Paper - The embedding of the embryo guinea-pig in the uterine wall and its nutrition at that stage of development
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Emrys-Roberts E. The embedding of the embryo guinea-pig in the uterine wall and its nutrition at that stage of development. (1910) J Anat Physiol. 44(Pt 2): 192-203. PMID 17232840
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The Embedding of the Embryo Guinea-pig in the Uterine Wall and its Nutrition at that Stage of Development
By E. Emrys-Roberts, M.B., Ch.B. (Vict.), M.D. (Liverpool),
Demonstrator of Pathology, University of Bristol; late Sub-Curator of the Museum of Pathology (Gynaecological section), University of Liverpool.
The study of the changes produced by the early embryo on the parts contiguous has, during recent years, attracted the attention of many observers. finer microscopy and the examination of very early human embryos, combined with a generous survey of the lower types, have gone far to alter considerably the views held by the older writers.
Chief among the discoveries made has been the action of the embryo upon the maternal wall during the process known as implantation.
For the purposes of tl1is investigation into the means by which the implantation of the early embryo is affected, guinea—pigs were made use of ; the research was carried out mainly upon the lines laid down by von Spee in his work on the implantation in the guinea-pig} and it is with a sense of gratitude that one acknowledges at the outset the work of this fine observer. Not only is the guinea-pig an easily accessible animal, but it has been shown that, in the method of implantation whereby its embryo attaches itself to the maternal wall, much may be learnt with regard to human embryonic implantation; attention, however, was confined to the stages more immediately connected with the process itself, and the further developments of the embryo were not followed up.
The methods employed to obtain the embryos at the required stage were those outlined by von Spee. These consisted in removing the uterine horns at a definite period, i.e. the sixth to the seventh day after coitus, transferring them to the fixing reagent, and then proceeding by serial sections to cut each horn from one end to the other. This was necessarily a laborious and trying undertaking; each individual section had to be examined, since there was no microscopic indication as to the site of the embryo. Indeed it has happened that many thousands of sections have been examined fruitlessly.
1 “Die Implantation des Meersehweincheneies in die Uteruswa.nd,” Zeit. fur Morph. uml An.thrap., Bd. iii., Heft 1, S. 130-182
The first series was fixed in Miiller’s solution ; latterly Mann’s ﬂuid was used: of the two the latter gave the better results. The sections in all instances were stained with heematoxylin and eosin.
It is unnecessary to dwell at any length upon the anatomy of the uterus of the guinea-pig (see Plate 1., fig. 1).
Typically bicornuate, the horns are covered for the greater part of their periphery with peritoneum. The muscular layers, outer longitudinal, inner circular, play no part microscopically in our investigation. It is with the inner lining of the uterus that we are chieﬂy concerned. This is the portion in which the uterine glands are situated: relatively tl1ick—it is the same thickness as the muscular layers co1nbined—it is arranged in oval fashion around an elongated slit, the lumen, whose axis lies in the plane of the peritoneal attaclnnent. The uterine glands are convoluted, especially at their bases, opening by long mouths on to the surface, where their epithelium—— cylindrical, ciliated, and usually of the thickness of one cell——is continuous with that lining the lumen. The connective tissue stroma is dense and is composed of polyhedral cells. In the neighbourhood of the bases of the glands the stroma is considerably reinforced by muscle fibres derived from the circular muscle layer, and it is undoubtedly a fair inference to suppose that the presence of these fibres materially assists the secretory ﬂow of the glands. The lumen, then, is a narrow slit lined by a ciliated cylindrical epithelium one layer deep, with an underlying stroma of dense connective tissue, and it is to the lumen that our attention will be presently directed.
Our next concern is the embryo itself. It descends the Fallopian tube until it reaches the lumen of the uterus, in company usually with one or more embryos, and, since the act of coitus has been noted and the sow has been removed from possibilities of further intercourse, its fellow embryos are approximately of the same age. The individual sites of implantation are located at different areas, and not aggregated in one spot. VVhat subtle force determines this procedure it is not possible to say, nor is it possible to explain why the combined action of the increased secretion of the uterine glands and of the ciliated epithelium of the lume11 does not entirely sweep the embryos out of the uterus altogether.
It has not come within the scope of my investigation to determine the successive positions of the embryos in the course of their descent from day to day : I can only furnish evidence of their position at and about that date —-the sixth to the seventh day—at which it was expected that implantation would be brought about.
F or the present we will confine our attention to the microscopic appearance of the embryo and the uterine wall at and about the time of implantation. 194 Dr E. Emrys—Rol)erts
The embryo has attained to that stage at which the inner cell mass is still undifl'erentiated, and, except at the area in direct contact with the maternal epithelium, is completely surrounded by the zona pellucida. In section it usually appears oval in shape, with its long axis in tlie direction of the axis of the lumen of the uterine horn. Its site of implantation with regard to the lumen itself is variable; it seems to have no predilection for any particular position; here one’s experience does not quite coincide with that of Von Spec, who states that the majority of implantations occur at the antimesometrial end of the uterine lumen, which he likens to the fundus of the human uterus. I11 a series of twelve embryos one is found close to the mesometrial end of the lumen, four are nearer the mesometrial end, other six are nearer the opposite end, while one is situate in the middle of the lumen; at least seven seem to lie in the orifices of the glands. It seems, therefore, that the position of the embryo within the lumen is a matter of chance, and, since the stroma is of nearly the same thickness at the ends as at the sides of the lu111e11, there would appear to be no vital necessity for the implantation to take place at any particular site; at the same time there is no disposition on the part of the embryo to avoid implantation in a gland orifice. As before stated, the uterine horns were examined for embryos at varying periods between the sixth and the seventh day after coitus.
Embryo No. 1 (Plate I., fig. 2).—In No. 1 we have an example of an embryo lying free within the uterine lumen with its long axis parallel to that of the lumen. The zona pellucida with which it is surrounded has, owing to the character of the fixing reagent, become attenuated. The embryo itself is seen to be composed of an undiﬂ'erentiated mass of large cells. Though not in direct apposition, the epithelium lining the uterine lumen in the immediate neighbourhood of the embryo shows destructive effects, the result of the action of the outermost cells of the embryo; the individual cells are shorter, and ill defined, and their nuclei have become irregular in outline. This example represents in my series the earliest evidence of destructive action on the part of the embryonic cells. Further specimens will demonstrate the later results of this action—the disintegration and final disappearance of the uterine epithelium. The size of embryo No. 1 would suggest section through it near one of the poles ; had the series been complete, it might have been possible to show actual contact and implantation in the sections further on in the series.
Embryo N0. 2 (Plate H., fig. 3).—Here it will be seen that contact has been established with a further destruction of the epithelium. The site of implantation was the side of the lumen, nearer the mcsomctrial end. At one spot in the series of this particular implantation the surface of the Embedding of the Embryo Guinea—Pig in the Uterine Wall 195
embryo is in direct apposition to the underlying connective tissue stroma,
"elsewhere the degenerated and broken-down cells of the epithelium
intervene between the embryo and the stroma. The embryonic area of contact is not confined to the segment of the circumference which first came into apposition with the uterine epithelium, but has involved almost half of the circumference. An examination of the stroma immediately underlying the site of implantation shows marked degeneration of the nuclei and cells of the connective tissue with the formation of intercellular
P1-IOTOMICROGRAPH N o. 1.—A very high magnification of embryo No. 3, showing the structure of the embryo. The zona pellucida is lost at the area of contact with the uterine epithelium. The cell-niass is undifferentiated. The demarcation between the embryo and the maternal tissues is easily recognisable for the greater part of the area of implantation; at the upper margin the distinction is not so well maintained. The disintegrated state of the uterine epithelium is demonstrated.
areas of lymphatic oedema. The long axis of the_embryo corresponds to that of the lumen.
E'mb'ryo N 0. 3 (Plate II., fig. 4, photomicrograph No. 1).—The embryo, almost spherical in outline, has gained an implantation into the mouth of a gland, near the mesometrial end of the lumen. The free surface shows the zona pellucida which is lost at the surface of the attachment.
No differentiation of the embryonic cells into outer and inner cell masses has taken place‘. Near the antimesometrial pole the uterine epithelium has been entirely destroyed, but, owing to the nature of the site of implantation, a well-defined layer of epithelium, belonging to the orifice of the uterine gland, intervenes between the implantation area and
the underlying connective tissue stroma. Sections further in the series ‘
show that, where the gland-mouth epithelium has ceased, the embryo has established direct contact with the connective tissue stroma, with the
formation of vacliolations in the" immediate vicinity.
Embryo No. 4 (Plate III., fig, 5, -photomicrographs Nos. 2 and 3).The embryo, lying with its long axis parallel with the long axis of the lumen, has aﬂixed itself to the uterine wall at a spot in the centre of the lumen. The whole of the inner half of its circumference is in direct contact with the uterine wall; surrounding the‘ free surface of the circumference is seen the zona pellucida, reduced in the fixing process to a thin line, which becomes lost as soon as direct contact is established. The uterine epithelium has almost, but not quite, disappeared at the site of implantation. The underlying -connective tissue stroma shows some vacuolation.
PHOTOMICROGRAPH No. 2.—-A high-power view of embryo No. 4. It shows the destruction of the ‘uterine epithelium at the site of implantation, together with a. slight degree of vacuo ation of the connective tissue stroma.
Embryo N 0. 5 (Plate III., fig. 6, photomicrographs Nos. 4 and 5) shows an implantation near the mesometrial end of the uterine lumen. The embryo Embedding of the Embryo Guinea—Pig in the Uterine Wall 197
lies with its long axis parallel to that of the lumen, complete destruction of the uterine epithelium has resulted in the direct apposition of the embryo to the underlying connective tissue stroma, small vacuolated areas are seen at the line of apposition, while larger areas are seen deeper down in the connective tissue stroma. The zona pellucida is seen on the outer surface of the embryo, but has disappeared at the line of contact. H
A study of the preceding specimens clearly brings out the essential phenomena associated with the implantation of the embryo in the uterine horn of the guinea—pig. The embryo in its course down the lumen of the
PEOTOMICROGRAPH No. 3.—A very high magnification of embryo No. 4. It shows the zona pellucida continuous over the whole of the free surface of the embryo, which is itself a mass of undifferentiated cells. The uterine epithelium has been destroyed until only a. thin layer of disorganised cells separates the embryo from the subjacent connective tissue stroma, which shows a degree of vacuolation.
uterine horn eventually comes to a standstill, the choice of site -appearing to be determined by no obvious consideration. _ There does not, however, seem to be the propensity for implantation at the antimesomet-rial end of the lumen exhibited by the majority of Von Spee’s embryos. Implantations in gland orifices are not to be Wondered at, considering the‘ frequent occurrence of these structures in the uterine lumen. The process of implantation is accompanied by the loss of the zona pellucida covering the embryo, this structure being invariably lost along the line of attachment, resulting in the direct apposition of embryonic cells to those of the uterine epithelium.
The action of the embryonic cells upon the uterine cells is seen to be one of disintegration and destruction ; the maternal cells first encountered—tl1e cells of the uterine epithelium—are destroyed in a manner that leaves no doubt as to this action: they entirely disappear and their place is occupied by the embryo. When the connective tissue stroma is reached the process is repeated, and the embryo appears to sink down’ into the substance of the uterine Wall. Maternal cells are to be seen exhibiting all stages of this process of disintegration, and, When the stroma has been reached, the
PHOTOMICROGRAPH No. 4.—A high magnification of embryo No. 5. It shows the destruction of the uterine epithelium in a very marked manner.
connective tissue cells for a variable radius show vacuolations and degenerative changes, which show that the action of the embryonic cells is capable of effecting destructive changes at a distance. This observation is amply borne out by a study of the figures accompanying von Spee’s monograph. .
What the exact nature of the action of the embryonic cells is it is diﬂicult to determine. It may be supposed, for the sake of argument, that the embryo accomplishes implantation by reason of an assimilative action on the part of its outermost cells, comparable to the methods by which the amoeba is capable of assimilating its nutrition. Were this so, we should be prepared to find elongated processes from the embryonic cells extending to the uterine cells, in the manner of amoebic processes, there coalescing and at the same time enveloping portions of maternal protoplasm. Also we should expect to see the maternal tissues retaining full vigour except at the area in immediate contact with the cells of the embryo. On the other hand, it may be suggested that implantation results from a digestive action on the part of the embryonic cells: from a digestive secretion or enzyme produced by these cells reacting upon the maternal tissues. In support of this contention We have the demonstration in embryo No. 1 of the commencing
PHOTOMICROGRAPH No. 5.—A very high magnification of embryo No. 5, showing the embryo, a mass of undifierentiated cells, surrounded on its free surface by zona pellucida, in direct contact with the stroma of connective tissue cells ; near the upper pole a cell of the embryo is seen protruding into the uterine epithelium. Vacuolation of the connective tissue stroma is seen.
disintegration of the uterine epithelium, although the embryo is, in this particular section, still at an appreciable distance from the uterine wall; and also the demonstration, in embryos Nos. 2, 4, 5, and 6, of vacuolated areas in the maternal tissues the vicinity of the implantation sites, suficiently remote from the embryonic cells to lead us to believe that the action of these cells is by no means confined to the tissues in the immediate vicinity.
Although, in the photomicrographs Nos. 4, 5, and 6 of embryos Nos. 5 and 6, there is to be seen a cell in each of them protruding into the uterine tissues, this occurrence cannot be held in any way to support the assumption that the nutrition is obtained by assimilation. Whatever may be the explanation of this phenomenon, which appears to be suﬂiciently rare to attract attention, it certainly does not coincide with the expectations outlined with regard to the process by which direct assimilation is accomplished. On the whole, I am of opinion that the Weight of evidence from the histological standpoint is in favour of attributing to the embryonic cells a,
PHOTOMICROGRAPH No. 6.—A high magnification of embryo No. 6. It shows the destruction of the epithelium at the orifice of the gland, with vacuolated areas intervening between the embryo and the uterine tissues. One of the cells of the embryo, undergoing mitosis, is seen extending into the uterine epithelium and having around it an area of vacuolation.
digestive action upon the maternal tissues, resulting from the production of a digestive secretion by the outermost cells of the embryo.’
Whilst studying thesections prepared. for the purpose of this investigation, I noted that the embryo of the guinea—pig underwent remarkable changes while still free within the lumen of the uterus. That the embryo grew from the single cell into the mass of.cells that we have seen making up its structure, was suﬂiciently striking to lead me into an inquiry into the nature of the metabolic changes involved in this development. I conceived the necessity for Water and salts, and also for proteid. These I surmised might befound in the secretion of the uterine glands. To prove their presence in so small an organ as the uterus of the guinea-pig was difficult, so I had recourse to the uteri of cows and of bitches, and an examination established the presence of these constituents, both in the resting and also in the pro—oestral stages.‘
My next endeavour was to ascertain if possible the methods by which these substances were incorporated iI1to the organism of the embryo. The water and salts presented no insuperable difficulty, since it is generally agreed that these substances are capable of absorption as such, without having to undergo preliminary changes of constitution; but with regard to the proteid a difficulty presented itself—the diﬂiculty of believing the embryo to be capable of absorbing proteid in an unchanged condition. My next step was to supplement the impossible task of gauging the proteid changes, if any, undergone in the minute and unworkable uterus of the guinea—pig, by studying the development of an embryo under the most favourable conditions. In choosing the chick I was guided by the fact that the white of the egg represented the mammalian uterine secretion, while the limits of the shell permitted any changes that might have taken place to be carefully examined.
I was able to demonstrate, on and after the sixth day of incubation, the presence in the white of the egg, surrounding the growing embryo, of albumoses and peptones, proving that in the chick the proteid in the white of the egg underwent a process of preliminary digestion on the part of the chorionic cells? This discovery was very encouraging, and suggested the probability that the decision I had come to from the microscopical examination of the embryo, viz. that the process of implantation was to be attributed to a digestive action on the part of the embryonic cells, was a correct one. That the embryo of the guinea-pig, during the period that it remained free within the lumen of the uterus, reacted in a similar manner upon the proteid of the uterine secretion, would seem also to possess an element of verisimilitude, especially since it is more than probable that the action of the embryonic cells upon the maternal wall did not develop suddenly, but existed as a potential force before implantation took place. In the Jommal of Amcfmny (mrl I’}1.g/si,olo,r/3/, vol. xxxviii. p. 333,“ we learn that, in the implantation of the embryo in the ferret, the cells of the uterine lumen, at the side opposite to the site of implantation, undergo very serious changes of a degenerative charaeter—-changes that can only be explained on the grounds that the embryonic cells secrete an enzyme of a proteolytic nature, whose action is by no means confined to the area of contact. At the same time it is manifest that, both in the ferret and in the guinea-pig,
1 I’r0c. Roy. Soc, 1908. 2 Ibid. “ 1lI1l’ITL’m»al'I.Il/L (hm and l)z:[i2n‘e'nt'£at7'm1 of I’luce71ta, Arthur Robinson.
the maternal tissues do not possess any substance capable of resisting such proteolytic action during the early stages of placentation. That such a resisting substance is evolved later must be patent, otherwise the embryo would completely destroy the uterine wall, and eventually enter the peritoneal cavity. I have not studied the further stages in these types of mammals, but my study of human placentation, based upon the examination of Very many placentae from one a few days old, found iii a decidual cast of the uterus, to those at full—term, leads me to believe that the so-called Nitabuch’s layer, a layer of broken—down maternal and embryonic cells together with fibrin, existing between the foetal villi and maternal tissues, has a definite function, viz. that of the preservation of the maternal tissues from the further action of the embryonic cells. For this reason I termed the layer the “protective layer.” My study of the physiological results attendant upon early pregnancy has suggested to me that, in addition to the “protective layer,” there is also produced by the maternal organism, for the resistance of the action of the foetal cells, a substance in the blood, acting as an antienzymic body, so as, eventually, to neutralise completely the foetal products. The manifestation of the enzymic intoxication may well be the pernicious vomiting which not unusually occurs i11 early pregnancy and the insanity which sometimes follows. If this be so the artificial production of an antibody to this enzyme should not be difficult.
I am now engaged in conducting experiments with this aim in view, and, with the hope that I may obtain an antibody capable of withstanding the action of the enzyme produced by the foetal cells, and by so doing to restore the normal balance which should exist between the foetal and maternal elements. So far as I am aware the suggestion here made is entirely new, and I am hopeful that this research into the physiology of the generative system, which, for several years, has had for its objective the demonstration of a tangible relation between the presence of the foetus and its effect upon the mother, and also the evolution of a therapeutic reagent whereby the disorders arising from the presence of the foetus might be combated, will lead to valuable results.
My attention has been drawn to an abstract of a memoir by Dr Teacher and Dr Bryce? embodying the descriptive demonstration of an extremely young human ovum found in a decidual cast of the uterus. The conelusion drawn by these authors, that the plasmodium destroys the decidua, probably by enzyme action, is quite in keeping with the results I have already tabulated, and bears out the conclusion I arrived at while studying a very similar specimen. A minute description of the embryo discovered by myself in a deeidual cast of the uterus will appear in due course.
‘Frorm. Ifny. Soc., 1905, Proceedings of the Royal Society of Medz'c2'71e, vol. i., No. 9, p. 264. Jour. of Anal. and Phy., Jam. 1910.
In conclusion, I have to express my sense of gratitude for the unfailing kindness and sympathy of all with whom I have been brought into contact. Especially I wish to thank Mr Walter Heape, F.R.S., Mr Frank J. Cole, B.Sc., Professor Sherrington, Professor Briggs, Professor Benj. Moore, and Dr H. E. Roaf, all of whom have, from time to time, given me much needed help and encouragement.
Explanation of Plates
Fig. 1. A semi-diagrammatic view of the section through the uterine horn of the guinea-pig, containing embryo No. 2. The sulci on the outer surface have resulted from the contraction of the fibres of the longitudinal muscle layer, after excision; their presence is of no importance. The site of implantation is seen to be at the side of the lumen near the mesometrial end.
fig. 2. A drawing of embryo No. l. The section has been made through it near one of its poles. It demonstrates commencing degeneration of the uterine epithelium.
fig. 3. A drawing of embryo No. 2, showing the destruction of the uterine epithelium and degeneration and vaeuolation of the underlying connective tissue stroma. fig. 4. A drawing of embryo No. 3, where implantation has occurred in a gland-mouth. The zone of uterine epithelial cells intervening between the degenerated surface epithelium and the connective tissue stroma represents the epithelium lining the mouth of the gland.
fig. 5. A drawing of embryo No. 4, showing the destructive effects of the process of embedding. The uterine epithelium is destroyed, and the connective tissue stroma presents a degenerated appearance with areas of vacuolation.
fig. 6. A drawing of embryo N 0. 5. Implantation is seen resulting in complete destruction of the maternal tissues. At the upper pole of the embryo a cell is seen protruding; elsewhere the conditions resemble those seen in fig. 5.
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