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The effects of estradiol and progesterone on the growth and differentiation of the quail oviduct Boogaard, Connie 1975

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THE EFFECTS OF ESTRADIOL AND PROGESTERONE ON THE GROWTH AND DIFFERENTIATION OF THE QUAIL OVIDUCT by CONNIE BOOGAARD B.A., C a l i f o r n i a S t a t e , F u l l e r t o n A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRlTlgty J&OLUMBIA May, 1975 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of o a /o 9 • The University of Brit ish Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 7M<vt3£>t /f?S~ (7 ABSTRACT The e f f e c t s of e s t r a d i o l and progesterone treatment on the growth and d i f f e r e n t i a t i o n of the q u a i l oviduct have been i n v e s t i g a t e d , i n terms of morphology, growth, and, biochemical d i f f e r e n t i a t i o n . H i s t o l o g i c a l l y , e s t r a d i o l induced the formation of tu b u l a r gland c e l l s by 5 days of treatment, and e p i t h e l i a l d i f f e r e n t i a t i o n by 10 days of treatment. Progesterone treatment appeared to enhance e p i t h e l i a l d i f f e r e n t i a t i o n s l i g h t l y . The treatment a l s o enhanced the growth of the o v i d u c t , as i n d i c a t e d by increases i n s i z e , weight, and the content of the t i s s u e c o n s t i t u e n t s , p r o t e i n , RNA and DNA. E s t r a d i o l treatment a l s o induced the synt h e s i s of two s p e c i f i c p r o t e i n s , ovalbumin and lysozyme. However, e s t r a d i o l treatment f a i l e d to induce syn-t h e s i s of the p r o t e i n a v i d i n . Only progesterone induced the synt h e s i s of t h i s p r o t e i n . Withdrawal from treatment f o r ten days caused a l o s s of c e l l s and of t i s s u e c o n s t i t u e n t s . This l o s s was greater i n those b i r d s withdrawn from estrogen plus progesterone treatment than i n those withdrawn from estrogen treatment. I n some cases, s p e c i f i c p r o t e i n s were a l s o absent i n oviducts from b i r d s withdrawn from treatment. TABLE OF CONTENTS Page Ab s t r a c t i Table of Contents i i L i s t of Figur e s i i i L i s t of Tables i v L i s t of P l a t e s v I n t r o d u c t i o n to L i t e r a t u r e Review 1 L i t e r a t u r e Review ' 2 I n t r o d u c t i o n 16 M a t e r i a l s and Methods 17 Res u l t s 21 H i s t o l o g y 21 Assays of T o t a l Growth 46 Assays of S p e c i f i c P r o t e i n s 59 Summary of R e s u l t s 70 D i s c u s s i o n , O u t l i n e 76 D i s c u s s i o n of the R e s u l t s of t h i s Experimentation 77 D i s c u s s i o n of the Response as a Phenomenon of Induction 80 The Nature of the C e l l u l a r Response to Estrogen 82 The Nature of the C e l l u l a r Response to Progesterone 95 Events a General Model W i l l Have to E x p l a i n 101 Comparison of t h i s Induction Phenomenon to Embryonic Induction 102 Conclusion 105 References 107 Appendices 121 LIST OF FIGURES Figure 1. 2A. 2B. 3. 4. 5 • 7 • 8 • 9. Proposed Time Sequence of Estrogenic Responses, w i t h Influences of Progesterone Diagram of the Chick Oviduct I l l u s t r a t i o n of a Cross-Section through the Oviduct Magnum a f t e r Treatment w i t h Estrogen and Progesterone The V a r i a t i o n i n Wet Weight as a Function of Treatment The V a r i a t i o n i n P r o t e i n Content as a Function TXX \/'cL IT 1 cL 1z XX H_XX l^i^^V. XX t £i xx t 9. s SL Jt1XIXX C t IL )^XX f T XT^^ SL1111^^ XI d ' X V r x X T 1 3. 't L^^ )XX 2LXX X3^ T^ L )^XX t€1XX t d>S 3. '^XIXX^ ^ t L^^ )XX )^ f XT3-tXfl^ X^X *t * » • • • • 111 3_ xi t n. o xx P r o f i l e o f Ov^cLlbuxiiixx oxx ScprxcLcis^c G ^ 2 0 0 • • F u n c t i o f o f T r i i t m e n t Z y m e C ° n C e n t r a t i ° n 3 3 * Proposed C o n t r o l of Ovalbumin Synthesis Page 11 12 12 49 49 51 51 61 66 84 LIST OF TABLES Table Page I . The Content and Concentration of Various Tissue Components of the Oviduct at D i f f e r e n t Stages of Treatment 53 I I . Summary of the Weights, and the Presence or Absence of S p e c i f i c P r o t e i n s i n the Oviduct Magna of B i r d s Withdrawn from Treatment 68 LIST OF PLATES P l a t e page l a and l b . C r o s s - s e c t i o n of an Oviduct from a 10-day o l d b i r d which had re c e i v e d c o n t r o l treatment f o r 5 days l c . C r o s s - s e c t i o n of an oviduct from a 15-day o l d b i r d which had re c e i v e d c o n t r o l treatment f o r 10 days. 24 24 2a and 2b. C r o s s - s e c t i o n of an oviduct from a 20-day o l d b i r d which had re c e i v e d c o n t r o l treatment f o r 15 days 26 28 34 3a and 3b. Cross-section, of an oviduct from a 30-day o l d b i r d which had re c e i v e d control.treatment f o r 15 days, followed by 10 days without treatment 4a and 4b. C r o s s - s e c t i o n of an oviduct magnum from a b i r d t r e a t e d f o r 5 days w i t h e s t r a d i o l 32 5a and 5b. C r o s s - s e c t i o n of an oviduct magnum, from a b i r d t r e a t e d f o r 10 days w i t h e s t r a d i o l 6a and 6b. C r o s s - s e c t i o n of an oviduct magnum from a b i r d t r e a t e d f o r 15 days w i t h e s t r a d i o l 36 7a and 7b. C r o s s - s e c t i o n of an oviduct magnum from a b i r d t r e a t e d f o r 10 days w i t h e s t r a d i o l and 5 days w i t h progesterone • 38 8a and 8b. C r o s s - s e c t i o n of an oviduct magnum from a b i r d withdrawn from estrogen plus progesterone treatment f o r 10 days 42 9a, b, c. Cro s s - s e c t i o n s of oviduct magna from b i r d s w i t h -drawn from estrogen p l u s progesterone treatment f o r 10 days 44 10a. Ouchterlony p l a t e demonstrating the r e a c t i o n of a n t i - c h i c k ovalbumin w i t h c h i c k ovalbumin, q u a i l ovalbumin, and magnum e x t r a c t from b i r d s withdrawn from estrogen treatment 63 10b. Ouchterlony p l a t e demonstrating the r e a c t i o n of a n t i - c h i c k ovalbumin w i t h c h i c k ovalbumin, q u a i l ovalbumin, and s e r i a l d i l u t i o n s of a magnum e x t r a c t from a b i r d t r e a t e d f o r 10 days w i t h estrogen 63 P l a t e P a g e 10c. Ouchterlony p l a t e demonstrating the r e a c t i o n of a n t i - c h i c k ovalbumin w i t h c h i c k ovalbumin, q u a i l ovalbumin, and a magnum e x t r a c t from a yz^:™"-™^..r^.*^r. ^ INTRODUCTION The mature oviduct of a s e x u a l l y a c t i v e chicken comprises a tube n e a r l y 80 cm i n l e n g t h , whose f u n c t i o n i t i s to secrete the mainly proteinaceous m a t e r i a l that w i l l be in c l u d e d i n the o v i p o s i t e d egg, as w e l l as the s h e l l that surrounds i t . Since t h i s i s , then, a s e c r e t o r y organ, i t lends i t s e l f n i c e l y to s t u d i e s which r e q u i r e both a morphological and a biochemical marker f o r d i f f e r e n t i a t i o n , and has been s t u d i e d e x t e n s i v e l y i n t h i s context. Recently, a great deal of research has attempted to e s t a b l i s h i n d e t a i l the response of the c h i c k oviduct to e s t r a d i o l and progesterone. Work on that system has i n d i c a t e d that treatment of an immature c h i c k w i t h e s t r a d i o l causes dramatic growth and d i f f e r e n t i a t i o n of the oviduct t i s s u e . I t was f u r t h e r reported that withdrawal of estrogen a f t e r such treatment r e s u l t s i n the l o s s of f u n c t i o n i n g of the n e w l y - d i f f e r e n t i a t e d c e l l s of the o v i d u c t , but that the c e l l s themselves remain. That i s , the c e l l s are s t a b l e to a l o s s of hormone. This c e l l s t a b i l i t y i s a very marked divergence from the s i t u a t i o n i n mammals, where t h i s i s known not to occur. I t i s p o s s i b l e that t h i s c h a r a c t e r i s t i c i s unique to f o w l , or to the avian group. However, b r i e f i n i t i a l r e p o r ts had i n d i c a t e d that i n q u a i l , the c e l l s of the oviduct are l o s t during the lengthy r e s t i n g p e r i o d (54), thus i n d i c a t i n g that the s t a b i l i t y of the c h i c k oviduct c e l l s i n the absence of hormone i s c h a r a c t e r i s t i c of that s p e c i e s . A d i f f e r e n c e as marked as the l i f e or death of the c e l l s i n the absence of hormone i s s u r p r i s i n g i n species of the same order, and so i t was decided to i n v e s t i g a t e i n greater d e t a i l the response of the q u a i l oviduct to s t e r o i d hormones. This i n i t i a l study was ther e f o r e undertaken to e s t a b l i s h the major responses of the q u a i l oviduct to s t e r o i d hormones, i n order to serve as a base f o r f u r t h e r experimentation. LITERATURE REVIEW H i s t o r i c a l L i t e r a t u r e The oviduct of avian species, l i k e i t s counterpart, the mammalian g e n i t a l t r a c t , has long been known to be under the con t r o l of sex hormones. As early as 1928, Riddle and Tange (1) demonstrated that estrogenic extracts from the ovary or placenta of sows would induce growth of the oviduct of immature pigeons, and noticed that, per unit dose, t h i s growth was not as marked as that observed i n the uterus of mammals. Two years, l a t e r , i n 1930, Juhn and Gustavson (2) performed the same type of experiment using a human placental extract on fowls, and observed a 10-fold increase i n oviduct weight. Histo-l o g i c a l examination demonstrated that both growth and d i f f e r e n t i a t i o n took place: the mucosal folds increased i n s i z e and the tubular gland c e l l s developed. Increased v a s c u l a r i z a t i o n was also noted, and i t was possible to induce these same e f f e c t s i n the rudimentary Mullerian ducts of males and females at any stage of post-embryonic development (3, 4). This response has also been demonstrated i n sparrows (5), and i n fowl and turkey given synthetic estrogens o r a l l y (6), as well as i n chicks given e s t r a d i o l (7). In 1944, Hertz and S e b r e l l (8) noted that a dietary deficiency of panto-thenic acid, a f o l i c acid precursor, i n h i b i t e d the response of the oviduct to estrogen and i n 1949 (9) i t was demonstrated that a purine analog could do the same thing. Both these treatments are now known to i n t e r f e r e with DNA synthesis, i n d i c a t i n g that such synthesis i s necessary f o r the growth response. Gradually i t became of i n t e r e s t to many researchers to investigate the e f f e c t s of the d i f f e r e n t sex steroids s i n g l y and i n combination with each other. In 1940, two groups i n v e s t i g a t e d the e f f e c t s of hormones other than estrogens. W i t s c h i and Fugo (10) demonstrated that estrogens s t i m u l a t e the oviducts and r i g h t rudimentary ducts of s t a r l i n g s , and that progesterone d i d not c o n t r i b u t e to the response, although testosterone could. I n t h i s s p e c i e s , the females have p a i r e d vasa d e f e r e n t i a which respond to androgens, and are known to secrete considerable amounts of testosterone i n the breeding season. The authors th e r e f o r e thought i t probable that testosterone c o n t r i b u t e d to the normal development of the o v i d u c t , at l e a s t i n t h i s s p e c i e s . Androgen has a l s o been shown to augment the o v i d u c a l response to estrogen i n chicks (11), and i n n i g h t herons (12). I n t h i s l a t t e r case, however, i t was a l s o shown that only estrogens can cause h y p e r p l a s i a (12). Subsequently, a marked dichotomy of r e s u l t s appeared, some i n d i c a t i n g synergism and some antagonism i n the o v i d u c a l response to estrogen i n combina-t i o n w i t h other s t e r o i d s . I n 1947, Hertz et a l (13) noted that progesterone was a n t a g o n i s t i c to the o v i d u c a l e s t r o g e n i c response; that i s , a marked i n h i b i t i o n was noted when the two were given simultaneously, although proges-terone d i d not have an a n t a g o n i s t i c e f f e c t on the response of serum l i p e m i a and calcemia to estrogen, i n d i c a t i n g that the antagonism was s p e c i f i c to the ovid u c t . This i n h i b i t i o n by progesterone of the estrogen-induced hypertrophy of the oviduct of immature chi c k s was noted a l s o by P h i l l i p s et a l i n 1952 (14). On the other hand, i n 1951, Mason (15) demonstrated that progesterone alone caused no weight increase i n the c h i c k o v i d u c t , but when given w i t h estrogen, caused a marked hypertrophy, which was greater than that caused by estrogen alone. He po s t u l a t e d that progesterone was necessary f o r normal f u n c t i o n i n g of the o v i d u c t . In 1956, Breneman (16) noted the same e f f e c t of progesterone, and found the s y n e r g i s t i c e f f e c t f o r testosterone as w e l l . However, he a l s o noted that antagonism was observed w i t h low doses of proges-terone. These f i n d i n g s were supported by those of Mason i n 1952 (15), which demonstrated that estrogen and progesterone may act s y n e r g i s t i c a l l y or a n t a g o n i s t i c a l l y i n c h i c k o v i d u c t , depending on the r e l a t i v e r a t i o s of both. Estrogen always caused an increase i n weight, whereas progesterone alone had no such e f f e c t , but the grea t e s t increases were always found when the two were combined i n unequal amounts. In 1950, Hertz (17) noted that estrogen caused an increase i n serum b i o t i n l e v e l , and that an a n t i - b i o t i n f a c t o r present i n the egg-white was made i n the oviduct under the i n f l u e n c e of the f u l l y f u n c t i o n a l ovary. Chicks that had been c a s t r a t e d and t r e a t e d w i t h estrogen produced no a n t i -b i o t i n , whereas those t r e a t e d w i t h both estrogen and progesterone d i d , thus demonstrating that progesterone was f u n c t i o n a l i n causing s e c r e t i o n . This e f f e c t on s e c r e t i o n was a l s o noted by Brant and Nalbandov i n 1952 (18), who demonstrated that although estrogen alone caused growth of the o v i d u c t , there were few s e c r e t o r y granules. Again, androgen or progesterone alone had no e f f e c t , but i n combination w i t h estrogen, these s t e r o i d s caused an increase i n s i z e greater than that r e s u l t i n g from estrogen alone, and s e c r e t o r y granules were abundant. Brant and Nalbandov concluded that the simultaneous presence of two hormones was e s s e n t i a l f o r albumin granule production, but d i d not demonstrate which s t e r o i d was normally the a c t i v e second hormone. In 1956 (19), they continued t h e i r s t u d i e s by demonstrating that progesterone and testosterone act s y n e r g i s t i c a l l y w i t h estrogen i n the s e c r e t i o n of albumin. They saw no a n t a g o n i s t i c e f f e c t , but i n these experiments the estrogen was administered by p e l l e t i m p l a n t a t i o n a few days before progesterone or t e s t o -sterone was given. (The s i g n i f i c a n c e of t h i s w i l l be apparent l a t e r . ) By implanting a s t r i n g of beads i n t o the o v i d u c t , they demonstrated that none of the hormones alone could cause s e c r e t i o n of the albumin. When the combination of estrogen plus progesterone or estrogen plus testosterone was used, a la r g e s e c r e t i o n was apparent. This was more pronounced w i t h estrogen plus proges-terone than w i t h estrogen plus t e s t o s t e r o n e . They concluded that the f u l l s e c r e t o r y p o t e n t i a l of the oviduct r e q u i r e s 2 hormones, although the second may be e i t h e r progesterone or testo s t e r o n e . Estrogen and testosterone acted s y n e r g i s t i c a l l y to cause n i t r o g e n r e t e n t i o n (20), which was not accomplished by e i t h e r s t e r o i d alone. S i m i l a r -l y , i t was demonstrated that estrogen and progesterone caused a r i s e i n the fre e r i b o f l a v i n content of the magnum (21), an increase which d i d not occur i n response to estrogen or progesterone alone. I t th e r e f o r e appeared that both hormones are e s s e n t i a l to some responses. However, the controversy over whether progesterone acted a n t a g o n i s t i c a l l y or s y n e r g i s t i c a l l y w i t h estrogen continued. In 1954, T u l l n e r e t a l (22) showed that both progesterone and allopregnane-21-ol-3,20-dione acetate could antagonize estrogen. This l a t t e r compound, a n o n - c o r t i c o i d , was the greatest i n h i b i t o r . They a l s o demonstrated that such c o r t i c o i d s as d e s o x y c o r t i c o s t e r -one i n h i b i t e d the estrogenic response of the o v i d u c t . Since the allopregnane-21-ol-3,20-dione acetate and desoxycorticosterone d i d not act as progestagens i n any of the assays used, the authors concluded that "the i n h i b i t i o n of the oviduct response i n estrogen-treated chicks may be c l e a r l y d i s s o c i a t e d from other b i o l o g i c a l a c t i o n s of progesterone". They thus separated the a n t i -e s t r o g e n i c e f f e c t s from the p r o g e s t a t i o n a l p r o p e r t i e s of progesterone. In 1963, Dorfman and Dorfman (23) reported that the a n t a g o n i s t i c or syn-e r g i s t i c e f f e c t of progesterone depended on both the c o n c e n t r a t i o n r a t i o and the dose. These r e s u l t s supported those of Mason i n 1952 (15), and Breneman i n 1956 (16) . Progesterone was found to be s y n e r g i s t i c to low doses of estrogen, but a n t a g o n i s t i c to high doses of estrogen. Pregne-5-ene-3,20-dione acetate was s y n e r g i s t i c w i t h estrogen, but had no e f f e c t alone. D e s o x y c o r t i -costerone was a l s o s y n e r g i s t i c w i t h estrogen, although i t was i n h i b i t o r y at low doses. The authors noted that i t had been suggested (22) that the anta-g o n i s t i c e f f e c t of progesterone was independent of i t s p r o g e s t a t i o n a l proper-t i e s . Having observed the i d e n t i c a l a c t i o n of c o r t i c o i d s , they suggested that the s y n e r g i s t i c e f f e c t of progesterone on the estrogenic response may a l s o be independent of i t s p r o g e s t a t i o n a l p r o p e r t i e s . Such a suggestion r a i s e s the question of why progesterone would be ex-pected to act as a progestagen i n a species which does not become pregnant; t h i s i s not n e c e s s a r i l y a r h e t o r i c a l question when the amount of d i s c u s s i o n s t i l l devoted to t h i s phenomenon i s considered (24). D e f i n i t e p r o g e s t a t i o n a l e f f e c t s of progesterone i n t h i s non-mammalian system have yet to be demonstrat-ed. At any r a t e , Dorfman and Dorfman concluded that the type of response to progesterone, whether a n t a g o n i s t i c or s y n e r g i s t i c , w i l l depend on the i n t e n -s i t y of the e s t r o g e n i c response r a t h e r than on the r a t i o of the two hormones to each other (24). Thus, at a maximal estrogen response, progesterone appeared to be a n t a g o n i s t i c , whereas at minimal estrogen responses, i t appeared to be s y n e r g i s t i c . A comparison of these e a r l y s t u d i e s w i t h each other i s hindered by a number of problems which appear q u i t e c l e a r l y i n h i n d s i g h t . The very e a r l i e s t s t u d i e s used crude e s t r o g e n i c e x t r a c t s , demonstrating only that they c o n t a i n -ed estrogen, and not exc l u d i n g the p o s s i b i l i t y that they contained p r o g e s t e r -one or other hormones as w e l l . Of the s t u d i e s which immediately followed those, estrogens were chosen by somewhat random methods, si n c e the b i o l o g i c a l -l y a c t i v e s t e r o i d was not known. Some s t u d i e s t h e r e f o r e u t i l i z e d a l pha-e s t r a d i o l , which i s r e l a t i v e l y i n a c t i v e ; others used estrone, s t i l l others used s t i l b o e s t r o l or one of i t s d e r i v a t i v e s . These a l l have d i f f e r e n t potencies, and i t i s known that t h e i r e f f e c t s , e s p e c i a l l y i n combination w i t h progesterone, w i l l vary. The a c t u a l dose of a c t i v e agent t h e r e f o r e d i f f e r e d i n these s t u d i e s . More r e c e n t l y , i n s t u d i e s concerning combination e x p e r i -ments, the method i n which the two hormones were administered i s a l s o r e l e v a n t , since p e l l e t i m p l a n t a t i o n would have a d i f f e r e n t e f f e c t on the serum estrogen l e v e l than i n j e c t i o n . Because of the same c o n s i d e r a t i o n , the time course of i n j e c t i o n s would be important, i n experiments where the hormones were i n j e c t e d . However, i n s p i t e of these problems, some t a n g i b l e conclusions can s t i l l be drawn from t h i s work: (1) estrogen s t i m u l a t e d the growth and d i f f e r e n t i a t i o n of the o v i d u c t ; (2) a second s t e r o i d acted to increase s e c r e t i o n ; (3) the e f f e c t of the second hormone on the response to estrogen would depend on the r e l a t i v e doses of the two hormones, and on the s t a t e of the oviduct at the time of a d m i n i s t r a t i o n of the second s t e r o i d . Most of these e a r l y s t u d i e s , then, were concerned w i t h the o v e r a l l growth response of the oviduct to estrogens alone, or estrogens and other hormones i n combinations. Due to the r e l a t i v e l y u n refined nature of the techniques a v a i l a b l e at the time, the e f f e c t s of these hormones on s p e c i f i c p r o t e i n s y n t h e s i s were not i n v e s t i g a t e d . However, one p r o t e i n had been known to e x i s t due to i t s b i o l o g i c a l a c t i v i t y of b i n d i n g to the v i t a m i n b i o t i n . When i n c l u d e d i n the d i e t of animals or the growth medium of micro-organisms such as yeast, the p r o t e i n caused death to the organism. With t h i s a b i l i t y to assay f o r a s p e c i f i c p r o t e i n , researchers could i n v e s t i g a t e the e f f e c t s of various hormones on the production of t h i s p r o t e i n . Such s t u d i e s occurred concomitantly w i t h those hormone s t u d i e s already mentioned, and should be seen i n that l i g h t . A v i d i n was f i r s t named by Eakin et a l (25) as the p r o t e i n f a c t o r i n egg-white that caused egg-white i n j u r y to a d u l t hens and r a t s fed on a d i e t of raw egg-white. A d d i t i o n of b i o t i n to the d i e t prevented or cured egg-white i n j u r y (26). B i o t i n accumulated i n the y o l k of the egg (27), under es t r o g e n i c c o n t r o l (28), which may e x p l a i n , i n r e t r o s p e c t , why the a v i d i n does not cause i n j u r y to the embryo. I n 1942, Hertz and S e b r e l l (29) demonstrated that a v i d i n was present i n the egg-white of turkey, duck, goose and hen eggs, i n the oviducts of hens and f r o g s , and i n the e g g - j e l l y of two species of Rana. They suggested that a v i d i n was a s e c r e t o r y product of oviducts of b i r d s and amphibians, and that i t may p l a y an important part i n embryonic development (29). Although no f u r t h e r r e p o r t s have e l u c i d a t e d i t s r o l e i n embryonic development, a v i d i n has r e c e n t l y been shown to be present a l s o i n oviducts of pigeons (30) and l i z a r d s (31). In 1943, Frapps et a l (32) demonstrated that a v i d i n was found i n a l l l e v e l s of the magnum of chic k o v i d u c t , but not i n the magna of non-laying hens. They concluded that a v i d i n production was a s s o c i a t e d , e i t h e r d i r e c t l y or i n d i r e c t l y , w i t h the complete reproductive f u n c t i o n of the ovary. They continued t h e i r s t u d i e s by i n v e s t i g a t i n g the e f f e c t s of hormones on a v i d i n production (33). Using immature c h i c k s , they demonstrated that the production of a v i d i n depended on the s y n e r g i s t i c a c t i o n of s t i l b o e s t r o l and progesterone, although they o c c a s i o n a l l y found a p o s i t i v e response to s t i l b o e s t r o l alone. Since at that time the i d e n t i t y of the hormones secreted by the b i r d ovary was i n doubt, and s i n c e i n lower v e r t e b r a t e s such as amphibians testosterone could a l s o induce o v u l a t i o n , they concluded only that the production of a v i d i n r e q u i r e d some other s t e r i o d i n a d d i t i o n to estrogen. This other s t e r o i d , they p o s t u l a t e d , could have been of e x t r a - o v a r i a n o r i g i n (32). In l i g h t of l a t e r developments i n t h i s area (24), that c o n c l u s i o n showed amazing i n s i g h t . I n 1944 (34), Hertz et a l demonstrated that both testosterone and desoxy-c o r t i c o s t e r o n e a c e t a t e , as w e l l as progesterone, could induce a v i d i n produc-t i o n i n c h i c k s that had been p r e - t r e a t e d w i t h estrogen. They recognized the i n t e r c h a n g e a b i l i t y of these s t e r o i d s i n other f u n c t i o n , such as the mainten-ance of l i f e i n adrenalectomized animals and the p r e c i p i t a t i o n of endometrial b l e e d i n g i n the monkey. They ther e f o r e questioned which of these agents might play the a c t i v e r o l e i n inducing a v i d i n s y n t h e s i s i n v i v o (34). In 1949 (35), Hertz et a l f u r t h e r demonstrated that s t i l b o e s t r o l t r e a t -ment caused a 5 - f o l d increase i n the b i o t i n a c t i v i t y i n the serum, but no a v i d i n formation occurred i n the o v i d u c t . Since a d m i n i s t r a t i o n of estrogen and progesterone caused both an increase of b i o t i n a c t i v i t y i n the serum and of a v i d i n content i n the o v i d u c t , they thought that a v i d i n remained i n the t i s s u e of the o v i d u c t , and d i d not c i r c u l a t e (17). More r e c e n t l y , the system was looked at i n greater d e t a i l , due to the increased s e n s i t i v i t y of some techniques, and the development of new ones. Most of these l a t e r s t u d i e s centered around the changes i n t o t a l or s p e c i f i c protein content of the oviduct due to hormone administration. In 1960, Brown and Jackson (36) reported that there were no s t r i k i n g differences i n the compositions of the organ i n d i f f e r e n t age groups, although the oviduct of the broody hen had a s l i g h t l y lower protein content. In 1961, Kalman and Opsahl demonstrated that estrogen caused an increased incorpora¬t i o n of C- -leucine into protein (37). In 1965, Oades and Brown (38) performed electrophoresis of the water-soluble proteins of the oviduct magnum a f t e r estrogen treatment, and demonstrated a r e l a t i v e increase i n albumin A2 and A3 and the post-albumins i n the treated oviduct. There was a lower con-tent of water-soluble magnum proteins i n broody hens and i n hens treated with estrogen only,than i n hens treated with LH or estrogen plus LH. However, there was a large increase i n the water-soluble magnum proteins i n oviducts from hens treated with estrogen plus progesterone or estrogen plus t e s t o s t e r -one. This upheld the e a r l i e r work which demonstrated that a second s t e r o i d , when given with estrogen, acted to increase secretion. An increase i n alk a -l i n e phosphatase a c t i v i t y was demonstrated i n 1965 by Pande et a l (39), and an increase i n oviducal glycogen i n 1969 by C e c i l et_ a l (40), as a r e s u l t of estrogen action. Recent History Even more recently, a number of studies (41-53) have investigated exten-s i v e l y the response of the oviduct to estrogen and progesterone, both during primary and secondary stimulation. The re s u l t s of these i n v e s t i g a t i o n s form a comprehensive p i c t u r e of the oviducal response. The events of primary stimulation are summarized i n Figure 1 and Figure 2. E s t r a d i o l causes the i n i t i a l dispersion of the stroma of the ti s s u e , r e s u l t i n g Figure 1. Proposed Time Sequence of Estrogenic Responses, w i t h Influences of Progesterone Estrogen StJoma Hyperemia I n v a s i L by c e l l s from blood Water I m b i b i t i o n 1 , Stromal s w e l l i n g Stromal D i s p e r s i o n -Estrogen E p i t h e l i u m Quiescent C e l l s Progenitor Tubular Gland C e l l s E, P , / 'or EP » A c t i v e C e l l s (Others) to P r o t o d i f f e r e n t i a t e d State, w i t h Ovalbumin Synthesis blocks Non- 4r F u n c t i o n i n g C e l l s ~~ Hormone + Hormone Further DNA Synthesis, to Tubular Gland C e l l s enhances C e l l - S p e c i f i c P r o t e i n ~ Synthesis; Ovalbumin, ^ Lysozyme, Conalbumin, Ovomucoid E (lengthy treatment), P, or EP (P enhances) E p i t h e l i a l D i f f e r -e n t i a t i o n to C i l i a t e d C e l l s and Goblet C e l l s only A v i d i n S ^ t h e s i s by Goblet C e l l s See t e x t f o r e x p l a n a t i o n . E = Estrogen; P = Progesterone; EP = Estrogen + Progesterone Fig. 2a Chick Oviduct JL. Infundibulum Magnum Isth- shell gland Vagina white^  protein )^ Fig. 2b Magnum (cross section) Adapted from oka & Schimke (Ret. 48) Stroma Undifferentiated Ciliated Prolonged ~ ^ ^ § & C Goblet or Progesterone J l s i r f ^ e c r e t o r y cells Immature Tubular gland cells. containing egg-white proteins I • 11 i n hyperemia and water i m b i b i t i o n (41). Perhaps as a r e s u l t of the hyperemia, mononuclear c e l l s from the blood invade the stroma, g i v i n g r i s e to the p o s s i b i l i t y of c e l l - c e l l i n t e r a c t i o n s (41). Concomitantly, or soon t h e r e a f t e r , the e p i t h e l i a l c e l l s l i n i n g the lumen d i f f e r e n t i a t e (41) as f a r as a proto-d i f f e r e n t i a t e d s t a t e (42), i n which indentations i n t o the stroma appear, but are not yet completely formed (43). These p r o t o d i f f e r e n t i a t e d t u b ular gland c e l l s c o n t a i n s e c r e t o r y granules, and ovalbumin i s present i n the oviduct magnum (43). At t h i s stage, microfilaments become e s s e n t i a l i n the budding o f f of these c e l l s to form t u b u l a r gland c e l l s (44). E i t h e r estrogen, or estrogen and progesterone together, can induce the d i f f e r e n t i a t i o n of the oviduct magnum to t h i s stage; however, only estrogen can continue the d i f f e r -e n t i a t i o n of these p r o t o d i f f e r e n t i a t e d c e l l s to tu b u l a r gland c e l l s (43). The presence of progesterone together w i t h estrogen abolishes the subsequent d i f f e r e n t i a t i o n of these p r o t o d i f f e r e n t i a t e d c e l l s to tubular gland c e l l s (43) and leads i n s t e a d to e p i t h e l i a l d i f f e r e n t i a t i o n of c i l i a t e d c e l l s and goblet c e l l s (43, 45, 46, 47). The process of budding o f f to form t u b u l a r gland c e l l s continues f o r a number of days, depending on dose. A f t e r i t i s complete, the remaining e p i t h -e l i a l c e l l s d i f f e r e n t i a t e to become e i t h e r c i l i a t e d c e l l s or se c r e t o r y goblet c e l l s (41, 45, 48). Progesterone enhances t h i s type of d i f f e r e n t i a t i o n (47). I f progesterone i s given w i t h estrogen, so that t u b u l a r gland c e l l d i f f e r e n t i a -t i o n i s a r r e s t e d a t the p r o t o d i f f e r e n t i a t e d stage, then c e l l s can be seen i n the e l e c t r o n microscope which c o n t a i n both s e c r e t o r y granules and c i l i a (43). As the tu b u l a r gland c e l l s d i f f e r e n t i a t e , ovalbumin concentration increases 300-fold i n the course of 15 days of treatment (49). The c o n c e n t r a t i o n of lysozyme, another p r o t e i n made i n the tubular gland c e l l s , a l s o increases i n t h i s p e r i o d (45, 48). The e f f e c t of progesterone on t h i s process i s a complex one, depending p r i m a r i l y on the time at which i t i s administered. Progesterone w i l l i n h i b i t a l l the processes that r e q u i r e DNA s y n t h e s i s (45, 48), and so w i l l a b o l i s h the growth of the oviduct i f i t i s given concomitantly w i t h estrogen from the beginning of treatment (45, 48). This r e d u c t i o n i s the r e s u l t of a b o l i s h i n g the tubular gland c e l l d i f f e r e n t i a t i o n , s i n c e these c e l l s form the vast bulk of the d i f f e r e n t i a t e d oviduct magnum. However, i f the onset of progesterone treatment i s delayed somewhat, so that estrogen has had an opportunity to induce t u b u l a r gland c e l l s , then those c e l l s that have d i f f e r e n t i a t e d w i l l contined s y n t h e s i z i n g ovalbumin and lysozyme, even a f t e r progesterone t r e a t -ment has begun (45, 48). Indeed, progesterone has a s y n e r g i s t i c e f f e c t on the f u n c t i o n i n g of the tubular gland c e l l s (45, 48). Progesterone has the opposite e f f e c t on the d i f f e r e n t i a t i o n of the e p i t h e l i a l c i l i a t e d c e l l s and s e c r e t o r y goblet c e l l s : i t enhances t h e i r d i f f e r e n t i a t i o n g r e a t l y (47). In a d d i t i o n , progesterone has an a c t i o n on t h i s t i s s u e which estrogen does not have: the i n d u c t i o n of the s y n t h e s i s of the p r o t e i n a v i d i n i n the e p i t h e l i a l goblet c e l l s (50). This p r o t e i n i s present i n highest amounts when progesterone i s administered part-way through estrogen treatment: e i t h e r too long or too short a pre-treatment w i t h estrogen i s i n h i b i t o r y (49). Upon estrogen withdrawal, a decrease i n a l l t i s s u e components i s seen. This i s l e s s marked i n the case of DNA content (48), but w i t h time the c e l l s are a l s o l o s t (51, 52), contrary to e a r l i e r r e p o r t s (45, 48). Thus, mainten-ance of the d i f f e r e n t i a t e d s t a t e of the oviduct r e q u i r e s continued s t i m u l a t i o n by the hormone. Readministration of estrogen to c h i c k s withdrawn from treatment r e s u l t s i n a qu i c k e r , greater response w i t h a much shor t e r time l a g i n i n d u c t i o n of s p e c i f i c p r o t e i n s y n t h e s i s (48, 53). The remaining t u b u l a r gland c e l l s resume f u n c t i o n i n g , accounting f o r the s h o r t e r time l a g (48). Estrogen and progesterone both s t i m u l a t e s p e c i f i c p r o t e i n s y n t h e s i s , but the response to progesterone i s l e s s than that to estrogen (48, 53). When both are given together, the response i s greater than the response to e i t h e r alone, i n d i c a t i n g that the two hormones act s y n e r g i s t i c a l l y i n enhancing p r o t e i n s y n t h e s i s (48), although they act a n t a g o n i s t i c a l l y w i t h regard to c e l l d i v i s i o n . I t has therefore been suggested that c e l l p r o l i f e r a t i o n and c e l l f u n c t i o n i n g are s t i m u l a t e d by estrogen i n d i f f e r e n t manners, such that proges-terone i n t e r f e r e s w i t h the one process, but not the other (45, 48). The ovi d u c t represents an e x c e l l e n t example of t i s s u e growth and d i f f e r -e n t i a t i o n i n response to s t e r o i d s t i m u l a t i o n . I t has biochemical markers that can be used to measure the e f f e c t of e i t h e r estrogen or progesterone, and the considerable growth produces l a r g e t i s s u e masses f o r biochemical a n a l y s i s . I t represents a t i s s u e which responds to two indu c e r s , where the a c t i o n s of both are known (at l e a s t i n p a r t ) , and where the a c t i o n of the second depends to a la r g e extent on the a c t i o n of the f i r s t . The oviduct t h e r e f o r e represents an e x c e l l e n t system i n which to study the biochemical processes and p o s s i b l e c o n t r o l mechanisms of s t e r o i d - i n d u c e d gene a c t i v i t y i n d i f f e r e n t i a t i o n . Two major l a b o r a t o r i e s have been i n v e s t i g a t i n g these processes: one d i r e c t e d by P a l m i t e r , i n v e s t i g a t i n g the r e a d m i n i s t r a t i o n of estrogen to chicks withdrawn from treatment, and one d i r e c t e d by O'Malley, i n v e s t i g a t i n g the r o l e of progesterone i n inducing a v i d i n s y n t h e s i s . INTRODUCTION The response of the c h i c k oviduct to estrogen and progesterone has t h e r e f o r e been e s t a b l i s h e d i n d e t a i l . The work on that system i n d i c a t e d that treatment of an immature c h i c k w i t h e s t r a d i o l caused dramatic growth and d i f f e r e n t i a t i o n of the oviduct t i s s u e . However, one of the most s t r i k i n g d i f f e r e n c e s reported was t h a t , contrary to the case i n mammals, the c e l l s of the c h i c k oviduct were not l o s t upon withdrawal from hormone treatment. In another species (Cortunix c o r t u n i x japonica- ) of the same order ( G a l l u s ) , e a r l y r e p o r t s had i n d i c a t e d that the c e l l s of the oviduct were l o s t during the lengthy r e s t i n g period (54, 55). This has a l s o been more r e c e n t l y confirmed (56). Although t h i s i s now known to be the case i n chicks as w e l l (51, 52), at the time t h i s study was begun, i t had not yet been demonstrated. Since a d i f f e r e n c e as marked as the l i f e or death of the c e l l s i n the absence of hormone i s a s t r i k i n g d i f f e r e n c e i n species of the same order, i t was decided to i n v e s t i g a t e i n greater d e t a i l the major responses of the q u a i l oviduct to estrogen and progesterone. This i n i t i a l study then, was designed to be an i n t r o d u c t o r y and e x p l o r a -tory base f o r f u r t h e r s t u d i e s . I t t h e r e f o r e attempted to e s t a b l i s h the q u a i l ' s response to estrogen and progesterone i n terms comparable to the c h i c k system already s t u d i e d . Thus, I decided to i n v e s t i g a t e the e f f e c t s of e s t r o -gen and progesterone on the h i s t o l o g i c s t r u c t u r e of the o v i d u c t , w i t h p a r t i c u l a r reference to gland c e l l formation and e p i t h e l i a l c e l l d i f f e r e n t i a -t i o n . In a d d i t i o n , I i n v e s t i g a t e d the e f f e c t s of these s t e r i o d s on the growth of the organ, as measured by wet weight, p r o t e i n , RNA and DNA content, as w e l l as t h e i r e f f e c t s on s p e c i f i c p r o t e i n s y n t h e s i s , w i t h p a r t i c u l a r reference to ovalbumin, lysozyme and a v i d i n s y n t h e s i s . MATERIALS AND METHODS Treatment of the Animals Japanese q u a i l of the s t r a i n Coturnix coturnix japonica . were obtained from the Poultry Science Department, U. B.C., on the f i r s t day a f t e r hatching. They were maintained i n e l e c t r i c a l l y heated brooders, fed on 28% Turkey Starter, and were kept on a schedule of 8 hours l i g h t , 16 hours darkness. At f i v e days of age, a 15-day i n j e c t i o n plan was begun. Each b i r d received 0.2 mg d a i l y of e s t r a d i o l i n 0.1 ml of sesame o i l subcutaneously i n the back of the neck. Control birds received the vehic l e only. On the tenth day of treatment the experimental animals were divided into two groups, one of which received progesterone (0.2 mg i n 0.1 ml of sesame o i l d a i l y ) , while the other continued to receive e s t r a d i o l for the remaining 5 days of treatment. This treatment was then followed by ten days of hormone withdrawal. Five to nine animals from each group were s a c r i f i c e d by ether-anaesthesia on the f i r s t , f i f t h , tenth, f i f t e e n t h , and twenty-fifth days of treatment, and t h e i r o v i -ducts were excised and f i x e d i n glutaraldehyde; or weighed and then frozen at -70°C, to be homogenized l a t e r f o r the assays. When tissue was excised for the c e l l - s p e c i f i c p rotein assays, only the magnum portion of the oviduct was used; i n cont r o l animals t h i s was estimated by using only the upper h a l f of the oviduct. To t a l Growth Assays Five to nine oviducts from each of the indicated time groups were thawed and reweighed, and homogenized i n d i s t i l l e d water by four 45-second bursts of a S o r v a l l Omni-Mixer, at a speed s e t t i n g of 5, interrupted by one-rminute cooling periods. DNA and RNA were e x t r a c t e d from the homogenate by the method of Schneider (57). DNA was assayed by the diphenylamine r e a c t i o n (58), w i t h c a l f thymus DNA (Calbiochem) as the standard. RNA was assayed by the o r c i n o l r e a c t i o n (59), w i t h yeast RNA (Calbiochem) as the standard. P r o t e i n was estimated by the method of Lowry et a l (60), w i t h c r y s t a l l i n e bovine serum albumin as standard. S p e c i f i c P r o t e i n Assays Oviduct magna were thawed and weighed, and homogenized i n 0.01 M sodium phosphate b u f f e r , pH 7.0, w i t h 0.015 M NaCl, to give a 2% (w/v) homogenate, f o r the assays of ovalbumin, lysozyme, and a v i d i n . The homogenates were c e n t r i f u g e d i n a Beckman p r e p a r a t i v e u l t r a c e n t r i f u g e f o r 60 minutes at 105,000 x g, and the supernatant was recovered. Q u a i l ovalbumin was p a r t i a l l y p u r i f i e d from q u a i l egg-white by the method described by Kabat and Meyer (61) f o r the p u r i f i c a t i o n of ovalbumin from chicken egg-white. Egg-white was d i l u t e d w i t h an equal volume of d i s t i l l e d water, and a volume of 100% sa t u r a t e d ammonium sulphate s o l u t i o n equal to the t o t a l volume was added, to b r i n g the mixture to 50% s a t u r a t i o n . A f t e r 2 hours at room temperature, the p r e c i p i t a t e from t h i s was removed by c e n t r i f u g a t i o n , and the supernatant adjusted to pH 4.6 w i t h 0.2 M H 2S0 4. Saturated ammonium sulphate was then added by the drop w i t h s t i r r i n g u n t i l a permanent opalescence was reached. The mixture was allowed to stand f o r 2 days, and the c r y s t a l s c o l l e c t e d by c e n t r i f u g a t i o n . The ovalbumin thus obtained was r e c r y s t a l l i z e d twice from 50% saturat e d ammonium sulphate, recovered by c e n t r i f u g a t i o n , and d r i e d i n a d e s s i c a t o r . I t was l a t e r d i s s o l v e d i n homogenizing b u f f e r , a p p l i e d to a Sephadex G-200 column, 60 cm x CH I and e l u t e d w i t h 0.02'M T r i s - H C l , pH 7.0". Immunological c r o s s - r e a c t i v i t y of q u a i l and c h i c k ovalbumin (Calbiochem) and the presence or absence of ovalbumin i n the homogenates of oviducts from various stages of hormonal treatment was e s t a b l i s h e d by the use of Ouchterlony m i c r o - d i f f u s i o n t e s t s . This was performed as described by Work and Work (62) w i t h 1% agar i n phosphate-buffered s a l i n e (8.9%), pH 6.9. The s l i d e s were washed i n s a l i n e and d i s t i l l e d water, then d r i e d and s t a i n e d w i t h amido black. Lysozyme was assayed by the method of Litwack (63). The a c t i v i t y was measured by the change i n transmittance at 645 my at 25°C, i n a Unicam SP 1800 Spectrophotometer. The r e a c t i o n mixture c o n s i s t e d of 1 ml of 0.066 M potas-sium phosphate b u f f e r , pH 6.2, w i t h 0.1% NaCl; and Micrococcus- l y s o d e i k t i c u s , 20 mg dry weight/100 ml of b u f f e r s o l u t i o n . 0.1 ml of enzyme s o l u t i o n or ov i d u c t e x t r a c t was added. The r e a c t i o n mixture was read against a blank of d i s t i l l e d water. A c t i v i t y i s expressed as micrograms of lysozyme present, and i s based on the a c t i v i t y of p u r i f i e d chicken egg-white lysozyme ( C a l b i o -chem) as standard. The lower l i m i t of s e n s i t i v i t y of t h i s assay was 2 ygm/ml. A v i d i n was assayed by the method of Korenman and O'Malley (64). The assay i s based on the b i n d i n g of a v i d i n to C 1 4 - c a r b o n y l b i o t i n (45 mCi/mmole; Amersham Searle) . To 0.5 ml of C 1 4 - b i o t i n (1.35 x 10~ 3 yg/ml) was added 0.1 ml of a v i d i n standard (0.54 units/mg, N u t r i t i o n a l Biochemicals) of concen-t r a t i o n s v a r y i n g between 0.5 yg/ml to 2.0 yg/ml, d i s s o l v e d i n 0.2 M ammonium carbonate, or 0.1 ml of oviduct e x t r a c t . A f t e r 15 minutes at room temperature, 1 ml of ammonium carbonate c o n t a i n i n g 10 mg bentonite was added. A f t e r an a d d i t i o n a l 5 minutes, the mixture was t r a n s f e r r e d to a 3 ml p l a s t i c Luer-Lok syringe (B-D), w i t h a Swinnex f i l t e r holder ( M i l l i p o r e ) attached to the end. Pressure was then a p p l i e d w i t h the plunger, and the mixture was f i l t e r e d through the M i l l i p o r e f i l t e r (0.45 y pore s i z e ) . The f i l t e r was washed twice with about 1.8 ml of 0.2 M ammonium carbonate, then removed and transferred to a s c i n t i l l a t i o n v i a l , dissolved i n 10 ml of Bray's s o l u t i o n (65) and counted for 10 minutes i n an Isocap 300 l i q u i d s c i n t i l l a t i o n counter (Nuclear Chicago) on Channel 5. Background was approximately 30 cpm, and e f f i c i e n c y was about 85%. Histology The f r e s h l y excised tissue was f i x e d i n Bouin's or i n 2.5% g l u t a r a l d e -hyde i n 0.1 M phosphate buffer, pH 7.2, for at l e a s t 24 hours, then dehydrated through a s e r i e s of alcohols (50-100%), and three changes of acetone. The ti s s u e was embedded i n JB-4 P l a s t i c from Polysciences, Inc. (butoxy-ethanol-g l y c o l methacrylate) as follows: three changes (15 minutes each) i n Solution A; f i n a l polymerization i n Solution A and B (42:1), overnight at room tempera-ture. One to two micron sections were cut using glass knives, and stained at 45°C with Harris's hemotoxylin and eosin. The t h i n p l a s t i c sections afforded only l i t t l e contrast, and so were photographed using a phase contrast objective, thus giving the resultant pictures an unusual and often f l u i d RESULTS I. H i s t o l o g y A. C o n t r o l Animals The oviducts from c o n t r o l b i r d s appear to undergo p r o g r e s s i v e changes during the treatment p e r i o d , p r i m a r i l y i n the stroma. These i n c l u d e changes i n the p a t t e r n of c e l l arrangement, i n nuclear morphology, i n s i z e and i n v a s c u l a r i z a t i o n . As can be seen i n oviducts from b i r d s which had served as c o n t r o l s f o r 5 days ( r e f e r to P l a t e 1 ) , the stromal c e l l s were very densely packed against the e p i t h e l i u m . At the base of each f u t u r e mucosal f o l d , the c e l l s were much l e s s densely packed together, and there appeared to be greater i n t e r c e l l u l a r space. The e p i t h e l i u m was a simple columnar e p i t h e l i u m . Each i n d i v i d u a l f u t u r e mucosal f o l d t h e r e f o r e had the appearance of a primordium, such as a limb bud. By f i f t e e n days of c o n t r o l treatment, t h i s p a t t e r n of stromal c e l l arrangement had changed ( r e f e r to P l a t e 2). The stromal c e l l s had dispersed n o t i c e a b l y , and were no longer densely packed a g a i n s t the e p i t h e l i u m . They appeared to be uniformly d i s t r i b u t e d throughout the length and width of the mucosal f o l d . However, the c e l l s i n the center of the f o l d s appear to have n u c l e i which are longer and t h i n n e r than those c l o s e r to the e p i t h e l i u m . These long, t h i n n u c l e i appeared to be o r i e n t e d along the v e r t i c a l a x i s of the f o l d . This gives an o v e r a l l appearance of streaming, as i f the c e l l s were being sprayed out from a fountainhead at the center and base of each f o l d . Between the f o l d s , near the periphery of the ov i d u c t , the long t h i n n u c l e i are h o r i z o n t a l l y o r i e n t e d , so that the streaming appeared to be continuous from the base and center of one f o l d to the base and center of the next. At the very periphery of the o v i d u c t , a l l of the long t h i n n u c l e i are h o r i z o n t a l -l y o r i e n t e d , thus g i v i n g the impression that a stream of c e l l s o r i e n t e d i n a c i r c u l a r manner surrounds the o v i d u c t , w i t h p e r p e n d i c u l a r l y o r i e n t e d i n t e r r u p -t i o n s i n the streaming at r e g u l a r i n t e r v a l s . These i n t e r r u p t i o n s form the base and center of the f o l d s . The major d i f f e r e n c e s , then, between the oviducts from f i v e - and f i f t e e n -day c o n t r o l s , are the s i z e and the dense packing of the stromal c e l l s against the e p i t h e l i u m i n the five-day c o n t r o l o v i d u c t s . By f i f t e e n days, the s i z e of the o v i d u c t s had approximately doubled, the mucosal f o l d s had enlarged somewhat, and the stromal c e l l s had dispersed throughout the area of the f o l d s . The e p i t h e l i u m remained a simple columnar e p i t h e l i u m . By 25 days of treatment, or 30 days of l i f e , v a s c u l a r i z a t i o n can be seen to have increased ( r e f e r to P l a t e 3). Blood v e s s e l s were present only i n the mesenteries and at the very periphery of the oviduct i n the f i v e - d a y c o n t r o l s , and at the very base of the mucosal f o l d s i n the ten- and f i f t e e n - d a y c o n t r o l s . In the oviducts from the twenty-five-day c o n t r o l b i r d s , c a p i l l a r i e s could be seen extending from the base of the f o l d s up i n t o the center of the f o l d s . Thus v a s c u l a r i z a t i o n , when i t occurs, f o l l o w s the "stream" of the c e l l p a t t e r n . The changes which occur i n the c o n t r o l oviducts t h e r e f o r e resemble those of any embryonic primoridum i n response to i t s inducer (70) This i n c l u d e s the i n t i a l stromal packing against the e p i t h e l i u m , the stromal c e l l rearrange-ment, and the beginning of v a s c u l a r i z a t i o n . These observations are at v a r i a n c e w i t h those of Fertuck and Newstead (66), who have reported that t u b u l a r gland c e l l s are present i n the oviduct by 30 days of age. However, O i s h i and Lauber (67, 68) have demonstrated t h a t , i n P l a t e l a . C r o s s - s e c t i o n of an oviduct from a 10-day-old b i r d , which had r e c e i v e d c o n t r o l treatment f o r 5 days, x 168. Note arrangement of stromal c e l l s (S) densely packed against e p i -thelium (E), and v a s c u l a r i z a t i o n (BV) i n mesenteries surround-i n g o v i d u c t . Fixed i n Bouin's and embedded i n wax; 5 microns. P l a t e l b . Same, x 680. Note simple columnar e p i t h e l i u m (E), w i t h c e l l s packed together c l o s e l y enough to appear p s e u d o - s t r a t i f l e d . P l a t e l c . C r o s s - s e c t i o n of an oviduct from a 15-day-old b i r d which had r e c e i v e d c o n t r o l treatment f o r 10 days, x 672. The same arrangement of e p i t h e l i a l c e l l s and stromal c e l l s as described above appears at t h i s time. F i x e d i n g l u t a r a l d e -hyde and embedded i n p l a s t i c . 1 micron. P l a t e 2a. C r o s s - s e c t i o n of an oviduct from a 20-day-old b i r d , which had r e c e i v e d c o n t r o l treatment f o r 15 days, x 168. Note d i s p e r s i o n of stromal c e l l s ( S), throughout area of mucosal f o l d s , v a s c u l a r i z a t i o n (BV) at the base of the f o l d s , simple columnar e p i t h e l i u m (E). F i x e d i n glutaraldehyde, and embedded i n p l a s t i c . 1 micron. P l a t e 2b. Same, x 672. Note o r i e n t a t i o n of stromal c e l l n u c l e i at center of f o l d s along the v e r t i c a l a x i s of the f o l d . E p i t h e l i u m (E); stroma (S), and blood c e l l s (BC). P l a t e 3a. Cr o s s - s e c t i o n of an oviduct from a 30-day-old b i r d , which had r e c e i v e d c o n t r o l treatment f o r 15 days, followed by no treatment f o r 10 days, x 168. Note same arrangement of stromal c e l l s (S) as i n 15-day c o n t r o l b i r d s ( P l a t e 2). Fixed i n glutaraldehyde and embedded i n p l a s t i c . 1 micron. P l a t e 3b. Same, x 672. Note appearance of blood v e s s e l s (BV) i n the mucosal f o l d s . the short day length used i n t h i s study (8L/16D), gonadal growth i s adversely a f f e c t e d ( r e l a t i v e to 24L/0D) by reducing, v i a the p i n e a l , gonadotrophin s e c r e t i o n (67, 68, 69; see a l s o 164). Eroschenko and Wilson have a l s o demonstrated that r e g r e s s i o n of a d u l t " q u a i l oviducts and gonads can be induced by t h i s short day length, r e l a t i v e to 16L/8D (56). The study by Fertuck and Newstead (66) d i d not i n d i c a t e the day length used; however, the discrepancy between those r e s u l t s and these could be explained by a d i f f e r e n t day l e n g t h . The changes described here have a l s o been reported by Kohler et a l (41) to be the i n i t i a l r e s u l t s of estrogen treatment on the c h i c k o v i d u c t . Thus i t would appear that estrogen i s present i n the immature q u a i l at a l e v e l capable of inducing the e a r l y changes i n the stroma, but not d i f f e r e n t i a t i o n of the t u b u l a r gland c e l l s . B. Treated Animals Very d e f i n i t e changes took place w i t h i n f i v e days of estrogen treatment, as can be seen i n P l a t e 4. The oviduct had grown tremendously i n s i z e , and the mucosal f o l d s enlarged g r e a t l y to f i l l most of the lumen. These f o l d s were f i l l e d w i t h tubular gland c e l l s c o n t a i n i n g e o s i n o p h i l i c granules. The glands, which secrete the egg-white p r o t e i n s (50, 66, 43, 71), a r i s e by budding o f f of the e p i t h e l i u m , and the glands open i n t o the lumen. P o i n t s of entry i n t o the lumen could be seen i n . t h e mucosal f o l d s . The simple columnar e p i t h e l i u m appeared to be very s l i g h t l y c i l i a t e d i n some p l a c e s , but no other d i s t i n c t evidence of d i f f e r e n t i a t i o n was seen i n the e p i t h e l i u m . Occasional c i l i a t e d c e l l s a l s o occur i n mammalian u t e r i , even i n the absence of hormone (72). By 10 days of treatment ( r e f e r to P l a t e 5 ) , the oviduct s i z e was g r e a t l y increased. Tubular gland c e l l s c o n t a i n a great abundance of granules. These granules s t a i n w i t h v a r y i n g i n t e n s i t y , and i t has been suggested (66) that t h i s r e f l e c t s v a r y i n g concentrations of the egg-white p r o t e i n s . Nucleated blood c e l l s were present as w e l l , and e p i t h e l i a l d i f f e r e n t i a t i o n had occurred. The p s e u d o - s t r a t i f l e d columnar e p i t h e l i u m contained some c e l l s which had i n t e n s e l y - s t a i n e d , b a s a l l y - s i t u a t e d n u c l e i , and others that had p a l e , c e n t r a l l y - p l a c e d n u c l e i . C i l i a were apparent continuously along the e p i t h e l i -um, so that i t was not p o s s i b l e to determine from these s e c t i o n s alone which c e l l s they belonged to. However, i t i s l i k e l y they belonged to the c e l l s w i t h c e n t r a l l y - p l a c e d n u c l e i , s i n c e t h i s i s the case i n c h i c k o v i d u c t , and si n c e the other c e l l s are thought to be s e c r e t o r y . In a l l e p i t h e l i a l c e l l s , the n u c l e i were rounded and elongated. By 15 days of estrogen t r e a t m e n t , ( r e f e r to P l a t e 6 ) , the b a s a l l y - s i t u a t e d n u c l e i of the s e c r e t o r y e p i t h e l i a l c e l l s were no longer elongated, but appeared to be pushed against the basement membrane. In some cases these c e l l s seemed to be i n v o l v e d i n the process of s e c r e t i o n . The c e n t r a l l y -placed n u c l e i of c i l i a t e d c e l l s were rounded, and the c i l i a were much more prominent. Oviducts t r e a t e d f o r 10 days w i t h estrogen and then 5 days w i t h proges-terone ( r e f e r to P l a t e 7) o c c a s i o n a l l y contained many unstained areas, suggesting that s e c r e t i o n of some of the granules i n t o the lumen had occurred. The granules were so abundant and so d a r k l y - s t a i n i n g that i t was d i f f i c u l t to l o c a t e the n u c l e i of the tubular gland c e l l s . In some oviducts both the c i l i a t e d c e l l s w i t h c e n t r a l n u c l e i and the secr e t o r y c e l l s w i t h b a s a l n u c l e i had i n t e n s e l y - s t a i n e d , compressed n u c l e i . In other o v i d u c t s , only the b a s a l n u c l e i of the s e c r e t o r y c e l l s were compressed, w h i l e the c e n t r a l n u c l e i of P l a t e 4a. C r o s s - s e c t i o n of an oviduct magnum from a b i r d t r e a t e d f o r 5 days w i t h estrogen, x 168. Note the t u b u l a r glands (TG), v a s c u l a r i z a t i o n (BV). Fi x e d i n glutaraldehyde and embedded i n p l a s t i c . 1 micron. P l a t e 4b. Same, x 800. Note o c c a s i o n a l c i l i a t e d c e l l s (C), p o i n t s of entry of t u b u l a r glands i n t o the lumen Carrows), and presence of e o s i n o p h i l i c granules (G) i n t u b u l a r gland c e l l s . P l a t e 5a. Cr o s s - s e c t i o n of an ovid u c t magnum from a b i r d t r e a t e d f o r 10 days w i t h estrogen, x 168. Note abundance of glands and granules, and increase i n s i z e and v a s c u l a r i z a t i o n . Fixed i n glutaraldehyde and embedded i n p l a s t i c . 1 micron. P l a t e 5b. Same, x 800. Note v a s c u l a r i z a t i o n (BV) near e p i t h e l i u m CE), abundance of c i l i a (C) on e p i t h e l i u m , and presence of two c e l l types i n ep i t h e l i u m . Granules i n tubular gland c e l l s s t a i n w i t h many d i f f e r e n t i n t e n s i t i e s . P l a t e 6a. C r o s s - s e c t i o n of an oviduct magnum from a b i r d t r e a t e d f o r 15 days w i t h estrogen, x 168. Compare w i t h P l a t e 4a f o r an i n d i c a t i o n of increase i n s i z e over the treatment p e r i o d . F i x e d i n glutaraldehyde and embedded i n p l a s t i c . 1 micron. P l a t e 6b. Same, x 1010. Note d a r k l y - s t a i n i n g b a s a l n u c l e i of goblet c e l l s (G) and profuse c i l i a (C) of ep i t h e l i u m . Granules of the t u b u l a r gland c e l l s appear to s t a i n w i t h same i n t e n s i t y . P l a t e 7a. C r o s s - s e c t i o n of oviduct magnum from a b i r d t r e a t e d f o r 10 days w i t h estrogen, f o l l o w e d by 5 days of progesterone, x 168. Note unstained areas. F i x e d i n glutaraldehyde and embedded i n p l a s t i c . 1 micron. P l a t e 7b. Same, x 672. Note compressed appearance of n u c l e i and datfker-staining cytoplasm of c i l i a t e d c e l l s (C), and goblet c e l l s (GC) i n process of s e c r e t i o n . Lumen = L. the c i l i a t e d c e l l s were not. Others have a l s o reported that the c i l i a t e d c e l l s become extremely compressed between the se c r e t o r y c e l l s , because the l a t t e r become engorged w i t h s e c r e t o r y products (56) . C i l i a appeared i n some cases to be q u i t e long and dense, and d a r k l y - s t a i n i n g . C. Animals Withdrawn from Hormone Treatment Oviducts of animals withdrawn from estrogen plus progesterone treatment were smaller than those withdrawn from estrogen treatment. Of the oviducts withdrawn from estrogen plus progesterone treatment ( r e f e r to P l a t e s 8 and 9 ) , some were l a r g e r than others, although there were few i f any h i s t o l o g i c a l d i f f e r e n c e s between these two s i z e s of o v i d u c t s . V a s c u l a r i z a t i o n was s t i l l e v i dent, although somewhat lessened, and there were fewer and smaller t u b u l a r gland c e l l s . There were no d i s t i n c t granules i n any of the tu b u l a r gland c e l l s , but some contained an e o s i n o p h i l i c m a t e r i a l , whereas others appeared empty, i n that t h e i r centers were e n t i r e l y unstained. There appeared to be more s i n g l e c e l l s i n the stroma which were not organized i n t o t u b ular gland c e l l s . The p s e u d o - s t r a t i f l e d e p i t h e l i u m appeared to be i n a d i f f e r e n -t i a t e d s t a t e : i t was p r o f u s e l y c i l i a t e d , and contained c e l l s w i t h c e n t r a l , l i g h t n u c l e i , and some w i t h b a s a l l y - s i t u a t e d , d a r k l y - s t a i n i n g n u c l e i . The n u c l e i of a l l the e p i t h e l i a l c e l l s were rounded and elongated, r a t h e r than the dark, compressed n u c l e i of the estrogen plus progesterone t r e a t e d oviduct e p i t h e l i u m . There were no evidences of s e c r e t i o n s i n any of the e p i t h e l i a l c e l l s ( r e f e r to P l a t e 8b). Those oviducts withdrawn from estrogen plus progesterone treatment which were l a r g e r , appeared h i s t o l o g i c a l l y to be q u i t e s i m i l a r to the smaller ones j u s t described ( r e f e r to P l a t e 9). These showed greater v a s c u l a r i z a t i o n , and i n a d d i t i o n had l a r g e areas of the mucosal f o l d s which contained n e i t h e r c e l l s nor blood v e s s e l s . They a l s o contained many tubular glands w i t h un-s t a i n e d centers. Although the general appearance of these seemed l e s s h e a l t h y than the s m a l l e r o v i d u c t s , i t should be pointed out that these were f i x e d i n Bouin's r a t h e r than glutaraldehyde. Bouin's i s g e n e r a l l y a poorer f i x a t i v e (76). Oviducts from animals withdrawn from estrogen treatment were a l l l a r g e r than those withdrawn from estrogen plus progesterone treatment. These had s t i l l g reater v a s c u l a r i z a t i o n than those withdrawn from progesterone treatment. In a d d i t i o n , there were l a r g e unstained areas i n the center of the f o l d s , as w e l l as some tu b u l a r glands which were empty-appearing. These o v i d u c t s had the same general appearance, but contained many more tu b u l a r glands, and fewer t u b u l a r glands w i t h unstained centers. E p i t h e l i a l d i f f e r e n t i a t i o n i n these o v i d u c t s withdrawn from estrogen treatment d i d not d i f f e r s i g n i f i c a n t l y from that described f o r oviducts withdrawn from estrogen plus progesterone treatment. I t i s evident from these h i s t o l o g i c a l r e s u l t s that the q u a i l o viduct e x h i b i t s the three c h a r a c t e r i s t i c s common to developing systems: growth, morphogenetic movement, and c e l l d i f f e r e n t i a t i o n . These changes compare to those reported to occur i n the c h i c k oviduct as a r e s u l t of estrogen and progesterone treatment. Estrogen i s known to cause, i n i t i a l l y , changes i n the stroma, d i s p e r s i n g stromal c e l l s , and to cause water i m b i b i t i o n and hyperemia (41). The v a s c u l a r i z a t i o n of the region i s followed by blood c e l l i n v a s i o n of the stroma (41). A f t e r 2 to 3 days, t u b u l a r gland c e l l s begin to bud o f f from the e p i t h e l i u m (41, 45, 48). A p r o t o - d i f f e r e n t i a t e d s t a t e (42) has been P l a t e 8a. C r o s s - s e c t i o n of an oviduct magnum from a b i r d t r e a t e d f o r 10 days w i t h estrogen, 5 days w i t h progesterone, and then withdrawn from treatment f o r 10 days, x 550. Note empty-appearing tubular gland (arrows), blood c e l l s (BC) and g e n e r a l l y unorganized appearance of stroma. E p i t h e l i u m r e t a i n s c i l i a (C). Fixed i n glutaraldehyde and embedded i n p l a s t i c . 1 micron. P l a t e 8b. Same, x 1606. C i l i a t e d (C) 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 (E) remains d i f f e r e n t i a t e d i n t o two c e l l types. Although b a s a l l y -s i t u a t e d , d a r k l y - s t a i n i n g n u c l e i are a c h a r a c t e r i s t i c of s e c r e t o r y goblet c e l l s , no evidence of s e c r e t i o n can be seen. N u c l e i are elongated, and no longer compressed. Note i n v a g i n a -t i o n of e p i t h e l i u m (arrow) which may have been former opening of a t u b u l a r gland c e l l i n t o lumen (L). P l a t e 9a. C r o s s - s e c t i o n of an oviduct magnum from a b i r d t r e a t e d f o r 10 days w i t h estrogen, 5 days w i t h progesterone, and then withdrawn from treatment f o r 10 days, x 168. This oviduct was one of the smaller ones from the group. Note empty-appearing t u b u l a r glands (arrow), v a s c u l a r i z a t i o n (BV) and presence of some tubular gland c e l l s w i t h e o s i n o p h i l i c c e n t e r s . Fixed i n glutaraldehyde, embedded i n p l a s t i c . 1 micron. P l a t e 9b. Same, x 168. This was one of the l a r g e r oviducts from the group. Legend as above. Note d i f f e r e n c e i n s i z e , i n comparison to 9a. Fixed i n Bouin's, embedded i n p l a s t i c . 1 micron. P l a t e 9c. Same as 9b, x 550. Note c i l i a t e d c e l l s (C) i n pseudo-s t r a t i f i e d e p i t h e l i u m , and e p i t h e l i a l i n v a g i n a t i o n (arrow), perhaps re p r e s e n t i n g former s i t e of entry of tubular gland i n t o lumen ( L ) . noted by P a l m i t e r and Wrenn (43), w i t h incomplete i n d e n t a t i o n s of the e p i t h e -l i a l c e l l s i n t o the stroma. These p r o t o - d i f f e r e n t i a t e d c e l l s continue* to form t u b u l a r gland c e l l s when estrogen i s present (43). M i c r o f i l a m e n t s are e s s e n t i a l f o r t h i s process of budding (44), which i s l a t e r accompanied by glandular m i t o s i s as w e l l (45). Budding continues f o r a number of days, and when i t i s complete, the l u m i n a l e p i t h e l i a l c e l l s d i f f e r e n t i a t e to form c i l i a t e d c e l l s and goblet s e c r e t o r y c e l l s (43, 45, 46, 47). The former con-t a i n c e n t r a l l y - l o c a t e d , p a l e l y - s t a i n i n g n u c l e i , and the l a t t e r c o n t a i n dark, a p i c a l l y - s i t u a t e d n u c l e i (41, 45) as reported above f o r q u a i l . Progesterone treatment has been shown by Oka and Schimke to prevent the d i f f e r e n t i a t i o n of t u b u l a r gland c e l l s (45). However, that work demonstrat-ed that the e f f e c t s of progesterone depended on the stage of d i f f e r e n t i a t i o n at which i t was administered (45). Thus, i f progesterone was given concomit-a n t l y w i t h estrogen from the beginning of treatment, i t a b o l i s h e d the t u b u l a r gland c e l l d i f f e r e n t i a t i o n and t h e r e f o r e the growth of the o v i d u c t . However, i f the onset of progesterone treatment was delayed u n t i l l a t e r stages of estrogen treatment, i t had l e s s i n h i b i t o r y e f f e c t , s i n c e i t d i d not i n t e r f e r e w i t h the t u b u l a r gland c e l l s once they were d i f f e r e n t i a t e d (45) . This l a t t e r f a c t would e x p l a i n why progesterone i n the present study demonstrated no antagonism: i t i s begun only a f t e r prolonged estrogen treatment, at a time when tubular gland c e l l d i f f e r e n t i a t i o n may already be complete. Progesterone i s known to enhance e p i t h e l i a l d i f f e r e n t i a t i o n i n chicks (47, 48). This i s the case even i f estrogen i s not given f i r s t (47). This r e s u l t was not i n d i c a t e d i n the present work w i t h q u a i l , perhaps because prolonged estrogen treatment has the same e f f e c t (41, 45, 48). Thus, i n t h i s study, the 15-day estrogen treatment and the 10-day estrogen, 5-day progester-one treatment experiments d i d not show great d i f f e r e n c e s i n e p i t h e l i a l d i f f e r -e n t i a t i o n , but s m a l l d i f f e r e n c e s d i d e x i s t . In the estrogen-plus-progesterone t r e a t e d o v i d u c t s , the n u c l e i of both the s e c r e t o r y c e l l s and the c i l i a t e d c e l l s were very compact and d a r k l y - s t a i n i n g , not o v a l . In some cases, the cytoplasm as w e l l as the n u c l e i of the c i l i a t e d c e l l s was compressed between the s e c r e t o r y c e l l s . In t i s s u e withdrawn from treatment, smaller oviducts w i t h fewer t u b u l a r gland c e l l s were seen, which supports the e a r l y i n d i c a t i o n s of S t r o t t (54) and of F i t z g e r a l d (55), and the more recent i n v e s t i g a t i o n s of Eroschenko and Wilson (56) on r e g r e s s i n g q u a i l o v i d u c t s . Those reported r e s u l t s i n d i c a t e d that c e l l s are l o s t during r e g r e s s i o n , a time when estrogen i s diminished i n the animal due to decreased gonadotropin r e l e a s e (67, 68, 69). The e p i t h e -l i a l c e l l s appear to remain d i f f e r e n t i a t e d s i n c e they are reported by Eroschenko and Wilson (56) to r e t a i n c i l i a f o r up to 3 weeks a f t e r o v i d u c a l r e g r e s s i o n i s induced i n a d u l t animals by r e d u c t i o n of d a y l i g h t . In c h i c k s , withdrawal from hormone treatment was i n i t i a l l y reported to r e s u l t i n no l o s s of c e l l s , but only a r e d u c t i o n of c e l l s i z e (45, 48). However, Yu et a l (51, 52) have reported that DNA content i s g r e a t l y reduced during r e g r e s s i o n i n c h i c k o v i d u c t , and more r e c e n t l y , P a l m i t e r (77) has reported that t u b u l a r gland c e l l s make up 80% of the c e l l s of a t r e a t e d o v i d u c t , but only 20% of the c e l l s of an o v i d u c t withdrawn from treatment. Therefore i t appears that the c h i c k oviduct a l s o l o s e s c e l l s upon hormone withdrawal. I I . Assays of T o t a l Growth: Weight, P r o t e i n , RNA and DNA Content Estrogen caused a dramatic increase i n the growth of the o v i d u c t s , r e -s u l t i n g i n a 700-fold increase i n wet weight (Figure 3) from 2.5 mg on the f i r s t day of treatment to an average value of 1.7 gm a f t e r 15 days of estrogen treatment. In b i r d s r e c e i v i n g progesterone r a t h e r than estrogen f o r the l a s t 5 days of treatment, the average o v i d u c a l weight reached 2.0 gm. The greatest r a t e of increase was between days 5 and 10 of treatment; the response slowed somewhat between days 10 and 15 of treatment. Ten days a f t e r withdrawal from estrogen treatment, the wet weight had decreased only s l i g h t l y . This d i f f e r e n c e i s not s i g n i f i c a n t (p = 0.04). However, oviducts which had r e c e i v e d 10 days of estrogen treatment followed by 5 days of progesterone treatment, l o s t about 85% of t h e i r wet weight i n the same 10-day withdrawal p e r i o d , thus demonstrating a f a r greater decrease than the oviducts from b i r d s withdrawn from estrogen treatment. S i m i l a r changes were seen i n the p r o t e i n (Figure 4) and RNA (Figure 5) content of the o v i d u c t s . The g r e a t e s t increase was always between 5 and 10 days of treatment. The increase observed when progesterone replaced estrogen from 10 to 15 days of treatment may be s i g n i f i c a n t f o r the change i n RNA con-tent (p = 0.01), but i s not s i g n i f i c a n t f o r the change i n p r o t e i n content (p = 0.04). The concentration of p r o t e i n and RNA per u n i t weight d i d not increase as g r e a t l y as the t o t a l amount of each component, due to the con-comitant increase i n wet weight.(see Table I ) . The c o n c e n t r a t i o n of RNA remained f a i r l y constant throughout treatment, whereas the p r o t e i n concentra-t i o n showed l e s s v a r i a n c e and a g r e a t e r i n c r e a s e . The changes i n RNA and p r o t e i n c o n c e n t r a t i o n throughout treatment were not s i g n i f i c a n t . However, t o t a l p r o t e i n and t o t a l RNA content increased approximately 1000-fold and 500-fold r e s p e c t i v e l y over c o n t r o l values i n the 15 days of estrogen treatment, i n d i c a t i n g again the very r a p i d growth of the o v i d u c t . Figure 3. The V a r i a t i o n i n Wet Weight as a Function of Treatment Legend: o , treatment w i t h e s t r a d i o l . • , treatment w i t h progesterone. t withdrawn from treatment. , c o n t r o l o v i d u c t s . Bars represent standard e r r o r . 00 Figure 4. The V a r i a t i o n i n P r o t e i n Content as a Function of Treatment. Legend as above. E Fjg^Time vs. Wet Weight 3 r -Treatment-Withdrawal 0 Fig.4- Time vs Protein Content 500 -Treatment —Withdrawal I 400 o 300 i i •t i 0 5 10 15 20 25 Day of Treatment 200 o &L 100 E 0 .0 1 J. i 0 5 10 15 20 25 Day of Treatment 4^ CD Figure 5. The V a r i a t i o n i n Oviduct RNA Content as a Function of Treatment. Legend as i n Figure 3. Figure 6. The V a r i a t i o n i n Oviduct DNA Content as a Function of Treatment. Legend as i n Figure 3. u, o 30 r 25 F i g ! • Time vs RNA Content RgJ. :Time vs DNA Content , n -Treat ment W it hd rawal 12 r $ o •120 O < 1 5 § 1 0 en £ 5 0 •Treatment — Withdrawal K v -•f 1.... ..1 0 5 10 15 20 25 Day of Treatment 0 5 10 15 20 25 Day of Treatment CJI A f t e r 10 days of withdrawal from estrogen treatment, approximately one-t h i r d of the p r o t e i n content, and one-half of the RNA content of the oviduct were l o s t . In c o n t r a s t to t h i s , i n the oviducts that had recei v e d estrogen plus progesterone treatment, approximately 90% of both the RNA and the p r o t e i n content was l o s t a f t e r 10 days of withdrawal. This greater r a t e of decrease a f t e r withdrawal from estrogen plus progesterone corresponds to the greater decrease i n the wet weight of the organ when withdrawn from estrogen p l u s progesterone treatment r a t h e r than from treatment w i t h estrogen only. The changes i n the DNA content (Figure 6) as a r e s u l t of treatment v a r i e d from the other t i s s u e components i n 2 ways: 1) This i s the only t i s s u e com-ponent not increasedby estrogen plus progesterone treatment r e l a t i v e to continued estrogen treatment. The DNA content of estrogen-plus^progesterone t r e a t e d o v iducts v a r i e s only s l i g h t l y from the 10-day estrogen value, i n d i c a t i n g that progesterone does not s t i m u l a t e DNA s y n t h e s i s , at l e a s t not at t h i s p o i n t i n the d i f f e r e n t i a t i o n of the t i s s u e . The co n c e n t r a t i o n of DNA a c t u a l l y decreased s i g n i f i c a n t l y w i t h progesterone treatment (see Table I ) . 2) Upon withdrawal from estrogen treatment, a l o s s of approximately 50% of the DNA content was noted, as i n RNA content, but a l o s s of only two-thirds of the DNA content was noted i n the oviducts withdrawn from estrogen plus progester-one treatment. This i s i n con t r a s t to the l o s s of 90% of the RNA and p r o t e i n content, and 85% of the wet weight i n these same o v i d u c t s , i n d i c a t i n g that l e s s DNA was l o s t than other components. The conc e n t r a t i o n of DNA a c t u a l l y increased a f t e r withdrawal from progesterone, whereas i t decreased a f t e r w i t h -drawal from estrogen (see Table I ) . This c o n c e n t r a t i o n increase may be an i n d i c a t i o n of the greater l o s s of other components, such as wet weight and p r o t e i n . Table I. Summary of the V a r i a t i o n s i n the Content and Concentrations of Tissue Constituents i n the Oviduct at D i f f e r e n t Stages of Treatment. No. i n sample Treatment P r o t e i n Mg/oviduct Protein/wt Mg/gm RNA Mg/oviduct RNA/wt Mg/gm DNA Mg/oviduct DNA/wt Mg/gm 7 5dE 41.5±1.3 98.5213.24 4.96+0.38 11.8610.52 2.4910.09 6.01+0.19 9 lOdE 185.0819.48 137.2613.13 15.7210.86 11.73+0.56 8.18+0.27 7.86+0.18 9 15dE 309.33H7.77 188.82115.18 20.43+0.95 12.4010.68 11.81±0.52 7.22+0.56 4 10dE5dP 402.00±60.76 201.51135.96 25.06+1.51 12.24+0.34 9.7010.46 4.75+0.19 5 15dE10dO 204.80116.92 147.9913.16 12.73±1.59 9.0810.32 5.37+0.36 3.9710.37 5 10dE5dP10dO 48.48±9.13 155.2318.15 2.8210.37 9/3010.52 2.93+0.33 9.7410.40 Co n t r o l s : OdC 0.0229 91.6 0.0222 8.9 0.0255 10.2 5dC 0.342 83.2 0.0443 10.8 0.0408 9.95 lOdC 0.715 110.0 0.0485 7.46 0.067 10.4 15 dC 0.757 85.2 0.0690 7.75 0.94 10.6 25dC 2.00 88.8 0.214 9.56 0.258 11.5 C o n t r o l oviducts a l s o increased i n these components during the treatment p e r i o d , although to a very s l i g h t extent. This may be due to very s l i g h t , i n i t i a l estrogen-induced changes, or i t may be the r e s u l t of the general growth of the animal. These r e s u l t s are comparable to those reported f o r the e f f e c t of estrogen on c h i c k oviduct (41, 45, 46, 48, 51, 52). Estrogen treatment f o r 10 days caused an increase i n a l l parameters i n c h i c k oviduct a l s o , but there i s a s l i g h t d i f f e r e n c e between the.chick oviduct work and the data presented here f o r the q u a i l , i n the variance observed i n the response. There i s somewhat l e s s variance i n the work of Oka and Schimke (45), who used only the magnum p o r t i o n of the chick oviduct f o r t o t a l growth assays, and i n the work of Yu et a l , who used whole o v i d u c t s . Yu et a l (46, 51, 52) reported the value and standard d e v i a t i o n f o r DNA and RNA concentrations (as percent of dry matter) f o r pooled samples of whole oviducts to be 1.8±0.1% f o r DNA and 2.3±0.1% f o r RNA. These represent a smaller v a r i a n c e than that reported here, but the r e s u l t s are not s t r i c t l y comparable, s i n c e r e p r e s e n t a t i o n i n t h i s t h e s i s i s percent of wet weight. No variance i s i n d i c a t e d i n t h e i r paper f o r t o t a l DNA content per ovi d u c t . This s l i g h t d i f f e r e n c e i n variance can probably be a t t r i b u t e d to d i f f e r e n c e s i n experimental procedures, such as time schedule of i n j e c t i o n s , dose, and species d i f f e r e n c e s . Progesterone appeared to exert no a n t a g o n i s t i c e f f e c t i n t h i s system, as has been reported f o r c h i c k s . However, as mentioned e a r l i e r , the e f f e c t of progesterone on the est r o g e n i c response depends on the stage of d i f f e r e n t i a t i o n at which i t i s administered (45, 48, 43). The means by which progesterone i n h i b i t s the est r o g e n i c growth response i s by a b o l i s h i n g t u b u l a r gland c e l l d i f f e r e n t i a t i o n (43), s i n c e these c e l l s make up the vast bulk of the magnum. However, i f progesterone i s given a f t e r these c e l l s are already present, i t does not i n h i b i t , but a c t u a l l y enhances t h e i r f u n c t i o n i n g (45, 48). Thus i f progesterone i s given a f t e r estrogen i n d u c t i o n , one would expect to see an increase i n such t i s s u e components as weight, p r o t e i n , and RNA content, but not DNA content, as progesterone i n h i b i t s formation of new t u b u l a r gland c e l l s . The r e s u l t s presented here support that p r e d i c t i o n . The d i f f e r e n c e s between oviducts t r e a t e d f o r 15 days w i t h estrogen, and those t r e a t e d f o r 10 days w i t h estrogen, 5 days progesterone are s m a l l . In the amount of t o t a l t i s s u e c o n s t i t u e n t s , the d i f f e r e n c e s between these two groups are not s i g n i f i -cant (weight, p = 0.014; RNA, p = 0.022; DNA, p = 0.03; p r o t e i n , p = 0,239). In the concentrations of t i s s u e c o n s t i t u e n t s , oviducts from these two groups are not d i f f e r e n t i n p r o t e i n c o n c e n t r a t i o n (p = 0.703), or RNA c o n c e n t r a t i o n (p = 0.84), but are s i g n i f i c a n t l y d i f f e r e n t i n DNA c o n c e n t r a t i o n (p,>= 0.002)'. This would be due not only to the r e l a t i v e l y greater synthesis of DNA i n estrogen t r e a t e d o v i d u c t s , but a l s o to the r e l a t i v e l y greater s y n t h e s i s of p r o t e i n and RNA i n the estrogen plus progesterone t r e a t e d o v i d u c t s . This i s i n keeping w i t h the known e f f e c t s of progesterone i n chick o v i d u c t , i n i n h i b i t i n g new tubular gland c e l l s y n t h e s i s and i n enchancing the f u n c t i o n i n g of those c e l l s already present. Withdrawal of hormone leads to a l o s s of a l l components, but DNA was l o s t to a l e s s e r extent than any other t i s s u e component. A greater l o s s occurred i n - a l l components a f t e r withdrawal from estrogen plus progesterone treatment than a f t e r withdrawal from estrogen. This may be due to 1) a f t e r r e c e i v i n g progesterone i n s t e a d of estrogen f o r the l a s t 5 days of treatment, these oviducts have a c t u a l l y been withdrawn from estrogen f o r 5 e x t r a days; or 2) progesterone may be a c t i n g i n such a manner as to make the t i s s u e l e s s s t a b l e to a l o s s of hormone. I f oviducts withdrawn from estrogen plus pro-gesterone treatment are a c t u a l l y regressed to a greater extent because they have been withdrawn from estrogen treatment 5 e x t r a days, then t h i s i m p l i e s that progesterone i s l e s s e f f i c i e n t as a l o n g - a c t i n g s t i m u l a t o r than i s estrogen. This may be due to: 1) Estrogen could p r e c i p i t a t e out at the s i t e of i n j e c t i o n , whereas progesterone, being more s o l u b l e , would not. This would create a "depot" of estrogen, but not of progesterone, which would be l o s t i n the 5 days of progesterone treatment before withdrawal. 2) Progester-one may be metabolized more e f f i c i e n t l y than estrogen. This i s known to be the case i n n e a r l y a l l animals (78). 3) Estrogen could be a s s o c i a t i n g w i t h l i p i d s i n the serum, which would create another type of "depot" (165). Progesterone, being more p o l a r , may not. This has been reported i n r a t s (79). In t h i s c o n s i d e r a t i o n , i t i s r e l e v a n t that estrogen induces serum l i p e m i a i n chicks (80, 73, 170). The e f f e c t s of progesterone on serum l i p e m i a are not known. On the other hand, i f progesterone i s a c t i n g to make the t i s s u e l e s s s t a b l e to a l o s s of hormone, t h i s could be e i t h e r by inducing greater meta-b o l i s m of estrogen and of i t s e l f , or by inducing a s p e c i f i c serum b i n d e r , which would reduce the amount of a c t i v e s t e r o i d . The r e s u l t s of t h i s study do not a l l o w a choice between these p o s s i b i l i -t i e s . A l l of them are l i k e l y . These r e s u l t s do i n d i c a t e however, that those oviducts from b i r d s withdrawn from estrogen treatment are at an e a r l i e r stage of withdrawal than those from b i r d s withdrawn from estrogen p l u s progesterone treatment. The r e s u l t s reported here support those of Yu et a l (51) f o r the c h i c k o v i d u c t , and Eroschenko and Wilson f o r the q u a i l oviduct (56). The study of Eroschenko and Wilson reported only h i s t o l o g i c a l changes, but i n d i c a t e d that tubular gland c e l l s were l o s t during r e g r e s s i o n of a d u l t o v i d u c t s . The r e -port of Yu et a l (51) e s t a b l i s h e d that the DNA content of the c h i c k o v i d u c t v a r i e s from an average of 11,456 yg i n a developing oviduct to 83,087 yg i n a l a y i n g hen, to 17,486 yg i n a r e g r e s s i n g o v i d u c t . These are contrary to the e a r l y reports of Oka and Schimke (45, 48), that the c e l l s remained during hormone withdrawal, but ceased f u n c t i o n i n g . More r e c e n t l y , P a l m i t e r has a l s o i n d i c a t e d t h a t , contrary to e a r l y r e p o r t s , the percentage of tubular gland c e l l s i n oviducts withdrawn from treatment i s only 20% as compared to 80% a f t e r 6 days of r e a d m i n i s t r a t i o n of estrogen (77). Thus i t appears that i n the absence of hormone, not only c e l l f u n c t i o n i n g , but e v e n t u a l l y the c e l l s themselves are l o s t . The p a t t e r n of l o s s of the various t i s s u e components a f t e r withdrawal was not i n v e s t i g a t e d i n d e t a i l i n t h i s work, but i n the study of Oka and Schimke (48), f o r c h i c k o v i d u c t , two d i f f e r e n t patterns were seen f o r the l o s s of components on hormone withdrawal. When hormone was withdrawn p r i o r to day 10 of treatment, each component continued to increase u n t i l what would have been day 10 of treatment, then d e c l i n e d . I f withdrawal began on day 10 or l a t e r , t h i s continued increase d i d not occur, but an immediate decrease was seen i n each component. The authors a t t r i b u t e d the continued increase to a p r e c i p i t a t i o n of estrogen at the s i t e of i n j e c t i o n , but d i d not e x p l a i n why i t d i d not occur a f t e r day 10, which presumably would have allowed f o r even more estrogen to p r e c i p i t a t e . I t may be that the treatment was inducing a serum b i n d i n g p r o t e i n a f t e r day 10, which would more e f f e c t i v e l y remove f r e e estrogen from the c i r c u l a t i o n , and make the l o s s of hormone to the t i s s u e more immediate. A l t e r n a t i v e l y , a new type of metabolism may have been induced. Thus, the p a t t e r n of l o s s of t i s s u e components on withdrawal may depend on the length of treatment p r i o r to withdrawal, as w e l l as the s t e r o i d used during treatment. Withdrawal from estrogen plus progesterone treatment has not been r e p o r t -ed i n other s t u d i e s . However, the r e s u l t s presented here concerning the withdrawal from estrogen treatment are at variance w i t h those reported by Oka and Schimke (48). Those authors reported that 12 days of withdrawal a f t e r 10 days of estrogen treatment r e s u l t s i n a f a r greater l o s s of wet weight (76% decrease) and RNA content (82% decrease) than DNA content (24% decrease). In t h i s study, approximately the same amount of DNA, RNA and p r o t e i n were l o s t during withdrawal from estrogen treatment. The greater l o s s of weight and RNA i n t h e i r study compared to t h i s one may be due to the s h o r t e r treatment and the longer withdrawal p e r i o d . However, that does not e x p l a i n why, i n t h e i r study, l e s s DNA was l o s t than other components, whereas i n t h i s study, DNA was l o s t to the same extent as the other t i s s u e components. I t may be p o s s i b l e to e x p l a i n t h i s discrepancy by observing the p a t t e r n of withdrawal more c l o s e l y . Oka and Schimke (48) demon-s t r a t e d that i n i t i a l l y , DNA i s l o s t at a r a t e s i m i l a r to lysozyme and wet weight, but that w i t h i n 5 days a f t e r the beginning of withdrawal, the r a t e of l o s s of DNA had slowed g r e a t l y . In t h i s study, estrogen was administered f o r 15 days, and i t i s probable that s t i m u l a t i o n continues beyond that time as a r e s u l t of the "depot" e f f e c t discussed e a r l i e r . These oviducts would there-fore be i n an e a r l i e r stage of withdrawal than those of Oka and Schimke. I n i t i a l l y , the r a t e of l o s s of DNA i s s i m i l a r to that of weight or lysozyme, but the r a t e slows g r e a t l y a f t e r about 5 days of withdrawal (48). I f i t i s assumed that the oviducts from b i r d s withdrawn from estrogen treatment i n t h i s study are i n an e a r l i e r stage of withdrawal, due to the l e n g t h i e r treatment, than those of Oka and Schimke, then these r e s u l t s are compatible w i t h those. This same assumption would e x p l a i n why oviducts from b i r d s withdrawn from estrogen treatment l o s t about the same amount of DNA as RNA or p r o t e i n , whereas those oviducts from b i r d s withdrawn from estrogen plus progesterone treatment l o s t l e s s DNA than RNA or p r o t e i n . I I I . S p e c i f i c P r o t e i n s Ovalbumin Ovalbumin p u r i f i e d as described under M a t e r i a l s and Methods was demon-s t r a t e d to be impure by Sephadex G-200 g e l f i l t r a t i o n (see Figure 7 ). I t was there f o r e not u s e f u l i n preparing a standard f o r the q u a n t i t a t i o n of ant i g e n -antibody p r e c i p i t a t e s . Although i t i s approximately the same molecular weight as ch i c k ovalbumin, as judged by the e l u t i o n p r o f i l e on Sephadex G-200, these two p r o t e i n s are not completely immunologically i d e n t i c a l , as can be seen from the Ouchterlony p l a t e ( r e f e r to P l a t e 10a). This precludes the use of c h i c k ovalbumin to construct a standard curve. In a d d i t i o n , as seen i n P l a t e 10a and b, the antibody p r e p a r a t i o n i s impure; there are two p r e c i p i -t a t i n g s p e c i e s , one of which i s p a r t i a l l y i d e n t i c a l w i t h c h i c k ovalbumin. The second p r e c i p i t a t i n g species has not been i d e n t i f i e d . That t h i s second species i s not simply an a r t i f a c t of conc e n t r a t i o n i s evident by the f a c t that i t can be d i l u t e d out by s e r i a l d i l u t i o n , to a p o i n t where the f i r s t species i s s t i l l e v ident, but not the second (see P l a t e 10b). Therefore, by demonstration of p a r t i a l i d e n t i t y w i t h c h i c k ovalbumin, and i d e n t i t y w i t h q u a i l ovalbumin, ovalbumin has been detected i n a l l t r e a t e d oviduct magna, i n a l l of the magna from animals withdrawn from estrogen treatment, and i n one-half of the magna withdrawn from estrogen plus proges-Figure 7. E l u t i o n P r o f i l e of Ovalbumin on Sephadex G-200. 1.5 ml of ch i c k or q u a i l ovalbumin (4 mg/ml) was a p p l i e d to a column of height 60 cm, and diameter, 1.5 cm, and e l u t e d w i t h T r i s b u f f e r at a flow r a t e of 0.1 mg/min. F r a c t i o n s of v 2.1 ml were c o l l e c t e d and absorbance at 280 nm was deter-mined f o r each f r a c t i o n . Fig.7. Elution Profile of Ovalbumen on Sephadex G-200 0 2 4 6 8 10 12 14 16 18 20 22 24 Fraction Number P l a t e 10a. Ouchterlony p l a t e demonstrating the r e a c t i o n of a n t i - c h i c k ovalbumin a n t i b o d i e s w i t h c h i c k ovalbumin (CO), q u a i l o v a l -bumin (QO), and magnum e x t r a c t (ME) from b i r d s withdrawn from estrogen treatment. 5 y l of Antibody p r e p a r a t i o n (1 yg antibody protein/ml) was a p p l i e d to the center w e l l , and 5 y l each of c h i c k ovalbumin (100 yg/ml) q u a i l ovalbumin (500 yg/ ml) or oviduct e x t r a c t (1%) were a p p l i e d to the appropriate outer w e l l s . P l a t e 10b. Ouchterlony p l a t e demonstrating the r e a c t i o n of a n t i - c h i c k ovalbumin w i t h c h i c k ovalbumin, q u a i l ovalbumin, and magnum e x t r a c t from b i r d s t r e a t e d f o r 10 days w i t h estrogen. Legend and p r o t o c o l as above. S e r i a l d i l u t i o n s : 1) 2% ex-t r a c t ; 2) 1% e x t r a c t ; 3) 0.2% e x t r a c t ; 4) 0.1% e x t r a c t . P l a t e 10c. Ouchterlony p l a t e demonstrating the r e a c t i o n of a n t i - c h i c k ovalbumin w i t h c h i c k ovalbumin, q u a i l ovalbumin, and a magnum e x t r a c t from a b i r d withdrawn from estrogen p l u s pro-gesterone treatment. Legend and p r o t o c o l as above. This i s an example of a negative r e a c t i o n . ME QO CO terone treatment ( r e f e r to P l a t e 10c) . No ovalbumin was detected i n any of the c o n t r o l o v i d u c t s . The lower l i m i t of the assay was 25 yg ovalbumin/ml. Lysozyme The lower l i m i t of s e n s i t i v i t y of the assay f o r lysozyme i s 2 yg/ml (see standard curve i n appendix). The readings at the lower l i m i t of the standard curve are t h e r e f o r e subject to a great deal of e r r o r . Concentration of the homogenate would have r e s u l t e d i n higher readings s u b j e c t to l e s s e r r o r , but t h i s was not p o s s i b l e , due to the very s m a l l s i z e of the c o n t r o l oviduct magna. In r e c o g n i t i o n of t h i s problem, i t i s concluded only that a trend i s seen i n lysozyme concentrations (see Figure 8) appearing (given the l i m i t a t i o n s mentioned), f i r s t on day 10 of estrogen treatment, and perhaps i n c r e a s i n g when progesterone i s s u b s t i t u t e d f o r estrogen between days 10 and 15 of treatment. Lysozyme was present i n one-half of the oviducts withdrawn from estrogen treatment, but i n none of the oviducts withdrawn from progesterone treatment. Estrogen t h e r e f o r e induced the s y n t h e s i s of the egg-white p r o t e i n s , ovalbumin and lysozyme, i n q u a i l o v i d u c t . This has been reported to be the case i n chicks as w e l l (45, 48), and the s y n t h e s i s of these p r o t e i n s has been l o c a l i z e d i n the t u b u l a r gland c e l l s (71). In c h i c k s , only estrogen can induce the s y n t h e s i s of these p r o t e i n s during primary s t i m u l a t i o n , whereas progesterone i s a l s o able to induce t h e i r s y n t h e s i s during secondary s t i m u l a -t i o n (53). In t h i s study, estrogen f o l l o w e d by progesterone treatment appears to cause a r e l a t i v e l y g r e a t e r lysozyme syn t h e s i s than continued estrogen treatment. However, due to the problems inherent i n the lysozyme assay, Figure 8. V a r i a t i o n s i n Lysozyme Concentration of Oviduct Magna as a Function of Treatment. Legend as i n Figure 3. Fig.8_:Time vs.Lysozyme Concentration Day of Treatment which would cause a smaller than a c t u a l measurement at the lower end of the s c a l e , t h i s i n t e r p r e t a t i o n of the data presented here would probably not be v a l i d . An e a r l i e r genetic study of avian p r o t e i n s i n d i c a t e d that p r o t e i n s e l e c -t r o p h o r e t i c a l l y s i m i l a r to ovalbumin and lysozyme could be obtained from q u a i l egg-white (85), and a s i m i l a r , more recent study (141) has i n d i c a t e d that estrogen i n j e c t i o n can induce the presence of ovalbumin i n the oviducts of r e s t i n g b i r d s . These r e s u l t s support those s t u d i e s . The r e s u l t s reported here as the e f f e c t s of withdrawal of treatment are at variance w i t h those reported f o r c h i c k o v i d u c t . I t has been shown that withdrawal of hormone r e s u l t s i n r a p i d c e s s a t i o n of s y n t h e s i s of c e l l - s p e c i f i c p r o t e i n s , and that a f t e r 10 days of estrogen treatment and 10 days of w i t h -drawal, the content of ovalbumin and lysozyme i n the c h i c k oviduct has dimin-ished (45, 48). Yu et a l have a l s o demonstrated that during r e g r e s s i o n , hen o v i d u c t magna do not make these p r o t e i n s (52). I t was t h e r e f o r e s u r p r i s i n g to f i n d that of the q u a i l oviduct magna withdrawn from estrogen treatment, a l l contained ovalbumin, and one-half contained lysozyme. Of the magna withdrawn from estrogen p l u s progesterone treatment, one-half contained o v a l -bumin, and none contained lysozyme. This i n f o r m a t i o n i s presented i n Table I I . There are two p o s s i b l e explanations f o r t h i s r e s u l t : e i t h e r some b i r d s were r e g r e s s i n g f a s t e r than others, or some of the b i r d s had begun estrogen s e c r e t i o n n a t u r a l l y . The l a t t e r would normally be the case i n b i r d s of t h i s age, as evidenced both by the stromal changes noted i n c o n t r o l b i r d s , and the r e p o r t of Fertuck and Newstead (66) that tubular gland c e l l s are present at 30 days of l i f e i n untreated q u a i l . I f t h i s was the : case, then as soon a.s the estrogen l e v e l f e l l to a p o i n t low enough to r e l e a s e the hypothalamus from Table I I Summary of Weights, and the Presence or Absence of S p e c i f i c P r o t e i n s i n the Oviduct Magna of B i r d s Withdrawn from Treatment. Magna withdrawn from estrogen treatment Wt. 0.57 0.77 0.67 0.33 0.17 0.26 Oval. + + + + + + Lys. + + + Magna withdrawn from estrogen p l u s proges-terone treatment Wt. 0.25 0.20 0.24 0.21 0.10 0.12 0.10 Oval. + + + Lys. 0.14 i n h i b i t i o n i n the experimental q u a i l , o v a r i a n growth and estrogen s e c r e t i o n should have begun. Although the l e v e l of estrogen produced by the animal would have been extremely low as compared to the amount i n j e c t e d , i t i s a w e l l known f a c t that the secondary s t i m u l a t i o n of an oviduct r e q u i r e s f a r l e s s hormone, and gives a f a s t e r and greater response than the primary s t i m u l a t i o n (53). This suggested small d i f f e r e n c e i n estrogen s e c r e t i o n i n h a l f of the oviducts withdrawn from estrogen treatment would t h e r e f o r e have created a marked d i f f e r e n c e i n both s i z e and t i s s u e components, i n c l u d i n g c e l l - s p e c i f i c p r o t e i n s , as compared to those o v i d u c t s withdrawn from treatment which had not begun estrogen s e c r e t i o n . There i s no way of knowing from these s t u d i e s whether the ovalbumin present i n the oviducts withdrawn from estrogen t r e a t -ment and the lysozyme found only i n the l a r g e r h a l f of these oviducts was newly ma*or simply not yet degraded. However, the f a c t that the b i r d s w i t h l a r g e r oviducts a l s o had o b v i o u s l y l a r g e r o varies i n d i c a t e s that they may have begun estrogen s e c r e t i o n , r a t h e r than there being two d i f f e r e n t r a t e s of r e g r e s s i o n to e x p l a i n t h i s dichotomy. Although the oviduct magna withdrawn from estrogen plus progesterone treatment a l s o f e l l i n t o two groups, as mentioned, i t should be noted that both of these two groups were much smaller i n terms of wet weight, than those withdrawn from estrogen treatment. This r e s u l t i s not s u r p r i s i n g , i f one r e c a l l s that the withdrawal from estrogen plus progesterone treatment r e s u l t e d i n a f a r greater l o s s of a l l components of organ growth than d i d the w i t h -drawal from estrogen treatment. None of the oviduct magna withdrawn from estrogen plus progesterone treatment contained lysozyme, and only the l a r g e r h a l f of them contained ovalbumin. A v i d i n A v i d i n was present only i n those magna which had r e c e i v e d progesterone treatment, not i n the oviduct magna from b i r d s r e c e i v i n g estrogen treatment only, or the t i s s u e from b i r d s withdrawn from treatment. The average concen-t r a t i o n of a v i d i n was 60.9±19.5 yg/gm wet weight. The p o s s i b i l i t y of the exi s t e n c e of an i n h i b i t o r of a v i d i n - b i o t i n b i n d i n g was i n v e s t i g a t e d by adding a known amount of a v i d i n and an excess of b i o t i n to the homogenates of estrogen-treated o v i d u c t s . An enhancement of b i n d i n g (approximately 20% increase) was found under these circumstances, as compared to when the a v i d i n and b i o t i n were incubated i n simple aqueous s o l u t i o n . A v i d i n has long been known to be under the c o n t r o l of estrogen plus some other s t e r o i d i n chicks (32), although the nature of the i n v i v o second agent remains i n question (24). A v i d i n has been demonstrated to be present i n the goblet c e l l s of the magnum e p i t h e l i u m (50) only a f t e r progesterone or a n t i -estrogen a d m i n i s t r a t i o n to estrogen-treated chicks (50). In t h i s r e s p e c t , the r e s u l t s reported here support the data f o r the ch i c k : a v i d i n i s present only i n oviduct magna of b i r d s t r e a t e d w i t h estrogen plus progesterone; even pro-longed estrogen treatment does not induce the sy n t h e s i s of t h i s p r o t e i n i n q u a i l t i s s u e . IV. Summary of Results The r e s u l t s reported here can be summarized as f o l l o w s : Estrogen g r e a t l y enhances the growth and d i f f e r e n t i a t i o n of the q u a i l o v i d u c t . W i t h i n 5 days of treatment, the mucosal f o l d s have grown to f i l l the enlarged lumen, and there are abundant t u b u l a r gland c e l l s i n these. V a s c u l a r i z a t i o n i s evident. By 10 days, t u b u l a r gland c e l l s have g r e a t l y increased i n number, and are engorged w i t h granules, and d i f f e r e n t i a t i o n of e p i t h e l i a l c e l l s i s evident. By 15 days of treatment, the granules of the t u b u l a r gland c e l l s have become so l a r g e and numerous as to obscure the n u c l e i of the c e l l s , and e p i t h e l i a l c e l l d i f f e r e n t i a t i o n i s q u i t e pronounced. Growth, as measured by i n c r e a s e s i n wet weight, RNA, DNA, and p r o t e i n content, has increased g r e a t l y i n t h i s p e r i o d . The egg-white p r o t e i n s of ovalbumin and lysozyme are a l s o induced by t h i s estrogen treatment. The e f f e c t s of progesterone, when added a f t e r 10 days of estrogen t r e a t -ment, are a s m a l l increase i n e p i t h e l i a l c e l l d i f f e r e n t i a t i o n , and a s m a l l r e l a t i v e increase i n a l l t i s s u e components except DNA. The d i f f e r -ence between 15 days of estrogen treatment and 10 days estrogen, 5 days pro-gesterone treatment i s s m a l l ; t h i s i s b e l i e v e d to be due to the prolonged e s t r o g e n i c s t i m u l a t i o n . However, the progesterone treatment d i s p l a y s a d e f i n i t e antagonism f o r the continued DNA s y n t h e s i s seen between 10 and 15 days estrogen treatment. This could be accounted f o r by the a n t i e s t r o g e n i c e f f e c t s of progesterone, since these a f f e c t c e l l p r o l i f e r a t i o n , but not c e l l f u n c t i o n . Ovalbumin and lysozyme were a l s o present during continued proges-terone treatment. In a d d i t i o n , progesterone induced the appearance of the p r o t e i n a v i d i n . Withdrawal of hormone treatment l e d to a l o s s of a l l t i s s u e components. This l o s s was greater a f t e r withdrawal from estrogen plus progesterone t r e a t -ment than a f t e r withdrawal from estrogen treatment, i n d i c a t i n g that hormone i s necessary f o r maintenance of the d i f f e r e n t i a t e d s t a t e , as w e l l as f o r the l i f e of the c e l l s . Oviducts withdrawn from both treatments f e l l i n t o two groups, one group being about twice the weight of the other, and c o n t a i n i n g some of the s p e c i f i c p r o t e i n s . The smaller oviducts i n the group withdrawn from estrogen plus progesterone contained no ovalbumin or lysozyme. I t i s b e l i e v e d that t h i s dichotomy represents the r e s u l t of resumed or i n i t i a t e d estrogen syn t h e s i s by the animals which had l a r g e r o v i d u c t s . I t i s at t h i s time that d i f f e r e n t i a t i o n of the oviduct normally occurs, and although t h i s would be a very low amount of estrogen s e c r e t i o n , i t i s well-documented that the secondary response r e q u i r e s f a r l e s s hormone than the primary response (53) . At a l l times during treatment, the ovaries or t e s t i s of the t r e a t e d b i r d s were notably s m a l l , probably due to a decreased gonadotrophs output. In b i r d s withdrawn from hormone treatment, the gonads were of l a r g e r s i z e i n those b i r d s w i t h l a r g e r o v i d u c t s , thus i n d i c a t i n g again that these may have begun estrogen s e c r e t i o n . An a n a l y s i s of the oviduct system i s o b v i o u s l y complicated by the f a c t that more than one s t e r o i d i s i n v o l v e d i n the d i f f e r e n t i a t i o n of t h i s t i s s u e . From these r e s u l t s , and those reported elsewhere, the e f f e c t s of estrogen can be summed as f o l l o w s : 1) stromal s w e l l i n g and d i s p e r s i o n , v a s c u l a r i z a t i o n , and blood c e l l i n v a s i o n ; 2) d i f f e r e n t i a t i o n of progenitor c e l l s to p r o t o d i f f e r e n t i a t e d c e l l s and t u b u l a r gland c e l l s , w i t h concomitant s y n t h e s i s of ovalbumin and lysozyme; 3) a f t e r prolonged treatment, the d i f f e r e n t i a t i o n of c i l i a t e d c e l l s and goblet c e l l s ; 4) continued presence of estrogen i s necessary f o r support of the d i f f e r e n t i a t e d s t a t e ; withdrawal leads to l o s s of d i f f e r e n t i a t i o n and of c e l l s . Response #1: Stromal changes occur i n i t i a l l y , without the occurrence of increased v a s c u l a r i z a t i o n ; that appears to happen l a t e r , a f t e r the edema and d i s p e r s i o n of the c e l l s . Thus c e l l - c e l l i n t e r a c t i o n which might a r i s e from i n v a s i o n of the stroma by blood c e l l , i s not a f a c t o r i n the e a r l y stromal changes, although i t may be a f a c t o r i n the l a t e r changes and i n d u c t i o n of tu b u l a r gland c e l l s . Response #2: The d i f f e r e n t i a t i o n of the progenitor c e l l s to tubular gland c e l l s occurs a f t e r v a s c u l a r i z a t i o n . Therefore, c e l l - c e l l i n t e r a c t i o n s may be in v o l v e d (41, 45), e i t h e r w i t h c e l l s from the blood, or w i t h the stromal c e l l s themselves. By t h i s time the stromal c e l l s are swollen and disp e r s e d and have migrated out to the periphery. The f a c t that ovalbumin syn t h e s i s appears to be concomitant w i t h and not subsequent to morphological d i f f e r e n -t i a t i o n (43) i n d i c a t e s that the genes f o r both of these are expressed s i m u l -taneously r a t h e r than s e q u e n t i a l l y . This i n d i c a t e s that morphologic d i f f e r -e n t i a t i o n i s not a necessary p r e r e q u i s i t e to biochemical d i f f e r e n t i a t i o n . Response #3: Very l i t t l e i s known about the e p i t h e l i a l d i f f e r e n t i a t i o n to c i l i a t e d c e l l s and goblet c e l l s , except that i t normally does not occur u n t i l a f t e r prolonged estrogen treatment (45, 48), that i s , a f t e r t u b u l a r gland c e l l s have stopped budding o f f . These processes may th e r e f o r e be mutually e x c l u s i v e . However, t h i s i s not i n d i c a t e d by the f a c t t h a t , i f estrogen and progesterone are given together, p r o t o d i f f e r e n t i a t e d c e l l s can d i f f e r e n t i a t e i n t o other e p i t h e l i a l c e l l types c o n t a i n i n g both s e c r e t o r y granules and c i l i a (43). Response #4: Withdrawal leads to l o s s of d i f f e r e n t i a t i o n and of the c e l l s . This may be due to the f a c t that estrogen supports the v a s c u l a r i z a t i o n of the t i s s u e . This would not, however, e x p l a i n the more immediate l o s s of c e l l f u n c t i o n i n g seen on withdrawal of the hormone i n chi c k oviduct (48), nor would i t e x p l a i n the s e q u e n t i a l nature of c e l l death, s i n c e tubular gland c e l l s are l o s t w h i l e e p i t h e l i a l c e l l s s t i l l remain d i f f e r e n t i a t e d . The e f f e c t s of progesterone can be summed as f o l l o w s : 1) Antagonism of estrogen-induced c e l l p r o l i f e r a t i o n . 2) Synergism w i t h estrogen-induced c e l l f u n c t i o n i n g . 3) Enhancement of e p i t h e l i a l c e l l d i f f e r e n t i a t i o n to c i l i a t e d c e l l s and goblet c e l l s . 4) Induction of a v i d i n s y n t h e s i s . Response #1, the antagonism to the e s t r o g e n i c response, occurs at the p r o t o -d i f f e r e n t i a t e d t u b ular gland c e l l stage, where continued d i f f e r e n t i a t i o n of these c e l l s to t ubular gland c e l l s i s a b o l i s h e d , and t h e r e f o r e the growth of the t i s s u e i s i n t e r f e r e d w i t h (43). I t i s evident i n t h i s study as an i n t e r f e r e n c e w i t h the continued DNA synthesis noted between 10 and 15 days of estrogen treatment. Obviously, then, i f progesterone i s given a f t e r the tubular gland c e l l s have d i f f e r e n t i a t e d , or n e a r l y so, t h i s response w i l l not be so evident as when i t i s given at the beginning of treatment. Response #2: The s y n e r g i s t i c response w i t h estrogen-induced c e l l f u n c t i o n i n g (45, 48), noted i n c h i c k o v i d u c t , i s seen i n the q u a i l as a s l i g h t increase i n a l l t i s s u e components except DNA. Both t h i s response and response #4 are measures of the e f f e c t of progesterone on p r o t e i n s y n t h e s i s , but a v i d i n syn-t h e s i s i s d i f f e r e n t i n that i t occurs i n d i f f e r e n t c e l l s from those which c a r r y on the bulk of the p r o t e i n s y n t h e s i s , and i t has an absolute requirement f o r progesterone i n d u c t i o n (50), which the s y n t h e s i s of the other p r o t e i n s does not. The mechanism of progesterone synergism w i t h estrogen-induced s p e c i f i c p r o t e i n s y n t h e s i s i s b e l i e v e d to be v i a an increased message t r a n s c r i p t i o n , f o r some of these p r o t e i n s (97). Response #3: The enhancement of e p i t h e l i a l d i f f e r e n t i a t i o n to c i l i a t e d c e l l s and goblet c e l l s may be r e l a t e d to the a b o l i t i o n of the tu b u l a r gland c e l l d i f f e r e n t i a t i o n , s i n c e these do not normally occur simultaneously. That i s , the simple a b o l i t i o n of the one may be the enhancement of the other. This enhancement of e p i t h e l i a l c e l l d i f f e r e n t i a t i o n can be seen even i n oviducts not t r e a t e d i n i t i a l l y w i t h estrogen (47); but other than t h i s there i s no inf o r m a t i o n a v a i l a b l e on the mechanism of a c t i o n of progesterone i n enhancing e p i t h e l i a l d i f f e r e n t i a t i o n . Response #4: A v i d i n s y n t h e s i s occurs i n the e p i t h e l i a l goblet c e l l s of the ch i c k oviduct (50), and i s known to r e q u i r e progesterone or an a n t i - e s t r o g e n f o r i t s i n d u c t i o n . The amount of a v i d i n s y n t h e s i s i s al s o known to be dependent on the length of estrogen treatment given before progesterone treatment i s begun (49). A l l of these responses lead towards increased growth and f u n c t i o n i n g of the t i s s u e except the f i r s t response to progesterone, the antagonism of estrogen-induced c e l l p r o l i f e r a t i o n . I t may ther e f o r e be wise to use cauti o n i n a t t r i b u t i n g t h i s l a t t e r response to the same mechanism of a c t i o n as the others. DISCUSSION: AN OUTLINE A. D i s c u s s i o n of R e s u l t s of t h i s Experimentation B. The Response as a Phenomenon of Induction 1. The Nature of the C e l l u l a r Response to Estrogen: P a l m i t e r ' s Model a. The Evidence b. D i s c u s s i o n of the Model c. Comparison to Other Steroid-Responsive Systems 2. The Response to Progesterone: O'Malley's Model a. The Evidence b. D i s c u s s i o n of the Model 3. Events a General Model W i l l Have to E x p l a i n C. Comparison of t h i s Induction Phenomenon to Embryonic Indu c t i o n 1. The Inducer 2. The Competence of the Responding C e l l s D. A B r i e f Assessment of the State of the F i e l d DISCUSSION A. The R e s u l t s of t h i s Experimentation The r e s u l t s reported here i n d i c a t e that dramatic growth takes p l a c e , and that gene expression i s r a d i c a l l y a l t e r e d , as a r e s u l t of estrogen treatment. This i s evident both i n the appearance of new c e l l types and of new p r o t e i n s . This gene expression appears to have a temporal or s e q u e n t i a l c h a r a c t e r i s t i c as w e l l , s i n c e the stromal changes occur f i r s t , then t u b u l a r gland c e l l s appear, and e p i t h e l i a l d i f f e r e n t i a t i o n occurs l a s t . The temporal nature of the gene expression i s a l s o i n d i c a t e d by the f a c t that ovalbumin syn t h e s i s i s induced before lysozyme s y n t h e s i s . I t i s a l s o apparent from these r e s u l t s that the inducer does not simply t r i g g e r an already pre-programmed c e l l , which then proceeds on i t s own i n accordance w i t h i t s programming. This i s evident from two observations: 1) the two s t e r o i d s have d i f f e r e n t e f f e c t s , i n d i c a t i n g s p e c i f i c i t y i n the inducer, and 2) withdrawal of hormone leads to a l o s s of gene expression. This l a t t e r i s i n d i c a t e d by l o s s of c e l l s i n oviducts withdrawn from treatment, as w e l l as l o s s of ovalbumin and lysozyme i n some of these o v i d u c t s . In a d d i t i o n , although the e p i t h e l i u m remains c i l i a t e d , i t c l e a r l y changes from i t s appearance at the time of treatment. This gene expression i s t h e r e f o r e dependent on the continued presence of the inducer. Such a very high amount of sy n t h e s i s of c e l l - s p e c i f i c p r o t e i n s (as judged by h i s t o l o g y as w e l l as biochemistry) over so sustained a length of time, would imply that the inducer a c t i v a t e s or enhances the a c t i v i t y of the genes i n v o l v e d i n t h i s s y n t h e s i s . Other p o s s i b i l i t i e s , such as masked messenger ( t r a n s l a t i o n c o n t r o l ) , or a c t i v a t i o n of a p r e - p r o t e i n molecule, seem i m p l a u s i b l e when the magnitude of the response i s considered. I t t h e r e f o r e seems probable that the new gene expression seen here as a r e s u l t of estrogen treatment i s the r e s u l t of gene a c t i v a t i o n , or greatly-enhanced t r a n s c r i p t i o n of genes already a c t i v e . These r e s u l t s would t h e r e f o r e support any model f o r the mechanism of estrogen a c t i o n which inc l u d e s these p o s s i b i l i t i e s . Such a model would be that of P a l m i t e r and co-workers, whose o r i g i n a l idea i t was to examine the i n d u c t i o n of one p r o t e i n (ovalbumin) by one s t e r o i d (estrogen), and to t e s t the p o s s i b i l i t y of c o n t r o l at each of the steps i n the i n d u c t i o n process (89). That i s , i n v e s t i g a t i o n s were c a r r i e d out to determine i f c o n t r o l was exerted at the l e v e l of message t r a n s c r i p t i o n , message proc e s s i n g , t r a n s p o r t to cytoplasm, t r a n s l a t i o n of message or message degradation. The r e s u l t s of P a l m i t e r ' s i n v e s t i g a t i o n s supported a model i n which estrogen acts v i a gene a c t i v a t i o n r a t h e r than by way of a masked messenger or the a c t i v a t i o n of a p r e - p r o t e i n . The r e s u l t s reported i n t h i s t h e s i s are i n keeping w i t h that model. The response that has been reported here to progesterone when i t i s given a f t e r estrogen treatment i s t h r e e - f o l d i n nature: the i n d u c t i o n of a v i d i n s y n t h e s i s , the support of continued growth and f u n c t i o n i n g , and the i n h i b i t i o n of continued DNA s y n t h e s i s . Thus i t i s c l e a r that the response to progesterone encompasses not only unique a c t i o n , but a c t i o n s y n e r g i s t i c to estrogen a c t i o n , and a c t i o n opposed to estrogen a c t i o n . Whether or not a l l three of these types of a c t i o n s are mediated by the same mechanism i s not known. Taken s e p a r a t e l y , each a c t i o n i s of s m a l l enough magnitude that i t could be explained i n a number of ways. The induc-t i o n of a v i d i n s y n t h e s i s , f o r example, could be explained by new gene a c t i v i t y , by a masked messenger hypothesis, or by an a c t i v a t i o n of a pre-p r o t e i n . Work on the chick oviduct which uses a v i d i n i n d u c t i o n as a marker f o r progesterone a c t i o n has l e d to a model of progesterone a c t i o n i n v o l v i n g new gene t r a n s c r i p t i o n (90). These r e s u l t s are compatible w i t h that model, but can n e i t h e r support nor c o n t r a d i c t i t . The a c t i o n s of progesterone that are s y n e r g i s t i c w i t h estrogen a c t i o n (that i s , the enhancement of tu b u l a r gland c e l l f u n c t i o n i n g ) could be e x p l a i n -ed by enhanced t r a n s c r i p t i o n of es t r o g e n - a c t i v a t e d genes, or enhanced proces-s i n g of messenger from these genes, or enhanced t r a n s l a t i o n of messenger, or decreased degradation of messenger or of p r o t e i n . Work on the c h i c k oviduct which i n v e s t i g a t e s the a c t i o n of progesterone i n inducing the synth e s i s of the tubular gland c e l l p r o t e i n s i n d i c a t e s that progesterone, when given alone, on 2° s t i m u l a t i o n , i s able to i n i t i a t e new gene a c t i v a t i o n (71), and when i t i s given w i t h estrogen, to enhance t r a n s c r i p t i o n of some of the messenger RNAs in v o l v e d (97). The increased s y n t h e s i s of p r o t e i n s and a l l t i s s u e components except DNA reported here support that hypothesis. However, pro-gesterone was not given together w i t h estrogen, but f o l l o w i n g estrogen. Thus i t i s not p o s s i b l e to i n d i c a t e whether the response to progesterone i s one of i n i t i a t i n g gene a c t i v i t y , or simply continuance of es t r o g e n - a c t i v a t e d t r a n s -c r i p t i o n . Since estrogen presence i s necessary f o r continued t r a n s c r i p t i o n , i t seems l i k e l y that both of these p o s s i b i l i t i e s are o c c u r r i n g . The a c t i o n of progesterone that i s a n t i - e s t r o g e n i c i n nature i s the i n h i b i t i o n of DNA s y n t h e s i s . In the ch i c k o v i d u c t , t h i s i s the cause of the a b o l i t i o n of tubular gland c e l l d i f f e r e n t i a t i o n and the growth response. There has been no e l u c i d a t i o n of the mechanism of t h i s a c t i o n . I t has been so proposed that estrogen s t i m u l a t e s c e l l p r o l i f e r a t i o n i n a d i f f e r e n t manner than c e l l f u n c t i o n i n g , such that progesterone can i n t e r f e r e w i t h the f i r s t process, but not the second. The r e s u l t s reported here are compatible w i t h such a .hypothesis, but can n e i t h e r support nor c o n t r a d i c t i t . There are a number of p o s s i b l e mechanisms by which t h i s a c t i o n could be mediated. P r o -gesterone may i n t e r f e r e w i t h one or many es t r o g e n - a c t i v a t e d genes, which code f o r a p r o t e i n or p r o t e i n s e s s e n t i a l f o r t h i s process; a l t e r n a t i v e l y , terone or a progesterone-induced gene product may i n t e r f e r e w i t h the f u n c t i o n -in g of such a p r o t e i n . That i s , t h i s may be the r e s u l t of a nuclear or a cytoplasmic a c t i o n of progesterone, of the progesterone—receptor complex, or of a product induced by i t . B. The Response as a Phenomenon of Induc t i o n The changes that are reported i n t h i s t h e s i s c l o s e l y p a r a l l e l those known f o r the ch i c k o v i d u c t , and i t i s suggested that so great a response as observ-ed i n both b i r d s must encompass the expression of many d i f f e r e n t genes. Most of the work on estrogen and progesterone-induced d i f f e r e n t i a t i o n of oviduct t i s s u e has been d i r e c t e d toward an explanation of t h i s gene expression. I t i s t h e r e f o r e r e l e v a n t to examine at t h i s p o i n t the i n d u c t i o n of oviduct d i f f e r e n t i a t i o n , to consider the work done on s i m i l a r systems, and to c r i t i -c a l l y examine the models of s t e r o i d a c t i o n that have been proposed to e x p l a i n t h i s e f f e c t . The r e s u l t s reported here support or are compatible w i t h both the model f o r estrogen a c t i o n , and the model f o r progesterone a c t i o n i n ch i c k o v i d u c t . The f i r s t , based on P a l m i t e r ' s work, hypothesizes that estrogen a c t s a t the l e v e l of t r a n s c r i p t i o n , to independently induce a c t i v a t i o n of many genes (89). The model f o r progesterone a c t i o n , based on O'Malley's work, i n d i c a t e s that progesterone binds to i t s r e c e p t o r , enters the nucleus, and there binds to a c i d i c chromatin p r o t e i n s , so a c t i v a t i n g new t r a n s c r i p t i o n (90, 91), Both models or hypotheses th e r e f o r e have at base an a c t i v a t i o n of new t r a n s c r i p t i o n , although they are based on e n t i r e l y d i f f e r e n t data. Both P a l m i t e r ' s hypothesis f o r estrogen a c t i o n and O'Malley's f o r proges-terone a c t i o n on c h i c k oviduct are extensions of the general model f o r s t e r o i d a c t i o n (86-88). This general model i s : that the s t e r o i d enters the c e l l , binds to a cytoplasmic receptor p r o t e i n , enters the nucleus, and there acts i n some as yet unknown way to a l t e r t r a n s c r i p t i o n . Such receptor p r o t e i n s have been demonstrated f o r estrogen, progesterone and t e s t o s t e r o n e i n c h i c k oviduct (139, 74, 75, 129-132), and f o r estrogen i n q u a i l oviduct (140). This mechanism of a c t i o n i s g e n e r a l l y thought to be a p p l i c a b l e to a l l cases of s t e r o i d i n d u c t i o n i n a l l organisms (86-88). The two s p e c i f i c cases mentioned here, of estrogen and progesterone i n c h i c k o v i d u c t , are of r e l e -vance both because they represent l e a d i n g work i n the f i e l d , and because they r e l a t e d i r e c t l y to the problem presented here - the e x p l a n a t i o n of c e l l u l a r response to a s t e r o i d inducer i n the avian o v i d u c t . These models w i l l t h e r e f o r e be examined i n terms of the evidence on which each i s based, the c o n t r o v e r s i a l aspects of each, and how each compares to models f o r s t e r o i d a c t i o n i n mammalian systems. The c h a r a c t e r i s t i c s of t h i s i n d u c t i o n phenomenon w i l l a l s o be compared to those of embryologic i n d u c t i o n phenomenon, i n an e f f o r t to understand the common denominators of these processes. 1. The Nature of the C e l l u l a r Response to Estrogen: P a l m i t e r ' s Model When d i s c u s s i n g the response of c e l l s to the inducers, care must be taken to d i s t i n g u i s h between the c e l l types and between the d i f f e r e n t stages of treatment as w e l l . Two major c l a s s i f i c a t i o n s e x i s t : the primary or i n i t i a l response, and the secondary response, which i s the response of a t i s s u e that has been p r e v i o u s l y t r e a t e d , withdrawn from hormone treatment, and then s t i m u l a t e d a second time. The primary response of the c h i c k oviduct to e s t r o -gen has been o u t l i n e d i n the i n t r o d u c t i o n , and o u t l i n e d i n d e t a i l i n the r e s u l t s s e c t i o n of t h i s t h e s i s . The major work on the a c t i o n of estrogen on the oviduct has made use of the secondary response, since that i s somewhat more s p e c i f i c , because i t omits a l a r g e p a r t of the growth response, and involves p r i m a r i l y a r e t u r n to f u n c t i o n i n g of c e l l s already present. The knowledge gained has been the work of P a l m i t e r and co-workers. I t was the o r i g i n a l i d e a of t h i s group to examine the i n d u c t i o n of one p r o t e i n (ovalbumin) by one s t e r o i d (estrogen), and to t e s t the p o s s i b i l i t y of c o n t r o l at each of the steps i n the i n d u c t i o n process (89). That i s , i n v e s t i g a t i o n s were c a r r i e d out to determine i f c o n t r o l was exerted at the l e v e l of message t r a n s c r i p t i o n , message processing, t r a n s p o r t of message to cytoplasm-;' t r a n s -l a t i o n of message, and message degradation. This work w i l l be examined f i r s t i n terms of the evidence that has been reported, second i n terms of the model that the author presents to account f o r the evidence, and then i n terms of a comparison of t h i s model and evidence to other s t e r o i d - r e s p o n s i v e systems. a. The Evidence The r e s u l t s obtained by P a l m i t e r and others concerning the e f f e c t of estrogen and other s t e r o i d s on oviducts withdrawn from hormone are summarized i n the diagram on the f o l l o w i n g page. These r e s u l t s i n c l u d e the f o l l o w i n g : The secondary response i s f a s t e r than the primary one; f o l l o w i n g a l a g p e r i o d of 3 hours, ovalbumin i s detected and begins i n c r e a s i n g i n amount (53). The beginning of t h i s response, Phase I , i s represented by an o r g a n i z a t i o n of polysomes from e x i s t i n g monosomes, r a t h e r than as a r e s u l t of new rRNA synthe-s i s . This phase occurs i n the f i r s t 12 hours,: and can be e l i c i t e d by e i t h e r estrogen or progesterone, or both together (53). The second phase (Phase I I ) i s represented by continued increases i n a l l c o n s t i t u e n t s , w i t h rRNA synt h e s i s c o n t r i b u t i n g new ribosomes to the pool of o l d and new ribosomes from which polysomes are drawn (53). Phase I I can be e l i c i t e d by estrogen o n l y , not by progesterone, but progesterone w i t h estrogen does not i n h i b i t the response, r a t h e r , they act s y n e r g i s t i c a l l y (53). Ovalbumin i s apparently synthesized and r e l e a s e d at the same r a t e as other p r o t e i n s , but i t s s y n t h e s i s cannot be i n i t i a t e d i n v i t r o (53). Oval-bumin message was i s o l a t e d from ovalbumin-synthesizing polysomes by immuno-adsor p t i o n to anti-ovalbumin a n t i b o d i e s , and was incubated w i t h l a b e l l e d precursors and v i r a l reverse t r a n s c r i p t a s e , to make a l a b e l l e d gene f o r ovalbumin (92, 93). Although c o n s i s t i n g of only 200 n u c l e o t i d e s , i t was nevertheless p o s s i b l e to use t h i s "probe" to h y b r i d i z e to c e l l u l a r DNA, and demonstrate that i n the i n d u c t i o n of ovalbumin, gene a m p l i f i c a t i o n does not occur (93). Ovalbumin message was l a t e r i s o l a t e d from t o t a l average polysomal RNA (94), and reverse t r a n s c r i p t a s e from a v i a n m y e l o b l a s t o s i s v i r u s was used i n the same manner (95). The product of that r e a c t i o n was the same sm a l l s i z e , but was used to demonstrate, by h y b r i d i z a t i o n , that 93% of h y b r i d i z a b l e Figure 9. Proposed C o n t r o l of Ovalbumin Synthesis. 7) 8) 9) 10) 11) 12) Tubular Gland Cell Cytoplasm a n d C i ) 2) 3) 4) occur. One copy per Gene a m p l i f i c a t i o n does not TsllH E l a t i o n at a r a t a of 28 molecules/min/ - 1 ^ " 1 1 , a f t e r a 3-hour l a g by de novo 5) 6) ^ ^ ^ i i ^ ^ ^ of ge„es already a c t i v e . Other messages synthesized at d i f f e r e n t r a t e s . I f other s t e r o i d s are present, t h e i r s y n e r g i s t i c e f f e c t i s mediated v i a increased messenger accumula-t i o n , not decreased degradation, by m u l t i p l e c o n t r o l of a s i n g l e r a t e - l i m i t i n g s t e p , or by a d d i t i o n a l r a t e - l i m i t i n g steps. Processing i s extremely r a p i d , or absent; no s t a b l e pre-message e x i s t s ; no s t a b l e pre-message i s stored i n withdrawal. Transport to the cytoplasm i s not a r a t e - l i m i t i n g s t e p ; there i s no nuclear p o o l of message, so r e -a c t i v a t i o n of a tr a n s p o r t to cytoplasm i s not o c c u r r i n g . I n i t i a t i o n of p r o t e i n s y n t h e s i s i s not r a t e - l i m i t i n g ; there i s no cytoplasmic pool of t r a n s l a t a b l e o «A . f\ * 1 r~S 4~ l-» I s*. n o n - t r a n s l a t a b l e message. T r a n s l a t i o n i s enhanced by inc r e a s e d r a t e s of i n i t i a t i o n and elo n g a t i o n of polypeptide chains; t h i s i s not s p e c i f i c f o r ovalbumin. There i s no evidence of a c y t o p l a s m i c r e p r e s s o r . 93% of the message i s i n the polysomes. The s y n e r g i s t i c e f f e c t s of progesterone or testosterone w i t h estrogen are not exerted a t t r a n s l a t i o n . Estrogen may s t a b i l i z e the message, s i n c e i t i s degraded upon withdrawal; n u c l e i from t i s s u e withdrawn from treatment c o n t a i n one copy per nucleus. oo E = Estrogen; R, = Cytoplasmic r e c e p t o r . m a t e r i a l was i n the polysomes, 5% i n the nucleus, and 2% i n the non-polysomal cytoplasm. This i n d i c a t e d that there was no pool of n o n - t r a n s l a t e d messenger i n the cytoplasm, e i t h e r i n t r a n s l a t a b l e or n o n - t r a n s l a t a b l e form, thus supporting e a r l i e r s t u d i e s which had assayed messenger by a c t i v i t y i n a c e l l -f r e e system (96) (see a l s o r e f . 137, 122, 138). Two percent of the nuclear h y b r i d i z a t i o n was a high molecular weight species w i t h a sedimentation c o e f f i c i e n t of 30S (the message i s 16S-18S) (95). I f t h i s very small amount i n d i c a t e s a primary t r a n s c r i p t i o n product, then r a p i d conversion to a t r a n s l a t a b l e form i s i n d i c a t e d ; not v i a a s t a b l e pre-message (95). The appearance of message i n the polysomes without accumulation i n the nucleus f i r s t , i n d i c a t e s that estrogen i s not r e a c t i v a t i n g a t r a n s p o r t to the cytoplasm (95). The c l o s e c o r r e l a t i o n of the k i n e t i c s of ovalbumin s y n t h e s i s and the appearance of h y b r i d i z a b l e and t r a n s l a t a b l e messenger, together w i t h the l a c k of a s i g n i f i c a n t pool of messenger i n the non-polysomal cytoplasm, i n d i c a t e that the i n i t i a t i o n of p r o t e i n synthesis i s not a l i m i t i n g f a c t o r (95). The estimated l e v e l of message i n the c e l l s during periods of hormone withdrawal was one molecule per nucleus, so that i t was not p o s s i b l e to demon-s t r a t e c o n c l u s i v e l y whether or not s y n t h e s i s occurred during periods of hormone withdrawal; that i s , whether r e - a d m i n i s t r a t i o n caused enhancement of e x i s t i n g t r a n s c r i p t i o n , or de novo t r a n s c r i p t i o n (95), The l o s s of messenger a c t i v i t y on withdrawal of hormone i n d i c a t e d that continued estrogen treatment was necessary f o r continued presence of messenger; the authors considered i t p o s s i b l e that the message was unstable i n the absence of estrogen (96). Other p r o t e i n s - ovalbumin, conalbumin, ovomucoid, and lysozyme - are a l l made i n the same t u b u l a r gland c e l l s , f o l l o w i n g the secondary a d m i n i s t r a t i o n of estrogen (71). Progesterone can a l s o induce a l l four p r o t e i n s , although i n lower amounts, and w i t h d i f f e r e n t time courses (71). Progesterone causes p r e f e r e n t i a l synthesis of conalbumin r e l a t i v e to ovalbumin, and the ovalbumin synthesis begins more r a p i d l y , but reaches a steady-state at a lower l e v e l (71). Conalbumin i s p r e f e r e n t i a l l y synthesized at lower doses' of estrogen, and conalbumin and ovomucoid are both s e l e c t i v e l y enhanced by progesterone and estrogen together, and by t e s t o s t e r o n e and estrogen together, r e l a t i v e to estrogen alone. This occurs i n s p i t e of the f a c t that t e s t o s t e r o n e i s i n a c t i v e alone (71). This s e l e c t i v e increase i s not due to messenger s t a b i l i -z a t i o n r e l a t i v e to estrogen treatment only, as judged by decay i n the presence of actinomycin-D (71). The increase i n p r o t e i n synthesis on secondary s t i m u l a t i o n by estrogen was p a r t i a l l y the r e s u l t of an i n c re a s e i n the r a t e of e l o n g a t i o n of p o l y -peptide chains i n t i s s u e from t r e a t e d animals, as compared to that i n animals withdrawn from hormone, and p a r t i a l l y the r e s u l t of an i n c r e a s e i n the r a t e of i n i t i a t i o n of p r o t e i n synthesis (97). I n i t i a t i o n i s r a t e - l i m i t i n g i n t i s s u e that has been withdrawn from hormone, whereas e l o n g a t i o n i s r a t e - l i m i t i n g i n the t r e a t e d t i s s u e . The s y n e r g i s t i c e f f e c t s of progesterone w i t h estrogen, and of t e s t o s t e r -one w i t h estrogen, r e l a t i v e to estrogen treatment alone, on ovomucoid and conalbumin s y n t h e s i s , are not due to an increase i n r a t e s of i n i t i a t i o n or e l o n g a t i o n (97). They are t h e r e f o r e b e l i e v e d to r e f l e c t an i n c r e a s e i n the concentration of t r a n s l a t a b l e messenger f o r these p r o t e i n s (97). The " s u p e r i n d u c t i v e " e f f e c t of actinomycin-D was demonstrated to be due to enhanced t r a n s l a t i o n , not to increased messenger, which r u l e d out the p o s t u l a t e d cytoplasmic repressor of Tomkins (99), at l e a s t i n t h i s system (99, 96, 98). Enhanced t r a n s l a t i o n was t e n t a t i v e l y a t t r i b u t e d to the r e l a t i v e -l y long h a l f - l i f e of the message, which would t h e r e f o r e be i n c r e a s i n g l y able to compete f o r r a t e - l i m i t i n g f a c t o r s , as other messengers died out (98). The s y n e r g i s t i c e f f e c t of testosterone and estrogen together r e l a t i v e to estrogen treatment alone was shown to extend to rRNA s y n t h e s i s , and to an increase i n RNA polymerase a c t i v i t y , which was considered to be the r e s u l t of more growing chains, r a t h e r than increased elongation r a t e (100). This supports the c o n c l u s i o n that t e s t o s t e r o n e and estrogen together cause an increase i n messenger con c e n t r a t i o n r e l a t i v e to estrogen alone, and i n d i c a t e s that testosterone s e l e c t i v e l y enhances some est r o g e n - a c t i v a t e d genes (100), and t h e r e f o r e does not act e n t i r e l y by the same mechanism that estrogen or proges-terone do, s i n c e these l a t t e r s t e r o i d s a l s o i n i t i a t e gene a c t i v i t y . The i n i t i a l r a t e of ovalbumin message accumulation, and the steady-state r a t e of accumulation, as measured by e s t i m a t i o n of i t s s y n t h e s i s and h a l f - l i f e , were c a l c u l a t e d to be 22 and 34 molecules per minute per t u b u l a r gland c e l l , which P a l m i t e r b e l i e v e s to be c l o s e enough to represent a s i n g l e value of 28 molecules per minute per c e l l , i m p l y i n g a simple, s i n g l e - s t e p model of con-s t a n t r a t e increase (77). He a l s o notes, though, t h a t the time l a g of 3 hours between s t e r o i d a d m i n i s t r a t i o n and message accumulation speaks against a simple one-step model (77). From comparison w i t h other systems, i t was c a l c u -l a t e d that a s i n g l e gene can support t h i s r a t e of t r a n s c r i p t i o n even i f only 30-50% of the products of t r a n s c r i p t i o n reach the cytoplasm (77). The data i n d i c a t e c o n t r o l at the l e v e l of s y n t h e s i s or a c t i v a t i o n of messenger, and suggest vast d i f f e r e n c e s i n the r a t e s of s y n t h e s i s or a c t i v a t i o n of d i f f e r e n t messengers (77). The ovalbumin messenger appears to be heterogeneous, by i t s p a r t i a l b i n d i n g to M i l l i p o r e f i l t e r s (92, 94), and by the broad band range produced on acrylamide g e l e l e c t r o p h o r e s i s , covering a range of molecular weight of 100,000 (94). The p r e p a r a t i o n from which t h i s ovalbumin message i s i s o l a t e d i s t o t a l average polysomal RNA, p u r i f i e d by sucrose sedimentation and g e l e l e c t r o p h o r e s i s , r a t h e r than by immunoadsorption. The immunoprecipitation of the product of t r a n s l a t i o n i n a c e l l - f r e e system does not a l l o w a demonstra-t i o n that the p r e p a r a t i o n i s pure, although c a l c u l a t i o n s based on the percent-age of messengers that are excluded by the procedure, and the percentage of messenger that i s ovalbumin messenger, i n d i c a t e that the p r e p a r a t i o n must be h i g h l y s e l e c t e d f o r ovalbumin message (94). The molecular weight of the message i s 875,000, as judged by e l e c t r o p h o r e s i s , and 550,000 as judged by sucrose sedimentation (94). This corresponds to 2640 and 1670 n u c l e o t i d e s r e s p e c t i v e l y , of which 1161 w i l l be t r a n s l a t e d to ovalbumin (94). At l e a s t part of the l a r g e u n t r a n s l a t e d p o r t i o n i s represented by polyA; of the remainder, i t i s thought that t h i s i s t r a n s c r i b e d w i t h the message (95). I t has most r e c e n t l y been shown that the message does not undergo any s i g n i f i c a n t change i n molecular weight a f t e r i t s i n i t i a l t r a n s c r i p t i o n , i n d i c a t i n g that processing i s i n s i g n i f i c a n t or absent (95, 163). This p o i n t s more d i r e c t l y to the process of t r a n s c r i p t i o n as the c o n t r o l p o i n t i n the i n d u c t i o n of t h i s p r o t e i n . P a l m i t e r f e e l s that the i n d u c t i o n of the four major p r o t e i n s mentioned i s not coordinated by a s i n g l e r e g u l a t o r y f a c t o r (71) because: 1) conalbumin sy n t h e s i s does not d i m i n i s h f a s t e r than ovalbumin s y n t h e s i s , which would be expected from the s h o r t e r h a l f - l i f e of i t s messenger (although he notes that t h i s may be due to the gradual drop i n estrogen to the l e v e l where i t p r e f e r e n t i a l l y induces conalbumin s y n t h e s i s ; 2) d i f f e r e n t s t e r o i d s have s e l e c t i v e e f f e c t s on the i n d u c t i o n of the p r o t e i n s , which are not at the l e v e l of message degradation or t r a n s l a t i o n ; 3) there are d i f f e r e n c e s i n estrogen th r e s h o l d l e v e l r e q u i r e d f o r conalbumin i n d u c t i o n r e l a t i v e to ovalbumin i n d u c t i o n ; and 4) other s t e r o i d s e l i c i t only p a r t of the t o t a l response; that i s , they do not e l i c i t rRNA s y n t h e s i s , DNA s y n t h e s i s , e t c e t e r a (71). He t h e r e f o r e does not f e e l that the response to other s t e r o i d s can be both coordinated and s e l e c t i v e . Although he thi n k s i t l i k e l y that each p r o t e i n i s independently regulated (71, 101), no mention i s made of the p o s s i b i l i t y of a few intermediates, or one f o r each s t e r o i d . Although the s i n g l e r a t e of accumulation of message speaks f o r a simple, one-step i n d u c t i o n of ovalbumin message, P a l m i t e r f e e l s that the 3-hour time l a g i n v o l v e d would a l l o w f o r any event i n a c y c l e of t r a n s c r i p t i o n - t r a n s l a t i o n -feedback events, and so speaks against a simple one-step model (77). b. D i s c u s s i o n of the Model A model of coordinated response due to a s i n g l e intermediate f a c t o r i s not favoured by P a l m i t e r , due to the p r e f e r e n t i a l s y n t h e s i s of conalbumin at low doses, i t s longer-thari-expected r a t e of degradation, and the s e l e c t i v e e f f e c t s of other s t e r o i d s , i n c l u d i n g the l a c k of e f f e c t of these on p e r i p h e r a l events such as rRNA s y n t h e s i s , DNA s y n t h e s i s , e t c e t e r a (71). P a l m i t e r notes that the p r e f e r e n t i a l s y n t h e s i s of conalbumin at low doses, and i t s longer-than-expected r a t e of decay on withdrawal of hormone, may be r e l a t e d i n that the s l o w l y f a l l i n g l e v e l s of estrogen at withdrawal may p r e f e r e n t i a l l y enhance conalbumin s y n t h e s i s (71). He suggests a study i n the presence of an a n t i -estrogen (71), but to my knowledge, no f u r t h e r s t u d i e s along that l i n e have been conducted. I t i s of i n t e r e s t here, that conalbumin has a l s o been noted i n the blood of immature, non-treated b i r d s (52, 38, 102), and i s t h e r e f o r e not thought to be under s t r i c t e s t r o g e n i c c o n t r o l (102). The three-hour time l a g speaks against a simple one-step model of e s t r o -gen a c t i o n (77), but cannot i n d i c a t e i n any way whether the responses are coordinated or independent. I t would only allow time f o r intermediate a c t i o n s ; t h i s could be i n keeping w i t h both p o s s i b i l i t i e s . Such intermediate a c t i o n s could i n c l u d e any imaginable sequence of events, i n c l u d i n g changes i n n u c l e a r i o n c o n c e n t r a t i o n s , which are thought to be a c o n t r o l l i n g element i n ecdysone-induced p u f f i n g patterns i n Chironomous s a l i v a r y gland chromosomes C103, 104). Such a p o s s i b i l i t y should be viewed i n the l i g h t of the known e f f e c t s of i o n concentrations on s t e r o i d receptors (105). The s e l e c t i v e e f f e c t s of other s t e r o i d s , i n c l u d i n g the l a c k of e f f e c t on p e r i p h e r a l events, may speak f o r independent c o n t r o l , but these e f f e c t s are nevertheless not at v a r i a n c e w i t h a model of coordinated c o n t r o l , such as the Britten-Davidson model of gene a c t i v i t y i n e a r l y development (106). In that case, the a c t i o n of other s t e r o i d s alone or i n combination w i t h estrogen, could be s e l e c t i v e , but a l s o coordinated. I f the B r i t t e n - D a v i d s o n model were to be a p p l i e d to s t e r o i d i n d u c t i o n , then one would expect, using the Redundant Receptor Model, that a l l genes a c t i v a t e d by estrogen have a receptor r e g i o n to bind the a c t i v a t o r RNA; that i s , they would a l l have a common sequence as s o c i a t e d w i t h them. I f the M u l t i p l e I n t e g r a t o r Model i s used i n s t e a d , t h i s would not be the case; there would be d i f f e r e n t a c t i v a t o r RNAs f o r d i f f e r e n t genes, or groups of genes. Thus i t i s p o s s i b l e that a s i n g l e e n t i t y , or a few such e n t i t i e s , could mediate the p l e i o t r o p i c responses, and s t i l l a l l o w f o r the s e l e c t i v e e f f e c t s of other s t e r o i d s . This model does not account f o r the p r e f e r e n t i a l s y n t h e s i s of conalbumin at lower doses of estrogen. I t does r e q u i r e t h a t each s t e r o i d operate by a d i f f e r e n t intermediate. A model r e q u i r i n g the independent i n d u c t i o n of the c e l l - s p e c i f i c p r o t e i n s would account f o r the a v a i l a b l e data regarding t h e i r i n d u c t i o n at d i f f e r e n t doses and d i f f e r e n t s t e r o i d combinations, but such a model i s i n t e l l e c t u a l l y d i s p l e a s i n g when extended to the o b v i o u s l y coordinated responses of the primary s t i m u l a t i o n , or even to the complete secondary response. I t may be that more than one model should be considered; that i s , i t may be that there i s more than one mechanism of a c t i o n i n v o l v e d . Whether these responses are independently or c o o r d i n a t e l y c o n t r o l l e d bears d i r e c t l y on the nature of the model. P a l m i t e r has pointed out (77) that i f there i s a s i n g l e r a t e - l i m i t i n g c o n t r o l step i n the i n d u c t i o n of each of the four p r o t e i n s , then i t must be subject to m u l t i p l e r e g u l a t i o n , o t h e r -wise one must p o s t u l a t e a d d i t i o n a l r a t e - l i m i t i n g steps to account f o r the various e f f e c t s of d i f f e r e n t s t e r o i d s . He f e e l s , however, that the genes f o r each of these p r o t e i n s may be independently c o n t r o l l e d by the s t e r o i d ( s ) (71, 101). I f t h i s model were extended to the primary response, one would imagine that a model of coordinated c o n t r o l would r e q u i r e measurably l e s s estrogen bound i n the nucleus than a model r e q u i r i n g that each gene be i n d i v i d u a l l y a c t i v a t e d by estrogen. Such an i n v e s t i g a t i o n , however, would be hampered by the assumption that there i s no i n t e r m e d i a t e ( s ) , and by the d i f f i c u l t i e s i n v o l v e d i n e s t i m a t i n g nuclear b i n d i n g . The i m p l i e d assumption to t h i s p o i n t i n the d i s c u s s i o n has been that the process being c o n t r o l l e d i s t r a n s c r i p t i o n . Recent work demonstrating the absence of any precursor f o r ovalbumin message supports t h i s assumption (163). Again, i n t e l l e c t u a l l y , a model of estrogen a c t i o n r e l y i n g on the indepen-dent a c t i v a t i o n of each gene by estrogen or i t s intermediate i s d i s p l e a s i n g , since i t does not e x p l a i n the c o o r d i n a t i o n evident i n the primary response, when d i f f e r e n t i a t i o n , d i v i s i o n , and s p e c i f i c p r o t e i n s y n t h e s i s are a l l induced. One would expect these responses to occur i n an o r d e r l y sequence i n time, but such a sequence has not been e s t a b l i s h e d i n d e t a i l , due p o s s i b l y to the high doses of estrogen used, which preclude a r e s o l u t i o n of the f i n e r aspects of the r e s p o i i S G . The model of independent gene r e g u l a t i o n , however, has only been suggested f o r the r e g u l a t i o n of the egg-white p r o t e i n s . I f a general model f o r s t e r o i d a c t i o n i s assumed, then i t would have to e x p l a i n not only the primary and secondary a c t i o n s of estrogen, but those of progesterone as w e l l , which i n c l u d e the d i s t i n c t a n t i - e s t r o g e n i c e f f e c t s of progesterone when i t i s given concomitantly w i t h estrogen i n the primary s t i m u l a t i o n . c. Comparison to Other Steroid-Responsive Systems Because t h i s secondary response of oviduct t i s s u e i s not d i r e c t l y compar-able to e i t h e r the f u l l growth and d i f f e r e n t i a t i o n response of the oviduct or of mammalian t i s s u e s , nor to the simple response of a l t e r a t i o n of a c e l l u l a r metabolic p a t t e r n seen w i t h the c o r t i c o i d s , i t i s d i f f i c u l t to know what system to use f o r a comparison to the three-hour time l a g . On primary s t i m u l a t i o n , the time l a g i s 24 hours f o r synthesis of ovalbumin; on secondary s t i m u l a t i o n , t h i s occurs a f t e r only 3 hours, although rRNA s y n t h e s i s does not occur u n t i l 12 hours a f t e r s t i m u l a t i o n . The synt h e s i s of s p e c i f i c p r o t e i n before rRNA s y n t h e s i s (53) has been reported i n other systems as w e l l (107). I t should a l s o be noted that t h i s avian system i s u s e f u l f o r the same reason that i t i s anomalous: f o r the presence of biochemical markers f o r s t e r o i d a c t i o n . Mammalian t i s s u e s which grow and d i f f e r e n t i a t e i n response to estrogen and progesterone do not make luxury p r o t e i n s f o r export. At l e a s t one s p e c i f i c p r o t e i n , a peroxidase, has been reported to be induced by e s t r o -gen i n mammalian systems; however, t h i s i s not a t i s s u e - s p e c i f i c p r o t e i n (108). These two major d i f f e r e n c e s , that ovalbumin i s a luxury p r o t e i n made f o r export, and that t h i s secondary response i s n e i t h e r one of f u l l growth and d i f f e r e n t i a t i o n nor a simple metabolic response, make t h i s phenomenon hard to c l a s s i f y , and d i f f i c u l t to compare. The very e a r l y responses to s t e r o i d s measured i n mammalian responsive t i s s u e s are d e f i n i t e l y not l e a d i n g to the synthe s i s of luxury p r o t e i n s , and are l i k e l y to be f u n c t i o n a l i n the cont i n u -a t i o n of the response. This d i f f e r e n c e i n b i o l o g i c a l f u n c t i o n i s great enough to warrant c a u t i o n i n assuming a d i r e c t comparison between the two types of systems. P a l m i t e r s t a t e s that the time l a g allows f o r any i n a c y c l e of t r a n s c r i p -t i o n - t r a n s l a t i o n - f e e d b a c k events (77), and i n v e s t i g a t i o n s of the e a r l y responses of other t i s s u e s to s t e r o i d s are i n agreement w i t h t h i s p o s s i b i l i t y . A s i m i l a r s i t u a t i o n occurs i n chick l i v e r , where the synthesis of the p r o t e i n p h o s v i t i n i s induced by estrogen treatment 12 hours a f t e r primary i n j e c t i o n , or 6 hours a f t e r secondary i n j e c t i o n (110). During the f i r s t 2 hours, there i s an i n c o r p o r a t i o n of l a b e l i n t o n u c l e a r p r o t e i n s , and the accumulation of two s p e c i f i c p r o t e i n s was noted (107) . One of these was a b a s i c , n o n h i s t o n e p r o t e i n , and the other was an a c i d i c p r o t e i n , both of low molecular weight (26,000 and 20,000 r e s p e c t i v e l y ) . The increase i n these two p r o t e i n s was thought to be due to transport from the cytoplasm, r a t h e r than to new synthe-s i s (107). An a l t e r a t i o n of nuclear non-histone c o n s t i t u e n t s has a l s o been noted i n the e f f e c t s of C o r t i s o l on r a t l i v e r (81), the e f f e c t of estrogen on r a t uterus (82), and i n numerous other systems (133). In mammalian systems, where the type of response measured i s of a s l i g h t l y d i f f e r e n t nature, there i s s t i l l some evidence of a time l a g i n the response. In r a t uterus, the e a r l y responses, which, are not d i r e c t l y a s s o c i a t e d w i t h growth, can be e l i c i t e d by e s t r a d i o l or e s t r i o l (109). The growth can be e l i c i t e d only by e s t r a d i o l , or, i f repeated i n j e c t i o n s are given, by e s t r i o l as w e l l (156). Anderson et^ al f e l t that growth could be c o r r e l a t e d w i t h the amount of receptor-estrogen complex i n the nucleus at 6 hours, a f t e r the i n i t i -a l i n j e c t i o n , but not at 1 or 3 hours. That i s , the a c t i o n of the complex i n the nucleus at 6 hours i s d i f f e r e n t from i t s a c t i o n i n the nucleus at 1 or 3 hours (109). I t i s h i g h l y u n l i k e l y that there i s any d i f f e r e n c e i n the complex i t s e l f between 1, 3 and 6 hours, and t h e r e f o r e , a d i f f e r e n c e i n the nucleus would be i n d i c a t e d , e i t h e r i n the q u a l i t y , q u a n t i t y or arrangement of i t s c o n s t i t u e n t s . I t t h e r e f o r e appears u n l i k e l y that the i n d u c t i o n of ovalbumin message i s the r e s u l t of an i n i t i a l d i r e c t a c t i v a t i o n of the genes by the e s t r a d i o l -receptor complex. Regardless of whether the gene i s a c t i v a t e d independently of other genes or c o o r d i n a t e l y w i t h them i t s a c t i v a t i o n i s not l i k e l y the i n i t i a l a c t i o n of the receptor-estrogen complex i n the nucleus. In summary, the i n d u c t i o n of ovalbumin s y n t h e s i s occurs at the l e v e l of t r a n s c r i p t i o n of the messenger f o r ovalbumin, and t h i s may be the c o n t r o l p o i n t f o r the i n d u c t i o n of the other major p r o t e i n s as w e l l . I f t h i s i s the only c o n t r o l p o i n t i n the i n d u c t i o n of s p e c i f i c p r o t e i n s y n t h e s i s , then i t must be subject to m u l t i p l e r e g u l a t i o n , to account f o r the e f f e c t s of other s t e r o i d s . A l l of the e f f e c t s of a l l of the s t e r o i d s could not be mediated by a s i n g l e intermediate f a c t o r , s i n c e t h i s would not account f o r the s e l e c t i v e e f f e c t s of various s t e r o i d s , nor the d i f f e r e n t e f f e c t s of one s t e r o i d at d i f -f e r e n t doses. Therefore, two p o s s i b i l i t i e s remain: e i t h e r each gene i s inde-pendently c o n t r o l l e d by each s t e r o i d (whether by i t s e l f , or through an i n t e r -mediate f a c t o r f o r each gene), or each s t e r o i d operates by one or a few intermediates ( s p e c i f i c f o r that s t e r o i d ) , and the gene a c t i o n i s coordinated. There i s as yet no evidence to d i s t i n g u i s h between these p o s s i b i l i t i e s . The data document q u i t e n i c e l y the events beginning w i t h the i n d u c t i o n of ovalbumin messenger t r a n s c r i p t i o n , but cannot i n d i c a t e what happens p r i o r to that event. 2. The Response to Progesterone: O'Malley's Model The i n v e s t i g a t i o n s i n t o the response of oviduct t i s s u e to progesterone have centered p r i m a r i l y around the i n d u c t i o n of the syn t h e s i s of the p r o t e i n a v i d i n i n response to a progesterone i n j e c t i o n a f t e r estrogen treatment has been given. The work of O'Malley's group has centered around the i n d u c t i o n of t h i s p r o t e i n , and the e l u c i d a t i o n of a p o s s i b l e mechanism of a c t i o n f o r progesterone. This work w i l l a l s o be reviewed f i r s t i n terms of the evidence presented, then the model that has been proposed to account f o r i t , and l a s t , i n terms of comparison to other systems. a. The Evidence The i n d u c t i o n of a v i d i n r e q u i r e s new p r o t e i n s y n t h e s i s (111, 112), and may or may not r e q u i r e new RNA s y n t h e s i s (111, 112, 113); the s t u d i e s using actinomycin-D give v a r y i n g r e s u l t s . The phenomenon of " s u p e r i n d u c t i o n " occurs here (113), as w e l l as w i t h ovalbumin, but i n t h i s case i t i s p o s s i b l e to induce a v i d i n s y n t h e s i s i n v i t r o (111-113), contrary to the s i t u a t i o n w i t h ovalbumin (99). In i t s a c t i o n on the ch i c k o v i d u c t , progesterone a f f e c t s the sy n t h e s i s of r a p i d l y - l a b e l l e d nuclear RNA, and the RNA polymerase a c t i v i t y which would cause i t (113, 114), but the changes are s m a l l , and the use of whole n u c l e i precludes the d i s t i n c t i o n between amounts of enzyme, enzyme a c t i v i t y , and/or changes i n template a v a i l a b i l i t y (114). RNA-DNA h y b r i d i z a t i o n s t u d i e s (116, 117) i n d i c a t e that oviducts t r e a t e d w i t h estrogen and progesterone cont a i n a l l the RNA species present i n estrogen-t r e a t e d o v i d u c t s , plus some RNA species that are not present i n the l a t t e r case (115, 116, 117). Other i n t e r p r e t a t i o n s of t h i s data i n c l u d e the p o s s i b i l -i t y of the s t e r o i d s t a b i l i z i n g a r a p i d l y turning-over minor p o p u l a t i o n of RNA (116), which has been mentioned a l s o as a p o s s i b l e e x p l a n a t i o n f o r the e f f e c t of estrogen on ovalbumin message (95, 97). Gene a m p l i f i c a t i o n , although u n l i k e l y , could a l s o account f o r t h i s data. There i s no evidence that the proposed new RNA being produced i s a v i d i n message, and i t i s l i k e l y not, s i n c e the short i n c u b a t i o n time used s e l e c t s f o r redundant sequences, and sin c e goblet c e l l s of the magnum p o r t i o n make up only a s m a l l percentage of the whole oviducts that were used. O'Malley and McGuire a l s o p o i n t out that r a p i d l y - l a b e l l e d nuclear RNA i s u s u a l l y rRNA or precursor rRNA (111). However, t h i s i s not l i k e l y the case here, s i n c e progesterone i s unable to induce Phase I I of the secondary response; that i s , i t i s unable to induce new rRNA syn t h e s i s (53). An i n v e s t i g a t i o n of the e f f e c t s of progesterone on the polysome p r o f i l e i n d i c a t e s a negative e f f e c t of progesterone on polysome formation (118), but i n t e r p r e t a t i o n of these s t u d i e s i s hindered by the presence of an a r t i f a c t i n the technique used; i . e . , the l a c k of use of detergent before c e n t r i f u g a t i o n . In the absence of detergent, only 10-20% of the polysomes w i l l be recovered, due to a p e l l e t i n g of polysomes bound to endoplasmic r e t i c u l u m under these c o n d i t i o n s (119). T r a n s l a t a b l e messenger f o r a v i d i n has been demonstrated to be present only a f t e r progesterone i n d u c t i o n (120, 121). RNA from oviducts t r e a t e d w i t h estrogen and -progesterone has the a b i l i t y to induce a v i d i n synthesis i n an estrogen-treated o v i d u c t , when i t i s i n j e c t e d i n t o the lumen of the oviduct (157, 123). That t h i s i s a d i r e c t t r a n s l a t i o n of that RNA i s evident from cross-species t r a n s f e r s using immunologically n o n - c r o s s - r e a c t i v e a v i d i n molecules (124). The n e c e s s i t y f o r both estrogen and progesterone i n the i n d u c t i o n of a v i d i n s y n t h e s i s complicates the a n a l y s i s of i n d u c t i o n by adding the p o s s i b i l i -t y that estrogen may induce the pre-message, which would then be a c t i v a t e d by progesterone. Such a s i t u a t i o n would be analogous to the masked messenger of amphibian oocytes (125). I f t o t a l l y untreated o v i d u c t s can synthesize a v i d i n i n response to progesterone, then t h i s would not be the case. One such i n c i d e n t has been reported (111), but i s at variance w i t h most other s t u d i e s (24, 49). The matter i s confused f u r t h e r by the rep o r t that a v i d i n s y n t h e s i s i s under the c o n t r o l of estrogen i n l i z a r d s , w i t h progesterone and testosterone a c t i n g s y n e r g i s t i c a l l y w i t h estrogen i n t h i s regard (126). An e v o l u t i o n a r y switch from estrogen to progesterone i n d u c t i o n , would of course confer the advantage of arranging a supply of b i o t i n i n the y o l k before the b i o t i n - b i n d i n g p r o t e i n i s made, but such a s w i t c h , again, could occur at the t r a n s c r i p t i o n a l or p o s t - t r a n s c r i p t i o n a l l e v e l . Receptor p r o t e i n s f o r progesterone have been demonstrated i n c h i c k o v i -duct i n cytoplasm and n u c l e i (127), which have sedimentation c o e f f i c i e n t s of 4S i n the presence of s a l t , and 8S and 5S i n i t s absence (127). Only t e s t o -sterone and 5-a-pregnane-3,20-dione could compete w i t h progesterone f o r b i n d -i n g to the receptor (127); these s t e r o i d s a l s o induce a v i d i n s y n t h e s i s (128, 24). By f r a c t i o n a t i n g and r e - i n c u b a t i n g the components of chromatin, and exchanging these components, the authors demonstrated a greater b i n d i n g of the progesterone-receptor complex to the a c i d i c chromatin p r o t e i n s than to the histones (129). A more extensive f r a c t i o n a t i o n of the a c i d i c chromatin p r o t e i n s , and r e - i n c u b a t i o n , was attempted i n order to f u r t h e r i d e n t i f y the nature of the a c i d i c p r o t e i n s r e s p o n s i b l e f o r b i n d i n g . A number of assays were employed to demonstrate the 3-dimensional s t r u c t u r a l f i d e l i t y of these h y b r i d chromatins (130). I f f r a c t i o n a t e d chromatin components incubated i n v i t r o a c t u a l l y r e t u r n to t h e i r o r i g i n a l 3-dimensional s t r u c t u r e , then chromatin may be a self-assembly s t r u c t u r e . However, i t i s not necessary to assume the 3-dimensional s t r u c t u r a l f i d e l i t y of the h y b r i d chromatins to accept that the complex binds to the a c i d i c p r o t e i n s . A f u r t h e r i n v e s t i g a t i o n of the receptors i n d i c a t e d that there are two components (131), w i t h the same s t e r o i d s p e c i f i c i t y and hormone b i n d i n g k i n e t i c s (131), but that Component A binds to DNA, whereas Component B does not, and that Component B binds to chromatin, whereas Component A does not (132). b. D i s c u s s i o n of the Model O'Malley's model, then, i s one i n which the progesterone-receptor complex enters the nucleus and binds to the a c i d i c chromatin p r o t e i n s , thus a c t i n g at the "nuclear or t r a n s c r i p t i o n a l l e v e l of p r o t e i n s y n t h e s i s , r e s u l t i n g i n new gene t r a n s c r i p t i o n s and eventuating i n a v i d i n s y n t h e s i s " (116). This model, i f i t i s to be a general model f o r progesterone a c t i o n , would have to account f o r the e a r l y e f f e c t s of progesterone as w e l l . Although the p o s s i b i l i t i e s f o r c o o r d i n a t i o n of the responses are obvious i n t h i s model, there i s as yet no evidence to i n d i c a t e that a v i d i n s y n t h e s i s r e s u l t s from new t r a n s c r i p t i o n , nor that such proposed new t r a n s c r i p t i o n i s the r e s u l t of the b i n d i n g of the progesterone-receptor complex to a c i d i c chromatin p r o t e i n s . A p o s s i b l e causal r e l a t i o n s h i p between the b i n d i n g to a c i d i c chromatin p r o t e i n s and any of the other a c t i o n s of progesterone remains to be demonstrated (133). Nevertheless, the f o l l o w i n g c o r r e l a t i o n s are evident: 1) A f t e r 6 days of d i e t h y l s t i l b e s t r o l (DES) treatment, chromatin of n u c l e i of t u b u l a r gland c e l l s becomes condensed, and was described as "heterochromatin" by.Kohler et a l (50). At that dosage of DES, the peak of c e l l p r o l i f e r a t i o n occurred at 6 days; t h e r e a f t e r , c e l l p r o l i f e r a t i o n ceased and f u r t h e r growth was a t t r i b u t e d to hypertrophy (50). 2) An i n j e c t i o n of progesterone to t r e a t e d c h i c k s had g r e a t e s t e f f e c t i n inducing a v i d i n synthe-s i s a t 6 days of treatment. Greater or l e s s e r estrogen treatment i n h i b i t e d t h i s response to progesterone (49). 3) A c i d i c chromatin p r o t e i n s of the ... nucleus are reported to change b i p h a s i c a l l y during treatment, i n c r e a s i n g i n amount and v a r i e t y during the f i r s t 6 days of treatment, and decreasing t h e r e a f t e r (134). 4) The b i n d i n g of the progesterone-receptor complex to the a c i d i c chromatin p r o t e i n v a r i e s i n the same b i p h a s i c manner (135). The c o r r e l a t i o n of changes i n a c i d i c chromatin p r o t e i n s and chromatin condensation has been noted i n other systems, and i s discussed at length by LeSturgeon et a l (136). However, any d i s c u s s i o n of the e f f e c t s of these changes on a v i d i n s y n t h e s i s remains s p e c u l a t i v e . Again, i t should be noted that chromatin from oviducts w i l l probably not r e f l e c t to any appreciable extent the chromatin of the goblet c e l l s i n which a v i d i n i s made. Thus, there i s a problem i n using a v i d i n s y n t h e s i s as a marker f o r progesterone a c t i o n : namely, i t i s s p e c i f i c to c e l l s which represent a s m a l l percentage of the t o t a l t i s s u e , and these c e l l s g e n e r a l l y d i f f e r e n t i a t e a f t e r t u b u l a r gland c e l l s , which are the major bulk of the t i s s u e (50, 45, 58). The d i f f i c u l t y w i t h t h i s model, as opposed to that of P a l m i t e r , i s that i t i n d i c a t e s only the very i n i t i a l events of s t e r o i d a c t i o n , but does not c o r r e l a t e them w i t h the l a t e r changes. Spelsberg i s of the o p i n i o n that receptor p r o t e i n s f o r s t e r o i d s are i n t r a c e l l u l a r gene r e g u l a t o r s (133), and that t h i s c o n t r o l a c t i o n i s v i a the p r o t e i n r a t h e r than the s t e r o i d ; that i s , he proposes that the s t e r o i d only acts to t r a n s p o r t the p r o t e i n s i n t o the nucleus, where the p r o t e i n s a c t i v a t e or derepress the responsive genes. He i s a l s o of the o p i n i o n that there are other p r o t e i n s of a s i m i l a r nature i n c e l l s . He points out t h a t , f o r v i r t u a l -l y every s t e r o i d - r e s p o n s i v e system, an a c t i v a t i o n of t r a n s c r i p t i o n has been p o s t u l a t e d , f o l l o w i n g a b i n d i n g to a nuclear c o n s t i t u e n t , although the nature of the nuclear c o n s t i t u e n t i m p l i c a t e d has v a r i e d w i d e l y . He i n d i c a t e s 1) that the reported b i n d i n g to histones can be accounted f o r by contamination w i t h a c i d i c nuclear p r o t e i n s ; 2) that the reported b i n d i n g to a c i d i c chromatin p r o t e i n s may be hindered by the use of proteases that simply d i g e s t the receptor p r o t e i n s and r e l e a s e the l a b e l l e d s t e r o i d , r a t h e r than by d i g e s t i n g the nuclear acceptor s i t e , and r e l e a s i n g the complex; or 3) that e x t r a c t i o n procedures may e x t r a c t the receptor (an a c i d i c p r o t e i n ) , r a t h e r than the nuclear acceptor s i t e . He s t a t e s t h e r e f o r e , that i d e n t i f i c a t i o n of the nuclear acceptor i s not p o s s i b l e (using i n v i v o systems) by the present tech-niques, and he f u r t h e r p o i n t s out that b i n d i n g to DNA gives g e n e r a l l y non-s p e c i f i c r e s u l t s , indeed that even d i g e s t i o n by nucleases give v a r y i n g r e s u l t s . He has t h e r e f o r e pursued an i n v i t r o a n a l y s i s , but such i n v i t r o work i s hindered by the f a c t that i t r e l i e s on f r a c t i o n a t i o n under denaturing c o n d i t i o n s , and r e c o n s t i t u t i o n by r e - i n c u b a t i o n . Nevertheless, although the 3-dimensional s t r u c t u r a l f i d e l i t y of such chromatin i s i n doubt, i t i s not necessary to assume the 3-dimensional s t r u c t u r a l f i d e l i t y of the chromatin i n order to accept that the complex may b i n d to the a c i d i c chromatin p r o t e i n s . This model i s t h e r e f o r e q u i t e f e a s i b l e . 3. Events a General Model W i l l Have to E x p l a i n Any model f o r s t e r o i d a c t i o n on c h i c k oviduct that i s a general model w i l l have to account f o r the f o l l o w i n g : 1) The s e q u e n t i a l nature of the response. That i s , why do the stromal c e l l s respond f i r s t , and the p r o g e n i t o r tubular gland c e l l s second? I f t h i s i s a s e n s i t i v i t y to a lower estrogen t h r e s h o l d , how i s i t mediated? By higher c o n c e n t r a t i o n of the receptor, or by a nuclear c o n s t i t u e n t , or i s i t simply that the changes undergone by the stromal c e l l s produce some c o n d i t i o n necessary f o r the response of the progenitor tubular gland c e l l s ? L i k e w i s e , why do the c i l i a t e d c e l l s and goblet c e l l d i f f e r e n t i a t e l a s t ? 2) The 3-hour time l a g i n the secondary s t i m u l a t i o n , and the 24-48 hour time l a g i n the primary s t i m u l a t i o n . A simple b i n d i n g of the r e c e p t o r -s t e r o i d complex to any nuclear c o n s t i t u e n t and subsequent a c t i v a t i o n of t r a n s -s c r i p t i o n does not account f o r t h i s time l a g , i f no intermediates e x i s t . 3) The dose-effect of estrogen. The f a c t that a lower dose of estrogen p r e f e r e n t i a l l y enhances conalbumin syn t h e s i s i s hard to r e c o n c i l e to a model of simple b i n d i n g of r e c e p t o r - s t e r o i d complex to a n u c l e a r c o n s t i t u e n t and a c t i v a t i o n of t r a n s c r i p t i o n . 4) The s e l e c t i v e e f f e c t s of other s t e r o i d s , both when they are given alone, and i n combination w i t h estrogen. This would i n c l u d e both the s e l e c -t i v e and s y n e r g i s t i c e f f e c t s of progesterone and t e s t o s t e r o n e w i t h estrogen, and the a n t i - e s t r o g e n i c e f f e c t s of progesterone w i t h estrogen when t h i s i s administered at the beginning of treatment. C. Comparison of t h i s Induction Phenomenon to Embryonic Indu c t i o n The type of i n d u c t i o n discussed here e x h i b i t s d i f f e r e n c e s from the normal embryonic i n d u c t i o n , both i n c o n s i d e r a t i o n of the inducer, and of the c e l l ' s response. 1. The Inducer The s p e c i f i c i t y of the inducer i n t h i s case i s very exact, and the v a r i -able parameters are known. This i s not the case i n embryonic i n d u c t i o n , where i n most cases the inducer i s not only unknown, but may a l s o be k i l l e d , heat-t r e a t e d , or s u b s t i t u t e d by simple s a l t s o l u t i o n s and s t i l l induce some response. 2. The Competence of the Responding C e l l s The d i f f e r e n c e s extend a l s o to the c e l l ' s competence to respond. In the s t e r o i d - i n d u c e d d i f f e r e n t i a t i n g systems discussed here, there appears to be no time l i m i t to the competence of the t i s s u e to respond. In other s i t u a t i o n s , as r a t uterus, there i s a time l i m i t before which the c e l l s do not respond f u l l y , but there appears to be no subsequent time l i m i t (148, 149, 150). In s t i l l other systems of s t e r o i d - i n d u c t i o n , such as neonatal androgenization, there i s a very d e f i n i t e , circumscribed time p e r i o d before and a f t e r which the s t e r o i d has no e f f e c t (158, 159). This l a t t e r s i t u a t i o n i s , of course, the normal case i n embryonic i n d u c t i o n . From an embryological p o i n t of view, the d i f f e r e n c e between i n d u c t i o n and competence i s the d i f f e r e n c e between the c o n t r o l by the inducer over the c e l l ' s a c t i o n s , and the c o n t r o l of the c e l l over i t s own response to the inducer. A great d e a l of work has centered around the c o n t r o l of the inducer over the c e l l ' s a c t i o n s . The f o l l o w i n g question i s a l s o of i n t e r e s t : What c o n t r o l does the oviduct c e l l have over i t s own response? Such a c o n t r o l might p o s s i b l y be mediated at any of the p o i n t s i n v e s t i g a t e d f o r the c o n t r o l of the c e l l - s p e c i f i c p r o t e i n s . Obviously, the c e l l u l a r c o n c e n t r a t i o n of the receptor (139) could be a c o n t r o l p o i n t i n the response of the c e l l to the s t e r o i d . However, much evidence i n d i c a t e s that the s t e r o i d determines the con c e n t r a t i o n of i t s own receptor, or the co n c e n t r a t i o n of the receptor f o r other s t e r o i d s (142, 89-91). In t h i s manner, estrogen induces the production of progesterone receptor i n guinea p i g uterus, and progesterone may i n a c t i v a t e i t s own receptor (143-145). In c h i c k o v i d u c t , i t has been reported that estrogen enhances the receptor f o r progesterone (146), although t h i s i s not a s e l e c t i v e a c t i o n (146, d i s c ) . Progesterone has been shown to enhance the co n c e n t r a t i o n of the estrogen receptor (147). These observations p o i n t to an important d i f f e r e n c e between the experimental and the n a t u r a l s i t u a t i o n s : the presence of two or more hormones simultaneously, and the i n t e r a c t i o n s of these. The simple f a c t that the receptor i s present, however, does not mean that the hormone w i l l a c t . In r a t ute r u s , the competence to respond to inducer i s developed step-wise, over a period of 30 days, although the receptor i s present at b i r t h , able to bind e s t r a d i o l , and enter and bind i n the nucleus (148, 149, 150). This would i n d i c a t e that a d d i t i o n a l f a c t o r s aire necessaxy f o r each new step, and so i n d i c a t e the p o s s i b i l i t y of a d d i t i o n a l c o n t r o l steps. This p o s s i b i l i t y i s a l s o evident i n r e c e p t o r - c o n t a i n i n g mutants of immunocytes that are r e s i s t a n t to g l u c o c o r t i c o i d a c t i o n , even though the receptors bind g l u c o c o r t i c o i d s and enter and bind i n the nucleus (151). In other systems development of competence i s r e f l e c t e d i n a l t e r a t i o n s of the type of b i n d i n g observed i n the target t i s s u e s . This has been reported f o r DES b i n d i n g i n r a t b r a i n (152, 160), androgen b i n d i n g i n r a t epididymis (153, 161), and g l u c o c o r t i c o i d b i n d i n g i n lung and other t i s s u e s (154). Another d i f f e r e n c e between t h i s type of i n d u c t i o n and that seen commonly i n embryonic systems i s that the c e l l s never become incapable of responding to the inducer when i t i s present, and never become capable of continued f u n c t i o n i n the absence of the inducer. This i s p a r t i a l l y r e l a t e d to the absence of a c r i t i c a l time p e r i o d as discussed e a r l i e r . However, re g a r d l e s s of the f a c t that the c e l l s do not stop responding, t h e i r response changes i n the course of treatment. Kohler et a l have reported that f o r the f i r s t 6 days of treatment, the growth response i s p r i m a r i l y by h y p e r p l a s i a (41); t h e r e a f t e r , i t i s p r i m a r i l y represented by c e l l u l a r hypertrophy. There i s , t h e r e f o r e , a po i n t at which the response changes, at which the c e l l s respond by f u n c t i o n i n g but no longer by d i v i d i n g . This change i n response may r e s u l t from a change induced by the e s t r a d i o l treatment, o r , i n the n a t u r a l s i t u a t i o n , i t may be-induced by other sources, such as other c e l l s , or other s t e r o i d s . The absence of continued f u n c t i o n of the oviduct t i s s u e i n the absence of inducer i s a l s o a notable exception to normal embryonic i n d u c t i o n . The f a c t that continued s t i m u l a t i o n by estrogen i s necessary f o r continued t r a n s c r i p -t i o n , and that other s t e r o i d s do not have the same e f f e c t , although they have s i m i l a r e f f e c t s , preclude the p o s s i b i l i t y t hat the inducer simply a c t s to t r i g g e r a gross c o n t r o l , w i t h the c e l l then a c t i n g . t o r e g u l a t e i t s e l f . In the oviduct system, the inducer o b v i o u s l y exerts a f i n e c o n t r o l as w e l l . The recent observation, that the v a g i n a l and u t e r i n e c e r v i c a l e p i t h e l i a l c e l l s of n e o n a t a l l y estrogenized female mice can p r o l i f e r a t e and d i f f e r e n t i a t e without estrogen, and are l e s s s e n s i t i v e to exogenous estrogen.(155) i s r e l a t e d to these c h a r a c t e r i s t i c s of the n e c e s s i t y f o r continued estrogen presence and the i n a b i l i t y to not respond .in the presence of the inducer.. S i m i l a r s t u d i e s have been performed on n e o n a t a l l y androgenized. rodents (83, 84). Although no equivalent of the neonatal c r i t i c a l p e r i o d has been e s t a b l i s h e d i n c h i c k s , i t i s evident, i n rodents a t l e a s t , that the a c t i o n of the s t e r o i d on i t s t a r g e t t i s s u e a t d i f f e r e n t times i n development can be q u i t e d i f f e r e n t . In t h i s respect i t i s reminiscent of Paul Weiss' statement, "Hormone a c t i o n i s probably not a one-shot operation; the agent i n v o l v e d more l i k e l y operates a t d i f f e r e n t times, repeatedly, producing d i f f e r e n t r e s u l t s , depending on the time a t which i t works" (162). Obviously, the t o t a l a c t i o n of the hormone on a t i s s u e throughout the l i f e of the t i s s u e i s a. more complex* matter than the simple i n d u c t i o n to d i f f e r e n t i a t i o n a t the time of puberty.. D. A B r i e f Assessment of the State of the F i e l d • The research that has been discussed here appears to have proceeded to the l i m i t of the present technology. Minor advances may yet be made, such as the i d e n t i f i c a t i o n of the non-translated p o r t i o n of the message.. However, the major advances i n t h i s area w i l l r e q u i r e a more complete knowledge of two areas: the s t r u c t u r e of chromatin, and the process of t r a n s c r i p t i o n . Only when these areas are e l u c i d a t e d w i l l i t be p o s s i b l e to measure an a l t e r a t i o n of these by the hormone treatment. Perhaps i n r e c o g n i t i o n of t h i s interdependency, the 1975 Conference of the So c i e t y f o r Developmental B i o l o g y w i l l address i t s e l f to two areas: the mechanism of s t e r o i d , a c t i o n and the process o f . t r a n s c r i p -t i o n . 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(1973) "Chromatin-sterone and t h e ^ n d u c t i o n of Egg W h i t e S r o t e i n S y n t h e s i s ^ n ^ C h i c k Magnum". C e l l D i f f e r e n t i a t i o n 2: 163. 140. M U l l e r , W. E. G., Totsuka, A. and Zahn, R. K. (1974) " A s s o c i a t i o n of an E s t r a d i o l Receptor w i t h the DNA-Dependent RNA Polymerase I from Immature Q u a i l Oviduct". Biochem. Biophys. Acta 366: 224. 141. Baker, C. M. A. and Manwell, C. (1975) "Molecular B i o l o g y of Avi a n P r o t e i n s . X I I . P r o t e i n Polymorphism i n the Stubble Q u a i l C o t u r n i x  p e c t o r a l i s - And a B r i e f Note on the In d u c t i o n of Egg-White P r o t e i n . Synthesis i n Wild B i r d s by Hormone". Comp. Biochem. P h y s i o l . 50B: 142. C l a r k , J . H. and G o r s k i , J . (1970) "Ontogeny of the Estrogen Receptor During E a r l y U t e r i n e Development". Science 16: 76. 143. Milgrom, E., Atger, M. and B e a u l i e u , E.-E. (1970) "Progesterone i n Uterus and Plasma. IV. Progesterone Receptors i n Guinea P i g Uterus and C y t o s o l " . S t e r o i d s 16: 741. During the E s t r u s Cycle and Impl a n t a t i o n i n the Guinea-Pig". Endocr. 90: 1071. 145. Milgrom, E., T h i , L., Atger, M. and B e a u l i e u , E.-E. (1973) "Mechanisms Re g u l a t i n g the Concentration and the Conformation of Progesterone Receptors i n the Uterus". J . B i o l . Chem. 248: 6366. O'Malley, B. W. , Rosenfeld, G. C , Comstock, J . P. and Means. A. R. (1972) "I n d u c t i o n of S p e c i f i c T r a n s l a t a b l e Messenger RNAs by Oestrogen and ^ " ^ i ^ r s s i ^ X ^ ^ : F i f th K a r o l i n s k a I n s t i t u t e t , Stockholm. 147. P a v l i k , E. J . and Coulson, P. B. (1974). "The E f f e c t of Estrogen and So c i e t y f o r C e l l B i o l o g y , San Diego, Nov. 1974. 148. Somjen, D., Kaye, A. M. and Lindner, H. R. (1973) "Postnatal Develop-ment of the Uterine Response to Estradiol-17B i n the Rat". Dev. B i o l . 31: 409. 149. Somjen, D., Somjen, G., King, R. J . B., Kaye, A. M. and Lindner, H. R. (1973) "Nuclear Binding of 0estradiol-17B and Induction of P r o t e i n Synthesis i n the Rat Uterus during Postnatal Development". Biochem. J . 136: 25. 150. Kaye, A. M., Sheratzky, D. and Lindner, H. R. (1972) " K i n e t i c s of DNA Synthesis i n Immature Rat Uterus: Age Dependence and E s t r a d i o l Stimulation". Biochim. Biophys. Acta 261: 475. 151. Tomkins, G. (1973) "Molecular Biology of Steroid Hormones", i n Strategies for the Control of Gene Expression, A. Kohn and A. Shadkey, eds., Plenum Press, New York. 152. Plapinger, L. and MacEwen, B. S. (1973) "Ontogeny of Estradiol-Binding Sites i n Rat B — -'- T A * " * J - 1 - " i n Cytosol and rain. I. Appearance of Presumptive.Adult Receptors i N u c l e i " . Endocr. 93: 1119. 153. Calandra, R. S., Poderta, E. J . , Rivarola, M. A. and Blaquier, J . A. (1974) "Tissue Androgens and A n d r o p h i l l i c Proteins i n Rat Epididymis during Sexual Development". Steroids 24: 507. 154. Giannopoulous, G. (1974) "Variations i n the Levels of Cytoplasmic G l u c o c o r t i c o i d Receptors i n Lungs of Various Species at D i f f e r e n t Developmental Stages". Endocr. 94_: 450. 155. Shyamala, G., Mori, T. and Bern, H. A. (1974) "Nuclear and Cytoplasmic Oestrogen Receptors i n Vaginal and Uterine Tissue of Mice Treated Neonatally with Steroids and P r o l a c t i n " . J . of Endocr. 63: 275. 156. Anderson, J . A., Peck, E. J . , J r . and Clark, J . H. (1973) "Nuclear 157. Tuohimaa, P., Segal, S. J . and Koide, S. S. (1972) "Induction of Avidin Synthesis by RNA Obtained from Chick Oviduct". Proc. Nat. Acad. S c i . 69: 2814. 158. Gorski, R. A. (1971) "Gonadal Hormones and the P e r i n a t a l Development of Neuroendocrine.. Function", i n F r o n t i e r s i n Neuroendocrinology, L. M a r t i n i and W. F. Ganong, eds., Oxford Univ. Press, New York. 159. DeMoor, P., Verhoeven, G. and Heyns, W. (1973) "Permanent E f f e c t s Foetal and Neonatal Testosterone Secretion on Steroid Metabolism and Binding". D i f f e r e n t i a t i o n ! : 241. of 165. 167. tes i n Rat Brain. I I . C h a r a c t e r i s t i c s of a 160. Plapinger, L., MacEwen, B. S. and Clemens, L. B. (1973) "Ontogeny of E s t r a d i o l Binding S i t - — — " ----- • • ' - 7 Neonatal t h « ^ ^ « 161. Roth, G. S. u.*/<U • Age-Kexated Changes In SnPrifnV r,1„oo C Orticoid Neonatal, Binding Macromolecule^^Endocr!' 9 3 ^ 1 1 2 9 (1974) "Age-Relate Changes i n S p e c i f i c Glucoc Binding, by Steroid Responsive Tissues of Rats". Endocr. 94: 82 162. Weiss, P. (1959) "Ontogeny of Selected Endocrine Receptors- The 1 6 3 - M c K 1 ^ ; l i ^ ^ ^ i J ^ l 2 ^ Z r s ^ L as Polysomal Ovalbumin Messenger". Proc. Nat. Acad. S c i . 71: 4327. 164. Gibson, W. R., F o l l e t t , B. K. and G l e d h i l l , B. (1975) "Plasma Levels of L u t e i n i z i n g Hormone i n Gonadectomized Japanese Quail Exposed to Short or.to Long Davlengths". J . Endorr. fi4: R7 '• y c 64: 87. Dresser-Weist, L. and Zwintz, C. (1974) "Differences i n the Binding Capacity f o r Steroid Hormones of Sera from Embryonic and Adult Rats". Endokrinol. 64: 33 166. F e l l , P. D., Glasser, S. R., Toft, D. 0. and O'Malley, B. W. (1972) "Progesterone Binding i n Mouse and Rat Uterus". Endocr. 91: 738. Faber, L. E., Sandman, M. L. and. Stanely, H. E. (1972) . "Progesterone Binding Proteins of the Rat and Rabbit Uterus". J . B i o l . Chem. 247: 5648. 168. Rao, B. R., Weist, W. G. and A l l e n , W. M. (1973) "Progesterone Receptor i n Rabbit Uterus. I. Characterization and E s t r a d i o l 17-B Augmenta-t i o n " . Endocr. 92: 1229. 169. Dische, Z. (1955) "Color Reactions of Nucleic Acid Components", Chapter 9, Vol. I, of The Nucleic Acids, E. Chargoff and J . N. David-son, eds., Academic Press, New York. 170. Jones, I. C , Bellamy, D., Chan, D. K. 0., F o l l e t t , B. K., Henderson I W., P h i l l i p s , J . G. and Smart, R. S. (1972) " B i o l o g i c a l Actions of S t e r o l * Hormone* inNon-Mammalian Vertebrates", Chapter 8 i n Steroids i n Non-mammalian Vertebrates, D. R. I d l e r , ed., Academic Press, N.Y. 120a APPENDICES Protein Assay 0.5 ml of sample or standard + 5.0 ml of Solution C 10 min at room temperature + 0.5 ml of F o l i n ' s reagent, d i l u t e d to 1 N 30 min at room temperature Read absorbance at 660 Solution C = 50:1 of Solution A:Solution B (v/v) Solution A = 2% Na 2C0 3 (w/v) i n 0.1 N NaOH Solution B = 0.5% CuS0 4'5H 20 (w/v) i n 1% NaK Tartrate (w/v) This procedure follows that of Lowry et a l (60). Extraction Procedure f o r Ribonucleic Acid Assay 1 ml of homogenate or standard I + 2.5 ml cold 10% TCA (w/v) I centrifuge, keep p e l l e t 4 resuspend i n 2.5 ml 10% cold TCA I centrifuge, keep p e l l e t resuspend i n 5 ml 95% ETOH 1 centrxfuge, keep p e l l e t resuspend i n 5 ml 95% ETOH I centrifuge, keep p e l l e t I resuspend i n 2 ml 0.1 N NaOH 1 heat 45 minutes at 80 C •I. C O O l 0T1 X C e i . + 0.5 ml cold 10% TCA I centrifuge save slpernatant f o r r i b o n u c l e i c acid assays This procedure follows the Reference of Schneider (57) with the exception thcit the p e l l e t W3S f i n s l l y resuspended i n 2 nil of 0 1 N N9.OH 3 n d h e 3 t e d for \^5 minutes 3 .t 8 0^C r3 .ther th.3tx bein^ resuspended i n 2 ml of 1 0 N No.OH 3 . d l e t st3.nd f o r 20 hours 3 1 37°C ttien xieutr3 l i zed \\^itti 0 ^ 6 H 3_ RNA Assay 2.5. ml F e C l 3 (0.04% (w/v) i n HCl) + 0.5 ml o r c i n o l reagent (50 mg orcinol/ml i n 95% ETOH) 1 + 1.0 ml of standard or sample •1 + H 20 to 5.0 ml t o t a l i heat 30 min i n b o i l i n g water bath J' cool on i c e read at 665 This procedure follows the basic procedures of Mejbaum (59), Dische (169), and Schneider (57), with the following exceptions: 1) the F e C l 3 i s 0.04% i n con. HCl, rather than 0.1% (Mejbaum, dissolved i n o r c i n a l reagent) or 0.5% (Schneider, dissolved i n o r c i n o l reagent); 2) o r c i n o l reagent i s 50 mg/ml, rather than 100 mg/ml ( a l l others); 3) the reaction was allowed to proceed f o r 30 min at 100°C, rather than 20 min (Dische and Schneider), or 40 min (Mejbaum); sample volumes were also a l t e r e d s l i g h t l y . Mejbaum 0.1% F e C l 3 i n 100 mg orcinol/ml 95% ETOH Dische z \ Schneider 0.5 ml of 0.1% FeCl 3-6H 20 0.5 gm F e C l 3 3 ml sample + 0.3 ml o r c i n o l 40 min at 100°C read at 670 nm added to 100 ml con. HCl 6% o r c i n o l i n 95% ETOH 1.5 ml sample + 3.0 ml. acid reagent + 0.2 ml o r c i n o l . ' 20 min 100°C read at 665 nm i n 100 ml con. HCl; 1 gm orcinol/100 ml acid reagent 0.2 ml sample + 1.3 ml H 20 + 1.5 ml reagent 20 min at 100°C read at 660 nm DNA Assay: Extraction Procedure 1 ml homogenate or standard 2.5 ml cold 10% TCA centrifuge resuspend xn. 2 «5 ml cold 10% TCA (w/v) 1 centrifuge, keep p e l l e t + 2.0^ml 5% PCA (v/v) heat at 70° f o r 20 min 1 cool, use supernatant This procedure follows that of Schneider, with the exception that i t omits extraction with 95% ETOH, since t h i s was determined to have no e f f e c t on the f i n a l values obtained. DNA Assay: Diphenylamine Reaction 1.0 ml I + I"°  m l r e a g e n t 30°C 20 hours read at 600 nm Reagent i s 1.5 gm diphenylamine i n 100 ml g l a c i a l a c e t i c . a c i d , with 1.5 ml con. H 2S0 4. At the time of use, 0.1 ml of acetaldehyde (16 mg/ml) i s added to 20 ml of the above reagent, and t h i s was used i n the assay. This assay follows the diphenylamine reaction according to the procedure of Burton (58). Ovalbumin Ouchterlony Plates Preparation of Agar Medium: To 1 gm of agar, 5 ml of borate buffer, 95 ml of s a l i n e (8.5 gm/1) was added enough sodium azide to make 0.1% azide. The borate buffer consisted of: Boric acid (6.184 gm) , borax (Na 2B 40 7•10H 20; 9.536 gm), NaCl (4.348 gm) and water, to make one l i t e r . The pH was adjusted to 8.4-8.5. The immunodiffusion was c a r r i e d out on large microscope s l i d e s that had been cleaned, rinsed twice with a l c o h o l , and twice with ether. 2 ml of agar was added to each s l i d e and allowed to s o l i d i f y . Holes were punched i n the agar 1 cm apart, the samples were applied, and allowed to d i f f u s e f o r 48 hours. The volume of sample applied was 5 m i c r o l i t e r s ; the antibody protein was d i l u t e d to 1 mg of antibody protein/ml, and t h i s was used, i n the d i f f u s i o n p l a t e s . A f t e r 48 hours, the plates were washed i n 0.9% s a l i n e f o r 2-3 days, then i n d i s t i l l e d water for 3 hours, then dried i n open a i r . They were stained i n amido black for \ hour, then washed i n 2% a c e t i c a c i d f o r 30 min. This procedure follows that of Work and Work (62). Lysozyme Assay Preparation of B a c t e r i a l C e l l s : inoculum of Micrococcus.lysodeikticus • grow i n l i q u i d medium at 30° f o r 48-72 hours, i n a shaker 1 . c o l l e c t by c en. t n f i l i a t i o n vo luxn.es of d i s t i l l e d x^ateir I c o l l e c t by ce n t r i f u g a t i o n wash 3 times with cold acetone c o l l e c t by ce n t r i f u g a t i o n wash twice with cold ether 1 c o l l e c t by c e n t r i f u g a t i o n I dry by a i r and suction on a sintered glass funnel store i n a desiccator at 5 The l i q u i d medium used w s: 0.5% Bacto-peptone, 0.5% NaCl, 0.3% beef extract, and 0.1% yeast extract. This procedure follows the procedure of Litwack (63). Lysozyme Assay b a c t e r i a l c e l l suspension i n cuvette 1 ml of transmission adjusted to 50% at 645 nm i I read % transmission every 30 sec I . . A c t i v i t y , change i n % transmission between 30 and 60 seconds of reaction C e l l Suspension: 20 mg of prepared c e l l s of Micrococcus l y s o d e i k t i c u s i n 90 ml of 0.15 M phosphate buffer, pH 6.2, with 10 ml 1% NaCl. This procedure follows that of Litwack (63), with the exception that 20 instead of 25 mg of cells/100 ml buffer were used. The re a c t i o n was read against a d i s t i l l e d water blank. As can be seen from the standard curve on the next page, the lowest measurable amount was 2 yg/ml. This i s believed to be due to the p a r t i c u l a t e nature of the substrate. A v i d i n Assay Reagents and .Standards are d i l u t e d i n 0.2 M Ammonium Carbonate 0.5 ml of C 1 4 B i o t i n I + 0.1 ml of sample or standard l e t s 13.1*1. d 10 mi xi 9. t TC oom temp G I T S , tut IT 6 i l e t stand 5 min 1 transfer, to M i l l i p o r e F i l t e r , 0.45 y pore si z e I r i n s e twice with ammonium carbonate I transfer f i l t e r to s c i n t i l l a t i o n v i a l I dissolve with 10 ml of Bray's s o l u t i o n (65) I count This procedure i s e s s e n t i a l l y that of Korenmann and O'Malley (64). Background i n counting was approximately 28-30 cpm; and e f f i c i e n c y was about 85%. Quench curve f o r co r r e c t i o n to DPM i s shown on following page, along with standard curve. 100 r QUENCH CURVE 

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