the nonreplicated region. Actually, six of the 17 molecules are in accord with the relation as shown in Fig. 10. In the remainder, the contour lengths of the denaturation loops are shorter by as much as 50% than expected. This could have resulted from the formation of a number of smaller loops which were too small to be detected or, alternatively, from a failure of some of the denaturation loops to MATSUMOTO, KASAMATSU, PIK6, AND VINOGRAD Mitochondrial DNA Replication Schematic diagrams illustrating branch migration in covalently closed replicative intermediates. Thick and thin lines represent progeny strands and parental strands, respectively. Fig. 8 A, Cairns' type replicative intermediate. In Fig. 8 B, d and e represent the lengths of the branch-migrated linear duplex segments; a and a', the replicated regions remaining after branch migration; cl and c2, duplex segments in the nonreplicated region; and f, the length of the denatured loop. Fig. 8 C illustrates the partial melting at the fork regions.
spread out fully on the electron microscope specimen grid. The molecules which have undergone branch migration are clearly replicative intermediates because, as indicated in Fig. 8 B, the lengths a and a' were equal to each other and the sum ((a' + a)/2, cl, c2, and f/2) was equal to the monomer length. The extent of replication, ((a + a')/2 + d + e) 100/(monomer length), varied from 16 to 68% among the 17 molecules analyzed as shown in the inset in Fig. 10. It may be calculated from the inset that approximately one-third of the replicated regions in these molecules had branch migrated.
At the present time the driving force for the double-branch migration or, alternatively, for the formation of the denaturation loops is not understood, nor can we account for the fact that only one-fifth of the large replicating forms underwent branch migration.

DISCUSSION
Because of the large number of mitochondria in sea urchin oocytes, we have been able to obtain relatively clean preparations of mtDNA with simple preparative procedures and without DNase treatment of the mitochondria. Our results show that the replicative intermediates of oocyte mtDNA are similar to those found in mouse L cells (8,9,16) and in rat liver and rat hepatoma cells (22,23). However, there are several differences as outlined below.
The frequency of D-loop molecules in total oocyte mtDNA, 4-7%, is the lowest reported so far. A substantial loss of D loops is considered to be unlikely because of the apparent low rate of nicking during isolation. The low frequency of D-loop molecules suggests that the relative dura-tion of this holding stage in mtDNA replication is shorter in these cells. Another difference from the model derived from the study of mtDNA replication in L cells is in the pattern of duplex synthesis on the displaced strand. In sea urchin mtDNA duplex synthesis is initiated in nearly all molecules with an expansion of 15-20% as against approximately 60% expansion in L cells (16). During further expansion, the rate of duplex synthesis keeps pace wi{h the rate of expansion (Fig. 3). About 25% of expansions contain multiple duplex segments (Fig. 4), indicating that duplex synthesis takes place with multiple initiations. Cairns' forms, varying in size from D-loop to 0.8 genome size, are frequent: about 25% of Exp-D molecules in a total mtDNA preparation from freshly isolated oocytes and about 80% of larger replicative intermediates in a preparation obtained from cultured oocytes. The frequency of Cairns' forms is considerably higher than that observed in other systems, namely, about I% of Exp-D molecules of mtDNA in L cells (16) and 3-8% of all doubleforked molecules in normal rat liver and rat hepatoma cells (23). It is clear that the degree to which displacement and duplex syntheses are synchronized may vary in different systems, for reasons which are not understood at present. The low frequency of gapped circles in the oocyte DNA is consistent with the high frequency of Cairns' forms. The gapped circles have been postulated to arise upon separation of fully expanded replicating intermediates before duplex synthesis has been completed (16).
The results of isotope labeling experiments (Fig.  5) show that isolated oocytes take up and incorporate [3H]thymidine into mtDNA. The observation that Exp-D molecules are located primarily in the intermediate region of an EthBr-CsC1 gradient  Table II) indicates that these molecules are covalently closed as has been shown for replicating mtDNA in L cells (15) and for SV40 DNA (17). These findings imply that displacement synthesis is accompanied by nicking-closing cycles.
The duration of a complete cycle of replication of mtDNA in S. purpuratus oocytes is difficult to estimate since the time needed for the completion of oocyte growth is not known. However, on the basis of studies of oogenesis in wild S. purpuratus females (7), the duration of oocyte growth from 20 to 80 #m (mature) size can be roughly estimated at 3-4 wk, giving a doubling time for oocyte volume of about 4 days. Since about 10 14% of mtDNA in a total oocyte preparation (Table I) are replicating forms (exclusive of D-loop molecules), the replication time of a mtDNA molecule can be estimated at about 10-14 h. The corresponding overall rate of replication (about 0.007/Lm/min) is similar to that estimated for rat liver mtDNA (23) but is considerably slower than the 0.1 /Lm/min calculated for mtDNA in L cells (16).
At the termination of oogenesis, sea urchin eggs undergo nuclear maturation and are stored in the ovary until spawning and fertilization. Nearly all the mtDNA recovered from maure, unfertilized eggs orS. purpuratus and L. pictus were present in the form of covalently closed, clean circular duplexes, A loss of D loops during isolation of the mtDNA is considered highly unlikely, since special care was taken in the preparative procedures and spreading for electron microscopy was carried out as expeditiously as possible, without any intervening storage of the DNA. We conclude that mtDNA in these eggs is stored as clean duplex circles. Electron microscope analyses of mtDNA from sea urchin embryos indicate that the mtDNA remains in this form throughout early development at least until the early pluteus stage (Matsumoto and PikG, unpublished observations). However, this observation is not generally true for animal eggs, since over 80% of mtDNA molecules in unfertilized frog eggs contain D loops (Hallberg, personal communication). The reason for this difference is unknown but it may be related to differences in the state of nuclear and cytoplasmic maturation in these eggs. The sea urchin egg is fertilized after nuclear maturation has been completed whereas in vertebrate eggs fertilization takes place at the second maturation metaphase.