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The effects of hormonal and early surgical bursectomy on the thymic development of the chick embryo Dixon, Dennis Kent 1977

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THE EFFECTS OF HORMONAL AND EARLY SURGICAL BURSECTOMY ON THE THYMIC DEVELOPMENT OF THE CHICK EMBRYO by DENNIS KENT DIXON B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF POULTRY SCIENCE We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August, 1977 •fcT) Dennis Kent Dixon, 1977. MASTER OF SCIENCE xn In presenting th is thes is in p a r ti a l ' fu1fiIment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n , of th is thes is for f inanc ia l gain sha l l not be allowed without my wr i t ten pe rm i ss ion. Department of P o u l t r y S c i e n c e The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 August 9, 1977 I ABSTRACT Hormonal bursectomy s i g n i f i c a n t l y reduces thymocyte numbers i n 18 day embryonic thymuses when i t i s c a r r i e d o u t on o r b e f o r e t h e f i f t h day o f i n c u b a t i o n . E a r l y embryonic s u r g i c a l bursectomy (at 72 hours i n ovo) s i m i l a r l y reduces thymocyte numbers. When hormonal bursectomy and e a r l y embryonic s u r g i c a l bursectomy a r e combined, t h e e f f e c t s appear to, be a d d i t i v e p r o d u c i n g a n e a r l y t w o - f o l d d e p r e s s i o n o f thymocyte numbers.. Such r e s u l t s s u ggest t h a t h o r -monal bursectomy, on o r b e f o r e t h e f i f t h day o f i n c u -b a t i o n , and e a r l y s u r g i c a l bursectomy a f f e c t thymocyte numbers by two d i f f e r e n t ..mechanisms. I t i s p o s t u l a t e d t h a t t h e e f f e c t s o f hormonal bursectomy p r i o r t o the f i f t h day o f i n c u b a t i o n r e p r e s e n t a t e s t o s t e r o n e -thymus i n t e r a c t i o n . . T h i s i n t e r a c t i o n appears t o r e s u l t i n an i n t e r f e r e n c e w i t h the i n i t i a l p o p u l a t i o n o f t h e thymus by m i g r a t i n g stem c e l l s , r e s u l t i n g i n a r e d u c t i o n o f t h e numbers o f thymocytes. E a r l y s u r g i c a l bursectomy would presumably c i r c u m v e n t such an i n t e r -a c t i o n . I t i s suggested t h a t e a r l y s u r g i c a l bursectomy does i n d e e d reduce thymocyte numbers by removing a p o t e n t i a l b u r s a l c o n t r i b u t i o n t o thymocyte numbers. Based on t h e e v i d e n c e o f t h i s s t u d y and o t h e r i n v e s t i -g a t i o n s , i t i s suggested t h a t the b u r s a c o n t r i b u t e s s i g n i f i c a n t l y t o a secondary i n f l o w o f t h y m i c stem c e l l s o c c u r i n g l a t e r i n the embryonic p e r i o d . i i i TABLE OF CONTENTS Page I . INTRODUCTION 1 I I . LITERATURE REVIEW 1 A. The B u r s a o f F a b r i c i u s 1 1. B u r s a l S t r u c t u r e and Ontogeny. . . . 1 2. Bursectomy and B u r s a l F u n c t i o n . . . 5 B. The Thymus 12 1. Thymic S t r u c t u r e and Ontogeny. . . . 12 2. The P r i m a r y E f f e c t s o f Thymectomy and Thymic F u n c t i o n . . . ... . . ... 15 C. The D i s t r i b u t i o n o f B u r s a l and Thymic C e l l s and Lymphoid C e l l T r a f f i c 18 D. Some E f f e c t s o f Bursectomy on Thymic Development 22 I I I . THE EFFECTS OF HORMONAL BURSECTOMY ON THYMIC DEVELOPMENT 2 5 A. M a t e r i a l s and Methods 2 5 B. R e s u l t s and D i s c u s s i o n 27 IV. THE EFFECTS OF EARLY EMBRYONIC SURGICAL BURSECTOMY ON THYMIC DEVELOPMENT 34 A. M a t e r i a l s and Methods 34 B. R e s u l t s and D i s c u s s i o n 36 V. THE EFFECTS OF COMBINED HORMONAL AND EARLY EMBRYONIC SURGICAL BURSECTOMY ON THYMIC DEVELOPMENT 41 A. M a t e r i a l s and Methods 41 B. R e s u l t s and D i s c u s s i o n 42 V I . GENERAL DISCUSSION AND CONCLUSIONS . . . . . 49 V I I . SUMMARY. . 56 V I I I . LITERATURE CITED 57 i v LIST OF TABLES TABLE Page I . E f f e c t s o f hormonal bursectomy a t v a r i o u s i n c u b a t i o n s t a g e s on t h e th y m i c development o f 19 day c h i c k embryos 29 I I . Summary o f e f f e c t s o f hormonal bursectomy on the: .thymic development o f 19 day c h i c k embryos 30 I I I . E f f e c t s o f SBx a t t h r e e days i n ovo on t h e th y m i c development o f 18 day c h i c k embryos . . 37 IV. E f f e c t s o f SBx, HBx and SBx + HBx on the body w e i g h t s , s p l e e n w e i g h t s and spleen:., body w e i g h t r a t i o s o f 18 day c h i c k embryos. . . 4 4 V. E f f e c t s o f SBx, HBx and SBx + HBx on the thym i c development o f 18 day c h i c k embryos. . . 4 6 V LIST OF FIGURES F i g u r e Page 1. Cross s e c t i o n s of 19 day embryonic c h i c k thymus hormonally bursectomized at a) two days of i n c u b a t i o n and b) a t nine days of i n c u b a t i o n . H & E s t a i n . M a g n i f i c a t i o n 90x . 32 2. Crpss s e c t i o n s of 18 day embryonic c h i c k thymus a) s u r g i c a l l y bursectomized a t three days i n ovo and b) c o n t r o l . H & E S t a i n . M a g n i f i c a t i o n 90x 39 3. Cross s e c t i o n of an 18 day embryonic c h i c k thymus s u r g i c a l l y bursectomized a t three days i n ovo and hormonally bursectomized a t f o u r days of i n c u b a t i o n . H & E s t a i n . M a g n i f i c a t i o n 90x 47 v i ACKNOWLEDGEMENTS I would l i k e to take th i s opportunity to thank some of the many people whose valuable assistance enabled the successful completion of th i s thes i s . I .am grate fu l to Dr. R .C. Fitzsimmons whos generously provided me with the f a c i l i t i e s to carry out my research and enthus ias t ic encouragement during d i f f i c u l t times. I would also l i k e to thank the members of my committee for t h e i r patience and construct ive advice. A spec ia l note of appreciat ion goes to Dr. C W . Roberts who p a t i e n t l y ass i s ted me with the s t a t i s t i c a l analys i s of the data and offered much encouragement and worldly advice. I am also indebted to Ms. Gale Dawson for her kind cooperation and expert typing of t h i s manuscript. I. INTRODUCTION Since the d i s c o v e r y o f the bursa of F a b r i c i u s and the thymus, there has been c o n s i d e r a b l e r e s e a r c h i n t o t h e i r s t r u c t u r e and ontogeny, w i t h p a r t i c u l a r i n t e r e s t i n t h e i r r o l e s i n immunological mechanisms. Such r e s e a r c h , e s p e c i a l l y i n the c h i c k embryo, has y i e l d e d much i n f o r m a t i o n concerning the em b r y o l o g i c a l o r i g i n s o f t h e i r v a r i o u s c e l l u l a r components. T h e i r seemingly separate and d i s t i n c t c o n t r i b u t i o n t o immunological r e a c t i o n s and the development o f immunological mechanisms has been a l s o r e v e a l e d . In view of t h i s , the o b j e c t i v e s o f t h i s t h e s i s are to review and d i s c u s s the f o l l o w i n g : a) the ontogeny and f u n c t i o n of the bursa and thymus, b) t h e i r developmental i n t e r n -r e l a t i o n s h i p s , c) the r a t i o n a l e f o r undertaking t h i s i n v e s t i g a t i o n and d) the s i g n i f i c a n c e o f the r e s u l t s i n terms of our understanding o f the ontogeny of these two organs and the r e l a t i o n s h i p s t h a t e x i s t between them d u r i n g t h e i r embryonic development. I I . LITERATURE REVIEW A. The Bursa of F a b r i c i o u s 1. B u r s a l S t r u c t u r e and Ontogeny The bursa of F a b r i c i u s was d i s c o v e r e d i n 1621 and 2 named a f t e r i t s d i s c o v e r e r , Hieronymus F a b r i c i u s (1). I t i s p r i m a r i l y a lymphoid o r g a n , p e c u l i a r t o t h e f o w l , t h a t d e v e l o p e s from a d i v e r t i c u l u m o f the c l o a c a l -c o l o n j u n c t i o n i n t o a b l i n d o v a l s a c . I t i s connected by a s h o r t s t a l k t o t h e d o r s a l r e g i o n o f t h e c l o a c a (1,.2). A more r e c e n t i n v e s t i g a t i o n (3), u s i n g l i g h t and e l e c t r o n m i c r o s c o p y t e c h n i q u e s , has c a r e f u l l y d e t a i l e d t h e d e v e l o p -m e n t a l s t r u c t u r e s and e v e n t s as o u t l i n e d below. The e a r l i e s t appearance o f t h e b u r s a l a n l a g e , a t f o u r days i n c u b a t i o n , i s a group o f v e s i c l e s i n t h e mesenchyme l y i n g between t h e u n r u p t u r e d u r o d e a l membrane and t h e c l o a c a l e p i t h e l i u m . A t t h e s e v e n t h day o f i n c u b a t i o n , t h e b u r s a i s a h o l l o w sac w h i c h l i e s c e p h a l a d , d o r s a l t o th e c l o a c a and i s s t i l l s e p a r a t e d from t h e c l o a c a . I t i s l i n e d on i t s l u m i n a l s u r f a c e w i t h a two t o f o u r c e l l l a y e r o f p s e u d o s t r a t i f i e d e p i t h e l i u m and i s surrounded by a l o o s e network o f u n d i f f e r e n t i a t e d mesenchymal c e l l s . D u r i n g t h e t e n t h day, t h e b u r s a l lumen e n l a r g e s and t h e p r o x i m a l p o r t i o n becomes c o n t i n u o u s w i t h t h e c l o a c a , f o r m i n g t h e b u r s a l d u c t . A t a p p r o x i m a t e l y t h e same t i m e , t h e m i t o t i c r a t e s o f t h e e p i t h e l i a l and mesenchymal c e l l s i n c r e a s e . The r e s u l t i n g p r o l i f e r a t i o n forms t h e l o n g -i t u d i n a l f o l d s o r p l i c a e w h i c h p r o j e c t i n t o the lumen o f the b u r s a , w h i c h i s s t i l l l i n e d on t h e l u m i n a l f a c e w i t h e p i t h e l i u m . B e f o r e t h e n i n t h day o f i n c u b a t i o n , t h e b u r s a l c e l l t y p e s a r e a l i n i n g o f e p i t h e l i a l c e l l s , u n d i f f e r e n t i a t e d mesenchymal c e l l s , and e n d o t h e l i a l c e l l s w hich l i n e t h i n -3 walled vascular channels. At the beginning of the n inth day, there are a few h e t e r o p h i l i c granulocytes and large p r imi t ive mononuclear c e l l s with basophi l i c cytoplasm, thought to be lymphobalsts, present i n the mesenchyme. By the end of the ninth day, and with increased frequency during the tenth day, lymphoblast presence i s noted wi th in the epithel ium forming nodular s t ructures . During the 12th and 13th days, the nodules increase i n s ize due to lymphoblast p r o l i f e r a t i o n and by 18 days the lymphoid f o l l i c u l a r structure has matured, rendering the bursa a s tructure p r i m a r i l y composed of densely packed lymphoid f o l l i c l e s . During the l a s t e r stages of f o l l i c l e formation, there i s a rearrangement of the e p i t h e l i a l c e l l s wi th in the f o l l i c l e in to a s ingle l ayer that surrounds c e n t r a l l y located lymphoid c e l l s , forming the medullary c e l l s of the mature f o l l i c l e s . The c o r t i c a l layer of . lymphoid c e l l s appears to be derived from those that were subadjacent to the epi thel ium. The c o r t i c a l and medullary c e l l s are thus separated by an e p i t h e l i a l layer and a c l o s e l y associated c a p i l l a r y . In the adult b i r d , there i s breed v a r i a t i o n of the time at which the bursa reaches maximum s i z e , and has been reported as 10 to 12 weeks of age i n Barred' crosses , 4.5 to 6 weeks i n White Leghorns and 8 to 11 weeks i n Rhode Is land Reds (4). The bursa begins to involute and regress with the advent of sexual maturity , a process which has been a t t r ibuted to t e s t i c u l a r and adrenal 4 hormones (1, 2, 4). The p r e c i s e o r i g i n of b u r s a l lymphocytes has been c o n t r o v e r s i a l . T h e i r d i f f e r e n t i a t i o n from v a r i o u s c e l l types has been supported by a v a r i e t y o f h i s t o l o g i c a l and experimental evidence. Most of the e a r l y work, based on h i s t o l o g i c a l changes i n the d e v e l o p i n g bursa, has supported the i n s i t u d i f f e r e n t i a t i o n of the e a r l y e p i t h e l i a l c e l l s i n t o lymphoid c e l l s (2, 5, 6, 7) under the i n d u c t i v e i n f l u e n c e of the ectoderm (5) oh the mesenchymal c e l l s (8). Others have supported the d i f f e r e n t i a t i o n o f mesenchymal c e l l s (9, 10) i n t o lymphocytes. However, v a r i o u s s t u d i e s u t i l i z i n g chromosomal markers to analyse the degree o f c e l l u l a r chimerism i n p a r a b i o t i c embryos (11, 12, 13), g r a f t e d t i s s u e s (12) and n a t u r a l twins (13), a l l found a mixture of lymphocyte types i n the bursa from both donor and host. The d i f f e r e n t c e l l types were of v a r y i n g percentages, depending on the time o f g r a f t i n g . T h i s evidence, together w i t h c h i m e r i c r e c o n s t i t u t i o n of s u b - l e t h a l l y i r r a d i a t e d c h i c k embryos (14), lends s t r o n g support to the i d e a t h a t b u r s a l lymphocytes a r i s e from blood borne p r e c u r s o r c e l l s which migrate i n t o the bursa, p r o l i f e r a t e and mature i n t o b u r s a l lymphocytes. T h i s model has s i n c e r e c e n t l y been confirmed h i s t o l o g i c a l l y and the l a r g e mononuclear c e l l w i t h b a s o p h i l i c cytoplasm, seen i n the surrounding mesenchyme p r i o r t o the n i n t h day of i n c u b a t i o n , has been i d e n t i f i e d as the m i g r a t i n g stem c e l l (3). Experimental c o n f i r m a t i o n of t h i s model 5 has a l s o been e l e g a n t l y demonstrated by i n t e r s p e c i e s ' g r a f t i n g o f b u r s a l rudiments from the c h i c k embryo, a t v a r i o u s stages o f development, i n t o the Japanese q u a i l embryo (15, 16). The r e s u l t i n g c h i m e r i c g r a f t s i n d i c a t e t h a t a wave of m i g r a t i n g stem c e l l s e n t e r the bursa a t approximately nine days of i n c u b a t i o n and begin t h e i r p r o l i f e r a t i o n . The bursa a l s o appears capable of a t t r a c t i n g m i g r a t i n g stem c e l l s even when p l a c e d i n he t e r o -t o p i c l o c a t i o n s . 2. Bursectomy and B u r s a l F u n c t i o n The r o l e played by the bursa i n immunological f u n c t i o n s was i n i t i a l l y d i s c o v e r e d by a c c i d e n t . G l i c k e t a l (17) r e p o r t e d t h a t chickens t h a t had been s u r g i c a l l y bursectomized a t two weeks of age were found t o have a di m i n i s h e d p r e c i p i t i n response to Salmonella typhimurium. L a t e r Rao e t a l (18), Meyer e t a l (19) and Warner and Burnet (6) found they c o u l d i n h i b i t the development of the embryonic bursa by i n j e c t i n g an a p p r o p r i a t e amount of 19-n o r t e s t o s t e r o n e i n t o the a l l a n t o i c c a v i t y a t f i v e days of i n c u b a t i o n . I t was a l s o subsequently r e p o r t e d t h a t such "hormonal bursectomy" at f i v e days of i n c u b a t i o n would impair antibody p r o d u c t i o n to a much g r e a t e r degree than s u r g i c a l bursectomy c a r r i e d out on ne o n a t a l b i r d s (20). Many subsequent s t u d i e s confirmed t h i s obser-v a t i o n by comparing the e f f i c a c y of a v a r i e t y of doses, t i m i n g of a d m i n i s t r a t i o n , routes of a d m i n i s t r a t i o n and type 6 of s t e ro id employed. Androgens with a completely reduced s t e ro id nucleus were the most e f fec t ive i n i n h i b i t i n g bursa l development (21) and were more e f fec t ive than estrogenic compounds (22). Dosages as small as 0.8 mg. of 19-nortestosterone, administered p r i o r to the ninth day of incubat ion, completely i n h i b i t e d bursal lymphoid development (23). I t was also found that dipping eggs i n solut ions of testosterone proprionate i n e t h y l a lcohol could i n h i b i t bursa l development (24) and concentrations of 12.8 mg. per 1.0 ml. could e f f e c t i v e l y abol i sh the presence of serum antibodies (25). The timing of administrat ion of an i n h i b i t o r y dose of testosterone i s also of c r i t i c a l importance i n achieving ab la t ion of the bursa, with re su l t s ranging from com-plete i n h i b i t i o n of the development of the e p i t h e l i a l sac to only p a r t i a l i n h i b i t i o n of the lymphoid f o l l i c l e s (23). I t seemed apparent that the mechanism by which testosterone i n h i b i t s bursa l development i s int imate ly associated with the sequence of events during the develop-ment of the bursa. Subsequent studies proved t h i s to be the case. Mueller et a l (26) found that 4 mg. of 19-nortestosterone was much more e f fec t ive i n i n h i b i t i n g bursa l development when administered on the 12th day of incubation than on the 13th day. S i m i l a r l y , Ackerman and Knouff (8, 27) found an increase i n the a lka l ine phosphatase a c t i v i t y of the subep i the l i a l mesenchyme coincident with lympho-epi thel ia l nodule formation 7 (between 12 and 15 days of i n c u b a t i o n ) . When t e s t o s t e r o n e was a d m i n i s t e r e d p r i o r to the 12th day,.and as l a t e as the 11th day, there was an i n h i b i t i o n o f a l k a l i n e phosphatase a c t i v i t y as w e l l as b u r s a l development. They concluded t h a t the presence of a l k a l i n e phosphatase a c t i v i t y marks an i n d u c t i v e step, mediated by the mesenchyme, t h a t i s necessary f o r the normal development of the bursa. Furthermore, t h i s step i s ap p a r e n t l y blocked by .the presence o f t e s t o s t e r o n e . F u r t h e r evidence t h a t t e s t o s t e r o n e f u n c t i o n s i n b l o c k i n g development of b u r s a l e p i t h e l i a l elements, and t h e i r u l t i m a t e a t t r a c t i o n f o r m i g r a t i n g lymphocyte p r e c u r s o r s , was presented by Moore and Owen (12). They found t h a t by g r a f t i n g b u r s a l rudiments from hormonally bursectomized donors i n t o normal hosts or from normal donors i n t o hormonally bursectomized hosts ( g r a f t e d a f t e r 12 days i n c u b a t i o n ) , the g r a f t s developed normally. These g r a f t s a l s o contained, n e a r l y a f u l l compliment o f .host lymphocyte types. T h i s , coupled w i t h the f a c t t h a t g r a f t s from a hormonally bursectomized donor p l a c e d i n t o a bursectomized host f a i l e d t o develop, i n d i c a t e d t h a t t e s t o s t e r o n e seems to i n h i b i t the p r o l i f e r a t i o n and not the a v a i l a b i l i t y o f stem c e l l s f o r normal b u r s a l development (12). From these s t u d i e s , and many o t h e r s , the irole the bursa p l a y s i n immunological f u n c t i o n has become i n c r e a s i n g l y apparent. In 1962, Warner and Szenberg (28, 29) were f i r s t t o p o s t u l a t e the d i s t i n c t c o n t r i b u t i o n s 8 of the bursa and thymus by a d i s s o c i a t i o n o f t h e i r .. immunological responsiveness. They r e p o r t e d t h a t hormonal bursectomy at 12 day s of i n c u b a t i o n produced an i n h i b i t i o n o f antibody responses w i t h no e f f e c t on g r a f t - v e r s u s - h o s t (GvH) r e a c t i o n s . There was a l s o a r e d u c t i o n i n the numbers of s p l e e n i c nodules as w e l l as a s l i g h t impairment o f homograft r e j e c t i o n i n bursectomized b i r d s . There was a marked impairment o f both GvH a c t i v i t y and homo-g r a f t r e j e c t i o n i n b i r d s t h a t demonstrated an atrophy o f the thymic c o r t e x f o l l o w i n g hormonal bursectomy. In another study, impairment o f homograft r e j e c t i o n was a l s o shown f o r b i r d s s u r g i c a l l y bursectomized a t hatch or hormonally bursectomized as embryos (30). In c e r t a i n e x p e r i m e n t a l l y induced auto-immune d i s e a s e s , such as experimental a l l e r g i c e n c e p h a l o m y e l i t i s , neonatal s u r g i c a l bursectomy had no e f f e c t on the s u c c e p t i b i l i t y t o the d i s e a s e , nor d i d i t impair delayed h y p e r s e n s i t i v i t y r e a c t i o n s (31). Thymectomy, on the other h a n d , . s i g n i f i c a n t l y impaired both a c t i v e and p a s s i v e i n d u c t i o n of the d i s e a s e (31). Neonatal s u r g i c a l bursectomy was a l s o shown to g r e a t l y decrease c i r c u l a t i n g l e v e l s of antibody t o human re d blood c e l l s (32). These b i r d s consequently d i s -p l a y e d a l a c k of p r o l i f e r a t i n g plasmacytes i n the spl e e n . S i m i l a r l y , n eonatal b i r d s s u r g i c a l l y bursectomized and s u b - l e t h a l l y x - i r r a d i a t e d were shown to possess l i t t l e o r no serum a n t i b o d i e s , c e l l - b o u n d immunoglobulins, no primary antibody response to B r u c e l l a abortus and no plaque 9 forming c e l l s t o sheep red blood c e l l s ( 3 3 ) . I t was concluded t h a t the plasma c e l l l i n e was absent f o l l o w i n g t h i s treatment. Cooper e t a l (34) then f o l l o w e d the e f f e c t s of s u r g i c a l bursectomy a t v a r i o u s times d u r i n g development on a number of immunological parameters i n an e f f o r t to determine the p r e c i s e i n f l u e n c e of the bursa on the development of immunological responses. They found t h a t s u r g i c a l bursectomy a t one day p o s t hatch reduces the primary antibody response and the number of germinal c e n t e r s i n the spleen. I t has l i t t l e e f f e c t on the numbers of c i r c u l a t i n g plasmacytes or on the l e v e l s of t o t a l serum IgG or IgM. S u r g i c a l bursectomy c a r r i e d out on the 19 day embryo g r e a t l y reduced the number of germinal c e n t e r s i n the spleen, i n h i b i t e d the primary response completely, reduced the l e v e l s of IgG but had l i t t l e e f f e c t on IgM l e v e l s or the number of c i r c u l a t i n g plasma c e l l s . When bursectomy was c a r r i e d out on the 17 day embryo, the number of germinal c e n t e r s and the c i r c u l a t i n g l e v e l s of IgM and IgG were g r e a t l y reduced. T h i s treatment a l s o u s u a l l y e l i m i n a t e d both the primary and the secondary antibody response. They t h e r e f o r e concluded t h a t the bursa i s the s i t e r e s p o n s i b l e f o r the development of germinal c e n t e r c e l l s , plasma c e l l s and the a b i l i t y to produce a n t i b o d i e s . The (sequential e f f e c t s of s u r g i c a l bursectomy on antibody p r o d u c t i o n and c e l l l i n e s , i n d i c a t e d the t i m i n g of developmental steps i n the maturation of the humoral d i v i s i o n of the immune response. They added t h a t 10 the bursa may induce the d i f f e r e n t i a t i o n of two c e l l l i n e s 7 each capable of IgM or IgG production/ or i t induces the switch over from IgM to IgG synthesis i n plasma c e l l s . The concept of a bursa l mediated switch, , f rom IgM production to IgG production, has gained much favor l a t e l y . A d d i t i o n a l support for t h i s notion has been provided by the fact that treatment of the developing., embryo with heterologous serum prepared s p e c i f i c a l l y against chicken IgM, followed by surg ica l . bursectomy at hatch, produces b irds that are t o t a l l y and permanently devoid of any traces of immunoglobulins (35). However, a technique developed i n our lab (36) for removing presumptive bursa l tissue,, thereby preventing bursa l presence at any time during development, has produced b i rds capable of synthesiz ing IgM, a l b e i t i n small quant i t i e s . This may be interpreted as evidence for a secondary non-bursal s i t e for developing humoral competence as some authors have suggested (37, 38). C e r t a i n l y , more research i s required to determine the e x c l u s i v i t y of the bursa 's r o l e i n humoral development. As the importance of the presence of the bursa for development of the humoral response has become increas ing ly apparent, further inves t iga t ions were made of the c h a r a c t e r i s t i c s and functions of the i n d i v i d u a l bursa l lymphocytes. Phys ica l changes are seen i n bursa l lymphocytes during t h e i r development. Peterson and Good (39), and l a t e r Sherman and Auerbach (40), u t i l i z e d Coulter 11 counters to f i n d c h a r a c t e r i s t i c changes i n b u r s a l c e l l volumes d u r i n g the course o f embryonic development. They d e t e c t e d a g r a d u a l decrease i n mean c e l l volumes d u r i n g b u r s a l maturation w i t h a major r e d u c t i o n a l s h i f t o c c u r r i n g between 18 days:of i n c u b a t i o n and a few days pa s t h a t c h i n g . R e l a t i v e l y l a r g e c e l l ; types p e r s i s t throughout l i f e , however, when compared to thymic lymphocytes. H i s t o l o g i c evidence a l s o confirms the l a r g e c e l l types i n the bursa and t h a t they possess a s i g n i f i c a n t l y g r e a t e r abundance of polysomes (41). B u r s a l lymphocytes a l s o show an i n c r e a s e d 32 3 r a t e of uptake of B and t r i t i a t e d thymidine ( H-Tdr) over thymic lymphocytes i n v i t r o (42, 43), as w e l l as i n v i v o (44). In .addition, c o r t i c a l b u r s a l lymphocytes showed a 3 g r e a t e r i n c o r p o r a t i o n o f H-Tdr than medullary lymphocytes (44). T h i s , . c o u p l e d w i t h the f a c t t h a t medullary lymphocytes d i s p l a y s u r f a c e immunoglobulins w h i l e c o r t i c a l c e l l s do not, has l e d these authors t o suggest t h a t medullary lymphocytes r e p r e s e n t a. more mature form of b u r s a l c e l l and t h a t c o r t i c a l c e l l s are the immature p r o l i f e r a t i n g c e l l s . S p e c i f i c a n t i s e r a perpared a g a i n s t b u r s a l or thymic c e l l a n tigens has served to c o n f i r m the f u n c t i o n s of b u r s a l c e l l s i n the p e r i p h e r a l c i r c u l a t i o n and s p l e e n . A n t i - b u r s a l c e l l serum (ABS) has been shown to be c y t o t o x i c ( i n the presence o f complement) to g r e a t e r than 90% of b u r s a l c e l l lymphocytes, while anti-thymus c e l l serum (ATS) i s t o x i c to g r e a t e r than 90% of thymic lymphocytes, but l e s s than 12% of b u r s a l c e l l s (45). 12 In t h i s same study, approximately 3 0% of the spleen c e l l s reacted p o s i t i v e l y with ABS and a s i m i l a r percentage were p o s i t i v e for ant i -chicken immunoglobulin serum, i n d i c -at ing spleen c e l l s bearing the bursa l antigens also synthesize immunoglobulins. The combined presence of bursal c e l l antigens and c e l l surface immunoglobulin was also demonstrated by Wick et a l . (46) as we l l as c e l l surface antigen common to both thymus and bursa l c e l l s . ABS and ATS has also been employed to confirm the spleen and per iphera l blood c e l l s responsible for ce r t a in types of immune responses. ABS, i n the presence of complement, was able to i n h i b i t plaque formation to red blood c e l l s but not graft-versus-host react ion (47, 48). thought to be mediated by thymic lymphocytes. B. The Thymus 1. Thymic Structure and Ontogeny In the adult b i r d , the thymus consis t s of 14 lobes i n t o t a l , of which there are 7 lobes associated along each of the r i g h t and l e f t jugular ve ins . Payne (1) reviews i t s s tructure as being s imi l a r to the mammalian thymus. Each lobe i s p a r t i a l l y separated by connective t i s sue and c l e a r l y d iv ided into an outer zone (cortex) of densely packed small lymphocytes with scattered r e t i c u l a r c e l l s and an inner zone.designated the medulla. The medulla consis t s of less densely packed lymphocytes, 13 r e t i c u l a r c e l l s , and f o c i of e p i t h e l i a l c e l l s denoted H a s s a l l ' s c o r p u s c l e s . Thymic r e g r e s s i o n occurs a t the onset of s e x u a l m a t u r i t y and can be induced a r t i f i c i a l l y by the a d m i n i s t r a t i o n of c o r t i c o i d s (49). I t i s c h a r a c t e r i z e d by a l o s s o f c o r t i c a l s t r u c t u r e l e a v i n g o n l y medullary t i s s u e w i t h few lymphocytes (49). During embryogenesis, the thymus i s the f i r s t lymphoid s t r u c t u r e t o appear. Venzke (50) and Hammond (51) d e s c r i b e i t s development as e p i t h e l i a l cords growing out from the t h i r d and f o u r t h pharyngeal pouches along the j u g u l a r v e i n on the f i f t h day of i n c u b a t i o n . By 5 1/2 days, the e p i t h e l i a l cords no longer m a i n t a i n c o n t a c t w i t h pharyngeal e p i t h e l i u m and l i e surrounded by mesenchyme. Large lymphocytes appear i n the mesenchyme by 7 1/2 days and by 8 1/2 days lymphocytes are p r e s e n t w i t h i n the thymic anlage. From an e m b r y o l o g i c a l s t a n d p o i n t , many of the i d e a s and c o n t r o v e r s y t h a t concerned the development of the bursa are r e l e v a n t to d i s c u s s i o n s of thymic development. E a r l y work i n v o l v i n g the i n v i t r o c u l t u r e of mouse thymic rudiments supported the n o t i o n t h a t lymphocytes were formed from an i n t r i n s i c c e l l source, such as mesenchymal i n d u c t i o n o f e p i t h e l i a l c e l l s (52, 53). T h i s i d e a was proposed e a r l i e r (5) and r e c e i v e d support from h i s t o l o g i c a l data from the c h i c k embryo (54) and mammalian s t u d i e s (55, 56). T h i s q u e s t i o n o f lymphocyte o r i g i n was s e t t l e d u s i n g the same methods employed i n i n v e s t i g a t i n g b u r s a l c e l l o r i g i n s . 14 P r e l i m i n a r y work by H a r r i s and Ford (57), u s i n g chromosome markers and thymus g r a f t i n g techniques, suggested an a f f e r e n t stream o f c e l l s from the host which populated donor g r a f t s i n mice. L a t e r , Moore and Owen (11, 14, 58), u s i n g the same techniques they had f o r b u r s a l s t u d i e s , concluded t h a t thymic rudiments were populated by blood borne lymphocyte p r e c u r s o r s t h a t p r o l i f e r a t e d and d i f f e r e n t i a t e d i n t o thymic lymphocytes. S i m i l a r l y , u s i n g the technique o f g r a f t i n g thymic rudiments of v a r i o u s ages onto the c h o r i o a l l a n t o i c membrane of the c h i c k embryo, Owen and R i t t e r (59) p l a c e d the time of stem c e l l m i g r a t i o n to the thymus a t between the seventh and n i n t h day of i n c u b a t i o n . T h i s has a l s o been confirmed by i n v i t r o c u l t u r e of c h i c k thymus (6 0). Perhaps the most d e f i n i t i v e study a n a l y s i n g the p r e c i s e o r i g i n and c e l l u l a r c o n t r i b u t i o n s . o f the v a r i o u s thymic elements, as w e l l as the t i m i n g o f events i n thymic development, has been c a r r i e d out by Le Douarin and J o t t e r e a u (61, 62). Using the same techniques of i n t e r s p e c i e s . g r a f t i n g as they d i d i n s t u d y i n g b u r s a l development, they have demonstrated u n e q u i v o c a l l y the migratory stem c e l l o r i g i n of the thymic lymphocyte. Moreover, they have confirmed t h a t the f i r s t d e t e c t a b l e lymphoid elements of the embryonic thymus a r e the -cells w i t h a l a r g e nucleus and a s t r o n g l y b a s o p h i l i c cytoplasm, as suggested by Moore and Owen (58) and s i m i l a r to t h a t seen i n the bursa (15, 16). I t can a l s o be concluded from t h e i r study t h a t at a p r e c i s e stage 15 o f i t s h i s t o g e n e s i s t h e t h y m i c r u d i m e n t r e c e i v e s a r a p i d i n f l o w o f stem c e l l s . I t b e g i n s i n t h e l a t t e r h a l f o f t h e s e v e n t h day o f i n c u b a t i o n and i s o f r e l a t i v e l y s h o r t d u r a t i o n , l a s t i n g a p p r o x i m a t e l y 36 h o u r s . F o l l o w i n g t h i s p e r i o d , stem c e l l i n f i l t r a t i o n o c c u r s a t a v e r y s l o w r a t e o r i s n o n - e x i s t e n t . A t some t i m e p r i o r t o h a t c h i n g o r i n e a r l y n e o n a t a l l i f e . , a s u c c e s s i v e wave o f stem c e l l s a p p e a r i n t h e p e r i p h e r a l c o r t e x . They b e g i n p r o l i f e r a t i o n .while l y m p h o c y t e s f r o m t h e f i r s t c e l l wave l e a v e t h e thymus. They p r o g r e s s i v e l y i n v a d e t h e whole thymus f r o m . t h e e x t e r n a l t o t h e i n t e r n a l c o r t e x and f i n a l l y t h e m e d u l l a . They c o n c l u d e f r o m t h e i r i n v e s t i g a t i o n s t h a t t h e thymus i s r e p o p u l a t e d a t l e a s t t w i c e d u r i n g e m b r y o n i c l i f e by s u c c e s s i v e waves o f m i g r a t o r y s t e m c e l l s and t h a t e a c h wave i s o f r e l a t i v e l y s h o r t d u r a t i o n . T h e s e stem c e l l s a r e a v a i l a b l e t o t h e thymus t h r o u g h o u t e m b r y o n i c l i f e and i t i s t h e thymus t h a t - t r i g g e r s m i g r a t i o n and a t t r a c t s stem c e l l s t o i t . The thymus a l s o has t h e c a p a b i l i t y t o a t t r a c t s tem c e l l s i n h e t e r o t o p i c l o c a t i o n s and a c r o s s s p e c i e s b a r r i e r s ( 6 2 ) . 2. The P r i m a r y E f f e c t s o f Thymectomy and T hymic F u n c t i o n I n i t i a l s t u d i e s o f t h e a v i a n thymus c o n c e r n e d i t s p o s s i b l e e n d o c r i n e r o l e i n s u c h p h y s i o l o g i c a l f u n c t i o n s as g r o w t h , egg p r o d u c t i o n and i t s r e l a t i o n s h i p t o o t h e r e n d o c r i n e g l a n d s , i n p a r t i c u l a r t h e t h y r o i d , a d r e n a l s and 16 gonads (1). Experimental evidence, however, f a i l e d to support such a n o t i o n . With d i s c o v e r i e s concerning the mammalian thymus, to g e t h e r w i t h r e v e l a t i o n s of the bursa's r o l e i n the iiraiune response, there was renewed i n t e r e s t i n a v i a n thymic f u n c t i o n . I t was apparent from mammalian s t u d i e s t h a t the thymus was r e s p o n s i b l e f o r the a b i l i t y t o r e j e c t t i s s u e g r a f t s , f o r the p r e v e n t i o n o f c h r o n i c wasting d i s e a s e and f o r the c e l l s p a r t i c i p a t i n g i n g r a f t -v e r s u s - h o s t * (GvH) r e a c t i o n s (63). The thymus was a l s o shown t o c o n t r i b u t e immunocompetant smal l lymphocytes thought to p a r t i c i p a t e i n c e l l u l a r immune responses and m a i n t a i n immunological memory (6 3). Much of the subsequent experimental evidence concerning the a v i a n thymus has presented a. s i m i l a r p i c t u r e . J a n k o vic and I s v a n e s k i (31) found t h a t neonatal thymectomy prevented the i n c i d e n c e of experimental a l l e r g i c e n c e p h a l o m y e l i t i s as w e l l as i m p a i r i n g the delayed h y p e r s e n s i t i v i t y r e a c t i o n t o s p i n a l cord l i p i d s and t u b e r c u l i n . I t i s a l s o of i n t e r e s t to note t h a t b i r d s showing thymic c o r t i c a l damage f o l l o w -i n g hormonal bursectomy a l s o demonstrate impairment of d e l a y e d h y p e r s e n s i t i v i t y (29). Thymectomy has l i t t l e o r no e f f e c t on antibody p r o d u c t i o n t o most antigens (32, 64, 65). I t a l s o r e s u l t s i n an absence of s m a l l lymphocytes t h a t normally surround the Scweigger-Seidel sheaths, nodules and a r t e r i e s of the spleen, comprising.the d i f f u s e lymphocytic areas of the white pulp (64). P e r i p h e r a l s m a l l lymphocytes are decreased g r e a t l y i n thymectomized 17 b i r d s and t h i s i s c o r r e l a t e d w i t h an i n c r e a s e i n the time r e q u i r e d t o r e j e c t s k i n g r a f t s (64, 66). As i n mammalian s t u d i e s , thymic lymphocytes have a l s o been found to be r e s p o n s i b l e f o r the GvH r e a c t i o n i n the c h i c k (42, 47, 48, 49). However, while c y t o t o x i c ATS can d e s t r o y the GvH potency of-thymic c e l l s , i t does not i n h i b i t responsiveness to mitogens such as phytohemagglutinin (47). In a phenomena s i m i l a r to the GvH, the mixed lymphocyte r e a c t i o n (MLR), i t has a l s o been demonstrated t h a t the responding c e l l s are thymic lymphocytes, and i n p a r t i c u l a r , medullary thymocytes (64). Thymocytes undergo p h y s i c o - c h e m i c a l changes d u r i n g the course of development, as d i s c u s s e d e a r l i e r w i t h b u r s a l lymphocytes, t h a t can a l s o be r e l a t e d t o immunological m a t u r i t y and f u n c t i o n . Warner (49) n o t i c e d t h a t c o r t i s o n e induced atrophy of. the thymic c o r t e x and i n c r e a s e d GvH potency of thymic c e l l suspensions. He concluded t h a t t h i s treatment i n c r e a s e d the c o n c e n t r a t i o n of medullary thymocytes and t h a t these c e l l s are mature GvH competent c e l l s w h i l e c o r t i c a l c e l l s r e p r e s e n t immature p r o l i f e r a t i n g c e l l s . With the same techniques they had used f o r b u r s a l c e l l a n a l y s i s , Peterson and Good (39) and Sherman and Auerbach (40) found t h a t a r e d u c t i o n a l s h i f t i n mean c e l l volumes of thymocytes o c c u r r e d between the 12th and 18th days of i n c u b a t i o n . T h i s was i n t e r p r e t e d to be the p r o d u c t i o n of s m a l l e r more mature thymocytes. Along t h i s l i n e o f i n v e s t i g a t i o n , p r o b a b l y the most compre-hensive s e r i e s of s t u d i e s has been performed by Droege e t a l 18 (86-74). D i s c o v e r i n g a c e r t a i n amount of h e t e r o g e n e i t y i n thymic lymphocytes, they have been able to c l a s s i f y thymocytes i n t o v a r i o u s sub-populations based on e l e c t r o p h o r e t i c m o b i l i t y (68,69), d i f f e r e n t i a l c e n t r i f u g a t i o n (70, 71) and a combination of these two p l u s c e l l s i z e d i s t r i b u t i o n a n a l y s i s (72, 73). Such three-way a n a l y s i s of thymocytes has d i f f e r e n t i a t e d three b a s i c sub-populations i n the thy-mus (72). The thymocyte p o p u l a t i o n which i s the l a r g e s t , l e a s t dense and the f a s t e s t moving e l e t r o p h o r e t i c a l l y i s thought to r e p r e s e n t the mature.medullary s m a l l lymphocyte (72). I t appears to be GvH competent, c o r t i s o n e r e s i s t a n t and predominates i n the thymus o f s e x u a l l y mature b i r d s whose c o r t e x has i n v o l u t e d (73). The remaining two p o p u l a t i o n s , whose f u n c t i o n s are not c l e a r , c o r r e l a t e p r o p o r t i o n a t e l y w i t h the d i v i d i n g and n o n - d i v i d i n g compartments o f the c o r t e x (73). During the course o f development, there i s a s h i f t i n the p r o p o r t i o n s of sub-p o p u l a t i o n s from the predominantly s m a l l e s t , most dense p o p u l a t i o n , to t h a t of the mature c e l l s seen i n the a d u l t thymus (72). A s i m i l a r d i f f e r e n t i a t i o n of sub-p o p u l a t i o n s has a l s o been demonstrated f o r the mouse thymus (74). C. The D i s t r i b u t i o n o f B u r s a l and Thymic C e l l s and Lymphoid C e l l T r a f f i c The p r e v i o u s d i s c u s s i o n has demonstrated t h a t the bursa and thymus export c e l l s t o the s p l e e n and p e r i p h e r a l c i r c u l a t i o n . A survey of the l i t e r a t u r e . ' . i l l u s t r a t e s .that 19 t h i s has been confirmed by the use of many d i f f e r e n t techniques and t h a t b u r s a l and thymic c e l l s are prese n t i n other lymphoid t i s s u e s as w e l l . Thorbecke e t a l (75) noted the presence o f plasma c e l l s . i n the o u t e r p o r t i o n s o f the thymic medulla i n germfree, as w e l l as c o n v e n t i o n a l b i r d s . S i m i l a r l y , Tamminen e t a l (76) separ-ated b u r s a l c e l l s capable o f responding to B r u c e l l a  abortus from thymic c e l l s capable of "GvH by d i f f e r e n t i a l c e n t r i f u g a t i o n o f a d u l t thymus c e l l suspensions. However, perhaps the most u s e f u l technique f o r d e t e c t i n g the presence of b u r s a l o r thymic c e l l s has been the use of s p e c i f i c a n t i s e r a prepared a g a i n s t thymic or b u r s a l antigens (ATS and A B S , r e s p e c t i v e l y ) , coupled w i t h a f l u o r e s c e n t marker. Such techniques, as p r e v i o u s l y d i s c u s s e d , have d e t e c t e d the presence o f approximately 8%.bursal c e l l s i n the thymus and 13% thymus c e l l s i n the bursa of fou r week o l d b i r d s (77) . There was a l s o a c e r t a i n percentage of thymus and b u r s a l c e l l s showing.neither a n t i g e n , termed n u l l c e l l s (77) , and the presence o f common antigens to both thymus and b u r s a l c e l l s (46). A l b i n i and Wick (78), i n an e x t e n s i v e survey o f lymphatic t i s s u e s from chickens 1 day to 24 weeks of age, found the. f o l l o w i n g percentages o f thymic and b u r s a l c e l l s . The bursa ranged from 70 t o 90% p o s i t i v e f o r ABS and 0 to 7% p o s i t i v e f o r ATS. Thymic t i s s u e ranged from 80 to 100% ATS p o s i t i v e and 0 to 20% ABS p o s i t i v e . ABS and ATS p o s i t i v e c e l l s were approximately equal at 5 weeks:.;of age i n 20 the c e c a l t o n s i l s , with*a predominance of ABS p o s i t i v e c e l l s w i t h i n c r e a s i n g age. The spleen and p e r i p h e r a l b l o o d showed a s l i g h t l y g r e a t e r percentage of ATS than ABS p o s i t i v e c e l l s . F i n a l l y , u s i n g s i m i l a r t echniques, G l i c k e t a l (79) r e c e n t l y demonstrated t h a t c e l l s b e a r i n g s u r f a c e immunoglobulins (mature b u r s a l c e l l s ) never exceeded 11% i n the s p l e e n or 4% i n the thymus, up t o 8 weeks of age. Bone marrow a l s o . d i s p l a y e d the presence of medium and s m a l l lymphocytes, b e a r i n g s u r f a c e immuno-g l o b u l i n , a t 9.5% and. 8.8% r e s p e c t i v e l y . The p r e v i o u s d i s c u s s i o n s concerning the o r i g i n s of b u r s a l and thymic lymphocytes and t h e i r subsequent d i s t r i b u t i o n i n v a r i o u s lymphoid t i s s u e s serves to i l l u s t r a t e t h a t there appears to be a m i g r a t i o n of lym-phoid c e l l s w i t h i n the embryo and a d u l t . Such m i g r a t i o n has been i n v e s t i g a t e d by more d i r e c t experimental means and the reader i s r e f e r r e d to L i n n a e t a l (8 0) f o r an e x c e l l e n t review and c r i t i c a l e v a l u a t i o n of techniques. The primary s i t e f o r hematopoiesis and the o r i g i n a t i n g source of lymphoid stem c e l l s i s thought by many authors t o be the b l o o d i s l a n d s of the y o l k sac (1). L o c a l i z e d l a b e l l i n g of y o l k sac c e l l s of 16 day o l d embryos, by r a d i o i s o t o p e s , has demonstrated the m i g r a t i o n o f y o l k sac c e l l s t o the bursa and thymus w i t h i n 24 hours (81). In a d d i t i o n , y o l k sac c e l l s b e a r i n g b u r s a l and thymic c e l l a ntigens have been d e t e c t e d as e a r l y a t 5 to 7 days of i n c u b a t i o n (82). These s t u d i e s suggest -21 t h a t not o n l y are the c e l l s o f the y o l k sac r e s p o n s i b l e f o r p o p u l a t i n g the bursa and thymus but t h a t these c e l l s may be committed to these organs very e a r l y i n development. B u r s a l c e l l s a l s o migrate from the bursa to o t h e r lymphoid t i s s u e s as i m p l i e d from the p r e v i o u s d i s c u s s i o n s and was f i r s t v e r i f i e d by. Woods and L i n n a (83) i n 1965. By l a b e l l i n g b u r s a l c e l l s o f a d u l t c h i c k e n s , they d i s c o v e r e d l a b e l l e d c e l l s i n the spleen, i n t e s t i n a l lymphoid t i s s u e , l i v e r and the thymus. Such m i g r a t i o n was a l s o shown to be i n h i b i t e d by bursectomy f o l l o w i n g l a b e l l i n g . . T h i s was confirmed i n a l a t e r study (84) which i n d i c a t e d t h a t the g r e a t e s t m i g r a t i o n was to the spleen, f o l l o w e d by the duodenum, bone marrow and thymus, i n d e c r e a s i n g l e v e l s . However, b u r s a l l a b e l l i n g i n the 18 day embryo demonstrated t h a t there was a g r e a t e r r a t e of m i g r a t i o n to thymus, r a t h e r than to the spleen (86, 87) and to the bone marrow (88). These types of c e l l s were a l s o shown to possess antibody r e s t o r a t i o n c a p a b i l i t i e s i n bursectomized (88) or bursectomized-i r r a d i a t e d b i r d s (87). A s e r i e s of s t u d i e s by Toivanen e t a l (89-92) has c l a s s i f i e d these c e l l s as postembryonic b u r s a l stem c e l l . They have found such c e l l s t o be immunocompetent, capable of r e s t o r i n g antibody s y n t h e s i s i n bursectomized. .hosts, p r i m a r i l y to migrate from the bursa f o l l o w i n g maturation to the p e r i p h e r a l lymphoid t i s s u e s and to i n c r e a s e i n numbers w i t h age and b u r s a l 22 i n v o l u t i o n . By the same token, thymic c e l l s are a l s o migratory. In a d d i t i o n t o the e x i s t i n g evidence, Warner (42) found t h a t thymic c e l l s home to the spleen i n g r e a t e r percentages than do b u r s a l c e l l s . Noting t h a t the thymus precedes the bursa i n development and d e t e c t i n g the presence o f thymic c e l l s i n the bursa, he a l s o suggested t h a t the thymus may i n p a r t "seed" the bursa with c e l l s between 12 and 16 days of i n c u b a t i o n (93). Back (94) concluded t h a t the thymus i s a l s o r e s p o n s i b l e f o r c o n t r i b u t i n g the m a j o r i t y of lymphocytes p o p u l a t i n g the i n t e s t i n a l e p i t h e l i u m . Thymic lymphocytes are a l s o found t o home to the bone marrow f o l l o w i n g a n t i g e n i c s t i m u l a t i o n i n a d u l t b i r d s and not the spleen, c e c a l t o n s i l s o r bursa (95). D. Some E f f e c t s o f Bursectomy on Thymic Development In view o f the g e n e r a l l y accepted n o t i o n t h a t the bursa and thymus r e p r e s e n t and c o n t r i b u t e t o separate d i v i s i o n s of the immune system, i t i s i n t e r e s t i n g to c o n s i d e r the i m p l i c a t i o n s o f c e l l u l a r m i g r a t i o n s between these two lymphoid organs. The preceeding d i s c u s s i o n has presented evidence that, m i g r a t i o n does e x i s t , however the magnitude and f u n c t i o n a l p r o p e r t i e s of such m i g r a t i n g c e l l s remains t o be demonstrated. Our p a r t i c u l a r i n t e r e s t s l i e w i t h the b u r s a l c o n t r i b u t i o n t o thymic development, which stems from e a r l i e r 23 work i n our l a b (36) t h a t demonstrated an i n h i b i t i o n of thymic development f o l l o w i n g s u r g i c a l bursectomy at 70 hours i n ovo. C e r t a i n l y , s t u d y i n g thymic development i n the absence of the bursa i s one approach t h a t may be taken to determine b u r s a l i n f l u e n c e . However, much of the e a r l y work p r e s e n t s , a t b e s t , c o n t r o v e r s i a l evidence t o support a r e l a t i o n s h i p between bursectomy and thymic development. Hormonal bursectomy, performed p r i o r to the 13th day of i n c u b a t i o n , produces v a r y i n g degrees of thymic c o r t i c a l atrophy ranging from normal development to complete c o r t i c a l atrophy (18,19, 26, 28, 29). Other r e p o r t s c l a i m no e f f e c t on the thymus regard-l e s s o f the dose and time of t e s t o s t e r o n e a d m i n i s t r a t i o n (21, 23.)., S t i l l o t h e r r e p o r t s c l a i m i n c o n s i s t e n t r e s u l t s w i t h estrogens or adrogens (22). The predominant c r i t e r i a used f o r e v a l u a t i o n o f the thymus has been h i s t o l o g i c a l evidence; however,Sherman and Auerbach (40) n o t i c e d a s h i f t t o l a r g e r mean c e l l volumes, i n the thymus o f embryos t r e a t e d w i t h t e s t o s t e r o n e . Perhaps the most s t r i k i n g evidence f o r the presence of a bursa-dependent p o p u l a t i o n i n the thymus i s g i v e n by Zucker e t a l [ (73). Using the same techniques they had f o r d e l i n e a t i n g thymocyte sub-populations, they d e t e c t e d a d e f i c i e n c y i n one thymic sub-population of a d u l t b i r d s t h a t were n e o n a t a l l y bursectomized. They a l s o estimated t h i s b u r s a l -dependent p o p u l a t i o n to comprise approximately 15% of the t o t a l thymic lymphocyte p o o l . 24 Such e n c o u r a g i n g , a l b e i t c o n t r o v e r s i a l , e v i d e n c e has l e d us t o attempt t o q u a n t i t a t e t h e e f f e c t s o f hormonal, e a r l y s u r g i c a l and combined h o r m o n a l - e a r l y s u r g i c a l bursectomy on t h e growth and development o f t h e thymus i n . t h e c h i c k embryo. 25 I I I . THE EFFECTS OF HORMONAL BURSECTOMY ON THYMIC DEVELOPMENT A. M a t e r i a l s and Methods F e r t i l e eggs were ob t a i n e d from White Leghorn stock maintained on the P o u l t r y Farm a t the U n i v e r s i t y of B r i t i s h Columbia. A l l eggs were incubated under standard c o n d i t i o n s i n a Jamesway Model 252 Incubator. Groups of 24 eggs were s u b d i v i d e d i n t o two equal groups f o r hormonal bursectomy (HBx) and c o n t r o l . A l l eggs were candled p r i o r t o treatment and o n l y v i a b l e embryos were r e t a i n e d . Hormonal bursectomy was c a r r i e d out on separate groups a t 2, 3, 4, 5, 6, 7, 9, 10 and 15 days of i n c u b a t i o n . Each hormonal bursectomy was performed i n the f o l l o w i n g manner. Two hol e s were made i n the e g g s h e l l w i t h the p o i n t o f an 18 gauge needle. The f i r s t hole was made i n t o the a i r c e l l , w h i l e the second hole was made on the s h e l l ' s l a t e r a l aspect, thus c r e a t i n g an a r t i f i c i a l a i r c e l l over the embryov Each egg was i n j e c t e d through the l a t e r a l h o l e with 2 mg. of t e s t o s t e r o n e p r o p i o n a t e ( 4 ANDROSTIN - l i p - OL - 3 - ONE -* PROPIONATE) i n 0.1 ml. of commercial corn o i l c a r r i e r . C o n t r o l eggs r e c e i v e d 0.1 ml of c a r r i e r o n l y . The ho l e s were s e a l e d w i t h drops of melted p a r a f f i n and the eggs * Sigma Chemicals 26 r e p l a c e d i n the i n c u b a t o r . Samples of the egg contents of embryo f a t a l i t i e s were c u l t u r e d on M u e l l e r - H i n t o n agar (DIFCO) c o n t a i n i n g 5% d e f i b r i n a t e d sheep's blood, as a r o u t i n e examination f o r b a c t e r i a l contamination i n t h i s and the subsequent two experiments. A l l eggs were examined a f t e r 19' days of incuba-t i o n . R e p r e s e n t a t i v e t i s s u e samples were f i x e d i n Bouins f i x a t i v e , embedded i n p a r a f f i n , s e r i a l l y s e c t i o n e d and s t a i n e d w i t h hematoxylin and e o s i n f o r h i s t o l o g i c a l examination. Thymocytes were q u a n t i t a t e d a c c o r d i n g to the method of S a l l s t r o m and Aim (96) . A c c o r d i n g l y , a l l seven r i g h t thymic lobes were d i s s e c t e d f r e e of the j u g u l a r v e i n and p l a c e d i n 1 ml of a s o l u t i o n c o n t a i n i n g one p a r t Dulbecco's phosphate b u f f e r e d s a l i n e (DPBS), * one p a r t Hank's balanced s a l t s o l u t i o n (HBSS) and 1% (w/v) bovine serum albumin (BSA) + The thymus t i s s u e was then f i n e l y minced w i t h s c i s s o r s and g e n t l y homogenized by a . loose f i t t i n g t e f l o n t i s s u e homogenizer. T i s s u e fragments and l a r g e c e l l aggregates were allowed to s e t t l e f o r two minutes;and the supernatants t r a n s f e r r e d t o 10 ml g l a s s tubes. A f t e r c e n t r i f u g a t i o n a t 500 x g f o r 10 minutes at room temperature, the supernatants were d i s c a r d e d and the c e l l p e l l e t resuspended i n 1 ml of DPBS/HBSS/BSA s o l u t i o n . Thymocyte numbers were determined by count, *GIBC0 +Sigma Chemicals 27 u s i n g an Improved Neubauer Hemocytometer. A minimum -2 3 volume o f 2 x 10 mm was counted. B u r s a l p e r s ence was d e t e r m i n e d by g r o s s i n s p e c t i o n and v e r i f i e d by h i s t o l o g i c a l e x a m i n a t i o n . Embryo age was e s t i m a t e d u s i n g methods a c c o r d i n g t o Hamburger and H a m i l t o n ( 9 7 ) . D i f f e r e n c e s between t r e a t m e n t and c o n t r o l means were t e s t e d by t h e S t u d e n t ' s t - t e s t . The d i f f e r e n c e between means,and w i t h i n ^ t r e a t r a e n t s ' o v e r t i m e was t e s t e d by a n a l y s i s o f v a r i a n c e . A l l l e v e l s o f s i g n i f i c a n c e were chosen a t P £ 0.05. B. R e s u l t s and D i s c u s s i o n A t o t a l o f 84 eggs were h o r m o n a l l y b u r s e c t o m i z e d a t , t h e v a r i o u s s t a g e s mentioned and a t o t a l o f 85 eggs were used as c o n t r o l s , d i v i d e d t o c o r r e s p o n d t o t h e t r e a t -ment groups. The average m o r t a l i t y f o r hormonal bursectomy and c o n t r o l was 2 6.2% and 21.6%, r e s p e c t i v e l y . I n no case was b a c t e r i a l c o n t a m i n a t i o n p r e s e n t i n embryo f a t a l i t i e s . I n a p p a r e n t agreement w i t h t h e l i t e r a t u r e p r e v i o u s l y d i s c u s s e d (6, 18, 19, 26, 28, 2 9 ) , t h e r e i s c o n s i d e r a b l e v a r i a t i o n i n t h e degree o f t h y m i c c o r t i c a l development, as seen h i s t o l o g i c a l l y . T h i s s e r v e s t o emphasize the d i f f i c u l t y one e n c o u n t e r s i n a t t e m p t i n g t o q u a n t i t a t i v e l y a s s e s s t h e e f f e c t s o f hormonal b u r -sectomy on thymic development by h i s t o l o g i c a l p r o c e d u r e s . I n a l l t r e a t m e n t groups, w i t h t h e e x c e p t i o n o f t h o s e b u r s e c t o m i z e d on day 15, t h e b u r s a was p a t e n t l y a b s e n t. 28 Those hormonally bursectomized on day 15 demonstrated burs presence, but w i t h a somewhat r e t a r d e d gross appearance. Q u a n t i t a t i v e l y however, thymic development f o r the v a r i o u s treatment groups p r e s e n t s an i n t e r e s t i n g . . p i c t u r e . • (Table. I) Mean thymocyte numbers were s i g n i f i c a n t l y l e s s than t h e i r c o n t r o l means i n groups bursectomized between 2 and 5 days of i n c u b a t i o n , and not s i g n i f i c a n t l y d i f f e r e n t when bursectomized a t days 6 through 15. There was no s i g n i f i -cant d i f f e r e n c e found w i t h i n the hormonal bursectomy means o r the c o n t r o l means between days 2 and 5. T h i s i n d i c a t e s t h a t the e f f e c t of hormonal bursectomy on thymocyte numbers i s u n i f o r m l y the same when i t i s c a r r i e d out by day 5 or e a r l i e r (Table I I ) . T h i s seems to be i n disagreement w i t h p r e v i o u s l y r e p o r t e d h i s t o l o g i c a l f i n d i n g s (98) and may r e f l e c t the d i f f i c u l t i e s i n a s s e s s i n g h i s t o l o g i c a l evidence q u a n t i t a t i v e l y . What i s i n t e r e s t i n g i s t h a t t h i s d e p r e s s i o n o f thymocyte numbers does not occur when HBx i s per-formed a f t e r the f i f t h day of i n c u b a t i o n , r e g a r d l e s s of whether the bursa i s p r e s e n t or not. T h i s i s somewhat p a r a d o x i c a l i n view o f the proposed model which suggests t h a t there i s a s i g n i f i c a n t b u r s a l c o n t r i b u t i o n t o thymocyte numbers. Perhaps the h i s t o l o g i c a l appearance of the thymuses from embryos t h a t demonstrate depressed thymocyte numbers has some b e a r i n g on the r e s u l t s . Although the degree of c o r t i c a l atrophy i s somewhat v a r i a b l e and s i m i l a r h i s t o l o g i c a l l y to p r e v i o u s l y 29 TABLE I EFFECTS OF HORMONAL BURSECTOMY AT VARIOUS INCUBATION STAGES ON THE THYMIC DEVELOPMENT OF 19 DAY CHICK EMBRYOS HORMONAL BURSECTOMY CONTROL Days Incubated at time of treatment Number of Embryos Mean Thymocyte Count Number of Thymocyte Count X10 (+ S.E.) Embryos XI0 (+ S.E.) 2 8 3.52 + 0. 38* 5 5.43 + 0. 33 3 9 3.60 + 0.50* 7 6.24 + 1.00 4 9 4.50 + 0.55* 8 7.28 + 0. 86 5 10 4. 02 + 0.57* 9 7.47 + 0. 82 6 9 7.67 + 2. 62 9 5.85 + 1. 01 7 10 5.54 + 0. 94 9 5.23 + 1. 00 9 8 6 .22 + 0.93 3 4. 46 + 0. 91 10 4 3.45 + 0. 51 2 3.77 + 0. 91 15 5 5.03 + 0. 45 6 4. 00 + 1.63 +Right Thymic Lobes Only * S i g n i f i c a n t d i f ferences , treated vs. control s at P i 0.05 30 TABLE SUMMARY OF EFFECTS OF ON THE THYMIC DEVELOPMENT II HORMONAL BURSECTOMY OF 19 DAY CHICK EMBRYOS Days Incubated Pooled Mean Thymocyte Count + ^ t f " 6 ° f (X10 7 + S.E.)  Treatment Hormonal Bursectomy Control 2 through 5 4.01 + 0.26* 6.77 + 0.43 6 through 15 5.92 + 0.49 4.99 + 0.50 +Right thymic lobes only *Treated vs. control, s i g n i f i c a n t l y d i f f e r e n t at P & 0.05 31 r e p o r t e d r e s u l t s (6, 28, 29) ( F i g . 1), i t s appearance resembles the i n v o l u t e d c o r t e x seen a t sexual m a t u r i t y (1). Perhaps t h i s i s not s u r p r i s i n g s i n c e i n c r e a s e d l e v e l s of androgens at puberty have been i m p l i c a t e d as one o f the causes of the i n v o l u t i o n process (1, 63, 99). However, i t i s of i n t e r e s t t o c o n t r a s t t h i s morphology wit h the thymic morphology seen f o l l o w i n g SBx (vide i n f r a ) which does not have an i n v o l u t e d appearance. Why an i n v o l - . uted c o r t e x i s not induced c o n s i s t e n t l y by HBx a f t e r day f i v e i s s t i l l u n c l e a r . However, the data would appear to support a d i r e c t e f f e c t o f t e s t o s t e r o n e on the thymus i n s t e a d o f the e f f e c t being mediated by the absence of a b u r s a l i n f l u e n c e on thymic development. Because o f the s p e c i f i c times which t e s t o s t e r o n e a f f e c t s thymocyte numbers, i t might be expected t h a t t e s t o s t e r o n e d i r e c t l y i n h i b i t s some stage i n the i n d u c t i o n and development o f the thymic primordium. These e m b r y o l o g i c a l processes occur on o r before the f i f t h day of i n c u b a t i o n (50, 51, 61, 62) and may subsequently i n h i b i t or delay lymphocyte p r o l i f e r a t i o n . Support f o r t h i s n o t i o n i s c e r t a i n l y e q u i v o c a l . For example, i t has been r e p o r t e d t h a t there i s . n o e f f e c t of t e s t o s t e r o n e on the a l k a l i n e phosphatase a c t i v i t y of the thymus s i m i l a r t o t h a t seen c o i n c i d e n t w i t h the i n h i b i t i o n of lymphocyte p r o l i f e r a t i o n i n the bursa (98). A l s o , i s o l a t e d thymic endothelium i s capable of i n d u c i n g mesenchymal t i s s u e t o p a r t i c i p a t e i n thymic h i s t o g e n e s i s as e a r l y as the 15 32 Figure 1. Cross sections of 19 day embryonic chick thymus hormonally bursectomized at a) two days of incubation and b) at nine days of incubation.. H & E s t a i n . Magni f icat ion 90x. 33 somite stage (62), w e l l i n advance of the t e s t o s t e r o n e i n h i b i t i o n of thymic development r e p o r t e d here. L a s t l y , except f o r v a r y i n g degrees of c o r t i c a l atrophy, the thymuses of t r e a t e d embryos r e p r e s e n t a r e l a t i v e l y , n o r m a l h i s -t o l o g i c a l p i c t u r e , i n d i c a t i n g no t e s t o s t e r o n e i n h i b i t i o n o r malformation of the non-lymphocytic thymic c e l l u l a r elements. In view of t h i s , i t would appear more l i k e l y t h a t a t some time p r i o r t o the f i f t h day of i n c u b a t i o n , t e s t o s t e r o n e a l t e r s the thymic primordium in.some way t h a t i s beyond d e t e c t i o n by the methods employed to date. Such an a l t e r a t i o n may l e a d t o a p a r t i a l i n h i b i t i o n o f the thymus 1 r e p o r t e d a b i l i t y to a t t r a c t the i n i t i a l wave of i n v a d i n g stem c e l l s (62). T h i s mechanism would le a d to a s i g n i f i c a n t decrease i n the number of c e l l s a v a i l a b l e f o r p r o l i f e r a t i o n and r e s u l t i n an over a l l r e d u c t i o n i n the number of thymocytes, c o n s i s t e n t w i t h the o b s e r v a t i o n s made here. The exact mechanisms by which t e s t o s t e r o n e i n f l u e n c e s thymocyte numbers however c e r t a i n l y r e q u i r e s f u r t h e r i n v e s t i g a t i o n . IV. THE EFFECTS OF EARLY EMBRYONIC SURGICAL BURSECTOMY ON THYMIC DEVELOPMENT A. M a t e r i a l s and Methods S u r g i c a l bursectomy was c a r r i e d out a c c o r d i n g to Fitzsimmons e t a l (36). A c c o r d i n g l y , f e r t i l e White Leghorn eggs were incubated f o r 3 days under the c o n d i t i o n s de-s c r i b e d p r e v i o u s l y . The eggs were p l a c e d s m a l l end up f o r one hour p r i o r t o the o p e r a t i o n . Each egg's s u r f a c e was swabbed w i t h 70% e t h a n o l p r i o r t o opening - and the e n t i r e procedure was maintained under s t e r i l e c o n d i t i o n s . A s m a l l hole was made i n the narrow end of the egg [ (opposite the a i r c e l l ) and c a r e f u l l y e n l a r g e d w i t h f i n e f o r c e p s (to about 1 cm i n diameter) to expose the whole embryo. Embryos were staged a c c o r d i n g to Hamburger and Hamilton (97) and o n l y those of stage 17-18 were used. Operations on a c c e p t a b l e embryos were c a r r i e d out under a d i s s e c t i o n miscroscope (mag. 12x). The embryo was f l o a t e d to the top o f the hole by the a d d i t i o n of an a p p r o p r i a t e amount o f albumen taken from f r e s h eggs incubated, under the same c o n d i t i o n s as the experimental eggs. A c a t a r a c t k n i f e was used to make the s m a l l s l i t i n the c h o r i o n p e r p e n d i c u l a r to the embryonic a x i s and j u s t p o s t e r i o r to the l e g buds. The t i p of the k n i f e was i n s e r t e d to one s i d e of the m i d l i n e and the opening was extended to the o p p o s i t e s i d e . The amnion was c u t i n the same manner. Care was taken not t o puncture the v i t e l l i n e c i r c u l a t i o n , the y o l k sac or the 35 d e v e l o p i n g a l l a n t o i s . The t i p of the t a i l was then drawn out to l i e on top o f the c h o r i o n . A loop o f f i n e c h i l d s ' h a i r , p r e v i o u s l y s t o r e d i n 75% e t h a n o l w i t h 0.05% t i n c t u r e of i o d i n e , war. r i n s e d i n 70% e t h a n o l , then i n s t e r i l e c h i c k r i n g e r , and passed over the f r e e end of the- t a i l . I t was p o s i t i o n e d j u s t caudal to the l e g buds and the knot t i g h t e n e d s u f f i c i e n t l y to l i g a t e the p a i r e d d o r s a l a o r t a e , w i t h care not to cut i n t o the t i s s u e . The l i g a t e d t a i l p o r t i o n and f r e e ends of the h a i r were trimmed wi t h i r i s s c i s s o r s and.removed. A 2 cm square of p a r a f f i n s h e e t i n g ( P a r a f i l m - F i s h e r S c i e n t i f i c ) was p l a c e d over the hole and cemented to the s h e l l by s p a r i n g l y p a i n t i n g over the edges w i t h melted p a r a f f i n (m.p. 70°C) . Sham-operations were c a r r i e d . , out i n the same manner w i t h the e x c e p t i o n t h a t t h e . t a i l s were not l i g a t e d or cut i n any way. A f t e r s e a l i n g and i n s p e c t i o n s f o r s e a l i n t e g r i t y , a l l eggs were r e p l a c e d i n the i n c u b a t o r , l a r g e end up, and incubated f o r an a d d i t i o n a l 15 days. Untouched eggs were maintained as p o s i t i v e c o n t r o l s . When the accumulated i n c u b a t i o n time had reached 18 days, a l l eggs were removed and examined i n the same f a s h i o n as t h a t d e s c r i b e d f o r hormonal bursectomy. The d i f f e r e n c e i n means between the v a r i o u s treatments was e v a l u a t e d by A n a l y s i s of V a r i a n c e (P^.0.05). 36 B. R e s u l t s and D i s c u s s i o n s A t o t a l o f 1260 eggs were r e a d i e d f o r s u r g i c a l bursectomy w i t h a t o t a l o f 218 deemed v i a b l e f o r f u r t h e r i n c u b a t i o n f o l l o w i n g surgery. Of these, 28 s u r v i v e d to 18 days o f i n c u b a t i o n w i t h an a s s o c i a t e d m o r t a l i t y of 87.2%. S i m i l a r l y , 79 of 600 eggs were incubated as Sham-operated, wi t h 17 s u r v i v i n g (80.1% m o r t a l i t y ) ; 103 out o f 116 c o n t r o l s s u r v i v e d , w i t h a m o r t a l i t y o f 11.3%. B a c t e r i a l contamination was found to be pre s e n t i n 52.4% of the f a t a l i t i e s , w i t h Staphylococcus e p i d e r - m i d i s the predominant microorganism. I t was necessary, due to the hi g h m o r t a l i t y , to p o o l the r e s u l t s o f thr e e separate t r i a l s where . . r e p r e s e n t a t i v e s of.each treatment group were a v a i l a b l e and s u i t a b l e f o r a n a l y s i s . Spleen t o body weight r a t i o s were not s i g n i f i c a n t l y d i f f e r e n t f o r the three groups t e s t e d ( P ^ 0.05). Mean t o t a l thymocyte numbers were t a b u l a t e d (Table I I I ) , and i t was found t h a t s u r g i c a l bursectomy s i g n i f i c a n t l y depressed thymocyte numbers over t h a t o f sham-operated and c o n t r o l embryos. Sham-o e p r a t i o n o f embryos d i d not s i g n i f i c a n t l y reduce mean thymocyte numbers over c o n t r o l s . These r e s u l t s are i n agreement w i t h p r e v i o u s l y r e p o r t e d h i s t o l o g i c a l f i n d i n g s (36) where s u r g i c a l bursectomy seemed t o i n h i b i t thymic development. S i m i l a r l y , i t was found t h a t h i s t o l o g i c a l l y the thymuses o f SBx embryos were somewhat s m a l l e r than 37 TABLE I I I EFFECTS OF SBx AT THREE DAYS IN OVO ON THE THYMIC DEVELOPMENT OF 18 DAY CHICK EMBRYOS TREATMENT No. o f Thymocyte* Embryos+ Count p e r ml. X 1 0 7 (+ S.E.) SBX 6 1.65 + 0.18** SHAM - CONTROLS 5 2 . 9 8 + 0 . 6 6*** CONTROLS 7 3 . 2 1 + 0 . 4 8 + P o o l e d d a t a from s e p a r a t e t r i a l s * R i g h t Thymic Lobes o n l y ** S i g n i f i c a n t d i f f e r e n c e s , t r e a t e d Vs. s h a m - c o n t r o l s and c o n t r o l s a t P ^  0.05 *** No s i g n i f i c a n t d i f f e r e n c e s , s h a m - c o n t r o l s v s . c o n t r o l s a t P < 0.05 38 t h a t o f c o n t r o l s ( F i g u r e 2) b u t t h e r e were no g r o s s m o r p h o l o g i c a l d e f e c t s . Q u a n t i t a t i v e l y , t h i s e x p e r i m e n t i n d i c a t e s t h a t t h e l e v e l o f i n h i b i t i o n i s a p p r o x i m a t e l y 55% o f n o r m a l t h y m o c y t e numbers. I t a l s o i n d i c a t e s t h a t t h e t r a u m a o f t h e o p e r a t i o n i s n o t c o n t r i b u t i n g s i g n i f i c a n t l y t o t h e r e d u c t i o n i n t h y m o c y t e numbers o b s e r v e d f o l l o w i n g s u r g i c a l b u r s e c t o m y , a t l e a s t as f a r as s h a m - o p e r a t i o n s i m u l a t e s s u c h . t r a u m a . I t a p p e a r s t h e r e f o r e t h a t n o r m a l p r o l i f e r a t i o n and d e v e l o p m e n t o f t h y m o c y t e numbers i s d e p e n d e n t on b u r s a l p r e s e n c e . T h i s does n o t seem c o m p a r a b l e t o t h o s e f i n d i n g s p r e s e n t e d on t h e e f f e c t s o f h o r m o n a l b u r s e c t o m y . However, as r e p o r t e d e a r l i e r , t h e e f f e c t s o f h o r m o n a l b u r s e c t o m y may be a d i r e c t e f f e c t o f t e s t o s t e r o n e on t h e thymus. S u r g i c a l b u r s e c t o m y w o u l d c i r c u m v e n t any unknown o r i n d i r e c t . e f f e c t s o f t e s t o s t e r o n e and p o s s i b l y r e p r e s e n t a c l e a r e r p i c t u r e o f b u r s a l i n f l u e n c e on t h y m i c d e v e l o p m e n t . One p o s s i b l e i n t e r p r e t a t i o n i s t h a t t h e e f f e c t s o f h o r m o n a l and e a r l y s u r g i c a l b u r s e c t o m y r e p r e s e n t two d i f f e r e n t m o d e l s a f f e c t i n g t h y m i c d e v e l o p m e n t . As m e n t i o n e d , t e s t o s t e r o n e may i n h i b i t , i n some way, t h e e a r l y p o p u l a t i o n o f t h e t h y m i c p r i m o r d i u m by t h e i n i t i a l wave o f i n v a d i n g stem c e l l s . On t h e o t h e r hand, t h e s u r g i c a l r e m o v a l o f b u r s a l t i s s u e a t an e a r l y enough s t a g e may remove a p o t e n t i a l c o n t r i b u t i o n o f l y m p h o c y t e s f r o m t h e b u r s a . T h i s b u r s a l c o n t r i b u t i o n may c o m p r i s e p a r t o f t h e s e c o n d a r y wave o f stem c e l l s t h a t 39 F i g u r e 2. C r o s s s e c t i o n s o f 18 day embryonic c h i c k thymus a) s u r g i c a l l y b u r s e c t o m i z e d a t t h r e e days i n  ovo and b) c o n t r o l . H & E s t a i n . M a g n i f i c a t i o n 90x. 40 repo p u l a t e the thymus some time before hatching. Indeed, a r e p o p u l a t i o n has been demonstrated (62), but the o r i g i n o f these stem c e l l s has not been i n v e s t i g a t e d . I f the above i n t e r p r e t a t i o n i s t r u e , one would expect a compounding e f f e c t o f combined e a r l y s u r g i c a l and hormonal bursectomy i n d e p r e s s i n g thymocyte numbers over t h a t of e i t h e r s u r g i c a l or hormonal bursectomy i n d i v i d u a l l y . Such reasoning was the r a t i o n a l e f o r attempting the t h i r d . e x p e r i m e n t d e s c r i b e d i n the f o l l o w i n g s e c t i o n . 41 V. THE EFFECTS OF COMBINED HORMONAL AND EARLY EMBRYONIC SURGICAL BURSECTOMY ON THYMIC DEVELOPMENT A. Mater ia l s and Methods Groups of 12 0 f e r t i l e White Leghorn eggs were incubated under the condi t ion described prev ious ly . 60 eggs were removed at 3 days of incubat ion, candled, v iab le embryos were s u r g i c a l l y bursectomized according to the method described i n the preceding sec t ion , and replaced i n the incubator. At 4 days of incubat ion, a l l eggs were candled and one-half of the v i a b l e , s u r g i c a l l y bursectomized group and one-half of the previous ly untouched eggs were hormonally bursectomized. This procedure produced four groups of eggs: those that were s u r g i c a l l y bursectomized at 3 days incubat ion; those that were s u r g i c a l l y bursectomized at 3 days and hormonally bursectomized at 4 days incubat ion; those that were hormonally bursectomized at 4 days; and those that were completely untouched serving as po s i t i ve contro l s . A l l eggs were examined at 18 days of incubation using the previous methods with the fol lowing modif icat ions . Spleen and body weights were taken for a l l embryos examined. Body weights were taken without inc lud ing the weights of the yolk sac or extra-embryonic membranes. In add i t ion , 42 thymus tissue was gently homogenized i n phosphate . buffered saline (PBS) and the c e l l suspension was f i l t e r e d through two layers of tissue placed i n a small funnel. The f i l t e r was washed with 3 ml of PBS and coll e c t e d with the homogenate. The remainder of the procedure and s t a t i s t i c a l analysis were l e f t unchanged. B. Results and Discussion In t h i s experiment a t o t a l of 80 eggs were incubated as s u r g i c a l l y bursectomized, .40 as combined s u r g i c a l l y and hormonally bursectomized, 29 as hormonally bursectomized and 2 0 as controls. The associated mortality for each group was 86.1%, 91.1%, 34.5% and 8.3% respectively. B a c t e r i a l contamination was pre-sent i n 30.4% of the embryo f a t a l i t i e s , again with Staphylococcus epidermidis being the predominant microorganism. Out of three separate t r i a l s , only one t r i a l was successful i n that representatives of each treatment group were viable and suitable for analysis. Only embryos showing no apparent abnormalities were analysed. In the group s u r g i c a l l y bursectomized, one embryo was not included as being representative of that group due to an incomplete removal of the bursa. In f act, the thymocyte count for that embryo approached that of control values (eg. 9.25 x 10 ). S i m i l a r l y , 43 one embryo o f t t h e combined s u r g i c a l and hormonal bursectomy group was not i n c l u d e d f o r the same reason. Although i t i s not p o s s i b l e t o t e l l i f i t was mis-operated (the bursa w i l l be absent due to t e s t o s t e r o n e ) , c i r c u m s t a n t i a l evidence i n d i c a t e s t h i s may be so. I t was c o n s i d e r a b l y l a r g e r than the other members of i t s group, the c l o a c a and anal s p h i n c t e r were as w e l l developed as those of the hormonally bursectomized group (these are absent or p o o r l y formed i n s u r g i c a l l y bursectomized embryos) and i t s thymocyte count was s l i g h t l y h i g h e r than those of the hormonally bursectomized group (eg. 7.95 x 10 ). The mean body weights, spleen weights and spleen to body weight r a t i o s are presented i n Table IV. I t i s i n t e r e s t i n g . to note t h a t s pleen weights are the h i g h e s t i n the s u r g i c a l l y bursectomized group. T h i s appears to be the o p p o s i t e of what occurs i n the neonatal c h i c k f o l l o w i n g e a r l y s u r g i c a l bursectomy, as r e p o r t e d p r e v i o u s l y (36). Perhaps the d e p r e s s i o n of spleen w e i g h t s . i n the neonate found in. t h a t study i s due to the a d d i t i o n a l s t r e s s these b i r d s experience i n n e o n a t a l l i f e as a r e s u l t of bursectomy. I t i s a l s o i n t e r e s t i n g t o note t h a t the spleen to body weight r a t i o i s s i g n i f i c a n t l y h i g h e r i n the s u r g i c a l l y , bursectomized group over t h a t of the combined s u r g i c a l and hormonal bursectomy, hormonal bursectomy and c o n t r o l groups as i l l u s t r a t e d i n Table IV. The data seems t o suggest TABLE IV EFFECTS OF SBx, HBx AND SBx + HBx ON THE BODY WEIGHTS, SPLEEN WEIGHTS AND SPLEEN: BODY WEIGHT RATIOS OF 18 DAY CHICK EMBRYOS Treatment No. of Embryos Body Weight Spleen Weight Spleen: Body Weight r a t i o (g., + S.E.) (g., + S.E.) (X10-4, + S.E.) SBx 5 20. ,19 + 1. , 35 .0162 + . 0008 8. .12 + 0. , 52* HBx 11 27. ,08 + 0. , 82 . 0125 + .0007 4. .63 + 0. ,28 SBX..+ HBx 2 15. .84 + 1. ,12 . 0102 + .0024 6. .37. 1. , 07 C o n t r o l s 9 28. .16 +• 0. , 81 . 0142 + . 0007 5. .06 + 0. ,22 * S i g n i f i c a n t d i f f e r e n c e , SBx vs. HBx, SBx + HBx and c o n t r o l s a t 0.05 45 t h a t t e s t o s t e r o n e may have a d e p r e s s i n g e f f e c t on s p l e e n t o body w e i g h t r a t i o s t h a t o f f s e t s t h e e f f e c t s o f s u r g i c a l bursectomy i n t h e combined treatment group. The s i g n i f i c a n c e o f such d a t a i s n o t a t t h i s t i m e c l e a r . The apparent d i s c r e p a n c y o f t h i s d a t a w i t h t h e f i n d i n g s o f t h e p r e v i o u s e x p e r i m e n t f o r s u r g i c a l l y b u r s e c t o m i z e d embryos c o u l d be accounted f o r by p o s s i b l e s a m p l i n g e r r o r s i n combining t h e t h r e e t r i a l s i n t h e o r i g i n a l e x p e r i m e n t . The e f f e c t s o f t h e s e v a r i o u s t r e a t m e n t on thymocyte numbers, however, p r e s e n t an i n t e r e s t i n g p i c t u r e (Table V ) . The combined e f f e c t s o f hormonal and s u r g i c a l bursectomy produce an approximate 85% r e d u c t i o n i n thymocyte numbers from t h a t o f c o n t r o l embryos and a p p r o x i m a t e l y a 65% r e d u c t i o n from t h a t o f s u r g i c a l o r hormonal bursectomy a l o n e . W h i l e t h e r e i s no s i g n i f i c a n t d i f f e r e n c e i n thymocyte numbers between h o r m o n a l l y o r s u r g i c a l l y b u r s e c t o m i z e d embryos, e i t h e r i s c a p a b l e o f s i g n i f i c a n t l y r e d u c i n g thymocyte numbers by a p p r o x i m a t e l y 57%. H i s t o l o g i c a l l y , t h e thymus from t h e combined t r e a t m e n t group appears t o have no a b n o r m a l i t i e s o f t h y m i c elements ( F i g u r e 3 ) . The main d i f f e r e n c e s l i e i n t h e f a c t t h a t t h e y a r e g r e a t l y reduced i n s i z e , w i t h an o b v i o u s d e f i c i e n c y i n i t s c o r t i c a l complement o f c e l l s . The thymuses of t h e s u r g i c a l l y b u r s e c t o m i z e d group appear t o p r e s e n t an a s p e c t somewhat i n t e r -mediate t o t h e combined t r e a t m e n t and c o n t r o l groups ( F i g u r e 2 ) . 46 TABLE V EFFECTS OF SBx, HBx AND SBx + HBx ON THE THYMIC DEVELOPMENT OF 18 DAY CHICK EMBRYOS Treatment No. of Embryos T o t a l Thymocyte Count X10 6 (+ S.E.) + SBx + HBx 2.13 + 0.13* SBx HBx 11 6.25 + 0.76** 6.07 + 0.29*** CONTROLS 14.44 + 1.16 + Right Thymic Lobes on l y * S i g n i f i c a n t d i f f e r e n c e , SBx + HBx vs. SBx, HBx and c o n t r o l s a t P 4 0.05 ** S i g n i f i c a n t d i f f e r e n c e s , SBx and HBx vs. C o n t r o l s a t P < 0. 05 *** No s i g n i f i c a n t d i f f e r e n c e s , SBx vs. HBx a t P ^ 0.05 47 Figure 3. Cross sect ion of an 18 day embryonic chick thymus s u r g i c a l l y bursectomized at three days i n ovo and hormonally bursectomized at four days of incubat ion. H & E s t a i n . Magni f ica t ion 90x. 48 The s i g n i f i c a n c e o f t h e s e f i n d i n g s appears t o be i n terms o f s u p p o r t f o r t h e model p o s t u l a t e d i n the p r e v i o u s e x p e r i m e n t . The compounding e f f e c t s o f combining e a r l y s u r g i c a l and hormonal bursectomy would t e n d t o suggest t h a t each t r e a t m e n t , viewed s e p a r a t e l y , may mediate i t s e f f e c t s by d i f f e r e n t mechanisms. The f a c t t h a t t o g e t h e r t h e y do not c o m p l e t e l y i n h i b i t thymocyte numbers c o u l d be e x p l a i n e d by a s m a l l m i g r a t i o n o f stem c e l l s w h i c h o c c u r s o u t s i d e t h e t i m e s o f major stem c e l l i n f l u x . Many o t h e r i n t e r p r e t a t i o n s are p o s s i b l e ; an a d d i t i o n a l r e v i e w o f s u p p o r t i n g o r r e f u t i n g e v i d e n c e may h e l p t o e s t a b l i s h i t s v a l i d i t y . 49 V I . GENERAL DISCUSSION AND CONCLUSIONS To b r i e f l y summarize t h e e x i s t i n g e v i d e n c e p r e v i o u s l y d i s c u s s e d c o n c e r n i n g t h y m i c d e v e l o p m e n t , i t i s known t h a t t h e thymus i s p o p u l a t e d , b y an i n f l u x o f m i g r a t i n g stem c e l l s . T h i s i n f l u x i s o f r e l a t i v e l y s h o r t d u r a t i o n and o c c u r s a r o u n d t h e end o f t h e s e v e n t h day o f i n c u b a t i o n . A s i m i l a r m i g r a t i o n o f stem c e l l s a l s o ' o c c u r s sometimes i n t h e l a t e e m b r y o n i c l i f e , com-p l e t e l y r e p l a c i n g t h e e x i s t i n g t h y m o c y t e p o p u l a t i o n , w h i c h i s e m i g r a t i n g t o p e r i p h e r a l s i t e s ( 6 2 ) . A t l e a s t some o f t h e t h y m o c y t e s p r e s e n t i n t h e thymus a r e d e r i v e d f r o m c e l l u l a r t r a f f i c f r o m t h e b u r s a i n t h e embryo (82, 85) and i n t h e a d u l t (69, 70, 73, 8 3 ) . C h a r a c t e r i z a t i o n o f t h y m i c s u b - p o p u l a t i o n s i n d i c a t e s t h a t t h e r e i s a b u r s a l - d e p e n d e n t s u b - p o p u l a t i o n , c o m p r i s i n g 15% o f t h e t o a l t h y m i c l y m p h o c y t e p o o l , t h a t i s a b s e n t f o l l o w i n g n e o n a t a l s u r g i c a l b u r s e c t o m y ( 7 3 ) . I n f a c t , t h e 18 day e m b r y o n i c thymus c o n s i s t s m a i n l y o f c e l l s c h a r a c t e r i s t i c o f t h e b u r s a l - d e p e n d e n t s u b - p o p u l a t i o n ( 6 9 ) . I n c o n t r a s t , t h e thymuses o f a d u l t b i r d s whose b u r s a has i n v o l u t e d c o n t a i n t h e same t h y m i c s u b - p o p u l a t i o n s as t h o s e o f j u v e n i l e b i r d s w h i c h h ave b e e n n e o n a t a l l y b u r s e c t o m i z e d . , (70) . . F u r t h e r e v i d e n c e o f b u r s a l i n f l u e n c e 50 on thymic development has been suggested by thymic c o r t i c a l atrophy induced by hormonal bursectomy (18, 19, 26, 28, 29) and early surgical bursectomy (36). Although t h i s evidence has been somewhat controversial, the data presented i n th i s study presents aspects of the e f f e c t s of hormonal and surgical bursectomy which suggest a possible explanation for the observed r e s u l t s . The model that has been suggested, based on the obser-vations made i n t h i s investigation, appears to be compatible with the evidence suggesting a bursal c e l l u l a r contribution to thymocyte numbers. That i s , the depression of thymocyte numbers following hormonal bursectomy appears to be mediated by a d i r e c t influence of t e s t -osterone on the thymic primordium at five days of incubation and may be explained as a testosterone induced i n h i b i t i o n of the i n i t i a l population of the thymus by migrating stem c e l l s . Early surgical bursectomy, on the other hands, would circumvent any effects of a testosterone-thymus in t e r a c t i o n . Such a model could suggest more accurately the s i g n i f i c a n t reduction i n thymocyte numbers. Interestingly enough, th i s reduction i s s i m i l a r i n magnitude to the ef f e c t s seen following hormonal bursectomy on or before the f i f t h , day of incubation. If i n fact early surgical bursectomy removes a po t e n t i a l contribution of c e l l s from the bursa to thymocyte numbers, one might postulate that hormonal and surgical bursectomy mediate t h e i r e f f e c t s by two 51 s e p a r a t e mechanisms. Hormonal bursectomy reduces thymocyte numbers by t h e mechanism d i s c u s s e d above, and s u r g i c a l bursectomy may f u n c t i o n by removing t h e b u r s a l c o n t r i b u t i o n t o t h e second r e p o p u l a t i o n o f t h e thymus w h i c h o c c u r s l a t e r i n embryonic l i f e (62). T h i s would appear t o be c o n s i s t e n t w i t h t h e f i n d i n g s t h a t b u r s a l - d e p e n d e n t s u b - p o p u l a t i o n s predominate i n 18 day embryonic thymuses (69). S i m i l a r l y , i f two s e p a r a t e mechanisms a r e i n o p e r a t i o n , one would e x p e c t an a d d i t i v e e f f e c t i n r e d u c i n g thymocyte numbers by com-b i n i n g hormonal w i t h e a r l y embryonic s u r g i c a l bursectomy. The r e s u l t s o f t h e t h i r d e x p e r i m e n t v e r i f y such an a d d i t i v e e f f e c t . Combined hormonal and s u r g i c a l bursectomy n e a r l y d o u b les t h e r e d u c t i o n i n thymocyte numbers produced by e i t h e r o f t h e t r e a t m e n t s i n d i v i d u a l l y . Such r e s u l t s l e n d f u r t h e r s u p p o r t t o t h e n o t i o n t h a t two s e p a r a t e mechanisms a r e i n v o l v e d and t h a t e a r l y s u r g i c a l bursectomy may be a v a l i d approach t o d e l i n e a t i n g t h e b u r s a l c o n t r i b u t i o n t o thymocyte numbers. However, i f the r e s u l t s o f e a r l y s u r g i c a l bursectomy a r e p r e d i c a t e d s o l e l y on t h e absence o f t h e b u r s a , i t s t i l l remains t o r e c o n c i l e t h e f a c t t h a t t h e r e was no d e p r e s s i o n i n thymocyte numbers when hormonal bursectomy was c a r r i e d o u t a f t e r f i v e days o f i n c u b a t i o n . I n a d d i t i o n t o t h o s e p o s s i b i l i t i e s d i s c u s s e d e a r l i e r , s e v e r a l a d d i t i o n a l i n t e r p r e t a t i o n s are p o s s i b l e . I t c o u l d be s u ggested t h a t t e s t o s t e r o n e may somehow mask th e 52 e f f e c t s of bursal absence. The evidence would not appear to support t h i s idea due to the fact that testosterone does not mask the e f f e c t s of bursal absence i n the combined treatment group. Another possible interpre-t a t i o n i s that testosterone may act d i r e c t l y on the stem c e l l s , i n h i b i t i n g t h e i r migratory a b i l i t y i n some way. However, there appears to be no s i g n i f i c a n t evidence for an embryological event coincident with the f i f t h day incubation of thymocyte numbers that might possibly explain t h i s mechanism for testosterone action. S i m i l a r l y , stem c e l l s have been shown to be capable of populating thymic grafts throughouththe embryonic period from as early as three days of incubation (62). In view of t h i s a v a i l a b i l i t y , i t seems unlikel y that the period when testosterone exerts i t s e f f e c t s (eg. 2-5 days of incubation) i s p a r t i c u l a r l y c r i t i c a l for stem c e l l s and t h e i r migratory a b i l i t i e s . S i m i l a r l y , stem c e l l s have no d i f f i c u l t y i n populating normal bursal rudiments grafted to the ch o r i o a l l a n t o i c membranes of HBx treated hosts (12). Perhaps a more plausible explanation of t h i s anomaly may be based on c e l l u l a r migrating mechanisms. Although hormonal bursectomy i n h i b i t s bursal development com-pl e t e l y and induces some cloa c a l abnormalities, the urodeal region and i t s associated vascular network may not be disrupted s u f f i c i e n t l y to impede normal c e l l u l a r migration patterns. Consequently, c e l l s may be capable of migrating through t h i s region and subsequently to the' thymus, even though they have not resided i n the bursa l microenvironment. On the other hand, ex t i rpa t ion of the t a i l bud during ear ly surg i ca l bursectomy completely ablates the cloaca and normal vascular networks. This may s i g n i f i c a n t l y a l t e r normal c e l l u l a r migration patterns and prevent homing to the thymus from t h i s region. F i n a l l y , one might in te rpre t the re su l t s '.as i n d i c a t i n g the absence of a bursal humoral factor that inf luences thymic development. The presence of a bura l humoral factor w a s . f i r s t reported by G l i c k (100) and has been widely supported as being capable of re s tor ing antibody synthesis and increas ing the number of sp lenic f o l l i c l e s i n hormonally or s u r g i c a l l y bursectomized b i rds (32, 101-104). Such a factor was not found by some authors (105, 106) and discounted due to funct ional de f i c i enc ie s of bursa implants (106). However, Lymphocyte St imulating Hormone (LSH), normally found i n the thymus, i s present.. i n bursa l t i s sue at f ive times the l e v e l that ex i s t s i n chicken thymus t i s sue (99). LSH i s known to increase the s ize of the spleen and lymph nodes i n mice, as we l l as increase the lymphocyte to polymorph rat io , of the per iphera l blood (99). S i m i l a r l y , a c i r c u l a t i n g serum factor has been found to be e s sent i a l for the p r o l i f e r a t i o n of thymocytes during the i n v i t r o cul ture of embryonic chick thymus t i s sue (107). F i n a l l y , a recent inves t iga t ion has demonstrated the existence of a bursa l extract 54 c a p a b l e o f i n d u c i n g B c e l l d i f f e r e n t i a t i o n i n immature bone marrow c e l l s and has b e e n d e n o t e d b u r s o p o i e t i n ( 1 0 8 ) . A l t h o u g h t h e e v i d e n c e f o r t h e e x i s t e n c e o f a b u r s a l f a c t o r ( s ) i s c o n v i n c i n g , t h e r o l e i t may p l a y i n t h y m o c y t e p r o l i f e r a t i o n i s q u e s t i o n a b l e b a s e d on t h e e v i d e n c e p r e s e n t e d h e r e . S i n c e t h e e x i s t i n g d a t a i n d i c a t e s i t i s a b s e n t f o l l o w i n g e i t h e r h o r m o n a l o r s u r g i c a l b u r s e c t o m y , one w o u l d e x p e c t i t s a b s e n c e t o a f f e c t t h y m o c y t e numbers whenever t h e b u r s a was a b s e n t . T h i s i s i n a p p e a r e n t c o n t r a d i c t i o n w i t h t h e r e s u l t s o b s e r v e d i n t h e hormonal' b u r s e c t o m y e x p e r i m e n t . I n a d d i t i o n , t h e r e s h o u l d n o t be an a d d i t i v e e f f e c t s i m i l a r t o t h a t s e e n f o l l o w i n g c o m b i n e d s u r g i c a l and h o r m o n a l b u r s e c t o m y . C e r t a i n l y , f u r t h e r i n v e s t i g a t i o n i s n e c e s s a r y t o r e s o l v e t h e s e a p p a r e n t p a r a d o x e s . A l t h o u g h b u r s a l f a c t o r ( s ) may i n f l u e n c e t h y m i c d e v e l o p m e n t i n some way, i t s s i g n i f i c a n c e w o u l d a p p e a r t o be l e s s t h a n t h o s e mechanisms p o s t u l a t e d h e r e . I n c o n t r a s t , T e o d o r c z y k and P o t w o r o w s k i (109) have r e c e n t l y d e m o n s t r a t e d t h a t a b u r s a l s u b - p o p u l a t i o n c o u l d be i n d u c e d t o d i f f e r e n t i a t e as T - c e l l s by a s o l u b l e thymus f a c t o r . They have c o n c l u d e d t h a t t h i s o f f e r s f u r t h e r s u p p o r t t o t h e n o t i o n o f a b u r s a l c e l l u l a r c o n t r i b u t i o n t o t h y m i c d e v e l o p m e n t . I n c o n c l u d i n g , i t w o u l d a p p e a r t h a t t h e s u g g e s t i o n t h a t h o r m o n a l and e a r l y s u r g i c a l b u r s e c t o m y m e d i a t e t h e i r e f f e c t s on t h y m o c y t e numbers by s e p a r a t e 55 mechanisms i s s u p p o r t e d by t h e d a t a p r e s e n t e d . T h i s w o u l d a l s o seem t o be c o n s i s t e n t w i t h e x i s t i n g d a t a r e g a r d i n g b u r s a l c e l l m i g r a t i o n t o t h e thymus and t h y m i c l y m p h o p o i e s i s . The o b s e r v a t i o n s made h e r e w o u l d i n d i c a t e t h a t t h e c e l l u l a r c o n t r i b u t i o n o f t h e b u r s a t o t h e thymus may be o f p r o p o r t i o n s l a r g e r t h a n t h a t p r e v i o u s l y c o n s i d e r e d ( 7 3 ) . C e r t a i n l y t h e p r e c i s e e x t e n t and n a t u r e o f t h e b u r s a l c e l l u l a r c o n t r i b u t i o n r e q u i r e s f u r t h e r v e r i f i c a t i o n . The i m p l i c a t i o n s o f b u r s a - d e r i v e d c e l l s i n t h e thymus p o s e i n t e r e s t i n g q u e s t i o n s a s t o t h e i r f u n c t i o n . F o r example, Droege (110) h a s r e c e n t l y d e m o n s t r a t e d t h a t a t l e a s t one. f u n c t i o n o f a b u r s a l - d e p e n d e n t t h y m i c s u b - p o p u l a t i o n i s t o a c t a s a s u p p r e s s o r c e l l o f a n t i b o d y p r o d u c t i o n and c e l l - m e d i a t e d immune r e s p o n s e s . He s u g g e s t s t h a t t h e s e c e l l s may p l a y a p i v o t a l r o l e i n t h e d e v e l o p m e n t o f s e l f - t o l e r a n c e and t h e r e g u l a t i o n o f t h e immune r e s p o n s e . Such d i s c o v e r i e s c e r t a i n l y open e x c i t i n g new a r e a s o f i n v e s t i g a t i o n and may f u r t h e r c h a l l e n g e t h e n o t i o n t h a t t h e b u r s a and thymus r e p r e s e n t c o m p l e t e l y dechotomous b r a n c h e s o f t h e immune s y s t e m . 56 VI I . SUMMARY Hormonal b u r s e c t o m y , p r i o r t o t h e s i x t h day o f i n c u b a t i o n , and e a r l y s u r g i c a l b u r s e c t o m y p r o d u c e s i m i l a r r e d u c t i o n s i n t h y m o c y t e numbers. C o m b i n i n g t h e s e t r e a t m e n t s r e s u l t s i n n e a r l y a t w o - f o l d r e d u c t i o n i n t h e number o f t h y m o c y t e s . T h e s e r e s u l t s s u g g e s t t h a t ! t h e i r i n d i v i d u a l e f f e c t s a r e a d d i t i v e and t h a t t h e y a r e m e d i a t e d v i a s e p a r a t e mechanisms. 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