Paper - Early Development and placentation in arvicola (microtus) amphibius (1922)
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Early Development and Placentation in Arvicola (Microtus) amphibius, with Special Reference to the Origin of Placental Giant Cells
By G. S. Sansom, B.Sc.
Honorary Research Assistant, Department of Anatomy, University College, London.
This work was begun in 1913 at the suggestion of Professor J. P. Hill, partly on. material belonging to him and partly on that collected by myself. At that time we had no early stages in our possession and it was not until the Spring of 1919 that I was able to obtain a sufficiently complete series.
The water voles, in the wild state, were killed during the months of March and April when their first breeding season occurs. The ovaries and uteri were, in every case, removed immediately after death and placed in fixing fluids. Of the latter, the following gave the most satisfactory results:
(i) Bouin’s picro-formol acetic. (ii) Alcohol sublimate-acetic mixture, consisting of 60 parts absolute alcohol, saturated with Hg(Cl,, 30 parts chloroform, 10 parts glacial acetic acid.
One of the finer cytological fixatives would have been most valuable but, owing to the nature of development in these rodents, the embryo is shut off from the uterine lumen by a considerable thickness of compact tissue and a fixing agent of great penetrative power was essential.
The ovaries and uteri were cut in serial sections, some at 8 some at 10. Where, as in later stages, a complete series was not necessary, some sections were cut at 54, thickness.
Throughout the work I am very greatly indebted to Professor J. P. Hill for much kind assistance, advice and encouragement.
I am also indebted to Mr F. J. Pittock of University College for advice on photographic matters connected with my preparation of the photomicrographs.
The early development and placentation of Microtus amphibius does not appear to have been worked out hitherto, but with the material now available it is possible to form a clear view of the general course of events.
In the allied species of voles, Arvicola agrestis and Arvicola arvalis, a certain amount of work has already been published, but my own observations are not in. complete agreement with those of Disse as regards the uterine changes and origin of the giant cells, which latter form such a conspicuous feature in the placentae of these rodents. Kupffer and Biehringer have described for the Field Vole and Water Vole respectively, the so-called “inversion of the germ layers,” the reason for, and significance of, this “inversion” is now so well understood that I shall not do more than refer to this aspect of development in the water vole. Considerable space, however, is devoted to a description of the origin and significance of the giant cells which play such an important réle in the placentation of Microtus.
CHANGES IN THE UTERINE TISSUES PRIOR TO ATTACHMENT
Externally little change is visible to indicate the presence of early developmental stages. A number of voles killed during the breeding season exhibited localised dilatations of the uteri, which suggested the presence of blastocysts, but examination of the ovaries and Fallopian tubes in section indicated that the uteri were not pregnant. _In some cases the ovaries contained ripe follicles with undischarged ova, in others corpora lutea were present, but the eggs were still situated in the Fallopian tubes. Moreover, the ovary in the water vole is surrounded by a tough closed capsule which obscures the details and renders difficult the determination of the condition of the. ovarian follicles when examined in the fresh or fixed state.
These localised dilatations of the uteri are indicative of changes undergone by the maternal tissues preparatory to the attachment of the embryos, hence it would appear that the sites of attachment of the blastocysts to the uterine mucosa are predetermined before the eggs reach the uteri at all.
Examination of these special areas of the uterus was rendered more difficult owing to the fact that after fixation and embedding the dilatations were no longer recognisable. Examination of many complete series of sections through uteri possessing these localised swellings demonstrated that the latter were due to a considerable increase in size and number of the superficial blood vessels. These vessels contract on fixation far more than the conjunctive tissue, hence it comes about that the uterus, which in the fresh state exhibited marked dilatations, appears after fixation to be of a more or less uniform diameter throughout, and those areas where the swellings occurred are. only recognisable from the hypervascularity of the outer layers.
This increase in vascularity of the uterus is chiefly noticeable in the external layer of longitudinal muscle fibres and between it and the inner circular layer, where large blood sinuses occur. It seemed probable that these localised areas indicated the future sites of attachment of the eggs, and with a view to ascertaining the causes which conditioned the attachment at certain points and the fairly uniform spacing out of the embryos in the uteri, a large number of serial sections of uteri in which these local dilatations occurred, but in which the eggs had not reached the uteri, were examined in detail. The results were mainly negative; no definite changes could be detected either in the shape of the uterine lumen, or in the character of the lining epithelial cells, which marked off these areas as predetermined spots for the attachment of the eggs.
It is of course possible that these dilatations indicated the places where embryos were attached during a preceding pregnancy. From material collected, as this was, in the wild state, it is not possible to determine whether a female had already had young.
Many workers have studied this question which, however, still remains unanswered. Widakowich noted that the mucosa became more vascular before the attachment of the embryos occurred, but he apparently made no reference to the presence of local dilatations. He states, however, that cilia appear on certain areas of the uterine epithelium shortly after copulation and that they disappear after a short time. Other workers have confirmed this observation.
I have not detected cilia either in the Fallopian tubes or uterine lumen of Microtus but it is possible that transitory ciliated areas occur.
It is not easy to imagine how cilia can cause the attachment of the eggs, but it is quite conceivable that they might prevent it. If that were the case one would expect to find large tracts of uterine epithelium provided with cilia and localised areas on the antimesometrial side devoid of them, the latter areas being the appointed places of attachment.
An alternative arrangement for arresting the eggs in certain regions of the uteri, and one moreover for which I have some slight evidence, is that in which the uterine epithelium contracts around the egg, closing above it temporarily until the remaining eggs are all attached. One can imagine that certain areas of the uterine epithelium on the antimesometrial side become hypersensitive, these hypersensitive areas corresponding to those of hypervascularity, and that they react to the presence of eggs in the lumen by closing above them. If such a condition obtains, then the eggs nearest the Fallopian tubes become enclosed first, the remainder passing over the enclosed eggs and becoming arrested in turn as they reach the successive sensitive areas.
The eggs while in the Fallopian tube are often crowded together, two or more often occurring in the same section, hence it is obvious that some such mechanism must exist in the uterus to prevent several embryos from lodging in one fold of the epithelial wall, for if such were to occur, it would be impossible for them all to reach maturity and examination of the ovaries of females in late stages of pregnancy reveals that there are no corpora lutea in excess of the number of developing embryos, hence one can assume that eggs do not become attached to the uterine epithelium so close together that one or more fail to develop. —
An examination of the uterine epithelium in the neighbourhood of the blastocyst shown in Pl. XXV, fig. 1 reveals the fact that the latter is lying in a crypt or groove of the antimesometrial uterine lumen. Study of the serial sections in this region indicates that this groove was, at the time of preservation, or shortly prior thereto, shut off from the main lumen mesometrically to it. In fig. 1 a break is seen in the uterine epithelium on the right side, from which three or four cells have disappeared, while on the left side, there is, attached to the intact epithelium, a triangular mass consisting of these missing cells. This clearly indicates that these epithelial layers were at one time in contact. The condition here figured is traceable in the sections over a distance of nearly -4 mm.
One may therefore conclude that the crypt containing the blastocyst, becomes temporarily separated off from the remainder of the uterine lumen by the close adherence of the uterine epithelium forming its lips.
It might be suggested that this closure of the uterine lumen was an artefact, brought about by mechanical pressure after death. This, however, seems very improbable, for if such were the case, one would expect to find signs of lateral compression of the uterus in this region. No indications of such compression are recognisable; the diameter of the uterus in the region where this adhesion occurs is almost precisely the same as in other areas and the serous coat exhibits no signs of injury.
There is, moreover, collateral evidence that this adhesion was initiated prior to death, for the epithelial cells in that region are more columnar than those of the neighbouring portions of the epithelium. Pl. XXYV, fig. 1.
SEGMENTATION OF THE OVUM
As has been described in a previous publication, the oocyte of Microtus gives off the first polar body while it is still in the Graafian follicle and this polar body divides mitotically into two. The outer wall of the follicle which has, by this time, become exceedingly thin, then ruptures and the secondary oocyte, surrounded by its zona and the cells of the corona radiata, passes into the fluid filled cavity of the capsule into which the fimbriated end of the Fallopian tube opens.
Unfortunately no stages of fertilisation were obtained, but from the fact that numerous sperms occur in the ovarian capsule it seems probable that fertilisation occurs therein. The oocyte, invested in its zona, then gives off the second polar body and passes into the Fallopian tube.
The first cleavage occurs in a plane at right angles to the plane of separation of the polar bodies and results in the formation of two blastomeres, which are apparently identical in size and character.
The average measurement of the two-celled egg is 054mm. x -045 mm.; the thickness of the zona is -008 mm. The latter, under minute examination, shows no signs of radial canals. In some of the two-celled stages in my possession there are indications of the presence of degenerating polar bodies lying underneath the zona in the cleavage plane, but in the majority of cases they are not recognisable.
No stages were obtained showing the completion of the second cleavage, but an ovarian segmenting egg, which has been described in a paper dealing with parthenogenetic cleavage in the water vole, shows the four blastomeres arranged in a cross shaped manner which is known to be typical of these rodents.
The second cleavage therefore probably results in the formation of the typical cross-shaped arrangement of the four cells, after which stage the segmentation appears to occur irregularly, for in the eight-celled stage, which measures :074 x -053 mm. there is no definite arrangement of the blastomeres. The zona is still intact and the cells appear similar in size and character, though had more precise cytoplasmic fixatives been employed, it is possible that some differentiation between the cells would have been recognisable. Unfortunately none of these eggs are really well preserved as they were fixed in situ in the Fallopian tubes.
Several stages with 12 or 13 cells were obtained. These morulae vary in size somewhat, the largest measuring -074 x -056mm. and the: smallest -069 x -056 mm. It will be seen therefore that during these early cleavages the egg has not materially increased in size. In the best preserved egg of this stage thirteen nuclei are present, one of which is situated in a central cell, which is, however, exposed to the surface on one side, thus suggesting a condition of epibole. This egg lies free in the upper portion of the uterus near the junction of the latter with the Fallopian tube; it has not yet reached its site of attachment. The zona is still present but appears to be in process of degeneration.
FORMATION OF THE BLASTOCYST
This irregular segmentation continues and leads to the formation of a more or less spherical blastocyst, having the form of a thin walled vesicle, at one side of which is a globular mass of cells projecting into the cavity (PI. XXV, figs. 1 and 2). This mass of cells constitutes the embryonal knot and denotes, what I shall call, the upper pole of the egg, i.e. that portion which will become directed towards the mesometrium. The thin wall of the vesicle is the Trophoblast or extra-embryonal ectoderm; it is apparently at this stage quite distinct from, though in intimate contact with, the cells of the embryonal knot, at the upper pole of the egg.
The blastocyst shown in fig. 1 measures -08 x -07 x -07 mm. of which the inner cell mass, or embryonal knot, measures -05 x -04 x ‘04mm. The one shown in fig. 2 measures -09 x 075 x -06mm., the embryonal knot °05 x -06 x -04 mm. These blastocysts have apparently reached their definite sites of attachment to the uterus, but they are not yet correctly orientated although they are attached by a mucous secretion to the epithelium. These stages correspond in development to Sobotta’s mouse egg at the end of the fourth day or Huber’s rat egg of the fifth day after fertilisation.
The blastocyst represented in fig. 2 shows signs of differentiation of the cells of its embryonal knot into an outer layer of more or less uniform granular cells enclosing two rather larger cells, with pale staining less granular, cytoplasm. This apparent differentiation of the embryonal knot is not recognisable in the blastocyst shown in fig. 1, which is cut almost horizontally, the next section of the series passing through the embryonal knot above the level of the blastocyst cavity.
It is, however, possible that the two pale-staining cells visible in Pl. XXYV, fig. 2, are simply the products of a recent cell division. The two daughter cells resulting from a division are often rather different in appearance to neighbouring cells which have not so recently divided. The appearance of the nuclei in this case does not afford much guide.
The trophoblastic cells constituting the outer wall of the vesicle are somewhat elongated and flattened. Their cytoplasm is pale-staining and coarsely granular with ragged free surfaces.
At this time the uterine tissues exhibit, very characteristic changes. The uterine lumen is wide and simple in character, the convolutions and diverticula, which are present prior to pregnancy, having disappeared in these regions where the blastocysts are located. The uterine glands are large but simple in character and their epithelium has undergone marked changes, the cells, normally columnar, are more cubical and the nuclei appear spaced out in a peculiar manner (Pl. XXV, fig. 1). This histological change is also recognisable in the uterine epithelium itself, but more especially in that portion of the anti-mesometrial wall neighbouring on the blastocysts. There the epithelial cells are very pale-staining and their nuclei small and widely separated, the cytoplasm around them appearing quite colourless and free from granularity.
In view of the fact that these cells are destined, at an early date, to degenerate and disappear, one might suppose that these histological changes were the preliminary processes leading up to that degeneration, but an examination of other sections in this, or in similar, series shows that tracts of uterine epithelial cells with identical characters occur in several other places, even on the mesometrial side of the lumen; it would appear therefore that these changes are in no wise conditioned by the presence of blastocysts in the uterus and that they do not necessarily indicate those regions in which the uterine epithelium is destined to disappear.
The connective tissue cells of the mucosa exhibit no marked change, but leucocytes are present in considerable numbers and capillaries are more numerous. As regards vascularity, however, by far the greatest change is noticed in the superficial layers of the uterus. Numerous and extensive blood vessels occur in the outer muscular coat. These vessels have contracted very considerably during fixation and subsequent treatment, with the result that the outer wall of the uterus has in most cases been thrown into numerous folds.
BLASTOCYST STAGES AND THEIR RELATIONS TO THE DECIDUAL CAVITY
The blastocyst, having reached its definitive position on the antimesometrial side of the uterine lumen, becomes orientated with its upper pole, containing the embryonal cell mass, directed towards the mesometrium. The Development and Placentation in Arvicola amphibius 339
cells of the uterine epithelium, around the sides and lower pole of the blastocyst, flow together with the formation of a symplasma layer. This breaks down and disappears, with the result that the egg comes to lie in a narrow cleft, the implantation crypt, bounded laterally and antimesometrially, by the stroma cells of the uterine mucosa.
Text-fig. 1 illustrates this stage. The blastocyst lies in the implantation crypt, the opening of which into the narrow uterine lumen is closed by a plug of necrotic tissue, consisting of degenerating red blood corpuscles, leucocytes
and portions of symplasma derived from the uterine epithelium. The blastocyst, which has the form of a slightly flattened sphere, measuring -11 mm. in transverse diameter and_.-06 mm. in the future long diameter, consists of an outer trophoblastic layer, which is in contact at the mesometrial pole with a rounded mass of compact cells, which constitutes the embryonal knot. The _eavity of the blastocyst is fairly extensive and contains several pale-staining cells which are mostly in close contact with the lower surface of the embryonal knot. These cells represent the future entoderm.
The outer wall of the vesicle appears to have contracted away from the uterine tissue during fixation or subsequent treatment. Its cells possess digitiform processes which during life no doubt penetrated into the spaces between the stroma cells. These irregular cells, constituting the outer wall of the blastocyst, serve to attach the latter to the maternal tissue lining the implantation crypt, they are usually regarded as purély trophoblastic in character and as being formed from the original trophoblastic wall of the unattached blastocyst. There is, however, a strong probability that in Arvicola amphibius the outer wall of the blastocyst at this time is not a purely embryonal structure, but that certain maternal cells have become incorporated in it.
Later, when dealing with the subject of giant cells, I shall produce evidence in support of this view, which appears at first sight improbable.
The cells of the mucosa surrounding the implantation cavity have lost their normal character, their nuclei are crowded together and cell outlines are in places unrecognisable. Numerous leucocytes are present, both in the superficial and deeper layers. The capillaries: have increased in number to an enormous extent, particularly in the lateral and antimesometrial portions of the mucosa. A few of the endothelial lining cells of these capillaries are enlarged and stain very deeply.
The next stage in development is represented in text-fig. 2. The blastocyst measures -074 x -09 mm. and roughly corresponds to the six day rat embryo figured by Huber. The embryonal knot is distinctly separated from the covering layer of trophoblast. It has the form of a very compact ovoid mass of rather pale-staining cells measuring -053 x -04mm. At the mesometrial end the covering trophoblast forms a curved disc, the margins of which bend upwards towards the mesometrium. It is in intimate contact with, but everywhere recognisable from the embryonal ectoderm and its constituent cells, which are cubical in character, stain more deeply than those of the latter. The margins of this trophoblastic plate extend outwards and upwards in contact with the uterine tissues of the implantation crypt, which communicates with the uterine lumen, mesometrially to it, by a comparatively narrow channel filled with necrotic tissue, degenerating epithelial cells and blood corpuscles, This trophoblastic plate is the primordium of the Trager of Salenka, or Ectoplacental Cone of Duval.
The parietal trophoblastic wall of the vesicle is continuous with the ectoplacental plate at its margins and extends around the implantation cavity in very intimate contact with the maternal tissue. It apparently consists of rather large irregular cells united by strands, but, as I have already stated, there is reason to believe that some at least of these cells are of maternal origin. ,
The entoderm has the form of a continuous layer of elongated, spindleshaped cells, loosely investing the lower surface of the embryonal ectodermal mass. This layer of cells constitutes the visceral or inner yolk sac wall; between it and the ectoderm there are present one or two isolated cells which are probably entodermal cells which have not become incorporated in the layer. :
The differentiation of the uterine tissues has proceeded still further; the vascularisation of the outer, muscular layers has resulted in the formation of numerous and extensive blood sinuses between the longitudinal and circular muscle layers. From these sinuses many capillaries extend into the mucosa and give the latter, with the exception of that portion immediately surrounding the uterine lumen, a loose spongy character. The uterine glands are greatly reduced, both in size and number.
This uterus, in the fresh state, exhibited well marked localised dilatations, corresponding, presumably, to the sites of attachment of embryos, but after ‘sectioning, the variations in diameter, between the embryonal and interembryonal regions, were quite insignificant. The plications in the outer wall of the uterus indicated the extent of the contraction during fixation and subsequent treatment.
A considerable advance in embryonal development is shown in text-fig. 3 Here the blastocyst measures -11 x -11 mm., of which the embryonal ectodermal mass measures :04 x -04mm. The ectoplacental trophoblast or Trager has thickened at its edges, where it is in contact with the walls of the implantation cavity, and appears to be in a state of active proliferative growth. The 342 G. S. Sansom.
ectodermal mass has the form of an almost spherical solid structure composed of rather pale-staining cells. The entoderm, in this stage, forms a closed vesicle, consisting of elongated, deeply-staining spindle-shaped cells with ovoid nuclei. The upper wall of this entodermal vesicle is applied to the lower surface of the ectodermal mass and constitutes the visceral yolk-sac wall; it is continuous with the parietal yolk-sac wall which is at this time intact, the yolk-sac cavity being shut off from the cavity of the blastocyst. This outer yolk-sac entoderm is destined to disappear as an intact layer at an early date, though its cells continue to divide and remnants of the layer persist throughout the whole gestation period.
The peripheral or parietal wall of the vesicle consists, over its extent, of a single layer of large cells with very vacuolated cytoplasm united by fine strands. The nuclei of these cells are, on the average, far larger than those of the ectoplacental trophoblast and their cytoplasm is prolonged into irregular processes which pass into the decidual tissue surrounding the implantation cavity. These cells appear to be actively phagocytic, for many red blood corpuscles and dark-staining granules are present in them.
Similar cells, at the antimesometrial end of the early blastocyst, have been described by Sobotta, Duval and Melissinos in the Mouse, and by Widakowich Development and Placentation in Arvicola amphibius 343
and Huber in the Rat. Considerable discussion as to their significance has taken place; I shall revert to this question when describing the origin of the placental giant cells.
The next stage in development is represented in text-fig. 4. The blastocyst now measures :16 mm. in length and -11 mm. in diameter. The embryonal ectodermal mass measures -048 x -048 x 06mm. The ectoplacental trophoblast
has the form of a slightly curved plate of deeply-staining cells, considerably thicker than in the preceding stage. Marginally it is continuous with a fine structureless membrane which is in contact with the wall of the implantation cavity. This membrane, which represents the parietal trophoblastic wall of the blastocyst, is Reichert’s membrane. In intimate union with this membrane are large cells with vacuolated cytoplasm, similar to those described in the last stage.
The embryonal ectoderm has the form of an almost spherical solid mass of pale-staining cells in contact with, but sharply marked off from, the ectoplacental trophoblast. The increase in thickness of the latter over its middle region has forced the embryonal ectoderm downwards into the cavity of the blastocyst, with the result that the underlying layer of entoderm has become invaginated. This visceral or splanchnic entoderm is mostly in close contact with the lower surface and sides of the embryonal ectoderm. It consists of a single layer of very regular, dark-staining, spindle-shaped cells with ovoid nuclei. At the point of junction of the ectoplacental trophoblast and embryonal ectoderm this layer is reflected downwards into the cavity of the blastocyst as the parietal entoderm, but it no longer constitutes a continuous membrane. Over the lateral walls of the cavity it is complete and its cells similar in character to those of the visceral layer, but: around the lower pole of the blastocyst it has already broken down and is only represented by a few scattered cells. These cells, however, continue to divide and form an attenuated layer in contact with Reichert’s membrane.
The maternal tissues have undergone further changes. The cells of the mucosa around the implantation cavity have increased in size and assumed the character of typical decidual cells. They are compactly grouped together and cell outlines are in many places -unrecognisable. Numerous capillaries are present in these masses of decidual cells, and their endothelial lining cells are often large and darkly staining. The implantation cavity is still in open continuity with the uterine lumen, but the epithelium of the latter shows signs of degeneration over a considerable area at the antimesometrial end, the cell walls having disappeared with the formation of a symplasma layer, which is destined to degenerate at an early date.
Between the stages already described and that represented by text-fig. 5 there is, unfortunately, a considerable gap in the material, but a stage which we possess of the Field Vole, the development of which appears to agree closely with that of the Water Vole, serves to bridge the gap fairly well.
The blastocyst in question has the form of an elongated hollow cylinder measuring ‘55 mm. in length and -15 mm. in diameter. It corresponds roughly with that represented by Huber’s fig. 27c of the eight day rat embryo.
One can distinguish three areas of this egg cylinder. First a hemispherical, cup-shaped, region at the antimesometrial end, constituted by the embryonal ectoderm. The cells forming the wall of this deep cup, which appears almost horse-shoe shaped in vertical section, are very closely crowded together and are in a state of active proliferation, numerous mitoses being present. The lips of this cup are in continuity with, but fairly sharply defined from, the extraembryonal ectoderm which constitutes the middle region of the egg cylinder. There the cells are less crowded together than in the embryonal region and mitoses, though present, are far less numerous. At the mesometrial end this layer passes rather abruptly into the tissue of the Trager, which is, by this time, a cone-shaped structure of considerable size, composed of rather large pale-staining cells with almost spherical nuclei. The cells of the Trager are irregularly disposed in groups, between which are present masses of maternal blood corpuscles. The Trager projects upwards into the still persistent uterine lumen the epithelium of which has disappeared in the antimesometrial portion,
while over the remainder, with the exception of a small area at the mesometrial end, it has broken down with the formation of a symplasma layer. Mingled with the cells of the advancing edge of the Trager are a number of large cells united by cytoplasmic filaments. The spaces between these cells, and the cytoplasm of the cells themselves, contain maternal blood corpuscles, which also occur amongst the trophoblastic cells of the Triger. These cells, which are in reality small giant cells, are held by some workers to be trophoblastic in origin, but as will be seen later they are actually of maternal origin.
The parietal trophoblastic wall of the egg cylinder is continuous at the mesometrial end with the cells of the Trager, but it has the form of a thin structureless membrane in intimate contact with the walls of the implantation cavity. This membrane—Reichert’s membrane—has adherent to its inner surface a few scattered isolated cells, mostly fusiform in shape, which represent the parietal yolk-sac entoderm.
The egg cylinder itself is clothed externally by a layer of splanchnic or visceral entoderm. Over the embryonal ectodermal cup it has the form of a single layer of fusiform or ovoid cells, constituting the embryonal entoderm, whilst over the extra-embryonal ectoderm it is composed of very large columnar cells, in which the nuclei are situated basally. The cytoplasm of these cells is very coarsely granular and in places vacuolated, but I cannot detect the haemoglobin granules which Sobotta described and figured in the outer portions of the cells of the visceral entoderm in the Mouse. As this layer approaches the mesometrial end of the cylinder the cells become more cubical in character and at the junction of the extra-embryonal ectoderm and the Trager it disappears as a continuous membrane and is only represented by the scattered cells in contact with Reichert’s membrane, referred to above. _ As regards the maternal tissues, a considerable advance in differentiation has taken place. All the cells of the mucosa antimesometrially and laterally to the implantation cavity have become converted into typical decidual cells with large vesicular, pale-staining, nuclei. These masses of decidual cells are penetrated by numerous extensive blood sinuses, the endothelial cells of which are large and deeply-staining. Around the walls of the implantation cavity and extending upwards therefrom, in contact with the symplasma layer formed from the degenerating uterine epithelium, is a belt of deeply-staining spindle-shaped cells with dark elongated nuclei. These cells I regard, for reasons which I shall state later, as the endothelial cells of maternal capillaries, the lumina of which have disappeared owing to apposition of the vessel walls.
Coming nowto the stage of Arvicola amphibius represented by text-fig. 5, we find the blastocyst completely embedded in maternal tissue, the uterine lumen being restricted to a very narrow passage, with fine radial canals, at the mesometrial side of the decidual swelling. It measures -3 mm. in length and -15 mm. in breadth and is of the cylindrical form, characteristic of rodents with inversion of the germinal layers.
The ectoplacental cone is now greatly enlarged and consists of a mass of rather pale-staining cellular tissue and is of irregular shape, being prolonged into digitiform processes which extend upwards into the decidual tissue mesometrially to it. The nuclei of these trophoblastic cells are smaller than those of the maternal cells and react to stains in a rather different manner. The Trager forms the roof of a cavity of considerable size, the primitive amniotic cavity, which later becomes subdivided into the amniotic cavity proper and the ectoplacental or false amniotic cavity. The embryonal ectoderm now has the form of a deep cup, the lip of which is continuous with the tissue of the Trager, its walls being composed of narrow columnar cells which increase in number towards the lower pole, where the layer is considerably thicker. Externally, this embryonal ectoderm is invested by the splanchnic yolk-sac entoderm, which consists of a single layer of long columnar cells with pale-staining vacuolated cytoplasm. The inner ends of these cells are often densely granular, but here again, I am unable to detect the presence of haemoglobin granules. At the junction of the embryonal ectoderm and the trophoblast, the entoderm is reflected back, but it almost immediately loses its character as a distinct cell layer and is only represented by a few scattered cells lying in contact with the outer wall of the vesicle formed by Reichert’s membrane. This now has the form of an exceedingly thin structureless ‘membrane, in intimate contact with, and difficult to distinguish from, the maternal tissues lining the implantation cavity. These tissues have by this time undergone marked changes. An intense cell proliferation has taken place around the implantation cavity with the result that the decidual swelling has become converted into a compact tissue, composed of large cells, with rather pale-staining nuclei, subdivided by numerous fine blood channels lined by endothelium. The lumina of these capillaries are often exceedingly narrow, indeed the endothelial walls are frequently so closely apposed that the lumen appears completely obliterated. The nuclei of these endothelial cells are long and narrow, stain intensely dark with haematoxylin, whilst the cell cytoplasm stains deeply with eosine. These narrow capillaries, with their modified endothelial lining, form a very well marked annular zone around the implantation cavity, and a belt of these cells constitutes the actual lining of the cavity around the antimesometrial end and lateral regions of the embryo. They are apparently fused with Reichert’s membrane, with the result that the latter is scarcely recognisable.
It will be seen from the above that this early blastocyst of Microtus differs somewhat from the corresponding stage of the Mouse and Rat. In the latter forms, the ectodermal cylinder is completely invested, except at the upper pole, by the splanchnic yolk-sac entoderm, whereas in Microtus amphibius the junction of the embryonal ectoderm and the trophoblast, i.e. the region from which the amnion folds will later arise, marks the limit of upward extension of the yolk-sac entoderm. In later stages, however, this condition no longer obtains. The visceral entoderm gradually extends in a mesometrial direction, so that a similarity with the typical murine condition is re-established.
The next stage available is represented in Pl. XXV, fig. 3. The blastocyst measures 1-4 mm. in length and -63 mm. in diameter, excluding the trophoblast of the Trager, the limits of which are difficult to determine for purposes of measurement. The blastocyst now exhibits the form typical for rodents with inversion; it contains three extensive cavities: the one nearest the mesometrium is purely trophoblastic and constitutes the false amniotic or ectoplacental cavity. Its roof is continuous with the tissue of the Trager; its floor, together with a layer of mesoderm, constitutes the roof of the extra-embryonal coelom, the so-called “chorion.” Its lateral walls are clothed externally with a layer of splanchnic entoderm, the visceral yolk-sac wall having, by now, extended upwards almost to the junction of the Trager with the lateral walls of the ectoplacental cavity. The middle and largest cavity of the blastocyst is the extra-embryonal coelom, which is lined by an exceedingly fine layer of fusiform mesodermal cells. Its roof, as already described, consists of a layer two or three cells thick of cubical trophoblastic cells, lined internally with mesoderm, and constitutes the “chorion.” Its lateral walls are clothed with columnar entodermal cells, while the floor, which is extremely thin, is constituted by the amnion, consisting of two closely apposed fine layers of ectoderm and mesoderm. The embryonal ectoderm has the form of a thick curved’ plate, the medullary plate, composed of elongated columnar cells. The visceral entoderm in which the vesicle is invested is not of the same character throughout. Below the embryonal ectoderm the entodermal cells are partly cubical, partly fusiform and constitute a thin compact layer. In the middle region of the egg cylinder, around the extra-embryonal coelom, the entoderm consists of very tall columnar cells, in which the nuclei are situated basally. The cytoplasm of these cells is vacuolated, coarsely granular, and prolonged into irregular processes. This layer gradually thins down as it approaches the Trager, and before reaching the latter it is reflected back as the parietal yolksac wall, which is represented by isolated cells, lying in contact with Reichert’s membrane. The latter is continuous with the tissue of the Trager at its margins and forms a very distinct lining to the implantation cavity; it has already increased somewhat in thickness as compared with the previous stage and would appear to have a protective function. It prevents the passage of maternal blood, particularly leucocytes, into the yolk-sac cavity, and limits the destructive and phagocytic activities of the giant cells or megalokaryocytes to the maternal tissues. It persists throughout the gestation period and increases very considerably in thickness in later stages.
The uterine swellings in which the embryos are situated are at this stage of considerable size. They are egg-shaped, the narrow end, in which the embryo is located, being antimesometrial in position. They average 4 mm. in diameter and 5-5 mm. in length (Pl. XXV, figs. 8 and 4).
The original uterine lumen on the mesometrial side has almost disappeared, being represented by an exceedingly fine passage, but its former connection with the implantation cavity is usually marked by an extensive blood sinus, which serves to assist the rapid penetration of the foetal trophoblast of the Trager. The decidual tissue is everywhere extremely vascular. On the mesometrial side, the cells are small and densely crowded together, with numerous large blood sinuses lined by endothelium. On the antimesometrial side the cells are large and pale-staining, having the character of typical decidual cells; blood sinuses are equally abundant, but far smaller than those at the mesometrial end. The implantation cavity itself is considerably larger than the embryonal formation, the space between Reichert’s membrane and the decidua being occupied by extensive blood lacunae, enclosed in the meshes of a fine network, the strands of which are united by very large cells with denselystaining nuclei. The origin and nature of these cells is discussed later.
I do not propose to describe the further development of the embryo, or the origin of the mesoderm, as detailed accounts of the development of other rodents are available and from the rather limited number of stages of Microtus at hand, it is not possible to follow the development in detail. I shall therefore proceed to describe the placentation, and origin of the placental giant cells.
PLACENTATION AND ORIGIN OF THE GIANT CELLS
The general character of the placentation in Arvicola amphibius does not appear to differ markedly from that of the Rat or Mouse, and the ripe placenta is very similar. In all three forms the blastocyst comes to lie excentrically in a crypt on the antimesometrial side of the uterine lumen. The uterine epithelium surrounding it degenerates and disappears, with the result that the trophoblastic wall of the vesicle is brought into direct contact with the stroma of the uterine mucosa. The cells of the latter immediately around the blastocyst become destroyed by the agency of giant cells, while those more remote become converted into decidual cells. The ectoplacental trophoblast at the mesometrial end of the blastocyst thickens rapidly and grows forwards through the uterine tissues which thus become penetrated by a coarse network of trophoblastic cells, which destroys the endothelial lining of the maternal blood vessels. The blood extravasations thus formed become enclosed in trophoblastic tissue. At a later stage the mesodermal allantois fuses with the “chorionic” mesoderm underlying the trophoblast and when vascularisation of the allantois is initiated, the allanto-chorionic mesenchyme, carrying foetal capillaries, extends into the trophoblast, which by this time has the form of a complex system of lamellae, honeycombed with narrow channels in which the maternal blood circulates. In this way the definitive allantoic placenta is established.
Although the placentation of Arvicola amphibius is similar to that of other murine rodents, there are considerable differences in detail.
Before describing the development of the placenta, however, it seems desirable to give an account of the origin of the giant cells.
Origin of the Giant Cells.
A very characteristic feature of Rodent placentae is the presence of numerous large cells, Riesenzellen, Megalokaryocytes or Giant Cells as various authors have designated them. They occur abundantly in the Rat, Mouse and Field Vole but do not appear to attain the number or dimensions that they do in the placenta of the Water Vole.
If one examines a section through the fairly late placenta, such as is represented in Pl. XXXII, fig. 27, one sees that the margins of the placental dise consist almost exclusively of these giant cells, while very many others occur in the decidual tissues, particularly in the region just beyond the limit of penetration of the trophoblast, that is to say, in the area which has been termed the “‘Umlagerungszone.” These giant cells make their appearance soon after the egg has become attached to the uterine tissues, and a considerable time before the allantoic outgrowth is recognisable. In stages corresponding to that represented in Pl. XXV, fig. 8, one finds a few large isolated giant cells of very considerable size embedded in the decidual tissues, not necessarily adjacent to the implantation cavity, in fact often close to the muscularis.
The question as to the origin of these cells has occupied the attention of a number of workers but the conclusions arrived at are by no means in agreement.
According to Jenkinson, Duval, Sobotta and Hubrecht, these giant cells are of foetal origin, being derived from the trophoblast. These writers affirm that certain cells of the parietal trophoblast of the early blastocyst, become detached, penetrate the maternal tissues, and grow in size by ingulfing decidual cells and maternal blood.
According to Disse, who worked on Arvicola arvalis, the giant cells are purely maternal in derivation. He states that they appear prior to the attachment of the egg to the uterine epithelium and that their origin from maternal tissue is therefore beyond question. He ascribes their formation to some stimulus, derived from the fertilised egg, but in no wise limited to the immediate neighbourhood of the egg, since large giant cells and symplasma masses occur deep down in the uterine tissues, far from the implantation cavity. The course of events according to Disse is as follows: Some stimulus from the fertilised egg causes certain maternal cells to flow together with the formation of symplasma masses, while others increase in size, by ingulfing decidual cells and symplasma masses already formed, until they come into contact with the walls of maternal capillaries. They destroy the endothelial walls and pass into the lumen of the vessels, whence they are carried to the implantation cavity and deposited on the wall thereof. In this way, Disse explains the presence of the giant cell network which surrounds the embryonal formation in the earlier stages and which persists around the margin of the placenta throughout the whole period of gestation.
Disse supports his statements with numerous figures representing these Riesenzellen ingulfing decidual cells, symplasma masses and maternal blood. He also shows them in contact with, and, according to him, destroying, by phagocytosis, the endothelial walls of maternal capillaries and ultimately lying free in the lumen of the vessels.
Otto Grosser, in his more recent description of the placenta of the Rat, expresses agreement with the observations of Duval and Sobotta. He states that cells from the transitory trophoblastic wall of the vesicle penetrate the mucosa and grow into giant cells, which serve to enlarge the implantation cavity by destroying the decidual cells which constitute its wall. According to him, the superficial layer of the trophoblast of the Trager also becomes, in later stages, converted into giant cells and constitutes the Umlagerungszone. He says: —
“In etwas spateren Stadien (figs. 118 and 119) ist diese Veriinderung der peripheren Tragerzone gleichfalls kenntlich; von Degeneration der Zellen ist aber nur mehr wenig zu sehen, die Zellen sind in intensiver resorbierender Tatigkeit begriffen und vielfach mit aufgenommenen miitterlichen Blutkérperchen beladen. Die Zellen sind bedeutend grésser als die tibrigen Trophoblastelemente, sie wandeln sich in die Riesenzellen alterer Placenten um.” He holds, however, that a certain number of giant cells are of maternal origin being formed from decidual cells which, under the influence of the invading trophoblast, flow together with the formation of symplasma masses: “‘ Unter seinem (Trager) Einfluss, der vielleicht anfanglich mehr in einer Fermentwirkung wie in einem aktiven Vordringen der Zellen besteht, gehen die.oberflachlichen Deciduaschichten zugrunde, zum Teil nach Symplasmabildung (miitterliche Riesenzellen).”’
Jenkinson, in his account of the histology and physiology of the placenta of the Mouse, also supports the trophoblastic origin of the giant cells.
All these workers are of opinion that the giant cells are essentially migratory in character. Sobotta, Duval, Jenkinson and Grosser maintain that they migrate from the trophoblastic wall of the vesicle into the decidua, while Disse, on the other hand, maintains that they are formed in the decidua remote from the blastocyst and migrate to the wall of the implantation cavity.
My own observations on the placenta of Arvicola amphibius do not confirm either of these theories. I am in agreement with Disse as regards the maternal character of these giant cells but differ from him as to their time of first appearance and as to their origin. There is, moreover, no evidence that these cells in the placenta of Microtus are migratory.
In the stage represented by text-fig. 5 the implantation cavity is surrounded by an annular zone of tissue which, owing to its peculiar reaction to stains, is sharply differentiated from the remainder. It consists of a belt of maternal capillaries, the lumina of which are greatly reduced, with the result that the endothelial walls are almost in apposition. The cytoplasm of these endothelial cells stains intensely with eosine, while the nuclei stain black with haematoxylin.
Capillaries with such a modified endothelial lining are present in many parts of the decidual swelling, but they reach their maximum number in the area surrounding the lateral walls of the implantation cavity.
It is these endothelial cells, I maintain, which give origin to the giant cells or megalokaryocytes of the Microtus placenta.
Figs. 5-16 (Pls. XX VI, XXVII and XXVIII) are reproductions of photomicrographs illustrating the successive stages in giant cell formation. They are not selected from progressive developmental stages, since it is unnecessary to do so, for. the transformation of endothelial cells into giant cells is a continuous process, which lasts until the placenta is fully formed. It is moreover a rapid metamorphosis, for quite large giant cells are present in early stages such as PL XXV, fig. 3.
Pl. XXVI, fig. 5 represents a portion of the decidual capsule around the embryo shown in text-fig. 5. The dark coloured strands are endothelial cells lining maternal capillaries, the lumina of which are greatly reduced. The cells are somewhat enlarged and their cytoplasm stains intensely with eosine.
In Pl. XXVI, fig. 6 is shown, under a higher magnification, one of these capillaries traversing typical decidual tissue. It will be seen that the endothelial lining is quite intact but that many of the cells have undergone characteristic changes. Their nuclei stain intensely dark and are considerably enlarged. The cytoplasm is increased in amount and appears fibrillar.
In Pl. X XVI, fig. 7 is seen an enlarged capillary or sinus, the endothelial wall of whichis everywhere recognisable yet in which certain cells have increased enormously in size. The nuclei are large and vesicular with irregular black chromatin masses and fine reticulum. The cytoplasm is finely fibrillar in character with very irregular ragged free surfaces.
In Pl. XXVI, fig. 8 is shown a similar stage. The cells though profoundly changed are still unmistakably endothelial. The deeply-staining cytoplasm, which formed the characteristic annular zone around the implantation cavity in text-fig. 5, is clearly visible.
A later stage is represented in Pl. XXVII, fig. 9; here the cells have increased in thickness and their outer surfaces are in more intimate contact with the surrounding decidual cells; it is in fact difficult to determine their limits, for their cytoplasm is prolonged into irregular processes which pass between the neighbouring cells.
In the next stage, Pl. X XVII, fig. 10, the cells have increased in size still further and are unmistakably giant cells, yet they still retain their endothelial character.
In Pl. XXVIL, fig. 11, but for the fact that the enlarged cells form a more or less continuous layer, enclosing a space filled by maternal blood, one would hardly recognise them as transformed endothelial cells. Their cytoplasm is vacuolated and in many cases contains fragments of decidual cells and blood corpuscles. ;
The maximum development is shown in Pls. XXVII and XXVIII, figs. 12 and 18. Their cytoplasm is coarsely fibrillar, vacuolated and contains much foreign matter. The cells remain quite distinct from one another, they do not form a syncytium or symplasma, the cell outlines between neighbouring giant cells being clearly defined, but where their cytoplasm comes into contact with decidual tissue it appears to be actively phagocytic, ingulfing the decidual cells and symplasma masses formed therefrom. Numerous brown granules are present around the periphery of some of these cells (Pl. XXVIII, fig. 14). They possibly represent a haemoglobin derivative, for numerous red blood corpuscles in various stages of degeneration are present in the cytoplasm. These cells are actively phagocytic, their cytoplasm usually containing large vacuoles and fragments of maternal cells. The fixatives employed have not permitted me to determine the occurrence of fat and glycogen.
The giant cells apparently attain their maximum size in the outer regions of the uterine tissues (Pl. XXVIII, fig. 15), where one occasionally finds isolated cells measuring as much as -3 mm. in length, quite close to the muscularis. It is, however, around the embryonal formation that the giant cells are most abundant (Pls. XXVIII, XXIX and XXXII, figs. 16, 17 and 27). Here they constitute a vascular network, the meshes of which contain maternal
- blood. This network makes its appearance quite early. In the stage represented by Pl. XXV, fig. 3 the blastocyst is already surrounded by a belt of tissue which consists almost entirely of giant cells united by strands enclosing maternal blood. These large blood lacunae are therefore not extravasations, as they are commonly called, since their containing walls are of purely endothelial origin. As no injections were carried out I am unable to determine whether the blood, during life, circulates through the meshes of this network, but there is no reason to suppose that it does not. The maximum development of this vascular network is shown in Pl. XXVIII, fig. 16, and Pl. XXXII, fig. 27. Its innermost layer comes into contact with Reichert’s membrane and the cytoplasmic filaments of its individual cells come into actual organic continuity therewith. The outer layer of giant cells is in contact with the maternal decidual cells which, under the influence of these phagocytic cells, flow together to form irregular, deeply-staining, symplasma masses. Pl. XXIX, figs. 17 and 18 show the formation of such symplasma from decidual cells in proximity to the megalokaryocytes. Ultimately these symplasma masses are ingulfed and absorbed by the giant cells, as shown in Pl. XXIX, figs. 19 and 20, with the result that, all the decidual tissue around the lateral walls and antimesometrial end of the embryonal formation, is replaced by giant cells. As the embryo increases in size, this giant cell network becomes stretched and its constituent cells become very attenuated, so that in later stages, such as that represented by Pl. XXXII, fig. 25, the yolk-sac splanchnopleur is only separated from the regenerated uterine epithelium by a thin layer composed of Reichert’s membrane and the flattened giant cells. Around the margin of the placental disc, however, this stretching process is strictly limited, with the result that the giant cell network persists in this region and its cells increase in size; hence in later stages the giant cells are most conspicuous in the placental margin (Pl. XXXII, fig. 25 and Pl. XXXII, fig. 27).
The function of these giant cells would appear to be twofold. In the first place they serve, by their destruction and absorbtion of the maternal tissues, and by their resulting growth in size, to surround the implantation cavity with an exceedingly vascular spongy tissue, which permits and facilitates expansion of the embryo and which, constituting as it does the maternal portion of the yolk-sac placenta, also facilitates the nutrition of the embryo. In the second place, these cells serve as purely destructive agents which ingulf maternal tissues. As the embryo increases in size, a very considerable amount of maternal tissue must be removed from the lateral and antimesometrial portions of the decidua and there is probably a decided advantage in employing, for this phagocytic purpose, the same agents as are employed for increasing the vascularity of the implantation cavity wall, and agents, the life of which is moreover strictly limited.
With regard to those giant cells which are sometimes found close to the muscularis, and which attain a diameter of -3 mm. (Pl. XXVIII, fig. 15), it is not clear what useful purpose they can serve, but their position may be more or less accidental, as we have no knowledge of. those processes which condition the conversion of normal endothelial cells into megalokaryocytes. An isolated endothelial cell of some superficial blood vessel, if it once started its metamorphosis into a giant cell, might be expected to grow more rapidly than one surrounded by other giant cells, for in the latter case the food supply would of necessity be more limited. There is, as I have already, stated, no evidence that these giant cells are migratory and I regard the presence of these isolated cells as an indication that migration does not take place, for if it did, these cells would probably make their way to the wall of the implantation cavity. The presence of giant cells in the placental labyrinth itself also points to the fact that they are not migratory. The advance of the foetal trophoblast into the maternal tissues is aided by the presence of giant cells which destroy the decidual cells and increase the vascularity of the tissues. These giant cells are surrounded by the trophoblast and apparently remain at their places of origin even when the allanto-chorionic villi penetrate the latter.
Although Disse studied Arvicola arvalis it is of interest to note that many of the figures which accompany his paper might have been drawn from my own preparations. His figs. 7 and 17 which, according to him, represent giant cells destroying by phagocytosis the walls of maternal blood lacunae prior to entering the blood stream, can be interpreted as large isolated endothelial cells. The fact that the endothelial lining is absent where in contact with the Riesenzellen indicates that the latter are derived from constituent cells of that endothelium.
Although Hubrecht later maintained that giant cells were of trophoblastic origin, yet his observations on Erinaceus support my view, for he remarked on the thickened endothelium of the maternal vessels and stated that it gave origin to a layer of bulky cells, with conspicuous nuclei, surrounding the blastocyst and enclosing spacious blood lacunae. The large individual units of this layer he called Deciduofracts. Finally, he suggested a possible homology between the Deciduofracts of Erinaceus and the giant cells of Rodents.
If this explanation which I have put forward of the origin of placental giant cells is applicable to other types such as the Field Vole, Rat and Mouse, it is remarkable that the earlier workers did not consider its possibility. Duval, in his account of the Rodent placenta, noticed that many sinuses containing maternal blood were lined by large cells, which he interpreted as trophoblastic.
According to him the trophoblast creeps up the walls of the maternal vessels destroying the endothelial lining and forming a pseudo-endothelium of trophoblastic origin which he termed the “couche plasmodiale endovasculaire.”’ Jenkinson, in his more recent account of the Mouse placenta, criticised this statement as follows: ‘“‘It seems to me obvious that his (Duval’s) ‘endovascular plasmodium’ is nothing else but the lining of a trophoblastic sinus in the upper, glycogenic, portion of the placenta. In other words he is absolutely correct in attributing an embryonic origin to the cells which form the lining of these cavities, totally incorrect in regarding the cavities themselves as maternal.” Jenkinson himself, apparently, had no doubt that these cells were trophoblastic, for he states: “‘The glycogenic tissue is traversed by.sinuses leading into the sinuses which pass directly through the placenta, and, like these, lined by a pseudo-endothelium of flattened trophoblastic cells. These cells are always larger than the endothelial cells of the maternal tissues (figs. 35 and 39); they frequently project boldly into the lumen of the sinus and in fact may become almost large enough to deserve the name of megalokaryocytes.”’
Maximow, on the other hand, does not agree with this view for he states that the “couche plasmodiale endovasculaire”’ of Duval is simply an hypertrophied condition of the endothelial cells, which, according to him, later become infiltrated with leucocytes and destroyed.
It is of interest to note that in certain pathological conditions analogous changes, in the character of endothelial cells, occur.
Dr J. A. Murray has drawn my attention to the Report of the Tuberculosis Commission for 1911, in which Eastwood describes and figures certain changes in endothelial cells under the influence of tubercle bacilli. The report, which is exceedingly lengthy, contains the following passages:
“In early infections it is constantly found that the endothelial cells of the affected area become swollen. This change must be attributed to some diffusible irritant associated with the presence of bacilli; it is not confined to the endothelial cells with which bacilli are actually in contact. Swollen endothelial cells often become detached and pass into the fluid contained within the endothelial lining. They then present the appearance of what are sometimes called "macrophages" (p. 279).
“Associated with the swelling up of those endothelial cells which are in direct contact with bacilli, there are changes in the nucleus. The nucleus generally stains more darkly than the normal, and the material taking this stain often stains diffusely and tends to escape into the surrounding protoplasm” (p. 279).
“Whilst these nuclear changes are going on there is also to be observed a dissolution of continuity in the protoplasmic outline and a tendency of the ‘endothelial protoplasm to fuse with the protoplasm of contiguous parenchymatous or other cells” (p. 280).
‘“‘A further stage in the process which is frequently observed is that these groups of nuclei continue, for a time, to multiply; they then form large groups which, taken in conjunction with their protoplasmic environment, are termed giant cells” (p. 168, sect. (3)).
In connection with the above it is perhaps significant that the cytoplasm 356 G. S. Sansom
of the placental giant cells stains diffusely with basic dyes, e.g. haematoxylin, and tends to fuse with the cytoplasm of decidual cells in contiguity with it. Pl. XXVII, figs. 9, 10, 11, and 12.
It appears therefore that some stimulus derived from the fertilised egg causes the endothelial cells of the capillaries, in the embryonal regions of the uterus, to grow into phagocytic giant cells. It is probably correct to attribute this stimulus to the embryo and not to the corpus luteum, since the endothelial cells in the inter-embryonal areas retain their normal character.
There remains for discussion the question of the significance of the large cells which are present at the antimesometrial end of the early blastocyst, referred to on page 842. The interpretation of these cells is by no means easy.
Sobotta described these cells at the antimesometrial end of the 6 days blastocyst of the Mouse and held that they were giant cells derived from the parietal trophoblast and that they served for the attachment of the blastocyst to the uterine tissues and, by their phagocytic activity, brought about extravasations of blood for the nourishment of the embryo.
Melissinos, on the other hand, maintained that the Cells in question owed their apparent size to the fact that, through the shrinkage of the blastocyst during preservation, they are seen in sections in surface view and not in profile. Widakowich also noted these cells and accepted the explanation of their appearance put forward by Melissinos. Huber, in his more recent description of the development of the Albino Rat, supported this interpretation.
In Arvicola amphibius I am unable to accept the view that the size of these cells is apparent and not real, for the following reasons:
(a) The nuclei are considerably larger in all dimensions than those of the trophoblastic cells in other regions, measuring -015 x -02 x -02 mm., as compared with -01 x -008 x -01 mm.
(b) The cells in this region of the blastocyst remain the apparent largest throughout the whole series of sections, whereas if their apparent size were due merely to superficial cutting, they might well be expected to appear of normal dimensions in other sections.
(c) In horizontal sections through the implantation cavity, they still appear larger than the remainder.
Hitherto all workers are agreed that these large antimesometrial cells are trophoblastic; their identity does not appear to have been questioned. I venture to suggest, however, that they are maternal in origin, and that they are true giant cells, derived from endothelial cells in precisely the same way as the large megalokaryocytes of the placenta.
The question of the derivation of these cells is intimately bound up with a second one, namely, the mode of origin and growth of Reichert’s membrane, and it must be admitted at the outset that it is almost impossible to demonstrate clearly, either by drawings or photomicrographs of sections of early blastocysts, that these cells are of maternal origin, since they frequently appear to be actually constituent cells of the vesicle wall. But this structureless membrane, whether it be regarded as a product of the parietal trophoblast of the early vesicle, or as a basement membrane laid down by the cells of the parietal entoderm, in later stages comes into most intimate connection with the giant cells and is then, undoubtedly, no longer a purely embryonal structure, for it increases enormously in thickness in that region where the giant cell network persists, i.e. around the margin of the placental disc, and processes of the giant cells fuse with it and are identical in appearance with it (Pl. XXX, fig. 28).
Moreover maternal leucocytes collect in large numbers outside this membrane, which undoubtedly serves to prevent the passage of leucocytes into the yolk-sac cavity, yet a certain number are actually embedded in its substance. This is strong evidence that the increase in thickness of this membrane is brought about through the agency of giant cells, for it is difficult to conceive how leucocytes, which are initially outside the membrane, can become encapsuled in it, if the latter is being added to on its inner surface. If, therefore, we admit that this membrane is partly the product of giant cells, it is not unreasonable to suggest that cells found in structural continuity with it in early stages, are actually maternal giant cells, and further that they are phagocytic endothelial cells.
If one examines the decidual tissue close around the antimesometrial end of the early blastocyst, one finds numerous capillaries, many of the endothelial cells of which are undergoing the initial changes which ultimately lead to the formation of giant cells. This change is probably due, as suggested above, to some stimulus derived from the egg, therefore one would expect to find it most evident in those endothelial cells which come into actual contact with the wall of the blastocyst. It seems exceedingly probable therefore, that these large cells, which form part of the outer wall of the early blastocyst and which have given rise to the view that some at least of the placental giant cells are of trophoblastic origin, are themselves of maternal origin.
With regard to the origin of the giant cells in the placenta of the Mouse.
I am of opinion that the giant cells arise in the same manner as in Microtus, and that the suggested homology between these cells and the Deciduofracts of Erinaceus is a true one. Although Hubrecht’s description of the placentation of the Hedgehog, in which he makes this suggestion, was published 33 years ago, no worker seems to have confirmed it. This is remarkable because the work of Disse in 1906 and of Pujiula in 1908 established the fact that giant cells were derived from maternal tissues, whereas up till then most authorities were of opinion that the Deciduofracts of Erinaceus were trophoblastic in origin.
I have had no opportunity of examining the placenta of Erinaceus, but judging from Hubrecht’s very detailed description and excellent figures, it seems clear that a change in the character of the endothelial cells occurs similar to that which Has been described herein for Microtus. It should be noted that, in the Mouse, the change in character of the endothelial cells in early stages, corresponding to text-figs. 4 and 5, is far less marked than in Microtus; this is probably in correlation with the fact that the placental giant cells are far less numerous in the Mouse. In the Field Vole however, they are at least as marked as in the Water Vole. I hope at some future date to describe the origin of the giant cells in the Field Vole.
DEVELOPMENT OF THE PLACENTA
Placentation is initiated on the foetal side by the outgrowth and active proliferation of the trophoblast covering the upper pole of the blastocyst, the ectoplacental cone or Trager, and on the maternal side by the conversion of the uterine epithelium mesometrially to the egg, into a symplasma layer which rapidly disappears, with the result that the trophoblast is brought into contact with the sub-epithelial tissue. The latter is exceedingly rich in capillaries, the endothelial cells of which, in the neighbourhood of the implantation cavity, undergo those preliminary changes which lead ultimately to the formation of phagocytic giant cells. These endothelial cells often lose continuity with one another, with the result that blood extravasations take place into the uterine lumen mesometrially to the embryo. The foetal trophoblast grows rapidly upwards into this extravasation, losing, as it does so, its original compact pyramidal form and assuming the character of a coarse cellular network, enclosing in its meshes maternal blood.
At the same time a rapid metamorphosis of endothelial cells into giant cells takes place around the lateral walls and antimesometrial end of the implantation cavity. The giant cells thus formed ingulf the neighbouring decidual cells and enlarge the implantation cavity which in this way comes to be surrounded by a network of giant cells, the strands of which are of endothelial origin, and the meshes of which are filled with maternal blood. This belt of highly vascular tissue comes into intimate contact with the outer wall of the blastocyst, which consists of a fine structureless membrane derived from the original trophoblastic wall of the vesicle. Strands from the giant cells fuse with this membrane and are indistinguishable from it. The Trager, which is at first cone shaped in vertical section, extends rapidly in the lateral direction; thus increasing the area of attachment to the maternal tissues. Its penetration is greatly assisted by the activity of the giant cells, which not only ingulf and resorb the decidual cells but which also increase the vascularity of the tissue into which the trophoblast will extend. The development has now reached the stage represented by Pl. X XV, fig. 3, in which it will be seen that the blastocyst is surrounded by extensive blood lacunae the limiting walls of which are formed by the endothelial giant cells. The section shown in this figure does not pass through the Trager.
_In the meantime the primordium of the allantois makes its appearance as an almost solid outgrowth of mesodermal cells at the level of the junction of the amnionic fold with the medullary plate (Pl. XXV, fig. 3). As development proceeds the allantois increases in size and grows upwards through the extraDevelopment and embryonal coelom as a pear-shaped structure, the distal half of which is vesicular in character, i.e. it contains a fluid filled cavity, whilst the proximal portion consists of loosely packed stellate mesenchymatous cells. Pari passu with this growth of the allantois, the extra-embryonal coelom increases markedly in size, apparently under the influence of internal pressure, with the result that the chorionic roof is foreed upwards until it comes into contact with the overlying trophoblast of the Traiger (Pl. X XV, fig. 4).
This contact between the ‘‘chorion” and trophoblast is first initiated over the middle region as the result of which the ectoplacental or false amniotic cavity becomes restricted to an annular space, the external diameter of which corresponds roughly with the diameter of the Trager at its base. The “chorion” consists of a single layer of more or less cubical trophoblastic cells invested on its lower surface by an exceedingly attenuated mesodermal membrane in which the nuclei are widely separated and filiform in character. The trophoblastic cells of the ‘‘chorion” are apparently identical in character with and, after fusion has taken place, indistinguishable from, those of the Trager.
At the stage of development represented by Pl. XXV, fig. 3 the trophoblast is purely cellular, there is no differentiation into a cytotrophoblast and syncytiotrophoblast; moreover, it does not appear to be actively phagocytic; owing to the activity of the endothelial giant cells, the decidual tissue mesometrially to the Trager is largely resorbed with the result that the advancing edge of the trophoblast is preceded by a belt of giant cells and maternal blood. It should be noted, however, that this belt of giant cells, which probably corresponds to the ‘“‘zone of ingrowth” or ‘“‘ Umlagerungszone” of the Rat, is not itself migratory. It is formed de novo in successive regions, always slightly in advance of the ingrowing trophoblast, which latter therefore comes to enclose in its meshes, not only maternal blood, but also endothelial giant cells. In these early stages many of the trophoblastic lacunae possess an endothelial lining, which soon, however, degenerates, though deeply-staining masses which represent the giant cell nuclei are recognisable for a considerable time. In the lateral regions and around the antimesometrial side of the embryonal formation similar changes in the maternal tissues are in progress. Immediately outside Reichert’s membrane there is established a vascular giant cell network containing maternal blood. The decidual tissues adjacent to this zone become converted into multinucleate symplasma masses (Pl. X XIX, fig. 17) which are ingulfed and resorbed by the giant cells (Pl. X-XIX, fig. 20). Similar symplasma masses are formed from the decidual cells mesometrially to the “zone of ingrowth.”
An embryo at this stage possesses five pairs of mesodermal somites. The allantois measures 1-5 mm. in length and -45 mm. in diameter at its distal vesicular end. The vesicular portion is at present quite devoid of blood vessels, but the presence of foetal vessels in the embryonal mesenchyme, adjacent to the attachment of the allantoic stalk, indicates that vascularisation will shortly take place.
Unfortunately no stages were obtained in which the first union of the allantois with the “chorion” was taking place, but it seems probable that by the time the vesicular portion has reached the trophoblast its vascularisation is completed. In the next developmental stage available it is seen that the area of allanto-chorionic union is almost as wide as the area of the decidua into which the trophoblast has penetrated. This wide area of union is doubtless correlated with, if not actually conditioned by, the vesicular character of the allantois, for it is clear that a more rapid flattening can take place in the case of a vesicle than in that of a solid sphere.
Immediately this layer of vascular allantoic mesenchyme has come into contact with the chorionic mesenchyme, the formation of foetal villi sets in. It appears, as Jenkinson has pointed out for the Mouse, that the overlying chorionic trophoblast is not merely invaginated into the trophoblast of the Trager by the upgrowth of the allantoic mesenchyme, but that it takes an active part in the process of ingrowth. In the case of the Mouse, however, the trophoblast of the Trager is already syncytial, constituting the -“‘ plasmoditrophoblast,” whereas in Microtus the trophoblast retains its cellular character until the penetration of the allantoic villi has set in. In these early stages of placental formation it is therefore possible to distinguish three regions in the foetal portion of the placenta. Firstly an irregular zone of actively proliferating cellular trophoblast beyond the region of penetration of the allantois. Secondly a zone of syncytial trophoblast into which the foetal villi have penetrated. Thirdly a zone of cellular trophoblast, which Jenkinson calls the “cytotrophoblast,” which is simply the floor of the original ectoplacental cavity. This cellular trophoblast very soon becomes perforated by the allantoic villi, with the result that the latter come into direct contact with the syncytial layer. The ingrowth of the allantoic villi results in the formation of a series of thin trophoblastic syncytial lamellae, containing maternal blood, separated by mesenchymatous villi carrying foetal capillaries. In this way the definitive allantoic placenta is established (Pl. XXXII, fig. 27).
From now onwards throughout the gestation period the changes in the placenta are purely of the nature of an elaboration of pre-existing structure.
In vertical sections through the placental dise of stages similar to, and more advanced than, that represented by figs. 25 and 27 three well defined zones are recognisable. The zone nearest the mesometrium consists of typical decidual tissue penetrated by wide blood lacunae, the endothelial lining cells of which are in various stages of conversion into giant cells, those nearest the ingrowing trophoblast being usually considerably more advanced in their metamorphosis, with the result that the decidual tissue in this region becomes converted into a syncytium which is resorbed by the giant cells. The middle zone of the placental disc consists of the actively proliferating cellular trophoblast, in which are embedded numerous giant cells and masses of maternal blood.
The third zone is constituted by the placental labyrinth consisting of trophoblastic syncytial lamellae honeycombed with channels, in which the maternal blood circulates, alternating with mesenchymatous villi carrying allantoic capillaries. Attached to the syncytial walls of the lamellae are numerous small giant cells, which have apparently remained at their places of origin on the vessel walls and become surrounded by the trophoblastic syncytium.
It seems clear that the ‘“‘Umlagerungszone” of Grosser, which according to him is formed by the conversion of the superficial layer of the trophoblast into giant cells, is, in Microtus, represented by the irregular layer of giant cells formed from the endothelial cells lining the maternal blood sinuses in proximity to the ingrowing trophoblast.
According to Duval, the trophoblast creeps up the walls of these maternal sinuses, destroys the endothelial lining and forms a pseudo-endothelium of syncytial trophoblast which he termed the “couche plasmodiale endovasculaire.” Jenkinson’s interpretation of Duval’s figures representing this endovascular plasmodium, differs somewhat. He agrees with Duval that the cavities are lined by trophoblast, but maintains that they are not maternal sinuses. He considers that they are simply spaces in the foetal trophoblast containing extravasated maternal blood.
There is little doubt that in Microtus these sinuses are maternal and that their containing walls are modified endothelial cells.
As development proceeds the proliferation of the cellular trophoblast fails to keep pace with the ingrowth of the foetal villi, with the result that the placental labyrinth increases in thickness at the expense of the middle layer. In the nearly ripe placenta the cellular trophoblast, which formerly constituted the middle zone, is only represented by comparatively small islands of tissue surrounded by giant cells, blood lacunae and decidual cells.
THE YOLK-SAC PLACENTA
The yolk-sac placenta in Microtus, as in other rodents with inversion of the germ layers, plays a subsidiary réle in the nutrition of the embryo. Owing to the character of development, the yolk-sac cavity early comes into open continuity with the cavity of the blastocyst (text-fig. 4). At this time the entoderm cells constituting its persistent visceral wall are ovoid and simple in character, but by the time the blastocyst has reached the “‘egg-cylinder” stage they exhibit a differentiation which indicates that they are engaged in resorbtive activity (text-fig. 5).
The entodermal cells around the embryonal ectoderm first become columnar, their cytoplasm exhibits vacuolation, their free surfaces being granular and apparently engaged in absorbing nutrient fluid from the yolk-sac cavity. As already stated, there is no evidence in the case of Microtus that these cells contain haemoglobin. No maternal blood can reach the yolk-sac cavity, which remains throughout gestation shut off from the maternal tissues by Reichert’s membrane, and any substance absorbed by the entodermal cells of the yolk-sac splanchnopleur must have passed by osmosis through this membrane.
It is of interest to note that these entodermal cells attain their maximum size and activity, below the embryonal ectoderm, quite early in development (text-fig. 5), whereas at a rather later stage, such as is represented in Pl. XXV, fig. 3, they have become reduced in size in this region and form a layer of cubical or flattened cells, which do not appear to be absorbtive at all, whilst on the other hand the cells covering the extra-embryonal coelom and ectoplacental cavity have become tall and columnar.
At a time when the embryo possesses about five somites, vascularisation of the yolk-sac splanchnopleur sets in, and concurrently therewith there are developed folds and villi, in that portion of the splanchnopleur which is adjacent to the placenta (Pl. XXYV, fig. 4).
As development proceeds these entodermal folds increase in size and complexity and omphalopleural vessels penetrate into them (Pl. XXX, fig. 22).
These villous folds attain their maximum size over the margins of the placental disc, whereas on the antimesometrial side the entoderm has the form of a simple layer of columnar cells in close contact with Reichert’s membrane. Apparently, this villous portion of the yolk-sac splanchnopleur is concerned with the absorbtion of nutritive material, which has diffused into the yolk-sac cavity from the annular zone of vascular giant cell tissue, which encircles the placenta proper.
At a stage corresponding to that shown in Pl. XXXI, fig. 25, the uterine lumen has been reformed on the antimesometrial side of the capsularis, and the yolk-sae splanchnopleur is only separated from the uterine cavity by a comparatively thin layer, consisting of Reichert’s membrane, the network of attenuated and degenerating giant cells, and a thin layer of circular muscle fibres. As the embryo increases in size the capsularis becomes stretched still. further, until, in very late stages, it is represented by a thin membrane composed of the now flattened cells of the yolk-sac splanchnopleur closely applied to Reichert’s membrane.
' According to Jenkinson and Grosser, this layer ruptures a considerable time before birth, with the result that the yolk-sac splanchnopleur becomes exposed to the uterine lumen. In Arvicola amphibius this does not occur, for in the latest stage which I possess, where the uterine swellings measure 16 mm. in diameter and the placenta 138mm. x 3:5 mm., the splanchnopleur and Reichert’s membrane are still intact.
Owing to the fact that the Water Vole material was collected in the wild state, it is not possible to ascertain the age of this foetus, but it is clear from its size and condition that it is close on full time.
It is worthy of note, therefore, that Reichert’s membrane persists throughout the gestation period, or at all events, until parturition is imminent.
- The development of Microtus amphibius is of the excentric type. It agrees in general outlines with that of the Mouse and Rat.
- Theimplantation cavity early becomes surrounded by a belt of maternal capillaries, the endothelial cells of which exhibit characteristic changes.
- These endothelial cells become phagocytic giant cells. The decidual tissue adjacent to them breaks down with the formation of symplasma masses, which are resorbed by these giant cells.
- All the placental giant cells are of maternal endothelial origin. The trophoblast does not contribute to their formation, as has been described in the Mouse and Rat.
- The ectoplacental trophoblast does not appear to be actively phagocytic. Its penetration is assisted by the giant cells, which destroy the decidual tissue in its line of advance.
- Around the antimesometrial end, and lateral walls, of the implantation cavity, the giant cells form a vascular network in contact with Reichert’s membrane. This network serves to facilitate the rapid stretching of the capsularis which results from growth of the embryo.
- The trophoblast of the Trager retains its cellular character until the penetration of the allanto-chorionic mesenchyme has set in, and even then the formation of syncytio-trophoblast is limited to that portion in contact with, or adjacent to, the allantoic villi.
- The ripe placenta is of the discoidal, haemo-chorialis type, the maternal blood circulating in syncytial trophoblastic lamellae, subdivided by foetal mesenchymatous villi, carrying allantoic capillaries.
- The yolk-sac splanchnopleur does not come into contact with maternal tissues until parturition, or very shortly prior thereto. It is always separated therefrom by the persistent Reichert’s membrane.
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DESCRIPTION OF PLATES XXV—XXXII
Fig. 1. Transverse section through a uterus containing an unattached egg. The characteristic change in the character of the uterine epithelial cells is clearly visible. x 200.
Fig. 2. An early stage similar to the above. Localised changes in the epithelial cells of the uterine lumen at the antimesometrial end are visible. x 200.
Fig. 3. Longitudinal vertical section through a uterus containing an early blastocyst. The section does not pass through the Trager. x 15.
Fig. 4. Transverse section through a uterus containing an embryo with about five somites. The ectoplacental cavity is obliterated, except around its margins, by the union of the “chorion” with the trophoblast. The vesicular character of the allantois is well shown. x 15.
Fig. 5. Section through the maternal tissue near the implantation cavity, in a stage corresponding to text-fig. 5 or fig. 21. The deeply stained strands are endothelial cells lining narrow maternal capillaries. x 150.
Fig. 6. A higher power view of the same tissue traversed by a fine capillary, the endothelial cells of which are beginning their metamorphosis into giant cells. x 360.
Fig. 7. Section through the placenta, showing a large maternal blood vessel. The endothelial cells are greatly enlarged. x 360.
Fig. 8. Section through the decidual tissue in which may be seen a maternal capillary, the lining of which has undergone hypertrophy. x 360.
Fig. 9. Section through the decidua showing a maternal vessel. Three endothelial cells have become giant cells. Their outer surfaces, which abut against the decidual cells, have fused therewith. x 360. Plate XXV
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Journal of Anatomy, Vol. LVI, Parts 3 & 4
Plate XXX Journal of Anatomy, Vol. LVI, Parts 3 & 4 Plate XXXI Journal of Anatomy, Vol. LVI, Parts 3 & 4 Plate XXXII Fig. Fig.
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Development and Placentation in Arvicola amphibius 365
10. Section through the decidual tissue, showing a large maternal blood sinus, the endothelial cells of which are typical giant cells yet retain their endothelial character. x 360.
11. Section through the decidual tissue, showing a small maternal blood vessel, the endothelial cells of which have become typical giant cells. x 360.
12. Section through the placenta at a stage rather earlier than that shown in fig. 25. The character of the giant cell network near the margin of the placenta is clearly indicated. The spaces between the cells are occupied by maternal blood. x 230. .
13. Section through the fairly late placenta near its margin. The giant cell network has attained its maximum development. x 220.
14. Section through a similar region to the preceding one, showing the brown granules around the periphery of the giant cells. x 220.
15. Section through the antimesometrial side of the decidual swelling close to the muscularis mucosae. An isolated giant cell measuring -3 mm. in length is lying amongst the cells of the mucosa. x 150. .
16. Section through the wall of the implantation cavity, showing the structure of the giant cell network and its intimate connection with Reichert’s membrane, on the right. To the left may be seen maternal symplasma formed from the decidual cells under the influence of the giant.cells. x 220.
17. Section through the wall of the implantation cavity. On the right is Reichert’s membrane attached to the inner (right) surface of which are the isolated cells of the parietal yolk-sac entoderm. On the left of the giant cells is the decidual tissue, the portions of which nearest the giant cells are forming a symplasma. x 220.
18. Section through the wall of the implantation cavity. On the right is the giant cell network, containing maternal blood, on the left the decidual tissue, which is becoming converted into a symplasma. x 220.
19. Section through the wall of the implantation cavity, showing a large giant cell in process of ingulfing maternal symplasma. x 220.
20. Section through the implantation cavity wall showing a large giant cell in the cytoplasm of which are masses of maternal symplasma. x 200.
21. Longitudinal section through the egg-cylinder of the Field Vole—Arvicola agrestis. x 100.
22. Section through the yolk-sac splanchnopleur of the late embryo, near its attachment to the placenta. Compare figs. 25 and 27. x 230.
23. Section through the placental margin showing the growth in thickness of Reichert’s membrane and its intimate connection with the placental giant cells. Embedded in the membrane are maternal leucocytes, while adherent to its inner (upper) surface are the cells of the parietal yolk-sac wall. The spaces between the giant cells are filled with maternal blood. x 380.
24. Section through the margin of the late placenta. At the right hand top corner is the outer wall of the uterus with the regenerated uterine epithelium. To the left of the latter is the newly formed uterine lumen, bounded on the inside by the capsularis, which consists of a layer of giant cells in contact with Reichert’s membrane, which attains its maximum thickness in this region. Adherent to its inner surface are the cells of the parietal entoderm, while embedded in it are maternal leucocytes. The large cavity shown in the figure is the yolk-sac cavity. x 200.
25. Transverse section of the uterus containing a fairly late embryo. The section passes through the allantoic stalk and yolk stalk. The reformed uterine lumen is visible at the antimesometrial end. The capsularis is now comparatively thin. x 12.
26. The centre of the placental disc of the same specimen. The general character of the placental labyrinth, and its relations to the cellular trophoblast immediately above it, are well shown. x36.
27. The margin of the placental disc of the same specimen, showing the structure of the giant cell network in this region. The membrane limiting the placenta on its lower surface is Reichert’s membrane. Attached to the latter are the entodermal cells of the parietal yolk-sac wall. In the lower portion of the figure is the yolk-sac splanchnopleur. The cavity above it is the yolk-sac cavity, that below it is the extra-embryonal coelom. x 36.
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