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23. Four cells of the granulosa of the same specimen, showing ring-like vacuoles in the cytoplasm like those seen in lutein  
23. Four cells of the granulosa of the same specimen, showing ring-like vacuoles in the cytoplasm like those seen in lutein  


cells. Mallory's stain. X600.  
cells. Mallory's stain. X600.
 
 
 
==CONTRIBUTIONS TO EMBRYOLOGY, NO. 6.==
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM OF THE
HUMAN EMBRYO.
 
 
 
By Charles R. Essick.
With three plates.
 
 
 
95
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM OF THE
HUMAN EMBRYO.
 
 
 
By Charles R. Essick.
 
 
 
INTRODUCTORY.
The human embryo, during the seventh week of development, possesses two bilaterally
symmetrical cavities in the substance of the corpus striatum. Since one lies nearer the
midline than the other, I have called them cavum mediale corporis striati and cavum
laterale corporis striati. The former is deeply placed in the striate body, while the latter
occurs nearer the surface of the brain, at times separated from the pia mater by a layer of
nervous tissue only a single cell in thickness. In all of the embryos in the collection of the
Carnegie Institution of Washington, ranging from 15 to 20 mm., crown-rump measurement,
one or both o: these cavities are present, while above these measurements they appear with
diminishing frequency; so that in all of the specimens under 15 mm. (measurement in for-
malin) and all over 24 mm. evidences of them are lacking.
 
Undoubtedly the ephemeral character of these cavities and their similarity to artifacts
account for the fact that they have heretofore escaped notice. The study of human
embryological material can not always be followed in specimens that have been fixed alive ;
more often one must be content with tissue that has begun to macerate. It is very natural,
then, to regard a rent in tissue as a fault in technique, especially in the central nervous sys-
tem, where shrinkage is one of the most difficult things to avoid in preparing and mounting
serial sections.
 
THE CELL-CONTENT OF THE STRIATE CAVITIES.
Inasmuch as the identification of both mesial and lateral cavities is based on their
contents, it may be well to describe the peculiar cells, foreign to the nervous tissue, which
occupy both cavities in large numbers whenever found in the striate body. They must be
regarded as the best proof of the normal occurrence of spaces in the human brain. From
the moment one of these striate cavities makes its appearance as an irregular break in the
continuity of the nervous tissue, until its complete disappearance, it is inhabited by large
amceboid cells having the power of phagocytosis. Study of the whole embryo and its
membranes shows that these cells are not peculiar to the cavities; for the same cellular
elements may be seen in the mesenchymal spaces, especially when of loose texture. Enor-
mous numbers may be found in the younger chorionic villi; along the umbilical cord many
wander near the surface; multitudes are present everywhere in the loose mesenchyme
around the central nervous system, especially near the base of the fore-brain; they occur
with less frequency along the aorta and in the mesentery.
 
These widely distributed cells have been figured by many observers— usually in con-
nection with the formation of the blood. Hofbauer, Grosser, and Minot have illustrated
the cells in the chorionic villi of human embryos of the first months of development. Hof-
bauer (5, p. 28) first called attention to specific round cells appearing in the human placenta
toward the end of the fourth week of pregnancy. Describing in detail these "vakuolaren
Zellen," he called attention to the many points of similarity to the plasma cell— i. c, the
eccentric nucleus with its rich chromatin network and its surrounding clear zone.
 
 
 
98 TRANSITORY CAVITIES IN THE CORPUS STRIATUM.
 
According to Grosser (3, p. 224) : "Regel-massig finden sich in Zottenstroma bei Eiern
des ersten Schwanger-schaftsmonate, grosse, protoplasma-reiche Zellen, auch die in letzter
Zeit wieder Hofbauer aufmerksam gemacht hat, deren Bedeutung aber noch unklar ist."
 
These two authors are at a loss to explain the existence of the cells, while Minot (8,
p. 498 ff.) regards them as erythrocytes which have wandered into the mesenchyma, and,
remaining there, have swollen by imbibition and are undergoing degeneration by vacuoliza-
tion of their protoplasm. In figure 361 this author has illustrated "degenerating blood-cells"
in the chorion of embryo 350 — an embryo included in this study. Such an interpretation
finds little or no support from my observations. The vacuolization of the protoplasm sug-
gests degeneration, but such an hypothesis is overwhelmed by many other factors pointing
to a very active life — i. e., mitosis, phagocytosis of foreign material, evidences of amoeboid
movement, and lack of hemoglobin. Then, too, his figure 361 gives a false impression of
the relative size of a normal erythrocyte and these large phagocytic cells (cf. figs. 13, 15,
18, 22, and 31). The former may easily be accommodated in the body of the latter, and
only the largest red cell that one can find shows any approach to the size of the phagocyte.
Most observers have regarded these colorless cells as the ancestors of the blood elements, at
least whenever they are found in the body of the embryo itself. O. van der Stricht (11)
includes under the class of phagocytes "leucoblasts" which correspond exactly to these cells,
so widely distributed through the whole ovum. Saxer (10) derives them from the common
vascular and blood anlagen, which he differentiates from connective-tissue elements. From
these primary "Wanderzellen" of Saxer descend a series of cell-forms, including giant cells,
wandering cells of I, II, and III orders, erythrocytes, and colorless cells. The Wander-
zellen persist into adult life and continue their blood-forming powers in the bone marrow
and adenoid tissues.
 
Maximow (6) holds that the embryonic ancestor of this cell is the mesenchyme out of
whose network it is gradually separated to become a free-moving cell. With the exception
of the extremities and gill-arches, he finds these "Wanderzellen" everywhere in the loose
mesenchyme, "allermeisten sich in Kopfmesenchym in der Nahe der Gehirnwand, vornehm-
lich an ihrer ventrolateralen Seite befinden."
 
For a long time, morphologically similar cells have been recognized in the adult as
playing an important role in inflammation. It has recently been shown that these probably
have no relationship to the normal blood elements. Metchnikoff (7) has given the cells
of this class the name of "macrophage." This term has been adopted here because of its
application by recent observers' to a group of cells normally inhabiting the connective
tissues, serous cavities, and hematopoietic organs. The remarkable peculiarities of the
adult cells characterize them even in the very young embryo. This will be seen from the
following description, for although they are wholly unlike the young and incompletely differ-
entiated tissue in which they are found, they correspond with striking exactness to those of
the adult. A very clear physiological relationship between the widely distributed adult cells
has been demonstrated by Dr. Evans (2) by means of vital stains; his results have led me
to include these cells in the same class because of their morphological characteristics.
 
While Hofbauer's use of stains is hardly analogous to their introduction as vital dyes,
it is interesting to note that by teasing living chorionic villi in solutions of neutral red, he
found (p. 124), "Wahrend alles (darunter auch das Syncytium) ungefarbt blieb oder einen
leichten Stich in Rosa annahm, treten die ' vakuolaren' Zellen der Zotte tief saturiert gefarbt
und beherschen die Bildflache." Plato (9) showed that cells engaged in phagocytosis are
particularly prone to stand out brilliantly in supravital stains because of the deep tingcing
of their phagocytized contents. Such evidence would account for Hofbauer's finding.
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM. 99
 
Several characteristics, easily recognized, permit the inclusion in the same tissue class
of the large mononuclear cells found so extensively distributed in the membranes of the
embryo, in the umbilical cord, in the mesenchymal spaces, and in the cavities in the corpus
striatum.
 
The average cell-body of the younger embryo measures from 10 to 13/t, while in the
older specimens it ranges from 10 to 19/x. This increase in size accompanies roughly the
growth of the embryo, although there are always smaller cells belonging to this group
(figs. 19, 25, 30). Thus it may be seen that the largest red cell is several times smaller
than the largest macrophage, and the former may be easily accommodated inside the latter.
Comparison of the measurement of many cells from chorion and cavum corporis striati
shows them to be of the same size, when one is fortunate enough to get the membranes and
embryo mounted on the same slide; even when mounted separately there is very little
discrepancy in favor of cells in either position.
 
The shape of the cell depends largely on its environment. When free it tends to assume
a spherical form (figs. 13, 17, 18, 20, 21), but in places where it can find support it sends
out long processes which are undoubtedly pseudopodia (figs. 15, 22). To obtain speci-
mens of cells with extended pseudopodia the fixation must be prompt; failure to procure
such pictures of moving cells is best explained on this basis. The shape is often irregularly
oval, due to the ingestion of foreign bodies, particularly erythrocytes (figs. 26, 27). At
times the macrophages grow in clumps of from 20 to 50, forming a mulberry-like mass.
This may occur in either medial or lateral cavity of the corpus striatum, and is well illus-
trated in figure 12 for the cavum laterale. Here the cells have becope flattened by the
pressure of their neighbors and in section seem to be forming a loose membrane the elements
of which are polygonal in outline.
 
The cells have a limiting membrane, very thin but definite; although it is almost
impossible to defend the view that the phenomenon illustrated in figure 12 is not an instance
of protoplasmic bridges connecting several cell-bodies. Large numbers of cells are extremely
vermcose (figs. 23, 25, 26). Examination of the cavities where these cells are found
reveals a great deal of debris simulating protoplasm. This is undoubtedly coagulated
proteid which has accepted the counterstain and has precipitated on the surface of the
macrophages. Figures 13, 18, 19, and 20 represent more faithfully the delicate cell-mem-
brane when the cell has withdrawn all of its pseudopodia.
 
The protoplasm is finely granular and is delicately arranged throughout the cell-body
in anastomosing strands which give an appearance of lacework. These trabecular vary
greatly in thickness and surround vacuoles within the cytoplasm. In the younger cells
these vacuoles are usually small, but at times they may be larger than the cell-nucleus
itself. Where the protoplasmic strands reach the cell-membrane they reinforce it in an
irregularly shaped mesh, such as may be seen in the illustrations of cells from chorion or
brain. This appearance is brought about by the extreme vacuolization which extends
throughout the whole cell, and as a result the finely granular protoplasm is heaped up in the
interstices between the vacuoles and the cell-wall. Great numbers of macrophages may be
found with foreign material included within their cell-bodies; the power of phagocytosis is
one of the most interesting phenomena exhibited by these cells. Although cells containing
ingested matter are rarely found in the chorionic villi, the cells everywhere in the body of
the embryo may contain phagocytized matter. The erythrocyte forms the foreign material
most frequently recognized within the phagocytes. An extravasation of red corpuscles
in the young embryo has occurred in most of the material studied; this is probably due to
the fact that it was subjected to high pressure in abortion. The presence of large numbers
 
 
 
100 TRANSITORY CAVITIES IN THE CORPUS STRIATUM.
 
of erythrocytes free in the tissue-spaces is not unfortunate, since they have stimulated these
macrophages to unusual activities whenever it occurred in their neighborhood. Embryo
406 furnishes many examples of red cells ingested by these large mononuclears in the cavi-
ties of the corpus striatum. Figure 26 is particularly interesting, as it illustrates a remark-
able phenomenon, namely, the division of a cell with a large mass of foreign material in its
cytoplasm. One of the asters is found on the edge of the cell, while the other' lies beneath
the ingested nucleated red blood-cell, and could not be drawn without confusing the picture.
Figure 27 shows the beginning digestion of the erythrocyte. Here the large macro-
phage from the cavum laterale corporis striati contains two smooth spheroidal bodies,
stained decidedly pink, yet not as deeply colored as the erythrocyte contained in the
dividing cell (fig. 26). Erythrosin has a marked affinity for cells containing hemoglobin,
and it here reveals these inclusions as two homogeneous globules. There is no evidence
of a nucleus in either of these two hemoglobin-containing fragments. Embryo 74 contains
cells which show the last stages of erythrocyte digestion (fig. 28). Here the macrophages
contain a few granules of brown pigment— not very unlike the large phagocytic cells known
as "Herzfehlerzellen," in the sputum of patients suffering from chronic passive congestion
of the lungs. It is very certain that long after the embryonic heart has stopped beating
and erythrocytes have been forced out of the capillaries, these huge amoeboid cells continue
to be actively engaged in the ingestion of foreign bodies. Even up to the time of fixation,
apparently, these cells are functioning, if mitotic division may be used as an index of a living
cell. It is easy to understand how some of these red cells may be reduced to brown pigment
granules when one considers the length of time which often elapses between the separation
of the placenta from the uterine wall and the fixation of the specimen. Careful, prolonged
search has been made to determine the presence of nervous tissue in the bodies of the
phagocytic cells inhabiting the corpora striata. In no instance was it possible to identify
with certainty any of the inclusions as neuroblasts.
 
Most of the cells contain a single nucleus, for the most part eccentrically placed. It
is large, varying from 4 to 6m in the younger embryo and from 5 to 8m in the older, and has
a distinct nuclear membrane. The rich chromatin network with its many enlargements is
beautifully shown in the specimens stained with hemotoxylin. Many of the cells have
nucleoli attaining at times a diameter of 2 M . These are then the center of a radiation
of chromatin threads. The nucleus may be irregular in shape, as shown in figures 12, 13,
and 28, but in most instances it has a smooth contour. The irregular nuclei belong to cells
which have begun to show signs of degeneration. As they grow older they gradually lose
their power of staining and finally disappear. The eccentricity of the nucleus is a most
striking peculiarity. One commonly sees the nucleus in direct apposition to the cell-
membrane. Perhaps the only instance of a lack of similarity between the macrophages of
the embryo and those of the chorionic villi is to be found in the method of regeneration.
 
Everywhere in the mesenchymal spaces, and especially in the cavities in the corpus
striatum, it is not difficult to find mitotic figures in these phagocytes (figs. 24, 25, 26);
in contrast to this one very rarely comes upon a dividing macrophage in the chorionic villi.
Figure 16 is an illustration of such an instance. It must be noted that all of the charac-
teristics described for the protoplasm are retained during mitosis— loose texture, vacuoliza-
tion, and engorgement with foreign particles. Cells with two nuclei have been observed
in the brain cavities, and the peculiar protoplasmic strands which sometimes bridge the gap
between them are illustrated in figure 29. The size of the nuclei, as well as the presence of
a nucleolus, in only one of them, makes it probable that we are dealing with direct division
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM. 101
 
in these cells, although there is no indication of a constriction of the cell-body. An oddly
shaped nucleus has been pictured in figure 30 ; here the form is concavo-convex, with the
convex surface near the cell-membrane. This illustration is inadequate in so far as it gives
one the impression of a kidney-shaped nucleus.
 
Unfortunately, not all of the material at my disposal has been exhausted in an effort
to establish a life-history for these macrophages. Many puzzling questions have been
presented by the distribution of the cells; yet a few general conclusions may be drawn from
these observations. With few exceptions, in the youngest human embryos the preservation
has precluded a careful cytological study; and often it is with reluctance that one admits
the failure to discover these cells in positions where one expects to find them.
 
The chorionic villi in the very youngest ova seem to be devoid of the large vacuolated
cells first noted by Hofbauer. Embryos of 2 mm. (No. 391 in this collection) possess a few
of these cells sparsely scattered about the villi and a few in the chorion itself, but none in
the body stalk nor in the embryo. In the larger specimens parts of the villi are entirely
free from these independent cells, which are found in large numbers in other portions of
the villi, chorion, and even the body stalk (No. 186, 3.5 mm.; No. 164, 3.5 mm., and No.
463, 3.9 mm.), but the mesenchyme around the aorta or brain shows none of the typical
large vacuolated cells so common in the membranes. After the embryo has passed 5 mm.
these cells may be seen in the mediastinum and around the brain; the ease with which they
are found in the loose mesenchyme increases with the growth of the embryo. As noted
by Hofbauer, they gradually disappear from the villi as the mesenchyme takes on a more
fibrillar character. Hence in the older portions of the chorionic villi — i. e., those in which
the larger vessels and denser framework exist — relatively few of these phagocytic cells may
be found, even in the younger embryos. In the later stages they disappear from the pla-
centa entirely.
 
Certain embryos contain a much larger number of macrophages in their placentae than
others of a similar stage of development. This peculiarity, as we shall see, holds true for
the cavities in the corpus striatum, where there is the same tendency to extreme variation
in the number of the cells present. The presence of large numbers of macrophages in one
locality is not necessarily accompanied by a similar increase in the other; thus in embryo 350
the placenta (fig. 14) contains very many cells, while the cavum mediale corporis striati con-
tains only a few (fig. 2) ; in embryo 22, on the other hand, the conditions are exactly reversed.
 
In order to determine the origin of these cells more accurate methods of investigation
than the mere study of routine serial sections must be employed. Hofbauer's attention
was directed to their mode of origin by Marchand; these workers believed them to be
descendants of the connective-tissue elements. A section taken from embryo 800 might
be given in support of their view. The character of the cells of the layer of Langhans is not
very unlike that of the younger macrophages, and it is possible that the macrophages of the
villi may arise from that layer directly. The cells, when in the body of the embryo proper,
apparently preserve their specificity and give rise to younger generations by mitotic division.
 
These cells, then, whose presence in the striate cavities argues strongly for the functional
existence of bilateral spaces, should be considered as of the type of extra-vascular phago-
cytes. They correspond in morphology to the Hofbauer cells of the young chorionic villus,
to the Wanderzellen of Maximow and Saxer, and to the macrophages of later writers.
 
 
 
102
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM.
 
 
 
THE OCCURRENCE OF THE TWO STRIATE CAVITIES.
 
In searching through the literature for illustrations of these cavities onty one could be
found which unquestionably had reproduced either of the spaces in the corpus striatum.
His (4, p. 124, fig. 83) has published a photomicrograph of the fore-brain of a 16 mm.
human embryo Se, in which the two halves of the brain were not sectioned symmetrically,
so that different levels appear on the two sides of the median line. Fortunately, on the
left side the cavum laterale corporis striati occurred in the plane of section. It is roughly
piriform, the pointed extremity being directed toward the cerebral ventricle, while the blunt
end approaches the surface of the striate body. This illustrationof the lateral cavity is very
similar in its position and size to those illustrated in figure 7, which is photographed from
embryo 409, a specimen of exactly the same length as Se. The lateral cavity usually develops
before the medial, so that it is safe to predict that the His embryo would show another
cavity more deeply placed in the striate body if the plane of section had not been so oblique.
 
The accompanying list of embryological material from the Carnegie Institution of
Washington includes all the specimens from 15 mm. to 23 mm. (crown-rump length) which
permit of histological study. With but one exception, evidences of one or both sets of
cavities have been found in the corpus striatum.
 
 
 
Crown-rump
 
 
Collection
 
 
< i own-rump
 
 
Collection
 
 
Crown-rump
 
 
Collection
 
 
length in mm.
 
 
No.
 
 
length in mm.
 
 
No.
 
 
length in mm.
 
 
No.
 
 
14
 
 
144
 
 
16
 
 
74
 
 
20
 
 
349
 
 
15
 
 
71!l
 
 
17
 
 
290
 
 
20
 
 
128
 
 
15
 
 
350
 
 
17
 
 
576
 
 
20
 
 
240
 
 
15.2
 
 
423
 
 
17.2
 
 
424
 
 
20
 
 
22
 
 
15 5
 
 
390
 
 
IS
 
 
432
 
 
21
 
 
400
 
 
10
 
 
43
 
 
18.5
 
 
431
 
 
23
 
 
453
 
 
16
 
 
400
 
 
19
 
 
293
 
 
23
 
 
382
 
 
10
 
 
400
 
 
19
 
 
229
 
 
24
 
 
455
 
 
 
The cavities in the corpus striatum fall into two groups — one appearing very near the
lateral surface of the brain, the other occupying a medial position within its substance.
Generally each is but a single cavity, and all of the smaller diverticula communicate with
the main cavity. Either, however, may be represented by multiple cavities wholly uncon-
nected with one another, yet so close as to be separated only by a thin partition of nervous
tissue. As it lies deep in the striate body, the medial cavity tends to be more smooth-walled
than the lateral. The latter is frequently traversed by numerous blood-vessels having a
thin layer of neuroblasts adhering to their walls; small strands of nerve-cells stretch like
spider-webs from one wall to another. In addition to the cavum mediale and cavum
laterale an intermediate group is present in one specimen, situated midway between the
two. In all of the specimens studied the medial group appears in younger embryos than
does the lateral, and disappears first in some of them. There are embryos of 24 mm. in
which both cavities are still well developed.
 
No direct communication between these cavities and the vascular system could be
made out. In none of the four injected embryos included in this study (No. 390, 15.5 mm. ;
No. 424, 17.2 mm.; No. 460, 21 mm.; No. 382, 23 mm.) was there any tendency for the
injected mass to flow out into these spaces, although vessels containing granules of injection
coursed directly through the cavity. In one instance (embryo 460) a small amount of
India ink backed into the cavum laterale from an extensive subpial extravasation. When
erythrocytes have been found in these spaces the cavity has shared in a generalized extrava-
sation of formed elements throughout the entire head.
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM. 103
 
THE CAVUM MEDIALE CORPORIS STRIATI.
 
If one neglect embryo 144, on account of its being given its measurement after it was
mounted on the slide, whereas all of the other specimens were measured before embedding,
we find that the cavum mediale makes its earliest appearance in embryos of 15 mm. (No. 7 1 !)
of this series). Deep in the substance of the striate body, in close proximity to a blood-
vessel, may be found a slight separation of the nervous tissue. Although not unlike an
artifact, it exists in exactly the same place on the two halves of the brain — in other words,
it is bilaterally symmetrical. In the transverse sections of this embryo it appears, under
low magnification, as a less dense area 150m long and 65^ broad. A single 4C> section
includes the major portion of the entire space, and it is terminated abruptly in the sections
on each side of it. Closer observation reveals strands of neuroblasts bridging the gap, here
a single cell stretching across the cleft, there a group of young neuroblasts extending from
one wall to the other. In the irregular fissure thus produced are found many macrophages,
their cell-bodies almost filling the space formed by the separation of nerve-cells. Unfortu-
nately, a careful cytological study is not possible, owing to the thickness of the section and
the dense stain. No formed elements could be made out in the bodies of these large phago-
cytes, although it seems likely that an extensive destruction of nervous tissue is taking place.
The macrophages are not found outside of the space {i. e., among the cells making up the
adjacent nervous tissue), but lie among the nerve-cells which bridge the newly forming
cavity. Were it not for the presence of these foreign cells, the earliest beginnings of the
cavum mediale could not be easily determined from the material at hand, since this cavity
differs in no other respect from a host of similar spaces found about the blood-vessels in the
brain of this embryo, produced by too rapid interchange of fluids in the preparation for
serial sections.
 
The further elaboration of the mesial cavity is furnished by embryo 423 (15.2 mm.),
embryo 350 (15 mm.), and embryo 406 (16 mm.). In all of these specimens the tissue of
the central nervous system is still intact in the region of the future lateral cavity. The
cleft, which was barely demonstrable in No. 719 (15 mm.), has enlarged so that a complete
separation of the nervous tissue has resulted. The vessel which was noted in the youngest
embryo now lies exposed in the wall of the cavity, or even courses through it unsupported
save for a few nerve-cells. Already the appearances of rupture of the nervous tissue have
almost entirely disappeared and the protoplasmic strands which bridged the gap at its for-
mation are wanting. Though not perfectly smooth in contour, as figure 2 illustrates, it has
the appearance of being punched out of the section, so that healthy neuroblasts end abruptly
at the boundary of the space. Occasionally one finds strands of cells bridging the mesial
cavity at all periods of its existence. Such bridges are, then, comparatively large and are
composed of many nerve-cells. The tissue making up the walls of the cavity is not in any
sense a membrane, but appears to be composed of normal looking, undeveloped nerve-cells
showing the same unbounded relation to one another as is found in the brain elsewhere.
The edge of the space does not show the condensation of protoplasm such as is seen on the
surface of the brain, where the cells form the external limiting membrane.
 
From the time of their first appearance, the cells contained in these cavities vary con-
siderably in number as one compares different specimens. At times almost the entire
mesial cavity is taken by macrophages which lie in close apposition to one another (fig. 8) .
For no apparent reason, the cell-content of the mesial cavity (and the same is true for the
lateral cavity) varies considerably, but in general one finds a greater number of macrophages
compared to the volume of the cavity in the mesial than in the lateral cavity.
 
 
 
104 TRANSITORY CAVITIES IN THE CORPUS STRIATUM.
 
Very quickly the mesial cavity reaches its maximum size. Thus in embryo 74 (16
mm.) the dimensions are roughly 195/* by 300m by 416m, but these dimensions are not con-
stant in other specimens. There seems to be no relationship between the length of the
embryo and the volume of the mesial cavity. Its method of disappearance seems to be by
gradual contraction of its walls, so that there appears to be a tendency for the older speci-
mens to have smaller mesial cavities. Thus in embryo 460 (21 mm.) the cavum mediale has
vanished entirely from the left side and is but 96m hi its greatest dimension in the right corpus
striatum. Neither the shape nor complexity of the space is characteristic. Usually, how-
ever, the cavity, with its smooth walls, assumes a spherical, ovoid, or lentiform shape.
Many times diverticula extend from the main cavity, so that what appear to be multiple
cavities in a single section, when traced through the series, turn out to communicate with one
another. Figure 2 from embryo 350 shows such a condition. Finally there may be a
duplication of these mesial cavities, so that two or more unconnected cavities may be found
where normally only one appears. When this occurs each cavity is smaller than usual,
but such a variation is not necessarily repeated on the opposite side of the brain. These
cavities have but one distinguishing mark — namely, their position. As far as could be
determined from series cut in the three planes, this space occupies a middle position in the
striate body and is about as far from the cerebral ventricle as the surface of the brain. In
figure 11 a geometrical projection gives its relation to the brain as well as to the lateral
cavity. In the embryos of this collection no remnants of a mesial cavity appear when
the length of 24 mm. has been exceeded.
 
THE CAVUM LATERALE CORPORIS STRIATI.
 
Farther laterally in the corpus striatum a second splitting of the nervous tissue makes
its appearance soon after the medial one is evident. This space, which I have called the
cavum laterale corporis striati, could not be made out in specimens 719, 350, 406, and 423.
Embryo 144 1 (14 mm.) furnishes the earliest observed stage in the formation of the cavum
laterale. An illustration (fig. 3) of the left side of this embryo gives a clear idea of its
position in the brain. At the time of its appearance it occupies a deep position in the
striate body just beneath the deeply stained ependymal zone. The separation of the ner-
vous elements measures 270m by 26m, and may be traced through four sections — i. e., 160m
of tissue. Its fellow on the other side of the brain has the same measurements. A drawing
(fig. 4) of this less dense region brings out characters quite similar to those already
described for the beginning mesial cavity. A narrow, elongated slit appears among the
young nerve-cells which bridge over it with numerous thin filaments of protoplasm. In
places the cell-body with its nucleus seems to be suspended midway between the diverging
walls, as if undecided as to which it will follow. To the right, a small blood-vessel [bv)
extends from one wall to the other. Moving among the strands of nervous tissue may be
seen several large macrophages (ma) with their typical eccentric nuclei.
 
Like the cavum mediale, the cavum laterale reaches its full development very soon after
its appearance. Its growth proceeds at the expense of the tissue lying between it and the
surface of the brain, and usually halts only after a single layer of nerve-cells is interposed
between it and the pia mater. All the transitional stages between the earliest evidences of
a splitting of nervous tissue and such an appearance as is shown in the photomicrograph
(fig. 5) have been found in the series of embryos here studied. The space does not
always extend so close to the surface as this specimen would indicate. In every instance
 
'Measured from sections after mounting.
 
 
 
TRANSITORY CAVITIES IN THE CORPVS STRIATUM. L05
 
where good histological pictures were present there were no evidences of an anatomical
opening under or through the pia mater. By studying the drawing (fig. 6) of a higher
magnification the character of the walls and the behavior of the nervous tissue surrounding
the cavity are more clearly made out. Roughly hour-glass in section, one finds the cavity
spreading out just under the surface of the brain and having a narrow constriction where it
adjoins the ependymal zone. Other embryos show an even more exaggerated narrowing
than embryo 431 (18.5 mm.). Everywhere the contour is rough and broken by projecting
masses of nerve-cells. Especially near the surface of the brain numerous delicate anasto-
mosing processes of nerve-cells traverse the space. Five blood-vessels (br) are included in
this section, coursing from one wall to another. The characteristic phagocytic cells (ma) are
present here in relatively larger numbers than in most of the lateral cavities. It is unusual
to find macrophages so numerous as to form a morula-mass, except in the cavum mediale.
Single cells are wandering among the protoplasmic strands along the edges of the cavity.
A large amount of debris is present, floating about in the space, whether merely coagulated
proteid or degenerated formed elements could not be determined. This cavity was the
largest seen, measuring 50G> in depth from the brain surface and 350^ in average diameter.
Like the mesial, the lateral cavity varies considerably in shape, volume, and contents.
The size does not increase pari passu with the growth of the embryo, except within the wide
limits of its appearance and disappearance, both of which are remarkably sharp. In
volume the lateral cavity is many times greater than the mesial, and owing to the irregu-
larity of its walls has no characteristic shape. The diverticula are usually large and the
lateral cavity is but seldom represented by several independent spaces. Here one usually
finds delicate strands of nerve-cells stretching for a considerable distance across the cavity
in a manner that suggests the growing embryonic nerve-cells on artificial media. This
lends a roughened surface to the wall as contrasted with the smoother cavum mediale.
Although the lateral cavities in embryo 431 (fig. 6) have many more macrophages per
unit volume than the medial cavities of embryo 350 (fig. 2), in the main one finds but few
cells in any one section of the lateral cavity.
 
For observing the relations of the cavities to one another a fortunate section through
embryo 409 (16 mm.) was photographed with both low and high power magnification
(figs. 7, 8, 9). The transverse section includes all four cavities, two on each side (fig. 7),
and between the cavum mediale and laterale appears the dark mass of neuroblasts con-
stituting the earliest nuclear anlagen in the striate body. Comparison of the magnified
photomicrographs of. the right mesial (fig. 8) and right lateral cavity (fig. 9) reveals
the characteristics of both spaces as found in the study of the entire series of embryos.
Most striking, perhaps, is the macrophage-content, In the mesial cavity almost the enl ire
space is occupied by these phagocytes, while it is with difficulty that one makes out the five
macrophages hidden among the delicate protoplasmic network making up the boundaries
of the lateral space. The cavum laterale, as it lies just beneath the surface of the brain,
presents a group of unconnected areas in cross-section, an appearance due to the processes
sent out by the nerve-cells lying more or less within the cavity itself. Traced serially, t hey
all communicate, forming a complex honeycombed system. In marked contrast to this
is the smooth-walled cavum mediale, the rounded mesial wall of which is made up of
embryonic nerve-cells arranged along the contour as regularly as if retained by a limiting
membrane. The lateral boundary of this space is made up of a large engorged vessel (bv)
which lies exposed in the cavity wall. It is very common to find a vessel standing out
in relief in the mesial cavity, and rarely even passing through it,
 
 
 
106 TRANSITORY CAVITIES IN THE CORPUS STRIATUM.
 
THE DISAPPEARANCE OF THE CAVITIES.
 
The inoccupation of both cavities by nervous tissue and their consequent disappearance
is apparently as rapid as are their appearance and growth. Embryo 460 (21 mm.) shows
an extremely small cavity in the medial position on the right side and a complete absence
on the left. In no instance could evidence of the mesial cavity be found after it had ceased
to exist as a break in the nervous tissue, i. e., in embryos greater than 24 mm. crown-
rump length. An extremely interesting picture is furnished by embryo 453 (23 mm.),
inasmuch as it constitutes a stage immediately following the replacement of nervous tissue
in the cavum laterale. Close to the surface of this brain, where in younger stages one is
wont to find the lateral cavities of the striate body, a considerable collection of macrophages
lies among the nerve-cells. There is no longer any break in the nervous tissue, but, in a
very circumscribed area, these phagocytes may be made out where the walls fused with
one another. Large vacuolated cells are present in symmetrical positions on both sides
of the brain and are undoubtedly beginning to migrate from the nervous tissue. The
mesenchyme without the brain contains many of these same cells, which are present in
greater numbers near the site of the former cavities. It is not improbable that the macro-
phages which we see here distributed among the nerve-cells would have crept out to join
their brothers outside of the external limiting membrane.
 
Three specimens included in this list are worthy of notice, inasmuch as they constitute
the extreme variations which have occurred. In the first, embryo 240, although its meas-
urement is recorded as 20 mm., no sign of either space can be made out. The tissue is
fairly well preserved and mounted. We are most likely dealing with a precocious or
delayed development of the cavity. On the other hand, we may have a condition analogous
to that obtaining for the pig. In this animal but two laboratory specimens, measuring
19 mm., showed an undoubted cavity in the corpus striatum (the mesial one) containing
macrophages. Many serial sections of pig embryos from 10 mm. to 25 mm. were searched
for evidences of either cavity, but in order to separate possible artifacts — which often occur
in the central nervous system — from a true cavity, a typical vacuolated cell with eccentric
nucleus was sought within the cleft. In all probability the existence of this cavity is of
extremely short duration in this animal.
 
Embryos 128 (20 mm.) and 455 (24 mm.) showed a most remarkable degree of vacuoli-
zation of the striate body. The former embryo has a complicated cavity in the corpus
striatum, which lies just under the ependymal zone between the mesial and lateral cavities.
I have called this the cavum intermedium corporis striati, but it is evidently an anomaly.
It is unconnected with either the medial or lateral cavity, which are well developed. The
latter specimen shows a remarkable reduplication of the medial cavity. The region occu-
pied by the medial group is very much larger, being extended laterally in the ependymal
zone. There are so many small and independent spaces in the nervous tissue that it would
be tedious to count them. In most of these cavities are found one or more macrophages,
and their presence eliminates artifacts due to rapid dehydration.
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM. 107
 
DISCUSSION OF THE OCCURRENCE OF THESE CAVITIES.
One naturally inquires as to the role of these sudden breaks in the continuity of the
nervous tissue coincident with the appearance of a foreign cell which proves to he a common
element distributed in various parts of the embryonic body and young chorion, and which
is in all respects the counterpart of the macrophage of the adult. Equally mysteriously,
after persisting during the time in which the embryo grows '.) mm., there is just as abrupt
an obliteration of these spaces and a complete disappearance of the phagocytic cells from
the corpus striatum.
 
Before suggesting a' probable reason for their existence, the possibility of an artifact
must first be dismissed, since, in its early beginning, each cavity has the appearance of a
shrinkage space. A large cavity having been observed occupying the center of the corpus
striatum of a chick which has been incubated for 10(H hours, a specimen of similar age was
carefully carried through graded alcohol in order to obviate as much shrinkage as possible.
This chick was 121 hours old and measured 13 mm. in 40 per cent alcohol. It was fixed
in modified Bouin's fluid, passed through 1 to 2 per cent grades of alcohol, and cut into 15m
sections in paraffin. Figure 10 is a photomicrograph from this specimen, and the degree of
shrinkage may be judged by examining the retina. There is only the slightest tendency
toward separation of the layers— and this is perhaps the most difficult organ to maintain
perfectly without previously opening the eyeball to facilitate free interchange of fluids.
Not one of the blood-vessels in the brain shows the slightest perivascular space, but each
has a fully distended lumen. The medial cavity, illustrated on both sides, lies between the
ependymal and nuclear zones. Contained within these complicated spaces are cells which
are undoubtedly the macrophages, and which exist in very small numbers compared to the
volume of the cavity. In all respects, except for the superficial position, the cavum corporis
striati in the chick has the characteristics of the cavum laterale of the human embryo;
i. e., relatively few cells, roughened walls, and large, complicated lumen. There is no other
cavity nearer the surface in either of the two specimens studied. No attempt has been made
to follow the development in the chick, yet here is an excellent opportunity to compare the
behavior of the macrophages in a vitally stained embryo.
 
Concerning the part played by these cavities, several possibilities have suggested them-
selves, but none are without objection. First, the spaces may be places where macrophages
are formed during the short period of embryonic growth, just as the macrophage formation
in the fiver of the adult is limited to the endothelium. Glia cells have been described by
Alzheimer (1) as forming macrophages ("Kornchenzellen") in the adult, and we may be
dealing with such a transformation here. No such transition forms giving weight to this
view were observed. If these spaces serve as foci of regeneration, then the size of the cavum
laterale seems to be out of all proportion to the number of cells contained in it .
 
More likely we are dealing with an actual tissue destruction in the embryo of this
early age. Both right cavities have been illustrated topographically (fig. 11). It will
be noticed that this is the region about which a great shifting of tissue must take place to
form the island of Reil and the temporal lobe. This mass movement might result in the
destruction of earlier connections, and the macrophages are present simply to take away the
debris formed by the rapid shifting of one portion of the brain over another. Two facts.
however, are out of accord with this hypothesis ; the formation of the Sylvian fissure has
its inception in embryos of the third month and then progresses over a very long period; in
other places, where extreme sliding of tissue, like the pontine formation, is present, one
never finds this extreme grade of destruction.
 
 
 
108
 
 
 
TRANSITORY CAVITIES IN THE CORPUS STRIATUM.
 
 
 
A destruction due to other causes is possible; for example, the imperfect circulation
of perivascular cerebro-spinal fluid which may be forming in larger quantities than can be
absorbed. A dual source for cerebro-spinal fluid has been shown by Weed (12) as being
derived from the choroid plexus and the nervous tissue via the perivascular spaces. Unpub-
lished work, undertaken by him in the Anatomical Laboratory of the Johns Hopkins
University, shows many interesting facts concerning the circulation of cerebro-spinal fluid
in the pig embryo.
 
The various inconsistencies, as well as the lack of uniformity in structure, could be
explained on the assumption of a slow accumulation of fluid about the vessels of the corpus
striatum for a short time and its disappearance as soon as adequate pathways in the menin-
ges are established. Such an hypothesis of an accumulation of fluid in the striate body
gains support from many of these recorded findings. The invariable association of per-
forating blood-vessels with the cavities; the development of the spaces before the meningeal
differentiation has proceeded far; the presence of coagulated protein-like contents; the
bridging of the spaces by tissue strands resembling those of the adult perivascular canals —
these are factors of possible consequence in this suggested origin. From this viewpoint,
the accumulation of the intrinsic fluid of the nervous tissue resulted in the initial tissue
destruction; further distention with more extensive tissue destruction and invasion of
macrophages occurred; then, with a possible drainage outward of this fluid accumulation,
the pressure was relieved and the space rapidly filled with the nervous tissue.
 
SUMMARY.
 
The observations here presented indicate that there occur in the corpus striatum of the
human embryo between 15 and 20 mm. in length, two bilaterally symmetrical cavities.
The presence in these cavities of cells morphologically similar to the macrophages of the
adult and to the Hofbauer cells of the embryonic chorion argues strongly in favor of a func-
tional significance. The lateral of these cavities is larger and less completely filled with
macrophages than is the mesial. No satisfactory explanation of their presence can be
definitely advanced.
 
 
 
BIBLIOGRAPHY.
 
 
 
1. Alzheimer, A. Beitrage zur Kcnntnis der pathologi-
 
schen Neuroglia und ihrer Beziehungen zu den
Abbauvorgangen im Nervengewebe. Histol. u.
histopat.hol. Arbeit, lid. Grosshirnrinde (Nissl-
Alzheimer), Jena, 1910, in, 401-562, 8 pi.
 
2. Evans, H. M. The macrophages of mammals. Amer-
 
ican Journ. Phys., 1915, xxxvn, 1-16.
 
3. Grosser, O. Vergleichende Anatomie und Entwick-
 
lungsgeschichte der Eihaute und Plazenta. 8°,
Leipzig and Wien., W. Braumliller, 1909.
 
4. His, W. Die Entwicklung des menschlichen Gehirns
 
wahrend der ersten Monate. 8°, Leipzig, S. Hirzel,
1904.
 
5. Hofbauer, J. Grundziige einer Biologie der mensch-
 
lichen Plazenta. 8°, Leipzig and Wien., W. Brau-
mliller, 1905.
 
6. Maximow, A. Untersuchungen iiber Blut und Binde-
 
gewebe. Arch. f. mikr. Anat., Bonn, 1909, lxxiii,
444-561, 5 pi.
 
7. Metchnikoff, E. Lecons sur la pathologie comparee
 
de l'inflammation. 8°, Paris, G. Masson, 1892.
Also, transl.: 8°, London, Kegan Paul, etc., 1893.
 
 
 
10
 
 
 
11
 
 
 
12
 
 
 
S. Minot, C. S. Die Entwicklung des Blutes. Handb.
 
d. Entwcklngsgesch. d. Menschen (Keibel <fe Mall),
 
Leipzig, 1911, n, 483-517. Also, transl.: Man.
 
Human Embryol. (Keibel & Mall), Philadelphia,
 
1912, ii, 498-534.
9. Plato, J. Ueber die vitale Farbbarkeit der Phagocyten
 
des Menschen und einiger Saugethicre mit Neutral-
roth. Arch. f. mikr. Anat., Bonn, 1900, lvi, 868-
 
917, 1 pi.
Saxer, Fr. Die Entstehung der rothen und weissen
 
Blutkorperchen. Anat. Hefte, 1 Abt., Wiesbaden,
 
VI, 347-532, 8 pi.
Van der Stricht, O. Nouvelles recherches sur la genese
 
des globules rouges et des globules Wanes du sang.
 
Arch. d. biol., Gand, 1892, xn, 199-344, 6 pi.
Weed, L. H. The dual source of cerebro-spinal fluid.
 
Journ. Med. Research, Boston, 1914-15, xxxi
 
(n. s., xxvi), 93-111, 6 pi.
 
 
 
,- • . -
 
 
 
 
 
 
 
 
 
 
6
 
1. Photomicrograph. Embryo 350 (/5 mm.). Cross-section 75 - I - 5. X 8 The cavum mediale corporis striali (c m) appears in symmetrical
 
positions on the two sides of the brain. That on the right side is given at a higher magnification in Figure 2.
 
2. Photomicrograph. Cavum mediale dextrum. X I44. The position is indicated in Figure I .
 
3. Photomicrograoh. Embryo I44 (14 mm.). Sagittal section 4-1-2. X 5. The earliest appearance of the cavum laterale sinistrum (c/)
 
is indicated here and illustrated at a higher magnification in Figure 4.
 
4. Drawing of the cavum laterale corporis striati. X 500. Macrophages (m a) may be ■teen among the strands of protoplasm bridging the
 
cavity, b v, blood-vessel The r>ns ; tion of this drawing is indicated in Figure 3. (Drawn by J. F. DiJinch.)
5 Photomicrograph. Embryo 43 ! (18.5 mm.). Sagittal section 12-1-1. X 7'<f. The very large cavum laterale sinistrum (c/) is
 
shown in its relation to the nia mater and the ventricle of the brain A more highly magnified drawing is given in Figure 6.
b Drawing of the cavum laterale sinistrum. X '90 A single layer of nerve-cells separates the space from the pia mater (p m). Large
 
numbers of macrophages (m a), a detailed drawing of which is given in Figure 12. are present Rlood-vessels (b v) traverse the cavity.
nded by a few nerve-cells. The position of this drawing is indicated in Figure 5. (Drawn hi/ J F. Didusch.)
 
 
 
 
• #
 
 
 
 
&L
 
 
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9
 
 
 
 
 
 
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fciiv*
 
 
 
 
 
 
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7. Photomicrograph. Embryo 409 ( /6 mm.). Cross-section 10-2-5. X 10. This figure illustrates the symmetrical positions occupied
 
by the lateral (c /) and mesial (c m) cavities. Larger reproductions of them are shown in Figures 8 and 9.
 
8. Photomicrograph of the cavum mediale corporis striati. X 200. The smooth-walled space is parked with macrophages. An engorged
 
blood-vessel (b v) forms its lateral boundary. The position of this illustration is indicated in Figure 7.
 
9. Photomicrograph of the cavum laterale corporis striati X200. Some of the nerve-cells are growing across the cavity. The position is
 
shown in Figure 7.
 
10. Photomicrograph of a section through the corpus striatum of a chicle (121 hours incubation). X 7'<. The cavities (<: s) in the
 
striate body are unusually large.
 
11. Geometrical projection of a model made at 50 diameters. Embryo 460 (21 mm.). X6J£. The cavum laterale (c / ) and cavum
 
mediale (c m) are shown on the right side.
 
12. Macrophages indicated in Figure 6 X 1000. (Drawn by J. F. Vidusch.)
 
 
 
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13. Photomicrograph o( macrophages in cavum mediale. X 1000.
 
14. Photomicrograph of chorionic villus of Embryo 350. X 410.
15-17. Macrophages from the chorionic villi. X 1000.
 
18-30. Macrophages from the cava corporis striati. X 1000.
 
31. Normal erythrocytes from a 15 mm. human embryo. X 1000.
 
 
 
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Latest revision as of 13:41, 11 August 2020

CONTRIBUTIONS TO EMBRYOLOGY, NO. 5.

THE CORPUS LUTEUM OF PREGNANCY, AS IT IS IN SWINE.


By George W. Corner. With three plates.


CONTENTS.


Introduction

Breeding habits of the sow

General features of the corpus luteum

Histology of the corpus luteum of the sow .

Cytology of the lutein cell

Life history of the lutein cell of pregnancy . .


PAGE.

71 72 72 73 75 79


Previous observers on the corpus luteum at vari- ous stages of pregnancy 80

Characteristics of the corpus luteum at the various

stages of pregnancy 81


PAGE.

Criteria of age of the corpus luteum 82

Theoretical considerations 84

Distinction between the corpus luteum of preg- nancy and of ovulation 85

Influence of pregnancy on the structure and func- tion of the Graafian follicles 86

Abnormalities of the lutein tissue 88

Migration of the ovum in the sow 90

Conclusions 92

Literature cited 93


THE CORPUS LUTEUM OF PREGNANCY, AS IT IS IN SWINE.


INTRODUCTION.

At the instance of Professor Mall, I have undertaken the studies to he presented in this paper, in the hope of gaining such knowledge as would enable us to draw up a standard description of the corpus luteum throughout its history. If we knew the exact appearance of the human corpus luteum at all its stages, especially during pregnancy, we should have an instrument of the greatest value in the solution of many complicated problems of embryology and physiology, and even of clinical gynecology and organotherapy. Since the path of such a research as I have here begun is as yet unblazed, and because studies of the human tissues are so hampered by pathological changes, faulty histories, and difficulties of collection and preservation, for the beginning we have limited ourselves to the ovary of the domestic sow, and have started out with the simple hope of obtaining an answer to one question: What is the appearance of the corpus luteum at each and every stage of preg- nancy? To this query as simple an answer has been found, but the work has led in such unexpected directions and into such interesting by-ways that the reader will no doubt feel as much disappointment in reading as the author has in writing a paper which answers one question, only to raise a score as yet unsolved.

During the months of October 1913 to May 1914 I have collected from the hundreds of sows' uteri and adnexa obtained at the slaughter-house adjacent to this laboratory, 128 pairs of ovaries from pregnant animals, the embryos being in each case examined and measured. The youngest embryo studied had 5 somites, and its age was estimated at about 15 days. The specimen is preserved as pig embryo No. 10 in the embryological collection in this laboratory. The oldest fetus in my series was 290 mm. long, or just about at term. In this paper, where lengths are given, they represent careful crown-rump measurements. Since the age-length ratio of embryos of the pig has never been fully worked out, the ages given are only approximate, and were obtained, for the younger stages, by comparison with Kernel's lists and figures (1897), and for the older stages from a table found in Strange- ways' "Veterinary Anatomy," and said to have been compiled from the works of Gurlt, Leyhs, Franck, and others. The entire uteri and adnexa were received at the laboratory within an hour after killing, generally within a few minutes, and while yet warm. The fixing agent used was 10 per cent aqueous solution of formalin (40 per cent formaldehyde). The preparations were made by cannulating the ovarian artery, or the uterine, in which latter case it was advisable to tie off a few branches. The blood-vessels of the ovary were then injected with the formalin solution heated to 40° to 50° centigrade, and were placed in the same fluid. In a few cases where injection was not possible or not successful, the cor- pora were sliced with a razor-blade and the ovaries immersed in the warm formalin. For the study of the fat-content of the tissue, slices were cut from certain of the fresh corpora lutea and fixed in 2 per cent osmic acid for 24 hours. In all cases the ovaries were numbered consecutively, the right and left ovaries being indicated, the embryo pigs were measured, and the number of them in each horn of the uterus recorded.

The specimens were left in the formalin 24 to 48 hours, the solution having been allowed to cool an hour or two after immersing the specimens. After fixing they were washed in running water 24 hours, then dehydrated by passing through graded alcohols for 24 hours each (30, 40, 50, 60, 65, 70, 75, 80 per cent). The whole ovaries were then placed in


72 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

80 per cent alcohol for preservation, and blocks cut from them for sectioning were put for 24 hours in each of the following fluids: 85, 90, 95, 98 per cent alcohol, equal parts 98 per cent alcohol and ether, celloidin solutions 2, 4, 6, 8, 10, 12, 14 per cent. Finally the celloidin blocks were hardened with chloroform. As a routine, from one corpus luteum of each ovary or pair of ovaries two sections were made, 10 or 15 microns in thickness (the sections often passed through two adjacent corpora lutea), and in addition sections were made of any unusual or puzzling structure. Of these two routine sections one was stained with eosin and Ehrlich's hematoxylin, the other with Mallory's triple connective-tissue stain (acid fuchsin, phosphomolybdic acid, aniline blue, and orange G) . The latter staining method is particularly valuable, since it not only gives beautiful pictures of the connective tissue, but also of those very structures of the lutein cells which are most important for the present study.

BREEDING HABITS OF THE SOW. The females of the wild swine are monestrous and give birth to one litter a year, accord- ing to Kaeppeli (1908). But in domestication the sow undergoes an estrous cycle lasting 2 to 4 weeks, generally 3 weeks (Kaeppeli, Fleming: Veterinary Obstetrics). If the animal becomes pregnant, she may conceive again 5 weeks after delivery. The period of gestation is 16 to 17 weeks, usually between 116 and 120 days. Farmers commonly arrange to have litters produced in the spring and autumn, but in the material received at this laboratory, from stock raised for slaughter, fetuses are found in all stages of development, without great regard to the time of year.

The domestic sow is a very prolific animal, sometimes giving birth to as many as 19 pigs in one litter. Wentworth (1914) mentions an extreme case in which a sow gave birth to 23 pigs in one litter. John Hunter kept a record of the total progeny of one sow, in order to have a control for another, one of whose ovaries he had removed. During her life the normal animal gave birth to 13 litters, numbering in all 162 pigs, making an average of 12.5 pigs for each birth. In animals sent to the city for slaughter such high figures do not occur, for several reasons. The animals are sold young, often while bearing their first litters, which are commonly small in number; or if they are multipara, they are sold for the very reason that they are not profilic breeders. Moreover, the stock sent to the slaugh- ter-house is the general product of the country-side, not always from selected droves. My records show that 6 is the commonest number of pigs in one litter, the extremes being 1 and 10.

GENERAL FEATURES OF THE CORPUS LUTEUM. On examining the ovary of the pregnant sow, the first objects to strike the eye are the extraordinarily prominent corpora lutea, which project nearly all their volume from the surface of the* ovary. In each ovary of the domestic sow I have found from 1, or none, to 10 recent corpora lutea, and in both ovaries from 1 to 16, most commonly 8. For the reasons which I have given, these figures are lower than would be found in well-bred, selected stock. It will be noticed also that the pigs are commonly fewer than the corpora lutea; in other words, that frequently not all the ova expelled at one time develop into pigs. The corpora lutea are generally ovoid or spherical in form, with outside diameters of 8 to 10 mm., most often about 10x10x10 mm. The full size is attained when the fetuses are about 10 mm. long, and does not change until retrogression of the corpus luteum is well advanced after delivery ; in other animals the corpus luteum is said to increase slowly in size through- out pregnancy. (For instance, the bat, as studied by Van der Stricht, 1912.) The custom-


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 73

ary name, corpus luteum, does not apply to swine, for the color of the cut surface of the fresh organ is a light pinkish gray, which only changes to yellow in the old corpora.

Besides the 1 to 10 corpora lutea in a given ovary, which experience shows mark the site of the ovulation which led to the pregnancy, there are often a number of smaller cor- pora lutea, which are bright yellow in color, and on section prove to consist mainly of dense connective tissue. In some ovaries there is still another older generation, dating from the second previous ovulation, which are seen as small white bodies nearly buried in the ovary. The remains of corpora lutea older than the second previous generation are not usually visible on gross examination, except as minute opaque bodies tucked in between the follicles and appearing in sections as the familiar hyaline areas. Since the corpus luteum remains as a distinct structure through the two succeeding ovulations, it follows that during the sexually active period of life the ovary should never be free from corpora lutea. This was true of the specimens examined by Kaeppeli (1908), but in our material there are many which contain nothing but follicles and what are apparently corpora lutea several periods old. Either these are atretic follicles, resembling corpora lutea, the sexually active period not having begun, or else it is not uncommon for the sow to pass an cestrual period without ovulation.

If, then, the corpus luteum is still evident after two successive ovulations following its formation, it becomes important to the progress of our study to know whether there is any likelihood of confusing the corpora lutea oi two different generations. When two or more generations are present, by microscopical study we always find such marked histo- logical differences that there is no possibility of confusion, but on naked-eye examination it is not uncommon to find corpora lutea apparently intermediate, in size and color, between the two obviously different groups present. I have submitted all such cases, 8 in number, to microscopical study, which showed that in 3 cases corpora lutea markedly smaller than their mates presented the same histological appearances. A typical instance is given by a specimen in which there were two corpora lutea of pregnancy 8 to 9 mm. in diameter, and another of the same histological appearance only 4 mm. in diameter. In another case it was necessary to section every corpus luteum of both ovaries to find how many of them were recent. The moral is that critical points regarding the age of corpora lutea can not be decided without microscopical evidence.

Important also are the questions as to whether the corpora lutea of both ovaries, dating from the same ovulation, present the same histological appearance, and whether all the corpora of one ovary, dating from the same ovulation, are alike. To answer these questions, I have examined sections taken from corpora lutea of both ovaries of 19 sows. and a larger number taken from two or more corpora lutea of single ovaries, with the simple result that all the corpora lutea of the same pregnancy, in both ovaries are in cytological structure absolutely identical. Sometimes the central cavity of the follicle lingers longer than in its mates, or there is a minor difference in size or other gross characteristic, but in microscopic structure never.

HISTOLOGY OF THE CORPUS LUTEUM OF THE SOW. It is necessary, for the sake of clearness, to discuss briefly the perennial question of the ovary — that of the origin of the lutein cells— which above almost all other anatomical questions is notable for radical differences of opinion among the many capable investigators who have attacked it. The reader will recall that there are three views as to the source of the lutein cells of the mammalian corpus luteum. The first suggestion was that of von


74 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

Baer (1827), who believed that the lutein cells are derived from the connective tissue about the follicle, the theca interna; and the second is that of Bischoff (1842), who concluded that the cells come from the epithelial layer of the follicle, the membrana granulosa. The chief arguments in favor of the first view are that, firstly, as the Graafian follicle ripens, the theca cells show marked changes; they swell in volume, become rounded, and in short come to resemble the lutein cell very closely (see below, p. 86, and figures 16 and 18) ; secondly, the membrana granulosa of large follicles is often degenerated, and is believed to be cast off at the time of rupture; thirdly, such follicles as do not rupture are filled up by proliferation of the theca interna, and in this process of atresia become very much like corpora lutea. It is interesting to note that the latter view happens to have for prominent defenders those who have worked most carefully upon the sow's ovary, Benckiser (1884), Jankowski (1904), and Clark (1898). Great doubt has been cast upon it, however, by the careful and patient work of Sobotta (1896 ff.) and his followers, who maintain the origin of the lutein cells from the granulosa alone, and do not admit the participation of theca cells except to form the connective tissue reticulum.

A third theory, due I believe to Schron (1863), but upheld recently by Rabl (1898), Seitz (1896), Leo Loeb (1906), R. Meyer (1911), Van der Stricht (1912), and others, is that both layers of the follicle form lutein cells. The upholders of this view do not deny one of the arguments of their opponents, that regarding the changes of the theca interna during the ripening process, but they think it is not permissible to draw conclusions from the resemblances between follicular atresia and corpus luteum formation. Moreover, it is not true that the granulosa is cast off from normal follicles before or at the time of rupture, as is well shown in figure 1. Many of those who believe that the theca cells take part in lutein-cell formation describe some of the "theca lutein cells," as they call them, remaining for a time in little groups about the periphery of the corpus luteum and along the septa of connective tissue which penetrate it.

I think it may be said with fairness that most of those who have really devoted them- selves to accurate studies of the corpus luteum with modern methods maintain the origin of the lutein cells either from the granulosa or from both layers of the follicle. With only a few specimens of very early corpora lutea at hand, I can not presume to enter this contro- versy, except to mention that in the sow the granulosa undoubtedly persists after rupture of the follicle, and becomes vascularized by vessels from the theca interna. After this there is a gap in my series until the corpus luteum becomes solid. However, from some new points in the histology of the corpus luteum, which I am about to mention, it seems ! that in the sow, at least, there is a fourth possibility to be reckoned with by the investigator who is to clear up this baffling question — namely, that the granulosa and perhaps part of the theca enter into the formation of true lutein cells, while some of the cells of the theca interna remain as distinct cells of special nature in the fully formed corpora lutea.

It is apparent at first glance at a well-prepared section of the corpus luteum of the sow that it is not the simple structure composed of a parenchyma of lutein cells and a frame- work of connective tissue which is figured in the manuals of histology. The lutein cells are indeed the chief element, but lying between them are other cells of at least two sorts, which can be found at all stages of pregnancy. For want of better terms I shall denote them as additional cells of the corpus luteum, types 1 and 2.

Type 1 (fig. 2, b). — About the periphery of the corpus luteum, and along the septa of connective tissue which penetrate it, in many sections one sees small groups of cells whicli have the following characteristics: round or oval nuclei containing a moderate amount of


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 75

chromatin (more than the true lutein cells), and smoothly staining, very finely granular cytoplasm. The cells are round, or slightly elongated in one direction, and are smaller than the lutein cells, having a diameter of 15 to 20 microns. Now, if we examine the figures of some upholders of the compromise or joint-origin theory of the corpus luteum, for instance, those of R. Meyer (1911), we find that they show cells of this same appearance in this same situation, which are described in the text as cells of the theca interna, not yet changed into lutein cells or connective-tissue cells. Are these cells which I have described in the pig identical with the so-called theca lutein cells? The evidence for this view lies in the facts that they are present in the early stages of pregnancy, are then very distinct from the ordinary lutein cells, and lie mostly in the position in which the theca lutein cells are described. Against it is the fact that when the corpus luteum undergoes certain changes to be described later as distinctive of the latter part of pregnancy, these cells seem to increase in number, they follow the growing connective-tissue reticulum into all parts of the corpus luteum, and they come to resemble the true lutein cells very closefy; indeed, at times it is almost impossible to distinguish them; whence it might be inferred that the two sorts of cells are different only in state of activity. The origin of these cells must be left without further definite statement, although we shall come back to them later, as they form part of our criteria for determining the age of the corpus luteum by microscopical examination. Type 2 (fig. 3, b). — These are cells of varying form and size, generally rather smaller than the lutein cells, ranging in shape from spindles to branching and spherical forms, having a cytoplasm which stains deeply with eosin, and takes a dark brown or purple color with Mallory's stain, but sometimes takes instead the aniline blue ingredient of the mixture. Very often the CNdoplasm is filled with minute vacuoles which appear as bright, highly refractile granules about 1 to 2 microns in diameter. No matter what fixing reagent and stain is used, they remain vacuoles, and may even be found in osmic-acid material. The same vacuoles may occasionally be found in the lutein cells. I have not seen these cells in fresh teased preparations. They are not numerous; in one section from a very few to a hundred may be seen scattered among the hundreds of lutein cells, but they are not found in all ovaries, although they are easily seen, because of their dark stain and the fact that their cytoplasm is usually slightly shrunken, and hence they stand out distinctly. They seem to have been observed previously only by Delestre, who saw them, if I interpret his description aright, in the corpora lutea of cows (1910). As to the nature of these cells, it is difficult to say whether they represent a modification of the lutein cells or of the connec- tive tissue. There seem to be transitions in both directions. But there is perhaps a clue in the specimen illustrated in figure 1, of a very recently ruptured follicle, in which the layer of theca interna cells nearest the granulosa is partly composed of cells similar in size, shape, appearance, and staining reactions to the cells in question. The significance of the cells is totally obscure to me.

CYTOLOGY OF THE LUTEIN CELL.

The lutein cell possesses the elementary structures found in all animal cells. The nucleus, which in the active stages of the corpus luteum is round, vesicular, and poor in chromatin, has one or more large nucleoli. The cytoplasm, at certain stages to be specified below, is differentiated into two portions: an inner dense zone, the endoplasm, which in fixed specimens appears to be very finely granular, and an outer zone, the exoplasm. This latter zone, whether studied in fresh preparations or in carefully fixed sections, is of the most astonishing complexity. It is so full of granules and globules of diverse substances


76 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

that the nucleus of the fresh cell can sometimes hardly be seen. Some of these granules are mitochondria; this may very easily be confirmed by making a preparation of fresh teased cells with Janus green; this dye gives a vital stain of the mitochondria, as shown by Bensley (1911). Other globules are fatty and are selectively stained, for the purpose of analyzing the cell elements, by any of the usual methods for neutral fats, such as Sudan III and osmic acid. The nature of the fatty substance of the lutein cell, as found in the horse, cow, and sow, has been studied microchemically by Cesa-Bianchi (1908), who thinks it is lecithin; the existence of this substance in the corpora lutea had previously been declared by Loisel (1904) on the basis of test-tube analyses. A striking contrast to the statements just made is offered by the report of J. W. Miller (1910) upon his studies of the human ovary, in which he states emphatically that there is no substance in the fresh corpus luteum of preg- nancy which gives the reactions of the neutral fats, and that such reactions are obtained only during the puerperium; that is to say, in retrogressive corpora lutea. On the other hand, the lutein cell of the cat is often so honeycombed with fat that the vacuoles resulting from the action of alcohol and ether obscure the finer structure of the cell, so that I found it impossible to use some cats' ovaries which were at hand, in studies to corroborate the results of this research.

The most comprehensive description of the fatty inclusions in the lutein cells is that of Van der Stricht (1912), who worked with the ovaries of several species of bats. He finds the cells of young corpora lutea loaded with osmic-blackening material, but that there is a gradual decrease in amount of this substance until the latter part of pregnancy, when globules again begin to be deposited. It is his opinion that the early deposit of fat is epithelial in nature, representing a secretion of the corpus luteum, but that the later reappear- ance of presumably fatty material is a sign of senescence of the tissue. I shall detail my findings in the sows' lutein cells later; it suffices to say here that my preparations agree fully with those of Van der Stricht, in spite of the great difference in zoological position of the animals used.

In celloidin sections of corpora lutea from animals containing fetuses less than 40 mm. long, fixed with such common solutions as formalin, absolute alcohol, and bichloride of mercury, in which neither the fat nor mitochondria are preserved; and in osmic acid prep- arations, in which the fat is distinguished by its intensely black color, we find the peripheral part of every cell, the exoplasm, occupied by a most curious maze of clear spaces, of protean form, alternating with rings and rod-shaped masses of cytoplasmic material, which give the cell an almost indescribable but striking appearance (fig. 4).

Since these peculiar structures are found to be important guides in following the his- tory of the lutein cell, it will be advisable to review briefly the conclusions of other investi- gators regarding similar appearances in other cells. In 1898 Golgi reported that in prepara- tions of nerve cells from the central nervous system, made by a method of chrome-silver impregnation, he found the metallic substance deposited in the form of a network which lay in the cytoplasmic portion of the cell. This structure he called the "Apparato reticulare interne" About the same time Nelis and Holmgren both described net-like or strand-like appearances in the cytoplasm of nerve cells, which were seen as unstained areas in sections colored by dyes. Nelis called these structures "etat spiremateux du protoplasm" and Holmgren applied the term "Saftkanalchen" and later "trophospongium" to the essentially similar structures which he had found.

As these canals began to be discovered in nerve cells of all kinds, and then in cells of many other organs, their importance was recognized, and the many questions connected


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 77

with them were vigorously discussed. Golgi denied the identity of his reticular apparatus with the trophospongium, but Holmgren affirmed it, at least in part. The latter view has taken the ascendant. Ram6n y Cajal believes them to be identical (1908). Cowdry (1912) , from the study of specimens prepared by the methods advocated by both discoverers, points out the great similarity of the two phenomena.

The functional importance assigned to the reticular apparatus by different authors is varied. It seems well established that the appearances are not artifacts, but are present during life. Some have thought them excretory channels, others circulatory paths for cell juices. Holmgren thinks they are often entered by processes of the connective-tissue cells and that they are the morphological expression of vital processes such as the formation of secretion droplets and the like. According to this view the system of canaliculi is an unstable, constantly changing structure, like any other physiologically active organ, a view which explains the variability of the histological appearance. Most investigators have conceived the reticular apparatus to be a constant component of the cells in which it is found, but von Bergen (1904), who studied the canaliculi in a great variety of cells from many organs, maintains the transitory nature of the structure. This latter opinion is certainly borne out by the study of the lutein cells, as will be shown later.

Cohn (1903), in a careful paper on the histology of the corpus luteUm, states that he was unable to see the trophospongium in the lutein cell, although he had followed the methods of Holmgren; but Vastarini-Cresi (1904), in a short note, mentions, without describing, certain preparations which he had demonstrated at Naples in 1904. Cesa-Bianchi (1908) describes and figures lutein cells of swine which show appearances resembling the tropho- spongium as observed in other cells. By the Golgi method Riquier (1910) demonstrated an intracellular reticular apparatus in the lutein cells of the cow and, what is very important in connection with the findings to be described in this paper, he showed that the structures undergo marked changes with the advancing age of the cell.

Now, if we take a well-prepared section of the corpus luteum of a pregnant sow, whose fetuses are perhaps 100 mm. long, and stain it with any strong cytoplasmic stain (Mallory's does excellently), careful study of the lutein cells shows that the cytoplasm contains un- stained areas which are roughly concentric to the nucleus, and which appear to form canal-like paths in the cell (fig. 8). If we take younger corpora lutea, we find the canalic- ular apparatus growing more and more complex. It assumes the form of wide V-shaped spaces, long clefts, and circles in the cytoplasm, so extensive that the nucleus is surrounded by only a narrow zone of endoplasm (fig. 7). But it is in the corpora lutea of pregnancies under 30 mm. that the highest development of the exoplasm is found (figs. 4 and 5). Here the exoplasm is occupied by a most curious and elaborate system of vacuoles, almost every one of which in turn contains a spherule of substance which, although it takes the same stain as the cytoplasm, yet has a more hyaline appearance, and is seen on section as a bright ring. Within many of the spherules is found another and tiny vacuole.

Curiously enough, the only investigator who has previously described this remarkable state of the lutein cells is F. Cohn (1903), who is mentioned above as denying the existence of the trophospongium in lutein cells. He observed all the appearances which have just been mentioned, in the corpus luteum of the rabbit at the height of its development, and undertook certain microchemical studies on their nature. He found that the innermost tiny vacuole contains a substance which blackens with osmic acid, presumably fat. In the sow's corpus luteum (fig. 6, a) I have been able to confirm this observation of Cohn, but not his other observation, namely, that the outermost vacuole, or clear space about the


78 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

hyaline ring, is stainable with one of the methods for demonstrating myelin, Plessen and Rabinowitz's modification of Weigert's hematoxylin. I have seen the ring-like structures in tissue fixed with formalin, osmic acid, saturated mercuric chloride, Zenker's fluid, and absolute alcohol. Although I have not made an extensive search for these formations in various species, I have looked through a number of specimens prepared for other purposes which chanced to be at hand in the laboratory, and found the ring-like bodies in corpora lutea of the human species, the dog, and the cat. Add to these Cohn's original description in the rabbit, and we have seen the peculiar exoplasmic rings in the corpora lutea of ungu- lates, carnivora, rodents, and primates — surely a general distribution among the higher mammalia. Van der Stricht, in his long paper on secretory appearances in the corpora lutea of bats (1912), does not mention them, but the lutein cells of the bat (as are often those of the cat) are heavily loaded with fatty matter which would obscure the other details of the cytoplasm.

To avoid making an unproved assumption, in this paper I shall refer to these forma- tions merely as exoplasmic formations, but I think there can be very little doubt that they are really one stage of the canalicular apparatus of Golgi-Holmgren, although granules within the canaliculi have never been described in other cells. In the first place, they can be traced back directly through all transitions from cells containing the classical form of trophospongium (figs. 4 to 10). In the second place, ring-like canaliculi are not new. Cattaneo (1914), in a paper on the canalicular apparatus of ovarian ova, figures appearances not unlike those under discussion, except that he shows no bodies within the canaliculi; and Bensley (1910) has shown very similar spaces in the root-tip cells of the onion. The ring- like structures are so beautiful and so striking that one wonders why they have not been emphasized long ago by some of those who have studied the ovary. Still, they require the best of fixation, and in the usual eosin stain are not as visible, as with Mallory's connective- tissue stain and others which give a strong coloring of the cytoplasm. Yet I think they have been seen before and misunderstood. For instance, J. G. Clark (1898) states that the lutein cells of swine become full of vacuoles and their cytoplasm becomes shrunken, but he was probably actually describing the exoplasmic vacuoles, which we shall see to be evidences of cellular activity, not of senescence.

There is still another important histological element of the lutein cell to be considered, namely, certain granules of the endoplasmic zone which were described by Cesa-Bianchi in 1908. He found, in the perinuclear portions of the cell, a large number of densely packed granules about 1 micron in diameter. To demonstrate them, since the multitude of cytoplasmic structures makes them difficult to see in the living cell, he fixes the tissue in Zenker's fluid or saturated mercuric chloride, sections it in paraffin, and stains with any one of a number of staining combinations, especially hemalum-safranin light green, iron hematoxylin, and Mann's complicated methyl-blue-eosin stain. I have repeated the Zenker safranin light green method, but I find that the granules show up fairly well in formalin tissue stained with hematoxylin and eosin, and very well with Mallory's triple connective-tissue stain; indeed, I had observed them in my preparations before I knew of Cesa-Bianchi's work. In tissue fixed with osmic acid they appear as dark-gray granules against the light-gray endoplasm (fig. 9, c). With Mallory's stain they generally take the orange constituent of the mixture and appear as bright round bodies against a blue-gray background (fig. 7, c) ; but when the sections take the aniline blue ingredient of the stain more strongly— as they sometimes do, with the result that the nucleus becomes blue— it is interesting to note that the endoplasmic granules also stain blue. Cesa-Bianchi's figures,


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 70

which are rather diagrammatic, show the granules clustered closely about the nucleus, bul I generally find them more or less diffusely scattered through the endoplasm. When the endoplasm reaches to the periphery, as it does in the later stages, then consequently the granules are seen throughout the cell. As to their nature and function we can only guess. As they are not dissolved from fixed tissues by ether, alcohol, or water, and because they are preserved by mercury salts, Cesa-Bianchi thinks they are of albuminous nature. I see no reason to think otherwise, although one hesitates to speculate on the chemical nature during life of minute objects which one does not see until they have been through •">(• to 70 changes of reagents. It has impressed me strongly that the staining reactions of the endoplasmic granules are the same as those of the peculiar rings found in the exoplasm, Now, in the life-history of the lutein cell, as we shall see later, the exoplasm recedes as tin' endoplasm becomes wider, and the granules seem to increase in proportion to the amount of the endoplasm. It occurs to me that possibly the two structures are but stages in the same process. Cesa-Bianchi presumes the granules to be connected with the supposed phenomena of internal secretion of the corpus luteum, and that his findings support the theory of an internal secretion; a view based of course on analogy, but one which must be kept in mind.

In the corpus luteum of the sow I do not observe the yellow granules of pigment which are said to be plainly visible in the corpus of the cow, and concerning which the remarkable discovery has recently been made by Escher (1913) that in chemical composition the sub- stance is identical in all reactions with the lipochrome body carotin, found in carrots and green leaves.

LIFE HISTORY OF THE LUTEIN CELL OF PREGNANCY.

In the earliest corpus luteum of known age of pregnancy in my series of sections, the lutein cells are fully formed and contain the peripheral ring-apparatus in a high state of development. In discussing them at an earlier stage we tread upon debatable ground. In a specimen to be described at length (p. 88, figs. 21, 22, 23), as an illustration of the interesting anomaly known as partial accessory lutein-cell formation, the cells of the granu- losa of the follicle in question, at the point where the supposed lutein-cell formation is taking place, are swollen, possess vesicular nuclei, and contain in their cytoplasm very definite canals having within them a few minute hyaline rings. The resemblance to the fully formed lutein cell is striking.

In corpora lutea of pregnancy of 20 to 25 mm., all the lutein cells are of about the same size, are rounded in outline, and possess round vesicular nuclei relatively rich in chromatin. The endoplasmic zone is almost nil, being crowded out by the great development of the exoplasmic formations. The latter are composed of the ring-like structures previously described, in a high stage of perfection. At this time the cells contain considerable quan- tities of osmic-blackening substance, which almost fills some of the cells with good-sized globules of varying magnitude, besides occurring, as shown by Cohn, in tiny droplets at the centers of the spherical structures of the peripheral vacuolar apparatus (fig. 6, a).

As the cells grow older the following changes take place, as shown in figures 4 to 15:

(1) The nuclei become paler by reason of the lessening of the chromatin.

(2) The ring-like formations give place to less elaborate forms; that is, to simple clefts and V-shaped spaces in the cytoplasm, arranged concentrically to the nucleus. In corpora lutea of old pregnancies (150 to 290 mm.) no sign of the once elaborate structure is to be found in most cells.


80 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

(3) The fat content changes, at first decreasing. In the cells of the type most abun- dant at the middle of pregnancy, in which the endoplasmic zone occupies half or more of the cytoplasmic area, the globules of osmic-blackening material are found in amount much less than at previous stages, and clustered at the margin between exoplasm and endoplasm. Toward the end of pregnancy, when the fetuses are 200 mm. or more in length, the lipoid material has practically disappeared from the cell, being found, if present, as a few globules at the periphery. But still later, in the corpora lutea of pregnancies of 270 mm. or longer, many cells contain one or two globules twice as large as those previously seen, and which are often observed in ordinary sections as large vacuoles, sometimes as large as the nucleus, in the cytoplasm. This later deposition of fat I take to be a sign of senility of the tissue, whereas the large amount of lipoid in the early lutein cell would seem to be correlated with physiological activity. I have already mentioned the similar conclusion of Van der Stricht regarding the corpora lutea of the bat. It is very interesting to note that the occurrence of fatty substances in the connective-tissue cells and in those cells which I have called type 1 is in inverse ratio to that of the lutein cells. At 20 mm. only relatively few and very small osmic-blackening granules are found in the spindle cells, but as pregnancy advances fat is deposited in larger and larger masses until at 250 mm. the nuclei of nearly all the interstitial connective- tissue cells are hidden by the large, closely packed fat globules.

(4) Another change with advancing age of the fetuses is in the chromatic granules of the endoplasm, which appear to grow larger and more apparent, reaching sometimes a diameter of 2 microns.

Throughout all this change the size of the lutein cell remains practically the same.

PREVIOUS OBSERVERS ON THE CORPUS LUTEUM AT VARIOUS STAGES OF PREGNANCY.

While it has been recognized in a vague way by some authors that the corpus luteum is not the same at all stages of pregnancy, others hold that after the organ is formed it maintains its structure in the same state until delivery or at least until retrogression sets in, as, for instance, Ravano (1907) and R. Meyer (1911). At any rate, the attempt has never been made to trace the changes carefully and to relate the corpus to the stage of the fetus at all times of pregnancy. In general, investigators have recognized merely three stages of the corpus luteum, namely formation, maturity, and retrogression. Robert Meyer would divide the first stage into two, proliferation and vascularization. Cesa-Bianchi, on the basis of his work on the evidences of internal secretion in the corpus luteum of swine, mares, and cows, mentioned above (1908), finds three stages of the lutein cells. In the first or prepara- tory period, the lutein cells are of relatively small dimensions (15 to 20 microns), with clear and regular contour, round or oval form, nucleus almost central, with rich chromatic net and marked nucleolus, protoplasm homogeneous or uniformly granular, not presenting granules, vacuoles, or inclusions. This description does not fit the stage of formation as I find it in the sow, but it is not easy to say at what time of pregnancy Cesa-Bianchi places his first stage, as he does not give full measurements. His second period is characterized by the presence of abundant granulations in the cytoplasm. The lutein cells are 30 to 40 microns in diameter; in form they are irregularly polyhedral, with excentric nuclei which are vesicular, poor in chromatin, and possess numerous nucleoli. The cytoplasm presents two zones: the endoplasm, directly encircling the nucleus and filled with the chromatic granules described by him; and the exoplasm, presenting vacuoles of varying size. The third period is characterized by the presence in the cytoplasm of numerous drops of fat.


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 81

In 1910 Delestre published, from the Clinique Baudeloquo, the results of his studies in this direction, using the cow as the subject of his work, on account of the fact that the term of pregnancy is very nearly that of the human, being 284 to 285 days. Unfortunately his youngest corpus luteum of pregnancy dated from about 2\ months, the snout-rump length of the calf being 12 cm., so that he missed the very stage at which the exoplasmic apparatus is at its height, if the sow's corpora lutea are like the cow's. At this stage he describes four cellular elements of the corpus luteum: (1) The lutein cells, presenting no particular feature. (2) Cells somewhat larger than the lutein cells, having lightly staining protoplasm. I do not know what these may be, as I have not seen them in the sow. (3) Plasmodial masses with 5 or 6 nuclei, destined to form young lutein cells. I have not seen these in the sow. (4) Cells which, if I interpret the description rightly, are like those mentioned above as additional cells of the corpus luteum, type 2. Delestre states that at 4 months the plasmodial masses have disappeared, giving rise to young lutein cells, which are smaller than adult lutein cells, and are grouped, several together, in one mesh of the connective tissue. There are now two sorts of lutein cells, one containing fat, the other free of fat. At 4^ months the number of cells containing fat is augmented and the young cells are rare. At 5 months all the young cells have disappeared and the adult features predominate, most of the cells containing fat. At this time signs of degeneration appear, in the form of vagueness of outline of some of the cells, poor staining reaction, and dried-up skeleton-like nucleus.

Delestre gives a comparative table of the characteristics of the corpus luteum at the beginning and end of pregnancy, which in most particulars applies equally well to the sow, although I can not confirm his account of the formation of young lutein cells from plasmodial masses, nor the description of degenerating cells at such an early time. With these simple outlines of the life-history of the corpus luteum as described by previous investigators, and with the extensive cytological data now at hand, we have the tools for the main part of our task, which is to relate the changing anatomy of the corpus luteum to the stages of advancing pregnancy.

CHARACTERISTICS OF THE CORPUS LUTEUM AT THE VARIOUS STAGES OF PREGNANCY.

I had hoped that the gross characteristics of the corpus luteum would be of help in the problem at hand, but I have been forced to the conclusion that the differentiation of corpora lutea of varying ages is only to be made with the oil-immersion lens. The corpus luteum of the youngest pregnancy in my series is already solid, so we gain nothing from the size of the central cavity, as we might in other species, such as the human, in which the cavity lingers longer. The blood-vascular system changes little during pregnancy, since there is the same amount of tissue to be supplied. The color is variable and gives no dis- tinction of age. Sometimes in early corpora lutea the rapid increase in volume of lutein tissue causes a bulging of the inner substance of the corpus luteum through the point of rupture, the so-called "Propf" of the German writers. If we find an ovary in which the corpora lutea possess "Propfen," or several of the corpora contain slight remains of the former central cavity in the form of small spaces containing old blood, then probably the ovulation was fairly recent; but, on the other hand, in pregnancies of the first month the corpora lutea are often solid and homogeneous.

We must resort, then, to the immersion lens and a study of finer histological details. Because it will make the results more convincing, I shall mention briefly the method by


82


THE CORPUS LUTEUM OF PREGNANCY IN SWINE.


which the material was studied. As stated in the introduction, all the specimens had been prepared in the same way. All the specimens from pregnancies of 20 to 25 mm. were taken together and carefully studied, then all those from pregnancies of 50 to 55 mm., and so forth; and in this way was obtained an idea of the nature of the corpus luteum at a series of stages from an early period to term. Next the specimens of intervening length were examined, and finally from these observations it was found possible to divide the life of the corpus luteum into a number of definite periods according to the length of the fetus. When I was sure of being on firm ground, I began to go through box after box of sections, registering a diag- nosis of the age of each corpus luteum, and then looking it up in the records. With increas- ing experience it became possible to place the specimen in its proper period practically every time, never varying from the correct stage by more than one of the seven periods of preg- nancy. Of course these periods into which I have found it possible to rank my specimens are purely empirical, and represent lines which I have drawn at the limits of my ability to distinguish the stages of the cytological changes. I had at first several more periods inter- polated between those given, but found that I could not regularly distinguish them. In short, it can generally be told, by examination of a section of a corpus luteum, on the basis of distinguishing characteristics to be given below, in which one of the following periods of pregnancy the specimen falls:


Preparatory period

Exoplasmic development, first part. . Exoplasmic development, seeond part..

Transition period

Endoplasmic development, first part Endoplasmic development, second part Beginning retrogression


Length of


Less than 20 20 to 30 30 to 55 55 to 140 140 to 170 170 to 220 220 to 290


Needless to say, the corpus luteum, being an organic body, presents considerable variety of structure, so that these periods overlap, and pass gradually into each other, in such a way that, besides the specimens which fall near a border-line and hence are naturally difficult to place, a small number give an appearance which experience shows is normal to corpora lutea of older or younger age, just as in a series of embryos a few will exceed or fall behind the others in development.

CRITERIA OF AGE OF THE CORPUS LUTEUM. First period: preparation.— Pigs less than 20 mm. long, duration of pregnancy less than 25 days (fig. 4). The corpus luteum of pregnancy does not attain full size until the twentieth day or thereabouts, but variation in size is so marked that no dependence is to be placed on dimension as a sign of early corpora lutea. On section the following features may be noted : The tissue is very densely packed, so that the nuclei of the slender connective- tissue cells seem relatively numerous. The septa of connective-tissue fibers are rather marked, but are narrow and compressed, and still preserve the radial arrangement in which they invaded the space. Between them are packed the lutein cells, which are polymorphic, but usually elongated in the direction of the radius of the corpus luteum; they are much more varied in size than in the succeeding stages. The nuclei are more chromatic than they will be found later. The ring-forms occur in every lutein cell, and are so large and


THE CORPUS LUTEUM OF PREGNANCY IX SWINE. 83

numerous that there is practically no endoplasm. Fat is present in greal quantity in the lutein cells, as in the next stage, under which it will be described. In the interstitial con- nective-tissue cells of the corpus luteum there is a moderate quantity of fat in fine granules.

Second period: height of exoplasmic development. Pigs 20 to 30 nun. long, approximate duration of pregnancy 25 to 30 days (fig. 5). The striking feature of this stage is that all the lutein cells are in the same state. All tend to be rounded in outline, all are of the same size, and show a uniform development of the exoplasmic apparatus, with ring-forms in every cell. The endoplasm is still very limited in amount. The nucleus is not so chro- matic as in the previous period. Osmic-blackening substance is present in considerable amount in the lutein cells, in some cases almost filling the cells with globules of diameters varying from 2 to 5 microns. In the most typical cases the globules are thickly clumped at one end of the cell, or at both, but there is often a cluster of fat globules about the nucleus; indeed, at this stage the distribution of fat is almost general, at least in the lutein cells. However, the connective-tissue cells show little fat; in osmic preparations they show at most a few small black granules (fig. 6).

Third period: height of exoplasmic development, second part. — Pigs 30 to 55 mm. long, approximate duration of pregnancy 30 to 40 days (fig. 7). The previous stage passes insensibly into this, in which in many cells the exoplasmic structure is more varied in form; some cells contain the ring-forms (fig. 7, a), others present irregular channels and clefts (fig. 7, b). The endoplasmic zone about the nucleus can now be seen, and in a very few cells reaches to the periphery, in which case there are no peripheral spaces in the cytoplasm. In the endoplasm are seen the chromatic granules described by Cesa-Bianchi (fig. 7, c).

Fourth period: transition. — Pigs 55 to 140 mm. long, approximate duration of preg- nancy 40 to 75 days (fig. 8). The ring-structures are disappearing, and become rare in the latter half of this stage. I have seen them but once or twice in ovaries from pregnancies above 140 mm., and therefore set that as the upper limit of the period. It is noted that the progressive changes of the cells are a little earlier at the periphery of the corpus luteum than at the center, and hence estimates of the age of the corpus luteum must be based upon a study of the whole area, giving more weight to the state of the peripheral tissue. In the earlier half of this period one sees an occasional cell without any exoplasmic clear areas or channels; in other words the entire cell, from nucleus to border, is occupied by the homo- geneous endoplasm. Such cells become fairly common toward the latter half of the period, and in some fields of the lens may form half of the lutein cells. Fat has decreased in quan- tity in the lutein cells, where it is found chiefly at the border between endoplasm and exo- plasm. In the lutein cells it occurs in globules of varying size (fig. 9, a), but in the con- nective-tissue cells in smaller granules only (fig. 9, b).

Fifth period: endoplasmic development. — Pigs 140 to 170 mm. long, approximate dura- tion of pregnancy 75 to 105 days (fig. 10). A marked change has taken place in the corpus luteum, although it is not strikingly shown in the figure — a change which has for its most obvious feature a great increase in the diversity of the cells. Although the ring- forms have disappeared from all but a rare cell or two, yet many cells show considerable peripheral canalization. Next to such a cell may be a lutein cell which shows only endo- plasm (fig. 10, a). Moreover, there has been an increase in the amount of connective tissue, so that in many sections the lutein cells are somewhat spread apart. Between them, adding to the diversity of appearance, lie the several kinds of cells which we have mentioned as members of this complicated structure, namely, the branching or spindle cells of the connective tissue, with their product, the reticular fibrils; the darkly staining cells which


84 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

I have called type 2; and, most notable of all, many cells of that type which I have called additional cells of the corpus luteum, type 1, which, with their nuclei, more chromatic than that of the lutein cell, and their homogeneous cytoplasm, resemble the "theca lutein cells" of other writers (fig. 10, b). With the increase of the connective tissue, penetrating between and spacing apart the true lutein cells, these smaller, homogeneous cells are found much more generally scattered through the corpus luteum than before. In osmic prepara- tions they are often loaded with large densely-packed black granules (fig. 11, a). Others of the interstitial cells of the corpus luteum, of all types, contain only fine, diffusely scattered black granules (fig. 11, b). In the lutein cells themselves the amount of osmic-staining matter is progressively lessening; the globules are found in a narrow zone at or near the periphery of the cell. I think that most of the vacuoles seen at the border of the cell in Mallory's material at this stage are due to the fatty substance, and that the lesser number only are due to remains of the once extensive peripheral canalicular system.

Sixth period: endoplasmic development, second part. — Pigs 170 to 220 mm. long, approxi- mate duration of pregnancy 105 to 110 days (fig. 12). The diversity of staining reaction is less marked than in the preceding stage, and the diversity of size of the cells is less notice- able, because in the first place the lutein cells have lost the peripheral vacuolization, while, secondly, the smaller interstitial cells of the corpus luteum have grown in size, and therefore the two types of cells so resemble each other that it is difficult to distinguish them. Fat is very slight in amount and limited to the periphery of the lutein cells, where a few granules are found. The interstitial cells of the corpus luteum are often loaded with large fat globules (fig. 13).

Seventh period: beginning retrogression. — Pigs 220 to 290 mm. long, approximate dura- tion of pregnancy 110 days to term (fig. 14). The only difference between this stage and the preceding is that here the lutein cells frequently contain one or more fat globules larger than any previously seen, which are found near the periphery of the cell, and at first in cells near the periphery of the corpus luteum (figs. 14, 15). The oldest corpus luteum of my series (290 mm.) contains many such vacuoles.

THEORETICAL CONSIDERATIONS CONCERNING THE LIFE-HISTORY OF THE CORPUS LUTEUM.

I have purposely omitted theoretical discussion from the foregoing description, because I do not wish false analogy or faulty deduction to belittle the value of these observations as criteria for determining the age of lutein cells of pregnancy. I may be entirely wrong in ascribing to the cells which I have called type 1 an independent existence and a classifica- tion different from the true lutein cells, and in thinking that they resemble the theca lutein cells of the descriptions of Rabl, Cohn, and R. Meyer. Those who believe that the origin of the lutein cells from the theca interna alone is incontrovertibly established will simply say that, as all the cells of the corpus luteum arise from one source, what I have described are merely stages or variants of one type of cell. On the other hand, those who adhere to the now widely accepted view that both layers of the follicle enter into the forma- tion of the lutein cells should see no difficulty in my tentative proposition that, in the sow at least, the theca cells perhaps persist partly as distinct cells throughout pregnancy. For, as I have mentioned, Rabl, Cohn, and R. Meyer have thought them to persist well into pregnancy, and Van der Stricht indeed thinks that they persist throughout pregnancy as cells of internal secretion, perhaps less active than the true lutein cells.


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 85

As to the lutein cell itself, the present findings, that the cells are apparently in a high state of organization from early pregnancy to term, would seem to give some strength to the contention that the corpus luteum has a function of importance in pregnancy. Empiri- cally it has been shown clearly that extracts and powders of corpus luteum have a beneficial effect in certain disturbances of the reproductive system in women (see Burnam (1912) for full account and literature to date) ; and it has been found that extracts of corpus luteum when injected into dogs promptly cause a lowering of the blood-pressure. None of those who have worked in this field, so far as I know, have tried the effects of corpora lutea of definite age. There is, however, certain evidence that the corpus luteum during the first half of pregnancy has a different potency than in the second half, namely, the fact dis- covered by Fraenkel (1903) and confirmed by Marshall and Jolly (1905), Niskoubina (1909), Dick and Curtis (1912), and others, that extirpation of the corpus luteum during the first half of pregnancy is almost invariably followed by abortion. This fact, which seems so true for guinea-pigs and rabbits, has been flatly denied to hold good in animals by Daels (1908), and for human patients by Essen-Moller (1904), Graefe (1905), Flatau (1907), and Sokoloff (1913), while Puech and Vanverts (1913) propose the compromise statement that the corpus luteum is useful in an accessory way, but not absolutely necessary to the embedding and early development of the human embryo. Unfortunately for our present discussion, the experiment has not been tried on sows, which are not convenient laboratory animals. I agree with Cohn and Van der Stricht that the microscopic appearance of the lutein cell seems to favor the theory of an internal secretion of the corpus luteum, and I would add that the corpus luteum, while apparently in a high state of activity from the beginning to the end of pregnancy, shows greatly different forms of cell- structure in the earlier and later months. We may hope that this hint will be put to further test by experiment and clinical observation.

DISTINCTION BETWEEN THE CORPUS LUTEUM OF PREGNANCY AND OF OVULATION. The history of the corpus luteum for 350 years has been a curious mingling of truth and error. Discovered by Volcherus Coiter in 1573, the corpus luteum was thought by Regner de Graaf to be an evidence of pregnancy or of previous child-bearing. Abernethy and Sir Astley Cooper swore away the innocence of a dead woman in a court of law because one of her ovaries was found at post-mortem to contain a corpus luteum. About the earlier decades of the nineteenth century it became known that every ovulation is followed by the formation of a corpus, and then arose the still unsettled discussion as to whether the corpus luteum of ovulation can be distinguished from the corpus of pregnancy. Interesting accounts of the earlier phases of this question are found in Dalton's Prize Essay of the Philadelphia Academy of Sciences for 1851, and in Taylor's "Medical Jurisprudence." Next the microscope was called to aid, with little effect. De Sin6ty (1877) believed the two sorts of corpora to be exactly alike. The same statement is made by Ravano (1907). Marshall (1910) sums up his opinion in the statement that the two kinds of corpora are in the earlier stages identical, and otherwise essentially similar. Niskoubina (1909) thinks that the corpora lutea of pregnancy of rabbits contain more fat than the corpora of ovula- tion, but Fenger, working on the ovaries of cows, analyzed hundreds of corpora lutea, and found the fat content equal (1914). Miller (1914), in the latest publication upon this subject, holds that the corpora lutea of pregnancy (human) may be distinguished from those of ovulation by the absence of fat-reaction, of colloid degeneration, and of deposition of


86 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

calcium. It is plain that the divergent opinions upon the question are due to incomplete knowledge of the corpus luteum at all its stages. Meanwhile the clinicians are beginning to put the matter up to the anatomists anew, for instance, with such statements as that of Dannreuther (1914), that the dried corpus luteum of pregnancy is much more efficient therapeutically than the corpus luteum of ovulation.

I have sections of ovaries from about 25 uteri which did not contain embryos, many of them having been carefully examined by another investigator who was searching for early embryos. No doubt a few of them are from pregnant animals after delivery, so that the corpora lutea may not all be those of ovulation. At any rate, it can be said with certainty that the presence of embryos in the uterus is to be diagnosed by microscopic examination of the ovary. The difference between the corpus luteum of pregnancy and that of ovulation is like the difference between an army and a mob. In the corpus luteum of pregnancy there is a regularity of structure which is lacking in thai of ovulation. For instance, in the latter we find cells having highly developed exoplasm side by side with others having a smooth undifferentiated cytoplasm and large fat-vacuoles in them (fig. 17). The fat-vacuoles of the connective-tissue cells are usually much larger and more numerous than in the corpus luteum of pregnancy, so that the sections are riddled with holes. It appears likely, though it can not be proven, since we have no way of determining the age of a non-pregnant corpus luteum from the slaughter-house, that retrogression sets in before the corpus is fully formed, and affects the cells in an irregular way, so that they do not all progress through the stages of their life history at one time. Whether or not there are differences of note in the first 15 days I can not say, as I have no data for corpora lutea of pregnancy in the first two weeks. After this period the two sorts of corpora lutea can be distinguished from each other.

INFLUENCE OF PREGNANCY ON THE STRUCTURE AND FUNCTION OF THE GRAAFIAN FOLLICLES.

It has been known for a long time that pregnancy interrupts the function of the corpus luteum; that in the presence of an embryo in the uterus there is normally no ovulation and therefore no formation of new corpora lutea. In animals which undergo rut or men- struation these phenomena are also interrupted by pregnancy.

To understand the causes underlying the non-occurrence of ovulation during preg- nancy, we must know the life-history of the normal follicle. Fortunately this is a subject upon which there is substantial agreement between all observers. As is well known, the fully formed resting follicle consists of three layers: the membrana granulosa, of presumably epithelial origin; the theca interna, probably of mesenchymal origin; and the theca externa, of mesenchymal origin. The exact histological characteristics of the three layers vary slightly in different animals, but in the pig the granulosa is composed of 3 to 10 layers of rounded cells (the lowest layer, next the theca, is columnar) with round, darkly staining nuclei and little cytoplasm (fig. 16, a). The theca interna is composed of 5 to 10 layers of short, spindle-shaped cells, with moderate amount of cytoplasm and round, or oval, nuclei, not so chromatic as those of the granulosa (fig. 16, b). The theca externa is made up of definitely spindle-shaped cells of connective-tissue derived from the ovarian stroma (fig. 16, e).

Now, one of two fates awaits the unripe follicle in the course of time. It may never attain ripeness, but instead may undergo degenerative changes leading to obliteration.


THE CORPUS LUTEUM OF PREGNANCY IX SWINE. 87

The process of destruction, which is called atresia, consists of the degeneration of the granulosa cells, shrinkage and disappearance of the ovum, and filling of the cavity by enlarged cells of the theca interna, so that the atretic follicle resembles a corpus luteum, and like the latter is finally replaced by a hyaline scar. On the other hand, the follicle may proceed to ripeness, in which case the granulosa tends to range its cells in rather marked layers, and the cells of the theca interna increase in number, show mitoses, become polyg- onal, swell in size, and have deposited in them numerous fat granules, so that altogether they show a notable resemblance to lutein cells (fig. IS, b). Next, through causes not yet understood, the follicle bursts, the ovum is extruded, and a corpus luteum is formed. Should rupture not occur, atresia may take place in the ripe as well as the unripe follicle. As one of the causes of the non-ovulation during pregnancy, it has been pointed out, especially by De Sinety (1877), Stratz (1898), and Schulin (1881), that atresia of the follicles tends to be very marked during pregnancy. Sandes has confirmed this in the marsupial Dasyurus (1903). In the pig it is certainly true. I have not a sufficiently large number of non-pregnant ovaries to make a numerical comparison, but it can be stated that a great part of the larger follicles in pregnant animals are degenerating. Yet in very many ovaries of pregnancy fairly large intact follicles are found. Very interesting in this con- nection is the observation of Pearl and Surface (1914) that ovulation in the domestic fowl can be inhibited by the administration of corpus-luteum extracts.

It has been suggested by some that the follicles do not grow to full size during preg- nancy, and that hence the crop of follicles which gave rise to the embryos in uU ro is not followed by another crop until pregnancy is terminated. This view has been studied by Leo Loeb (1906). He reports that in many of his animals the follicles went on to full size during pregnancy, but that they did not rupture, as they would in the absence of pregnancy. It is unfortunate that microscopic studies were not made, for, as pointed out by Robert Meyer, in the human ovary mere size of the Graafian follicles is a faulty criterion of their state; those which appear large and ripe may in fact be in a stage of cystic atresia.

The diameter of the ripe follicle of the sow is said by Kaeppeli to be 5 to 8 mm., and in this statement he is followed by Schmaltz (1911), but I believe that normal ripe follicles may grow much larger than 8 mm. Eenckiser (1884) included in his studies on the origin of the corpus luteum a perfectly normal ripe follicle 1 1 mm. in diameter. I collected 24 pairs of ovaries at random, without selection except to exclude specimens containing cor- pora lutea and cystic formations. The largest follicles in each ovary varied in diameter from 1 to 10 mm. ,On microscopical examination even the largest follicle of these ovaries proved to be normal and ripe. The lower limit of ripeness is difficult to set. I have seen 3 mm. follicles which were apparently ripe; and a recent corpus luteum of ovulation may be as small as 5 mm., showing that the follicle from which it proceeded must have been no larger. In the hundreds of ovaries I have examined I have seen many such large follicles, and I have no doubt that had they been examined microscopically they would have proved normal.

As a contrast to this, in my entire series of ovaries from pregnant sows no follicles were found having an outside diameter of more than 6 mm. with two exceptions, and I did not find ripe follicles in any ovary of the series. The conclusion is simple: in pregnancy the Graafian follicles may attain or maintain a size at which, in the absence of pregnancy, ripening and rupture might occur. But they do not usually attain the larger diameters reached by follicles in the non-pregnant ovary; and when they do happen to reach large size, they are found to be either unripe or atretic.


88 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

Fellner (1909) believes that in the human ovary a few follicles may remain ripe or become ripe during pregnancy. Ravano (1907) has taken the still more advanced stand- point, based on the study of ovaries from 60 pregnant women who came to the operating table or to post-mortem during pregnancy. He found many follicles "approaching ripe- ness," and also thinks that he had three cases in which ovulation had occurred during pregnancy. These were merely cases in which two corpora lutea were found without twin pregnancy ; and the microscopical details given are so slight that I do not think these cases are sufficient evidence on which to base such a statement as Ravano's that ovulation and corpus luteum formation subsequent to that which gave rise to the fetus occur in 5 per cent of all human pregnancies. In spite of these cases I retain the opinion that the rupture of a Graafian follicle in an ovary containing a corpus luteum of pregnancy during term is an extremely rare occurrence. Keller (1913) did not find a case in 24 human cases, Fellner ( 1909) in 13 cases, nor Seitz (1905) in 36. Ruge (1913) studied 106 human ovaries without finding a freshly ruptured follicle, and he thinks the presence of a recent corpus luteum excludes the possibility of the rupture of a follicle. The case which I have to report seems to be one of those classic exceptions which prove a rule.

The uterus of specimen No. 70 contained three fetuses whose crown-rump length measured 70 to 75 mm. The left ovary contained no corpora lutea; the right ovary con- tained 8 prominent corpora lutea about 11 mm. in diameter and a number of follicles measuring 5 mm. in diameter. Between one of the corpora lutea and one of the follicles was found a body of ovoid shape about 2 by 2 by 3 mm. in size, which presented on its sur- face a crater-like scar covered by a few tags of fibrin, resembling exactly the healing point of rupture of an ordinary corpus luteum, but only about 0.5 mm. in diameter (fig. 19, a). A section of this structure showed that it was indeed a recently ruptured follicle (fig. 20). The granulosa was several layers thick, of normal appearance, and intact except at its point of rupture, where it and also the theca were replaced by a recent scar. The granulosa was rather wavy in contour (fig. 20, a), and beneath it lay the theca interna, thicker than usual (fig. 20, b). If I interpret the appearance aright, neither layer showed signs of con- version into lutein cells. I believe that this unusual specimen is due to an accidental rupture of a Graafian follicle, probably by pressure between its neighboring growing follicle and corpus luteum.

ABNORMALITIES OF THE LUTEIN TISSUE.

An interesting anomaly of the ovary is partial accessory lutein-cell formation, to which attention was called by R. Meyer (1913) . This consists of the formation, during pregnancy, of lutein cells in atretic follicles or even in ripe follicles, which in some human ovaries is said to go so far that a full corpus luteum may be formed which is finally indistinguishable from the true corpus luteum proper to the pregnancy, which is found in the same ovary or its mate. This phenomenon, at least in its advanced state, must be very uncommon. In the sow I have seen a case of what is apparently the same partial lutein formation in a normal ripe follicle, not during pregnancy, but in the presence of a corpus luteum of ovulation.

The right ovary of specimen No. 136 contains 4 corpora lutea 5 mm. in diameter, which appear on section to be young corpora lutea of ovulation. In addition there is a follicle 10 mm. in diameter, to be described. The left ovary contains 3 corpora similar in gross appearance to those of the right ovary, except that one of them is slightly cystic. On section of the large follicle in the right ovary, it is found that over almost the entire circum-


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 89

ference the wall presents, as would be expected, the structure of a normal unripe follicle. At one point, however (fig. 21, a), for a space of 3 mm. the whole theca interna is doubled or tripled in thickness, its cells show the appearance of ripeness, and have begun to invade the granulosa (fig. 22, a). The granulosa cells, in turn, are 20 to 30 microns in diameter and many of them have vesicular nuclei with prominent nucleoli; they are filled with fat- vacuoles, and some of them have in their cytoplasm the ring-formations of young lutein cells. In short, the granulosa cells are turning into lutein cells (fig. 23).

Seitz (1905) has emphasized the transformation of theca-interna cells of atretic follicles into lutein-like cells as a physiological phenomenon in pregnancy. While I do not consider this process as uniformly present in the sow, as Seitz maintains it is in the human, still we do occasionally find follicles lacking the granulosa, but with the theca interna cells full of fat-vacuoles and enlarged just as in ripe follicles. It is hard to see how it is to be decided whether these were normal ripe follicles at the beginning of pregnancy, which are merely becoming atretic, or whether they were formerly atretic follicles in which the formation of theca-lutein cells is taking place, as Seitz would have it.

Here we come to the border-line between normal and pathological anatomy of the ovary. While my specimens of ovarian cysts were not collected with a view to formal study, still I shall risk presenting a few incidental observations in the hope of calling atten- tion to the unusually promising opportunity offered by the sow's ovary for the study of the pathology of the Graafian follicle and the corpus luteum. Cysts are extremely common, are often multiple, and are frequently found in ovaries containing normal corpora lutea of ovulation, so that comparisons can be made. It needs only time and patience to collect a great number, and the result would undoubtedly give us explanations of much that is obscure concerning these tumors.

In glancing over the 13 specimens of ovarian cysts lined by lutein cells which I have collected, it at once appears that they fall into two classes. First, there are cases in which one of a crop of corpora has merely retained its original cavity beyond the normal time, the cavity has become fixed, and may have become much enlarged. Second, there are cystic cavities in the ovary which are lined by lutein cells, but which we can not as surely say came from the corpora lutea, as they may merely represent atretic follicles in which lutein-cell formation has taken place. One's view of the origin of these cells will depend entirely upon the view of the origin of the lutein cells of normal corpora. As will have been gleaned from this paper, the writer has tentatively come to the belief that the true lutein cells are derived from the granulosa cells, but that the theca interna gives rise to cells resembling lutein cells, which seem in part to remain as special cells in the fully formed corpus luteum, but most of which are lost, either by reverting to connective-tissue cells of the ordinary kind or by becoming themselves genuine lutein cells. On this theory, we should have the following kinds of lutein-cell cysts:

I. Arising in corpora lutea which have remained cystic or have undergone cystic degeneration. II. Arising in follicles:

a. Accessory lutein-cell formation from both layers of normal follicles, as described by

Meyer.

b. Accessory lutein-cell formation from the theca alone, as in atretic follicles.

It is of clinical importance to know something about the life-history of lutein-cell cysts— for instance, whether such a cyst may persist and by its activity inhibit ovulation, as in certain women, in whom persistent corpora lutea seem to cause sterility. Now, the


90 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

lutein cells in cystic tumors are not often as well preserved as in normal tissue, but I believe that in all the specimens in hand the lutein cells are of the same age as those of the normal cor- pora in the same ovary. I have not seen lutein-cell cysts containing active-looking cells of different age from the corpora lutea present. Presumably, then, the lutein cells of cysts are as transient as normal lutein cells, and in cases where the cystic formation persists, the lutein cells lining it probably revert or disappear, so that finally the cysts can not be distinguished from an old Graafian follicle cyst.

In the 128 specimens from pregnant sows of which I have record, there was only one case of lutein-cell cyst. In the same number of non-pregnant ovaries, containing corpora lutea of ovulation, we should find 5 or 10 lutein-cell cysts. For comparison, I have looked up the cases indexed as corpus-luteum cysts in the records of the gynecological service of the Johns Hopkins Hospital. There were 19 cases available, all discovered at operation for other conditions. One patient had passed the menopause (61 years) ; the others were from 16 to 42 years.

8 had never been pregnant.

4 had their last child or miscarriage 6 or more years before.

5 had their last child or miscarriage 22 months to 4 years before. 1 had miscarried 4 months before, at the second month.

1 had miscarried 2 weeks before, at the sixth week.

Apparently, then, the corpus luteum of pregnancy is a comparatively less frequent seat of cysts than that of non-pregnant animals, a fact in accord with the general rule that abnormal alterations are less frequent in organs exercising a needed function.

MIGRATION OF THE OVUM IN THE SOW.

.Since the uterus of the sow is bifid nearly its whole length, it is very easy to count the number of fetuses on each side. When this is done, it is often observed that there are more pigs on one side of the uterus than there are corpora lutea in the corresponding ovary. In a series of 117 uteri, I found 28 in which there was 1 more pig than corpora lutea on a given side, 13 cases with 2 more pigs than corpora lutea on one side, and 2 in which the pigs num- bered 3 more than the corpora lutea of the corresponding ovary.

This phenomenon may be explained in three ways: (1) corpora lutea may not have formed at the site of the follicle which gave rise to the supernumerary pigs; (2) there may have been two or more ova in one follicle, which therefore produced two or more embryos, but only one corpus luteum; (3) one or more ova may have crossed the abdominal cavity and entered the opposite tube (external migration), or passed down to the junction of the uterine horns on its own side and into the opposite tube from below (internal migration).

(1) The failure of a corpus luteum to form at the site of a ruptured follicle is not unthinkable, but must be very rare. In hundreds of sows' uteri I have never seen a case of pregnancy without corpora lutea; nor do I know of any really unexceptionable cases reported from the human or any other of the mammalia which give birth to a single infant, in which such a phenomenon would be more easily detected. Fellner (1909) has mentioned one case in a woman who died on the day of delivery, Ravano (1907) cites four, also from women prac- tically at term, and Miller (1914) one from a case of eclampsia. Although these cases are of interest otherwise, the absence of a corpus luteum at term can not be taken as evidence that one had not been present earlier in pregnancy, especially since all these cases were complicated by disease.


THE CORPUS LUTEUM OF PREGNANCY IN SWINE. 91

(2) In the sow, however, I have found a uterus in which there were T> fetuses, hut only 4 corpora lutea in both ovaries. Such a case, if our first hypothesis he excluded, can be explained only by the second, namely, that two of the pigs originated from a polyovular Graafian follicle, and this latter explanation is based on anatomical facts. Follicles con- taining more than one ovum have been known for many years, and have been recorded from man, the cat, dog, sow, certain bats, and other animals. Ancel (1903) thinks they are especially common in the dog, in which he has found follicles with 2, 3, 4, 5 ova. Arnold (1912), in an interesting report with review of the literature, describes a human case in which a large proportion of follicles were polyovular, some of them having as many as 18 ova. The anatomical origin of the condition is still obscure. The question arises whether the phenomenon is sufficiently common to account for the fact that in swine 37 per cent of uteri have more pigs in one horn than there are corpora lutea in the corresponding ovary. Schmaltz (1911) states that polyovular follicles are seen in almost every preparation of the sow's ovary, sometimes having as many as 6 ova, 4 to a follicle being frequent, but he thinks that such follicles are usually doomed to become atretic and rarely attain maturity. In sections I personally have found only 2 polyovular follicles, one of them with 2, and the other with 3 ova, and these follicles were both small and undeveloped. The members of a class in histology were requested to watch for the condition when studying the ovary of the sow, and one of the students found and showed me 2 ova which he had discovered in a large ripe follicle. The conclusion is that some of the 43 cases under discussion may have been due to the fertilization of ova from polyovular follicles, but that the latter certainly do not occur frequently enough to explain all the cases, since for this it would be necessary for at least one follicle to be polyovular in one-third of all the groups of follicles maturing at once.

(3) On the other hand, migration of the ovum is a well-attested fact, in the human subject, and has been produced experimentally in animals by Leopold (1880), who excised one ovary and the opposite tube, and found that the animals could still become pregnant. Howard A. Kelly performed the same operation in a human patient, for the cure of disease, and the woman later became pregnant. Other interesting examples have been mentioned by J. Whitridge Williams, who found the corpus luteum in one ovary and the embryo in the opposite tube, in 5 out of 30 carefully recorded cases of extra-uterine pregnancy. These are all cases of external migration. The occurrence of internal migration has never been conclusively proven. The frequency of external migration of the ovum is difficult to esti- mate in the human subject, since it is demonstrable only in the presence of some abnormal condition, such as the lack of one tube and the opposite ovary, a bicornate uterus, an extra- uterine pregnancy, or the like. Mayrhofer (1876) estimates tentatively that migration must occur at least once in every ten ovulations in the human female.

That it can take place in the sow I think is proven by four uteri of my series, in each of which one ovary contained no corpora lutea, and yet in two of the cases the side of the uterus corresponding to the ovary without corpora lutea contained 1 fetus, in the other two cases 3 fetuses. In all four cases the total number of corpora lutea in both ovaries accounted for the total number of pigs in both sides of the uterus. In all likelihood we can explain most of our cases by external migration. The point which I wish to bring out is not that migra- tion is a possibility — that has been shown many times — but I want to emphasize its great frequency. For every time it can be demonstrated in the sow, there must be many more times when it can not be detected; for instance, when ova migrate from both ovaries, and hence the condition is not apparent. It is a very conservative estimate if we suppose that one or more ova migrate in 50 per cent of all ovulations in the sow. In a sense, the term


92 THE CORPUS LUTEUM OF PREGNANCY IN SWINE.

migration is a misnomer, for the ovum does not make any great journey to reach the oppo- site tube. I have not had the opportunity to study the relations of the pelvic organs in an adult sow, but in large fetuses the ovaries may be quite close to each other; I have even found them touching. It is likely that the fimbria? of the Fallopian tubes, which in the sow are widely spreading and patulous, frequently reach both ovaries, and hence make it a matter of chance which tube receives a given ovum.

The question of the migration of the ovum has two aspects of immediate practical interest. To the gynecological surgeon it is important to know that the extirpation of one tube and the opposite ovary will not necessarily cause sterility. For the biologist the subject brings up certain questions concerning the determination of sex, which will be mentioned briefly. It is a very ancient theory that the production of two sexes of offspring and their practically equal distribution in number are due to the formation of males by one ovary, of females by the other, the left ovary usually being honored by the ascription of female-producing potencies. This simple hypothesis is capable of proof or disproof by numerous methods, which have not been wanting a trial, the most recent of such experi- ments being those of G. H. Parker (1914), who adopted a very neat and ingenious plan of experimentation. The method was to take an animal possessing a notably bifid uterus, namely, the sow, and count the number of unborn pigs on each side with reference to sex. Parker recognized the possible disturbing effect of migration of the ovum, though I think to a far less degree than the facts warrant; and therefore he took large litters, and omitted from his counts the pigs in the middle of the uterine horns, which may be thought most likely to be the mixed product of the uterine horns, and counted only the two fetuses next to each ovary and the two next the junction of the horns. The result, from the tabulation of 1 ,300 pairs of fetuses, gave an almost absolutely equal distribution of the sexes on the two sides. Now, at first sight it might seem that the great frequency of migration of the ovum, which I have shown to exist, renders Parker's conclusions false, since they are based on the general assumption that the embryos on one side of the uterus come from the corresponding ovary. But, on the other hand, if it is true, as I believe, that the embryos in either horn of the uterus are an inextricable mixture of the products of the two ovaries, then Parker's figures can only be explained by the far-fetched assumption that exactly 50 per cent of the ova migrate, or else that both sexes are equally represented in each ovary; the latter being of course the conclusions already reached by Parker.

CONCLUSIONS.

In the foregoing paper the writer has given an account of the histology of the corpus luteum of the domestic sow, remarking the presence of cells differing from the typical lutein cells.

A description is given of the canalicular apparatus and granules found in the lutein cells, and it is shown that these structures undergo progressive changes during the course of pregnancy.

The microscopic appearance of the corpus luteum is described as it varies with the advance of pregnancy.

The corpus luteum of pregnancy is distinguished from that of ovulation by the more regular and uniform morphology of the former, and the greater infiltration of fat in the latter.

During pregnancy the Graafian follicles do not undergo the process of ripening, or change of the theca interna which is preparatory to rupture.

External migration of the ovum is a normal and very frequent occurrence in the sow.


THE CORPUS LUTEUM OF PREGNANCY IN SWINE.


93


LITERATURE CITED.


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VON Baer, C. E. 1827. Dc ovi mammalium et hominis genesi. 4°, Lipsise, L. Voss, 1827.

Benckiser, A. 1884. Zur Entwickelungsgeschichtc des Corpus luteum. Arch. f. Gynaek., Berlin, 1884, xxin, 350-366, 1 pi.

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von Bischoff, T. L. W. 1842. Entwickelungsgeschichte des Kaninchen-Eies. 4°, Braunschweig, F. Vieweg & Sohn, 1842.

Birnam, C. F. 1912. Corpus luteum extract, with sugges- tions as to its use in gynecologic practice. Journ. Am. Med. Assn., Chicago, 1912, lix, 698-703.

Cattaneo, D. 1914. Richerche sulla struttura del ovario dei mammiferi. Arch. ital. di anat. e di embriol., Firenze, 1914, xn, 1-34.

Cesa-Bianchi, D. 1908. Di alcune particolarita di strut- turi e dei fenomeni di secrezione del corpo luteo. Internat. Monatschr. f. Anat. u. Physiol., Leipzig, 1908, xxv, 1-43.

Clark, J. G. 1S98. The origin, growth, and fate of the corpus luteum as observed in the ovary of the pig and man. Johns Hopkins Hosp. Rep., Baltimore, 1898, vn, 181-221.

Cohn, F. 1903. Zur Histologic und Histogenese des Cor- pus luteum. Arch. f. mikr. Anat., Bonn, 1903, lxii, 745.

C'oiter, V. 1573. Anatomicajexercitationesobservationes- que variae. fol. Noribergise, 1573.

In his: Extcrnarum et internarum principalium (etc.)

Cowdry, E. V. 1912. The relations of mitochondria and other cytoplasmic constituents in the spinal gan- glion cells of the pigeon. Internat. Monatschr. f. Anat. u. Phvsiol., Leipzig, 1912-13, xxix, 473-504, 3 pi.

Daels, F. 1908. On the relations between the ovaries and the uterus. Surg., Gynec. and Obst., Chicago, 1908, vi, 153-159. '

Dalton, J. G, Jr. 1851. On the corpus luteum of men- struation and pregnancy. Trans. Am. Med. Assn., Philadelphia, 1851, iv, 549-644, 4 pi. Also: Re- print, 8°, Philadelphia, T. K. & P. C. Collins.

Dannreither, W. T. 1914. Corpus luteum organo- therapy in clinical practice. Journ. Am. Med. Assn., Chicago, 1914, lxii, 359-362.

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Dick, G. F. and A. H. Curtis. 1912. Concerning the function of the corpus luteum, and some allied prob- lems. Surg., Gynec. and Obst., Chicago, 1912, xv, 588-593.

Escher, H. H. 1913. Ueber den Farbstoff des Corpus luteum. Zeitschr. f. physiol. Chem., Strassburg, 1913, lxxxiii, 198-211.

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Fellner, O. 0. 1909. Zur Histologic des Ovariums in der Schwangerschaft. Arch. f. mikr. Anat.. Bonn. 1909. lxxiii, 288-305.

Fe.vger, F. 1914. Distinction between the corpus luteum of ovulation and the true corpus luteum "f preg- nancy. Journ. Am. Med. \--n . Chicago, 1914, i.xn, 1249-1250.

Flatai. S. 1907. Ueber Ovariotomie wahrend der Schwangerschaft. Arch. f. Gynaek., Berlin, 1907, lxxxii, 452-471.

Fraenkel, L. 1903. Die Funktion des Corpus luteum. Ar.h. f. Gynaek., Berl., 1903, lxviii, 438-545, 1 pi.

Golgi, C. 1898. Sur la structure des cellules nerveuses des ganglions spinaux. Arch, ital de biol., Turin, 1S98 99. xxx, 60-71, 278-288.

de Graaf, R. 1672. De mulierum organis generationi inservientibus tractatus novus. 8°, Lugduni Bat. (Leyden), ex off. Hackiania, 1672.

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1. The wall of a recently ruptured Graafian follicle, showing the granulosa intact. Hematoxylin and eosin. X "• '00.

(6) Granulosa, (c) Theca interna.

2. Additional cells of corpus luteum, type I. Mallory*s stain. X 400.

(a) Lutein cells. (A) Additional cells, type I.

3. Additional cells of corpus luteum. type 2. Mallory's stain. X 650.

(a) Lutein cell. (A) Additional cells, type 2.

4. Corpus luteum, first period. Mallory's stain. X 650.

5. Corpus luteum. second period, Mallory's stain. X 650.

6. Corpus luteum, second period, osmic acid stain. X 650.

(a) Droplet of fat inside one of the peripheral vacuoles.

7. Corpus luteum. third period. Mallory's stain. X 650.

(a) Lutein cell containing ring-form of canaliculi. (A) Lutein cell containing cleft-like canalicular apparatus, (c) Chromatic granules.

8. Corpus luteum. fourth period. Mallory's stain. X 650.

9. Corpus luteum, fourth period, osmic acid stain. X 650.

(a) Fat-globules in a lutein cell. (A) Fat-globules in a connective tissue cell, (c) Chromatic granules.





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10. Corpus luteum, fifth period. Mallory's stain. X 650.

(a) Lutein cell showing no peripheral vacuoles. (A) Additional cell, type I . I I. Corpus luteum, fifth period, osmic acid stain. X 650.

(a) "Additional" cell containing large fat-globules.

(A) Connective tissue cell containing minute fat-granules.

12. Corpus luteum, sixth period, Mallory's stain. X 650.

13. Corpus luteum, sixth period, osmic acid stain. X 650.

14. Corpus luteum, seventh period, Mallory's stain. X 650.

15. Corpus luteum, seventh period, osmic acid stain. X 650.

16. Wall of unripe Graafian follicle, hematoxylin and eosin. X 400.

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1 7. Corpus luteum, of ovulation (non-pregnant sow), hematoxylin and eosin. X 650.

18. Wall of ripe Graafian follicle, hematoxylin and eosin. X 400. (A) Theca interna.

19. Specimen No. 70, showing a Graafian follicle ruptured during pregnancy. Hematoxylin and eosin. X 3.

(a) The ruptured follicle. (A) An unruptured follicle, (c) A corpus luteum of pregnancy.

20. Wall of follicle shown in Figure 19. Hematoxylin and eosin. X- ca. 100.

(a) Granulosa. (A) Theca interna.

21. Specimen No. 136, a Graafian follicle showing partial accessory lutein-cell formation. Hematoxylin and eosin. X 3.

(a) Point at which the accessory lutein formation occurs.

22. Showing the area marked a. Fig. 21, enlarged 70 diameters. Hematoxylin and eosin. X 70.

23. Four cells of the granulosa of the same specimen, showing ring-like vacuoles in the cytoplasm like those seen in lutein

cells. Mallory's stain. X600.

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