Talk:Book - Contributions to Embryology Carnegie Institution No.62

CONTRIBUTIONS TO EMBRYOLOGY, No. 62. FURTHER EXPERIMENTAL STUDIES ON FETAL ABSORPTION.

III. THE BEHAVIOR OF THE FETAL MEMBRANES AND PLACENTA OF THE GUINEA- PIG TOWARD TRYPAN BLUE INJECTED INTO THE MATERNAL BLOOD- STREAM.

IV. THE BEHAVIOR OF THE PLACENTA AND FETAL MEMBRANES OF THE RABBIT TOWARD TRYPAN BLUE INJECTED INTO THE MATERNAL BLOOD-STREAM .

By George B. Wislocki, Of the Anatomical Laboratory, Johns Hopkins Medical School. With one plate.

III. THE BEHAVIOR OF THE FETAL MEMBRANES AND PLACENTA OF THE GUINEA-PIG TOWARD TRYPAN BLUE INJECTED INTO THE MATERNAL BLOOD-STREAM

Our knowledge of the behavior of vital dyes in the pregnant animal rests upon the careful observations of Goldman (1909) upon mice and rats. He observed that when vital dyes, such as trypan blue and pj^rrhol blue, were injected into the maternal circulation, they were not transmitted through the placenta to the fetus. The barriers which prevent the dye from entering the fetus are the chorionic ecto- derm of the placenta and the epithelium of the viteUine membrane. In the rat and mouse the latter constitutes the outermost fetal covering. He also noted the interesting fact that the placenta acts as an attraction center for vital dyes, since, with the initiation of pregnancy in an animal which has been vitally stained, the dye is to a great extent given up by the liver, spleen, and other tissues in which it is stored and is conveyed by the blood-stream to the developing placenta, where it is immediately absorbed by the chorionic epithelium and the endodermal cells of the vitelUne membrane.

The present studies, which are a continuation of a series of investigations on fetal absorption (Wislocki, 1920), concern the behavior of the placenta and fetal membranes of the guinea-pig and rabbit toward trypan blue. Although similar in many respects to the observations of Goldmann on the rat and mouse, there are differences sufficiently great to give interest to a description of the process of vital staining in these rodents.

The guinea-pigs used in these experiments were stained by the technique usually employed in administering vital dyes. There was no opportunity to study the placenta and fetal membranes in the early stages of their development, and the following description applies only to the period from the time the fetus reaches a length of about 15 mm. up to term.

In an animal killed at the end of a week after sLx injections of dye-stuff the maternal tissues were found to be deeply stained. Each fetus was completely surrounded by its vitelline membrane, which was stained dark blue, the color being deepest in a villous zone adjacent to the placenta and fading somewhat towards the antimesometral pole. The amniotic fluid was usually colorless but sometimes showed a sUght tinge in deeply stained animals. The amnion, when stripped away from the vitelline membrane and examined in salt solution, as a rule was seen to be uncolored; in rare instances, however, it showed a blue tint, attributable to large and repeated injections of the dj^e. The fetus and umbilical cord likewise were usually entirely unstained, although a barely perceptible blue coloration of the fetus was noted in several instances, associated with a bluish tinge in the amniotic fluid and membrane.

Goldmann noticed in the mouse and rat the staining of the amniotic membrane and fluid, but never any of the fetus. He believed that the dye in the amnion must be derived from the neighboring vitelhne membrane and this, he thought, was an argument in favor of the origin of the amniotic fluid, in part at least, from the maternal blood. It is more probable that the trypan blue occasionally seen in the amniotic fluid of rodents first enters the fetal blood-stream through both the vitelline membrane and the placenta. A trace of the dye in the fetal blood might readily escape detection, whereas, as soon as it had diffused from there into the colorless amniotic fluid it would attract attention. That it immediately enters the amnion from the vitelline membrane, without first entering the fetal circulation, seems improbable, for the vitelline membrane is well vascularized by the omphalo- mesenteric vessels and, should a dye diffuse from its epithelium through the base- ment membrane, it would necessarily be swept into these large vascular channels before it could enter the amnion.

The outside of the placenta appeared blue and on gross section the entire organ was found similarly stained with the exception of an area about 5 by 3 mm., which occupied a central position between the placental labyrinth and decidua (fig. 1) . As will be shown later, this white tissue consists of chorionic villi possessing a peculiar arrangement and designated by Duval as "the roof of the central excavation."

In many rodents, such as the rat, mouse, and guinea-pig, the vitelline mem- brane, as described by Duval and Sobotta, becomes the outermost fetal covering and persists throughout intrauterine life. Its epithelial surface comes in direct contact with the uterine wall and serves to nourish the fetus by the absorption of embrj^otrophe and the assimilation of extravasated maternal blood. This is more readily accomplished by the development of numerous villi which project into the uterine space and are covered by a single layer of cylindrical, phagocytic epithelial cells. This layer stains very rapidly and deeply with trypan blue (figs. 1 and 2). A single injection suflfices, after a few hours, to cause the cytoplasm of the cells to become heavily laden with fine blue granules. One is justified in speaking of the cells covering the villi as an attraction center for vital dyes, for nowhere else in the body are the dye-stuffs so rapidly taken up.

The villi show their greatest development near the line of attachment to the placenta, while towards the equator of the vitelline membrane they become progressively lower. Finally they disappear, so that the membrane at its anti- mesometral pole is perfectly smooth. During this transition the epithelium changes from a cyUndrical to a cuboidal type. The latter is probably less phagocytic than the former, for the cuboidal cells contain relatively few trypan-blue granules.

The \alli cease suddenly near the attachment of the vitelhne membrane to the placental surface (figs. 1,2), and the cylindrical, vitally staining epithelium changes abruptly to a delicate, flattened type which fails to absorb the dye. This un- stained, flattened tissue completely lines the angle (sinus entodermaticus) formed by the membrane at its attachment to the placenta. As the vitelline membrane (ectoplaccntal endoderm) spreads out laterally over the surface of the placenta, its epitheUum again undergoes a change (fig. 2). The cells prohferate, forming in many places small tufts or clusters, the older cells of which gradually become constricted off by new cells arising from the basement membrane.

Duval noted the remarkable papillary hyperplasia of the epithelium in this region but did not ascribe to it any function. These cells do not stain vitally as do those of the neighboring villi, and therefore their function possibly is not one of phagocytosis or absorption. It is interesting to note that the greatest proliferation of the epithelium occurs during the middle of pregnancy and that towards term it ceases. At birth the vitelline membrane in this region has been reduced to a single layer of columnar or cuboidal epithehal cells resting upon a simple basement membrane.

We shall now consider the vitally stained placenta. From a knowledge of the behavior of the placenta of the mouse and rat towards trypan blue, we should expect to find the dye abundantly present in the chorionic epithelium. We should look for it particularly in the giant cells and syncytium of fetal origin which invade and destroy the maternal tissue and attach the placenta to the uterine wall, and also in the delicate epithelial snyncytium of the labyrinth which covers the viUi and forms a barrier between the maternal blood-stream and the fetal capillaries.

To the unaided eye the placenta appears quite blue, with the exception of a tiny flattened area near its center, which always remains unstained (figs. 1, 4). On microscopic examination one is surprised to find very little of the dye stored within the chorionic epithelium. If the animal has not been deeplj^ stained one may fail to find any at all. In a well-stained guinea-pig, it can be distinguished as a fine blue tracery in the chorionic epithelium of the placental labyrinth (fig. 3) . The stain is faint, however, as compared with 'ts exhibition in the same cells of the mouse and rat.

In the giant cells and syncj^ium of fetal origin which invade the decidua and form an irregular boundary between the fetal and maternal tissues, commonly referred to as the central zone or "Umlagerungszone," no trypan-blue granules are found. This is surprising, since in the injected mouse and rat these cells are filled with blue pigment throughout gestation. Possibly the fetal syncji^ium stains \dtally in the guinea-pig in younger stages, when the ectoplacental cone is sending out syncytial roots and is burrowing its way into the decidua. However, in the youngest fetus (15 mm.) observed in these experiments no dye was found, nor was any found in the syncj' tium which, in the guinea-pig, di^ddes the placental labyrinth into numerous lobules, the so-called interlobular syncytium.

We have already mentioned that the roof of the central excavation, a structure peculiar to the placenta of the guinea-pig, stands out in the gross as an unstained area. When the mesoderm containing allantoic vessels invades the ectoplacenta, the latter is virtually hollowed out and a central core of mesodermal tissue is formed. This has been termed by Duval the central excavation. Part of this mesoderm finally invades the central zone, hollows out the masses of trophoblast situated there, spreads out over a broad area, and sends into the decidua a number of villous processes covered by several layers of ectodermal cells. This conspicuous structure, the function of which is unknown, has been designated by Duval as the roof of the central excavation. It persists throughout gestation but the decidual tissue which it embraces between its slender, finger-like processes gradually degenerates and, as term approaches, becomes converted into a homogeneous substance. The villi themselves, together with the vessels supplying them, appear to degenerate to a great extent. It is interesting to note that this same area is conspicuously blue when the vital dye is injected into the fetal instead of into the maternal blood- stream (figs. 4, 5).

The behavior of the surface of the placenta, just beneath the vitelline mem- brane, is also noteworthy. The proliferation of the epithelium of the cctoplacental endoderm has already been described and need not be referred to further. Very earlj' in the development of the placenta a layer of fetal syncytium comes to lie directly beneath the membrane. The periphery of this syncytium, however, very soon undergoes a change. It shapes itself into two or three layers of ectodermal cells with large round nuclei surrounded by a wide zone of vacuolated cytoplasm. Duval called attention to these cells on the surface of the placenta of the guinea-pig and referred to them as giant cells. In a section through the border of the placenta, at about the middle of pregnancy, one sees the vitelline membrane beneath which are layers of these giant cells, beneath which in turn is a layer of border syncytium containing large lacunae, and finally the placental labyrinth (fig. 6) . In an animal which has been stained with trypan blue the ectoplasm of the giant cells is filled with fine granules of the dye (fig. 6) . As the end of gestation approaches, these cells become shrunken and in part disintegrate and disappear.

In the decidua serotina groups of free macrophages, containing varying amounts of trypan blue in their cytoplasm, were encountered. Many of these wan- dering cells appeared to be undergoing degeneration. They were similar to those described by doldmann in the mouse and rat, which were thought by him to be important in the transfer of nutrient material, such as glycogen, from the tissues and vessels of the decidua to the fetal ectoderm. In the myometrium and serosa, in the neighborhood of the placental attachment, hundreds of similarly stained cells were observed. Goldmann believed that in the mouse and rat many of these are derivatives of the serous cells covering the peritoneal surface of the uterus. This may also be the case in the guinea-pig, for the peritoneal cells were con- spicuously loaded with the dye.

One further aspect of the behavior of trypan blue is worthy of description. In several of the guinea-pigs used in these experiments large injections of trypan blue were administered, resulting in mild inflammatory changes in the placenta, to judge from a poor staining reaction on the part of the syncytium and the presence of numerous polymorphonuclear leucocytes in the sinusoids. It was observed that the polymorphonuclear leucocytes in the maternal blood-spaces contained numer- ous tiny granules of trypan blue within their cytoplasm (fig. 7). It has been the general belief that none of the elements of the circulating blood normally stain with vital dyes. Downey (1917), however, has attempted to show that the pol3'morpho- nuclear leucocyte is capable of ingesting vital dye under certain conditions. In the circulating blood conditions are unfavorable, he claims, for phagocytosis of any kind to occur, and he bases his argument on the failure of the polymorphonuclear leucocytes, while moving rapidlj^ to take up bacteria, vital dye, or any other par- ticulate matter. Downey has shown experimentally that in the areas where the velocity of the blood-stream is diminished, or outside of the blood-vessels, the leucocytes stain with vital dyes, and he believes that this staining is quite compar- able to that seen in macrophages. The question is still debatable whether or not his assumption is correct and whether the staining should be considered as physio- logical rather than as an indication of cell injury.

The placentae in which the staining of the polymorphonuclear leucocytes was observed showed evidence of shght inflammatory changes, but there was nothing in the appearance of the leucoc}i;es themselves to condemn them as moribund. Further evidence is necessary, however, before it can be decided definitely whether or not true vital staining of polymorphonuclear leucocytes ever occurs.

In conclusion, a few observations upon the fate of a fine suspension of carbon particles, such as one finds in India ink, injected into the blood-stream of the preg- nant guinea-pig, will be described. Several animals were injected and killed on the second, fourth, and sixth day thereafter, respectively. In these animals the liver and spleen appeared very black while the color of the other abdominal organs was almost normal. On opening the thorax the lungs were seen to contain considerable carbon. The bone-marrow was also conspicuously black, and traces of carbon were visible to the unaided eye in many other tissues, such as the ovaries, pancreas, omentum, diaphragm, and mesentery. In the uterus, placenta, or vitelline membrane, however, no carbon was recognizable. The fetuses appeared perfectly normal, the amniotic fluid clear and colorless. Under the microscope particles of carbon were found in great abundance in the liver, spleen, and lungs, where they had been phagocytosed by endothelial and connective-tissue cells. In the omentum, mesentery, bone-marrow, and diaphragm, similar but less numerous particles were encountered; in the placenta and fetal membranes, on the other hand, there was not even a trace of carbon.

In order to determine whether the cells of the vitelline membrane refuse under all circumstances to take up carbon particles, a small quantity of ink was injected directly into the uterine cavity in the region of the villi. Microscopic examination of the villi after this experiment showed that the cells covering them did not take up a single particle of carbon into their cytoplasm, although they were seen actively engaged in the absorption of hemoglobin. It appears, therefore, that the endoder- mal cells of the vitelUne membrane and the chorionic epitheUum of the placental labyrinth are unable to phagocytize particles as coarse as those of india ink, although they are capable of absorbing a substance as finely dispersed as trypan blue.

SUMMARY

1. Trypan blue, injected into the pregnant guinea-pig, stains the placenta and the vitelline membrane but does not enter the amniotic fluid or the fetus, except in traces.
2. A zone of the vitelline membrane covered by villi absorbs the dye very rapidly and in large amount, and may well be termed an attraction center for the dye. The portion of the viteUine membrane covering the placenta, the ectoplacental endoderm, fails to stain vitally.
3. Trypan blue is absorbed and stored to a slight extent by the ectoderm covering the chorionic villi of the placental labyrinth. It is not taken up at all by the interlobular syncytium or by that part of the ectoderm, composed of giant cells and syncytium, which occupies the central zone (Urnlagerungszone).
4. The dye appears in the form of fine granules in the layer of giant cells described by Duval, situated beneath the ectoplacental endoderm.
5. It is also taken up by a host of free macrophages which are found throughout the decidua serotina and the uterine wall.
6. Carbon particles injected into the maternal blood-stream are not phagocytosed by any cells of the placenta or fetal membranes.

IV. BEHAVIOR OF THE PLACENTA AND FETAL MEMBRANES OF THE RABBIT TOWARD TRYPAN BLUE INJECTED INTO THE MATERNAL BLOOD-STREAM

Successive doses of trypan blue were administered intravenously to six pregnant rabbits, after which the animals were killed. Gestation was more than half completed at the time the last injection was done, and therefore no observations were made during the early stages of development.

On opening the abdomen the usual distribution of color was observed. The conspicuous uterus, which ordinarily contained from 6 to 12 fetuses, was deeply stained, both on surface view and on section. The placentae were all dark blue. The vitelUne, or outermost fetal membrane, was dark blue, the color being deepest in an equatorial zone between the mesometral and antimesometral poles of the fetal sac. The stain faded gradually toward the antimesometral pole, while in the opposite direction it ceased abruptly in the region of the terminal sinus. A band of unstained membrane — the chorion Iseve — consequently remained between the terminal sinus and the border of the placenta.

The vitelhne membrane, loosely attached to the amniotic membrane by strands of mesoderm, was easily stripped, revealing the dehcate amnion inclosing the amni- otic fluid. The amnion itself was faintly stained and the amniotic fluid contained traces of the dye.

When large and repeated doses of trypan blue are injected into the maternal blood-stream, the amniotic fluid may become quite deeply stained. In such cases the fetus also shows .a light staining. In the less deeply stained animals, in which the amniotic fluid contains only a trace of dye, a hardly appreciable staining of the fetus may be observed. Such a staining as this might easily escape detection. It is the writer's belief that the fetus probably contains at all times as much trypan blue as the amniotic fluid, but that the dye, when present only in traces, is less readily visible in the fetal tissues than in the amniotic fluid. The escape of traces of dj^e from the maternal into the embryonic circulation and the amniotic fluid appears to impair in no way the vitality of the fetus.

The observation that soluble dye-stuffs of various kinds, injected into the maternal blood-stream, can be detected frequently in traces in the amniotic fluid but less often in the fetal tissues, has led a number of investigators to conclude that fluid diffuses directly from the maternal blood-stream into the amniotic fluid, and that it is not necessary for the substances to enter first the fetal circulation; nor are they supposed to reach the amniotic fluid through the placenta, but are thought to enter it directly through the fetal membranes. One theory uses these observations to prove that the amniotic fluid is in whole or in part a transudate from the maternal vessels.

Leaving the human being out of consideration, the question of the possibility 'in animals (from observations on which the claim is based) of the formation of the amniotic fluid in this way meets with several grave objections. None of the observers working on rodents have taken into consideration the fact that the amniotic sac is completely surrounded by the vitelline membrane. This membrane possesses an unbroken epitheUal surface whose cells are concerned chiefly in the transfer of products from the embryotrophe to the fetal circulation. Furthermore, it is richly vascularized and consequently the amniotic fluid is completely surrounded by fetal blood-vessels which would make it difficult to conceive of dye-stuffs diffus- ing through the membrane without first entering the fetal circulation. Moreover, from the present observations and those on the guinea-pig recorded in the study just preceding, it appears probable that dyes such as trypan blue, when administered in large doses, stain the fetus as well as the amniotic fluid. The portal of entry of these traces of dye could be either the placenta or the vitelline membrane, or both, but it appears likely that, whichever route the dye pursues, it first enters the fetal blood-stream.

In the human being it is theoretically conceivable that fluid may diffuse directly from the maternal blood-stream into the amniotic fluid. The wall of the amniotic cavity contains no fetal vessels and the chorion is intimately fused with the uterine mucosa in which prominent maternal vessels are visible. If fluid escapes from these vessels it would seem quite possible that some of it might diffuse through the amniotic membrane directly into the amniotic sac.

It is of interest to consider the differences which exist in various animals in regard to the staining of the amniotic fluid after injection of a vital dye into the maternal blood-stream. Goldmann (1909) first noted the staining of the amniotic fluid in the rat and mouse after the administration of vital dyes. The author has observed the passage of traces of trypan blue into the amniotic fluid of the guinea- pig and rabbit. In similar experiments on cats (Wislocki, 1920), however, the amniotic fluid, as well as the allantoic fluid, remained unstained. The conclusion to be drawn from these observations is that the fetal membranes and placenta; of rodents are slightly permeable to ultra-microscopic particles, such as trypan blue, while those of carnivorous animals are impermeable.

Romer (1905) made parallel observations upon the permeability of the mem branes and placenta in different classes of animals. He injected tetanus antitoxin into pregnant sheep, cows, rabbits, guinea-pigs, and human beings. He observed that the antitoxin passed readily from mother to fetus in the human, that it was transmitted only occasionally in the guinea-pig and rabbit, and that transmission never occurred in sheep or cows. What is the explanation of this striking difference in behavior? Romer concluded from his experiments that the more heterogeneous the substance the more readily is it transmitted by the placenta. Thus tetanus antitoxin, which was derived from the horse, was extremely heterogeneous for man but only slightly so for the cow and sheep. Another explanation suggests itself, both for Romer 's results and for the passage of traces of trypan blue through some placenta; and not through others. We must recall that Grosser (1909) has classified placenta? according to the degree of union which exists between the fetal and maternal tissues. Thus, in the pig, cow, sheep, etc., the simplest types, there occurs merely an apposition of the chorionic ectoderm to the unbroken surface of the uterus. In carnivora, such as the cat and dog, which represent the next highest type, the chorionic ectoderm has invaded the uterine mucosa so that the placenta is made up of maternal blood-vessels intimately surrounded by fetal tissue. In rodents and man, the highest types, a still more intimate union of fetal and maternal tissues occurs. Here the maternal blood eventually flows in channels completely lined by fetal ectoderm, and hence only a layer of syncytium and delicate connective tissue separates the maternal blood from the fetal capillaries. It is apparent that in these animals (rabbit, guinea-pig, mouse, rat, and man) in which tetanus antitoxin or traces of trypan blue have been found in the fetus or the amniotic fluid, an extremely thin barrier of cells separates the maternal from the fetal blood-stream. In the cat, sheep, and cow, on the other hand, where the union is less intimate and numerous cells intervene between the two circulations, the transmission of antitoxin and trypan blue has not been observed.

The vitelline membrane of the rabbit is composed of a single layer of columnar cells, resting on a basement membrane beneath which are numerous blood-vessels. The surface of the membrane at its antimesometral pole is smooth, while in the neighborhood of the terminal sinus, where it ends in a ragged edge, it is covered by villi which project into the semi-fluid embryotrophe imprisoned between them and the uterine wall. In the injected animal the viteUine membrane is deep blue on gross appearance, and microscopically granules of trypan blue are found in large numbers in the columnar cells composing the membrane (fig. 8). These large granules, usually 20 or more to a cell, are scattered throughout the cytoplasm. The single oval or round nucleus contains none of the dye. No granules are visible in the basement membrane or beneath it in the walls of the fetal vessels. The amniotic membrane contains no trace of dya.

In the rabbit the labyrinth comprises the bulk of the placenta during the second half of pregnancy. Septa of fetal connective tissue, containing branches of the umbilical vessels, divide it roughly into lobules. The umbiUcal vessels break up within the lobules into capillaries which are completely lined by endothelial cells and are accompanied by deUcate supporting strands of mesoderm. The space between the capillaries is occupied by a syncytium composed of fetal ectoderm, the meshes of which surround innumerable tiny spaces in which the maternal blood circulates. In consequence of this arrangement the maternal and fetal blood- streams are separated by only a thin layer of syncytium and the delicate endo- thehum of the fetal capillaries. In fact, in many places even the syncytium appears to be absent, so that the maternal blood-cells come in direct contact with the fetal endothelium, an arrangement no doubt greatly facihtating the interchange of substances.

Trypan blue was found abundantly present in the placental labyrinth as aggregations of tiny granules throughout the syncytium (fig. 9). None of it was seen in the cells forming the fetal capillaries. Beneath the labyrinth, and uniting it to the uterine wall, lies a layer of giant cells. These cells, which early in gestation play a prominent part in the growth of the placenta and the nutrition of the fetus, are reduced during the latter half of gestation to a relatively inconspicuous layer. In these vitally stained rabbits the cytoplasm of these cells became filled with numerous tiny blue granules, showing that the cells, even with approaching maturity, possess the power to assimilate materials brought to them. Beneath the layer of giant cells lies the outermost or decidual layer of the placenta, composed of decidual cells, many of which were found to be undergoing degeneration. This layer is traversed by conspicuous maternal vessels which pass to and from the labyrinth. Trypan blue was present only in the cells which were degenerating and whose cytoplasm and nuclei, as a result, stained diffusely. The uterine musculature was noteworthy because of the number of vitally stained macrophages visible in its connective-tissue septa.

SUMMARY

1. Trypan blue injected into the maternal blood-stream of the pregnant rabbit

stains the placenta and vitelline membrane.

1. The dye passes in traces from the maternal into the fetal circulation, staining the fetus and amniotic fluid very slightly.

1. The dye is stored in the form of granules in the cells of the vitelline membrane and in the syncytium and giant cells of fetal origin in the placenta.

CONCLUSIONS

Our knowledge concerning the behavior of the placenta and fetal membranes toward colloidal dyes injected into the blood-stream may be summed up as follows:

Finely dispersed colloids, such as trj^pan blue and pyrrhol blue, when injected intravenously into the pregnant animal, reach the placenta and are there absorbed and stored in the form of granules in the chorionic ectoderm. In the mouse and rat the dye passes in traces into the amniotic fluid but fails to stain the fetus (Goldmann) . In the guinea-pig and rabbit it likewise passes in traces to the amni- otic fluid, but in addition it faintly stains the fetus. In the cat vital dyes are not transmitted, even in traces, to the amniotic fluid or to the fetus (Wislocki, 1920). This variation in behavior may be explained on comparative anatomical grounds, differences of architecture making the placenta of carnivora less permeable than that of rodentia. Vital dyes are also absorbed and concentrated into granules in the cj^toplasm of the cells of the vitelline membrane, which in rodentia forms the outermost fetal covering. Granules of dye are deposited in a structure pecuhar to carnivora, termed in the cat the "brown border," which is a modified portion of the chorionic membrane bordering the placenta.

REFERENCES CITED

Downey, H., 1917. Reactions of blood and tissue cells to acid colloidal dyes under experimental con- ditions. Anat. Record, vol. 12, p. 429.

, 1918. Further studies on the reactions of blood and

tissue cells to acid colloidal dyes. Anat. Record, vol. 15, p. 103.

GoLDMANN, E. E., 1909. Die au.ssere und innere Sekretion des gesunden und kranken Organismus im Lichte der "\-italen Fiirbung." Beitr. z. klin. Chir., vol. 64, p. 192.

Grosser, O., 1909. Vergleichende Anatomie und Entwick- lungsgeschichte der Eihaute und der Placenta. Vienna.

, 1904. Weitere Studien zur Frage der Intrau- terinen und extrauterinen Antitoxiniibertrag- ung von der Mutter auf ihre Nachkommen. Beitr. z. exper. Therap., Heft 9.

G. B., 1920. Experimental studies on fetal absorption. I. The \itally stained fetua. II. The beha\nor of the fetal membranes and placenta of the cat toward colloidal dyes in- jected into the maternal blood-stream. Con- tributions to Embryology, vol. 11, Carnegie Inst. Wash. Pub. 274.

DESCRIPTION OF PLATE

FlQ. 1

Fio. 2

FiQ. 3. FiQ. 4. Fig. 5.

Fig. 6. Fig. 7. Fig. 8. Fig. 9.

p. I., Placental labjTinth.

r. c. e., Roof of the central excavation.

3. endo., Sinus endodermaticus.

V. m.. Vitelline membrane.

Gros.9 specimen showing appearance of the vitelline membrane and placenta of a fetal guinea-pig, measuring 18 mm. after injection of trypan blue into the maternal blood-stream. The deeply stained villous portion of the vitelline membrane is readily distinguishable, as is also the unstained roof of the central excavation.

Section through vitelline membrane of guinea-pig at its attachment to the surface of placenta. The char- acter of the epithelium covering the various portions of the vitelline membrane is clearly shown. The endodermal cells clothing the villi are loaded with trypan-blue granules. The curious papillary hyper- plasia of the ectoplacental endodorm is well illustrated.

Section through placental labyrinth of guinea-pig, showing tracery of trypan blue in the chorionic epithelium after repeated administration of the dye.

Specimen showing distribution of trypan blue in the placenta of guinea-pig after injection of the dye into maternal blood-stream. Note that the only unstained area is the roof of the central excavation.

Specimen showing the distribution of trypan blue in the placenta of guinea-pig, after injection of the dye mto the fetal circulation. Note that when the dye reaches the placenta through the fetal vessels, the roof of the central excavation becomes stained, but none of the dye diffuses into the decidua.

Section through the surface of the placenta of guinea-pig (about the middle of pregnancy) showing the vitally stained giant cells beneath the ectoplacental endotlerm.

Section of the placental labyrinth of guinea-pig showing polymorphonuclear leucocytes containing granules of trypan blue.

Section through the viteUine membrane of the rabbit, showing the columnar cells covering the villi filled with granules of trypan blue.

Section from labyrinth of rabbit's placenta, showing syncytium in which numerous tiny granules of trypan blue are visible.