Re: ASTER (All Version) 2011 TRiViUM 64 Bit
Achmat Das
The history of the early development of the bird's egg has been obscure because of the difficulty of obtaining abundant material in a close series of consecutive stages. On account of the regular egg laying habits of pigeons and because they breed readily in confinement, they offer invaluable material to the student of bird embryology.
The pigeon lays two eggs. The first is laid about 4.30 or 5.00 o'clock in the afternoon. About eight o'clock in the evening of the same day the second egg leaves the ovary and is fertilized at that time. It is laid about 1.00 or 1.30 p. m. of the second day following the time of laying of the first egg; that is, it is laid about forty -one hours after fertilization. It is evident that after the first egg is laid, the pigeon may be killed and the second egg obtained in any desired stage.
These facts were published by Dr. E. TI. Harper ('04) in his thesis on The Fertilization and Early Development of the Pigeon's Egg." In regard to ovulation, Dr. Harper says (p. 352), "In all cases observed, this has taken place between seven and nine o'clock." In this paper I shall refer to eight o'clock in the evening as the ai)proximate time of fertilization, although the exact time for any particular egg is not known.
For this research, one hundred and forty-four eggs have been obtained, covering the period required for the egg to pass through the oviduct. Of these, there is an egg for every hour, with more abundant material at critical stages. The present thesis refers especially to the first fifteen hours after fertilization. Problems in later oviducal development are reserved for publication at some future time.
My thanks are dne to Professor Whitman and other members of the Department of Zoology for a fellowship and an assistantship, which has made it possible for me to carry on this research. Professor F. R. Lillie, who suggested the problem, has followed the work with helpful interest, and I am particularly indebted to him. Professor W. L. Tower has given indispensable help in the technique of photography, and Professor C. M. Child has helped me with some literature. Figs. 5, 6, 9, 11 and 14 are the work of Mr. Kenji Toda.
I. Methods. Following the methods of workers who have preceded me, the blastoderm has been killed and hardened on the yolk and the orientation marked with a bristle. Immediatelv after a window has been
made through the shell, a bristle is inserted in the side of the yolk toward the blunt pole of the shell. Later (usually when the egg is in 70 per cent alcohol) a iive-sided piece, including the blastoderm, is cut out from the yolk. One side of the five-sided area is perpendicular to the chalazal axis and is toward the large pole of the egg. Two sides are parallel to each other and to the chalazal axis, and the last two sides meet in a sharp angle pointed toward the small pole of the egg. Fig. 1 explains this orientation, the anterior side of the blastoderm being toward the point of the arrow. This fivesided block is easily seen in the paraffin cake for orientation in cutting. In some eggs there is little difference between the blunt and sharp poles of the shell, and in these the orientation is uncertain after the egg has been taken out of the oviduct. While the egg
is passing tliroiigh tlie oviduct, the small end is directed posteriorly. I have taken the precaution to indicate by a pencil mark on the shell the orientation of the egg in the oviduct. Or, if it was obtained before the formation of the shell, the orientation was marked with a bristle inserted into the yolk before the egg was removed from the oviduct.
As for killing fluids, I have used Kleinenberg's picro-sulphuric acid (strong solution) plus ten per cent acetic acid more than any other. Flemming's fluid is good for surface views, but not for material which is to be sectioned.
Formalin (three or five per cent) has been favorable for killing those eggs that were to be used as whole mounts. With Conklin's hsematoxylin stain they present considerable differentiation in different regions of the blastoderm.
In photograi^hing the eggs, an arc light has ])een used for illumination. Sometimes the eggs have been removed from the shell and albumen and placed in a dish of salt solution. Others have been photographed in formalin after having been, removed from the egg envelopes. In a few cases the egg was left in the shell, through which a window was made in order to expose the blastoderm. Of course, when the egg is in this position, it is difficult to get sufficient light directed down into the egg and onto the blastoderm. One cannot be sure that such a photograph shows all of the accessory cleavage, and other details. The magnification was obtained by photographing through a microscope with ISTo. 1 Leitz ocular, and No. 2 Leitz objective.
1 pb, first polar body; 2 pb, second polar body; ^, male pronucleus; 9 , female pronucleus ; sn, supernumerary sperm nuclei ; mp, marginal periblast ; cp., central periblast ; wy, white yolk ; n P, nucleus of Pander.
Fig. 6) and were described by liim as follows: "The slightly oval disc has a greater diameter of 3.5 mm. It is divided into two zones quite clearly distinguished in opacity, the outer zone being due to the abrupt thinning out of the fine granular matter of the disc." Tliese two areas are the blastodisc and the periblast. In an egg obtained at 11 p.m., about three hours after fertilization, the blastoderm was 2.96 mm. in transverse and 3.08 mm. in longitudinal diameter. These measurements were taken on the living egg. Fig. 45 shows the surface view of such an egg.
The central transverse section of another egg gives the diameter of the blastodisc about 2.5 mm. This egg was taken from the oviduct at 11.30 p.m. (three and one-half hours after fertilization). In the sections I found the male and female pronuclei and a number of supernumerary sperm nuclei. The latter had migrated peripherally from their place of entrance and occupied a circle at the inner margin of the periblast. Several nuclei were peripheral to those at the inner margin of the periblast. They are probably not nuclei of the original migration, but may be sisters to those just central to them, if we may suppose that a nuclear division has taken place since the migration into the periblast. Fig. 2 shows a central transverse section of this egg, but the nuclei were in several successive sections. See also a diagram traced from this figure, Fig. 3.
The distribution of the sperm nuclei, their disappearance, and later the distribution of the periblast nuclei are illustrated in seven charts. The figure lettered A in each chart represents the surface view of the egg. The orientation is the same for all the surface views presented in this thesis. The anterior side of the blastoderm is away from the observer, and the axis of the future embryo is ill a diagonal position, as indicated in Fig. 1. These fig-ures (A of the charts) were drawn from the living eggs in salt solution to the same scale as nearly as possible in free hand work. \Vlien a dotted circle occurs in A or B of any chart, it represents the apparent peripheral limit of the marginal periblast. The supernumeraiy sperm nuclei, and later the periblast nuclei, may migrate peripherally to this distance. How much further the cytoplasm of the periblast extends I do not know : for I cannot demonstrate the existence of
cytoplasm in the mass of yolk granules except by the presence of nuclei and except in a few places where cytoplasmic islands and strands appear. The periblastic zone, as shown by a dotted circle in the charts, appears in the surface view of the living eggs at certain
stages and is shown in several photographs. Fig. B is a diagram reconstructed from a st-iidy of transverse sections, and Fig. C is a diagram of a central transverse section. The sperm nuclei and the periblast nuclei represented in Fig. B of the several charts are
in superficial positions in the marginal periblast, but those within the circle bounding the blastodisc are in the region recognized as the central periblast. They are therefore deep and are not confused with the nuclei of primary cleavage. The following characters are used in the charts :
Chart I. From a study of the sections, then, it becomes evident that before the appearance of the first cleavage plane the supernumerary sperm nuclei migrate into the periblast and occupy a circle which is later characterized by accessory cleavage. This is illustrated in Chart I. A central transverse section of the same egg is shown in Fig. 2.
Chart II A represents the surface view of an egg in the fourcelled stage. It was taken from the oviduct at 2 a.m., or about six hours after fertilization. The blastodisc is incompletely divided into four blastomeres which are continuous at their outer margins. They are also continuous below with the yolk, see Chart II C and Fig. 4.
The blastodisc is surrounded by the periblast, the peripheral limit of which as it appears in surface view, is indicated here by a dotted circle. At the inner margin of the periblast there is an incomplete zone of accessory cleavage. Where the accessory cleavage does not appear, the blastomeres are continuous peripherally with the periblast. This fact will be noted again in the description of later stages. (Chart III A, Figs. 9, and 10 A and B.)
In the sections fewer sperm nuclei were found than in the maturation stage, which indicates that a varying number enter the egg (Harper '04, p. 362). A number of sperm nuclei had migrated in superficial positions to the outer margin of the zone which the periblast presents in surface view and others were in the central periblast. Although the central periblast is not at this stage separated from the blastomeres, it may be identified with the finely granular region below the blastodisc, and the thickness of the latter
is determined by the depth of the vertical cleavage plane. The latter measures 0.12 mm. in this egg, and the first horizontal cleavage (Fig. 5) which marks the position of the future segmentation cavity occurs later at the same depth. Compare Figs. 2, 3, 4, 5, 6, and 11,
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