Mitochondrial DNA synthesis in oocytes of Xenopus laevis

414

Preliminary

notes

cells to participate in intercellular transfer of three unrelated categories of small molecule. This suggests that intercellular transfer of different classes of small molecule is mediated by systems which have at least some elements in common, and lends genetic support to the view, based on physical studies [3, 17, 181, of the permeable junction or gap junction as an array of hydrophilic pores, specific for substrate only in terms of size, forming a direct link between the cytoplasms of cells in contact. We are grateful to Professors J. H. Subak-Sharpe and J. A. Pateman for their constant support and encouragement, to Dr B. Car&t for permission to include the results of some of his enzyme assays and to Drs C. MacDonald, E. Buultjens and S. Gaunt for helpful discussions. This work was supported by grant no. SP1341 from the Cancer Research Campaign to Professors J. H. Subak-Sharpe and J. A. Pateman.

References

18. Sheridan, J D, Hammer-Wilson, M, Preus, D & Johnson, R G, J cell biol76 (1978) 532. 19. Hooper, M L & Slack, C, Dev biol55 (1977) 271. 20. Fogh, J & Fogh, H, Proc sot exp biol med 117 (1964) 899. 21. Chen, T R, Exp cell res 104 (1977) 255. 22. Slack, C, Morgan, R H M, Carritt, B, Goldfarb, P S G & Hooper, M L, Exp cell res 98 (1976) 1. 23. Hooper, M L, Carritt, B, Goldfarb, P S G & Slack, C, Somat cell genet 3 (1977) 313. Received August 3 1, 1978 Revised version received November 2, 1978 Accepted November 7, 1978

Printed m Sweden Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/79/04414-05$02.00/O

Mitochondrial DNA synthesis in oocytes of Xenopus laevis ANDREW C. WEBB and CATHERINE J. CAMP, Department of Biological Sciences, Wellesley College Welleley, MA 02181, USA

1. Subak-Shame. J H. Burk. R R & Pitts. J D. Heredity 21 (1%6) 342. 2. Subak-Sharpe, J H, Burk, R R & Pitts, J D, J cell sci 4 (1%9) 353. 3. Pitts, J D, The developmental biology of plants and animals (ed C F Graham & P F Wareing) p. %. Blackwell, Oxford, UK (1976). 4. Pitts, J D & Burk, R R, Nature 264 (1976) 762. 5. Fentiman, I, Taylor-Papadimitriou, J & Stoker, M. Nature 264 (1976) 760. 6. Gaunt, S J & Subak-Sharpe, J H, Exp cell res 120(1979) 307. 7. Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117(1978) 195. 8. y;;s, J D & Simms, J W, Exp cell res 104 (1977)

Summary. Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35kO.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28kO.02 pgloocytelh. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [3H]dTTP was calculated to be 0.43+0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.

9. Corsaro, C M & Migeon, B R, Nature 268 (1976) 737. 10. Martin, G R & Evans, M J, Proc natl acad sci US 72 (1975) 1441. 11. Gilula, N B, Reeves, 0 R & Steinbach, A, Nature 235 (1972) 262. 12. Loewenstein, W R, Cold Spring Harbor symp quant biol40 (1975) 49. 13. Carritt, B, Goldfarb, P S G, Hooper, M L & Slack, C, Exp cell res 106 (1977) 71. 14. Rieske, E, Schubert, P & Kreutzberg, G W, Brain res 84 (1975) 365. 15. Pitts, J D & Finbow, M E, Intercellular communication (ed W C De Mello) p. 61. Plenum, New York (1977). 16. Carritt, B, Cytogenet cell genet 19 (1977) 44. 17. Makowski, L, Caspar, D L D, Phillips, W C & Goodenough, D A, J cell biol74 (1977) 629.

Mitochondria are one of the many components of oocytes that are accumulated and stored during oogenesis for subsequent use by the developing embryo. For example, a mature, ovulated oocyte ofXenopus laevis, the South African clawed frog, contains a 105-fold enrichment of mitochondria compared with a somatic cell and 3OCL500times as much mitochondrial DNA (mtDNA) as chromosomal DNA [l]. In recent years much information has been compiled on the accumulation and synthesis of

Exp Cell

Res

119 (1979)

Preliminary

mtDNA in embryonic systems [l-4], but comparatively little is known concerning mtDNA activity during oogenesis. A series of six stages through which the Xenopus oocyte progresses have been morphologically characterized by Dumont [5]. During stage 1 the mitochondrial aggregate present in the mature oogonium [6] condenses to form the Balbiani body [7, 81. Ultrastructural and autoradiographic studies have suggested that expansion of the Balbiani body during pre-vitellogenic growth (stages l-3) is accompanied by an increase in the mitochondrial population [7, 81. This increase in organelle number is accompanied by essentially a linear accumulation of mtDNA during this initial period of oogenesis [9]. With the onset of active yolk accumulation, the Balbiani body disaggregates [7] at about the same time accumulation of mtDNA ceases [9]. One question which this phenomenon raises is whether the observed cessation of mtDNA accumulation, at a time when the oocyte is only about half-grown, is due to either a steady-state synthetic system, similar to that proposed for mtRNA by Webb et al. [lo], or alternatively to the termination of mtDNA synthesis from mid-oogenesis (stage 34) until day 2 of embryonic development. In order to distinguish between these two possibilities, incubation of isolated mitochondria in the presence of [3H]dTTP [l l] has been employed to determine the relative synthetic rates of mtDNA in stage 3 and stage 6 oocytes ofxenopus. Materials

and Methods

Isolation of oocyte mitochondria. Stage 3 and 6 oocytes [S] were isolated manually after CohagenasePronase (Calbiochem, Grade B) digestion of ovarian fragments from unstimulated Xenopus laevis (Snake Farm, Fish Hoek, RSA) as previously described [9]. The method of mitochondrial isolation was essentially that of Gause et al. [ 121,with about 2 000 oocytes being used in each preparation. Contaminating yolk and pigment granules were removed by isopycnic sucrose

notes

415

gradient centrifugation [I], and the purified mitochondria were resuspended in 0.4 ml of 80 mM sucrose, 10 mM EDTA, 30 mM Tris-HCI (pH 7) and kept at 5°C until used in the incorporation experiments. The quantitative estimation of protein was performed [ 131 on duplicate 10 ~1 samples of each mitochondrial suspension, using a bovine serum albumin (BSA) standardization curve. In vitro labeling of mtDNA. The method of Gause et al. [ 121was adapted to label Xenopus oocyte mtDNA in vitro with [methyl-3H]thymidine 5’-triphosphate (r3H]dTTP, SchwarzlMann, 50 Cilmmole). Incubation of 500 ~1 assays containing 2.5-10 mg/ml mitochondrial protein was performed at 20°C in a shaking water bath (300 rpm) for a period of 2 h. The final ethanol-washed TCA pellet was dissolved in 250 ~1 of NCS tissue solubilizer (Amersham/Searle) by hearing at 50°C for 60 min. After cooling, raxoactivity was determined by addition of 5 ml of PPO-toluene (4 g/l) scintillation fluid. The amount ‘of f3HldTTP incorporation into mtDNA was assessed as being the total TCA-insoluble radioactivity per assay minus the nonspecific TCA-precipitable radioactivity of the incorporation mixture without mitochondrial protein, or in the presence of an equivalent amount of BSA. Isolation of tntDNA. Preparations of DNA from labeled mitochondria were made using the method of Borst et al. [14]. The ethanol-precipitated nucleic acid was dissolved in 1 ml of 0.1 mM Tris-HC1 (pH 7) containing 5 mM MgCl,. To determine the authenticity of acid-insoluble radioactivity as incorporation into mtDNA, the nucleic acid sample was subjected to digestion at 37°C for 30 min in the presence of 100~a/ ml pancreatic DNase I (Worthington). Upon completion of the incubation period both DNase-digested and control samples were further deproteinized with chloroform-isoamyl alcohol and TCA precipitated. Over 90% of the radioactivity associated with isolated mtDNA was found to be DNase sensitive. In ovo labeling of mtDNA. The kinetics of [3H]dTTP incorporation into stage 6 oocyte mtDNA was determined as already reported for mtRNA using [3H]GTP [lo]. Twenty to thirty manually defolliculated, white-banded Xenopus oocytes were used for each time point, each oocyte being micro-injected with approx. 4 pmoles of [3H]dTTP (5.2~105 dpm, 50 Cilmmole) and cultured at 20°C in sterile Ringer’s solution. Batches of oocytes were cultured for 2, 4, 6, 8, 12, 15 and 18 h as well as a “zero-time” point. DNA was extracted from isolated mitochondria as nreviously described [9]. The ethanol-precipitated miDNA was resuspended in 0.01 M Tris-HCI (pH 8), 0.01 M EDTA and unincorporated [3H]dTTP removed by passage over a Sephadex G-25 column equilibrated with the same buffer. Fractions were pooled, corrected for recovery [see lo] and [3H]dTTP incorporation determined by TCA precipitation. Mitochondrial DNA polymerase activity. The activity of DNA polymerase associated with mitochondria extracted either from whole ovary or isolated oocytes was assayed essentially as described by Mikhailov & Gause [3], except that denatured calf thymus DNA (100 pg/ml) was provided as a templateprimer and 5 FM [3H]dTTP (ICN, 80 Cilmmole) was included in the incubation medium at a specific activity of 1 Cilmmole. Exp Cd Res 119 (1979)

416

Preliminary

notes

Table 1. In vitro synthesis of DNA by mitochondria isolated from stage 3 and stage 6 Xenopus oocytes a Oocyte stageb 3 6 6 6 6 6

Animal A A B C D

[3H]dTTP incorporation (pmolhg proteinc/h)

Synthetic rate (pgloocytelh)”

0.24k0.01e 0.02~0.009’ 0.018 0.021 0.025 0.021+0.003’

2.35f0.28’ 0.28f0.01’ 0.25 0.30 0.28 0.28&0.028

I

I

I

I

2

6

10

14

18

3-

l-

a See Materials and Methods for details. * See ref. [5]. c Mitochondrial soluble protein. d See Results for calculations. e Mean values (*SD.) taken from four analyses of animal A. ’ Mean values (?S.D.) taken from six separate determinations on animal A. u Mean (+S.D.) of all values for stage 6 oocytes given in table 1.

Results

I

time (hours); ordinate: dpmloocyte (x10-2). Representative kinetics of in ovo r3H]dTTP incorporation into mtDNA isolated from Xenopus stage 6 oocytes (see Materials and Methods for details). In this particular experiment, each oocyte was microinjected with 5.2~ lo6 dpm r3H]dTTP (approx. 4 pmoles). The specific activity of the mitochondrial r3H]dTTP pool 4-6 h after microinjection was considered to have equilibrated with the cytoplasmic dTTP pool (see Results) at about 5x 104dpm/pmole. It can be seen from the curve that once linear, the rate of [3H]dTTP incorporation is about 19 dpm/oocyte/h, corresponding to a rate of mtDNA synthesis of some 0.54 pg/ oocytelh.

Fig. I. Abscissa:

Mitochondria isolated from Xenopus stage 6 oocytes incorporate 0.021 pmoles r3H]dTTP/mg mitochondrial protein/h into pressed on a per oocyte basis, the mitoTCA-precipitable material. This represents chondria exhibit almost an g-fold decrease roughly an order of magnitude decrease in in the incorporation rate (table 1) from the the incorporation rate of mitochondria ex- time at which the Balbiani body (mitotracted from stage 3 oocytes (table 1). Using chondrial aggregate) of the developing oocyte disaggregates (stage 3) through to the published value of 10 ,ug mitochondrial protein per stage 6 Xenopus oocyte [l], completion of the growth phase (stage 6). these incorporation data can be readily conIn calculating synthetic rates of mtDNA verted to [3H]dTTP/oocyte/h. However, based on these incorporation data it was asthe lack of an empirical value for mito- sumed that the base composition of Xenochondrial protein content of stage 3 oocytes pus mtDNA is identical with that of the necessitates that an estimation be made (i) total mtRNA population [l]. Thymine utilizing the experimentally determined would thus constitute 24 mole-percent of mtDNA content in the stage 3 oocyte of the DNA molecule, and for every pmole of 3.0 ng [9] and (ii) assuming that a constant r3H]dTTP incorporated as dTMP, 1415 pg ratio of mtDNA to mitochondrial protein is of mtDNA would be synthesized. The remaintained throughout Xenopus develop- sults in table 1 show that the stage 6 oocyte ment [ 11.Under these conditions, an homo- synthesized mtDNA at a rate of 0.28 pglh genate of 143 stage 3 oocytes would yield compared to 2.35 pg/h for the stage 3 1 mg of mitochondrial protein. When exoocyte. By comparison, the kinetics of [3H]Exp Cell Res 119 (1979)

Preliminary

Table 2. DNA polymerase chondrial extracts a

activity

in mito-

Source of mitochondria

[3H]dTTP incorporation at 20°C (nmol/mg mitochondrial protein/h)

Whole ovary Stage 3 oocytes Stage 6 oocytes

5.2f0.44* 4.5f0.36 1.6t0.36

a For incubation conditions, consult Materials and Methods. * Values are means (fS.D.) from three analyses.

dTTP incorporation by the intact Xenopus stage 6 yielded a rate of mtDNA synthesis in ovo of 0.43t0.08 pg/oocyte/h (n=4). In calculating this rate, the cytoplasmic dTTP pool of the stage 6 oocyte was taken to be 7 pmoles [15] and assumptions were made [see lo] concerning’ the relative specific activities and equilibration of the mitochondrial and cytoplasmic pools. Typically, as shown in fig. 1, an initial lag phase of 4-6 h precedes linear [3H]dTTP incorporation reflecting similar kinetics of mitochondrial dTTP equilibration to those reported for GTP [lo]. Clearly one factor that could substantially affect rates of DNA biosynthesis within mitochondria at different stages of oogenesis is the level of DNA polymerase activity in the organelle. Table 2 summarizes data that suggest the level of mitochondrial DNA polymerase activity in stage 3 oocytes to be about 3-fold higher than that found in the fully-grown stage 6 oocytes. The observed enzyme activity was found to be strictly dependent upon the presence of a DNA template (4-7% of control) and strongly inhibited by the addition of 50 pg/ ml ethidium bromide (Sigma) to the incubation mixture (3-5% of control). It is apparent that an elevated mitochondrial DNA polymerase activity during the earlier

notes

417

stages of Xenopus oogenesis could in part account for the more rapid synthesis of mtDNA in stage 3, compared with stage 6 oocytes. Discussion

The results of this study (see table 1) indicate that the pattern of mtDNA accumulation duringXenopus oogenesis observed by Webb & Smith [9] can be attributed to a reduction in the mtDNA synthetic rate of the later stage oocytes rather than a “steady-state” situation where the rate of degradation equals the rate of synthesis. The slightly higher in ovo rate of mtDNA synthesis obtained during this study for the stage 6 oocyte suggests that the in vitro rates may in fact be minimum values and not represent the true intrinsic rates of mtDNA synthesis attributable to these developmental stages. The experimental evidence gathered in this and previous studies onxenopus oogenesis indicate that prior to stage 4 of development, immature oocytes are active in mitochondrial proliferation [8], thereby contributing to the observed accumulation of mtDNA [9] and Balbiani body growth [7]. Assuming that the rate of mtDNA synthesis remains constant during these early stages, and utilizing the published data on growth rates for Xenopus oocytes [16], it would require a theoretical synthetic rate of 34 pg/oocyte/h to account for the observed increase of mtDNA from stage 2 to stage 4 [9]. Since mtDNA synthesis is dependent upon mtDNA polymerase activity, any inhibition in the synthesis, activity or availability of the polymerase due to the in vitro isolation procedure would account for the comparatively low value of 2.35 pg/ oocyte/h obtained (table 1) for the stage 3 oocytes. For example, it is possible that optimum DNA polymerase activity may be Exp Cell Res 119 (1979)

418

Preliminary

notes

dependent upon the spatial arrangement of the Balbiani body and germinal vesicle, since it is known that mtDNA polymerase is coded for on the nuclear genome [ 171. The inevitable disruption of the Balbiani body during the mitochondrial isolation procedure may consequently depress the polymerase activity. In a similar vein, it was of considerable interest to find there to be a marked depression in DNA polymerase activity associated with mitochondria from stage 6 compared to stage 3 oocytes (table 2). There is, of course, a naturally occurring dispersion of the Balbiani body at or around stage 3 of Xenopus oogenesis [.5, 71. Furthermore, there is a precedent for potential fluctuations in DNA polymerase activity, albeit cytoplasmic, during Xenopus oogenesis. For example, replication of calf-thymus DNA following injection into stage 3 oocytes was found to be 2-fold greater than that observed at stage 6 [ 181. Once the replication of mtDNA molecules has been completed by stage 4 of Xenopus oocyte growth, the low synthetic rate measured in this investigation could account for the production of solely D-loop configurations, which are known to be present in 76% of the mtDNA molecules isolated from a mature oocyte [19]. If Dloop synthesis commences in stage 4 oocytes, a theoretical synthetic rate of some 0.30 pgloocytelh would be required to account for the observed frequency of Dloops in full-grown oocytes. Interestingly, the rates of synthesis obtained in the present study for mtDNA in stage 6 oocytes are perfectly consistent with this calculation. In accordance with the present finding, Mikhailov & Gause [3] have observed a transition from a state of active mtDNA synthesis in the young oocyte of the loach Exp

Cd

Res I19 (1979)

to one of relative quiescence in the older oocyte. Furthermore, the rate of mtDNA synthesis in both loach [3] and sea urchin [4] embryos is accelerated during the initial stages of embryogenesis. These observations show remarkable agreement with the experimental findings on Xenopus in this and previous studies [ 1, 81 implying that a similar pattern of mtDNA synthetic activity may operate in several developing systems. The authors gratefully acknowledge the assistance of Dr Jim Reynhout during the microinjection experiments. This study was supported in part by a Faculty Award made to A. C. W. by Wellesley College.

References I. Chase, J W & Dawid, I B, Dev bio127 (1972) 504. 2. Piko, L, Dev biol21 (1970) 257. 3. Mikhailov, V S & Gause, G G Jr, Dev biol 41 (1974) 57. 4. Gadaleta, M N, Nicotra, A, Del Prete, M G & Saccone, C, Cell differ 6 (1977) 85. 5. Dumont, J N, J morphol 136(1972) 153. 6. Al-Mukhtar, K A K &Webb, A C, J embryo1 exp morph0126 (1971) 195. I. Balinsky, B I & Devis, R J, Acta embryo1 morphol exp 6 (1963) 55. 8. Al-Mukhtar, K A K, Ph.D thesis, University of Southampton, UK (1970). 9. Webb, A C & Smith, L D, Dev biol56 (1977) 219. IO. Webb, A C, LaMarca, M J & Smith, L D, Dev biol 45 (1975) 44. 11. Gause, G G Jr & Mikhailov. V S. Biochim biouhvs acta 324 (1973) 189. 12. Gause, G G Jr, Dolgilevich, S M, Fatkullina, L G & Mikhailov, V S, Biochim biophys acta 312 (1973) 179. 13. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 14. Borst, P, Ruttenberg, G J C M & Kroon, A M, Biochim biophys acta 149(1967) 140. IS. Woodland, H R & Pestell, R Q W, Biochem j 127 (1972) 597. 16. Scheer, U, Dev biol30 (1973) 13. 17. Borst, P, Ann rev biochem 41 (1972) 333. 18. Gurdon, J B & Speight, V A, Exp cell res 55 (1969) 253. 19. Hallberg, R L, Dev biol38 (1974) 346. Received September 7, 1978 Revised version received November I, 1978 Accepted November 6, 1978