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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 t h e Department o f ZOOLOGY  We a c c e p t t h i s t h e s i s as conforming t o t h e required 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 B r i t i s h 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. of  It  is understood that copying or publication  this thesis for financial gain shall not be allowed without my  written permission.  Department of  o a /o 9 •  The University of B r i t i s h Columbia 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  7M<vt3£> /f?S~ t  (7  ABSTRACT The e f f e c t s o f e s t r a d i o l and p r o g e s t e r o n e treatment on the growth and d i f f e r e n t i a t i o n o f the q u a i l o v i d u c t have been i n v e s t i g a t e d , i n terms o f morphology, growth, and, b i o c h e m i c a l 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 t u b u l a r gland c e l l s by 5 days o f t r e a t m e n t , 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 o f t r e a t m e n t . P r o g e s t e r o n e t r e a t m e n t appeared  t o enhance e p i t h e l i a l  differentiation  slightly. The t r e a t m e n t a l s o enhanced t h e growth o f t h e o v i d u c t , as i n d i c a t e d by i n c r e a s e s i n s i z e , w e i g h t , and t h e c o n t e n t o f t h e 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 t r e a t m e n t a l s o i n d u c e d t h e s y n t h e s i s o f 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 t o induce s y n -  t h e s i s of the p r o t e i n a v i d i n . this  Only p r o g e s t e r o n e i n d u c e d the s y n t h e s i s o f  protein. W i t h d r a w a l from t r e a t m e n t f o r t e n days caused a l o s s o f c e l l s and o f  tissue constituents.  T h i s l o s s was g r e a t e r i n those b i r d s withdrawn  from  e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t than i n those withdrawn from e s t r o g e n treatment.  I n some c a s e s , s p e c i f i c p r o t e i n s were a l s o absent i n o v i d u c t s  from b i r d s withdrawn  from t r e a t m e n t .  TABLE OF CONTENTS Page Abstract  i  T a b l e of C o n t e n t s  i i  L i s t of Figures  i i i  L i s t of Tables  iv  L i s t of P l a t e s  v  I n t r o d u c t i o n t o L i t e r a t u r e Review  1  L i t e r a t u r e Review  '2  Introduction  16  M a t e r i a l s and Methods  17  Results  21  Histology  21  A s s a y s 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 o f R e s u l t s  70  Discussion, Outline  76  D i s c u s s i o n o f the R e s u l t s o f t h i s E x p e r i m e n t a t i o n  77  D i s c u s s i o n of t h e Response as a Phenomenon of I n d u c t i o n  80  The N a t u r e o f the C e l l u l a r  Response t o E s t r o g e n  82  The N a t u r e o f the C e l l u l a r  Response t o P r o g e s t e r o n e  95  E v e n t s a G e n e r a l Model W i l l Have t o E x p l a i n  101  Comparison of t h i s I n d u c t i o n Phenomenon t o Embryonic I n d u c t i o n  102  Conclusion  105  References  107  Appendices  121  LIST OF FIGURES Page  Figure 1.  Proposed Time Sequence of E s t r o g e n i c Responses, w i t h Influences of Progesterone  11  2A.  Diagram o f the C h i c k O v i d u c t  12  2B.  I l l u s t r a t i o n o f a C r o s s - S e c t i o n t h r o u g h the O v i d u c t Magnum a f t e r Treatment w i t h E s t r o g e n and P r o g e s t e r o n e  12  3.  The V a r i a t i o n i n Wet Weight as a F u n c t i o n o f Treatment  4.  The V a r i a t i o n i n P r o t e i n Content as a F u n c t i o n  49  49 5•  7• 8•  9.  l^i^^V.  XX t £i xx t  9. s  SL Jt1XIXX C t IL ^)XX  ' 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  oxx  ScprxcLcis^c  TXX  111  \/ 'cL IT 1 cL 1z  3_ xi t n. o xx  XX H_XX  Profile  of  Ov^cLlbuxiiixx  Functiof o f Triitment  Z y m e  C  °  n  C  e  n  t  r  a  t  i  °  n  Proposed C o n t r o l o f Ovalbumin S y n t h e s i s  3 3  f  T XT^^ SL1111^^ XI d XT3-tXfl^^XX *t * » • • • •  G ^ 2 0 0 •• *  51 51 61  66 84  LIST OF TABLES Table I.  II.  Page The Content and C o n c e n t r a t i o n o f V a r i o u s T i s s u e Components o f t h e O v i d u c t a t D i f f e r e n t Stages of Treatment  53  Summary of t h e W e i g h t s , and t h e P r e s e n c e o r Absence of S p e c i f i c P r o t e i n s i n t h e O v i d u c t Magna of B i r d s Withdrawn from Treatment  68  LIST OF PLATES Plate l a and l b .  lc.  2a and 2b.  3a and 3b.  4a and 4b. 5a and 5b. 6a and 6b. 7a and 7b.  8a and 8b.  9a, b, c.  10a.  10b.  p  C r o s s - s e c t i o n of an O v i d u c t from a 10-day o l d b i r d w h i c h had r e c e i v e d c o n t r o l t r e a t m e n t f o r 5 days  age  24  C r o s s - s e c t i o n o f an o v i d u c t from a 15-day o l d b i r d w h i c h had r e c e i v e d c o n t r o l t r e a t m e n t f o r 10 days.  24  C r o s s - s e c t i o n o f an o v i d u c t from a 20-day o l d b i r d w h i c h had r e c e i v e d c o n t r o l t r e a t m e n t f o r 15 days  26  C r o s s - s e c t i o n , of an o v i d u c t from a 30-day o l d b i r d w h i c h had r e c e i v e d c o n t r o l . t r e a t m e n t f o r 15 days, f o l l o w e d by 10 days w i t h o u t t r e a t m e n t  28  C r o s s - s e c t i o n o f an o v i d u c t 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  C r o s s - s e c t i o n o f an o v i d 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 e s t r a d i o l  34  C r o s s - s e c t i o n o f an o v i d u c t 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  C r o s s - s e c t i o n o f an o v i d 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 e s t r a d i o l and 5 days w i t h progesterone  •  38  C r o s s - s e c t i o n o f an o v i d u c t magnum from a b i r d withdrawn from e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t f o r 10 days  42  C r o s s - s e c t i o n s o f o v i d u c t magna from b i r d s w i t h drawn from e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t f o r 10 days  44  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 o v a l b u m i n , q u a i l o v a l b u m i n , and magnum e x t r a c t from b i r d s withdrawn from e s t r o g e n t r e a t m e n t  63  Ouchterlony p l a t e demonstrating a n t i - c h i c k ovalbumin w i t h c h i c k o v a l b u m i n , and s e r i a l d i l u t i o n s from a b i r d t r e a t e d f o r 10 days  63  the r e a c t i o n of ovalbumin, q u a i l o f a magnum e x t r a c t with estrogen  Plate 10c.  P a  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 o v a l b u m i n , q u a i l o v a l b u m i n , and a magnum e x t r a c t from a  yz^:™"-™^.. ^.*^r. r  g  ^  e  INTRODUCTION The mature o v i d u c t of a s e x u a l l y a c t i v e c h i c k e n 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 t o s e c r e t e t h e m a i n l y  proteinaceous  m a t e r i a l t h a t w i l l be i n 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 t h e 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  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 for  itself  and a b i o c h e m i c a l  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  marker  context.  R e c e n t l y , a g r e a t d e a l o f r e s e a r c h has a t t e m p t e d t o e s t a b l i s h i n d e t a i l the response o f the c h i c k o v i d u c t t o e s t r a d i o l and p r o g e s t e r o n e .  Work on  t h a t system has i n d i c a t e d t h a t t r e a t m e n t o f an immature c h i c k w i t h  estradiol  causes d r a m a t i c  I t was  growth and d i f f e r e n t i a t i o n o f t h e o v i d u c t t i s s u e .  further reported that withdrawal  of estrogen  a f t e r such t r e a t m e n t r e s u l t s i n  the l o s s o f f u n c t i o n i n g o f t h e n e w l y - d i f f e r e n t i a t e d c e l l s o f t h e o v i d u c t , b u t t h a t t h e c e l l s themselves remain. hormone.  That i s , the c e l l s a r e s t a b l e t o a l o s s of  T h i s c e l l s t a b i l i t y i s a v e r y marked d i v e r g e n c e  i n mammals, where t h i s i s known n o t t o o c c u r .  I t i s p o s s i b l e that  c h a r a c t e r i s t i c i s unique t o f o w l , o r t o t h e a v i a n group. initial  from t h e s i t u a t i o n this  However, b r i e f  r e p o r t s had i n d i c a t e d t h a t i n q u a i l , the c e l l s o f t h e o v i d u c t a r e l o s t  d u r i n g the l e n g t h y r e s t i n g p e r i o d ( 5 4 ) , thus i n d i c a t i n g t h a t t h e s t a b i l i t y o f the c h i c k o v i d u c t c e l l s i n the absence o f hormone i s c h a r a c t e r i s t i c o f t h a t species.  A d i f f e r e n c e as marked as t h e l i f e o r death o f t h e c e l l s i n t h e  absence of hormone i s s u r p r i s i n g i n s p e c i e s o f t h e same o r d e r , and so i t was decided  t o i n v e s t i g a t e i n g r e a t e r d e t a i l t h e response o f t h e q u a i l o v i d u c t t o  s t e r o i d hormones.  T h i s i n i t i a l s t u d y was t h e r e f o r e undertaken t o e s t a b l i s h  the major responses o f t h e q u a i l o v i d u c t t o s t e r o i d hormones, i n o r d e r t o serve as a base f o r f u r t h e r  experimentation.  LITERATURE REVIEW  Historical Literature The  o v i d u c t of a v i a n s p e c i e s , l i k e  i t s counterpart,  the mammalian g e n i t a l  t r a c t , has l o n g been known to be under the c o n t r o l o f sex hormones.  As  e a r l y as 1928, R i d d l e and Tange (1) demonstrated t h a t e s t r o g e n i c e x t r a c t s from the ovary or p l a c e n t a o f sows would induce pigeons,  growth o f the o v i d u c t o f immature  and n o t i c e d t h a t , per u n i t dose, t h i s growth was n o t as marked as  t h a t observed i n the uterus  o f mammals.  Two years, l a t e r , i n 1930, Juhn and  Gustavson (2) performed the same type o f experiment u s i n g a human p l a c e n t a l e x t r a c t on f o w l s , and observed a 1 0 - f o l d i n c r e a s e i n o v i d u c t w e i g h t .  Histo-  l o g i c a l examination demonstrated t h a t both growth and d i f f e r e n t i a t i o n  took  place:  the mucosal f o l d s i n c r e a s e d i n s i z e and the t u b u l a r g l a n d  developed. induce  Increased  these  cells  v a s c u l a r i z a t i o n was a l s o noted, and i t was p o s s i b l e t o  same e f f e c t s i n the rudimentary M u l l e r i a n ducts o f males and  females a t any stage o f post-embryonic development  (3, 4 ) . T h i s response has  a l s o been demonstrated i n sparrows ( 5 ) , and i n fowl and t u r k e y g i v e n s y n t h e t i c estrogens  orally  ( 6 ) , as w e l l as i n c h i c k s g i v e n e s t r a d i o l ( 7 ) .  In 1944, H e r t z thenic acid, a f o l i c to e s t r o g e n do  and S e b r e l l (8) noted t h a t a d i e t a r y d e f i c i e n c y of pantoacid precursor, i n h i b i t e d  the response o f the o v i d u c t  and i n 1949 (9) i t was demonstrated t h a t a p u r i n e  the same t h i n g .  Both these  analog  treatments a r e now known to i n t e r f e r e  DNA s y n t h e s i s , i n d i c a t i n g t h a t such s y n t h e s i s i s n e c e s s a r y  could with  f o r the growth  response. Gradually  i t became o f i n t e r e s t  to many r e s e a r c h e r s  t o i n v e s t i g a t e the  e f f e c t s o f the d i f f e r e n t sex s t e r o i d s s i n g l y and i n combination w i t h  each  other.  I n 1940, two groups i n v e s t i g a t e d t h e e f f e c t s o f hormones o t h e r  estrogens.  than  W i t s c h i and Fugo (10) demonstrated t h a t e s t r o g e n s s t i m u l a t e the  o v i d u c t s and r i g h t r u d i m e n t a r y ducts o f s t a r l i n g s , and t h a t p r o g e s t e r o n e d i d not c o n t r i b u t e t o t h e r e s p o n s e , a l t h o u g h  testosterone could.  In this species,  the females have p a i r e d v a s a d e f e r e n t i a w h i c h respond t o androgens, and a r e known t o s e c r e t e c o n s i d e r a b l e amounts o f t e s t o s t e r o n e i n t h e b r e e d i n g The  season.  a u t h o r s t h e r e f o r e thought i t p r o b a b l e t h a t t e s t o s t e r o n e c o n t r i b u t e d t o  the normal development o f t h e o v i d u c t , a t 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 t o augment the o v i d u c a l response t o e s t r o g e n  i n chicks  ( 1 1 ) , and i n n i g h t herons ( 1 2 ) .  I n t h i s l a t t e r c a s e , however, i t was a l s o  shown t h a t o n l y e s t r o g e n s can cause h y p e r p l a s i a  (12).  S u b s e q u e n t l y , a marked dichotomy o f r e s u l t s appeared, some i n d i c a t i n g s y n e r g i s m and some antagonism i n the o v i d u c a l response t o e s t r o g e n t i o n with other s t e r o i d s .  i n combina-  I n 1947, H e r t z e t a l (13) n o t e d t h a t p r o g e s t e r o n e  was a n t a g o n i s t i c t o t h e o v i d u c a l e s t r o g e n i c r e s p o n s e ; t h a t i s , a marked i n h i b i t i o n was n o t e d when the two were g i v e n s i m u l t a n e o u s l y ,  although  proges-  terone d i d n o t have an a n t a g o n i s t i c e f f e c t on t h e response o f serum l i p e m i a and c a l c e m i a oviduct.  to estrogen,  i n d i c a t i n g t h a t t h e antagonism was s p e c i f i c t o the  T h i s i n h i b i t i o n by p r o g e s t e r o n e o f the e s t r o g e n - i n d u c e d  hypertrophy  of t h e o v i d u c t o f immature c h i c k s was n o t e d a l s o by P h i l l i p s e t a l i n 1952 (14). On the o t h e r hand, i n 1951, Mason (15) demonstrated t h a t p r o g e s t e r o n e a l o n e caused no w e i g h t i n c r e a s e i n t h e c h i c k o v i d u c t , b u t when g i v e n w i t h estrogen, estrogen  caused a marked h y p e r t r o p h y , w h i c h was g r e a t e r than t h a t caused by alone.  He p o s t u l a t e d t h a t p r o g e s t e r o n e was n e c e s s a r y f o r normal  f u n c t i o n i n g of the o v i d u c t .  I n 1956, Breneman (16) noted the same e f f e c t o f  p r o g e s t e r o n e , and found the s y n e r g i s t i c e f f e c t f o r t e s t o s t e r o n e as w e l l . However, he a l s o n o t e d t h a t antagonism was observed w i t h low doses o f p r o g e s terone.  These f i n d i n g s were s u p p o r t e d by those o f Mason i n 1952 ( 1 5 ) , w h i c h  demonstrated  t h a t e s t r o g e n and p r o g e s t e r o n e may a c t s y n e r g i s t i c a l l y o r  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 o f b o t h . E s t r o g e n always caused an i n c r e a s e i n w e i g h t , whereas p r o g e s t e r o n e a l o n e had no such e f f e c t , b u t the g r e a t e s t i n c r e a s e s were always found when t h e two were combined i n unequal amounts. I n 1950, H e r t z (17) n o t e d t h a t e s t r o g e n caused an i n c r e a s e i n serum b i o t i n l e v e l , and t h a t an a n t i - b i o t i n f a c t o r p r e s e n t i n t h e egg-white was made i n t h e o v i d u c t under the i n f l u e n c e o f t h e f u l l y f u n c t i o n a l o v a r y . C h i c k s t h a t had been c a s t r a t e d and t r e a t e d w i t h e s t r o g e n 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 b o t h e s t r o g e n and p r o g e s t e r o n e d i d , thus d e m o n s t r a t i n g t h a t p r o g e s t e r o n e was f u n c t i o n a l i n c a u s i n g 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 n o t e d by B r a n t and Nalbandov i n 1952 ( 1 8 ) , who demonstrated  t h a t a l t h o u g h e s t r o g e n a l o n e caused growth o f t h e o v i d u c t , t h e r e  were few s e c r e t o r y g r a n u l e s .  A g a i n , androgen o r p r o g e s t e r o n e a l o n e had no  e f f e c t , b u t i n c o m b i n a t i o n w i t h e s t r o g e n , these s t e r o i d s caused an i n c r e a s e i n s i z e g r e a t e r than t h a t r e s u l t i n g from e s t r o g e n a l o n e , and s e c r e t o r y g r a n u l e s were abundant.  B r a n t and Nalbandov c o n c l u d e d t h a t t h e s i m u l t a n e o u s  presence o f two hormones was e s s e n t i a l f o r albumin g r a n u l e p r o d u c t i o n , b u t d i d n o t demonstrate which s t e r o i d was n o r m a l l y t h e a c t i v e second hormone. I n 1956 ( 1 9 ) , they c o n t i n u e d t h e i r s t u d i e s by d e m o n s t r a t i n g t h a t p r o g e s t e r o n e and t e s t o s t e r o n e a c t s y n e r g i s t i c a l l y w i t h e s t r o g e n i n the s e c r e t i o n o f albumin. They saw no a n t a g o n i s t i c e f f e c t , b u t i n t h e s e experiments t h e e s t r o g e n was  a d m i n i s t e r e d by p e l l e t i m p l a n t a t i o n a few days b e f o r e p r o g e s t e r o n e o r t e s t o s t e r o n e was g i v e n .  (The s i g n i f i c a n c e o f t h i s w i l l be apparent l a t e r . )  i m p l a n t i n g a s t r i n g o f beads i n t o t h e o v i d u c t , they demonstrated the hormones a l o n e c o u l d cause s e c r e t i o n o f the a l b u m i n .  By  t h a t none o f  When t h e c o m b i n a t i o n  o f e s t r o g e n p l u s p r o g e s t e r o n e o r e s t r o g e n p l u s t e s t o s t e r o n e was used, a l a r g e s e c r e t i o n was a p p a r e n t .  T h i s was more pronounced w i t h e s t r o g e n p l u s p r o g e s -  t e r o n e than w i t h e s t r o g e n p l u s t e s t o s t e r o n e . They c o n c l u d e d t h a t t h e f u l l s e c r e t o r y p o t e n t i a l o f t h e o v i d u c t r e q u i r e s 2 hormones, a l t h o u g h t h e second may be e i t h e r p r o g e s t e r o n e o r t e s t o s t e r o n e . E s t r o g e n and t e s t o s t e r o n e a c t e d s y n e r g i s t i c a l l y t o cause n i t r o g e n r e t e n t i o n ( 2 0 ) , which was n o t a c c o m p l i s h e d by e i t h e r s t e r o i d a l o n e . l y , i t was demonstrated  Similar-  t h a t e s t r o g e n and p r o g e s t e r o n e caused a r i s e i n t h e  f r e e r i b o f l a v i n c o n t e n t o f t h e magnum ( 2 1 ) , an i n c r e a s e which d i d n o t o c c u r i n response t o e s t r o g e n o r p r o g e s t e r o n e a l o n e .  I t t h e r e f o r e appeared  that both  hormones a r e e s s e n t i a l t o some r e s p o n s e s . However, t h e c o n t r o v e r s y o v e r whether p r o g e s t e r o n e a c t e d 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.  I n 1954, T u l l n e r e t a l (22)  showed t h a t b o t h p r o g e s t e r o n e and a l l o p r e g n a n e - 2 1 - o l - 3 , 2 0 - d i o n e a c e t a t e c o u l d antagonize estrogen. inhibitor.  T h i s l a t t e r compound, a n o n - c o r t i c o i d , was t h e g r e a t e s t  They a l s o demonstrated  t h a t 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 t h e e s t r o g e n i c response o f t h e o v i d u c t .  Since the allopregnane-  2 1 - o l - 3 , 2 0 - d i o n e a c e t a t e and d e s o x y c o r t i c o s t e r o n e d i d n o t a c t as progestagens i n any o f t h e a s s a y s used, t h e a u t h o r s c o n c l u d e d t h a t " t h e i n h i b i t i o n o f t h e o v i d u c t response i n e s t r o g e n - t r e a t e d c h i c k s 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 actions o f progesterone".  They thus s e p a r a t e d t h e 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 p r o g e s t e r o n e . I n 1963, Dorfman and Dorfman (23) r e p o r t e d t h a t the a n t a g o n i s t i c o r s y n e r g i s t i c e f f e c t of p r o g e s t e r o n e depended on b o t h the c o n c e n t r a t i o n r a t i o the dose. i n 1956  These r e s u l t s s u p p o r t e d those o f Mason i n 1952  (16) . P r o g e s t e r o n e was  a l s o s y n e r g i s t i c w i t h e s t r o g e n , a l t h o u g h i t was  Desoxycorti-  i n h i b i t o r y at  The a u t h o r s noted t h a t i t had been s u g g e s t e d (22) t h a t the a n t a -  g o n i s t i c e f f e c t of p r o g e s t e r o n e was ties.  Pregne-5-ene-3,20-dione  s y n e r g i s t i c w i t h e s t r o g e n , but had no e f f e c t a l o n e .  c o s t e r o n e was low doses.  ( 1 5 ) , and Breneman  found t o be s y n e r g i s t i c t o low doses of  e s t r o g e n , but a n t a g o n i s t i c t o h i g h doses o f e s t r o g e n . a c e t a t e was  and  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 -  Having observed the i d e n t i c a l a c t i o n o f c o r t i c o i d s , they suggested  the s y n e r g i s t i c e f f e c t of p r o g e s t e r o n e on the e s t r o g e n i c response may  that  a l s o be  independent o f 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 s u g g e s t i o n r a i s e s the q u e s t i o n of why p r o g e s t e r o n e would be exp e c t e d to a c t as a p r o g e s t a g e n i n a s p e c i e s which does n o t become p r e g n a n t ; 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 q u e s t i o n when the amount o f d i s c u s s i o n s t i l l devoted t o t h i s phenomenon i s c o n s i d e r e d ( 2 4 ) .  Definite progestational  e f f e c t s of p r o g e s t e r o n e i n t h i s non-mammalian system have y e t to be ed.  demonstrat-  At any r a t e , Dorfman and Dorfman c o n c l u d e d t h a t the type of response to  p r o g e s t e r o n e , whether a n t a g o n i s t i c o r 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 o f 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 o t h e r ( 2 4 ) .  Thus, a t a maximal e s t r o g e n r e s p o n s e , p r o g e s t e r o n e  appeared  t o be a n t a g o n i s t i c , whereas a t m i n i m a l e s t r o g e n r e s p o n s e s , i t  appeared  to be  synergistic.  A comparison o f these e a r l y s t u d i e s w i t h each o t h e r i s h i n d e r e d by a number o f problems w h i c h 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 v e r y 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 , d e m o n s t r a t i n g o n l y t h a t they c o n t a i n ed e s t r o g e n , and n o t e x c l u d i n g t h e p o s s i b i l i t y t h a t they c o n t a i n e d p r o g e s t e r one o r o t h e r hormones as w e l l .  Of t h e s t u d i e s w h i c h i m m e d i a t e l y f o l l o w e d  t h o s e , e s t r o g e n s were chosen by somewhat random methods, s i n c e t h e b i o l o g i c a l ly  a c t i v e s t e r o i d was n o t 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 p h a -  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 ; o t h e r s used e s t r o n e , s t i l l used s t i l b o e s t r o l o r one of i t s d e r i v a t i v e s .  These a l l  others  have d i f f e r e n t  p o t e n c i e s , and i t i s known t h a t t h e i r e f f e c t s , e s p e c i a l l y i n c o m b i n a t i o n w i t h progesterone, w i l l vary. in  these s t u d i e s .  The a c t u a l dose o f a c t i v e agent t h e r e f o r e d i f f e r e d  More r e c e n t l y , i n s t u d i e s c o n c e r n i n g c o m b i n a t i o n  experi-  ments, t h e method i n which t h e two hormones were a d m i n i s t e r e d i s a l s o r e l e v a n t , s i n c e 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 t h e serum e s t r o g e n l e v e l than i n j e c t i o n .  Because o f t h e same c o n s i d e r a t i o n , t h e time c o u r s e o f  i n j e c t i o n s would be i m p o r t a n t , i n experiments where the hormones were i n j e c t e d . However, i n s p i t e o f these problems, drawn from t h i s work: of  some t a n g i b l e c o n c l u s i o n s can s t i l l be  (1) e s t r o g e n s t i m u l a t e d t h e growth and d i f f e r e n t i a t i o n  t h e o v i d u c t ; (2) a second s t e r o i d a c t e d t o i n c r e a s e s e c r e t i o n ; (3) t h e  e f f e c t o f t h e second hormone on t h e response t o e s t r o g e n would depend on t h e r e l a t i v e doses o f t h e two hormones, and on t h e s t a t e o f t h e o v i d u c t a t t h e time o f a d m i n i s t r a t i o n o f t h e second  steroid.  Most o f these e a r l y s t u d i e s , t h e n , were concerned w i t h t h e o v e r a l l  growth  response o f t h e o v i d u c t t o e s t r o g e n s a l o n e , o r e s t r o g e n s and o t h e r hormones in  combinations.  Due t o t h e r e l a t i v e l y u n r e f i n e d n a t u r e o f t h e t e c h n i q u e s  a v a i l a b l e a t t h e t i m e , t h e e f f e c t s o f t h e s e 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 n o t i n v e s t i g a t e d .  However, one p r o t e i n had been known t o e x i s t  due t o i t s b i o l o g i c a l a c t i v i t y o f b i n d i n g t o t h e 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 o f a n i m a l s o r the growth medium of m i c r o - o r g a n i s m s such as y e a s t , the p r o t e i n caused death t o the organism. assay f o r a s p e c i f i c p r o t e i n , r e s e a r c h e r s  With t h i s a b i l i t y  c o u l d i n v e s t i g a t e the e f f e c t s of  v a r i o u s hormones on the p r o d u c t i o n o f 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 a l r e a d y mentioned, and s h o u l d  seen i n t h a t  light.  A v i d i n was  to  be  f i r s t named by E a k i n et a l (25) as the p r o t e i n f a c t o r i n  egg-  w h i t e t h a t caused egg-white i n j u r y t o a d u l t hens and r a t s f e d on a d i e t of raw egg-white. i n j u r y (26).  A d d i t i o n of b i o t i n to the d i e t p r e v e n t e d or cured B i o t i n accumulated i n the y o l k of the egg  c o n t r o l ( 2 8 ) , w h i c h may  e x p l a i n , i n r e t r o s p e c t , why  egg-white  ( 2 7 ) , under e s t r o g e n i c  the a v i d i n does not  cause  i n j u r y to the embryo. I n 1942,  H e r t z and S e b r e l l (29) demonstrated t h a t a v i d i n was  the egg-white of t u r k e y , duck, goose and hen and  i t may  They suggested t h a t  a s e c r e t o r y p r o d u c t of o v i d u c t s o f b i r d s and a m p h i b i a n s , and  p l a y an i m p o r t a n t  in  eggs, i n the o v i d u c t s o f hens  f r o g s , and i n the e g g - j e l l y of two s p e c i e s of Rana.  a v i d i n was  present  p a r t i n embryonic development ( 2 9 ) .  that  A l t h o u g h 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 t o be p r e s e n t lizards  a l s o i n o v i d u c t s of p i g e o n s (30)  (31).  I n 1943,  Frapps e t a l (32) demonstrated t h a t a v i d i n was  found i n a l l  l e v e l s o f the magnum o f c h i c k o v i d u c t , but not i n the magna of hens.  and  They c o n c l u d e d t h a t a v i d i n p r o d u c t i o n was  or i n d i r e c t l y , w i t h the complete r e p r o d u c t i v e continued  non-laying  associated, either directly  f u n c t i o n of the o v a r y .  They  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 o f hormones on a v i d i n  production  (33).  U s i n g immature c h i c k s , they demonstrated t h a t the  o f 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 o f s t i l b o e s t r o l and although  production  progesterone,  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.  S i n c e a t t h a t time the i d e n t i t y o f the hormones s e c r e t e d by the b i r d 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  c o u l d a l s o induce  o v u l a t i o n , they concluded  ovary  testosterone  o n l y t h a t the p r o d u c t i o n  a v i d i n r e q u i r e d some o t h e r s t e r i o d i n a d d i t i o n t o e s t r o g e n .  This  of  other  s t e r o i d , they p o s t u l a t e d , c o u l d have been o f e x t r a - o v a r i a n o r i g i n ( 3 2 ) . l i g h t o f l a t e r developments i n t h i s a r e a  In  ( 2 4 ) , t h a t c o n c l u s i o n showed  amazing i n s i g h t . I n 1944  (34), H e r t z e t a l demonstrated t h a t b o t h t e s t o s t e r o n e and  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 p r o g e s t e r o n e ,  desoxy-  c o u l d induce a v i d i n produc-  t i o n i n c h i c k s t h a t had been p r e - t r e a t e d w i t h e s t r o g e n .  They r e c o g n i z e d  the  i n t e r c h a n g e a b i l i t y o f these s t e r o i d s i n o t h e r f u n c t i o n , such as the m a i n t e n ance o f l i f e i n a d r e n a l e c t o m i z e d b l e e d i n g i n the monkey.  animals  and  the p r e c i p i t a t i o n of  They t h e r e f o r e q u e s t i o n e d w h i c h o f these  might p l a y the a c t i v e r o l e i n i n d u c i n g a v i d i n s y n t h e s i s i n v i v o I n 1949  endometrial agents  (34).  ( 3 5 ) , H e r t z e t a l f u r t h e r demonstrated t h a t s t i l b o e s t r o l  treat-  ment caused a 5 - f o l d i n c r e a s e i n the b i o t i n a c t i v i t y i n the serum, but a v i d i n f o r m a t i o n o c c u r r e d i n the o v i d u c t . and p r o g e s t e r o n e of a v i d i n c o n t e n t  S i n c e a d m i n i s t r a t i o n of  no  estrogen  caused b o t h an i n c r e a s e o f b i o t i n a c t i v i t y i n the serum and i n the o v i d u c t , they thought t h a t 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 More r e c e n t l y , the system was  (17).  l o o k e d a t i n g r e a t e r d e t a i l , due  i n c r e a s e d s e n s i t i v i t y of some t e c h n i q u e s , and  the development o f new  Most o f these l a t e r s t u d i e s c e n t e r e d around the changes i n t o t a l o r  to the ones. specific  p r o t e i n c o n t e n t o f the o v i d u c t  due to hormone  administration.  In 1960, Brown and Jackson (36) r e p o r t e d  t h a t there were no s t r i k i n g  d i f f e r e n c e s i n the compositions o f t h e organ i n d i f f e r e n t age groups, a l t h o u g h the o v i d u c t  o f the broody hen had a s l i g h t l y  lower p r o t e i n c o n t e n t .  Kalman and Opsahl demonstrated t h a t e s t r o g e n caused an i n c r e a s e d t i o n o f C-  -leucine into protein  (37).  I n 1965,  performed e l e c t r o p h o r e s i s o f the w a t e r - s o l u b l e  proteins  and A3 and the post-albumins i n the t r e a t e d o v i d u c t .  estrogen only,than  magnum p r o t e i n s  of the o v i d u c t magnum i n albumin A2  There was a lower con-  i n broody hens and i n hens t r e a t e d w i t h  i n hens t r e a t e d w i t h LH o r e s t r o g e n p l u s LH.  there was a l a r g e i n c r e a s e  incorpora¬  Oades and Brown (38)  a f t e r e s t r o g e n treatment, and demonstrated a r e l a t i v e i n c r e a s e  tent of water-soluble  i n the w a t e r - s o l u b l e  magnum p r o t e i n s  However, i n oviducts  from hens t r e a t e d w i t h e s t r o g e n p l u s p r o g e s t e r o n e o r e s t r o g e n p l u s one.  I n 1961,  testoster-  T h i s upheld the e a r l i e r work which demonstrated that a second s t e r o i d ,  when g i v e n w i t h e s t r o g e n ,  acted  to increase s e c r e t i o n .  An i n c r e a s e  i n alka-  l i n e phosphatase a c t i v i t y was demonstrated i n 1965 by Pande e t a l (39), and an i n c r e a s e  i n o v i d u c a l glycogen i n 1969 by C e c i l et_ a l (40), as a r e s u l t o f  estrogen a c t i o n .  Recent H i s t o r y Even more r e c e n t l y , a number o f s t u d i e s sively  the response o f the o v i d u c t  primary and secondary s t i m u l a t i o n .  t o e s t r o g e n and p r o g e s t e r o n e , both  response.  events o f primary s t i m u l a t i o n a r e summarized i n F i g u r e  E s t r a d i o l causes the i n i t i a l  during  The r e s u l t s o f 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 o f the o v i d u c a l The  (41-53) have i n v e s t i g a t e d e x t e n -  1 and F i g u r e 2.  d i s p e r s i o n o f the stroma o f the t i s s u e ,  resulting  F i g u r e 1.  Proposed Time Sequence of E s t r o g e n i c Responses, w i t h I n f l u e n c e s of Progesterone  Estrogen  Estrogen  StJoma  Epithelium  Progenitor Tubular Gland C e l l s  1 , Stromal swelling  I n v a s i L by c e l l s from blood  » Active Cells  Quiescent C e l l s  Water Imbibition  Hyperemia  E,  Stromal Dispersion-  'or  (Others)  P,/ EP  to P r o t o d i f f e r e n t i a t e d S t a t e , w i t h Ovalbumin Synthesis  E (lengthy treatment), P, or EP (P enhances)  blocks F u r t h e r DNA S y n t h e s i s , to Tubular Gland C e l l s enhances NonFunctioning Cells  See  Hormone  4r ~~  +  ~ ^  Hormone  Cell-Specific Protein S y n t h e s i s ; Ovalbumin, Lysozyme, Conalbumin, Ovomucoid  EP = E s t r o g e n +  only Avidin S^thesis by G o b l e t C e l l s  text for explanation.  E = Estrogen; P = Progesterone;  Epithelial Differe 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  Progesterone  F i g . 2a  Chick Oviduct  I•  Infundibulum  Magnum  JL.  11  Isth- shell gland Vagina  white^ protein^) F i g . 2b  Magnum (cross section)  Adapted from oka & Schimke (Ret. 48)  Undifferentiated  Stroma Immature  Ciliated  Prolonged ~ ^ ^ § & C Goblet or Progesterone J l s i r f ^ e c r e t o r y cells Tubular gland cells. containing egg-white proteins  i n hyperemia and w a t e r i m b i b i t i o n ( 4 1 ) .  Perhaps as a r e s u l t o f t h e hyperemia,  mononuclear c e l l s from t h e b l o o d invade t h e stroma, g i v i n g r i s e t o t h e p o s s i b i l i t y of c e l l - c e l l interactions  (41).  C o n c o m i t a n t l y , o r 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 t h e lumen d i f f e r e n t i a t e (41) as f a r as 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 ( 4 2 ) , i n w h i c h i n d e n t a t i o n s i n t o t h e stroma appear, b u t a r e n o t y e t c o m p l e t e l y formed ( 4 3 ) .  These p r o t o d i f f e r e n t i a t e d t u b u l a r g l a n d  c e l l s c o n t a i n s e c r e t o r y g r a n u l e s , and ovalbumin magnum ( 4 3 ) .  i s present i n the oviduct  A t t h i s s t a g e , m i c r o f i l a m e n t s become e s s e n t i a l i n t h e budding  o f f o f these c e l l s t o form t u b u l a r g l a n d c e l l s e s t r o g e n and p r o g e s t e r o n e  (44).  Either estrogen, or  together, can induce the d i f f e r e n t i a t i o n o f the  o v i d u c t magnum t o t h i s s t a g e ; however, o n l y e s t r o g e n can c o n t i n u e t h e d i f f e r e n t i a t i o n o f 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 t o t u b u l a r g l a n d c e l l s ( 4 3 ) . The presence o f p r o g e s t e r o n e  t o g e t h e r w i t h e s t r o g e n a b o l i s h e s t h e subsequent  d i f f e r e n t i a t i o n o f t h e s e p r o t o d i f f e r e n t i a t e d c e l l s t o t u b u l a r g l a n d c e l l s (43) and l e a d s i n s t e a d t o 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 o f c i l i a t e d c e l l s and g o b l e t cells  (43, 45, 46, 4 7 ) . The p r o c e s s o f budding o f f t o form t u b u l a r g l a n d c e l l s c o n t i n u e s f o r a  number o f days, depending on dose.  A f t e r i t i s complete,  the remaining  epith-  e l i a l c e l l s d i f f e r e n t i a t e t o become e i t h e r c i l i a t e d c e l l s o r s e c r e t o r y g o b l e t cells  ( 4 1 , 45, 4 8 ) . P r o g e s t e r o n e enhances t h i s type o f d i f f e r e n t i a t i o n ( 4 7 ) .  I f progesterone  i s g i v e n w i t h e s t r o g e n , so t h a t t u b u l a r g l a n d c e l l  differentia-  t i o n i s a r r e s t e d a t t h e p r o t o d i f f e r e n t i a t e d s t a g e , then c e l l s c a n be seen i n the e l e c t r o n m i c r o s c o p e which c o n t a i n b o t h s e c r e t o r y g r a n u l e s and c i l i a ( 4 3 ) . As t h e t u b u l a r g l a n d c e l l s d i f f e r e n t i a t e , ovalbumin 3 0 0 - f o l d i n t h e course o f 15 days o f treatment lysozyme,  (49).  concentration increases The c o n c e n t r a t i o n o f  a n o t h e r p r o t e i n made i n t h e t u b u l a r g l a n d c e l l s , a l s o i n c r e a s e s i n  t h i s p e r i o d (45, 4 8 ) . The e f f e c t o f p r o g e s t e r o n e on t h i s p r o c e s s i s a complex one, p r i m a r i l y on the time a t which i t i s a d m i n i s t e r e d . a l l the p r o c e s s e s t h a t r e q u i r e DNA  depending  Progesterone w i l l  s y n t h e s i s (45, 4 8 ) , and so w i l l  inhibit  abolish  the growth of the o v i d u c t i f i t i s g i v e n c o n c o m i t a n t l y w i t h e s t r o g e n from the b e g i n n i n g of treatment  (45, 48).  T h i s r e d u c t i o n i s the r e s u l t o f a b o l i s h i n g  the t u b u l a r g l a n d 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 v a s t b u l k of  the d i f f e r e n t i a t e d o v i d u c t magnum.  However, i f the o n s e t of  progesterone  treatment i s d e l a y e d somewhat, so t h a t e s t r o g e n has had an o p p o r t u n i t y to induce t u b u l a r g l a n d c e l l s , then those c e l l s t h a t have d i f f e r e n t i a t e d w i l l c o n t i n e d s y n t h e s i z i n g ovalbumin ment has begun (45, 48).  and lysozyme, even a f t e r p r o g e s t e r o n e  treat-  Indeed, p r o g e s t e r o n e has a s y n e r g i s t i c e f f e c t  the f u n c t i o n i n g of the t u b u l a r g l a n d c e l l s  on  (45, 48).  P r o g e s t e r o n e has the o p p o s i t e e f f e c t on the d i f f e r e n t i a t i o n o f 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 g o b l e t c e l l s : d i f f e r e n t i a t i o n g r e a t l y (47).  I n a d d i t i o n , p r o g e s t e r o n e has an a c t i o n on  t i s s u e w h i c h e s t r o g e n does not have:  i n h i g h e s t amounts when p r o g e s t e r o n e  inhibitory  this  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 g o b l e t c e l l s  treatment:  i t enhances t h e i r  (50).  This p r o t e i n i s present  i s a d m i n i s t e r e d part-way through  estrogen  e i t h e r too l o n g or too s h o r t a p r e - t r e a t m e n t w i t h e s t r o g e n i s (49).  Upon e s t r o g e n w i t h d r a w a l , a decrease T h i s i s l e s s marked i n the case of DNA  i n a l l t i s s u e components i s seen.  c o n t e n t ( 4 8 ) , but w i t h time the c e l l s  a r e a l s o l o s t (51, 5 2 ) , c o n t r a r y t o e a r l i e r r e p o r t s (45, 4 8 ) .  Thus,  mainten-  ance o f the d i f f e r e n t i a t e d s t a t e o f the o v i d u c t r e q u i r e s c o n t i n u e d s t i m u l a t i o n by the hormone.  R e a d m i n i s t r a t i o n o f e s t r o g e n to c h i c k s w i t h d r a w n from  treatment  r e s u l t s i n a q u i c k e r , g r e a t e r response w i t h a much s h o r 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, 5 3 ) . g l a n d c e l l s resume f u n c t i o n i n g , a c c o u n t i n g Estrogen  and p r o g e s t e r o n e  the response t o p r o g e s t e r o n e  The r e m a i n i n g  tubular  f o r the s h o r t e r time l a g ( 4 8 ) .  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 i s l e s s than t h a t t o e s t r o g e n  (48, 5 3 ) . When  b o t h a r e g i v e n t o g e t h e r , the r e s p o n s e i s g r e a t e r than the response to e i t h e r a l o n e , i n d i c a t i n g t h a t the two hormones a c t 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 synthesis (48), although division.  they a c t a n t a g o n i s t i c a l l y w i t h r e g a r d t o c e l l  I t has t h e r e f o r e been suggested t h a t 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 a r e s t i m u l a t e d by e s t r o g e n i n d i f f e r e n t manners, such t h a t p r o g e s terone i n t e r f e r e s w i t h the one p r o c e s s , b u t not the o t h e r  (45, 4 8 ) .  The o v i d u c t r e p r e s e n t s an e x c e l l e n t example o f 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 t o s t e r o i d s t i m u l a t i o n .  I t has b i o c h e m i c a l markers t h a t  can be used to measure the e f f e c t o f e i t h e r e s t r o g e n o r p r o g e s t e r o n e ,  and t h e  c o n s i d e r a b l e growth produces l a r g e t i s s u e masses f o r b i o c h e m i c a l a n a l y s i s . I t r e p r e s e n t s a t i s s u e w h i c h responds t o two i n d u c e r s , where the a c t i o n s o f b o t h a r e known ( a t l e a s t i n p a r t ) , and where the a c t i o n o f the second depends t o a l a r g e e x t e n t on the a c t i o n of t h e f i r s t .  The o v i d u c t t h e r e f o r e r e p r e s e n t s  e x c e l l e n t system i n w h i c h to study the b i o c h e m i c a l p r o c e s s e s  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 . 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 p r o c e s s e s : 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 o f e s t r o g e n from t r e a t m e n t , progesterone  Two  one d i r e c t e d by  t o c h i c k s withdrawn  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 t h e r o l e o f  i n inducing a v i d i n synthesis.  an  INTRODUCTION  The  response of the c h i c k o v i d u c t to e s t r o g e n  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 .  and p r o g e s t e r o n e  The work on t h a t system i n d i c a t e d t h a t  t r e a t m e n t of an immature c h i c k w i t h e s t r a d i o l caused d r a m a t i c d i f f e r e n t i a t i o n of the o v i d u c t t i s s u e . d i f f e r e n c e s r e p o r t e d was  However, one  and  of the most s t r i k i n g  from hormone t r e a t m e n t .  In  ( C o r t u n i x c o r t u n i x j a p o n i c a - ) of the same o r d e r ( 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 t h a t the c e l l s of the o v i d u c t were l o s t d u r i n g  l e n g t h y r e s t i n g p e r i o d (54, 55). (56).  growth  t h a t , c o n t r a r y to the case i n mammals, the c e l l s of  the c h i c k o v i d u c t were not l o s t upon w i t h d r a w a l another species  has  A l t h o u g h t h i s i s now  a t the time t h i s s t u d y was  T h i s has a l s o been more r e c e n t l y  the  confirmed  known to be the case i n c h i c k s as w e l l (51, 5 2 ) , begun, i t had not y e t been demonstrated.  S i n c e a d i f f e r e n c e as marked as the l i f e o r 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 s p e c i e s of the same o r d e r , i t was  decided  to i n v e s t i g a t e i n g r e a t e r d e t a i l the major r e s p o n s e s of the q u a i l  oviduct to estrogen This i n i t i a l  and  study  progesterone. t h e n , was  t o r y base f o r f u r t h e r s t u d i e s . q u a i l ' s response to e s t r o g e n system a l r e a d y s t u d i e d .  d e s i g n e d to be an i n t r o d u c t o r y and  explora-  I t t h e r e f o r e attempted t o e s t a b l i s h the  and p r o g e s t e r o n e i n terms comparable t o the  Thus, I d e c i d e d  chick  t o 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 p r o g e s t e r o n e 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 tion.  to g l a n d c e l l f o r m a t i o n  and e p i t h e l i a l c e l l  differentia-  I n 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 w e i g h t , p r o t e i n , RNA  and DNA  c o n t e n t , 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 r e f e r e n c e ovalbumin, lysozyme and a v i d i n s y n t h e s i s .  to  MATERIALS AND METHODS  Treatment  o f the Animals  Japanese  q u a i l o f the s t r a i n C o t u r n i x c o t u r n i x j a p o n i c a . were o b t a i n e d  from the P o u l t r y S c i e n c e Department, U. B . C . , on the f i r s t  day a f t e r h a t c h i n g .  They were m a i n t a i n e d i n e l e c t r i c a l l y heated b r o o d e r s , f e d on 28% Turkey S t a r t e r , and were kept on a s c h e d u l e o f 8 hours l i g h t , 16 hours At  f i v e days of age, a 15-day i n j e c t i o n p l a n was begun.  darkness.  Each b i r d  received  0.2 mg d a i l y o f 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.  C o n t r o l b i r d s r e c e i v e d the v e h i c l e o n l y .  On the t e n t h day o f  treatment the e x p e r i m e n t a l animals were d i v i d e d i n t o two groups, one o f which received progesterone  (0.2 mg i n 0.1 ml o f sesame o i l d a i l y ) , w h i l e the o t h e r  c o n t i n u e d t o r e c e i v e e s t r a d i o l f o r t h e remaining 5 days o f treatment. treatment was then f o l l o w e d by t e n days o f hormone w i t h d r a w a l .  This  Five to nine  animals from each group were s a c r i f i c e d by e t h e r - a n a e s t h e s i a on the f i r s t , fifth,  t e n t h , f i f t e e n t h , and t w e n t y - f i f t h days o f treatment, and t h e i r  ovi-  ducts were e x c i s e d and f i x e d i n g l u t a r a l d e h y d e ; or weighed and then f r o z e n a t -70°C, t o be homogenized l a t e r f o r the a s s a y s .  When t i s s u e was e x c i s e d f o r  the c e l l - s p e c i f i c p r o t e i n a s s a y s , o n l y t h e magnum p o r t i o n of t h e o v i d u c t was used; i n c o n t r o l animals t h i s was e s t i m a t e d by u s i n g o n l y the upper h a l f of the o v i d u c t .  T o t a l Growth Assays F i v e to n i n e o v i d u c t s from each of the i n d i c a t e d and reweighed,  time groups were thawed  and homogenized i n d i s t i l l e d water by f o u r 45-second  bursts of  a S o r v a l l Omni-Mixer, a t a speed s e t t i n g o f 5, i n t e r r u p t e d by one-rminute cooling periods.  DNA and RNA were e x t r a c t e d from t h e homogenate by t h e method o f S c h n e i d e r (57).  DNA was assayed by t h e d i p h e n y l a m i n e r e a c t i o n ( 5 8 ) , w i t h c a l f thymus  DNA (Calbiochem) as t h e s t a n d a r d .  RNA was a s s a y e d by the o r c i n o l r e a c t i o n  ( 5 9 ) , w i t h y e a s t RNA (Calbiochem) as t h e s t a n d a r d .  P r o t e i n was e s t i m a t e d by  the method o f Lowry e t a l ( 6 0 ) , w i t h c r y s t a l l i n e b o v i n e serum albumin as standard.  S p e c i f i c P r o t e i n Assays O v i d u c t 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 N a C l , t o g i v e a 2% (w/v) homogenate, f o r t h e a s s a y s o f o v a l b u m i n , 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 a t 105,000 x g, and t h e s u p e r n a t a n t was r e c o v e r e d . 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 d e s c r i b e d by Kabat and Meyer (61) f o r t h e p u r i f i c a t i o n o f ovalbumin c h i c k e n egg-white.  from  Egg-white was d i l u t e d w i t h an e q u a l volume o f d i s t i l l e d  w a t e r , and a volume o f 100% s a t u r a t e d ammonium s u l p h a t e s o l u t i o n e q u a l t o t h e t o t a l volume was added, t o b r i n g t h e m i x t u r e t o 50% s a t u r a t i o n .  A f t e r 2 hours  a t room t e m p e r a t u r e , 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 t h e s u p e r n a t a n t a d j u s t e d t o pH 4.6 w i t h 0.2 M H S 0 . 2  4  S a t u r a t e d ammonium  s u l p h a t e 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 o p a l e s c e n c e was  reached.  The m i x t u r e was a l l o w e d t o s t a n d f o r 2 days, and t h e 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 o b t a i n e d was r e c r y s t a l l i z e d  t w i c e from 50% s a t u r a t e d ammonium s u l p h a t e , r e c o v e r e d by c e n t r i f u g a t i o n , and dried i n a dessicator.  I t was l a t e r d i s s o l v e d i n homogenizing  a p p l i e d t o a Sephadex G-200 column, 60 cm x T r i s - H C l , pH 7.0".  buffer,  C H I and e l u t e d w i t h 0.02'M  Immunological c r o s s - r e a c t i v i t y o f q u a i l and c h i c k ovalbumin  (Calbiochem)  and the presence o r absence o f ovalbumin i n the homogenates of o v i d u c t s from v a r i o u s s t a g e s of hormonal t r e a t m e n t was e s t a b l i s h e d by the use of O u c h t e r l o n y micro-diffusion tests.  T h i s was performed as d e s c r i b e d by Work and Work (62)  w i t h 1% agar i n p h o s p h a t e - b u f f e r e d 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 w a t e r , then d r i e d and s t a i n e d w i t h amido b l a c k . Lysozyme was  assayed by the method of L i t w a c k ( 6 3 ) .  The a c t i v i t y  was  measured by the change i n t r a n s m i t t a n c e a t 645 my a t 25°C, i n a Unicam SP Spectrophotometer. sium phosphate  The r e a c t i o n m i x t u r e c o n s i s t e d o f 1 ml o f 0.066 M p o t a s -  b u f f e r , pH 6.2, w i t h 0.1% N a C l ; and Micrococcus- l y s o d e i k t i c u s ,  20 mg d r y weight/100  ml o f b u f f e r s o l u t i o n .  o v i d u c t e x t r a c t was added. of  d i s t i l l e d water.  0.1 ml of enzyme s o l u t i o n o r  The r e a c t i o n m i x t u r e was  read a g a i n s t a blank  A c t i v i t y i s e x p r e s s e d as micrograms of lysozyme p r e s e n t ,  and i s based on the a c t i v i t y o f p u r i f i e d c h i c k e n egg-white lysozyme chem) as s t a n d a r d . A v i d i n was  1800  The lower l i m i t o f s e n s i t i v i t y o f t h i s a s s a y was  assayed by the method o f Korenman and O'Malley  a s s a y i s based on the b i n d i n g of a v i d i n t o C - c a r b o n y l b i o t i n 1 4  Amersham S e a r l e ) .  (Calbio-  To 0.5 ml of C - b i o t i n (1.35 x 1 0 ~ 1 4  3  (64).  2 ygm/ml. The  (45 mCi/mmole;  yg/ml) was  added  0.1 ml o f a v i d i n s t a n d a r d (0.54 u n i t s / m g , N u t r i t i o n a l B i o c h e m i c a l s ) o f concent 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  c a r b o n a t e , o r 0.1 ml o f o v i d u c t e x t r a c t .  A f t e r 15 minutes a t room t e m p e r a t u r e ,  1 ml o f ammonium c a r b o n a t e c o n t a i n i n g 10 mg b e n t o n i t e was added. a d d i t i o n a l 5 m i n u t e s , the m i x t u r e was  A f t e r an  t r a n s f e r r e d t o a 3 ml p l a s t i c  Luer-Lok  s y r i n g e (B-D), w i t h a Swinnex f i l t e r h o l d e r ( M i l l i p o r e ) a t t a c h e d t o the end. P r e s s u r e was  then a p p l i e d w i t h the p l u n g e r , and the m i x t u r e was  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 ) .  filtered  The f i l t e r was washed t w i c e  w i t h about 1.8  ml o f 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 , d i s s o l v e d i n 10 ml of Bray's s o l u t i o n (65) counted f o r 10 minutes i n an Isocap 300 Chicago) on Channel 5. was  about  Background was  and  l i q u i d s c i n t i l l a t i o n counter a p p r o x i m a t e l y 30 cpm,  and  (Nuclear  efficiency  85%.  Histology The  f r e s h l y excised  hyde i n 0.1  t i s s u e was  M phosphate b u f f e r , pH  through a s e r i e s of a l c o h o l s t i s s u e was  embedded i n JB-4  f i x e d i n Bouin's 7.2,  (50-100%), and  A;  i n S o l u t i o n A and  at  One  to two  three changes of acetone.  P l a s t i c from P o l y s c i e n c e s ,  follows:  ture.  Inc.  three changes (15 minutes each) i n S o l u t i o n  45°C w i t h H a r r i s ' s h e m o t o x y l i n and c o n t r a s t , and  The  (butoxy-ethanol-  B  (42:1), o v e r n i g h t  a t room tempera-  m i c r o n s e c t i o n s were cut u s i n g g l a s s k n i v e s ,  afforded only l i t t l e  glutaralde-  f o r a t l e a s t 24 hours, then dehydrated  g l y c o l m e t h a c r y l a t e ) as f i n a l polymerization  or i n 2.5%  eosin.  The  thin plastic  and  stained  sections  so were photographed u s i n g a phase  o b j e c t i v e , thus g i v i n g the r e s u l t a n t p i c t u r e s an u n u s u a l and  often  contrast  fluid  RESULTS  I.  Histology  A.  C o n t r o l Animals The o v i d u c t s from c o n t r o l b i r d s appear t o undergo p r o g r e s s i v e changes  d u r i n g the treatment p e r i o d , p r i m a r i l y i n t h e stroma.  These i n c l u d e changes  i n t h e p a t t e r n o f c e l l arrangement, i n n u c l e a r morphology, i n s i z e and i n vascularization. As can be seen i n o v i d u c t s from b i r d s w h i c h had s e r v e d as c o n t r o l s f o r 5 days ( r e f e r t o P l a t e 1 ) , t h e s t r o m a l c e l l s were v e r y d e n s e l y packed a g a i n s t the e p i t h e l i u m .  A t t h e base o f each f u t u r e mucosal f o l d , t h e c e l l s  were much l e s s d e n s e l y packed t o g e t h e r , and t h e r e appeared t o be g r e a t e r 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 s i m p l e 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 t h e appearance o f a p r i m o r d i u m , such as a l i m b bud. By f i f t e e n days o f c o n t r o l t r e a t m e n t ,  t h i s p a t t e r n of stromal  cell  arrangement had changed ( r e f e r t o P l a t e 2 ) . The s t r o m a l c e l l s had d i s p e r s e d n o t i c e a b l y , and were no l o n g e r d e n s e l y packed a g a i n s t the e p i t h e l i u m .  They  appeared t o be u n i f o r m l y d i s t r i b u t e d throughout t h e l e n g t h and w i d t h o f t h e mucosal f o l d .  However, t h e c e l l s i n t h e c e n t e r o f t h e f o l d s appear t o have  n u c l e i which a r e l o n g e r and t h i n n e r than those c l o s e r t o t h e e p i t h e l i u m . These l o n g , t h i n n u c l e i appeared t o be o r i e n t e d a l o n g t h e v e r t i c a l a x i s of the f o l d .  T h i s g i v e s an o v e r a l l appearance o f s t r e a m i n g , as i f t h e c e l l s  were b e i n g sprayed  out from a f o u n t a i n h e a d  a t the c e n t e r and base o f each f o l d .  Between t h e f o l d s , near t h e p e r i p h e r y o f t h e o v i d u c t , t h e l o n g t h i n n u c l e i a r e h o r i z o n t a l l y o r i e n t e d , so t h a t t h e s t r e a m i n g appeared t o be  continuous  from the base and c e n t e r o f one  f o l d t o the base and c e n t e r o f the n e x t .  At  the v e r y p e r i p h e r y o f the o v i d u c t , a l l o f the l o n g t h i n n u c l e i a r e 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 i m p r e s s i o n t h a t a s t r e a m 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 s t r e a m i n g a t r e g u l a r i n t e r v a l s . base and c e n t e r of the  These i n t e r r u p t i o n s form the  folds.  The major d i f f e r e n c e s , t h e n , between the o v i d u c t s from f i v e - and  fifteen-  day c o n t r o l s , a r e the s i z e and the dense p a c k i n g of the s t r o m a l c e l l s a g a i n s t the e p i t h e l i u m i n the f i v e - d a y c o n t r o l o v i d u c t s . o f the o v i d u c t s had a p p r o x i m a t e l y  doubled,  By f i f t e e n days, the s i z e  the mucosal f o l d s had  enlarged  somewhat, and the s t r o m a l c e l l s had d i s p e r s e d throughout the a r e a of the  folds.  The e p i t h e l i u m remained a s i m p l e columnar e p i t h e l i u m . By 25 days o f t r e a t m e n t , o r 30 days o f l i f e , v a s c u l a r i z a t i o n can be seen to have i n c r e a s e d ( r e f e r to P l a t e 3 ) . mesenteries  B l o o d v e s s e l s were p r e s e n t o n l y i n the  and a t the v e r y p e r i p h e r y of the o v i d u c t i n the f i v e - d a y c o n t r o l s ,  and a t the v e r y base o f the mucosal f o l d s i n the t e n - and f i f t e e n - d a y c o n t r o l s . In  the o v i d u c t s from the t w e n t y - f i v e - d a y  seen e x t e n d i n g  c o n t r o l b i r d s , c a p i l l a r i e s c o u l d be  from the base o f the f o l d s up i n t o the c e n t e r of the  Thus v a s c u l a r i z a t i o n , when i t o c c u r s , f o l l o w s the "stream" The  folds.  of the c e l l p a t t e r n .  changes w h i c h o c c u r i n the c o n t r o l o v i d u c t s t h e r e f o r e resemble  o f any embryonic p r i m o r i d u m i n response t o i t s i n d u c e r  (70)  those  This includes  the i n t i a l s t r o m a l p a c k i n g a g a i n s t the e p i t h e l i u m , the s t r o m a l c e l l  rearrange-  ment, and the b e g i n n i n g o f v a s c u l a r i z a t i o n . These o b s e r v a t i o n s a r e a t v a r i a n c e w i t h those of F e r t u c k and Newstead (66),  who  have r e p o r t e d t h a t t u b u l a r g l a n d c e l l s a r e p r e s e n t i n the o v i d u c t by  30 days o f age.  However, O i s h i and Lauber (67, 68) have demonstrated t h a t , i n  Plate l a .  C r o s s - s e c t i o n o f an o v i d u c t 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  arrangement o f s t r o m a l c e l l s  f o r 5 days,  x 168.  (S) d e n s e l y packed a g a i n s t  t h e l i u m ( E ) , and v a s c u l a r i z a t i o n (BV) i n m e s e n t e r i e s  Plate lb.  Note epi-  surround-  ing oviduct.  F i x e d i n Bouin's and embedded i n wax; 5 m i c r o n s .  Same, x 680.  Note s i m p l e columnar e p i t h e l i u m ( E ) , w i t h  cells  packed t o g e t h e r c l o s e l y enough t o appear p s e u d o - s t r a t i f l e d .  Plate l c .  C r o s s - s e c t i o n o f an o v i d u c t from a 1 5 - d a y - o l d b i r d w h i c h 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 o f e p i t h e l i a l c e l l s and s t r o m a l c e l l s as d e s c r i b e d above appears a t t h i s t i m e . hyde and embedded i n p l a s t i c .  Fixed i n glutaralde-  1 micron.  P l a t e 2a.  C r o s s - s e c t i o n o f an o v i d u c t from a 20-day-old had r e c e i v e d c o n t r o l treatment dispersion  of s t r o m a l c e l l s  folds, vascularization  P l a t e 2b.  Same, x 672.  x 168.  Note  ( S ) , throughout a r e a of mucosal  (BV) a t the base o f the f o l d s , s i m p l e  columnar e p i t h e l i u m ( E ) . embedded i n p l a s t i c .  f o r 15 days,  b i r d , which  Fixed i n glutaraldehyde,  and  1 micron.  Note o r i e n t a t i o n o f s t r o m a l c e l l n u c l e i a t  c e n t e r of f o l d s a l o n g the v e r t i c a l a x i s o f the f o l d . E p i t h e l i u m ( E ) ; stroma ( S ) , and b l o o d c e l l s  (BC).  P l a t e 3a.  C r o s s - s e c t i o n o f an o v i d u c t from a 3 0 - d a y - o l d b i r d , w h i c h had r e c e i v e d c o n t r o l t r e a t m e n t f o r 15 d a y s , f o l l o w e d by no t r e a t m e n t f o r 10 days, stromal c e l l s  Same, x 672. the mucosal  Note same arrangement o f  (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  P l a t e 3b.  x 168.  and embedded i n p l a s t i c .  1 micron.  Note appearance o f b l o o d v e s s e l s (BV) i n folds.  the s h o r t day affected  l e n g t h used i n t h i s study  (8L/16D), g o n a d a l growth i s a d v e r s e l y  ( r e l a t i v e t o 24L/0D) by r e d u c i n g , v i a the p i n e a l , g o n a d o t r o p h i n  s e c r e t i o n (67, 68, 69; see a l s o 164).  Eroschenko and W i l s o n have a l s o  demonstrated t h a t r e g r e s s i o n of a d u l t " q u a i l o v i d u c t s and gonads can be by t h i s s h o r t day  l e n g t h , r e l a t i v e to 16L/8D ( 5 6 ) .  Newstead (66) d i d not i n d i c a t e the day between those r e s u l t s and The  The  study by F e r t u c k  l e n g t h used; however, the  length.  changes d e s c r i b e d h e r e have a l s o been r e p o r t e d by K o h l e r e t a l  i t would appear t h a t e s t r o g e n  t r e a t m e n t on the c h i c k o v i d u c t .  i s present  Treated  (41) Thus  i n the immature q u a i l a t a l e v e l  c a p a b l e o f i n d u c i n g the e a r l y changes i n the s t r o m a , but not of the t u b u l a r g l a n d  and  discrepancy  these c o u l d be e x p l a i n e d by a d i f f e r e n t day  to be the i n i t i a l r e s u l t s of e s t r o g e n  B.  induced  differentiation  cells.  Animals  Very d e f i n i t e changes took p l a c e w i t h i n f i v e days of e s t r o g e n as can be seen i n P l a t e 4. the mucosal f o l d s e n l a r g e d  The  o v i d u c t had  treatment,  grown tremendously i n s i z e ,  g r e a t l y to f i l l most of the lumen.  and  These f o l d s  were f i l l e d w i t h t u b u l a r g l a n d 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 g r a n u l e s .  The  g l a n d s , w h i c h s e c r e t e the egg-white p r o t e i n s (50, 66, 43, 7 1 ) , a r i s e by budding off  of the e p i t h e l i u m , and  the g l a n d s open i n t o the lumen.  P o i n t s of  entry  i n t o the lumen c o u l d be seen i n . t h e mucosal f o l d s . The  s i m p l e columnar e p i t h e l i u m appeared t o be v e r y 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 o t h e r d i s t i n c t e v i d e n c e of d i f f e r e n t i a t i o n was epithelium.  Occasional  the absence o f hormone  seen i n the  c i l i a t e d c e l l s a l s o o c c u r i n mammalian u t e r i , even i n (72).  By 10 days of t r e a t m e n t ( r e f e r to P l a t e 5 ) , the o v i d u c t s i z e was  greatly  increased. granules  T u b u l a r g l a n d c e l l s c o n t a i n a g r e a t abundance o f g r a n u l e s .  These  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) t h a t  this r e f l e c t s varying concentrations b l o o d c e l l s were p r e s e n t  o f t h e egg-white p r o t e i n s .  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 o c c u r r e d .  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 c o n t a i n e d  some c e l l s w h i c h 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 o t h e r s centrally-placed nuclei.  Nucleated  t h a t had p a l e ,  C i l i a were apparent c o n t i n u o u s l y a l o n g t h e e p i t h e l i -  um, so t h a t i t was n o t p o s s i b l e t o d e t e r m i n e from t h e s e s e c t i o n s a l o n e w h i c h c e l l s they b e l o n g e d t o . However, i t i s l i k e l y they b e l o n g e d t o t h e c e l l s with centrally-placed nuclei, since this  i s t h e case i n c h i c k o v i d u c t , and  s i n c e t h e o t h e r c e l l s a r e thought t o be s e c r e t o r y .  In a l l epithelial  cells,  the n u c l e i were rounded and e l o n g a t e d . By 15 days o f e s t r o g e n  treatment,(refer 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 o f 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 l o n g e r e l o n g a t e d , b u t appeared t o be pushed a g a i n s t t h e basement membrane.  I n some cases these  c e l l s seemed t o be i n v o l v e d i n the p r o c e s s o f s e c r e t i o n .  The c e n t r a l l y -  p l a c e d n u c l e i o f c i l i a t e d c e l l s were rounded, and t h e c i l i a were much more prominent. Oviducts terone  t r e a t e d f o r 10 days w i t h e s t r o g e n  and then 5 days w i t h p r o g e s -  ( r e f e r t o P l a t e 7) o c c a s i o n a l l y c o n t a i n e d many u n s t a i n e d  suggesting  t h a t s e c r e t i o n o f some o f t h e g r a n u l e s  areas,  i n t o t h e lumen had o c c u r r e d .  The g r a n u l e s were so abundant and so d a r k l y - s t a i n i n g t h a t i t was d i f f i c u l t to l o c a t e t h e n u c l e i o f t h e t u b u l a r g l a n d c e l l s .  I n some o v i d u c t s b o t h t h e  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 t h e s e c r 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 .  I n other o v i d u c t s , only the b a s a l  n u c l e i o f t h e s e c r e t o r y c e l l s were compressed, w h i l e t h e c e n t r a l n u c l e i o f  P l a t e 4a.  C r o s s - s e c t i o n o f an o v i d u c t 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 o g e n ,  x 168.  v a s c u l a r i z a t i o n (BV).  Fixed i n glutaraldehyde  in plastic.  P l a t e 4b.  Same, x 800.  Note the t u b u l a r glands (TG), and embedded  1 micron.  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), points of  e n t r y o f t u b u l a r glands i n t o t h e lumen Carrows), and presence of e o s i n o p h i l i c g r a n u l e s  (G) i n t u b u l a r g l a n d  cells.  P l a t e 5a.  C r o s s - s e c t i o n o f an o v i d 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 e s t r o g e n , and  g r a n u l e s , and i n c r e a s e 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  P l a t e 5b.  x 168. Note abundance o f g l a n d s  and embedded i n p l a s t i c .  1 micron.  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 o f c i l i a  (C) on e p i t h e l i u m , and presence o f two  c e l l types i n e p i t h e l i u m .  Granules i n tubular gland  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 .  cells  P l a t e 6a.  C r o s s - s e c t i o n o f an o v i d u c t 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 o g e n ,  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 o f i n c r e a s e i n s i z e over t h e t r e a t m e n t p e r i o d . Fixed i n glutaraldehyde  P l a t e 6b.  Same, x 1010. cells  and embedded i n p l a s t i c .  1 micron.  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 o f g o b l e t  (G) and p r o f u s e c i l i a  (C) o f e p i t h e l i u m .  Granules  o f t h e t u b u l a r g l a n d c e l l s appear t o s t a i n w i t h same intensity.  P l a t e 7a.  C r o s s - s e c t i o n o f o v i d 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 e s t r o g e n , f o l l o w e d by 5 days o f x 168.  Note u n s t a i n e d a r e a s .  embedded i n p l a s t i c .  P l a t e 7b.  Same, x 672.  F i x e d i n g l u t a r a l d e h y d e and  1 micron.  Note compressed appearance o f n u c l e i and  datfker-staining cytoplasm goblet c e l l s  progesterone,  of c i l i a t e d c e l l s  (GC) i n p r o c e s s o f s e c r e t i o n .  ( C ) , and Lumen = L.  the c i l i a t e d c e l l s were n o t . c e l l s become e x t r e m e l y  Others have a l s o r e p o r t e d t h a t t h e c i l i a t e d  compressed between t h e s e c r e t o r y c e l l s , because t h e  l a t t e r become engorged w i t h s e c r e t o r y p r o d u c t s  (56) .  C i l i a appeared i n some  cases t o be q u i t e l o n g and dense, and d a r k l y - s t a i n i n g . C.  Animals Withdrawn from Hormone Treatment O v i d u c t s o f animals w i t h d r a w n from e s t r o g e n p l u s p r o g e s t e r o n e  were s m a l l e r t h a n those w i t h d r a w n from e s t r o g e n t r e a t m e n t . withdrawn from e s t r o g e n p l u s p r o g e s t e r o n e some were l a r g e r than o t h e r s , a l t h o u g h  treatment  Of the o v i d u c t s  ( r e f e r t o P l a t e s 8 and 9 ) ,  t h e r e 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 o f o v i d u c t s . evident, although  treatment  V a s c u l a r i z a t i o n was  still  somewhat l e s s e n e d , and t h e r e were fewer and s m a l l e r  tubular gland c e l l s .  There were no d i s t i n c t g r a n u l e s  i n any o f t h e t u b u l a r  g l a n d c e l l s , b u t some c o n t a i n e d an e o s i n o p h i l i c m a t e r i a l , whereas o t h e r s appeared empty, i n t h a t t h e i r c e n t e r s were e n t i r e l y u n s t a i n e d . to be more s i n g l e c e l l s i n t h e stroma w h i c h were n o t o r g a n i z e d gland c e l l s .  There appeared into tubular  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 t o be i n a d i f f e r e n -  tiated state:  i t was p r o f u s e l y c i l i a t e d , and c o n t a i n e d c e l l s w i t h  central,  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 . n u c l e i o f a l l t h e e p i t h e l i a l c e l l s were rounded and e l o n g a t e d , the dark, compressed n u c l e i o f t h e e s t r o g e n p l u s p r o g e s t e r o n e epithelium. cells  There were no evidences  The  r a t h e r than treated oviduct  of s e c r e t i o n s i n any o f t h e e p i t h e l i a l  ( r e f e r t o P l a t e 8b). Those o v i d u c t s w i t h d r a w n from e s t r o g e n p l u s p r o g e s t e r o n e  treatment  which  were l a r g e r , appeared h i s t o l o g i c a l l y t o be q u i t e s i m i l a r t o t h e s m a l l e r ones j u s t described  ( r e f e r t o P l a t e 9 ) . These showed g r e a t e r 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 o f the mucosal f o l d s w h i c h c o n t a i n e d n e i t h e r c e l l s nor b l o o d v e s s e l s . stained centers.  They a l s o c o n t a i n e d many t u b u l a r g l a n d s w i t h un-  A l t h o u g h the g e n e r a l appearance o f 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 s h o u l d be p o i n t e d out t h a t these were f i x e d i n Bouin's r a t h e r than g l u t a r a l d e h y d e .  Bouin's i s g e n e r a l l y a poorer  fixative  (76). O v i d u c t s from a n i m a l s withdrawn  from e s t r o g e n t r e a t m e n t were a l l l a r g e r  than those withdrawn from e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t . s t i l l g r e a t e r v a s c u l a r i z a t i o n than those withdrawn  These had  from p r o g e s t e r o n e t r e a t m e n t .  In a d d i t i o n , t h e r e were l a r g e u n s t a i n e d areas i n the c e n t e r o f the f o l d s , as w e l l as some t u b u l a r g l a n d s which were empty-appearing.  These o v i d u c t s had  the same g e n e r a l appearance, but c o n t a i n e d many more t u b u l a r g l a n d s , and fewer t u b u l a r glands w i t h u n s t a i n e d c e n t e r s . these o v i d u c t s withdrawn  Epithelial differentiation in  from e s t r o g e n treatment d i d not d i f f e r  from t h a t d e s c r i b e d f o r o v i d u c t s withdrawn  significantly  from e s t r o g e n p l u s p r o g e s t e r o n e  treatment. I t i s e v i d e n t from t h e s e h i s t o l o g i c a l r e s u l t s t h a t the q u a i l o v i d u c t e x h i b i t s the t h r e e c h a r a c t e r i s t i c s common t o d e v e l o p i n g systems: morphogenetic  movement, and c e l l d i f f e r e n t i a t i o n .  growth,  These changes compare t o  those r e p o r t e d to o c c u r i n the c h i c k o v i d u c t as a r e s u l t o f e s t r o g e n and progesterone treatment.  E s t r o g e n 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 s t r o m a l c e l l s , and to cause w a t e r i m b i b i t i o n and (41). of  The v a s c u l a r i z a t i o n o f the r e g i o n i s f o l l o w e d by b l o o d c e l l  the stroma  (41).  hyperemia invasion  A f t e r 2 to 3 days, t u b u l a r g l a n d c e l l s b e g i n to bud o f f  from the e p i t h e l i u m (41, 45, 4 8 ) .  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 o f an o v i d 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 e s t r o g e n , 5 days w i t h p r o g e s t e r o n e , and then w i t h d r a w n from t r e a t m e n t f o r 10 days,  x 550.  appearing tubular gland (arrows), blood c e l l s g e n e r a l l y u n o r g a n i z e d appearance o f stroma. retains c i l i a in plastic.  P l a t e 8b.  Same, x 1606.  (C).  Note empty(BC) and Epithelium  F i x e d i n g l u t a r a l d e h y d e and embedded  1 micron.  Ciliated  (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 t y p e s .  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 g o b l e t c e l l s , no e v i d e n c e o f s e c r e t i o n can be seen. N u c l e i a r e e l o n g a t e d , and no l o n g e r compressed.  Note  invagina-  t i o n o f e p i t h e l i u m (arrow) w h i c h may have been former opening of  a tubular 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 o f an o v i d u c t magnum from a b i r d 10 days w i t h e s t r o g e n , 5 days w i t h p r o g e s t e r o n e , w i t h d r a w n from treatment  f o r 10 days,  t u b u l a r glands  and then  x 168. T h i s  was one o f the s m a l l e r ones from t h e group. appearing  treated f o r  oviduct  Note empty-  ( a r r o w ) , v a s c u l a r i z a t i o n (BV) and  presence o f some t u b u l a r g l a n d c e l l s w i t h e o s i n o p h i l i c centers. 1  P l a t e 9b.  F i x e d i n g l u t a r a l d e h y d e , embedded i n p l a s t i c .  micron.  Same, x 168. group.  T h i s was one o f t h e l a r g e r o v i d u c t s from t h e  Legend as above.  Note d i f f e r e n c e i n s i z e , i n  comparison t o 9a. F i x e d i n B o u i n ' s , 1  P l a t e 9c.  embedded i n p l a s t i c .  micron.  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 ( a r r o w ) , perhaps r e p r e s e n t i n g former s i t e o f e n t r y o f t u b u l a r i n t o lumen ( L ) .  gland  n o t e d by P a l m i t e r and Wrenn ( 4 3 ) , w i t h i n c o m p l e t e 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  form t u b u l a r g l a n d c e l l s when e s t r o g e n i s p r e s e n t ( 4 3 ) . essential  f o r t h i s p r o c e s s o f budding  g l a n d u l a r m i t o s i s as w e l l when i t i s complete,  (45).  c e l l s continue*  M i c r o f i l a m e n t s are  ( 4 4 ) , which i s l a t e r accompanied by  Budding c o n t i n u e s f o r a number o f days,  tain centrally-located,  Progesterone  palely-staining  nuclei  The  former  con-  n u c l e i , and the l a t t e r c o n t a i n d a r k ,  (41, 45) as r e p o r t e d above f o r q u a i l .  treatment has been shown by Oka and Schimke t o p r e v e n t  the d i f f e r e n t i a t i o n o f t u b u l a r g l a n d c e l l s ( 4 5 ) . ed t h a t the e f f e c t s at w h i c h i t was  and  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 g o b l e t s e c r e t o r y c e l l s (43, 45, 46, 47).  apically-situated  to  However, t h a t work  demonstrat-  o f p r o g e s t e r o n e depended on the s t a g e o f d i f f e r e n t i a t i o n  administered (45).  Thus, i f p r o g e s t e r o n e was  given  concomit-  a n t l y w i t h e s t r o g e n from the b e g i n n i n g o f t r e a t m e n t , i t a b o l i s h e d the t u b u l a r g l a n d 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 . if  the onset of p r o g e s t e r o n e  t r e a t m e n t was  delayed u n t i l l a t e r stages of  e s t r o g e n t r e a t m e n t , 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 w i t h the t u b u l a r g l a n d c e l l s once they were d i f f e r e n t i a t e d f a c t would e x p l a i n why antagonism:  progesterone  However,  (45) .  interfere  This l a t t e r  i n the p r e s e n t study demonstrated  no  i t i s begun o n l y a f t e r p r o l o n g e d e s t r o g e n t r e a t m e n t , a t a time  when t u b u l a r g l a n d c e l l d i f f e r e n t i a t i o n may Progesterone  a l r e a d y be  complete.  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 c h i c k s  (47, 48).  T h i s i s the case even i f e s t r o g e n i s not g i v e n f i r s t  r e s u l t was  not i n d i c a t e d  (47).  This  i n the p r e s e n t work w i t h q u a i l , perhaps because  p r o l o n g e d e s t r o g e n treatment has the same e f f e c t  (41, 45, 48).  Thus, i n t h i s  s t u d y , the 15-day e s t r o g e n treatment and the 10-day e s t r o g e n , 5-day p r o g e s t e r -  one  t r e a t m e n t e x p e r i m e n t s d i d not show g r e a t d i f f e r e n c e s i n e p i t h e l i a l  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 .  I n the  differ-  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 o f b o t h the s e c r e t o r y c e l l s and c e l l s were v e r y compact and d a r k l y - s t a i n i n g , not o v a l .  the  ciliated  I n some c a s e s ,  c y t o p l a s m 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  the  compressed between  the s e c r e t o r y c e l l s . I n t i s s u e w i t h d r a w n from t r e a t m e n t , g l a n d c e l l s were seen, w h i c h s u p p o r t s and of F i t z g e r a l d ( 5 5 ) , and Wilson  s m a l l e r o v i d u c t s w i t h fewer t u b u l a r  the e a r l y i n d i c a t i o n s of S t r o t t  the more r e c e n t i n v e s t i g a t i o n s o f Eroschenko  (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 .  to decreased gonadotropin  i s diminished  r e l e a s e (67, 68, 69).  The  l i a l c e l l s appear t o remain d i f f e r e n t i a t e d s i n c e they a r e r e p o r t e d Eroschenko and W i l s o n  and  Those r e p o r t e d r e s u l t s i n d i c a t e d  t h a t c e l l s a r e l o s t d u r i n g r e g r e s s i o n , a time when e s t r o g e n the a n i m a l due  (54)  in  epitheby  (56) to r e t a i n c i l i a f o r up t o 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 i n d u c e d i n a d u l t a n i m a l s 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 t r e a t m e n t was  i n i t i a l l y reported  r e s u l t i n no l o s s of c e l l s , but o n l y a r e d u c t i o n of c e l l s i z e (45, However, Yu e t a l (51, 52) have r e p o r t e d t h a t DNA  content  to  48).  i s g r e a t l y reduced  d u r i n g 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  r e p o r t e d t h a t t u b u l a r g l a n d 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 o n l y 20% o f the c e l l s of an o v i d u c t withdrawn from Therefore  treatment.  i t appears t h a t the c h i c k o v i d u c t a l s o l o s e s c e l l s upon hormone  withdrawal.  II.  Assays o f T o t a l Growth:  Weight, P r o t e i n , RNA  E s t r o g e n caused a d r a m a t i c  and DNA  Content  i n c r e a s e 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 7 0 0 - f o l d i n c r e a s e i n wet w e i g h t ( F i g u r e 3) from 2.5 mg  on  the  f i r s t day o f t r e a t m e n t to an average v a l u e of 1.7 treatment.  gm a f t e r 15 days o f e s t r o g e n  I n b i r d s r e c e i v i n g p r o g e s t e r o n e r a t h e r than e s t r o g e n f o r the l a s t  5 days o f t r e a t m e n t , the average o v i d u c a l w e i g h t reached 2.0 gm.  The  g r e a t e s t r a t e of i n c r e a s e was between days 5 and 10 o f t r e a t m e n t ; the  response  slowed somewhat between days 10 and 15 o f t r e a t m e n t . Ten days a f t e r w i t h d r a w a l from e s t r o g e n t r e a t m e n t , the wet w e i g h t decreased o n l y s l i g h t l y .  T h i s d i f f e r e n c e i s not s i g n i f i c a n t  had  (p = 0.04).  However, o v i d u c t s which had r e c e i v e d 10 days of e s t r o g e n t r e a t m e n t f o l l o w e d by 5 days o f p r o g e s t e r o n e  t r e a t m e n t , l o s t about 85% o f t h e i r wet w e i g h t i n  the same 10-day w i t h d r a w a l p e r i o d , thus d e m o n s t r a t i n g a f a r g r e a t e r decrease than the o v i d u c t s from b i r d s w i t h d r a w n from e s t r o g e n  treatment.  S i m i l a r changes were seen i n the p r o t e i n ( F i g u r e 4) and RNA c o n t e n t of the o v i d u c t s . days of t r e a t m e n t .  The g r e a t e s t i n c r e a s e was  always between 5 and  The i n c r e a s e observed when p r o g e s t e r o n e  from 10 to 15 days of t r e a t m e n t may  The  be s i g n i f i c a n t f o r the change i n RNA  c o n c e n t r a t i o n o f p r o t e i n and RNA  10  replaced estrogen  t e n t (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 (p = 0.04).  ( F i g u r e 5)  con-  content  per u n i t w e i g h t d i d not  i n c r e a s e as g r e a t l y as the t o t a l amount of each component, due t o the conc o m i t a n t i n c r e a s e i n wet w e i g h t . ( s e e Table I ) . remained f a i r l y c o n s t a n t throughout  The c o n c e n t r a t i o n of  t r e a t m e n t , whereas the p r o t e i n c o n c e n t r a -  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 . p r o t e i n c o n c e n t r a t i o n throughout t o t a l p r o t e i n and t o t a l RNA  RNA  The  changes i n RNA  t r e a t m e n t were not s i g n i f i c a n t .  However,  content increased approximately 1000-fold  5 0 0 - f o l d r e s p e c t i v e l y over c o n t r o l v a l u e s i n the 15 days o f e s t r o g e n i n d i c a t i n g a g a i n the v e r y r a p i d growth of the o v i d u c t .  and  and treatment,  F i g u r e 3.  The V a r i a t i o n i n Wet Weight as a F u n c t i o n of Treatment Legend:  o  , treatment w i t h e s t r a d i o l .  •  , treatment w i t h t  withdrawn from  progesterone. treatment.  , control oviducts. Bars represent standard  error. 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 F u n c t i o n of Treatment. Legend as above.  3  Fjg^Time vs. Wet Weight  Fig.4- Time vs Protein Content  -Treatment-Withdrawal  500 -Treatment —Withdrawal  r  E  I  400  o 300 i  0  •t  0  200  o  &L  i  100  i  5 10 15 20 25 Day of Treatment  4^ CD  E  00  .0  i 1  J.  5 10 15 20 25 Day of Treatment  F i g u r e 5.  The V a r i a t i o n i n Oviduct RNA Legend as i n F i g u r e  F i g u r e 6.  Content as a F u n c t i o n of Treatment.  3.  The V a r i a t i o n i n Oviduct DNA Legend as i n F i g u r e 3.  Content as a F u n c t i o n of Treatment. u, o  F i g ! • Time vs RNA Content  R g J . :Time vs DNA Content , -Treat ment W it hd rawal 12 r $ n  o  30 r •Treatment — Withdrawal 25  K  •120  v  O  <  CJI 15  §10 en  £ 5 -•f  00  1....  ..1  5 10 15 20 25 Day of Treatment  0  5 10 15 20 25 Day of Treatment  A f t e r 10 days o f w i t h d r a w a l from e s t r o g e n t r e a t m e n t , a p p r o x i m a t e l y onet h i r d o f t h e p r o t e i n c o n t e n t , and o n e - h a l f o f t h e RNA c o n t e n t o f t h e o v i d u c t were l o s t .  I n c o n t r a s t t o t h i s , i n t h e o v i d u c t s t h a t had r e c e i v e d e s t r o g e n  plus progesterone  t r e a t m e n t , a p p r o x i m a t e l y 90% o f b o t h t h e RNA and t h e p r o t e i n  c o n t e n t was l o s t a f t e r 10 days o f w i t h d r a w a l .  T h i s g r e a t e r r a t e o f decrease  a f t e r w i t h d r a w a l from e s t r o g e n p l u s p r o g e s t e r o n e decrease  corresponds  to the greater  i n the wet w e i g h t o f t h e o r g a n when withdrawn from e s t r o g e n p l u s  progesterone  t r e a t m e n t r a t h e r than from treatment w i t h e s t r o g e n o n l y .  The changes i n the DNA c o n t e n t ( F i g u r e 6) as a r e s u l t o f treatment from the o t h e r t i s s u e components i n 2 ways:  1) T h i s i s t h e o n l y t i s s u e com-  ponent n o t i n c r e a s e d b y e s t r o g e n p l u s p r o g e s t e r o n e continued estrogen treatment.  varied  treatment r e l a t i v e t o  The DNA c o n t e n t o f e s t r o g e n - p l u s ^ p r o g e s t e r o n e  t r e a t e d o v i d u c t s v a r i e s o n l y s l i g h t l y from t h e 10-day e s t r o g e n v a l u e , i n d i c a t i n g t h a t p r o g e s t e r o n e does n o t s t i m u l a t e DNA s y n t h e s i s , a t l e a s t n o t 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 c o n c e n t r a t i o n o f  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 p r o g e s t e r o n e t r e a t m e n t  (see Table I ) .  2) Upon w i t h d r a w a l from e s t r o g e n t r e a t m e n t , a l o s s o f a p p r o x i m a t e l y 50% o f the DNA c o n t e n t was n o t e d , as i n RNA c o n t e n t , b u t a l o s s o f o n l y t w o - t h i r d s o f the DNA c o n t e n t was noted i n t h e o v i d u c t s w i t h d r a w n from e s t r o g e n p l u s p r o g e s t e r one  treatment.  T h i s i s i n c o n t r a s t t o t h e l o s s o f 90% o f t h e RNA and p r o t e i n  c o n t e n t , and 85% o f t h e wet w e i g h t i n t h e s e same o v i d u c t s , i n d i c a t i n g t h a t l e s s DNA was l o s t than o t h e r components.  The c o n c e n t r a t i o n o f DNA  actually  i n c r e a s e d a f t e r w i t h d r a w a l from p r o g e s t e r o n e , whereas i t decreased a f t e r w i t h d r a w a l from e s t r o g e n (see Table I ) .  T h i s c o n c e n t r a t i o n i n c r e a s e may be an  i n d i c a t i o n o f the g r e a t e r l o s s o f o t h e r components, such as wet w e i g h t and protein.  T a b l e I . Summary o f t h e V a r i a t i o n s i n t h e Content and C o n c e n t r a t i o n s o f T i s s u e C o n s t i t u e n t s i n t h e O v i d u c t a t D i f f e r e n t Stages of Treatment. Protein/wt Mg/gm  RNA Mg/oviduct  RNA/wt Mg/gm  DNA Mg/oviduct  DNA/wt Mg/gm  Treatment  Protein Mg/oviduct  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  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  No. i n sample  Controls:  C o n t r o l o v i d u c t s a l s o i n c r e a s e d i n these components d u r i n g t h e treatment p e r i o d , although to a very s l i g h t extent. i n i t i a l estrogen-induced  T h i s may be due t o v e r y  slight,  changes, o r i t may be t h e r e s u l t o f the g e n e r a l  growth o f t h e a n i m a l . These r e s u l t s a r e comparable t o those r e p o r t e d f o r t h e e f f e c t o f e s t r o g e n on c h i c k o v i d u c t ( 4 1 , 45, 46, 48, 51, 5 2 ) . E s t r o g e n treatment  f o r 10 days  caused an i n c r e a s e i n a l l parameters i n c h i c k o v i d u c t a l s o , b u t t h e r e i s a s l i g h t d i f f e r e n c e between t h e . c h i c k o v i d u c t work and the d a t a p r e s e n t e d for  the q u a i l , i n the v a r i a n c e observed  i n the r e s p o n s e .  here  There i s somewhat  l e s s v a r i a n c e i n t h e work o f Oka and Schimke ( 4 5 ) , who used o n l y t h e magnum p o r t i o n o f t h e c h i c k o v i d u c t f o r t o t a l growth a s s a y s , and i n t h e work o f Yu et a l , who used whole o v i d u c t s .  Yu e t a l (46, 51, 52) r e p o r t e d t h e v a l u e and  s t a n d a r d d e v i a t i o n f o r DNA and RNA c o n c e n t r a t i o n s for RNA.  (as p e r c e n t o f d r y m a t t e r )  p o o l e d samples o f whole o v i d u c t s t o be 1.8±0.1% f o r DNA and 2.3±0.1% f o r These r e p r e s e n t a s m a l l e r v a r i a n c e than t h a t r e p o r t e d h e r e , b u t t h e  r e s u l t s a r e not s t r i c t l y p e r c e n t o f wet w e i g h t . content per oviduct.  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 No v a r i a n c e 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  T h i s s l i g h t d i f f e r e n c e i n v a r i a n c e c a n p r o b a b l y 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 a s time  schedule  o f i n j e c t i o n s , dose, and s p e c i e s d i f f e r e n c e s . Progesterone  appeared t o e x e r t 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 r e p o r t e d f o r c h i c k s . progesterone  However, as mentioned e a r l i e r , t h e e f f e c t o f  on t h e e s t r o g e n i c response depends on the stage o f d i f f e r e n t i a t i o n  a t which i t i s a d m i n i s t e r e d  ( 4 5 , 48, 4 3 ) . The means by w h i c h  progesterone  i n h i b i t s the e s t 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 g l a n d  cell  d i f f e r e n t i a t i o n ( 4 3 ) , s i n c e these c e l l s make up the v a s t b u l k o f t h e magnum.  However, i f p r o g e s t e r o n e i s g i v e n a f t e r t h e s e c e l l s are a l r e a d y p r e s e n t , i t does not i n h i b i t , b u t 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, 4 8 ) .  Thus  i f p r o g e s t e r o n e i s g i v e n a f t e r e s t r o g e n i n d u c t i o n , one would expect t o see an i n c r e a s e i n such t i s s u e components as w e i g h t , p r o t e i n , and RNA but not DNA cells.  c o n t e n t , as p r o g e s t e r o n e i n h i b i t s f o r m a t i o n o f new  The r e s u l t s p r e s e n t e d h e r e s u p p o r t t h a t p r e d i c t i o n .  content,  tubular gland  The  differences  between o v i d u c t s t r e a t e d f o r 15 days w i t h e s t r o g e n , and those t r e a t e d f o r 10 days w i t h e s t r o g e n , 5 days p r o g e s t e r o n e a r e s m a l l .  I n 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 a r e not c a n t ( w e i g h t , p = 0.014; RNA,  p = 0.022; DNA,  p = 0.03;  signifi-  p r o t e i n , p = 0,239).  I n the c o n c e n t r a t i o n s o f t i s s u e c o n s t i t u e n t s , o v i d u c t s from these two groups are  n o t 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), o r RNA c o n c e n t r a t i o n  (p = 0.84), but a r e 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)'.  T h i s would be due not o n l y to the r e l a t i v e l y g r e a t e r s y n t h e s i s o f DNA  in  e s t r o g e n 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 g r e a t e r s y n t h e s i s o f p r o t e i n and RNA  i n the e s t r o g e n p l u s p r o g e s t e r o n e t r e a t e d o v i d u c t s .  This i s  i n k e e p i n g w i t h the known e f f e c t s o f p r o g e s t e r o n e i n c h i c k o v i d u c t , i n i n h i b i t i n g new  t u b u l a r g l a n d 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 a l r e a d y p r e s e n t . W i t h d r a w a l o f hormone l e a d s to a l o s s o f a l l components, but DNA was to a l e s s e r e x t e n t than any o t h e r t i s s u e component.  lost  A greater loss occurred  i n - a l l components a f t e r w i t h d r a w a l from e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t than a f t e r w i t h d r a w a l from e s t r o g e n .  T h i s may be due to 1) a f t e r r e c e i v i n g  p r o g e s t e r o n e i n s t e a d o f e s t r o g e n f o r the l a s t 5 days o f t r e a t m e n t , these o v i d u c t s have a c t u a l l y been w i t h d r a w n from e s t r o g e n f o r 5 e x t r a days; or 2) p r o g e s t e r o n e 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 t o a l o s s o f hormone.  I f o v i d u c t s withdrawn  from e s t r o g e n p l u s p r o -  g e s t e r o n e t r e a t m e n t a r e a c t u a l l y r e g r e s s e d t o a g r e a t e r e x t e n t because they have been withdrawn  from e s t r o g e n t r e a t m e n t 5 e x t r a days, then t h i s  implies  t h a t p r o g e s t e r o n e 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.  T h i s may  be due to: 1) E s t r o g e n c o u l d p r e c i p i t a t e out a t the s i t e  of i n j e c t i o n , whereas p r o g e s t e r o n e , b e i n g more s o l u b l e , would n o t .  This  would c r e a t e a "depot" o f e s t r o g e n , but n o t of p r o g e s t e r o n e , w h i c h would be l o s t i n the 5 days o f p r o g e s t e r o n e treatment b e f o r e w i t h d r a w a l . one may  be m e t a b o l i z e d more e f f i c i e n t l y than e s t r o g e n .  the case i n n e a r l y a l l animals ( 7 8 ) .  T h i s i s known to be  3) E s t r o g e n c o u l d 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 c r e a t e a n o t h e r type of "depot" P r o g e s t e r o n e , b e i n g more p o l a r , may  2) P r o g e s t e r -  not.  (165).  T h i s has been r e p o r t e d i n r a t s ( 7 9 ) .  I n 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 t h a t e s t r o g e n i n d u c e s serum l i p e m i a i n c h i c k s (80, 73, 170).  The e f f e c t s o f p r o g e s t e r o n e on serum l i p e m i a a r e not  known. On the o t h e r hand, i f p r o g e s t e r o n e i s a c t i n g to make the t i s s u e  less  s t a b l e t o a l o s s o f hormone, t h i s c o u l d be e i t h e r by i n d u c i n g g r e a t e r metab o l i s m o f e s t r o g e n and o f i t s e l f , o r by i n d u c i n g a s p e c i f i c serum b i n d e r , w h i c h would reduce t h e amount of a c t i v e s t e r o i d . The r e s u l t s o f t h i s s t u d y do not a l l o w a c h o i c e between t h e s e p o s s i b i l i ties.  A l l o f them a r e l i k e l y .  o v i d u c t s from b i r d s withdrawn  These r e s u l t s do i n d i c a t e however, t h a t those from e s t r o g e n t r e a t m e n t are a t an e a r l i e r  s t a g e of w i t h d r a w a l than those from b i r d s withdrawn progesterone  from e s t r o g e n p l u s  treatment.  The r e s u l t s r e p o r t e d here s u p p o r t those o f Yu e t a l (51) f o r the c h i c k o v i d u c t , and Eroschenko  and W i l s o n f o r the q u a i l o v i d u c t ( 5 6 ) .  The s t u d y o f  Eroschenko and W i l s o n  r e p o r t e d o n l y h i s t o l o g i c a l changes, but i n d i c a t e d t h a t  t u b u l a r g l a n d c e l l s were l o s t d u r i n g r e g r e s s i o n o f a d u l t o v i d u c t s . p o r t of Yu e t a l (51) e s t a b l i s h e d t h a t the DNA  content  v a r i e s from an average of 11,456 yg i n a d e v e l o p i n g a l a y i n g hen,  t o 17,486 yg i n a r e g r e s s i n g o v i d u c t .  the e a r l y r e p o r t s of Oka hormone w i t h d r a w a l ,  and  The  of the c h i c k o v i d u c t  o v i d u c t to 83,087 yg i n These are c o n t r a r y  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 f t e r 6 days of r e a d m i n i s t r a t i o n o f e s t r o g e n  (77).  80%  Thus i t appears t h a t i n  the absence o f hormone, not o n l y 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  was  also  gland  c e l l s i n o v i d u c t s w i t h d r a w n from t r e a t m e n t i s o n l y 20% as compared t o  The  to  Schimke (45, 4 8 ) , t h a t the c e l l s remained d u r i n g  i n d i c a t e d t h a t , c o n t r a r y to e a r l y r e p o r t s , the p e r c e n t a g e of t u b u l a r  themselves are  re-  cells  lost.  p a t t e r n o f l o s s of the v a r i o u s t i s s u e components a f t e r  withdrawal  n o t 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 ( 4 8 ) , 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 p a t t e r n s were seen f o r the l o s s of components on hormone w i t h d r a w a l . day 10 o f treatment,  When hormone was  each component c o n t i n u e d  have been day 10 of treatment, t h e n d e c l i n e d . or l a t e r , t h i s c o n t i n u e d was  w i t h d r a w n p r i o r to  to i n c r e a s e u n t i l what would I f withdrawal  began on day  i n c r e a s e d i d not o c c u r , but an immediate decrease  seen i n each component.  a p r e c i p i t a t i o n of e s t r o g e n  The  a u t h o r s a t t r i b u t e d the c o n t i n u e d  i n c r e a s e to  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  i t d i d not o c c u r a f t e r day 10, w h i c h presumably would have a l l o w e d more e s t r o g e n  10  t o p r e c i p i t a t e . I t may  be t h a t the treatment was  why  f o r even  inducing a  serum b i n d i n g p r o t e i n a f t e r day 10, w h i c h 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 t o 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 m e t a b o l i s m may  Thus, the p a t t e r n of l o s s o f t i s s u e components on w i t h d r a w a l  have been may  induced.  depend on  the  l e n g t h o f treatment p r i o r to w i t h d r a w a l , as w e l l as the s t e r o i d used d u r i n g treatment. Withdrawal  from e s t r o g e n p l u s p r o g e s t e r o n e  ed i n o t h e r s t u d i e s .  treatment has n o t been r e p o r t -  However, the r e s u l t s p r e s e n t e d h e r e c o n c e r n i n g the  w i t h d r a w a l from e s t r o g e n treatment a r e a t v a r i a n c e w i t h those r e p o r t e d by and Schimke ( 4 8 ) .  Those a u t h o r s r e p o r t e d t h a t 12 days of w i t h d r a w a l a f t e r  10 days of e s t r o g e n treatment r e s u l t s i n a f a r g r e a t e r l o s s o f wet (76% decrease) and RNA  c o n t e n t (82% decrease) t h a n DNA  I n t h i s s t u d y , a p p r o x i m a t e l y the same amount o f DNA, d u r i n g w i t h d r a w a l from e s t r o g e n  weight  c o n t e n t (24% d e c r e a s e ) .  RNA  and p r o t e i n were l o s t  treatment.  The g r e a t e r l o s s o f w e i g h t and RNA may  Oka  i n t h e i r s t u d y compared t o t h i s  be due to the s h o r t e r treatment and the l o n g e r w i t h d r a w a l p e r i o d .  t h a t does not e x p l a i n why,  i n t h e i r s t u d y , l e s s DNA  components, whereas i n t h i s s t u d y , DNA o t h e r t i s s u e components.  I t may  was  However,  l o s t than o t h e r  l o s t to the same e x t e n t as the  be p o s s i b l e to e x p l a i n t h i s d i s c r e p a n c y by  o b s e r v i n g the p a t t e r n o f w i t h d r a w a l more c l o s e l y . s t r a t e d t h a t i n i t i a l l y , DNA  was  one  Oka and Schimke (48) demon-  i s l o s t a t a r a t e s i m i l a r to lysozyme and  wet  w e i g h t , but t h a t w i t h i n 5 days a f t e r the b e g i n n i n g o f w i t h d r a w a l , the r a t e o f l o s s of DNA  had slowed g r e a t l y .  I n t h i s s t u d y , e s t r o g e n was  administered for  15 days, and i t i s p r o b a b l e t h a t s t i m u l a t i o n c o n t i n u e s beyond t h a t time as a r e s u l t o f the "depot" e f f e c t d i s c u s s e d e a r l i e r .  These o v i d u c t s would t h e r e -  f o r e be i n an e a r l i e r s t a g e of w i t h d r a w a l than those o f Oka and Schimke. I n i t i a l l y , the r a t e of l o s s o f DNA  i s s i m i l a r to that of weight or  but the r a t e slows g r e a t l y a f t e r about 5 days o f w i t h d r a w a l ( 4 8 ) .  lysozyme, If i t is  assumed t h a t the o v i d u c t s from b i r d s withdrawn from e s t r o g e n treatment i n t h i s s t u d y are i n an e a r l i e r s t a g e of w i t h d r a w a l , due to the l e n g t h i e r  treatment,  than those o f Oka and Schimke, then t h e s e r e s u l t s a r e c o m p a t i b l e w i t h t h o s e . T h i s same assumption would e x p l a i n why o v i d u c t s from b i r d s withdrawn from e s t r o g e n t r e a t m e n t l o s t about t h e same amount o f DNA as RNA o r p r o t e i n , whereas those o v i d u c t s from b i r d s withdrawn from e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t l o s t l e s s DNA than RNA o r p r o t e i n . III.  Specific Proteins  Ovalbumin Ovalbumin p u r i f i e d as d e s c r i b e d under M a t e r i a l s and Methods was demons t r a t e d t o be impure by Sephadex G-200 g e l f i l t r a t i o n  (see F i g u r e 7 ) . I t was  t h e r e f o r e n o t u s e f u l i n p r e p a r i n g a s t a n d a r d f o r the q u a n t i t a t i o n o f a n t i g e n antibody p r e c i p i t a t e s .  A l t h o u g h i t i s a p p r o x i m a t e l y the same m o l e c u l a r  w e i g h t as c h i c k o v a l b u m i n , 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 a r e n o t c o m p l e t e l y i m m u n o l o g i c a l l y i d e n t i c a l , as can be seen from t h e O u c h t e r l o n y p l a t e ( r e f e r t o P l a t e 10a). o f c h i c k ovalbumin t o c o n s t r u c t a s t a n d a r d c u r v e . P l a t e 10a and b,  T h i s p r e c l u d e s t h e use  I n a d d i t i o n , as seen i n  t h e a n t i b o d y p r e p a r a t i o n i s impure; t h e r e a r e two p r e c i p i -  t a t i n g s p e c i e s , one o f w h i c h 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 s p e c i e s has n o t been i d e n t i f i e d . That t h i s second s p e c i e s i s n o t s i m p l y an a r t i f a c t o f c o n c e n t r a t i o n i s e v i d e n t by t h e f a c t t h a t i t can be d i l u t e d o u t by s e r i a l d i l u t i o n , t o a p o i n t where the f i r s t s p e c i e s i s s t i l l e v i d e n t , b u t n o t t h e second (see P l a t e 1 0 b ) . T h e r e f o r e , by d e m o n s t r a t i o n o f p a r t i a l i d e n t i t y w i t h c h i c k o v a l b u m i n , and i d e n t i t y w i t h q u a i l o v a l b u m i n , ovalbumin has been d e t e c t e d i n a l l  treated  o v i d u c t magna, i n a l l o f t h e magna from a n i m a l s withdrawn from e s t r o g e n t r e a t m e n t , and i n o n e - h a l f o f the magna withdrawn from e s t r o g e n p l u s p r o g e s -  F i g u r e 7.  E l u t i o n P r o f i l e o f Ovalbumin on Sephadex G-200. 1.5 m l o f c h i c k o r q u a i l ovalbumin (4 mg/ml) was a p p l i e d t o a column o f h e i g h t 60 cm, and d i a m e t e r , 1.5 cm, and e l u t e d w i t h T r i s b u f f e r a t a f l o w r a t e o f 0.1 mg/min.  Fractions of  v 2.1 ml were c o l l e c t e d and absorbance a t 280 nm was mined f o r each f r a c t i o n .  deter-  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 o v a l b u m i n (CO), bumin (QO),  and magnum e x t r a c t  from e s t r o g e n t r e a t m e n t .  quail oval-  (ME) from b i r d s w i t h d r a w n  5 y l of Antibody preparation  (1 y g  a n t i b o d y p r o t e i n / m l ) was a p p l i e d t o t h e c e n t e r w e l l , and 5 y l each o f c h i c k ovalbumin (100 yg/ml) q u a i l ovalbumin (500 y g / ml)  or oviduct extract  (1%) were a p p l i e d t o t h e a p p r o p r i a t e  outer w e l l s .  P l a t e 10b. O u c h t e r l o n y p l a t e d e m o n s t r a t i n g t h e r e a c t i o n o f a n t i - c h i c k o v a l b u m i n w i t h c h i c k o v a l b u m i n , 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 Legend and p r o t o c o l as above.  estrogen.  Serial dilutions:  1) 2% e x -  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 o v a l b u m i n , q u a i l o v a l b u m i n , and a magnum e x t r a c t from a b i r d w i t h d r a w n from e s t r o g e n p l u s p r o gesterone treatment.  Legend and p r o t o c o l as above.  i s an example o f a n e g a t i v e r e a c t i o n .  This  ME  QO  CO  terone treatment  ( r e f e r to P l a t e 10c) . No ovalbumin was  the c o n t r o l o v i d u c t s .  The  lower l i m i t o f the assay was  d e t e c t e d i n any o f 25 yg  ovalbumin/ml.  Lysozyme The lower l i m i t o f s e n s i t i v i t y of the assay f o r lysozyme i s 2 yg/ml (see s t a n d a r d curve i n a p p e n d i x ) .  The r e a d i n g s a t the lower l i m i t of the  s t a n d a r d curve are t h e r e f o r e s u b j e c t t o a g r e a t d e a l o f e r r o r .  Concentration  of the homogenate would have r e s u l t e d i n h i g h e r r e a d i n g s 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 t o the v e r y s m a l l s i z e o f the c o n t r o l o v i d u c t  magna. I n r e c o g n i t i o n o f t h i s problem,  i t i s c o n c l u d e d o n l y t h a t a t r e n d i s seen  i n lysozyme c o n c e n t r a t i o n s (see F i g u r e 8) a p p e a r i n g mentioned),  ( g i v e n the l i m i t a t i o n s  f i r s t on day 10 o f e s t r o g e n t r e a t m e n t , and perhaps i n c r e a s i n g  when p r o g e s t e r o n e  i s s u b s t i t u t e d f o r e s t r o g e n between days 10 and 15 of  treatment. Lysozyme was  p r e s e n t i n o n e - h a l f o f the o v i d u c t s w i t h d r a w n from e s t r o g e n  t r e a t m e n t , but i n none of the o v i d u c t s w i t h d r a w n from p r o g e s t e r o n e  treatment.  E s t r o g e n 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 quail oviduct.  T h i s has been r e p o r t e d to be  the  case i n c h i c k s as w e l l (45, 4 8 ) , 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 g l a n d c e l l s ( 7 1 ) .  I n c h i c k s , o n l y e s t r o g e n can  i n d u c e the s y n t h e s i s o f these p r o t e i n s d u r i n g p r i m a r y s t i m u l a t i o n , whereas progesterone t i o n (53).  i s a l s o a b l e to i n d u c e t h e i r s y n t h e s i s d u r i n g secondary s t i m u l a I n t h i s s t u d y , e s t r o g e n f o l l o w e d by p r o g e s t e r o n e  treatment  appears  t o cause a r e l a t i v e l y g r e a t e r lysozyme s y n t h e s i s t h a n c o n t i n u e d e s t r o g e n treatment.  However, due to the problems i n h e r e n t i n the lysozyme a s s a y ,  F i g u r e 8.  V a r i a t i o n s i n Lysozyme C o n c e n t r a t i o n o f O v i d u c t Magna as a F u n c t i o n o f Treatment.  Legend as i n F i g u r e 3.  Fig.8_:Time vs.Lysozyme Concentration  Day of Treatment  which would cause a s m a l l e r than a c t u a l measurement a t 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 o f the d a t a p r e s e n t e d here would p r o b a b l y n o t  be  valid. An e a r l i e r g e n e t i c s t u d y o f a v i a n p r o t e i n s i n d i c a t e d t h a t 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 t o ovalbumin  and lysozyme c o u l d be o b t a i n e d  q u a i l egg-white ( 8 5 ) , and a s i m i l a r , more r e c e n t s t u d y (141) has t h a t e s t r o g e n i n j e c t i o n can induce the presence o f ovalbumin of  resting birds.  from  indicated  i n the o v i d u c t s  These r e s u l t s s u p p o r t those s t u d i e s .  The r e s u l t s r e p o r t e d here as the e f f e c t s o f w i t h d r a w a l of treatment at  v a r i a n c e w i t h those r e p o r t e d f o r c h i c k o v i d u c t .  are  I t has been shown t h a t  w i t h d r a w a l 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 o f s y n t h e s i s o f  cell-specific  p r o t e i n s , and t h a t a f t e r 10 days of e s t r o g e n treatment and 10 days of w i t h d r a w a l , the c o n t e n t o f ovalbumin i s h e d (45, 4 8 ) .  and lysozyme i n the c h i c k o v i d u c t has  Yu e t a l have a l s o demonstrated  o v i d u c t magna do n o t make these p r o t e i n s ( 5 2 ) . to  t h a t d u r i n g r e g r e s s i o n , hen I t was  therefore surprising  f i n d t h a t o f the q u a i l o v i d u c t magna withdrawn from e s t r o g e n  a l l c o n t a i n e d ovalbumin,  and o n e - h a l f c o n t a i n e d lysozyme.  withdrawn from e s t r o g e n p l u s p r o g e s t e r o n e bumin, and none c o n t a i n e d lysozyme.  dimin-  treatment,  Of the magna  treatment, one-half contained o v a l -  T h i s i n f o r m a t i o n i s p r e s e n t e d i n Table I I .  There a r e two p o s s i b l e e x p l a n a t i o n s 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 o t h e r s , o r some o f the b i r d s had begun e s t r o g e n secretion naturally.  The l a t t e r would n o r m a l l y be the case i n b i r d s o f t h i s  age, as e v i d e n c e d b o t h by the s t r o m a l changes noted i n c o n t r o l b i r d s , and  the  r e p o r t o f F e r t u c k and Newstead (66) t h a t t u b u l a r g l a n d c e l l s are p r e s e n t a t 30 days o f l i f e i n u n t r e a t e d q u a i l . the e s t r o g e n l e v e l f e l l  I f t h i s was  the : c a s e , then as soon a.s  t o a p o i n t low enough t o r e l e a s e the hypothalamus from  Table I I Summary o f W e i g h t s , and t h e P r e s e n c e o r Absence o f S p e c i f i c P r o t e i n s i n t h e O v i d u c t Magna o f B i r d s Withdrawn from Treatment.  Magna withdrawn from estrogen plus progesterone treatment  Magna withdrawn from estrogen treatment Wt.  Oval.  Lys.  Wt.  Oval.  0.57  +  +  0.25  0.77  +  +  0.20  +  0.67  +  +  0.24  +  0.33  +  0.21  +  0.17  +  0.10  0.26  +  0.12 0.10 0.14  Lys.  i n h i b i t i o n i n the e x p e r i m e n t a l q u a i l , o v a r i a n growth and e s t r o g e n s e c r e t i o n s h o u l d have begun.  A l t h o u g h the l e v e l o f e s t r o g e n produced by t h e a n i m a l  would have been e x t r e m e l y 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 t h a t the secondary s t i m u l a t i o n o f an o v i d u c t r e q u i r e s f a r l e s s hormone, and g i v e s a f a s t e r and g r e a t e r response than the p r i m a r y s t i m u l a t i o n (53).  T h i s s u g g e s t e d s m a l l d i f f e r e n c e i n e s t r o g e n s e c r e t i o n i n h a l f o f the  o v i d u c t s withdrawn from e s t r o g e n t r e a t m e n t would t h e r e f o r e have c r e a t e d a marked d i f f e r e n c e i n b o t h s i z e and t i s s u e components, i n c l u d i n g p r o t e i n s , as compared to those o v i d u c t s withdrawn n o t begun e s t r o g e n s e c r e t i o n .  There i s no way  cell-specific  from t r e a t m e n t w h i c h  had  o f knowing from these s t u d i e s  whether the ovalbumin p r e s e n t i n the o v i d u c t s withdrawn from e s t r o g e n t r e a t ment and the lysozyme found o n l y i n the l a r g e r h a l f of these o v i d u c t s was newly m a * o r s i m p l y n o t y e t degraded.  However, the f a c t t h a t the b i r d s w i t h  l a r g e r o v i d u c t s a l s o had o b v i o u s l y l a r g e r o v a r i e s i n d i c a t e s t h a t they may  have  begun e s t r o g e n s e c r e t i o n , r a t h e r than t h e r e b e i n g 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.  A l t h o u g h the o v i d u c t magna withdrawn from e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t a l s o f e l l i n t o two groups, as mentioned,  i t s h o u l d be n o t e d t h a t  b o t h o f these two groups were much s m a l l e r i n terms o f wet w e i g h t , t h a n those withdrawn from e s t r o g e n t r e a t m e n t .  T h i s 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 t h a t the w i t h d r a w a l from e s t r o g e n p l u s p r o g e s t e r o n e t r e a t m e n t r e s u l t e d i n a f a r g r e a t e r l o s s of a l l components of organ growth than d i d the w i t h d r a w a l from e s t r o g e n t r e a t m e n t .  None o f the o v i d u c t magna withdrawn  from  e s t r o g e n p l u s p r o g e s t e r o n e treatment c o n t a i n e d lysozyme, and o n l y the l a r g e r h a l f o f them c o n t a i n e d ovalbumin.  Avidin A v i d i n was p r e s e n t o n l y i n those magna w h i c h had r e c e i v e d treatment,  progesterone  n o t i n t h e o v i d u c t magna from b i r d s r e c e i v i n g e s t r o g e n  o n l y , o r t h e t i s s u e from b i r d s w i t h d r a w n from treatment.  treatment  The average concen-  t r a t i o n o f a v i d i n was 60.9±19.5 yg/gm wet w e i g h t . The p o s s i b i l i t y o f the e x i s t e n c e o f an i n h i b i t o r o f 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 a d d i n g a known amount o f a v i d i n and an e x c e s s o f b i o t i n to the homogenates o f e s t r o g e n - t r e a t e d o v i d u c t s . (approximately  An enhancement o f b i n d i n g  20% i n c r e a s e ) was found under these c i r c u m s t a n c e s ,  t o when t h e a v i d i n and b i o t i n were i n c u b a t e d  as compared  i n s i m p l e aqueous s o l u t i o n .  A v i d i n has l o n g been known t o be under t h e c o n t r o l o f e s t r o g e n p l u s some other s t e r o i d i n chicks (32), although remains i n q u e s t i o n (24).  t h e n a t u r e o f t h e i n v i v o second agent  A v i d i n has been demonstrated t o be p r e s e n t  g o b l e t c e l l s o f t h e magnum e p i t h e l i u m (50) o n l y a f t e r p r o g e s t e r o n e estrogen a d m i n i s t r a t i o n to estrogen-treated chicks (50). r e s u l t s reported here support  the data f o r the c h i c k :  quail  IV.  or anti-  I n t h i s respect, the  a v i d i n i s present  i n o v i d u c t magna o f b i r d s t r e a t e d w i t h e s t r o g e n p l u s p r o g e s t e r o n e ; l o n g e d e s t r o g e n treatment  i n the  only  even p r o -  does n o t induce t h e s y n t h e s i s o f t h i s p r o t e i n i n  tissue.  Summary o f R e s u l t s The  r e s u l t s r e p o r t e d h e r e can be summarized as f o l l o w s :  enhances t h e growth and d i f f e r e n t i a t i o n o f t h e q u a i l o v i d u c t . of t r e a t m e n t ,  t h e mucosal f o l d s have grown t o f i l l  t h e r e a r e abundant t u b u l a r g l a n d c e l l s i n t h e s e .  Estrogen  greatly  W i t h i n 5 days  t h e e n l a r g e d lumen, and Vascularization i s evident.  By 10 days, t u b u l a r g l a n d c e l l s have g r e a t l y i n c r e a s e d i n number, and a r e  engorged w i t h g r a n u l e s , and d i f f e r e n t i a t i o n o f e p i t h e l i a l c e l l s i s e v i d e n t . By 15 days o f t r e a t m e n t , the g r a n u l e s o f the t u b u l a r g l a n d 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 o f the c e l l s , and 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. i n wet w e i g h t , RNA, period.  DNA,  The egg-white  epithelial  Growth, as measured by i n c r e a s e s  and p r o t e i n c o n t e n t , has i n c r e a s e d g r e a t l y i n t h i s  p r o t e i n s o f ovalbumin and lysozyme a r e a l s o  induced  by t h i s e s t r o g e n t r e a t m e n t . The e f f e c t s of p r o g e s t e r o n e , when added a f t e r 10 days o f e s t r o g e n t r e a t ment, a r e a s m a l l i n c r e a s e 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 i n c r e a s e i n a l l t i s s u e components e x c e p t DNA.  The  differ-  ence between 15 days o f e s t r o g e n t r e a t m e n t and 10 days e s t r o g e n , 5 days p r o gesterone t r e a t m e n t 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 p r o l o n g e d estrogenic stimulation. d e f i n i t e antagonism  However, the p r o g e s t e r o n e t r e a t m e n t d i s p l a y s a  f o r the c o n t i n u e d DNA  days e s t r o g e n t r e a t m e n t .  s y n t h e s i s seen between 10 and  15  T h i s c o u l d 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 p r o g e s t e r o n e , s i n c e 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 function.  cell  Ovalbumin and lysozyme were a l s o p r e s e n t d u r i n g c o n t i n u e d p r o g e s -  terone treatment.  I n a d d i t i o n , p r o g e s t e r o n e i n d u c e d the appearance of the  protein avidin. W i t h d r a w a l o f hormone treatment l e d to a l o s s o f a l l t i s s u e components. T h i s l o s s was  g r e a t e r a f t e r w i t h d r a w a l from e s t r o g e n p l u s p r o g e s t e r o n e  treat-  ment than a f t e r w i t h d r a w a l from e s t r o g e n t r e a t m e n t , i n d i c a t i n g t h a t hormone i s n e c e s s a r y f o r maintenance o f 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 of  the c e l l s .  O v i d u c t s withdrawn  from b o t h t r e a t m e n t s f e l l i n t o two  life  groups,  one group b e i n g about t w i c e the w e i g h t o f the o t h e r , and c o n t a i n i n g some o f the s p e c i f i c p r o t e i n s .  The s m a l l e r o v i d u c t s i n the group withdrawn  from  estrogen plus progesterone  c o n t a i n e d no ovalbumin o r lysozyme.  I t i s believed  t h a t t h i s dichotomy r e p r e s e n t s t h e r e s u l t of resumed o r i n i t i a t e d s y n t h e s i s by t h e a n i m a l s w h i c h had l a r g e r o v i d u c t s .  estrogen  I t i s a t t h i s time t h a t  d i f f e r e n t i a t i o n o f the o v i d u c t n o r m a l l y o c c u r s , and a l t h o u g h t h i s would be a v e r y low amount o f e s t r o g e n s e c r e t i o n , i t i s well-documented t h a t t h e secondary response r e q u i r e s f a r l e s s hormone than t h e p r i m a r y response  (53) .  A t a l l times d u r i n g t r e a t m e n t , the o v a r i e s o r t e s t i s o f the t r e a t e d b i r d s were n o t a b l y s m a l l , p r o b a b l y due to a decreased g o n a d o t r o p h s o u t p u t . I n b i r d s w i t h d r a w n from hormone t r e a t m e n t , t h e gonads were o f 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 a g a i n t h a t t h e s e may have begun e s t r o g e n s e c r e t i o n . An a n a l y s i s o f t h e o v i d u c t system i s o b v i o u s l y c o m p l i c a t e d by the f a c t t h a t 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 From these r e s u l t s , and those r e p o r t e d e l s e w h e r e ,  the e f f e c t s o f e s t r o g e n can  be summed as f o l l o w s : 1)  s t r o m a l 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 b l o o d cell  2)  invasion;  d i f f e r e n t i a t i o n of p r o g e n i t o r c e l l s to  protodifferentiated  c e l l s and t u b u l a r g l a n d c e l l s , w i t h concomitant ovalbumin 3)  and  synthesis of  lysozyme;  a f t e r p r o l o n g e d t r e a t m e n t , the d i f f e r e n t i a t i o n o f c i l i a t e d c e l l s and g o b l e t c e l l s ;  4)  c o n t i n u e d presence o f e s t r o g e n i s n e c e s s a r y f o r s u p p o r t o f the d i f f e r e n t i a t e d s t a t e ; w i t h d r a w a l l e a d s t o l o s s o f d i f f e r e n t i a t i o n and o f c e l l s .  tissue.  Response #1:  S t r o m a l changes o c c u r i n i t i a l l y , w i t h o u t t h e o c c u r r e n c e o f  i n c r e a s e d v a s c u l a r i z a t i o n ; t h a t appears t o happen l a t e r , a f t e r t h e edema and d i s p e r s i o n o f 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 w h i c h might a r i s e from  i n v a s i o n o f t h e stroma by b l o o d c e l l , i s n o t a f a c t o r i n t h e e a r l y s t r o m a l changes, a l t h o u g h 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 o f tubular gland  cells.  Response #2:  The d i f f e r e n t i a t i o n o f t h e p r o g e n i t o r c e l l s t o t u b u l a r g l a n d  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 .  T h e r e f o r e , c e l l - c e l l i n t e r a c t i o n s may be  i n v o l v e d ( 4 1 , 4 5 ) , e i t h e r w i t h c e l l s from t h e b l o o d , o r w i t h t h e s t r o m a l c e l l s themselves.  By t h i s time t h e s t r o m a l c e l l s a r e s w o l l e n and d i s p e r s e d and  have m i g r a t e d out t o the p e r i p h e r y .  The f a c t t h a t ovalbumin s y n t h e s i s  appears t o be concomitant w i t h and n o t subsequent t o m o r p h o l o g i c a l  differen-  t i a t i o n (43) i n d i c a t e s t h a t the genes f o r b o t h o f these a r e e x p r e s s e d t a n e o u s l y 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  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 t o biochemical Response #3:  a f t e r p r o l o n g e d e s t r o g e n treatment c e l l s have stopped budding o f f .  progesterone  differ-  differentiation.  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 t o  c i l i a t e d c e l l s and g o b l e t c e l l s , except  exclusive.  simul-  t h a t i t n o r m a l l y does n o t o c c u r  until  (45, 4 8 ) , that i s , a f t e r t u b u l a r gland  These p r o c e s s e s may t h e r e f o r e be m u t u a l l y  However, t h i s i s n o t i n d i c a t e d by t h e f a c t t h a t , i f e s t r o g e n and a r e g i v e n t o g e t h e r , 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 o t h e r 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 b o t h s e c r e t o r y g r a n u l e s and c i l i a (43). Response #4:  Withdrawal  l e a d s t o l o s s o f d i f f e r e n t i a t i o n and o f t h e c e l l s .  T h i s may be due t o t h e f a c t t h a t e s t r o g e n s u p p o r t s t h e v a s c u l a r i z a t i o n o f the tissue.  T h i s would n o t , however, e x p l a i n t h e more immediate l o s s o f c e l l  f u n c t i o n i n g seen on w i t h d r a w a l o f t h e hormone i n c h i c k o v i d u c t ( 4 8 ) , nor would i t e x p l a i n the s e q u e n t i a l n a t u r e o f c e l l d e a t h , s i n c e t u b u l a r g l a n d c e l l s a r e 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  differentiated.  The e f f e c t s o f p r o g e s t e r o n e can be summed as f o l l o w s : 1)  Antagonism  2)  Synergism w i t h e s t r o g e n - i n d u c e d c e l l  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 t o c i l i a t e d c e l l s goblet  4)  of e s t r o g e n - i n d u c e d c e l l  proliferation. functioning. and  cells.  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 .  Response #1,  the antagonism  to the e s t r o g e n i c r e s p o n s e , o c c u r s a t the p r o t o -  d i f f e r e n t i a t e d t u b u l a r g l a n d c e l l s t a g e , where c o n t i n u e d d i f f e r e n t i a t i o n o f these c e l l s t o t u b u l a r g l a n d 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 of  the t i s s u e i s i n t e r f e r e d w i t h ( 4 3 ) .  i n t e r f e r e n c e w i t h the c o n t i n u e d DNA estrogen treatment.  growth  I t i s e v i d e n t i n t h i s s t u d y as an  s y n t h e s i s n o t e d between 10 and 15 days o f  O b v i o u s l y , t h e n , i f p r o g e s t e r o n e i s g i v e n a f t e r the  t u b u l a r g l a n d c e l l s have d i f f e r e n t i a t e d , o r n e a r l y so, t h i s response w i l l  not  be so e v i d e n t as when i t i s g i v e n a t the b e g i n n i n g o f t r e a t m e n t . Response #2: (45,  The s y n e r g i s t i c response w i t h e s t r o g e n - i n d u c e d c e l l  functioning  4 8 ) , n o t e d 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 i n c r e a s e  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 o f the e f f e c t of p r o g e s t e r o n e on p r o t e i n s y n t h e s i s , but a v i d i n s y n t h e s i s i s d i f f e r e n t i n t h a t i t o c c u r s i n d i f f e r e n t c e l l s from those which  carry  on the b u l k o f the p r o t e i n s y n t h e s i s , and i t has an a b s o l u t e requirement f o r p r o g e s t e r o n e i n d u c t i o n ( 5 0 ) , w h i c h the s y n t h e s i s o f the o t h e r p r o t e i n s does not.  The mechanism of p r o g e s t e r o n e s y n e r g i s m w i t h e s t r o g e n - i n d u c e d  specific  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 t o be v i a an i n c r e a s e d message t r a n s c r i p t i o n , f o r  some o f these p r o t e i n s ( 9 7 ) . Response #3: and  The enhancement o f 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 t o c i l i a t e d  g o b l e t c e l l s may be r e l a t e d t o t h e a b o l i t i o n o f t h e t u b u l a r g l a n d  d i f f e r e n t i a t i o n , s i n c e these do n o t n o r m a l l y  occur simultaneously.  cells  cell  That i s ,  the s i m p l e a b o l i t i o n o f the one may be t h e enhancement o f t h e o t h e r .  This  enhancement o f 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 o v i d u c t s not t r e a t e d i n i t i a l l y w i t h e s t r o g e n  ( 4 7 ) ; b u t o t h e r than t h i s t h e r e i s no  i n f o r m a t i o n a v a i l a b l e on the mechanism o f a c t i o n o f p r o g e s t e r o n e i n enhancing epithelial  differentiation.  Response #4:  A v i d i n synthesis occurs  chick oviduct  ( 5 0 ) , and i s known t o r e q u i r e p r o g e s t e r o n e o r an a n t i - e s t r o g e n  for i t s induction.  i n the e p i t h e l i a l g o b l e t c e l l s o f the  The amount o f a v i d i n s y n t h e s i s i s a l s o known t o be  dependent on the l e n g t h o f e s t r o g e n  treatment given before  progesterone  t r e a t m e n t i s begun ( 4 9 ) .  A l l o f these responses l e a d towards i n c r e a s e d growth and f u n c t i o n i n g o f the t i s s u e except the f i r s t response to p r o g e s t e r o n e , the antagonism of estrogen-induced in attributing others.  cell proliferation.  I t may t h e r e f o r e be w i s e t o use c a u t i o n  t h i s l a t t e r response t o the same mechanism o f a c t i o n as t h e  DISCUSSION:  AN OUTLINE  A.  D i s c u s s i o n o f R e s u l t s of t h i s E x p e r i m e n t a t i o n  B.  The Response as a Phenomenon of I n d u c t i o n 1.  2.  3. C.  D.  The N a t u r e o f the C e l l u l a r Response t o E s t r o g e n :  P a l m i t e r ' s Model  a.  The E v i d e n c e  b.  D i s c u s s i o n o f the Model  c.  Comparison t o Other S t e r o i d - R e s p o n s i v e Systems  The Response t o P r o g e s t e r o n e : a.  The E v i d e n c e  b.  D i s c u s s i o n o f the Model  O ' M a l l e y ' s Model  E v e n t s a G e n e r a l Model W i l l Have t o E x p l a i n  Comparison o f t h i s I n d u c t i o n Phenomenon to Embryonic I n d u c t i o n 1.  The Inducer  2.  The Competence o f the Responding C e l l s  A B r i e f Assessment o f the S t a t e o f t h e F i e l d  DISCUSSION A.  The R e s u l t s of t h i s The  Experimentation  r e s u l t s r e p o r t e d h e r e i n d i c a t e t h a t d r a m a t i c growth t a k e s p l a c e , and  t h a t gene e x p r e s s i o n 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 o f e s t r o g e n  treatment.  T h i s i s e v i d e n t b o t h i n the appearance of new  proteins.  c e l l types and o f new  T h i s gene e x p r e s s i o n appears t o 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 s t r o m a l changes o c c u r f i r s t ,  then t u b u l a r g l a n d  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 o c c u r s l a s t .  The  cells  temporal nature  of  the gene e x p r e s s i o n i s a l s o i n d i c a t e d by the f a c t t h a t ovalbumin s y n t h e s i s i s induced b e f o r e lysozyme s y n t h e s i s . I t i s a l s o a p p a r e n t from these r e s u l t s t h a t the i n d u c e r does not  simply  t r i g g e r an a l r e a d y pre-programmed c e l l , w h i c h then proceeds on i t s own accordance w i t h i t s programming.  T h i s i s e v i d e n t from two  in  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 i n d u c e r , and 2) w i t h d r a w a l  of hormone l e a d s to a l o s s of gene e x p r e s s i o n .  T h i s l a t t e r i s i n d i c a t e d by l o s s o f c e l l s i n o v i d u c t s w i t h d r a w n from  treatment,  as w e l l as l o s s o f 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 , a l t h o u g h 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 a t the time o f  treatment.  T h i s gene e x p r e s s i o n i s t h e r e f o r e dependent on the c o n t i n u e d p r e s e n c e of the i n d u c e r .  Such a v e r y h i g h amount of s y n t h e s i s of  cell-specific  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 b i o c h e m i s t r y ) over so s u s t a i n e d a l e n g t h of time, would i m p l y t h a t the i n d u c e r a c t i v a t e s o r 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  m o l e c u l e , seem i m p l a u s i b l e when the magnitude of the response i s I t t h e r e f o r e seems p r o b a b l e t h a t the new r e s u l t of e s t r o g e n  considered.  gene e x p r e s s i o n seen here as a  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 a l r e a d y a c t i v e . These r e s u l t s would t h e r e f o r e s u p p o r t any model f o r the mechanism of estrogen  a c t i o n w h i c h i n c l u d e s these p o s s i b i l i t i e s .  Such a model would  t h a t of P a l m i t e r and c o - w o r k e r s , whose o r i g i n a l i d e a i t was i n d u c t i o n of one p r o t e i n (ovalbumin) by one  be  t o examine the  s t e r o i d ( e s t r o g e n ) , 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 a t each of the s t e p s i n the i n d u c t i o n p r o c e s s 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  a t the l e v e l of message t r a n s c r i p t i o n , message p r o c e s s i n g ,  t r a n s p o r t to  c y t o p l a s m , t r a n s l a t i o n of message o r message d e g r a d a t i o n .  The  P a l m i t e r ' s i n v e s t i g a t i o n s s u p p o r t e d a model i n w h i c h e s t r o g e n a c t i v a t i o n r a t h e r than by way pre-protein.  The  (89).  r e s u l t s of a c t s v i a gene  of a masked messenger or the a c t i v a t i o n o f a  r e s u l t s r e p o r t e d i n t h i s t h e s i s are i n k e e p i n g w i t h t h a t  model. The  response t h a t has been r e p o r t e d h e r e t o p r o g e s t e r o n e 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 n a t u r e :  a v i d i n s y n t h e s i s , the s u p p o r t of c o n t i n u e d i n h i b i t i o n of c o n t i n u e d DNA  synthesis.  growth and  the i n d u c t i o n of  f u n c t i o n i n g , and  the  Thus i t i s c l e a r t h a t the response  to p r o g e s t e r o n e encompasses not o n l y 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 e s t r o g e n  a c t i o n , and a c t i o n opposed to e s t r o g e n a c t i o n .  Whether or not a l l t h r e e o f these types of a c t i o n s a r e m e d i a t e d by same mechanism i s n o t known.  the  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 t h a t i t c o u l d be e x p l a i n e d i n a number o f ways.  The  t i o n of a v i d i n s y n t h e s i s , f o r example, c o u l d be e x p l a i n e d by new  gene  induc-  a c t i v i t y , by a masked messenger h y p o t h e s i s , protein.  or by an a c t i v a t i o n o f a p r e -  Work on t h e c h i c k o v i d u c t w h i c h uses a v i d i n i n d u c t i o n as a marker  f o r p r o g e s t e r o n e a c t i o n has l e d t o a model o f p r o g e s t e r o n e 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 a r e c o m p a t i b l e w i t h t h a t model,  but can n e i t h e r s u p p o r t n o r 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  ( t h a t i s , the enhancement o f t u b u l a r g l a n d c e l l f u n c t i o n i n g ) c o u l d be e x p l a i n ed by enhanced t r a n s c r i p t i o n o f e s t r o g e n - a c t i v a t e d  genes, o r enhanced p r o c e s -  s i n g o f messenger from these genes, o r enhanced t r a n s l a t i o n o f messenger, o r decreased degradation  o f messenger o r o f p r o t e i n .  Work on t h e 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 synthesis of the t u b u l a r g l a n d c e l l p r o t e i n s i n d i c a t e s t h a t p r o g e s t e r o n e , when g i v e n  alone,  on 2° s t i m u l a t i o n , i s a b l e t o i n i t i a t e new gene a c t i v a t i o n ( 7 1 ) , and when i t i s given w i t h estrogen, RNAs i n v o l v e d  (97).  t o enhance t r a n s c r i p t i o n o f some o f the messenger  The i n c r e a s e d s y n t h e s i s o f p r o t e i n s and a l l t i s s u e  components e x c e p t DNA r e p o r t e d h e r e s u p p o r t t h a t h y p o t h e s i s . g e s t e r o n e was n o t g i v e n t o g e t h e r w i t h e s t r o g e n ,  However, p r o -  but f o l l o w i n g estrogen.  Thus  i t i s n o t p o s s i b l e t o i n d i c a t e whether the response t o p r o g e s t e r o n e i s one o f i n i t i a t i n g gene a c t i v i t y , o r s i m p l y c o n t i n u a n c e o f e s t r o g e n - a c t i v a t e d cription.  trans-  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 t h a t b o t h o f these p o s s i b i l i t i e s a r e 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 o f DNA s y n t h e s i s .  I n the c h i c k o v i d u c t , t h i s i s the cause o f t h e  a b o l i t i o n o f t u b u l a r g l a n d c e l l d i f f e r e n t i a t i o n and t h e growth r e s p o n s e . There has been no e l u c i d a t i o n o f t h e mechanism o f t h i s a c t i o n .  I t has been  so proposed t h a t e s t r o g e n 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 t h a t p r o g e s t e r o n e can i n t e r f e r e w i t h t h e f i r s t p r o c e s s , b u t n o t the second.  The r e s u l t s r e p o r t e d here a r e c o m p a t i b l e w i t h  such a . h y p o t h e s i s , b u t can n e i t h e r s u p p o r t n o r c o n t r a d i c t i t .  There a r e a  number o f p o s s i b l e mechanisms by which t h i s a c t i o n c o u l d be m e d i a t e d .  Pro-  g e s t e r o n e may i n t e r f e r e w i t h one o r many e s t r o g e n - a c t i v a t e d genes, which code for  a protein or proteins 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 ,  t e r o n e o r a p r o g e s t e r o n e - i n d u c e d gene p r o d u c t may i n t e r f e r e w i t h t h e f u n c t i o n ing  o f such a p r o t e i n .  That i s , t h i s may be t h e r e s u l t o f a n u c l e a r o r a  c y t o p l a s m i c a c t i o n o f p r o g e s t e r o n e , o f the p r o g e s t e r o n e — r e c e p t o r complex, o r of  B.  a p r o d u c t i n d u c e d by i t .  The Response a s a Phenomenon o f I n d u c t i o n The changes t h a t a r e r e p o r t e d 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  for  the c h i c k o v i d u c t , and i t i s suggested t h a t so g r e a t a response as o b s e r v -  ed i n b o t h b i r d s must encompass t h e e x p r e s s i o n o f many d i f f e r e n t genes. of  Most  t h e work on e s t r o g e n and p r o g e s t e r o n e - i n d u c e d d i f f e r e n t i a t i o n o f o v i d u c t  t i s s u e has been d i r e c t e d toward an e x p l a n a t i o n o f t h i s gene e x p r e s s i o n . I t i s t h e r e f o r e r e l e v a n t t o examine a t t h i s p o i n t t h e i n d u c t i o n o f o v i d u c t d i f f e r e n t i a t i o n , t o c o n s i d e r the work done on s i m i l a r systems, and t o c r i t i c a l l y examine the models o f s t e r o i d a c t i o n t h a t have been proposed t o e x p l a i n this effect. The r e s u l t s r e p o r t e d here s u p p o r t o r a r e c o m p a t i b l e w i t h b o t h t h e model for  e s t r o g e n a c t i o n , and t h e model f o r p r o g e s t e r o n e a c t i o n i n c h 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, h y p o t h e s i z e s t h a t e s t r o g e n a c t s a t t h e l e v e l o f t r a n s c r i p t i o n , t o i n d e p e n d e n t l y i n d u c e a c t i v a t i o n o f many genes ( 8 9 ) .  The model f o r p r o g e s t e r o n e  a c t i o n , based on O'Malley's work, i n d i c a t e s t h a t  p r o g e s t e r o n e b i n d s to i t s r e c e p t o r , e n t e r s the n u c l e u s , and t h e r e b i n d s t o a c i d i c c h r o m a t i n 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, 9 1 ) ,  models o r hypotheses t h e r e f o r e have a t base an a c t i v a t i o n o f new  Both  transcription,  a l t h o u g h they a r e based on e n t i r e l y d i f f e r e n t d a t a . Both P a l m i t e r ' s h y p o t h e s i s f o r e s t r o g e n a c t i o n and O'Malley's f o r p r o g e s t e r o n e a c t i o n on c h i c k o v i d u c t a r e e x t e n s i o n s o f the g e n e r a l model f o r s t e r o i d a c t i o n (86-88).  T h i s g e n e r a l model i s :  t h a t the s t e r o i d e n t e r s the  cell,  b i n d s to a c y t o p l a s m i c r e c e p t o r p r o t e i n , e n t e r s the n u c l e u s , and t h e r e a c t s i n some as y e t unknown way  to a l t e r t r a n s c r i p t i o n .  have been demonstrated f o r e s t r o g e n , p r o g e s t e r o n e  Such r e c e p t o r p r o t e i n s and t e s t o s t e r o n e i n c h i c k  o v i d u c t (139, 74, 75, 129-132), and f o r e s t r o g e n i n q u a i l o v i d u c t (140). T h i s 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 t o 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). mentioned h e r e , of e s t r o g e n and p r o g e s t e r o n e  The  two s p e c i f i c  cases  i n c h i c k o v i d u c t , are o f r e l e -  vance b o t h because they r e p r e s e n t 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 t o the problem p r e s e n t e d here - the e x p l a n a t i o n of c e l l u l a r response will  t o a s t e r o i d i n d u c e r i n the a v i a n o v i d u c t .  These models  t h e r e f o r e be examined i n terms of the e v i d e n c e on which each i s based,  the c o n t r o v e r s i a l a s p e c t s of each, and how  each compares t o models f o r  s t e r o i d a c t i o n i n mammalian systems. The to  c h a r a c t e r i s t i c s o f t h i s i n d u c t i o n phenomenon w i l l a l s o be compared  those o f e m b r y o l o g i c  i n d u c t i o n phenomenon, i n an e f f o r t to understand  common denominators o f these  processes.  the  1.  The N a t u r e o f t h e C e l l u l a r Response t o E s t r o g e n :  P a l m i t e r ' s Model  When d i s c u s s i n g t h e response o f c e l l s t o t h e i n d u c e r s , c a r e must be t a k e n to d i s t i n g u i s h between t h e c e l l types and between t h e d i f f e r e n t s t a g e s o f t r e a t m e n t 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  r e s p o n s e , and t h e secondary r e s p o n s e , w h i c h i s t h e response o f a t i s s u e t h a t has been p r e v i o u s l y t r e a t e d , withdrawn s t i m u l a t e d a second t i m e .  from hormone t r e a t m e n t , and then  The p r i m a r y response o f t h e c h i c k o v i d u c t t o e s t r o -  gen has been o u t l i n e d i n t h e 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 t h e results section of this thesis.  The major work on t h e a c t i o n o f e s t r o g e n on  the o v i d u c t has made use o f t h e secondary r e s p o n s e , s i n c e t h a t 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 o f t h e growth r e s p o n s e , 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 a l r e a d y present. The knowledge g a i n e d has been the work o f P a l m i t e r and c o - w o r k e r s . was  It  t h e o r i g i n a l i d e a o f t h i s group t o examine t h e i n d u c t i o n o f one p r o t e i n  (ovalbumin) by one s t e r o i d ( e s t r o g e n ) , and t o t e s t t h e p o s s i b i l i t y o f c o n t r o l at  each o f t h e s t e p s i n t h e i n d u c t i o n p r o c e s s ( 8 9 ) .  That i s ,  investigations  were c a r r i e d o u t t o determine i f c o n t r o l was e x e r t e d a t t h e l e v e l o f message t r a n s c r i p t i o n , message p r o c e s s i n g , t r a n s p o r t o f message t o cytoplasm-;' t r a n s l a t i o n o f message, and message d e g r a d a t i o n . T h i s work w i l l be examined f i r s t i n terms o f t h e e v i d e n c e t h a t has been r e p o r t e d , second i n terms o f t h e model t h a t t h e a u t h o r presents to account f o r the e v i d e n c e , and then i n terms o f a comparison o f t h i s model and e v i d e n c e t o other steroid-responsive  a.  systems.  The E v i d e n c e  The r e s u l t s o b t a i n e d by P a l m i t e r and o t h e r s c o n c e r n i n g t h e e f f e c t o f  e s t r o g e n and o t h e r s t e r o i d s on o v i d u c t s w i t h d r a w n from hormone a r e summarized i n the diagram on the f o l l o w i n g page. The secondary  response  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 :  i s f a s t e r than the p r i m a r y one;  following a lag  p e r i o d o f 3 h o u r s , ovalbumin i s d e t e c t e d and b e g i n s i n c r e a s i n g i n amount ( 5 3 ) . The b e g i n n i n g of t h i s response, Phase I , i s r e p r e s e n t e d by an o r g a n i z a t i o n o f 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 sis.  rRNA s y n t h e -  T h i s phase o c c u r s i n the f i r s t 12 hours,: and can be e l i c i t e d by  estrogen or progesterone,  or b o t h t o g e t h e r (53).  either  The second phase (Phase I I )  i s r e p r e s e n t e d by c o n t i n u e d i n c r e a s e s i n a l l c o n s t i t u e n t s , w i t h rRNA s y n t h e s i s c o n t r i b u t i n g new  ribosomes to the p o o l of o l d and new  polysomes a r e drawn (53).  ribosomes from w h i c h  Phase I I can be e l i c i t e d by e s t r o g e n o n l y , n o t  p r o g e s t e r o n e , b u t p r o g e s t e r o n e w i t h e s t r o g e n does not i n h i b i t the r a t h e r , they a c t s y n e r g i s t i c a l l y  by  response,  (53).  Ovalbumin i s a p p a r e n t l y s y n t h e s i z e d and r e l e a s e d a t the same r a t e as o t h e r 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 bumin message was  (53).  Oval-  i s o l a t e d from o v a l b u m i n - s y n t h e s i z i n g polysomes by immuno-  a d s o r p t i o n t o a n t i - o v a l b u m i n a n t i b o d i e s , and was  incubated with l a b e l l e d  p r e c u r s o r s and v i r a l r e v e r s e 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, 9 3 ) .  A l t h o u g h c o n s i s t i n g o f o n l y 200 n u c l e o t i d e s , i t was  n e v e r t h e l e s s p o s s i b l e t o 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, demonstrate t h a t i n the i n d u c t i o n of ovalbumin,  and  gene a m p l i f i c a t i o n does n o t  o c c u r (93). Ovalbumin message was  l a t e r i s o l a t e d from t o t a l average p o l y s o m a l  ( 9 4 ) , and r e v e r s e 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 the same manner ( 9 5 ) . but was  The p r o d u c t o f t h a t r e a c t i o n was  RNA  used i n  the same s m a l l s i z e ,  used to demonstrate, by h y b r i d i z a t i o n , t h a t 93% of h y b r i d i z a b l e  F i g u r e 9.  P r o p o s e d C o n t r o l o f Ovalbumin S y n t h e s i s . i)  Gene a m p l i f i c a t i o n does n o t o c c u r .  One copy p e r  TsllH  E l a t i o n a t a r a t a o f 28 m o l e c u l e s / m i n / " 1 1 , a f t e r a 3-hour l a g by de novo ^ ^ ^ i i ^ ^ ^ o f e„es already a c t i v e . 3) Other messages s y n t h e s i z e d a t d i f f e r e n t r a t e s . 4) I f o t h e r s t e r o i d s a r e p r e s e n t , 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 i n c r e a s e d messenger accumulat i o n , n o t decreased d e g r a d a t i o n , 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 , o r by a d d i t i o n a l r a t e - l i m i t i n g steps. 5) P r o c e s s i n g i s e x t r e m e l y r a p i d , o r a b s e n t ; 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 s t o r e d i n withdrawal. 6) T r a n s p o r t t o t h e c y t o p l a s m i s n o t a r a t e - l i m i t i n g s t e p ; t h e r e i s no n u c l e a r p o o l o f message, s o r e a n d C a c t i v a t i o n o f a t r a n s p o r t t o cytoplasm i s n o t occurring. I n i t i a t i o n o f p r o t e i n s y n t h e s i s i s n o t r a t e - l i m i t i n g ; t h e r e i s no c y t o p l a s m i c p o o l o f t r a n s l a t a b l e o n o n«A - t r a n s l a t4~a b l e message. T r a n s l a t i o n i s enhanced by i n c r e a s e d r a t e s o f i n i t i a t i o n and e l o n g a t i o n o f p o l y p e p t i d e c h a i n s ; t h i s i s not s p e c i f i c f o r ovalbumin. There i s no e v i d e n c e o f a c y t o p l a s m i c r e p r e s s o r . 93% o f t h e message i s i n t h e polysomes. The s y n e r g i s t i c e f f e c t s o f p r o g e s t e r o n e o r t e s t o s t e r o n e w i t h e s t r o g e n a r e n o t e x e r t e d a t t r a n s l a t i o n . E s t r o g e n may s t a b i l i z e t h e message, s i n c e i t i s degraded upon w i t h d r a w a l ; n u c l e i from t i s s u e w i t h d r a w n from t r e a t m e n t c o n t a i n one copy p e r n u c l e u s .  Cytoplasm  2)  -  1  ^  g  Tubular Gland  7)  .  8) 9) 10) 11) 12)  f\  * 1 r~S  l-» I  E = Estrogen;  Cell  s*.  R, = C y t o p l a s m i c  receptor.  oo  m a t e r i a l was cytoplasm.  i n the polysomes, 5% i n the n u c l e u s , and 2% i n the non-polysomal T h i s i n d i c a t e d t h a t t h e r e was  no p o o l of n o n - t r a n s l a t e d messenger  i n the c y t o p l a s m , e i t h e r i n t r a n s l a t a b l e o r n o n - t r a n s l a t a b l e form,  thus  s u p p o r t i n g e a r l i e r s t u d i e s w h i c h had assayed messenger by a c t i v i t y i n a f r e e system (96) (see a l s o r e f . 137, Two  122,  cell-  138).  p e r c e n t o f the n u c l e a r h y b r i d i z a t i o n was  s p e c i e s w i t h a s e d i m e n t a t i o n c o e f f i c i e n t of 30S  a high molecular  weight  (the message i s 16S-18S) ( 9 5 ) .  I f t h i s v e r y s m a l l amount i n d i c a t e s a p r i m a r y t r a n s c r i p t i o n p r o d u c t ,  then  r a p i d c o n v e r s i o n 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 p r e message ( 9 5 ) .  The appearance o f message i n the polysomes w i t h o u t  accumulation  in  the n u c l e u s f i r s t , i n d i c a t e s t h a t e s t r o g e n 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 c y t o p l a s m The  (95).  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 o f 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, t o g e t h e r w i t h the l a c k o f a s i g n i f i c a n t p o o l o f messenger i n the non-polysomal c y t o p l a s m ,  indicate  t h a t the i n i t i a t i o n o f p r o t e i n s y n t h e s i s i s not a l i m i t i n g f a c t o r ( 9 5 ) . The e s t i m a t e d l e v e l o f message i n the c e l l s d u r i n g p e r i o d s of hormone w i t h d r a w a l was  one m o l e c u l e per n u c l e u s , so t h a t i t was  not p o s s i b l e t o 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 o c c u r r e d d u r i n g p e r i o d s o f hormone w i t h d r a w a l ; t h a t 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 , o r de novo t r a n s c r i p t i o n ( 9 5 ) ,  The l o s s of messenger a c t i v i t y  on w i t h d r a w a l of hormone i n d i c a t e d t h a t c o n t i n u e d e s t r o g e n treatment n e c e s s a r y f o r c o n t i n u e d presence p o s s i b l e t h a t the message was  was  o f messenger; the a u t h o r s c o n s i d e r e d i t  u n s t a b l e i n the absence of e s t r o g e n (96).  Other p r o t e i n s - ovalbumin,  conalbumin,  ovomucoid, and lysozyme - a r e a l l  made i n the same t u b u l a r g l a n d 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 f o u r p r o t e i n s , a l t h o u g h  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 s y n t h e s i s o f conalbumin r e l a t i v e t o o v a l b u m i n , and the o v a l b u m i n s y n t h e s i s b e g i n s more r a p i d l y , but reaches a s t e a d y - s t a t e a t a lower (71).  Conalbumin i s p r e f e r e n t i a l l y s y n t h e s i z e d a t lower doses' of  and conalbumin and ovomucoid a r e b o t h s e l e c t i v e l y enhanced by and e s t r o g e n  t o g e t h e r , and by t e s t o s t e r o n e and e s t r o g e n  estrogen alone. i n a c t i v e alone  level  estrogen,  progesterone  together, r e l a t i v e to  T h i s o c c u r s i n s p i t e of the f a c t t h a t t e s t o s t e r o n e i s (71).  T h i s s e l e c t i v e i n c r e a s e i s not due  z a t i o n r e l a t i v e to e s t r o g e n  treatment  to messenger s t a b i l i -  o n l y , as judged by decay i n the p r e s e n c e  of a c t i n o m y c i n - D ( 7 1 ) . The was  i n c r e a s e i n p r o t e i n s y n t h e s i s on secondary s t i m u l a t i o n by  estrogen  p a r t i a l l y the r e s u l t o f an i n c r e 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 -  p e p t i d e c h a i n s i n t i s s u e from t r e a t e d a n i m a l s , as compared t o t h a t i n  animals  w i t h d r a w n 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 o f i n i t i a t i o n o f p r o t e i n s y n t h e s i s (97).  Initiation i s rate-limiting i n tissue  t h a t has been w i t h d r a w n 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  one w i t h e s t r o g e n ,  r e l a t i v e t o e s t r o g e n treatment  conalbumin s y n t h e s i s , a r e not due e l o n g a t i o n (97).  w i t h e s t r o g e n , and o f t e s t o s t e r a l o n e , on ovomucoid  and  to an i n c r e a s e i n r a t e s of i n i t i a t i o n or  They a r e t h e r e f o r e b e l i e v e d t o r e f l e c t an i n c r e a s e i n the  c o n c e n t r a t i o n o f 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 ( 9 7 ) . The  " s u p e r i n d u c t i v e " e f f e c t o f a c t i n o m y c i n - D was  demonstrated t o be  to enhanced t r a n s l a t i o n , not t o i n c r e a s e d messenger, w h i c h r u l e d out  the  due  p o s t u l a t e d c y t o p l a s m i c r e p r e s s o r o f Tomkins ( 9 9 ) , a t 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  relative-  l y l o n g h a l f - l i f e of the message, w h i c h would t h e r e f o r e be i n c r e a s i n g l y a b l e 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 o t h e r messengers d i e d out The estrogen  (98).  s y n e r g i s t i c e f f e c t o f t e s t o s t e r o n e and e s t r o g e n t o g e t h e r r e l a t i v e t o treatment  i n c r e a s e i n RNA  a l o n e was  shown t o e x t e n d t o rRNA s y n t h e s i s , and  polymerase a c t i v i t y , w h i c h was  considered  to  to be the  result  of more growing c h a i n s , r a t h e r than i n c r e a s e d e l o n g a t i o n r a t e (100). supports  the c o n c l u s i o n t h a t t e s t o s t e r o n e and e s t r o g e n  an  This  t o g e t h e r cause an  i n c r e a s e i n messenger c o n c e n t r a t i o n r e l a t i v e t o e s t r o g e n a l o n e , and i n d i c a t e s t h a t t e s t o s t e r o n e s e l e c t i v e l y enhances some e s t 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 a c t e n t i r e l y by the same mechanism t h a t e s t r o g e n or p r o g e s 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 o f ovalbumin message a c c u m u l a t i o n ,  r a t e of a c c u m u l a t i o n ,  and the  steady-state  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  half-life,  were c a l c u l a t e d t o be 22 and 34 m o l e c u l e s per minute p e r t u b u l a r g l a n d  cell,  w h i c h P a l m i t e r b e l i e v e s to be c l o s e enough t o r e p r e s e n t a s i n g l e v a l u e of  28  m o l e c u l e s per minute per c e l l , i m p l y i n g a s i m p l e , s i n g l e - s t e p model of cons t a n t r a t e i n c r e a s e (77).  He a l s o n o t e s , though, t h a t the time l a g o f 3 h o u r s  between s t e r o i d a d m i n i s t r a t i o n and message a c c u m u l a t i o n s i m p l e one-step model (77).  From comparison w i t h o t h e r systems, i t was  l a t e d t h a t a s i n g l e gene can s u p p o r t 30-50% of the p r o d u c t s  speaks a g a i n s t a calcu-  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 o n l y  o f t r a n s c r i p t i o n r e a c h the c y t o p l a s m  (77).  The  data  i n d i c a t e c o n t r o l a t the l e v e l o f s y n t h e s i s or a c t i v a t i o n o f messenger, and suggest v a s t 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 h e t e r o g e n e o u s , 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 on a c r y l a m i d e  (92, 9 4 ) , and by the broad band range produced  g e l e l e c t r o p h o r e s i s , c o v e r i n g a range of m o l e c u l a r  100,000 (94).  The  weight of  p r e p a r a t i o n from w h i c h t h i s ovalbumin message i s i s o l a t e d  i s t o t a l average p o l y s o m a l RNA,  p u r i f i e d by sucrose  e l e c t r o p h o r e s i s , r a t h e r than by immunoadsorption.  sedimentation The  and  gel  immunoprecipitation  o f the p r o d u c t 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 demonstrat i o n t h a t the p r e p a r a t i o n i s p u r e , a l t h o u g h age o f messengers t h a t are e x c l u d e d  c a l c u l a t i o n s based on the  by the p r o c e d u r e , and  percent-  the p e r c e n t a g e of  messenger t h a t i s ovalbumin messenger, i n d i c a t e t h a t 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 ( 9 4 ) .  The m o l e c u l a r  message i s 875,000, as judged by e l e c t r o p h o r e s i s , and sucrose  sedimentation  (94).  w e i g h t of  the  550,000 as judged by  T h i s c o r r e s p o n d s to 2640 and  1670  nucleotides  r e s p e c t i v e l y , of w h i c h 1161 w i l l be t r a n s l a t e d to ovalbumin ( 9 4 ) .  At l e a s t  p a r t 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 r e p r e s e n t e d  the  by p o l y A ; of  r e m a i n d e r , i t i s thought t h a t t h i s i s t r a n s c r i b e d w i t h the message ( 9 5 ) . has most r e c e n t l y been shown t h a t the message does not undergo any change i n m o l e c u l a r  It  significant  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  p r o c e s s i n g i s i n s i g n i f i c a n t o r absent (95, 163).  T h i s p o i n t s more d i r e c t l y  t o the p r o c e s s 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  this  protein. P a l m i t e r f e e l s t h a t the i n d u c t i o n of the f o u r major p r o t e i n s mentioned i s not c o o r d i n a t e d 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  s y n t h e s i s does not d i m i n i s h f a s t e r than o v a l b u m i n s y n t h e s i s , w h i c h would be e x p e c t e d from the s h o r t e r h a l f - l i f e of i t s messenger ( a l t h o u g h he n o t e s t h a t t h i s may  be due  t o the g r a d u a l drop i n e s t r o g e n  t o the l e v e l where i t  p r e f e r e n t i a l l y i n d u c e s 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 , w h i c h are not a t the of message d e g r a d a t i o n  level  or t r a n s l a t i o n ; 3) t h e r e are d i f f e r e n c e s i n e s t r o g e n  t h 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) o t h e r s t e r o i d s e l i c i t o n l y p a r t of the t o t a l r e s p o n s e ;  t h a t i s , they do not e l i c i t rRNA s y n t h e s i s , DNA He  t h e r e f o r e does not  coordinated  synthesis, etcetera  (71).  f e e l t h a t the response t o o t h e r s t e r o i d s can be b o t h  and s e l e c t i v e .  i s independently regulated of a few i n t e r m e d i a t e s ,  A l t h o u g h he t h i n k s i t l i k e l y t h a t each p r o t e i n (71, 101), no mention i s made of the  o r one  possibility  f o r each s t e r o i d .  A l t h o u g h the s i n g l e r a t e of a c c u m u l a t i o n 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 t h a t the 3-hour time lag  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 o f  transcription-translation-  feedback e v e n t s , and so speaks a g a i n s t a s i m p l e one-step model b.  (77).  D i s c u s s i o n of the Model  A model o f c o o r d i n a t e d  response due  not f a v o u r e d by P a l m i t e r , due  to a s i n g l e intermediate  factor i s  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 a t  low doses, i t s l o n g e r - t h a r i - e x p e c t e d  r a t e of d e g r a d a t i o n ,  and  the s e l e c t i v e  e f f e c t s o f o t h e r 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 t h e s e on p e r i p h e r a l e v e n t s 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  t h a t the p r e f e r e n t i a l s y n t h e s i s of conalbumin a t low doses, and t h a n - e x p e c t e d r a t e of decay on w i t h d r a w a l of hormone, may the s l o w l y f a l l i n g l e v e l s of e s t r o g e n a t w i t h d r a w a l may conalbumin s y n t h e s i s e s t r o g e n ( 7 1 ) , but  (71).  i t s longer-  be r e l a t e d i n t h a t  p r e f e r e n t i a l l y enhance  He s u g g e s t s a s t u d y i n the p r e s e n c e o f an  t o my knowledge, no f u r t h e r s t u d i e s a l o n g  anti-  t h a t l i n e have  been conducted.  I t i s o f i n t e r e s t h e r e , t h a t conalbumin has a l s o been n o t e d  i n the b l o o d of immature, n o n - t r e a t e d  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 a g a i n s t a s i m p l e one-step model of e s t r o -  gen a c t i o n ( 7 7 ) , but cannot i n d i c a t e i n any way c o o r d i n a t e d or independent.  whether the r e s p o n s e s are  I t would o n l y a l l o w time f o r i n t e r m e d i a t e a c t i o n s ;  t h i s c o u l d be i n k e e p i n g w i t h b o t h p o s s i b i l i t i e s . c o u l d i n c l u d e any i m a g i n a b l e  Such i n t e r m e d i a t e a c t i o n s  sequence of e v e n t s , 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 , w h i c h a r e thought to be a c o n t r o l l i n g element i n ecdysoneinduced p u f f i n g p a t t e r n s i n Chironomous s a l i v a r y g l a n d chromosomes C103,  104).  Such a p o s s i b i l i t y s h o u l d be viewed i n the l i g h t of the known e f f e c t s o f i o n c o n c e n t r a t i o n s on s t e r o i d r e c e p t o r s  (105).  The s e l e c t i v e e f f e c t s o f o t h e r 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 p e r i p h e r a l e v e n t s , may  on  speak f o r independent c o n t r o l , but these e f f e c t s are  n e v e r t h e l e s s not at v a r i a n c e w i t h a model o f c o o r d i n a t e d c o n t r o l , such as B r i t t e n - D a v i d s o n model o f gene a c t i v i t y i n e a r l y development (106). case, the a c t i o n o f o t h e r s t e r o i d s a l o n e or i n c o m b i n a t i o n c o u l d be s e l e c t i v e , but a l s o c o o r d i n a t e d .  with  the  In that  estrogen,  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 e x p e c t , u s i n g the Redundant R e c e p t o r Model, t h a t a l l genes a c t i v a t e d by e s t r o g e n have a r e c e p t o r t o b i n d the a c t i v a t o r RNA; a s s o c i a t e d w i t h them.  region  t h a t i s , they would a l l have a common sequence  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 c a s e ; t h e r e 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 o f genes.  Thus i t i s p o s s i b l e t h a t a s i n g l e e n t i t y , o r a  few such e n t i t i e s , c o u l d mediate the p l e i o t r o p i c f o r the s e l e c t i v e e f f e c t s o f o t h e r s t e r o i d s .  responses,  and s t i l l  allow  T h i s 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 a t l o w e r doses of e s t r o g e n .  I t does  r e q u i r e t h a t each s t e r o i d o p e r a t e by a d i f f e r e n t i n t e r m e d i a t e . 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 would account f o r the a v a i l a b l e d a t a r e g a r d i n g  proteins  t h e i r induction 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 c o m b i n a t i o n s , 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 c o o r d i n a t e d  responses of  p r i m a r y s t i m u l a t i o n , o r even t o the complete secondary r e s p o n s e . t h a t more than one model s h o u l d be c o n s i d e r e d ;  t h a t i s , i t may  the  I t may  be t h a t  be 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 i n d e p e n d e n t l y or c o o r d i n a t e l y c o n t r o l l e d b e a r s d i r e c t l y on the n a t u r e of the model.  P a l m i t e r has  p o i n t e d out  (77)  t h a t i f t h e r e 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 s t e p i n the i n d u c t i o n of each of the f o u r p r o t e i n s , then i t must be s u b j e c t t o m u l t i p l e r e g u l a t i o n , o t h e r w i s e 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 s t e p s t o account f o r the v a r i o u s 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 . f o r each of these p r o t e i n s may (71, 101).  He  f e e l s , however, t h a t the genes  be i n d e p e n d e n t l y c o n t r o l l e d by the s t e r o i d ( s )  I f t h i s model were extended t o the p r i m a r y r e s p o n s e , one  imagine t h a t a model of c o o r d i n a t e d  would  c o n t r o l would r e q u i r e measurably l e s s  e s t r o g e n bound i n the n u c l e u s than a model r e q u i r i n g t h a t 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 e s t r o g e n .  Such an i n v e s t i g a t i o n , however, would  hampered by the assumption t h a t t h e r e i s no i n t e r m e d i a t e ( s ) , and by d i f f i c u l t i e s involved i n estimating nuclear The  the  binding.  i m p l i e d assumption t o t h i s p o i n t i n the d i s c u s s i o n has been t h a t  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 d e m o n s t r a t i n g  absence of any p r e c u r s o r  be  the  the  f o r ovalbumin message s u p p o r t s t h i s assumption  (163).  A g a i n , i n t e l l e c t u a l l y , a model o f e s t r o g e n a c t i o n r e l y i n g on t h e i n d e p e n dent a c t i v a t i o n o f each gene by e s t r o g e n o r i t s i n t e r m e d i a t e i s d i s p l e a s i n g , s i n c e i t does n o t e x p l a i n t h e c o o r d i n a t i o n e v i d e n t i n t h e p r i m a r y r e s p o n s e , 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 a r e a l l  induced.  One would e x p e c t these responses t o o c c u r i n an o r d e r l y sequence i n t i m e , but such a sequence has n o t 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 t o t h e h i g h doses o f e s t r o g e n used, which p r e c l u d e a r e s o l u t i o n o f t h e f i n e r a s p e c t s o f the  r e s p o i i S G .  The model o f independent gene r e g u l a t i o n , however, has o n l y  been suggested f o r t h e r e g u l a t i o n o f 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, t h e n i t would have t o e x p l a i n n o t o n l y the p r i m a r y and secondary a c t i o n s o f e s t r o g e n , b u t t h o s e o f p r o g e s t e r o n e as w e l l , which i n c l u d e t h e 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 o f p r o g e s t e r o n e when i t i s g i v e n c o n c o m i t a n t l y w i t h e s t r o g e n i n the p r i m a r y s t i m u l a t i o n .  c.  Comparison  t o Other S t e r o i d - R e s p o n s i v e Systems  Because t h i s secondary response o f o v i d u c t t i s s u e i s n o t d i r e c t l y compara b l e t o e i t h e r t h e f u l l growth and d i f f e r e n t i a t i o n response o f the o v i d u c t or o f mammalian t i s s u e s , n o r t o t h e s i m p l e response o f a l t e r a t i o n o f a c e l l u l a r m e t a b o l i c 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 t o know what system t o u s e f o r a comparison t o t h e t h r e e - h o u r time l a g .  On p r i m a r y  s t i m u l a t i o n , t h e time l a g i s 24 hours f o r s y n t h e s i s o f o v a l b u m i n ; on secondary s t i m u l a t i o n , t h i s o c c u r s a f t e r o n l y 3 h o u r s , a l t h o u g h rRNA s y n t h e s i s does n o t o c c u r u n t i l 12 hours a f t e r s t i m u l a t i o n .  The s y n t h e s i s o f s p e c i f i c p r o t e i n  b e f o r e rRNA s y n t h e s i s (53) has been r e p o r t e d i n o t h e r systems as w e l l (107). I t s h o u l d a l s o be n o t e d t h a t t h i s a v i a n system i s u s e f u l f o r t h e same r e a s o n t h a t i t i s anomalous:  f o r t h e p r e s e n c e o f b i o c h e m i c a l markers f o r  steroid action.  Mammalian t i s s u e s w h i c h grow and d i f f e r e n t i a t e i n response  to e s t r o g e n and p r o g e s t e r o n e do n o t make l u x u r y p r o t e i n s f o r e x p o r t .  At least  one s p e c i f i c p r o t e i n , a p e r o x i d a s e , has been r e p o r t e d t o be i n d u c e d by e s t r o gen i n mammalian systems; however, t h i s i s n o t a t i s s u e - s p e c i f i c p r o t e i n ( 1 0 8 ) . These two major d i f f e r e n c e s , t h a t ovalbumin i s a l u x u r y p r o t e i n made f o r e x p o r t , and t h a t t h i s secondary response i s n e i t h e r one o f f u l l growth and d i f f e r e n t i a t i o n n o r a s i m p l e m e t a b o l i c r e s p o n s e , make t h i s phenomenon h a r d t o c l a s s i f y , and d i f f i c u l t t o compare.  The v e r y e a r l y responses t o s t e r o i d s  measured i n mammalian r e s p o n s i v e t i s s u e s a r e d e f i n i t e l y n o t l e a d i n g t o t h e s y n t h e s i s o f l u x u r y p r o t e i n s , and a r e l i k e l y t o be f u n c t i o n a l i n the c o n t i n u a t i o n o f the response. to w a r r a n t  T h i s 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 g r e a t enough  c a u t i o n i n assuming a d i r e c t comparison between t h e two types o f  systems. P a l m i t e r s t a t e s t h a t t h e time l a g a l l o w s f o r any i n a c y c l e o f 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 ( 7 7 ) , and i n v e s t i g a t i o n s o f t h e e a r l y responses o f o t h e r t i s s u e s t o s t e r o i d s a r e i n agreement w i t h t h i s  possibility.  A s i m i l a r s i t u a t i o n o c c u r s i n c h i c k l i v e r , where t h e s y n t h e s i s o f t h e p r o t e i n p h o s v i t i n i s i n d u c e d by e s t r o g e n t r e a t m e n t 12 hours a f t e r p r i m a r y o r 6 hours a f t e r secondary i n j e c t i o n (110).  injection,  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 o f 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 t h e a c c u m u l a t i o n o f two s p e c i f i c p r o t e i n s was n o t e d (107) .  One o f t h e s e was a b a s i c ,  nonhistone  p r o t e i n , and the o t h e r was an a c i d i c p r o t e i n , b o t h o f low m o l e c u l a r w e i g h t (26,000 and 20,000 r e s p e c t i v e l y ) .  The i n c r e a s e i n t h e s e two p r o t e i n s was  thought t o be due t o t r a n s p o r t from t h e c y t o p l a s m , r a t h e r than t o new s y n t h e sis  (107).  An a l t e r a t i o n o f n u c l e a r n o n - h i s t o n e c o n s t i t u e n t s has a l s o been  noted i n t h e e f f e c t s o f C o r t i s o l on r a t l i v e r  ( 8 1 ) , t h e e f f e c t o f e s t r o g e n on  rat uterus  ( 8 2 ) , and i n numerous o t h e r systems  (133).  I n mammalian systems, where the type o f response measured i s of a d i f f e r e n t nature,  t h e r e i s s t i l l some e v i d e n c e of a time l a g i n the  response.  I n r a t u t e r u s , the e a r l y r e s p o n s e s , which, are not d i r e c t l y a s s o c i a t e d 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  Anderson et^ al  amount of r e c e p t o r - e s t r o g e n  with  growth can  e l i c i t e d o n l y by e s t r a d i o l , o r , i f r e p e a t e d i n j e c t i o n s are g i v e n , by as w e l l (156).  slightly  be  estriol  f e l t t h a t growth c o u l d be c o r r e l a t e d w i t h  complex i n the n u c l e u s a t 6 h o u r s , a f t e r the  a l i n j e c t i o n , but not a t 1 or 3 h o u r s .  the initi-  That i s , the a c t i o n of the complex i n  the n u c l e u s a t 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 n u c l e u s a t 1 or 3 hours (109).  I t i s h i g h l y u n l i k e l y t h a t t h e r e 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 h o u r s , and  t h e r e f o r e , a d i f f e r e n c e i n the  n u c l e u s 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 its  constituents. I t t h e r e f o r e appears u n l i k e l y t h a t 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 r e c e p t o r complex.  estradiol-  R e g a r d l e s s of whether the gene i s a c t i v a t e d i n d e p e n d e n t l y  of o t h e r 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 i n i t i a l a c t i o n of the r e c e p t o r - e s t r o g e n  complex i n the  the  nucleus.  I n summary, the i n d u c t i o n of ovalbumin s y n t h e s i s o c c u r s a t 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 o v a l b u m i n , 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 o t h e r major p r o t e i n s as w e l l .  I f t h i s i s the  o n l y c o n t r o l p o i n t i n the i n d u c t i o n o f 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 s u b j e c t 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 steroids.  other  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 c o u l d 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 v a r i o u s 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 a t d i f f e r e n t doses.  T h e r e f o r e , two p o s s i b i l i t i e s remain:  p e n d e n t l y c o n t r o l l e d by each s t e r o i d  e i t h e r each gene i s i n d e -  (whether by i t s e l f ,  o r t h r o u g h an i n t e r -  mediate f a c t o r f o r each gene), o r each s t e r o i d o p e r a t e s by one o r a few i n t e r m e d i a t e s ( s p e c i f i c f o r t h a t s t e r o i d ) , and t h e gene a c t i o n i s c o o r d i n a t e d . There i s as y e t no e v i d e n c e t o d i s t i n g u i s h between t h e s e p o s s i b i l i t i e s .  The  d a t a document q u i t e n i c e l y the e v e n t s b e g i n n i n g 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 , b u t cannot i n d i c a t e what happens p r i o r t o t h a t e v e n t .  2.  The Response t o P r o g e s t e r o n e :  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 o f o v i d u c t t i s s u e t o p r o g e s t e r o n e have c e n t e r e d p r i m a r i l y around 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 response t o a p r o g e s t e r o n e i n j e c t i o n a f t e r e s t r o g e n t r e a t m e n t has been g i v e n . of  The work of O'Malley's group has c e n t e r e d around the i n d u c t i o n  t h i s p r o t e i n , and the e l u c i d a t i o n o f a p o s s i b l e mechanism o f a c t i o n f o r  progesterone.  T h i s work w i l l a l s o be r e v i e w e d f i r s t i n terms o f the e v i d e n c e  p r e s e n t e d , then the model t h a t has been proposed t o account f o r i t , and  last,  i n terms o f comparison t o o t h e r systems. a.  The E v i d e n c e  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, 1 1 2 ) , and may  o r may n o t r e q u i r e new RNA s y n t h e s i s (111, 112, 1 1 3 ) ; the s t u d i e s u s i n g  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 "  o c c u r s h e r e (113), as w e l l as w i t h ovalbumin, b u t 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 synthesis i n v i t r o  ovalbumin ( 9 9 ) .  (111-113), c o n t r a r y t o t h e s i t u a t i o n w i t h  I n i t s a c t i o n on t h e c h i c k o v i d u c t , p r o g e s t e r o n e a f f e c t s t h e s y n t h e s i s of r a p i d l y - l a b e l l e d n u c l e a r RNA, and t h e RNA polymerase a c t i v i t y w h i c h would cause i t (113, 114), b u t t h e changes a r e s m a l l , and t h e u s e o f whole n u c l e i precludes  t h e d i s t i n c t i o n between amounts o f enzyme, enzyme a c t i v i t y , and/or  changes i n t e m p l a t e 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 t h a t o v i d u c t s w i t h e s t r o g e n and p r o g e s t e r o n e c o n t a i n a l l t h e RNA s p e c i e s p r e s e n t t r e a t e d o v i d u c t s , p l u s some RNA s p e c i e s t h a t a r e n o t p r e s e n t case (115, 116, 117).  treated  i n estrogen-  i n the l a t t e r  Other i n t e r p r e t a t i o n s o f t h i s data i n c l u d e t h e p o s s i b i l -  i t y o f 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 t u r n i n g - o v e r minor p o p u l a t i o n o f RNA (116), w h i c h 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 t h e e f f e c t of e s t r o g e n  on ovalbumin message (95, 97).  u n l i k e l y , c o u l d a l s o account f o r t h i s d a t a .  Gene a m p l i f i c a t i o n , a l t h o u g h There i s no e v i d e n c e t h a t t h e  proposed new RNA b e i n g produced i s a v i d i n message, and i t i s l i k e l y n o t ,  since  the s h o r t 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 s i n c e g o b l e t c e l l s o f t h e magnum p o r t i o n make up o n l y a s m a l l p e r c e n t a g e o f t h e whole o v i d u c t s t h a t were used.  O'Malley and McGuire a l s o p o i n t o u t t h a t  r a p i d l y - l a b e l l e d n u c l e a r RNA i s u s u a l l y rRNA o r p r e c u r s o r  rRNA (111).  However,  t h i s i s n o t l i k e l y t h e case h e r e , s i n c e p r o g e s t e r o n e i s u n a b l e t o i n d u c e Phase I I o f t h e secondary r e s p o n s e ; t h a t i s , i t i s u n a b l e t o i n d u c e new rRNA synthesis  (53).  An i n v e s t i g a t i o n o f t h e e f f e c t s o f p r o g e s t e r o n e on t h e 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 o f p r o g e s t e r o n e on polysome f o r m a t i o n  (118), b u t  i n t e r p r e t a t i o n o f these s t u d i e s i s h i n d e r e d by t h e p r e s e n c e o f an a r t i f a c t i n the t e c h n i q u e  used; i . e . , t h e l a c k o f use o f d e t e r g e n t  before c e n t r i f u g a t i o n .  I n t h e absence o f d e t e r g e n t , o n l y 10-20% o f t h e polysomes w i l l be due  recovered,  t o a p e l l e t i n g o f polysomes bound t o endoplasmic r e t i c u l u m under  conditions  these  (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 t o be p r e s e n t only a f t e r progesterone  i n d u c t i o n (120, 1 2 1 ) . RNA from o v i d u c t s t r e a t e d w i t h  e s t r o g e n a n d p r o g e s t e r o n e has t h e a b i l i t y t o induce a v i d i n s y n t h e s i s i n an -  e s t r o g e n - t r e a t e d o v i d u c t , when i t i s i n j e c t e d i n t o t h e lumen o f t h e o v i d u c t (157, 1 2 3 ) . 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 o f t h a t RNA i s e v i d e n t from cross-species t r a n s f e r s using immunologically molecules  non-cross-reactive a v i d i n  (124).  The n e c e s s i t y f o r b o t h e s t r o g e n and p r o g e s t e r o n e a v i d i n synthesis complicates t y t h a t e s t r o g e n may induce by p r o g e s t e r o n e .  i n the i n d u c t i o n of  t h e a n a l y s i s o f i n d u c t i o n by a d d i n g t h e p o s s i b i l i t h e pre-message, w h i c h would t h e n be a c t i v a t e d  Such a s i t u a t i o n would be analogous t o t h e masked messenger  of amphibian oocytes  (125).  I f t o t a l l y u n t r e a t e d o v i d u c t s can s y n t h e s i z e  a v i d i n i n response t o p r o g e s t e r o n e , i n c i d e n t has been r e p o r t e d  then t h i s would n o t be t h e case.  One such  (111), b u t i s a t v a r i a n c e w i t h most o t h e r s t u d i e s  (24, 4 9 ) . The m a t t e r i s confused  f u r t h e r by t h e r e p o r t t h a t a v i d i n s y n t h e s i s i s  under t h e c o n t r o l o f e s t r o g e n i n l i z a r d s , w i t h p r o g e s t e r o n e  and t e s t o s t e r o n e  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  An e v o l u t i o n a r y  s w i t c h from e s t r o g e n  to progesterone  (126).  i n d u c t i o n , would o f c o u r s e  confer the  advantage o f a r r a n g i n g a s u p p l y o f b i o t i n i n t h e y o l k b e f o r e the b i o t i n - b i n d i n g p r o t e i n i s made, b u t such a s w i t c h , a g a i n , c o u l d o c c u r a t 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  level.  Receptor p r o t e i n s f o r p r o g e s t e r o n e have been demonstrated  i n chick  ovi-  duct i n c y t o p l a s m and n u c l e i ( 1 2 7 ) , w h i c h have s e d i m e n t a t i o n c o e f f i c i e n t s o f 4S i n the p r e s e n c e of s a l t , and 8S and 5S i n i t s absence (127).  Only  testo-  s t e r o n e and 5-a-pregnane-3,20-dione c o u l d compete w i t h p r o g e s t e r o n e f o r b i n d i n g to the r e c e p t o r (127); t h e s e s t e r o i d s a l s o i n d u c e 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 o f c h r o m a t i n , and exchanging these components, the a u t h o r s demonstrated a g r e a t e r b i n d i n g of the p r o g e s t e r o n e - r e c e p t o r complex t o the a c i d i c c h r o m a t i n p r o t e i n s than t o the h i s t o n e s (129).  A more e x t e n s i v e f r a c t i o n a t i o n of the a c i d i c  p r o t e i n s , and r e - i n c u b a t i o n , was  chromatin  attempted i n o r d e r to f u r t h e r i d e n t i f y the  n a t u r e 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 a s s a y s were  employed t o demonstrate the 3 - d i m e n s i o n a l s t r u c t u r a l f i d e l i t y o f these h y b r i d c h r o m a t i n s (130).  I f f r a c t i o n a t e d c h r o m a t i n components i n c u b a t e d 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 - d i m e n s i o n a l s t r u c t u r e , then c h r o m a t i n may  be a s e l f - a s s e m b l y s t r u c t u r e .  However, i t i s n o t n e c e s s a r y t o assume the  3 - d i m e n s i o n a l s t r u c t u r a l f i d e l i t y o f the h y b r i d c h r o m a t i n s to a c c e p t t h a t the complex b i n d s t o 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 o f the r e c e p t o r s i n d i c a t e d t h a t t h e r e a r e  two  components ( 1 3 1 ) , 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 kinetics  (131), but t h a t Component A b i n d s to DNA,  whereas Component B does n o t ,  and t h a t Component B b i n d s t o c h r o m a t i n , whereas Component A does not (132).  b.  D i s c u s s i o n o f the Model  O'Malley's model, t h e n , i s one i n which the p r o g e s t e r o n e - r e c e p t o r complex e n t e r s the n u c l e u s and b i n d s t o the a c i d i c c h r o m a t i n p r o t e i n s , thus a c t i n g a t  the " n u c l e a r o r  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 e v e n t u a t i n g i n a v i d i n s y n t h e s i s " (116).  T h i s model,  i f i t i s t o be a g e n e r a l model f o r p r o g e s t e r o n e a c t i o n , would have t o account for  the e a r l y e f f e c t s o f p r o g e s t e r o n e as w e l l . A l t h o u g h 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 a r e o b v i o u s  i n t h i s model, t h e r e i s as y e t no e v i d e n c e r e s u l t s from new  to i n d i c a t e t h a t a v i d i n s y n t h e s i s  t r a n s c r i p t i o n , n o r t h a t such proposed new  transcription i s  the r e s u l t of the b i n d i n g of the p r o g e s t e r o n e - r e c e p t o r complex to a c i d i c c h r o m a t i n p r o t e i n s . A p o s s i b l e c a u s a l 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 c h r o m a t i n p r o t e i n s and any o f the o t h e r a c t i o n s o f remains t o be demonstrated are evident:  (133).  progesterone  N e v e r t h e l e s s , the f o l l o w i n g c o r r e l a t i o n s  1) A f t e r 6 days o f d i e t h y l s t i l b e s t r o l (DES)  treatment,  of n u c l e i of t u b u l a r g l a n d c e l l s becomes condensed, and was "heterochromatin" by.Kohler et a l (50).  d e s c r i b e d as  At t h a t dosage o f DES,  the peak o f  c e l l p r o l i f e r a t i o n o c c u r r e d a t 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 and f u r t h e r growth was progesterone  a t t r i b u t e d to h y p e r t r o p h y  to p r o g e s t e r o n e  Greater or l e s s e r estrogen treatment (49).  ceased  2) An i n j e c t i o n o f  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 i n d u c i n g a v i d i n  s i s a t 6 days o f t r e a t m e n t . t h i s response  (50).  chromatin  synthe-  inhibited  3) A c i d i c c h r o m a t i n p r o t e i n s of the  ...  n u c l e u s a r e r e p o r t e d to change b i p h a s i c a l l y d u r i n g t r e a t m e n t , i n c r e a s i n g i n amount and v a r i e t y d u r i n g the f i r s t 6 days of t r e a t m e n t , and d e c r e a s i n g t h e r e a f t e r (134).  4) The b i n d i n g o f the p r o g e s t e r o n e - r e c e p t o r complex to the  a c i d i c c h r o m a t i n 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 o f changes i n a c i d i c c h r o m a t i n p r o t e i n s and  chromatin  c o n d e n s a t i o n has been noted i n o t h e r systems, and i s d i s c u s s e d a t l e n g t h by LeSturgeon  e t a l (136).  However, any d i s c u s s i o n of the e f f e c t s o f t h e s e  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 . t h a t c h r o m a t i n from o v i d u c t s w i l l p r o b a b l y  Again,  i t s h o u l d be n o t e d  not r e f l e c t to any  appreciable  e x t e n t the c h r o m a t i n of the g o b l e t c e l l s i n w h i c h a v i d i n i s made.  Thus, t h e r e  i s a problem i n u s i n g a v i d i n s y n t h e s i s as a marker f o r p r o g e s t e r o n e 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 w h i c h r e p r e s e n t  a s m a l l p e r c e n t a g e of  t o t a l t i s s u e , and t h e s e 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 c e l l s , w h i c h a r e the major b u l k of the t i s s u e (50, 45, The  the  gland  58).  d i f f i c u l t y w i t h t h i s model, as opposed to t h a t of P a l m i t e r , i s t h a t  i t indicates  o n l y the v e r y 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 t h a t r e c e p t o r p r o t e i n s f o r s t e r o i d s a r e  i n t r a c e l l u l a r gene r e g u l a t o r s ( 1 3 3 ) , and  t h a t 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 ; t h a t i s , he proposes t h a t the s t e r o i d o n l y a c t s to t r a n s p o r t the p r o t e i n s i n t o the n u c l e u s , where the p r o t e i n s a c t i v a t e o r d e r e p r e s s the r e s p o n s i v e  genes.  o t h e r p r o t e i n s of a s i m i l a r n a t u r e  He i s a l s o o f the o p i n i o n t h a t t h e r e in cells.  are  He p o i n t s 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 o f the n u c l e a r 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 .  the  He i n d i c a t e s  the r e p o r t e d b i n d i n g to h i s t o n e s can be accounted f o r by c o n t a m i n a t i o n a c i d i c nuclear proteins; p r o t e i n s may  nature 1)  that  with  2) t h a t the r e p o r t e d b i n d i n g to a c i d i c c h r o m a t i n  be h i n d e r e d by the use o f p r o t e a s e s  that simply digest  the  r e c e p t o r 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 n u c l e a r a c c e p t o r p r o c e d u r e s may  s i t e , and r e l e a s i n g the complex; o r  e x t r a c t the r e c e p t o r  nuclear acceptor  site.  3) t h a t e x t r a c t i o n  (an a c i d i c p r o t e i n ) , r a t h e r than the  He s t a t e s t h e r e f o r e , t h a t i d e n t i f i c a t i o n of  the  nuclear acceptor niques,  i s not p o s s i b l e ( u s i n g i n v i v o systems) by the p r e s e n t  and he f u r t h e r p o i n t s out t h a t b i n d i n g to DNA  g i v e s 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 , i n d e e d t h a t even d i g e s t i o n by n u c l e a s e s g i v e results.  He has  work i s h i n d e r e d  tech-  varying  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 by the f a c t t h a t i t r e l i e s on f r a c t i o n a t i o n under  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,  denaturing  although  the  3 - d i m e n s i o n a l s t r u c t u r a l f i d e l i t y of such c h r o m a t i n i s i n doubt, i t i s not n e c e s s a r y to assume the 3 - d i m e n s i o n a l s t r u c t u r a l f i d e l i t y of the c h r o m a t i n i n order  to a c c e p t t h a t the complex may  b i n d t o the a c i d i c c h r o m a t i n p r o t e i n s .  T h i s 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 G e n e r a l Model W i l l Have t o 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 o v i d u c t t h a t i s a g e n e r a l  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 n a t u r e of the r e s p o n s e .  c e l l s respond f i r s t , and  the p r o g e n i t o r  That i s , why  t u b u l a r g l a n d c e l l s second?  i s a s e n s i t i v i t y t o a l o w e r e s t r o g e n t h r e s h o l d , how concentration  i s i t mediated?  stromal If this By  higher  of the r e c e p t o r , or by a n u c l e a r c o n s t i t u e n t , or i s i t s i m p l y  t h a t the changes undergone by the s t r o m a l  c e l l s produce some c o n d i t i o n  n e c e s s a r y f o r the response of the p r o g e n i t o r why  do the  tubular gland c e l l s ?  Likewise,  do the c i l i a t e d c e l l s and g o b l e t 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 p r i m a r y s t i m u l a t i o n .  A s i m p l e b i n d i n g of the  receptor-  s t e r o i d complex to any n u c l e a r 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 i n t e r m e d i a t e s 3)  The  dose-effect  of e s t r o g e n .  The  exist.  f a c t t h a t a lower dose of e s t r o g e n  p r e f e r e n t i a l l y enhances conalbumin s y n t h e s i s i s h a r d to r e c o n c i l e t o a model o f s i m p l e 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 o f o t h e r s t e r o i d s , b o t h when they a r e g i v e n  a l o n e , and i n c o m b i n a t i o n w i t h e s t r o g e n .  T h i s would i n c l u d e b o t h 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 o f p r o g e s t e r o n e  and t e s t o s t e r o n e w i t h e s t r o g e n ,  and the a n t i - e s t r o g e n i c e f f e c t s o f p r o g e s t e r o n e a d m i n i s t e r e d a t the b e g i n n i n g of  C.  w i t h e s t r o g e n when t h i s i s  treatment.  Comparison o f t h i s I n d u c t i o n Phenomenon to Embryonic I n d u c t i o n The  type of i n d u c t i o n d i s c u s s e d h e r e 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 , b o t h i n c o n s i d e r a t i o n of the i n d u c e r , and o f the  cell's  response. 1.  The  Inducer  The  s p e c i f i c i t y o f the i n d u c e r i n t h i s case i s v e r y e x a c t , and the v a r i -  a b l e parameters a r e known.  T h i s i s not the case i n embryonic i n d u c t i o n , where  i n most cases the i n d u c e r i s not o n l y unknown, but may  a l s o be k i l l e d ,  heat-  t r e a t e d , o r s u b s t i t u t e d by s i m p l e s a l t s o l u t i o n s and s t i l l induce some 2.  response.  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 r e s p o n d .  I n 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 d i s c u s s e d h e r e , t h e r e appears t o be no time l i m i t t o the competence o f the t i s s u e to respond.  In other  situations,  as r a t u t e r u s , t h e r e i s a time l i m i t b e f o r e w h i c h the c e l l s do not respond f u l l y , but t h e r e appears t o be no subsequent time l i m i t  (148, 149, 150).  s t i l l o t h e r systems o f s t e r o i d - i n d u c t i o n , such as n e o n a t a l  In  androgenization,  t h e r e i s a v e r y d e f i n i t e , c i r c u m s c r i b e d time p e r i o d b e f o r e 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).  T h i s l a t t e r s i t u a t i o n i s , o f c o u r s e , the  normal case i n embryonic i n d u c t i o n . From an e m b r y o l o g i c a l p o i n t o f v i e w , t h e d i f f e r e n c e between i n d u c t i o n and competence i s t h e d i f f e r e n c e between the c o n t r o l by the i n d u c e r over t h e c e l l ' s a c t i o n s , and the c o n t r o l o f the c e l l over i t s own response t o the inducer.  A g r e a t d e a l o f work has c e n t e r e d around t h e c o n t r o l o f the i n d u c e r  over the c e l l ' s a c t i o n s .  The f o l l o w i n g q u e s t i o n i s a l s o o f i n t e r e s t :  c o n t r o l does the o v i d u c t c e l l have over i t s own response?  What  Such a c o n t r o l  might p o s s i b l y be mediated a t any o f 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 o f the c e l l - s p e c i f i c p r o t e i n s . O b v i o u s l y , t h e c e l l u l a r c o n c e n t r a t i o n o f t h e r e c e p t o r (139) c o u l d be a c o n t r o l p o i n t i n the response o f t h e c e l l t o t h e s t e r o i d . evidence  However, much  i n d i c a t e s t h a t t h e s t e r o i d determines t h e c o n c e n t r a t i o n o f i t s own  r e c e p t o r , o r the c o n c e n t r a t i o n o f t h e r e c e p t o r f o r o t h e r s t e r o i d s (142, 89-91). In  t h i s manner, e s t r o g e n i n d u c e s  the p r o d u c t i o n o f p r o g e s t e r o n e  guinea p i g u t e r u s , and p r o g e s t e r o n e In for  receptor i n  may i n a c t i v a t e i t s own r e c e p t o r  (143-145).  c h i c k o v i d u c t , i t has been r e p o r t e d t h a t e s t r o g e n enhances the r e c e p t o r progesterone  Progesterone  (146), a l t h o u g h t h i s i s n o t a s e l e c t i v e a c t i o n (146, d i s c ) .  has been shown t o enhance t h e c o n c e n t r a t i o n o f the e s t r o g e n  r e c e p t o r (147).  These o b s e r v a t i o n s p o i n t to an i m p o r t a n t d i f f e r e n c e between  the e x p e r i m e n t a l and the n a t u r a l s i t u a t i o n s :  t h e p r e s e n c e o f two o r more  hormones s i m u l t a n e o u s l y , and t h e i n t e r a c t i o n s o f t h e s e . The s i m p l e f a c t t h a t the r e c e p t o r i s p r e s e n t , however, does not mean t h a t the hormone w i l l a c t . I n r a t u t e r u s , the competence t o respond t o i n d u c e r i s developed  s t e p - w i s e , over a p e r i o d o f 30 days, a l t h o u g h the r e c e p t o r i s  p r e s e n t a t b i r t h , a b l e t o b i n d e s t r a d i o l , and e n t e r and b i n d i n t h e n u c l e u s (148, 149, 1 5 0 ) . T h i s would i n d i c a t e t h a t a d d i t i o n a l f a c t o r s aire for  necessaxy  each new s t e p , and so i n d i c a t e t h e p o s s i b i l i t y o f a d d i t i o n a l c o n t r o l s t e p s .  T h i s p o s s i b i l i t y i s a l s o e v i d e n t i n r e c e p t o r - c o n t a i n i n g mutants of immunocytes t h a t a r e r e s i s t a n t t o g l u c o c o r t i c o i d a c t i o n , even though t h e r e c e p t o r s b i n d g l u c o c o r t i c o i d s and e n t e r and b i n d i n the n u c l e u s  (151).  In o t h e r systems development o f 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 o f the type o f b i n d i n g observed for  i n the target t i s s u e s .  T h i s has been r e p o r t e d  DES b i n d i n g i n r a t b r a i n (152, 1 6 0 ) , androgen b i n d i n g i n r a t e p i d i d y m i s  (153, 1 6 1 ) , and g l u c o c o r t i c o i d b i n d i n g i n l u n g and o t h e r t i s s u e s  (154).  A n o t h e r d i f f e r e n c e between t h i s type o f i n d u c t i o n and t h a t seen commonly i n embryonic systems i s t h a t t h e c e l l s n e v e r become i n c a p a b l e o f r e s p o n d i n g to  the i n d u c e r when i t i s p r e s e n t , and never become c a p a b l e o f c o n t i n u e d  f u n c t i o n i n t h e absence o f t h e i n d u c e r .  This i s p a r t i a l l y r e l a t e d to the  absence o f a c r i t i c a l time p e r i o d as d i s c u s s e d e a r l i e r .  However, r e g a r d l e s s  o f the f a c t t h a t the c e l l s do n o t s t o p r e s p o n d i n g , t h e i r response the c o u r s e o f t r e a t m e n t . of treatment,  changes i n  K o h l e r e t a l have r e p o r t e d t h a t f o r t h e f i r s t 6 days  t h e 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 ( 4 1 ) ; t h e r e a f t e r ,  i t i s p r i m a r i l y r e p r e s e n t e d by c e l l u l a r h y p e r t r o p h y .  There i s , t h e r e f o r e , a  p o i n t a t w h i c h the response  changes, a t w h i c h t h e c e l l s respond by f u n c t i o n i n g  but no l o n g e r by d i v i d i n g .  T h i s change i n response may r e s u l t from a change  i n d u c e d by t h e e s t r a d i o l t r e a t m e n t , 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  -  i n d u c e d by o t h e r s o u r c e s , such as o t h e r c e l l s , o r o t h e r s t e r o i d s . The absence o f c o n t i n u e d f u n c t i o n o f t h e o v i d u c t t i s s u e i n t h e absence o f i n d u c e r i s a l s o a n o t a b l e e x c e p t i o n t o normal embryonic i n d u c t i o n . t h a t c o n t i n u e d s t i m u l a t i o n by e s t r o g e n i s n e c e s s a r y  f o r continued  The f a c t transcrip-  t i o n , and t h a t o t h e r s t e r o i d s do n o t have t h e same e f f e c t , a l t h o u g h 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 h a t the inducer simply a c t s to t r i g g e r a g r o s s c o n t r o l , w i t h t h e 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 .  I n the  o v i d u c t system, t h e i n d u c e r o b v i o u s l y e x e r t s a f i n e c o n t r o l as w e l l . The r e c e n t o b s e r v a t i o n , t h a t t h e v a g i n a l and u t e r i n e c e r v i c a l  epithelial  c e l l s o f n e o n a t a l l y e s t r o g e n i z e d 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 w i t h o u t e s t r o g e n , and a r e l e s s s e n s i t i v e t o exogenous e s t r o g e n . ( 1 5 5 ) i s r e l a t e d to  t h e s e c h a r a c t e r i s t i c s o f t h e n e c e s s i t y f o r c o n t i n u e d e s t r o g e n presence and  the i n a b i l i t y t o n o t respond . i n t h e presence o f t h e inducer.. have been performed  Similar studies  on n e o n a t a l l y androgenized. r o d e n t s (83, 8 4 ) . A l t h o u g h no  e q u i v a l e n t o f t h e n e o n a t a l 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 e v i d e n t , i n r o d e n t s a t l e a s t , t h a t t h e 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 this  r e s p e c t i t i s r e m i n i s c e n t of P a u l Weiss' s t a t e m e n t , "Hormone a c t i o n i s p r o b a b l y not a one-shot  o p e r a t i o n ; t h e agent i n v o l v e d more l i k e l y o p e r a t e s a t d i f f e r e n t  t i m e s , r e p e a t e d l y , p r o d u c i n g d i f f e r e n t r e s u l t s , depending i t works" (162).  on the time a t w h i c h  O b v i o u s l y , t h e t o t a l a c t i o n o f the hormone on a t i s s u e  throughout t h e l i f e of t h e t i s s u e i s a. more complex* m a t t e r than t h e s i m p l e i n d u c t i o n t o 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 o f t h e S t a t e o f t h e F i e l d • The r e s e a r c h t h a t has been d i s c u s s e d here appears  l i m i t o f the p r e s e n t t e c h n o l o g y .  t o have proceeded  t o the  M i n o r advances may y e t be made, such as t h e  i d e n t i f i c a t i o n o f t h e n o n - t r a n s l a t e d p o r t i o n o f the message.. However, t h e major advances i n t h i s a r e a w i l l r e q u i r e a more complete knowledge o f two a r e a s : the s t r u c t u r e o f c h r o m a t i n , and t h e p r o c e s s o f t r a n s c r i p t i o n .  Only when t h e s e  a r e a s a r e 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 t o measure an a l t e r a t i o n o f t h e s e by the hormone t r e a t m e n t .  Perhaps i n r e c o g n i t i o n of t h i s i n t e r d e p e n d e n c y , the  1975 Conference of the S o c i e t y f o r Developmental B i o l o g y w i l l a d d r e s s t o two a r e a s : tion.  itself  the mechanism of s t e r o i d , a c t i o n and the p r o c e s s o f . t r a n s c r i p -  When these a r e u n d e r s t o o d , the phenomenon of c e l l d i f f e r e n t i a t i o n  be on i t s way  to being u n r a v e l l e d .  may  REFERENCES  1.  R i d d l e , 0. and Tange, M. (1928) " S t u d i e s on the P h y s i o l o g y o f Reproduct i o n i n B i r d s " . Am. J . P h y s i o l . 87_: 97.  2.  Juhn, M. and Gustavson, R. G. (1930) "The P r o d u c t i o n o f Female G e n i t a l S u b s i d i a r y C h a r a c t e r s by I n j e c t i o n s o f Human P l a c e n t a l Hormone i n Fowls". J . Exp. 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Chem. 247: 51.  132.  Schrader, W. T., T o f t , D. 0. and O'Malley, B. W. (1972) "Progesterone B i n d i n g Components o f C h i c k O v i d u c t . V I . I n t e r a c t i o n o f P u r i f i e d Progesterone-Receptor Components w i t h N u c l e a r C o n s t i t u e n t s " . J . B i o l . Chem. 247: 2401.  133.  S p e l s b e r g , T. C.  (1974)  "The Role o f N u c l e a r A c i d i c P r o t e i n s i n B i n d i n g  New York. 134.  S p e l s b e r g , T. C , M i t c h e l l , W. M., C h y t i l , F., W i l s o n , E. M. and O'Malley, B. W. (1973) "Chromatin of the D e v e l o p i n g C h i c k O v i d u c t : Changes i n the A c i d i c P r o t e i n s " . B i o c h . Biophys. A c t a 312: 765.  135.  S p e l s b e r g , T. C , S t e g g l e s , A. W. and O'Malley, i n Chromatin — — " Development". it". B i o c h . Biophys. A c t a 254: 129. n  136.  J  LeStourgeon, W. M., T a t t e n , R. and E o r e r , A. (1974). "The N u c l e a r A c i d i c P r o t e i n s i n C e l l P r o l i f e r a t i o n and D i f f e r e n t i a t i o n " , Chapter 6, p. 159, o f A c i d i c P r o t e i n s o f the Nucleus, I . L. Cameron and J e t e r , J . R., J r T T ^ d s . Academic" P r T s I T New York.  137.  Comstock, J . P., R o s e n f e l d , G. C , O'Malley,  Acad. S c i . 69:  B. W. and Means, A. R.  2377.  138.  Means, A. R., Comstock, J . P., R o s e n f e l d , G. C. and O'Malley, B (1972) "Ovalbumin Messenger RNA o f C h i c k O v i d u c t : P a r t i a Il ' c h a r a c t e r i z a t i o n , E s t r o g e n Dependence, and T r a n s l a t i o n I n V i t r o " . Proc. Nat. Acad. S c i . 69: 1146.  139.  P a l m i t e r , R. D., C a t l i n , G. H. and Cox, R. F.  (1973)  "Chromatin-  s t e r o n e and t h e ^ n d u c t i o n o f 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., T o t s u k a , A. and Zahn, R. K. (1974) " A s s o c i a t i o n o f an E s t r a d i o l R e c e p t o r w i t h t h e DNA-Dependent RNA P o l y m e r a s e I from Immature Q u a i l O v i d u c t " . Biochem. B i o p h y s . A c t a 366: 224.  141.  B a k e r , C. M. A. and M a n w e l l , C. (1975) " M o l e c u l a r B i o l o g y o f A v i a n P r o t e i n s . X I I . P r o t e i n Polymorphism i n t h e S t u b b l e 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 t h e I n d u c t i o n o f Egg-White P r o t e i n . S y n t h e s i s i n W i l d 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 o f t h e E s t r o g e n D u r i n g E a r l y U t e r i n e Development". S c i e n c e 16: 76.  143.  M i l g r o m , E., A t g e r , M. and B e a u l i e u , E.-E. (1970) " P r o g e s t e r o n e i n U t e r u s and Plasma. I V . P r o g e s t e r o n e R e c e p t o r s i n Guinea P i g U t e r u s and C y t o s o l " . S t e r o i d s 16: 741.  Receptor  D u r i n g t h e E s t r u s C y c l e and I m p l a n t a t i o n i n t h e G u i n e a - P i g " . Endocr. 90: 1071. 145.  M i l g r o m , E., T h i , L., A t g e r , M. and B e a u l i e u , E.-E. (1973) "Mechanisms R e g u l a t i n g t h e C o n c e n t r a t i o n and t h e C o n f o r m a t i o n o f P r o g e s t e r o n e R e c e p t o r s i n t h e U t e r u s " . J . B i o l . Chem. 248: 6366.  146.  O'Malley, B. W. , R o s e n f e l d , G. C , Comstock, J . P. and Means. A. R. (1972) " I n d u c t i o n o f 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  Karolinska Institutet, 147.  s  s  i  ^  X ^ ^ :  F  i  f  th  Stockholm.  P a v l i k , E. J . and C o u l s o n , P. B.  (1974).  "The E f f e c t o f E s t r o g e n and  S o c i e t y f o r C e l l B i o l o g y , San D i e g o , Nov. 1974.  148.  Somjen, D., Kaye, A. M. and L i n d n e r , H. R. (1973) " P o s t n a t a l D e v e l o p ment of the U t e r i n e Response t o E s t r a d i o l - 1 7 B i n the R a t " . Dev. B i o l . 31: 409.  149.  Somjen, D., Somjen, G., K i n g , R. J . B., Kaye, A. M. and L i n d n e r , H. R. (1973) "Nuclear B i n d i n g o f 0 e s t r a d i o l - 1 7 B and I n d u c t i o n of P r o t e i n S y n t h e s i s i n the Rat U t e r u s d u r i n g P o s t n a t a l Development". Biochem. J . 136: 25.  150.  Kaye, A. M., Sheratzky, D. and L i n d n e r , H. R. (1972) " K i n e t i c s of DNA S y n t h e s i s i n Immature Rat U t e r u s : Age Dependence and E s t r a d i o l Stimulation". Biochim. Biophys. A c t a 261: 475.  151.  Tomkins, G. (1973) " M o l e c u l a r B i o l o g y of S t e r o i d Hormones", i n S t r a t e g i e s f o r the C o n t r o l of Gene E x p r e s s i o n , A. Kohn and A. Shadkey, eds., Plenum P r e s s , New York.  152.  P l a p i n g e r , L. and MacEwen, B. S. (1973) "Ontogeny of E s t r a d i o l - B i n d i n g * - - " Receptors S i t e s i n Rat B r—a -'i n . I . Appearance * of" Presumptive.Adult i n C y t o s o l and id N u c l e i " . Endocr. 93: 1119. T  A  J  1  153.  C a l a n d r a , R. S., Poderta, E. J . , R i v a r o l a , M. A. and B l a q u i e r , J . A. (1974) " T i s s u e Androgens and A n d r o p h i l l i c P r o t e i n s i n Rat E p i d i d y m i s d u r i n g Sexual Development". S t e r o i d s 24: 507.  154.  Giannopoulous, G. (1974) " V a r i a t i o n s i n the L e v e l s o f C y t o p l a s m i c G l u c o c o r t i c o i d R e c e p t o r s i n Lungs o f V a r i o u s S p e c i e s a t D i f f e r e n t Developmental Stages". Endocr. 94_: 450.  155.  Shyamala, G., M o r i , T. and Bern, H. A. (1974) "Nuclear and Cytoplasmic Oestrogen R e c e p t o r s i n V a g i n a l and U t e r i n e T i s s u e o f Mice T r e a t e d N e o n a t a l l y w i t h S t e r o i d s 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 C l a r k , J . H.  157.  Tuohimaa, P., S e g a l , S. J . and Koide, S. S. (1972) " 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 by RNA Obtained from C h i c k O v i d u c t " . P r o c . Nat. Acad. S c i . 69: 2814.  158.  G o r s k i , R. A. (1971) "Gonadal Hormones and the P e r i n a t a l Development o f Neuroendocrine.. F u n c t i o n " , 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. P r e s s , New York.  159.  DeMoor, P., Verhoeven, G. and Heyns, W. (1973) "Permanent E f f e c t s of F o e t a l and N e o n a t a l T e s t o s t e r o n e S e c r e t i o n on S t e r o i d Metabolism and B i n d i n g " . D i f f e r e n t i a t i o n ! : 241.  (1973)  "Nuclear  160.  P l a p i n g e r , L., MacEwen, B. S. and Clemens, L. B. (1973) "Ontogeny of E s t r a d i o l B i n d i n g S ites t - in — " Rat — Brain. I I . C----h a r a c t e r i•s t•i c ' s o f- a Neonatal Btihn d « i ^ n ^g « Macromolecule^^Endocr!' Neonatal, 93^1129 7  161.  Roth, G. S. u.*/<U (1974) "Age-Related • Age-Kexated Changes Changes iIn n SSnPrifnV p e c i f i c Glucoc r,1„oo rticoid Binding, by S t e r o i d Responsive T i s s u e s o f R a t s " . Endocr. 94: 82  162.  Weiss, P.  163  -  CO  M  c  K  1 ^ ;  (1959)  "Ontogeny o f S e l e c t e d E n d o c r i n e Receptors-  l i ^ ^ ^ i J ^ l 2 ^ Z  as Polysomal Ovalbumin Messenger".  P r o c . Nat. Acad.  The  r s ^ L 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 L e v e l s of L u t e i n i z i n g Hormone i n Gonadectomized Japanese Q u a i l Exposed to Short o r . t o Long D a v E n d o r r . 64: fi4: R7 y l e n g t h s " . J . Endocr. 87. '•  165.  D r e s s e r - W e i s t , L. and Zwintz, C. (1974) " D i f f e r e n c e s i n the B i n d i n g C a p a c i t y f o r S t e r o i d Hormones of Sera from Embryonic and A d u l t R a t s " . E n d o k r i n o l . 64: 33  166.  F e l l , P. D., G l a s s e r , S. R., T o f t , D. 0. and O'Malley, B. W. (1972) "Progesterone B i n d i n g i n Mouse and Rat U t e r u s " . Endocr. 91: 738.  167.  Faber, L. E., Sandman, M. L. and. S t a n e l y , H. E. (1972) . "Progesterone B i n d i n g P r o t e i n s o f the Rat and R a b b i t U t e r u s " . J . B i o l . Chem. 247: 5648.  168.  Rao,  169.  D i s c h e , Z. (1955) " C o l o r R e a c t i o n s of N u c l e i c A c i d Components", Chapter 9, V o l . I , of The N u c l e i c A c i d s , E. C h a r g o f f and J . N. D a v i d son, eds., Academic P r e s s , 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 A c t i o n s of S t e r o l * Hormone* inNon-Mammalian V e r t e b r a t e s " , Chapter 8 i n S t e r o i d s i n Non-mammalian V e r t e b r a t e s , D. R. I d l e r , ed., Academic P r e s s , N.Y.  B. R., Weist, W. G. and A l l e n , W. M. (1973) "Progesterone Receptor i n R a b b i t U t e r u s . I . C h a r a c t e r i z a t i o n and E s t r a d i o l 17-B Augmentation". Endocr. 92: 1229.  120a  APPENDICES  P r o t e i n Assay  0.5 ml of sample or standard  + 5.0 ml of S o l u t i o n C  10 min a t room  temperature  + 0.5 ml of F o l i n ' s r e a g e n t , d i l u t e d  to 1 N  30 min a t room temperature  Read absorbance a t 660  S o l u t i o n C = 50:1 o f S o l u t i o n A : S o l u t i o n B (v/v) S o l u t i o n A = 2% N a C 0 2  (w/v) i n 0.1 N NaOH  3  S o l u t i o n B = 0.5% CuS0 '5H 0 (w/v) i n 1% NaK T a r t r a t e 4  T h i s procedure f o l l o w s  2  t h a t of Lowry e t a l ( 6 0 ) .  (w/v)  E x t r a c t i o n Procedure f o r R i b o n u c l e i c A c i d  Assay  1 ml of homogenate o r s t a n d a r d  I + 2.5 ml c o l d 10% TCA (w/v)  I c e n t r i f u g e , keep  pellet  4 resuspend i n 2.5 m l 10% c o l d TCA  I c e n t r i f u g e , keep  pellet  resuspend i n 5 ml 95% ETOH  1 c e n t r x f u g e , keep  pellet  resuspend i n 5 ml 95% ETOH  I c e n t r i f u g e , keep  pellet  I heat 45 minutes a t 80 C resuspend i n 2 ml 0.1 N NaOH  COOl  1 •I.  0T1 X C e  i. + 0.5 ml c o l d 10% TCA  I  centrifuge save s l p e r n a t a n t f o r r i b o n u c l e i c a c i d a s s a y s  T h i s p r o c e d u r e f o l l o w s the Reference o f Schneider thcit the p e l l e t ^\5 minutes let  W3S  3 . t 8 0^C  f i n s l l y resuspended  i n 2 nil o f 0 1 N N9.OH  r 3 . t h e r th.3tx b e i n ^ resuspended  st3.nd f o r 20 hours 3 1 37°C  (57) w i t h t h e e x c e p t i o n 3nd he3ted  i n 2 ml o f 1 0 N No.OH  ttien xieutr3 l i zed \\^itti 0 ^ 6  H 3_  for 3. d  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 o r c i n o l / m l i n 95% ETOH)  1  + 1.0 ml o f standard or sample  i J'  + H 0 •1 t o 5.0 ml t o t a l 2  heat 30 min i n b o i l i n g water bath c o o l on i c e read a t 665  T h i s procedure Schneider  f o l l o w s the b a s i c procedures  o f Mejbaum  (57), w i t h the f o l l o w i n g e x c e p t i o n s :  (59), D i s c h e  1) the F e C l  3  (169), and  i s 0.04% i n con.  HCl, r a t h e r than 0.1% (Mejbaum, d i s s o l v e d i n o r c i n a l reagent)  or 0.5%  ( S c h n e i d e r , d i s s o l v e d i n o r c i n o l r e a g e n t ) ; 2) o r c i n o l reagent  i s 50 mg/ml,  r a t h e r than 100 mg/ml ( a l l o t h e r s ) ; 3) the r e a c t i o n was a l l o w e d  t o proceed f o r  30 min a t 100°C, r a t h e r than 20 min (Dische and S c h n e i d e r ) , o r 40 min (Mejbaum); sample volumes were a l s o a l t e r e d Mejbaum 0.1% F e C l  3  slightly. z \ Schneider  Dische i n 100 mg  o r c i n o l / m l 95% ETOH  0.5 ml o f 0.1% F e C l - 6 H 0 3  2  0.5 gm F e C l  added t o 100 ml con. H C l  100 ml con. H C l ;  6% o r c i n o l i n 95% ETOH  1 gm o r c i n o l / 1 0 0 ml acid  3 ml sample + 0.3 ml o r c i n o l  1.5 ml sample + 3.0 ml. a c i d  read a t 670 nm  ' 20 min 100°C read a t 665 nm  reagent  0.2 ml sample reagent  + 0.2 ml o r c i n o l 40 min a t 100°C  in  3  .  + 1.3 ml H 0 2  + 1.5 ml reagent 20 min a t 100°C read a t 660 nm  DNA  Assay:  Extraction  Procedure  1 ml homogenate or standard 2.5 ml c o l d 10%  TCA  centrifuge resuspend  xn. 2 «5 ml c o l d 10% TCA  (w/v)  1  c e n t r i f u g e , keep p e l l e t + 2.0^ml 5% PCA heat a t 70°  1  c o o l , use  (v/v)  f o r 20  min  supernatant  T h i s procedure f o l l o w s t h a t of S c h n e i d e r , w i t h the e x c e p t i o n t h a t i t omits e x t r a c t i o n w i t h 95% ETOH, s i n c e 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  1.0  I  +  ml  I"°  m l  30°C 20  r e a g e n t  hours  read a t 600  Reagent i s 1.5 H S0 . 2  4  Reaction  gm diphenylamine  nm  i n 100 ml g l a c i a l a c e t i c . a c i d , w i t h 1.5 ml  At the time of use, 0.1 ml of a c e t a l d e h y d e  20 ml of the above r e a g e n t , and  t h i s was  T h i s assay f o l l o w s the diphenylamine Burton (58).  (16 mg/ml) i s added to  used i n the assay.  r e a c t i o n a c c o r d i n g to the procedure of  con.  Ovalbumin O u c h t e r l o n y P l a t e s  P r e p a r a t i o n of Agar Medium: To 1 gm of agar, 5 ml of b o r a t e b u f f e r , 95 ml of s a l i n e added enough sodium a z i d e t o make 0.1% Boric acid water,  (6.184 gm) ,  borax  to make one l i t e r .  been c l e a n e d , r i n s e d was  2  4  7  was  The b o r a t e b u f f e r c o n s i s t e d o f :  ( N a B 0 • 1 0 H 0 ; 9.536 gm),  The pH was  The immunodiffusion was  azide.  (8.5 gm/1)  2  NaCl  (4.348 gm)  and  a d j u s t e d to 8.4-8.5.  c a r r i e d out on l a r g e microscope s l i d e s t h a t had  twice w i t h a l c o h o l , and  twice w i t h e t h e r .  added to each s l i d e and a l l o w e d to s o l i d i f y .  2 ml of agar  Holes were punched i n the  agar 1 cm a p a r t , the samples were a p p l i e d , and a l l o w e d to d i f f u s e f o r 48 h o u r s . The volume o f sample a p p l i e d was diluted plates.  to 1 mg  5 m i c r o l i t e r s ; the a n t i b o d y p r o t e i n  o f a n t i b o d y p r o t e i n / m l , and  t h i s was  was  used, i n the d i f f u s i o n  A f t e r 48 hours, the p l a t e s were washed i n 0.9%  s a l i n e f o r 2-3  then i n d i s t i l l e d water f o r 3 hours, then d r i e d i n open a i r .  They were  s t a i n e d i n amido b l a c k f o r \ hour, then washed i n 2% a c e t i c a c i d f o r 30 T h i s procedure f o l l o w s t h a t of Work and Work (62).  days,  min.  Lysozyme Assay  Preparation of B a c t e r i a l  Cells:  inoculum o f M i c r o c o c c u s . l y s o d e i k t i c u s grow i n l i q u i d medium a t 30° i n a shaker  •  f o r 48-72 hours,  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 o f d i s t i l l e d x^ateir  I  c o l l e c t by c e n t r i f u g a t i o n wash 3 times w i t h c o l d  acetone  c o l l e c t by c e n t r i f u g a t i o n  wash twice w i t h c o l d e t h e r  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 s u c t i o n on a s i n t e r e d g l a s s f u n n e l  s t o used r e i nwas: a d e s i0.5% c c a t oBacto-peptone, r at 5 The l i q u i d medium  0.5% NaCl, 0.3% beef  and 0.1% y e a s t e x t r a c t . T h i s procedure f o l l o w s the procedure o f L i t w a c k ( 6 3 ) .  extract,  Lysozyme Assay  1 ml of b a c t e r i a l c e l l  suspension i n cuvette  t r a n s m i s s i o n a d j u s t e d to 50% a t 645  nm  i  I I ..  read % t r a n s m i s s i o n every 30 sec  Activity,  Cell  Suspension:  20 mg  change i n % t r a n s m i s s i o n between 30 and 60 seconds of r e a c t i o n  of p r e p a r e d c e l l s of M i c r o c o c c u s l y s o d e i k t i c u s i n  90 ml of 0.15  M phosphate  b u f f e r , pH 6.2,  w i t h 10  ml  1% N a C l .  T h i s procedure f o l l o w s t h a t o f L i t w a c k i n s t e a d of 25 mg  (63), w i t h the e x c e p t i o n t h a t  of c e l l s / 1 0 0 ml b u f f e r were used.  The r e a c t i o n was  20 read  a g a i n s t a d i s t i l l e d water b l a n k .  As can be seen from the standard curve on the next page, the lowest amount was of  2 yg/ml.  the s u b s t r a t e .  This i s believed  to be due  measurable  to the p a r t i c u l a t e n a t u r e  Avidin  Reagents and .Standards  are diluted  0.5 ml o f C  1 4  Assay  i n 0.2 M Ammonium Carbonate  Biotin  I + 0.1 ml of sample or standard let  s 13.1*1. d  i  10 m i xi  9. t  TC oom  temp G I T S , tut IT 6  l e t stand 5 min  1  transfer, to M i l l i p o r e F i l t e r ,  I  rinse  T h i s procedure  twice w i t h ammonium  filter  dissolve count  w i t h 10 ml of Bray's  i s essentially  curve f o r c o r r e c t i o n  that  size  carbonate  transfer  i n c o u n t i n g was a p p r o x i m a t e l y  curve.  I I I  0.45 y pore  to s c i n t i l l a t i o n  vial  s o l u t i o n (65)  o f Korenmann and O'Malley ( 6 4 ) . Background  28-30 cpm; and e f f i c i e n c y was about 85%. Quench  t o DPM i s shown on f o l l o w i n g  page, a l o n g w i t h  standard  100 r  QUENCH CURVE  

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