The Early Development of the Cat (Felis Domestica)

The Early Development of the Cat (Felis domestica).

By J. P. Hill, D.Sc., F.R.S.,

and Margaret Tribe, D.Sc. (From the Department of Anatomy (Embryology and Histology), University College, London).

With Plates 24-9 and 12 Text-figures.

CONTENTS. PAGE INTRODUCTION ...... 514 MATERIAL AND METHODS ...... 516 CHAPTER I.—THE OVUM or THE CAT ...... 518 1. Maturation and Ovulation ...... 51S 2. Structure of the Ovum 520 3. Fertilization 523 4. Remarks on Fertilization ...... 531 CHAPTER II.—THE PROCESS OF CLEAVAGE ..... 533 1. Description of Cleavage ...... 533 2. Remarks on Cleavage ...... 550 3. Yolk-elimination (Deutoplastnolysis) ..... 553 CHAPTER III.—FORMATION OF THE BLASTOCYST .... 554 1. Late Morula and Early Blastocyst Stages from the Uteius . 554 2. Formation of the Didermic Blastocyst .... 559 CHAPTER IV.—DISCUSSION ...... 574 1. Mode of Formation of the Blastocyst .... 574 2. History of Embryonal Knot and Covering Trophoblast . 579 3. Prochordal Plate 589 LIST OF REFERENCES ...... 596 EXPLANATION OF PLATES • 600 NO. 272 M m 514 J. P. HILL AND MARGARET TRIBE

INTRODUCTION. OUR knowledge of the early development of the Cat, as indeed of the Carnivora in general, is singularly incomplete. Th. L. W. Bischoff, in his classical monograph ' Entwicklungs- geschichte des Hunde-Eies ' published in 1845, provided the first account of the development of a Carnivore from the unsegmented egg onwards and, considering the date of his work, his achievement was really a remarkable one. In addition to later embryonic stages, he described and figured a series of segmenting ova and blastocysts of the Dog, with such fidelity and accuracy that his account is, even to-day, of the greatest interest and value. He recognized and figured the germinal vesicle of the full-grown ovarian ovum and the refractive granules (' Dotter ') present in the cytoplasm of the latter. He observed the persistence of part of the discus proligerus round the unsegmented tubal egg, and noted its thick zona and the numerous sperm-heads imbedded in it. He figured the two-celled egg with what are evidently two polar bodies situated in the plane of cleavage. He described and figured the three-celled and four-celled stages, his figures •of the latter showing quite accurately the characteristic cross- shaped arrangement of the blastomeres. He figured three later tubal eggs, said to be composed of eight, ten, and eighteen blastomeres respectively, and he also illustrated and described a series of morulae and blastocysts from the uterus. He recognized the delicate membranous wall of the blastocyst lying inside the zona and also ' den runden und gleichmassig dunkeln Fruchthof ', and since he found that the latter is situated in the former, he termed this vesicular stage ' die Keimblase '. No further contribution to our knowledge of early Carnivore development appears to have been made until 1876. In that year E. A. Schafer (now Sir E. Sharpey Schafer) gave an account (45) of the structure of blastocysts of the Cat, possessing a bilaminar embryonal area, one-sixtieth of an inch (0-4 mm.) in diameter. In 1897 Bonnet (14), in his paper on the develop- THE EARLY DEVELOPMENT OF THE CAT 515

ment of the Dog, described and figured (his figs. 1-3, Taf. 80-1) three eggs of the Cat, one unfertilized, a second with two pronuclei, and a third divided into nine blastomeres of unequal size. He obtained no early material of the Dog, and begins his description of the development with blastocysts 1-5x1-2 mm. in diameter. In 1911 there appeared a valuable paper by R. van der Stricht (55) entitled ' Vitellogenese dans l'ovule de chatte '. This paper, as is indicated by the title, is primarily concerned with providing a detailed account of the growth of the ovarian ovum, including the process of vitellogenesis in the Cat, but it also includes most valuable chapters dealing with maturation, fertilization, and the early stages of cleavage, though the cleavage process is not followed in detail beyond the three- celled stage. It also contains important data relating to breeding habits, oestrus, and ovulation. In the same year there appeared yet another paper, that of W. H. Longley (37), likewise dealing with the phenomena of maturation and ovulation, the structure of the ovum and the first cleavage division in the Cat. The most recent contribution to our knowledge of Carnivore development we owe to 0. van der Stricht. In two papers (53, 54), published in 1923, he has described the structure of a series of blastocysts of the Dog ranging from 0-20 to 1-5 mm. in diameter, and has traced the history of the inner cell-mass. His observations, which are referred to later, whilst agreeing in most respects with our own, show that there are certain very interesting differences in the details of the differentiation of the inner cell-mass in the two forms. We are greatly indebted to Professor van der Stricht for affording us the opportunity of examining his preparations of these blastocysts and also of earlier rnorula stages which he has not yet described. We desire to express our thanks to Mr. F. J. Pittock for invaluable help in the preparation of the material, the microphotographs and models, to Mr. T. L. Poulton for the drawings of the models represented in Text-figs. 1-8, to Miss M. Rhodes for figs. 5-9 (PI. 25), and to Mr. W. Pilgrim for figs. 1-4 M m2 516 J. P. HILL AND MARGARET TRIBE

(PI. 24). To the Government Grant Committee of the Boyal Society we are indebted for grants in aid of our work.

MATERIAL AND METHODS. The material which forms the basis of this paper has all been obtained from cats of unknown origin, which came into our hands shortly after death. Its collection was begun in 1909, with the object primarily of obtaining demonstration material for teaching purposes, and has involved the examination of some hundreds of females, only a small proportion of which yielded early stages. We have rejected for the purposes of this paper all eggs which appeared abnormal or whose state of preservation was unsatisfactory. The material that remains forms a fairly graded series extending from the fertilized egg through early and late cleavage stages up to the completed blastocyst in which the definitive embryonal area is established. Although our material, more especially of the early cleavage stages, is not abundant enough to enable us to settle conclusively certain problems, e. g. the significance of the ' corps enigmatique ', and the very interesting question of the lineage of the central cells which furnish the embryonal knot of the early blastocyst, it is nevertheless sufficiently complete to enable us to give what we hope is a fairly adequate account of the cleavage process and the formation of the blastocyst and its constituent layers in Felis, and thus to provide for the first time a connected account based on modern methods of the early ontogeny of a Carnivore. Owing to the convoluted character of the Fallopian tubes, their small diameter and relatively thick walls, the Cat is not altogether an ideal subject from which to obtain cleavage stages. In cases where the condition of the corpora lutea and the absence of uterine swellings indicated the presence of eggs in the tubes, our normal procedure was to dissect away the folds of the peritoneum supporting the tube so as to enable the latter to be straightened out. It was then cut through, close to its entrance to the uterus, and perfused with the fixing fluid by means of a pipette provided with a strong rubber bulb. THE EARLY DEVELOPMENT OF THE CAT 517

In this way the ova were washed out into a crystal dish, and were afterwards searched for under the higher powers of a Zeiss—Greenough binocular microscope. We have also employed the method advocated by R. van der Stricht and other workers (cf. Longley, p. 143) of expressing the contents of the straightened-out tube on to a slide, by firmly stroking the tube (preferably cut into two or more segments) along its length with the back of a scalpel. We regard this as a very useful method. As fixatives we have employed the fluids of Plemming and Hermann as well as picro-nitro-osmic acid, with satisfactory results. In order to prevent loss during imbedding, we fastened each egg to a thin slice of brain-cortex by means of a thin solution of photoxylin (0-5 to 1 per cent.). Sections were cut by the Minot rotary microtome, mostly at 6 /x, and were stained with Heidenhain's iron haematoxylin. We have prepared wax-plate models of a number of cleavage stages, which are illustrated in Text-figs. 1-8. These have proved invaluable more especially in tracing out the lineage of the central cells of the morula from which the embryonal knot is derived, and in demonstrating the occurrence of a process of overgrowth or epiboly, whereby the central cells come to be completely enclosed by the future trophoblastie cells. Migration of Eggs.—In view of recent observations (cf. Corner, 19) on the migration of ova from one uterus into the other in the Pig, we take this opportunity of recording a case in a cat which came under our notice in collecting the material described in this paper. In this particular cat (9.9.5.12) one ovary showed no visible corpora lutea, but the related uterus possessed two uterine swellings, one low down at the junction of the uterus Avith the vagina and the other higher up. The former swelling contained an abnormal embryo, the latter a normal one. The other ovary showed three or four corpora lutea, and the uterus a single uterine swelling. We think this case affords strong presumptive evidence of migration. 518 J. P. HILL AND MARGARET TRIBE Duplication of Uterine Lumen.—We also venture to record here an interesting case of duplication of the uterine lumen. In Cat 27.1.10 one of the uteri possessed two lumina, which commenced close to its upper extremity and continued through its entire length quite separate and distinct and at its lower end opened by separate openings into the

CHAPTER I.—THE OVUM OF THE CAT.

1. MATURATION AND OVULATION. WE give here a short resume of the observations of E. van der Stricht (55) and Longley (37) on the phenomena of maturation and ovulation, supplemented by our own. B. van der Stricht states that oestrus in cats generally lasts from two to three days (? after the first copulation, Longley). At the beginning of oestrus (first day), as a general rule, the intra-ovarian ovum (primary oocyte) has completed its growth, i.e. it has attained the stage of the full-grown ovarian ovum with a peripherally situated germinal vesicle. During the second clay (beginning or end) maturation sets in, and in the course of some hours the first polar body is extruded and the second polar mitotic figure established (stage of the mature ovarian ovum or secondary oocyte). Provided copulation has previously taken place ovulation now follows, and the ovum, still surrounded by the corona radiata of the discus proligerus (which persists for a variable period), is received into the Fallopian tube and is there fertilized, the time of fertilization practically coinciding with the end of oestrus (end of second or third day). Longley states that of a series of ten females killed at periods ranging from twenty-three to fifty hours after pairing, six had ovulated. The ova are said to pass very rapidly through the segment of the tube succeeding the ostium abdominale into its proximal third next the uterus, where, according to B. van der Stricht, fertilization usually appears to be effected though there are exceptions. B. van der Stricht records obtaining a three-celled egg from the middle third of THE EABLY DEVELOPMENT OF THE CAT 519 the tube, and Bonnet a nine-celled egg from the same region as well as an egg with two pronuclei, about 1-5 cm. from the ostiuni uterinum. Cleavage, as our own observations show, is completed in the uterine segment of the tube, and the eggs do not pass into the uterus until about the stage when the morula is becoming converted into the early blastocyst through the appearance of the beginnings of the blastocyst cavity. As in other mammals, consequent on the penetration of the sperm into the ovum, the second meiotic division is completed and the second polar body given off. In the absence of fertilization, the second polar division is not completed (cf. our eggs 1 and 2, referred to below). The ovum of the Cat thus agrees with that of the great majority of other mammals in that the first meiotic division is completed and the second initiated whilst it is still enclosed in its Graafian follicle in the ovary. In this respect it differs from the ovum of the Dog, which, as 0. van der Stricht has shown, is shed in the condition of a full-grown ovarian ovum, prior to the completion of the first meiotic division. He thinks that the same holds true for the human oocyte, though A. Thomson (56) believes he has been able to observe both meiotic divisions and the elimination of both polar bodies in the intra-ovarian ovum. We agree with 0. van der Stricht that the observations of Thomson stand in need of confirmation. It appears to be well established that ovulation in the Cat is normally induced by copulation (de Winiwarter and Sain- mont (58), Ancel and Bouin (2), Longley (37), E. van der Stricht (55)). Nevertheless it would occasionally seem to happen spontaneously. Bonnet (14) records finding an unfertilized egg in the tube ' bei einer langere Zeit in Einzelhaft gehaltenen und nicht belegten Katze '. We ourselves have obtained from each of two cats a single tubal egg (eggs 1 and 2) unfertilized and devoid of any trace of sperms. In egg 1, the first polar body is distinguishable though its limits are not very clearly defined, and 0028 mm. distant from it there is present in the egg-cytoplasm a group of chromosomes belonging to the second polar mitotic figure, the 520 J. P. HILL AND MARGARET TRIBE spindle-fibres of which are not visible. In egg 2 the first polar body has apparently divided. E. van der Stricht (55) records and figures the division of the first polar body in a ripe ovarian egg, and we record it as probable in our eggs 2 and 4. Longley (37) states that division of the first polar body ' is not of very common occurrence, especially in normal eggs '. The second polar mitotic figure is represented by an equatorial plate of small granular chromosomes, situated in the peripheral cytoplasm 0-05 mm. distant from the first polar body. No spindle- fibres are visible, but from the position of the equatorial plate it is evident that in this case the axis of the second polar figure lies tangentially to the surface and not at right angles thereto as is usual. No doubt these eggs are somewhat abnormal, having probably been some little time in the tubes before fixation, but they serve to demonstrate that spontaneous ovulation can occur in the absence of copulation and that when fertilization is not effected the second polar body is not separated. 2. STRUCTURE OP THE OVUM. The unsegmented tubal egg of the Cat appears in section not quite spherical but ovalish in outline. It varies in sectional diameter from 0084 to 011 x0-09 mm. (average of 0095 x 0082 mm. in thirteen eggs measured). The ovum itself (exclusive of the zona) varies in section from 0-069 x 0-57 mm. to 0-09 x 0-08 mm. in diameter (average of 0-0S2 x 0-07 mm.). Longley, with reference to the ovarian ovum of the Cat, states that ' the liAang cat's egg has a diameter ranging from 0-135 to 0-15 mm. and is surrounded by a zona 0012 to 0015 mm. in thickness '. We have no measurements either of full-grown ovarian or tubal ova in the fresh state^ but we have records of two tubal eggs which, measured in the fixing fluid, had diameters of 0153 mm. and 0-136 mm. respectively, whilst four eggs, in Hermann's fluid, measured 0-119x0-11 mm. in diameter, so that there is actually a considerable variation in the size of the ova. The zona varies in thickness in unsegmented ova from 00018 to 0006 mm. It increases in thickness during the sojourn of the egg in the tube, and reaches its THE EARLY DEVELOPMENT OF THE CAT 521

maximum (0-012 mm.) in late morula and early blastocyst stages which have recently passed into the uterus. It remains intact, though of course greatly thinned out, until long after the blastocyst has become didermic. Robinson (43) states that in the Ferret, ' the zona pellucida persists until shortly after the formation of the primitive streak in the embryonic area, but it disappears in the region of the trophoblast at an earlier period'. In the Dog he states it ' does not disappear till after the formation of the primitive streak '. As already noted, the cells of the corona radiata remain adherent to it for a varying period after ovulation, and even after the cells have been lost the basal syncytial layer of the corona seems to persist outside the zona proper, but we are uncertain whether it is eventually lost or is incorporated in the zona. However that may be, when the zona has attained its maximum thickness it appears as a clear, perfectly homogeneous and resistant membrane, which no doubt plays an important r61e in the mechanism of development, since, besides acting as a protection and support for the segmenting egg, it may con- ceivably act the part of a semipermeable membrane and thus prevent the too rapid absorption of fluid and consequent too rapid expansion of the early blastocyst. The structure of the ovum has been described in detail by E. van der Stricht, our own observations being largely confirmatory of his. The ovum of the Cat is provided with a considerable amount of deutoplasm mainly in the form of fat-globules of variable size, large, medium, and small. They are less abundant than in the egg of the Dog where they are of medium size, but more numerous than in the ovum of Cavia where they are small (0. van der Stricht, 52). In the full-grown ovarian ovum, with a peripheral germinal vesicle, E. van der Stricht shows that there is present, as in the preceding growth-stages, a superficial, thin zone of cytoplasm especially rich in mitochondria, ' une zone corticale mitochondriale plastique (de 0. van der Stricht) ', situated immediately below the egg-membrane and devoid of fat- globules. Within this investing zone, the cytoplasmic body 522 J. P. HILL AND MARGARET TRIBE of the ovum contains more or less abundant fat-globules, with mitochondria distributed in the interspaces between them. He distinguishes two groups of eggs according to the amount and disposition of the fat-globules. The first group comprises eggs, relatively rich in fat, the globules being mainly aggregated in the central region and towards one pole, so that the egg possesses a definite polarity, the plastic pole being less rich in fat than the deutoplasmic. In such eggs there is often present, below the superficial layer, a relatively broad zone in which are distributed small fat-globules. The second group comprises eggs in which the fat-globules are relatively less abundant and are mainly grouped in the central region, between which and the superficial layer is a broad zone usually poor in fat except at its periphery, where numerous small globules are generally present. At this stage there is in this group of eggs no obvious polarity. Nevertheless in eggs of both groups during maturation and fertilization stages, polarity, in the distribution of the fat-globules is perfectly definite, though it bears no constant relation to the place of expulsion of the polar bodies. These latter may lie adjacent to each other at the plastic pole as in our egg 9, or at the deutoplasmic as in egg 4, or they may occupy an intermediate position between the two as in egg 5, or again they may lie remote from each other on opposite surfaces of the egg as in egg 7. This variable position of the polar bodies is largely determined by the position originally taken up by the germinal vesicle in the full-grown ovum, that position bearing no constant relation to the deutoplasmic accumulation within the egg (Longley). In view of the well-known experimental work of Eusso (44) on the ova of the Babbit, E. van der Stricht is inclined to believe that eggs of the first group are destined to produce females, those of the second males, but as to that we offer no opinion. Our own observations on eggs at the stage of fertilization are confirmatory of those of R. van der Stricht so far as concerns the polar distribution of the fat-globules, i. e. in any given egg it is possible, usually without any great difficulty, to satisfy oneself that one hemisphere is richer in THE EARLY DEVELOPMENT OF THE CAT 523 . fat than the other, but we should hesitate to accept his grouping of the eggs into two clear-cut sets. We agree that some eggs have relatively little fat and others much more, but these, we hold, are but the extremes of a variable series. Lastly, in connexion with the structure of the ovum, it remains to be mentioned that E. van der Stricht has described the presence in the periphery of the ovum of one or two rounded or ovalish bodies of a size comparable with that of a mammalian red blood corpuscle, and with a structure recalling that of a typical ' vitelline body ' and which he has termed ' corps enigmatique '. They are formed, he says, at the expense of small safraninophil granules that appear in the young oocyte. He regards them as in some way taking part in the formation of the ' plastic vitellus ', and as being also perhaps of the nature of germ-cell determinants. We have also encountered this enigmatical body in our material and record its possible presence in two of the central cells of our sixty-three- celled cleavage stage, but we have not been able to follow it into later stages and are unable to offer any suggestion as to its significance. 3. FERTILIZATION. Comprised in our material are eleven eggs derived from five cats : Cat I, eggs 1 and 2 ; Cat II, eggs (3) and 4 ; Cat III, eggs 5, 6, 7, and (8) ; Cat IV, eggs 9 and 10 ; Cat V, egg 11. These eggs fall into three groups : Group 1. Differentiation of pronuclei, egg 1. Group 2. Pronuclei remote from each other, eggs (3) and 6. Group 3. Pronuclei approximated "I eggs 2, 5. 11, or in contact / 4, 7, 9, 10, (8). Eggs 3 and 8 contain three pronuclei. Group 1. Egg 1 (A. 4.4.17 egg A). Diameter, 0-091 x 0-088 mm. Ovum, 0084 x 0-081 mm. Zona, 0-002 mm. in thickness, surrounded by cells of corona radiata. Pig. 1, PI. 24. This egg is worthy of notice since it shows an interesting phase in the differentiation of the 3 pronucleus. As may be 524 J. P. HILL AND MARGARET TRIBE seen in fig. 1, PL 24, the £ pronucleus lies shortly below the superficial mitochondrial zone and has reached the condition of a membranate vesicular nucleus, but is still small (0-008 x 0-007 mm.) with a shape very much like that of a short blunt- nosed bullet. The chromatin is massed like a cap in its blunt apex, and appears to be in process of differentiation to form a spireme thread. Behind the cap are two ovoidal granules in contact with each other, whilst the remainder of the nuclear space is occupied by a pale-staining reticulum. In contact with the apex of the pronucleus is a granular area of cytoplasm, free from fat-globules, which is possibly the sperm-sphere, but it has not been possible to detect the sperm-centriole. Unfor- tunately the section which contained the main body of the $ pronucleus is the only one missing in the series, but the position of the pronucleus is recognizable in the succeeding section shortly below the second (?) polar body which occupies a distinct bay in the surface of the ovum, adjacent to the deutoplasmic pole. In close proximity to the polar body are several sperm-heads which seem to have penetrated the zona and are therefore supernumerary, but they are similar to those which are found imbedded in the latter. This egg shows particularly well the superficial mitochondrial zone of the egg-cytoplasm described by R. van der Stricht and the mitochondria which are distributed in the cytoplasmic framework supporting the fat-globules. Fat-globules, some of them reaching a large size, are present in abundance throughout the body of the ovum, with the exception of an area extending through three or four sections on one side marking the plastic pole. There is no intermediate zone intervening between the mitochondrial zone and the more internal fat-laden region, the latter abutting directly on the former. Group 2. Egg 6 (B. 14.17.5.12). Diameter, 0-1x009 mm. Ovum, 009 x0-08 mm. Zona, 00048 mm. thick, homogeneous with numbers of sperm-heads imbedded in it. Figs. 5 and 6, PL 25. This egg (fig. 5) corresponds closely with egg 94 described TP EARLY DEVELOPMENT OF THE CAT 525 and figured by E. van der Strioht (p. 452 and figs. 78-82). The two pronuclei, appearing as large clear spheres, are fully constituted and are indistinguishable except for a very slight difference in size. They occur towards one side of the egg, both appearing in one section (fig. 5), and are separated from each other by an area of cytoplasm 0026 mm. in width in which are present numerous fat-globules. On their outer sides the pronuclei abut on the peripheral plastic zone of cytoplasm containing fine fat-globules in no great numbers. The fat-globules in the body of the ovum are abundant and are mainly massed in the central region and towards one pole (fig. 6), whilst they are sparse and small in the peripheral plastic zone in which there are also present small light-staining homogeneous masses. Polarity in respect of the distribution of the fat-globules is unmistakable. The polar bodies are not distinct, but the ' enigmatical body ' is clearly seen in fig. 5 lying close below the zona and enclosing a central granule. In this egg, as in egg 94 of E. van der Stricht, the central plastic zone in which the pronuclei are eventually found situated, is not yet differentiated. Its formation must involve the shifting of the centrally situated fat-globules to a more peripheral position. It is further worthy of note that the pronuclei, as in egg 43 of E. van der Stricht (fig. 43, PI. xv), are so placed that a line joining their centres is approximately at right angles to the polar axis of the egg. Later on, when they have acquired their definitive position in the central plastic region, they lie in that axis, instead of at right angles to it. Group 3. Egg 2 (A. 4.4.17 B).. Diameter, 0-091 x 0-076 mm. Ovum, 0-087x0-073 mm. Zona thin, 00018 mm., with the remains of the corona radiata. The two pronuclei are situated in the less fat-rich hemisphere some distance below the egg-surface. They differ somewhat in size, structure, and position. The smaller of the two is oval, lies towards the centre of the egg, and is, we consider, the ? pronucleus. It measures 0012x0009 mm. in diameter, 526 J. P. HILL AND MARGARET TRIBE and possesses a subcentral spherical karyosome between which and the nuclear membrane is a chromatin-reticulum or rather a spireme thread segmented into five or six pieces which are roughly centred on the karyosome internally and spread out at their peripheral ends on the inner surface of the nuclear membrane. The larger of the two pronuclei (the 3) is spherical and has a diameter of 0-014 mm. It is rather less deeply situated than the smaller, and is separated from it by a distance of 0-016 mm. It contains one large and two smaller karyosomes, and in close relation to the inner surface of the nuclear membrane, on the side next the smaller pronucleus, are numbers of small chromatin granules and one larger mass. Immediately adjacent to this pronucleus there occurs a minute ovalish body, 0-008 x 0-002 mm. in diameter, containing a central granule. This body we regard as the sperm-centrosphere. As in the companion egg 1, the cytoplasmic structure is well preserved. The superficial mitochondrial zone is less marked than in egg 1, but the mitochondrial formations in the remainder of the cytoplasm are even more abundant. The fat-globules are numerous and of large size and are mainly located in one hemisphere, the pronuclei being situated in the other. The polar bodies, situated close together, are seen in surface view : one is larger and possesses two chromatin clumps, the other appears to be divided into two unequal parts.

Egg 5 (14.17.5.12 A). Diameter, 0-1 x 0-084 mm. Ovum, 0086x0072 mm. Zona, 0-004 mm., with numbers of sperm-heads imbedded in it. Fig. 7, PI. 25. The pronuclei are approximated but not in actual contact. They are large and vesicular, with one or two karyosomes and a distinct linin reticulum with scanty chromatin. Of the two, one (fig. 7) is smaller (0-021 mm. in diameter) and situated nearer the polar bodies ; we regard it as the ? pronucleus ; the other (g) is larger (0028 mm.) and rather more superficially situated. Both lie in the plastic hemisphere. The fat-globules (of medium to small size) are larger and more abundant towards one pole (fig. 7) ; towards the opposite or plastic

528 J. P. HILL AND MARGARET TRIBE

The fat-globules are small, fairly numerous, and mostly aggregated in one hemisphere, leaving the other 'hemisphere in which the pronuclei are situated relatively free. The two polar bodies lie adjacent to each other at the plastic pole.

Egg 10 (15.23.5.12 B). Diameter, 0-088 x 0-069 mm. Ovum, 0079x0057 mm. Zona, 00036 mm. Kg. 9, PI. 25. It is noteworthy that eggs 9 and 10 from Cat IV are both small. The two pronuclei lie in contact in the central region of the egg. They differ in size. The smaller (the ?) is approximately central and is spherical, with a large central and one or two smaller karyosomes and peripheral chromatin granules. Immediately outside the nuclear membrane is a very distinct centrosome granule. The larger pronucleus (the d) is ovalish and is situated nearer the plastic pole. It possesses several karyosomes and chromatin masses located mainly on the side nearest the smaller pronucleus. The nuclear membrane appears indistinct on the side nearest the plastic pole. There is an unmistakable polarity in the disposition of the fat-globules. They are less numerous, but many of them reach a much larger size than in egg 9, and they are mainly located in one hemisphere, though they extend up around the central region in which the pronuclei are situated. Plastic and deutoplasmic poles are readily recognizable, the region of the plastic pole being almost free from fat. The polar bodies and ' corps enigmatique ' were not observed.

Egg 4 (13.17.5.12 A). Diameter in fixative, 0-102 x 0-11 mm.; in section, 009x0089 mm. Ovum, 0069x0057 mm. Zona with remains of corona radiata attached and with sperm-heads imbedded in it. The ovum has contracted away from the zona, leaving a large penivitelline space. The two pronuclei lie in contact in the central region of the ovum which is free from fat-globules. They are large and vesicular and differ slightly in size. The larger one is ovalish in form and possesses a coarse reticulum with one large and THE EARLY DEVELOPMENT OF THE CAT 529 numerous smaller karyosomes which tend to be collected on the side nearest the other pronucleus. The latter, slightly the smaller of the two, is spherical and possesses a coarse reticulum with numbers of small karyosomes, again tending to be grouped on the side nearest the other pronucleus. The fat-globules are small and not very numerous and are most concentrated towards one pole, that adjacent to which are situated the polar bodies. The latter lie in proximity to one another and comprise a smaller one with a trilobate clump of chromatin and a larger, possibly divided into two.

Egg 11 (9.5.19). Diameter, 009x0076 mm. Ovum, 0084x0069 mm. Zona, 0-003 mm., with sperm-heads imbedded in it. Fig. 2, PI. 24. This well-fixed egg is at the stage just preceding the formation of the first cleavage spindle. The egg-cytoplasm consists of a very narrow finely granular and more deeply staining superficial zone, which is thicker at the plastic pole than elsewhere, and a coarsely granular ground-mass in which the fat- globules are situated. The fat-globules, which are not very abundant, are mainly grouped towards one pole of the egg, below and especially round the pronuclei which are subcentral in position. The pronuclei are approximated but not in actual contact and are of unequal size, the larger measuring 0-024 x 0-021 mm., and the smaller 0-018 x 0-016 mm. in diameter. The larger pronucleus (the

Amongst our fertilization stages we have met with two eggs in each of which there is a supernumerary $ pronucleus, as in egg 96 of B. van der Stricht. Egg 8 (D. 14.17.5.12). Diameter, 0-1 mm. Ovum, 0-084 x 0-079 mm. Zona (exclusive of outer follicular invest- ment), 00036 mm. There are two pronuclei, a larger (0-021 x 0-019 mm.) and a smaller (0-016 x 0-014 mm.), which lie in contact towards one side of the ovum, and which correspond to the pronuclei of the normal egg, whilst a supernumerary pronucleus is situated shortly below the surface at the opposite side of the egg and is of practically the same size as the larger one of the other pair. The fat-globules are most abundant in the region of the egg between the accessory and the paired pronuclei, and round the latter are also fairly numerous whilst they are less numerous in the region occupied by the supernumerary pronucleus, as in egg 96 of E. van der Stricht. Polarity in the distribution of the fat-globules is therefore indicated, the pair of pronuclei THE EARLY DEVELOPMENT OF THE CAT 531 lying in the more deutoplasmic region. The polar bodies are apparently normal. Egg 3 (13.17.5.12 B). Diameter in fixative, 0-10 mm., enclosed by remains of corona radiata in which are imbedded numbers of sperm-heads. This egg is very similar to the preceding. There are two pronuclei, a larger and a smaller lying in contact shortly below the surface on one side and a supernumerary one of about the same size as the larger one of the pair but less rich in chromatin, situated superficially at the opposite side of the egg. The two polar bodies lie in contact, and possess one and two angular masses of chromatin respectively, the one with two chromatin masses having a diameter of 0009 x 0007 mm. The special interest of these eggs is that they show that the larger of the two pronuclei in normal eggs is the male. This is in agreement with the statement of Lams (36) that in Cavia the

4. REMARKS ON FERTILIZATION. Our knowledge of the phenomena of maturation and fertilization in the monodelphian mammals is now extensive, thanks to the work of O. van der Stricht (50, 52), H. Lams (35, 36), Sobotta (47), and others. 0. van der Stricht (52) has recently given a most valuable resume of his own investigations and those of his pupils, to which we would refer the reader. Our observations on the fertilization process in the Cat are in the main in agreement with those of R. van der Stricht. Contrary to his statement, however, we are able to demonstrate that the pronuclei, once they have reached their definitive condition, are not identical but differ in size and in position, the cj pronucleus being always larger and always more superficially situated, nearer the upper or plastic pole, than the ? pronucleus. This is in agreement with the conclusion of Lams (36) for the pronuclei in Cavia as above stated, and of O. van der Stricht (50) for those of Vesperugo. 532 J. P. HILL AND MARGARET TRIBE

The pronuclei, after they have enlarged and assumed their characteristic vesicular form, migrate inwards and come to lie in the central plastic region of the egg which is more or less free from large fat-globules ; if fat-globules are originally present in this central region as in our egg 6 and egg 94 of R. van der Stricht, they would seem to undergo displacement towards the deutoplasmic pole, but apart from this they maintain their original polar distribution during the fertilization process, no reversal of polarity such as 0. van der Stricht (50) and Lams (36) have described for Vesperugo and Cavia occurring in the Cat. The pronuclei, once they have reached the central plastic region, come to lie in close proximity or in actual contact, and acquire a disposition such that the larger (<$) pronucleus is situated nearer the plastic pole, the smaller (?) pronucleus nearer the deutoplasmic pole. The line joining their centres marks, as R. van der Stricht has emphasized, the definitive axis of the egg and the plane of the first cleavage, and it coincides as near as may be with the polar axis. Although the egg of the Cat presents an unmistakable polarity owing to the greater abundance of fat-globules at the lower pole as compared with the upper, there is a considerable amount. of variation and irregularity in their distribution in different eggs, with the result that it is often difficult to determine precisely the position of the polar axis, but once the pronuclei have acquired their final position the definitive egg-axis is readily determinable. Like R. van der Stricht, we have never been able to observe the tail of the spermatozoon in the egg-cytoplasm. In both the Cat and the Dog, as 0. van der Stricht points out, the sperm-tail is very delicate and often difficult to see outside the ovum ; he records, however, that he has been able to demonstrate its presence in several ova of the dog. Only in one ovum (egg 2) have we been able to observe the sperm-centrosphere, whilst in another (egg 10) we record the presence of what we regard as the egg-centrosonie (centriole) adjacent to the ? pronucleus. The observations of 0. van der Stricht (50) and Lams (36) on the process of fertilization in THE EARLY DEVELOPMENT OF THE CAT 533

Vesperugo and Cavia, where the sperm-tail remains adherent to the central corpuscle of one of the poles of the first cleavage spindle demonstrate that in these mammals ' le ou les sper- matocentres persistent et participent a l'edification des deux spheres attractives definitives de la premiere etoile-mere de l'oeuf en division ' (52, p. 15).

CHAPTEK II.—THE PKOCESS OP CLEAVAGE.

1. DESCRIPTION OF CLEAVAGE. OUR material of the early cleavage stages is scanty, but such as it is, it supplements that of R. van der Stricht. The latter was not able to observe the first cleavage mitotic figure. We have been a little more fortunate, having obtained from one cat three eggs in the phase of the first cleavage, but unfortunately in two of them the spindle is not well preserved and it is impossible to determine its axis. The third egg (egg 12, B. 16.5.19) is worthy of a brief description.

Egg 12. Diameter, 0-093x0-084 mm. Ovum, 0-079 x 0074 mm. Zona about 0003 mm. Fig. 3, PI. 24. The ovum is incompletely divided into two halves by a deep circular groove extending in more than half-way towards the centre (fig. 3). In each half there is a small membranate nucleus, the nuclei differing in character. That on the right (0-008 x 0-006 mm. in diameter) is oval and possesses one karyosome and peripheral chromatin granules; that on the left is slightly larger (0-009x0007 mm.) and more deeply staining than the other, it possesses several karyosomes, and its contour is somewhat irregular. In the centre, about mid-way between the two nuclei, remains of the spindle-fibres with their intermediary thickenings (rsp.) are distinctly visible. Polarity in the disposition of the fat-globules is not obvious, but it may be noted that the right half appears to be richer in fat than the left. The two polar bodies lie in the plane of division, one, the larger (measuring 0-007 x 0-0048 mm.), lies peripherally in 534 J. P. HILL AND MARGARET TRIBE contact with the left half; the other, smaller, lies opposite the groove.

Two-celled Egg in Division. Egg 13 (A. 15.5.19 (a)). Diameter, 0-009 mm. Fig. 4, PI. 24. This egg is one of three and is belated as compared with its companions which are in the four-celled stage. The two blastomeres are of unequal size ; the larger one (on the left in fig. 4) has a diameter of 0-07 x 0-04 mm. and is richer in fat-globules than the other which measures 0-06 x 0-03 mm. The larger blastomere possesses an equatorial plate of clumped chromosomes, practically centrally situated and with remains of the asters on opposite sides, but the spindle- fibres are not preserved. The mitotic figure has been cut obliquely, and it is not possible to determine with certainty the plane of division, though vertical division approximately at right angles to the plane of the first cleavage is suggested. The smaller blastomere possesses a membranate nucleus of a distinctive lobed appearance situated subcentrally towards the plastic pole. Its nuclear membrane is very delicate and irregularly sinuous or lobed. The nuclear space is occupied by a dense spireme or group of chromosomes except peripherally, below the nuclear membrane, where the reticulum appears to have become swollen and vesicular. Adjacent to the nucleus is what seems to be an extra-nuclear group of chromatin granules as well as a small accessory nuclear body, whilst a second similar body is present towards the plastic pole. We agree with E. van der Stricht that these nuclear bodies are to be regarded as lobes of the nucleus which for some unknown reason have become separated off. In this same blastomere there is present at the deutoplasmic pole a minute spherical body with a deeply staining membrane enclosing a lighter area in which are situated two central granules. This is probably the ' corps enigmatique ' which E. van der Stricht thinks is characteristic of this particular blastomere with the lobed nucleus, but we find a similar body with one central granule situated at the plastic pole of the other blastomere. As regards the polarity of the blastomeres, in both the fat- THE EABLY DEVELOPMENT OF THE CAT 585 globules are more abundant towards one pole, the deutoplasmic pole which corresponds in the two. They extend up peripherally towards the opposite or plastic pole, but leave that, as well as the central region, relatively free. The superficial groove between the two blastomeres, coincident with the plane of cleavage, is occupied by a granular material which may possibly have been eliminated by the blastomeres, and, if so, is to be regarded as deutoplasmolytic in nature. It is clear from the condition of the nuclei in this egg that the larger blastomere in Avhich the equatorial plate is already differentiated would have divided before the other ; this is in agreement with E. van der Stricht's conclusion that the blastomere with the regular (non-lobed) nucleus divides before that with the lobed nucleus. Longley (37) figures a two-celled egg (his fig. 13, PI. iv) in which the nuclei of the two blastomeres are seen to differ in size, one is larger and spherical in outline, the other is smaller and irregularly ovalish. A polar body is shown lying in the peripheral groove between the blastomeres. The two-celled egg of the Dog, according to 0. van der Stricht (52) is very similar, apart from the form and disposition of the mitochondria, to that of the Cat. The blastomeres exhibit a distinct polarity in the distribution of the fat-globules, whilst their nuclei, excentrically situated towards their upper poles, differ in form, one being distinctly lobed, the other, more regular. The two polar bodies lie adjacent to the lower poles of the blastomeres. E. van der Stricht lays emphasis on the facts that the two blastomeres of the two-celled egg differ in the form of their nuclei, in the time of their division, sometimes in the number of the larger rnitochondrial formations occurring in them, and always in the presence of the ' corps enigmatique ' in the blastomere with the lobed nucleus (but in regard to the latter compare above). He apparently attaches considerable importance to these differences, and points out that they afford confirmation of the conclusion reached by E. van Beneden in the case of the rabbit that the first two blastomeres play perfectly distinct roles in the formation of the constituent parts 536 J. P. HILL AND MARGARET TRIBE of the blastocyst. We need only say here that that conclusion is not in accordance with our observations which lead us to think that the parent cell of the inner cell-mass is not separated until the next cleavage, i. e. is one particular cell of the second cleavage generation, the other three cells furnishing the trophoblast. Two three-celled eggs have been described by E. van der Stricht (55, p. 462). The blastomere of the two-celled stage with the regular nucleus has divided, whilst the other with the lobed nucleus and the ' corps enigrnatique ' is still undivided, its nucleus being at the beginning of the prophase and containing a spireme-thread. It is clear that the two divisions of the second cleavage are not synchronous but successive. If the first cleavage plane, which passes through the plastic and deutoplasmic poles of the ovum be regarded as vertical, then according to E. van der Stricht's observations the first of these two divisions of the second cleavage is also vertical and at right angles to the first cleavage plane. Assuming this determination to be correct, it follows, from our own observations on the four-celled stage, that the plane of the second of these divisions must be horizontal (equatorial), since in the four-celled stage the blastomeres are in pairs so arranged as to form a cross-shaped group, the plane of division of the one pair being at right angles to that of the other pair. That being so, it necessarily follows frorn the polar distribution of the fat-globules in the blastomeres of the two-celled stage as described by E. van der Stricht and by us, that one of the four blastomeres of the four-celled stage should be poorer in fat-globules and one richer in the same than the other three. Four-celled Eggs. E. van der Stricht had available two four-celled eggs, but the arrangement of the blastomeres is not described. We have examined four such eggs, two from one cat, egg 14 (A. 15.5.19 b) and egg 15 (A. 15.5.19 c), and two from another, egg 16 (3.6.19 a) and egg 17 (3.6.19/3). THE EARLY DEVELOPMENT OF THE CAT 537 Egg 14. Diameter about 009 mm. Two views of the model of this egg are shown in Text-fig. 1 a and b. The four blastomeres are readily grouped into two

TEXT-PIG. 1.

Two views (a and &) of model of egg 14, four-celled stage. x about 300. pairs (1 & 2) and (3 & 4), so arranged as to form an interlocking cross-shaped group. Pair 1 & 2 occupied one hemisphere and pair 3 & 4 the other, the plane of division between 1 & 2 cutting that between 3 & 4 obliquely. The blastomeres are approximately of the same size, but one pair is richer in fat-globules than the other. Apart from this no one blastomere can be said to be specially poor or specially rich in fat-globules.

TEXT-FIG. 2.

Two views (a and b) of model of egg 16, four-celled stage. x about 300.

Egg 15. Text-fig. 2 shows two views (a and b) of the mode, of this egg, which is noteworthy on account of the inequality in the size of the blastomeres. 538 J. P. HILL AND MARGARET TRIBE Blastomeres 1 & 2 and 3 & 4 form pairs, the plane of division between 1 & 2 being at right angles to that between 3 & 4, so that there is an obvious cross-shaped arrangement, less regular than in egg 14. Blastomeres 3 & 4 together are considerably larger than those of the other pair, pointing to inequality in the two-celled stage, and the facts that blastomere 4 is the largest and 2 the smallest point to inequality in both divisions of the second cleavage. Blastomere 4 is fairly rich in large fat-globules, whilst 3 has very few, from which we may conclude that the plane of division between these two blastomeres is that of the horizontal second cleavage.

TEXT-FIG. 3.

Two views of model of egg 16, four-celled stage, x about 300.

Egg 16. Diameter, 0-08 x 0-072 x 0-072 mm. Blastomeres 1 & 2 and 3 & 4 form pairs so arranged as to form a perfectly definite cross (Text-fig. 3, a and b), the plane of separation of 3 & 4 being at right angles to that of 1 & 2. Blastomeres 1 & 2 extend round so as to envelop 3 & 4 laterally. What appears to be a polar body lies in the plane of division between 1 & 2 and may mark the vertical axis. If so, then the division between 3 & 4 again marks the horizontal second cleavage plane. The blastomeres apart from slight differences in size appear to be similar, though possibly blastomere 3 is somewhat richer in fat-globules than 4. THE EARLY DEVELOPMENT OF THE CAT 539

Egg 17. Diameter, 0-088 x 074 x 0-054 mm. This egg is from the same cat as the preceding egg but is smaller. Blastomeres 2 & 4, which are large, and 1 & 3, which are smaller and differ in size (1 being larger than 3), seem to form pairs of very unequal size. They are arranged to form a rather irregular cross, the plane of division between 1 & 3 being at right angles to that of 2 & 4. A minute oval body in blastomere 3 is probably the ' corps enigmatique '. In all these four-celled eggs there is present in the superficial grooves between the blastomeres, and between the latter and the zona, a granular material which is possibly deutoplasmolytic. Seven-celled Eggs. We have available for description two seven-celled eggs. Egg 18 (13.5.19) and egg 19 (17.31.1.13). E. van der Stricht records obtaining four eggs (from the Fallopian tube 0-5 cm. from its uterine opening) with seven or eight blastomeres, and he figures sections of two of those with seven blastomeres (his photo. 87, 88, 89). This seven-celled condition results from the fact that three of the blastomeres of the four-celled egg have undergone the third cleavage divisions, whilst one has remained undivided. The fact that this stage is represented in the collections of both E. van der Stricht and ourselves may be taken to indicate that the belated division of one of the blastomeres of the four-celled stage is a normal occurrence, and accordingly the question arises as to its significance. We suggest that possibly this particular blastomere may turn out to be the mother-cell of the two central cells of later cleavage stages, from which the inner cell-mass or embryonal knot originates. Egg 18 (13.5.19). Diameter inside zona, 0-082 x 0069 x 009 mm. An excellently preserved egg but exceptional in that the zona is separated into two layers, the intervening space containing coagulum in which are sperms and sperm-heads. The seven blastomeres are arranged as shown in the figure of the model (Text-fig. 4 a and b). There are six smaller 540 J. P. HILL AND MARGARET TRIBE blastomeres (1-6) and one larger (7), measuring 0036 x 0-055x0-05 mm. This, an undivided blastomere of the four- celled stage, is situated at one pole of the oval egg, and is overlapped by blastomeres 6, 3, and 5 and to a very slight extent by 4. Blastomere 3 is in the telophase of division, the two groups of chromosomes being separated by the thickness of one section (figs.. 10 and 11, PI. 26), and is superficially grooved round its mid-region (Text-fig. 4fc), i.e. this blastomere is in process of completing the fourth cleavage division. The blastomeres all contain numerous larger and smaller fat-vacuoles and, in the superficial grooves between the blasto-

TEXT-FIG. 4.

Two views (o and 6) of model of egg 18, seven-celled stage. Blasto- mere 7 is regarded as the parent central cell, x about 300. meres as well as in places between the blastomeres and the zona, there is present a granular material in which are small spherical masses, the whole probably deutoplasmolytic. A polar body (00096 x 0-007 in diameter) is present in the superficial groove between blastomeres 1 & 2, and rather deeply situated in blastomere 6, is a possible ' corps enigmatique '. There has evidently been a certain amount of shifting of the blastomeres as is indicated by the overlapping of blastomere 7 by the other cells, and accordingly it is very difficult to make out the grouping of the blastomeres in pairs. The following grouping is suggested as a possibility, the Eoman numerals indicating the blastomeres of the four-celled stage : I II III IV 7 + 3&5 + 1&6 + 2&4 THE EARLY DEVELOPMENT OF TflE CAT 541 The plane of division between 7 and 3 & 5 is approximately at right angles to that between 1 & 6 and 2 & 4. Egg 19 (17.31.1.13). Diameter, 0-1 xO-09 xO-08 mm., distinctly larger than the preceding. Two views of the model of this egg are shown in Text-fig. 5 a and b and a sectional view in fig. 20, PI. 27. The undivided blastomere (5) (Text-fig. 5 b) is large, measuring 0-072 x 0-069 x 0-045 mm. in diameter. Blastomeres 1, 2, 6, 7, and 4 lie in contact with its margin, but there is no definite overlapping. Blastomere 3 lies on the opposite side of the egg to 5 (Text- fig. 5 a), and is separated from it centrally by a small space.

TEXT-FIG. 5.

Two views (a and 6) of model of egg 19, seven-celled stage. Blasto- mere 5 is regarded as the parent central cell, x about 300.

Blastomeres 6 & 7 are larger than either of the other pairs, G exceeding 7 in size. Blastomeres 3 & 4 are approximately of the same size as 1 & 2. Here again it is difficult to determine the grouping, but we suggest the following : I II III IV 5 + 6&7 + 1&2 + 3&4 Under this grouping the plane of division between 5 and 6 & 7 is very oblique to that between 1 & 2 and 3 & 4. In the central space, as well as in the superficial grooves between the blastomeres, there is present a considerable amount of finely granular material. 542 J. P. HILL AND MARGARET TRIBE The relations of the paired blastomeres to the undivided blastomeres in these two eggs, and more especially in 18, distinctly suggest the possibility of the occurrence in succeeding stages of a process of epiboly whereby the smaller blastomeres come to surround the larger one. In this connexion it is worthy of note that in the Pig, Assheton (4) states that one of the blastomeres in a nine-celled stage ' is far larger than any of the others ' (p. 335, and fig. 14, PL 26). He further states (p. 337): ' In the four-celled stage there is always a slight difference in size, but at the eight- to twelve-segment stage the difference is most marked, and recalls the great difference which we find in many molluscan ova. In no case, however, have I found any difference perceptible except in size. The smaller segments form a cap upon the larger.' He thinks it' extremely probable ' that a process of epiboly takes place, though in consonance with his well-known views he holds that ' it is really the hypoblast which grows round the epiblast ' (p. 338).

Sixteen-celled Eggs. Three eggs of this stage are available for description, all obtained from one cat (8.9.5.12). They are of very great interest inasmuch as the two constituent parts of the future blastocyst are now clearly distinguishable. The future inner cell-mass or embryonal knot is represented by two centrally placed cells, the future trophoblast by fourteen peripheral blastomeres which enclose the central cells either partially or completely.

Egg 20 (8 C). Diameter inside zona, 009 x 0-069 x 0-07S mm. Zona thin, 00024 mm. Four views of the model of this egg are shown in Text-fig. 6 a, b, c, d, and a combined view of two sections in fig. 21, PL 27. The egg, now in the stage of a morula, consists of fourteen peripheral or trophoblastic cells and two centrally placed cells which are all but completely enclosed by the former. The peripheral blastomeres are spherical and are in contact only over small contiguous areas of their surfaces. They contain THE EARLY DEVELOPMENT OF THE CAT 543 fat-globules in fair abundance (fig. 21, PI. 27) and are similar though they differ somewhat in size, e. g. blastomere 2 measures 0028 x 0-024 x 0-018 mm. in diameter, blastomere 13, 0-028 x 0026x0026 mm. The two central blastomeres (Text-fig. 6 d, 12 & 5), in spite of their very different destiny, do not appear to differ from the

TEXT-FIG. 6.

Four views, o, 6, c, d (section), of model of egg 20, sixteen-eelled stage. Blastomeres 5 & 12 are the central cells, x about 300. peripheral in any recognizable respect except position. They are here nearly completely enclosed, but it is an interesting and significant fact that the outer end of central blastomere 12 is visible in the model (Text-fig. 6 c) through the gap at one pole of the mornla, bounded by blastomeres 13, 15, 14, and 16. Text-fig. 6 d, representing the cut surface of a vertical section of the model, illustrates this relation as well as the position of the central blastomeres (5 & 12), and affords conclusive evidence 544 J. P. HILL AND MARGARET TRIBE in favour of the occurrence of epiboly, i. e. of a process of overgrowth whereby the central cells become secondarily enclosed by the peripheral. The two central cells differ slightly in size, blastomere 12 measuring 0-024x0-031 xO-24 mm., and 5, 0-018 x 0-028 x 0-024 mm. They are thus of very much the same average size as the peripheral cells. As concerns their origin we think their relations to each other and to the peripheral cells indicate pretty definitely that they have arisen by the slightly unequal division of a single parent cell which we suggest is the large blastomere of the seven-celled stage, representing an undivided blastomere of the four-celled stage. If our suggestion is accepted then the constitution of the morula might be represented as follows : I II III IV [1X2] + [4] + [3 + (1X2)] + [3 + (1X2)]=2C + 14 Y Y Y Y Y Y 3rd 4th 4th 5th 4th 5th cleavage generation From this lineage it follows (1) that the central cells ought to be larger than the peripheral, and (2) that four of the peripheral cells ought to be smaller than the remaining ten since they belong to a later cleavage generation, viz. the 5th. So far as we have observed neither of these conditions appears to be realized in this egg as the result perhaps of inequalities in the antecedent divisions. There is of course another alternative, and that is to assume that normally an eight-celled stage occurs in the Cat and that the products of division of one of the blastomeres simply become overgrown and enclosed by the fourteen blastomeres arising from the division of the other seven. In this case the constitution of the morula would be as follows : I II III IV 1x2 + 1x2 + 1x2 + 1x2=8 blastomeres (lx2C) + (l x2) + 4 +4 + 4 =20 + 14 i.e. the sixteen blastomeres would all belong to the fourth THE EARLY DEVELOPMENT OF THE CAT 545 cleavage generation, and the parent cell of the inner cell-mass would belong to the third cleavage generation instead of to the second as in the first interpretation. Unfortunately no detailed description of an eight-celled egg is available, and a more precise determination of the lineage of the single formative cell must await the result of further observation. Egg 21 (8 A). Diameter (inside zona), 0-091 x 0084 x 0-09 mm. TEXT-FIG. 7.

Four views, a, b, c, d (section), of model of egg 21, sixteen-celled stage. Blastomeres 1 and 16 are the central cells, x about 300. In this morula (Text-fig. 7 a, b, c, d) the peripheral cells form a rather more continuous enveloping layer than in the preceding. There is one undoubted central cell (Text-fig. Id, 16) completely, enclosed and measuring 0-03 x 0-026 x 0-024 mm. in diameter. It is thus of approximately the same size as central blastomere 12 of the preceding egg, and is too small to give origin by division to two centrals of the size of those in that NO. 272 O 0 546 J. P. HILL AND MARGARET TRIBE egg. One of its ends, however, is in contact with a large blastomere (1) measuring 0-03 x 0036 x 0-084 mm., which projects at one pole of the morula, being surrounded by five of the peripheral blastomeres (Text-fig. 7 a and c (2, 3, 4, 5, 6). Enclosed between these blastomeres, it extends inwards to terminate in contact with central cell (16) (Text-fig. 7 d). We regard this blastomere (1) as likewise a potential central cell (the sister-cell of 16) which has not yet become completely enclosed by the overgrowth of the surrounding peripheral cells. Egg 22 (8 B). Diameter (inside zona), 0096 x 0-079 x 0-078 mm. This morula is very similar to the preceding. There is again one undoubted central cell measuring 0-033 x 0-028 xO-03 mm. in diameter. This central cell comes into contact with a blastomere (16) which projects at the surface and whose base is surrounded by five peripheral cells, as in the case of blastomere 1 of the preceding egg. Curiously enough, its measurements are identical with those of its presumed sister-cell, the undoubted central. Here again we think that the epibolic process is less advanced than in egg 20. In these sixteen-celled eggs a good deal of granular coagulum is present around and between the blastomeres. Twenty-two- to Thirty-one-celled Eggs. The four morulae included here are all characterized by the presence of two central cells, completely enclosed by a single layer of trophoblastic cells to whose division the increase in the number of the blastomeres is due. The epibolic process has accordingly been completed. Egg 23 (22.5.19 A). Diameter, 0-10x0-084 mm. Morula, 0-09 x 0-07 x 0-08 mm. Zona, 0-007 mm., now markedly thickened. This morula consists of twenty-two cells, two central cells completely surrounded by twenty trophoblastic. The cells are compactly arranged, without intercellular spaces between them, and entirely fill the space inside the very thick zona (figs. 12 and 13, PI. 26). THE EARLY DEVELOPMENT OF THE CAT 547

On the assumption that the two centrals are derived from a single blastomere of the four-celled stage, the constitution of this morula may be expressed as follows : I II III IV (1 x 2 0) + (1 x 2 x 2 x 2) + (1 x 2 x 2 x 2) + (1 x 2 x 2) i.e. the morula consists of two cells of cleavage generation 3 (the two centrals), sixteen of cleavage generation 5, and four of cleavage generation 4. It follows that four trophoblastic cells should be larger than the remainder, and from our measurements we actually find that three at least are distinctly larger than the others. The trophoblastic cells vary in diameter from 003 x 0-043 x 0-031 mm. (blastomere 14) to 0-018 x 0019 x 0-016 mm. (blastomere 13). They are rich in fat-globules, mostly of large size. Blastomeres 2 & 5 contain each a small accessory nucleus, and in 5 in addition there is present the ' corps enigmatique ' deeply placed to the inner side of the nucleus (fig. 12, 5. c.e.). Blastomeres 10 & 11 are sister-cells, recently formed as indicated by their small deeply staining nuclei, whilst blastomeres 16 & 18 are in process of division. Blastomeres 5 & 13 (figs. 12 & 13), though markedly unequal in size, follow on in the series in apparent continuity and probably also form a pair. The two central cells lie in contact and are of unequal size, central cell 6 (fig. 12) measuring 0-02S x 0-026 x 0-024 mm. in diameter, central cell 12 (fig. 13) measuring 0024 x 0019 x 0-018 mm. They are less rich in fat-globules than the trophoblast cells. The relations of blastomeres 12 & 13, as seen in fig. 13, rather suggest that these blastomeres might be sister- cells, the products of tangential division but, as stated above, we think it more probable that blastomeres 5 & 13 are sister-cells.

Egg 24 (22.15.19 B). Diameter, 0-098x0-091x0-078 mm. Morula, 0-084 x 0-076 x 0-07 mm. Zona, 0-006 mm. This very fine morula, from the same cat as the preceding, consists of twenty-three blastomeres, two centrals (11 & 13), and twenty-one trophoblastic. Three views of the model of OO2 548 J. P. HILL AND MARGARET TRIBE this egg are shown in Text-fig. 8 a, b, and c, whilst three con- secutive sections are figured in figs. 14-16, PL 26. As compared with the preceding egg the trophoblast cells here form a more regular enclosing layer around the centrals, their characteristic radial arrangement around the latter being well shown in Text-fig. 8 c and fig. 14.

Three views, a, b, c (section) of model of egg 24, twenty-three-celled stage. Blastonieres 11 and 13 are the central cells, x about 300.

Blastomeres 1 & 2 are sister-cells, the division having but recently been completed as is evidenced by the small size of their nuclei and the still recognizable remains of the spindle. In blastomere 17 is a ' corps enigmatique ' superficially placed, whilst adjacent to the large nucleus is a small accessory one. The two central cells again differ in size (Text-fig. 8 c and figs. 14—16, c.c 11 & 13). Central cell 11, the larger of the two, measures 0036 x 0033 x 003 mm. and possesses a membranate nucleus containing a segmented spireme, indicative of early division. Central cell 13 is much smaller, measuring 0-022 x THE EARLY DEVELOPMENT OF THE CAT 549

0-021x0-018 mm. Its nucleus is in the resting condition. In the cytoplasm of both are numbers of large fat globules. A polar body with three chromatin masses (fig. 15, Pb.) lies in contact with the zona in the superficial groove between blastomeres 8 & 12. Egg 25 (19.11.2.13). Diameter (in fixative), 0-1 x 0-09 mm. Zona thin, partially separated. The morula consists of twenty-four cells, two central, and twenty-two trophoblastic (fig. 27, PI. 27). One of the centrals is slightly larger than the other and possesses two nuclei, a larger and a smaller. Egg 26 (16.19.12.12). Diameter (in fixative), 0-127 x 0-1 mm. This morula consists of thirty-one cells, two central and twenty-nine trophoblastic. The trophoblastic cells do not form such a regular layer as in the preceding morula, the cells being more spherical in form. The two central cells are in contact and are similar except that one is slightly larger than the other. E. van der Stricht (55) gives two microphotographs of sections of a thirty-celled egg (figs. 92 and 93, PI. xix) in which already several central cells appear to be present. It is noteworthy that in all of these four morulae (eggs 23-6) one of the central cells is larger than the other. It is tempting to suggest that the one (the larger since it appears to divide first (egg 24)) gives origin to the embryonal ectoderm, the other to the entoderm, but we have no evidence to offer in support of this suggestion. Fifty-nine- to Sixty-three-celled Eggs. Egg 27 (27.1.10 B). Diameter, 040 x 0-09 mm. Morula, 0-076 x 0-072 x 0-08 mm. Zona very thick, 0-012 mm. Egg 28 (27.1.10 A). Diameter, 0-10 x 0-098 mm.-. Zona also thick, 0-0096 mm. Fig. 26, PI. 27, and fig. 17, PI. 26. These two morulae, from the same cat, are essentially similar, differing only in the number of the blastomeres. It has been a very difficult task to make an accurate count of the blasto- 550 J. P. HILL AND MARGARET TRIBE meres, but we estimate that egg 27 consists of ± 59 cells of which 10 are centrals and 49 trophoblastic, whilst egg 28 consists of ± 63 cells of which S are centrals and 55 trophoblastic. The blastomeres are not so compactly grouped as in the preceding morulae, the central cells, in particular, being loosely arranged with obvious interspaces between them. The trophoblast consists of a single connected layer of ovoidal cells, clearly marked off from the centrals (figs. 26 and 17). They contain fat-globules and vary somewhat in size, their average diameter in egg 28 being 0-015 x 0-014 mm. One of them in the latter egg is in process of division. The trophoblast in both these eggs has over considerable parts of its extent shrunk away from contact with the zona. The central cells are spherical or cuboidal in form, and on the average are smaller than the trophoblastic cells ; in egg 27 they vary in diameter from 0-012 to 0-018 mm., but we have not observed any staining or other difference between the larger and the smaller cells. They contain fat-globules and do not appear to differ in any essential respect from the trophoblastic cells. In two of them in "egg 28 there is present a small spherical body with a light centre and a darker-staining periphery, suggestive of the ' corps enigmatique '. In egg 27 one of the centrals is in process of division. Unfortunately our records do not state whether eggs 27 and 28 were derived from the tube or the uterus ; if from the latter we think the fact would have been noted. R. van der Stricht (55) states that he obtained morulae of about twenty- eight to thirty blastomeres from the uterine third of the tube.

2. EEMARKS ON CLEAVAGE. Our observations on the cleavage of the fertilized ovum of the Cat may be summarized as follows. The first cleavage which we assume to be vertical divides the ovum into two blastomeres which may be equal or somewhat unequal. One of the two possesses a curiously lobed nucleus, the other a normal one. The second cleavage is effected by two successive divisions, THE EARLY DEVELOPMENT OF THE CAT 551

the blastomere with the normal nucleus dividing before the other and in a vertical plane with resulting production of a three-celled stage, the other dividing later in the horizontal (equatorial) plane, at approximately right angles to the first. As the result the blastomeres of the four-celled stage are grouped in two pairs so arranged as to form a cross-shaped figure. This is apparently the usual arrangement of the blastomeres at this stage in the Monodelphia, having been recorded for a considerable number of forms, Dog and Eabbit (Bischoff), Rhinolophus (van Beneden and Julin), Vespertilio (van Beneden, 11, fig. 6, but not for the eggs shown in figs. 4 and 5), Erinaceus (Kunsemiiller), Macacus nemestrinus (Selenka). Assheton (6) states that he has seen four-segments stages of rabbit, pig, dog, ferret, and hedgehog, and has always found the pairs ' crossways '. The third cleavage is represented in our material by two seven-celled eggs, three of the blastomeres of the four-celled stage having divided, the fourth remaining undivided. In one of these eggs (18), the large blastomere is already to some slight extent overlapped by the smaller. Our next cleavage stage comprises three eggs composed of sixteen blastomeres. In all these we are able to recognize a grouping into two more centrally situated blastomeres and fourteen peripheral blastomeres which more or less completely surround the former. The peripheral cells we interpret as the future trophoblast, and the two central cells as the parent cells of the inner cell-mass. The central cells, we suggest, are derived by the division of the large blastomere of the seven-celled stage, i.e. from one of the blastomeres of the four-celled stage or alternatively by the division of one of the blastomeres of the eight-celled stage ; unfortunately no description of an eight- celled egg of Pelis is available. On the basis of the former suggestion the sixteen-celled stage would comprise two central blastomeres belonging to the third cleavage generation, ten blastomeres of the fourth generation, and four of the fifth generation. On the alternative suggestion, the sixteen blastomeres would all be of the same cleavage generation, viz. the fourth. 552 J. P. HILL AND MARGARET TRIBE

We conclude that the parent formative cell from which the central "cells and eventually the inner cell-mass are derived, is one particular blastomere either of the four-celled or eight- celled stage, i. e. it belongs either to the second or to the third cleavage generation. Such a ' precocious segregation ' of the blastomeres into two groups, with determinate destinies, has now been recorded for a number of mammals, for the marsupial, Dasyurus (Hill, 24), where it is effected at the fourth cleavage, for Didelphys (Hartman, 23, Hill, 25), which is the only case definitely known to us amongst the mammals in which the segregation is effected at the first cleavage,1 and by various observers for Monodelphia. Hubrecht (28) has shown that the early morula of Tupaia, composed of about twelve or more cells, consists of a single lightly staining central cell completely enclosed by a single layer of peripheral cells. In the Sheep, Assheton (5) records that in two out of four eight-celled stages, ' one of the segments differs from all the others, from which he concludes that the differentiation occurs at the third generation, whilst in the Pig, as already noted (p. 542), he states that one of the blastomeres in a nine-celled stage ' is far larger than any of the others '. In the case of Erinaceus, Baurneister (7) describes the morula of about sixteen cells as consisting of a peripheral layer enclosing one or several central cells, whilst in Xantharpya, Kohlbrugge (33) figures, but oddly enough does not describe, sections of early morulae in which two categories of cells are evident. In his figs. 5 and 6 a single central cell appears, surrounded in the one case by five and in the other by seven peripheral cells ; in fig. 7 two centrals are visible, surrounded by ten peripheral; and in fig. 8, five centrals surrounded also by ten peripheral. This early separation of the blastomeres into two groups respectively formative or embryonal and non-formative or trophoblastic is a phenomenon which Ave believe with van Beneden will be found to be characteristic of all the Mono- delphia without exception, though the precise time of separation of the two groups probably varies somewhat in different 1 If we accept the observations of van Beneden (8), the same must hold good also for the Rabbit. THE EARLY DEVELOPMENT OF THE CAT 553 forms. And that a process of overgrowth or epiboly actually does occur whereby the formative cell or cells become enclosed by the trophoblastic is, we think, conclusively demonstrated for the Cat by the text-figures of the models of early morulae that we present in this paper. The occurrence of such a process was originally described by E. van Beneden (8) for the Babbit in 1875, and subsequently by van Beneden (10) and Duval (20) for Vespertilio and by Assheton (5) for the Sheep, and it is doubtless also of universal occurrence in the Monodelphia though evidently not always easy to demonstrate, judging from the accounts of E. van Beneden himself and of Assheton. Succeeding these sixteen-celled eggs, we describe four morulae composed of twenty-two, twenty-three, twenty-four, and thirty- one blastomeres respectively, in all of which the epibolic process has been completed, there being present two central cells of unequal size, completely enclosed by a single layer of trophoblastic cells to whose division the increase in the number of the blastomeres is due. Active division of the trophoblast cells proceeds, and at the same time the central cells also begin to divide. R. van der Stricht figures sections of a thirty-celled morula in which already several central cells are present, and we describe two morulae with about fifty-nine and sixty-three blastomeres respectively, the former possessing ten central cells and the latter eight. Continued division of both the central and trophoblastic cells finally leads to the stage of the completed morula represented by our morula 29, in which the space inside the trophoblast is filled by a central group of cells representing the future inner cell-mass or embryonal knot and formed by the division of the two central cells of earlier stages.

3. YOLK-ELIMINATION (Deutoplasmolysis). This phenomenon, the elimination from the fertilized ovum or from the blastomeres during early cleavage stages of surplus deutoplasmic material, was first described by 0. van der Stricht (50) for Vesperugo under the name of ' deuto- plasmolyse ' and independently by one of us (24) for Dasyurus. 554 J. P. HILL AND MARGARET TRIBE

It has now been recorded for a number of other mammals.. Mouse (Lams and Doorme, 35), Cavia (Lams, 36), Dog (0. van der Stricht), Didelphys (Hartman, Hill), Cat (E. van der Stricht). The latter observer states that most of the eggs he examined in stages of cleavage (two-, three-, and many- celled) exhibited very evident signs of deutoplasmolysis, the fragments of deutoplasm, often in process of liquefaction, lying generally in the neighbourhood of the polar body. In several of the cleavage stages we have studied, we have recorded the presence of a granular material, occasionally as in egg 18 containing small spherical masses, around the blastomeres. This material, or some of it at least, is very possibly deutoplasmolytic, but we have not observed it in actual process of elimination, so that it is difficult to judge how much of it represents surplus material, how much coagulum, the result of fixation. The elimination of this surplus material is possibly the means by which the nucleo-cytoplasmic ratio normal for the particular ovum is established, as suggested by G. Levi,1 but the necessity for its occurrence is, we would emphasize, the outcome of the phylogeny of the Therian ovum, i.e. of the fact that it has been derived from such a meroblastic yolk-laden ovum as is found in the existing Monotremes and Eeptiles, and is not to be regarded simply as one of the ' manifestations dynamiques de la fecondation ' as Brachet (16) would seem to suggest, though the act of fertilization may set the process in operation just as it does cleavage.

CHAPTER III.—FORMATION OP THE BLASTOCYST.

1. LATE MORULA AND EARLY BLASTOCYST STAGES FROM THE UTERUS. THE material available for the study of these stages, of especial interest for the elucidation of the mode of formation of the blastocyst, consists of (a) a series of five eggs from the uteri 1 "Studi sulla grandezza delle cellule" III, 'Bicerohe di Biologia dedicate al Prof. A. Lustig'. Firenze, 1914. THE EARLY DEVELOPMENT OF THE CAT 555 of Cat 5.1.10, labelled A-E, three from one uterus, two from the other, and ranging from the solid morula, through morulae in which the blastocyst-cavity is appearing to the early blastocyst, and (b) two uterine eggs from Cat 4.6.19. We have not found it possible to make an accurate count of the blastomeres in these eggs. Morula 29 (5.1.10 E). Diameter, 0-128 x 0-10 mm. Diameter of morula, 008 x0-072 mm. Zona, 0-0096 mm. Pig. 22, PL 27. The morula is ovoidal in section (fig. 22) and is separated by a space from the very thick zona. Such a space occurs in all the eggs of this series, though it is quite narrow in blastocyst 82. Whether it is the result of contraction during fixation or represents a fluid-filled space in the living egg, we are unable to determine. We have drawings of some of the morulae of this series, made with the aid of the camera lucida, whilst they were still in the fixative (picro-nitro-osrnic acid), in which the space is clearly shown, and in this connexion it is worthy of note that Assheton (3) states in the case of the Eabbit that ' up to the moment of the beginning of the [blastocyst] cavity . . . the embryo may often be found to be slightly retracted from the zona radiata in the fresh state ' (p. 131). The morula consists of an outer rather more deeply staining layer of trophoblast, more or less clearly marked off from the somewhat lighter-staining mass of central or embryonal cells. The trophoblast appears as a single layer of large cuboidal to more flattened cells with large nuclei and sparse fat-globules. The central mass fills the space inside the trophoblast and is composed of large polyhedral or polygonal cells, also with large nuclei and containing fat-globules. Between the cells, as also between them and the trophoblast, are well-marked intercellular spaces, some cleft-like, others irregular, no doubt filled with fluid in the living egg. There is already evidence of the commencing degeneration of some of the central cells. In the section following that represented in fig. 22, three such cells with small degenerating nuclei are met with immediately 556 J. P. HILL AND MARGARET TRIBE below the trophoblast. In one the cell-body is indefinite, the nucleus is about half the diameter of that of the normal central cell and has its chromatin massed on one side of the nuclear membrane ; in a second the cell-body is again ill-defined, and the nucleus is still smaller, darkly staining, and pycnotic ; in a third the small cell-body contains a correspondingly reduced bilobed and pycnotic nucleus. One trophoblastic and one central cell are in process of division.

Morula 30 (5.1.10D). Diameter, 0-12 x 010 mm. Diameter of morula, 0-076 x 0069 mm. Zona, 0-012 mm. Figs. 23 and 24, PI. 27. This morula is very similar to the preceding. The central cells, however, are rather more loosely arranged, the intercellular spaces being more marked, whilst there is unmistakable evidence of cytolysis of certain of the cells (fig. 24). On the right in the figure is to be seen a cell with a quite degenerate deeply staining nucleus, whilst towards the bottom left of the figure are two quite degenerate cells with shrivelled deeply staining nuclei. In the section preceding that represented in fig. 24 occurs a small pycnotic nucleus lying free in a space apparently formed by vacuolation of the cell-body. TAVO of the trophoblastic cells are in mitosis. Sparse fat-globules are present in the trophoblast, but they appear to have largely disappeared from the central cells.

Morula 81 (5.1.10C). Diameter, 0-12x0-11 mm. Diameter of morula, 0084x0072 mm. Zona, 0-012 mm. up to 0-014 mm. Fig. 25, PI. 27. This morula generally resembles the preceding two, but on one side, between the trophoblast and the central cells, there is now present a continuous somewhat irregular space extending through six sections and containing remnants of a delicate cytoplasmic reticulum and, in fig. 25 towards the left, a small degenerate nucleus. This space is the beginning of the blastocyst-cavity. We regard it as being formed partly by the THE EARLY DEVELOPMENT OF THE CAT 557 breaking down of certain of the central cells, partly by the flowing together of fluid-filled intercellular spaces. The central cells are separated here and there by irregular intercellular clefts, but on the whole are more compactly arranged than in morula 30. Fat-globules are present both in the trophoblast and the central cells, in the latter in fair numbers. One of the trophoblast cells is in mitosis.

Blastocyst 32 (5.1.10B). Diameter, 0-108x0-105 mm. Diameter of blastocyst, 0-079 x 0075 mm. Zona, 0-012 mm. Pig. 28, PI. 28. In this egg the blastocyst-cavity is represented by two spaces of unequal size which extend up on one side of the blastocyst from the lower pole to just past the equator, the sectional plane being apparently transverse to the polar diameter. The spaces lie between the trophoblast and the mass of central cells (fig. 28), and appear empty except for the presence of traces of cytoplasmic detritus adjoining the central cells and, in the larger, of a very degenerate cell-remnant. The central cells are separated here and there by intercellular spaces and appear similar except that certain of them have more eosinophil cell-bodies than the others. There is no evidence of degeneration in the main body of the central cells. Two central cells and one trophoblastic are in mitosis

Blastocyst 33 (5.1.10 A). Diameter, 012x010 mm. Diameter of blastocyst, 0-08 x 0-067 mm. Zona variable up to 0-012 mm. Fig. 29, PI. 28. The blastocoele is now a continuous cavity situated in the lower hemisphere of the blastocyst and measuring 0024 x 0043 x 0-048 mm. It is bounded^ below and at the sides by the trophoblast and above by the' mass of central cells. The blastocyst stage is thus definitely established. It consists of the investing trophoblast in the form of a single layer of cells, attached to the inner surface of which, and occupying the upper 558 J. P. HILL AND MARGARET TRIBE hemisphere is the embryonal knot or inner cell-mass formed by the central cells of earlier stages. The trophoblastic cells are now rather more flattened in form, especially where they bound the blastocoele. Four of them are in process of division. In one, in addition to the normal nucleus and in contact with it, there is present a quite small accessory nucleus. In the blastocyst-cavity there is present what appears to be a mass of cytoplasm about the size of a central cell but non- nucleated ; and on one side, in contact with the trophoblast, is a vacuolated and much degenerate cell with a pale-staining remnant of the nucleus. Further evidence is thus afforded of the occurrence of degeneration amongst the central cells. The embryonal knot consists for the most part of large cuboidal cells separated here and there and from .the trophoblast by intercellular spaces. They appear similar in their cytological characters except that some few of them tend to have more definitely contoured cell-bodies and stain rather more deeply with eosin than the others. Two such cells are seen at the lower border of the inner cell-mass in fig. 29, and shortly above them is another definitely contoured cell with an eosinophil cell-body. We suggest that possibly these eosinophil cells are entodermal cells, and that the process of segregation or delamination which results eventually in the formation of a connected layer of entoderm has already commenced. A somewhat remarkable feature, the significance of which is not clear, is the occurrence in seven of the central cells of a small accessory nucleus adjacent to the normal nucleus and exhibiting obvious signs of degeneration, such nuclei being small and in most cases deeply staining and pycnotic. A number of cells both trophoblastic and central are in division.

We append here a brief mention of the two uterine eggs from Cat 4.6.19, one of them, A (34), being in the morula stage, the other, B (85), an early blastocyst. THE EARLY DEVELOPMENT OF THE CAT 559

Morula 34. Diameter, Oil x 009 mm. Morula, 0-076 x 0-069 mm. Zona. 0-012 mm. This morula in its stage of development is very similar to morula 31. What we take to be the commencing blastocyst- cavity is an irregular cleft-like space which reaches the trophoblast at one end and extends up obliquely between the central cells to terminate near the equator. Certain of the central cells stain rather more deeply and possess more definitely contoured cell-bodies than the others, and may possibly be the entodermal mother-cells. One such cell is in mitosis. Blastocyst 35. Diameter, 0-11 x 0-10 mm. Blastocyst, 008 x 0076 mm. Zona, 0-012 mm. This blastocyst appears to be slightly in advance of blastocyst 33, inasmuch as its trophoblastic cells are distinctly more flattened and the blastocyst-cavity is more extensive, but the central cells are much less compactly arranged than in 33, and we question whether the specimen is quite normal. We mention it here because it provides unmistakable evidence of the presence of two varieties of central cells, viz. (a) definitely contoured cells, ovoidal or spherical, with relatively small nuclei and voluminous light-staining cell-bodies, often vacuolated and frequently with one or two minute accessory nuclei, and (b) smaller cells of irregular form with relatively large nuclei and less voluminous, more darkly-staining cell-bodies. Variety (a) appears to be more numerous than (b), otherwise we should have been inclined to regard it as entodermal, but since neither corresponds with the presumed entodermal mother-cells in morula 34 and blastocyst 33, and since we are not satisfied that the specimen is quite normal, we do not venture to suggest which of the two is ectodermal and which entodermal in significance.

. 2. FORMATION OF THE DIDERMIC BLASTOCYST. Blastocyst 36 (6 A. 6.5.12). Diameter in fixative, 0-25 mm. ; in section, 0-225 mm. Zona, 0-0036 to 0-0048 mm. Figs. 30 and 32, PI. 28. This blastocyst is, unfortunately, separated by a considerable 560 J. P. HILL AND MAHGAEET TRIBE gap from blastocyst 33. In the interval marked growth has taken place, the diameter of the blastocyst having practically been trebled, whilst the constituent cells of the inner cell-mass have become segregated to form the embryonal ectoderm and the definitive entoderm. The wall of the blastocyst at this stage is trilaminar over the small area at the embryonal pole corresponding to the site of the inner cell-mass and unilaminar over the rest of its extent, where it is formed of the trophoblast alone. In all these later blastocysts, owing to the pressure exerted by the fluid filling the blastocyst-cavity, the blastocyst wall lies closely applied during life to the inner surface of the zona. The trophoblast is now in the form of an attenuated layer of flattened cells which is somewhat thicker immediately around the embryonal primordium than elsewhere, whilst over the latter, where it constitutes the covering trophoblast (Eauber's layer), it is already extremely thin, though still perfectly continuous (figs. 30 and 32, PI. 28). The embryonal primordium appears in section as a lenticular mass (0-096 x 0-099 mm. in diameter) situated at the upper pole, in close contact with the covering trophoblast. Its two constituent parts, the embryonal ectoderm and the entoderm, are now perfectly distinct. The embryonal ectoderm consists of a mass of cells, somewhat loosely arranged, with poorly defined outlines and relatively large nuclei. They contain numbers of small fat-globules. The entoderm is represented by a more or less connected layer of plump fusiform cells, closely investing the under-surface of the embryonal ectoderm and conterminous therewith. The cells possess definitely contoured bodies, and their cytoplasm and nuclei stain rather more deeply than those of the ectoderm, the nuclei being on the average smaller than those of the latter. They contain but few fat-globules. At its periphery the entoderm is in places attached to the trophoblast, but it has not yet begun to spread below the latter (figs. 30 and 32). Over the margin of the embryonal ectodermal mass, the thicker trophoblast surrounding the latter thins out to form the THE EARLY DEVELOPMENT OF THE CAT 561 extremely attenuated covering trophoblast, the nuclei of which are small and flattened (fig. 80, c.tr.). It is worthy of note that the zona is perceptibly thicker over the upper polar region (0-0048 nim.) than over the lower (0-0036 mm.), an indication of greater growth-activity in the latter as compared with the former.

Blastocyst 37 (6B. 6.5.12). Diameter, 0-33x0-32 mm. Diameter embryonal ectoderm, 0-072 x 0069 mm. Fig. 31, PI. 28. This blastocyst differs in some slight details from the preceding and is a little more advanced. The trophoblast over the upper polar region is distinctly thicker than the corresponding area in 36, and is asymmetrical with reference to the embryonal primordium, the latter lying just within its margin. The mass of embryonal ectoderm has now assumed a more rounded form, appearing in fig. 31 as a spherical mass of cells, whilst the nuclei show a tendency to become arranged round the periphery bordered by the entoderm. The cells contain numbers of fair-sized fat-globules and several of them are in mitosis. The entoderm, composed where it underlies the ectodermal mass, of a single layer of fusiform to oblong cells, is now thickened round the periphery of the mass, so as to fill up the angle between it and the trophoblast. The cells are here cuboidal, in places two deep, and some of them are in division (fig. 31). 'Since this thickening directly underlies the trophoblast we may take it that we have here the first step in the peripheral growth of the entoderm to line the blastocyst-cavity. The entoderm contains sparse fat-globules, and such also occur in the thicker trophoblast. The covering trophoblast is thick over the periphery of the ectodermal mass, but becomes markedly attenuated directly over the central part of the latter, its nuclei being small, ovoidal to flattened, and deeply staining. It appears to be continuous right across in the section figured (fig. 31), but in NO. 272 P p 562 J. P. HILL AND MARGARET TRIBE the next section there is a minute surface indentation which possibly marks an interruption in its continuity. In the next two sections continuity of the layer is also doubtful, but in the fourth succeeding that figured it is undoubted. The zona over the lower hemisphere is just half the thickness (0-0024 mm.) of that over the upper.

Bias to cyst 3 8 (14.1.10 D). Diameter, 0-35x0-33 mm. Diameter embryonal ectoderm, 0-072 x 006 x 004 mm. in thickness. Zona over embryonal primordium, 00036 mm., over lower hemisphere, 0-033 mm. Figs. 33 a, 33 b, 33 c, PI. 28. This blastocyst, of slightly greater diameter than the preceding, is distinctly more advanced in development. The mass of embryonal ectoderm is still of a rounded form but is now more flattened on its upper surface, and on this there is present centrally a slight but definite depression, roofed over by the zona alone (fig. 33 a). The depression is limited to the section figured with a trace in the next following. Over it the covering layer has now definitely disappeared, and the peripheral trophoblast has become attached round its margin. It contains traces of a granular material, possibly the degenerate remains of the covering layer. An important advance in the differentiation of the ectodermal mass is now seen in the assumption by its constituent cells of a fairly definite columnar arrangement, the cells being disposed radially to the curved floor of the surface depression, with their nuclei in two rows, a basal and a more central. Some of the cells are in mitosis. We think the surface depression is simply the outcome of this assumption by the ectodermal cells of a columnar arrangement. It has only a transitory existence and is not to be regarded as of the nature of a primitive amniotic cavity. The entoderm invests the convex under-surface of the ectodermal mass as a continuous layer of more or less flattened fusiform cells, but an important advance is seen in the fact that it has now spread out for a short distance (0-072 mm.) THE EAELY DEVELOPMENT OF THE CAT 563

below the trophoblast surrounding the embryonal primordium (figs. 83 a, 33 b, and 33 c). Small fat-globules are sparsely present in the embryonal ectoderm, the entoderm and the trophoblast around the embryonal primordium. The latter portion of the trophoblast in this blastocyst is only slightly thicker than the remainder, and the zona is also more uniform in thickness than in 37.

Blastocyst 39 (18A). Diameter, 0-297 mm. Diameter embryonal ectoderm, 0-072x0-72 mm. This blastocyst, the smallest of three from one cat, generally resembles the preceding. The mass of embryonal ectoderm is plano-convex in form, being more flattened and thinner than in 38, whilst the surface depression is shallower and more extensive. In both this and the preceding blastocyst, curiously enough, the depression is eccentric in position. The peripheral extension of the entoderm is just beginning.

Blastocyst 40 (18 C). Diameter, 0-34x0-32 mm. Diameter embryonal ectoderm, 0-048 x 0062 mm. The embryonal ectodermal mass is exceptionally small and has the form of a concavo-convex disc, its surface depression being deeper and the columnar arrangement of its cells better marked than in 39. The entoderm has extended for a distance of 006 mm. beyond the margin of the embryonal ectoderm. The zona and trophoblast, over an area of the upper hemisphere in which the embryonal primordium is again excentrically situated, are much thicker than over the lower hemisphere.

Blastocyst 41 (14.1.10 C). Diameter, 0-32 mm. Diameter embryonal ectoderm, 0-072 x 0-06 mm. x 0-03 mm. in thickness. This blastocyst essentially resembles 88. The surface depression on the embryonal ectoderm is shallow and confined to two sections. The entoderm is just beginning to extend peripherally. p p 2 564 J. P. HILL AND MARGARET TRIBE

Blastocyst 42 (18B). Diameter, 0-32. mm. Diameter embryonal ectoderm, 0-072 x 0076 mm. x 0-03 mm. thick. Fig. 34, PI. 28. This excellent blastocyst shows a distinct advance on the preceding stages in that the embryonal ectoderm now forms a definite slightly curved plate or embryonal shield, thicker centrally than marginally and composed of columnar cells (fig. 34). It is separated from the zona by a shallow but wide depression (0-048 x 0-004 mm. in diameter), at the periphery of which the trophoblast passes into continuity with the embryonal ectoderm. The entoderm consists of a continuous layer of flattened cells, which tend to be thicker below the margin of the ectodermal plate than centrally. It extends out below the trophoblast for a maximum distance of about 007 mm. The zona and" trophoblast are again thickest over a considerable area of the upper hemisphere, the embryonal primordium being excentric in relation to it. Fat-globules are present in fair abundance in the cells of the ectodermal plate and more scantily in the entoderm and thickened trophoblast.

Blastocyst 43 (11.2.10). Diameter, 0-34 x 0-3 mm. Diameter embryonal ectoderm, 0096 x 0-09 mm. . x 0-026 mm. in thickness. Fig. 35, PI. 29. This blastocyst essentially resembles the preceding but the embryonal shield has increased in area and become slightly thinner, whilst the entoderm, which is composed of a thin layer of flattened cells and appears to be uniform throughout, has now spread peripherally over about the upper third of the extent of the trophoblast. • The zona is, exceptionally, a shade thicker over the lower hemisphere than over the upper, whilst the trophoblast is pretty uniform over its extent. THE EARLY DEVELOPMENT OF THE CAT 565

Blastocyst 44 and 45 (29.4.12, 8 A and B). 3 A. Diameter, 0-52x0-47 mm. Shield-ectoderm, 009 x 0-099 mm. 3B. Diameter, 0-59x0-57 mm. Shield-ectoderm, 0-09 x 0-10 mm. x 0-024 mm. thick. Fig. 37, PI. 29, and Text- fig. 9. • These two blastocysts are very similar, 3 B being the better of the two. The most interesting feature in this stage is the presence of a locally thickened area of entoderm underlying the anterior portion of the embryonal shield, which we identify as the protochordal plate of Hubrecht (27) and which we shall speak of as the prochordal plate. As the result of the differentiation of this plate the embryonal area acquires, as 0. van der Stricht (54) has pointed out for the embryonal area of the Dog's blastocyst, a recognizable bilateral symmetry and also a recognizable antero-posterior axis (coincident with the line of symmetry), since the plate always lies nearer what is the anterior margin of the embryonal shield. In the case of blastocyst 3 B our serial sections appear to be pretty accurately transverse to the antero-posterior axis. The shield-ectoderm forms a plate almost but not quite circular, its antero-posterior diameter being a trifle shorter than its transverse. The upper surface of the plate is practically flat, its under surface convex. It is composed of a single layer of columnar cells, thickest centrally and becoming thinner towards the margins where it becomes continuous with the trophoblast, the junction between the two being perfectly definite (fig. 37). A graphic reconstruction of the prochordal plate in 3 B is shown in Text-fig. 9, from which it will be seen that it takes the form of a transversely oval area, situated excentrically below the central region of the embryonal shield and nearer the anterior than the posterior margin of the same. It begins 0-018 mm. behind the anterior margin of the shield and extends back for a distance of 0048 mm., attaining a maximum width of 0-07 mm. The entoderm forming the plate, has a thickness 566 J. P. HILL AND MARGARET TRIBE of 0007 mm. as compared with 0005 mm. for that underlying the hinder third of the shield. It is composed of small, almost, cubical cells with close-set ovalish nuclei (fig. 37) as described and figured (fig. 30, PI. xxxvii) by Hubrecht (27) for Sorex, and is thus readily distinguishable from the remaining entoderm in which the cells are larger and more flattened, their nuclei consequently being set farther apart. The plate lies below the thicker central part of the shield-ectoderm, and thins out on each side below the marginal region of the same. TEXT-FIG. 9.

Graphic reconstruction of shield-ectoderm and prochordal plate of blastocyst 45. x 500. The entoderm has made further progress in its peripheral extension and now reaches the equator of the vesicle. The trophoblast over the upper hemisphere is still distinctly thicker than that over the lower, the former having a thickness of 0008 mm. the latter of 0-0048 mm. Blastocysts 46 and 47 (1.5.12.4 A and B). 4 A. Diameter, 0-52 mm. Shield-ectoderm, 0-108 x 0-108 mm. 4B. Diameter, 0-56 mm. Shield-ectoderm, 010 x 0-108 mm. These blastocysts differ in no essential respect from the preceding, but the peripheral extension of the entoderm is not so great. A graphic reconstruction of the prochordal plate in 4 A is shown in Text-fig. 10. THE EARLY DEVELOPMENT OF THE CAT 567

It is essentially similar to that of the preceding blastocyst, appearing as a transversely oval area underlying the central region of the embryonal shield but more approximated to the anterior margin of the same. The peripheral embryonal entoderm around the plate is somewhat thicker than the extra- embryonal. TEXT-FIG. 10.

Graphic reconstruction of shield-ectoderm and prochordal plate of blastocyst 46. x 500. Blastocysts 48 and 49 (11.3.10 A and B). Diameter, A, 0-5x0-48 mm. B, 0-6x0-5 mm. Shield- ectoderm, A, 0-11 x 0-12 mm. ; B, 0-12 x 013 mm. The only feature in these blastocysts calling for comment is the position of the prochordal plate. It is now distinctly excentric in position and underlies the anterior two-thirds or thereabouts of the embryonal shield, much as in Text-fig. 11. Peripherally the entoderm has extended to the equator of the vesicle. Blastocyst 50 (29.4.12.3 C). Diameter, 0-59x0-54 mm. Embryonal shield, antero- posterior diameter, 0-12 mm. ; transverse diameter, 0-10 mm. Fig. 18, PI. 26. This vesicle is interesting in possessing a broad equatorial 568 J. P. HILL AND MARGARET TRIBE band of specially thickened trophoblast, suggestive at first sight of the precocious development of the zonary band of thickened placental trophoblast characteristic of much later stages but probably only indicative of the commencement of the general thickening of the trophoblast all over its extent which is already completed in the next older blastocysts. The cells composing the band are cubical, with spherical nuclei, and vary in thickness from 0-007 to 0-009 mm. Over the lower pole the trophoblast cells are flattened and attenuated, whilst round the embryonal shield they are somewhat thicker, but even so are less than half as thick as those of the zonary band. The shield-ectoderm and the underlying prochordal plate are seen in longitudinal section in fig. 18. The plate occupies much the same position as in the preceding blastocyst. It begins shortly behind the anterior margin of the shield-ectoderm and extends back for a distance of 0-08 mm. It is composed of cubical cells with close-set oval or spherical nuclei and attains a thickness of about 0-008 mm. The marginal embryonal entoderm around the plate has a thickness of about 0-005 mm. It continues out a short distance beyond the margin of the shield before passing into the thin extra-embryonal entoderm of the vesicle wall. The latter entoderm now extends a little below the equator. The zona over the upper hemisphere has a thickness of 0-0024 mm., and is slightly thicker than that of the lower hemisphere. Blastocysts 51, 52, and 53 (11.3.15 A, B, C). A. Diameter, 1-25x0-86 mm. Embryonal shield, 0-49 x 0-33 mm. x 0027 mm. in thickness. Zona about 00018 mm. B. Diameter 1-4x1 mm. Embryonal shield, 0-41 x 0-3 mm. C. Diameter 1 x 0-8 mm. Embryonal shield, 0-28 x 0-29 mm. x 0-027 mm. thick. Zona, 0002 mm. . Figs. 36 and 3S. PI. 29. These three blastocysts, the latest of the Cat that we describe, are now bilaminar throughout, the entoderm having spread so as to form a complete lining to the blastocyst-cavity. THE BAELY DEVELOPMENT OF TJIE CAT 569

From the measurements given above it will be seen that in A and B the embryonal shield has increased in size, most markedly along its antero-posterior axis, and so from being approximately circular has assumed an oval form. • It will also be observed that in these two blastocysts one diameter is greater than the other so that they are no longer spherical but ovalish in form, and are thus on the way to assume the citron-shape characteristic of the later blastocysts of both the Cat and Dog. According to Bonnet (14), the long axis of the embryonal area in the Dog lies at right angles to the long axis of the vesicle, but we are unable to say what the relation is in these vesicles. In C, the smallest of the three, growth has also taken place but to a lesser extent and more uniformly, the blastocyst being still approximately spherical and the shield circular. A longitudinal section through the embryonal shield and prochordal plate of C is represented in fig. 38, PI. 29. The embryonal shield appears as a flat plate of fairly uniform thickness, intercalated in the trophoblast and composed of columnar cells, the nuclei of which in the central region are arranged in two irregular rows. A feature of interest in these blastocysts is the general increase which has taken place in the thickness of the trophoblast. It now consists of a uniform layer, 0-009 mm. in thickness, composed of cubical to oblong cells with convexly pro- jecting outer surface (fig. 36, PI. 29). The prochordal plate (Text-fig. 11 and fig. 38) occupies the same position as in the immediately preceding blastocysts. It begins in front below the anterior margin of the shield- ectoderm and extends back below the anterior two-thirds of the same for a distance of 0-19 mm. It has a thickness of 0012 mm. as compared with 0-007 mrn- for the embryonal ectoderm around it, both being thicker than the corresponding parts in blastocyst 50. As in the latter, the embryonal entoderm continues out for a short distance beyond the margins of the shield before passing into the attenuated extra-embryonal entoderm of the vesicle wall (fig. 36). 570 J. P. HILL AND MARGARET TRIBE In his paper on the structure of the blastocyst of the Cat, Schafer (45) described the presence of a thin structureless membrane on the upper surface of the embryonal entoderm which he termed the membrana limitans hypoblastica and regarded as of entodermal origin. We have observed indica- tions of this same membrane in these blastocysts, and consider that it is simply the upper cell-membrane of the entoderm

TEXT-FIG. 11.

Graphic reconstruction of shield-ectoderm and prochordal plate of blastocyst 53. x 250. which has become artificially separated (compare also in this connexion Weysse (57), Assheton (5, p. 353), and Keibel (32)).

We conclude our description of the bilaminar blastocyst with a brief account of an excellently preserved blastocyst of the Dog, for which we are greatly indebted to our friend Dr. J. A. Murray, Director of the Imperial Cancer Research Fund. It differs in certain interesting respects from the comparable stage in the Cat. THE EARLY DEVELOPMENT OF THE CAT 571

Blastocyst 54. Dog. Diameter about 1-75 mm. Em- bryonal shield, 0-27 x 026 mm. Pigs. 89 and 40, PI. 29 ; fig. 19, PI. 26 ; and Text-fig. 12. As in our oldest cat blastocysts, the blastocyst wall is bilaminar over its extent. The trophoblast, composed of large, flattened cells, is, however, extremely attenuated as compared with that of the Cat, whilst the extra-embryonal entoderm is also very thin (fig. 40). The blastocyst of the Dog differs, as is well known, from that of the Cat in the presence externally to the zona of a gelatinous layer produced into villous processes (fig. 19). This layer was first described and figured by Bischoff (13) and later by Bonnet (14), who termed it the ' prochorion ' and showed that it served for the temporary attachment of the blastocyst. He regarded it as formed by a secretion of the uterine glands, but more recently 0. van der Stricht (53) has stated that it is derived from the superficial layer of the zona. The shield-ectoderm is approximately circular and appears as a well-defined flat plate which is somewhat thinner marginally where it becomes continuous with the trophoblast than elsewhere. It is composed of columnar cells, the nuclei of which tend to be arranged in two irregular strata.; some of them are in process of division. The most interesting structure in this blastocyst is undoubtedly the prochordal plate, a graphic reconstruction of which is shown in Text-fig. 12. As in our earlier cat blastocysts, it underlies the central region of the shield-ectoderm but nearer the anterior than the posterior margin of the same. It begins about 0-048 mm. behind the anterior margin of the shield and extends back for a distance of about 0-14 mm. with a width of about 0-10 mm. It differs markedly in its sectional appearance from that of our oldest cat blastocysts since it consists of a somewhat irregular layer of cells of variable form (branching, cubical, or fusiform) and with rounded or ovalish nuclei, smaller than those of the ectoderm. Its under surface presents an even contour but its upper surface is quite irregular, many of the cells being produced into branched and anastomos- 572 J. P. HILL AND MARGARET TRIBE ing processes which terminate on the under surface of the shield-ectoderm (figs. 39 and 19). A number of the cells are in mitosis, the axes of the spindles being at right angles or oblique to the plane of the entoderm (fig. 19), whilst here and there detached cells are met with lying free between the plate and the shield-ectoderm (figs. 89 and 19). These cells have evidently been proliferated from the plate, and we conclude

TEXT-PIG. 12.

Graphic reconstruction of shield-ectoderm and prochordal plate of blastocyst 54 (Dog). x 250. accordingly that the proliferation of mesenchyme from the prochordal plate has already commenced at this relatively early stage. Behind the prochordal plate the embryonal entoderm underlying the hinder portion of the shield is quite thin and inactive-looking (fig. 40, PI. 29). Bonnet (14) apparently failed to recognize any localized thickening of the entoderm in the earliest blastocyst of the Dog he was able to examine (diameter 1-5 x 1-2 mm., embryonal shield 0-16 x 0-08 mm.), and even in blastocysts of his Group III THE EARLY DEVELOPMENT OF THE CAT 573

(with diameters of 1-8-2-5 x 1-5-2 mm., the embryonal shield varying from 0-25x2 mm. to 0-4x0-35 mm.) he bases his orientation of the embryonal area on the fact that the long axis of the latter lies at right angles to the greater diameter of the blastocyst, and states that it is impossible to recognize with certainty the future anterior and posterior ends. It is not until the blastocyst has attained a diameter of 4-5x3 mm. (with an embryonal area 0-65x0-75 mm. in diameter and a primitive streak 0-3 mm. in length) that he describes under the designation of the ' Erganzungsplatte des Urdarmstranges ' a very short somewhat thickened area, of the entoderm which underlies the anterior margin of the embryonal shield and is joined by the cranial end of the head process (' Urdarmstrang '). This area is actively proliferating mesoderm, and is recognized by Bonnet as identical with Hubrecht's protochordal plate. 0. van der Stricht (54), in a preliminary account of his observations on a series of early blastocysts of the Dog, states that in blastocysts 1-1-5 mm. in diameter the flattened entodermal cells ' sooner or later become columnar in the future cephalic half of the embryonal area. In this way the latter acquires its bilateral symmetry.' This thickened area undoubtedly corresponds with the area we have identified as the prochordal plate in the cat blastocyst. His statement (53, p. 494) that it has nothing to do with the formation of mesoderm is probably explained by the fact that he had not studied in detail blastocysts over 1-5 mm. in diameter. E. van Beneden also clearly recognized the existence of this thickened area of entoderm in Vespertilio and at a remarkably early period. In his posthumous paper of 1911 (11), in the description of fig. 56 representing a section through an early blastocyst at the stage when the entoderm is already present in the upper hemisphere and the embryonal ectoderm is still in the form of a solid undifferentiated mass, special attention is called to the fact that below one-half of the ectodermal mass the lecithophoral layer is composed of very flattened cells, whilst below the other half the cells are almost cubical, 574 J. P. HILL AND MARGARET TRIBE

and it is emphasized that this difference in thickness confers on the embryonal region of the blastocyst a bilateral symmetry. The same localized thickening is followed through older blastocysts up to the appearance of the primitive streak and head-process (fig. 68), and it is conclusively demonstrated that the thickening underlies the cranial part of the shield-ectoderm, that the primitive streak differentiates from the more caudal region of the same, underneath which the lecithophoral layer is thin, and that the head-process extends forwards, at first as a free process, as far as the hinder margin of the thickening. Later on, definite continuity is established between the two.

CHAPTEE IV.—DISCUSSION.

1. MODE OF FORMATION OF THE BLASTOCYST. THE blastocyst of the Cat, like that of other Monodelphia, is derived from the completed morula as the result of the appearance at one of its poles of a fluid-filled cavity situated between the central cells destined to form the embryonal knot or inner cell-mass and the peripheral layer (trophoectoderm, trophoblast). The precise mode of origin of this cavity is a matter of some little interest, though but few observers have discussed it in any detail. It is worthy of note (1) that the zona attains its maximum thickness (0012 mm.) in the late morulae and early blastocysts described in the preceding pages, and (2) that the morula shows no appreciable increase in size during the period the blastocyst-cavity is in process of appearing. The progressive increase in thickness of the zona is no doubt simply due to the fact that up till now the egg has undergone no marked increase in diameter, but whether the presence of a very thick zona has any significance in relation to blastocyst- formation is a question not easy to answer, though it is con- ceivable that it tends to inhibit the too rapid diffusion of fluid into the egg when the blastocyst-cavity is in process of formation and, once that cavity has appeared, the too rapid expansion of the blastocyst. THE EARLY DEVELOPMENT OF THE CAT 575

The facts that the morula shows no appreciable increase in size during the formation of the blastocyst-cavity, and that the cells of the embryonal knot are no more compactly arranged after than before the appearance of that cavity, render it clear that no mere flowing together of inter- or intra-cellular spaces or vacuoles is a sufficient explanation of its origin in Pelis. The only other alternative mode of formation is by the cytolysis of certain of the central cells, and of that there is evidence in our eggs 29, 30, 31, 32, and 33. The evidence derivable from study of these eggs leads us to conclude that the blastocyst-cavity in Pelis arises partly by the degeneration of certain of the central cells, partly by the running together of fluid-filled intercellular spaces. Once the cavity has appeared its subsequent enlargement is brought about by the active expansion of the enclosing layer of trophoblast, with accompanying absorption of fluid from the uterine lumen. Growth is at first most marked over the lower hemisphere, since the embryonal knot, closely applied to the thin and inactive covering trophoblast at the upper pole, acts as a hindrance to expansion in this region. This is shown by the fact that in our early enlarging blastocysts, the zona, though very much thinner than in the late morulae, is thickest over the upper hemisphere, and also by the fact that the trophoblast is thicker immediately around the embryonal knot than elsewhere. Kohlbrugge (33), in describing the formation of the blastocyst in the Bat, Xantharpya amplexicaudata, clearly recognized that the first appearance of the blastocyst-cavity in the form of a series of small isolated cavities which subsequently run together is the result of the breaking down of certain of the central cells. He says, ' Es kann dies nur durch Zellenschwund geschehen, da das Ei ja immer noch von der festen Zona umgeben ist, die dabei prall gefiillt ist' (p. 14). His figures 9—12 illustrate the process and show that during the period when the blastocyst-cavity is forming the egg does not increase in size. In the Mouse, Sobotta (47) describes the cavity of the 576 J. P. HILL AND MARGARET TRIBE blastocyst as arising by the confluence of a number of irregular spaces which appear between the cleavage-cells, whilst Melis- sinos (38), also in the Mouse, Huber (26) in the Eat, and Hubrecht (29) in Tarsius, state that it arises as a single intercellular space. In the Eabbit, Assheton (3) figures the first appearance of the blastocyst-cavity in a morula of seventy- seven hours (fig. 21, PI. 15) as a curved intercellular cleft situated between the trophoblast of the lower polar region and the cells of the inner cell-mass. He admits that the question of how the cavity is actually produced is one of very great difficulty, but thinks that ' the first cause which produces the cleft that subsequently enlarges into the cavity of the blastodermic vesicle may be a more active growth of the outer layer of cells '. He adds ' undoubtedly there is after this time a more active growth of the outer layer of cells '. No mention is made of intracellular vacuoles or of cell-degeneration, but in the Pig (4) he is inclined to think that the cavity owes its origin to the running together of such intracellular vacuoles, so also in the Ferret, and ' less clearly so in the Sheep ' (6). E. van Beneden (10) attached great theoretical significance to the precise mode of formation of the blastocyst-cavity, and in his preliminary paper of 1899 on the early development of Vespertilio murinus, he considers the process in some' detail. His conclusions as set forth in that paper may be summarized as follows : (1) Intracellular vacuoles appear in those cells of the inner cell-mass, situated in one hemisphere of the morula, which in subsequent stages come to limit the blastocyst-cavity; (2) the protoplasmic walls separating these vacuoles are then gradually withdrawn into the cells on what will be the convex surface of the mass of embryonal ectoderm, and thus the vacuoles run together to form the single continuous blastocyst-cavity, the process first beginning on the side where the vacuoles adjoin the enveloping layer of trophoblast. The cells in which these vacuoles are formed, by ' un veritable acte de secretion ', give origin to the layer which is by most embryologists termed the THE EARLY DEVELOPMENT OF THE CAT 577 entoderm or hypoblast, but which in van Beneden's view is not the true entoderm, and so he terms it the ' lecithophore ' and includes with it the fluid filling the blastocyst-cavity, since this fluid is a secretory product of the lecithophoral cells. The yolk-entoderm of other Vertebrates may for practical purposes be taken as the equivalent of the ' lecithophore '. In his posthumous paper of 1911 (11), edited by A. Brachet, emphasis is laid on the presence in the inner cell-mass as well as in the blastocyst-cavity of cells in process of degeneration,' de noyaux en chromatolyse dans un protoplasme en voie de degeneres- cence ' (p. 38 ; cf. figs. 89, 44, 45, 46), and it is stated, ' il semble done que la formation de la cavite blastodermique, bien que resultant essentiellement d'un processus d'elaboration et de secretion des cellules, ne s'acheve pas sans que certaines de celles-ci n'y trouvent la mort ' (p. 38). To this mode of formation of the blastocyst-cavity by the flowing together of intracellular vacuoles, van Beneden, on theoretical grounds, attached great importance since he believed it demonstrated the correctness of his view of the homology of the blastocyst-cavity of the mammal with the sub-germinal cavity of the sauropsidan egg, extended by further accumulation of fluid so as to include the whole of the yolk. In the case of both cavities he believed that the fluid filling them resulted from a definite process of intracellular secretion. In the present connexion the essential point in van Beneden's interpretation is that he regards the cells in which the vacuole3 appear as lecithophoral cells, i.e. as cells which in the broad sense are entodermal or, more strictly, yolk-entodermal. In any case the cells in question form the layer which clothes the under surface of the embryonal ectodermal mass in the early blastocyst, and is by the majority of writers termed the entoderm. Now van Beneden's interpretation presupposes a precocious segregation of these cells in one hemisphere of the morula, and of that no evidence is forthcoming from what we know of other mammals. Indeed, such evidence as we possess concerning the origin of the entoderm in the Mammalia is directly opposed to the occurrence of any such early segregation. NO. 272 Q q 578 J. P. HILL AND MARGARET TRIBE (1) The observations of one of us on the origin of the entoderm in the marsupial Dasyurus demonstrate that the unilaminar formative or embryonal region of the blastocyst- wall is composed of two varieties of cells, viz. a more numerous series of larger, lighter-staining cells, destined to form the embryonal ectoderm, and a less numerous series of smaller, more deeply staining cells which eventually migrate inwards to form the entodermal lining of the vesicle. (2) In Didelphys. Hartman (23) has obtained entirely comparable results. (3) The observations of Patterson (42) on the formation of the entoderm in Tatusia show that the inner cell-mass of the blastocyst likewise consists of two types of cell, viz. larger, lighter-staining cells destined to form the embryonal ectoderm and smaller more darkly staining cells, with sharply denned outline which at first are evenly distributed amongst the former and which later migrate down to the deep surface of the inner cell-mass and eventually separate off to form the entoderm. (4) The observations of 0. van der Stricht on the origin of the entoderm in the Dog show that the entodermal cells only make their appearance subsequently to the disappearance of Eauber's layer and the intercalation of the embryonal knot in the trophoblast, i. e. long after the formation of the blastocyst-cavity. Whilst we are prepared to accept van Beneden's description •of the formation of the blastocyst-cavity in Vespertilio by the confluence of intracellular vacuoles, accompanied by the actual breaking down of certain of the central cells, we do not think that his interpretation of the vacuolated cells as exclusively lecithophoral is fully established. At the same time we must confess that we attach little or no phylogenetic importance to the precise way in which the blastocyst-cavity arises in the mammals, since its mode of formation differs in the three subclasses and may very well vary in its details within the limits of the Monodelphia, even though it is an homologous •cavity throughout. THE EARLY DEVELOPMENT OF THE CAT 579

2. HISTORY OF EMBRYONAL KNOT AND COVERING TROPHOBLAST. Our observations show that the embryonal knot furnishes, as in other Monodelphia, the embryonal ectoderm and the entoderm of the vesicle, segregation of the two commencing shortly after the appearance of the blastocyst-cavity and being completed relatively early. Our material is not sufficiently extensive to enable us to trace the origin of the entoderm in detail, but we have produced some evidence indicative of the presence in the embryonal knot of two varieties of cells which we interpret as the parent cells of the embryonal ectoderm and entoderm respectively, and which we regard as entirely comparable with the two types of cell observed by one of us (24) in the formative region of the blastocyst-wall in Dasyurus, by Hartman (23) in that of Didelphys, and by Patterson (42) in the embryonal knot of Tatusia. In these three mammals the entodermal mother-cells reach their final position on the under surface of the layer or mass formed by the segregation of the other variety (embryonal ectoderm) as the result of a process of active migration, and we see no reason to suppose that the same process does not occur in the Cat. Already in our blastocyst 36 (0-25 mm. in diameter) the entoderm appears as a more or less connected layer underlying the mass of embryonal ectoderm, and from there it gradually extends by its own growth to form a complete lining to the blastocyst-cavity. The embryonal ectoderm in the just-mentioned blastocyst has the form of a lenticular mass of cells which is in the closest apposition with the already very attenuated covering trophoblast (Eauber's layer). It soon assumes a more rounded form and at the same time its cells undergo rearrangement and take on a columnar form. This results in the formation of a slight depression on its surface and the disappearance of the covering layer in the region of the depression, the trophoblast now becoming connected with the embryonal ectoderm round the margin of the same. In this way the embryonal ectoderm becomes intercalated in the trophoblast and so exposed at the 580 J. p. HILL AND MARGARET TRIBE surface. As the vesicle grows, so the embryonal ectoderm expands to form an approximately circular disc, the shield- ectoderm or ectoderm, of the embryonal area, composed of a layer of columnar cells and in continuity marginally with the trophoblast. 0. van der Stricht's observations (53, 54) show that in the Dog the sequence of events in the region of the embryonal knot is somewhat different from what we describe in this paper for the Cat. According to his account, in blastocysts 0-2 to 0-3 mm. in diameter, the embryonal knot is still undifferentiated, and appears as a convex mass attached to the inner surface of the trophoblast. As the result of the increasing pressure of the fluid filling the blastocyst-cavity, the knot gradually becomes flattened out, and in vesicles 0-3 to 0-4 mm. in diameter it ' compresses and gradually repels laterally, outside the embryonal pole, the overlying cells of Eauber's layer. Even- tually the knot is intercalated within the unilaminar layer of the blastocyst which is now everywhere monodermic. This stage recalls that of Marsupials ' (J. P. Hill). In other words, in the Dog it is the embryonal knot itself, according to 0. van der Stricht, which expands and becomes intercalated in the trophoblast, whereas in the Cat the knot first undergoes differentiation into its two constituent parts and then the embryonal ectoderm, without preliminary expansion, becomes intercalated in the trophoblast, Rauber's layer at the same time disappearing. According to O. van der Stricht, it is not until the blastocyst has reached a diameter of 0-7 to 0-8 mm., the embryonal knot having meantime become more flattened and thinner, that' a few small cells seem to be forced or compressed towards its inner surface, next the blastocyst-cavity ; these give rise to the lecithophore or hypoblast which is now virtually laid down '. O. van der Stricht has made the very interesting observation that the entoderm cells which first appear are capable of passing back into the knot if the internal pressure be reduced by accidental puncturing of the wall of the living blastocyst, a valuable confirmation of the migratory powers of the entoderm to which attention was first called by one of THE EARLY DEVELOPMENT OF THE CAT 581 us, in the case of the entoderm mother-cells in Dasyurus. In blastocysts 1 to 1-5 mm. in diameter, van der Stricht states that the entoderm forms a continuous layer below the embryonal ectoderm and has also extended out to line the trophoblast, the vesicle-wall eventually becoming didermic throughout. The relatively late appearance of the entoderm in the Dog is a noteworthy difference from the Cat. Much more important, however, is the fundamental agreement in the development of these two Carnivores, which is to be found in the fact that the embryonal ectoderm becomes freely exposed at the surface and intercalated in the trophoblast as the result of the thinning out and eventual disappearance of Eauber's layer, no closed ecto-trophoblastic or (so-called) primitive amniotic cavity being formed. From the descriptive part of this paper it seems clear that the thinning and eventual rupture of Rauber's layer is not directly due to the expansion of the embryonal ectoderm to form the shield-ectoderm, as is apparently the case, e.g., in the Rabbit, since this expansion only takes place after the disappearance of the covering layer. If we are justified in ascribing the attenuation of the covering layer to a mechanical cause, then we think an efficient cause is to be found in the close attachment (amounting almost to fusion) of the embryonal ectoderm to the covering layer, which no doubt prevents the growth of this area of the trophoblast during the expansion of the vesicle, whilst the embryonal mass probably also exerts a certain amount of pressure on the covering layer, as 0. van der Stricht suggests, such pressure resulting from the gradually increasing tension of the fluid in the blastocyst-cavity, consequent on the growth of the blastocyst-wall and the imbibition of fluid from the uterine lumen. But the actual rupture of the layer and the concomitant appearance of the slight depression on the surface of the embryonal ectoderm seen in blastocyst 38 (fig. 33 a) are, we think, related phenomena, the result of the commencing assumption by the ectodermal cells of a columnar arrangement. This surface depression, we would emphasize, is a mere transitory formation with, in our opinion, 582 J. P. HILL AND MARGARET TRIBE no significance other than that just indicated, and is in no sense to be regarded as a vestigial representative of the so-called primitive amniotic cavity seen in certain other mammals (Pteropus, Galeopithecus, Tatusia, Cheiroptera, higher Primates, &c). If now we turn for a moment to the consideration of the developmental history of the embryonal knot and its covering layer in the Monodelphia generally, we find that the knot, whilst in all cases giving origin to the embryonal ectoderm (including probably also the amniotic ectoderm) and the entire entoderm of the vesicle, varies considerably in the details of its differentiation, and that the fate of its covering layer is also very variable. During the assumption by the embryonal ectodermal mass of its definitive epithelial form, the covering layer may, as in the Cat, disappear, with the result that the shield- ectoderm becomes intercalated in the trophoblast and exposed at the surface or it may persist either temporarily or per- manently, in which case a cavity arises either between it and the shield-ectoderm or actually in the embryonal ectoderm itself. The cavity so arising may have merely a temporary existence or, on the other hand, it may persist to form the definitive amniotic cavity. The variations just indicated may be grouped as follows : Group I.—The shield-ectoderm becomes intercalated in the trophoblast and exposed at the surface as the result of the thinning out and disappearance of the covering trophoblast. No closed cavity is formed during the differentiation of the shield-ectoderm. The arnnion arises by fold-formation. Lepus, Felis, Cam's, Ovis, Sorex. Group II.—During the differentiation of the shield- ectoderm a transitory (ecto-trophoblastic) cavity appears between the latter and the covering trophoblast, the shield- ectoderm forming a more or less curved or even Y-shaped plate. When the shield-ectoderm flattens out, the covering trophoblast is ruptured, and the ectoderm at the same time becomes exposed and intercalated in the trophoblast as in Group I. The amnion develops by fold-formation. THE EARLY DEVELOPMENT OF THE CAT 583

Tupaia, Talpa, Tarsius, Sus. In Cervus (Keibel, 32) a temporary cavity generally appears in the embryonal ectodermal mass itself, though sometimes the covering layer ruptures before the appearance of such a cavity as in Group I. Group III.—An ecto-trophoblastic cavity arises as in Group II (Vespertilio) or the cavity appears in the embryonal ectodermal mass, its roof soon opening out (Erinaceus) or disappearing (Miniopterus). The shield-ectoderm never becomes exposed and the amnion is formed by fold-formation below the persisting covering layer, the ecto-trophoblastic cavity becoming secondarily enclosed to form the amniotic cavity. Arvicola and Mus spp. may also be included here : a primitive amniotic cavity arises as in Group IV, but it later opens into an ectoplacental cavity situated in the ectoplacental trophoblast and the amnion is formed by secondary closure. Group IV.—The covering trophoblast persists and the shield-ectoderm never becomes exposed. A cavity (the primitive amniotic cavity, sensu stricto) arises in the embryonal ectodermal mass, which persists to form the definitive amniotic cavity, its floor being constituted by embryonal ectoderm and its roof by amniotic ectoderm. Mesoderm and coelom penetrate later between the latter and the trophoblast, thus producing amnion on the inside and chorion on the outside. Pteropus, Cavia, Tatusia, Xantharpya, Galeopithecus, higher Primates. The question of amnion-formation in the mammals generally is outside the scope of this paper, but it is so intimately bound up with the history of the embryonal knot and its covering layer that some brief reference to current views is here necessary. For a detailed exposition the reader is referred to the papers of Hubrecht (28, 30, 31), van Beneden (10), Assheton (6), and Da Costa (17, 18). Hubrecht, in his dissertation on the phylogeny of the amnion and the significance of the trophoblast published in 1895, was, we think, the first to draw attention to the occurrence of two types of amnion-formation in the Monodelphia, viz. (a) by the closure of amniotic folds and (b) by the persistence of 584 J. P. HILL AND MARPARET TRIBE a, cavity which appears already completely closed in the blastocyst, to form the definitive amniotic cavity. He formulated the view that the latter type of ' closed amnion-formation ' is the primitive one for the Amniota as a whole, and that the more familiar mode of amnion-formation by the closure of amniotic folds has been secondarily derived from the ' closed ' type as the result of coenogenetic modification (' als einen bedeutend cenogenetisch modificierten Entwickelungsprozess ') (28, p. 24). Hubrecht's views on this question are based on his conception of the trophoblast as of the nature of a larval envelope, the homologue of the ' Deckschicht' of the amphibian embryo, a conception which has met with much adverse criticism from various writers (cf. Hill, 24) and which we do not propose to consider further here, though we acknowledge the value and stimulating character of Hubrecht's contributions to our knowledge of amnion-formation. In 1899 E. van Beneden (10), in his classical paper on the early development of Vespertilio, also supported the view that the ' closed ' method of amnion-formation is more primitive than that by fold-formation, not for the Amniota as a whole, however, but only within the limits of the Monodelphia. This conclusion he reached on quite other grounds than those advanced by Hubrecht, to whose .theoretical views he was, as is well known, strongly opposed. He points out that between the types of development represented by the Primates and the Rodents with inversion on the one hand, and by the Rabbit and Shrew on the other, all transitional stages exist, and he proceeds to inquire which of these modes of development is to be regarded as the more primitive for the Monodelphia. ' II y a de serieuses raisons de penser ', he writes (p. 331), ' que le developpement du Lapin, considere pendant longtemps comme typique pour les Mammi- feres placentaires, est au contraire le terme extreme d'une serie cenogenetique ; que le mode ancestral de developpement des Mammiferes placentaires se rapprochait beaucoup de ce que nous observons chez les Rongeurs a feuillets renverses et que c'est, par alteration progressive de ce processus primitif, THE EAELY DEVELOPMENT OF THE CAT 585 que revolution en est arrivee a s'accomplir cornrne chez le Lapin et les Musaraignes.1 Van Beneden termed the cavity formed either between the embryonal ectoderm and the trophoblast or actually in the mass of embryonal ectoderm itself the primitive amniotic cavity irrespective of whether it per- sisted to form the definitive cavity or not, and on the view that the occurrence of such a cavity is the ancestral condition for the Monodelphia he regarded the temporary cavity as the vestigial representative of the persisting one. Assheton (5, 6) also accepted the view that the closed typo of amnion-formation was the more primitive. He writes (6, p. 267), ' this means that the rabbit, sheep or dog type of amnion formation has been derived from a type like that seen in Cavia, whose ancestors had the sauropsidan mode of amnion formation'. More recently Da Costa (17), in giving an account of the development of the amnion in the Bat, Miniopterus, has reviewed the whole question, and without hesitation declares himself in favour of the views of Hubrecht and van Beneden, but without adding any arguments of weight in its favour. He says (18, p. 327), ' je considere comme primitive l'amnio- genese par creusement du bouton embryonnaire et la formation de plis comme seeondaire. Cette opinion, qui a ete celle de Hubrecht et de van Beneden, me semble inattaquable, du moins au point de vue ontogenetique.' He distinguishes the former type as a ' Schizamnios', the latter as a 'Plectamnios'. Van Beneden bases his view on the following considerations (10, p. 331) : (1) the formation of a cavity in the mass of embryonal ectoderm has been observed in all the orders of Monodelphia, from the Ungulates to the Primates ; (2) the occurrence even in the Babbit of an embryonal knot; (3) Rauber's layer is obviously in the Babbit a vestigial structure, homologous to a part of the ectoplacenta of other mammals, and in the Babbit and the Shrew plays neither a functional nor a phylogenetic role. We must confess we are not impressed by any one of these considerations. As concerns (1) our own observations and those 58G J. P. HILL AND MARGARET TRIBE of 0. van der Stricht show that in two representatives of the order Carnivora, Felis and Canis, no closed cavity appears during the differentiation of the embryonal ectoderm, and in any case we are of opinion that the formation of a closed cavity at this period in the development of the blastocyst is of no phylogenetic significance whatever and is simply a by-product, so to say, of that peculiar adaptive condition, viz. the complete enclosure of the formative cells by the trophoblast which is characteristic of all the Monodelphia. Its presence or absence and its permanent or transitory character are evidently conditioned by a variety of circum- stances, e.g. the relation of the blastocyst to the uterus, the character of the covering layer and the mode and rate of expansion of the embryonal ectoderm, and we are inclined to think that its appearance can be shown to be related to the assumption by the constituent cells of the embryonal ectodermal mass of their definitive epithelial form, a rearrangement which in a number of cases (e. g. Talpa, Vespertilio) is apparently accompanied by the degeneration of some of the ectodermal cells. The case of Vesperugo noctula described by 0. van der Stricht (48, 51) appears to form an exception, since here the ecto-trophoblastic cavity only appears after the embryonal ectoderm has spread out below the covering trophoblast in the form of an epithelial layer, but in an earlier stage, when the embryonal ectoderm is in process of differentiation, van der Stricht states that there are present, between the latter and the already attached placental trophoblast, ' souvent des fentes, des espaces irreguliers, en quelque sorte virtuels, autour desquels sont seriees les cellules epitheliales de l'epiblaste embryonnaire' (51, p. 7). These spaces disappear as the embryonal ectoderm spreads out in close contact with the placental trophoblast, but there can be little doubt they represent the ecto-trophoblastic cavity which here becomes temporarily obliterated owing to some exceptional condition of pressure inside the blastocyst of this particular species. Consideration (2) is discussed by van Beneden at some length, and the view is expressed that ' l'amas interne, massif, THE EARLY DEVELOPMENT OF THE CAT 587 des Mammiferes actuels doit son origine a la substitution d'un processus cenogenetique a une invagination primitive de la tache embryonnaire ' (p. 332). Although it is nowhere expressly so stated, van Beneden would appear to have regarded the so-called primitive amniotic cavity as in some sense a reappear- ance of the hypothetical invagination cavity, and consequently those mammals in which such a cavity arises and persists to form the definitive amniotic cavity are more primitive than those in which the embryonal ectoderm becomes directly exposed and intercalated in the trophoblast as in the Babbit and Cat. One of us (24) has already discussed the significance of the enclosure of the embryonal knot by the trophoblast in the Monodelphia, and we need only say here that whilst we are at one with van Beneden in regarding the internal position of the embryonal cells in the Monodelphia as a purely adaptive phenomenon, we are unable to picture to ourselves an ancestral developmental stage involving an actual invagination of the embryonal area within an enveloping layer. There is no evidence of such a stage in ontogeny, and we think that the Monodelphian condition was attained quite suddenly as a direct advance on such an antecedent condition as is seen in the blastocyst of existing marsupials. As concerns consideration (3) we think the statement that the covering layer plays no functional role either in the Rabbit or Shrew goes too far, and we venture to suggest that it has a functional value in completing the wall of the expanding blastocyst and in providing a place of attachment for the inner cell-mass, but we agree it has no phylogenetic value since it is essentially a structural adaptation evolved within the limits of the Monodelphian mammals. Whilst we are quite prepared to accept the view that the early history of the embryonal knot in the Eabbit is atypical for the Monodelphia so far as concerns its unique mode of expansion, we cannot agree that in respect of the disappearance of the covering layer and the absence of a primitive amniotic cavity the Rabbit and Shrew are less primitive than Mus and 588 J. P. HILL AND MARGARET TRIBE Cavia. Indeed, we would maintain that the type of development seen in these Eodents with inversion and that exhibited by the higher Primates, Pteropus, &c, far from being primitive, represent the culmination of • specialization along separate and independent lines of developmental adaptation. Just as the palaeontological evidence demonstrates parallelism in the evolution of skeletal characters in the most diverse orders of the Mammalia, so we hold the embryological evidence testifies to the existence of parallel and ipso facto independent lines of ontogenetic development. In spite of the weight of authority in favour of the opposing view, we hold fast to the conclusion, which is of course not new, indeed may be regarded as old-fashioned, that the type of development seen in our Group I is to be regarded as the most primitive for the Monodelphia. That view appears to us to be supported by the following considerations : (1) it is the type of development which most closely approximates to the conditions obtaining amongst the lower Mammalia, Canis, as 0. van der Stricht has shown, actually realizing, after the disappearance of Eauber's layer, the marsupial condition where the formative (embryonal) cells are from the first spread out at the surface to form the upper hemisphere of the blastocyst. (2) It is the simplest and most direct type of development, the embryonal ectoderm in attaining its definitive form and its ancestral surface-position simply bringing about the degeneration, with resulting complete disappearance of Eauber's layer. (3) The development of the amnion by fold- formation is clearly the ancestral method for the Monodelphia, seeing it is the sole method met with in the Sauropsida, the Monotremes, and the Marsupials. The 'closed' method of amnion-formation as in our Group IV, as well as that by fold- formation below the persisting Eauber's layer as in Group III are, in our opinion, secondary specializations which have been acquired by various Monodelphia in adaptation to the conditions of intra-uterine development, and we would lay particular emphasis on the fact that in all such cases the blastocyst either becomes directly attached to the uterine lining as the THE EARLY DEVELOPMENT OF THE CAT 589 result of the proliferative activity of the trophoblast over at least the embryonal pole (i. e. over an area including the covering trophoblast), or is actually imbedded or in process of becoming imbedded in that lining. AVe know of no recorded instance of closed amnion-formation in a blastocyst which reaches the embryonal shield stage whilst still free in the uterine lumen. Supporters of the primitiveness of the ' closed ' amnion would read the series from Group IV to I. We prefer to start with Group I and to regard the others as independent lines of specialization.

3. PROCHORDAL PLATE. It is outside the scope of this paper to deal at any length with the prochordal plate and in particular to consider in detail its later history, but in view of the fact that its presence has now been demonstrated in embryos of all classes of Verte- brates and of its great intrinsic interest, some brief discussion of its significance may not be out of place. As already indicated, Hubrecht in 1890 was the first to direct special attention to the thickened patch of entoderm which in the blastocyst of the mammal underlies the anterior region of the shield-ectoderm, and with which the anterior extremity of the head-process later on makes connexion, and to apply to it the name of protochordal plate. In his paper on the development of the germinal layers of Sorex, Hubrecht (27) states that in blastocysts 0-8 to 1 mm. in diameter, in which the shield-ectoderm is differentiated and the hypoblast completely lines the blastocyst-cavity, he invariably finds that the hypoblast is thickened just below the anterior margin of the embryonic shield over an area comprising some five or six dozen cells. The cells over this area are thicker and more massive and the nuclei are much more closely packed (fig. 30, PI. xxxvii) than over the remainder of the hypoblast. He states further (p. 508), ' this patch of modified hypoblast-cells has at the beginning an oval shape, with the long axis perpendicular to that of the embryonic shield. Part of this patch will develop into the anterior portion of the notochord ; for this reason 590 J. P. HILL AND MABGARET TRIBE

I will call it the protochordal plate.' Hubrecht reserved for a future publication fuller consideration of the history of this plate including its participation in the formation of the wall of the fore-gut and pharyngeal membrane as well as in that of the heart, and though, unfortunately, he never carried that intention into full effect, he deals in some detail with the protochordal plate in his papers on Tarsius (29) and on the early ontogenetic phenomena in mammals (30). It is worthy of note, however, that in none of his papers did he ever produce any definite evidence for his statement that it gives origin to the anterior extremity of the chorda. He was apparently still of that opinion when he wrote his Tarsius paper (v. p. 79), though it is clear, as Assheton (6) and Brachet (15, p. 518) have pointed out, that it is incompatible with the theoretical views he expounds in the same paper, according to which the chorda is restricted to the notogenetic region of the body of the embryo (chordal region of head and the trunk), whilst the cephalogenetic region comprising the most anterior part of the head is prechordal. It is to this prechordal region that the protochordal plate belongs, and accordingly it should not, a priori, produce chorda. Hubrecht's views on the significance of the plate are referred to later. In 1901 Bonnet (14 a) gave a detailed account of the protochordal plate in the Dog, terming it, as already noted, the ' Erganzungsplatte des Urdarmstranges ' because he believed it produced the same derivatives as the ' Urdarm ', viz. mesoderm, chorda, and gut. Already in 1889 he had concluded from his observations on the Sheep that the anterior extremity of the chorda is derived from a ' Chorda-entoblast' area in the roof of the fore-gut and he re-affirms this conclusion for the Dog, but his evidence, we think, is insufficient to settle the question finally. He points out that in embryos with sixteen pairs of somites the anterior portion of the chorda reaches close up to the dorsal insertion of the primitive pharyngeal membrane (oral plate), and actually terminates in what he regards as the last remnant of the ' Erganzungsplatte ', viz. the ' Praoral- Entodermtasche' (Seessel's pocket). It thus extends over THE EARLY DEVELOPMENT OF THE CAT 591 the area occupied in earlier stages by the ' Erganzungsplatte ', and he accordingly concludes that this anterior part of the chorda is formed from the latter plate pari passu with its reduction from behind forwards. It is, however, possible that the anterior portion in question is formed simply by the growth of the chorda itself, its original connexion with the plate or a derivative of the same being maintained during the process of elongation, and until that possibility is definitely excluded we must hold Bonnet's contention unproven. Apart from this question Bonnet's observations clearly demonstrate that the plate gives origin, by proliferation, to cephalic mesoderm, that it forms the upper part, at all events, of the entoderm of the oral plate, and that it furnishes the wall of the pre-oral gut- rudiment (Seessel's pocket) which in the Dog is small and inconstant. Under the name ' Praoral-Entodermtasche ' Bonnet includes, besides Seessel's pocket, a median mass of cells (clearly shown in his fig. 67 representing a frontal section through the head of an embryo of sixteen somites) which he regards as forming part of the wall of the latter pocket. In this mass the chorda terminates, and from it grow out laterally two solid cellular buds which Bonnet identifies as the vestiges of the premandibular head-cavities of other Vertebrates. This mass is clearly the homologue of the median plate of cells which Oppel in 1890 described under the name of ' Praechordalplatte ' in Anguis and from which he showed well-developed premandibular cavities take their origin, and it is also identical in its relations with the irregular plate-like mass of cells described and figured by K. M. Parker (41, fig. 5) in an embryo of Perameles nasuta under the name of the prechordal plate. This mass is in continuity with the distal extremity of Seessel's pocket, whilst it is directly continuous behind with the chorda. Dr. Parker shows that the mass in question is the remnant of the more extensive prochordal plate of earlier stages, from which the primordia of the premandibular head-cavities take origin as solid lateral outgrowths (see especially her figs. 1 and 2), quite similar to the buds described and figured by 592 J. P. HILL AND MARGARET TRIBE Bonnet, though it is a significant fact that they appear in a relatively earlier developmental stage in Perameles than in the Dog. The fact that the premandibular cavities are related to or take their origin from a median mass of cells (very variously designated by different observers) which forms part of, or is connected with, the anterodorsal wall of the fore-gut and with which the chorda is fused, has been recorded by a large number of observers, including van Wijhe 1882, Hoffmann 1886, Oppel 1890 (' Praechordalplatte'), Platt 1891, Rex 1897 (' inter- epitheliale Zellmasse'), Chiarugi 1898 (' massa cellulare mediana '), Corning 1899 (' terminate Zellmasse'), Dorello 1900 (' ammasso cellulare ' or ' endodermale '), Salvi 1903, 1905 (' massa entodermica preorale ' or ' massa cellulare '), Dohrn 1905 (' chorda-entoblast'), Filatoff 1907 (' Zwischenplatte der ersten Somiten '), K. M. Parker 1917, and Adelmann 1922 (prechordal plate). The relationship of the premandibular somites to such a median cell-mass has thus been widely recognized, but Bonnet has the merit of having been the first to show that this mass is a derivative of the protochordal plate. Dr. K. M. Parker (41), in the work on early marsupial embryos already referred to, reached the same conclusion, and has given excellent figures of the mass in embryos of Perameles and Dasyurus which, following Oppel, she terms the prechordal plate. Dr. Parker's results are in general agreement with those of Bonnet except that she obtained no evidence in support of the contention that the protochordal plate furnishes chorda. In particular she has been able to demonstrate that the premandibular somites, which in a number of marsupials are quite well developed (E. A. Fraser, 21), ' arise (in Perameles) from a prechordal plate which represents a derivative of the anterodorsal wall of the fore-gut, which is formed from the protochordal plate '. In Chrysemys, Brachet (15) recognizes the protochordal, ' ou plus exactement prechordale ', plate as the narrow band of yolk-entoderm (' endoblaste vitellin ') below the anterior margin of the shield-ectoderm. When the anterior extremity THE EARLY DEVELOPMENT OF THE CAT 593

of the embryo becomes delimited, part of it, he states, becomes extra-embryonal, the remainder furnishes the end-wall of the fore-gut as well as part of its floor. It does not take part in the formation of the chorda : at the most it plays a role in the constitution of the premandibular mesoderm. The most recent contribution to our knowledge of the prochordal plate is the interesting paper of Adelmann (1). He has failed, however, to realize that the diffuse cellular mass in which the chorda terminates anteriorly, and which he terms the prechordal plate, is not the protochordal plate of Hubrecht but only a secondary derivative of it. He also is convinced like Brachet that neither in Squalus nor in the chick does the prechordal plate contribute to the chorda, whilst with reference to Squalus he reaches the very interesting conclusion that the portion of the prechordal mesoderm immediately anterior to the chorda gives origin not only to the premandibular but also to the anterior head-cavities, whilst its lateral and posterior portions give rise to the anterior parts of the mandibular cavities. A firm believer in the so-called ' blastopore ' theory, he concludes (p. 88) that the prechordal plate represents pre- . axial mesoderm ' which is formed at the primitive dorsal or cranial lip of the blastopore '. We think, however, that the author is on surer ground when he says (p. 68), ' accepting Hubrecht's definition [of gastrulation], it follows that the material for the prechordal plate is laid down early in development during the process of gastrulation '. In his paper on the early developmental stages of Manis, van Oordt (39) discusses at some length the question whether or not the prochordal plate contributes to the formation of the anterior end of the chorda, and though he thinks that his own observations ' certainly are a support for the view of Bonnet', he considers ' it is not proved that the anterior part of the notochord arises from it' (the protochordal plate), and for that reason he employs the designation ' prochordal plate ' instead of protochordal plate to indicate the area of the entoderm in question. We follow van Oordt in this usage, since the designation NO. 272 E r 594 J. P. HILL AND MARGARET TRIBE prochordal plate simply signifies a particular area of entoderm which is situated in front of the primordium of the chorda (the head-process), and which precedes it in time and does not imply, as does the term protochordal plate, that it has a necessary genetic relationship to the chorda. Our present knowledge of the prochordal plate may be summarized as follows. The prochordal plate is a localized area of entoderm which early becomes recognizable in that part of the embryonal primordium which is destined to form the anterior region of the head of the vertebrate embryo, and with which the primordium of the chorda early becomes continuous. It gives origin to the following: (a) cephalic mesenchyme, (b) part at least of the lining of the fore-gut, in particular the whole or at least part of the entoderm of the oral plate (primitive pharyngeal membrane), (c) Seessel's pocket (preoral gut-rudiment), (d) a median mass or plate of mesoderm (the so-called prechordal plate of certain authors) with which the chorda remains for a time in continuity and from which the premandibular somites take their origin. Whether or not the plate participates, either directly or by way of the prechordal mass of mesoderm, in the formation of the anterior extremity of the chorda is a question not yet conclusively settled, though the balance of opinion appears to be against any such participation, whilst the contention of Hubrecht that it furnishes the pericardial coelom and theheart- endothelium is not in accord with what we know of the origin of these structures in other mammals. Occurring as it does in the embryos of all Vertebrates, from the Pishes to the Mammals, it is clear this prochordal plate represents a very ancient heritage of the Vertebrates, though, ap Hubrecht complained years ago, students of vertebrate mor- phology have largely failed to take account of its existence. If now, finally, we proceed to inquire what is the phylogenetic significance of this remarkable patch of entoderm, we think the most fertile suggestion is that put forward in certain papers of Hubrecht. Towards the end of his monograph on cleavage and germ-layer formation in Tarsius (29, p. 80), THE EARLY DEVELOPMENT OF THE CAT 595

Hubrecht, in criticizing Bonnet's substitution of the designation ' Erganzungsplatte ' for his own term, protochordal plate, makes the following statement, without further explanation or comment: ' es sich hier gar nicht um eine " Erganzung " handelt desjenigen was mehr nach hinten in der Region der Notogenesis Vorgeht, sondern umgekehrt um das primordiale, das altere Entoderm, welches direct aus dem zwei- blattrigen Gastrulastadium heriiber geliefert wurde ! ' (spaced type ours). Six years later, in his paper on the early ontogenetic phenomena in mammals (30, p. 63), he returns to the same idea, and in reproducing a figure of Legros (PI. Y, fig. 120) illustrating a longitudinal section of a late gastrular or rather post- gastrular stage of Amphioxus in which blastopore-closure is well advanced, he writes : ' In it we see the region marked pp. singled out by Legros as part of the original entoderm of the wide-mouthed gastrula, which in Amphioxus is formed—in contradistinction to all other Vertebrates—by invagination and not by delamination '. The region which Hubrecht here designates by the letters pp, signifying protochordal plate, is part of the so-called primary entoderm which is formed by the invagination of the primitive entodermal plate of the flattened or bun-shaped blastula, and which in the post-gastrular stage constitutes the antero-ventral wall of the archenteron, as contrasted with the remainder of the entoderm, the secondary entoderm so-called, which is laid down during the closure of the blastopore, as the result of the growth-activity of the blasto- poric lip. As is well known through the work of Lwoff, Legros, Cerfontaine, MacBride, and others, the primary entoderm is distinguishable from the secondary not only by its position but by the fact that it is composed, at all events at first, of larger cells, richer in yolk-granules. Now Hubrecht's view is that the protochordal plate in the Craniata is the representative of the invaginated or primary entoderm of Amphioxus, and although the germ of this interpretation occurred to him so far back as 1902 it is a remarkable fact that he not only took no pains to substantiate it but so R r 2 596 J. P. HILL AND MARGARET TRIBE failed to lay emphasis on it, even in his paper of 1908, that it would appear to have escaped the notice of all subsequent workers in this field. As a matter of fact, we oursslves only came across the paragraphs we have quoted above after we had written the first draft of this section, and indeed with some surprise, since one of us has for years past advocated just this very view in his lectures and had looked upon it as Jtn original

On this question we are glad to find ourselves ir. complete agreement with our late friend, the illustrious Dutch embryo- logist. With him, we believe that the prochordal plate of the Craniate embryo is the representative of that ps,rt of the primary entoderm of the Amphioxus gastrula which bounds the anterior end of the archenteron. It is from this region of the archenteron that tie anterior eoelomic sacs of Amphioxus take their origin, and if we accept the homology of these eoelomic sacs with the premandibular head-cavities of the Craniata, as maintained by Goodrich (22), then it necessarily follows that the entodermal areas from which they arise are also homologous. The prochordal plate is thus an integral constituent of that most anterior region of the head which forms noi; only the extreme anterior end of the embryo but also its phylo genetically oldest part, and which has been termed by Hubrecht the cephalogenetic, and by Brachet the acrogenetic, region of the embryonic body. The prochordal plate is accordingly a structure which carries us right back to the dawn of vertebrate evolution. LIST OF EBFERBNCES. 1. Adelmann, H. B.—" The Significance of the Prechordal Plate: an Interpretative Study ", ' Amer. Journ. Anat.', vol. 31, 1922. 2. Ancel, P., et Bouin, P.—" Sur la fonction du corps jaune ' , ' C. R. Soc. Biol.', torn. 68, 1909. 3. Assheton, R.—" A Re-investigation into the early Stages of the Development of the Rabbit", ' Quart. Journ. Micr. Sci.', vol. 37, 1894. 4. "The Development of the Pig during the First Ten Days", ibid., vol. 41, 1899. THE EARLY DEVELOPMENT OF THE CAT 597

5. " The Segmentation of the Ovum of the Sheep, &c", ibid. 6. " Professor Hubreoht's Paper on the Early Ontogenetic Pheno- mena in Mammals ", &c, ibid., vol. 54, 1910. 7. Baumeister, T.—-"Die Entwicklungsvorgange am Keime des Igels, Erinaceus europaeus, L.", etc., 'Zeitschr. wiss. Zool.', Bd. 105, 1913. 8. van Beneden, E.—"La maturation de l'ceuf, la f^condation et les premieres phases de developpement embryonnaire des Mammiferes, d'apres des recherches faites chez le Lapin ", ' Bull, de l'Acad. des Sciences de Belgique', 1875. 9. et Julin, C.—"Observations sur la maturation, etc., de l'ceuf chez les Cheiropteres ", ' Arch, de Biol.', torn. 1, 1880. 10. "Recherches sur les premiers stades de developpement du Murin (Vespertilio murinus) ", ' Anat. Anz.', Bd. 16, 1899. 11. "Recherches sur l'embryologie des Mammiferes. I. De la segmentation, de la formation de la cavite blastodermique et do l'embryon didermique chez le Murin ", ' Arch, de Biol.', torn. 26, 1911. 12. Bischoff, T. L. W.—' Entwicklungsgeschichte des Kaninchen-Eies ', Braunschweig, 1842. 13. ' Entwicklungsgeschichte des Hunde-Eies', Braunschweig, 1845. 14. Bonnet, B..—" Beitrage zur Embryologie des Hundes ", ' Anat. Hefte ', 1. Abt., Heft 28/30, Bd. 9, 1897. 14 a. " Erste Fortsetzung ", ibid., Heft 51, Bd. 16, 1901. • 15. Brachet, A.—" Recherches sur l'embryologie des Reptiles. Acrogenese, Cephalogenese et Cormogenese chez Chrysemys marginata", ' Arch, de Biol.', torn. 29, 1914. 16. ' Traite d'Embryologie des Vertebres ', Paris, 1921. 17. Da Costa, A. C.—" Sur la formation de l'amnios chez les Cheiropteres (Miniopterus schreibersii) et, en general, chez les Mammiferes ", ' Mem. Soc. Portug. des Sci. Nat., Ser. biol.', no. 3, 1920. Cf. also ' C. R. Soc. Biol.', torn. 82, pp. 588 and 604, 1919. 18. " Sur les conditions de la formation de 1'amnios chez les Mammi- feres ", ' C. R. Soc. Biol.', torn. 86, p. 327, 1922. 19. Corner, G. W.-—"Internal migration of the Ovum", 'Johns Hopkins Hosp. Bull.', vol. 32, 1921. 20. Duval, M.—"Etudes sur l'embryologie des Cheiropteres", ' Journ. Anat. et Physiol.', torn. 31, 1895. 21. Fraser, E. A.—" The Head Cavities and Development of the Eye Muscles in Trichosurus vulpecula; with notes on some other Marsupials ", ' Proc. Zool. Soc, Lond.', 1915. 22. Goodrich, E. S.—" Proboscis Pores in Craniate Vertebrates, a suggestion concerning the Premandibular Somites and Hypophysis", ' Quart. Journ. Micr. Sci.', vol. 62, 1917. 598 J. P. HILL AND MARGARET TRIBE 23. Hartman, C. G.—" Studies in the development of the Opossum, Didelphysvirginiana, L.", ' Journ. Morph.', vol. 32, 1(119. 24. Hill, J. P.—" The early development of the Marsupialia, with special reference to the native cat (Daayurus viverrinus) ", ' Quart. Journ. Micr. Sci.', vol. 56, 1910. 25. " Some observations on the early development of Didelphys aurita", ' Quart. Journ. Micr. Sci.', vol. 63, 1918. 26. Huber, G. C.—" The Development of the Albino Rat, Mus norwegicus albinua ", ' Mem. Wistar Inst.', no. 5, 1915. 27. Hubrecht, A. A. W.—" Studies in Mammalian Embryology. II. The Development of the Germinal Layers of Sorex vulgaris ", ' Quart. Journ. Micr. Sci.', vol. 31, 1890. 28. " Die Phylogenese des Amnions und die Bedeutung des Tropho- blastes", ' Verhand. Kon. Akad. v. Wetensch A nsterdam', Dl. 4, 1895. 29. " Furchung u. Keimblattbildung bei Tarsius spectrum ", ' Ver- hand. Kon. Akad. v. Wetensch. Amsterdam', Dl. 8, 1902. 30. " Early Ontogenetio Phenomena in Mammals and their bearing on our Interpretation of the Phylogeny of the Vertebrates ",' Quart. Journ. Micr. Sci.', vol. 53, 1909. 31. " Friihe Entwicklungsstadien des Igels (Brinaceuseuropaeus, L.) u. ihre Bedeutung fur die Vorgeschiohte (Phylogenese) des Am- nions ", ' Zool. Jahrb.', Suppl.-Bd. 15, ' Spengel-Festschrift', Bd. 2, 1912. 32. Keibel, F.—" Die Entwickelung des Behes bis zur Anlage des Meso- • blastes ", ' Arch. Anat. u. Physiol., Anat. Abt.', 1902. 33. Kohlbrugge, J. H. F.—." Befruchtung u. Keimbildung bei der Fleder- maus ' Xantharpya amplexicaudata ' ", ' Verhand. Kon. Akad. v. Wetensch. Amsterdam ', Dl. 17, 1913. 34. Kunsemuller, M.—" Die Eifurchung des Igels (Erinaceus europaeus, L.) ", ' Zeitschr. wiss. Zool.', Bd. 85, 1906. 35. Lams, H., et Doorme, J.—" Nouvelles recherches sur la maturation et la fecondation de l'oeuf des Mammiferes ", ' Arch. Biol.', torn. 23, 1907. 36. Lams, H.—" fitude de l'ceuf de cobaye aux premier;; stades de l'embryogenese ", ibid., torn. 28, 1913. 37. Longley, W. H.—"The maturation of the egg and ovulation in the domestic cat", ' Amer. Journ. Anat.', vol. 12, 1911-1'!. 38. Melissinos, K.—" Die Entwicklung des Eies der Mause, etc.", ' Arch. Mikr. Anat.', Bd. 70, 1907. 39. Van Oordt, G. J.—" Early developmental stages of Manis Javanica, Dosm.", ' Verhand. Kon. Akad. v. Wetensch. Amsterdum ', Dl. 21, 1921. THE EARLY DEVELOPMENT OF THE CAT 599

40. Oppel, A.—" Ueber Vorderkopfsomiten u. die Kopfhohle von Anguis fragilis", ' Arch. Mikr. Anat.', Bd. 36, 1890. 41. Parker, K. M.—"The Development of the Hypophysis cerebri, Preoral Gut and related structures in the Marsupialia ", ' Journ. Anat.1, vol. 51, 1917. 42. Patterson, J. T.—" Polyembryonic development in Tatusia novem- cincta ", ' Journ. Morph.', vol. 24, 1913. 43. Robinson, A.—" Lectures on the early stages of the development of Mammalian ova and on the formation of the placenta in different groups of Mammals ", ' Journ. Anat. and Physiol.', vol. 38, 1903. 44. Busso, A.—" Studien iiber die Bestimmung des weiblichen Ge- schlechts ", Jena, 1909. Cf. ' R. Accad. d. Lincei', vol. 16. 45. Schafer, E. A.—" Description of a Mammalian Ovum in an early condition of Development", ' Proc. Roy. Soc.', vol. 24, 1875-6. 46. Selenka, E.—" Studien iiber die Entwickelungsgeschichte der Tiere ", ' Vergl. Keimesgeschichte der Primaten ', Heft 10. Wiesbaden, 1903. 47. Sobotta, J.—" Die Entwicklung des Eies der Maus, etc.", ' Arch. Mikr. Anat.', Bd. 61, 1903. 48. van der Stricht, 0.—" La fixation de Pceuf de chauve-souris a l'in- terieur de I'ut6rus ", ' Verh. d. Anat. Gesell. in Tubingen ', 1899. 49. " La structure de l'ceuf de chienne et la genese du corps jaune ", ' C. R. l'A8soc. des Anat. a Marseille ', 1908. 50. "La structure de l'ceuf des Mammiferes (chauve-souris, Ves- perugo noctula)", 3e partie, ' M6m. de l'Acad. roy. de Belgique ', 2e serie, torn. 2, 1909. 51. " Sur le m^canisme de la fixation de l'oeuf de chauve-souris (V. noctula) dans l'ut^rus ", ' C. R. l'Assoc. des Anat. a Paris', 1911. 52. "Etude compared des oeufs des Mammiferes, etc.", ' C. R. l'Assoc. d. Anat. a Gand ', 1922, 'Arch, de Biol.', torn. 33, 1923. 53. " L'evolution du blastocyste de chienne ",' C. R. l'Assoc. d. Anat. a Lyon', 1923. 54. —— " The Blastocyst of the Dog ", ' Journ. Anat.', vol. 58, 1923. 55. van der Stricht, R.—" Vitellogenese dans 1'ovule de chatte ", ' Arch. Biol.', torn. 26, 1911. 56. Thomson, A.—" The maturation of the human ovum ", ' Journ. of Anat.', vol. 53, 19]9. 57. Weysse, A. W.—" On the blastodermic vesicle of Sus scrofa ", ' Proc. Amer. Acad. Arts and Sci.', vol. 30, 1894. 58. de Winiwarter, H., et Sainmont, 6.—" Nouvelles recherches sur l'ovogenese et l'organogenese de l'ovaire des mammiferes (chat) ", ' Arch, de Biol.', torn. 24, 1908-9. 600 J. P. HILL AND MARGARET TRIBE

EXPLANATION OF PLATES 24-9. Illustrating Professor J. P. Hill's and Dr. M. Tribe's paper on ' The Early Development of the Oat (F e 1 i s d o m 3 s t i c a) '.

LIST OF COMMON REFERENCE LETTERS. bl.c, blastocyst-cavity; c.e, central cell; c.e. 'corps eni ^matique' ; c.r., corona radiata; c.tr., covering trophoblast; Dp., djutoplasmie pole; e.ect., embryonal ectoderm; e.dm., eliminated deutoplasm; ent., entoderm; f.gl., fat-globules ; i.c.m., inner cell-mass (embryonal knot); m.z., mitochondrial zone; mes., mesenchyme; Pb., polar bady; Pch., ' prochorion ' ; female pronucleus ; Pp., plastic pole ; p.pl., prochordal plate ; rsp., remains of fir.jt cleavage- spindle ; sh.ect., shield-ectoderm ; sp., sperm-heads ; tr., trophoblast; z.p., zona. FELIS DOMESTICA.

PLATE 24. Fig. 1.—Egg 1, section 14/1 ( x 700), showing persistent corsna radiata {c.r.), the zona {z.p.), sperm-heads (sp.), the mitochondrial zone (m.z.), and central region of ovum with fat-globule spaces and the

PLATE 25. Figs. 5 and 6.—Egg 6, sections (x 7C0). Fig. 5 (section 5/1) nearer plastic pole, showing

PLATE 26. Figs. 10 and 11.—Egg 18. Microphotographs (x640) of two adjacent sections, 8 & 9/3. Seven-celled egg. Figs. 12 and 13.—Egg 23.—Microphotographs ( x 640), fig. 12, section 2/2; fig. 13, section 4/2. Twenty-two-celled egg. c.c. 6 and c.c. 12, central cells; c.e., ' corps enigmatique'. Figs. 14, 15, 16.—Egg 24. Microphotographs (x 640) of three adjoining sections 1, 2 & 3/2. Twenty-three-celled egg. c.c. 11 and cc 13, central cells. Fig. 17.—Egg 28. Microphotograph, section 6/2 {x about 650). Fig. 18.—Blastocyst 50. Microphotograph (x just over 400), section 9/7. p.pl., prochordal plate. Fig. 19.—Blastocyst 54 (Dog). Microphotograph (x 383) of section through prochordal plate, showing cells of plate in process of division. pch., ' prochorion ' outside zona {z.p.).

PLATE 27. Fig. 20.—Egg 19. Combination fig. (x 640), section 7 & 8/2. Fig. 21.—Egg 20. Combination fig. (x640), section 4 & 5/3. c.c. I, central cell 12. Fig. 22.—Morula 29. Section 3/4 (x 640). c.c, central cells (inner cell- mass) ; tr., trophoblast. Figs. 23 and 24.—Morula 30, sections 2/2 and 6/2 (x640). i.c.m., inner cell-mass ; tr., trophoblast. Fig. 25.—Morula 31. Section 1/4 (x 640). bl.c, first appearance of blastocyst-cavity ; i.c.m., inner cell-mass ; tr., trophoblast. Fig. 26.—Egg 28. Section 4/2 (x 640). Sixty-three-celled egg. «.c, central cell; tr., trophoblast. Fig. 27.—Egg 25. Section 11/1 ( x 640). Twenty-four-celled egg. c.c, central cell. PLATE 28. Fig. 28.—Blastocyst 32. Section 6/2 (x 640). bl.c, blastocyst-cavity. Fig. 29.—Blastocyst 33. Section 10/3 ( x 640). bl.c, blastocyst-cavity; ent., entoderm cell (?); i.c.m., inner cell-mass ; tr,, trophoblast. Figs. 30 and 32.—Blastocyst 36. Sections 10/4 and 1/5 ( x 400). c.tr., covering trophoblast (Rauher's layer); e.ect., mass of embryonal ectoderm ; ent., entoderm. Fig. 31.—Blastocyst 37. Section 8/2 (x400). dr., covering trophoblast ; e.ect., mass of embryonal ectoderm ; ent., entoderm. Figs. 33 a, 33 6, 33 c—Blastocyst 38, sections 1/2, 16/1, and 2/2 (x 400). In fig. 33 a, which passes through the central region of the embryonal ectoderm (e.ect.), the covering trophoblast has disappeared and a slight depression is present on the surface of the ectodermal mass, the cells of which are now 602 J. P. HILL AND MARGARET TRIBE assuming a columnar arrangement. The covering trophobla3t (c.tr.) is still present over the more peripheral portion of the embryonal ectoderm (figs. 33 6 and 33 c). Kg. 34.—Blastocyst 42. Section 7/2 ( x 420). sh.ect., shield-ectoderm.

PLATE 29. Fig. 35.—Blastocyst 43. Section 4/4 (x 400). Fig. 36.—Blastocyst 52. Section (x550) through bilaminar wall of blastocyst, away from embryonal area, tr., trophoblast; int., extra- embryonal entoderm. Fig. 37.—Blastocyst 45. Section 14/3 ( x 400). p.pl., prochordal plate. Fig. 38.—Blastocyst 53. Section, 7.2.2 (x 300), along the antero- posterior axis of the embryonal area. • p.pl., prochordal plale;- sh.ect., shield-ectoderm; ent., entoderm.- Cards. Fig. 39.—Blastocyst 54 (Dog). SectioD, 12.1.3 (x400), through prochordal plate (p.pl.). mes., mesenchyme cells, proliferated iron, the plate. Fig. 40.—Blastocyst 54 (Dog). Section, 8.2.3 (x 400), passing through hinder region of embryonal area. QucutJoiirri. Mia: Sd, Vol.68,NA\ PL 24.

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