View source for Paper - On the first appearance of the renal artery, and the relative development of the kidneys and wolffian bodies in pig embryos (1905)

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=On the first appearance of the Renal Artery, and the Relative Development of the Kidneys and Wolffian Bodies in Pig Embryos =

By Eben C. Hill. From the Anatomical Laboratory of the Johns HopKins University.


==Introduction==
A review of the literature on the development of the {{kidney}}s and Wolffian bodies in {{pig}} embryos, shows but little concerning the blood supply and the relative size of these organs. Perhaps the most extensive study is found in Keibel's Normentafel, in which the anlages and subsequent development of these glands are traced histologically. Here, however, no measurements except those of the embryos are given. In the accounts of the embryology of the arterial system there is no work which shows the relative development of the blood supply to these two glands. At the suggestion of Dr. Pohlman, I measured these organs in pig embryos, and made a number of arterial injections. These injections demonstrate the first appearance and development of the renal artery and afford an opportunity to compare the blood supply of the kidney and Wolffian body in pig embryos ranging from 20 mm. to 75 mm. in length. The measurements show the relative size of these glands at various stages from 20 mm. to 132 mm.

==Material and Methods==
The abundance of material from the slaughter houses offered every facility for numerous measurements and repeated injections upon live pig embryos, while the valuable collection of human embryos belonging to Professor Mall afforded opportunity to compare the human fetal development with that of the pig. The pig embryo is especially adapted for the study of the Wolffian bodies because of the relatively larger development of this organ in the pig than in man or rabbit.

===Measurements===
As each uterus was opened the vertexbreech lengths of the embryos were made and measurements of the kidneys and Wolffian bodies were taken. These measurements were then averaged and the results were used in plotting the curves of their relative development. In cases where the growth of these organs was abnormal, their measurements were not included in the averages.

===Injections===
In making injections, India ink, diluted to one-third of its commercial strength, was used.' In certain instances, pure water formed a precipitate, when added to the



^Taguchi's method modified. Arch. f. Mikr. Anat., 1888.




commercial ink, but this could be easily remedied by the addition of a small amount of weak ammonia. Lamp-black solutions proved to be most unsatisfactory, because of the difficulty in obtaining solutions of similar consistency. Frequently, also, this injection mass would clog the needle of the sj'ringe. All injections were made through the hypogastric artery before the embryo was removed from its surrounding membranes. In this way the organism was protected from undue pressure and from injury in handling. During the operation, the embryo still attached to a portion of the uterine wall was immersed in warm water. A ligature was passed around the umbilical cord close to the wall of the uterus, thereby preventing infusion of the injection mass through the uterine tissues. The hypogastric vein was then pricked with a needle, thus reducing the pressure in the circulatory system consequent upon the entrance of the injection fluid. All injections were made with a fine hypodermic syringe by piercing the hypogastric artery close to the abdominal wall. To obtain an even flow it was necessary to have a perfectly adjusted syringe devoid of all capacity markings. With such a syringe an even pressure of about one drop in three seconds was maintained with little difficulty. By watching the femoral arteries the progress of the fluid was followed and an injection without extravasation was obtained. After the injection, the membranes were removed and a measurement of the body length was taken. Then an abdominal incision was made and the liver and portions of the intestines were dissected away, exposing the kidneys and Wolffian bodies. In the smaller stages it was necessary to displace the Wolffian body m order to obtain a measurement of the kidney, as at this time it is completely hidden by the larger gland. The positions of these organs relative to the vertebral column were also noted, though this was more easily discerned after clearing.

===Clearing===
To clear the specimens, the Schultz method, modified by Dr. Mall, was used. The specimen was first thrown into 95 per cent alcohol until completely shriveled. As a rule forty-eight hours were sufficient to accomplish this result. Then the upper portion of the embryo, just above the twelfth thoracic vertebra, was dissected off, leaving the Wolffian bodies and the kidneys attached to the body wall. Thus during the clearing in a 3 per cent solution of sodium hydroxide, which followed, the action of the reagent could be watched and controlled. This usually took from four to eight hours. When the tissues had become translucent in this medium the specimen was transferred to 20 per cent glycerine for a week or more, and was then placed in absolute glycerine where it became transparent. Upon complete clearing the vertebral column was shown quite prominently and its position relative to the kidneys and Wolffian bodies was noted. In this absolute glycerine the organs became so firm that the remaining portions of the body wall were removed and the specimen was then placed between watch crystals for microscopic study. Injections were also attempted through the liver while the embryo was alive, but the vascularization of the Wolffian bodies is such that venous injections were most unsatisfactory. The causes of this will be taken up later.



Table Showing The Length In Millimeters of the Kidneys and Wolffian Bodies of Pig Embryos.

The measurements were made from vertex to breech, and include all of the embryos in each uterus. In case of asymmetric development of these glands in any embryo averages were made of the lengths of both organs.

Relative Size of the Kidney and Wolffian Body at Different Stages.



Vertex-Breech.


Kidney. WoltBan Body.




20


1.2


7.3




21


1.2


7.3




20


1.1


7.4


Uterus 1


23


1.2


7.2




22


1.3


7.1




21


1.0


7.3




20


1.2


7.2




28


2.5


9.0




28


2.6


8.0


Uterus 2


29 27


2.5 2.4


8.5 9.2




28


3.0


7.0




29


2.T


8.7




30


2.8


8.6




32


3.2


8.8


Uterus 3


30 33


3.5 3.4


8.7 8.8




29


2.9


8.9




34


3.5


8.7




' 31


3.9


9.2




33


4.0


9.1


Uterus 4


33


3.8


9.0




35


3.7


9.1




. 33


4.1


8.9




- 38


5.0


10.0




39


5.5


9.5




38


5.5


10.0


Uterus 5


38


5.0


10.0




39


5.0


10.0




39


6.5 abnormally


8.3 abnormally




L


large.


small.




r 39


5.5


10.0




39


5.5


9.5


Uterus 6


38


5.7


9.5




39


5.5


10.0




40


5.6


10.2




f 40


5.8


10.0




41


5.9


10.0




40


5.9


11.0




42


5.9


11.0


Uterus 7

41 43


6.8 6.0


10.0




11.2




41


5.8


10.5




42


3.9


11.3




39


5.6


11.5




. 41


5.9


10.0




(- 49


8.0


11.5




48


7.0


12.0




48


6.5


11.1


Uterus 8


49 48


6.0 7.0


12.0




10.0




49


7.3


11.0




48


7.5


11.5




. 49


8.0


10.5




f 49 1 49


7.3


11.0




7.8


10.5


Uterus 9


49


6.8


12.0




1 48


8.0


10.0




1^ 50


7.8


10.5




• 57


9.2


11.5




57


9.0


11.4




56


9.2


11.2


Uterus 10


58


9.0


11.5




57


0.3


11.0




59


9.5


11.5




. 58


9.0


11.4



62



JOHNS HOPKINS HOSPITAL BULLETIN.



[No. 167.



Vertex-Breech. 60



Uterus


13

70 67 67

f 68 67 68


Uterus


14 ^


69

67

68

. 67

f 84.5 84.5 84.0


Uterus


15

85.0 84.5 84.0 85.0

85.0


Uterus


16


84.6 84.4

90.0 91.0


Uterus


17

90.0 89.5 90.1

94.3 94.5


Uterus


18


94.3 94.0 94.4 94.3

'112.2


Uterus


" 1


112.5 111.9 112.0

120.6


Uterus


20

121.0 120.8 120,7

132.4 132.6


Uterus


21

r


131.9 133.0 132.5

155.1


Uterus


22


156.9 157.2 155.5



Kidney. Wolffian Body.


9.2




11.5


10.6


abnormally


9.8 abnormally




large.


small.


9.5




11.4


9.4




11.5


9.4




11.5


9.3




11.0


9.5




11.7


9.0




11.3


9.5




11.0


9.7




10.5


11.5




11.5


11.5




11.0


11.0




12.0


11.5




11.0


11.4




11.5


11.5




11.0


11.7




11.8


11.5




12.0


11.5




11.4


11.4




11.5


11.2




11.2


11.5




11.7


11.6




11.4


14.4




10.0


14.5




10.0


15.0




10.0


14.6




9.5


14.2




11.0


U.O




11.0


17.2


abnormally


8.0 abnormally




large.


small.


14.5




10.2


14.7




10.0


14.3




10.5


15.0




9.7


15.3




9.5


15.0




9.7


14.6




10.0


15.0




9.6


15.7




9.2


15.9




9.0


15.6




9.0


15.5




9.8


15.7




8.7


15.6




8.9


18.3




7.1


18.4




7.0


18.0




7.1


18.0




6.7


19.4




5.6


19.6




5.4


19.5




5.5


19.5




5.5


20.0


At this stage the Wolffian


20.3


bodies have


become so atro

21.0


phled at the anterior end


20.5


that accurate measurements


21.0


are Impossible.


23.6






23.8






24.0






23.8







That a balancing of function exists between these two glands is suggested by the fact that an embryo having an unusually large kidney development has correspondingly small Wolffian bodies.

In the accompanying diagram, the curves are constructed from the foregoing measurements and represent the growth of the kidney and Wolffian body as compared with the general development of the embryos. In the case of the WolfiSan body it is impossible to properly depict the atrophy of this gland by a curve, because the degeneration which occurs mainly in the cephalic end is marked rather by a decrease in width than in length.

Embryological Development of the Wolffian Body and Kidney. — As is well knovra, the Wolffian body finds its anlage in the Wolffian duct which is developed as a solid mesoblastic cord of cells, lying close to the vertebral column in the abdominal cavity. This cord of cells acquires a lumen and penetrates the urogenital portion of the cloaca. Adjacent to the anterior end of this duct on the outer side lie the mesoblastic cells which later become the Wolffian body. The rapid growth of this gland from the 7 mm. stage to that of 10 mm. has been described by Allen." He states that between 7 mm. and 8 mm. the Wolffian body is almost doubled in size, while at 10 mm. it is half as broad again, and has also increased dorso-ventrally. MacCallum has found at this same stage of 8 mm. well-formed glomeruli and tubules throughout the whole length of the organ.' The Wolffian duct is also fully developed and according to Minot is larger proportionately at this stage than at any time later.

The Wolffian bodies are elongated structures attached to the mesentery close to the dorsal wall. The anterior and posterior ends curve toward the median line, though the curve is more exaggerated in the case of the former. The anterior end tapers slightly when the gland is at its fullest development, and it is in this portion that atrophy begins. The Wolffian duct lies as a ridge on the flat dorso-lateral surface of the gland and extending from the anterior end empties into the urogenital part of the cloaca. During its course across the organ it sends out at regular intervals tubules which are very much contorted and almost encircle the periphery of the organ. The arteries supplying the gland penetrate the dorso-medial portion and form with the distal end of these tubules glomeruli similar in structure to those of the true kidney, though slightly larger.'

The venous system in this gland consists of branching capillaries which follow the periphery and anastomose profusely in the midst of the organ. Injections consequently resulted in almost solid masses of black with no definite structure. The relation of the arteries and veins is easily discerned by sections and shows a vascular arrangement similar to that of the kidney. The arteries and veins in the glomeruli are sur


A study of the foregoing tables shows comparatively little variation in the body lengths of the embryos in each uterus. In the cases of abnormal development of the kidney, it is interesting to note the corresponding size of the Wolffian body.



' Allen, B. M., Embryonic Development of Ovary and Testis of Mammals, American Journal of Anatomy, Vol. Ill, No. 2.

MacCallum, J. B., Notes .on the Wolffian bodies of Higher Mammals, American Journal of Anatomy, Vol. I, No. 3.

MacCallum, op. cit.


Febeuaet, 1905.]



JOHNS HOPKINS HOSPITAL BULLETIN.



63



rounded by an epithelial membrane similar in structure to Bowman's capsule of the kidney, and the histological structure of the tubules is also quite similar. Each tubide begins at the glomerulus as a somewhat constricted tube and widens in the middle, narrowing again before reaching the duct. The space between the tubules, according to Minot, is lined with epithelium and gives evidence of a sinusoidal circulation. Kidney. — The kidney, as has been long known, arises as a tubular diverticulum from the Wolffian duct near its entrance into the cloaca. This broadens at its distal end, forming the renal pelvis, while the tubular portion becomes the ureter. When the embryo is 12 mm. in length the pelvis of the renal between the Wolffian body and kidneys is more easily illustrated.

In Figure 1 the kidney is moving in a cephalic direction toward its permanent position and has at this stage received no apparent blood supply. To ascertain that no injection had reached the organ it was completely mascerated and studied under high power. No suggestion of arterial supply was discovered and it is probable that the renal arteries in the pig embryo as in the human fetus do not penetrate the kidneys until they have reached their permanent position. It was supposed that these arteries might enter the kidneys earlier and grow i;p the aorta as the organ ascended, but injections


Diagram showing the relative size of the Itidney and Wolffian body as compared with the length of the embryo. Averages were made of the measurements of the embryos in each ulterus except ia certain cases of abnormal development, when only the average of those normally developed was taken.



anlage is between the fifth lumbar and second sacral vertebrae, just below the division of the dorsal aorta into the right and left hypogastric arteries."

From this stage in the development of the kidney and Wolffian body, which has already been so thoroughly worked out, the following figures trace the relative development of these glands and their blood supply.

It was found best in order to avoid confusion to omit from the drawing the adrenal glands, ureters and Wolffian ducts as well as the ovary or testis. In this way the comparison and examinations of sections show no vascularization until the kidney has reached its permanent position. That the absence of injection fluid in the kidney at this stage was not due to imperfect injection is evinced by the fact that the finest capillaries of the lower extremities and viscera are completely filled.



'Lewis, F. T., The Gross Anatomy of a 12 mm. pig, Vol. VII, Americal Journal of Anatomy.




As in the human embryo, rotation of the kidney occurs before the entrance of the blood supply. According to Pohlman, this rotation takes place in the human embryo at 14 mm.° My study of sections of pig embryos places the rotation of the kidneys in this genus between 12 and 15 mm. The vascularization of these glands in the human fetus, as has also been proved by Pohlman, talces place during the time the embryo is increasing in length from 25 mm. to 30 mm. In the pig embryo, the first appearance of the renal artery which I have been able to demonstrate, occurs at 28 mm.




° Pohlman, A. G., Concerning the embryology of Kidney Anomalies, American Medicine, Vol. VII, No. 25, pages 987-990.





The Wolffian bodies at this stage are highly vascularized and the glomeruli are fully developed.

The number of arteries leading from the aorta to these glands during the early stages is approximately the same, while the increase in size is proportional to the body growth.

Figure 2 shows the entrance of the renal artery at the earliest stage, though only a very slight arterial supply to the glomeruli was discernible. The Wolffian body is more highly vascularized and has broadened and lengthened.

In Figure 3 the glomeruli of the kidney have received their arterial supply while the Wolffian arteries have increased considerably in diameter. The organ has also increased in size. The heavy injection shown in the dorso-medial region indicates as in the first two figures the region of the glomeruli. It is around these that the tubules end. The position of the organs relative to the vertebrae is but slightly changed. At this stage the development of these vertebrae and the kidneys and Wolffian bodies seems quite proportional.

Figure 4 shows the rapid increase in the vascularization of the kidney and its relative growth. A slight atrophying of the anterior portion of the Wolffian body has begun, and the arterial supply of the glomeruli of this part of the gland is less extensive than in the posterior end, where the blood supply still continues to increase.

In Figure 5 the four anteriorly situated arteries to the Wolffian body have decreased in diameter and the accompanying glomeruli show consequent lack of blood supply. The posterior glomeruli on the other hand are still well vascularized, and there is no atrophy of the gland itself in this portion. The kidney manifests a further development, both in the number of its glomeruli and in its uniform growth.

In Figure 6 it was found to be impossible to separate the two glands so that we find the Wolffian body closely attached to the lower ventral border of the kidney. The atrophy of the anterior portion of the Wolffian body has become most evident, though this degeneration is more manifest in width than in length. The glomeruli in this part of the gland have practically no blood supply and several of the arteries have entirely disappeared. By this time the cortex of the kidney has become so thickened that only the bhirred outlines of the arteries are discernible.'

Figure 7 shows the complete disappearance of the anterior Wolffian arteries and the absence of glomeruli in this portion of the gland. Three of the posterior arteries still persist and supply the glomeruli of this part of the organ, though one of these seems ready to atrophy. In the kidney development there is little more to be noticed except a general increase in size and blood supply.


'Recent injections which were cleared in a saturated solution of sodium hydroxide instead of a 2% solution bring out quite distinctly the course and distribution of the renal arteries and the location of the glomeruli in the kidneys of pig embryos 68 mm. and 75 mm. in length. The general arrangement of these arteries and glomeruli is similar to that found in figure five.




Injection methods give no further information concerning the Wolffian body after this stage. It is well known, however, that the anterior portion continues to rapidly diminish in width and later in length, and that the remaining tubules are finally claimed by the testis or ovary.

In the male the Wolffian duct becomes the canal of the epididymis, the vas deferens and the common ejaculatory duct, and the remains of the gland itself become the vasa afferentia, ductuli aberrantes and the rudimentary paradidymis.

In the female, the duct becomes the longitudinal duct of the parovarium and the hydatids of Morgagni, while the tubules of the Wolffian body are transformed into the rudimentary tubules of the parovarium and of the paroophoron.

==Summary==

A. The renal artery penetrates the kidney when the embryo has attained a length of 28 mm. ; rotation of this gland having occurred between 12 mm. and 15 mm.

B. A balancing of function is suggested by a study of the measurements of these glands.

C. As the blood supply to the kidney increases there is a corresponding atrophying of the Wolffian arteries.

D. Atrophy of the Wolffian arteries is first evident when the embryo is 45 mm. in length.

E. As atrophy of the Wolffian body itself continues, the sex gland becomes more firmly attached to the posterior portion, and the remaining tubules and duct are claimed by the ovary or testis.


Before completing this article, I wish to thank Professor Keibel, of the University of Freiburg, for the privileges of his laboratory, in which a portion of this work was done.