The developmental endocrinolog of the spleey n in chick ...

/ . Embryol. exp. Morph. Vol. 24, 2, pp. 357-365, 1970 357
Printed in Great Britain

The developmental endocrinology of the spleen
in chick embryos

I. The pars distalis

By T. W. BETZ1

From the Department of Biology, Carleton University

SUMMARY

Partial decapitation ('hypophysectomy') of the chick embryo significantly reduces body
growth by 20 days (stage 46 —) of incubation as indicated by body weights, 60 % of normal;
the lengths of the toe and tibiotarsus, 80 % and 78 % of normal respectively; and liver growth,
47 % of normal, but the gall bladder was not apparently enlarged. It significantly increases
spleen growth to 82 % greater than normal, suppresses or retards white pulp differentiation
and splenic vasculogenesis but enhances red pulp development.

A single pars distalis gland placed as a chorioallantoic graft into operated embryos pre-
vents the development of these defects except for liver growth which, while improved, is still
subnormal. If the number of grafts is increased by one or two there is no change in the
amount of influence on growth and development of the chick embryo. This apparent regula-
tion occurs by some unknown mechanism even in the absence of the hypothalamus.

Thus body and liver growth is normally stimulated by the pars distalis but spleen growth
and red pulp differentiation are suppressed even though the gland stimulates splenic white
pulp histogenesis. The hypothalamus, epiphysis and pars nervosa (removed by partial de-
capitation) are not apparently involved in the developmental endocrinology of the spleen in
chick embryos.

INTRODUCTION

Partial decapitation results in splenomegaly (Case, 1951; Konigsberg, 1954;

Ebert, 1957; Betz, 1967, see review of Hinni & Watterson, 1963), which appears

on the 16th day of development (Vogel, 1956), becoming more pronounced as

development proceeds so that by 17 days of incubation the spleen has enlarged

50% as compared to normal (Case, 1951). By the 18th day the spleen is 47% to

56% larger than normal (Vogel, 1956; DeLanney, Ebert, Coffman & Mun,

1962). Ordinarily the increases in spleen and body weight are proportional

(Vogel, 1957) but in partially decapitated embryos the body weight increases

71 % between days 14 and 18 of incubation and the spleen 137 % over the same

period. Also the free cholesterol content per unit weight of the spleen declines

sharply during the same period (Vogel, 1957). Splenomegaly in 20-day-old

partially decapitated chick embryos was apparently prevented or alleviated by

1 Author's address: Department of Biology, Carleton University, Colonel By Drive,
Ottawa I, Canada.

23-2

358 T. W. BETZ

pars distalis grafts on the chorioallantois (Betz, 1967) but the extent of the
correction had not been analysed.

The results of this study demonstrate that pars distalis grafts restore the de-
velopment of the spleen of partially decapitated chick embryos to normal.

MATERIAL AND METHODS

The experimental design is summarized in Table 1. Fertile White Leghorn
eggs (Hyline strain 934E) were selected at random and incubated without turn-
ing at 37-2±0-5 °C and 6 0 ± 6 % relative humidity. The normal embryos for
group 1 received no further treatment. The embryos for groups 2-5 were
'hypophysectomized' by partial decapitation at 36-40 h of incubation (Betz,
1967). The operated embryos for groups 3-5 at 9 days of incubation received one,

Table 1. Experimental groups for the effects of pars distalis hormones and their
absence on the development of the spleen in (stage 46 — ) chick embryos

Group Treatment No. of Theoretical
specimens amounts of
no. Normal pars distalis
' Hypophysectomized' 15 hormones
1 ' Hypophysectomized' plus one 10
2 pars distalis graft 10 +
3 ' Hypophysectomized' plus two
pars distalis grafts 10 0
4 ' Hypophysectomized' plus three
pars distalis grafts 2 +
5
++

+++

two or three pars distalis glands respectively from 10-day-old donor embryos as
chorioallantoic grafts (Betz, 1967). After 20 days (at stage 46— : Hamburger &
Hamilton, 1951) of incubation (20x24h + 3 h for thermal equilibration) the
grafts and embryos were collected and fixed in Bouin's fluid (Betz, 1967).
Interference with albumen ingestion and consequent growth was minimized
by including only partial decapitates with an open esophagus (Betz, 1968). The
heads of all embryos were removed to make the bodies comparable (Betz, 1967,
1968). The fixed headless bodies were blotted and weighed to the nearest 0-1 g
and the lengths of the right third toe (Betz, 1968) and tibiotarsus were measured
to the nearest 0-5 mm. The spleens were cleaned of adherent tissues, blotted and
weighed to the nearest 001 mg. The livers were also weighed after the gall
bladders had been removed. The means and standard deviations were calculated
for the lengths of the tibiotarsus and third toe and for the weights of the body,
spleen and liver (Table 2). References to significant differences between the
means are based on the results of an F test at the 005 level of significance and
Duncan's new multiple range test at the 001 level of significance. The mean
weight of the spleen as a ratio of the bone lengths and of the organ and body

Development of the spleen: I 359

weights and the ratio of liver to body weight were calculated (Table 3). The experi-
mental values were compared to the normal values by expressing them as a
percentage of the normal values (Tables 2, 3). Representative spleens were pre-
pared for histological examination with an ordinary paraffin technique, serially
sectioned at 8 /t and stained with hematoxylin and eosin. Representative sections
were photographed.

RESULTS

The gross morphology of the embryos was defective in group 2 and appeared
normal in groups 3-5, except that they lacked the derivatives of the head parts
resected in the operation as described before (Betz, 1967, 1968); of endocrino-
logical importance the hypothalamus, epiphysis, pars distalis and pars nervosa
are missing. All of the grafts were of normal size and well vascularized as
reported by Betz (1967, 1968).

Table 2. The average lengths of the right tibiotarsus and third toe, and weights
of fixed headless body, liver and spleen as affected by various amounts of pars
distalis hormones and their absence in the five experimental groups

Group Tibio- Nor- Toe Nor- Body Nor- Liver Nor- Spleen Nor-
tarsus mal (mm) mal (gm) mal (mg) mal (mg) mal
(mm) (%) (%) (%) (%)
(%)

1 191* 100 20-4 100 14-9 100 448-0 100 6 0 100

±1-06 ± 1-54 ±2-83 ±71-57 ±1-27

2 14-9 78 16-3 80 89 60 212-9t 47 10-9 182

±1-60 ±1-82 ± 1 -25 ±47-60 ±2-81

3 18-5 97 20-6 101 13-4 90 331-4 74 7-3 122

±1-43 ±1-43 ±1-83 ±27-79 ±2-08

4 18 6 97 210 103 13-7 92 323-8 72 7-7 128

± 107 ±1-69 ±2-02 ± 66-40 ±2-25

5 190 99 200 98 12-5 84 282-4J 63 71 119

+0 + 2-12 + 2-76 + 51-55 + 3-81

* The underlined between-group means are not different from each other as indicated by Duncan's
new multiple range test at the 001 level of significance.

f Group 2 is significantly different from all but group 5.
X Group 5 is not significantly different from any but group 1.

Body growth (Table 2) as indicated by the differences in the body weights and
the lengths of the tibiotarsus and toe is significantly reduced in the absence of the
pars distalis and is not greater than normal if more than one pars distalis graft is
used. Tibiotarsus and toe growth are depressed to the same degree by partial
decapitation but body growth is more severely reduced.

Liver growth while depressed by' hypophysectomy' is only partially restored by
one pars distalis graft and two grafts are no more effective. In group 5 liver
growth is intermediate between group 2 and groups 3 and 4, probably due to

360 T. W. BETZ

the small size of the group and a consequent sampling error. Liver growth rela-
tive to body weight (Table 3) is not as severely depressed by ' hypophysectomy' as
indicated by the differences between the absolute and relative liver weights but
the degree of repair in groups 3, 4 and 5 is not apparently greater. The gall
bladder did not appear to be enlarged by 'hypophysectomy', in contrast to the
results of Konigsberg (1954) in chick embryos and by Nalbandov & Card
(1943) in growing chickens. The reason for this difference requires further study.

Table 3. The average spleen weight as a ratio of tibiotarsus and toe length, body
and liver weight and the ratio of liver to body weight in the five experimental groups

Spleen/ Normal Spleen/ Normal Spleen/ Normal Liver/ Normal
Group tibiotarsus (%) toe body (%) body

1 0-314 100 0-294 100 0-410 100 30-4 100
±0061 228 ±0-104 300 ±4-46 78
±0071 119 124 23-8* 82
0-672 126 1-226 139 ±4-24 78
2 0-744 237 ±0189 122 ±0-364 139 25_1 75
±3-86
±0-220 0-352 0-505 23^6
±0102 ±0136 ±307
3 0-394 125 22^8_
0-369 0-565 ±8-94
±0-112 ±0096 ±0-118

4 0-416 132 0-357 0-549
±0-153 ±0-182
±0-120

5 0-377 120

±0-371

* The underlined between-group means are not different from each other as indicated by Duncan's
new multiple range test at the 001 level of significance.

F I G U R E S 1-8

These are prints of photomicrographs of representative sections of the spleens of
20-day-old chick embryos (stage 4 6 - ) from the five experimental groups. They were
fixed in Bouin's fluid, embedded in paraffin, sectioned at 8/*, and stained with
hematoxylin and eosin. All are magnified x 400. The arrows point to a white pulp
follicle (light grey areas) surrounded by red pulp.

Figs. 1, 2. The normal range of variation in spleen histology (group 1). The follicles
are compact and crisply delimited from the red pulp.

Figs. 3, 4. The range of variation in spleens of untreated 'hypophysectomized' chick
embryos (group 2). The follicles are not apparent (Fig. 3) or reduced in number and
size and are more compact (Fig. 4).

Figs. 5, 6. The range of variation in spleens of 'hypophysectomized' chick embryos
with one pars distalis, chorioallantoic graft (group 3). The number and organization
of the follicles are within the normal range of variation. Compare with Figs. 1, 2.
Fig. 7. A representative spleen from 'hypophysectomized' chick embryos with two
pars distalis grafts (group 4). The histology is not appreciably different from
normal (Figs. 1, 2) or the spleens of the other treated 'hypophysectomized' embryos
(Figs. 5, 6, 8).

Fig. 8. A representative spleen from 'hypophysectomized' chick embryos with three
pars distalis grafts (group 5). The histology is not appreciably different from normal
(Figs. 1, 2) or the spleens of the other treated 'hypophysectomized' embryos (Figs.
5-7).

Development of the spleen: I 361

362 T. W. BETZ

' Hypophysectomy' results in a remarkable splenomegaly by the 20th day of
development. Spleen growth is reduced to normal by one pars distalis graft but
the addition of one or two grafts does not further inhibit growth. The enhance-
ment and suppression of spleen growth relative to the body is more pronounced.
If other parameters of body growth (tibiotarsus and toe length) are used instead
of body weight and differences between experimental groups do not change
appreciably except that the spleen growth caused by 'hypophysectomy' is
enhanced (Table 3).

Normally the spleen has well differentiated follicles of white pulp surrounded
by red pulp by the 20th day of incubation (Figs. 1, 2). Partial decapitation
suppresses or retards the differentiation of the white pulp (Figs. 3, 4) so that at
best the frequency and size of the follicles are severely reduced. Even under the
oil-immersion objective there are no signs of white pulp differentiation in the
more severely affected spleens (Fig. 3). The histological changes in red and white
cells induced by partial decapitation are not simply due to an altered staining
reaction of the cells as they can also be distinguished by structural differences.
In group 2 the small follicles are denser than normal and in groups 3, 4 and 5 the
follicles tend to be less dense than normal. In addition, the vascular development
is subnormal in that the diameter of the vessels is larger and they are deficient in
connective tissue. The spleens of 'hypophysectomized' embryos with 1, 2 or 3
pars distalis grafts are within the normal range of variation and are not different
from each other (Figs. 5-8).

DISCUSSION

It has been indicated that the endocrinological effects of partial decapitation
are due to adeno-hypophysectomy (Betz, 1970). In this experiment, perhaps
contrary to expectation, one pars distalis graft was just as effective as two or
three in preventing the defects induced by partial decapitation. Thus the amount
of influence the grafts have on the development and growth of these organs and
the body is seemingly regulated even in the absence of the hypothalamus. That
the organs are capable of additional response is indicated by their hypertrophy
after exogenous hormone treatment (Betz, 1970). Therefore the basis of apparent
regulation could be that even two additional glands as grafts simply do not
produce enough hormones to influence the spleen additionally. However, this
does not seem to be the case as one graft can fully restore the other defects pro-
duced by partial decapitation in chick embryos. Therefore it seems more probable
that the total hormone output of two or three healthy grafts has been regulated
in some way to give the same concentration and effect as produced by one graft.
Perhaps the grafts inhibit the hormonal output of each other, or perhaps
negative feedback inhibition by the endocrine end organ hormones (adrenal and
thyroid glands) is operative in regulating the amounts of hormones produced by
the grafts. The answer awaits analysis (see discussion of Betz, 1967, 1970). In
any event, the hypothalamus, epiphysis and pars nervosa are not apparently

Development of the spleen: I 363

involved in the developmental endocrinology of the spleen in chick em-
bryos.

The results demonstrate that spleen growth is normally inhibited by the pars
distalis. The gland stimulates red pulp histogenesis but inhibits white pulp
differentiation. Adrenocorticoids, especially the ll-/?-hydroxy derivatives, are
able to inhibit splenomegaly induced by a graft-versus-host (GVH) reaction
(Warner & Burnet, 1961). In the chick embryo corticoids begin to be released
from the adrenal glands probably by 12 days of incubation (Betz, 1970) and the
first signs of splenomegaly induced by partial decapitation appear on day 16 of
development (Vogel, 1956). Perhaps the growth of the spleen is normally in-
hibited by endogenous adrenocorticoids; the secretion of which (in amounts
sufficient to inhibit growth) depends on ACTH from the embryonic pars distalis
(Betz, 1970). Vogel's results (1956) may indicate that the peripheral titer of
corticoids first becomes high enough to inhibit spleen growth on the 16th day of
development or at least the spleen becomes sensitive to corticoid deprivation on
that day. The extent to which other hormones are involved in the growth and
histogenesis of the chick spleen awaits further research.

The vascular changes in the spleen in the groups of embryos may indicate that
the vascular development of the spleen in partially decapitated embryos is sub-
normal as occurs in other organs of these operated embryos (see review of
Hinni & Watterson, 1963) and is prevented by pars distalis grafts (Betz,
1967).

The splenomegaly induced by a GVH reaction (Van Alten, 1959; Van Alten &
Fennell, 1959) is paralleled by a proportionate hepatomegaly (Solomon, 1961)
which is dose-sensitive (Seto, 1966). However, splenomegaly induced by partial
decapitation is accompanied by a significant decrease in liver growth which for
some unknown reason is not completely alleviated by pars distalis replacement
therapy. The present results concur with those of Solomon (1961) in that there
is no apparent sexual dimorphism in splenomegaly or hepatomegaly, indicating
that sex steroids are probably not important in the in vivo development of the
spleen.

Ebert (1957) and DeLanney et al (1962) showed that splenomegaly induced
by a GVH reaction proceeds without modification or reduction in partially
decapitated embryos. In fact the spleen grows slightly more rapidly. It is tempt-
ing to suggest that this slight acceleration of spleen growth is due to a release
from adrenocorticoid inhibition in the operated embryos.

Splenomegaly induced by partial decapitation is different from that induced
by a GVH reaction in that (1) the growth rates of the spleen are different
(De Lanney et al. 1962), (2) there is no hepatomegaly (Seto, 1966), (3) the en-
larged spleens have subnormal white pulp differentiation while that of the red
pulp is enhanced, and (4) there are no signs of cystic development or necrosis
(Fennell, 1966).

Currently a study is being made of the effects of other hormones in addition

364 T. W. BETZ

to hydrocortisone on the developmental endocrinology of the spleen of chick
embryos.

RESUME

Vendocrinologie du developpement de la rate dans les
embryons de Poulet. I: La pars distalis

La decapitation partielle (' hypophysectomie') d'embryons de Poulet a 20 jours d'incubation
(stade 46 — ) reduit Ja croissance corporelle de facon significative: de 60% lorsqu'elle est
calculee par le poids du corps; de 80 et 78 % respectivement pour la longueur de l'orteil et du
tibiotarse; de 47 % pour la croissance du foie dont la vesicule biliaire n'etait apparamment pas
agrandie. Cette operation augmente de facon significative la croissance de la rate de 82 % par
rapport a la normale; elle supprime ou retarde la differenciation de la palpe blanche et de la
vasculogenese splenique mais accroit le developpement de la pulpe rouge.

Uneseule pars distalis hypophysaire placeeen greffechorioallantoidiennedansdesembryons
operes, empeche la realisation de ces deficiences sauf pour le foie dont la croissance, bien
qu'amelioree reste subnormale. L'augmentation du nombre de greffes d'une a deux unites n'a
pas d'influence sur la croissance et le developpement de I'embryon de Poulet. Cette regulation
apparente se manifeste meme en l'absence de Phypothalamus par un mechanisme qui reste
inconnu.

Done, la croissance du corps et du foie est normalement stimulee par la pars distalis mais la
croissance de la rate et la differenciation de la pulpe rouge sont supprimees bien que la pars
distalis stimule Phistogenese de la pulpe blanche splenique. L'hypothalamus, l'epiphyse et la
pars nervosa (enlevees par la decapitation partielle) ne sont pas apparamment concernees
dans l'endocrinologie du developpement de la rate chez I'embryon de Poulet.

This research was supported by a grant from the National Research Council of Canada. I
am particularly indebted to Mr D. L. Mallon for his expert technical assistance.

REFERENCES

BETZ, T. W. (1967). The effects of embryonic pars distalis grafts on the development of hypo-
physectomized chick embryos. Gen. comp. Endocr. 9, 172—186.

BETZ, T. W. (1968). The effects of embryonic pars distalis grafts and albumen on the growth of
chick embryos. /. Embryoi exp. Morph. 20, 431-436.

BETZ, T. W. (1970). The pars distalis and avian development. In Hormones in Development
(ed. E. J. W. Barrington & M. Hamburgh). New York: Appleton-Century-Crofts. (In the
Press).

CASE, J. F. (1951). The Effects of Decapitation on the Functional Development of the Adrenal
Cortex in the Chick Embryo. Doctoral Dissertation, The Johns Hopkins University.

DELANNEY, L. E., EBERT, J. D., COFFMAN, C. M. & MUN, A. M. (1962). On the chick spleen:
origin; patterns of normal development and their experimental modification. Carnegie
Jnstn Wash., 621. Contr. Embryoi. 37, 57-85.

EBERT, J. D. (1957). Annual report of the director of the Department of Embryology. Yb.
Carnegie Instn Wash. 56, 297-356.

FENNELL, R. A. (1966). Some histochemical observations on the effects of chorioallantoic
grafts on the spleen, bursa, and peripheral blood of chicken embryos. /. Morph. 118,149-166.

HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.

HINNI, J. B. & WATTERSON, R. L. (1963). Modified development of the duodenum of chick
embryos hypophysectomized by partial decapitation. /. Morph. 113, 381-426.

KONIGSBERG, I. R. (1954). The effects of early pituitary removal by 'decapitation' on
carbohydrate metabolism in the chick embryo. /. exp. Zool. 125, 151-169.

Development of the spleen: I 365

NALBANDOV, A. V. & CARD, L. E. (1943). Effect of hypophysectomy of growing chicks.
/. exp. Zool. 94, 387-413.

SETO, F. (1966). A correlated growth response of spleen and liver during the graft-versus-host
reaction in the chick embryo. Growth 30, 257-262.

SOLOMON, J. B. (196.1). The onset and maturation of the graft versus host reaction in chickens.
/. Embryol. exp. Morph. 9, 355-369.

VAN ALTEN, P. J. (1959). The effects of chorioallantoic grafts on the developing chick embryo.
II. Studies of adult antigens in the duodenum and spleen. /. Embryol. exp. Morph. 7,
476-486.

VAN ALTEN, P. J. & FENNELL, R. A. (1959). The effects of chorioallantoic grafts on the de-
veloping chick embryo. I. Studies on weight and histology of homologous and hetero-
logous tissues. /. Embryo/, exp. Morph. 7, 459-475.

VOGEL, N. W. (1956). Pituitary-Gonad Relationship in the Chick Embryo. Doctoral Dis-
sertation, Indiana University.

VOGEL, N. W. (1957). Free tissue cholesterol and growth in chick embryos hypophysectomized
by 'decapitation'. Anat. Rec. 127, 382.

WARNER, N. L. & BURNET, F. M. (1961). The influence of anti-inflammatory corticosteroids
on the growth of the chick embryo and the manifestations of the Simonsen phenomenon.
Aust. J. exp. Biol. med. Sci. 39, 235-248.

{Manuscript received 20 October 1969)