accumulation of mitochondrial dna during oogenesis in xenopus laevis

DEVELOPMENTAL BIOLOGY 56, 219-225 (1977)

Accumulation of Mitochondrial DNA during Oogenesis in Xenopus laevis

ANDREW C. WEBB’ AND L. DENNIS SMITH

Department of Biological Sciences, Purdue University, West Lafayette, Zndiana 47907

Received September 9,1976; accepted September 22,1976

The mitochondrial DNA (mtDNA) content of Xenopus laeuis oocytes at various stages of oogenesis has been determined by molecular hybridization with 3H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 pm in diameter (stage 1) contains 0.95 k 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500-750 Km diameter oocyte), by which time a steady-state level of 4.28 + 0.40 ng/oocyt.e is attained. Using the hybridization assay, the stage 6 (full-gr0wn)Xenopu.s oocyte contains 4.51 k 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termina- tion of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.

INTRODUCTION

It has been recognized for several years that the ovulated eggs of many animals are enormously enriched for 2 variety of organelles and macromolecules. This “store” of substances is sequestered within the egg during oogenesis, but the time of appearance of the various materials bears no consistent relationship to the progres- sion of oocytes through the stages of oogenesis. For example, ribosomal RNA (2% and 1% RNA), which constitutes the bulk of the total RNA in full-grown Xenopus Zaeuis oocytes, appears to accumulate lin- early in oocytes between 300 and 1000 pm in diameter (33). Yet 5s RNA, which forms an integral component of the mature ribo- some is not synthesized coordinately with 28s and 18s molecules, but rather is accumulated in 15 to 20-fold molar excess during previtellogenesis (18). Similarly, the content of poly(A)-containing RNA rises until the oocyte is about 300 pm in diame-

’ Present address: Department of Biological Sci- ences, ,Wellesley College, Wellesley, Massachusetts 02181.

ter and then remains at a maximum value for the remainder of oogenesis (32). Fi- nally, the micropinocytotic uptake of yolk protein (vitellogenin) commences in oocytes about 500 pm in diameter, reaches a maximum in oocytes with diameters around 900-1100 pm, and then declines (34).

The full-grown Xenopus oocyte is also 105-fold enriched for mitochondria, compared to somatic cells and contains some 300 times as much mtDNA as chromo- somal DNA (9). This large population of mitochondria appears to serve as the sole source of mitochondria for the developing embryo (15) since no net increase in the mtDNA content of embryos can be de- tected for about 2 to 3 days after fertilization (9). However, neither the time nor the sequence of mitochondrial accumulation within the oocyte has been fully eluci- dated.

Many ultrastructural observations (e.g., l-3) have already demonstrated that there are large numbers of mitochondria that form the bulk of the cytoplasmic structure

219

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0012-1606

220 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

known as the Balbiani body or “yolk nu- cleus,” as defined by early students of egg cytology (see 31). The origin of the Balbi- ani body has been traced back to a juxtanuclear mitochondrial aggregate within the Xenopus primordial germ cell and oogonium (2). This mitochondrial aggregate increases in size in previtellogenic oocytes, leading to suggestions that it represents the cytological manifestation of intense mitochondrial replication (2). In support of this hypothesis, high-resolution autoradiography has indicated considerable DNA synthesis in association with the mitochondrial aggregate during the initial stages ofxenopus oogenesis (1). However, the synthesis of mtDNA appears to termi- nate (2) with the time of Balbiani body disaggregation at the onset of vitellogenesis (3).

The present study was undertaken to determine if the accumulation of mtDNA correlates with the time during oogenesis when oocytes contain the Balbiani body, or whether mtDNA continues to accumulate after dispersal of the mitochondrial aggregate. For this purpose, we have chosen to utilize, with slight modification, the hybridization assay developed for the quantitation of mtDNA by Chase and Dawid (9). Our results indicate that the youngest previtellogenic oocytes examined (~250 pm in diameter) contain slightly less than 1 ng of mtDNA, and this content increases progressively until the oocyte reaches about 500-750 pm in diameter, by which time it has accumulated about 4.5 ng of mtDNA. This maximum value is maintained throughout the remainder of oocyte growth, oocyte maturation, and fertilization. Thus, attainment of the maximum mtDNA content appears to correlate fairly well with the onset of vitellogenesis and dispersal of the Balbiani body in Xenopus oocytes.

MATERIALS AND METHODS

Mitochondrial DNA. Xenopus mitochondria were isolated from whole adult

ovaries using essentially the procedure described by Chase and Dawid (9). DNA was extracted from the pelleted mitochondria by the Hirt (25) technique, followed by purification of closed circular mtDNA [Form I, Ref. (24)l in CsCl-EthBr gradients (26). Extensive dialysis, passage over Dowex 50-W (Bio-Rad) and equilib- rium centrifugation in CsCl gradients yielded the final preparation of highly purified mtDNA.

Plasmid DNA. Plasmid Co1 E, (strain JC 411) was prepared as described by Cle- well and Helinski (11). The DNA was labeled with [methyZ-3H]thymidine (20-50 Ci/mmole, Schwarz/Mann) to a specific activity of approximately 3 x lo4 cpm/pg (5), purified as for mtDNA, and then further characterized by velocity sedimentation in sucrose gradients (23).

14C-labeled mitochondria. The radioac- tive mitochondria used as a recovery marker during the isolation of Xenopus mitochondria were prepared from preswol- len BHK fibroblasts (BHK-21, clone 13) grown to confluence in Eagle’s MEM (Kansas City Biological) supplemented with 10% (v/v) fetal calf serum (Flow Lab- oratories) and containing 0.25 &i/ml of [14Clarginine monohydrochloride (318 mCi/mmole, Amersham-Serle). The mitochondrial pellet was resuspended in EDTA buffer and divided into loo-p.1 aliquots (approximately 1000 cpm), which were stored frozen at -20°C.

Complementary RNA. In vitro tran- scription of mtDNA was performed as previously described (30) in 250-500 ~1 of reaction mixture containing all four nucleoside triphosphates tritiated to a high specific activity (>lO Cilmmole), 5-20 pug of pure mtDNA, and saturating amounts of RNA polymerase (>lO units/pg of DNA) isolated from E. coli strain A-19 by the method of Burgess (8). Incorporation of labeled nucleotides was found to be essentially linear over the first 2 hr of incubation at 37°C. Recovery of the [3HlcRNA (sp act 106-10’ cpmlpg) was performed as de-

BRIEF NOTES 221

tailed by Graham and Skinner (21) with the exceptions that (i) RNA was extracted by shaking with an equal volume of 1:l (vl v) phenol/chloroform containing 0.1% (wl v) 8hydroxyquinoline and saturated with 0.1 M Tris-HCl (pH 81, (ii) the phenolic layer was reextracted with 50 n&f Tris- HCl (pH 8), (iii) the pooled aqueous phases were passed directly over a Sephadex G-50 (fine) column, and (iv) E. coli tRNA (Sigma) was used as carrier during the ethanol precipitations.

mtDNA-cRNA hybridization. Oocytes were dissociated from either adult or im- mature Xenopus ovaries by collagenase (Calbiochem, grade B) digestion and man- ually sorted into stages essentially accord- ing to the scheme proposed by Dumont (17). Numbers of oocytes ranging from 1000 stage 1 to 200 stage 6 were selected for each determination and any adhering fol- licle cells were removed by incubation in Pronase (Calbiochem, grade B) as previously described (35). Full-grown oocytes that were to undergo in vitro maturation were also treated as already reported (35).

After the addition of a sample of 14C- labeled BHK cell mitochondria to the extraction buffer, a crude mitochondrial preparation was obtained from each group of oocytes as described above. The mitochondria were then lysed by the addition of 8x SSC containing 0.5% (w/v) SDS. A sample of 3H-labeled plasmid DNA was added to each lysate before alkali denatur- ation in 0.1 x SSC followed by gravity loading in 4x SSC at 4°C (two to three passes) onto presoaked nitrocellulose filters (Schleicher and Schnell, type B-6, 25 mm). Filters were processed for hybridization more or less as described by Gillespie and Speigelman (201, except that they were preincubated for 1 hr in Denhardt mixture (16). Four “mini” filters (6.5 mm diameter) were cut from each dried large filter (19 mm effective loading diameter); two were counted immediately to provide recovery and filter retention data, while the other two were used for hybridization.

Control “mini” filters containing known amounts of highly purified mtDNA were constructed in a similar manner. The total mtDNA bound to a large filter (75-90%) was computed from “mini” filter radioactivity by appropriate area correction.

Prior to hybridization, the [3HlcRNA sample was resuspended in 8x SSC, passed three times through filters soaked for 30 min in 8x SSC containing 0.1% (w/ v) SDS (19), and then heated in a boiling water bath for 10 min (28) before mixing with an equal volume of formamide. Fil- ter-bound mtDNA was hybridized in sealed scintillation vials with subsaturat- ing amounts of [3H]cRNA in 4~ SSC/50% (v/v) formamide at 40°C for 16-20 hr (14). The filters were then washed with two changes of hybridization buffer at 40°C and two changes of 4x SSC at room temperature. The filters were further incu- bated for 30 min at 37°C in 2 x SSC containing 5 lLglm1 of boiled pancreatic and 10 units/ml of T, ribonucleases (lo), washed twice with 2x SSC at 37”C, and rinsed twice with SSC at room temperature. Fil- ter-bound radioactivity was determined by combustion of filters as described previously (27).

All data were corrected for nonspecific binding of [3HlcRNA, which was assessed for each experiment by inclusion of blank filters and also filters containing Esche- richia coli DNA (Sigma) in the same reassociation mixture. Control experiments with [3Hlplasmid DNA indicated that 5- 8% of the filter-bound DNA was lost during hybridization and subsequent processing.

RESULTS

Filters with known and unknown amounts of mtDNA were hybridized together in vials containing 3H-labeled cRNA as described in detail above. Control experiments have shown (see Fig. 1) that at least over a range of input mtDNA of lo-350 ng per filter, the hybridization of labeleli cRNA is directly proportional to the amount of DNA bound to the filter,


“r

I I I I I I 1 50 100 150 200 250 Xx) 350

MITCCHONDRIAL ONAIFILTER (ng)

FIG. 1. Relationship between [3H]cRNA hybridization and mtDNA bound to filters. Different amounts of Xenopus ovarian mtDNA were loaded onto filters (see Materials and Methods) either alone (x) or mixed with an excess of heterologous DNA before hybridization together in the same solution of VHlcRNA. Binding and retention of the DNA to filters was monitored individually (see Materials and Methods) in the mixed samples by the inclusion of L3H]plasmid DNA marker to each, whereas for ovarian mtDNA alone, it was taken to be 75% for all filters in the series. Xenopw nuclear DNA was prepared from erythrocytes as described by Brown and Weber (7). BHK cell mtDNA was extracted from unlabeled BHK cell mitochondria as described for the isolation of Xenopus mtDNA in Materials and Methods. X, Xenopus ovarian mtDNA alone; 0, Xenopus ovarian mtDNA with E. coli DNA; A, Xen- opus ovarian mtDNA with BHK cell mtDNA; 0, Xenopus ovarian mtDNA with plasmid DNA; 0, Xenopus ovarian mtDNA with Xenopus nuclear DNA.

and unaffected by the presence of heterologous DNAs. By reference to cRNA binding levels to standard filters loaded with known amounts of purified Xenopus mtDNA included during each hybridization reaction in the same vial, the quantity of mtDNA isolated from different stage oocytes was determined. The results from several experiments are summarized in Fig. 2.

Any estimation of changes in the abso- lute amounts of mtDNA during oogenesis will of necessity depend critically upon an accurate quantitation of recovery and/or loss of mtDNA at each step in the isolation procedure and subsequent assay.

The loss of mitochondria during their isolation from Xenopus oocytes was deter-

mined by the inclusion of 14C-labeled heterologous mitochondria in the homogeni- zation buffer. Since the BHK cell mitochondria used were labeled with P4Clarginine (see Materials and Methods) and we have no good data on the leaching- out of soluble proteins from isolated mitochondria, our estimate of 20-25% loss of mitochondria during extraction must be considered a maximum value or potential overestimate. This conclusion is further substantiated by our previous estimates based on cytochrome oxidase monitoring (351, which indicated a 15-20% loss of mitochondria during extraction. In addition, our use of crude mitochondrial lysates rather than purified DNA during the hybridization assay for mtDNA content may have been subject to overestimates due to some RNA-RNA reassociation, rather than exclusively DNA-RNA as assumed. We have further assumed that there is no variability in the proportion of Co1 E, DNA to mtDNA molecules which are al-

OOCYTE STAGE

I23 4 5 6 ,M UF

II II I I II"1 11

1 I1 1 I I I .I I I 2 4 6 8 IO 12 - M UF

MEAN OOCYTE DIAMETER (,“n~nlK)-~)

FIG. 2. Oocyte mtDNA content during oogenesis inxenopus laevis. The hybridization data have been corrected for losses during preparation of mtDNA (see Results) before being expressed as nanograms of mtDNA per oocyte. The number of separate determinations used to calculate the mean value (m) for the different oocyte stages appears encircled above each data point. The vertical confidence limits rep- resent the standard deviation of the values at each stage, whereas the horizontal limits reflect the size range of oocytes included in the determinations. M, oocytes induced to mature in vitro by exposure to progesterone; UF, unfertilized egg data (0) from Chase and Dawid (9).

BRIEF NOTES 223

kali sensitive in samples from different oocyte stages. Taking these factors into account, our estimates (see Fig. 2) of 4.51 f. 0.69 ng of mtDNA per stage 6 oocyte, and 4.4 ng for maturing oocytes, compare very favorably (within the statistical error of our own estimates) with the previously reported (9) direct determination for the unfertilized Xenopus egg of 3.8 ng.

It is clear from the data presented here that the accumulation of mtDNA by Xeno- pus oocytes is not a continuous process throughout the period of oogenesis. The smallest previtellogenic oocytes examined in this study were all grouped together as stage 1 oocytes with diameters less than 250 pm. We found that these oocytes each contained on average 0.95 ? 0.47 ng of mtDNA (Fig. 2). However, by this time, the prominent juxtanuclear mitochondrial aggregate (Balbiani body) has already un- dergone considerable enlargement (2, 3). Thus, the Xenopus oocyte entering its growth phase undoubtedly inherits consid- erably less than 1 ng of mtDNA from the oogonium. Beyond stage 1, there appears to be an almost linear increase in the content of mtDNA until oocytes reach about 500-750 pm in diameter (stage 41, after which time the amount of mtDNA remains more or less constant. In fact, considering the data of Chase and Dawid (9), this amount remains essentially constant through the remainder of oogenesis, oocyte maturation, ovulation, and fertilization until about stage 30 of embryonic development, when linear accumulation of mtDNA begins again, resulting in a dou- bling of embryo mtDNA content by stage 45 (swimming tadpoles).

DISCUSSION

One interpretation of our data might be that the plateau seen in Fig. 2 represents a steady-state level of mtDNA. This, of course, would necessitate continued synthesis of mtDNA throughout the terminal stage of Xenopus oogenesis, coupled with turnover of mtDNA, and possibly the mi-

tochondria, at a rate identical to the rate of synthesis. For example, assuming a half- life of about 1 week for mtDNA (9, 22), a synthetic rate of 16 pglhr would maintain a steady-state value of 4 ng of mtDNA in oocytes beyond the time they reached 500- 750 pm in diameter (i.e., the steady-state value equals the synthetic constant divided by the degradation constant). Natu- rally, a longer half-life would require a lower synthetic rate. In this respect, Chase and Dawid (9) have estimated a rate of mtDNA synthesis in postgastrula embryos (prior to stage 30) of 4-7 pglembryolhr, at a time when embryos also maintain a level of about 4 ng of mtDNA, with no net accumulation.

The above interpretation would imply no correlation between the presence of the Balbiani body and mitochondrial biogene- sis. Unfortunately, there is at present lit- tle direct evidence bearing on the question of mtDNA synthesis during oogenesis in Xenopus. Hallberg (24) has reported that mtDNA isolated from whole Xenopus ovaries contains measurable radioactivity after injection of females with 13Hlthymi- dine. However, all stages of oogenesis, in- cluding small oocytes actively accumulat- ing mtDNA, would have been included in the preparation. More recently, Brachet et al. (6) have reported that Xenopus oocytes undergoing maturation incorpo- rate 13H]thymidine into their mtDNA. Although neither of these studies on Xeno-

Pus allow quantitative estimates of mtDNA synthesis during oocyte growth, work on mitochondria isolated from loach oocytes (29), indicates that, at least in ui- tro , [3H]thymidine triphosphate is incor- porated into mtDNA at a rate of about 0.2 pmol/mg of mitochondrial protein/hr.

Recently, quantitative studies on mtDNA synthesis in Xenopus oocytes have been initiated. In this case, stage 6 oocytes were injected with [3Hldn’P and the kinetics of incorporation into mtDNA were determined (Webb and Camp, un- published data). Assuming the mitochon-


drial d!N’P precursor pool specific activity equals that of the cytoplasmic pool at equi- librium [see Ref. (35)] and utilizing the Woodland and Pestell (36) value for the d’M’P pool size in Xenopus stage 6 oocytes, a minimum rate of dlTP incorporation into mtDNA of 0.038 x lop2 pmol/oocyte/hr was calculated. Taking into account the base composition of Xenopus mtDNA (12, 13) this value for [3H]dlTP incorporation represents about 0.5 pg/oocyte/hr of mtDNA synthesis. This level of synthesis is clearly too low, by at least an order of magnitude, to maintain a steady-state level of mtDNA during oogenesis. Rather, it would appear that incorporation of ra- dioactive precursors into mtDNA, at least in stage 6 oocytes, reflects repair and/or limited turnover of the mitochondrial ge- nome. In connection with the latter possi- bility, two observations may prove rele- vant. First, Berk and Clayton (4) have shown that, in mouse L-cells, the displace- ment loop (D-loop) structure is unstable, turning over with a half-life of about 8 hr, and second, at least 76% of the mtDNA circle found in Xenopus unfertilized eggs contain D-loops (24).

The results of our study serve to rein- force the hypothesis that during early oogenesis in Xenopus, there is intense mtDNA synthesis and accumulation, which results in a dramatic increase in the oocyte mitochondrial population. Autora- diographic studies have indicated that considerable DNA synthesis occurs in association with the Balbiani body (1) and it has been suggested that this aggregation of mitochondria is a cytological manifestation of rapid, localized mitochondriogenesis (2). This correlation between the Balbi- ani body and mitochondrial replication is further supported by ultrastructural observations which indicate that the aggregate disperses when the oocyte reaches about 400 pm in diameter (2, 3). From our results, it is clear that, by this time, the bulk of the mtDNA has already been synthesized and accumulated. Thus, there ap-

pears to be a fairly good correlation between mitochondriogenesis and the presence of the Balbiani body. However, this correlation should not be construed to imply any mechanistic relationship. It is also clear from our data that some mtDNA accumulation can continue in oocytes beyond the time at which the Balbiani body disperses, as well as in developing embryos (9). In this respect, it would be of interest to determine the kinetics of mtDNA accumulation in axolotl oocytes, which do not possess a discrete mitochondrial aggregate during previtellogensis (1).

This work was supported by grants from the USPHS (No. HD 04229) and the NSF (No. GB 8741). The authors wish to thank Dr. W. Umbreit of Rut- gers University and Drs. Bill Cramer and Rob Allen of Purdue University for their generous gifts of E. coli strains A-19, JC411 and BHK cells, respectively. We are also indebted to Dr. Dale Graham for her valuable criticism of this manuscript during its preparation.

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accumulation of mitochondrial dna during oogenesis in xenopus laevis

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