Paper - The early development of the goat (1942)
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Other papers by this author
Amoroso EC, Barclay AE, Franklin KJ, Prichard MM. The bifurcation of the posterior caval channel in the eutherian foetal heart. J Anat. 1942 Apr;76(Pt 3):240-7. PMID 17104894
Amoroso EC, Franklin KJ, Prichard MM. Observations on the cardio-vascular system and lungs of an African elephant foetus. J Anat. 1941 Oct;76(Pt 1):100-11. PMID 17104876
Lawn AM, Chiquoine AD, Amoroso EC. The development of the placenta in the sheep and goat: an electron microscope study. J Anat. 1969 Nov;105(Pt 3):557-78. PubMed PMID: 5387511; PubMed Central PMCID: PMC1232191.
AMOROSO EC, BELL FR, ROSENBERG H. The relationship of the vasomotor and respiratory regions in the medulla oblongata of the sheep. J Physiol. 1954 Oct 28;126(1):86-95. PubMed PMID: 13212731; PubMed Central PMCID: PMC1365643.
AMOROSO EC, BELL FR, ROSENBERG H. The localization of respiratory regions in the ovine rhombencephalon. J Physiol. 1951 Mar;113(1):2p-3p. PubMed PMID: 14825241.
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The Early Development of the Goat (Capra hircus)
By E. C. Amoroso, W. F. B. Griffiths and W. J. Hamilton
From the Department of Histology and Embryology, Royal Veterinary College, London, and the Anatomy Department, St Bartholomew’s Hospital Medical College, London
1 Royal Veterinary College. 2 St Bartholomew’s Hospital Medical College.
Despite the vast amount of literature and study devoted to early mammalian development, there are few available references which deal with the early cleavage stages in the goat. So far as we can ascertain, only two observations have been made on the living goat egg; one by Krause (1837) who measured three ripe ovarian eggs, using a measurement open to doubt, but from which Hartman (1929) estimated the size of the living goat egg to be of the order of 140, a figure which is remarkably close to the average diameter of 145-3 for the ova measured in the present investigation; the other by Amoroso, Griffiths & Hamilton (19396) who described and figured tubal eggs in the one-cell, two-cell and four-cell stages. Assheton’s (1908) youngest of five specimens (copulation age 5 days 20 hr.) had already left the tube and is at a stage in development comparable to the oldest of the uterine eggs we describe. The only other contribution is that of Tsukaguchi (1912), who described several stages of the goat’s blastocyst (age not stated), the earliest of which corresponds closely to Assheton’s specimen of the sixth day. Like Assheton, Tsukaguchi does not describe or figure the segmenting stages. The present contribution attempts to complete the lacunae in our knowledge in both these respects.
Material and Methods
The material for that part of the investigation which is reported in this paper was obtained from a group of fifteen females, during thé breeding seasons from September 1937 to January 1940. Since the mutual behaviour of the nanny and billy alone constitutes a reliable indication of oestrus, the most: satisfactory criterion for its detection was the introduction of the male among the females at frequent intervals (from 4 to 6 hr.), and observing the willingness of the females to associate with the males. When a particular female was found to associate freely with the male she was removed from the flock and suitable precautions were taken to prevent mating until it was decided to breed from her. Oestrus was dated from the time the female would ‘stand’ and allow the male to mount to the time of last acceptance of the male. Since ovulation is said to occur towards the end of heat, the animals were bred as late in heat as was possible and killed at the times necessary for recovering eggs of the desired ages. ,
In securing the eggs the methods used were essentially those devised by Corner (1917) for obtaining pig eggs and adequately described by Clark (1934) for the sheep. The method employed is satisfactory, since in the great majority of cases the number of eggs recovered always corresponded with the number of freshly ruptured follicles. After recovery most of the eggs were photographed while fresh in isotonic saline and some after fixation. The intact eggs were photographed with a Leitz ‘Makam’ attachment, fitted in some cases with a Leitz ‘Ultropak’ illuminator (see Pl. 1, figs. 1, 5 and 9). Most photographs were taken at a magnification of x95 and then enlarged to x250. A few were, however, taken at x 200. A]l measurements were made from photomicrographs of known magnification.
The fixatives used were the solutions of Bouin, Flemming and Zenker, the latter with a little acetic acid. These were found satisfactory. Various procedures were tried for embedding the eggs, since it was essential to have a method sufficiently reliable to deal with the limited amount of material available. The method finally adopted was a modification of the method devised by Hill & Tribe (1924) for dealing with cat eggs. It will be described elsewhere. Serial sections 5 thick were cut and stained with Heidenhain’s iron haematoxylin or with Weigert’s haematoxylin followed by eosin.
In order to determine more accurately the spatial relationships of the blastomeres and to determine their volumes, a series of dissectable wax reconstruction models at a magnification of x 800 were made. These are illustrated in Text-figs. 1—4.
One-cell stage: first segmentation spindle
Two unsegmented eggs (14A and 4B) are represented in our material. One of these, 4B (PI. 1, figs. 1, 2), is at the stage of the first segmentation spindle and was recovered with its sibling, a two-cell egg, approximately 30} hr. after insemination. The other egg, 14A (Pl. 1, fig. 3), was washed from the left uterine horn together with another egg, more advanced in development, precisely 140 hr. after mating. It is perhaps a degenerate or.pathological egg which would not develop. These eggs are almost if not quite ‘spherical, and the diameters of the two, as measured on the photographs, are given in Tables 1 and 2. As is apparent from Table 2, the difference in the volumes of the vitelli of these two eggs is considerable. Egg 14A, having a post-coital age of 140 hr., has not developed to schedule, since normal eggs would be in the morula or early blastocyst: stages.
The zona peHucida of egg 4B (PI. 1, figs. 1, 2) has an inner diameter of 145, an outer of 170, and a thickness of 12-5. It appears quite homogeneous and is completely devoid of cumulus cells, although a number of spermatozoa can be seen embedded in it. The nearly spherical vitellus almost completely fills its cavity, leaving, however, an evident perivitelline space which, .at this stage, is free of any granular detritus. A polar body was not seen in the living egg but was plainly visible in the sectioned material. In the morphology of the vitellus itself the most conspicuous feature is the presence of fat granules of various sizes in its cytoplasm. These are embedded in .a somewhat dense mottled matrix, and can be best seen in the central region of the egg, where they are sufficiently concentrated to obscure the nucleus. The peripheral zone is clearer and has fewer granules; here they form a marginal ring immediately beneath the vitelline membrane and there is no trace of the polar accumulation of fat as described in some mammalian eggs.
The egg shown in PI. 1, fig. 8, is quite different in appearance and is illustrative of the fate of the unfertilized egg. The yolk spheres are larger and no longer discrete, being clumped towards the centre of the cell, thereby increasing the opacity in this region. The vitellus is shrunken, its volume having shrunk from 1,204,260 to 974,348 y3, almost 20%. The shrinkage is, however, uniform, since the egg retains its almost spherical outline. In the sectioned egg (PI. 2, fig. 24), the lacunae of the fat globules in the peripheral zone are larger than in the fertile egg and their distribution is patchy, as in degeneration.
The sectional series of the fertile egg 4B (Pl. 2, figs. 22, 28) reveals that this egg is in the phase of the first cleavage spindle, a stage which is reached just under 31 hr. after insemination. The cytoplasm of this egg, which is everywhere in contact with the zona, consists of a narrow homogeneous and more deeply staining superficial zone which is thicker at the pole adjacent to the polar bodies than elsewhere, and a reticular, more lightly staining central mass in which the nucleus is embedded. ‘The lacunae of the larger fat globules are not very abundant and are mainly grouped about the superficial zone. The nucleus which is in anaphase is practically centrally situated, and extends through three sections, two of which are shown in PI. 2, figs. 22, 23. The mitotic figure consists of two clumped masses of chromosomes still united by spindle fibres, with remains of the asters in the vicinity of the chromosomes. As the chromosomes are not all at the same level, it is impossible to be sure of their number. The two polar bodies which do not occur in the sections figured are adjacent to each other and lie in a distinct bay at the surface of the egg. Each possesses a single spherical mass of chromatin and they are so placed that a line joining their centre to the equatorial plate or midpoint of the spindle would cut the latter almost at right angles. It is suggested that division which is imminent would be at right angles to this line.
Table 1. Diameters and volumes of zonas in microns and cubic microns
(Table to be formatted)
Stage of eggs. Mod.: mean outer diameter of zona. Mid.: mean inner diameter of zona. Z.P.: thickness of zona. Vol.; corresponding volume.
Eggs Stage Mod. Mid. ZP. Vol. 14A 1-cell 164 143 10-5 1,531,112 4B 1-cell 173 145 14-0 1,596,256 4A 2-cell 174 145 14-0 1,596,256 1A 2-cell 175-3 152 11-65 1,838,778 5A 4-cell 176-6 156-6 10-0 - 2,007,035 5B 4-cell 163-5 145-0 9-25 1,596,256 2A 8-cell 178-3 152-6 12-8 1,855,043 2B 8-cell 178-3 152-6 12-8 1,855,043 14B 9-cell 168-0 142-0 13-0 1,499,214 12A 12-cell : 168-3 143-3 12-5 1,547,284 12C 13-cell 172-0 147-0 12-5 1,663,224 7A 32-cell 166-3 136-3 15-0 1,331,723 7B 30 +2-cell 167-6 138-0 14-8 1,376,055 7C 30-cell 163-3 139-0 12-0 1,406,187 13B 96-cell (EB) 168-3 143-3 12-5 1,547,284 8A 126-cell (B) 170-0 145-0 12-5 1,596,256 Average 170-4 145-3 12-5 1,615,188
Table 2. Diameters and volumes of vitelli in microns
(Table to be formatted)
Stage: stage in development of eggs. SMD.: mean diameter of smallest vitellus or cell. LMD.: mean diameter of largest vitellus or cell. AMD.: average mean diameter of vitelli or cells. S.Vol.: volume of smallest vitellus. L.Vol.: volume of largest vitellus. A.Vol.: average volume of vitelli.
Eggs Stage SMD. LMD. AMD. 8.Vol. L.Vol. A.Vol.
14A 1-cell _ _ 123-3 _ — 974,348 4B 1-cell —_ — 132-0 — _ 1,204,260 4A 2-cell 105-3. - 123-3 117-0 606,131 974,348 838,603 1A 2-cell 101-6 113-0 109-2 539,464 735,499 | 678,076 5A 4-cell 76-6 92-0 "86-8 234,414 407,721 338,882 5B 4-cell 73-3 88-3 83-3 207,903 362,935 304,831 2A 8-cell 66-6 73-3 70-8 153,980 _— 207,903 183,471 2B 8-cell 64-6 76-6 72-6 140,501 234,414 199,532
14B 9-cell 48-3 73-3 64-9 59,734 207,903 143,794
We have been fortunate in securing two tubal eggs, one (no: 4A) at the initiation, the other (no. 1A) at the completion of the two-cell stage. These eggs (PI. 1, figs. 4-6) were recovered 80} and 48 hr. after mating respectively, and in both the first cleavage is complete. Before discussing these eggs an interesting observation in one instance should be mentioned. The egg in Pl. 1, figs. 5, 6, was seen by one of us (W. F. B. G.) to complete its division from one to two cells in the interval which elapsed between recovery and. photography.
The two blastomeres resulting from the first division are not of equal size, but they are both alike in structure in the photographs. Our measurements of the sizes of the two cells and the inner and outer diameters of the zonas are given in Tables 1 and 2. The vitelli have much the same appearance as in the uncleaved egg, differing from it only in size and shape. They are both ovoid and are.in contact over a fairly wide area, with their longer diameters (PI. 1, fig. 5) parallel to the cleavage plane and the shorter at right angles to it. The spatial relationship of the constituent cells is well illustrated in the models of these eggs (Text-fig. 1 a, b). In Pl. 1, fig. 6, where the cells measure 180 x 110 and 108 x 100 p, the area of contact is 3817-23 2, while in the slightly older egg (PI. 1, fig. 4) with cells measuring 122 x 95 and 110 x 85 p, it is 8028-49 u?. In the living egg there is no suggestion of polarity in the two-cell stage, the distribution of yolk granules being almost the same as in the late one-cell stage. The cytoplasm of each shows the same concentration of yolk material in the centre and the fat appears to be equally distributed between the two cells. The nucleus remains obscured and a polar body with a single chromatin mass lies on the surface of the smaller cell in Pl. 1, fig. 6. In this figure, the polar body measures 15 x 10, and has a surface area of 561-9? and a volume of 1252-83. In the intact egg, it was not in contact with the zona and moved freely in the perivitelline space. The zona is free of cumulus cells although a number of spermatozoa are embedded in it.
The nuclei in the blastomeres of the early two-cell stage are illustrated on Pl. 8, figs.- 25-27. Both are fully constituted and alike in structure, appearing as large, smooth, clear spheres almost in the centre of each cell. Each possesses one nucleolus and peripheral chromatin granules; both lie in the pale staining, finely reticular zone of the cytoplasm. In the larger cell, the peripheral accumulation of homogeneous cytoplasm stretches round the circumference of the egg without, however, invading the region below the contact surfaces. In the smaller blastomere it is restricted to one pole, so that a distinct polarity is present in this cell. Fat globules (small to medium) are present in abundance throughout the body of the egg. In PI. 3, fig. 27, a polar body with a single chromatin mass and large fat vacuole lies in contact with the zona and smaller blastomere in the vicinity of the superficial groove between the two cells.
In our second egg of this stage the two blastomeres are again of unequal size (Pl. 1, fig. 4). Apart from this, there are no ‘obvious structural differences between the two cells, nor do the cells differ from those of the previous egg, except in regard to size and nuclear changes associated with their varying states of mitotic activity. The sectional series of this egg shows it to be more advanced in development. Both blastomeres (Pl. 3, figs. 28, 29) possess equatorial plates, with chromosomes clumped in metaphase. In the larger cell, that on the right in fig. 28, the mitotic figure has been cut transversely, whilst in the smaller, that on the left in fig. 29, the plane of section is parallel to the axis of the spindle. Accordingly, the plane of division of the larger blastomere would be at right angles to the plane of the first cleavage, while that of the smaller cell would be parallel to it. With the completion of this division, then, the . blastomeres of the four-cell stage should be grouped in two pairs, so arranged as to form a cross-shaped figure. As far as is known, these eggs constitute the first initial cleavage stages of the goat’s ovum to be recorded.
Text-fig. 1. Models of eggs nos. 4A and 1A (a, 6), two-cell stages and nos. 5A and 5B (c, d), four-cell stages. x600. P.B. polar body.
We have available in our collection two eggs (5A, 5B) at this stage of development, which were recovered from the fallopian tubes of a female killed 60 hr. after copulation. In these eggs the conditions foreshadowed by the position of the cleavage planes of the two-cell stage are established, and the four blastomeres are readily grouped into pairs of very unequal size, so arranged. as to form a rather irregular cross (PI. -1, figs. 7-9 and Text-fig. 1 c, d). Of the two pairs of cells in each specimen, pair 1 and 2 occupy one hemispheres and 8 and 4 the other, the place of division between 1 and 2 in each case being almost at right angles to that between 3 and 4.
Though considerably smaller, the blastomeres repeat the structural design of the one- and two-cell vitellus, but both specimens are noteworthy in that each shows some inequality in the distribution of fat, one pair being richer in fat globules than the other. Many of these globules reach a ‘much larger size than in previous stages, and are mainly located at opposite poles of the cells, though they extend up to the central region. The similarity in distribution of the fat, more especially.in regard to the occurrence of the larger globules, is strikingly illustrated in Pl. 1, figs. 7-9, a large globule appearing in one blastomere in each of the eggs. In the intact egg, a nucleus is visible in some of the cells. The zona is normal in appearance and a perivitelline space is present, but we did not observe any movement of the blastomeres. No polar body was visible.
Table 3. Relative volume of blastomeres calculated from wax-plate reconstructions
(Table to be formatted)
The cells have been arranged in descending order of magnitude to emphasize the striking fact that there is invariably one small cell.
Percentage of total volume represented by each blastomere Cell c A :
stage Egg A- B. C oD E F G@ H I J K L M 2 4A 5789 4210 — -— -—- ~ ~~ ~~ ~ ~ ~~ ~ =
1A 5600 4400 — ~—- — ~~ ~ ~ ~ ~~ ~ ~~ =
4 5A 30-40 27:20 2560 168 — — ~—~ — — ~—~ ~— ~ —
5B 29-60 2760 2630 1640 — — — ~~ ~ ~ ~~ +
8 2B 1617 12-74 12-25 12:25 12-25 1225 1225 980 — — — — —
2A 14:80 13-36 12-87 1237 1237 11-88 1138 1089 — — — — —
9 14B 17-51 14-60 12-40 1240 1094 875 875 730 730 — — — —
10 12B 14-70 12-25 11-27 10-78 10-78 980 833 833 7:84 588 — — — 12 12A 13-85 12:04 9-63 9-04 9:04 9:04 904 7:22 602 602 602 301 — 13 12C 11:50 10-25 960 960 960 833 7-69 641 6-41 5:75. 5-75 450 4-50
In the sectioned material, the quantitative differences in the distribution of the fat globules and the structural condition of the nuclei in the two eggs are shown in Pl. 3, figs. 30, 31. Apart from the varying distribution of the fat globules and the differences, in size of the cells, the only other structural changes are those associated with the varying states of division of the nuclei. In this latter respect, egg no. 5B, from the left tube, appears to be younger, since all its nuclei are in the resting phase, whilst in no. 5A from the opposite tube, the spindle of division has already appeared in the smallest cell (Pl. 3, fig. 30). In both eggs there is present in the interval between the two larger blastomeres remnants of the polar bodies, whilst between the zona and the superficial grooves between the cells, cytoplasmic detritus has accumulated.
The measurements of the volumes of the blastomeres of the four-celled eggs are summarized in Table 3 (5A and 5B). From these data, it is seen that the diversity in cell size is more striking than at the two-cell stage. In this connexion it will be recalled that Heuser & Streeter (1929) found in the four-cell stage of the ovum of the “pig differences in the size of the blastomeres which approximate very closely the figures given in Table 3.
Text-fig. 2. Two views of models of eggs no. 2B (a, 5), and no. 2A (c, d), eight-cell stages. 7 x 600. P.B. polar body. .
Eight-cell stage This stage is represented in our series by a litter of two eggs (nos. 2A and 2B) from the same tube, obtained 85 hr. after insemination. These two eggs, except for slight differences in their nuclear pattern, are essentially similar. The data recording the measurements of the living eggs are given in Table 1, and a photograph of one of them, no. 2B, is shown in Pl. 1, fig. 10. From these data and from the photograph it is manifest that the eggs have not altered appreciably in size, but the blastomeres are unquestionably smaller and more nearly spherical. In each egg the blastomeres are fairly closely adherent to one another and to the inner surface of the zona, where they are somewhat flattened; between them they share fifteen large and two small contact surfaces. Two of the cells in each egg are in contact with five others, while the remainder adhere to four of their neighbours. There seems to be little variation in the arrangement of the cells in the two specimens, and they conform to. the lowsurface tension type described by Lewis & Wright (1935) for mouse eggs.
The eight blastomeres are arranged as shown in the figures of the models (Textfig. 2 a, b, c and d), and the percentage of the total volume of each blastomere is given in Table 3 (2A and 2B). The arrangement of the cells as seen in the views of the models is surprisingly similar, and the measurements of the two eggs (Table 1, 2A and 2B) are actually identical. From the data in Table 3, it is seen that one cell appears to be constantly large (2 in the text-figure) and one small (7 in the text-figure), while the size of each of the other six cells lies somewhere between the two extremes. It will be noticed that these measurements for our eight-cell goat eggs approximate very closely those given by Gregory (1980) for the eight-cell rabbit egg.
In their sectional anatomy (Pl. 3, figs. 32-34) the cells retain many of the characteristics of the younger stages. The fat globules are as large as in the four-cell egg, and are as unevenly distributed, but the nuclei, hitherto smooth and membranate when at rest, have become lobed. This lobation is not shared equally by the two eggs, ‘the nuclei of which are all at rest: those in egg 2A (figs. 32, 33) are all bilobed and in general have their chromatin peripherally arranged, while those in 2B (fig. 34) are more spherical and possess several central chromatin masses. In both these eggs, in the central grooves between the blastomeres, there is present a considerable amount of cytoplasmic detritus in which are small granular spherical masses.
There is in our collection a single nine-cell egg (no. 14B) which was obtained with its sibling (no. 14A), an unfertile egg, 140 hr. after insemination. This uterine egg, though belated in development, does not appear in the photograph of the fresh specimen (Pl. 1, fig. 11) to differ from the preceding stages in any recognizable respect except size and number of blastomeres. Notwithstanding its unpunctuality it is worthy of a brief description, since there are signs of unmistakable polarity in respect of the position of the constituent cells, which are segregated into groups of larger and smaller cells occupying opposite poles (Pl. 1, fig. 11 and Text-fig. 3 a,b). In the models the larger cells are numbered 1, 3, 4 and 5. The volumes of these cells were determined and the results are summarized in Table 3 (14B).
When the sectioned material (Pl. 3, figs. 35, 36) is examined the constituent cells are less compactly arranged than in previous stages and all contain numerous larger and smaller fat vacuoles distributed equally in each cell; also in each cell a small crescentic cytoplasmic area, which is non-reticular, is seen. Sometimes this area is at the sides of the cell directed towards the periphery and is in contact with the zona pellucida: at other times it is towards the centre of the egg (see fig. 85). In some of the cells are lobed nuclei, and in the superficial groove between the larger blastomeres and the zona there is present what appears to be two masses of cytoplasm (P.B. in the models) about half the size of the smallest blastomere, but non-nucleated and devoid of chromatin. Since we are not satisfied that the egg is quite normal, we do not venture to suggest what is the nature of these bodies—whether cell remains or cytoplasmic extrusion.
Ten-, twelve- and thirteen-cell stages
Female no. 12 provided the three uterine morulae (12B, 12A and 12C) which are available for the description of these stages. They were recovered from an animal killed 98 hr. after a single service, no. 12B with ten cells and no. 12C with thirteen coming from the left uterine horn, whilst no. 12A was obtained from the right horn.
Text-fig. 3. Two views of models of eggs no, 14B (a, b), nine-cell stage and no. 12A (c, d), twelve-cell stage. x600. P.B., a, non-nucleated cytoplasmic bodies; P.B., c, polar bodies.
These three eggs differ from each other not only in the numbers but also in the arrangement of their component cells and are of unusual interest inasmuch as they provide evidence of qualitative and quantitative differences in the constituent blastomeres. They provide also some evidence which may explain the manner in which the central cell becomes enclosed. An undoubted central cell is present in no. 12C.
Unfortunately, however, we have no record of the living ten-cell vitellus. The absence of any photographs of the intact egg, however, may be taken as evidence that it differed but little in appearance from its litter mates. The living twelve- and thirteencelled eggs are very similar in appearance to those with nine cells (Pl. 1, fig. 12 and Pl..2, fig. 13).
Ten-cell stage. In the granular cytoplasm of the blastomeres of the sectioned egg (Pl. 4, figs. 37, 38) fat vacuoles of varying sizes are found concentrated around the margins -of the cells bordering the zona pellucida where the nuclei are situated. In some of these, however, the lacunae of the fat globules are larger and much more abundant than in others (e.g. that on the left in fig. 87), and in these instances tend to be more evenly distributed throughout the body of the cell. None of the nuclei are in mitosis; all, however, are irregular or bilobed, and a polar body with a large and distinct chromatin mass lies between the central borders of the cells. The occurrence of a ten-cell stage, with nuclei fully constituted and at rest, indicates that cleavage of the eight cells of the eight-cell stage is not always simultaneous, and two of the cells in this instance must be regarded as having divided much sooner than the rest.An inspection of Table 3 reveals an unmistakable disparity in the volumes of the constituent blastomeres of this egg (no. 12B). The largest cell, A, contains 14-7 % of the total volume of the cytoplasmic material of the egg, and is almost three times as . large as the smallest J, which contains 5-88 %. This disparity is even more striking than in the eight-cell stage. As yet, however, except for differences in size and a more liberal quota of fat globules in one cell, all the cells appear to be similar.
Twelve-cell stage. Three views of the model of egg no. 12A with twelve blastomeres are shown in Text-figs. 3c,d and 4a (section). This egg differs from the one last described in the following respects: (1) in addition to an increase in the number of its constituent cells, one large cell (5 in the models) is nearly completely enclosed, only the outer end of this cell being visible through a. gap in the side of the model (Text-figs. 3c, 4 a) bounded by blastomeres 1, 2, 3 and 4; (2) the inner half of blastomere 2, also a large cell (Text-fig. 4a), is completely enclosed from above, below, and on each side by the neighbouring cells, while the outer half projects as much beyond the periphery as the inner half projects into the centre of the egg. Unfortunately, however, the nuclei of these two cells (Pl. 4, fig. 39), as indeed of all the cells of this egg, are in the resting condition, and we are unable to state precisely what the axis of the mitotic figure would have been had these cells been allowed to divide. Nevertheless, in view of the condition of the nuclear divisions in some of the larger cells of later stages, we venture to suggest that these cells would divide in such a way that the inner of the two daughter cells would be completely enclosed; one cell being in advance of the other, a thirteen-cell stage would result. Such a condition is established in the next egg to. be described. As in the ten-cell stage the cells are similar in appearance (PI. 4, figs. 39, 40) and apart from size and form no cytological differences could be detected between them. The only differences which are sufficiently marked as to be interpreted as evidenve of the early differentiation of the trophoblast are those relating to differences in the size and form of the cells, those in the lower half of the figures being perceptibly smaller and more flattened than those in the upper half of the figures. A study of Table 3 (12A) reveals a discrepancy in the size of the blastomeres which is even more marked than in the last egg. There are two large cells, four small, and six of about medium size. The two largest, A and B, take up 13-85 and 12-04% of the total volume, while of the four smallest cells three (I, J and K) are equal in size and contain 6-02 % of the total volume, the smallest, L, having a volume percentage of 3-01. The remaining cells have percentage volumes of 9-63, 9-04 (four cells) and 7-22. From these data it is evident that blastomere A is more than four times as large as L, and B is exactly twice the size of I, J and K and three times as large as L. This discrepancy in the volumes of the individual cells is not without ontogenetic significance, and will be taken up when the thirteen-cell egg has been considered. ,
Thirteen-cell stage. This morula no. 12C, of slightly greater diameter than the preceding (Table 1, 12C), has one of its blastomeres completely enclosed (9 in Text-fig. 4 b) and is distinctly more advanced in development. PI. 2, fig. 18, shows its appearance in the fresh condition in saline, and a view of a section of the model of. this egg is shown in Text-fig. 4 b, while a section through the mid-plane of the ovum is illustrated in Pl. 4, fig. 41. In the intact condition it is very similar to the twelve-cell stage (cf. Pl. 1, fig. 12). The egg now consists of thirteen blastomeres, twelve superficial and one undoubtedly central (Text-fig. 4b and Pl. 4, fig. 41). On examination of the cut surface of the model, it is found that cell 9, having a percentage volume of 4-5, is - completely enclosed, and it is in contact with five peripheral cells (4, 5; 6, 8 and 10). Of these, cell 10 with a volume percentage of 9-60 projects deeply into the central mass of the egg and its base is almost completely surrounded as in the case of cell 2 in the twelve-cell egg no. 12B (Text-fig. 4a). From its size and relations it seems very likely that its inner end was destined to become enclosed when the cell divided. There is, however, no indication that division is impending, since all the cells of this egg have resting nuclei and we are, consequently, unable to state precisely what the results of division would be. In the sectioned egg (PI. 4, fig. 41), all the cells are alike in structure and have an appearance similar to the cells of the twelve-cell stage, except that the larger fat globules are more evenly distributed about the outer margin of the cells. The-cells are compactly arranged. The nuclei, however, are central and, in contrast to some of the resting nuclei found at earlier stages, are no longer lobed. As in the twelve-celled egg, although the blastomeres are histologically alike, they show considerable differences in size (Table 3, 12C); two cells (A and B) are larger than the others, two (L and M) are small, while the nine remaining may be arranged in five groups which form an evenly graded series between the two extremes. This disparity in cell size which is so marked a feature in the eggs of this litter is not without significance and, as has been shown by Heuser & Streeter (1929), points to the more active division on the part of some cells than others. It provides the first concrete evidence of the early differentiation of the trophoblast cells.
Fertile goat eggs soon after they have entered the uterus begin to show unmistakable signs of an increase in the number of central cells and the commencing formation of the cavity of the blastocyst. At the same time, some, but not all, of the peripheral cells begin to show evident signs of flattening out. There are three such eggs in our collection. They were all obtained from a single female precisely 120 hr. after copulation and are figured in the living state on Pl. 2, figs. 14-16. Two, nos. 7A and 7B, had just entered the uterine horn, while the third (no. 7C) was still at the tubo-uterine junction. Only 7A and 7C are available in the sectioned series, 7B having been lost during embedding. The living eggs exhibit varying degrees of compactness or adhesion of the cells to one another. In some (e.g. fig. 15), where the surface tension is presumably high, the blastomeres are spherical and project beyond the general level of the egg, while in others (e.g. fig. 16) there is the opposite extreme, for the cells are closely adherent and form a more compact mass. In Table 1 (7A, 7B and 7C) are given the measurements of the living egg, and it is to be noted that the mean inner diameter of the cavity of the zona of all these eggs is, exceptionally, slightly smaller than in the remaining eggs of the series, while the zona pellucida itself is, in the case of eggs 7A and 7B, perceptibly thicker.
Text-fig. 4. Models of goat eggs showing central cells, differentiation of formative cells (black with white stipple), trophoblast (stippled), and entoderm (cross-hatched), and origin of the blastocyst cavity. x c.460. @ (section), egg no. 12A, twelve-cell stage; b (section), egg no. 12C, thirteen-cell stage with central cell; c, d (cut surfaces), morula no. 7C, thirty-cell stage; e, f (cut surfaces), morula no. 74, thirty-two cell stage; g, h (cut surfaces), early blastocyst no. 13B, about ninety-six cells; i (section), blastocyst no. 8, about 126 cells. ‘
Of this series 7C is a spherical morula recovered -from the tubo-uterine junction and consists of a solid mass of thirty cells of varying sizes, which are separated by a large perivitelline space from the zona. Four of these are centrals, one of which is larger than the others and possesses a large chromatic nucleus (Pl. 4, fig. 44). The others are smaller and have perceptibly smaller nuclei (Pl. 4, figs. 42, 48). The blastomeres are not so compactly arranged as in the preceding stages, the central cells, in particular, being separated by obvious intercellular spaces. These cells have all welldefined outlines and are cuboidal or irregularly polygonal, and what we take to be the commencing blastocyst cavity occurs as a series of irregular cleft-like spaces between the centrally placed cells and peripheral cells. Two views of the cut surfaces of the model of this egg, showing the relationship of these spaces to the peripheral and central cells, are illustrated in Text-fig. 4c, d. Apart from size and position, the cells show no marked cytological differences, although those beginning to flatten on the periphery are obvious trophoblast cells.
These peripheral cells are clearly marked off from the centrals and vary greatly in size. The larger cells (shaded dark in the models) occupy one pole and are adherent in places to the centrals, while the smaller bound the incipient cavity and are found at the opposite pole of the cell. Some of these cells have numerous fatty globules which are responsible for the vacuolated appearance of the cells seen in Pl. 4, figs. 42-44, indicating the sectional anatomy of this egg. A number are in varying stages of mitosis. One cleavage spindle in a peripheral cell of average size (that in the upper right quadrant of the figure) which is of interest is shown in Pl. 4, fig. 44. One pole of the spindle, in anaphase, is directed towards the large central cell, the other towards the periphery, so that on the completion of the division two cells would result of which one would be a central and the other a peripheral. We are of opinion that central cells in this and in previous stages result from the radial division of these more peripherally lying cells.
Our next egg, no. 7A, a litter mate of the preceding, differs in appearance in the living state from its sibling. It shows in the sectional series (Pl. 4, figs. 45-48), as also in the photographs of the cut surfaces of the model (Text-fig. 4e, f), a definite advance in the degree of organization of its constituent cells. These have acquired more certain identifying characteristics which develop pari passu with the formation of the cavity of the blastocyst and can now be more readily separated into a group of larger cells occupying one pole (the shaded cells in the model), and a group of smaller cells which lie at the oppesite pole.
In this egg there are thirty-two cells; four centrals and twenty-eight peripherals. Of the latter, seven are large and form a compact group on the surface of the egg closely adherent to the central cells. The smaller peripheral cells are ovoid or slightly flattened, with large nuclei and sparse fat globules, and constitute a continuous investing layer over the lower hemisphere of the egg. The central cells are irregularly polygonal and vary in size (Pl. 4, figs. 45-48). The largest is in mitosis — in anaphase — with the axis of the spindle parallel to the group of seven large peripheral cells and would, on completing its division, yield two cells of which both would remain in contact with the larger surface cells. The blastocyst cavity is again represented by a series of broad intercellular spaces between the centrals and the smaller peripheral cells, and extends from the lower pole of the egg- to just past the equator (Text-fig. 4e, f). The flattening of the peripheral cells is more evident where they bound the cavity. :
During the sixth day the two parts of the egg, trophoblast and formative area, are morphologically completely differentiated. This stage is represented in our collection by a litter of three eggs obtained from a female 134 hr. after insemination. Of this . litter, however, only two eggs, 183A and 18B, are available for examination in the serial sections, the other having been lost in handling. These two eggs are essentially similar in appearance, differing only in the number of blastomeres and the extent of development of the blastocyst cavity. Pl. 2, figs. 17, 18, portray eggs no. 18C (the one which was lost) and no. 13B in the living state. As can be seen from the: photographs of the living egg, marked structural changes have taken place since the last stage, and a crescentic group of larger cells is present at one pole (Text-fig. 4 g, h) while a.sharply defined group encircling a central darker area (the cavity of the blastocyst) occupies the other pole. The egg thus appears definitely regulated -as to its axis, and we can identify with certainty the two groups of cells which are destined to form the formative area and the trophoblast and, coincident with the differentiation of the latter, the blastocyst cavity. It is noteworthy that in beth the eggs of this stage where the cavity-of the blastocyst is better defined, the blastocyst wall is closely applied to the inner surface of the zona and is not, as in the previous stage, separated from it by a considerable space.
Text-fig. 5. Tracings of photomicrographs ( x 320), of serial sections of egg no. 13 B, at 134 hr. post-coitum, showing the demarcation of the trophoblast (stippled) and the residual formative cells (black). The blastocyst cavity is represented by a series of fluid-filled intercellular spaces in the trophoblastic part of the egg. The cellular mass of the trophoblast which is adherent to the under-surface of the formative cells contributes to the formation of the entoderm.
Since it was possible, because of the clear-cut separation of the’ groups of cells, to orientate the egg as to the plane of cutting, the sectional series (Pl. 5, figs. 49-52) amply confirm the demarcation between the trophoblast cells and the larger formative cells at the embryonic pole. The series also provides evidence of the increase in size of the intercellular spaces between the trophoblastic elements. As these features are ° more evident when the sections are examined in series, serial tracings of egg no. 13B, in which the trophoblast is shown in stipple and the formative cells in black, are reproduced as Text-fig. 5. Examination of this series suffices to carry conviction of the completeness in the separation of the two groups of cells and the acquisition by the egg of its distinctive polarity.
Turning now to the actual developmental transformations of the blastocyst cavity, it is found to be perceptibly larger than in the antecedent stages and now has the form of a curved intercellular cleft between the trophoblast cells, so situated as to separate a single layer of trophoblast, which clothes the under-surface of the formative mass, from the main trophoblast at the lower polar region. As regards the manner of formation, there seems little doubt that its enlargement has been brought about by the confluence of the fluid-filled intercellular spaces of the earlier stages.
A count was made of the cells in both of the sectioned eggs of this stage, and their approximate constitution was as follows:
Egg 18A consists of eighty cells of which twelve constitute the formative area and sixty-eight are trophoblastic, while-egg 18B consists of ninety-six cells, of which nineteen are formative (some distinctly larger than others) and seventy-seven trophoblastic; and of the latter sixteen form the roof of the cavity of the blastocyst and are closely applied to the under-surface of the formative mass.
The didermic blastocyst
There is, unfortunately, a gap between the stage just described and our uterine blastocyst no. 8, which is the last of: the series. This egg was recovered 156 hr. after copulation. Pl. 2, figs. 19-21, portray the egg in its entirety, while Text-fig. 4 7 shows a cut surface of the model of the egg.
The blastocyst is now fully constituted and its polarity unmistakable. It consists of an outer investing layer of trophoblast composed of a single layer of loose flattened cells and an inner cell mass at the embryonic pole of many compact cells, the majority of which are rather larger and more irregular than those constituting the outer wall. The spherical zona is still intact and there are no indications of impending disintegration. It has an inner diameter of 145, an outer of 170 and a thickness of 12-5 p, measurements which are identical with those of our fertile unsegmented egg. The formative mass (Pl. 5, figs. 53-60) measures on the slides 0-098 x 0-038 x 0-075 mm. and has the form of a compact ovoidal disc composed of rather large, deeply staining, irregular cells; while overlying these are a few small irregularly shaped cells which blend with the flattened parietal trophoblast elements at the margins of the disc. They are the covering trophoblast. These covering cells cannot always be identified overlying the larger formative cells (see Pl. 5, figs. 58, 54), and it seems highly probable that the former are increased at the expense of cells and their descendants now forming a portion of the formative mass; int other words, they are in process of being differentiated off from the primitive large cells. In contact with the lower surface of the disc there are present, especially at one side, cells of a more elongated form and rather different appearance, which may be regarded with some confidence as the representatives of the future entoderm. It is noteworthy that this precocious segregation of cells from the under-surface of the disc is coincident with the complete differentiation of the cavity of the blastocyst and so with the trophoblast.
The primitive entoderm cells are very similar in appearance to the cells of the parietal trophoblast, and appearances such as those shown in PI. 5, figs. 56, 57, where a connecting strand of cytoplasm may be seen passing from a parietal cell to one on the under-surface of the disc, suggests a continuity of origin between the two. At the other end of the disc, Pl. 5, figs. 53, 54, it is impossible to be certain whether a cell is being incorporated into the disc or whether it is migrating therefrom. Here is evidence, then, of a contemporary origin of primitive entoderm and trophoblast and, by inference, an independent segregative origin of entoderm and formative cells. We venture to suggest, therefore, that these primitive entoderm cells are the direct descendants of some such cells of the trophoblast as those which roofed the incipient cavity in our last egg. Finally, when a count was made of the numbers of cells in this egg, its approximate constitution was as follows: (1) formative cells, 40; (2) primitive entoderm, 15; (3) covering trophoblast, 22; and (4) parietal trophoblast, 59.
Comments and Comparisons
Egg size For very few ungulates do we have adequate and comparable measurements of the living egg. The few available measurements are summarized in Table 4, while Tables 1 and 2 give the results of our own measurements on the sixteen goat eggs examined in the present investigation.
Table 4. Comparative measurements of living ova (ungulate)
(Table to be formatted)
Outside Inside . diameter of diameter of Thickness of
Animal Investigator zona in p zona in » zona in 4 Cow Evans & Miller (1935) 192 150-7 20-6 Hartman et al. (1931) 167 140 13-5
R. V. C. (unpublished) 162 137 12-5 Sheep Assheton (18982) 180 ~° 150 15-0 Allen et al. (1931) 167 143 12-0 Clark (1934) 175 - 147 14-0 R. V. C. (unpublished) 180 153 13-5 Goat Amoroso et al. (present investigation) 170-4 145-3 12-5 Amoroso et al. (19395) 170 145 12-5 Hartman (1929) _— 140 _ . Pig . Assheton (18985) . 164 132 16-0 Heuser & Streeter (1929) 160 130 15-0 R. V. C. (unpublished) 187 132 12-5 Horse Amoroso ef al. (19392) 158 131 13-5 Hartman (1929) _ 13%
The unpublished data is derived from measurements of the series of ova in the possession of one of us (E. C. A.).
The indications from these data (see Table 1) are that goat eggs, except for the transient but perceptible diminution in the diameter of the zona cavity in the late morulae (7A, 7B and 7C), do not vary considerably in size during the period when the ‘formative’ and ‘trophoblastic’ parts of the egg are in process of differentiation, and the average of 145-3 for our series of eggs is remarkably close to the average figure of 147-0 obtained by Clark (1934) for his series of sheep ova. Furthermore, it is evident that while the absolute size (see Table 4) of the mature ovum varies from species to species there is, in general (especially among the Pecora), a remarkable degree of similarity in the different eggs as measured by the inner diameter of the zona pellucida. In all of the investigations made upon the ungulates, only in the case of the pig and horse, animals with an epithelio-chorial placenta, is there any divergence in egg size from the tabulated mean for the Pecora. The associations demonstrated by the tabulated data cannot be accidental, and they cannot depend upon the size of the animal. It seems probable, however, that they may depend in some way upon the mechanism for implantation, and the relationship of egg size to placentation may be a primary one.
Zona pellucida and cumulus
From the photographs of the living egg and from Table 1 it is evident that the zona, pellucida in the goat remains fairly uniform in thickness during its tubal sojourn. Even as late as our blastocyst stage it retains many of the characteristics of the earlier stages, and it has almost the same dimensions and the same homogeneous appearance. How long it remains intact in the goat we are unable to state.
No corona radiata cells were attached to the zona in any of the goat eggs examined. This absence of adherent cumulus cells has also been recorded for the roe (Bischoff, 1854), sheep (Assheton, 1898a; McKenzie & Allen, 1933; Clark, 1934; McKenzie & Terrill, 1987; and others), cow (Hartman, Lewis, Miller & Swett, 1981; Evans & Miller, 1985), pig(Assheton, 18986; Corner & Amsbaugh, 1917; Corner, 1917; Heuser & Streeter, 1929), and horse (Amoroso et al. 1939a). Whether corona cells are still in position at the time of ovulation, disappearing soon thereafter, or whether the ovum. lies unattached to the surrounding cumulus just before the follicle ruptures, is at present uncertaifi. On the one hand, Webster (1921) refers to the flattened cumulus, about the size of a pin head, on the relatively enormous wall of the cow follicle, while Allen et al. (1930), on the other hand, have recorded a well-organized corona about an ovum from a large follicle from a human ovary. The persistence of a well-developed corona radiata surrounding the freshly ovulated egg occurs with regularity not only in the rabbit (Gregory, 1980; Pincus & Enzmann, 1932), guinea-pig (Squier, 1932), rat (Gilchrist & Pincus, 1932) and mouse (Lewis & Wright, 1985), but also in the cat (Hill & Tribe, 1924), dog (Evans & Cole, 1981), ferret (Hamilton, 1934) and man (Allen e¢ al. 1930). It is evident, therefore, that, while in many mammals the ova are shed surrounded by an apparently sticky cumulus, the ungulates appear to be exceptional, since very few or no follicle cells surround the egg soon after it is shed. The reasons for this species difference are not known, though the exceptional rate of tubal transit of opossum ova which enter the uterus in approximately 24 hr. has been attributed to their lack of surrounding cumulus (Pincus, 1936). While the absence of any adherent cumulus may be invoked as the cause of the acceleration of tubal transit in the opossum, and possibly also the pig, it is clear that we cannot associate this phenomenon causally with the duration of the tubal journey in the goat and sheep; for the eggs of the latter, which are usually devoid of adherent cumulus and have a shorter distance to travel, require a much longer time for the tubal passage than do the eggs of the pig.
In its morphology the goat egg is in all essentials similar to those of the other ungulates so far described. The cytoplasm of the egg is remarkably clear and contains fine fat granules evenly distributed throughout the egg. In this respect it resembles more closely the sheep egg than those of the pig and horse which are more opaque and possess coarser fat droplets. There are no indications of polarity in the uncleaved egg. The unicellular vitellus is spheroidal or hearly so, and does not fill the space of the zona. Within this perivitelline space a polar body is invarfibly present. In a few cases two were seen in the sectioned egg. The polar bodies are usually small, compact and spheroidal, though ovoid forms were encountered. They vary greatly in position in the different eggs, sometimes being found in the superficial grooves between the blastomeres, at other times buried deeply in the spaces enclosed by the central margins of the cells. There can be no doubt that this variation in position of the polar bodies is due to segmentation, pressure, and readjustment (Gregory, 1930). The almost universal occurrence of a single polar body (presumably the second, since the great majority of the eggs are fertile) in the segmenting goat egg points to the early disintegration of the first body. This is in agreement with the conclusions of Lewis & Wright (1985), who found that in more than 50% of their mouse eggs the first polar body had disappeared by the four-cell stage, while in only 25 % of the eggs were two bodies present in the eight-cell and morula stages. It is of some interest in this connexion’ to find that although Allen, McKenzie, Kennedy & Beare (1931) identified polar bodies in several sheep ova, neither Assheton (1898 a) nor Clark (1934) were able to find them; nor do. Green & Winters (1935) and McKenzie & Terrill (1987) figure them. In the two-cell egg of the cow described by Hartman et al. (1931) ‘neither polar body was differentiable’. In the goat a polar body is still identifiable in our late morulae, as indeed-in the blastocyst we describe.
Age, stage and location of eggs
Table 5 summarizes our observations on the age, state of development, and location of the several cleavage stages and early blastodermic vesicles. The ages giten refer to the post-coital age, and since we have no definite information of the time relation between copulation and ovulation, it will be readily understood that the ages are only approximate. Up to 30 hr. post-coitum the egg remains unsegmented, but is preparing to divide as is evidenced by the formation of the first cleavage spindle. It will be recalled that egg 4A (recovered 30} hr. post-ceitum) completed the division from the one-cell to the two-cell stage in the interval which elapsed between recovery and photography. In our other two-cell specimen recovered from the tube 48 hr. after copulation, division of the blastomeres is imminent. That the two-cell stage lasts about 18 hr. would séem, therefore, a permissible deduction. Eggs from 50-60 hr. postcoitum are in the four-cell stage, while those recovered between 60 and 85 hr. have completed the third cleavage while still in the fallopian tube. Older eggs, morulae, and early blastocysts from 98 hr. post-coitum, are at the tubo-uterine junction or have entered the uterus. Goat eggs, therefore, seem to enter the uterus some time between the close of the fourth day or the beginning of the fifth day after mating.
If we seek an orientation in comparative studies on the rate of cleavage of tubal eggs among the ungulates, we find many suggestive but few unequivocal data. This has been due in part to the fact that, of the forms studied, all ovulate spontaneously and there is, in consequence, some difficulty in timing ovulation. Nevertheless, for four species, pig (Heuser & Streeter, 1929), sheep (Clark, 1934), cow (Hartman et al. 1931) and goat (own data), we can construct approximate growth curves. These are presented in -Text-fig. 6. It is at once evident that there are striking differences in the rate of cleavage in the four species considered. Thus, while the goat and cow curves are the most regular, being almost straight lines, the sheep is the most precocious in its cleavage rate, and the pig falls somewhere between the two extremes. Further, whereas the first two cleavages occur earlier in the goat than in the pig, thereafter the rates of cleavage are reversed, the pig being the earlier to attain the sixteen-cell and subsequent stages. That this difference in the rate of segmentation is inherent in the eggs is supported by the studies of Castle & Gregory (1929) and Gregory & Castle (1981), on genetic races of rabbits which differ considerably in size, and in which they have shown that the cleavage rate is consistently more rapid in the large-sized race. That they are independent of tubal environment is rendered probable by the corroborative data from rabbit eggs cultivated in vitro (Lewis & Gregory, 1929), where divisions are well within the normal. periods.
Table 5. Age, stage and location of eggs
(Table to be formatted)
Time RE since , S Ot mating Cell _ Location of eggs in Female Egg hr. stage genital tract Remarks a> ee 4 4B 304 1 Tube First cleavage spindle ‘=< 4 4A 304 2 Tube > LIB 1 1A 48 2 Tube Second cleavage spindle — a 5 5A 60 4 Tube & h 5B 60 4 Tube One cell in division 4 . 2 2A 85 8 Tube All cells with resting nuclei 2B 85 8 Tube 12 12B 98 10 Uterus 12A 98 12 Uterus Formation of central cells 12C- “98 13 Uterus Central cells 7 TA 120° 3932 Uterus Morulae with central cells and 7B 120 30+2 Uterus commencing formation of cavity 7C 120 30 Tubo-uterine junction of blastocyst 13 13 A 134 80 Uterus Egg definitely regulated as to 13 B 134 96 Uterus axis. Formative area distinct 14 14A 140 1 Uterus : Degenerate 14B 140 9 Uterus Retarded 8 8A 156 126 Uterus Blastocyst
In addition to the varying rates of cleavage already noted, it must further be pointed out that ungulate eggs differ widely in the times of onset of visible differentiation. Thus, Heuser & Streeter (1929) found that differentiation of the trophoblast in the pig begins as early as 4 days 3} hr. after copulation, at which time the egg has left the tube and is in about the sixteen-cell stage. In the sheep, on the other hand, Clark (1934) detected a more precocious differentiation of the trophoblast in tubal eggs with sixteen cells at about 65-77 hr. after coitus. The eggs of the goat appear to begin their differentiation late in comparison with those of the sheep and pig; the really decisive evidence for this conclusion, however, is not yet at hand, and the intermediate stages between thirteen and thirty cells will be required for a definite solution of the problem. In the common stock rabbit Gregory (1980) reports that separation of the trophoblast from the rest of the egg is early, unquestionable signs of the change being found in the seventeen-celled stage 41-48 hr. after coitus. Castle & Gregory (1929) and also Gregory & Castle (1931) were further able to show that in spite of the inherent differences in the speed of segmentation in the large- and smallsized breeds the process of differentiation occurs at the same time. This dissociation of the fundamental embryogenic processes, however, has long been known, but to these authors must be given the credit of tracing it back as far as the morula stage. It may well be, then, that the more rapid rate of cleavage of the sheep egg as compared with those of the goat, cow, and pig depends on some such inherent mitotic intensity quite independent of differentiation rates.
Text-fig. 6. Comparative development of ova of the goat, sheep, cow and pig. Abscissa: time in hours after mating. Ordinate: number of cells. The goat, from data of present investigation. The sheep, from data of Clark (1934). The cow, from data of R.V.C, collection. The pig, from data of Heuser & Streeter (1929).
The wide discrepancies found among mammals in respect of the period of arrival of ova in the uterus have long been known, and their significance commented upon. Allen Thompson (1859) was among the earlier writers to comment upon the lack of correspondence between the time of the tubal journey and the length of gestation. That the rate of passage of ova through the tube is independent of the size of the animal and size of the egg seems well established. As we have already noted, it is independent of the length of the tube, except perhaps in the rabbit (Gregory, 1930), where it may be in some way thus correlated. Our own observations on goat eggs lead us to suspect, however, that in the ungulates at any rate, the time of arrival of the eggs in the uterus may, in some measure, depend upon the time at which the initial segregation of the formative and trophoblastic parts of the egg occurs. The collected data (Table 3 and Text-fig. 7) of the proportional volume constitution of our goat eggs, and more especially of 12 A, B and C (which had just entered the uterus), suggest inequalities in the size of the component cells and the possibility of the occurrence of more active division on the part of some cells than of others.
Text-fig. 7. Relative volumes of blastomeres of two-, four-, eight-, nine-, ten-, twelveand thirteen-cell stages.
This view appears to us to be supported by the data derived from the study of the cleavage process in the pig (Assheton, 1898b; Heuser & Streeter, 1929) and sheep (Assheton, 1898a; Clark, 1984). Thus, Heuser & Streeter report that in the case of the pig, at about the time the egg passes from the tube into the uterus, which it does between the close of the second day and the beginning of the third day, it is found in the four-cell stage. At this time the egg shows a distinct disparity in respect of cell size, and, correlated with this, a definite difference in the rate of subdivision of the constituent cells. ‘The property’, they state, ‘of more active cleavage is the first evidence that this egg shows of the sorting and localization of special substances.’
Assheton had already averred as early as 1898 that variability in respect of cell size in pig eggs was pronounced in the four-cell tubal egg, but more so in the later five-segment stage when the egg had just entered the uterine horn.. In the sheep he has also described an eight-cell tubal egg in which one segment differs most markedly in texture and in colour from all the remaining segments, while in another egg, also with eight cells, but recovered from ‘the upper end of one horn of the uterus of a sheep killed on the fourth day, one cell was slightly larger than any of the others’. Clark has also remarked upon this disparity in cell size of sheep ova just’ before they enter the uterus. He finds, however, that the discrepancy is sufficiently well marked only when the egg has reached the sixteen-cell stage.
Remarks on Cleavage
It is of some significance that certain striking similarities exist in the cleavage pattern of the eggs of the pig, sheep and goat. In the goat the two blastomeres from the first cleavage are unequal in size, but no qualitative differences between the two cells, as is described for the rat (Huber, 1915), dog (O. van der Stricht, 1928) and cat (R. van der Stricht, 1911; and Hill & Tribe, 1924) could be detected. Such a discrepancy in cell size has been reportéd for a number of mammals and would seem also to be the usual condition among the ungulates, having been recorded for the. pig (Heuser & Streeter, 1929), cow (Hartman et al. 1981) and sheep (Clark, 1984). That this difference is merely a matter of chance separation of cytoplasmic material in the progress of the initial cleavage is suggested by the work of one of us (W. J. H. 1934) for the ferret; the really decisive evidence for this conclusion is, however, not yet available. In the pig, on the other hand, Heuser & Streeter (1929), though they could find no demonstrable cytological differences between the two cells, tentatively suggested that they were probably not identical, and that with the completion of the first cleavage there had doubtless occurred some sorting of the constituent substances of the egg.
At the second cleavage the divisions are effected in two planes at right angles to . each other, and the two blastomeres divide at very nearly, but not quite, the same time, giving rise to a four-cell stage with the blastomeres so arranged as to form an interlocking cross-shaped figure. This cross arrangement of the blastomeres has been described in the pig (Assheton, 1898a; Heuser & Streeter, 1929), sheep (Clark, 1984), rabbit (Assheton, 1909; Gregory, 1930), hedgehog (Assheton, 1909), guinea-pig (Squier, 1982), mouse (Sabotta, 1924; Lewis & Wright, 1935), cat (Hill & Tribe, 1924), ferret (Assheton, 1909; Hamilton, 1984), dog (Bischoff, 1845; Assheton, 1909), monkey. (Lewis & Hartman, 1931). Macdonald & Long (1984) figure it for the rat and one of us (E. C. A.) has observed it in the cat, dog, pig, sheep and cow. In both of our living specimens the cross-arrangement was definite, but we did not observe any movement of the cells as described for the ferret (Hamilton, 1934). As pointed out by Hamilton the cross-arrangement of the spindles at the two-cell stage might lead us to expect a difference in the appearance of the cells at the four-cell stage. There is some evidence in the goat that this is so, for both our four-celled eggs are noteworthy in that each shows some inequality in the distribution of fat, one pair of cells being richer in fat globules than the other.
As has already been described, the nuclear conditions in the larger blastomere of the two-cell stage is only slightly in advance of that of the smaller cell. Consequently, in the goat the three-cell stage is abbreviated, the egg passing from the two- to the four-cell stage without the intervention of a pronounced three-cell stage. This fleeting condition of the three-cell stage has also been recorded for the ferret (Hamilton, 1934) and seems also to be true for the cat, for, though R. van der Stricht (1911) described two three-celled eggs, Hill & Tribe (1924) failed to recover this stage in their unique collection of tubal eggs.
Concerning the slight priority of the larger blastomere in the two successive divisions of the second cleavage of the goat egg, it is of some interest to find corroborative data in the motion-picture records of the living monkey egg (Lewis & Hartman, 1931). In the pig Heuser & Streeter (1929) also find that one of the blastomeres is more precocious in the onset of the second cleavage, giving rise to a three-cell egg. However, they do not state in‘which cell this priority is manifest, but point out that from this stage on the cells can be divided into those which show ‘more active cleavage’ and those which are ‘more deliberate’. These authors attach considerable importance to these differences in size and rate of division, as affording the first evidence of ‘the sorting and localization of special substances in the egg’, the more actively dividing cells being the precursors of the trophoblast, the more lethargic cells those which give rise to the embryo proper. So far the only visible evidence of differences in the cell groups of our four-cell eggs is a slight variation in the proportionate volume of the cells (see Table 3-and Text-fig. 7), and as far as our observations go we are inclined to think that the early dissociation of the cells, such as exists so constantly and characteristically in the pig, is effected at a later stage in the goat.
In ‘our material there are no intermediate stages between -those with four cells recovered 60 hr. post-coitum, and those with eight cells at 85 hr. after mating. Our third cleavage is represented by two eggs at the eight-cell stage, in both of which it is possible to recognize one cell which is constantly large, and one small.
The fourth cleavage is represented in our material by four eggs in the nine-, ten-, twelve- and thirteen-cell stage. An undoubted central cell is present in the twelvecell egg, and there are indications in the thirteen-cell specimen that the central cells ’ are produced by the radial division of some of the peripherals. In all our eggs of this cleavage we can recognize a polarity in respect of the position of the constituent cells" which are segregated into groups of smaller and larger cells occupying opposite poles, and which we interpret as indicative of the more active division on the part of some cells than of others. It will be recalled that this dissociation of the cells was observed in the pig and sheep, and it has also been recorded for the cat, but the time at which it occurs varies somewhat in the different species. Thus, in the pig it is effected early, at the second cleavage; in the sheep at the third or fourth; while in the cat, according to Hill & Tribe (1924), it may be as early as the second and not later than the third. That there is a relation, indeed a correlation, between the time when the egg acquires its distinctive polarity and the time of its arrival in the uterus has already been stressed. Hill & Tribe (1924) and Heuser & Streeter (1929) lay emphasis on this early dissociation of the blastomeres into two groups with determinate destinies. Moreover, Heuser & Streeter believe that the varying rates of division between the groups of cells is the really decisive factor. Furthermore, we believe with Streeter that the forces that appear to be responsible for their segregation are intrinsic and purely genetic rather than environmental. We cannot, however, agree with Hill & Tribe (1924) when they suggest that-the process of epiboly, which they believe they have conclusively demonstrated for the cat, may be of universal occurrence in the Monodelphia, ‘though evidently not always easy to demonstrate’. We question the speculation of Hill & Tribe, for which slight evidence is presented. We incline rather to the view (not new) that the central cells arise by radial division of some of the peripheral cells, further increase being aided by division of pre-existing cells. Indeed, if speculation on such meagre grounds is justified, the evident difficulty in demonstrating overgrowth might better be regarded as indicative of its absence.
With the completion of the fourth cleavage the egg is provided with the necessary requirements for the organization of the blastocyst, and by the time the morula consists of thirty-two cells a considerable degree of differentiation has been attained. The cells have now acquired more certain identifying characteristics, and marked functional changes are demonstrably associated with the differences which mark the early recognition of the trophoblast.
The two groups of cells which were recognizable earlier, though with less certainty, are now unmistakable. We have, on the one hand, the large, isolated, slowly dividing, primitive cells at the upper pole of the embryo; on the other hand, there are the smaller, more actively dividing cells which are well advanced in differentiation, and form an attenuated investing layer over the lower hemisphere of the egg. In contrast to these are the centrals. Some are large undifferentiated cells which repeat the structural design of the primitive cells, and with these they constitute a group. Others are smaller, further advanced in differentiation, and in places are separated by wide intercellular spaces from the flattened cells with which they thus present relations of varying intimacy. This distinction between the two types of centrals is stressed, because while at this stage they represent the closest associations of individuals, their further history shows that they are not equivalent groups. Moreover, the subsequent stages of development of the goat justify us in recognizing in the smaller cells trophoblastic elements, from which the parietal trophoblast and part of the entoderm will arise, and in the larger cells the formative elements which, after giving origin to covering trophoblast, are destined to lose their individuality and co-operate in the formation of the embryonic shield. Our goat morulae thus reproduce in all essentials the conditions described and figured by Heuser & Streeter (1929) for their pig eggs with sixteen to twenty cells (cf. their fig. 6 g, with our Text-fig. 4 g, h).
In our early blastocysts 18A, 18B and 18C with eighty to ninety-six cells, the continuous but independent mitotic rhythms of the trophoblast and formative cells have produced the characteristic segregation of the two parts of the egg. These eggs also provide evidence of the way the entire groups of cells exercise directive influences upon the further course of development of the blastocyst. In the eggs of this litter the blastocyst cavity appears in the form of a crescentic intercellular cleft in the trophoblastic part of the egg and is so situated as to separate a single layer of trophoblast which remains adherent to the mass of laggard cells from the main trophoblast— which may in places be several layers thick—at the lower polar region of the embryo. As a result, a large part of the main mass of trophoblast cells remains peripheral and gives rise to the parietal layer; the remainder, which are in contact with the formative cells, giving origin, we believe, to primitive entoderm and probably also to parietal cells. In the formation of the blastocyst cavity there is no evidence of liquefaction of the cells, the cavity being entirely intercellular and bounded on all sides by trophoblast. These findings are in general agreement with those of Assheton (1898 a, b) for the sheep and pig, and provide confirmation also of the views he advanced for the goat (1908, 1909). While he is inclined to think that the blastocyst cavity, which he regards as the archenteron in contradistinction to the segmentation cavity, owes its origin to the extension of intercellular vacuoles in the pig, it is ‘less clearly so in the sheep, and from general appearance still less in the goat’; he is certain, however, that ‘in each case it begins as a split amongst the hypoblast (trophoblast) cells’. He states. further ‘that there are in the sheep, pig, ferret, goat (Assheton, 1908, fig. 5) strands of protoplasm which connect the inner lining of the inner mass to the wall of the blastocyst, and this tends to show. that the inner lining of the inner mass is of common origin with the wall of the blastocyst; that is to say, the hypoblast and trophoblast are one’. Recently, Clark (1934) has examined the condition in the sheep, and has interpreted his results as supporting the view of Assheton regarding the hypoblastic origin of the trophoblast.
In their pig egg no. 248-8 with about twenty cells, Heuser & Streeter (1929) have figured and described the demarcation between the characteristic trophoblast cells enclosing the segmentation cavity and the residual primitive cells. Comparison of their figs. 5 and 6 h and figs. 26 and 27, pl. 1 with our Text-figs. 4g, h, 5, and Pl. 5, figs. 49-52, suffices to carry conviction of the essentially similar character of the underlying structural condition. In their fig. 6h of the cut surfaces of the model of this egg and their figs. 26 and 27, which are apparently consecutive serial sections through the same egg, they certainly show a layer of trophoblast stretching across the under-surface of the primitive mass; but this is not illustrated in the model (fig. 67) of their next succeeding stage, nos. 291-5; nor is it described in connexion with fig. 81, pl. 2, which is a section through the greatest thickness of the primitive mass of this embryo. What has become of this layer is not stated; but if we may judge from appearances such as are shown in their fig. 26, pl. 1 (no. 284-8), where a cell from the under-surface of the primitive mass appears to be in process of incorporation into the outer wall of the blastocyst cavity; and in fig. 82, pl. 2 from their egg no. 291-6, where ‘the first free entoderm mother cell’ with its thinned-out process is — nearly connected to the inner cell mass, it seems on the whole at least highly probable that:
- In the younger embryo no. 284-8, cells of the wall of the blastocyst are being increased at the expense of trophoblast cells now forming a part of the under-surface of the primitive mass.
- The cellular tissue, to which the first mother entoderm cell appears to be attached, could be regarded as simply a persisting portion of the cellular mass of the trophoblast which had lost its connexion with the outer layer through the development of the blastocyst cavity. That this layer would perhaps be a network of cells rather than a continuous sheet at the time when the blastocyst cavity is very rapidly expanding, as first suggested by Assheton, seems a permissible deduction.
- The accretion of material by the wall trophoblast would also be necessitated by the rapidly increasing size of the vesicle.
- On this basis the older blastocyst no. 291-6 would be immediately derivable from the prior condition which is found to exist in no. 284-8.
Heuser & Streeter do not interpret the changes observed by them in terms of the separate layer of trophoblast contributing to the primitive entoderm, but their facts readily fit themselves into that interpretation. They are inclined, however, to think that the trophoblast does contribute some entoderm, for they state: ‘When more mature entoderm cells make their appearance with. long processes, they are found adhering to all parts of the blastocyst wall. Often we could not say but that they were arising directly from the trophoblast. However, in view of what has gone before one must not run the danger of being misled by such appearances without more satisfactory proof.’
Returning now to our blastocyst we find that it has acquired its chief identifying characteristic, that is, the trophoblast constituting the larger part of the blastocyst is present as a continuous layer and forms a thin-walled fluid-filled vesicle. At the same time, the inner cell mass has the form of a concavo-convex disc consisting of large irregular formative cells closely invested over the greater part of its outer surface by the covering trophoblast, and on its inner surface by a tenuous membrane of flat primitive entoderm cells. It is evident that in this embryo we are again dealing with a mass of cells essentially similar in character to those already described in connexion with our earlier blastocyst. Furthermore, we are convinced that the covering trophoblast cells are derived from the larger formative cells, evidence of which is provided by the occurrence in our embryo no. 13B, of a few small cells superficially placed among the larger cells (see Pl. 5, figs. 51, 52). This finding is corroborated by the exact observations of Heuser & Streeter (1929) who reported the addition. of many cells from the inner cell mass to the trophoblast. We have found no evidence in any of our goat eggs of the ‘over-growth’ which Assheton (1898a) states is ‘quite unmistakable in the sheep’, of which there is a ‘distinct suggestion in the pig’, but of the occurrence of which he (1908), oddly enough, assumes in the goat. It is noteworthy, however, that when dealing with the earliest of the goat blastocysts which he describes, Assheton found no certain means of identifying the ectoderm cells (formative) which he believed to be ‘imbedded among or between the future trophoblast and hypoblast cells’. But if we may judge from his fig. | (Assheton, 1908), it seems clear that while the hypoblast is in the form of a continuous sheet, the covering trophoblast has not yet appeared over the whole extent of the surface of the inner mass. Indeed, Assheton himself provides the evidence. He states (p. 211) ‘the cells resemble those of the sheep’s blastocyst rather than the pig’s in being larger, more clearly delimited and more homogeneous’, a description which we believe applies more aptly to large undifferentiated formative cells rather than to trophoblast.
Concerning the inner lining cells, the facts previously alluded to indicate that this network of cells which attaches to the under-surface of the inner cell mass and resembles so closely the cells of the parietal trophoblast, is, at least in part, the remains of the original trophoblast lining, and we are led to believe that it represents the precocious beginnings of the entoderm. The above should not be interpreted, however, as a rejection of the hypothesis that these primitive entoderm cells may not in part be derived from the wall trophoblast, and there are appearances which support the latter view. But even if we assume that some of these cells are not all descendants of the original trophoblast layer which clothed the under-surface of the formative mass, but have a common origin from the parietal trophoblast, the validity of our case is in no way altered. We are compelled, therefore, to admit that the inner cell mass as here constituted is morphologically equivalent to much more than that portion formed at an earlier period and contains derivatives of both formative cells and trophoblast, i.e. covering trophoblast and entoderm, but as yet contains no true ectoderm, for it .
is only when the last trophoblast cells are given off that we can speak of the residual formative cells as ectoderm. Thus considered, this prior differentiation of the entoderm in contradistinction to the ectoderm must leave us ill content to ascribe to the latter the sharp individuality so long conceded to it.
- An account is given of the segmentation of the egg of the goat.
- The vitellus of the living unsegmented.egg is composed of fatty globulés embedded in a dense mottled matrix and almost completely fills the space.within the zona pellucida.
- No corona cells were attached to the egg in any of the stages examined.
- A polar body was present in the majority of the ova. Sometimes two were seen.
- Data are given of the diameters of the different eggs, relative volumes of the blastomeres, and the age stage and location of the eggs.
- The average size of the ovum of the goat as measured by the inner diameter of . the zona is 145-31 and the zona pellucida has an average thickness of 12-5 p.
- Up to the time of formation of the blastocyst cavity the eggs do not change appreciably in size.
- Fertile goat eggs enter the uterus sometime between the close of the fourth day and the beginning of the fifth day after mating; that is, at the time when the eggs have acquired their distinctive polarity. ,
- The goat is compared with the sheep, the cow and the pig as to the rate of cleavage as far as the blastocyst.
- In the fixed and sectioned unsegmented egg there is a more deeply staining cytoplasmic crescent around a more lightly staining central mass.
- The first cleavage spindle was found in an egg at 30} hr. post- coitum, and the process of division was observed at the same time in another egg of the same litter.
- The two blastomeres resulting from the first cleavage are of unequal size.
- The second cleavage takes place at 48 hr. post-coitum, and the spindles in the blastomeres are at right angles to each other, so that at the four-cell stage the blastomeres lie cross-wise.
- Four-cell stages were recovered from the tube 60 hr. post-coitum.
- The third and fourth cleavages are slow and require from 12 to 24 hr., the eight- and twelve-cell stages being found at 85 and 98 hr. post-coitum.
- At the thirteen-cell stage, which is reached about 98 hr. after mating, one blastomere is centrally placed.
- In the late morula, the commencing formation of the blastocyst cavity is indicated by the increase in size of the intercellular spaces between the centrally placed cells and the smaller trophoblast cells at one pole of the egg. Larger (formative) cells are found at the opposite pole.
- In the early blastocyst (i.e. during the sixth day) the two portions of the egg, trophoblastic and formative, are completely differentiated. The blastocyst cavity is situated among the smaller trophoblast cells which are capped by the larger formative cells.
- The trophoblast cells which roof the incipient cavity and are in contact with the deep surface of the formative cells participate in the formation of the entoderm.
- By the close of the seventh day the blastocyst has attained its chief characteristic, that of a spherical vesicle distended with fluid. Four types of cells are recognizable, formative cells, primitive entoderm cells, covering trophoblast, and parietal trophoblast.
It is with pleasure that the authors record their thanks to the many helpers who. have willingly given their services in different ways, and especially are thanks due to the following members of the Departments of Histology and Biology, Royal Veterinary College, where the work was carried out.
Mr R. L. Williams, not only for his help in making the models and determining their volumes, but also for his very careful drawings of Text-figs. 4-7. Mr R. H. Burgess for the care he took with the animals, and Mr J..E. Hancock for his invaluable help in taking the records of the onset and duration of heat, as well as for the preparation of the sections, which he shared with one of us (W. F. B. G.), and for the excellent photomicrographs which illustrate this paper. "We are also indebted to Mr A. K. Maxwell for the drawings of Text-figs. 1-8, and Mr A. S. Cox for much helpful advice during the experiments.
Finally, our obligations are due to the Central Research Funds Committee of the University of London and to the Research Grants Committee, Royal Veterinary College, for part of the expenses of the investigation:
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Explanation of Plates
All the photomicrographs are reproduced without retouching.
Fig. 1. A living unsegmented egg (no. 4B) at 303 hr. post-coitum, illuminated with incident lighting. x 250.
Fig. 2. The same egg as that shown in fig. 1, illuminated with transmitted light. x 250.
Fig. 3. A uterine egg (no. 14A) at 140 hr. post-coitum, illuminated with transmitted light. Observe the extensive perivitelline space and the greater opacity of the vitellus as compared with fig. 2. x 250.
Fig. 4. A living two-cell egg (no. 1 A) at 48 hr. post-coitum, illuminated with transmitted light and showing unequal blastomeres. x 250.
Figs. 5, 6. A living two-cell egg (no. 4A) at 303 hr. post-coitum obtained from the same fallopian tube as the one-cell stage shown in fig. 1. Fig. 5, illuminated with incident lighting and fig. 6, with transmitted light. Observe the polar body in fig. 6. x 250.
Figs. 7, 8. Living four-cell eggs (nos. 5A, 5B) at 60 hr. post-coitum, obtained from the same female but from opposite tubes and illuminated with transmitted light. Observe the cross arrangement of the blastomeres and the large fat globules in one of the cells. x 250.
Fig. 9. The same egg as that shown in fig. 8 illuminated with incident lighting. «x 250.
Fig. 10. A living eight-cell egg (no. 2B) at 85 hr. post-coitum, illuminated with transmitted light. x 250.
Fig. 11. A nine-cell uterine egg (no. 14B) at 140 hr. post-coitum, from the same uterine horn as the one-cell stage shown in fig. 3. Illuminated with transmitted light and showing differences in the size of the cells. x 250.
Fig. 12. A living twelve-cell egg (no. 12A) at 98 hr. post-coitum, illuminated with transmitted light. Observe the differences in size of the blastomeres. x 250.
Fig. 13. A living thirteen-cell stage (no. 12C) at 98 hr. post-coitum, from the same female, but the opposite tube as the twelve-cell stage shown in fig. 12. The smaller cells are compactly arranged and a polar body is seen in the upper part of the figure. x 250.
Fig. 14. A living uterine morula of about thirty cells (no. 7B) at 120 hr. post-coitum, illuminated with transmitted light. Observe the semi-compact arrangement of the cells and the extensive perivitelline space. x 250.
Fig. 15. A living morula of thirty cells (no. 7C) at 120 hr. post-coitum, from the same female as no. 7B, fig. 14, but from the tubo-uterine junction. Observe the loosely arranged blastomeres which form groups of larger and smaller cells at opposite poles of the egg. Note the polar body in the upper part of the figure. x 250.
Fig. 16. A living uterine morula of thirty-two cells (no. 7A) from the same horn as the morula shown in fig. 14. Observe the compact arrangement of the trophoblast cells and the loosely arranged larger formative cells. x 250.
Figs. 17, 18. Two living early uterine blastocysts (nos. 13C, 13B) from the same horn at 134 hr. postcoitum, illuminated with transmitted light. Observe the clear-cut demarcation between the larger loosely arranged formative cells and the compact trophoblastic cells surrounding the incipient blastocyst cavity. Note the complete correspondence of the two eggs. x 250.
Fig. 19. The living blastocyst (no. 8) at 156 hr. post-coitum, illuminated with transmitted light and photographed at the equator. The embryonic disc is separated by the large blastocyst cavity from the flattened parietal trophoblast. x 250.
Figs. 20, 21. The same blastocyst as that shown in fig. 19, but photographed with transmitted light a ata greater depth (fig. 20) and near the surface (fig. 21). x 200.
Figs. 22, 23. Egg 4B, at 30} hr. Two consecutive sections (1-2-1 and 1-2-2) through the egg, showing the first spindle cut longitudinally. x 500.
Fig. 24. Egg 14A. Section 1-1-14 ( x 500). Observe the large fat lacunae in the degenerate vitellus.
Figs. 25, 26, 27. Three consecutive sections (1-3-20, 2-1-1 and 2-1-2) ( x 500) through egg no. 4A. Twocell stage at 304 hr. Observe the polar body in fig. 27 and the darkly stained cytoplasmic crescent in each of the blastomeres.
Figs. 28, 29. Egg no. 1A. Two-cell stage at 48 hr. Two consecutive sections 1-3-5 and 1-3-4 ( x 500). In fig. 28 the chromosomes are cut transversely in the larger cell, while in fig. 29 they are cut longitudinally in the smaller cell.
Fig. 30. Egg no. 5A at 60 hr. Four-cell stage. Section 1-4-20 ( x 500). Two cells only are visible, one, the smaller is in metaphase.
Fig. 31. Egg no. 5B. Four-cell stage at 60 hr. Section 14-2 ( x 500), showing three of the four cells.
Figs. 32, 33. Egg no. 2A at 85 hr., at the eight-cell stage (sections 1-2-2 and 11-20) ( x 500). Five cells are visible in fig. 32; three are distinctly shown in fig. 33. The majority of the nuclei are lobed.
Fig. 34. Egg no. 2B at 85 hr. Eight-cell stage (section 1-1-22) ( x 500). Four cells are visible. None of the nuclei are lobed.
Figs. 35, 36. Two sections (1-1-11 and 1-1-14) of egg 14B at 140 hr. ( x 500). In fig. 35 the two nonnucleated cytoplasmic bodies are shown in the upper half of the figure.
Figs. 37, 38. Two sections (1-1-14 and 1-1-18) ( x 500) of egg no. 12B. Ten-cell stage at 98 hr. Five cells are shown in each of the figures. Observe the distribution of the fat lacunae, in each of the sections, the polar body in fig. 37 and the irregular nuclei in fig. 38.
Figs. 39, 40. Egg no. 12A at 98 hr. Twelve- cell stage (sections 2-1-6 and 2-1-9) ( x 500). Eight cells are visible. Nuclei resting.
Fig. 41. Egg no. 12C at 98 hr. Thirteen-cell stage (section 1-3-16) ( x 500). Seven cells only are shown in the photograph, one of which is central.
Figs. 42, 43, 44. Three consecutive sections (1-2-14 to 1-2-16) ( x 500) through morula no. 7C with thirty cells at 120 hr. Four central cells of different sizes are shown. Some of the peripheral cells are in mitosis.
Figs. 45, 46. Morula 7A with thirty-two cells at 120 hr. Sections 1-2-9 and 1-2-10 ( x 500). The ‘large central cell is in mitosis and is separated by wide intercellular spaces from the peripheral flattened trophoblast cells. Observe the polar body in fig. 46.
Figs. 47, 48. Two consecutive sections 1-2-4 and 1-2-5 ( x 500), through the opposite pole of the morula shown in figs. 45 and 46. The central cells are smaller and have resting nuclei. Wide intercellular spaces separate the cells.
Figs. 49-52. Early blastocyst (no. 13B) at 134 hr. Four consecutive sections 1-1-17 to 1-1-20 ( x 500). The demarcation between the trophoblast and the residual formative cells is complete. The blastocyst cavity is represented by a series of intercellular clefts in the trophoblastic portion of the egg.
Figs. 53-60. Blastocyst (no. 8) at 156 hr. Sections 1-2-9 to 1-2-16 ( x 500). The formative mass is now almost completely enclosed by covering trophoblast on its outer surface and primitive entoderm on its inner surface. Parietal trophoblast cells surround the blastocyst cavity below and on the sides.
Cite this page: Hill, M.A. (2021, April 10) Embryology Paper - The early development of the goat (1942). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_early_development_of_the_goat_(1942)
- © Dr Mark Hill 2021, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G