- Email: [email protected]
Fractionation of rat liver nuclei by sucrose gradient centrifugation
Experimental
Cell Research 49, 373-378 (1968)
FRACTIONATION
373
OF RAT LIVER NUCLEI
BY SUCROSE GRADIENT
CENTRIFUGATION
C. F. ALBRECHT Department of Chemistry including Biochemistry,
University of the Witwatersrand,
Johannesburg, and National Chemical Research Laboratory, Council for Scienti$c and Industrial Research, Pretoria, South Africa1
Received April 10, 1967
BECAUSE their
of the multiplicity
response
to the
studying
the influence
between
nuclei
from
of cell types
administration of carcinogens different
cells.
on the nucleus, A technique
of tetraploid
and diploid
rat liver
study
elaboration
of this technique
is an
tionate
stromal
in
biochemical
the
14C-6-erotic
and parenchymal
acid
activities
nuclei nuclei.
of the
and the
and
described
separation
[3]. The
present
one to partially
to illustrate
nuclei,
in
when
to be able to distinguish
enables
In order fractions
differences
it is desirable,
for the fractional
has been
different
in uiuo into the nuclear MATERIALS
in the liver
of carcinogens,
the
isolated
frac-
the differences incorporation
of
was studied.
AND METHODS
Animals.-Fed, female inbred albino rats weighing 150 g, having liver weights of about 5 g were used. Isolation of nuclei.-Rats were anaesthetised with ether fumes and perfused in situ with 50 ml of an ice-cold solution containing 0.25 M sucrose, 0.0016 M CaCl, and 0.9 per cent NaCl [6]. The liver was ground to a pulp in a chilled pestle and mortar and transferred with 30 ml of chilled 0.25 M sucrose solution containing 0.0033 M CaCl, [4], into a Dounce homogenizer and homogenized with 20 gentle strokes of the loose-fitting plunger. After straining through 6 layers of washed cheese cloth, the homogenate was centrifuged for 10 min at 800 g in an International refrigerated centrifuge. The pellet was resuspended in 100 ml of 2.2 M sucrose and centrifuged for 60 min at 50,000 g (about 24,000 rpm) in the No 30 rotor of the Spinco Model L Preparative Ultracentrifuge [a]. The pellets obtained were soaked for 15 min in 0.25 A& sucrose (10 ml per pellet) and then resuspended in a Dounce homogenizer with 5 strokes of the loose-fitting plunger. The nuclear suspension was then centrifuged again in the International centrifuge as described above, and the pellet obtained was resuspended in 1.5 ml of 0.25 M sucrose by using a Whirlimixer. The final suspension of nuclei was layered over a sucrose gradient for further purification and fractionation. Preparation of sucrose gradients.-Linear continuous sucrose gradients (30 ml of 0.5 M sucrose-25 ml of 1.0 M sucrose) were prepared in 100 ml Lusteroid International Centrifuge tubes by using a mixing apparatus of the type described by Britten
1 Present address. Experimenfal
Cell Research 49
374
C. F. Albrecht
Fig. i.-(n) Unfrktionated nuclei stained with dilute nkthylene lilue. A, Letraploid parenchymal; II, diploid parenchymal nucleus; and C, stromal nucleus. Y 600. (b) Tetraploid parenchymal nuclei from fraction 4. x 600. (c) Partially fractionated diploid parenchymal nuclei from fraction 15. x 600. (d) Stromal nuclei and a few damaged nuclei from fraction ‘21. < 600.
and Roberts [l] and modified by Stead, Hawtrey and Nourse [7]. The gradients were cooled in ice 2 h prior to use. The nuclear suspension was layered over the gradient which was then centrifuged for 10 min at 700 g (1000 rpm) in an International refrigerated centrifuge (Model PR-2). Immediately after centrifugation the gradient was collected into about 30 fractions (20 drops or about 1 ml each) by inserting a No 18G syringe needle into the base of the centrifuge tube. of OD,,, of nuclear fractions.PImmediately after collection, the Measurement nuclear fractions were diluted to 3 ml with 0.25 M sucrose and the OD,,, determined in a 1 cm cell. Microscopic examination.--The various nuclear fractions were studied in a Bright Experimental Cell Research 49
Fractionation
375
of rat liver nuclei
Line Hemacytometer under a Binocular Leitz Laborlux microscope at a magnification of x 430. For the determination of the different types of nuclei per fraction, nuclei were stained with a dilute solution of Methylene Blue in 0.25 iV sucrose. Nuclear counts were performed on 500 nuclei per fraction and a count variation of 5 per cent was considered a maximum. Classification of nuclei.-Nuclei were classified essentially according to the criteria adopted by Falzone, Barrows and Yiengst [3], i.e. tetraploid parenchymal, diploid parenchymal and stromal nuclei. Eye-micrometer measurements on 500 nuclei stained with dilute Methylene Blue showed three different mean diameter ranges viz. 10.5 ( +0.5) p, 7.6 ( kO.3) p, and 6.2 ( iO.2) p. These three nuclear diameters were interpreted to represent tetraploid parenchymal, diploid parenchymal and stromal nuclei respectively. These three different types of nuclei are illustrated in Fig. 1 a and as can be seen, the stromal nuclei have much smaller and less distinct nucleoli than are observed in the diploid or tetraploid parenchymal nuclei. A number of oval and oblong shaped nuclei with or without distinct nucleoli were also observed and classified as stromal nuclei. In vivo incorporation of W-6-erotic acid.-Rats were injected intraperitoneally with 5 PC of W-6-erotic acid (specific activity 44.5 mc/mM; Radiochemical Centre, Amersham), dissolved in 0.5 ml of distilled water, 1 h prior to sacrifice. In order to avoid self-absorbtion of radioactive counts the radioactively labelled nuclear fractions were diluted to give an OD,,, of 0.5 and 1 ml of each diluted fraction was then quantitatively transferred to Millipore filters and washed 3 times with 10 ml of ice-cold 5 per cent TCA. Fractions were then counted for radioactivity on a Packard Tri-Carb Liquid Scintillation counter for 10 min, corrected for background and expressed as counts/ min/OD,,,. RESULTS
After centrifugation the nuclei separate into two discreet bands about two cm apart half way down the gradient. Fig. 2 shows the OD,,, profile of the different fractions collected from the sucrose gradient. The two peaks A and B correspond respectively to the tetraploid and diploid nuclear fractions reported by Falzone, Barrows and Yiengst [3]. A drop of nuclear suspension from fractions 1-4 studied under the microscope (without coverslip) showed that these fractions contained small clumps of diploid nuclei bound loosely together. Fractions 22-32 contained damaged nuclei and many non-nuclear particles. Fractions 33 and 34 contained a mixture of nuclei that collect on the sides of the gradient tube during drop collection and are released into the last two fractions. Fractions 1-4, 22-32, and 33-34 were thus not studied further. The percentages of the three different types of nuclei per fraction are superimposed over the OD 26,,profile in Fig. 2. It can be seen that fractions 4-l 2 contain mostly tetraploid parenchymal nuclei, fractions 13-l 6 mostly 25
-
681804
Experimental
Cell Research
49
376
C. F. Albrecht
diploid parenchymal nuclei and fractions 18-21 mostly stromal nuclei. Fig. 1 a, b, c and d show the nuclei found in fractions 4, 15 and 21, respectively. The specific radioactivity of the different fractions in terms of in uiuo 14C-6-orotic acid incorporation is also indicated in Fig. 2. It can be seen that Clumped nuclei
Tetraploid
nuclei
Diploid
nuclei
Damaged
nuclei
and
cell debris
I.0
100%
05
50%
0.0
0% IO
15
20
25
30
34, Meniscus
Fig. 2.-Composition of nuclear fractions collected by sucrose gradient centrifugation. A, peak of tetraploid parenchymal nuclei; B, peak of diploid parenchymal and stromal nuclei. OD,,, (--), counts/min/OD,,, (---), percentage of tetraploid parenchymal nuclei (O-O), percentage of diploid parenchymal nuclei (a--o), percentage of stromal nuclei (A-A). Abscissa: Fraction numbers; ordinate: (left) ODzso “,@; (right) y0 different nuclei.
the highest counts (ca 1000 counts/min) are recorded from fractions containing the highest percentage of tetraploid parenchymal nuclei [4-S], while the counts recorded from fractions containing a high percentage of diploid parenchymal nuclei [14, 151 are slightly lower (ca 800-900 counts/min). Fractions containing high percentages of stromal nuclei have significantly lower counts. In order to get a measure of the activities of the stromal and parenchymal nuclei in the incorporation of 14C-6-orotic acid in viuo, the radioactivity of the various fractions was plotted against the percentage stromal nuclei present. As can be seen from Fig. 3 a straight line was obtained. Extrapolation of this indicates that a fraction containing 100 per cent stromal nuclei would have a specific activity of ca 100 counts/min compared with ca 1000 countsjmin for a fraction containing 100 per cent diploid or tetraploid parenchymal nuclei. Experimental
Cell Research 49
Fractionation
377
of rat liver nuclei
Fig. Y.-Linear relationship between the percentage of stromal nuclei per fraction and the speciin uiuo. (Fraction fic radioactivity of the fraction in terms of 14C-6-erotic acid incorporation numbers are indicated with each point on the graph.) Abscissa: Stromai nuclei in fraction; /fraction. orttinnte: C. P.M./OD260 111(1
DISCUSSION
The present work has also shown that it is possible partially to fractionate rat liver nuclei into fractions containing mainly parenchymal tetraploid, parenchymal diploid and stromal nuclei by sucrose gradient centrifugation.. The method used for fractionation is quicker than the gravity sedimentation technique of Falzone, Yiengst and Barrovvs [3], and also permits fractions to be collected so that in vivo and in vitro activities of different classes of nuclei can be studied. The present technique can also be used to gain some idea of the percentages of the different types of cells present in the liver under study. Calculations derived from the percentages of the different nuclei in each fraction and the CID,,, of the different fractions show that the total percentage of the different types of nuclei in fractions 4-21 are: tetraploid parenchymal-45 per cent, diploid parenchymal-30 per cent, and stromal nuclei-25 per cent. As far as parenchymal nuclei are concerned, there are thus 60 per cent tetraploid and 40 per cent diploid nuclei present in the Experimental
Cell Research 49
378
C. F. Albrecht
fractions studied. Nadal and Zajdela [5] have reported that isolated rat liver parenchymal cells from B-week-old rats (which is the approximate age of the rats studied), contain about 55 per cent tetraploid and 45 per cent diploid cells. This correlation would seem to suggest that the present technique can be used for the quantitative determination of parenchymal cell types present in the rat liver studied. In order to obtain an accurate determination, the clumped nuclei in fractons l-4 could also be included in the determination. The present technique could thus be used to obtain quantitative and qualitative information concerning the cellular populations and the biochemical activities of different types of nuclei in normal and pathological livers. SUMMARY
A technique for the partial fractional separation of tetraploid and diploid parenchymal and stromal rat liver nuclei using a linear, sucrose gradient and low speed centrifugation is described. In vivo incorporation of 14C-6erotic acid in different nuclear fractions was measured and it was shown that the diploid and tetraploid parenchymal nuclei had the same specific radioactivity whereas the stromal nuclei were only about 10 per cent as active. The technique can also be used to determine the percentages of the various kinds of nuclei in the liver being studied. Dr H. M. Schwartz and Dr J. B. Balinsky are thanked for helpful discussions and Mr L. Nourse for technical guidance. This project is supported by Grant No. 169/67/Cl from the Council for Scientific and Industrial Research.
REFERENCES 1. BRITTEN, R. J. and ROBERTS, R. B., Science 131, 32 (1960). 2. CHAUVEAU, J., MOULE, Y. and ROUILLER, C., Ezpt[ Cell Res. 11, 317 (1956). 3. FALZONE, J. A., BARROWS, C. H. and YIENGST, M. J., Exptl Cell Res. 26, 552 (1962). 4. MURAMATSU, M., SMETANA, K. and BUSCH, H., Cancer Res. 23, 510 (1963). 5. NADAL, C. and ZAJDELA, F., Exptl Cell Res. 42, 99 (1966). 6. REES, K. R. and ROWLAND, G. F., Biochem. J. 78, 89 (1961). 7. STEAD, R., NOURSE, L. D. and HAWTREY, A. O., S. Air. J. Med. Sci. 29, 79 (1964).
Experimental
Cell Research 49
Cell Research 49, 373-378 (1968)
FRACTIONATION
373
OF RAT LIVER NUCLEI
BY SUCROSE GRADIENT
CENTRIFUGATION
C. F. ALBRECHT Department of Chemistry including Biochemistry,
University of the Witwatersrand,
Johannesburg, and National Chemical Research Laboratory, Council for Scienti$c and Industrial Research, Pretoria, South Africa1
Received April 10, 1967
BECAUSE their
of the multiplicity
response
to the
studying
the influence
between
nuclei
from
of cell types
administration of carcinogens different
cells.
on the nucleus, A technique
of tetraploid
and diploid
rat liver
study
elaboration
of this technique
is an
tionate
stromal
in
biochemical
the
14C-6-erotic
and parenchymal
acid
activities
nuclei nuclei.
of the
and the
and
described
separation
[3]. The
present
one to partially
to illustrate
nuclei,
in
when
to be able to distinguish
enables
In order fractions
differences
it is desirable,
for the fractional
has been
different
in uiuo into the nuclear MATERIALS
in the liver
of carcinogens,
the
isolated
frac-
the differences incorporation
of
was studied.
AND METHODS
Animals.-Fed, female inbred albino rats weighing 150 g, having liver weights of about 5 g were used. Isolation of nuclei.-Rats were anaesthetised with ether fumes and perfused in situ with 50 ml of an ice-cold solution containing 0.25 M sucrose, 0.0016 M CaCl, and 0.9 per cent NaCl [6]. The liver was ground to a pulp in a chilled pestle and mortar and transferred with 30 ml of chilled 0.25 M sucrose solution containing 0.0033 M CaCl, [4], into a Dounce homogenizer and homogenized with 20 gentle strokes of the loose-fitting plunger. After straining through 6 layers of washed cheese cloth, the homogenate was centrifuged for 10 min at 800 g in an International refrigerated centrifuge. The pellet was resuspended in 100 ml of 2.2 M sucrose and centrifuged for 60 min at 50,000 g (about 24,000 rpm) in the No 30 rotor of the Spinco Model L Preparative Ultracentrifuge [a]. The pellets obtained were soaked for 15 min in 0.25 A& sucrose (10 ml per pellet) and then resuspended in a Dounce homogenizer with 5 strokes of the loose-fitting plunger. The nuclear suspension was then centrifuged again in the International centrifuge as described above, and the pellet obtained was resuspended in 1.5 ml of 0.25 M sucrose by using a Whirlimixer. The final suspension of nuclei was layered over a sucrose gradient for further purification and fractionation. Preparation of sucrose gradients.-Linear continuous sucrose gradients (30 ml of 0.5 M sucrose-25 ml of 1.0 M sucrose) were prepared in 100 ml Lusteroid International Centrifuge tubes by using a mixing apparatus of the type described by Britten
1 Present address. Experimenfal
Cell Research 49
374
C. F. Albrecht
Fig. i.-(n) Unfrktionated nuclei stained with dilute nkthylene lilue. A, Letraploid parenchymal; II, diploid parenchymal nucleus; and C, stromal nucleus. Y 600. (b) Tetraploid parenchymal nuclei from fraction 4. x 600. (c) Partially fractionated diploid parenchymal nuclei from fraction 15. x 600. (d) Stromal nuclei and a few damaged nuclei from fraction ‘21. < 600.
and Roberts [l] and modified by Stead, Hawtrey and Nourse [7]. The gradients were cooled in ice 2 h prior to use. The nuclear suspension was layered over the gradient which was then centrifuged for 10 min at 700 g (1000 rpm) in an International refrigerated centrifuge (Model PR-2). Immediately after centrifugation the gradient was collected into about 30 fractions (20 drops or about 1 ml each) by inserting a No 18G syringe needle into the base of the centrifuge tube. of OD,,, of nuclear fractions.PImmediately after collection, the Measurement nuclear fractions were diluted to 3 ml with 0.25 M sucrose and the OD,,, determined in a 1 cm cell. Microscopic examination.--The various nuclear fractions were studied in a Bright Experimental Cell Research 49
Fractionation
375
of rat liver nuclei
Line Hemacytometer under a Binocular Leitz Laborlux microscope at a magnification of x 430. For the determination of the different types of nuclei per fraction, nuclei were stained with a dilute solution of Methylene Blue in 0.25 iV sucrose. Nuclear counts were performed on 500 nuclei per fraction and a count variation of 5 per cent was considered a maximum. Classification of nuclei.-Nuclei were classified essentially according to the criteria adopted by Falzone, Barrows and Yiengst [3], i.e. tetraploid parenchymal, diploid parenchymal and stromal nuclei. Eye-micrometer measurements on 500 nuclei stained with dilute Methylene Blue showed three different mean diameter ranges viz. 10.5 ( +0.5) p, 7.6 ( kO.3) p, and 6.2 ( iO.2) p. These three nuclear diameters were interpreted to represent tetraploid parenchymal, diploid parenchymal and stromal nuclei respectively. These three different types of nuclei are illustrated in Fig. 1 a and as can be seen, the stromal nuclei have much smaller and less distinct nucleoli than are observed in the diploid or tetraploid parenchymal nuclei. A number of oval and oblong shaped nuclei with or without distinct nucleoli were also observed and classified as stromal nuclei. In vivo incorporation of W-6-erotic acid.-Rats were injected intraperitoneally with 5 PC of W-6-erotic acid (specific activity 44.5 mc/mM; Radiochemical Centre, Amersham), dissolved in 0.5 ml of distilled water, 1 h prior to sacrifice. In order to avoid self-absorbtion of radioactive counts the radioactively labelled nuclear fractions were diluted to give an OD,,, of 0.5 and 1 ml of each diluted fraction was then quantitatively transferred to Millipore filters and washed 3 times with 10 ml of ice-cold 5 per cent TCA. Fractions were then counted for radioactivity on a Packard Tri-Carb Liquid Scintillation counter for 10 min, corrected for background and expressed as counts/ min/OD,,,. RESULTS
After centrifugation the nuclei separate into two discreet bands about two cm apart half way down the gradient. Fig. 2 shows the OD,,, profile of the different fractions collected from the sucrose gradient. The two peaks A and B correspond respectively to the tetraploid and diploid nuclear fractions reported by Falzone, Barrows and Yiengst [3]. A drop of nuclear suspension from fractions 1-4 studied under the microscope (without coverslip) showed that these fractions contained small clumps of diploid nuclei bound loosely together. Fractions 22-32 contained damaged nuclei and many non-nuclear particles. Fractions 33 and 34 contained a mixture of nuclei that collect on the sides of the gradient tube during drop collection and are released into the last two fractions. Fractions 1-4, 22-32, and 33-34 were thus not studied further. The percentages of the three different types of nuclei per fraction are superimposed over the OD 26,,profile in Fig. 2. It can be seen that fractions 4-l 2 contain mostly tetraploid parenchymal nuclei, fractions 13-l 6 mostly 25
-
681804
Experimental
Cell Research
49
376
C. F. Albrecht
diploid parenchymal nuclei and fractions 18-21 mostly stromal nuclei. Fig. 1 a, b, c and d show the nuclei found in fractions 4, 15 and 21, respectively. The specific radioactivity of the different fractions in terms of in uiuo 14C-6-orotic acid incorporation is also indicated in Fig. 2. It can be seen that Clumped nuclei
Tetraploid
nuclei
Diploid
nuclei
Damaged
nuclei
and
cell debris
I.0
100%
05
50%
0.0
0% IO
15
20
25
30
34, Meniscus
Fig. 2.-Composition of nuclear fractions collected by sucrose gradient centrifugation. A, peak of tetraploid parenchymal nuclei; B, peak of diploid parenchymal and stromal nuclei. OD,,, (--), counts/min/OD,,, (---), percentage of tetraploid parenchymal nuclei (O-O), percentage of diploid parenchymal nuclei (a--o), percentage of stromal nuclei (A-A). Abscissa: Fraction numbers; ordinate: (left) ODzso “,@; (right) y0 different nuclei.
the highest counts (ca 1000 counts/min) are recorded from fractions containing the highest percentage of tetraploid parenchymal nuclei [4-S], while the counts recorded from fractions containing a high percentage of diploid parenchymal nuclei [14, 151 are slightly lower (ca 800-900 counts/min). Fractions containing high percentages of stromal nuclei have significantly lower counts. In order to get a measure of the activities of the stromal and parenchymal nuclei in the incorporation of 14C-6-orotic acid in viuo, the radioactivity of the various fractions was plotted against the percentage stromal nuclei present. As can be seen from Fig. 3 a straight line was obtained. Extrapolation of this indicates that a fraction containing 100 per cent stromal nuclei would have a specific activity of ca 100 counts/min compared with ca 1000 countsjmin for a fraction containing 100 per cent diploid or tetraploid parenchymal nuclei. Experimental
Cell Research 49
Fractionation
377
of rat liver nuclei
Fig. Y.-Linear relationship between the percentage of stromal nuclei per fraction and the speciin uiuo. (Fraction fic radioactivity of the fraction in terms of 14C-6-erotic acid incorporation numbers are indicated with each point on the graph.) Abscissa: Stromai nuclei in fraction; /fraction. orttinnte: C. P.M./OD260 111(1
DISCUSSION
The present work has also shown that it is possible partially to fractionate rat liver nuclei into fractions containing mainly parenchymal tetraploid, parenchymal diploid and stromal nuclei by sucrose gradient centrifugation.. The method used for fractionation is quicker than the gravity sedimentation technique of Falzone, Yiengst and Barrovvs [3], and also permits fractions to be collected so that in vivo and in vitro activities of different classes of nuclei can be studied. The present technique can also be used to gain some idea of the percentages of the different types of cells present in the liver under study. Calculations derived from the percentages of the different nuclei in each fraction and the CID,,, of the different fractions show that the total percentage of the different types of nuclei in fractions 4-21 are: tetraploid parenchymal-45 per cent, diploid parenchymal-30 per cent, and stromal nuclei-25 per cent. As far as parenchymal nuclei are concerned, there are thus 60 per cent tetraploid and 40 per cent diploid nuclei present in the Experimental
Cell Research 49
378
C. F. Albrecht
fractions studied. Nadal and Zajdela [5] have reported that isolated rat liver parenchymal cells from B-week-old rats (which is the approximate age of the rats studied), contain about 55 per cent tetraploid and 45 per cent diploid cells. This correlation would seem to suggest that the present technique can be used for the quantitative determination of parenchymal cell types present in the rat liver studied. In order to obtain an accurate determination, the clumped nuclei in fractons l-4 could also be included in the determination. The present technique could thus be used to obtain quantitative and qualitative information concerning the cellular populations and the biochemical activities of different types of nuclei in normal and pathological livers. SUMMARY
A technique for the partial fractional separation of tetraploid and diploid parenchymal and stromal rat liver nuclei using a linear, sucrose gradient and low speed centrifugation is described. In vivo incorporation of 14C-6erotic acid in different nuclear fractions was measured and it was shown that the diploid and tetraploid parenchymal nuclei had the same specific radioactivity whereas the stromal nuclei were only about 10 per cent as active. The technique can also be used to determine the percentages of the various kinds of nuclei in the liver being studied. Dr H. M. Schwartz and Dr J. B. Balinsky are thanked for helpful discussions and Mr L. Nourse for technical guidance. This project is supported by Grant No. 169/67/Cl from the Council for Scientific and Industrial Research.
REFERENCES 1. BRITTEN, R. J. and ROBERTS, R. B., Science 131, 32 (1960). 2. CHAUVEAU, J., MOULE, Y. and ROUILLER, C., Ezpt[ Cell Res. 11, 317 (1956). 3. FALZONE, J. A., BARROWS, C. H. and YIENGST, M. J., Exptl Cell Res. 26, 552 (1962). 4. MURAMATSU, M., SMETANA, K. and BUSCH, H., Cancer Res. 23, 510 (1963). 5. NADAL, C. and ZAJDELA, F., Exptl Cell Res. 42, 99 (1966). 6. REES, K. R. and ROWLAND, G. F., Biochem. J. 78, 89 (1961). 7. STEAD, R., NOURSE, L. D. and HAWTREY, A. O., S. Air. J. Med. Sci. 29, 79 (1964).
Experimental
Cell Research 49