Paper - The Organization and Cell-Lineage of the Ascidian Egg 1

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Conklin EG. The Organization and Cell-Lineage of the Ascidian Egg (1905) J. Acad., Nat. Sci. Phila. 13, 1.

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Conklin 1905 TOC: I. The Ovarian Egg | II. Maturation and Fertilization | III. Orientation of Egg and Embryo | IV. Cell-Lineage | V. Later Development | VI. Comparisons with A.mphioxus and Amphibia | VII. The Organization of the Egg | Summary | Literature Cited | Explanation of Figures

I. The Ovarian Egg

Much has been written on the egg envelopes and ovarian eggs of ascidians and I shall not here go over that ground in any detail. But in searching for the earliest different iations of the egg substance it is necessary to go back to the ovarian egg, and in so doing I have found some structures the real significance of which has not hitherto been appreciated.

1. Development of the Ovocyte

In a young ovocyte the cytoplasm stains uniformly and there is no trace of yolk or of test cells. Close around the nucleus is a granular mass which is deeply colored by plasma stains, the yolk nucleus or "yolk matrix" of Crampton (1899). As the egg grows, small spherules of yolk begin to appear in the vicinity of the yolk matrix, and this yolk gradually fills the central portion of the egg surrounding the nucleus, while the cytoplasm, which is free from yolk, occupies a peripheral position. Some of the follicle cells which surround the egg at this stage then invade the egg, thus forming the " test cells" which are located chiefly in the peripheral layer of cytoplasm. My observations as to the origin of these " test cells " agree with some of the most careful work, both ancient and modern, which has been done on this subject (Kowalevsky, 18G6, 1871; Seeliger, 1882; Van Beneden and Julin, 1886; Morgan, 1890; Floderus, 189G ; Bancroft, 1899).

The earliest appearance of polarity is found in the location of the yolk matrix on one side of the nucleus and in a slight eccentricity of the latter. I consider it very probable that the yolk matrix is derived from the attraction sphere of the last ovogonic mitosis, and that the chief axis of the egg represents the cell axis which passes through the centrosome and nucleus, and which, as I have previously shown (Conklin, 1902), is preserved in every cell throughout the cleavage of the egg and probably also in all later cell divisions. If this be true, the polarity of the egg is a differentiation which is carried over from generation to generation, and as this chief axis of the egg is identical with the gastrular axis, and bears a constant relationship to the principal axes of the embryo and adult, it will be seen that at least one important differentiation of an animal is predetermined (not predelineated) at all stages. Although this chief axis of the egg is usually recognizable at all stages by a slight eccentricity of the nucleus, it is often difficult to observe it after the disappearance of the yolk matrix. No other axial differentiations of the egg are recognizable until after the fertilization.

2. Test Cells and Chorion

In the fully formed ovarian eggs the test cells lie imbedded in a peripheral layer of clear protoplasm ; this layer stains intensely with plasma stains, and in the living eggs of Cynthia contains yellow pigment granules. In Cynthia the test cells are distributed singly and pretty uniformly in this peripheral layer (fig. 61), and the same is true of Ciona at an early stage in the formation of the ovocyte (figs. 168, 169), but in the fully formed ovarian egg of Ciona the test cells are found in little masses or "nests" of from three to six or eight cells each (fig. 170). A similar grouping of the test cells has been described by Morgan (1890) in an unidentified species of Clavellina. These cells are much smaller and more numerous than the test cells of Cynthia, and are evidently formed by division of the original test cells.

The test cells of Cynthia become quite large and contain yolk spherules, though they do not stain as densely as the yolk of the egg; in Ciona the test cells are very much smaller and do not contain these spherules. About the time that the ovarian eggs escape from the ovary the test cells are extruded from this peripheral layer of protoplasm, and the outlines of the egg, which up to this time have been irregular. become more nearly spherical. It is probable that the expulsion of the test cells and the assumption of the regular spherical form by the egg have a common cause in the increase of surface tension at this time.

At the time of the extrusion of the test cells I have observed in the ovarian eggs of Ciona a faintly-staining, homogeneous layer which lies inside the outer follicle cells and outside of the egg. The test cells lie on the inner border of this homogeneous layer; from its general appearance it is highly probable that the substance of which it is composed is extruded from the egg along with the test cells. This homogeneous material does not long persist as such but soon disappears and probably goes to form the chorion. At this time the egg undergoes considerable shrinkage in size, a distinct perivitelline space being formed, and the egg becoming regularly spherical [cf. figs. 171 and 172). It is evident that this is clue to the escape of fluid from the egg, probably the homogeneous substance described above.

In this connection a word or two as to the significance of the test cells may be permissible. The fact that in Cynthia they contain yolk and grow to a considerable size, and that spermatozoa not infrequently enter them (figs. 80 and 85 sn.), may be taken as evidence that these cells are rudimentan' eggs ; a view which is held by Floderus (1896). Bancroft (1899) and others.

3. Structure of Fully Formed Ovocyte

When first laid the living eggs of Cynthia are, exclusive of the egg envelopes, about 150 /< in diameter; those of Ciona are about the same size, but in Molgula they are much smaller, being about 100 /< in diameter. The very large germinal vesicle contains an abundant granular precipitate, an enormous nucleolus, and at wide intervals within the vesicle, but chiefly near the unclear membrane, a few deeply staining chromatic granules. These granules arc small at this time and it is difficult to determine their exact shape, though many of them appear to be V- or 5T-shaped; they are the bivalent chromosomes of the first maturation division. Close around the germinal vesicle and extending out nearly to the periphery of the egg is the yolk, which exists in the form of spherules, imbedded close together in the granular cytoplasm. Finally there is the peripheral layer of deeply staining protoplasm in which the test cells were formerly imbedded and which contains no yolk, but numerous refractive spherules much smaller than those of the yolk.

In the living eggs of Cynthia this peripheral layer is clear and transparent and contains uniformly but sparsely distributed yellow pigment, which seems to be associated with these small refractive spherules. This pigment is soluble in alcohol and hence cannot be observed in fixed and prepared material ; on the other hand, the alcohol in which large numbers of these eggs have been preserved, has the color of a solution of potassium bichromate. The test cells of Cynthia also contain yellow pigment granules which are gathered close around the nuclei of these cells. It is noticeable that most of the viscera of Cynthia contain this same yellow or orange pigment, the ovaries being especially highly colored. This pigment is much denser in some individuals than in others, and correspondingly one finds some ova in which there is little or none of the pigment, while in others it is very abundant. In general the animals which have little of the pigment in their viscera are those which produce eggs with little or no pigment, while those in which the viscera are deeply pigmented produce well-pigmented eggs. The central yolk mass of the living egg of Cynthia is of a slaty gray color, while the germinal vesicle is clear and transparent. Therefore, in the living egg of this species of ascidian, three areas can be distinguished with great clearness before the maturation divisions begin, the peripheral layer of protoplasm containing the yellow pigment, the central mass of srrav volk and the clear germinal vesicle.

In Ciona and Molgula also these three areas are distinguishable in the living egg before maturation, but not so clearly as in Cynthia. In Ciona the peripheral layer is nearly transparent, the yolk is a brownish red, while the germinal vesicle is also transparent. In Molgula both the peripheral layer and the germinal vesicle are transparent, while the yolk is gray, with a faint lilac tinge. A brief inspection of the eggs of Boltenia shows that in this genus the yolk is a bright red.

This peripheral layer of protoplasm, which is present in all the ascidian ova which I have studied, is, both in living and in stained material, the most striking feature of the egg before maturation and fertilization. It is surprising therefore that in spite of this fact it has received so little attention from those who have studied the ovarian history of the ascidian egg: in fact, with a single exception, I cannot be sure that it has ever been mentioned by any previous writer on this subject. In his paper on the origin of the test cells, Morgan (1890) figures and describes this "peripheral /.one of protoplasm" in an unidentified species of Clavellina from Green Turtle Cay, Bahamas. What may perhaps be the same layer was described bj' Kowalevsky (1866) as a "gelatinous layer," in which the test cells are at first imbedded ; later, about the time of the first cleavage, the test cells move to the periphery of this layer. In these respects this gelatinous layer of Kowalevsky resembles the peripheral layer of protoplasm, but in other respects the differences arc very great; for example, Kowalevsky says that this layer is formed by the activity of the follicle cells, that it closely surrounds the blastomeres during cleavage, and that it becomes the gelatinous mantle of the adult, while the cells within it (test cells) form the cells of the mantle. In none of these respects is this gelatinous layer like the peripheral layer of protoplasm described above, and I am in doubt whether Kowalevsky actually saw this layer of protoplasm or whether he is not describing the perivitelline space between the surface of the egg and the chorion. It is interesting to note that Kowalevsky calls especial attention to the yellow color of the test cells, though he nowhere indicates that he has seen any such yellow pigment in the egg itself. Kupffer (1870), on the other hand, concluded that the test cells were formed by free cell formation from the substance of the egg, because " die Zellen gleich Anfangs genau die Farbe des Dotters haben' '; however, he nowhere indicates that the peripheral layer of the egg differs in any way from the remainder. In not one of the many later papers on the ascidian egg can I find anv reference to this peripheral layer of protoplasm, except in that of Morgan already referred to. In view of the ease with which it can be seen, both in living and in stained material, and of the very important part which it takes in development, this is most remarkable and inexplicable. If due attention had been given to this feature of the ascidian egg, it is safe to say that some of the most conflicting accounts of ascidian embryology would never have been written.

A peripheral layer of protoplasm, entirely similar in structure to that of the ascidian egg, has been observed and described by Sobotta (1897) in the egg of Amphioxus. In this case Sobotta says that the peripheral layer gives rise in large part to the inner egg membrane, which forms about the time of the maturation, but even after the formation of this membrane a portion of this layer may remain at the periphery of the egg. Sobotta speaks of the desirability of observing this layer in the living egg, and from what I have seen in the ascidians I can but emphasize this suggestion. In the ascidians this layer does not disappear with the formation of the egg membrane, to which it contributes, but collects at the lower pole when the egg is fertilized; from Sobotta's figures I judge that the same thing happens in Amphioxus.

The colors of ascidian eggs deserve some notice at this place. In 1870 v. Kupffer observed in the living eggs of Ascidia canina {Ciona inlestinalis f) that in the early stages of the ovarian egg the ooplasm is yellow ; later, as the egg ripens, this color changes to a brownish red (Kupffer, 1870, p. 10). In the later stages of development this red color is limited entirely to the walls of the alimentary tract. In different animals and at different periods of the year Kupffer found that this color varied from a bright red to an orange tone (p. 17).

Van Beneden and Julin (1884, pp. 4 and 5) call attention to the fact that Corella parallelogrannna produces two kinds of eggs, one yellow the other gray in color. Both kinds of eggs develop normally and in the same manner and give rise to larva', the endoderm cells of which are colored yellow or gray. Clavellina rissoana also produces two differently colored kinds of eggs, one pure rose the other yellowish in tint. All eggs produced by the same individual have the same color.

In the species of Clavellina studied 1>\ Seeliger (1885) the color of the protoplasm surrounding the nuclei of the cleavage cells is yellow, as an inspection of his figures of the living eggs shows (v. his plate I).

These cases, taken in conjunction with my observations on the eggs of Cynthia, Ciona, Molgula and Bollema, show that the eggs of ascidians are frequently colored; these colors are usually found in the yolk, and in the later development pass into the endoderm cells. In Cynthia the peripheral layer is also colored, and this fact leads me to hope that some other ascidian may be found in which still other portions of the ooplasm may be differentially colored.

Conklin 1905 TOC: I. The Ovarian Egg | II. Maturation and Fertilization | III. Orientation of Egg and Embryo | IV. Cell-Lineage | V. Later Development | VI. Comparisons with A.mphioxus and Amphibia | VII. The Organization of the Egg | Summary | Literature Cited | Explanation of Figures

Conklin EG. The Organization and Cell-Lineage of the Ascidian Egg (1905) J. Acad., Nat. Sci. Phila. 13, 1.

Cite this page: Hill, M.A. (2020, February 26) Embryology Paper - The Organization and Cell-Lineage of the Ascidian Egg 1. Retrieved from

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