Developmentaland ComparativeImmunology,Vol. 14, pp. 457-463, 1990 Printed in the USA. All rights reserved.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
EFFECT OF THE PROGESTOGEN, ALLYLESTRIOL, ON THE BURSAL DEVELOPMENT OF THE CHICKEN Gy Soos, I. Olah, Zs. Kittner, I. Toro and B. Glick Sernmelweis UniversityMedical School, Budapest, Hungaryand Clemson University, Poultry Science DepartmentClemson,SC (Submitted March 1989;Accepted October 1989)
[3Abstract--Fertile eggs were dipped on the fifth day of incubation into varying concentrations of allylestriol (AE), a synthetic progesterone, and testosterone propionate (TP). Concentrations of AE higher than 0.1% inhibited hatchability. The AE inhibited the differentiation of the bursal mesenchyme and follicular development and subsequently resulted in bursectomy. The AE caused bursectomy in 40-50 times lower concentrations than did TP. An unusual lymphocyte accumulation occurred around the postcapillary venules in the bursal mesenchyme of AE-treated birds. This observation suggested that inhibition of mesenchyme differentiation may lead to a modification in bursal function. The AE modification of bursal development was compared to those produced by TP. We demonstrated that AE caused immunosuppression of the B-cell system.
pressed bursal weight (3). In mammals, progesterone but not estriol inhibited cytoxicity (4). Also the progesterone level in the fetal-maternal interface and maternal blood was high enough to exert immunosuppressive effects on T-lymp h o c y t e responsiveness in mammals (4,5,6). Since little is known concerning progesterone's role in B-cell development, we utilized the bursal model to compare the impact of a synthetic progesterone, allylestriol (AE), and TP on the B-cell system. Allylestriol impaired bursal development and suppressed antibody response to sheep red blood cells.
[3Keywords--Allyestriol; Bursa of Fabricius; B cells; Progesterone; Testosterone proprionate.
A pathogen-flee strain of Leghorns supplied the fertile eggs. Each of the AE-2, AE-.2, and A E - . I % groups included 40 eggs. Since these eggs failed to hatch, 3 lower concentrations of AE, .05, .04, and .025% were studied. The latter 3 treatments were replicated 6 x with 60 eggs in each replication. Testosterone-propionate treatments of 2.0, 1.0, and 0.5% included 60 eggs each. The pointed end of 5-day-old embryonated eggs was dipped for 5 s (3). The approximate amount of AE and TP transferred into the eggs was calculated by determining the loss of volume after dipping a constant number of eggs into 200 mL of solution. Since a reduction of approximately 10 mL occurred after dipping 50 eggs, one could estimate that a maximum of .2 mL solution en-
Chemical bursectomy of fertile eggs by testosterone (TP) is a useful method to study bursal function in B-cell differentiation and immunoglobulin synthesis (1). We have investigated the effect of TP on bursal development and concluded that TP impaired the differentiation of the bursal m e s e n c h y m e (2). Diethylstilbestrol, an estrogen, supAddress correspondence to Bruce Glick, 129 Poole Agriculture Center, Clemson University, Clemson, SC 29634-0379.
Materials and M e t h o d s
tered each egg. Thus, each egg received a maximum concentration of .1 mg of AE (.05% AE) or 4 mg of TP (2% TP). Histological examinations were made from 0.1; 0.05; and 0.025% AE and 2% TP-treated embryos. The tissue samples were taken every other day from day 7 of incubation to hatching. The bursae were fixed in 4% cacodylate buffered glutaraldehyde for 2 h. After rinsing in buffer, postfixation was made by 1% osmium tetroxide. The dehydration was
G. Soos et al.
followed by e m b e d d i n g in Polybed (Polysciences) and one-micron sections were stained with toluidine blue. E i g h t - w e e k - o l d c o n t r o l and AEtreated birds were injected intravenously with 0.5 mL of 2% sheep red b l o o d cells (SRBC) in saline. The chickens were bled on day 3, 5, and 7 after immunization. Total titers were determined by the microtiter procedure and IgG antibody by mercapto-ethanol treatment (7).
Figure 1, Fifteen-day-old allylestriol-treated bird's bursa. No sign of bud formation. The mesenchyme consisted of fibrocyte-like cells. Mag. × 180. Figure 2. AE-treated bird's bursa at hatch. The folds were developed but lacked follicular formation. One of the peripostcapillary infiltrations (arrow) was shown in Fig. 6. Mag. x 42.
Allyestriol in bursal development
Results Concentrations higher than 0.1% AE inhibited hatching. The majority of these chickens died between 17 and 19 days of incubation. Body weight of all the AE t r e a t e d and control birds was comparable on day 17 of embryonic life. Hatchability of the .05% AE group was slightly less than the control group. The bursa was not visible at hatch following exposure to .05 or .04% AE. Inspection with a stereo microscope revealed the presence of bursal tissue in a small percentage of these newly hatched chicks. Histological sections of these tissue remnants disclosed occasional follicles (Figs. 1-9). On the other hand, bursal tissue was usually present at hatch subsequent to the 0.025% AE treatment. These bursae appeared similar to controls but exhibited smaller bursal follicles. No antigen specific IgG was pro-
duced in the .05 or .04% AE-treated birds (Graph 1). The total agglutinin titers of the birds in the .025% AE group ranged between the titers of control and .05/.04% AE birds. Isotype classification was not determined for the .025% AE group. Up to I1 days of incubation, the bursal epithelial anlage, bursal lumen, and folds of the AE and control birds were similar. In normal birds, the mesenchymal cells differentiated into dark and light cells between 9 and 11 days of incubation (2). In AE-treated birds, the dark and light cells did not appear and the bursal m e s e n c h y m e consisted of similar cells (Fig. 1) which appeared to be fibrocytes. If the bursa existed at hatch, it was small and possessed within its lumen a viscous substance (Fig. 3, 4). These bursae possessed a few abortive follicles (Fig. 4) in which the dark cells contained very large, irregular shaped
Control AE treated
T :9'i~" [
IgMi IgG IgM
g :i ):"
Days after i.v. immunization by SRBC
Graph 1. Total and Class Specific Antibody Differences Between Control and .05% Allyleetriol (AE) Chickens.
G. Soos et al.
AE-treated bird's bursa at hatch. The lumen was filled with a viscous substance. The outlined area was shown on Fig. 4. Mag. x 42. Figure 4. Abortive follicles in the AE-treated chick, at hatch. Scattered dark cells can be observed in the epithelium (arrow). Mag. x160.
dense bodies (Fig. 5). In addition to the abortive follicles, lymphocytes accumulated around the postcapillary venules in the bursal mesenchyme (Fig. 6). These lymphocyte accumulations were unusual and appeared independent of the bursal epithelial anlage (Fig. 6), Thymus his-
tology appeared normal in all the AEtreated groups. If the bursal rudiment was present at hatch in TP-treated birds (Fig. 7), it possessed two types of follicles, normal (Fig. 8) and without epithelial reticulum (Fig. 9). The latter consisted of a lym-
Allyestriol in bursal development
Figure 5. In the abortive follicles the secretory cells were filled with large irregular dense bodies (arrow). Mag. x380. Figure 6. Lymphocytes accumulated around the postcapillary. Mag. x 320.
phocyte accumulation under the epithelium. The surface epithelium above this lymphoid accumulation was infiltrated by lymphoid cells. No follicular-associated epithelium was observed (Fig. 9).
Discussion We have observed that dark and light cells differentiated from the bursal mesenchyme between 9 and 11 days of embryonic life (2). The dark cells, which are the precursors of the secretory cells,
that is, dendritic cells, enter the surface epithelium of the bursa-inducing bud formation (2). The TP inhibited the differentiation of the bursal mesenchyme and follicular development (2,8). The effect of AE on the bursal mesenchyme is comparable with that of TP. However, one should realize that a few dark cells are formed in both AE and TP-treated birds, and therefore, an occasional follicle will appear after both treatments. Both treatments suppressed the development of IgG-antibody and thus may have interfered with isotype switching.
G. Soos et al.
Figure 7, TP-treated bird's bursa, at hatch. A few follicles developed close to the epithelium. The outlined follicles were shown on Figs. 8 and 9. Mag. x42. Figure 8. Normal follicle with epithelial reticulum and cortex formed around the medulla. TPtreated bird. Mag. ×380. Figure 9, Follicle without epithelium. The discontinuous basement membrane can be followed between the epithelium and follicle. Lymphocytes infiltrated the epithelium. No follicle-associated epithelium can be seen. Mag. x 520.
In the AE and TP-treated birds, lymphocyte infiltrations occurred around the postcapiUaries and under the surface epithelium, respectively. These lymphocyte infiltrations suggest that entry of blood-borne lymphocyte precursors into the bursal mesenchyme is not inhibited
by AE and TP treatment. During the formation of the peripheral lymphoid organs, mainly the cecal tonsils, lymphocyte infiltrations of the mucosal layer occurred around the postcapillaries and in close proximity of the epithelium (personal observation). There-
Allyestriol in bursal development
fore, if m e s e n c h y m a l differentiation is inhibited, that is, failure of formation o f dark cells from the bursal m e s e n c h y m e (2,8), the bursal m e s e n c h y m e m a y bec o m e the site of a peripheral lymphoid organ. In T P - t r e a t e d birds w h e n lymp h o c y t e infiltrations o c c u r r e d close to the epithelium, the a p p e a r a n c e o f the lympho-epithelial tissue resembled that of the cecal tonsil. After lymphoid infiltration, cells entered the surface epithelium, as o c c u r r e d in the cecal tonsil. T h e s e epithelial areas w e r e unlike the follicular-associated epithelium (FAE) of a normal bursa, The FAE of normal bursae lacked l y m p h o c y t e s but not secretory cells, that is, dendritic cells (8), These data intimate that the FAE m a y be dependent on the presence of secretory cells but not on the presence of lymphoc y t e s as c o n c l u d e d by B e e z h o l d et al. (9). The A E interfered in a d o s e - d e p e n dent m a n n e r with hatchability of chickens but not their development and growth. Up to 17 days of incubation, the A E c h i c k e n ' s growth was c o m p a r a b l e with that of the controls. H o w e v e r , after 17 days of e m b r y o n i c life the chickens
died if the A E concentration was higher than 0.1%. Since the chicken died 10-11 days after dipping, we conclude that the A E is not directly toxic to e m b r y o s . It is possible that death resulted from an interaction b e t w e e n the A E and the neuroendocrine system. T h e A E h a s an a l l y l r a d i c a l p r e v e n t i n g its m e t a b o l i s m b y the liver. T h e r e is no information a b o u t the A E metabolic p a t h w a y in chicken e m b r y o s . Progesterone is the precursor of testosterone. Therefore, it might be assumed that A E was converted to TP which res u l t e d in the b u r s e c t o m y . H o w e v e r , since a single progesterone moiety forms one TP molecule and 0.04 or 0.05% A E solutions exert a biological effect similar to 2% TP, o n e c o u l d c o n c l u d e t h a t p a t h w a y s other than TP are responsible for p r o g e s t e r o n e ' s influence on the Bcell system.
A c k n o w l e d g e m e n t s - - W e thank Gloria Freeman for typing the manuscript. This work was supported by a grant of the Hungarian Academy of Sciences and supported in part by the South Carolina Agricultural Experiment Station.
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6. Szekeres-Bartho, J.; Varga, E; Pacsa, R. S. Immunologic factors contributing to the initiation of labor-lymphocyte reactivity in term labor and threatened preterm delivery. Am. J. Obstetr. Gynecol. 155:108-112; 1986. 7. McCorkle, E; Glick, B. The effect of aging on immune competence in the chicken: antibodymediated immunity. Poultry Sci. 59:669-672; 1980. 8. Glick, B.; Olah, I. Contribution of a specialized dendritic cell, the secretory cell, to the microenvironment of the bursa of Fabricius, Avian immunology. In: Ewert, E L., Weber, W. T.; eds. New York: Alan R. Liss, Inc.; 1987:p. 54-66. 9. Beezhold, D. H.; Sachs, M. G.; Van Alten, E J. Lymphocyte requirement for the function development of follicle-associated epithelium. Dev. Comp. Immunol. 9:351-359; 1985.