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Differentiation of the chick oviduct in vitro Vanderhorst, Elizabeth Wilhelmina Maria 1972

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c>  DIFFERENTIATION OF-THE CHICK OVIDUCT i.H V-i-tro  by  ELIZABETH. W.M. B.Sc,  VANDERHORST  U n i v e r s i t y of B r i t i s h Columbia, 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  In the Department of P o u l t r y Science accept t h i s t h e s i s as conforming t o the required  THE UNIVERSITY OF BRITISH COLUMBIA August, 1972.  standard  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y  the U n i v e r s i t y  s h a l l make i t  I f u r t h e r agree  in p a r t i a l  freely  f u l f i l m e n t o f the of B r i t i s h  available  for  requirements  Columbia, I agree  for  that  r e f e r e n c e and s t u d y .  t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s of  this  representatives.  It  thesis for financial  i s understood that copying o r p u b l i c a t i o n gain s h a l l  written permission.  Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  not be allowed without my  ABSTRACT Immature c h i c k response  to  increases and  the  daily  in  injections  general  production  Three  types  These  results  appears  of  appearance  of  of  the  of  are  that  A  tissue  and as  serum well  (fetal  as  calf,  serum from  observed  i n the  specific  protein,  mesentary  or  failed  elicite  changes  i n the  from  ineffective  as  an  not  a l t e r the  oviduct  for  tissue  the  m e d i a was laying  DES  cultured  detectible The  injected  inducer  of  or  remains  estrogen.  to  DES.  The  follows. differen-  culture  or  conditions.  as the  serum)  changes  were  kidneys, oviduct and  i n s u l i n or  non-injected  DES  the  chemical homogenized  chicks  as  an  ovaries, tissue  was  differentiation in this for  of  conditions.  morphological of  DES  chick  production  above  with  results.  i n vivo  to  phosphorus  No  the  such  17 6 - e s t r a d i o l  the  of  hen,  in  addition  ovalbumin.  in vitro  chicks.  nor  ratios  supplemented with  tissues  Thus, with necessary  variety  associated  any  of  i n response  under the  Substituting did  a  histology  latter.  pituitaries  free  ovalbumin  injected  tissue  by  i n response  ovalbumin,  ureter  is indicated  induce the  cockerel, DES  tissue  Closely  to  culture  in  precipitable  ovalbumin  under  vivo  nitrogen:DNA  containing  tissue  standard  This  produced  oviduct  phosphorus  oviduct  DES.  protein  A t t e m p t s w e r e made t o tiation  differentiate in  immunologically  ovalbumin  in  of  oviduct  indicate  first  oviducts  system.  inducer  •  exception  of  estrogen  d i f f e r e n t i a t i o n of  unknown at  this  time.  any  factor(s)  immature  -  I l l  -  TABLE OF CONTENTS INTRODUCTION LITERATURE REVIEV7 GENERAL MATERIALS AND METHODS A. Culture Methods B. Disc Gel E l e c t r o p h o r e s i s C. Immunoelectrophoresis D. Sample P r e p a r a t i o n ; DNA and P r o t e i n Nitrogen A n a l y s i s E. Histology RESULTS A. A c t i o n of DES on chick oviducts i n v i v o B. D i f f e r e n t i a t i o n of chick oviducts i n v i t r o with DES i n media containing f e t a l c a l f , c o c k e r a l , or l a y i n g hen serum C. D i f f e r e n t i a t i o n of chick oviducts i n v i t r o with DES i n Medium 199 c o n t a i n i n g a high l e v e l (60%)of e i t h e r f e t a l c a l f or l a y i n g hen serum. D. D i f f e r e n t i a t i o n of c h i c k oviducts i n v i t r o c u l t u r e d on Medium 199 containing serum from DES i n j e c t e d chicks E. The maintenance of p a r t i a l l y d i f f e r e n t i a t e d oviducts i n v i t r o employing a medium containing serum from DES i n j e c t e d chicks F. Organogenetic i n t e r a c t i o n and the developing chick oviduct G. The e f f e c t of i n s u l i n i n a d d i t i o n t o DES i n the medium of oviduct organ c u l t u r e s H. The use of p i t u i t a r y homogenate i n Medium 199 with 2 0% estrogenated chick serum i n organ c u l t u r e s of chick oviducts I. The use o f homogenized mature oviducts from l a y i n g hens t o supplement the c u l t u r e media i n organ c u l t u r e s of chick oviducts J . The e f f e c t of 1 7 g - e s t r a d i o l on the immature chick oviduct DISCUSSION SUMMARY LITERATURE CITED  - iv 60  APPENDIX Table 1.  Table 2.  Table 3.  RF values of densitometry from d i s c gel e l e c t r o p h o r e s i s of homogenized oviduct samples of chicks treated with DES i n vivo  61  RF values of densitometry from d i s c gel e l e c t r o p h o r e s i s ' o f homogenized p a r t i a l l y d i f f e r e n t i a t e d oviduct samples c u l t u r e d with DES serum and c o n t r o l serum as w e l l as non-cultured p a r t i a l l y d i f f e r e n t i a t e d oviducts  63  P r o t e i n nitrogen/DNA r a t i o s of DES serum c u l t u r e d p a r t i a l l y d i f f e r e n t i a t e d chick oviducts  67  Formulae f o r determination and DNA  of p r o t e i n nitrogen  68  LIST OF FIGURES Diagram of incubating chamber Comparison of average oviduct wet weights of DES t r e a t e d and c o n t r o l chicks Immunoelectrophoresis of homogenized samples of oviducts stimulated with DES i n vivo Disc g e l e l e c t r o p h o r e s i s of homogenized samples of oviduct t i s s u e s f o l l o w i n g DES s t i m u l a t i o n i n vivo Disc g e l e l e c t r o p h o r e s i s and densitometer readings of homogenized samples of oviducts c u l t u r e d on Medium 19 9 supplemented w i t h f e t a l c a l f serum + DES Immunoelectrophoresis of homogenized samples of oviducts c u l t u r e d i n f e t a l c a l f serum H i s t o l o g i c a l sections of oviducts c u l t u r e d i n f e t a l c a l f serum with and without DES Disc g e l e l e c t r o p h o r e s i s of homogenized oviducts p r e v i o u s l y c u l t u r e d with or without DES serum Disc g e l e l e c t r o p h o r e s i s of c u l t u r e d and nonc u l t u r e d p a r t i a l l y d i f f e r e n t i a t e d oviduct tissues Immunoelectrophoresis of homogenized samples of oviducts co-cultured with surrounding t i s s u e s i n the presence and absence of DES  VI  LIST OF TABLES  Wet weights of t i s s u e s and gross weights of DES t r e a t e d and c o n t r o l chicks Oviduct p r o t e i n nitrogen/DNA r a t i o s of DES t r e a t e d and c o n t r o l chicks P r o t e i n nitrogen/DNA r a t i o s of DES t r e a t e d and c o n t r o l c u l t u r e d oviducts P r o t e i n nitrogen/DNA r a t i o s of DES t r e a t e d and c o n t r o l oviducts c u l t u r e d on Medium 199 plus 50% f e t a l c a l f and l a y i n g hen serum P r o t e i n nitrogen/DNA r a t i o s of DES chick serum and c o n t r o l chick serum t r e a t e d oviducts P r o t e i n nitrogen/DNA r a t i o s of DES and c o n t r o l serum t r e a t e d organ c u l t u r e s and non-cultured oviduct rudiments P r o t e i n nitrogen/DNA r a t i o s of oviducts coc u l t u r e d with surrounding t i s s u e s i n the presence or absence of DES P r o t e i n nitrogen/DNA r a t i o s of DES t r e a t e d and c o n t r o l oviducts incubated i n medium containing serum  - vii -  ACKNOWLEDGEMENTS The author wishes t o express her sincere a p p r e c i a t i o n t o Dr. R.C. Fitzsimmons who provided superv i s i o n , suggestions and the f a c i l i t i e s f o r t h i s study. She would also l i k e t o express her a p p r e c i a t i o n t o Dr. P.M. Townsley, Dr. R.J. Hudson and Dr. G. J a i n f o r t h e i r valuable d i s c u s s i o n s . Sincere thanks i s a l s o given t o Dr. D.B. Bragg, Dr. C R . Krishnamurti and Dr. W.D. K i t t s , the members of the author's  committee.  Thanks i s extended t o Mrs. Enid Stewart who typed t h i s t h e s i s , Miss Janet Gehring f o r v a l u a b l e suggestions as t o t h e s i s format, and Dr. D. S c h a l l e r who did a l l the photography f o r t h i s p r e s e n t a t i o n .  DEDICATION  THIS THESIS  IS DEDICATED TO MY MOTHER AND FATHER,  JACOBA AND IVO VANDERHORST,  I  INTRODUCTION The  a c t i o n of estrogen on i t s t a r g e t organ, the  uterus i n mammals and the oviduct i n b i r d s , has been studied q u i t e e x t e n s i v e l y i n the l a s t decade. been found that estrogen manifests  In mammals i t has  i t s e l f e a r l i e s t i n target  t i s s u e s by causing hyperemia, histamine  release and  an  accumulation of water, e l e c t r o l y t e s , as w e l l as plasma prot e i n s due to an increase i n c a p i l l a r y p e r m e a b i l i t y  (33).  I t l a t e r binds with receptors i n the cytoplasm of c e l l s  and  i s then t r a n s f e r r e d i n t o the nucleus where i t i s found bound to smaller p a r t i c l e s .  The hormone has been found to bind  with the chromatin by d i s p l a c i n g histones (34). increase i n RNA  and p r o t e i n synthesis occurs.  a l l of t h i s knowledge has been obtained  F i n a l l y , an Practically  from experiments  i n v o l v i n g i n vivo and i n v i t r o homogenate preparations. Studying u n d i f f e r e n t i a t e d target t i s s u e s i n a c u l t u r e system to determine the requirements f o r complete estrogen response has received l i t t l e a t t e n t i o n . The c u l t u r e of completely  undifferentiated uteri  and oviducts and subsequent development i n response to estrogen added to the c u l t u r e medium could provide a very u s e f u l t o o l f o r studying estrogen's mode of a c t i o n . plex environment of t a r g e t organs  The com-  i n i n t a c t animals as w e l l  as the unnatural conditions of t i s s u e homogenates would be alleviated. The purpose of t h i s study was to c u l t u r e u n d i f f e r e n t i a t e d oviducts i n v i t r o and t e s t various parameters which may  be necessary f o r estrogen i n d u c t i o n of p r o t e i n synthesis  LITERATURE REVIEW Differentiation ized morphologically s t r u c t u r e s and  o f the c h i c k o v i d u c t i s c h a r a c t e r -  by the development of s p e c i f i c  b i o c h e m i c a l l y by the s y n t h e s i s o f  cellular  specific  proteins. Kohler  et_ a l . (18)  found t h a t f o l l o w i n g the  i n j e c t i o n of 3-day o l d c h i c k s w i t h 5 mg (DES)  diethylstilbesterol  d a i l y , t h r e e d i s t i n c t t y p e s of e p i t h e l i a l c e l l s appeared.  These c e l l s d i f f e r e n t i a t e d primitive  from the homogenous a p p e a r i n g  c e l l s of the immature o v i d u c t mucosa.  This  layer  of c e l l s c o n s i s t s of p s e u d o s t r a t i f i e d columnar e p i t h e l i u m resting  upon a compact stroma of p o l y g o n a l  t u b u l a r gland  c e l l s and  p r o t e i n s , ovalbumin and  the g o b l e t  cells.  The  c e l l s produce the  avidin, respectively.  c e l l s form i n response t o e s t r o g e n ,  organized  specific  As the  tubular  bundles of  o  f i l a m e n t s 40-50 A i n d i a m e t e r appear at the l u m i n a l end the c e l l s .  These s t r u c t u r e s are not p r e s e n t i n u n d i f f e r e n -  t i a t e d o v i d u c t s and genesis  they may  be i m p o r t a n t agents i n morpho-  because o f t h e i r c o n t r a c t i l e  the b i o t i n b i n d i n g p r o t e i n , has estrogen  of  p r o p e r t i e s (36). A v i d i n ,  been n o t e d t o appear i n the  s t i m u l a t e d c h i c k o v i d u c t a f t e r a s i n g l e dose o f  p r o g e s t e r o n e (13, 22). developing  The  o v i d u c t are c i l i a t e d c e l l s concerned w i t h  T e r e n i u s (35) has of estrogen  t h i r d type of c e l l formed i n the  found e v i d e n c e f o r t h e  binding s i t e s i n target tissues.  o v i d u c t s from 7 t o 10 day  o l d c h i c k s t a k e up  motility.  existence  In_ v i t r o 173-estradiol  s p e c i f i c a l l y and t h i s r e v e a l s a s i m i l a r comparison t o mammalian estrogen t a r g e t t i s s u e s where estrogen has been found to be taken up by p a r t i c l e s i n the cytoplasm of r a t , c a l f , goat and mouse u t e r i , (7, 37). Marked p e r i v a s c u l a r edema i s an i n i t i a l response of the immature oviduct stroma to DES and t h i s i s accompanied by i n t e r s t i t i a l migration of mononuclear c e l l s .  M i t o s i s occurs  w i t h i n 24 hours and t u b u l a r gland c e l l s begin to develop at the 2nd, 3rd and 4th day of treatment.  Fine a p i c a l granules  appear i n the e p i t h e l i a l c e l l s at day 2 and ovalbumin can be detected immuno-chemically  by day 3 at which time a new  species of nuclear RNA ha.s been i d e n t i f i e d .  Ovalbumin granules  form w i t h i n the condensing vacuoles i n the g o l g i zone and are released i n t o the lumen of the gland a c i n i at about day 6 of treatment (17). Increased high molecular weight nuclear RNA  and  cytoplasmic t r a n s f e r RNA have been found i n oviduct t i s s u e s f o l l o w i n g estrogen a d m i n i s t r a t i o n to immature c h i c k s (6, 2 3). DES also causes a prolonged s t i m u l a t i o n of oviduct n u c l e a r . polymerase, the maximum rate of enzyme increase being between 12 and 24 hours post i n j e c t i o n .  This has been i n t e r p r e t e d as  r e f l e c t i n g an a l t e r a t i o n i n n u c l e a r t r a n s c r i p t i o n i n v o l v i n g genes governing the synthesis of ovalbumin, lysozyme  and  numerous other p r o t e i n s (24). I t has been shown by means of the nearest-neighbour a n a l y s i s that estrogen acts to a l t e r t r a n s c r i p t i o n and increase DNA-chrc>matin template a c t i v i t y  a f t e r 3 days of DES treatment but more d e f i n i t e l y by day 6 of s t e r o i d induced d i f f e r e n t i a t i o n (25).  The s t e r o i d promotes  the synthesis of d i f f e r e n t types of RNA  that a r i s e during  oviduct development and are not present  i n the u n d i f f e r e n -  t i a t e d gland. l a t e d and DES  The population of h y b r i d i z a b l e RNA stimulated animals d i f f e r s .  from unstimu  However, i t has  bee  pointed out t h a t caution must be e x e r c i s e d i n equating h y b r i d i z a b l e RNA same RNA  with mRNA f o r there i s no evidence that these  species are subsequently t r a n s l a t e d (12, 26). An i n c r e a s i n g number of hormones have been found  to be mediated by c y c l i c adenosine 3', 5'-monophosphate (29). This, compound, cAMP, has been shown to increase i n u t e r i of ovariectomized e s t r a d i o l (10). a c i d uptake was  r a t s f o l l o w i n g an intravenous Furthermore, i t was stimulated.  i n j e c t i o n of  found that u t e r i n e amino  However, Rosenfeld  et a l . (31)  have found that adenyl cyclase was not stimulated i n chick oviduct or r a t  prostate by estrogen or t e s t o s t e r o n e , suggest-  ing that growth and d i f f e r e n t i a t i o n of these t a r g e t t i s s u e s are not mediated by cAMP. Oka et a l . (21) have shown that estrogen  and  progesterone are s y n e r g i s t i c i n the production of lysozyme and ovalbumin.  Progesterone antagonizes  t u b u l a r gland c e l l s induced by  the formation  of  estrogen.  A two to three f o l d increase i n oviduct glycogen has been found i n a d d i t i o n t o the synthesis of s p e c i f i c  p r o t e i n when 1 7 S - e s t r a d i o l i s administered to immature c h i c k s . A l l f i v e anatomical parts of the o v i d u c t ,  (infundibulum,  magnum, isthmus, and uterovagina) have the same glycogen response.  There i s no change i n oviduct glucose, whereas,  i n the r a t increases i n u t e r i n e glycogen increases i n uterine glucose.  are p a r a l l e l e d by  However, i n the r a b b i t u t e r i n e  glycogen  synthesis i s not p a r a l l e l e d by increased  glucose  levels.  Glycogen synthesis i n both the chick and the r a b b i t  are not dependent on r i s i n g glucose l e v e l s (2).  t  A few s t u d i e s i n v o l v i n g t i s s u e c u l t u r e of chick oviducts have been reported.  O'Malley and Kohler (27)  examined 6 week DES treated monolayer oviduct c u l t u r e s obtained from DES t r e a t e d c h i c k s f o r ovalbumin i n d u c t i o n . A small amount of ovalbumin synthesis observed during the c o n t r o l period was followed by an increase of immunologically p r e c i p i t a b l e ovalbumin a f t e r the i n t r o d u c t i o n o f DES i n t o the c e l l cultures.  A peak was reached on the 10th day a f t e r which  a gradual d e c l i n e t o the base l i n e occurred even though exposure t o DES was continued.  They a l s o found that when DES  was added to oviduct monolayer c u l t u r e s obtained from 14'\dayold non-injected c h i c k s , the predominantly population of c e l l s transformed :morphological  fibroblastic  into e p i t h e l i a l cells.  The  changes to an e p i t h e l o i d type of c e l l appeared  to be r e v e r s i b l e , suggesting the c e l l s had not undergone spontaneous or v i r a l induced transformation associated w i t h change i n c e l l morphology.  Although  DNA synthesis d i d occur  a f t e r an i n i t i a l l a g phase, there was of ovalbumin synthesis.  A confluent  no report of i n i t i a t i o n l a y e r of f i b r o b l a s t l i k e  c e l l s appeared to be necessary f o r the above e f f e c t .  DES  added at the time of i n i t i a t i o n of the c u l t u r e s d i d not appear to increase the growth of e p i t h e l o i d c e l l s  (19).  Cohen et a l . (4) found an e l e v a t i o n of o r n i t h i n e carboxylase enzyme when oviducts  from immature chicks were  incubated as organ c u l t u r e s i n a medium containing serum and  5 ug DES/ml c u l t u r e medium.  occurred may  chick oviduct accomplished.  s t i m u l a t i o n which  favour an e a r l y step i n estrogen a c t i o n . However,  ovalbumin synthesis was The  The  chick  not  detected.  complete d i f f e r e n t i a t i o n of the  undifferentiated  i n a t i s s u e c u l t u r e system has not yet been The  object of t h i s study was  to c u l t u r e  u n d i f f e r e n t i a t e d oviduct t i s s u e in v i t r o and attempt to obtain estrogen induced d i f f e r e n t i a t i o n as evidenced by the product i o n of the s p e c i f i c p r o t e i n , ovalbumin.  GENERAL MATERIALS AND METHODS A population of bred White Leghorn chickens was the parent stock f o r the chicks used i n these experiments. Oviducts from chicks ranging i n ages from 3 days t o 3 weeks were used f o r i n v i t r o c u l t u r e work.  In the experiments  where chick serum was used t o supplement Medium 199, c h i c k s of the same age as those supplying the oviducts f o r c u l t u r e were bled. Tissue c u l t u r e Medium 199 and f e t a l c a l f serum were obtained from M i c r o b i o l o g i c a l A s s o c i a t e s ;  diethylstilbesterol  (DES) from Merk and Co.; 1 7 B - e s t r a d i o l from Sigma Chemical Co.;  r a b b i t antiovalbumin serum from N u t r i t i o n a l Biochemical  Co.; and agar from M i c r o b i o l o g i c a l A s s o c i a t e s . A.  Culture Methods DES was prepared f o r i n j e c t i o n by d i s s o l v i n g 2.5 gm  of the powder i n 17 ml n i n e t y - f i v e percent ethanol a f t e r which 33 ml of peanut o i l was added.  The i n j e c t e d dose f o r  a l l i n j e c t e d c h i c k s was 5 mg (0.1 ml o f the DES s o l u t i o n ) . When DES was added t o the c u l t u r e medium 5 pg was d i s s o l v e d i n 2 0 ml of ethanol and 1 ml of t h i s s o l u t i o n was added t o 50 ml c u l t u r e medium g i v i n g a concentration of 5 ug DES per ml of medium.  When 1 7 8 - e s t r a d i o l was used, 1.2 mg of the  hormone was.dissolved i n 10 ml ethanol and 0.25 ml o f the s o l u t i o n was added t o 30 ml c u l t u r e medium g i v i n g a concent r a t i o n of 1 ug per ml of medium.  Oviducts were removed from DES i n j e c t e d o r noni n j e c t e d chicks under s t e r i l e c o n d i t i o n s .  Tissues were  washed i n 2 changes of c u l t u r e Medium 199 before  being  placed on s t a i n l e s s s t e e l g r i d s i n disposable Flacon organ c u l t u r e dishes c o n t a i n i n g the media.  plastic  Five mis chick  Ringer s o l u t i o n was applied t o the absorbent d i s c around the centre w e l l t o provide a humid atmosphere i n the dishes. Sixteen of the c u l t u r e dishes were placed i n a p l a s t i c chamber containing a small amount o f water.  The chamber  was gassed c o n t i n u a l l y with 95% oxygen - 5% carbon d i o x i d e during the incubation period of 6 t o 7 days.  The gas flow  was 1.5 cubic feet per hour and the incubation temperature of the chamber was 37.5 + 0.5°C.  The c u l t u r e medium was  changed every 2 or 3 days. The a n t i b o t i c s used in.the c u l t u r e medium were p e n i c i l l i n and streptomycin.  A s o l u t i o n c o n s i s t i n g of 0.12  gm p e n i c i l l i n - K and 13.20 gm streptomycin  sulfate dissolved  i n 200 ml of d i s t i l l e d water was s t e r i l i z e d by m i l l i p o r e f i l t r a t i o n (0.45 u pore s i z e ) and stored frozen i n small vials.  One-tenth ml f o r every 10 ml o f medium was used. Blood was obtained from the b r a n c h i a l vein of  one year o l d hens and c o c k e r e l s .  Young female chicks  (ranging i n age from 17 t o 25 days) were bled from the jugular vein.  Blood was incubated  f o r one hour at  37.5 + 0.5°C a f t e r which the serum was c o l l e c t e d and m i l l i pore f i l t e r e d .  Adult chicken serum was always prepared  9.  gas gas  exhaust input  e l e c t r i c i a n s tape t o s e a l chamber plastic  chamber  inverted c e t r i plates t o support c u l t u r e dishes -i  FIGURE  1.  Diagram  of incubating  water  chamber.  10.  the day before an experiment was i n i t i a t e d i n a large enough quantity to supply the e n t i r e experiment;  whereas , chick  serum was prepared on the day o f c u l t u r e and on each day o f medium change. Cultures were terminated  by homogenizing each  oviduct i n 0.1ml phosphate b u f f e r i n 2 ml c o n i c a l c e n t r i f u g e tubes w i t h a small glass rod. u n t i l analyzed. MgCl  B.  2  They were stored at -10°C  The b u f f e r consisted of 0.07 M KC1 , 0.004 M  and .02 M phosphate b u f f e r (pH 7.1) (28).  Disc Gel E l e c t r o p h o r e s i s The procedure followed and reagents used f o r d i s c  g e l e l e c t r o p h o r e s i s were the same as those of Davis (5).  The  gel tubes throughout t h i s work were.0.5 cm i n diameter and 6.2 cm long.  Eight gels were run per r e s e r v o i r i n a current  of 40 milliamperes  f o r about one hour.  The oviduct samples  (about 10 mg each) were run i n d u p l i c a t e .  The ovalbumin  standard was prepared from f r e s h egg white as described by Kekwick and Cannan (16).  The procedure used f o r i t s  p u r i f i c a t i o n was the batch technique and column ion-exchange with carboxymethylcellulose  (30).  Approximately 10 ug o f  p u r i f i e d ovalbumin was applied t o each standard gels were scanned on a densicord  gel.  The  f o r i d e n t i f i c a t i o n of  p r o t e i n s i n the homogenate samples.  C.  Immunoelectrophoresis Standard macro-immunoelectrophoresis was c a r r i e d  out on 2 by 3 inch glass p l a t e s using a 2% agar i n sodium b a r b i t o l - h y d r o c h l o r i c a c i d b u f f e r (pH 8.3). The i n g r e d i e n t s are the same as o u t l i n e d by Hudson et a l . (14). To each s l i d e , d u p l i c a t e 10 yg a l i q u o t s of sample and a s i n g l e 5 yg a l i q u o t of standard  ovalbumin were a p p l i e d .  placed i n a current of 20 milliamperes 0.12 ml of r a b b i t antiovalbumin  f o r 45 t o 90 minutes.  serum was applied t o each of  the two troughs per s l i d e and incubated  D. ' Sample P r e p a r a t i o n ;  The s l i d e s were  at 21°C f o r 12 hours.  DNA and P r o t e i n Nitrogen  DNA determination  Analysis  was a m o d i f i c a t i o n of Schneider  et a l . (32) using diphenylamine t o develop the c o l o r .  The  p r o t e i n nitrogen a n a l y s i s was a m o d i f i c a t i o n of B r i t t and Herrmanns procedure ( 1 ) . A f t e r the desired a l i q u o t s of the oviducts homogenate had been removed f o r d i s c g e l and Immunoelectrop h o r e s i s , the samples were prepared f o r DNA and p r o t e i n nitrogen a n a l y s i s . Oviduct samples were washed i n 1'ml 95% ethanol, c e n t r i f u g e d at 2000 r.p.m. and decanted. by adding 1 ml of chloroform-ethanol  Fat was removed  (1:3) and incubating  samples at 70°C i n a water bath f o r 15 minutes. c e n t r i f u g a t i o n and decanting  After  the supernate, excess  chloroform was removed by a wash with ethanol followed by  a c o l d t r i c h l o r o a c e t i c a c i d wash t o remove RNA.  The DNA i n  the samples was extracted by incubating the p r e c i p i t a t e at 90°C i n two changes of t r i c h l o r o a c e t i c a c i d ( f i r s t 1 ml and then 0.5 ml).  The two DNA e x t r a c t s were combined and stored  at 8°C u n t i l DNA a n a l y s i s .  To the remaining p r e c i p i t a t e  0.02 ml of selenium c a t a l y s t and 0.2 ml H S 0 2  2 parts concentrated  H SO. ) were added. 0  4  (3 parts H 0: 2  Samoles were  incubated overnight i n a 9 6°C oven and then were placed i n a sand bath at 100°C.  The temperature was gradually r a i s e d  t o 280°C and samples were digested overnight. Those samples which turned t u r b i d were cleared by the a d d i t i o n of one or more drops of h^C^ s a f t e r which f u r t h e r incubation of one hour was necessary.  One ml o f d i s t i l l e d water was added t o  the samples and s u i t a b l e  a l i q u o t s (0.2, 0.1 or 0.05 ml) of  the samples were analyzed, with 5 ml d i l u t e d Nesslers s o l u t i o n (40 parts water t o 12 parts N e s s l e r s ) .  Quanti-  t a t i o n of p r o t e i n n i t r o g e n was done c o l o r i m e t r i c a l l y on a Beckman spectrophotometer at 500 X. T r i p l i c a t e n i t r o g e n standards of 20, 30 and 50 mg/ml were used t o estimate the unknowns. DNA was quantitated c o l o r i m e t r i c a l l y with diphenylamine.  One ml of the t r i c h l o r o a c e t i c a c i d e x t r a c t  was mixed with 2 ml of diphenylamine s o l u t i o n (a f r e s h s o l u t i o n containing 100 mg diphenylamine per 10 ml a c i d s o l u t i o n (400 parts g l a c i a l a c e t i c a c i d : 11 parts concent r a t e d H-SO.)) and incubated i n a b o i l i n g water bath f o r  13.  10 minutes.  Reading was done on the i c e cooled samples at  60 0 X on a Beckman spectrophotometer w i t h i n one hour the incubation period.  after  T r i p l i c a t e one ml samples of each  DMA standard at 20, 50 and 100 yg DNA/ml were prepared and treated i n the same manner as the t r i c h l o r o a c e t i c a c i d extracts.  The formulae f o r c a l c u l a t i n g the amount of p r o t e i n  nitrogen and of DNA i n each sample i s given i n Appendix V.  E.  Histology A standard technique f o r preparing the t i s s u e s and  f o r t h e i r subsequent s t a i n i n g was used as o u t l i n e d by Lynch et a l . (20). The thickness of the sections was 8 my and the s t a i n used as the hematoxylin-eosin s t a i n .  Erlicks  hematoxylin s o l u t i o n was prepared i n accord with Fords' method ( 8 ) .  14.  RESULTS A.  Action of DES on Chick Oviducts i n v i v o The purpose of the f i r s t experiment was t o induce  young chick oviducts t o d i f f e r e n t i a t e i n vivo and to measure the magnitude of t h i s response i n terms of organ wet weight and the synthesis of t o t a l p r o t e i n , DMA, and ovalbumin. Procedures: • Four day o l d chicks were i n j e c t e d d a i l y with 5  mg  DES  and were  sacrificed  every  3  or  4  A  days.  group  of chicks of the same hatch i n j e c t e d with 0.1 ml of c a r r i e r s o l u t i o n without DES supplied c o n t r o l oviducts. experimental  Control and  oviducts were stored i n 0.1 ml and 0.3 ml  buffer respectively.  The weights o f o v i d u c t s , l i v e r s and  chicks were recorded (Table 1 ) . The r e s u l t s of t h i s experiment i n d i c a t e that DES causes d i f f e r e n t i a t i o n of immature chick oviducts.  A  marked'increase i n wet weight of oviducts d i d occur a f t e r 3 d a i l y i n j e c t i o n s of DES.  No such increase was observed  i n the c o n t r o l s where oviduct weights remained below 0.1 gm (Figure 2). I n j e c t i n g 5 mg DES d a i l y i n t o immature p u l l e t s does have a detrimental e f f e c t on t h e i r growth r a t e .  After  14 such i n j e c t i o n s chicks showed an average body weight of 112 gm, whereas the c o n t r o l chicks weighed an average of 168 gm.  A f t e r 14 DES i n j e c t i o n s the l i v e r weight i s an  average of 7.0 gm and i s extremely f a t t y i n appearance i n contrast t o the l i v e r s of c o n t r o l chicks which averaged 5.2 gm and remained red i n c o l o r .  15.  TABLE 1.  WET WEIGHTS OF TISSUES AND GROSS WEIGHTS OF DES TREATED AND CONTROL CHICKS'.  Treatment  Age of Chicks at S a c r i f i c e i n Days 8^ 11 15 18 Weight Grams  OVIDUCT DES Injected Control Injected LIVER DES Injected Control I n i e c t e d CHICK DES Injected Control Injected  .43+.13 .01+0  1.40+.05 . 01+0  1.71+.16 .03+.01  2.54+.13 .05+.01  6.75+.74 4. 53+.52  7.00+.99 5.23+.19  98.00+8.09 112.56+8.12 147.00+2.89 168.00+9.60  a.  The 8-day o l d chicks had been i n j e c t e d 4 times. The 18-day o l d chicks had been i n j e c t e d 14 times.  b.  Each value represents the average weight of 3 chicks + standard d e v i a t i o n of the mean.  There synthesis (Table day  as  2).  The  This  average  value  No  appearance  occurs  after  use  that  only  as  identity  of the  for  the  fraction the  The  was  third  between The  no  disc and  after  i n the c o n t r o l  o f DES  found  by  could  as  fractions ratio and  e l u t e d a t pH 5.  These  was  4.55,  seen  i n Figure  be  by  the  separated  the  second  The  Confirmation  migrate  from  acetate components  of  the  phosphorus  t o p r o t e i n was  the  4.7 5  second  a t pH  indicate that  i s in their  1.99  first and  the d i f f e r e n c e  phosphorus  content.  2 atoms o f phosphorus  (A2)  per protein molecule;  ammonium  The  (Al) contains  contains and  3.  egg  f o r the second.  results  ovalbumins  values.  of  of phosphorus 0.93  14  purification  with  obtained  4  ovalbumin  electrophoresis the  paper  per  one  atom  of  the t h i r d  (A3)  contains  rate of electrophoretic migration  g e l e l e c t r o p h o r e s i s d e p e n d s on  last.  3.64  from  f o r the  filter  ovalbumin  A l would  t o 0.52  resin  fraction  phosphorus.  samples  to  protein  ratios  i o n exchange  molecule;  phosphorus  injection  of ovalbumin  molar  a t pH  oviduct  precipitable  homogeneous band.  the three  first  protein  one  first  sharply  observed  e t a l . (27)  on  appeared  analysis.  i n contrast  increases  one  3 types  Yet  1.88  f o r oviduct  of immunologically  of cellulose  buffers.  i n general  p r o t e i n nitrogen/DNA  ratio  c h a n g e was  Rhodes white  increase  c h i c k s was  injections. The  an  i n d i c a t e d by  injected  control.  was  the degree  through the g e l f i r s t ,  Rhodes e t a l . (27) a l s o f o u n d t h a t  A2  the  of  in  ionization  second  and  amount o f  A3  the  17.  three types of ovalbumin d i f f e r e d .  A l was the most concen-  t r a t e d and A3 the l e a s t concentrated. The gels of DES t r e a t e d o v i d u c t s i n t h i s experiment revealed the three types of ovalbumin (Figure 4).  I t was  found that during the 14 day i n j e c t i o n period ovalbumin type A3 appeared f i r s t followed by types A2 and A l i n that order (Figure 4).  I n i t i a l l y , then, the non-phosphorylated form of  ovalbumin appears as a r e s u l t of DES treatment. Gels of c o n t r o l oviduct samples d i d not contain the  three ovalbumins.  There were two f a i n t bands, one of  which migrated at about the same r a t e as A3. phoresis i n d i c a t e d that t h i s was not ovalbumin.  ImmunoelectroThe other  band migrated at a much slower r a t e and was probably conalbumin which can be eluted (Rhodes et a l . (27)).  by c e l l u l o s e exchange at pH 6  18.  3.00  2.75  2.50  Age  FIGURE 2.  of Chicks , Days  Comparison of average oviduct wet weights of DES t r e a t e d and c o n t r o l c h i c k s .  TABLE 2.  Treatment  OVIDUCT PROTEIN MITROGEN/DNA RATIOS OF DES TREATED AND CONTROL CHICKS.  3  Age o f Chicks at S a c r i f i c e i n Davs 8 11 15 " 18 PN/DNA R a t i o s  DES I n j e c t e d  1.88+.11  2.67+.14  Control I n j e c t e d .52+.43  .52+.37  5  3.00+.23  3.64+.07 .54+.06  a.  The 8-day o l d chicks had been i n j e c t e d 4 times The 18-day o l d chicks had been i n j e c t e d 14 times.  b.  Each value represents the mean r a t i o of 3 oviducts + standard d e v i a t i o n of the mean.  20  FIGURE 3.  Immunoelectrophoresis of homogenized samples of oviducts stimulated with DES i n v i v o , a. Represents a 14-day o l d oviduct exposed to one i n j e c t i o n and c. represents 15-day o l d oviduct exposed to two i n j e c t i o n s ; b. i n d i c a t e s standard ovalbumin.  FIGURE 4.  Disc g e l e l e c t r o p h o r e s i s o f homogenized samples o f o v i d u c t t i s s u e s f o l l o w i n g DES s t i m u l a t i o n in vivo.  B.  D i f f e r e n t i a t i o n of Chick Oviducts i n v i t r o with DES i n Media Containing F e t a l C a l f , Cockerel, or Laying Hen Serum. Sera  hen  were  used  contained tiation  from  fetal  i n the  immature  which chick  Procedures: experiments  identical Fetal the  with  calf,  first,  Oviducts 20%  Three  3 day  the  second  serum.  Each  third at  i n the  DES/ml c u l t u r e m e d i a ) . cultures by  disc gel  of  the  they  differen-  culture  The  experiments  supplemented sera  were  were  serum.  used  in  experiments r e s p e c t i v e l y . 37.5°C i n  or  A f t e r seven and  80%  consisted  presence  were t e r m i n a t e d  i f  undifferentiated oviducts  l a y i n g hen  experiment  cultured  for  d u p l i c a t e d organ  old chicks.  incubated  laying  determine  necessary  using  exception  and  the  oviducts.  c o c k e r e l , and  were  oviducts  are  were p e r f o r m e d  from non-injected  c o c k e r e l , and  c u l t u r e medium t o  substances  of  calf,  stored  e l e c t r o p h o r e s i s and  DNA  of  M e d i u m 199  and  two  groups  of  of  (5  absence days o f at  incubation  -10°C  and  DES  until  protein  yg the  analysis  nitrogen  quantitation. Disc from oviducts calf  electrophoresis of  c u l t u r e d on  serum r e v e a l e d  Densitometer that  gel  little  no  readings or  no  increase of these  199  gels  Gels  and  densitometer  cultured  on  M e d i u m 199  revealed  the  with  results.  homogenates  sepplemented  in protein  protein synthesis  treatment.  same  Medium  tissue  5b)  occurred  c o c k e r e l and  fetal  synthesis.  (Figure  readings  with  of  indicated  during  the  oviducts  l a y i n g hen  serum  The o v e r a l l averages of p r o t e i n nitrogen/DNA . r a t i o s between DES and c o n t r o l treatments d i d not d i f f e appreciably f o r t i s s u e s c u l t u r e d i n medium supplemented with f e t a l c a l f or c o c k e r a l serum (Table 3).  24 .  FIGURE 5a.  Disc g e l e l e c t r o p h o r e s i s and densitometer readings of homogenized samples of oviducts c u l t u r e d on Medium 19 9 supplemented with f e t a l c a l f serum + DES. Gel 1 was prepared from oviduct c u l t u r e d with DES and Gel 2 was prepared from c o n t r o l o v i d u c t . Gel 3 i s that of the standard ovalbumin.  25.  FIGURE 5b.  D i s c g e l e l e c t r o p h o r e s i s and densitometer r e a d i n g c f homogenized samples o f o v i d u c t s c u l t u r e d on Medium 199 supplemented with f e t a l c a l f serum + DES. Densitometer r e a d i n g o f g e l prepared from o v i d u c t c u l t u r e d on Medium c o n t a i n i n g DES.  26 .  FIGURE 5c.  D i s c g e l e l e c t r o p h o r e s i s and d e n s i t o m e t e r r e a d i n g s o f homogenized samples o f o v i d u c t s c u l t u r e d on Medium 199 supplemented w i t h f e t a l c a l f serum + DES. D e n s i t o m e t e r r e a d i n g o f g e l p r e p a r e d from s t a n d a r d ovalbumin.  TABLE 3.  PROTEIN NITROGEN/DNA RATIOS OF DES TREATED AND CONTROL CULTURED OVIDUCTS.  PN/DNA Ratios Serum  DES  b  a Control  Fetal calf  7.19 + .86 (8)  7.29 + .59 (6)  Cockerel  7.98 +1.02 (5)  7.66 +1.62 (4)  a.  Each value represents mean r a t i o + standard d e v i a t i o n of mean.  b.  Number of determinations used f o r c a l c u l a t i n g each value  28.  C.  D i f f e r e n t i a t i o n of Chick Oviducts i n v i t r o with DES i n Medium 19 9 Containing a High Level (50%) of e i t h e r F e t a l C a l f Serum or Laying Hen Serum. Various serum supplements as 2 0% of the c u l t u r e  medium did not r e s u l t i n d i f f e r e n t i a t i o n of oviducts t r e a t e d with DES  i n the previous experiment.  The purpose of t h i s  experiment was t o determine i f a higher l e v e l of serum would be more e f f e c t i v e . Procedures:  Three day o l d chick oviducts were  c u l t u r e d i n Medium 199 containing 50% serum.  Of the  20  oviducts c u l t u r e d 10 were incubated with f e t a l c a l f serum and 10 with l a y i n g hen serum.  Five c u l t u r e s from each group  were exposed to 5 ug DES per ml o f ' c u l t u r e medium, the remaining ones serving as the c o n t r o l . period was  7 days.  The  Cultures were terminated  incubation and  anlyzed  by Immunoelectrophoresis and d i s c g e l e l e c t r o p h o r e s i s one c u l t u r e of each treatment was  and  used f o r h i s t o l o g i c a l  preparations. U n d i f f e r e n t i a t e d oviduct c u l t u r e d i n Medium 199 containing high l e v e l s of e i t h e r f e t a l c a l f or l a y i n g hen serum d i d not d i f f e r e n t i a t e when exposed t o DES by both d i s c gel and Immunoelectrophoresis.  as i n d i c a t e d  Disc g e l  e l e c t r o p h o r e s i s i n d i c a t e d t h a t no increase i n amount or types of p r o t e i n synthesis occurred during the c u l t u r e period under the i n f l u e n c e of DES.  Immunoelectrophoresis  did not r e v e a l the synthesis of the s p e c i f i c p r o t e i n ovalbumin (Figure 6). P r o t e i n nitrogen/DNA r a t i o s , however, d i d r e v e a l  a s l i g h t but i n s i g n i f i c a n t increase i n t o t a l p r o t e i n synthesis i n DES t r e a t e d c u l t u r e s (Table 4 ) . H i s t o l o g i c a l sections of t i s s u e s c u l t u r e d with both f e t a l c a l f and l a y i n g hen serum are shown i n Figure There was no apparent d i f f e r e n c e i n the morphology of DES t r e a t e d oviducts compared with the c o n t r o l s .  30 .  FIGURE 6.  I m m u n o e l e c t r o p h o r e s i s o f homogenized samples o f o v i d u c t s c u l t u r e d i n f e t a l c a l f serum. S e c t i o n a. o f s l i d e was a p p l i e d w i t h o v i d u c t sample t h a t had been c u l t u r e d w i t h DES, w h i l e S e c t i o n c. was a p p l i e d w i t h the c o n t r o l . S e c t i o n b. c o n t a i n s p r e c i p i t i n bands o f standard ovalbumin.  TABLE 4.  PROTEIN NITROGEN/DNA RATIOS OF DES TREATED AND CONTROL OVIDUCTS CULTURED ON MEDIUM 19 9 PLUS 5 0% FETAL CALF AND LAYING HEN SERUM.  a PN/DNA Ratios Serum  DES  Control  Fetal calf  1.46 + .13  j.1.34 + .07  Laying hen.  1.71 + .15  1.44 + .13  a.  Each value represents the mean r a t i o o f 4 c u l t u r e s + standard d e v i a t i o n of the mean.  32.  FIGURE 7a.  H i s t o l o g i c a l sections of oviducts c u l t u r e d i n f e t a l c a l f serum w i t h and without DES. Oviduct c u l t u r e d with DES; magnification i s 40X.  33 .  FIGURE 7b.  H i s t o l o g i c a l s e c t i o n s of o v i d u c t s c u l t u r e d i n f e t a l c a l f serum w i t h and w i t h o u t DES. O v i d u c t c u l t u r e d w i t h DES; magnification i s 100X.  34 .  FIGURE 7c.  H i s t o l o g i c a l sections of oviducts c u l t u r e d i n f e t a l c a l f serum with and without DES. Oviduct c u l t u r e d without DES; magnification i s 40X.  D.  D i f f e r e n t i a t i o n o f C h i c k O v i d u c t s i n v i t r o c u l t u r e d on M e d i u m 1 9 9 c o n t a i n i n g S e r u m f r o m DES I n j e c t e d C h i c k s .  Duplicate over  time  chicks  experiments  t o t e s t whether  serum obtained  t h e same a g e a s t h o s e  would promote  estrogen  ( A a n d B) w e r e  which  from  donated  conducted DES  the  injected  oviducts  depending  differentiation i n  The f i r s t  o f two t r e a t m e n t s  cultures. Procedures: ted  of culturing oviducts  Medium from DES  199 a n d 2 0 % c h i c k  17 d a y o l d c h i c k s 4 hours  same e x c e p t with day  DES.  before  Non-cultured  Disc analysis  One b a n d  had been  band The  5).  injected with  control oviducts  analyzed  as  no n o t i c e a b l e  treated  o f 1 7 , 19 a n d 2 1  and  densitometry  increase  i n protein DES  i n a l l gels  tested  p r o t e i n nitrogen/DNA appeared  5 ug  well.  cultured with  as p r e v i o u s l y  obtained  f o r serum had n o t been  o f A3 i n t h e s t a n d a r d  oviducts  s e r u m was  on 80%  T h e c o n t r o l g r o u p was t h e  c o n s i s t e n t l y appears  ovalbumin  (Table  bled  by t h e o v i d u c t s  to that  treated  that  This  gel electrophoresis  revealed  synthesis  close  were  17 d a y o l d c h i c k s  serum.  bleeding.  the chicks  o l d chicks  from  consis-  slightly  gels. by  serum  with This  (Figure  an R f  8)  value  i s n o t an  Immunoelectrophoresis  ratios  o f t h e DES  higher  than  serum  the control  36  mmm  a.  FIGURE 8.  b.  e.  f.  D i s c g e l e l e c t r o p h o r e s i s o f homogenized o v i d u c t s p r e v i o u s l y c u l t u r e d with or without DES serum. The g e l s are as f o l l o w s : a. DES serum treatment b. C o n t r o l serum treatment c. Non-cultured c o n t r o l o v i d u c t from 17 day o l d chick d. Non-cultured c o n t r o l o v i d u c t from 19 day o l d chick e. Non-cultured c o n t r o l o v i d u c t from 21 day o l d chick f . Standard ovalbumin  TABLE 5.  PROTEIN NITROGEN/DNA RATIOS OF DES CHICK SERUM AND CONTROL CHICK SERUM TREATED OVIDUCTS.  PN/DNA Ratios  a  DES Serum  C o n t r o l Serum  Experiment A  0.98 + .11  0.88 + .07  Experiment B  1.21 + .13  1.14 + .15  a.  Each value represents the mean r a t i o of 6 c u l t u r e s + standard d e v i a t i o n of the mean.  38 . E.  The Maintenance of P a r t i a l l y D i f f e r e n t i a t e d Oviducts i n v i t r o Employing a Medium Containing Serum from DES I n j e c t e d Chicks. P a r t i a l l y d i f f e r e n t i a t e d oviducts obtained  from  17 day o l d chicks were c u l t u r e d on a medium c o n t a i n i n g serum from p r e v i o u s l y i n j e c t e d chicks of the same age t o determine i f t h e i r d i f f e r e n t i a t e d s t a t e would be Procedures:  maintained.  Chicks were i n j e c t e d with DES on the  12th, 14th and 15th day of age.  The p a r t i a l l y d i f f e r e n t i a t e d  oviducts were removed on the 17th day and the magnum p o r t i o n was cut i n t o 2 mm p i e c e s , each oviduct y i e l d i n g about 5 or 6 pieces.  Two pieces of each oviduct were c u l t u r e d separately  i n Medium 199 c o n t a i n i n g 20% chick serum (the chicks had been i n j e c t e d with 5 ug DES four hours p r i o r t o b l e e d i n g ) .  Two  more pieces were c u l t u r e d i n Medium 199 containing 20% chick serum obtained from c o n t r o l c h i c k s .  The  remaining  p o r t i o n s of each oviduct served as non-cultured c o n t r o l s . The t i s s u e s were c u l t u r e d 7 days and the c u l t u r e medium was changed every 3 days.  The c u l t u r e s were terminated and  analyzed as described i n the Methods Section. Disc g e l e l e c t r o p h o r e s i s and  densitometry  i n d i c a t e d that two of three ovalbumins (A2 and A3) were present i n both'the DES and non DES c u l t u r e d t i s s u e s as w e l l as the non-cultured  t i s s u e s (Figure 9).  (See Appendix  Table 2 f o r a complete record of Rf v a l u e s ) . The p r o t e i n nitrogen/DNA r a t i o s of the f i r s t t r i a l shows that there was more p r o t e i n i n the non-cultured  c o n t r o l oviducts than the c u l t u r e d ones (Table 6). was  s l i g h t l y l e s s p r o t e i n i n the DES  than the c u l t u r e d c o n t r o l s ; two t r i a l s DES  serum t r e a t e d c u l t u r e s  although the data v a r i e s i n the  does not appear to f u r t h e r stimulate  oviduct t i s s u e s i n v i t r o .  the  Appendix Table 3 shows separate  readings of oviduct t i s s u e s from each chick f o r the various treatments.  There  40.  a.  FIGURE 9.  b.  c.  d.  Disc g e l e l e c t r o p h o r e s i s of c u l t u r e d and nonc u l t u r e d p a r t i a l l y d i f f e r e n t i a t e d oviduct t i s s u e s . The gels are as f o l l o w s : a. b. c. d.  DES serum c u l t u r e treatment Control serum c u l t u r e treatment Non-cultured c o n t r o l Standard ovalbumin  H I  TABLE 6.  PROTEIN NITROGEN/DNA RATIOS OF DES AND CONTROL SERUM TREATED ORGAN CULTURES AND NON CULTURED OVIDUCT RUDIMENTS.  PN/DNA Ratios DES Serum Trial 1  1.50 + .21 ( 6 )  Trial 2  2.01 + .08 (6)  a  C o n t r o l Serum b  Non Cultured  2.07 + .25 (6)  3.3 + .16 (10)  2.07 + .32 (7)  1.6 + .16 (13)  a.  Each value represents the mean r a t i o + standard d e v i a t i o n of the mean.  b.  Number of determinations used f o r c a l c u l a t i n g each value.  F.  Organogeretic I n t e r a c t i o n and the Developing Chick Oviduct Tissue i n t e r a c t i o n s are v i t a l i n the d i f f e r e n t i a -  t i o n of many c e l l types.  The  f o l l o w i n g experiment was c a r r i e d  out to determine i f the t i s s u e s of the mesentery, u r e t e r , kidney or ovary provide i n vivo some inducing  stimulus  necessary f o r i n i t i a t i o n of d i f f e r e n t i a t i o n of immature chick oviducts. Procedures:  Each 4 day o l d chick oviduct  was  c u l t u r e d along with 2 mm pieces of kidney, the mesentery and u r e t e r as w e l l as a 1 mm ovary rudiment.  The o v i d u c t , u r e t e r ,  kidney and ovary are found i n close o p p o s i t i o n to each other i n v i v o , being held together by mesentery and the complete u n i t was kept i n t a c t as p o s s i b l e upon d i s s e c t i o n .  The  t i s s u e s were c u l t u r e d i n Medium 199 containing 20%  fetal  c a l f serum. DES  Twelve c u l t u r e s were set up, 6 of which had  (10 ug/ml c u l t u r e medium) added.  Medium was  every 2 days and the c u l t u r e s were terminated  changed  a f t e r 7 days  f o r a n a l y s i s as described i n the Methods Section. The data i n the above experiment shows no i n d i c a t i o n of there being any i n t e r a c t i o n between the immature oviduct and surrounding  tissues.  Disc g e l e l e c t r o p h o r e s i s  and densitomer readings revealed no new  protein synthesis.  No ovalbumin synthesis could be detected by Immunoelectrophoresis  (Figure 10) and p r o t e i n nitrogen/DNA r a t i o s were  s i m i l a r (see Table 7).  The morphology of the c u l t u r e d  t i s s u e s were s i m i l a r to those shown i n Figure 7, with no apparent d i f f e r e n c e s due to the  treatments.  43 .  FIGURE 10.  Immunoelectrophoresis of homogenized samples of oviducts co-cultured with surrounding t i s s u e s i n the presence and absence of DES. Section a. was applied with oviduct sample t r e a t e d with DES, while s e c t i o n c. was applied with the c o n t r o l oviduct sample. Section b. contains p r e c i p i t i n bands of standard ovalbumin.  TABLE 7.  PROTEIN NITROGEN/DNA RATIOS OF OVIDUCTS COCULTURED WITH SURROUNDING TISSUES IN THE PRESENCE OR ABSENCE OF DES.  PN/DNA Ratios DES 1.66  a.  + .26  Control 1.58 + .11  Each value represents the r a t i o of 3 c u l t u r e s + standard d e v i a t i o n of the mean.  G.  The E f f e c t of I n s u l i n i n A d d i t i o n to DES of Oviduct Organ Cultures.  i n the Medium  C e r i a n i (3) has found that i n s u l i n i s a r e q u i r e ment for.the d i f f e r e n t i a t i o n of mouse mammary gland i n v i t r o i n response to p r o l a c t i n , aldosterone, and progesterone. Because Medium 199 does not contain i n s u l i n , i n s u l i n added to the c u l t u r e medium along with DES  was  to determine i f  i t i s necessary i n the c u l t u r e system f o r DES-induced differentiation. Procedures:  Five-day o l d chicks were s a c r i f i c e d  and oviducts were c u l t u r e d on Medium 199 containing  20%  f e t a l c a l f serum and 10 ug DES ml.of c u l t u r e medium. I n s u l i n (0.05 mg/ml) was cultures.  added to both c o n t r o l and  The incubation period was  changed every 2 days. analyzed  experimental  6 days and the media was  The c u l t u r e s were terminated  and  as described i n the Methods Section. This experiment reveals no evidence that i n s u l i n  was  s t i m u l a t o r y to the t i s s u e s under the c u l t u r e c o n d i t i o n s .  The p r o t e i n nitrogen/DNA r a t i o s were s i m i l a r f o r both the c o n t r o l and t r e a t e d oviducts (Table 8).  Disc g e l e l e c t r o -  phoresis and densitometer readings d i d not r e v e a l p r o t e i n synthesis.  new  Ovalbumin synthesis was not detected i n  these c u l t u r e s by Immunoelectrophoresis. H i s t o l o g i c a l sections were s i m i l a r to those shown i n Figure 7. was no apparent change i n morphology of DES when compared to the c o n t r o l t i s s u e s .  There  treated tissues  TABLE 8.  PROTEIN NITROGEN/DNA RATIOS OF DES TREATED AND CONTROL OVIDUCTS INCUBATED IN MEDIUM CONTAINING INSULIN  PN/DNA R a t i o s DES 1.37 + .13 ( 2 )  a  Control b  1.29 + .14 (3)  a.  Each value represents the r a t i o of 3 c u l t u r e s + standard d e v i a t i o n of the mean.  b.  Number of determinations used f o r c a l c u l a t i n g each value.  47 . H.  The Use of P i t u i t a r y Homogenate i n Medium 19 9 with 2 0% Estrogenated Chick Serum i n Organ Cultures of Chick Oviducts. Factor(s) t o date have not been i s o l a t e d from  the p i t u i t a r y that stimulate d i f f e r e n t i a t i o n of o v i d u c t s . The object of t h i s experiment was t o determine i f homogenized p i t u i t a r i e s from DES and non-DES i n j e c t e d chicks woudl help initiate differentiation. Procedures:  Ten day o l d chicks were i n j e c t e d  with DES four hours p r i o r t o t h e i r s a c r i f i c e .  The chicks  were bled f o r serum and t h e i r p i t u i t a r i e s were removed. Oviducts from c o n t r o l chicks were put i n c u l t u r e with minced p i t u i t a r i e s and serum from DES t r e a t e d chicks (20% i n Medium 199).  One h a l f of the c u l t u r e s had c o n t r o l p i t u i t a r i e s and  the remainder had p i t u i t a r i e s from DES i n j e c t e d c h i c k s . The medium was changed every 2 days and the c u l t u r e s were terminated  a f t e r 7 days and t e s t e d f o r ovalbumin with  Immunoelectrophoresis. P i t u i t a r i e s from e i t h e r DES i n j e c t e d or c o n t r o l chicks d i d not enhance d i f f e r e n t i a t i o n oviducts i n v i t r o . Immunoelectrophoresis r e s u l t s were s i m i l a r t o the preceding two experiments (Figure 9) i n which no ovalbumin was detected.  I.  The Use of Homogenized Mature Oviducts from Laying Hens to Supplement the Culture Medium i n Organ Cultures of Chick Oviducts. Homogenized l a y i n g hen oviduct was  used as a  supplement i n Medium 199 with 20% f e t a l c a l f serum and  10  ug DES per ml of c u l t u r e medium to t e s t f o r p o s s i b l e f a c t o r s i n the mature oviduct which would, along with the  DES,  i n i t i a t e d i f f e r e n t i a t i o n of chick o v i d u c t s . The magnum p o r t i o n of an oviduct from a mature hen was removed under s t e r i l e c o n d i t i o n s and homogenized i n 5 ml Medium 199 at room temperature.  Ten  3-day-old  chicks were s a c r i f i c e d and oviducts were put i n t o c u l t u r e dishes with Medium 199 c o n t a i n i n g 20% f e t a l c a l f serum. One-tenth ml mature oviduct homogenate was of the c u l t u r e s . DES  added to each  Five of the c u l t u r e s were t r e a t e d with  and the remainder served as c o n t r o l s .  The medium was  changed once at 3 days and the c u l t u r e s were a f t e r 6 days of i n c u b a t i o n . f o r morphological  terminated  The c u l t u r e s were examined  i n d i c a t i o n s of d i f f e r e n t i a t i o n ,  by  means of the " h i s t o l o g i c a l procedure. H i s t o l o g i c a l sections of the c u l t u r e d t i s s u e s t r e a t e d with DES  d i d not i n d i c a t e any apparent  development over c o n t r o l t i s s u e s .  morphological  A l l the prepared sections  were s i m i l a r to those shown i n Figure 7.  J.  The E f f e c t of 17 3 - e s t r a d i o l on the Immature Chick Oviduct. The object of t h i s experiment was to determine  i f the n a t u r a l hormone would i n i t i a t e d i f f e r e n t i a t i o n that DES had not been able to mimic i n v i t r o . Procedures:  Four-day o l d chicks were s a c r i f i c e d  and oviducts were c u l t u r e d i n Medium 199 containing f e t a l c a l f serum with 1 ug/ml 1 7 3 - e s t r a d i o l . period was  6 days and the medium was  The  20%  incubation  changed every 2 days.  This experiment revealed no evidence that  173-  e s t r a d i o l was able to induce d i f f e r e n t i a t i o n of the c u l t u r e d oviduct t i s s u e s .  Ovalbumin synthesis was  detected by Immunoelectrophoresis.  These r e s u l t s were  s i m i l a r to those of preceding experiments where DES added to the media.  not  was  DISCUSSION F e t a l c a l f serum has been used as a supplement to Medium 199 by O'Malley et a l . (27) i n 6 week monolayer c u l t u r e s s t a r t e d from p a r t i a l l y d i f f e r e n t i a t e d oviducts of DES i n j e c t e d c h i c k s .  These c e l l s d i d respond by s y n t h e s i z i n g  ovalbumin when DES was added to the c u l t u r e system. Theref o r e , Medium 199 plus f e t a l c a l f serum does supply the required n u t r i e n t s f o r oviduct c e l l response to DES i f the c e l l s or t h e i r progenates have been p r e v i o u s l y exposed to DES i n vivo.  However, the r e s u l t s of t h i s study i n d i c a t e  that t h i s medium i s inadequate f o r the i n v i t r o d i f f e r e n t i a t i o n of untreated oviduct c e l l s . Chick, c o c k e r e l or l a y i n g hen serum d i d not initiate differentiation.  There are numerous blood c o n s t i t u -  ents i n the laying, hen that are a f f e c t e d by the i n v i v o l e v e l of estrogen.  There i s an increase i n c i r c u l a t i n g  lipids,  c h o l e s t e r o l , p r o t e i n s , g l o b u l i n s , albumins, plasma, i r o n , vitamins ( i . e . vitamin A, b i o t i n and r i b o f l a v i n ) , manganese and p r o t e i n bound calcium.  When only DES i s present i n the  c u l t u r e system, the c o n d i t i o n s under which the oviduct responds are very abnormal.  In the c u l t u r e system, there i s  no increase i n the above f a c t o r s as t h e i r increase i s dependent on other parts of the chicken's body.  For example,  the a c t i o n of estrogen on the hen's l i v e r i s r e s p o n s i b l e f o r lipemia and i t s a c t i o n on bone i s responsible f o r calcemia. The a d d i t i o n of l a v i n g hen serum t o the c u l t u r e svstem should  then f u r n i s h many n u t r i e n t s not present i n f e t a l c a l f , chick or c o c k e r e l serum.  However, the absence of p o s i t i v e data  leads one to suggest that f a c t o r s other than those prsent i n t h i s type of serum are involved i n the i n i t i a t i o n of differentiation. There was no confirmation that there are age dependent f a c t o r s necessary f o r i n d u c t i o n of d i f f e r e n t i a t i o n i n serum of young i n j e c t e d c h i c k s .  Once a hen has come i n t o  lay and the reproductive processes have been i n i t i a t e d , a f a c t o r or complex of f a c t o r s may no longer be necessary to maintain the d i f f e r e n t i a t e d oviduct.  O'Malley et a l . (27)  found that d i f f e r e n t i a t e d oviduct from chicks i n j e c t e d 12 times and then incubated i n monolayer c u l t u r e s kept t h e i r a b i l i t y to respond t o estrogen even a f t e r 6 weeks i n c u l t u r e . Once the c e l l s were induced to d i f f e r e n t i a t e they r e t a i n e d the capacity t o respond to DES over a considerable of time.  length  Serum from young i n j e c t e d chicks d i d not r e v e a l  the presence of a short l i v e d i n i t i a t i n g f a c t o r . When-serum obtained from chicks i n j e c t e d only once was added to the c u l t u r e medium without f u r t h e r a d d i t i o n of DES t o the medium there may not have been an adequate amount of p r o t e i n bound DES i n the serum. et a l .  However, Jensen  (15) found that 2 hours a f t e r the i n j e c t i o n of r a t s  with t r i t i a t e d estrone, t h e i r u t e r i contained  tritiated  e s t r a d i o l i n one tenth of the amount f o r the e s t r a d i o l c o n t r o l (estrone must f i r s t be o x i d i z e d to e s t r a d i o l t o be b i o l o g i c a l l y a c t i v e ) .  The maximum percentage dose of  e s t r a d i o l i n the uterus from subcutaneous o i l i n j e c t i o n o f estrone was found 4 hours a f t e r the i n j e c t i o n .  Therefore,  i f chicks were i n j e c t e d with DES 4- hours p r i o r t o being b l e d , there must be an adequate amount of p r o t e i n bound DES i n the serum p r o v i d i n g that DES acts i n the same manner as estrone. In the l a s t decade considerable progress has been made toward understanding i n t e r a c t i o n s between components of developing organ rudiments.  Organ rudiments can be  c u l t u r e d separately or i n combination.  F a i l u r e of rudiments  to continue developemnt i n i s o l a t i o n and renewal of development on recombination i s a t e s t f o r developmental action.  When t i s s u e s undergo a developmental  inter-  change f o l l o w -  ing the a s s o c i a t i o n , the i n t e r a c t i o n i s o f t e n r e c i p r o c a l . The p e r i o d of dependency on i n d u c t i o n i s of measurable duration.  Rudiments c u l t u r e d alone may lose the a b i l i t y  to i n t e r a c t with other t i s s u e s .  In c o n t r a s t , components  separated f o l l o w i n g a s u f f i c i e n t p e r i o d of a s s o c i a t i o n can continue development independently.  In a few instances  a c t i v e c e l l e x t r a c t s have been prepared from t i s s u e s that are respondible f o r c o n t i n u i n g development of neighbouring t i s s u e s (11).  I t was thought that perhaps, i n the f i r s t  steps of d i f f e r e n t i a t i o n with DES, the oviduct rudiment may r e q u i r e a f a c t o r or complex from t i s s u e s i n i t s close p r o x i m i t y ( i . e . kidney, ovary, u r e t e r , o r surrounding mesentery). These r e s u l t s show no i n d i c a t i o n o f there being any i n t e r a c t i o n between the immature oviduct and surrounding  tissues. I n s u l i n has been shown by C e r i a n i (3) t o be a requirement f o r the development of new born r a t mammary glan t i s s u e i n organ c u l t u r e . and progesterone  I n s u l i n , p r o l a c t i n , aldosterone  i n the c u l t u r e medium cause the synthesis  of a casein l i k e m a t e r i a l .  Without i n s u l i n the other  hormones are incapable of causing t h i s response. i s known t o a f f e c t u t i l i z a t i o n of glucose by and other pathways.  Insulin  glyconeogenesis  Pancreatectomy i n mammals i n h i b i t s the  u t i l i z a t i o n of glucose  (9).  Medium 199 used i n a l l the  preceding experiments contains no i n s u l i n , although some media such as Trowell T8 medium do contain 6 0 mg per l i t e r . These r e s u l t s r e v e a l no evidence that i n s u l i n i s i n v o l v e d i n the i n d u c t i o n of oviduct  rudiments.  P i t u i t a r i e s from DES or non-DES t r e a t e d c h i c k s d i d not enhance d i f f e r e n t i a t i o n of immature oviducts when added t o a c u l t u r e system i n which medium 19 9 was supplemented with 20% serum from DES t r e a t e d c h i c k s .  There would  be no FSH production by p i t u i t a r i e s of e i t h e r i n j e c t e d or non-injected c h i c k s .  A high l e v e l of the i n j e c t e d c i r c u l a -  t i n g hormone would i n h i b i t any FSH production i n c h i c k s . FSH i s not produced by p i t u i t a r i e s of immature non-injected chicks.  In a d d i t i o n t o FSH, the p i t u i t a r y may produce a  f a c t o r , h i t h e r t o u n i d e n t i f i e d that could a s s i s t DES i n the d i f f e r e n t i a t i o n of the immature o v i d u c t .  The r e s u l t s of  t h i s study do not r e v e a l the presence of an i n i t i a t i n g f a c t o r necessary f o r the d i f f e r e n t i a t i o n of the immature chick oviduct.  I t can be concluded that there are no f a c t o r s i n  the p i t u i t a r i e s e i t h e r stimulated with DES or not p r e v i o u s l y stimulated with DES which could i n i t i a t e the d i f f e r e n t i a t i o n process along with DES of the immature c u l t u r e d chick oviduct. H i s t o l o g i c a l sections do not r e v e a l any apparent development of the immature oviducts c u l t u r e d with mature hen oviduct homogenate and DES.  There are no f a c t o r s  evident i n the mature oviduct which could help i n i t i a t e differentiation  the  process.  Although DES mimics the a c t i o n of estrogen i t s chemical s t r u c t u r e i s very d i f f e r e n t from that of a l l the estrogenic hormones.  No p o s i t i v e r e s u l t s were obtained  when 1 7 8 - e s t r a d i o l was  added to organ c u l t u r e s of oviducts  on Medium 199 supplemented with 20% f e t a l c a l f serum.  This  suggests that 1 7 8 - e s t r a d i o l , although a n a t u r a l compound, does not i n i t i a t e d i f f e r e n t i a t i o n b e t t e r than DES the conditions o f these experiments.  under  55 .  SUMMARY Immature chick oviducts d i f f e r e n t i a t e i n vivo i n response to d a i l y i n j e c t i o n s of DES.  This i s i n d i c a t e d  by  increases i n general oviduct p r o t e i n nitrogen:DNA r a t i o s and the production of immunologically p r e c i p i t a b l e ovalbumin. Three types of ovalbumin are produced i n response to estrogen. These r e s u l t s i n d i c a t e that ovalbumin free of phosphorus appears f i r s t i n the oviduct t i s s u e i n response to DES. appearance of phosphorus containing  The  ovalbumin f o l l o v 7 s .  Attempts were made to induce the i n v i t r o d i f f e r e n t i a t i o n of oviduct t i s s u e under a v a r i e t y of c u l t u r e A standard c u l t u r e media was  supplemented with  and serum ( f e t a l c a l f , c o c k e r e l , l a y i n g hen, as w e l l as serum from DES  conditions.  injected chicks.  DES  or chick serum) No changes were  observed i n the t i s s u e h i s t o l o g y nor i n the production of  the  s p e c i f i c p r o t e i n , ovalbumin, under the above c o n d i t i o n s . C l o s e l y associated  t i s s u e s such as kidneys, o v a r i e s ,  mesentary or u r e t e r t i s s u e c u l t u r e d with the oviduct t i s s u e f a i l e d to e l i c i t e any d e t e c t i b l e morphological and changes i n the l a t t e r . p i t u i t a r i e s from DES  The  chemical  a d d i t i o n of i n s u l i n or homogenized  i n j e c t e d or non-injected chicks  was  i n e f f e c t i v e as an inducer of d i f f e r e n t i a t i o n i n t h i s system. S u b s t i t u t i n g 17 3 - e s t r a d i o l f o r DES  as an inducer  did not a l t e r the r e s u l t s . Thus, with the exception of estrogen  any  factor(s)  necessary f o r the i n vivo d i f f e r e n t i a t i o n of immature oviduct t i s s u e remains unknown at t h i s time.  56. LITERATURE CITED 1.  B r i t t , L.G. and Herrmann, H. 1959. P r o t e i n Accumulation i n E a r l y Chick Embryos Grown under D i f f e r e n t Conditions of Explanation. J . Embryol. ext>. Morph. 7: 66-72.  2.  C e c i l , A., Bitman, J . and Shaffner, C S . 1969. Oviducal Glycogen Content of Laying' and Nonlaying Kens and of E t r a d i o l Stimulated P u l l e t s . Proc. Soc. Exp. B i o l . Med., 131: 164-167.  3.  C e r i a n i , R.L. 1970. F e t a l Mammary Gland D i f f e r e n t i a t i o n In V i t r o i n Response t o Hormones. I I . Biochemical Findings Dev. B i o l . 21: 530-546.  4.  Cohen, S., O'Malley, B.W. and Stastny, M. Estrogenic Induction of Ornithine Decarboxylase In V i t r o and In Vivo, S c i . 17 0:  5.  David, B.J. 1964. Disc Gel E l e c t r o p h o r e s i s . I I . Method and A p p l i c a t i o n of Human Serum P r o t e i n s . Ann. N.Y. Acad. S c i . , 121: 404-427.  6.  Dingman, C.W. , Aronow, A., Bunting, S.L. , Peacock, A.C. and O'Malley, B.W. 1969. Changes i n Chick Oviduct Ribonucleic Acid Following Hormonal S t i m u l a t i o n . Biochem., 8: 489-495.  7.  Erdos, T. 1968. P r o p e r t i e s of a Uterine O e s t r a d i o l Receptor. Biochem. Biophys. Res. Commun., 32: 338-343.  8.  Ford, P. 1958. Advanced H i s t o l o g i c a l Technique. N.Y. Scholars L i b .  9.  Gorbman, A. and Bern, H.A. 1962. A Textbook o f Comparat i v e Endocrinology. John Wiley and Sons, Inc., New York.  10.  G r i f f i n , D.M. and Szego, CM. 1968. Adenosine 3', 5 Monophosphate S t i m u l a t i o n of Uterine Amino A c i d Uptake In V i t r o . L i f e S c i . , 7: 1017-1023.  11.  Grobstein, C. 1967. Mechanisms o f Organogenetic Tissue I n t e r a c t i o n . Nat. Can. I n s t . Mongr. , 26: 279-299 .  1  57 .  12.  Hahn , W.E., Church, R.B., Gorbman, A. and Wilmot, L. 1968. Estrone- and Progesterone-Induced Synthesis of New RNA Species i n the Chick Oviduct. Gen. Comp. E n d o c r i n o l . , 10: 438-442.  13.  H e r t z , R., Frapo, R.M. and S e b r a l l , W.H. 1943. Induction of A b i d i n Formation i n the Avian Oviduct by S t i l b e s t r o l Plus Progesterone. Proc. Soc. Exp. B i o l . Med., 52: 142-144.  14.  Hudson, R.J., Bandy, P.J. and K i t t s , W.D. Immunochemical Q u a n t i t a t i o n of Ovine Immunoglobulins. Am.  J . Vet. Res.,  31: 1231-1235.  15.  Jensen, E.V. and Jacobson, H.I. 1962. Basic Guides to the Mechanism of Estrogen A c t i o n . Rec. Progr. Hor. Res., 18: 387.  16.  Kekwick, R.A. and Cannan, R.K. XXXVI. The Hydrogen Ion D i s s o c i a t i o n Curve of the C r y s t a l l i n e Albumin of the Hens Egg.  17.  Kohler, P.0., Grumley, P.M. and O'Malley, B.W. 196 8. Estrogen-Induced l y s o d i f f e r e n t i a t i o n of the Ovalbumin S e c r e t i n g Glands of the Chick Oviduct. J . C e l l Bio. , 40: 8-27.  18.  Kohler, P.O., Grimley, P.M. and O'Malley, B.W. 1968. P r o t e i n Synthesis : D i f f e r e n t i a l S t i m u l a t i o n of C e l l S p e c i f i c P r o t e i n s i n E p i t h e l i a l C e l l s of Chick Oviduct. S c i . 60: 86-87.  19.  Kohler, P.O. and O'Malley, B.W. 1967. Estrogeninduced Morphologic Changes i n Monolayer Cultures of Immature Chick Oviduct. E n d o c r i n o l . 81: -1422-1427 .  20.  Lynch, M.J., Raphael, S.E., M e l l o r , L.B., Spare, P.D., H e l l s , P. and Inwood, M.J.H. Medical Laboratory Technology. W.B. Saunders Co., P h i l a d e l p h i a , 1963.  21.  Oka, T. and Schemke, R.T., 1969 . I n t e r a c t i o n of Estrogen and Progesterone i n Chick Oviduct Development. I I . E f f e c t s of Estrogen and Progesterone on Tubular Gland C e l l Function. J . C e l l B i o l . , 43: 123-137.  22.  O'Malley, B.W. 1969. Hormonal Regulation of N u c l e i c A c i d and P r o t e i n Synthesis. Trans. N.Y. Acad. S c i . , 478-502.  58 23.  O'Malley, B.W., Aronow, A., Peacock, A.C. and Dingman, C.W. 1968. E s t r o g e n Dependent I n c r e a s e o f T r a n s f e r RNA D u r i n g D i f f e r e n t i a t i o n o f C h i c k O v i d u c t . S c i . 162: 567-568.  24.  O'Malley, B.W., McGuire, W.L. and Korenman, S.G. 1967. Estrogen S t i m u l a t i o n o f Synthesis of S p e c i f i c P r o t e i n s and RNA Polymerase A c t i v i t y i n Immature Chick Oviduct. B i o c h i m . B i o p h y s . A c t a . , 145: 204-207.  25.  O'Malley, B.W. and M c G u i r e , W.L. 1968. S t u d i e s on t h e Mechanism o f E s t r o g e n - M e d i a t e d T i s s u e D i f f e r e n t i a t i o n : Regulation of Nuclear Transcription and I n d u c t i o n o f New RNA S D e c i e s . Proc. Nat. Acad. S c i . U.S.A., 60: 1527-1534.  26.  O'Malley, B.W., McGuire, W.L. and M i d d l e t o n , P.A. 1968. A l t e r e d Gene E x p r e s s i o n D u r i n g D i f f e r e n t i a t i o n : P o p u l a t i o n Changes i n H y b r i d i z a b l e RNA a f t e r S t i m u l a t i o n o f C h i c k O v i d u c t w i t h Oestrogen. Nature Lond., 218: 1249-1251.  27.  O'Malley, B.W. and K o h l e r , P.O. 196 7. Hormonal Induction of S p e c i f i c P r o t e i n s i n Chick Oviduct C e l l C u l t u r e s . B i o c h i m . Biophv. Res. Comm., 28: 1-7.  28.  O'Malley, B.W. and Korenman, S.G. 1967. S t u d i e s on the Mechanism o f Hormone I n d u c t i o n o f S p e c i f i c Protein. Immunological I d e n t i t y and K i n e t i c S t u d i e s o f A v i d i n S y n t h e s i z e d I n V i t r o by t h e Chick Oviduct. L i f e S c i . , 6: 1953-1959.  29.  Oye, I . 1969. Adenosine 3,'5'-Monophosphate ( C y c l i c AMP) as a Messenger M o l e c u l e i n Hormone A c t i o n . Nord. Med., 81: 97-101.  30.  Rhodes, M.B., A z a r i , R.P. and Feeney, R.F., 1958. A n a l y s i s F r a c t i o n a t i o n and P u r i f i c a t i o n o f Egg White P r o t e i n s v/ith C e l l u l o s e C a t i o n Exchange. J . B i o l . Chen., 230: 399-408.  31.  R o s e n f e l d , M.G. and O'Malley, B.W. 1970. S t e r o i d Hormones: E f f e c t s on A d e n y l C y c l a s e A c t i v i t y and Adenosine 3', 5'-Monophosphate i n Target T i s s u e s . S c i . , 168: 253-255. " :  32.  S c h n e i d e r , W.C. 1945. Phosphorus Compounds i n Animal Tissues. I . E x t r a c t i o n and E s t i m a t i o n o f Desoxypentose N u c l e i c A c i d and o f Pentose N u c l e i c A c i d . J . B i o l . Chem., 161: 293-303.  59. 33.  S p a z i a n i , E. and Szego, CM. 1958 . The Influence o f E s t r a d i o l and C o r t i s o l on Uterine Histamine o f the Ovariectomized Rat. E n d o c r i n o l . , 63: 669-678,  34.  Teng, C and Hamilton, H. 19 69. The Role o f Chromatin i n Estrogen A c t i o n i n the Uterus. I , The C o n t r o l of Template Capacity and Chemical Composition and the Binding of H E s t r a d i o l 176. Proc. Nat. Acad. S c i . U.S.A., 60: 1410-1417.  35.  Terenius, L. 1969. Oestrogen Binding S i t e s i n the Chicken Oviduct S i m i l a r t o those o f the Mouse • Uterus. Acta. E n d o c r i n o l . , 60: 79-90.  36.  Wrenn, J.T. and Wessels, N.K.  37.  Zimmering, P.E., Kahn , I . and Lieberman, S. 1970. E s t r a d i o l and Progesterone Binding t o a F r a c t i o n of Ovine Endometrial Cytoplasm. Ciochem. , 9: 2498-2506.  19 70. C y t o c h a l a s i n B: E f f e c t upon M i c r o f i l a m e n t s Involved i n Morphogenesis o f Estrogen Induced Glands o f Oviducts. Proc. Nat. Acad. S c i . U.S.A., 66: 904-9 08.  60.  A P P E N D I X  61.  TABLE 1. RF VALUES OF DENSITOMETRY FROM DISC GEL ELECTROPHORESIS OF HOMOGENIZED OVIDUCT SAMPLES OF CHICKS TREATED WITH DES In Vivo  Control Oviducts  Peaks A  3  Standard  . 69  3d  .71  1  2  . 77  A  l .83  ' .70  l 3d 3  A  d  0  . 72 .69  .76  .82  Standard  .68  .78  .83  3d  . 71  3  . 70  h h  .72  1  .66  I Z  17d  1  17d 17d  .77  .76  Standard  I J  2  A  l .83  .73  h  .72  A  .71  2  3 17d 3 d  Peaks A  Standard  J  Standard  3  Experimental Oviducts  1  .85  .74  .83  .90  .68  .73  .80  .71  .78  .68  1 II 2  .65  .70  .72  2  . 68  II  2  .66  .76  . 82  0  .64  J I  .66  . 71  .77  .69  .72  .79  .68  .75  . 81  .68  .73  .82  .67  .73  .79  Standard  . 68  .76  Standard  .69  .77  17d  3  .72  I I X  17d  3  .71  I1J  Standard  .70  17d  4  17d  . 81  3  3 . 82 Standard 1  1  III  2  .65  .71  .79  .68  III  2  .65  .71  .78  4  .72  I I X  17d  5  17d  5  .78  .85  I J  .65  .70  . 69  3 III  .68  .74  .85  .75  Standard  .66  . 74  .82  . 65  .73  . 80  .71  .72  .81  .69  .71  .73  .68  .73  .81  .69  .72  . 80  3  Standard  .70  19d  1  .76  19d  x  .73  2 iv  19d  2  .73  I V  19d  2  .72  3  I V  3  Standard  .70  21d  r  .72  21d  1  .72  2 21d 2 1 d  2  .74 . 72  .79  .85  I V  1  I V  2  .79  . 84  KEY TO TABLE 1  Roman numerals i n d i c a t e t h e t r e a t m e n t s as f o l l o w s : Treatment I  Age o f o v i d u c t i n days b e f o r e sacrifice 4  Number o f DES Injections 4  II  11  7  III  15  11  IV  18  14  S u b s c r i p t s ( i . e . 1^, 1^ e t c . ) . The roman numeral r e p r e s e n t s t h e t r e a t m e n t number and d i f f e r e n t subs c r i p t s r e p r e s e n t t h e d i f f e r e n t c h i c k s t h a t donated o v i d u c t sample i n a t r e a t m e n t . Identical subscripts r e p r e s e n t d u p l i c a t e d i s c g e l s o f samples o b t a i n e d from t h e same c h i c k . The c o n t r o l o v i d u c t s a r e l a b e l l e d w i t h t h e age o f t h e o v i d u c t i n days. The s u b s c r i p t s r e p r e s e n t t h e c h i c k s t h a t donated t h e o v i d u c t sample. I d e n t i c a l s u b s c r i p t s r e p r e s e n t d u p l i c a t e d i s c g e l s o f samples o b t a i n e d from t h e same control chick. Standard r e f e r s t o t h e s t a n d a r d p r e p a r e d o v a l b u m i n . One o r two s t a n d a r d d i s c g e l s were r u n p e r r e s e r v o i r .  TABLE 2 RF VALUES OF DENSITOMETRY FROM DISC GEL ELECTROPHORESIS OF HOMOGENIZED PARTIALLY DIFFERENTIATED OVIDUCT SAMPLES CULTURED WITH DES SERUM AND CONTROL SERUM AS WELL AS NON CULTURED PARTIALLY DIFFERENTIATED OVIDUCTS TREATMENTS 1.  Oviducts + DES Serum  Chick No. Standard  .  2.  Peaks A  3  A  2  A  l  .68 .78 .84  Standard  Oviducts + C o n t r o l Serum  Chick No.  3.  Peaks . 3 A  A  2  A  l  Non-Cultured 4. Injected Controls  Chick No.  Peaks A  3  A  2  A  l  l  Standard  . 69 .77 . 82  .68 .76 .82  . 72 .75 . 82  II  .71  I  .67 .73 . 84  . 71  IV  . 80  IV  .75  . 81  3d^  .76 .72  I  l .68 .74 . 82 Standard  .69 .77 .83  I  .67 .73 .81  3 d  . 71  3  d  2  IV  .73  Standard  .73 . 81 .87  3d  2  .70  IV  .70  Standard  .70 .79 . 84  3d  3  .72  Standard  .68 .78 .84  II  .68 .73 . 85 Standard  .69 .76 . 82  II  .75 . 79 . 87 Standard  . 69 .78 . 83 .71  IV  .77  .72  II  . 65 . 74 .81  IV  .75 . 80  .72  II  .71 . 74 . 8 3  .70 .75 .82  A  .70 .76 . 84  III  II  2  Standard  .72  .65 .69 .75  A  .69 .76 .82  I  II  3  .68 .76 . 81  .72  .68 .75 .82  A  Standard  II  Standard  Peaks  .70 . 77 .84  . 70 .75 . 81 . 80  Chick No.  Standard  III V  Non-Cultured,NonInjected Controls  .75 Standard  .73  . 86  3d-4 3d 4  .72  III  . 66 . 71 . 80 1 7 d  1  .66  III  . 61 . 67 .73  1  .68  17d  CO  TABLE  2  (Continued)  TREATMENTS  1.  Oviducts + DES S e r u m  2.  Oviducts + C o n t r o l Serum  Peaks Chick No.  A  A 3  A  A 2  A  A  l  Chick No.  A  3  A  A  2  A  A  A  .74  III  . 7 1 .76  . 84  III  . 74 . 8 1  Standard  .73  .86  III  .74  Standard  l  .83  Standard  h  i  c  No.  k A  A A  2  A  A  A l  h  i  c  k  No.  A  3  A  2  A  A  Standard  .70  .77  .82  17d  2  .68  Standard  .69  . 77 .83  17d  2  .64  .73  .77  .83  Standard  .68  .75  . 86  Standard  .69  IV  .82  3  IV .69  .76  . 82  VI  .70  .76  . 82  .72  .76  . 82  VI  .71  .76  . 84  . 71  V  .68  .76  V  . 72  .80  .72  . 74 . 84  V  .73  .81  .73  .76  V  .67  Standard  .68  . 77 . 83  Standard  .70.  . 76 .85  Non-Cultured,NonInjected Controls Peaks  C  . 76 . 84 . 85  4.  Peaks C  Standard . 74 . 82  Non-Cultured Injected Controls  Peaks  III  .81  3.  .72  .80  .69  A  A  .86  76  .83  .72  .82  .72  l  . 80  .70  .75  .65  .73  . 82  .71  .81  .73  .83 cn 4r  TABLE  2  (Continued)  TREATMENTS  Oviduct + DES S e r u m  Standard  A  3  .69  A  2  A  i  .76 .83  Chick No. Standard  A  3  .68  A  2  A  i  .74 . 8 1  Chick NO. Standard  A  3  .65  I  . 69  I  .69  I  . 71  •I  .68  I  .74  I  .70  II  . 70  II  . 70  II  .70  II  . 71  Standard  .69  III  .75  Standard  .76  .80  II  .70  II  .70  Standard  .67  . 80  III  . 69  III  .70  III  . 67  III  . 67  III  . 71  IV  . 72  IV  . 67  Standard  .72  IV  . 68  Standard  .68  . 8 1 .87  .68  .93  IV  . 71  IV  . 67  V  .67 . 78  V  .78  V  .73  V  .71  V  V  .68  VI  .73  . 68  VI  .67  VI  .76  Standard  .65 .69  Standard .72  Peaks  .78  VI  A  2  .72  A  l  Chick No.  A  3  A  2  A  l  . 78  . 7 1 . 79 . 59  .81  Standard  Non-Cultured,NonInjected Controls  Peaks  Peaks  Peaks Chick No.  Non-Cultured Injected Controls  Oviducts + C o n t r o l Serum  . 81 .88  . 69 .76  .83  .70  VI  .83  VII  . 71  VII  . 70  cn < cn  TABLE  2  (Continued)  a)  E a c h Roman n u m e r a l r e p r e s e n t s o n e of the f i r s t three treatments.  b)  The n o n - c u l t u r e d , n o n - d i f f e r e n t u a t e d o v i d u c t s a r e l a b e l l e d w i t h t h e age o f t h e o v i d u c t i n days. I d e n t i a l s u b s c r i p t s r e p r e s e n t d u p l i c a t e d i s c g e l s of samples o b t a i n e d from t h e same c h i c k .  c)  Standard r e f e r s to the run per r e s e r v o i r .  standard  chick which  prepared  donated  ovalbumin.  1 or  One  or  2 pieces  two  of  oviduct to  standard  disc  gels  each  were  cn cn  67. TABLE 3 PROTEIN NITROGEN/DNA RATIOS OF DES SERUM CULTURED PARTIALLY DIFFERENTIATED CHICK OVIDUCTS  DES-Serum  Trial 1  AVERAGE  Non-Cultured Controls  Ch i ck No.  PN/DNA  Chick No.  PN/DNA  Chick No.  I I II II III III IV IV V V  1.2 8  I I II II III III IV IV V V  1.19  I I II II III III IV IV V V  AVERAGE Trial 2  Control-Serum  1.65 1.46 1.56 .2.51 0.94 + .48  2.0 2. 32 2. 04 2 .10 1.71 1.92  2.01 + .08  2.56 2.02 2.93  2 .07 + .25  1.5 + .21 I I II II III III IV IV V V VI VI VII  1.85  I I II II III III IV IV V V VI VI VII  1.85 1. 57 3.43 1. 94 2. 39 2 . 64 2 .07 + .32  PN/DNA 2 . 48 2.69 3.62 3 .82 3 . 82 3.57 2.77 3 .33 3.87 3.05 3 .3 + .16  I I II II III III IV IV V V VI VI VII  1.29 . 87 1.24 1. 87 1.7 5 2 .72 2.03 2.18 1. 31 1.95 1.08 1. 89 . 65 1 .6 + .16  Each c h i c k donated 2 p i e c e s o f p a r t i a l l y d i f f e r e n t i a t e d o v i d u c t toward each t r e a t m e n t . C h i c k s were l a b e l l e d w i t h Roman numerals.  68  FORMULAE FOR DETERMINATION AND DNA.  1.  Protein  OF PROTEIN NITROGEN  Nitrogen  10 av.O.D. 10 yg N 10  X  O.D. observed of experimental s amp1e s  average p r o t e i n bound n i t r o g e n  X  v  o  l  u  m  e  f  a  Q  t  o  r  i n sample  volume f a c t o r was (1 i f .2 ml) (2 i f .1 ml) - (4 i f .05 ml)  2.  DNA  JLO  av.O.D. 10 yg DNA _^ 10  „  A  ~ , O.D. o f sample ^ i 5 observed n  amount o f DNA i n sample,  

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