The Golgi Apparatus in the Thyroid Gland of Amphibians, in its

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The Golgi Apparatus in the Thyroid Gland of
Amphibians, in its relation to
Excretion Polarity.
By
Eduard Uhlenhuth,
University of Maryland, Medical School.
With Plate 36.
IN 1923 Nassonov reported observations which suggested that
the Golgi apparatus is concerned in the elaboration of the
secretion droplets of gland-cells; he therefore concluded that
the Golgi apparatus is the secretory organ of the cell.
As Bowen (1929) has repeatedly and correctly emphasized,
it is necessary to distinguish carefully, in the activity of gland-
cells, between two processes, between secretion and excretion.
The former designates only that part of the activity of the cell
which ultimately leads to the manufacture of the secretion
products, while the latter relates to the removal of the secretion
products from the cell. Even assuming that the Golgi apparatus
plays an essential role in the manufacture of the secretion, such
a relation cannot, without further evidence, be considered as
indicating that excretion likewise is controlled by the Golgi
apparatus.
Those who support their claims for a participation of the Golgi
apparatus in the excretion of the secretory products from the
cell by referring to Nassonov's work do not fully appreciate
the bearing of Nassonov's discoveries. This author has not only
not demonstrated a relation between Golgi apparatus and
excretion, but has emphasized specifically that the mechanism
of excretion of the secretory products is not dependent on the
Golgi apparatus (1923, p. 179). In his first article (1923) he
states, p. 179: 'Der Mechanismus der Ausfuhrung des Sekretes
aus dem Zellkorper . . . hangt augenscheinlich nicht vom
Binnennetz ab.' Concerning the special problem of the excretion
polarity Nassonov made reference to this in only one place of
616 EDUARD UHLENHUTH
his first article (1923, p. 161). He says: 'Diese Orientierung des
Binnennetzes stets in der Eichtung zur Miindung des Sekretions-
stromes hin ist fur mich besonders interessant, da sie einen
weiteren indirekten Beweis fiir die Beteiligung des Binnennetzes
an der Sekretion liefert.' In none of his later articles, however,
has Nassonov made any further reference to this problem.
Going through the literature, in an attempt to utilize the
position of the Golgi apparatus, at either the apical or basal cell-
pole, in the interpretation of the secretion physiology of the
cell, one is struck by the complete absence of any experimental
data or any other well-founded evidence which would prove that
the Golgi apparatus migrates from one pole to the opposite when
the excretion polarity so changes, or that the excretion polarity
of a cell is determined by the position of the Golgi apparatus.
Nevertheless attempts are not wanting of using the position of
the Golgi apparatus as an indicator of the excretion polarity.
Courrier and Reiss (1922) seem to be the first ones who,
without presenting any evidence to substantiate their con-
clusions, infer that, in the parathyroids of young cats, the pole
at which the Golgi apparatus is located is that at which excre-
tion takes place. In a subsequent article Reiss (1922) employs
the same criteria in the case of the hypophysis.
That the position of the Golgi apparatus may sometimes be
reversed was noticed by the earlier students of the Golgi
apparatus, including its discoverer. Great caution, however, was
observed by them in formulating an interpretation. Golgi, in
1909, studied the reticular apparatus in the mucous cells of the
gastric mucosa and found a characteristic reversal of the position
of the apparatus; he attributed it, tentatively, to the action of
internal mechanical forces. At any rate it was obvious, even at
that early date, that the Golgi apparatus could hardly have
anything to do with secretion polarity, since the cells in question,
so far as known, excrete always through the same pole.
In 1926, Bowen for the first time warned against premature
conclusions regarding the position of the Golgi apparatus, and
showed that in the light of comparative studies there is little
ground for assuming that the position of the Golgi apparatus
corresponds to the excretion polarity of the cell. He found that
GOLGI APPARATUS IN THYEOID 617
the Golgi apparatus in the cells of the epididymis and vas
deferens of the cat may have either an apical or a basal position,
although the cells of both of these organs excrete only in an
apical direction. Eeferring to the role of the Golgi apparatus in
the secretory activity of the cell, he says (p. 409): '. .. it is clear
that the reversal is not associated with any particular functional
phase of the cell, for all stages in the secretory cycle are found
with reversed Golgi apparatus.' And in relation to the excretory
polarity he expresses himself in the following terms: 'I have
never seen anything which suggests that the reversal of polarity
as measured by the position of the Golgi apparatus has any
relation to the direction in which the secretory products are
extruded' (p. 409).
Similar observations have been made in many other cells
(Bowen's article of 1926 contains a brief review of the literature)
which so far have not been suspected of excreting through the
basal pole. Among the articles pertaining to this matter may
be mentioned one by Giroud (1928). This investigator studied
the cells in the intestinal wall and in the kidney tubules of
amphibians; in the latter cell-type the position of the apparatus
seems to vary with the species. Giroud attempts to explain the
reversal of the position of the Golgi apparatus in the cells of
the two organs studied by him, in terms of pressure changes
within the cell. It has been mentioned above that Golgi too
suggested mechanical forces as the cause of a reversed position
of the apparatus. Bowen (1926), however, considers the role
of mechanical forces, in the migration of the Golgi apparatus,
as doubtful, at least in the cells of the epididymis (p. 409).
Weatherford, in a recent article (1929), reports investigations
of the Golgi apparatus in the cells of the mammary gland. The
cells of this organ, according to our present knowledge, excrete
always at the apical pole. Nevertheless, the Golgi apparatus,
during certain secretory phases, migrates away from its strictly
apical position and comes to lie, in part, at the basal cell-pole.
THE GOLGI APPARATUS IN THE THYROID GLAND.
The Golgi apparatus in the epithelial cells of the thyroid gland
has been described by a considerable number of investigators.
618 EDUARD UHLBNHUTH
It should be mentioned, however, that heretofore the thyroid
gland was not a suitable object for the study of the physio-
logical role of the Golgi apparatus, since it has been only
recently that a fairly correct picture of the secreting thyroid
cell has become known. But even at present the structures of
the thyroid cell are still under discussion; the physiological
interpretation of the cytology of the thyroid is still in a stage
in which it needs to draw upon other well-established facts for
analysis rather than to be used as supporting evidence in the
investigation of other unsettled problems. In particular the
direction of the secretory current at different stages of the
secretion process is still a much-debated question; it would be
unwise to apply any of the current hypotheses relating to the
excretion polarity of the thyroid cell in the formulation of a
theory postulating a relation between the excretion polarity of
the cell and the position of the Golgi apparatus.
Cowdry (1922) was probably the first to study the position
of the Golgi apparatus in relation to the excretion polarity of
the thyroid cell. He found that in the thyroid of the guinea-pig,
in 1 cell out of every 500, the Golgi apparatus lies at the basal
side of the nucleus. On the strength of this observation he
claimed that the thyroid cells, under certain conditions, may
reverse the direction of their excreting activity and excrete
through the basal pole, but failed to offer any evidence showing
that there exists a relation between the position of the Golgi
apparatus and the excretion polarity of the cells. His article
is nevertheless often quoted as if he had presented evidence
proving that the position of the Golgi apparatus is reversed
whenever the secretion polarity of the cell is reversed.
Bowen, in his article referred to above (1926), takes a very
definite stand with regard to Cowdry's work. He says (p. 412):
'Cowdry's statement that "Apparently in the thryoid gland
alone is there variability in the position of the reticular material,
and in the thyroid gland alone have we clear evidence of
physiologic reversal in the direction of secretion" is thus hardly
in accord with the facts I think it unsafe to use this particular
case as evidence pro or con for any theory of secretory activities.'
More recently Ludford and Cramer (1929) investigated the
GOLGI APPARATUS IN THYROID 619
position of the Golgi apparatus in the cells of the thyroid gland.
In the thyroid of rats, the activity of which was raised to a
maximum by exposing the animals to low temperature, the
Golgi apparatus was always found at the apical pole. From
this observation the authors unfortunately concluded that a
normal thyroid, even when activated, excretes always through
the apical pole, into the lumen. In exophthalmic goitre glands,
especially of one white mouse, the Golgi apparatus in a number
of cells occupied a basal position; Ludford and Cramer take this
as an indication that in this pathological condition the excretion
polarity of the cells is reversed. Far-reaching conclusions upon
the causes of the toxic effects of the thyroid hormone in exoph-
thalmic goitre are based upon this supposed difference between
the excretion polarities of normal and exophthalmic goitre
thyroids. It will be shown presently that these conclusions are
premature, as the interpretation made by Ludford and Cramer
is obviously incorrect.
In 1926 Schwartzbach and the author of this article (34 and
35) found that a substance can be extracted from the pre-
hypophysis of cattle, which possesses the specific property of
activating the thyroid cells to a maximum degree, an observa-
tion which has since been verified in further studies (see under
Schwartzbach, Pigge, Uhlenhuth, Zurawski) made in our labora-
tory and has been confirmed by several other investigators
(Paal, 1931; Adams, 1932; Loeb, 1929; Aron, 1930). Similar
results may be obtained by grafting the anterior lobe (Ingram,
1928; Grant, 1930). Owing to this technique it is possible to
study the secretion mechanism of the thyroid cell with an
increased degree of accuracy. At present new concepts of the
secretory activity of the thyroid are in progress of formation.
Some of the most important problems centre around the
question, whether the thyroid cells are capable of excreting not
only through the apical, but also through the basal pole.
Madeleine Grant (1930), on the basis of indirect evidence, has
declared herself in favour of a bipolar excretion mechanism.
Zurawski and Uhlenhuth (1932), at a meeting of the American
Anatomical Association, demonstrated histological slides of the
thyroid of the Californian newt (Tirturus torosus), stimulated
620 EDUARD UHLENHUTS
by injections of thyroid activator, which lend themselves to
the interpretation that during the process of colloid release
(from the follicle into the blood sinusoids) the thyroid cells
excrete the liquefied cell-content through the basal pole. Never-
theless it appears to the writer of this article that in spite of its
highly suggestive character this evidence may not fully satisfy
a critical student of the secretion process of the thyroid, and
needs confirmation by further work in progress in our laboratory.
Yet the interpretation of the facts to be reported presently
hinges entirely on the question of the secretory polarity peculiar
to the thyroid cell in its different secretory phases. For this
reason it is necessary to state that, while the lack of a reversal
of the position of the Golgi apparatus in the thyroid cell to be
shown in this article is a valuable observation, the interpretation
given it here may be only of a temporary nature; its correctness
depends entirely on the correctness of the interpretation which
we have given to our histological pictures suggestive of basal
excretion.
The investigators whose work will be reviewed in the following
paragraphs hardly concerned themselves with an analysis of
the excretion polarity of the thyroid cell. For this reason the
interpretation of their observations will have to be weighed
with an even greater measure of caution.
Ingram (1928), using the grafting technique, was the first
to show that in the thyroid stimulated by the thyroid activator
the Golgi apparatus takes invariably an apical position. His
work is open to criticism, however, as he did not follow the
development of the secretion processes in chronologically
arranged series, nor did he state that, at the time of the death
of the animals, there were signs of basal excretion; his illustra-
tions do not convey the impression that the cells were really
active.
• More recently Wagschal (1931) in Max Aron's laboratory,
using our technique of stimulating the thyroid by the thyroid
activator, found that in guinea-pigs, too, the Golgi apparatus is
located invariably at the apex of the thyroid cells. The cells
illustrated in his article do not, however, appear to be highly
activated. Moreover, neither in Ingram's nor in Wagschal's
GOLGI APPAEATUS IN THYEOID 621
illustrations does the Golgi apparatus present the structure
which it does have, according to the work described here, in
really active cells.
At a meeting of the American Anatomical Association (1932)
the writer discussed and demonstrated preparations of the
thyroid of amphibians, stimulated to a high degree of function by
injections of thyroid activator, and impregnated by Nassonov's
osmic acid technique. In no case was the Golgi apparatus found
to occupy a basal position. At the same meeting Severinghaus
(1932), in discussing his work on the histological differentiation
of the cells of the hypophysis, mentioned that he had made
similar observations in the thyroids of warm-blooded animals,
activated by the same technique.
In this article the results presented in the preliminary paper
will be discussed in greater detail and data serving as evidence
in support of the conclusions will be submitted.
MATERIAL AND METHODS.
The adult California newt, Triturus torosus, was used
in the experiments.
As in previous experiments the anterior-lobe powder was pre-
pared in our own laboratory. The hypophyses were collected by
ourselves (in the slaughterhouse), the anterior lobes were
separated from the posterior lobes in not more than 40 minutes
after the death of the steers, taken home, cut with scissors into
small pieces, dried by an electric fan, ground to powder and
desiccated in the CaCl desiccator.
2
For injection the powder was extracted, by boiling slightly,
in an 0-5 per cent, solution of glacial acetic acid in a 30 per cent,
frog Ringer solution; the infusion was filtered and the filtrate
neutralized against lithmus paper by a N/5 NaOH solution to
a slightly acid end-point. After neutralization 0-5 c.cm. of the
liquid contained the extract of 100 mg. dried powder.
The injections were made every other day, into the peri-
toneal cavity. 0-5 c.cm. per animal was used at one injection.
The animals were always killed one day after the last injection.
The thyroids were removed in chloretone anesthesia, under
622 EDUARD UHLBNHUTH
30 per cent, frog Einger and dropped immediately into the
fixative.
For fixation two different methods were employed. In each
case the left thyroid was fixed in Zenker-glacial (without
NaSC>4); the right gland was fixed and impregnated with osmic
2
acid after the Kolatchev technique as modified by Nassonov.
For impregnation the thyroids were left in the osmic acid for
two days, at approximately 35° C. Ingram (1928) had found
two days just the right length of time to get satisfactory im-
pregnation in the thyroids of tadpoles; the same period of
immersion gave very excellent pictures of the reticular apparatus
in the salamander thyroid. For embedding paraffin was used.
The sections were cut at 7 microns.
THE GOLGI APPARATUS OF THE NORMAL AMPHIBIAN
THYROID, IN THE BESTING CONDITION.
In a recent article Charipper (1930) published a study of
the Golgi apparatus of the thyroid cells of the perennibranchiate
salamander Necturusmaculosus. In young specimens he
found the reticular apparatus to consist of a number of isolated
osmiophilic bodies, while later in the life of the individual it has
the shape of a network located at the apical side of the nucleus.
Apparently the Golgi apparatus of the amphibian thyroid shows
the same changes during the development of the individual and
of its organs as have been described by Da Fano (1922) for the
Golgi apparatus of the mammary gland of mammals. A similar
transformation from a stage of isolated pieces into a network is
known to take place during cleavage of the fertilized egg and
during development of nerve-cells (for a review of the literature
see Jacobs, 1927). A feature characteristic apparently for
Necturus was described by Charipper; the reticulum sends
a long filament to the apical surface of the cell where it ends in
a group of granules.
While the species used by Charipper is one which not only
does not metamorphose spontaneously, but has lost the ability
of responding with metamorphosis upon injections of thyroid-
hormone, the species Triturus torosus employed in our
experiment is one which undergoes normal metamorphosis.
GOLGI APPARATUS IN THYROID 623
Only one animal (a 3 of the series CCCXXXIII) proved suit-
able for the study of the Golgi apparatus in a truly dormant
thyroid gland. This animal represents the control of the experi-
mental animal CCCXXXIII b 12, to be described later on. The
newts of this series had been received from Oregon, in August
1931, at a season at which the thyroids of the newts are com-
pletely dormant; before being experimented on they were kept
in the laboratory for a period of nearly three months, a procedure
which in our experience ensures a thoroughly inactive gland.
The thyroid cells of this control newt were low cuboid or
flat, and did not contain either colloid droplets or Andersson
vacuoles. The intrafollicular colloid showed only few, small
vacuoles. The Golgi apparatus occupied an extreme apical
position (fig. 1, PI. 36). It consisted of an oval mass of a sub-
stance staining much darker than the rest of the cytoplasm.
In this mass (or possibly on it—a question which I will not
attempt to discuss here) were found deeply blackened bodies,
of the shape of loops, filaments, or large spherical or irregular
granules. This composition of the apparatus which will be seen
in many other illustrations of cells in practically any one of
the secretory phases, reminds one greatly of the differentiation
of the apparatus into true Golgi substance and idiosomatic sub-
stance described in the male germ-cells of invertebrates by
Bowen (1922) and by Beams and King (1932) in the nerve-cells
of Orthoptera. Jacobs (1927) states that this structure was
found so far only once, by Morelle, in vertebrates, and seems
inclined to doubt its existence in vertebrates.
In the resting thyroid cell the apical Golgi network does not
always represent the only element blackened by osmic acid.
As is shown in fig. 1, PI. 36, the cells contain, in addition to the
network, a varying number of greyish granules which at present
I consider of mixed nature representing mitochondria and
ordinary cell granules, furthermore a few deeply blackened
granules, often of irregular shape and of larger size than the
greyish granules. If these latter granules are a part of the Golgi
apparatus it would appear that even in the resting thyroid cell
the Golgi substance is not strictly condensed into one compact
network.
NO. 304 s s
624 EDUARD UHLENHUTH
THE GOLGI APPARATUS IN THE NORMAL ACTIVE THYROID
GLAND.
In a second series (CCCXXXIV) the conditions are of greater
complexity. For years we have observed in our laboratory that
adult newts received from Oregon during the winter months may
show highly active thyroids soon after arrival in the laboratory.
The newts used in series CCCXXXIV arrived in the laboratory
January 4, 1932, and exhibited signs of high sexual activity.
On the following day several pairs were found in copula. This
state of sexual activity, however, passed off in a week and the
animals exhibited a normal behaviour.
The animals of the series CCCXXXIV (a I to 4 and a 6 to 9)
to be discussed in this chapter represent the controls of the
animals CCCXXXIV b 2, 4, 6, and 7, which were injected with
thyroid activator and will be discussed later on. The controls
a 1-4 were not injected at all; the controls a 6-9 received injec-
tions of muscle extract prepared exactly like the anterior-lobe
extract. The muscle extract had no effect upon the thyroid
gland, as we have seen in previous articles.
Thyroids of a copulating male and female (a 1 and a 2), killed
in the act of mating one day after arrival, were found, contrary
•to expectations, in an entirely inactive state, representing the
extreme of a storage type. The follicles were of very consider-
able size and contained colloid which proved unusually hard,
when the specimens were sectioned for histological purposes;
the cells were greatly flattened out. Only Zenker and Champy
preparations were made. In both cases Andersson vacuoles were
absent, but in the thyroid of the female large numbers of intra-
cellular colloid droplets were found.
Two other animals (a 8 and a 4) were killed, one on the second,
the other on the third day after arrival. Their thyroids showed
a markedly increased activity. Especially conspicuous were the
large numbers of vacuoles in the intrafollicular colloid. Again
only Chmapy and Zenker preparations were made. In the male
the cells contained now some few Andersson vacuoles, but no
colloid droplets. In the female the number of Andersson
vacuoles was greater, and a moderate number of intracellular
colloid droplets was present.