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The effect of testosterone on the morphogenesis of the chick thymus.

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The Effect of Testosterone on the Morphogenesis
of the Chick Thymus '
JOHN E. KING? G. ADOLPH ACKERMAN AND RALPH A. KNOUFF
Department of Anatomy, The Ohio State University, Columbus, Ohio
ABSTRACT
Because of the inhibitory effect of testosterone on lympho-epithelial
nodule formation in the bursa of Fabricius, possibly through the suppression of alkaline phosphatase activity of the surrounding mesenchyme, there appears the possibility
of a comparable inhibitory effect on the thymus.
1. A single dose of one tenth milliliter (2.5 mg) of testosterone propionate in
sesame oil (Schering) was injected into chick eggs between the first and fifth days of
incubation, i.e., before and during the time of differentiation of the thymus from the
epithelial primordia and before the appearance of lymphoblasts. The thymuses of 195
chicks including 50 controls were examined after 18 days of incubation; grossly and
microscopically the thymus gland was relatively unaffected in all but a few chicks.
Because the thymus appeared unaffected by testosterone as opposed to its complete inhibitory effect on the bursa of Fabricius; and because of the apparent relationship of testosterone-labile alkaline phosphatase to epithelial nodule formation in the
bursa, further investigations were carried out on thymic differentiation and the possible role, if any, of alkaline phosphatase activity in the surrounding mesenchyme.
2 . Chick embryos between 5-10 days of incubation were fixed in 80% ethanol or
absolute acetone, embedded i n paraffin, serially sectioned and exposed to either
Gomori's glycerophosphate or Burstone's naphthol AS-MX phosphate procedures for
alkaline phosphatase activity. The mesenchyme surrounding the developing thymus
showed either a negative or a negligible reaction for alkaline phosphatase activity following these procedures. In contrast, the thymic primordium did exhibit slight phosphatase activity which appeared to be localized in the reticular-epithelial cells,
The difference i n demonstrable alkaline phosphatase activity of the capsule of
the thymus and of the subepithelial mesenchyme of the bursa of Fabricius suggests a
fundamental difference in the differentiation of the two organs in the embryonic chick.
The inhibitory effects of testosterone on tion, testosterone injected on the tenth to
the development of the bursa of Fabricius the thirteenth days of incubation causes a
in the embryonic chick may be summar- marked decrease in demonstrable alkaline
phosphatase activity of the sub-epithelial
ized in the following manner:
1. Testosterone injected into the allan- mesenchyme surrounding the developing
toic cavity of chick embryos on the fifth nodules in the bursa; a correlation beday of incubation, i.e., before the evolution tween testosterone-labile alkaline phosphaof the bursal primordium (Aspinal et al., tase and lympho-epithelial nodule forma61; Meyer et al., '59; Mueller et al., '60 tion has been suggested (Ackerman and
and Rao et al., '62) completely prevents the Knouff, '63).
Considering, then, on the basis of the
development of the bursa of Fabricius.
2. Testosterone injected after the evolu- similarities of the thymus and the bursa of
tion of the bursal primordium but before Fabricius, it was theorized that testosterthe differentiation of lymphocytic nodules one may have a similar effect on the thy(11-13 days of incubation) prevents the mus and that it may be possible to thymecdevelopment of these nodules (Rao et al., tomize a chick with testosterone. Earlier
reports have suggested that testosterone
'62).
3 . If injected after the formation of the does indeed reduce the size of the chick
lymphocytic nodules, i.e., (17 days), testos- thymus. Meyer et al. ('59) have shown
terone caused the transformation of the that 19-nortestosterone injected on the
nodules into epithelial lined crypts (Rao
1 Supported by Public Health Service Fellowship
5-fl-GI\iI-20,878-02 and research grant HE-04061-06
et al., '62).
( X E M ) from the National Institutes of Health,
It has been shown that in addition to in- Bethesda Maryland.
2 Pre-dbctoral Fellow, National Institute of General
hibiting lympho-epithelial nodule forma- Medical
Science.
ANAT. REC., 1 5 1 : 11-16.
11
12
JOHN E. KING, G . ADOLPH ACKERMAN AND RALPH A. KNOUFF
fifth day of incubation causes diminution
in the size of the chick thymus. Szenberg
and Warner ('62) have observed a decrease
in the size of the thymus of the chick
with the injection of testosterone on the
twelfth day of incubation. Others have
shown a reduction in chick thymus following the injection of 19-nortestosterone
(Papermaster and Good, '62).
It was the purpose of this investigation
to determine : (1) whether testosterone injected into the embryonic chick prior to
the evolution of the thymic primordium
from the pharyngeal pouches - i.e., prior
to five days of incubation - might result
in complete inhibition of thymic development and a suppression of lymphoblastic
transformation, and ( 2 ) whether the mesenchyme surrounding the thymic primordium showed demonstrable alkaline phosphatase activity; and if alkaline phosphatase were present, to determine whether
such phosphatase activity might be testosterone labile, and whether it played a role
here similar to that in the sub-epithelial
mesenchyme of the bursa of Fabricius.
MATERIALS A N D METHODS
1. On each of the first five days of incubation a separate series of white leghorn
chick eggs was injected with one tenth
milliliter (2.5 mg) of testosterone propionate (Schering) in sesame oil. One series
was injected on two consecutive days (second and third days of incubation). A series of controls was injected with one tenth
milliliter of sesame oil only. On the eighteenth day of incubation the thymus
glands of these chicks were examined
grossly, removed and fixed either in formalin-sublimate-acetic (FSA) or in cold
80% ethanol, cleared in benzene and embedded in paraffin. Alkaline phosphatase
activity was demonstrated in the ethanol
fixed tissues by means of the Gomori glycerophosphate (Pearse, '61) and the Burstone naphthol AS-MX phosphate - Fast
Blue RR (Burstone, '62) procedures. FSA
fixed tissues were subjected to routine hematoxylin and eosin stain. This study included a total of 145 experimental chicks
and 50 controls from four separate series.
2. At 12 hour intervals, from the fifth
to the tenth days of incubation a series of
untreated chick embryos was removed and
fixed in either cold 80% ethanol or in cold
absolute acetone. Serial paraffin sections
of the embryos were subjected either to the
Gomori alkaline phosphatase procedure
using a n incubation period of one half
hour or to the Burstone procedure with
four hours incubation. Serial sections of
FSA fixed embryos were stained with
routine hematoxylin and eosin and were
also examined. A parallel series of chick
embryos was given a single injection of
testosterone propionate (2.5 mg) on the
second, third, fourth or fifth days of incubation. Serial paraffin sections prepared
in the same manner as above were also
subjected to the procedures for alkaline
phosphatase activity and routine hematoxylin and eosin staining.
RESULTS
1. By the eighteenth day of incubation
the thymus appeared as a pair of sevenlobed cords extending from the lower jaw
to the anterior aperture of the thorax.
Most of the embryos treated with a single
injection of testosterone given from the
first to the fifth days of incubation showed
normal cellular and histological development; approximately one third of the
chicks presented a thymus decreased in
size or lacking in one or two thymic lobes.
In none of the individuals examined was
the size of the thymus markedly diminished. Those embryos treated with two
doses of testosterone, all failed to develop
beyond the day of the last injection.
Histologically the thymus of the eighteen day embryos has essentially reached
its definitive condition. The histological
appearance of the thymus of the embryos
treated with testosterone on the first, second, third and fourth days of incubation
was indistinguishable from that of the
controls. Embryos treated on the fifth day,
however, presented thymuses which appeared histologically to contain a somewhat greater number of lymphocytes in
the cortical zone than were found in the
cortex of the controls of the same age.
Distribution and intensity of demonstrable alkaline phosphatase activity in the
18 day chick thymus of both the controls
and the testosterone treated embryos were
identical; the most intense activity appeared in Hassall's corpuscles.
EFFECT OF TESTOSTERONE O N THE THYMUS
2. Upon examination of serial sections
of chick embryos removed at 12 hour intervals from the fifth to the tenth days of
incubation, we observed the development
of the thymic primordium from the time
of its evolution from the pharyngeal
pouches until the time of lymphoblastic
transformation. During this period we observed only minimal alkaline phosphatase
activity in the thymic primordium and in
the surrounding mesenchymal condensation (figs. 1-4). As the reticular-epithelial
cells of the thymic primordium developed,
they exhibited a slightly greater alkaline
phosphatase reaction than the surrounding
mesenchyme. Testosterone administered
during early embryonic development did
not appear to alter the alkaline phosphate
activity of the thymus or its surrounding
connective tissue capsule.
DISCUSSION
Morphogenetically and histogenetically
the thymus of 5 to 10 days development
and the bursa of Fabricius of 12 to 15 days
of development are relatively comparable
in the embryonic chick. The bursa between
12 to 15 days incubation exhibits a period
of proliferation of numerous epithelial
buds from the lining surface epithelium
which rapidly enlarge and undergo lymphoblastic transformation. Underlying
and encompassing each of these developing lympho-epithelial nodules is a connective tissue condensation shown to exhibit
intense alkaline phosphatase activity during this phase of embryonic development.
Alkaline phosphatase activity makes its appearance in the sub-epithelial mesenchyme
shortly before the initiation of nodule formation and becomes markedly diminished
after the fifteenth day following the
period of nodule formation and lymphoblastic transformation. Testosterone not
only inhibits epithelial nodule formation
and lymphoblastic development in the
bursa, but also inhibits alkaline phosphatase activity of the bursa1 mesenchyme.
The thymic primordium is bilateral, arising from both the third and fourth pharyngeal pouches on the fifth day of embryonic
development a time morphogenetically
comparable with the formation of epithelial
nodules in the bursa of Fabricius. The diffuse mesenchyme surrounding the epithe-
13
lial thymic primordium begins to condense, forming a distinct capsule by the
seventh day of development. Lymphoblastic transformation of the epithelial
cells of the thymus begins at this time and
is generalized by the tenth day of incubation. This phase of development corresponds closely to 13 to 15 days of incubation in the bursa of Fabricius. In contrast
to the intense phosphatase activity of the
mesenchyme surrounding the developing
lympho-epithelial nodules in the bursa only
slight alkaline phosphatase activity can be
demonstrated in the mesenchymal condensation of the developing thymus between
the fifth and tenth days of incubation in
the chick. Testosterone failed to alter even
this minimal phosphatase activity or appreciably inhibit thymic growth and development.
Although the actual inductive mechanism for the formation of epithelial nodules
and their subsequent lymphoblastic transformation in the bursa of Fabricius and
the formation of the epithelial thymic primordia from the pharyngeal pouches and
later lymphoblastic transformation in the
thymus are unknown, it appears likely that
distinct and independent mechanisms may
be in operation during these developmental
processes in the chick. The following evidence lends support to this view: ( 1 ) The
inductive role of connective tissue in inducing lymphoblastic transformation in the
embryonic thymus of the mouse has been
shown by Auerbach ('60, '61) in chimera
organ cultures. (2) The morphological
and histochemical studies of Ackerman
and Knouff ('63) indicated the close association of the sub-epithelial mesenchyme,
its alkaline phosphatase activity and vascularity with the formation and development of the epithelial nodules and their
lymphoblastic transformation in the bursa
of Fabricius of the embryonic chick. (3)
The suppression of alkaline phosphatase
activity by testosterone and a subsequent
inhibition of epithelial nodule formation
preventing the development of a lymphocytic organ suggests a causal relationship
between nodule formation and phosphatase activity. (4) The connective tissue condensation surrounding the developing thymus of the chick in contrast to the bursa
exhibits only minimal alkaline phosphatase
14
JOHN E. KING, G. ADOLPH ACKERMAN AND RALPH A. KNOUFF
activity from the time of the appearance of
the thymic primordium until lymphoblastic
proliferation has become well established.
(5) In contrast to the bursa, testosterone
given early during embryonic development
had little or no effect on the formation of
the thymic primordia, subsequent thymic
growth and development or lymphoblastic
formation.
LITERATURE CITED
Ackerman, G. A. 1962 Electron microscopy of
the bursa of Fabricius of the chick with particular reference to the lympho-epithelial nodules. J. Cell Biol., 13: 127-146.
Ackerman, G. A,, and R. A. Knouff 1959 Lymphocytopoiesis i n the bursa of Fabricius. Am.
J. Anat., 104: 163-206.
1963 Testosterone suppression of mesenchymal aIkaline phosphatase activity and
lympho-epithelial nodule formation in the
bursa of Fabricius i n the embryonic chick.
Anat. Rec., 146: 23-28.
Aspinall, R. L., R. K. Meyer and M. A. Rao 1961
Effect of various steroids on the development
of the bursa of Fabricius in chick embryos.
Endocrinol., 68: 944-949.
Auerbach, R. 1960 Morphogenetic interactions
in the development of the mouse thymus gland.
Develop. Biol., 2: 271-284.
1961 Experimental analysis of the
origin of cell types i n the development of the
mouse thymus. Develop. Biol., 3: 336-354.
Burstone, M. S. 1962 Enzyme Histochemistry.
Academic Press, New York.
Meyer, R. K., M. A. Rao and R. L. Aspinall 1959
Inhibition of the development of the bursa of
Fabricius in embryos of the common fowl by
19-nortestosterone. Endocrinol., 64: 890-897.
Mueller, A. P., H. R. Wolfe and R. K. Meyer
1960 Precipitin production in chickens. XXI.
Antibody production in bursectionized chicken
and in chicken injected with 19-nortestosterone on the fifth day of incubation. J. Immunol., 85: 172-179.
Papermaster, B. W., and R. A. Good 1962 Relative contributions of the thymus and the bursa
of Fabricius to the maturation of the lymphoreticular system and immunological potential
in the chicken. Nature, 196: 838-840.
Pearse, A. G. E. 1961 Histochemistry Theoretical and Applied. Little, Brown and Company,
Boston.
Rao, M. A,, R. L. Aspinall and R. K. Meyer
1962 Effect of dose and time of administration of 19-nortestosterone on the differentiation of lymphoid tissue in the bursa Fabricii of
chick embryos. Endocrinol., 70: 159-166.
Szenberg, A., and N. L. Warner 1962 Dissociation of immunological responsiveness in fowls
with a hormonally arrested development of
lymphoid tissues. Nature, 194: 146-147.
PLATE 1
EXPLANATION O F FIGURES
1 A section from the cervical region of a chick embryo of seven days
incubation showing minimal alkaline phosphatase activity i n the
mesenchymal condensation (arrows ) surrounding the thymus. Secondary non-enzymatic nuclear coloration is evident in the epithelial
cells of the thymus and other tissues. Gomori method for alkaline
phcsphatase, one-half hour incubation. x 350.
2
Section of a seven day chick embryo showing minimal alkaline phosphatase activity in the mesenchymal condensation i n contrast to the
epithelial cells of the thymic primordium (arrow). A slight reaction
also is demonstrable in the connective tissue immediately underlying
the epidermis ( e ) ; a n intense reaction is seen in the cells of the
dorsal root ganglion ( 8 ) . Burstone method for alkaline phosphatase
activity, four hours incubation. X 250.
3
Section showing the thymus of a chick embryo of eight days incubation. Note the thick mesenchymal condensation (arrows) showing
very little phosphatase activity i n contrast to the dorsal root ganglion
( 8 ) which shows a n intense reaction. Other tissues are non-reactive.
Gomori method for alkaline phosphatase activity, one-half hour incubation. x 250.
4
Section of a chick embryo of nine days incubation showing the lobulated thymic primordium. Secondary nuclear coloration of the thymic
cells obscures the moderate phosphatase reaction of the cytoplasm
of the reticular epithelial cells of the thymus (arrow). Gomori method
for alkaline phosphatase activity, one-half incubation. x 350.
EFFECT OF TESTOSTERONE ON THE THYMUS
John E. King, Adolph Ackerman and Ralph A. Knouff
PLATE 1
15
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