the quantitative measurement of electrolyte ...the equations used in the quantification of x-ray...

J. Cell Set. 3S, 67-85 (i979) 67Printed in Great Britain © Company of Biologists Limited 1979

THE QUANTITATIVE MEASUREMENT OF

ELECTROLYTE ELEMENTS IN NUCLEI

OF MATURING ERYTHROCYTES OF

CHICK EMBRYO USING ELECTRON-PROBE

X-RAY MICROANALYSIS

R. THERESA JONES, R. T. JOHNSON, B. L. GUPTA ANDT. A. HALLDepartment of Zoology, University of Cambridge, Downing Street,Cambridge, CBz jEjf, England

SUMMARY

Na, K and Cl measurements have been made on frozen sections of chick red blood cellsthroughout embryonic development, using electron-probe microanalysis. There is an apparentfluctuation in the levels of these elements during maturation, although the Na/K ratio remainsfairly constant. The nuclear Na concentration resembles that of the cytoplasm, rather than thatof the medium, at all stages. Inhibitor studies indicate that when cytoplasmic Na, K and Cllevels are altered, their corresponding nuclear levels are similarly affected. Additionally, themeasurements in nuclei isolated in anhydrous media from lyophilized cells have shown arte-factual accumulation of high Na, K, Ca and Mg.

INTRODUCTION

Chick red blood cells provide a suitable system for the study of terminal differ-entiation. During development of the chick embryo, 2 populations of red blood cellsare produced whose maturation is asynchronous: the first erythrocytes (primitiveseries) are made exclusively in extra-embryonic tissue, while the second population(definitive series) is produced in the yolk sac after about 4 days of development, andlater by the liver and spleen (Romanoff, i960; Lemez, 1964). By 8 days, although theprimitive series consists almost entirely of mature erythrocytes (Small & Davies, 1972),they constitute only 10 to 20% of the erythroid cells in the blood (Dawson, 1936);the remaining erythroblasts of the definitive series now begin maturation, but lesssynchronouslythan-theprimitive-red blood cells (Lemez, 1964).

The maturation of both primitive and definitive erythrocytes is marked by a seriesof striking morphological changes (Small & Davies, 1972) which are associated withthe almost complete genetic inactivation of the cells (Seligy & Neelin, 1970). Theproliferating erythroblasts from days 4 and 5 of embryonic development give rise tomaturing erythrocytes which are incapable of division; this quiescence is accompaniedby the loss of nuclear proteins (Dingman & Sporn, 1964; Gershey & Kleinsmith,1969), by cessation of DNA synthesis (both replicative and unscheduled) (Cameron &Prescott, 1963; Darzynkiewicz, 1971), by a marked reduction in RNA synthesis

68 R. T.Jones, R. T. Johnson, B. L. Gupta and T. A. Hall

(Cameron & Prescott, 1963; Madgwick, Maclean & Baynes, 1972; Zentgraf, Scheer &Franke, 1975) and by a loss of DNA supercoiling (Cook & Brazell, 1976). Despite this,both RNA and DNA polymerases are present (Scheintaub & Fiel, 1973; Longacre &Rutter, 1977) and it has been postulated that changes in the genetic activity of thesecells are related to altered patterns of association between regulatory proteins andDNA (e.g. Ruiz-Carrillo, Waugh, Littau & Allfrey, 1974). There is considerableevidence from prokaryotes that interactions between specific DNA sequences andregulatory molecules are determined by the ionic environment (Anderson, Nakashima& Coleman, 1975; Lin & Riggs, 1972). Thus, precise quantification of electrolyte levelswithin the cell nucleus may demonstrate the nature of the environment which controlsthe conformation and activity of chromatin (Lezzi & Gilbert, 1970; Rensing & Fisher,1975). Our aim has been to determine whether the maturation of the chick erythrocyteis associated with changes in the levels of Na and K in the nucleus and whether theselevels can be altered by means of inhibitors of the Na/K pumping systems located inthe plasma membrane (Hoffman, 1966; Glynn, 1968).

MATERIALS AND METHODS

Collection of tlte red blood cells

White Leghorn eggs were incubated at 39 °C. After various intervals red blood cells werecollected from embryos as follows: (1) the blood vessels of 4-day embryos were teased apartand the fluid collected; (2) the blood vessels, leading from the yolk sac to embryos of 6-12 daysof age, were cannulated using microsyringes; (3) blood was withdrawn directly from the heartof 15- to 18-day embryos, using a hypodermic syringe. These methods reduced contaminationof the blood cells by allantoic and amniotic fluids. The cells were collected and pooled from6-20 embryos from each age group, washed twice in Dulbecco's modification of Eagle'sminimal essential medium (Gibco) and resuspended in medium plus 10-20% (w/v) Dextran(mol.wt 230000; Sigma). Dextran was added to the medium to reduce ice-crystal damageduring quench-freezing, to improve section cutting at low temperature, and also to form asuitable matrix so that the sectioned medium could be used as a peripheral standard for thequantification of X-ray data (Gupta, Hall, Maddrell & Moreton, 1976; Gupta, Hall & Moreton,!977a)- Some of the cells were incubated in medium with either \o~* M ouabain or io~4 Mouabain plus io"4 M ethacrynic acid for 6-24 h at 39 °C. As the binding of ouabain to cellsdepends on the external K concentration (Boardman, Lamb & McCall, 1972; Baker & Willis,1970), the K in the medium was kept at the same level (8 mM) throughout inhibitor treatmentfor each age group. Control cells were incubated in Dulbecco's medium alone. The cells werewashed and resuspended in medium plus Dextran as before.

Preparation of sections of whole cells for microanalysis

A small droplet of the cell suspension was placed on the end of a copper rod of 1-5 mmdiameter. Each specimen was plunged into Freon 13 (monochlorotrifluoromethane) at — 181 °Cand stored in liquid-nitrogen until required. i-/im-thick frozen sections were obtained,mounted and stored, as described by Gupta et al. (1977a). The specimens were loaded onto themodified cold stage (— 170 CC) (Taylor & Burgess, 1977) of a JEOL JXA-50A electron-probemicroanalyser, where they were additionally protected by an anticontamination cap cooled byliquid-nitrogen.

The sections were examined in the microanalyser in the scanning transmission mode at anaccelerating voltage of 40-50 kV. A beam of 2-10 nA, focused to a diameter of 100-300 nm,was used either as a static probe or to scan a small rectangular area (raster) localized withincytoplasm or nucleus at magnifications of 5000 to 40000 times. Heterogeneity of electrolyteconcentration within subcellular compartments is more likely to be revealed by static probesthan by scanning rasters. Each field was analysed for 80 s real time. Although the spatial

Electrolytes in avian erythrocyte maturation 69

resolution is expected to be of the order of 300 nm (Hall, 1975; Gupta et al. 19770), the beamor specimen or both may drift during measurement, thereby producing anomalous results.This is of especial importance if the area to be measured lies at an interface, e.g. nucleus/cytoplasm or cytoplasm/medium. The centres of nuclear and cytoplasmic fields were thereforegenerally selected for study. Nevertheless, some data were collected from dense areas which layon the periphery of nuclei of older chick red blood cells, and which probably corresponded toregions containing condensed chromatin.

Characteristic X-rays for Na, Mg, K and Ca were recorded on diffracting spectrometers(Gupta et al. 1977 a) and the complete X-ray energy spectrum was obtained simultaneouslyfrom a Kevex Si (Li) energy dispersive spectrometer. The latter provided information aboutboth the characteristic X-rays from K, Cl, P and Fe, and the continuum or white radiationwhich was used as a measurement of local mass of the specimen under the beam (Gupta et al.1977a).

Ideally, microanalytical data should be obtained from sections in the hydrated state (Guptaet al. 1977a; Gupta, Berridge, Hall & Moreton, 1978a). Unfortunately, hydrated sections arepoor in contrast, and, as the cells under investigation were scattered widely throughout eachsection, they were difficult to resolve. Therefore, a compromise was reached whereby themajority of sections were examined after dehydration for 1-2 min under high vacuum in themicroanalyser column (Gupta et al. 1976), but a few sections were analysed in the hydratedstate for comparison. The equations used in the quantification of X-ray data to determineelemental concentrations are given by Gupta et al. (1977a, 1978a).

Preparation of isolated nuclei for microanalysis

A method for the isolation of nuclei by nonaqueous means which yields clean nuclei retainingcertain water-soluble constituents and high enzymic activities was selected to minimize extrac-tion and redistribution of free nuclear electrolytes (Kirsch et al. 1970; Gurney & Foster, 1977).Erythrocytes were collected from embryos as described and centrifuged in capillary tubes. Thetop half of each packed red cell column was discarded and the remaining cells placed in aplastic Petri dish which was immersed in Freon 13 chilled to its freezing point by liquid-nitrogen. The frozen samples were lyophilized at —40 °C over a PjOs trap for 4 days at io"3

torr (0-133 N m~'). Desiccated glycerol at approximately 2 °C was added directly to thelyophilized red cells while maintaining vacuum. Homogenization of the red cells and collectionof nuclei were carried out essentially as described by Kirsch et al. (1970). Droplets of nucleisuspended in glycerol were smeared at 2 °C over aluminized nylon film supported by Duraliumcollars (Gupta et al. 1977a, 1978a). Excess glycerol was removed and the nuclei were top-coated with a thin aluminium film prior to mounting into the microanalyser. X-ray data wererecorded as described above. Quantification of these data, using external Na, K, Ca and Mgstandards, is given in Appendix I (p. 79).

Interferometry

Red blood cells were collected from embryos after 4 days and 18 days of development asindicated above. They were examined with a Baker double refracting interference microscopeusing the shearing optical system and a half shade eye-piece (Baker, 1957), with monochromaticillumination (A = 546 nm). The retardation of light produced by nucleus and cytoplasm wasmeasured with respect to the bathing medium. The dry mass per unit volume of nucleus andcytoplasm was calculated according to Barer (1955).

RESULTS

Typical X-ray energy spectra taken from saline, nucleus and cytoplasm in sectionsare shown in Figs. 1-4. The concentrations of Na, K and Cl from such samples aregiven in mM kg"1 dry wt in Table 1.

Within the limitations of the technique, the average level of nuclear Na declines inthe population from 4 days to 6 and 8 days (P < o-i %), returning to its previous

R. T. Jones, R. T.Joknson, B. L. Gupta and T. A. Hall

Figs. 1-4. Typical X-ray energy spectra recorded with the Kevex energy-dispersivesystem. The horizontal axis of each spectrum is given in keV, the vertical axis is incounts (the full scale is given as FS = . . . above each spectrum). Al (from the speci-men holder and the substrate film), Na, P, S, Cl and K peaks are indicated. The peakat 3-6 keV consists of the Ca Ka, peak obscured by the K Kfl peak.Figs. 1, 2. Comparisons of X-ray energy spectra from frozen-hydrated sections(dotted spectra) with those from the same sections after dehydration within the micro-analyser column (bar spectra).

Fig. 1. A saline solution containing: Na, 60 mM; K, 80 m i ; Cl, 150 nrn; and Dex-tran 10% w/v (plus buffer, etc.).

Fig. 2. Cytoplasm of a red blood cell from a chick at the 6th day of embryonicdevelopment.

Fig. 3. Nucleus of a red blood cell from a 15-day chick embryo, after dehydration inthe microanalyser column. Note the large P peak.

Fig. 4. Cytoplasm of the same cell as in Fig. 3.

level by 12/13 days (P < o-i %) from which time it remains fairly constant. There isa peak in K concentration at 12/13 days (P < o-i %). Cl concentrations also fluctuateduring development; a substantial decrease from 4 days to 6-8 days (P < o-i %)follows the decline in both Na and K, but after this the level increases and remainsrelatively constant throughout the rest of development.

The average levels of Na in those areas of nucleus and cytoplasm which were

Electrolytes in avion erythrocyte maturation 71

measured are similar within the age groups, even at 6 and 8 days when electrolyteconcentrations have dropped. K, on the other hand, is similar in concentration innucleus and cytoplasm only at 4 and 6 days, but, later in development, the nuclearlevel increases and is significantly higher than that of the cytoplasm (P < o-i % for8 days, 15/17 days and 18 days). There is a decline in nuclear K at 15/17 days(P < o-i %) after which time the level remains at the value seen at 4 days. Thecytoplasmic K concentration does not change significantly from 15-18 days.

Table 1. Concentrations of Na, K and Cl in mM kg~x dry mass in maturing red bloodcells of chick embryos, calculated from microprobe measurements (mean ± 1 standarderror of the mean)

Nucleus Cytoplasm

Age and treatment Na K Cl Na K Cl

4-day* 120 ±16 346129 152123 12 188 ±19 327132 194119 9

6-day control* 42114 151116 2317 3 27112 113112 2213 3+ ouonly 9 I 4 12718 31I2 2 22 181 46 1+ ou and eth 169175 47I10 8 2 i i 8 2 — — — o

8-day** i8±4 268 ± 11 49 ± 7 7 i6±3 167119 Si±i2 3

12/13-day control* 115124 697144 14914 3 461 14 346I97 185I39 2

+ ou and eth 344l!5 147I31 123119 7 274I20 96121 188I40 4

15/17-day control** 74 i i2 254I14 9517 15 115116 150110 146111 13

18-day control* 123 116 346127 1101 18 10 115 120 1661 10 136130 9+ ouandeth 395115 30I2 184I25 10 364I23 21 14 3^3 ± 33 '3

Peripheral standard (known)

• Medium + 10% Dextran

** Medium + 20 % Dextran

Na K Cl1387 70 1141739 37 608

ou = Ouabain, io~* M; OU and eth = ouabain plus ethacrynic acid, io~* M each.

The amount of water in embryonic red blood cells taken from chicks of various agesand treatments is presented in Table 2. These data are subject to a number of technicaluncertainties (Gupta, 1978; Gupta et al. 1977 a, 1978 a; Gupta, Hall & Moreton19776), but probably provide a good approximation to the amount of water present.In an attempt to check these measurements, red blood cells taken from embryos of 4days and 18 days of age were studied further using an interferometric method (Baker,1957). The results are presented in Table 3. Both sets of data indicate that there is anoverall increase in nuclear dry mass per unit volume during maturation (Table 3(b)),and hence in the degree of hydration.

The dry mass determinations (and therefore the estimation of wet \vt and H2Ofractions) are necessarily carried out in the same regions as are used for X-ray quanti-fication of electrolytes; thus it is possible to convert electrolyte concentrations frommM kg"1 dry wt into approximate mM kg"1 wet wt concentrations (Appendix II,p. 81) or into mM I."1 H2O (Table 4) assuming that all the measured electrolyteelements are free and all water is solvent.

72 R. T.Jones, R. T.Johnson, B. L. Gupta and T. A. Hall

Table 2. Average water content of red blood cells of embryonic chicks, calculated from theratio of continuum X-ray counts produced by tlie medium to continuum X-ray countsproduced by nucleus or cytoplasm, in frosen-dehydrated sections

Age andtreatment

4-day control6-day control

+ OU

8-day control12/13-day control

+ ou and eth15/17-day control18-day control

+ ou and ethSummated data

% waterin medium+ Dextran

89-7#

897"89-7*8 2 - 3 "

897*89-7

#

82-3**

89-7*89-7*

Nucleus

79±475 ±966 ± 10

5 5 ± n75±768 ±760 ±9

62 ±569 ±668±3

n

13

33

7

31 0

8

1717

81

Cytoplasm

79 ±565 ±1168

5 6 ± i 973 ±1069 ±861 ±18

75 ±57° ±568±3

n

8

31

2

2

62

1 0

16

50

P

ns

nsns

ns

nsns

ns

nsns

ns

Values are expressed as % wet weight of the specimen (mean ± 1 standard error of the mean).• Medium + 10 % Dextran (w/v)

•• Medium + 20 % Dextran (w/v)ou = Ouabain, io~4 M; eth = ethacrynic acid, io~* M; ns = not significant.

Table 3. Dry mass data

(a) Dry mass of nucleus and cytoplasm in embryonic chick red blood cells, measured byinterferometry (mean ± 1 standard deviation)

Age,days Region of cell Retardation, nm

Dry mass,pg per /tm3

4 nucleus 25 731-6117-9 0-9410-02cytoplasm 25 627-7124-9 100 ±0-04

18 nucleus 25 7002 ±20-6 1-3010-04cytoplasm 25 633-4124-6 1-0710-03

(6) Comparison of dry mass values

Specimens

4-day nucleus v 4-daycytoplasm

4-day nucleus v 18-daynucleus

4-day cytoplasm v 18-daycytoplasm

18-day nucleus v 18-daycytoplasm

% Difference indry mass from

microprobe data

0

2 2

5

17

P

ns

1 /o

ns

ns

ns = Not significant.

% Difference indry mass per

unit volume frominterferometry

6

27

7

18

P(%)

< 01

< 01

< o-i

< 01


Table

Age andtreatment

4- Estimated

Na

concentrations

Nucleus

K

ofNa,

Cl

K

n

and Cl, m

Na

mM kg'1 wet

Cytoplasm

K

Wt

Cl n

4-day control*

6-day control*

+ ou

+ ou and eth

8-day control**

12/13-day control*

+ ou and eth

15/17-day control**

18-day control*

+ ou and eth

4° ±5(59 ±7)I7±6

(23 ±8)

3 ± i(5 ±2)52 ±23(78 ±35)

7 ± 2(13 ±7)48 ±10

(64 ±13)

(161 ±7)32±5

(54 ±8)45 ±6

(62 ± 8)

(191 ±7)

I I 2 ± 9(164 ±13)

62 ±7(83 ±9)40 ±3

(60 ±5)47 ±10

(7i ±15)105 ±4

(192 ±7)292 ± 18

(387 ±24)47 ±10

(69 ±15)i n ±6

(186 ±10)

I28± IO(i77 ±14)

IO± I

49 ±7(72 ±10)

9±3(12-±4)

10 ± 1(15 ±2)26 ±6

(39 ±9)

(35 ±7)63 ±2

(84 ±3)39±6

(58 ±9)41 ±3

(69 ±5)4 i ± 7

(57 ±10)65 ±8

(94 ±12)

12

Peripheral standard

• Medium + 10 % Dextran w/vNa

• • Medium+ 20% Dextran w/v

78±8 136 ± 13 8 i ± 8(iO4±io) (181 ± 17) (108 ± 10)

8±4(12 ±6)

7(10)

33 ±2(51 ±3)

60(88)

6 ± i(9 ±2)

15(22)

2 — — o

6±:

142 7(158) (8)130 6-6

(158) (8)Values in parentheses are in mM I""1 H,O. (Mean ±ou = Ouabain, io~4 M; OU and eth = ouabain plus

67 ±8(120±14)

3 i 8 ± s i32±37(35 ±7) ( I 8 I ± S I )

7 9 i ± 7 32±7(132 ±10) (46 ±10)

15 53±7 69±s(77 ±10) (100 ±7)

10 72 ±13 103 ±6(98 ±18) (140 ±8)

10 12618 7 ± i(i8o± 11)

K Cl117

(130)

107

(130)

20 ±5(36 ±9)

(97 ±21)64 ±13

(93 ± 19)67 ±5

(97 ± 7)86±i8

(117 ±26)i o 8 ± n

(i54±i6)

mM kg"1 wet wt(mM I.-1 H,O)

mM kg""1 wet wt(mM I.-1 H,0)

1 standard error of the mean),ethacrynic acid, io"1 M each.

13

13

Table 5. Average measured concentrations of Na, K, Ca and Mg in isolatednuclei from red blood cells of chick embryos

Age, days Na K Ca Mg

5 564 615 508 467 538 100

18 318 312 309

Values are given as mM kg"1 dry wt.

16582

130

In contrast to the data from sectioned erythrocyte nuclei the Na and K concentra-tions in nuclei isolated by a nonaqueous method are extremely high, and in nucleifrom older embryos, at least, they are very variable (Table 5).

Inhibitors of Na pump(s) such as ouabain and ethacrynic acid are known toincrease the concentration of cellular Na (Whittembury & Fishman, 1969; Lubovvitz

74 R. T. Jones, R. T. Johnson, B. L. Gupta and T. A. Hall

&Wittam, 1969; Leblanc&Erlij, 1969; Lamb & McCall, 1972; Kennedy & DeWeer,1976; Williams, Withrow & Woodbury, 1971; Hoffman & Kregenow, 1966; Pichon& Treherne, 1974; Glynn & Karlish, 1975; Kregenow, 1977). We have therefore

300

270

150

100

50

150

100

50

•_ I

1i ^£ ;

4 day 6 day 8 day 12/13 day 15/17 day 18 day

Fig. 5. Histogram of the wet weight data from red blood cells of 4- to 18-day chickembryos, A, nucleus; B, cytoplasm. Bars indicate one standard error from the mean.S, Na; ^ , K; m, Cl.

Table 6. Ratios of the characteristic X-ray counts from phosphorus to the continuum X-raycounts from the same fields of sectioned chick red blood cells

Age, days

468

12/13is/1718

Nucleus

1-7610-090-7610-11

085 ±008i-i4±o-i51-42 ±0-15i'77±o-n

n

1316

79

1946

ns =

Cytoplasm

1-47 ±0-14049 ±007o-68±o-340-37 ± 0 1 0

0-37 ± 0 0 60-35 ±003

Not significant.

n

9948

1436

P

nsnsns

< 0-2%< o-i%< o-i %

determined whether treatment of erythrocytes with these inhibitors results in changesin the concentration of cytoplasmic and nuclear Na and K. Ouabain treatment alone(io"4 M for 24 h in standard growth medium) had little effect on the concentrations ofany of the elements investigated in 6-day cells. Moreover, even in 6-day cells treatedwith both ouabain and ethacrynic acid (io"4 M), no statistically significant change in


nuclear Na was observed, although a decrease in nuclear K was found (P < 5 % ) .When older red blood cells were treated with ouabain and ethacrynic acid, the cyto-plasmic Na concentration increased dramatically (P < 0-2% at 12/13 days, P < o-i%at 18 days) with a concomitant decline in K (P < 5 % at 12/13 days, P < o-i % at18 days) (Tables 1 and 4). The nuclear levels of Na and K reflect a change in cyto-plasmic concentrations of these elements (for Na: P < o- i% at 12/13 days; for

JFig. 6. Scanning transmission image of a i-/im-thick section of a red blood cell froman 8-day-old embryonic chick, after dehydration in the column. Under the partialdark-ground conditions of imaging (Gupta et al. 1977a), the nucleus (n) appears lessdense than the cytoplasm (c). Some structure is visible in the nucleus, x 16000.

K: P < o-i% at 12/13 days and 18 days). Cl levels were unaffected by combinedinhibitor treatment at 12-13 days, but in older cells cytoplasmic Cl was greatlyaffected (P < 1 %) while nuclear Cl increased only slightly (P < 5 %).

The accuracy of the conversion of X-ray analytical results from rait kg"1 dry wtinto mM kg"1 wet wt depends upon various assumptions, e.g. (1) that the thickness ofthe section is even, (2) that there is no mass loss during data collection (Gupta et al.1977 a, 1978 a). However, the ratios of the elements present are independent of thesefactors. A histogram is therefore presented from which the ratios of Na, K and Cl innucleus and cytoplasm throughout erythrocyte maturation can readily be estimated(Fig. 5)-

Analysis of the characterisitic X-ray counts produced by phosphorus indicates thatits distribution between nucleus and cytoplasm changes during development. As thephosphorus present in cells is not 'free' in the same sense that electrolytes are, noattempt has been made to quantify the amount of phosphorus present. Instead, a

76 R. T. Jones, R. T. Johnson, B. L. Gupta and T. A. Hall

comparison has been made between the ratios of characteristic X-ray counts forphosphorus to continuum counts in nucleus and cytoplasm as these ratios shouldindicate the relative concentrations (Table 6). Up to 12-13 days, the nuclear andcytoplasmic phosphorus/continuum X-ray count ratios are not significantly differentwithin the age groups. In older cells, however, the values diverge, presumablyreflecting the decreased RNA metabolism in the cytoplasm.

Iron is found in nucleus and cytoplasm throughout erythrocyte maturation, inagreement with the fact that haemoglobin is distributed in both these compartments(Davies, 1961; Small & Davies, 1972).

DISCUSSION

The electrolyte measurements described in this work have been made largely onfrozen dehydrated erythrocyte sections, and as such are subject to the limitation thation translocation has occurred during dehydration. However, whenever parallelmeasurements have been made on hydrated sections, elemental concentrations werefound to be very similar, giving us confidence in the data. Moreover, microprobestudies from this laboratory (Gupta et al. 1976, 1977a, 1978a, b) and other labora-tories (Dorge et al. 1975; Somlyo, Shuman & Somlyo, 1977; Appleton & Newell,1977) on a variety of tissues have shown that if frozen sections are carefully dehydratedat very low temperatures, no translocation of diffusible electrolytes can be detected,except in spaces lacking an organic matrix. Dehydrated sections were generally chosenfor microprobe analysis because of superior image resolution (Fig. 6); nevertheless thespecific location of the static probe or scanning beam within a dehydrated nuclearsection could only be described rarely, and no conclusions can be drawn about thevariation in electrolyte concentrations and their possible association with subnuclearstructures. Most microprobe measurements were made in the central regions of thenucleus to avoid overlap with cytoplasm. Since the majority of condensed regions inthese nuclei are peripheral (Small & Davies, 1972) the measured electrolyte concentra-tions probably apply to central decondensed regions. These regions appear to have awater content similar to the younger, more active nuclei.

Although a considerable amount of data exists on cellular electrolyte levels, there hasbeen much disagreement about the nuclear ionic environment and in particular aboutthe amount of Na present. Microprobe analysis of sectioned erythrocytes from variousembryonic ages reveals that nuclear levels of Na, K and Cl are significantly differentfrom the external medium. K is elevated while Na and Cl are reduced, essentially inagreement with microprobe studies on other somatic cell nuclei (Gupta et al. 1976,1977a, 1978a; Gupta, Hall & Naftalin, 19786; Pieri et al. 1977; Cameron, Sparkes,Horn & Smith, 1977; Rick, Dorge, von Arnim & Thurau, 1978), with nuclear elec-trolyte levels in amphibian oocyte and fish neurons (Riemann, Muir & McGregor,1969; Dick, 1976; Century, Fenichel & Horowitz, 1970; Katzman, Lehrer & Wilson,1969), and also with measurements of electrochemical activities in a variety of cells(Palmer & Civan, 1977; Civan, 1978). Our studies reveal that in terms of concentra-tion, the dominant nuclear cation, measured in situ, is K rather than Na. There is also


an apparent anion deficit which may be compensated in part by the presence ofpolyanionic nuclear proteins (Bhavanandan & Davidson, 1975; Hunt & Oates, 1977).

The results are consistent with the hypothesis that the dramatic nuclear maturationin the developing erythrocyte is not associated with a major change in the total con-centrations of Na and K, despite the loss of considerable nuclear protein mass. Kremains the major cation throughout maturation and the nuclear Na/K ratio issimilar in primitive proliferating 4-day cells and in mature definitive 18-day erythro-cytes (Fig. 5). The apparent decline in nuclear ion levels at 6 days (100% primitiveseries) and 8 days (20% primitive series) may be artefactual in that only 3 and 7nuclei respectively were studied. It is also possible that the cells at this stage aresensitive to Dextran. However, the ratios of nuclear Na:K:Cl in each case are similarto those of the cytoplasm, and also to those from other stages of development (Fig. 5).If this decline is real, it suggests that the new definitive series is low in both nuclearand cytoplasmic ions, especially Na, at the start of maturation, and might thereforebe under considerable osmotic stress - a problem which, as yet, remains unresolved.Both Na and K levels are restored by 12/13 days (90% definitive series).

The data obtained from sectioned nuclei conflict with those from nuclei isolatedfrom chick erythrocytes albeit by techniques aimed at minimizing electrolyte loss andredistribution (Kirsch et al. 1970). The concentration of Na in the isolated nuclei isvariable, but in most cases, is increased up to 20 times the concentration in sectionedcells, although K levels are not raised to the same degree. Nuclei from a variety ofsources isolated by either the non-aqueous Behrens technique, or the glycerol pro-cedure, uniformly show extremely high levels of nuclear Na (Siebert & Langendorf,1970; Kirsch et al. 1970; this paper) which can be increased still further to 600 mM/kgdehydrated material by incubating the isolated nuclei in Na-containing salines (Itoh& Schwartz, 1957). We therefore consider these high levels to be artefactual, resultingfrom exposure of the nucleus during cell fractionation to extracellular fluids containinghigh Na, and not a reflexion that the nucleus in the intact cell is in direct communica-tion with the medium (see Moore & Morrill, 1976).

The electrolyte levels in nuclei can be changed profoundly in intact cells by theinhibition of membrane-associated Na-K ATPases. Simultaneous treatment of ery-throcytes with the inhibitors ouabain and ethacrynic acid results in massive increasesin nuclear Na to a level approaching that of the external medium. Our results suggestan absence of ouabain-sensitive sites in 6-day erythrocyte membranes; ethacrynicacid-sensitive sites appear to be present throughout maturation. The response oferythrocytes to Na/K pump inhibitors varies somewhat during maturation: erythro-cytes from older embryos accumulate more Na and show greater reduction in K levels,perhaps indicating that the number of membrane pumps and/or their efficiencydeclines with increasing maturation/ageing. A comparable result has been obtained byPalmer & Civan (1977) working with Chironomus salivary gland cells. After 'ageing'these cells by prolonged incubation in vitro, they found an increase in intracellular ionlevels. Pieri et al. (1977) find a similar gradual deterioration in the functions of theNa/K pump of ageing rat liver and brain cells. It is also possible to replace K by Naas the dominant nuclear cation if cells are incubated in medium lacking K (Gupta et al.

6 CEL 35


19780). What effects these changes would have on nuclear metabolism and whetherthese effects (if any) are reversible remain to be explored, although depletion of Karrests cella in division (Meeker, 1970).

The technical problems associated with electrolyte measurements in the sectionednuclei of single-cell suspensions do not yet permit precise statements to be madeabout the intranuclear localization of ions during erythrocyte maturation. There issome variability in electrolyte microanalytical data from these nuclei, which mayindicate that areas of condensed chromatin, like metaphase chromosomes (Cameronet al. 1977), have different electrolyte characteristics and dry mass from the rest of thenucleus.

Changes in the electrolyte levels of the medium lead to gross changes in chromatincondensation and deoxyribonucleoprotein (DNP) fibril dimensions (Hughes, 1952;Robbins, Pederson & Klein, 1970; Brasch, Seligy & Setterfield, 1971; Kellermayer &Jobst, 1970; Leake, Trench & Barry, 1972; Coutelle et al. 1974; Neelin, Mazen &Champagne, 1976) which suggests that DNP packing depends largely on modificationsof electrostatic interactions along the nucleohistone backbone, observations which aresupported by studies on the organization of isolated nucleoprotein in relation to ionicstrength (e.g. Bartley & Chalkley, 1973). Maximum contraction of nucleoprotein isobserved at a monovalent cation concentration of 0-15 to 0-2 M (Bradbury, Carpenter& Rattle, 1973; Bradbury, Danby, Rattle & Giancotti, 1975; Billet & Barry, 1974),presumably when electrostatic repulsion along the nucleohistone molecule is minim-ized. At low ionic strengths, electrostatic repulsion would elongate the molecule, whileat high ionic strengths histories are dissociated, which would also result in extensionof the nucleoprotein.

Moreover, there is clear evidence that histones are involved in chromatin condensa-tion, although the nature of Hi-DNA interactions in relation to histone modificationand changes in ionic strength (Renz & Day, 1976) are not fully understood. In avianerythrocytes it is possible that H5 is the most important regulatory histone since itaccumulates during maturation, in part replacing Hi (Sotirov & Johns, 1972), pro-hibits transcription and (disputedly) promotes condensation (Seligy & Neelin, 1970;Lurquin & Seligy, 1972; Bolund & Johns, 1973). However, the involvement of H5 orother histones in any of these processes will depend on the establishment of histone-DNA electrostatic and hydrophobic bonds, and histone-histone interactions whosestability will be related to the electrostatic charge density in the chromatin and there-fore to the ionic milieu of the nucleus. Thus the organization of the heterochromatinnuclear bodies of the erythrocyte (possible chromosome equivalents (Anderson &Norris, i960; Davies, 1961)) and DNP fibre dimensions, may depend primarily oncharge interactions which can be modified by variations of nuclear electrolyte levels.

The data presented in this paper are concerned with total monovalent electrolyteconcentrations. However, were all these electrolytes to be free in solution, the electro-lyte concentration would still not on average exceed 0-2 M, i.e. histone-DNA inter-actions could exist as envisaged in the literature. The abnormally high electrolytelevels observed in the isolated nuclei would result in nucleoprotein dissociation andmajor changes in chromatin organization. The concentrations of divalent cations in


erythrocyte nuclei remain to be determined; but it should be noted that they are farmore effective in promoting changes in DNP organization than are monovalent cations(Bradbury et al. 1973, 1975)- To gain a greater understanding of the distribution ofelectrolytes in nuclei, further information is needed concerning the amount andavailability of cation-binding sites in the nucleus (e.g. alkali cation-binding sites des-cribed by Besenfelder & Siebert, 1975; and the Mg- and Ca-binding sites of metaphasechromosomes (Cantor & Hearst, 1970; Steffensen, 1961)), and also the amount ofnuclear solvent water (Beall, Hazlewood & Rao, 1976).

We are especially grateful to Dr S. L. Schor, Dr A. M. Mullinger and Mr R. G. W. North-field for help in early stages of this investigation, which is supported by grants from the MedicalResearch Council and the Cancer Research Campaign. R. T. Johnson is a Research Fellow ofthe Cancer Research Campaign. The Biological Microprobe Laboratory was supported by agrant from the Science Research Council to Drs B. L. Gupta, T. A. Hall and R. B. Moreton.We thank Mrs Kate Barber and Messrs. A. J. Burgess, N. G. B. Cooper, M. J. Day and P. G.Taylor for technical assistance at various stages. Our gratitude is also due to Professor M. M.Civan for critically reading the manuscript.

APPENDIX I. QUANTIFICATION OF THE

X-RAY DATA FROM ISOLATED NUCLEI

The average energy loss of a 50-kV electron passing through an isolated nucleus isapproximately 1 keV, so that the nuclei may be regarded as 'thin' in the sense definedby Hall (1971). Quantification can then be based on the equation

Cx = kx{nx/W), (1)

where Cx is the mass-fraction of the element x (mM of element x per kg of specimen);kx is a coefficient of proportionality; nx is the background-corrected count obtainedfrom the characteristic X-rays of element x\ and W is the simultaneously obtainedbackground-corrected count of continuum X-rays from the specimen. In this studythe following values were used for the coefficients kx in the isolated nuclei:

ElementK

KNaCaMg

Detectorenergy-dispersivediffractordiffractordiffractordiffractor

Coefficient k

5053870860026704220

For the sectioned material in this report, the coefficients kx were obtained by usingthe peripheral medium around each section as the standard. In the case of the isolatednuclei there was no suitable peripheral medium and the coefficients were obtained asfollows:

(1) Potassium (both energy-dispersive and diffracting spectrometers): Standardswere frozen-dried i-/tm sections of quench-frozen Dextran/saline solutions, contain-ing 1000 mM of K per kg of Dextran.

6-2

80 R. T.Jones, R. T. Johnson, B. L. Gupta and T. A. Hall

(2) Sodium: Spectrometer sensitivity was calibrated in proportion to potassium, bycomparison of Na and K counts from frozen-dried i-/tm sections of quench-frozenDextran/saline solutions containing equimolar NaCl and KC1.

(3) Calcium: Spectrometer sensitivity was calibrated in proportion to potassium, bycomparison of Ca and K counts from dried droplets of solutions of equimolar CaCLand KC1.

(4) Magnesium: Spectrometer sensitivity was calibrated in proportion to sodium.Dried solutions of equimolar salts of Mg and Na proved to be unsuitable because ofinhomogeneity and segregation. Consequently the efficiency for Mg relative to Na wasdetermined from comparison of Mg and Na counts from bulk standards of pure Mgand NaCl respectively, via a set of estimated correction factors.

The ratio of Mg and Na count rates observed from the bulk standards, (Sv/Sx)b,can be expressed in the form

(5i//5Ar)6 = nM/nx nx/nx_c wM/tox fM/fX-C eM/ex, (2)

where nM/nx is the ratio of K-shell ionizations in pure bulk Mg and in (hypothetical)pure bulk Na; nx/nx_c is the ratio of sodium K-shell ionizations in pure bulk Na andin bulk NaCl; wy/wx is the ratio of the Mg and Na X-ray K-shell fluorescenceyields;/i)y//iV_c is the ratio of the X-ray self-absorption correction factors for Mgradiation in bulk Mg and for Na radiation in bulk NaCl; and eMlex is the ratio of theX-ray spectrometer efficiencies for the Mg and Na radiations.

The desired relative sensitivity, expressed in the form of the ratio of the signalintensities from a thin specimen containing Mg and Na in equimolar amounts, is

(SM/Sx)t = qjV/qx wM/wx eM/ex, (3)

where qu/qx is the ratio of the K-shell ionization cross-sections at the energy of theincident electrons.

The combination of equations (2) and (3) gives

(S,v/Sx)t = q^/q.w (S3l/SN)b nx/nM nx_c/nx fN_c/fM. (4)

The ratio of ionization cross-sections was obtained from the formula of Green &Cosslett (1961, p. 1210). At the operating voltage of 50 kV, the formula gives thevalue q3l/qx = 0779.

Observations on the bulk standard were done with a 20-kV electron beam. Theobserved value for (S^,/Sx)b was 8-45. At 20 kV, the ratio nN/n3/ according to theequation of Green & Cosslett (1961, p. 1211), uncorrected for electron backscatter, is1-41: the ratio nx_c/nx (equations of Hall, 1971, p. 228) is 0-412; the correction factorfor differences in backscattering (Hall, 1971, p. 215) is 0-99; and the product nx/n^r

nx_c/nx is hence 0-576. Finally the ratio of absorption corrections fX-c/fni (from theequation of Philibert, 1963, modified by Duncumb & Shields, 1966, as quoted by Hall,1971, p. 216) is 0-538. Substitution of these values into equation (4) gives the value2-04 for the relative sensitivity {Sv/Sx)t. Application of this factor to the sodiumcoefficient/Na in the Table gives the coefficient/JIg = 8600/2-04 = 4220.


APPENDIX II. ESTIMATION] OF LOCAL DRY-WEIGHT

FRACTIONS, AND CONVERSION OF MEASUREMENTS

FROM mM kg-1 DRY WEIGHT TO DIM kg-1 WET WEIGHT

Our preferred method for the measurement of local dry-weight fractions (Gupta,1978; Hall, 1978; Gupta et al. 1978 a) is based on the comparison of X-ray signalsfrom the selected region of the specimen before and after dehydration. Becausemeasurements on hydrated specimens were not needed for the main objectives of thepresent study, almost all of the sections were already dehydrated prior to X-rayanalysis. Therefore a different procedure was used to obtain the values listed inTable2.

The procedure is based on the assumptions that the mass per unit area is the samewithin the peripheral medium and within the selected region of the specimen in thesection in the fully hydrated state (i.e., when it is cut), and that the relative masses arenot affected by differential shrinkage during dehydration. These assumptions implythat

) = M(sp)/M(st), (5)

where f(l is the dry-weight fraction; 'st ' and 'sp ' refer respectively to the peripheralmedium and to the local region of the specimen; and M is mass per unit area afterdehydration. Since the corrected X-ray continuum count W is proportional to massper unit area, it follows that

/d(sp)=W(sp)/W(st) /d(st) (6)

and Table 2 is based on this equation.The elemental dry-weight concentrations in Table 1 were calculated from the

equation

= 7 wwhere Cx a is the concentration of element x in mM kg"1 dry weight. The wet-weight(or 'hydrated') concentrations of Table 4, CXik(sp), have been obtained from theequation

(8)

However, if the 2 factors on the right-hand side of this equation are replaced bytheir equivalents in equations (6) and (7), and it is noted that/d(st)Cxd(st) = Cx A(st),one sees that (8) is entirely equivalent to the equation

Cx,h(*p) = nx(sp)/nx(st) CXih(st) (9)

These equations show the real relationship between the dry-weight values of Table1 and the 'converted' wet-weight values of Table 4. Since the continuum signals Wappear in equation (7) but not in equation (9), the wet-weight values are actually freeof any of the uncertainties or errors associated with the continuum signal or its inter-pretation. But the wet-weight values rest on the assumptions of uniformity of section


thickness and shrinkage, while the dry-weight values are independent of theseassumptions.

Equation (9) is the basic equation used by Dorge et al. (1977).

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(Received 4 August 1978)

the quantitative measurement of electrolyte ...the equations used in the quantification of x-ray...

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