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The direct absorption of previously stained lipid by intact nuclei in liver cells of the rabbit.

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THE DIRECT ABSORPTION O F PREVIOUSLY
STAINED LIPID BY INTACT NUCLEI IN
LIVER CELLS O F THE RABBIT'
R. M. WOTTON' AND 8. S. LEVIN'
The Department of Zoology and Anatomy, The University of Mebraska,
Lincoln 8, Nebraska
TEN FIQURES
INTRODUCTION
Previously the relationship between cytoplasm and nucleus
in the cell has been studied emphasizing the nuclear influence
upon the cytosome. Pollister ('39), and Pollister and
Pollister ('43) have demonstrated this from one standpoint
by their observations on the association between the centromeres and centrioles in the gametes of certain snails. The
actual transfer, however, of substances across the karyotheca
of interphase nuclei in somatic cells has been noted very
seldom. Such an interaction between cytoplasm and nucleus
would depend in large measure upon the physical barrier
presented by the nuclear membrane. Callan and Tomlin ( '50)
have described the nuclear membrane as consisting of two
protein layers, with the outer one perforated by apertures
about 400 A in diameter. Anderson ('53) has suggested
a somewhat similar pattern consisting of a meshwork or
'The color reproduction in this paper was made possible by a grant from
the University Research Council of the University of Nebraska. Studies from
the Dopartment of Zoology, University of Nebraska, No. 293.
*The work presented in this paper was supported by a grant from the Pennsylvania Academy of Science for 1954 and 1955. P a r t of the study w w carried
out in the Department of Biological Sciences of the University of Pittsburgh,
Pittsburgh 13, Pa.
Department of Biological Sciences, the University of Pittsburgh, Pittsburgh
13, Pennsylvania.
155
156
R. M. WOTTON AND S. S. LEVIN
porous sheet covered with lipid for the envelope of nuclei
isolated from rat liver cells. Pollister, Gettner and Ward
('54) recently have described the appearance of small filaments passing through pores in the nuclear membrane of
certain amphibian ova. While these and other studies indicate
the possibility of contact between the cytoplasm and nucleoplasm, little evidence has been advanced to show the direct
passage of substances, readily visible under the light microscope, from the cytosome through the nuclear envelope into
the nucleus. Anderson ('53) has discussed the permeability
of the nuclear membrane toward macromolicules such as
enzymes, bovine serum albumin and partially hydrolyzed
gelatin. While these water soluble materials may be passed
across living nuclear membranes at the molecular level, they
are difKcult to follow microscopically. Zwemer ('33) and
Wotton and Martin ('53), however, have pointed out that
lipids are relatively easy to trace in histological preparations,
because of their characteristic staining reactions with Sudan
dyes and their physical properties of viscosity and plasticity.
The present observations upon the direct absorption of
previously stained cod liver oil by the nuclei of intact, hepatic
parenchymal cells in the rabbit form part of a study (to be
reported subsequently) in which the reaction of these cells
toward oil administered directly into the blood stream was
compared to that given by mouth.
PROCEDURE
Rabbits were selected as experimental animals for this
study, because of the ease with which oil could be introduced
directly into the blood stream. This was accomplished by
injecting with a hypodermic syringe measured amounts of
previously stained cod liver oil into the large ear veins. I n
this way the action of intestinal lipases upon the oil could be
avoided before its entry into the circulation. A total of 25
animals were used. These were divided into three groups
in such a way, that each group contained in addition to the
experimental animals which received oil through the ear vein
NUCLEAR ABSORPTION OF LIPID
15'7
and others which were given oil by stomach tube, controls
which received no oil. Our intention in dividing the experimental procedure into three complete parts wa,s to permit
us at yearly intervals to repeat twice in succession our
original study.
The stained cod liver oil was prepared by adding Sudan
IV in excess and thoroughly mixing with the oil by stirring.
The solution of bright red oil was allowed to stand for several
days, and then filtered through analytical grade filter paper
to remove the undissolved dye. I n this may cod liver oil was
marked for further identification in its passage throughout
the body of the rabbit (Zwemer '33, Wotton and Zwemer '39,
Zwemer and Wotton '44,and Wotton and Martin '53). As a
precautionary measure the colored oil was warmed before its
introduction into the blood stream by immersing for a time in
warm water the oil filled, hypoderlmic syringe. It was found
that adult rabbits could receive a dose of one to one and one
half ml of previously stained oil infused slowly into the ear
vein, without showing any signs of respiratory distress. Upon
the appearance of cyanosis in the lips of the animal injection
always was discontinued.
Animals were sacrificed at one to three hour intervals
following the intravenous or oral administration of colored
oil. Death was accomplished by compression of the thorax
to avoid the use of anaesthetics which might have a possible
fat solvent action in the body. Pieces of liver, heart, lung,
and kidney were preserved immediately in 10% pyridine
formalin (Burke '33). After 24 hrs fixation the tissues were
washed in running water to remove the formalin preparatory
to infiltration with gelatin. They were processed by the
gelatin embedding and frozen section technique proposed
by Zwemer ('33) for studying the lipids in corticoadrenal
and other cells.
Sections were spread on the surface of distilled water and
then picked up with microscope slides. A small amount of
1% gelatin solution was run under each section on the slide
to act as an adhesive agent. Slides were stained in Hansen's
158
El. M. WOTTON AND S. S. LEVIN
hematoxylin for nuclear detail, and in addition some of these
were counterstained subsequently in Romeis' Sudan I V
solution to intensify the color already present in the cod liver
oil. Glychrogel was used as a mounting medium (Wotton
and Zwemer '35, and Wotton '37).
OBSERVATIONS
Examination under the light microscope of slides containing sections of liver tissues revealed the presence of
previously stained lipid. This was recognized in the form
of droplets characteristically colored orange-red. Such cod
liver oil globules were seen in the lulmina of blood vessels,
liver sinusoids and within hepatic parenchymal cells. Those
cells most heavily loaded with stained liquid were observed in
areas surrounding branches of the hepatic portal vein.
Wotton and Zwemer ('39) have reported a similar distribution for fat in the liver of the cat.
The entrance of this previously stained cod liver oil into
liver cells was observed to occur in two ways. The most
commonly noted was the direct ingress of fat in characteristic
hour-glass form (Wotton and Zwemer '39, Zwemer and
Wotton '44, Wotton, Ellinger and Bartone '50, Wotton and
Village '51, Wotton and Martin '51, Wotton and Raskauskas
'54,and Wotton and Mosti '55) through the cell membrane
into the cytosome (figs. 1 and 2). I n addition other droplets
were seen in passage from fat containing Kupffer cells into
closely adjacent parenchymal cells (Murray and Freeman
'51, Friedman, Byers and Roseman '54, and Waddell, Geyer,
Clark and Stare '54).
Further microscopical study revealed the presence of one
o r more drops of Sudan IV colored oil in the nuclei of many
hepatic cells. When such nuclei were seen in amitotic division,
often the presence of a droplet of red fat was noted in one
lobe of the nucleus, while none was seen in the other portion.
Observations using the homogeneous immersion objective
showed in many instances a direct continuity between masses
of sudanophilic material within and without the nucleus (figs.
NUCLEAR ABSORPTION OF LIPID
159
2,3,5, to 10). In one case (fig. 2) a single drop of orange-red
oil may be seen in transition as one continuous aggregate
extending from the lumen of the sinusoid through the cytosome into the nucleus in one cell. Two constrictions may be
noted in this globule of previously stained cod liver oil
indicating the sites where it crosses the cell and nuclear membrane respectively. Once within the nucleus the red stained
oil assumed either a spherical shape (figs. 2, 3 and 5) or
spread out and occupied the spaces between the stained
chromatin material (figs. 7, 8, 9 and 10). Histologically no
difference could be detected in the appearance of the previously red stained oil before or after its entry into the nucleus. I n those slides made from the liver tissues of control
rabbits which had received no oil, stained fat was not seen
in the nuclei. At this time it seems worth-while to mention
that in an earlier study (Wotton and Martin '51) certain
nuclei in septa1 cells of the cat lung were observed to be ingesting previously stained lipid. While this was not reported
in that paper, because it was considered that further confirmation of this phenomenon would be desirable, it is included
here, for the sake of completeness.
1)I8cussIopJ
The factors controlling this process of lipid penetration
into the nucleoplasm are difEcult to determine. The structure
of the nuclear membrane certainly plays an important role
in permeability to lipids. That there is resistance offered
to the passage of fat by such a membrane is indicated by
the constrictions which oil globules exhibit at the points
where they enter the nucleus (figs. 2,5 and 10). This ingress
of previously stained cod liver oil through the nuclear membrane of hepatic cells follows a route comparable to that
observed for the passage of lipids across cytoplasmic membranes (Wotton and Zwemer '39, Zwemer and Wotton '44,
Wotton and Martin '51, Wotton and Village '51, and Wotton
and Mosti '55). In a similar way an aperture appears to be
formed in the karyotheca permitting oil to traverse this
160
R. M. WOTTON AND S. S. LEVIN
boundary. Frey-Wyssling ('53) in discussing the nature of
the plasmalemma considered it to be "a boundary layer in
which lipids accumulate", and Harvey ( '37) has shown that
elasticity similar to that exhibited by cell membranes is also
found on the surface of protein containing solutions. Collander ('37) indicates that lipid solubility and molecular size
control the permeability of nonelectrolytes (lipoid-sieve
theory). What function the submicroscopic porosities in the
nuclear membrane described by Anderson ('53) and others
may assume in this situation is not easy to assess. They may
merely serve for the transition of large water soluble molecules such as enzymes. The size of the apertures in the karyotheca, on the other hand, through which lipid has been seen
to enter the nucleus (figs. 2 to 10) is of a magnitude easily
detected by the oil immersion objective. A possible explanation for this phenomenon may be postulated on the basis of
a physical mechanism involving solution of the colored oil
in the cytosome with the lipid contained in the nuelear melmbrane (Anderson '53). Thus the increased concentration of
fatty material in a given region of the nuclear membrane or
envelope (Danielli '51) would lower considerably the surface
tension and the resistance of this membrane at this specific
site to the passage of previously stained cod liver oil. The
ultrastructure of the nuclear and plasma membranes may
differ markedly, but our studies, however, indicate that their
permeability toward lipids appears similar in both instances.
SUMMARY
Observations on hepatic, parenchymal cells of rabbits
which had received cod liver oil previously stained with
Sudan I V showed the presence of this fatty material within
the nuclei of many of these cells.
Drops of red colored oil in the cytoplasm were seen
connected directly through the nuclear (membrane t o similar
drops of oil within the nucleoplasm, indicating the ingress of
this material from the cytosome into the nucleus.
NUCLEAR ABSORPTION OF LIPID
161
It is considered that the physical solution of this red
colored cod liver oil with the lipid contained in the nuclear
membrane results in a localized concentration of surface
tension reducing substances which increase the permeability
of this barrier to lipids.
The similarity in the cytological picture presented by fats
passing through the nuclear membrane compared with that
seen for such substances crossing cell membranes suggests
a common, physical mechanism for both.
LITERATURE CITED
ANDERSON,
N. 0. 1953 On the nuclear envelope. Science. 117: 517-521.
BURKE,F. V. 1933 The p H of formalin-a
factor in fixation. Amcr. Jour.
Path. 9: 915-920.
CALLAN, H. F., AND 8. G. TOMLIN1950 Experimental studies on amphibian
oocyto nuclei, I. Investigation of the structure of the nuclear membrane by means of the electron microscope. Proc. Boy. SOC. B, 237:
367-378.
COLLAKDER,
R. 1937 Permeability. Ann. Rev. Biochem. 6: 1-18.
DANIELLI,J, F. 1951 Cell surface and physiology. In Cytology and Cell Physiology. Ed. by G. 11. Bourne. Oxford. Chap. IV, 153.
FEET-WYSSLINQ,
A. 1953 Submicroscopic morphology of protoplasm. Elsevier
Publishing Co., New Pork. 199.
FRIEDNIN,
M., 8. 0.BYERSAND R. H. RQSENMAN1954 XII. Demonstration of
the e~seiitialrole of the hepatic Reticulo-Endothelial cell (Kupffer
ccll) in the normal disposition of exogenously derived cholesterol.
Amer. Jour. Physiol. 177: 78-83.
HARVEY,E. N. 1937 Cytoplasm, surfacetension. Trans. Farady SOC. 3.7:
943-946.
MURRAY,
R. G., AND 8. FREEMAN
1951 The morphological distribution of intrnvenously injected fatty chyle and artificial fat emulsion in rats and
dogs. Jour. Lab. and Clin. Med. 38: 56-59.
POLLISTER,
A. W. 1939 Ccntrioles and centrosomas in the atypical spermatogenesis of vivipara. Proc. Nat. Acad. Sci. 26: 189-195.
POLLISTER,
A. W.,M. GETTNER
AND R. WARD1954 Il’ucleocytoplasmic interchange
h oocytes. Science. 220: 789.
POLLISTER,
A. W., AND P. F. POLLISTER
1943 The relation between centriole and
centromere in atypical spermatogenesis of viviparid snails. Annals
N. Y. Acad. Sci. 45: 148.
WADDELL,
W. R., R. P. GEPER, E. CLARKAND F. J. STARE
1954 Fnnction of the
Reticuloindothelial system in removal of emulsified fat from blood.
Amer. Jour. Physiol. 177: 90-94.
WOTTON,R. M. 1937 The application of Glychrogel mounting for trematodes.
Stain Tech. 12: 145-146.
162
R. M. WOTTON AND S. S. LEVIN
WOTTON,R. M., T. U. H. ELLINGEP
AND J. C. BARTONE
1950 A lipase reaction
i n phagocytes from the peritoneum of rats toward previously stained
fat. Anat. Rec. 107: 73-81.
WOTTONR. M., AND A. MARTIN,JR. 1951 The reaction of the lung in the cat
toward oil droplets i n the circulation. Anat. Bec. 110: 267-273.
1953 The role of the Golgi apparatus in f a t metabolism in
representative vertebrates. Proc. Penna. Acad. Sci. 27 : 277-285.
WOTTON,R.. M., AND M. E. MOSTI 1955 The direct absorption of previously
stained f a t in droplet form by the myocardium of the cat. Anat.
Rec. 122: 39-47.
WOTTON,R. M., AND M. RASKAUSKAS1954 Thc absorption of previously stained
lipid by planaria. Proc. Penna. Acad. Sci. 28: 247-251.
WOTTON,R. M.,AND P. VILLAGE1951 The transfer function of certain cells in
the wall of the Graafian follicle as revealed by their reaction in
previously stained f a t in the cat. Anat. Rec. 110: 121-127.
WOTTON,R. M., AND R. L. ZWEMER 1935 A note on “Glychrogel” mounting
solution. Stain Tech. 10: 21-22.
1939 Studies on direct and visible ingestion of f a t by differentiated body cells of the cat. Anat. Rec. Y5: 493-507.
ZWEMER,R. L. 1933 A method for studying adrenal and other lipoids by a
modified gelatin embedding and mounting technique. Anat. Rec. 57:
41-44.
ZWEMER,R. L., AND R. hf. WOTTON1944 Fat excretion in the guinea pig kidney.
Anat. Rec. 90: 107-114.
PLATES
PLATE 1
EXPLANATION OF FIGURES
1
Photomicrograph of n section of liver tissue showing the direct absorption
of previously stained cod liver oil into :t parenchymal cell. This is intlicatcd :It tip of upper arrow. Tip of lower arrow points to a n oil globule
i n a n hepatic sinusoid. 1200 X.
2
T i p of arrow indieatcw a drop of cod liver oil passing from the hepatic
siriuboid through the cytoqome into the nucleus of a liver cell in one continuous mass. Two constrictions may be seen, one at t h e site of entry
through the plasma mcmbraaic, and the othrr at the nuclear menibrane
respectively. 1200 x. Insert is a schematic sketch somewhat cnlarged t o
show smne structures 8s seen in the photomicrograph (cyt = rytosome, and
nuc = nucleus).
3
Tips of both arrows indicate previously stained cod liver oil i n nuclei of
liver cells. 1200 X.
4.
Large globule of previously stained cod liver oil in the nucleus of a liver
cell is indicated at tip of arrow. 1800 X.
5
Same nucleus as shown in figure 4, but a t :i differcnt love1 of focus. Tip
of arrow points to site of entrance of oil drop through the nuclear menibran? as seen from ahovt. 1800 X.
6
Same nucleus ns sliown in figure 5, b u t observrd by phase contrast microscopy. 1800 X.
7
Nucleus of liver cell showiiig entrance of cod liver oil into tlie nucleoplasni.
T i p of arrow indicates point of entry. 3600 X.
8 Similar t o figure 7, h u t showing a different nucleus. 3600 X,
9
Hepatic cells in which tho passage of previously stained cod liver oil across
the nuclear membrane can be seen a t tips of arrows. Note accumulation of
oil coating the outside of nucleus indicated at t i p of upper arrow.
Demonstrated by Anoptrnl (Wilska-Reiehert) phase contrast microscopy.
2400 X.
164
KUOLMAR BBSOKL’TION OB’ I,I€’IL)
R. M. WOTTOX A K I ) S. 6. LEVIN
165
NUCLEAR ABSORPTION OF L l P I D
PLATE 2
R. M. WOTTON A N D S. Y. LEVIN
EXPLANATION OF FIGURES
The optics employed in niaking the color photomicrograph shown in figure
10 consisted of a Reichert aprochromatic, oil immersion objective n. A. 1.3,
achromatic substage condensor n. A. 1.35 oiled t o the under side of the slide,
and an 8 x compensating eyepiece. Zeiss microscope filament lamp I11 was
used, without any color filters or ground glass screen, to obtain Koehler
type illumiiiation. Eastinail Kodaehronie film, type A. Exposure about 18
seconds.
1 0 Color photomicrograph showing the passage of previously, red stained,
cod liver oil into the nucleus of a hepatic parenchymal cell in the rabbit.
This is the same nucleus as shown in figure 8. 3600 X .
166
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staines, intact, nuclei, rabbits, direct, live, absorption, previously, lipid, cells
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