/. Embryo!, exp. Morph. Vol. 29, 1, pp. 245-251, 1973
245
Printed in Great Britain
The developmental endocrinology of the
spleen in chicken embryos
II. The pars distalis and splenomegaly
By R. B. GOLDBERG 1 AND T. W. BETZ2
From the Department of Biology, Carleton University
SUMMARY
Pars distalis-extract treatment of intact or 'hypophysectomized' chicken embryos resulted
in a splenomegaly, perhaps of a host-versus-graft nature, by 17 and 17-5 days of incubation.
Pars distalis grafts in intact embryos reduced spleen size, encouraged white-pulp follicle
formation, but 'suppressed' red-pulp development. Adult spleen extract and bovine serum
albumin did not change spleen sizes or histology appreciably in intact embryos.
Perhaps pars distalis hormones inhibit red-pulp development or stimulate white-pulp
follicles which may compete with red-pulp proliferation but encourage splenic vasculogenesis
alone or by stimulating adrenocorticoid, thyroid, and other hormone levels while other
extract factors cause splenomegaly.
INTRODUCTION
Partial decapitation of chicken embryos results in splenomegaly (82%
enlargement), with red-pulp hyperplasia but retarded white-pulp follicle development and vasculogenesis by 20 days of incubation (Betz, 1970). Single pars
distalis grafts restore splenic histogenesis and size (growth?) in 'hypophysectomized' chicken embryos, but one or two additional grafts are not different in
effect (Betz, 1970; Goldberg, 1971). Perhaps in reducing presumed adrenocorticoid inhibition 'hypophysectomy' amplifies splenomegaly (Betz, 1970).
Splenomegaly is also a response to chorioallantoic grafts of adult spleen with
mesenchymal differentiation of lymphoid hemocytoblasts to form erythroblasts
and inhibition of sinus, capillary and reticular development (see DeLanney,
Ebert, Coffman & Mun, 1962). Follicle tissue differentiates as lymphoid hemocytoblasts forming lymphocytes but in red pulp these cells form granular
leucocytes. Graft leucocytes produce antibodies against host proteins causing a
graft-versus-host (GVH) response. Enlargement involves immunologically
competent graft-cell colonization of host spleens, antibody damage, and a hostversus-graft (HVG) response of premature immunological competence and host
spleen cell proliferation (Simonsen, 1957; DeLanney et ah 1962; Mun & Burns,
1
Author's address: Department of Medical Biophysics, University of Toronto, Toronto
284, Ontario, Canada.
2
Author's address: Department of Biology, Carleton University, Ottawa, Ontario, Canada
K1S5B6 (for reprints).
246
R. B. GOLDBERG AND T. W. BETZ
1964; Ebert, 1964; Fennell, 1966; Sharon & Schwartz, 1970; Walker, Schoefl &
Lafferty, 1971). Splenomegaly by 'hypophysectomy' and HVG are histologically
distinct. Spleens of' hypophysectomized' embryos still respond to adult spleen
grafts (Ebert, 1957), showing the pars distalis is unnecessary for HVG responses.
We studied some effects of pars distalis extract and grafts, adult spleen extract
and bovine serum albumin (BSA), on splenic development in chicken embryos
to attempt separation of responses to protein hormones and other factors. Pars
distalis extract should have hormonally active and inactive proteins. Pars
distalis grafts should release only protein hormones. BSA should be only
hormonally inactive protein and adult spleen extract should be a potent HYG
inducer. Juvenile pars distalis extract was included for comparison.
MATERIALS AND METHODS
We analysed splenic responses to pars distalis extract and grafts in chicken
embryos from previous studies (Goldberg, 1971), and in intact embryos treated
with BSA and adult spleen extract.
Fertilized White Leghorn eggs (Hyline 934 F), selected at random, were incubated at 39 + 0-5 °C and 6 0 ± 5 % relative humidity and some embryos were
'hypophysectomized' by partial decapitation (see Goldberg, 1971) at stage 10
or 11 of Hamburger & Hamilton (1951). Shells were 'windowed' on day 2 of
incubation for access to the chorioallantois for treatment as before.
There were four experimental groups. In group 1, killed at day 17-5 of incubation, intact embryos received either 0-2 ml saline or 2-5, 5, or 10 gland equivalents
(GE) of adult pars distalis extract in 0-2 ml saline daily between days 12-5 and
16-5 of incubation. This group was repeated once. Asmall group similarly treated
with 5 and 10 GE cockerel pars distalis extract responded identically so data
were pooled. In group 2, killed at day 17, both intact and 'hypophysectomized'
embryos received saline, or 5 or 10 GE of extract (same regime), between days 12
and 16. One GE was the extract from one adult or two cockerel partes distales.
Unhealthy embryos were omitted before treatments. In group 3, each intact
embryo received three chorioallantoic grafts of embryonic chicken partes
distales - two 19-day-old glands at 9-5 days of incubation and one 20-day-old
gland at 10-5 days as before (Betz, 1967). From one to three grafts were found at
autopsy at 17-5 days of incubation. Data were pooled because responses to
grafts were indistinguishable. Treatments did not appreciably affect mortality.
Pars distalis extract preparation from frozen broiler chickens and fresh 1-dayold cockerels is as described by Goldberg (1971). These extracts in equivalent
concentrations were not different in effect. In group 4, killed at day 17 of
incubation, spleens from 11-5-month-old White Leghorn hens (PB-58) were
frozen, lyophilized, and saline extracted exactly as described for pars distalis
tissue by Goldberg (1971). Spleen extract and BSA (fraction V, Nutritional
Biochemicals) were given in amounts equal to the protein added in pars distalis
extract treatment (7 mg), using a duplicate regime.
Developmental endocrinology of the spleen
247
In all embryos decapitated between cervical vertebrae 5 and 6 the abdomen
was opened, sex recorded and bodies fixed in Bouin's fluid, decolorized in 70 %
ethanol saturated with lithium carbonate, and stored in 70% ethanol (Betz,
1967). Fixed spleens were dissected in 70% ethanol, blotted and weighed
(nearest 0-1 mg), then dehydrated in tertiary butyl alcohol and ethanol, embedded
in paraffin in vacuo, sectioned at approx. 8 jum, mounted on slides, deparaffinized
in xylol, hydrated in ethanol, stained with H & E, PAS-hematoxylin, or eosin
Y-orange G and toluidine blue (modified Dominici stain fide Gray, 1954).
Sections stained with H & E or PAS-hematoxylin were dehydrated in ethanol,
cleared in xylol, and mounted in Technicon. Sections stained in Dominici stain
were mounted directly in Kaiser's glycerol jelly (Humason, 1967) to preserve
metachromacy. Representative sections were photographed with Adox KB 14
film.
Means and standard deviations were calculated and means were compared
by Duncan's (1955) test at the 1 and 5% levels of significance.
RESULTS
Spleens in pars distalis extract-treated intact embryos by 17-5 days of incubation tended to be significantly heavier and enlarged mostly by red-pulp proliferation. Perhaps a slight increase in the proportion of white pulp occurs (Table 1,
Figs. 1-5). Pars distalis grafts in intact embryos significantly reduced spleen
weight and increased the number of small diffuse white-pulp follicles proportionately (Fig. 6). Splenomegaly was significant in pars distalis extract-treated
females (14-3, S.D. = ±4-7 mg), but males (9-7, S.D. = ±2-3 mg) did not significantly exceed controls of both sexes (8-2, S.D. = ±2-3); a problem for future
analysis.
Spleens of untreated ' hypophysectomized' embryos were enlarged insignificantly. With increased extract doses, weights became significantly greater.
Histologically, ' hypophysectomized' embryos were deficient in white pulp and
vasculogenesis (Figs. 3, 4). Extract treatment improved white-pulp development
and vasculogenesis but these parameters did not reach control levels, perhaps
because of splenomegaly (Fig. 5). BSA treatment did not increase spleen weight
significantly or alter histology. Adult spleen extract was ineffective in both
respects.
DISCUSSION
Splenomegaly may result from * hypophysectomy' or foreign proteins. A
secondary HVG response can be stimulated by a cell-free extract of a variety
of tissues (DeLanney et ah 1962). Microsomal fractions, apparently RNA
(Sharon & Schwartz, 1970), can stimulate splenomegaly by 17 days of incubation.
The pattern of response at 17 days, even with adult spleen grafts, usually does
not result in necrotic foci characteristic of full reaction. HVG splenomegaly is
also greater in females (Solomon, 1961). Enlargement of pars distalis extract-
248
R. B. GOLDBERG AND T. W. BETZ
Developmental endocrinology of the spleen
249
Table 1. Mean fixed spleen weights ( ± S . D . ) of intact and 'hypophysectomized''
chicken embryos with pars distalis extract and grafts at about 17 days of incubation
No. of
specimens
Group 1
17-5-day-old intact embryos
Group 2
17-day-old 'hypophysectomized'
embryos
17-day-old intact embryos
Group 3
17-5-day-old intact embryos
Group 4
17-day-old intact embryos
Treatment
Spleen
weight (mg)
Saline
2-5 GEf
5GE
10 GE
Saline
2-5 GE
5GE
10 GE
Saline
5 and 10 GE (cockerel
extract)
90 ±2-6
12-5 ±3-4
17-5 ±6-9**
12-2 ±2-7
7-8± 1-5
8-4± 1-3
10-6 + 4 0
8-8 ± 1-1
8-9 ±0-9
8
4
9
10
8
8
Saline
5GE
10 GE
Saline
5GE
10 GE
9-2 ±1-8
14-0 ±2-7*
19-4 ±7-7**
7-3 ±1-7
10-2 + 20
13-6 ±4-7**
9
10
Sham control
Pars distalis grafts (1-3)
8-7 ±2-0
5-6 ±2-3**
11
16
7
Saline
Adult spleen extract
Bovine serum albumin
7-9 ±2-6
7-6 ±2-4
9-3 ±2-4
15
6
5
6
4
3
3
4
3
6
14-0 ±3-4*
* Significantly different from control at 5 % level.
** Significantly different from control at 1 % level.
t 2-5 GE = gland equivalents of extract from 2-5 partes distales. Splenomegaly was
significant in extract-treated females but males did not exceed controls of both sexes.
FIGURES
1-6
Spleen histology in chicken embryos from experimental groups 1, 2 and 3. Bouin's
fixed, paraffin sections approx. 8 /*m, stain Dominici's. Scale line (Fig. 1) equals
100/mi.
Fig. 1. Spleen from saline control embryos. White-pulp (arrow) development more
than usual.
Fig. 2. Spleen from intact embryo given ten gland equivalents (GE) of pars distalis
extract. White pulp increased.
Figs. 3, 4. Spleens from untreated 'hypophysectomized' embryos to show range of
variation.
Fig. 5. Spleen from 'hypophysectomized' embryo given ten GE of extract. Slightly
more small white-pulp follicles and improved vasculogenesis.
Fig. 6. Spleen from pars distalis-grafted intact embryo. Number of small diffuse
white-pulp follicles (arrow) increased and vasculogenesis improved.
250
R. B. GOLDBERG AND T. W. BETZ
treated spleens in our study suggests a HVG type of response to some part of
the extract in agreement with DeLanney et ah (1962). Trophin (ACTH) stimulation of adrenal cortices should reduce spleen size as cortisone abolishes HVG
splenomegaly in rats and adrenalectomy enhances the onset and extent of
reaction (Kieffer & Ketchel, 1971). Adrenalectomy stimulates spleen 'growth'
in fetal rabbits (Beam, 1967), as does 'hypophysectomy' in chicken embryos
(Betz, 1970), presumably by reducing adrenocorticoid levels. The pars distalisadrenal axis is apparently established by 14-5 days of incubation in chicken
embryos (Woods, De Vries & Thommes, 1971). STH increases spleen growth
in rats (Fast, Garland, Thomson & Richards, 1970), which is coincident with
general growth stimulation. In pars distalis extract-treatments, ACTH may
mask STH effects. Thus, hormone activities of extract should decrease spleen
weight as indicated by pars distalis graft effects. Grafts may release protein
hormones but probably few if any other kinds of proteins. Apparently protein
trophic hormones do not also stimulate a HVG reaction. Spleen weight with
BSA treatment does not help to support the hypothesis that non-hormonal
extract components caused splenomegaly. All extracts producing splenomegaly
had some blood. Thus, leucocytic proteins might be effective agents, as Simonsen
(1957) indicated. But apparently adult spleen extract was ineffective in this
respect for unknown reasons.
Splenomegaly after 1-day-old cockerel pars distalis extract-treatment is
anomalous if the extract-produced splenomegaly and HGV reaction are related.
Day-old cockerels should be immunologically incompetent (Billingham, Brent
& Medawar, 1956), so extracts from them should not perhaps elicit HVG
responses. Although enlargement of these spleens was not less than that produced
by adult gland extract, it may be important because pars distalis grafts decreased
spleen weight.
Our interpretation of extract hormonal histological effects is subjective and
tentative, but appears consistent with maintenance of red-white pulp balance
by 'suppression' of red-pulp proliferation or stimulation of white pulp, presumably by adrenocorticoids and/or other hormones. Perhaps white pulp inhibits
red-pulp development. White-pulp development appears improved proportionately in spleens of pars distalis extract-treated intact and 'hypophysectomized'
embryos as does vasculogenesis, but the factors involved are not known. These
problems require further analysis.
Reasons for: similar responses to 5 and 10 GE cockerel-extract; to 1-3 grafts;
insignificant splenomegaly after 'hypophysectomy' in contradiction of other
data (Case, 1951 fide Betz, 1970); and inconsistent responses, between and
within treatment groups, to pars distalis extract are unknown.
We acknowledge support by a grant to T. W. Betz from the National Research Council
of Canada. R. B. Goldberg acknowledges his tenure of National Research Council, Ontario,
and Department of Biology Teaching Graduate Fellowships. Our thanks to D. L. Mallon,
J. F. Hughes and S. Skidd for technical assistance.
Developmental endocrinology of the spleen
251
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