1. Foetal tissues were transplanted into the pseudo-pregnant uterus of the rabbit, a suitable position for such grafts owing to the raised nutrition of the uterus occurring in the early stages of pseudo-pregnancy.

  2. Several tissues particularly large amounts of cartilage and small quantities of epithelial tissue persisted and grew, the former undergoing ossification.

  3. The growth of these foetal tissues in the pseudo-pregnant uterus did not cause the corpus luteum of pseudo-pregnancy to persist or the mammary gland to thicken as in pregnancy.

  4. Since—(a) the foetal tissues only grew where they were attached to the muscle layer of the uterus, although every effort was made to attach them to the mucosa; (b) the parts of the grafts lying in connection with the mucosa were absorbed rather than nourished; and (c) the introduction of living foetal tissue failed to cause any further development of the deciduomata which were produced by cutting the pseudo-pregnant mucosa—it is suggested that normally the attachment of the foetus is caused by the active erosive (digestive) agency of the foetal trophoblast, a function which is absent in the cells of the foetus itself.

  5. It seems probable that just as the small deciduomata are formed as a response to a cut so the large maternal placenta is formed as a response to the continuous erosion by the foetal trophoblast.

The primary object of the experiments described below was to determine whether the corpus luteum of pseudo-pregnancy in the rabbit could be made to persist, as in pregnancy, by grafting foetal tissues direct into the uterus, thus eliminating foetal membranes, fluids and placentae from the possible causes which determine the persistence of the corpus luteum during pregnancy ; and also whether the mammary gland could be caused to develop as in pregnancy by this means. The ultimate object was to determine the cause for the persistence of the corpus luteum, for this sometimes occurs in the cow without pregnancy, thereby causing sterility, and also to determine the factors which cause the pregnancy phase of the development of the udder. It may be stated at once that this primary object failed in that although the grafts took the corpus luteum did not persist nor did the mammary glands develop as in pregnancy, but since several other points of interest arose it was considered that it might be worth while publishing an account of the experiments.

In the rabbit the corpora lutea of pseudo-pregnancy (corresponding to the cyclic corpora lutea in other species) have a duration of about 16 days after which time they begin to atrophy, whereas the corpora lutea of pregnancy persist in large size until the end of pregnancy (32 days) (1). From about the 4th-ioth day of pseudopregnancy an incision in the uterine mucosa causes a growth of decidual tissue almost identical with that of the maternal placenta of pregnancy (2), such as was first described by Loeb (3) in the guinea-pig. The deciduomata so formed, however, do not continue to grow as the placenta does and with the atrophy of the corpus luteum at the 16th day they also begin to undergo regression in association with the uterine glands. It was hoped that the experiments would throw some light on the formation of the placenta and determine the cause of the difference between deciduomata and maternal placenta. The deciduomata are a growth response of the mucosa to damage of its tissues under the growth stimulating influence of the early stages of the corpus luteum, but they do not continue to undergo further growth as does the maternal placenta in which the growth is continued until the middle of pregnancy. Whether or no the introduction of foreign living tissue into the mucosa would cause further growth of the deciduomata was a point to be decided.

Another object in view was the study of the growth of foetal tissues, particularly of isolated bones under circumstances where their growth would be free from the usual stresses, strains and other influences of the surrounding foetal tissues.

The operations were performed in conjunction with Dr A. B. Appleton and with Dr S. A. Asdell and to them my thanks are due.

The does into which the foetal tissue was transferred were made pseudopregnant by coitus with a vasectomised buck. At the 7th day after sterile coitus the abdomen was opened along the linear alba, the uterine horns exposed, and a foetus or piece of the foetal tissue to be transplanted was inserted, usually one foetus or piece of tissue in each horn. The doe furnishing the graft was killed by breaking the neck and the foetus or parts were then cut out, with aseptic precautions, by one of us and handed to the other who inserted the graft into the uterus.

Three types of incision for inserting the graft in the uterus were made. In method (A) the uterus was slit longitudinally between the obmetral folds for the length of about one-third inch ; this resulted in it opening out to mushroom shape in this region by contraction of the muscular coat, thus exposing the mesometral folds of the mucosa (the normal site of placenta formation). These folds were then gently scraped with a scalpel so as to induce decidual formation and the tissue to be inserted was placed on the abrased surface, being kept in situ by a horse-hair ligature; generally, but not always, another suture was made drawing over the graft the cut edges of the muscular coat and so enclosing it within the uterus. It was found that the embryos of the 13 th day were very soft and difficulty was experienced in keeping all of them attached in situ, very often part breaking away and sliding over the surface of the uterus.

In method (B) a small incision was made in the muscular wall of the uterus which just penetrated to the mucosa and the graft was inserted in this pocket so that it would lie partly embedded in the mucosa and partly in the muscle coat, the outer muscular coat being drawn over it with horse-hair sutures to keep it in place.

In two cases (C) the bone graft was inserted into the cavity of the uterus (after the mucosa had been scraped) through a small hole in the muscle wall.

The operated animals were killed, some on the 16th day after coitus just before the time the corpus luteum of pseudo-pregnancy normally begins to atrophy in order to determine the progress of the graft up to this stage, while the remainder were killed at the 27th day after coitus for at this time in pseudo-pregnancy the corpora lutea and mammary glands are quite atrophic whereas in pregnancy the corpora lutea are large and the mammary glands have thickened. In some cases it was observed that the experiment had failed to cause the corpus luteum to persist before the animals were killed, since the does made a nest with fur between the 16th-20th days, as they often do at the end of pseudo-pregnancy.

EFFECT ON THE CORPUS LUTEUM

As mentioned above, there were in some cases symptoms in the living animal that the graft had not caused the corpus luteum to persist, the pseudo-pregnant nest being made between the 16th-20th day. It was also observed when the animals were killed that the corpora lutea resembled in size and appearance the corpora lutea of pseudo-pregnancy rather than those of pregnancy, being yellow or white (according to the colour of the fat of the doe) denoting an atrophic condition, rather than pinkish with a network of blood-vessels over them, as occurs in the pregnant condition at the 27th day. In order to make quite certain the diameters of the corpora lutea were measured; after the ovaries had been fixed in 10 per cent, formalin freehand sections were cut in water through the corpora lutea and the sections showing the largest diameter in each corpus luteum were picked out and measured under the microscope with an eyepiece micrometer. The sizes obtained in this way were compared with the sizes of a number of normal pregnant and pseudopregnant corpora lutea of similar stages and in every case they corresponded with the pseudo-pregnant and not with the pregnant, decrease in size from the 16th to 27th day being shown in every case (see Table I). Besides this anatomical test of their atrophy a physiological test was also made ; while the corpus luteum is active it inhibits ovulation after coitus (as, for example, up to the 16th day of pseudopregnancy or during the whole of pregnancy) (10), but as soon as atrophy of the corpus luteum begins ovulation occurs after coitus.

Table I.

Details of Experiments.

Details of Experiments.
Details of Experiments.

Several of the operated does to be killed at the 27th day were therefore mated on the 26th day and it was observed in all cases where this was done that a fresh ovulation had occurred when they were killed, the newly formed corpora lutea showing as small pink pimples on the surface of the ovary ; ova were also recovered in these cases from the Fallopian tubes (Table I). Some were also mated on the 15th and killed on the 16th day of pseudo-pregnancy but none of these had ovulated, this being normal for this stage of pseudo-pregnancy.

These results show that although foetal tissues may be developing in the uterus they fail to cause persistence of the corpora lutea ; no very large amount of foetal tissue was however present and it is just possible that the time between the 7th day, when the tissue was inserted, and the 16th, when the corpus luteum would normally atrophy, was not sufficient to allow of any influence appearing owing to the delay of growth caused by transplantation. It should be noted also that any influence of the foetal membranes, fluids and placentae has been excluded and that the tissue developed in the muscular coat of the uterus and not in the mucosa (see below) thus failing to give maternal placenta formation.

Not much value is attached to the negative results of this experiment for as certain only of the foetal tissues developed and not all of them these experiments do not exclude the possibility that some of the foetal tissues (which in these experiments did not develop) may be responsible for the persistence of the corpus luteum during pregnancy.

EFFECT ON THE MAMMARY GLAND

In pseudo-pregnancy the mammary gland is pinkish and the ducts grow laterally, reaching the maximum development of this phase about the 15th–16th day, after which time atrophy sets in and milk may be seen in the ducts. If, however, the doe is pregnant a second phase of growth—a growth in thickness due to swelling of the alveoli—begins after the 16th day and continues to the end of pregnancy. If the grafts made caused the second phase of growth to appear it would be expected that the glands of the does killed at the 27th day would weigh more than those killed at the 16th day, but this was not so (see Table I). The actual weights of the glands given are all high owing to the fact that does which had produced litters a short time previously were used for the operation; the time relations were, however, nearly similar in each case so that the results are comparable among themselves. As already noted above, not much value is attached to these negative results, for only certain of the foetal tissues were present in the grafts.

UTERINE REACTIONS

The pseudo-pregnant uterus would appear to be a very favourable site for the grafting of tissues, for in the early stages, under the influence of the corpus luteum, there is a large growth of uterine glands and the whole level of uterine nutrition is raised as can be seen by the bright pink colour and rounded appearance of the muscular coat. After the 16th day, however, when the corpus luteum begins to atrophy, the uterus follows suit and the glands become atrophic.

At the 7th day of pseudo-pregnancy the uterine mucosa responds to the stimulus of cutting or damage by the formation of decidual tissue ; the latter, however, begins to atrophy after the 16th day together with the glands of the mucosa (2).

The presence in the uterus of a graft of foetal tissue does not appear to have any influence in causing these deciduomata to persist longer or to cause further development in them, as can be seen from the results shown in Table I. In the does killed at the 16th day there was often quite a lot of decidual tissue, but at the 27th day only traces of it remained, that is, the presence of foreign living tissue in the uterus does not lead to a further development of the deciduomata into a placenta as it does in pregnancy.

Another point of great interest is the fact that the foetal grafted tissues only lived where they became attached to the muscle coat although every effort was made to implant them in the mucosa. In one case only (Fig. 7, No. 176, right) was a small piece of foetal tissue seen attached to the mucosa and this was being rapidly absorbed. Moreover, in the case of the bones where the femur was lying in com nection with the mucosa it was dead and undergoing absorption, but where it abutted on the muscular coat it was alive and growing. It would seem, therefore, that the mucosa does not nourish a foreign body with which it comes in contact but tends to absorb it. Evidence of this fact is also seen in the case of degenerate foetuses, such as occur frequently in the pig and rabbit (4); the tissues of these become mummified and absorbed from the uterus and do not become attached to it and grow.

It would appear that the uterine mucosa has not of itself any power of attaching substances to itself but that this function rests with the embryonic membranes; rather it has less function in this direction than such tissues as muscle and acts more as a preventer of attachments, i.e. when a blastocyst breaks down foetal tissues do not grow on the mucosa but are absorbed or shut off from nourishment, and it is only the active erosive agency of the trophoblast which enables them to obtain nourishment from the mucosa. This active erosive agency of the trophoblast causes the mucosa to respond by the formation of decidual tissue which in origin is merely scar tissue formed in an attempt to heal the wound. In the normal formation of the placenta in the rabbit the maternal epithelium is destroyed so that the foetal tissues lie next the maternal bloodstream (Mossman (5)).

By some it has been held that the pro-oestrum or the menstrual breakdown of the mucosa is necessary for the attachment of the foetal membranes but as is shown here, even if the mucosa is damaged by scraping and a living tissue is implanted in it, the latter does not grow but becomes absorbed by it just as degenerate foetuses are.

It would therefore appear probable that in those species (rabbit, pig, sheep, cow, etc.) where the ovum forms a free blastocyst occupying the cavity of the uterus the outer trophoblastic layer has an active erosive function in destroying the mucosa to form its attachment just as have the fertilised ova of those species (guinea-pig, man, etc.) which dissolve their way into the mucosa of the uterus at an early stage before developing foetal membranes.

It would appear that this erosive action of the outer trophoblastic layer of the foetal membranes continues for some time and is the cause of the development of the maternal placenta, in much the same way as deciduomata are formed from a cut in the mucosa but differing from them in that the erosive action is long continued. That this erosive action is a specific property of the plasmodial trophoblastic layer and is probably digestive in action is suggested by the fact that none of the foetal tissues implanted appeared to have any similar action. In a study of the mode of attachment in the foetal membranes of the cow (6) it was suggested that the digestive action of the trophoblast was the cause of attachment since the latter only occurred in areas free from glands where the nutritional requirements of the trophoblast were not satisfied by uterine milk, but no experimental evidence was then available. This view is contrary to that held by Jenkinson (7) who attributes the first sign of placental formation to crypt growth by the uterine epithelium and not to the functions of the foetal trophoblast.

FATE OF THE GRAFTED TISSUES

In all the femur grafts (see Table I) the cartilaginous epiphysial end where it came in contact with the muscular coat appeared to live and grow, but the shaft and other end which was embedded in the mucosa were absorbed. That this was a chance occurrence due to the position of the femur is suggested by the fact that in No. 167 (left), where the whole bone was lying on the outer side of the muscle, it appeared normal, whereas in No. 178 (right) (Fig. 2), where it was lying in the mucosa, the whole shaft was degenerate and both the epiphysial ends had fallen off. As will be seen by Fig. 1 (No. 171, right) and Fig. 3 (No. 178, left) the general form of the bone was maintained ; a tendency was sometimes observed, however, for the growth made at the ends to be rather thicker than usual. In many of the sections maternal blood was seen to be circulating within the foetal bony tissue.

Aron and Simon (8), who transplanted isolated embryonic bones of the guineapig subcutaneously in the adult, found that the epiphyses tended to become globular and the shaft to atrophy; they also found that when adjoining bones were transplanted together the shape of the epiphyses was much nearer normal, probably owing to pressure conditions.

In the grafts of whole foetuses of 13 and 15 days it was remarkable that certain of the tissues appeared to have a greater power of survival than others; cartilage in particular was always found in these grafts and increased in amount as the length of time the graft remained up to 20 days, the longest period investigated (compare Figs. 4 (No. 182), 5th day, and 5 (No. 174, right), 9th day, with Figs. 6 and 7 (No. 176, left and right), 20th day after the operation). In many of these pieces ossification was in progress and this was particularly noticeable in those killed 20 days after the operation (Figs. 6 and 7, No. 176, left and right).

Small cavities bordered by foetal epithelium were also present in the grafts in small amounts but the epithelial tissue did not appear to grow so well as the cartilage for it was not so evident on the 20th day (Fig. 6, No. 176, left) as it was on the 9th day (Fig. 5, No. 174, right) and still less than on the 5th day after the operation (Fig. 4, No. 182).

Nodules of lymphatic tissue were observed at the 5th day (Fig. 4, No. 182) and at the 20th day (Fig. 6, No. 176, left), while there also appeared to be a certain amount of undifferentiated or connective tissue of foetal origin in most of the grafts. In addition, No. 165, left (Fig. 8), contained a cyst-like body with every appearance of being formed from foetal tissue, possibly skin.

In both the kidney and skin grafts the tissues at the 9th day appeared moderately well preserved but by the 20th day they were completely atrophic and were rapidly being absorbed. It is evident that these tissues are less able to withstand transplantation in this situation than is tissue such as cartilage. In connection with the skin graft of No. 183 a cyst was developed containing a granular fluid; this liability of skin grafts to produce cysts has also been noted by Loeb (9), who also found in that the fate of kidney grafts was influenced by the place of transplantation.

In the ovary grafts (No. 180) some interstitial cells and possibly a few small oocytes remained at the 20th day, while some small cysts with granular centres were present in one of these grafts. Since the ovaries of the doe had not been removed is only to be expected from the work of Lipschütz (11) that the transplanted tissue would be undeveloped.

A detailed study of the tissues of the grafts is now being made in the laboratory of Prof. A. Peyron at the Institut Pasteur, Paris. The whole-foetus grafts appear to resemble in some respects the embryomata tumours that sometimes occur in man and the higher animals.

The much better survival of some tissues (cartilage) than of others (muscle) in these grafts corresponds with that which has been found in the cultivation of chick tissues outside the body and when implanted subcutaneously (Strangeways and Fell(12)). The retention of the bone form and the ossification of cartilage grafted suggest that this method might form a means of testing experimentally the factors which determine points of ossification and method of bone formation such as are at present known only from descriptions of the normal process (Fell (13)).

The experiments were performed at the Field Laboratories, Milton Road, Cambridge, in connection with the Institute of Animal Nutrition, and the expenses were largely defrayed out of a grant made to the Institute by the Ministry of Agriculture and Fisheries.

(1)
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Plate XV

Full descriptions of each are given in Table I under the number of each animal.

Fig. 1 No. 171, right. 22 day femur grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Growing cartilaginous head attached to muscle with bone shaft lying towards mucosa; remains of old decidual tissue in one fold of mucosa.

Fig. 2. No. 178, right. 22 day femur grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Atrophic shaft of bone lying between folds of mucosa.

Fig. 3. No. 178, left. 22 day femur grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Growing cartilaginous head attached to muscle with bone shaft lying towards mucosa. Mucosa atrophic without decidual formation.

Fig. 4. No. 182, left. 15 day whole foetus grafted; killed 12th day of pseudo-pregnancy. 2″ obj. and 1 eyepiece. Graft attached to muscle, on left cavities lined by foetal epithelium, in centre lymphatic tissue, small pieces of cartilage in upper left and lower right regions of graft. Mucosa in glandular stage of pseudo-pregnancy.

Fig. 1 No. 171, right. 22 day femur grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Growing cartilaginous head attached to muscle with bone shaft lying towards mucosa; remains of old decidual tissue in one fold of mucosa.

Fig. 2. No. 178, right. 22 day femur grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Atrophic shaft of bone lying between folds of mucosa.

Fig. 3. No. 178, left. 22 day femur grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Growing cartilaginous head attached to muscle with bone shaft lying towards mucosa. Mucosa atrophic without decidual formation.

Fig. 4. No. 182, left. 15 day whole foetus grafted; killed 12th day of pseudo-pregnancy. 2″ obj. and 1 eyepiece. Graft attached to muscle, on left cavities lined by foetal epithelium, in centre lymphatic tissue, small pieces of cartilage in upper left and lower right regions of graft. Mucosa in glandular stage of pseudo-pregnancy.

Plate XVI

Fig. 5. No. 174, right. 13 day whole foetus grafted; killed 16th day of pseudo-pregnancy. 4″ obj. and 1 eyepiece. Graft attached to muscle on each side of incision; several pieces of cartilage, one undergoing ossification. Little decidual tissue in one fold of the mucosa just below incision.

Fig. 6. No. 176, left. 13 day whole foetus grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Graft attached to outer muscular coat; several pieces of cartilage, one of bone and two undergoing ossification ; on upper right side cavities lined by epithelium ; nodule of lymphoid tissue in lower right corner.

Fig. 7. No. 176, right. 13 day whole foetus grafted; killed 27th day of pseudo-pregnancy. 4″ obj. and 1 eyepiece. Graft attached to outer side of muscle coat. Several pieces of cartilage and two of bone. Foetal tissue enclosed in one fold ofmucosa (left centre) quite atrophic and structureless.

Fig. 8. No. 165, left. 15 day whole foetus grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 1 eyepiece. Two pieces of cartilage and one of bone (low centre) attached to muscle coat; attached to this and running to opposite side of muscular incision a cyst wall possibly of foetal epidermal origin. One fold of mucosa (right) showing old decidual tissue.

Fig. 5. No. 174, right. 13 day whole foetus grafted; killed 16th day of pseudo-pregnancy. 4″ obj. and 1 eyepiece. Graft attached to muscle on each side of incision; several pieces of cartilage, one undergoing ossification. Little decidual tissue in one fold of the mucosa just below incision.

Fig. 6. No. 176, left. 13 day whole foetus grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 2 eyepiece. Graft attached to outer muscular coat; several pieces of cartilage, one of bone and two undergoing ossification ; on upper right side cavities lined by epithelium ; nodule of lymphoid tissue in lower right corner.

Fig. 7. No. 176, right. 13 day whole foetus grafted; killed 27th day of pseudo-pregnancy. 4″ obj. and 1 eyepiece. Graft attached to outer side of muscle coat. Several pieces of cartilage and two of bone. Foetal tissue enclosed in one fold ofmucosa (left centre) quite atrophic and structureless.

Fig. 8. No. 165, left. 15 day whole foetus grafted; killed 27th day of pseudo-pregnancy. 2″ obj. and 1 eyepiece. Two pieces of cartilage and one of bone (low centre) attached to muscle coat; attached to this and running to opposite side of muscular incision a cyst wall possibly of foetal epidermal origin. One fold of mucosa (right) showing old decidual tissue.