J. Phyeiol. (1973), 231, pp. 19-29 19With 1 plate and 4 text-ftgure8Printed in Great Britain

LOCATION OF INEXCHANGEABLE SODIUMIN THE NUCLEUS AND CYTOPLASM OF OOCYTES OF BUFO

BUFO EXPOSED TO SODIUM-FREE SOLUTIONS

BY D. A. T. DICK AND D. J. FRYFrom the Department of Anatomy, University of Dundee -z

Dundee DD1 4HN V::

(Received 30 June 1972)

SUMMARY

1. In oocytes exposed to Ringer solution in which Li substitutes for Na,13-62 % Na is inexchangeable with Li.

2. Nuclei of oocytes isolated by dissection in salt solutions swell irre-spective of the concentration or ionic components of the solution. Whenisolated in 4 %0 bovine albumin solutions, swelling is negligible.

3. When the nuclei of cells exposed to Li are isolated in 4 0 albuminsolution, less than 6% of the inexchangeable Na is found in the nucleus,while 36-88 % of it is found in the cytoplasmic fragments remaining afterremoval of the nucleus.

4. When a Li-exposed cell is crushed in a cellophane bag and dialysedagainst Ringer or Li-substituted Ringer, 86-92% of the inexchangeableNa diffuses out.

5. It thus appears that the inexchangeable Na is located almost entirelyin the cytoplasm and hardly at all in the nucleus, and is not bound tomacromolecules within the cell.

INTRODUCTION

Analysis of the kinetics of Na movements in toad oocytes (Dick & Lea,1964, 1965, 1967) has suggested that there exists a fraction of Na in theoocyte whose rate of exchange with external or internal Li is virtuallyzero, and whose rate of exchange with the remaining internal Na isreduced. Measurements of Na activity in the oocyte by means of Nasensitive microelectrodes (Dick & McLaughlin, 1969) have pointed to thesame conclusion. A likely site for this lithium-inexchangeable Na appearedto be the prominent oocyte germinal vesicle, or nucleus, particularly since,in mature frog oocytes, the nuclear Na concentration (per litre cell water)has been reported to be 2-3 times the cytoplasmic (Abelson & Duryee,1949; Naora, Naora, Izawa, Allfrey & Mirsky, 1962). The experiments

20 D. A. T. DICK AND D. J. FRY

described here were designed to show whether a significant amount of theLi-inexchangeable Na was present in isolated toad oocyte nuclei, orwhether the fraction was located principally in the cytoplasm.

METHODSPreparation of material

Immature oocytes of the toad Bufo bufo were isolated by trypsinization as de-scribed by Dick & Lea (1964), or by dissection under Ringer solution, and cells ofthe preferred size range (600-900 jum) selected. Nuclei were isolated from selectedoocytes by puncturing the cell membrane at an oblique angle with a tungsten needlesharpened by repeated dipping in fused NaNO2, followed by gentle squeezing of thecell with fine forceps on the side opposite the breach. By this method nuclei wereobtained clean and without any apparent damage. All manipulations of oocytes andnuclei were performed with a breaking pipette (Holter, 1943). With nuclei inparticular disturbance was kept to a minimum and with both cells and nuclei anycontact with an interface was avoided.The morphology of isolated cells and nuclei has been examined by electron micro-

scopy. Dick & Lea found that trypsinization produced no serious deformity of thecell membrane, and electron micrographs of isolated nuclei indicate that the nuclearisolation procedure causes no apparent morphological change (P1. 1).

TABLE 1. Composition of solutions used

Solution NaCl LiCl Na2HPO4 NaH2PO4 KCl K2HPO4 KH2PO4A 106-5 2-4 0-6 2-5 -B 109.5 1*2 0-2

All concentrations in mm. In addition both solutions contained CaCl2 2 mi,MgCl2 1 mm, and glucose 1 g/l.

SolutionsTable 1 shows the composition of the solutions used, with the exception of the

nuclear isolation media, which are described with the appropriate results. 24NaRinger solution was prepared by evaporating to dryness 24NaCl solution (Code SGS 1,Radiochemical Centre, Amersham) and adding a solution of the other constituentsto make a Ringer solution as shown in the Table. All chemicals used were A.R. grade(British Drug Houses), except the LiCl, for which A.R. grade was not available and'Laboratory Reagent' grade was used.The pH of all non-radioactive solutions, except the trypsin solution used for

isolating the oocytes and the nuclear isolation media, was adjusted to 7-4 +0-1(measured by a Pye portable potentiometric pH meter) with 200 mM-NaOH(200 mi-KOH for solution B). Buffered isolation media were adjusted to pH 6-8with 200 mm-KOH. Adjustment of radioactive solutions was avoided by addingseveral drops of 200 m-i-HCl to the 0 69 ml. 24NaCl solution before drying.

INEXCHANGEABLE Na IN TOAD OOCYTES

Volume measurements

Volumes were calculated from diameter measurements, using the formula

V -6 a+ )6 3

where a = minor axis, b = major axis when the oocytes or nuclei were notspherical.

Nuclear diameters were measured by means of a finely divided eyepiece grid,which allowed measurement of two diameters at right angles immediately afterisolation. Readings could be taken with an accuracy of + 2-5 /um. Diameters ofoocytes were measured in two directions at right angles by means of a filar eyepiecemicrometer, or with the eyepiece grid, as for the nuclei.

Measurement of Na loss to Li Ringer solutionSelected oocytes were loaded with 24Na by soaking them overnight (8-10 hr) in

radioactive Ringer solution, then washed in inactive Li Ringer solution and theirradioactivity determined using scintillation phosphor sandwiches as described inDick & Lea (1964). Successive counts of each cell were made at 1 hr intervals, thecell being returned to Li Ringer solution between each count. At the end of five orsix determinations, the cell diameter was taken and the nucleus isolated and countedseparately.

In the experiments where the radioactivity of the remaining cell constituents wasmeasured, nuclei were isolated in 100 1ad. isolation medium contained in the lowerhalf of a larger phosphor sandwich (Fig. 1 b, Dick & Lea, 1964). After removal of thenucleus for counting, the upper half of the sandwich was put in position and thewhole counted in the scintillation counter. In many cases the cytoplasm was washedbefore measurement of its radioactivity; the number of washes and the intervalbetween them varied, but the procedure for each wash was always to suck up halfthe solution with as little visible loss of cell material as possible, discard this, andrefill the phosphor sandwich base with fresh solution.

Flame photometry measurements

The Na content of groups of oocytes was measured by flame photometry asdescribed by Dick & Lea ( 1964), with the added precaution that each tube was testedfor zero deflexion when filled with distilled water before addition of sample.The method used for determining nuclear Na content was substantially the same.

As the nuclear membrane appears to be freely permeable to salts (see below) it wasnot considered practicable to attempt to measure the amount of Na not associatedwith macromolecules, but since the nucleus is known to swell rapidly in distilledwater and the membrane of the swollen nucleus allows the passage of large molecules(Macgregor, 1962) it was necessary to have only the briefest possible wash in dis-tilled water, or to avoid this altogether. Accordingly nuclei were washed four timesin the low Na isolation medium and once or not at all in distilled water before beingtransferred to the sample tube. Batches of five to twenty nuclei were used, all thenuclei of a batch being isolated within 2 min. Nuclear diameters were measured bymeans of an eyepiece graticule.

Cells from which the nucleus had been isolated with minimal visible loss of cyto-plasm were treated the same as the whole oocytes.

21

D. A. T. DICK AND D. J. FRYThe accuracy of the flame photometer measurements has been estimated at

±2% for high concentrations, but +5-10% at lower ones, where the scale de-flexion is low (Lea, 1964). This, however, applies only to measurements where blankreadings little more than zero are obtained. For nuclear measurements the blankreadings were necessarily higher and of the same order of magnitude as the samplereadings, and the error may be considerably higher.

Dialysue procedureAfter the last radioactivity measurement each oocyte was transferred to a sac of

Collodion membrane containing 200 1l. Li Ringer, and the sac closed by tighteninga cotton thread looped just below its opening, and tying it. The oocyte was rupturedby squeezing it between two smooth, flat, metal surfaces. Excess cotton and Collodionmembrane were trimmed away from the sac which was then placed in a small beakercontaining either Li Ringer or Ringer solution for 2j hr to allow dialysis to takeplace. The radioactivity in each sac was then determined.

Radioactive Ringer added to such sacs was shown to equilibrate with the sur-rounding solution, whereas the dye Evans Blue (mol. wt. approx. 1000) was retainedwithin the sacs and caused no discoloration of the surrounding medium until thesacs were pierced.

RESULTS

Osmotic properties of the i8Olated nucleusNuclei were isolated in a variety of salt solutions, including 150 mM-

LiCl, KC1, NaCl; 125 mM-KCl+25 mM-NaCl: Ringer solution; and LiRinger solution. In all cases swelling took place immediately and continuedfor several minutes. In KCl solutions, the initial rate of swelling was inde-pendent of the osmolarity of the external medium (Text-fig. 1), as was theequilibrium volume attained. In albumin solutions, on the other hand,there is a fairly good correlation between the initial rate of swelling perunit area and the concentration of protein in the medium. The resultsobtained with solutions of bovine serum albumin in Li Ringer at pH 6-8are shown in Text-fig. 2. If, instead of Li Ringer, the albumin solutionswere prepared with 0-15 M K2HPO4 at pH 6-8, which Battin (1959) foundto cause minimal change in the nuclear constituents of Triturus oocytes,similar results were obtained although the rates of swelling and shrinkagewere somewhat higher. No difference in the appearance of the nucleoplasmof the Bufo oocyte nuclei isolated in these two media could be detected,although changes could be produced by, for example, tripling the divalention concentration of the albumin-Li Ringer medium.

These results indicate that the membrane of the isolated nucleus isreadily permeable to salts but not to albumin. This also seems to be trueof the nuclear membrane of the intact oocyte (see Discussion), so that ifthere were any Li-inexchangeable Na in the nucleus it could not be retainedby the nuclear membrane but would presumably be associated with thenuclear macromolecules.

22

INEXCHANGEABLE Na IN TOAD OOCYTES 23

The location of the Li-ineclhangeable NaFrom the data shown in Fig. 2, 4% albumin in Li Ringer solution at

pH 6-8 seemed a suitable isolation medium for determining the Li-inexchangeable Na in the nucleus, and was used in most ofthe experiments.The time course of the loss of 2Na by two cells in Li Ringer is shown in

(6) I(7)1 (5)

I(6)6)

{(5)

I I I

100 200 300 400 500

Osmolarity (milliosmoles)

600

Text-fig. 1. Initial rate of swelling of oocyte nuclei isolated in KCI solutionsof varying osmolarity. Points and vertical bars show the mean +1 S.E. ofmean. n is shown in brackets beside each point.

+15

U)

I, +1 .0' +0U

L._

VI +0*5

3E E

0 .2

bO.

= -0.5

o -1 0

,x, -125.E

-2-0

4 5 6Albumin

concn. (%)

Text-fig. 2. Initial rate of swelling of oocyte nuclei isolated in Li Ringersolution containing varying concentrations of bovine serum albumin.Points and vertical bars show the mean +1 s.E. of mean. n = 5.

U 8

U 7U

*.I6

'U1U.__._ c' E. 4w E.r_ ,-4A-~~

o 2

co

&_ ICU

0

D. A. T. DICK AND D. J. FRY

Text-fig. 3: these oocytes are typical of those used. The radioactivity ofthe cell after 8 hr gives a good indication of the Li-inexchangeable Nacontent, but a more accurate estimate is obtained from the intercept ofthe graph ofNa efflux (determined by difference) against Na concentration(Text-fig. 4). As shown in Table 2, no correlation was found between theseestimates and the radioactivity present in the nuclei isolated from thesame cells. Nuclear Na contents were variable but uniformly low, and theradioactivity of the nucleus was in no case high enough to be identifiedwith any major proportion of the Li-inexchangeable Na.

80 - A BU

V,60 -

C

0

40 -

C

0U 20 -

z

O Al II I I I I0 200 400 600 0 200 400 600

Time (min)

Text-fig. 3. Loss of radioactive Na from two oocytes immersed in Li Ringersolution. A certain fraction of the Na exchanges with Li either slowly ornot at all. This is approximately 30% of the total Na in cell A and 20%in cell B.

In other experiments each nucleus was isolated in the lower half of oneofthe larger phosphor sandwiches, and cytoplasmic radioactivity measuredafter the nucleus had been removed for counting. As can be seen fromTable 3, the greater part of the cell radioactivity was found in the cyto-plasm. In the cases where only brief washes or none at all were given thecytoplasmic count averaged 73 % of the final cell count. Even after severallong washes, 46 % of the cell radioactivity, on average, still remained inthe cytoplasmic fragment.A good correlation was also found between the Na content, as determined

by flame photometry, of Li-soaked cells from which the nucleus had beenremoved and control cells. In all groups except one, nuclear Na concentra-tions were negligible (Table 4).

Dialysis of oocyte cytoplasmDialysis was used as a method of separating any Na which might be

associated with macromolecules in the oocyte from Na in free solution.

24

INEXCHANGEABLE Na IN TOAD OOCYTES

0-20

0-16

C8 0 120

xI

Z 008

0 04

0 50 10024Na content (counts/sec)

Text-fig. 4. Plot of Na efflux (obtained by difference) against Na contentin an isolated single oocyte kept in Li Ringer solution. The intercept on theabscissa gives the Li-inexchangeable Na, in this case approximately 30%of the total.

TABLE 2. Concentrations of inexchangeable and nuclear Na fromradioactivity measurements

InexchangeableInitial Na Na Nuclear Na

Cell (counts/sec) (counts/sec) (counts/sec)1 134 40 2-22 64 22 1-33 81 23 1.14 144 58 1-15 80 26 06 79 27 0-27 55 17 0-38 98 31 09 114 28 0-710 196 115 0-651 1 273 147 2-612 204 118 2-313 186 99 0-714 232 145 1.1

25

D. A. T. DICK AND D. J. FRY

TABLE 3. Concentrations of total, inexchangeable, cytoplasmic and nuclearNa from radioactivity measurements

Inex-changeable

Na(counts/

see)

12145221186277906384738277

Cyto-plasmicNa

(counts/sec)

8-16-6

4614135364614829265435

NuclearNa

(counts/sec)

0*300-50.40 5003-202-71-901-8

Dura-tion of

No. of washeswashes (mm)

0

0

0

230

0

0

0

2233

1

10101515

TABLE 4. Na concentrations of whole cells, nucleus, and cytoplasm measured byflame photometry

Na in control cells (group of 10 cells) 19-5 mmJnexchangeable Na after 13 hr in Na-free medium (solution B, Table 1) (group of

10 cells) 11-2mmNa in nucleus and cytoplasm after 13 hr in Na-free medium (groups of 7-9 cells)

Nuclei (mM)

0-00-50

3-60

0

1-1

Mean 0-7 mM

Cells after removalof the nucleus (mM)

5-57-9

14-97 0

11-713-1

Mean 10-0 mm

The decline in radioactivity of 24Na-loaded cells in Li-Ringer solution wasfollowed for 10 hr, and the oocytes then ruptured and dialysed againstLi Ringer or Ringer solution. After 2 9 hr in Li Ringer solution, the radio-activity of the dialysis sacs and their contents corresponded to only8+ 40% (mean and S.E. of ten observations) of the Li-inexchangeableradiosodium (as determined from the intercept of the graph of Na effluxagainst Na concentration). For the oocytes dialysed for a similar timeagainst Ringer, the comparable figure was 14 + 4 0 (mean and S.E. of nine

26

InitialNa

(counts/see)

62-972-1

124124142217206264255232245271241

Cell

123456789

10111213

INEXCHANGEABLE Na IN TOAD OOCYTES

observations): the two values do not differ significantly (P > 0.25). Fromthese results it seems unlikely that the Li-inexchangeable Na is associatedwith proteins or other macromolecules in the oocyte.

DISCUSSION

The fraction ofthe oocyte Na that appears to be virtually inexchangeablewith Li could be retained in some organelle whose membrane discriminatedbetween Na and Li, or be associated with some macromolecule possessingbinding sites with much greater affinity for Na than Li. The nucleus is themost prominent organelle, but the former mechanism can be ruled out inrelation to the nucleus if the osmotic properties of isolated nuclei give anyguide to the permeability of the nuclear membrane of the intact oocyte.Nuclei isolated from other amphibian oocytes (Triturws and Xenopusspecies) have also been found to be freely permeable to salts but not toprotein (Callan, 1949, 1952; Battin, 1959; Macgregor, 1962), and in thesespecies the electrical resistance of the nuclear membrane of the intact cellis negligible, indicating free passage of ions across the membrane (Kanno &Loewenstein, 1963). In frog oocytes, also, the results of injecting salt,albumin and polyvinylpyrrolidine solutions into the cytoplasm of other-wise intact cells indicate that the properties of the nuclei in these cells aresimilar to those of the isolated nuclei studied here (Harding & Feldherr,1959). It therefore seems reasonable to assume that, in intact Bufooocytes, inorganic ions such as Na and Li can pass freely across the nuclearmembrane.From the present results it also appears unlikely that nuclear macro-

molecules provide sites for Li-inexchangeable Na. Not only was little Nafound in the isolated nucleus after cells had been several hours in Lisolution, but also most of the remaining Na was shown to be still presentin the material left after nuclear isolation. The amount of Na left cor-related well with the quantity in the whole cell before nuclear isolation orthat in control cells. Although it is possible that Na was lost from nuclearbinding sites on isolation, it is hard to see how such escaping Na couldhave been retained in the cytoplasm when this had been subjected toseveral long washes, or to the washing procedure involved in the prepara-tion of a flame photometer sample. A cytoplasmic site for the Li-inex-changeable Na is also in keeping with the Na distribution found inautoradiographs of toad oocytes (Dick, Fry, John & Rogers, 1970). Thispresent Li-inexchangeable cytoplasmic fraction ofNa in immature oocytesmay also be related to the slowly exchangeable cytoplasmic fraction ofNadescribed in mature oocytes by Horowitz & Fenichel (1970).

There are several reports of binding of Na to various proteins (Carr,

27

D. A. T. DICK AND D. J. FRY

1956; Baker & Saroff, 1965: Lewis & Saroff, 1957), but it seems unlikelythat the Li-inexchangeable Na is bound similarly. The dialysis membraneused in these experiments was shown to retain substances with molecularweights greater than 1000, but did not retain the Li-inexchangeable Na.If binding to cytoplasmic macromolecules is important in this context,the integrity of the binding sites presumably depends on some structuralarrangement or some dialysable co-factor. The question of whether Na issequestered in cytoplasmic organelles remains open. On rupturing theoocyte the cytoplasm is scattered far more than it is during nuclearisolation, and the organelles probably become directly exposed to themedium. If their properties under these conditions were the same asin vivo, any Li-inexchangeable Na they contained would be expected to beretained by the dialysis sacs, but it would not be surprising if the perme-ability of the organelles were altered by extensive exposure to the solutionsused.From the results presented here it is reasonable to conclude that the

Li-inexchangeable Na is located in the cytoplasm of the oocyte; it seemsprobable that it lies in some organelle whose membrane has an effectiveimpermeability to Li, but it has not been possible to reach a firm con-clusion on this point.

This work was supported by a grant from the Medical Research Council. We wishto thank Mrs E. G. Dick for the electron micrographs and Mr D. Coldham for skilledtechnical assistance.

REFERENCES

ABELSON, P. H. & DuIYBEE, W. R. (1949). Radioactive sodium permeability andexchange in frog eggs. Biol. Bull. mar. biol. Lab. Woods Hole 96, 205-217.

BAxvu, H. P. & SAROFF, H. A. (1965). Binding of sodium ions to fl-lactoglobulin.Biochemistry, N.Y. 4, 1670-1677.

BArIN, W. T. (1959). The osmotic properties of nuclei isolated from amphibianoocytes. Expl Cell Res. 17, 59-75.

CALWAN, H. G. (1949). A physiological study of isolated nuclei and its implicationsregarding gene action. Hereditas 35, suppl. 547.

CAILAN, H. G. (1952). A general account of experimental work on amphibian oocytenuclei. Symp. Soc. exp. Biol. 6, 243-255.

CARa, C. W. (1956). Studies on the binding of small ions in protein solutions with theuse of membrane electrodes. VI. The binding of sodium and potassium ions insolutions of various proteins. Arch. Biochem. Biophys. 62, 476-484.

DicE, D. A. T. & LEA, E. J. A. (1964). Na fluxes in single toad oocytes with specialreference to the effect of external and internal Na concentration on Na efflux.J. Physiol. 174, 55-90.

DiCE, D. A. T. & LEA, E. J. A. (1965). Autoradiographic measurements of 22-sodium in the living oocyte. J. Physiol. 180, 178-185.

DIcE, D. A. T. & LEA, E. J. A. (1967). The partition of sodium fluxes in isolatedtoad oocytes. J. Physiol. 191, 289-308.

28

The Journal of Physiology, Vol. 231, No. 1 Plate 1

,,,.;e

Alb~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

N1

D. A. T. DICK AND D. J. FRY (Facing p. 29)

INEXCHANGEABLE Na IN TOAD OOCYTESDICE, D. A. T. & MCLAUGHLIN, S. G. A. (1969). The activities and concentrations ofsodium 4nd potassium in toad oocytes. J. Physiol. 205, 61-78.

DICE, D. A. T., FRY, D. J., JoHN, P. N. & RoGERs, A. W. (1970). Autoradiographicdemonstration of ihhomogeneous distribution of sodium in single oocytes in Bufobufo. J. Physiol. 210, 305-319.

FRY, D. J. (1968). Studies in ion and water fluxes across some cellular membranes.D. Phil. Thesis, Oxford.

HARDMIG, C. A. & FiFLDHFRa, C. (1959). Semipermeability of the nuclear membranein the intact cell. J. gen. Physiol. 42, 1155-1165.

HOLTER, H. (1943). Technique of the Cartesian diver. C. r. Tray. Lab. Carlsberg 24,399-478.

HoROwrIz, S. B. & F'ENICHEL, I. R. (1970). Analysis of sodium transport in theamphibian oocyte by extractive and radioautographic techniques. J. cell Biol. 47,120-131.

KANwo, Y. & LOEWENSTEIN, W. R. (1963).A study ofthe nucleus and cell membranesof oocytes with an intra-cellular electrode. Expl Cell Res. 31, 149-166.

LEA, E. J. A. (1964). Studies of ion and water fluxes in the cell membrane withparticular reference to single cell systems. D. Phil. Thesis, Oxford.

LImwIS, M. S. & SAROFF, H. A. (1957). The binding of ions to the muscle proteins.J. Am. chem. Soc. 79, 2112-2117.

MAcGREGOR, H. C. (1962). The behaviour of isolated nuclei. Expl Jell Be8. 26,520-525.

NAoRA, H., NAoRA, H., IZAWA, M., ALLEREY, V. G. & MIRSKY, A. E. (1962). Someobservations on differences in composition between the nucleus and cytoplasm ofthe frog oocyte. Proc. natn. Acad. Sci. U.S.A. 48, 853-859.

EXPLANATION OF PLATE

Electron micrographs of nuclei. N = nucleus.a, Nucleus isolated in 4% albumin solution.b, Part of the membrane of the same nucleus at higher magnification.c, Nucleus of intact cell (same magnification as in a).d, Higher power view of part of the membrane of the same nucleus (same magnifica-tion as in b).

29