INTRANUCLEOLAR BODIES IN NORMAL AND NEOPLASTIC
HUMAN TISSUE 1
ROBERT C. PAGE, M.D.
Fellow in Pathology, The Mayo Foundation
JAMES F. REGAN, M.D.
Fellow in Surgery, The Mayo Foundation,
WILLIAM CARPENTER MAcCARTY, M.D.
Section on Surgical Pathology, The Mayo Clinic
In a preliminary report (Regan, Page and MacCarty) intranucleolar bodies
in normal and malignant cells of human tissue were described. The results of
further study of these bodies are here presented.
Leydig, in 1852, in examining the ovum of Synapta digitata found a single
nucleolus which had a vacuole. Agassiz described vacuoles in the cells of the
turtle and called them" entosthoblasts " or " Valentini an vesicles." Balbiani
claimed that the nucleoli in the eggs of certain animals possessed contractile
vacuoles and Bohm confirmed this observation with regard to ova of other
small animals. Carlier observed a body, which he called the" endonucleolus,"
within the nucleoli of the cells of the pancreas of the hedgehog during hibernation, in which secretion had begun or had been in progress for some time.
In certain Protozoa Montgomery observed vacuoles situated usually at the
pole of the nucleolus nearest the nuclear membrane. As the nucleolus increased in size the vacuoles became larger and more numerous, usually fusing
to form one large vacuole. Montgomery concluded that vacuoles were extranuclear in origin and arose from the cytoplasm. While working with egg cells
of certain Metazoa he arrived at the opinion that small nutritive globules in
the nucleus, which originally were or came from yolk granules of the cytoplasm, penetrated the nucleolus and constituted the fluid vacuoles of the latter
structure. He called these vacuoles "assimilated nutritive globules." Occasionally he observed a vacuole which contained one or several small solid
bodies which stained like the ground substance of the nucleolus. These have
been termed "nucleololi." Montgomery believed that they were merely
loosened granules of the substance of the nucleolus which had come to be
within the vacuoles.
Macfarlane and his pupil Mann described nucleolini (granules within the
nucleolus) or nucleololi under the names of " endonucleolus" and "nucleolonucleus." Many structures described as nucleolini are in reality minute
vacuoles, which from their refractibility appear to be granules. Chambers,
working on the ova of Asterias, Arbacia, Echinarachnius, Cerebratulus, and
Fucus, described one or more vacuoles in the nucleolus. When the latter was
cut each part became round like a droplet. Abele noted vacuoles in the
nucleoli of the cells of Vicia amphicarpa, In an article by Goodpasture there
1
Submitted for publication October 15, 1937.
383
384
ROBERT C. PAGE, JAMES F. REGAN AND WILLIAM C. MACCARTY
is an illustration of a vacuolated nucleolus in a multipolar ganglion cell of a
rabbit which had been inoculated with the virus of herpes simplex.
Saguchi, working with pancreatic cells of Rana tcmporaria, described
minute granules in the nucleolus which stained brown to black with Cajal's
photographic method, after formalin fixation. He called them " argentophile
bodies" because of their affinity for silver salts. They could not be demonstrated by such fixatives as sublimate, formalin, trichloracetic acid, alcohol,
potassium bichromate and osmic acid, with various stains, but appeared only
as clear vacuoles. They occurred singly in some instances; in others several
were present. They were situated in most cases in the middle of the nucleolus. Where a large number of them were gathered together, the center of the
nucleolus often had an alveolar appearance. The granules were not infrequently seen near or closely applied to the periphery of the nucleolus. They
were not entirely homogeneous, but seemed to consist of a deeply staining
cortical layer and a more lightly staining internal part as seen in the Cajal
preparation.
Carleton, working on columnar epithelium of the eat's intestine, noticed
one or more intranucleolar granules when using Cajal's formol-silver nitrate
method for demonstrating the Golgi apparatus. He used the term " nucleolini" for these granules. In some instances each nucleolus contained as many
as five nucleolini but the vast majority of nucleoli had but one or two. The
fact that several nucleolini were sometimes found in a single nucleolus suggested that these bodies can undergo division. The nucleolinus in the frog
(Rana tcmporaria) was larger than in the cat; its shape was more variable;
and rarely were more than two nucleolini per nucleolus found. Carleton concluded that there seems to be little doubt that nucleoli are capable of multiplication and that the usual mode is by transverse fission. He thought it
probable that the fission of the nucleolus is shared by the nucIeolinus. The
latter was often seen to have a median constriction, and frequently two nucleolini were observed lying in the closest proximity within the nucleolus or
situated one toward each end of it. Carleton concluded further that there
does not seem to be any fixed stage in the process of nucleolar division at
which fission of the nucleolinus takes place. It frequently divided into two
within a nucleolus unprepared for multiplication. There seemed, nevertheless, to be a continuity of the substance of the nucleolinus during the division
of the nucleolus. The fact that in well impregnated pieces of epithelium,
when there were two nucleoli per nucleus, each contained a nucleolinus, was
strongly suggestive to him of the continuity of these bodies in daughter nuclei.
According to Farrerons the nucleolus is a homogeneous mass in which
argentophile spheres are found. When cells are stained with aniline dyes,
intranucleolar vacuoles are seen very clearly. There may be one or two and
these are very refractive; for this reason, they have been called the" refractive corpuscles of Lache." Their significance is unknown but some authors
identify them with the argentophile spherules of CajaI. Other authors believe that they are not solid corpuscles but are vacuoles, and have described
them as clear spaces varying in size and many times containing corpuscles
which have basophil reaction and which are of an excretory nature. Intravacuolar corpuscles have been described by numerous investigators. Far-
NORMAL AND NEOPLASTIC HUMAN TISSUE
385
rerons worked with brain tissue (Ammon's horn) which was fixed in formalin,
treated with zinc chloride to combat the reductive action of formalin, sectioned
in the frozen state, and stained with hematoxylin and eosin. He observed refractive corpuscles, which frequently were eosinophilic, in the interior of the
nucleolus. He also observed intranucleolar vacuoles which contained a
corpuscle. Marinesco observed small corpuscles in the interior of the nucleolus. Tello, with the aid of ultraviolet rays, found nucleolar vacuoles in
pyramidal cells of rabbits and human beings. These vacuoles appeared as
refractive, clear spaces of circular form. They were similar to those observed in stained cells.
More detailed information and a more extensive bibliography concerning
these structures may be obtained by consulting the articles cited.
FIG.
1.
PHOTOMICROGRAPH OF FRESH FROZEN SECTION SHOWING MAI.IGNA;I;T CELL WITH
INTRANUCLEOLAR BODY
(TERRY'S POI.YCHROME METHYLENE BLUE)
Courtesy of Dr. Wm. C. MacCarty.
METHOD OF STUDY
In the early part of this study numerous tissues-normal, neoplastic, and
inflammatory-which were removed surgically were studied microscopically
by means of fresh frozen sections (Wilson method) which were stained with
polychrome methylene blue (Terry) and by means of frozen sections which
were fixed with formalin and stained with hematoxylin and eosin. The observations in the preliminary report were all made on fresh frozen sections.
The following procedures were undertaken in order to study more in detail the morphology and chemical composition of intranucleolar bodies. Representative blocks were cut from 28 specimens (17 malignant tumors, 5 benign
tumors, 2 portions of normal tissue, and 4 inflammatory lesions) of tissue as
soon as they were brought from the operating room. One block was cut by
the frozen section technic and stained with polychrome methylene blue
(Terry). Another block was fixed with formalin and sections, which were
cut by freezing, were stained with hematoxylin and eosin. Four additional
blocks were cut from each specimen and fixed with the following solutions:
formalin, Zenker's solution, Helly's solution, and absolute alcohol.
386
ROBERT C. PAGE, JAMES F. REGAN AND WILLIAM C. MACCARTY
A portion of the block which was fixed with formalin was frozen, sectioned, and stained with scharlach R and sudan IV, while the remainder was
mounted in paraffin, sectioned and stained by the following methods: hematoxylin and eosin, Brown's modification of Gram's technic, a modified Galantha
silver impregnation method (formalin fixation instead of alcohol), and
Galantha's mucin method (11, 12).
The block of each specimen that was fixed with Helly's solution was used
to prepare sections which were stained by the Laidlaw technic (Pappenheimer
Q.
b
.1."".",.
~!~.""
'
t:fL~ ' I t '
~~'('~"
c
d
FIG. 2
a and b. Squamous-cell epithelioma of uterus, grade 4, showing variation in size and number
of intranucleolar bodies in nucleoli of same neoplasm (fresh frozen section; drawing X 1000).
c. Normal skin, showing nuclei containing one and two nucleoli, each with a single intranucleolar
body (fresh frozen section; drawing X 1000). d. Exophthalmic goiter after administration of
Lugol's solution; occasional intranucleolar body (fresh frozen section; drawing X 1000).
and Hawthorne). Sections of the blocks of tissue which were fixed in alcohol
were stained by the Best carmine technic. Sections prepared from blocks that
were fixed with Zenker's solution were stained with Giemsa's method. All
sections were carefully examined with the oil immersion objective.
The tissues studied were obtained from the following specimens: breast
(adenocarcinoma, chronic cystic mastitis, accessory breast and adenofibroma),
stomach (colloid adenocarcinoma, adenocarcinoma, benign pedunculate adenoma, gastric ulcer and duodenal ulcer), prostate gland (adenofibromatous
hyperplasia and adenocarcinoma), rhabdomyosarcoma of thigh, osteogenic
NORMAL AND NEOPLASTIC HUMAN TISSUE
387
sarcoma of femur, hemangio-endothelioma of the humerus, endometrium
(proliferative phase), squamous-cell epithelioma of thigh, melano-epithelioma
of rectum, adenocarcinoma of lung, papilloma of a rabbit (Shope virus tumor)
and normal skin.
RESULTS
Many names have been given by different observers to intranucleolar
bodies. The clear, non-staining areas or vacuoles within the nucleoli have
b·
d.
c
FIG. 3
a. Fibrocytes in a cystic ovary (fresh frozen section; drawing X 10(0). b. Papillary cystadenocarcinoma of ovary, grade 2 (fresh frozen section, drawing X 1000). c. Epithelial cells in
mucosa of chronically infected gallbladder (fresh frozen section, drawing X 10(0). d. Adenocarcinoma of breast, grade 3 (fresh frozen section, drawing X 10(0).
been called "assimilative nutritive globules," "refractive corpuscles," etc.,
whereas those which take the silver stain have received such designations as
" argentophile bodies" and" nucleolini." We do not intend to coin any new
terms, but for the purpose of discussion will designate the former as " refractive bodies" and the latter as " argentophile bodies."
In fresh frozen sections stained with Terry's polychrome methylene blue
the refractive bodies appear as white, unstained areas surrounded by nucleolar
substance which is stained dark blue (Figs. 1-6). They have a sharply defined border and are round or oval when small and few in number. When
they are large or when many are present they may appear flattened. In nor-
388
ROBERT C. PAGE, JAMES F. REGAN AND WILLIAM C. MACCARTY
mal tissues they occur infrequently and we have never seen more than one in a
nucleolus (Fig. 2 C, d). In cells of chronically infected tissue there are occasionally two refractive bodies in a single nucleolus (Fig. 3 c). In regenerating
epithelial cells of endometrium during the proliferative phase one or two refractive bodies are frequently seen (Fig. 4 a). In benign tumors the refractive bodies are small and are seen in only a few cells per field. When present
there are usually one or two in a nucleolus. When compared to those in normal tissues they are slightly larger and more frequent. In malignant cells,
refractive bodies are large and easily seen. They are present in many cells
per field, and from one to eight or more may be seen in each nucleolus. The
greatest numbers are found in cells of the most malignant tumors (grade 4).
Frequently they occupy practically the entire nucleolus and only a small
.•..•
...., A.,f,.:
.
........."
.•..~ .. ,
. ,;-or,
'. . ,. ;~.., . '.'.,::. . ·.e,;., .
-
.'~~~
(
.'
~> .,
. .'
.
'
,
..
1"M.~t'
•
',,1
"
'~.
{~.,
a.
~'.
'-:".
.f.'' ' '
e ·.
c
~
"\.
:~)
d
FIG. 4
a. Endometrium, proliferative phase, showing occasional single and double intranucleolar bodies
(fresh frozen section, drawing X 665). b. Squamous-cell epithelioma of hand, grade 2 j one
nucleus containing five nucleoli and intranucleolar bodies (fresh frozen section, drawing X 665).
c. Adenocarcinoma of uterus, grade 4; intranucleolar bodies giving alveolar appearance to nucleolus
(fresh frozen section, drawing X 665). d. Malignant brain tumor, medulloblastoma (fresh frozen
section, drawing X 665).
amount of darkly staining nucleolar substance is visible. This gives the nucleolus an alveolar appearance (Fig. 2 a, b; Fig. 3 b, d; Fig. 4 b, c, d; Fig. 5
a, b, c, d; Fig. 6 a, b, c, d). Occasionally one sees a darkly staining granule
within a refractive body.
In fixed frozen sections stained with hematoxylin and eosin, refractive
bodies are not seen as frequently or as easily as in fresh frozen sections stained
with Terry's polychrome methylene blue (Fig. 7). They are not as large or
as sharply defined in fixed frozen sections as in fresh frozen sections, and occasionally they have a slightly eosinophilic appearance. The relationship of
the number and size of refractive bodies in cells of normal tissues and of
benign and malignant tumors is the same as it is in fresh frozen sections
stained with Terry's polychrome blue.
NORMAL AND NEOPLASTIC HUMAN TISSUE
389
The intranucleolar bodies in paraffin sections stained with hematoxylin and
eosin appear smaller, are less frequently seen, and are more difficult to find
than they are in fresh and fixed frozen sections. Their outlines are hazy and
indistinct. The relationship as to size and number is the same as in the
preparations already described.
Brown's modification of Gram's stain was used to determine the presence
or absence of bacteria. In tissues which were stained by this method the
refractive bodies appeared approximately the same as they did in tissues which
were embedded in paraffin and stained with hematoxylin and eosin, but they
were seen less frequently. No bacteria were noted in the nucleoli. In sections stained for mucin, refractive bodies were occasionally seen, but no mucin
was observed in the nucleoli. Laidlaw's stain, which has been used to demon-
FIG. 5
a. Metastatic carcinoma in cervical lymph node from primary squamous-cell epithelioma of
lip, grade 2 (fresh frozen section, drawing X
grade 1 (fresh frozen section, drawing X 665).
and seven intranucleolar bodies (fresh frozen
colon, grade 3 (fresh frozen section, drawing X
665). b. Early adenocarcinoma of the stomach,
c. Adenocarcinoma of pancreas, grade 4; nucleolus
section, drawing X 665). d. Adenocarcinoma of
665).
strate inclusion bodies, failed to show their presence. Refractive bodies were
occasionally seen but presented their usual unstained appearance. No fat was
demonstrated in the nucleoli of frozen sections stained with scharlach Rand
sudan IV. An occasional refractive body was noted. In an attempt to determine the presence or absence of glycogen, sections were stained with Best's
carmine stain. A few intranucleolar bodies were seen in malignant cells but
no glycogen could be demonstrated in the nucleoli. Giemsa's stain did not
give additional information regarding these bodies. They were occasionally
seen.
Intranucleolar bodies are also demonstrable by silver precipitation methods (Carleton and Galantha). They appear as small, brown or black, round,
oval or rod-shaped granules. They are single or multiple. When large
numbers of granules are gathered together, clear areas often may be seen in
390
ROBERT C.
PAGE, JAMES
F.
REGAN AND WILLIAM
C. MAC CARTY
FIG. 6. PHOTOMICROGRAPHS OF FR~:SH FROZEN SECTIONS OF MALIGNANT TUMORS STAINED WITH
TERRY'S POLYCHROME METHYLENE BLUE, RETOUCHED BY ARTIST TO SHOW I:ooTRANUCLEOLAR
BODIES DISTINCTLY
a. Adenocarcinoma of breast, grade 3 (X 1105). b. Adenocarcinoma of breast, grade 4
( X 820). c. Adenocarcinoma of breast, grade 3 (X 1065). d. Adenocarcinoma of rectum, grade 2
(X 1065).
the center of the nucleolus, giving it an alveolar appearance (Fig. 8 a). With
Carleton's modification of Cajal's silver nitrate method of staining and an
aqueous solution of uranium nitrate as a fixative, these bodies were clearly
demonstrated. This method, however, did not produce as good sections as
did the modified silver technic of Galantha. The bodies appeared somewhat
smaller than when the Galantha method was used. In normal tissues from
NORMAL AND NEOPLASTIC HUMAN TISSUE
391
one to five argentophile bodies or granules are seen in practically all nucleoli;
the majority contain one or two. From one to five granules are also seen in
benign tumors but in the majority of nucleoli there are from three to five and
these are approximately the same size as in the normal tissue cells (Fig. 8 b,
c). Cells of malignant tumors contain many argentophile bodies. We have
counted as many as twenty-six in a single nucleolus; the usual number is from
six to twelve. There seems to be a relationship between the grade of malignancy and the number of argentophile bodies. These bodies are found in
the largest numbers in neoplasms of Broders' grades .3 and 4 (Figs. 8 a, d,
e, f).
COMMENT
By using formalin, absolute alcohol, Helly's and Zenker's solutions as
fixatives we were unable to demonstrate intranucleolar bodies with various
FIG.
7.
PHOTOMICROGRAPH OF FORMALIN-FIXED FROZEN SECTION, SHOWING MALIGNANT CELLS WITH
SIXGLE AXD MULTIPLE Il\'TRANUCLEOL\R BODIES (HEMATOXYLIN AND EOSIN)
Courtesy of Dr. Wm. C. MacCarty.
stains except as clear refractive vacuoles. This agrees with the work of
Saguchi on the pancreatic cells of the frog.
Some investigators are of the opinion that the argentophile bodies are
nothing more than small vacuoles in which silver has been precipitated at the
periphery. Cajal described argentophile bodies within the nucleoli of nerve
cells. He was of the opinion, however, that the refractive bodies were artefacts due to the reagents and to the autolytic phenomena which the nucleolus
suffers after its death before fixation.
We do not believe that the refractive bodies are artefacts. They are seen
with a high degree of constancy in both normal and pathologic tissue; they
are present in cells of tissues treated with many different fixatives and stains,
and they are seen, also, in fresh tissues, frozen, sectioned, and stained within
a few minutes after removal from the patient in the operating room.
Saguchi believed that the refractive bodies or vacuoles are the" negatives"
of the argentophile bodies. We are of the same opinion, though we have no
FlO. 8.
PHOTOMICROGRAPHS OF SECTIONS OF BLNIGN AND l\!1\J.lGNANT TUMORS STAlNW WITH
MODIFIED GALANTI1A SILVER TECIINIC
a. Adenocarcinoma of breast, grade 4, showing multiple argentophile bodies in nucleolus
(X 830). b. Adenoma of stomach showing several argentophile bodies in nucleoli (X 830).
c. Adenofibroma of breast showing several argentophile bodies in nucleoli (X 830). d. Adenocarcinoma of prostate gland, grade 3, showing many argentophile bodies in nucleolus (X 830).
e. Adenocarcinoma of prostate gland, grade 4, showing many distinct argentophile bodies in nucleolus (X 1320). f. Adenocarcinoma of breast, grade 4, showing large number of distinct argentophile bodies in nucleolus (X 1320).
392
NORMAL AND NEOPLASTIC HUMAN TISSUE
393
definite proof of this. The evidence for this opinion is that in cancer cells
the number of refractive bodies within the nucleoli is greater than in normal
cells. Certain other findings concerning argentophile and refractive bodies
in the nucleoli of cancer cells and of normal cells are relevant here. ( 1) A
greater number of argentophile bodies is found in cancer cells than in normal
cells. (2) On the other hand, the average number of argentophile bodies in
cancer cells is approximately double the number of refractive bodies in cells
of the same tumor. (3) The refractive bodies are larger in cancer cells than
in normal cells, whereas the argentophile bodies are the same size or, if anything, slightly smaller.
In an attempt to ascertain the nature of these refractive bodies, specific
stains were used. With the methods employed we were unable to obtain positive results. These bodies do not respond to tests for mucin, glycogen, fat,
bacteria, or inclusion bodies.
We have found intranucleolar bodies in all of the many and various types
of normal and pathologic human tissues we have studied. Nucleoli of normal tissues contain one and rarely two refractive bodies. In nucleoli of regenerative cells and cells of chronically inflamed tissue there are one to three.
Nucleoli of benign tumors contain one to three refractive bodies, whereas the
nucleoli of malignant tumors contain one to thirteen. In normal tissues the
refractive bodies are very small and can barely be seen with the oil immersion
objective in a few cells per field. They are very likely to be overlooked by the
casual observer or by one unacquainted with their appearance. They are
best seen in fresh frozen sections. With all forms of fixation they appear
smaller and are less distinct. Argentophile bodies are found in practically
every nucleolus in cells of normal and pathologic human tissue. In nucleoli
of normal cells an average of one or two bodies is seen and in nucleoli of regenerative cells an average of one to five. In nucleoli of benign tumors there
are usually three or four and in nucleoli of malignant tumors there are six to
twelve. The more malignant the tumor, the greater the number of argentophile bodies in the nucleoli. In many nucleoli the bodies are so close together that they have the appearance of a mass.
We do not know the nature of these structures or their significance. It
may be that they are concerned with the altered metabolism of rapidly growing cells of malignant tumors. They may be of some value in the differentiation of normal and malignant cells.
We present these findings with the hope that they will stimulate further
study of that neglected structure-the nucleolus.
SUMMARY
Intranucleolar bodies have been found in cells of normal tissues and benign
and malignant tumors by the use of general and specific stains. The intranucleolar bodies that appear as unstained areas or vacuoles are called "refractive bodies"; those which take the silver stain are called " argentophile
bodies." Refractive bodies are best seen in fresh frozen sections stained with
polychrome methylene blue (Terry, 23, 24). Refractive bodies are greater
in number and larger in cells of benign and malignant tumors than they are in
394
ROBERT C. PAGE, JAMES F. REGAN AND WILLIAM C. MACCARTY
normal cells. Argentophile bodies are present in greater numbers in cells of
benign and malignant tumors than they are in normal cells. The more malignant the neoplasm, the greater the number of intranucleolar bodies.
NOTE: We are greatly indebted to Miss K. McKay for the drawings and to Miss E.
de Galantha for technical assistance.
REFERENCES
1. ABELE, KARLIS: Compt. rend. Soc. de bioI. 92: 887-888, 1925.
2. AGASSIZ, L.: Quoted by Montgomery (IS).
3. BALBIANI, E. G.: Quoted by Montgomery (18).
4. BOHM, A. A.: Quoted by Montgomery (1S).
5. BRODERS, A. c.. J. A. M. A. 74: 656-664,1920.
6. CAJAL, RAYMOND: Quoted by Farrerons (10).
7. CARLETON, H. M.: Quart. ]. Micros. Sc. 64: 329-343, 1920.
S. CARLIER, E. W.: J. Anat. & Physiol. 30: 334-346, 1896.
9. CHAMBERS, ROBERT, JR.: Am. J. Physiol. 43: 1-12, 1917.
10. FARRERONS, F. X.: Bull. de la lnst. Catalan a de historia nat. 8: 26-36, 1925.
11. DE GALANTHA, ELENA: Am. J. Clin. Path. 5: 165-166,1935.
12. DE GALANTHA, ELENA: Am. J. Clin. Path. 6: 196-197, 1936.
13. GOODPASTURE, E. W.: Am.]. Path. 1: 1-9,1925.
14. LEYDIG, F.: Quoted by Montgomery (18).
15. MACFARLANE, J. M.: Quoted by Montgomery (IS).
16. MANN, G.: Quoted by Montgomery (18).
17. MARINESCO: Quoted by Farrerons (10).
18. MONTGOMERY, T. H., JR.: J. Morpho!. 15: 266-582, 1898-99.
19. PAPPENHEIMER, A. M., AND HAWTHORNE, J. J.: Am.]. Path. 12: 625-633, 1936.
20. REGAN, ]. F., PAGE, R. c., AND MACCARTY, W. c. Proc. Staff Meet. Mayo Clin. 12:
257-259,1937.
21. SAGUCHI, S.: Am. J. Anat. 26: 347-421, 1919-20.
22. TELLO: Quoted by Farrerons (10).
23. TERRY, B. T.: ]. Path. & Bact. 30: 573-575, 1927.
24. TERRY, B. T.: J. Lab. & Clin. Med. 14: 519-531,1929.
25. WILSON, L. B.: J. A. M. A. 45: 1737,1905.