The Yolk-sacs of Erinaceus europea and Putorius furo

Since in the rabbit maternal plasma proteins are found in the yolk-sac cavity, and some at least pass into the foetal circulation by way of the vitelline vessels (Brambell et al., 1952), a comparison with two such widely different forms as the hedgehog and ferret was thought to be of interest. The development and structure of the yolk-sacs, and the nature of their contents, in Erinaceus europea and Putorius furo are therefore described below.

The hedgehog material was collected in Caernarvonshire during the summers of 1949 and 1950. The first pregnancies were obtained in early May. Some of the hedgehogs and ferrets were killed by ethyl chloride anaesthesia, others by intravenous magnesium chloride. The ferrets were killed at stages of pregnancy between 16 and 21 days; the duration of pregnancy was timed from the commencement of copulation.

Ligatures were applied to the uterus at either side of each uterine swelling, and the swellings were then removed. Some were fixed in aqueous Bouin’s fluid and preserved in 70 per cent, alcohol. These were later sectioned and stained in Ehrlich’s haematoxylin and 1 per cent, aqueous eosin. The remainder were rapidly frozen in a tube containing isopentane cooled in a Thermos flask containing a mixture of 70 per cent, alcohol and solid carbon dioxide. The yolk-sac fluid was later dissected out as a globule of ice, which was then allowed to thaw (Brambell et al., 1949). The fluid was centrifuged and transferred to a tube for weighing. The amount of yolk-sac fluid obtained from some swellings was small, and in such cases the fluid from a number of swellings in the same uterus was pooled.

A small quantity of human thrombin solution was added to some of the yolk-sac fluid samples to determine the presence or absence of fibrinogen. The remainder of the yolk-sac fluid was used to determine the concentration of protein nitrogen. The method used was the simplified ultra-micro Kjeldahl technique described by Shaw & Beadle (1949). The protein nitrogen of the maternal serum was determined by micro Kjeldahl for comparison.

The placentation of Erinaceus europea has been described by Hubrecht (1889). Implantation is interstitial and antimesometrial.

The earliest stages examined were those of blastocysts which had already commenced implantation, the yolk-sac cavity of the smallest measuring 0·18 mm. across its greatest diameter. At this stage the decidua capsularis is being formed, but has not yet completely separated the implanting blastocyst from the uterine lumen.

The trophoblast of the blastocyst has already begun to extend into the surrounding maternal tissue, and is thickest at the antimesometrial pole where the embryonic plate will differentiate. Elsewhere it is two or three cells thick. Hubrecht described the appearance of lacunae within the trophoblast of the lateral walls of the blastocyst, and their subsequent formation in the mesometrial and antimesometrial walls. He attributed their formation to the very rapid enlargement of the blastocyst outpacing the proliferation of the trophoblast cells. In the earliest stages examined these lacunae, in which maternal blood is present, do not usually appear entirely within the trophoblast, but are formed, at first, between it and the adjacent decidua.

The blood-vessels in the decidual tissue adjacent to the blastocyst have increased in size and number, presumably to permit of an increased flow of blood to this region. They are lined by a distinct endothelium, the cells of which protrude into the lumina of the vessels. These enlarging endothelial cells will eventually form the trophospongia’. a region of maternal decidual tissue closely adjacent to the blastocyst (Hubrecht).

The blastocyst enlarges rapidly, and when the formation of the embryonic knob at the antimesometrial pole is completed the diameter of the yolk-sac has increased to 0·46 mm. The yolk-sac of the latest embryonic plate stage available, in which mesoderm has not differentiated, measures 2·0 mm. across its greatest diameter.

After the formation of the mesoderm, and the appearance in it of the coelom, the amniotic folds grow up into the cavity between the embryonic plate and the antimesometrial trophoblast. The mesoderm extends mesometrially, between the endoderm and the trophoblast, converting a part of the bilaminar omphalopleur into a trilaminar structure. At first the exocoel is not extensive, being confined to the margin of the embryonic area, but soon it enlarges and extends mesometrially to separate the vascular splanchnic mesoderm (area vasculosa) from the avascular somatic mesoderm.

The mesoderm continues to extend mesometrially between the endoderm and the trophoblast, and when the vascular yolk-sac placenta has attained its maximum development only the mesometrial third of the yolk-sac wall remains bilaminar. The trophoblast becomes converted into a spongy tissue in which numerous lacunae are formed. Maternal blood circulates in these lacunae which are continuous with the large blood-spaces which have been formed in the adjacent trophospongia. At certain scattered points some of the vessels of the area vasculosa, with their covering of mesenchyme, penetrate into the spongy trophoblast, and the omphaloidean villi which are thus formed bring the foetal blood into close proximity with the maternal blood present in the lacunae (Hubrecht).

The Plate, fig. A, shows a portion of the vascular yolk-sac placenta at its greatest development. At this stage the maternal and foetal circulations are generally separated from one another by two tissues—the trophoblast which is usually only one cell thick, and the foetal endothelium of the vitelline vessels. Thus in the hedgehog the omphaloidean placenta is haemochorial in character, and the vascular relations of the omphaloidean and allantochorionic placentae are similar.

The larger omphaloidean villi are formed in the antimesometrial region of the trilaminar omphalopleur, and they penetrate into the spongy trophoblast almost as far as the trophospongia. The somatic mesoderm cannot be discerned as a separate layer, but later in development it becomes noticeable in those regions of the trilaminar omphalopleur where omphaloidean villi have not been developed, as a single layer of squamous cells.

Later some of the large vitelline vessels, with their covering of endoderm and mesenchyme, protrude into the cavity of the yolk-sac. By increasing the internal surface area of the yolk-sac splanchnopleur they may facilitate absorption from the fluid contents.

The rapidly enlarging embryo causes the yolk-sac splanchnopleur to become partially inverted and concurrently brings about the withdrawal of the more antimesometrially situated vitelline vessels and their surrounding mesenchyme from their sites in the spongy trophoblast (Text-fig. 1). The extension of the exocoel, resulting in the separation of the yolk-sac splanchnopleur from the chorion and the withdrawal of the vascular mesodermal cores of the omphaloidean villi, is accompanied by the expansion of the allantois. The allantoic mesoderm extends mesometrially, increasing the area of the allanto-chorion. In the hedgehog no anastomoses are formed between the allantoic and vitelline circulations, such as occur in the rabbit (Duval, 1892), although these are closely adjacent to one another at some stages.

As the embryo enlarges the yolk-sac splanchnopleur is further invaginated and becomes thrown into folds, and its endodermal epithelium thickens progressively, the constituent cells becoming, at first, cuboidal and later low columnar. Their apical ends are rounded and their flat basal ends rest on a distinct basement membrane (Text-fig. 2). The mesenchyme which was previously of a loose texture, is now far more compact. In the latest stage available the yolk-sac appears, in transverse sections, as a slit-like crescentic cavity, and the much folded yolk-sac splanchnopleur has attained its greatest degree of differentiation.

Avascular yolk-sac placentation is initiated by the fusion of the trophoblast of the blastocyst with the uterine mucosa. In the early stages of pregnancy the entire yolk-sac wall was bilaminar and avascular, but with the formation of the mesoderm and the extension of the area vasculosa, the avascular yolk-sac placenta diminishes in extent. In Text-fig. 1 the avascular yolk-sac placenta is restricted to a small area at the mesometrial pole. Hubrecht has shown that in the hedgehog the mesometrial extension of the mesoderm is never completed, so that a small bilaminar area persists mesometrially throughout pregnancy.

Due to the rapid enlargement of the embryo the tissues of the uterine wall are stretched. At mid-pregnancy the mesometrial uterine wall is about 2·0 mm. thick, whereas in late stages its thickness has been reduced to about 0·7 mm.

Mesometrially the endodermal cells are squamous, and this form is maintained until late pregnancy, when they thicken considerably. However, in the latest stages examined these cells have, apparently, also been affected by stretching, for they have returned to their earlier squamous form.

In these late stages the trophoblast and inner region of the trophospongia are indistinguishable from one another, and together they form a compact narrow band of tissue (Plate, fig. B). The numerous blood lacunae which were present in these tissues at earlier stages have now completely disappeared.

In those regions of the trilaminar omphalopleur in which omphaloidean villi have not been developed, the somatic mesoderm can be distinguished from the splanchnic mesoderm even before the exocoel has extended to separate them. At first the somatic mesoderm is composed of squamous cells, but by mid-pregnancy these cells have thickened. At this stage a structureless hyaline layer makes its appearance between the somatic mesoderm and the trophoblast. Hubrecht briefly noted the time of its appearance and that it thickens in later stages and persists to term. It first becomes noticeable as a very thin layer, about 5μ in thickness, between the somatic mesoderm and trophoblast of the lateral walls of the conceptus, and is absent from the mesometrial wall of the yolk-sac until later. It increases in thickness as pregnancy proceeds and in the latest stage it is generally about 15μ thick at the mesometrial pole, but along the lateral walls of the conceptus it attains a thickness of up to 25-30μ (Plate, fig. B), and it extends up to the margin of the allantochorionic placenta. It is usually very closely adherent to the somatic mesoderm and trophoblast of the lateral walls and the endoderm and trophoblast of the mesometrial wall. However, at some points it has become detached from the underlying trophoblast. In the latest stages examined the somatic mesoderm of the lateral walls of the conceptus, like the endoderm of the mesometrial wall, has become stretched thin, and is again squamous.

In the ferret implantation is of the central type, the blastocysts remaining free in the uterus for some considerable time. At 9 days the blastocyst is still surrounded by its zona pellucida (Hamilton, 1934), and the trophoblast commences to fuse with the uterine epithelium at about days (Strahl & Ballman, 1915). By the 16th day the trophoblast is in contact with the uterine wall over almost the whole of its antimesometrial hemisphere, and blunt, finger-like trophoblastic villi have been formed which penetrate into the glandular mucosa. The greatest depth of penetration, 165μ, is attained in the region adjacent to the vascular splanchnic mesoderm. At the tips of the villi, and also along their sides, the epithelia of the uterine glands and the cells of the interglandular connective tissue are degenerating. The nuclei of the maternal cells thus affected are pycnotic and in various stages of disintegration.

Mesometrially the blastocyst is not in contact with the uterine epithelium, and in this region the uterine mucosa remains unchanged, being essentially similar in structure to the non-pregnant oestrus uterus. At the time of neural tube formation the area vasculosa has spread to cover the antimesometrial half of the blastocyst, and the trophoblast is generally two cells thick, except in the mesometrial region, where it is composed of a single layer of squamous cells.

The maternal tissues are not so highly vascular as in the hedgehog. The larger maternal blood-vessels pass radially in the connective tissue between the trophoblastic villi, and only at isolated points are they in close proximity to the trophoblast.

The structural changes which occur in the yolk-sac wall during the next 5 days of pregnancy have been described by Strahl & Ballman (1915). These changes are mainly associated with the increase in size of the trophoblastic villi and with the rapid extension of the allantois. The trophoblastic villi penetrate more deeply into the maternal tissue and become hollowed out. The cavities thus formed become partially filled with mesenchyme cells which are stellate and squamous in form and have been derived from the somatic mesoderm of the trilaminar omphalopleur (Plate, fig. C).

By the 18th day a large haematoma has been formed at the antimesometrial pole, and at days the mesoderm has completed its extension mesometrially and the yolk-sac placenta has attained its maximum development. The maternal and foetal blood circulations are separated from one another by the maternal endothelia, the trophoblastic ectoderm which is generally one cell thick, the mesenchyme, and the foetal endothelia. The trophoblastic villi have widened considerably, and at 19 days their mesenchymatous cores are still avascular, and they remain avascular until after the allantois has spread over them, when foetal blood-vessels derived from the vascular allantoic mesoderm can be discerned within them.

The volume of the yolk-sac increases up to about the 19th day, subsequently decreasing. At 21 days each yolk-sac yields about 0·16 ml. of fluid.

The arrangement of the embryonic membranes at 20 days is shown in Text-fig. 3. The allantois almost completely fills the exocoel, and the somatopleur and splanchnopleur are still in contact with one another at the mesometrial pole. By the 21st day the exocoel has extended to completely separate them, and the vascular wall of the yolk-sac is neither in contact with the uterine wall at any point, nor with the contents of the uterine lumen, and omphaloidean placentation is terminated.

Since the establishment of the omphaloidean placenta, which at the 16th day was mainly bilaminar, the yolk-sac membrane has undergone numerous structural changes. A thickening of the squamous endodermal epithelium is noticeable at 18- and 19-day stages, and by the 21st day the epithelium is composed throughout of cuboidal cells, the flattened basal ends of which rest on a basement membrane. The vitelline vessels are large, and are lined by a distinct endothelium (Text-fig. 4). The amount of undifferentiated mesenchyme present is small, and on the side adjacent to the exocoel the yolk-sac splanchnopleur is lined by a coelomic mesothelium, the nuclei of which, like those of the vascular endothelia, stain intensely in Ehrlich’s haematoxylin. A membrane of Reichert is not formed in the yolk-sac wall in the ferret.

Blood samples were collected from some of the hedgehogs and ferrets at autopsy and the concentration of protein nitrogen in the maternal serum was determined. In both the hedgehogs and the ferrets there were differences in the concentrations irrespective of the stage of pregnancy. Differences of a similar order existed in the sera of male hedgehogs which were tested. The protein concentration of the sera of the hedgehogs varied from 6·1 to 7·3 per cent., while that of the ferrets varied but slightly, with a mean value of 5·7 per cent.

The presence of a coagulum which stains faintly with aqueous eosin is noticeable in the yolk-sac cavity of hedgehogs in very early stages of pregnancy. In such stages, before the formation of the embryonic knob is completed, the coagulum appears evenly dispersed throughout the yolk-sac cavity. Later it is usually restricted to the periphery of the yolk-sac, being generally more concentrated along the inner margin of the partially inverted yolk-sac splanchnopleur.

Table 1 shows the concentration of protein nitrogen in the maternal sera of one non-pregnant and five pregnant hedgehogs and the concentration in the yolk-sac fluid of four of the pregnant animals.

The volume of fluid obtainable from the yolk-sacs at early stages is too small to be tested. As pregnancy proceeds the volume of the yolk-sac increases, and continues to increase beyond the stage at which the omphaloidean placenta attains its greatest development. Yolk-sac fluid was collected from the uterine swellings of twelve hedgehogs at different stages of pregnancy and tested for the presence of fibrinogen. Two late stages, H23 and H28, gave positive results; all the earlier stages gave negative results. The latest stage to give a negative result was H41 (Table 1), a stage of pregnancy comparable to that illustrated in Text-fig. 1, when the omphaloidean placenta has already passed its greatest development and is retrogressing.

In late stages the yolk-sac cavity is considerably reduced, and it is difficult to dissect out the frozen yolk-sac fluid from the swelling. The fluid obtained from such stages is rather similar in colour to that of the maternal serum. Care was taken to avoid contamination with tissue proteins and maternal blood. At or near term the fluid obtainable is too small to be tested.

At about mid-pregnancy the protein concentration of the yolk-sac fluid is approximately 3 per cent, of that of the maternal serum. This concentration increases towards the end of gestation; in H23 and H28 it is about 28 per cent, of that of the maternal serum (Table 1).

The yolk-sac fluid was collected from the uterine swellings of eleven ferrets at stages between the 16th and 21st days of pregnancy. In every case the reaction with thrombin was negative. In a number of animals the fluid contents of the exocoels were also tested, and these too gave negative results. In the ferret the volume of the yolk-sac increases up to about 19 days and subsequently decreases.

The yolk-sac fluid is an extremely dilute protein solution; in some animals a protein precipitate was unobtainable, and on the addition of trichloroacetic acid only a faint opalescence resulted. The concentration of protein nitrogen in the maternal sera of four of the ferrets was determined, and an attempt was made to estimate its concentration in the yolk-sac fluid of these animals (Table 2). The concentration in the yolk-sac fluid is so low that the results obtained can only be regarded as approximate. The highest protein nitrogen concentration, 0·10 mg. / ml., was obtained at days—about 1 per cent, of the concentration of the maternal serum.

In very early stages of pregnancy in Erinaceus the decidual tissue adjacent to the blastocyst becomes highly vascular and large blood-spaces are formed in the trophospongia, which is derived from the enlarged cells of the maternal endothélia. Lacunae are formed between the trophoblast and the adjacent decidua, and the maternal blood which circulates in these spaces is thus brought into close proximity with the embryonic tissues, and is separated from the cavity of the yolk-sac only by the bilaminar yolk-sac wall. No such complex organization of vascular channels occurs in the uterine tissue of Putorius.

Omphaloidean villi are developed in both forms, but whereas in the ferret they remain avascular until the allantoic mesoderm extends over them, in the hedgehog they are vascularized by vitelline vessels from the area vasculosa. The maternal and’foetal circulations are thus brought into close proximity over an increased surface area, and are separated from one another by two cellular layers, the trophoblast and the foetal endothelium. The avascular nature of the omphaloidean placenta of the ferret and its comparative inefficiency as an absorptive organ are probably reflected in the extremely low concentration of protein in the yolk-sac fluid. The highest concentration of protein nitrogen obtained was 010 mg./ml. at days compared with a concentration of 9·15 mg./ml. in the maternal serum. Fibrinogen was absent from the yolk-sac fluid of all the stages of pregnancy that were tested.

In Erinaceus the concentration of protein nitrogen in the yolk-sac fluid is considerably higher than that of the ferret. At mid-pregnancy a concentration of 0·35 mg./ml. was obtained, compared with 11·7 mg./ml. in the maternal serum, and fibrinogen was found to be present in the yolk-sac fluid at late stages of pregnancy. In such late stages the volume of the yolk-sac diminishes and the concentration of protein in the yolk-sac fluid increases. The increase in the concentration is probably due, in part, to the absorption of water from the yolk-sac fluid, but since this cannot entirely account for the higher concentrations there must be a continued entry of protein into the yolk-sac during the later stages of pregnancy.

In the rabbit implantation occurs on the 7th day, and by days a very close relationship has been established between the embryonic tissues and the maternal blood. As soon as the fusion areas are formed maternal blood capillaries grow towards them, and the maternal blood bathes the syncytial trophoblast (Parry, 1950). These fusion areas, which are suggested as the most obvious route of passage from the maternal blood into the yolk-sac cavity, reach their maximum size and vascularity at days. During the 7th and 8th days proteins enter the yolk-sac cavity from the maternal blood. It has been demonstrated experimentally that serum albumin enters the yolk-sac at a maximum rate at days, and that the rate of entry decreases to zero at 9 days. It was suggested that the further entry of serum albumin was prevented by a sudden change in the permeability of the membrane. There is a very rapid build up in the concentration of protein in the yolk-sac fluid between 7 and 9 days. This concentration reaches as high as 40-50 per cent, of that of the maternal plasma, and the fibrinogen which is of maternal origin is present in concentrations between 30 and 40 per cent, of that of the maternal blood (Brambell et al., 1949).

In Erinaceus a similar close relationship is established between the maternal blood and the embryonic tissues soon after implantation. At such early stages the yolk-sac cavity is minute, and it is not possible to test its fluid contents. However, at later stages the protein concentration of the yolk-sac fluid is comparatively low and fibrinogen is absent (Table 1). Thus in the rabbit and the hedgehog the early omphaloidean placenta is haemochorial in character, but despite this structural similarity the permeability of the membranes of the two forms must be very different, to account for the high concentration of protein in the yolk-sac fluid of the former, and the relatively low concentration in the yolk-sac fluid of the latter.

Later in development the omphaloidean placenta of the hedgehog is vàscularized by the vessels of the area vasculosa, and the foetal and maternal circulations are separated only by the trophoblast and the foetal endothelium. Thus in Erinaceus the foetal-maternal vascular relations of the omphaloidean and allantochorionic placentae are similar.

Fibrinogen is present in the yolk-sac fluid in late stages of pregnancy in the hedgehog. In such stages the omphaloidean placenta is rapidly retrogressing, and over its limited extent it is not so highly vascular as in earlier stages. The yolk-sac wall is in contact with the attenuated maternal tissues over the mesometrial pole only, and in this region a thick Reichert’s membrane has been formed, separating the endoderm from the trophoblast. It is unlikely, therefore, that entry is effected directly via the omphaloidean placenta since it is in such an advanced state of retrogression. Fibrinogen may enter the yolk-sac by way of the allantochorionic placenta, entering the foetal circulation and thence traversing the vascular endothelia of the vitelline vessels and the endodermal epithelium of the yolk-sac splanchnopleur; or more indirectly by traversing the intervening exocoel and passing through the epithelial layers of the yolk-sac splanchnopleur. Since both these routes are available at earlier stages of pregnancy, it seems unlikely that either is used. It would seem more reasonable to conclude that the fibrinogen is of foetal origin, and that it enters the yolk-sac cavity from the vitelline vessels of the yolk-sac splanchnopleur.

The structure of the yolk-sac splanchnopleur in the later stages of pregnancy in the hedgehog is very similar to that of the 24-day rabbit embryo (Morris, 1950). In both forms the endodermal epithelium is composed of columnar cells, and the mesenchyme, which becomes progressively more compact, remains undifferentiated except for the vitelline vessels which are formed within it. In the 21-day ferret embryo the cells of the endodermal epithelium are cuboidal and the mesenchyme layer is considerably thinner.

The homogenous membrane that is formed on the inner side of the trophoblast of the mesometrial hemisphere resembles the membrane of Reichert which is found in certain rodents, in the Common and Lesser Shrews (Brambell & Perry, 1945), and in the Indian Musk-Shrew, Crocidura (Sansom, 1937). The origin of the membrane in these forms is not clear. Sansom suggests that in Crocidura the trophoblast may be responsible for its growth, and that the endoderm of the bilaminar omphalopleur may also contribute to its formation. In Sorex Reichert’s membrane makes its appearance soon after the formation of the exocoel and before that of the allantois, and it is considered to be of trophoblastic origin (Brambell & Perry, 1945). In Erinaceus the membrane appears much later in development, but as in Sorex it is first distinguishable in the region of the extra-embryonic mesoderm, between the somatic mesoderm and the trophoblast of the lateral walls of the conceptus. Here it may possibly be formed by the thickening somatic mesodermal cells, or by the trophoblast, or both tissues may take part in its formation. In this region it is completely separated from the yolk-sac endoderm which can have no part in its formation. At the mesometrial pole it becomes noticeable slightly later in pregnancy, between the endoderm and the trophoblast of the bilaminar omphalopleur. These conditions are essentially similar to those which have been described in Sorex. Since there appears to be no sufficient reason for supposing that the membrane has a different origin in the different regions of the mesometrial hemisphere of the conceptus, it is reasonable to conclude that in Erinaceus, as in Sorex, Reichert’s membrane is trophoblastic in origin.

The yolk-sac in the ferret and in the hedgehog persists throughout pregnancy, but is considerably reduced in late stages. In Putorius the extension of the exocoel completely separates the yolk-sac splanchnopleur from the chorion, so that omphaloidean placentation is terminated at about 21 days. In Erinaceus the mesoderm does not complete its extension mesometrially, and although the omphaloidean placenta persists to term, the exocoel separates the vascular splanchnopleur from the somatopleur, so that the yolk-sac placenta in the very late stages is bilaminar and avascular.

 
• G

  Giant nucleus

 
• M

  Mesenchyme

 
• MB

  Maternal blood

 
• MV

  Maternal vessel

 
• R

  Reichert’s membrane

 
• S

  Somatic mesoderm

 
• T

  Trophoblast

 
• Tr

  Trophospongia

 
• U

  Uterine gland

 
• V

  Vitelline vessel.

The main part of this work was carried out at the Department of Zoology, University College of North Wales, during the tenure of an Agricultural Research Council Studentship, for which I wish to express my thanks. The author is much indebted to Professor F. W. R. Brambell, F.R.S., for his advice and encouragement, and also to Mr. W. A. Hemmings.