Observations upon the Fœtal Membranes of the Opossum and other Marsupials

Professor Owen established the following as the distinctive features of the Marsupial fœtal membranes: (1) A large subzonal membrane with folds fitting into the uterine furrows, but not adhering to the uterus and without villi. (2) A large and vascular yolk-sac partly flattened out over the inner surface of the subzonal membrane, and supplied by an artery and two veins. (3) An allantois of comparatively small size, not attached to the subzonal membrane, with a blood supply of two arteries and one vein. (4) An amnion closely investing the embryo and reflected over the base of the yolksac and allantoic stalks.

One sees at once that Professor Owen’s observations, valuable as they are, still leave us in doubt as to the real relationship existing between the foetal and maternal blood currents, which, after all, is the main question. By what process does the embryo, with little or no food yolk to draw upon, support life during the short but rapid period¹ of intrauterine growth, extending not over seventeen days in the opossum, and thirtyeight days² in the kangaroo?

My own observations partly confirm and partly contradict those of Professor Owen; they show that not only does the yolk-sac in the Marsupials perform the functions of the allantois in the placental Mammals, but that the method is the same, namely, by means of vascular villi developed upon the subzonal membrane over the attached or chorionic portion of the yolk-sac.

In the early part of March I had the good fortune to receive from one of my students³ a female opossum (Didelphys Virginiana), which was found to be in an early stage of pregnancy. After opening the animal I found that each horn of the uterus had a single swelling an inch and a half long and an inch in diameter. Upon laying one of these open, eight embryos were seen, lying in a row, partly enveloped in one or two long furrows. These furrows would extend along the lower internal wall of each uterus; if the animal were in its natural position they would then be horizontal—a fact the importance of which will appear later. The foetuses varied considerably in development, some being nearly twice as large as others. In the larger embryos there were two visceral clefts, the fœtal circulation was completely established, the fore limb was comparatively well developed, with the position of the toes faintly outlined, the hind limb was still bud-like. The tail extended somewhat beyond the hind limb; the cup of the eye was backward in development, presenting a horseshoe appearance, like that of a chick in the third day. Altogether by a comparison of the older embryos with some newly born opossums found upon another female, I conjecture that the embryos were about eight days old, and that the short period of intrauterine development was about half over. A fœtus of median size was detached by a slight pressure of the needle, and the subzonal membrane was found to be about 10 mm., or of an inch in diameter. Through this membrane the embryo could easily be seen. There was an opaque disc-like area on the subzonal membrane, and this was found to correspond to the partially adherent yolk-sac, which was spread over about one third of the inner surface of the membrane. When a portion of this area was seen in profile a large number of minute villi were at once noticed upon the surface of the subzonal membrane, which was smooth elsewhere. The yolk-sac, as in the kangaroos described by Professor Owen, had the figure of a cone, the base attached to the subzonal membrane and the apex at the umbilicus. At the edge of the area the yolk-sac was folded back upon itself, as in fig. 1 (woodcut). The umbilical stalk was wide. The attached area was covered with capillary vessels, and circumscribed by the sinus terminalis; this united near one edge of the disc, to form a single vitelline vein (Pl. XXXIII, fig. 1), and the vitelline arteries were either double or branched close to the embryo from a single trunk. They were difficult to distinguish.

The allantois was found in the various embryos in all stages of development, two of which are represented in Plate XXXIII, figs. 1 and 4). It arises, as in the Placentalia, just behind the umbilical stalk, and the mesoblast and hypoblast could be readily distinguished. In later stages it was a small sac with a wide stalk. In the embryos which were examined no blood-vessels could be detected, but they undoubtedly develop at a later period. Compared with the yolk-sac the allantois was extremely small, nor was it in contact with the subzonal membrane at any point. Still, no opinion could be formed as to its subsequent relations, for its development is evidently very rapid, and the embryos were in an early stage of growth.

The greatest interest naturally was directed to the villous area of the subzonal membrane. This could be separated with ease from the subjacent portion of the yolk-sac, revealing the rich capillary network of the latter. At this point a careful drawing of the foetus was made (Plate XXXIII, fig. 1), magnified about five diameters, and representing the proportions as nearly as could be done by the eye. The membrane was composed of a single layer of polygonal epithelial cells. When seen from above the villi appeared as rings of thickened epithelium of all sizes in profile (fig. 3); they were seen to be composed of a single layer of columnar cells, some of which were produced into minute processes. The villi varied considerably in height; they were hollow, and beneath them was a layer of flattened epithelium; whether the latter was derived from the subzonal membrane or had been torn off from the yolk-sac could not be ascertained. A portion of the villous area near the sinus terminalis, containing one of the vitelline arteries and a section of the vena terminalis (Plate XXXIII, fig. 2), shows that at this period there was no especial enlargement of the capillary vessels near the villi; in fact, none of the latter showed any trace of vascularity. Two facts, however, indicated that this would appear in a subsequent stage:—1. The villi were apparently beginning a similar line of development to that which they pursue over the attached allantoic area in the Placentalia. 2. The villous area in each foetus was in close contact with the uterine furrow, whereas the remainder of the subzonal membrane floated free in the uterine cavity. The word “attachment”would be incorrect in this connection, but the union with the uterine wall was sufficiently close to prevent separation, even when there was considerable motion in the water in which the uterus was placed.

By an unfortunate blunder in the laboratory one horn of the uterus containing the embyos in situ was thrown away, so that no satisfactory examination of the adjacent uterine wall could be made.

Although energetic efforts were made, no other pregnant females were captured,¹ so that my observations upon the opossum terminated at this period. I was quite convinced, however, that older Marsupial embryos would show vascular villi upon close examination.

The opportunity of completing and confirming the above results was due to the generous assistance of Professor Wilder, of Cornell University, and Professor Chapman, of the Jefferson Medical College of Philadelphia. The former most kindly placed at my disposal a quantity of Marsupial material, which he had procured from Australia; the latter allowed an examination of his valuable kangaroo foetus. To both of these gentlemen I wish to express my hearty acknowledgments.

Among Professor Wilder’s material was a fine foetus, which will be referred to as Specimen 2, because, although it was labelled “Removed from an Australian Marsupial,”the memorandum giving the generic name was lost. The fœtus was evidently not that of a kangaroo, but probably belonged to one of the smaller Australian Marsupials. Its external structure² as well as the character of its membranes left little doubt of this.

Specimen 2 is drawn natural size in fig. 5, and was believed to be in a somewhat advanced period of intra-uterine life. The embryo had well-developed fore limbs, in which the fingers were all distinct; the hind limbs, although much smaller, showed the division of the toes. The eyes were in a rudimentary condition, but the ear-pits could be plainly seen, while the mouth was large, with a much-protruding tongue. The tail was quite long. As a whole, the embryo in size and appearance resembled closely that of the oppossum at birth, except that the snout was. shorter, suggesting that the embryo belonged to one of the short-faced genera—Chironectis, Petaurus, Phalangista, or Phascolarctos.

Owing to the rupture of the subzonal membrane, as well as the removal of a portion of the yolk-sac, the precise relations of the membranes were difficult to determine. As far as they could be made out the whole was surrounded by a subzonal membrane, within which the yolk-sac was flattened out over a larger area than in the case of the opossum, a fact which was quite consistent with the advanced age of the embryo. The missing portion of ‘the yolk-sac was largely within the sinus terminalis, so that the extent and character of the attachment of the yolk-sac to the subzonal membrane could not be satisfactorily ascertained. The latter was carefully examined, and soon a number of low villi were discovered upon it without the aid of the glass; they were distributed over an area to which a highly vascular portion of the yolk-sac was adherent, which was, however, just without the limits of the sinus terminalis; what their distribution was within the limits circumscribed by the sinus terminalis could not be be followed, owing to the removal of the latter. In fig. 5 their position is indicated by a number of dots (v); as the figure represents the inner view of the yolk-sac, the villi were of course upon the lower surface, their position being more plainly shown in the woodcut (fig. 2, v). The villi are shown in fig. 6 as they appeared in profile under a low objective. They were considerably lower than the subzonal upgrowths of the opossum, so that the term villus cannot be given them very accurately. Upon separating the subzonal layer from the yolk-sac, the former was seen to be composed of somewhat flattened cells, which, over the summits of the villi (fig. 6, 5), had a truly squamous character, being quite transparent. The separation of the subzonal membrane did not leave the surface of the yolk-sac smooth as in the opossum, but covered with apparently solid papillæ, derived from the yolk-sac epithelium. Each of these was found to be provided with a single dilated capillary vessel branching over its summit (fig. 6, a). These papillæ, therefore, with their subzonal caps, recalled at once the simplest form of allantoic villi, which Professor Turner represents¹ as consisting of a vascular core raised upon the surface of the allantois and covered with a layer of pavement epithelium derived from the subzonal membrane.

The allantois in Specimen 2 consisted of a highly vascular sac, with quite a long narrow stalk, which was attached to the embryo just behind the umbilical stalk; its distal surface was covered with capillary vessels ramifying in all directions; the number of main blood-vessels supplying the allantois was not ascertained. This allantois was proportionately larger than in the advanced kangaroo foetus described by Professor Owen; in other respects it had the same appearance. A more important difference was seen in the fact that this sac had a disc-like area of attachment to what was apparently a portion of the subzonal membrane composed of pavement cells. This feature, if confirmed by later observations, is a highly important addition to our knowledge of the fœtal membrane of the Marsupials. Unfortunately, owing to the torn condition of the subzonal membrane, this point cannot be considered by any means certainly established.

The uncertainty which existed as to the generic and specific character of Specimen 2, and the difficulty of a positive determination of the relation of its membranes, made an examination of the kangaroo fœtus very desirable. According to Professor Chapman’s record, the mother was killed fourteen days after impregnation. The embryo was, however, in an earlier stage of development than that of the opossum;² the visceral clefts were still very distinct; the fore limb was elongated, but the hind limb was a mere bud. The yolk-sac,however, was spread over the inner surface of the subzonal membrane very much as in the older opossum embryos, while the allantois was a small sac supplied by two arteries.

The line of attachment of the yolk-sac to the subzonal membrane was marked, as described by Professor Chapman, by the sinus terminalis, and this, as in the opussum, seemed to give rise to a single vein near the edge of the disc; there was a single artery. The fœtus therefore closely resembled the earlier opossums, and it was very gratifying to discover minute villi all over the attached area of the yolk-sac, thus confirming the previous observations. These villi were so minute that it is not at all surprising that they were overlooked by previous observers. I have not yet had an opportunity of examining them closely; their external size and appearance was similar to those found in Specimen 2, although they were in an early stage of development. Beneath them the disc formed by the yolk resembled closely that of the opossum, and it was quite evident that at a later period they would resemble in internal structure those found in Specimen 2.

I think we may now regard the following facts, in respect to the fœtal membranes of the Marsupials as fairly well established.

1. That the yolk-sac at an early stage spreads over the inner surface of the subzonal membrane, forming a disc-like chorion, which in the kangaroo and opossum is bounded by the sinus terminalis. This chorion may become extensive in the later stages. The subzonal epithelium then gives rise to hollow conical upgrowths of columnar cells. From the epithelium of the yolk-sac there arise papillæ, which become vascular, while the subzonal cells become very much flattened. The rudimentary villi thus formed, in the early oposssum and kangaroo embryos, are thickly distributed over the area surrounded by the sinus terminalis, but in other forms they may extend beyond this area.

2. The allantois arises in the same way as in the Placentalia at quite an early stage of development, and soon becomes vascular. In the kangaroo, if it unites with the subzonal membrane at all, it is only in the later period of gestation. In the opossum it develops rapidly, so that a brief union with the subzonal membrane before birth is not improbable. In the unknown Marsupial (see Specimen 2) this union seems actually to have taken place.

3. The amnion, as in the Placentalia, in all cases invests the embryo.

4. One or two long furrows are formed along the lower internal border of the uterus in the kangaroo and opossum. In close contact with one of these in the opossum is placed the villous chorionic disc of each of the numerous foetuses; the remaining portions of the subzonal membrane are free. The embryo is undoubtedly retained in this position throughout intra-uterine life. During this period the opossum is known to keep remarkably quiet, so that the uterus is little disturbed, and is most of the time in a horizontal position.¹ The presence of foetal villi is strong evidence by analogy of the presence of minute crypts on the inner wall of the uterus.

It is an undoubted inference from the above facts that in the early stages of Marsupial development the vessels of the yolksac not only are the channels for conveying the maternal nutriment to the fœtus, but that this function is performed by capillaries distributed in low villi, and separated from the maternal structures, whatever the arrangement of the latter may be, by an extremely thin layer of subzonal epithelium. It is evident that these villi are altogether similar in structure to those which are found over the allantoic chorion of the pig;² the difference is merely one of degree. The rudimentary mechanism is sufficient to support the rapid growth of the embryo opossum, which at birth is completely equipped with all the necessary respiratory and digestive apparatus acquired during an intra-uterine period barely exceeding two weeks.³ This could not be effected if the absorbent villous area were shifting about from one part of the uterus to another. This fixity of position must have been an important step towards the establishment of an allantoic placenta.

Although we may now be reasonably certain of the early condition of the foetal membranes in the Marsupials, it must be borne in mind that all the latter part of their history is still a blank, and that the allantois in the later stages may enter into very important relations with the subzonal membrane. Balfour, with his usual discernment, suggested a probable condition among the primitive Placentalia,¹ in which the yolk-sac and allantois shared the placental function. I think it is not improbable from the evidence given by Specimen 2, and from the rapid growth of the allantois in the opossum, that this condition may yet be found among the Marsupials. The fact that no fœtal membranes are brought forth at birth has, I believe, been correctly attributed to the very tortuous vaginal passage through which the young pass in their descent.

The evolution of the placenta is an interesting subject of speculation, which it is tempting to review, now that we have more light upon the functions of the yolk-sac.

1. In the low reptilian forms which preceded the Mammals there was undoubtedly a substitution of the viviparous for the oviparous mode of reproduction,by the gradual reduction of the food yolk and the retention of the embryo in the uterus. The whole character of Mammalian development points unquestionably to the former presence of a mass of food yolk in the ovum, and there is every reason to suppose that the loss of this source of supply was gradually and pari passu compensated by the substitution of the maternal nutrition, so that the embryos were partly nourished by the yolk, partly by a feeble absorption of nutriment from the uterus through the contiguous umbilical vessels, the allantois retaining solely its reptilian functions.

2. With the diminution of food yolk came an increasing absorption of maternal nutriment through the chorion of the yolk-sac, upon which villi gradually appeared. The Marsupials may fall into this or the following class.

3. A condition in which the allantois and yolk-sac shared the placental function.

4. The primitive Placentalia (Balfour), in which the yolksac formed a large false chorion, and the allantois formed a small discoid placenta, and in which the maternal parts were not deciduous.

I hope during the spring of 1884 to be able to follow out the membranes of the opossum to the later stages. At present, owing to the hurried preparation of this paper, some valuable drawings have been omitted, and the study of the kangaroo was not so complete as I desired, nor have I been able to refer to all the authorities upon the subject, as I hope to do in a later paper.

May 14th, 1883.