Paper - The beginning and development of function in the suprarenal medulla of pig embryos (1922)
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Weymann MF. The beginning and development of function in the suprarenal medulla of pig embryos. (1922) Anat. Rec. 24(5): -294.
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The beginning and development of function in the suprarenal medulla of pig embryos
Morie F. Weymann
Department of Anatomy, Washington University School of Medicine
Since the beginning of intensive study of the endocrine glands, these organs have held an important place in the interest of students of growth and differentiation. It has been amply demonstrated that among some of the lower vertebrates endocrine function plays a significant role in the regulation of development and metamorphosis, and there are indications that in the mammals it is no less important. In the case of man, it has been suggested that variation in physical type is largely dependent on variation in endocrine activity during growth. WTiat part the glands of internal secretion play in regulating development will probably have to be determined largely by experimentation, but before conclusive experiments can be carried out it will be necessary to have a knowledge of the normal function of the endocrine glands during the embryonic period. A search of the literature shows that a number of attempts have been made to determine whether or not these glands function in the embryo, and that considerable evidence has accumulated on this point. The present work was undertaken with the view of making a study of a single gland in some convenient form in order to determine, if possible, when and how it normally begins to function.
On reviewing what is known of the development of the endocrine glands and considering the feasibility of available tests for function, the suprarenal medulla was selected as best suited for an initial study of this kind. The pig, while not well suited for experimentation, was nevertheless selected because of the abundance of easily available material and the completeness of our knowledge of its development.
Other investigators have commonly rehed on biological and chemical tests of the extract from embrj^onic suprarenals in their attempts to determine the presence or absence of function in these glands. Lewis ('16) states that Moore and Purinton were unable to find evidence of adrenaUn in the foetal suprarenals of man by the FeClg and blood-pressure tests. Svehla ('00) also reported similar results with human material, but he did find adrenalin present in glands from foetal dogs, as did Langlois and Rehns ('99) in those from sheep foetuses. Lewis was unable by chemical and biological tests to demonstrate it in human foetal suprarenals, but by the uterine strip test he got positive indications of its presence in extracts from the suprarenal gland of two full-term stiU-born infants. Lewis also tried out the color test of Folin and Denis, but concluded that it is useless in the study of foetal suprarenals, because foetal tissues in general are rich in mic acid, which gives the same blue color reaction as epinephrin.
Fenger ('12) found epinephrin by the iodic-acid method in the extract of suprarenals from pig embryos of seventy days; also in that from beef embryos of three months, and sheep embryos of three to four months. McCord ('15) showed, by the uterinestrip method, evidence of epinephrin in the suprarenals of beef embryos as early as the 113-mm. stage. Biological tests could not be carried out on younger embryos because the suprarenals were not large enough to furnish suflSicient extract, even when many were used. Langlois and Rehns believed they were able to demonstrate an effect of the suprarenal extract from sixtyday sheep embryos upon the blood pressure of a dog.
Thus it may be seen that there is considerable evidence in the literatm-e that the foetal suprarenals do function, and that they begin to function at an early period. But since there are some possible uncertainties with the biological tests, it seems desirable that the results obtained by them should be checked and supplemented b}^ a thoroughly reliable microchemical test. In the chrome reaction of the medullary cells, discovered by Henle ('65) and interpreted chemically by Ogata and Ogata ('17), we seem to have at our disposal just such a test. Since the time of Henle it has been known that suprarenals fixed in dichromate solution show a yellow-brown color in the cytoplasm of the medullary cells. This was for a time thought to be a complex adrenalin clu-omate compound. But the recent work of Ogata and Ogata has fxirnished convincing evidence that it is a simple inorganic salt, chromium dioxide. It is formed by the reducing action of adrenaUn on the dichromate. Kingsbury ('11) has presented e\-idence pointing in the same direction. He finds that the precipitates formed by various fixing fluids acting on the fresh suprarenal medulla are identical with those produced by the same fluids acting in v-itro on adrenalin.
The most critical of the Ogatas' experiments have been repeated and corroborated by the writer. It is found that if commercial adrenahn is added to IMiiller's fluid in a test-tube, a yellow-brown precipitate slowly forms. Under the microscope it has the same color as is revealed in the cji;oplasm of the medullary chromaffin' cells after fixation in MuUer's fluid. Chemical analj'sis of this precipitate shows it to be CrOg. As the precipitate is very soluble in alkali, and to a less degree in mineral acids, it was thought adidsable to see if these reagents would affect it in the same way in an albuminous solution as in the chromaffin cells themselves. To test this point, a series of sections from a suprarenal gland fixed in Miiller's fluid was mounted on slides in the usual way, while on another set of sUdes was placed a thin coating of the precipitate suspended in albumen fixative. The sUdes were numbered, and one of each set subjected to each of the several treatments mdicated in the accompanying table.
Haematoxylin and eosin stain
H. and e. stain with ammonia water
H. and e. with NBt and also acid alcohol
H. and e. with 2X HCl for 10 min.
H. and e. with 2X NaOH for 3 min.
H. and e. with 2X NaOH for 10 min.
H. and e. with 2N NaOH for 15 min.
' The term chromaffin seems to have been first applied to cells which show the Henle reaction by Kohn ('98).
Thus it is seen that the precipitate on the slide surrounded by coagulated albumen behaves in much the same manner with regard to solvents as that in the chi'omaffin cells. The fact that the acid did not affect the granules in the tissue as much as those in the artificial coagulum may very probably be explained by the more intimate relation of the precipitate to the natural protoplasm than to this artifically prepared albumen mixture. These supplementary experiments tend to support the evidence of the Ogatas that the chromaffin substance which appears in the cells is identical with the precipitate that is produced by adrenalin in the test-tube. Therefore the appearance of the Henle reaction in the suprarenal medulla has been considered by the writer as coincident with the appearance of epinephrin in this gland.
Prentiss and Arej^'s Textbook of Embryology contains the statement that the chrome reaction is an indication of the beginning of function of the gland. The data on which this statement is based are not mentioned in the book, and the writer has not been able to find a specific statement to this effect elsewhere in the literatiu-e.
The most satisfactory data with reference to the chrome reaction was found in an article by Wiesel ('01) on the development of the suprarenal medulla. Here it is stated, under his description of a 51-mm. pig, that "die in Chromsalzen geharteten Organe dieses Stadiums geben noch keine chromaffine Reaktion," which would seem to indicate that his next stage, a 63-mm. embryo, showed the brown color. In the description of the 63mm. size he makes no mention of chrome reaction. If the stage at which Wiesel first found the reaction were at 63 mm., then one might infer that the assumption of function occurs between 51 and 63 mm. But the writer finds that the reaction occurs in stages younger than these. This fact may have been easily overlooked by one not particularly interested in this phase of the problem.
In the series studied here embryos of 218, 142, 85, 75, 65, 55, 45, 40, 30 and 24 nun. were used. Other sizes were studied, but the above suffice to show the steps in the assumption of function by the suprarenal medulla. There were always several individuals of each size sectioned to check the results, and, when preparing glands from smaller embrj^os, an older suprarenal of a stage known to show the chrome reaction was always run through in the same container, in order to preclude the possibility of drawing erroneous conclusions on the basis of fortuitous variation in technique. The most satisfactory chrome salt for fixing was found to be the potassium dichromate in Muller's fluid. In the larger embryos the suprarenals were dissected out, the small embryos were sectioned in toto.
The method was as follows: After being fixed in ^Miiller's fluid for two days, the specimens were washed in running water for one hoiu" and then hardened in 70 per cent alcohol for two days more. They were then dehydrated, sectioned by the paraffin method, and stained lightlj' with neutral haematoxylin and eosin. In the younger stages sonie were stained only with haematoxylin in the hope of finding fainter chrome reactions, but this did not help as the faintest reaction could be distingmshed over a light eosin stain. Washing the specimens for twenty four hours was also tried to see if the precipitate could be washed or dissolved out in that time, but there seemed to be no tendency for this to occur.
In the following account of the supradenal of the pig at diff'erent ages no attempt is made to trace the development of the gland as a whole or to enter into the details of its histology, attention being concentrated on the appearance and development of the chromaffin reaction.
24-mm. stage. There is an accumulation of cells, apparently all of the same tj^^e, medial to the Wolffian body in the position of the future suprarenal gland. These cells, which are rather polyhedral, with no special cytoplasmic differentiation and round nuclei, have a tendency to be arranged in cords with open spaces between them. Their subsequent history shows them to be cortical cells. There is no evidence of medullary cells in the gland at this time. In the sympathetic ganghon region is a rather large group of cells which are quite embryonic in type and give no trace of a chromaffin reaction.
30-mm. stage. At this stage cells similar in appearance to those in the sympathetic ganglion are seen in the mesenchyme between the site of the suprarenal medulla and the ganglion. There is no brown color in their cA'toplasm and the nuclei are rather large and loosely reticular. A few small clumps of these cells appear on the periphery of the suprarenal anlage. The appearance would lead one to suspect migration from the ganglion.
Jfi-mm. stage. Beginning with this stage, the suprarenals were dissected out with a liberal amount of surrounding tissues, but the sympathetic ganglion was not included. Cells resembling those of the gangUon extend from its direction toward the gland. Several groups on the periphery of the suprarenal anlage appear to be penetrating between the cortical cells, and there is a difference in structure of the cells in any one group, some tending to become further differentiated as indicated by a decrease in size of the nucleus, which at this time becomes more compact and more deeply staining. There is the faintest suggestion of a chrome reaction, that is, a yellow-brown color, in the cytoplasm of these latter cells.
45-mm. stage. In this stage a few of the cells which seem to come from the sympathetic system are inside the cortical mass and numerous groups are on the periphery, with others scattered along toward the sympathetic ganglion region (fig. 1). In the cells with the darker, more deeply staining nuclei there is a faint, but definite, yellow-brown color in the cytoplasm (fig. 2). This color cannot be observed in mesenchymal or renal cells in the neighborhood. Some of these chromaffin cells are found in groups which are at some distance from the cortical anlage in the direction of the sympathetic ganglion. As these clumps of cells increase in size and as mitotic figm'es (fig. 3) are seen among them, the assumption is justified that they are multiplying as well as maturing.
55-mm. stage. Groups of medullary cells are evident in the periphery and toward the center of the cortical mass. About half of the medullary cells show the yellow-brown chrome reaction, which is of a somewhat deeper color than in those of the 4:5-nim. stage. The color also varies in the individual cells within a group, being apparentlj' deeper in those which, judged by their nuclei, are most differentiated.
60 mm. stage. There is little change from the oo-mm. stage except that the chrome reaction is more marked and more general, and some groups of medullary cells are situated more deeply in the cortical mass.
65-mm. stage. The reaction is present in the majority- of medullary cells. Islands of these latter are scattered throughout the cortex. The relative amoimt of medulla is less than in later stages. Mitotic figiu-es are seen in certam chromaffin cells.
75-mm. stage. Chromaffin cells are for the most part iu the middle portion of the gland cortex, with some peripheral and some central. The reaction is more marked, and is now of about the same iatensity in all the medullary cells.
8o-mm. stage. In this stage the medulla is not j'et entire]}central. AH medullary cells show a definite chromaffin reaction, but the maximum intensity has not yet been reached. Those on the periphery are as dark as those which are central in position. The nuclei of the chromaffin cells are not so dense as in the earlier stages, but do not have the loose network characteristic of the primitive undifferentiated cells of stages before the first appearance of the chromaffin reaction.
14^-mm. stage. AU medullary cells are central except one group seen on the periphery of the gland and a few strands running iato the cortex. The intensitj- of the reaction in the indi\'idual cell has now reached its maximmn (fig. 4) and is not darker in older stages.
218-mm. stage. ChromaflBn cells are aU cen trail j^ placed constituting a tj-pical suprarenal medulla. The density' of color is the same as in the 142-nim. stage. The individual cells have fairly abundant cytoplasm with rather dark-staining granules and spherical or oval nuclei. Many of the nuclei appear to have a brown tint, but careful studj' of the preparations shows that when this appearance is observed it is to be attributed wholly - to the brown granules in the overlying cytoplasm. The cortical and tissue cells show no brown color in the cytoplasm.
The observations recorded in the preceding descriptions may now be recapitulated in a few paragraphs which, it may be recalled, refer only to suprarenals of pig embrj^os that have been treated according to the technique indicated at the beginning of this paper.
1. The earliest indication of the chromaffin reaction that can be detected appears in embryos of about 40 nmi. In embryos of 45 mm. the reaction has become verj- definite, from which fact it appears that it is at about this time that medullarj^ cells begin to produce, or at least to store, adrenalin.
2. In early stages the reaction is observed in cells which are not at the site of the future suprarenal gland. This observation presents no difficulty if one accept the view that the medullary cells reach their definitive position by migration from the sympathetic — a view wliich is clearly set forth by TMiitehead ('02) and bj' Wiesel, who states that there is no longer any doubt that the suprarenal medulla "stammt einzig und allein vom Sympaticus und dessen Ganglien" (loc. cit., p. 141).
3. Assumption of the ability to give the chromaffin reaction occurs concomitantly with modification in the histological appearance of the cell, involving changes in the size and staining reaction of the nuclei. The suspicion might have arisen that these changes, including the chromaffin reaction, indicate degeneration of certain cells in each group, but no e%"idence was found that such is really the case.
4. Since cells that show the reaction are found in process of mitosis, it is clear that the function begins in a relatively undifferentiated condition of the cells, which are both multiplying and specializing as they approach their definitive position.
5. The fact that in the intermediate stages cells in the medullary region and those at the periphery of the cortex may show the same color intensitj- gives further evidence that the function of a cell is not wholly dependent on its position. The more mature cells, as indicated by the chromaffin reaction, are not necessarily the first to reach their destination.
6. The assumption of the function which is indicated by a chromaffin reaction takes place gradually in the medullary cells of embryos from 40 mm. to 75 mm. in length. After the latter stage apparenth' all the medullar}- cells show the reaction. Its intensity in individual cells increases from the 40-mm. to the 142-nmi. stage.
In summary, the development of function by the medullary cells of the suprarenal gland may be briefly stated as follows: The groups of embryonic cells found in the 24-mm. embryo between the sympathetic ganglia and the anlage of the suprarenal cortex give no indication of specific function. At between 40 and 45 mm. certain of them begin to show a faint chromaffin reaction in their c}-toplasm, their nuclei at the same time becoming denser, smaller, and more deeply staining. As the embryo grows these medullary cell groups penetrate the cortical anlage and finally occupy its central portion at the 142-mm. stage. During this process mitosis continues, all the cells gradually assume the capacity for a darker constant chromaffin reaction, the relative amount of c\i;oplasm increases, and the cells become from one and one-half to two times their original size. The nuclei are no longer as dense as when the cells first began to exhibit the chromaffin reaction, but are smaller, darker, and more granular thaih in the imdifferentiated cell.
These findings tend to confirm and supplement the results, so far as they go, of other workers who have used different methods and material. They fix more closely than has been done before the time at which one of the mammalian endocrine glands begins to function in a definite manner, and show that not all the medullary cells of the suprarenal gland begin their activity at the same time or in the same place. Of especial interest is the indication that the functioning of the medullary cells is dependent upon some intrinsic factor in the cell itself rather than upon its position with, reference to other tissues. Since no evidence of medullary function was found in embryos under 40 mm., at which time the principal organs and systems are well established, it does not seem probable that any primary malformations can be traced to distm-bances in the epinephrin production of the embrj'^o itself.
The writer is indebted to Dr. C. H. Danforth for suggesting the problem and for criticism of the work.
Fenger, F. 1912 On the presence of active principles in the thyroid and suprarenal gland? before and after birth. Jour. Biol. Chem., vol. 11, pp. 489-492.
Henle, J. 1865 f^ber das Gewebe der Nebenniere und der Hypophyse. Zeitschr. f . rationelle Med., Bd. 24, S. 143-152.
KiXGSBURT, B. F. 1911 The term 'chromaffin system' and the nature of the 'chromaffin reaction.' Anat. Rec, vol. 5, pp. 12-16.
KoHN, Alfred 1898 Ueber die Nebenniere. Prag. med. Wochenschr., Jahrg. 23, S. 193-195.
L.WGLOis, J. P., AND Rehns, J. 1899. Les capsules surrenales pendant la periode foetale. Compt. Rend. Soc. Biol., T. 51, pp. 146-147.
Lewis, J. H. 1916 The presence of epinephrin in human foetal adrenals. Jour. Biol. Chem., vol. 24, pp. 249-254.
McCoRD, C. P. 1915 The occurrence of pituitrin and epinephrin in foetal pituitary and suprarenal glands. Jour. Biol. Chem., vol. 33, pp. 435-438.
Og.\t.\, T., and Og.'^t.v, a. 1917 Henle's reaction of the chromaffin cells in the adrenals and the microscopic test for adrenalin. Jour. E.xper. Med., vol. 25, pp. 807-817.
SvEHLA, K. 1900 Experimentelle Beitriige zur Kenntniss der innern Secretion des Thymus, der Schilddriise und der Nebennieren von Em bryonen und Kindern. Arch. Exp. Path. u. Pharm., Bd. 53, S. 321-341.
Whitehead, R. H. 1902 The histogenesis of the adrenal in the pig. Am. Jour. Anat., vol. 2, pp. 349-360.
WiESEL, J. 1901 Uber die Entwicklung der Nebenniere des Schweines, besonders der Marksubstanz. Anat. Hefte, Bd. 16, S. 117-148.
PLATE I Explanation op figures
1 Section of the suprarenal gland from a 45-mm. pig embryo fixed in Miiller's fluid and stained with neutral haematoxylin and eosin. Magnified about X 40. The specimen shows, to the right, strands of chromaffin cells at and near the hilus and to the left the cortical part of the gland. The square indioate.s the region shown in figure 2, the circle that shown in figure 3.
2 Higher magnification of the region marked by a square in figure 1. The darker cells with small dense nuclei are the ones in which the chrome reaction is first definitely evident. The embryonic character of the other cells may be noted. This group of cells while near the cortex is not surrounded by it. Magnification about X 700.
Explanation of figures
3 Area within the circle marked on figure 1, selected to show mitosis in a cell which already exhibits the chromaffin reaction. Magnification about X960.
4 A region of the suprarenal gland of a 142-mm pig embryo fixed in Midler's fluid and stained with neutral haematoxylin and eosin. In this section the medullary cells show the chromaffin reaction at its height. The granules in the cytoplasm are of a yellowish brown color. Magnification about X 700.
Cite this page: Hill, M.A. (2020, May 28) Embryology Paper - The beginning and development of function in the suprarenal medulla of pig embryos (1922). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_beginning_and_development_of_function_in_the_suprarenal_medulla_of_pig_embryos_(1922)
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