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Acid mucopolysaccharides in the development of the Pacific great skate, Raja binoculata McConnachie, Peter Ross 1965

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AGID OF  MUCOPOLYSACCHARIDES  THE  PACIFIC  GREAT  I N THE  SKATE,  DEVELOPMENT  RAJA BINOCULATA  by  PETER B.Sc,  A  ROSS  University  THESIS THE  MCCONNACHIE  o f B r i t i s h  Columbia,  1962  SUBMITTED I N PARTIAL FULFILMENT REQUIREMENTS FOR MASTER in  OF  THE DEGREE  OF  OF  SCIENCE  the Department  of  ZOOLOGY  We  accept  required  THE  this  thesis  as conforming  t o the  standard  U N I V E R S I T Y OF JULY,  BRITISH 19 65  COLUMBIA  In p r e s e n t i n g  this  thesis  in p a r t i a l  fulfilment  of  the requirements f o r an advanced degree at the U n i v e r s i t y o f British  Columbia,  available  for  I agree that  the L i b r a r y s h a l l  r e f e r e n c e and s t u d y .  make i t  I f u r t h e r agree that  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  for  freely per-  scholarly  purposes may be granted by the Head o f my Department o r by his  representatives*  cation of t h i s  thesis  without my w r i t t e n  Department o f  It  permission.  ~^-^*-cr^/^x^<u-e^  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada Date  \y  ^  i s understood that copying o r p u b l i -  for financial  —  Columbia  gain shall  -  not be allowed  -  i  -  ABSTRACT  Histochemical acid, and  chondroitin sulphate  heparin,  which  great  skate  (Raja  characterize present  the situation  in  the adult.  neutral  polysaccharides  acidic  acid  with  was  smooth  mental  significance  occurred  i n a  (17-18  from;  neutral  mm.  localization from  early  form  f o r this similar  2.  polysaccharides (hyaluronic  acid  i n later  a  and  acid) t o ,  mucopoly-  stages,  development.  hyaluronic some  compound.  appeared tissue  o f some  develop-  Hyaluronic  acid  i n lesser  i n close  acid  adjacent  suggestive  form  embryos)  intracellular  through,  sulphated  o f cartilage  layer  1.  i n neurulating stages  quantity between  i n a  Pacific  leading  ranged  i n cleaving stages  strongly acidic  layers  acid  mucopolysaccharides  examined  n e u r u l a t i n g embryos  considerable  as  to  the events  observed  processes  i n areas  In  neurulae  stages  (chondroitin sulphates)  particularly  also  series o f i n order  mucopolysaccharides  c e l l  extracellular  saccharides  embryos  classed  mucopolysaccharides  of extracellular  associated  to a  B,  prehatching.  progression  combination  in  Embryonic  t o immediate A  compounds  and t o study  of acid  f o r hyaluronic  chondroitin sulphate  applied  binoculata)  specific  histochemically the acid  to  weakly  were  i n t h e embryos  cleavage  A/C,  are biological  mucopolysaccharides,  3.  treatments  quantity  i n  association with  post  - ii -  developing gut and mesonephros. Results of h i s t o l o g i c a l t e s t s i n immediate prehatching embryos agreed w i t h p r e v i o u s l y reported biochemical analyses of shark s k i n s and c a r t i l a g e s i . e . c h o n d r o i t i n sulphate B occurred p r i m a r i l y i n the s k i n and c h o n d r o i t i n sulphate A/c were a major component of the c a r t i l a g e matrix.  -  TABLE  i i i  OF  -  CONTENTS PAGE  ABSTRACT TABLE  i  OF C O N T E N T S  i  i  i  LIST  OF T A B L E S  v  LIST  OF F I G U R E S  v i  ACKNOWLEDGEMENT  v i i i  INTRODUCTION Purpose o f t h e Research literature review.  and general 1  MATERIALS  7  METHODS  9  RESULTS Cleavage  stage  15  2 - 4 mm.  embryo  17  6-7 mm.  embryo  19  1 7 - 1 8 mm. 4  embryo  33  cm. e m b r y o  37  7 cm. e m b r y o  49  14  53  cm. embryo  DISCUSSION Specificity Embryonic  o f staining  development  mucopolysaccharides SUMMARY LITERATURE  methods  o f  63  acid 66 76  CITED  78  -  i v -  PAGE ADDITIONAL APPENDIX  REFERENCES  A Structure  APPENDIX  82  of acid  mucopolysaccharides.  84  B Histochemical  methods employed.  86  -  LIST  v  OF  -  TABLES  TABLE  PAGE  I  Staining  results  i n cleaving  II  Staining  results  i n 2-4  mm.  embryos.  III  Staining  results  i n 6-7  mm.  embryos.  IV  Staining  results  i n 1 7 - 1 8 mm.  V  Measurements notochord  of anterior  of 4  cm.  embryos.  portion  25  & 36  40  Staining  VI  Staining results i n trunk regions of 7 cm. e m b r y o s . Comparison o f s t a i n i n g results i n head a n d t a i l r e g i o n s o f 7 cm. e m b r y o s .  52  Staining results i n skin o f 1 4 cm. e m b r y o s .  57  VIII  IX  X  G e n e r a l summary treatments.  cm.  20  of  embryo.  i n 4  16  Va  VII  results  embryos.  embryos.  and  of results  of  45  50  cartilage  staining  Diagram o f metabolic steps i n biosynthesis of chondroitin sulphates. f r o m K e n t , P . W. Some B i o c h e m i c a l aspects of Sulphated Mucopolysaccharides, Biochemical Society S y m p . No 2 0 , Cambr. U n i v . P r e s s , 1961.  67  75  26  -  LIST  v i -  OF  ILLUSTRATIONS  FIGURE  PAGE  1.  PAS  gradient  2.  ECM  i n 2-4  3.  Smooth  4.  ECM  5.  Smooth i n 6-7  6.  7.  8.  9.  mm.  layer  i n 6-7  mm.  i n c l e a v i n g embryos. embryos. form  ECM  21  i n 2-4  mm.  embryo.  embryo.  material  i n 6-7  ECM  forms 23  mm.  embryo 28  E f f e c t o f s u l p h a t i o n on cytoplasmic b a s o p h i l i a i n t h e 6-7 mm. embryo. Pyronin embryo.  22 22  l a y e r and 'pseudopod' mm. embryo.  AMPS l i k e midgut.  18  staining  of  ECM  30  i n t h e 6-7  mm. 32  Hyaluronic acid associated with mesonephric t u b u l e s i n t h e 1 7 - 1 8 mm. embryo.  34  Hyaluronic a c i d a s s o c i a t e d w i t h gut tube development i n t h e 1 7 - 1 8 mm. embryo.  34  11.  General morphology  38  12.  Notochord  12a.  Notochord i n the mid 4 cm. e m b r y o .  10.  13.  14.  15.  16.  sheath  of  of  the 4  the 4  cm.  cm.  trunk  embryo.  embryo.  region  of  41 the 42  4 cm. e m b r y o n o t o c h o r d a n t e r i o r end.  150  4 cm. e m b r y o n o t o c h o r d a n t e r i o r end.  350  4 cm. e m b r y o n o t o c h o r d a n t e r i o r end.  1500  4 cm. e m b r y o n o t o c h o r d a n t e r i o r end.  2200  microns  from 42  microns  from 43  microns  from 43  microns  from 44  -  v i i -  FIGURE  16a.  17.  18.  19.  .20.  21.  PAGE  Metachromacy o f peripheral mesenchyme s u r r o u n d i n g 4 cm. e m b r y o n o t o c h o r d .  44  Glycogen i n branchial 4 cm. e m b r y o .  48  Effect of diastase filament glycogen. Hyaluronic embryo.  acid  branchial 48  i n neural  Skeletal elements o f 14 cm. e m b r y o .  tube  of vertebral  of  7  cm.  23.  Irregular Alcian embryo c a r t i l a g e  column 54  Skin and underlying embryo. Intestinal  25. to 30.  of  51  22.  24.  on  filaments  epithelium  tissues  of  14  cm. 55  of  14  cm.  blue staining matrix.  embryo. of  14  55  cm. 59  Effect of diastase on A l c i a n blue staini n g o f 1 4 cm. e m b r y o c a r t i l a g e m a t r i x .  59  Effect of various histochemical treatments on s t a i n i n g o f s k i n and c a r t i l a g e matrix o f 14 cm. e m b r y o .  60, 61, 62  — viii -  ACKNOWLEDGEMENT  I wish t o express a p p r e c i a t i o n t o Dr. Peter Ford and t o Dr. C. V. Finnegan f o r the guidance, support and encouragement I have r e c e i v e d d u r i n g the course o f t h i s study. T h i s r e s e a r c h was supported i n p a r t by funds from the N a t i o n a l Research C o u n c i l o f Canada and the U n i t e d Fishermen and A l l i e d Worker's Union.  -  1  -  INTRODUCTION  Existing identification sections acid  histochemical  shown  and considerable  i n adult  that  AMPS  information (AMPS)  organisms.  i s available on the  However,  can be c h a r a c t e r i z e d  a n d i th a s n o t y e t b e e n  events  leading  specific  i th a s n o t y e t been s p e c i f i c a l l y i n  shown  to the situation  can be a s c e r t a i n e d  i n tissue  constitution of  embryos  adults  permit the  o f acid mucopolysaccharides  mucopolysaccharide  tissues  techniques  that  o f AMPS  the chain  o f  localization i n  histochemically  during  embryonic  development. Previous Elasmobranch AMPS Ford,  are present 196 4 ) .  investigate throughout Raja  yolk  s t a l k s have t o permit  Accordingly,  the embryonic  binoculata,  problem  i n this indicated  such  a  study  the problem  by histochemical  Before this  observations  means  describing  i ti s n e c e s s a r y  that  (great)  o f embryo  sufficient  (McConnachie and s e l e c t e d was t o  t h e AMPS  development  the Pacific  laboratory  occurring  o f the Elasmobranch, skate.  t h e methods  used  to  approach  to discuss  some  o f the  (Abbreviations used. AMPS- a c i d m u c o p o l y s a c c h a r i d e s , ABPAS- combined A l c i a n - b l u e - p e r i o d i c a c i d S c h i f f s t a i n f o r a c i d m u c o p o l y s a c c h a r i d e s , HA- H y a l u r o n i c a c i d , C-S-A C h o n d r o i t i n s u l p h a t e A, C-S-B - C h o n d r o i t i n s u l p h a t e B, C - S - C - C h o n d r o i t i n s u l p h a t e C, C P B - c e t y l pyridinium ' bromide)  - 2 -  a v a i l a b l e i n f o r m a t i o n on the nature of AMPS, t h e i r occurrence, and the techniques which have been used i n their  identification. Mucopolysaccharides  were f i r s t d e f i n e d i n  1938  as p o l y s a c c h a r i d e s c o n t a i n i n g hexosamine o c c u r r i n g f r e e or  as e s t e r s o f s u l p h u r i c a c i d .  Acid  mucopolysaccharides,  i n a d d i t i o n to hexosamine, have hexuronic a c i d as a component and occur f r e e or as sulphate e s t e r s . which are i n c l u d e d i n the a c i d mucopolysaccharide  second  Compounds group  are h y a l u r o n i c a c i d ; the c h o n d r o i t i n sulphates A, B and  C;  k e r a t o s u l p h a t e and h e p a r i n . H y a l u r o n i c a c i d i s a polymer of glucosamine  N-acetyl-D-  and D - g l u c u r o n i c a c i d u n i t s which occurs as a  free disaccharide i n synovial f l u i d ,  Wharton's j e l l y  and  v i t r e o u s humours and i s a component o f c a r t i l a g e m a t r i x . (Pearse, 1961;  Shaw and M a r t i n , 1962;  Dorfman, 1963).  The c h o n d r o i t i n sulphates are one o f the main a c i d mucopolysaccharide  components of c o n n e c t i v e t i s s u e s  and p a r t i c u l a r l y o f c a r t i l a g e m a t r i x .  In mammalian  c a r t i l a g e , c h o n d r o i t i n sulphates A and C may up to 40% of the dry weight, and are thought proteins  account f o r  (Bloom and Fawcett,  to occur i n c o v a l e n t l i n k a g e w i t h  (Dorfman, 1963).  cartilage  C h o n d r o i t i n sulphate B occurs  i n a o r t a s and i s the major s u l p h a t e d AMPS of s k i n 1963), C-S-A  1962)  and C are polymers of  (Dorfman,  N-acetyl-D-galactosamine  and D - g l u c u r o n i c a c i d u n i t s , d i f f e r i n g one  from another  only  - 3 -  i n the p o s i t i o n o f t h e i r sulphate groups. polymer o f N-acetyl-D-galactosamine  C-S-B i s a  and D - i d u r o n i c a c i d .  I t i s n o t l a b i l e t o the a c t i o n o f t e s t i c u l a r h y a l u r o n i d a s e as a r e C-S-A and C (Walker,  1961).  Keratosulphate, f i r s t i s o l a t e d from cornea, i s now  known to occur i n c a r t i l a g e .  With i n c r e a s i n g age i t  r e p l a c e s the c h r o n d r o i t i n sulphates i n human c o s t a l c a r t i l a g e and i n r a b b i t nucleus pulposus Davidson  and Small, 1963).  Keratosulphate i s a polymer o f  equal amounts o f N-acetyl-D-glucosamine, sulphate. response  Little  (Dorfman, 19 63;  D-galactose and  i s known o f the g l y c o s i d i c l i n k a g e s and  to hyaluronidase of keratosulphate. Heparin, u n l i k e the e x t r a c e l l u l a r  connective  t i s s u e AMPS, i s c e l l u l a r and i s found a s s o c i a t e d w i t h s t r o n g l y b a s i c p r o t e i n i n mast c e l l s Schiller,  1963).  (Spicer, 1963;  Heparin i s a polymer o f D-glucosamine  and D-glucuronic a c i d w i t h 3 sulphates per d i s s a c c h a r i d e unit  (Walker,  1961).  S t r u c t u r a l and chemical data p e r t a i n i n g to the above mentioned AMPS a r e presented i n Appendix A. Recent i n v e s t i g a t i o n s have shown t h a t AMPS have a wide d i s t r i b u t i o n i n d i f f e r e n t animal c l a s s e s and tissues  (Anno, Seno and Kawaguchi, 19 62;  Einstein,  1963; Kato and S i r 1 i n ,  S p i c e r , 1960 and 1963;  Zugibe,  Szabo and Roboz-  1963; Mathews, 1962;  1963).  To date, o n l y a few  i n v e s t i g a t i o n s have examined the s y n t h e s i s o f AMPS i n embryonic m a t e r i a l .  Such s t u d i e s have used i s o t o p e  -  incorporation (Ozello  and  Spicer,  and  possible  blocking  to  behaved  and  the  use  techniques  different  molar  for  and  that  Kelly,  blocking  (1964)  1963;  Azure  A  in  a  ratio  would  hyaluronic  of  acid  disaccharide  The  tissue  specific  of  orange  i n  and  at  at  e l e c t r o l y t e s .  flourescent  staining  varying  pH  acid,  the  tissue  technique  suggested  anionic  that  levels.  sections s i m i l a r  to  dyes  such  precipitates with  AMPS  sites  on  electrostatic upon  anionic  containing  unit w i l l  could  dissociation  hyaluronic  heparin  AMPS  which  detergents  inorganic a  that  Scott.  depend  the  of  showed  their  metachromatic  between  i t w i l l  values  and  polyanions  to  unblocking  and  form  (1963)  developed  and  (1963)  molecules.  that  i n  make  sophisticated  ammonium  Acridine  and  Bloom  Szirmai as  some AMPS  more  complex  concentrations  sulphates,  a  1:1  as  differentiation  through of  Scott  according  AMPS u s i n g  allows  pK  systems  techniques  extractions  further,  quaternary  unblocked  chondroitin  the  c e l l  Schiller,  i n histochemical  enzyme  make  Bloom  Saunders  means  with  possible.  p r e c i p i t a t e d by  dye  of  histochemically  selectively  and  1964;  d i f f e r e n t i a t e between  Kelly,  This  Prockop,  developments  differentiation  method  1964;  techniques  1963).  sections,  be  -  enzyme-assay  Bambry,  New i t  4  bind  the  groups  pH  cationic  disaccharide  nature  of  of  medium  the  involved.  units  dye-binding and  Thus  only  one  carboxyl  group  one  dye  molecule  at  per  neutral  pH.  - 5 -  Lowering  the pH w i l l decrease  the c a r b o x y l group d i s c u s s i o n  and below pH 2 dye-binding i s e x t i n g u i s h e d .  Chondroitin  sulphate i n c o n t r a s t , c o n t a i n s both a c a r b o x y l and a sulphate group.  A t pH 2, the sulphate group i s s t i l l s t r o n g l y  d i s s o c i a t e d and the d i s a c c h a r i d e w i l l b i n d one dye molecule. On t h i s b a s i s , Azure A a t pH 1-2 binds to sulphate  groups.  Yamada (1963b, 1964) s t u d i e d the s t a i n i n g r e a c t i o n s o f a r t i f i c i a l and n a t u r a l s u l p h a t e d p o l y s a c c h a r i d e s w i t h A l c i a n b l u e , A l c i a n blue-PAS and Azure A s t a i n s u s i n g b l o c k i n g techniques o f m e t h y l a t i o n - d e m e t h y l a t i o n and acetylation-deacetylation.  He suggested  that A l c i a n blue  s t a i n i n g o f AMPS i s n o t n e c e s s a r i l y dependent on the a c i d groups i n them and t h a t i s might be a d v i s a b l e t o examine t h e i r a c i d i t y by o t h e r s t a i n i n g p r o p e r t i e s such as t h i a z i n e dye metachromasia a t d i f f e r e n t pH l e v e l s . The s t u d i e s o f S p i c e r (1960, 1963) concerned the g r e a t v a r i e t y o f AMPS i n rodent e p i t h e l i a l mucins and made a comparison o f mast c e l l s t a i n i n g i n d i f f e r e n t rodent s p e c i e s . He used ABPAS, Azure A a t d i f f e r e n t pH l e v e l s , m e t h y l a t i o n demethylation,  and s u l p h a t i o n treatments.  a d i v e r s i t y o f AMPS s t a i n i n g responses was d i f f i c u l t .  He found so g r e a t  that c l a s s i f i c a t i o n  He suggested a c l a s s i f i c a t i o n o f AMPS based  on r e l a t i v e a c i d i t y , a z u r o p h i l i a and a l c i a n o p h i l i a .  He  a l s o found t h a t mast c e l l s a r e s t r o n g l y a z u r o p h i l i c even a t pH 0.5 and are n o n - r e a c t i v e w i t h A l c i a n b l u e .  This  l a c k o f a l c i a n o p h i l i a coupled w i t h s t r o n g a z u r o p h i l a i s c h a r a c t e r i s t i c o f h i g h l y s u l p h a t e d AMPS.  -  Enzyme the  as  identification However,  (Pearse,  amylases  (20  minutes  to  and,  prevent  diastase  Thus and  have  of  many  aiding  acid,  Zugibe,  1961).  sulphation  glycogen  (Yamada,  a  sequence  depend  information  on  on  relative  specific  and  G-S-C  Malt  diastase*  of  glycogen  treatments, 1962).  Malt  metachromacy  removed  (Pearse,  o f enzyme coupled  acidity  AMPS  d i s t i l l e d  and i f  cartilage matrix be  quite  solution i n  e l e c t r o l y t e extractions  which  C-S-A  before  basophilia w i l l that  histochemical  i s  f o r the removal 1%  1961).  treatments with  could  staining provide  identification  localization.  *  A n a l y t i c a l malt Corporation.  diastase,  f o r  i t nonspecific.  f r a c t i o n s however,  i s prolonged,  inorganic  of  used  mentioned  hyaluronidase  temperature,  sulphation  i t i s possible  definite  used  particularly,  cytoplasmic  treatments  1961;  widely  (19 60)  considered  hyaluronic  been  a t room  means  testis  Walker,  contains  incubation and  of  not been  Spicer  possible  hydrolyzing  and  water)  a  o f AMPS b u t  1961;  have  o f AMPS.  the action  specific,  -  extractions  identification  hyaluronidase  6  National  Biochemical  and  MATERIALS  In  this  elasmobranch, a  Raja  cartilaginous  anticipated January  and  G-S-B  presence  study Seno  used,  demonstrated  available  skin  the presence  since,  1962) have  shown  and the sting  galactosamine  of  hyaluronic On  development  the presence  shark  fractions  basis  G-S-C  and possibly  adult  stages. cases  in  sea water  be  from  less  o f Raja  hyaluronic  were  o f Georgia  a t 12°C.  from  A  protein  comparative  cartilages o f both  (Anno,  glucosamine  a constant  predictable  involvement involvement  studied. that would  i n cartilage  obtained by trawl a t 16 fathoms large  works  Squalus  AMPS lead  a n d C-S-B i n s k i n  acid  acid  as the  o f G-S-C,  binoculata  acid  and Meyer  Further  i t was a n t i c i p a t e d  o f hyaluronic  the Straits  indicated  i n the cartilages  i n embryos  in  and squid  1962) t h e presence  acid  Egg  could  o f hyaluronic  cartilage.  sulphates and a  this  presence  as i t i s  o f Seno  r a y Dasaytis and, i n a  bovine,  and Kawaguchi,  chondroitin  o f the  i n egg cases  o f two s p e c i e s as w e l l  o f C-S-C i n s k e l e t a l  o f whale,  embryos  c o n c e n t r a t i o n o f AMPS  biochemical investigations  of  the  were  sulphated keratosulphate i n cartilages  acanthis  and  a high  development,  September.  i n shark  (Mathews, and  binoculata,  fish,  through  have  o f AMPS  a n d i ti s r e a d i l y  The (1963)  study  t o  and  i nt h e  o f f Tsawassen  and were  s t o c k was h e l d  maintained  because  the  - 8  developmental external empty.  stage  could  appearance The  of  times  months  b u t i s n o t known  excess  of  months.  G i l b e r t , 1960;  Clark,  R.  i s slow  binoculata  obtaining  recently  developmental  n o t be  the case  incubation  15  -  and many  of rays  f o r Raja  ranges  binoculata  I960, 19 63;  1919)  The  rate  and  there  of  i s no  eggs  and  from  cases  (Tewinkel,  f e r t i l i z e d  stages.  determined  the  were  from  9-15  a n d may Libby  and  development  d i f f i c u l t y a  be  of  i n  succession of  i n  METHODS  Fixation  Acid fixation state  mucopolysaccharides  because  they  and because  o f their  Formalin 1960, in  Both  Zugibe  fixation  (Pearse,  change  o f AMPS w e r e  that  large  bromide  AMPS  AMPS w o u l d  these  o f AMPS.  break  as both  be p r e c i p i t a t e d .  anionic  groups groups,  the salt Kelly,  of  fixatives  to  be t h e b e s t  Morphological  used  A)  1963; Spicer,  effective  only  i n combination  with 1963).  alcoholic and acidic because  t h e former  and the latter Both  authors  with  protein However,  may  suggested  chloride  o r  formalin  bound such  AMPS ionic  o f AMPS.  I f staining  an a c i d i c  s t a i n i n g medium  would and  free  p r e c i p i t a t i o n  i s to  involve  i s required  linkage. Bloom  and Scott  (1963)  t o p r e c i p i t a t e AMPS  fixative detail  formalin  (SeeAppendix  (Yamada,  e.g. cetylpyridinium  fixation  anionic  neutral  solutions  (CPC o r CPB) i n c o m b i n a t i o n  improve  blocks  that  hydrated  1963; Szirmai,  n o t recommended  nature  cations  a n d a n y AMPS  special  i n a highly  nature.  frequently  stated  i n aqueous  the ionic  ionic  1961; Zugibe,  and Szirmai  reversible  i n tissues  require  1964) b u t i s p r o b a b l y  p r e c i p i t a t i n g protein protein  to  i s used  1963; Lhotka,  that  is  occur  (AMPS)  was  f o rb o t h  and large  and found  nucleic  comparable amounts  compared  acid  t o that  a  number  CPC-formalin  a n d AMPS.  obtained  o f amorphous  with  ground  -  tissue  substance  were  as  described  modified  by  Kelly,  -  retained.  Accordingly, formalin  10  the by  fixative  Williams  Bloom  and  s e l e c t e d was  and  Scott  Jackson  CPB-  (1956)  and  (1963).  Staining  AMPS p o s s e s s for  combination with  are  carboxyl,  hydroxyl at  any  free  anionic  site.  by  the  pH  Azure  pH  4.0  both  are  at  i o n i z e d and  sites.  With  ionized  and  depending (Zugibe,  on  Azure  Many e.g.  which  the  a of  A  1.5  of  of  f a c t o r s , the  most  charges acid,  on  Azure  A.  bind  and  only  dye  Spicer,  at  both  groups  are  or  not  sulphate  groups  1963). of  Gresyl  the  occurs.  surface  i n  This  on  a  i s  number  density  (Pearse, shows  thiazine  violet,  depending  Pearse,  i n  groups  occur  and  members  blue,  molecule  Thus,  considerably  substrate to  These  available i s  sulphate  carboxyl  molecules  according  are  can  metachromasia  dye  one  sulphate  important being the  as  c e r t a i n conditions,  sites  are  Toluidine  dye  such  i s decreased  dyes  of  available  binding  cationic  phenomenon  Hyaluronic  dye  1963;  A,  sites  s t a i n i n g medium.  carboxyl  pH  binding  w i l l  Which  Szirmai,  Azure  dyes  dyes  the  and at  of  under  proportion  polymerization  negative  of  binding  the  1963;  group  free  dye  and,  Cationic  determined A  number  cationic  sulphate  groups.  a  no  of  1961). metachromasia  -  in  dilute  adjacent about to  aqueous  and  s o l u t i o n s because  carboxyl  groups  10.3 X w h i c h  occur.  11 -  i n the polysaccharide  i snot s u f f i c i e n t l y  Polysaccharides  carboxyl  the distance  groups  close  chain i s  f o r dye-stacking  containing alternate  i . e . Chondroitin  between  sulphate,  sulphate  would  have  o an  interchange  stable  metachromasia Two  the  beta  beta the  distance  would  (violet)  form  form  f o rt h e purpose  attached  only  intensity  pH  their  study,  stain  stained with acidity  Alternately,  presence  o f  form. was  and the relative  Azure  o f acidic  a t p H 1.5 o r 4.0  s e c t i o n s were  reaction o f Lison  A magenta  A  due t o c a r b o x y l o r stained by  as adapted  AMPS a r e shown b y A l c i a n  blue  by  staining  by the r e do r pink  (blue-red t o purple)  polysaccharides.  a t PAS  reaction indicates  (SeeAppendix  Bf o r  procedures.) Blocking  both  The  significance  o r absence  2.7 a n d n e u t r a l p o l y s a c c h a r i d e s  staining.  Azure  A The  the  were  blue-PAS  (1960).  staining  form.  a n d t h e gamma  o f this  relative  groups.  Alcian  Spicer  recognized,  o f metachromasia.  examine  the  form  t o t h e presence  Sections  sulphate  can be  a n d t h e r e d o r gamma  (orthochromatic)  moderate,  expected.  i s due t o t h e simultaneous  However,  to  be  a n d some  states o f metachromasia  o r v i o l e t  alpha  o f 4 and 6 A  staining  r e a c t i o n s were  and Alcian  blue-PAS  methylation o f acid  used  i n conjunction  (ABPAS)  techniques.  and demethylation  groups  with  sequence  by hydrolyzing sulphate  blocks esters  -  and  forming  methyl  hydrolyzes only  methyl  (Yamada,  esters esters  (Yamada,  group  in  and  with  This  carboxyl thus  groups.  restores  Demethylation  carboxyl  deacetylation blocks restores  technique  ABPAS  groups  hydroxyl  i s more  staining which  amino  and  staining  useful  involves  only  i n hydroxyl  groups  particular. Sulphation  groups  and  fications  prolonged  malt  Hyaluronic and  Bentley, On  were  diastase  C-S-A  1963); and  some  i n 0.3  sections  M  NaCl  sulphates  extractions  were  Bloom On  Saunders*  and some  (1964)  identification sulphates  with  both  s t a i n i n g  polysaccharide  (Pearse,  1962).  and,  to  e s t e r i -  with degradation  remove  1961;  Shackleford  1964).  chondroitin  Kelly,  used  (Yamada,  hyaluronidase  C-S-C  sulphate  i n t e r p r e t e d as  t o remove.glycogen  d i f f e r e n t i a l  s u b s t i t u t e d f o r enzyme  removed  ester  groups  f o r p a r t i a l  Culling,  acid,  hydroxyl  e x t r a c t i o n s were  incubation, 1961;  to produce  are generally  of carbohydrate  treatments;  (Pearse,  i s used  the results  Enzyme  is  and  s t a i n i n g and  1963b).  conjunction  on  -  1963b).  Acetylation hydroxyl  12  extractions.  and both  a r e removed  carried  salt  Hyaluronic  hyaluronic i n 0.6  out according  extractions  M  acid  NaCl.  acid  and  the  These  t o the method  of  Scott. sections, Acridine  orange  of hyaluronic  and heparin  particularly  technique  acid,  i n parallel  i n older  the  was  used f o r  chondroitin  sections.  embryos,  -  A l l in  Appendix  of  -  the preceding  techniques  are  described  B.  The of  13  preceding  the following  developmental  (a)  Cleavage  (b)  2-4  mm.  early (c)  6-7  head  fold  mm.  embryo,  somitic,  17-18  mm.  cm.  embryo,  embryo,  s t i l l  pectoral  appearing,  embryo,  external anal  hindgut  area. forming  double  head  i n  complete.  branchial filaments of pectoral fins  being 1.4-1.6  not present  but pigmentation dimorphism  of  cm.,  on  epidermis  apparent  but  present.  pectoral width  split,  cm.  i n progress,  5.5  cm.,  branchial filaments, ocelli  membrane  apparent  forming.  of pectoral fins  s t i l l  i n  torsion  movements  and mesonephros  ocelli  head  hypomeric  islands  circulation  sexual  membrane  f i r s t  b r a n c h i a l f i l a m e n t s 1.5  width  fins  and  spontaneous  increasing,  cm.  blood  out growth  resorbed,  14  foregut,  and  i n midgut  intestine  extraembryonic 7 cm.  foregut  mesoderm,  f i r s t  embryo,  length,  (g)  short  splanchnic  apparent  t a i l ,  anal  and g a s t r u l a e .  neurulating stage,  formed,  complete,  (f)  embryo,  i n progress,  4  embryos  neurula.  extraembryonic  (e)  applied to  blastulae  torsion  in  were  stages:  stages,  mesoderm (d)  treatments  last  prehatching  no present, stage.  -  Embryos were CPB-formalin through  f o r 48  ethanol,  sectioned  at  8  14  -  removed  hours  cleared  microns  at  from  room  their  eggs,  temperature,  i n xylene, paraffin  and  examined  by  the  fixed  i n  dehydrated  embedded,  techniques  described. Between stage  were  2  examined.  sections  were  made  sections  from  an  several stages  and  had  one  embryonic  only  embryos  of  each  Frequently, parallel  from  treatments. which  7  specimen region  T h i s was 2  or  3  so  could  useful  i n  available  developmental series  that be  of  adjacent  subjected to  developmental embryos.  -  15  -  RESULTS  Cleavage  The azurophilia Details of  the  of  and  results  acid  staining  at  are  i n  The  A  shown  staining  the  at  presence  this  1.5  i s  which  of  shows  yolk are  which  formed  increased  I. 4  to  a  an  r e a c t i v i t y . l a b i l i t y  hyaluronidase  s l i g h t amount by  the  of  absence  absence  does on  not  free  normally  of  a  are  possibly  on  oligosaccharides  although the  some A l c i a n magenta  incubation  exception  portions  of  strongly  reactivity. staining  i s  On due  than  pink  and  this to  was  s l i g h t  hydroxyl  If  sulphate  to  PAS  present  the  did  of  restored  apparent  groups  of  which  r e a c t i v i t y to  account  affect  that  neutral  PAS  neutral  a l l staining  alcianophilia i n  i t i s  indicates  Diastase  not  presence  abolished  f o l d  polysaccharides).  coloration.  saponification basis  5  groups  (neutral due  to  this  hydroxyl  staining  supporting  some  cells  of  largely  Acetylation  of  stain.  a l c i a n - o p h i l i a but  further  polysaccharides.  was  blue  rather  reduced  reactivity,  density  staining  3  hydroxyl,groups,  of  ABPAS  high  azurophilia  presence  the  pH  supported  the  for  Table  l i t t l e  AMPS.  i n  esters  show v e r y PAS  Sulphation even  embryos  considerable  and pH  stage  contrast,  suggestes  hyaluronic  acidic  i n  s l i g h t Azure  digestion  of  cleavage  Stage  with  ventral a l l  PAS  almost a l l polysaccharides  TABLE CLEAVAGE  Superficial portions of cells  TREATMENT Azure A p H 4.0  0+  I  S T A G E S - B L A S T U L A TO (No. o f e m b r y o s 4 )  GASTRULA  Deep portions of cells  Cleavage furrows  Cytoplasm adjacent to furrow  0+  0++  0+  0+++  0+++  0+++  0+++++  ABP+++  ABP+++  ABP++++  PAS+  PAS+++  PAS++++  PAS++++  PAS+  Yolk  Disc gradient Uppermost Lowermost  0±  Azure A p H 1.5 Hyalase A.A p H 4.0 Sulphate A.A  p H 4.0  ABPAS Diastase ABPAS  0+++ AB+  PAS+  Acetylate ABPAS Acetylate Saponify ABPAS  AB+  PAS++  PAS+++  AB+ ABP+++  +  +++ AB+  PAS++++  PAS++++  PAS++  0 = an orthochromatic r e a c t i o n ; AB = A l c i a n b l u e reaction; PAS = S c h i f f r e a c t i o n ; A B P = c o n c u r r e n t A l c a i n B l u e a n d S c h i f f reaction; - = negative reaction; + = positive reaction t h e n u m b e r o f +'s b e i n g p r o p o r t i o n a l t o t h e i n t e n s i t y ; +. = d o u b t f u l reaction.  +++  -  as  the  hydroxyl  azurophilia materials  i s  a  groups.  suggestive  reflection  gradient of  of  fixation  1961).  gradient  persists after  This  glycogen  boundaries  (membranes)  any  stained in  i s more  the  bottom  than  i n the  the  the  about  microns  i n the  slight of  acidic  of  In  that  those i n  processes This  was  of  this  alcian surface  was  that  1.  i t can w e l l i s  be  at  the  not  be  due  to  the  c e l l  c e l l s .  The  portion  than  blue  deep  the  i s more  connection,  the  superficial heavily  i t was  apparent  s t a i n i n g alone  occurred  cells  yolky.  occurred  Because  to  cytoplasmic so  i s  adjacent  disc  of  which  tissues  the  were  i . e . the and Few  top  very  0.025  granular  nuclei  i n mitotic  were figures  diameter.  Extracellular  embryos.  and  glycogen  i t may  part  2-4  c e l l  but  stained  top.  of  treatment  heavily  cytoplasm  and  chyme  the  amount  s t a i n i n g density  s t a i n i n g was  observed 20  small  i n Figure  diastase  and  uppermost  A l l because  shown  greatest  control slides  only  that  s t a i n i n g occurred  ventral boundaries  c e l l  and  the  polymerization  The  and  PAS  i s  fixation.  lateral  of  means  polymerization  (Pearse,  called  This  blocks  present. A  half  -  a c e t y l a t i o n - s a p o n i f i c a t i o n treatment  restores  of  17  mm.  Embryo  s t a i n i n g associated  appeared designated  i n the as  2-4  mm.  with head  extracellular  mesenfold  material  mm.  -  F i g u r e 1. gradient,  x  Cleavage 42.  18  stage,  -  ABPAS  showing  PAS  - 19 -  (ECM)  a n d was  membrane neural  like  3 ) .  found  from  ectoderm  Azure  A  results  labile  to hyaluronidase,  amount  (See Table  thin  material)  are  involved  neutral higher is  b u t do  concentration  shown b y  same  type  ABPAS  indicate that This  than  of  mesoderm  PAS  i s shown  were most cells,  so  limited found  (perinotochordal a  low  con-  do  not  support  carboxyl i n the  also  groups  methylation-  indicate  acidic and  AMPS.  that  This  deacetylation  Polysaccharide  prevalent  less  i n very  groups,  i n a l l tissues i n  any weakly  staining.  a  o f AMPS was  some  are present  (Figures  and carboxyl  results  results  ECM  was  possibly being  acid.  ABPAS  of  t h e ECM  the notochord as  of the  mesoderm  only  the a c e t y l a t i o n blockade  notochord  (neutral)  i n epidermal  i n endoderm c e l l s  cells and  least  c e l l s .  6-7  In complete.  This  sequence.  concentrations  in  and present  recognized  polysaccharides  restoration  and  I I ) .  o r basement  strands  containing hydroxyl  i n staining.  demethylation  Thin  indicated that  of hyaluronic  entirely,  sheet  the ventral surface  surrounding  and was  centration this  AMPS  sheet  thin  to the underlying  acidic  a  a  and epidermis.  weakly  in  as  s t r u c t u r e on  ectoderm  extended 2,  also  t h e 6-7  The  mm.  mm.  Embryo  embryo,  hypomere has  the neural  separated  into  tube i s somatic  and  TABLE.I I 2 - 4 mm. (No.  TREATMENT  Ectoderm dorsal ventral  Azure A p H 4.0  0++ 0++  ECM  0+-+++  HEAD FOLD EMBRYO o f embryos 4)  . Mesoderm  Perinot. Notoch.  Endoderm  0++  0+++ 0++  0++  Yolk  Azure A p H 1.5 Hyalase Azure A p H 4.0 O  Diastase Azure A p H 4.0  0+ 0+  Me t h y l a t e Azure A p H 4.0 Methylate Saponify Azure A p H 4.0  0+++ 0+  0+  0+  0+ 0+  0+  ABP+++++  ABP++  ABP++ PAS++++  PAS+++  PAS+  AB+-+++  PAS++  PAS++ PAS+++  PAS+++  PAS+  0+  0+ 0++  ABPAS  ABP++ PAS++++  Hyalase ABPAS  PAS+ PAS++++  Acetylate ABPAS  0+  0+-+++  0+-++  AB+ AB+  AB+-+  AB+ AB+  AB+  ABPAS  PAS+++  PAS++  PAS+++  PAS++  PAS+  Methylate ABPAS  PAS+ PAS++  AB++  PAS+  ABP++ PAS++  PAS++  PAS+  Methylate Saponify ABPAS  ABP+ ABP++  AB+++  PAS++  ABP+++ PAS+++  PAS+++  PAS+  Acetylate  Sapo.nify  - 21 -  F i c r u r e 2. 2 - 4 mm. cellular extension  embryo showing form. Azure A  ECM o f t h e pH 4.0, x 200.  - 22 -  F i g u r e 3. P o r t i o n o f 2-4 mm. embryo showing smooth l a y e r form o f ECM (S) around.notochord (N) and on v e n t r a l s u r f a c e o f n e u r a l ectoderm. ABPAS, x 340.  F i g u r e 4. 6-7 mm. embryo showing e x t e n t o f ECM ( E ) . ABPAS, x 46.  -  23  -  F i g u r e 5. High power v i e w o f m i d - t r u n k portion o f 6-7 mm. embryo showing smooth l a y e r form o f ECM ( F ) a r o u n d n o t o c h o r d (N) a n d s u b n o t o c h o r d a l rod (S), and 'pseudopod f o r m o f ECM (P) e x t e n d ing from subnotochordal rod to i n t e s t i n e (I) and l a t e r a l s p l a n c h n i c mesoderm. A z u r e A pH 4.0, x 1230. 1  -  and  splanchnic  The  ECM  is  found  and  i s much  any  adjacent  1.2 than  show  the  6-7  a  c e l l  not  sheath  of  ECM  i n  thickness.  and  mm.  extent  higher  is  shown by A  results  mm.  stage  the  around  much  where  Figure  of  and  was  stage  4  ECM  and  mesoderm  present  but  polymerization the  f i r s t  s t a i n i n g of A  clearly  thicker,  i t and  i n  was 5  the  (Table than  pH  treatments AMPS as  AMPS.  This  and  strong  metachromasia  sulphated  AMPS,  i s  i s a  major  at  weak pH  C-S-A  staining,  blocking  sulphated  supported  or  AMPS  a  state  stages.  This i n  and  a l l ECM i s  C-S-A  of  the  that  the  C-S-C.  the  acid  a  of  weakly pH  1.5  that  with  some  minor methylation  groups  are  hyaluronidase  s t a i n i n g which or  be  ECM  being  s t r o n g l y by  by  presence  I t may  C-S-C  azurophilia indicating  extraction  i n  azurophilia at  component  blockade  for  i s  i s  considerable  4.0.  This  responsible  this  metachromasia  as  component. of  and  i n d i c a t e the  w e l l  shown by  probably i s  of  at  material  i n earlier  appearance  acidic  acid  III)  that  ECM.  strongly acidic  hyaluronic  show  a l l extracellular  i n nature,  Azure  the  midgut.  somatic  was  sheath) 4  4  the  between  between  h a l f micron form  the  processes  the  AMPS  of  by  i n  i n  development  entirely  some  A  than  of  embryo.  of  Azure  with  length  than  Staining stage  the  (perinotochordal  embryonic  mm.  over  tissue but  microns,  less  -  extensive  mesoderm.  notochord  about  more  associated  splanchnic the  components  24  I t  indicates i s notable  that that  TABLE I I I 6-7 mm.  EMBRYO  ECM  TREATMENT Azure A pH 4.0  M++  Azure A p H 1.5  0+  AZURE A CHEMICAL AND ENZYME (No. o f e m b r y o s 7)  Ectoderm  Neural ectoderm  Mesoderm  0+  0+-++  0++  TREATMENTS  Perinot. notoch. M-++  Syncytial Nuclei Endoderm 0+++ 0+  0+  Methylate A.A p H 4.0 to  M++-+++  Sulphate A.A p H 4.0  M++-+++  Sulphate A . A p H 1.5  0+-++  Acetylate A.A p H 4.0  0+-M+  Acetylate Saponify A . A p H 4.0  -  0+++  Hyalase A.A p H 4.0  -  0++++*  Diastase A.A p H 4.0  0+++*M+  0+-+  0+ 0+  0+  0+-++  0++ 0+-+  0++  M+ 0+  0+  0+  ~0+  Hyalase Diastase A.A p H 4.0 R.N.Aase A.A p H 4.0  *  M+++ 0++-M++  Orthochromatic  material  i sf o u n d  l i n i n g  anterior  midgut lumen  only.  TABLE ALCIAN  TREATMENT  BLUE  P A S CHEMICAL AND ENZYME  ECM  I I I CONT. TREATMENTS AND S P E C I A L  Ectoderm  Neural ectoderm  Mesoderm  TECHNIQUES Syncytial Perinot. Nuclei notoch. Endoderm  ABP+  ABP++  ABP+  AB+++++ ABP+  ABP++ PAS++  PAS+  PAS+  AB+++ PAS+  AB++ PAS++  AB++++  ABP++ PAS++  AB+++++  AB++ PAS++  AB+++++ PAS+  AB++ PAS++  AB++++++  AB++++  +++ +  ++ +  ABPAS AB++++ Hyalase ABPAS  AB++  Diastase ABPAS  AB++++  PAS+  PAS+  PAS+  Hyalase Diastase ABPAS  AB+  PAS+  PAS+  PAS+  R.N.Aase ABPAS  AB++++  Sulphate ABPAS  AB++++  PAS+  PAS+  PAS+  Pyronin  R.N.Aase Pyronin  ++++  +  +  ++++  +  +  +  +++ +  + +  ++++  ++  +++  +++  ++++ +++  ++ +  Saunders  1  Saunders 2  +  +  Saunders 3  Saunders Saunders Saunders  1 s t a i n s f o rh y a l u r o n i c a c i d . 2 s t a i n s f o rc h o n d r o i t i n sulphates 3 stains f o r heparin.  and heparin.  -  a  strong  orthochromatic  anterior and  midgut  after  material  Acridine  be  some  in  ABPAS  sheath  results  A  l i t t l e also  contains  some  the- o t h e r areas  phates,  which  probably  a  the  sheath which  sheath  results  as  and  i s about  equal  sheath  t h e two extraction b u t has  a t pH  C-S-C.  regions  with  1.5  demonstrable  Acridine  i n Azure  mm. A  does n o t .  suggest  of chondroitin  stage  that sul-  extractions  C-S-A  that  and/or  i n sufficient orange.  s t a i n i n g i n t h e 6-7  i n t h e 2-4  Saunders  I t i s suggested  i n ECM  Azure  material  a n d t h e ECM  of hyaluronidase  contains  Cytoplasmic as dense  perinotochordal  alcianophilia.  aimilar proportions  and/or  absence  o f PAS s t a i n i n g  alcianophilia  sulphates A  this  possibly  and diastase  ECM  That  polysaccharides.  indicate that  Azure  to stain  not  i t s  d i f f e r e n c e between  i s not y e t present  concentration  by  6)  i n t h e same m a n n e r b u t t h e  on the basis  material  consecutive  any d i f f e r e n c e as both  chondroitin  C-S-A  and  hyaluronidase  results  hand,  ECM  neutral  considerable  contain  are  C-S-C  suggest  to treatments orange  both  both  a  I t may  The a b s e n c e  o r no  do n o t s u g g e s t  Acridine  On  l i p i d .  of the  extraction  (Figure  i s inferred  staining.  on perinotochordal  results  respond  a n d ABPAS  consecutive  effect  t h e lumen  extraction.  show t h a t  o r removes  l i n i n g  hyaluronidase  i n nature  o f bound  results  blocks  A  orange  form  areas.  no  and diastase  contain  These  reaction  extraction, but not after  i s n o t AMPS  in  -  persists after  diastase  hyaluronidase  27  mm.  embryos i s  i n ABPAS  treatments.  With  treatments Azure  A  - 28  -  F i g u r e 6. AMPS l i k e m a t e r i a l (A) l i n i n g v e n t r a l p o r t i o n o f lumen o f a n t e r i o r midgut o f 6-7 mm. embryo. Azure A pH 4.0 f o l l o w i n g h y a l u r o n i d a s e d i g e s t i o n , x 230.  -  a l l  cytoplasmic  labile as  and  RNA-protein. azurophilia hypomeric endoderm stain. which weak  among  pattern  c e l l s . to  p a r t i a l l y  show  neural  weak  mesoderm  epidermis  Cytoplasmic  cytoplasmic  expected  to  i s RNA  of  i n  and  epidermis cells  neural and  alcianophilia  azurophilia but  and  cytoplasmic  do  p e r s i s t a n t PAS  i n endoderm, and  This  tube  notochord  some  RNAase  i n amount  the  and  labile,  due  intensely stained,  stained  results  i n hypomeric  diastase.  i s variation  are  weakly  i s moderate  notochord  to  t i s s u e s , as  mesoderm  ABPAS  labile  basophilia i s There  are  -  staining i s hyaluronidase  p a r t i a l l y  cytoplasmic  29  does  and not  reactivity tube,  very  absent i s  i n  not  i n  a  similar  occur  i n  endoderm. The that  neural  similar less  Saunders  ectoderm,  amounts  and  endoderm  agreement w i t h The of  the  is  l i k e l y  material  cells  the  RNAase  that  this  nuclear The  than  least,  indicates contain  cells  which  i s  i n  the  yolk  contain general  results.  hyaluronic  i n  acid  according  However,  e x t r a c t i o n and  due  nucleoprotein  cytoplasmic  a z u r o p h i l i a (Figure treatments  their  syncytium to  to or  the  azurophilia  sulphation.  azurophilia i s  abolishes  acid  cells  epidermal  n u c l e i found  AMPS o r  pyronin  notochord  acid,  treatments.  by  other  and  contain  preceding  contain  sulphation  affect  hyaluronic  ABPAS  abolished  more  as  and  mesoderm  syncytial  midgut  Saunders  of  reaction for hyaluronic  some  I t  acidic  nucleic  s t a i n i n g but  i s  acid  does  not  7).  were  included  for  this  -  30  -  F i g u r e 7. P o r t i o n o f t r u n k o f 6-7 mm. embryo showing absence of cytoplasmic s t a i n i n g w i t h A z u r e A p H 4.0 following sulphation, x 330.  developmental RNA-protein observed pyronin  stage  only  participation  previously  and  active  mast  cells  (Chen,  1963).  RNAase  incubation  involved  this  Results  i n s y n c y t i a l nuclear  but  n o t a t a l l i n ECM  appears  small but  t o be  of  a  material, sheath  used  f o r  treatment RNA  or  green-  membrane  immunilogically  before  substances and  RNA-protein f a i r l y to a  perinotochordal  cells  and  after  was  extensively lesser  sheath  acid  part  o f ECM  and/or  and  possibly The  i s much  embryo  and  degree  staining  i n mesoderm  to be  and  hyaluronic and  C-S-C  than  of  a the  ECM  cytoplasmic i n  cytoplasmic the  neural  ectoderm,  negligible.  perinotoare  part  reduced  the  i s greater  i n e p i t h e l i a l  appears  embryo,  demonstrable.  mm.  content  mm.  C-S-A  content  of hyaluronic  AMPS c o n t e n t  6-7  material  t o t h e 2-4  polysaccharide  endoderm  and basement  stains AMPS-like  consierable  polysaccharide  comparison  c e l l s .  methyl  extensive  pyroninophilia  i n the  not yet definitely  content  that  and  summary,  sheath  part  neutral  i s also  or  8). In  chordal  showed  RNA  I t was  that  pyroninophilia  and  (Figure  staining.  RNA  of this  showed  possible  laboratory  pyronin  i n cytoplasmic  on  substance  stain  where  check  i n ECM  and  i n ground  structures,  a  i n this  s t a i n i n g f o r DNA  pyroninophilia  acid  as  neutral ectoderm  notochord  and  - 32 -  F i g u r e 8. 6-7 mm. embryo showing ECM (E) s t a i n i n g w i t h p y r o n i n . S l i d e was incubated i n RNAase and s t a i n e d w i t h methyl-green p y r o n i n f o r n u c l e i c a c i d s , x 2 68.  -  17-18  In not  present  embryo.  the  diameter  for  a  small  mesoderm  but  ECM  of  sheath mm.  as  AMPS  such  alone  i s no  surrounding  and  e.g.  the  AMPS  are  6-7  mm.  same  2-3  times  present  except  epidermis  basement  present  basement  the  longer  definite are  i n the  although  mesoderm  content  i s a  shown  i s about  stage  structures,  there  extracellular  extent  However,  organ  addition,  Embryo  and  6-7  -  embryo  amount between  of  mesoderm  mm.  form  i n the  region.  structures  mm.  notochordal  as  mid-trunk  17-18  i n the  The  thickness  In  the  33  and  membrane  the  membrane  between  heart  i n  like  endoderm  gut  and  tube.  involving  developing  mesonephric  tubule. AMPS a r e cells and  which  are  trunk  are  packed  mesoderm The  layers pH and  of  4.0. the  labile  pH  other to  staining  indicated  i n close loosely  i n comparison  notochordal like  1.5  there  slightly  pH  1.5  sheath  are  to  the  2  layers,  extraction to  the  at  this  group  staining  pH  4  but  i n  the  2  or  3  thin  to  could  sulphated  layers  at  are  indicates  (C-S-A  present  carboxyl  packed  orthochromatic  Both  which  C-S-A/C  of  one  i s possibly  staining  close  sheath  i s metachromatic  Some h y a l u r o n i c a c i d intense  notochordal  consists  metachromatic.  i s due  the  mesenchyme  10).  material which  hyaluronidase at  i n quantity i n  proximity to  ( F i g u r e s 9,  matrix At  now  that  and/or account also  AMPS.  C-S-C). for  indicate The  - 34 -  F i g u r e 9. 17-18 mm. embryo showing H y a l u r o n i c a c i d i n basement membrane form around mesonephric t u b u l e s (MT) and i n f i b r o b l a s t s (F) p e r i p h e r a l t o notochord (N). ABPAS, x 240.  F i g u r e 10. H y a l u r o n i c a c i d i n basement membrane form around gut tube rudiment (G). ABPAS, x 220. 17-18 mm. embryo.  -  35  -  acetylation-saponification  treatments  any  neutral  participation  and  the methylation-saponification  taken  as  Results  polysaccharide  an  indication  are given The  a t pH  4,  and f a i l e d  that  are primarily  to  they  the acetylation  same  as  to stain  groups The  be  sheath  i n notochord  predominant  reaction,  the  PAS  saponification Azure  in  the sheath  pH  4,  and by  the absence digestion.  acetylation  methylation  that  weakly  this  Both r e a c t i v i t y The  Azure  A  trunk  to Azure  mesoderm  The  support  acidic  stains  that  staining. neutral by the  blocking  and  staining.  weakly  acidic  AMPS  the metachromacy a t pH  resistance loss  1.5  and,  after  of alcianophilia and  i s hyaluronic  indicate  acid.  and endoderm show  intensely  a t  of alcianophilia  the suggestion  and t o ABPAS  more  a n d PAS w h i c h  by  i s not the  i s inferred  o f PAS  material  mesoderm A  a n d some  of staining  and the p a r t i a l  following  f o r some  suggests  cells  response  indicate  the methylation  staining  were  indicates  The  sequences  cells  restoration  mesenchyme  hyaluronidase to  A  be  hyaluronidase  which  and only  of acidic  polysaccharides  1.5  to  acid.  responsible  presence  may  structures  a t pH  and methylation  may  staining  staining.  labile  hyaluronic  i n the notochord  carboxyl  group  like  completely  digestion  i n  suggest  IV.  b a s e m e n t membrane  metachromatic  to  treatment  o f sulphate  i n Table  f a i l  less  than  t h e mesenchyme.  than  endoderm  i s the reverse  with  o f what occurred  i n  TABLE I V 17-18  mm.  EMBRYO  (No.o f embryos  2)  TREATMENT  No t o c h .  Notoch. sheath  Sheath mesenchyme  Trunk mesoderm  Trunk endoderm  Endomesod, base. membr,  Azure A p H 4.0  0+  M++  0+++-M++  0+++-M+  0+  M+++  Azure A p H 1.5  M+-0+  0+  Hyalase A.A p H 4.0 Acetylate A.A p H 4.0 Acetylate Saponify A.A p H 4.0 ABPAS Acetylate ABPAS  M++  0+ PAS+++  PAS+++  0++-M+  M+  0++  0++  0++  0++  M++  ABP++++  AB++++  PAS++  PAS+  AB++++  ABP++++  AB++++  PAS+  PAS+  AB++++  Acetylate Saponify ABPAS  PAS+++++  PAS++  AB++++  ABP++  ABP+  AB++++  Methylate ABPAS  PAS+  AB++  AB++  PAS+  PAS+  AB+++  Methylate Saponify ABPAS  PAS++++  PAS++  PAS+  PAS+  PAS+  PAS+++  -  the  6-7 mm.  trunk  embryos.  mesoderm  37  This  -  suggests  associated with  some AMPS  content i n  the derivation  membrane  structures.  I t i s notable  mesoderm  and endoderm  the acetylation-saponification  sequence  induced  alcianophilia  that  o f basement  which  i n both  trunk  was n o t p r e s e n t i n  control  sections.  This  i s i n agreement w i t h  Yamada*s  (1963b)  suggestion  that  alcian  may  groups  other  than  carboxyl  4  In organ duct  i swell  i s present  with  collecting  tubules.  pancreatic  rudiment  closed is  completely  v i s i b l e  this  (fibroblasts),  content. definite 12, most  12a)  i n i t i a l  large  lobes  are present.  and trunk  This  anterior  Wolffian  tubules  joining and a i s  o f the spiral  stage,  s t i l l  valve  AMPS w e r e  restricted  the associated sheath  mesenchyme  trunk  No b a s e m e n t  mesoderm  however  i s complex;  i s not present  o f the notochord.  membrane  displayed  demonstrated w i t h i n i t .  complexity 2 mm.  definite  o f l i v e r  The s t r u c t u r e o f t h e s h e a t h l a y e r s may b e  development o f  The oesophagus  mesoderm.  associated with  units.  (Figure 11).  developmental sheath,  A  mesonephric  but the beginning  the notochordal  structures  30-40  involve  Embryo  established.  i n the intestine At  to  cm.  Two  staining  and sulphate  t h e 4 cm. e m b r y o ,  systems  blue  AMPS  three (Figures i nt h e  The a n t e r i o r  end o f  - 38 -  F i g u r e 11. General morphology o f 4 cm. embryo. S e c t i o n through p e c t o r a l f i n s . ABPAS, x 170.  -  the  notochord  same  level  diameter 1 . 8 mm. of  posteriad  begins  its to  from  extension  dorsad  (Table  V ) .  greatest extent the floor  hyaluronic This  acid  suggests  porated  into  0.45  mm.  inner  three  and  following  A  Neutral  staining  A  o f this  o f AMPS  (Table V a ) .  area  s l i g h t l y  I t reaches i s adjacent  13-16a).  The  and A c r i d i n e orange  elements  o f the  structure  i s present  layer  epithelium.  This  digestion.  are indicated although  incor-  chondrocranium.  i s the outer  and reduces  as indicated  AMPS  staining.  are t o b e  end o f the notochord.  hyaluronidase markedly  i s minimal.  and  a t pH 4 a n d s l i g h t l y  polysaccharides  constituent amount  Azure  A  end  t o b e C-S-A/C a n d  layered sheath  a n d i s a squamous  with  (anterior  materials which  o f the sheath  stained  Azure  Azure  the anterior  surface  1.5  i s shown  the parachordal  from  notochord  matrix  lateral  the notochord (Figures  area  o f AMPS  a maximum  i s laterad  after  precartilage  The  a matrix  o f the hindbrain.  only  maximum  approximately  a s 0 ) a n d b y 4 mm.  matrix  by both  u n t i l  a t 2 . 2 mm.  of the hindbrain  i n this  not reach  i s a distinctive  with  reaches  i s taken  the floor  There  cells  the notochord  of this  toward  material  the hindbrain a t the  of structure  a t 0 . 4 5 mm.,  the notochord  The  under  as t h e e p i p h y s i s and i t does  and complexity  extension  -  forward  p e r i p h e r a l mesenchyme  which  of  i swell  39  The  o f the i s densely  s t a i n e d a t pH Diastase  PAS s t a i n i n g  decreases  significantly.  t o be t h e major  there  may b e a  by A c r i d i n e orange  4  slight  results  TABLE MEASUREMENTS OF  Distance from Ant. End  Maximum Width  ANTERIOR  PORTIONS  Maximum Height  V OF  NOTOCHORD OF  Maximum e x t e n t of peripheral Mesenchyme AMPS (lateral)  4  cm.  EMBRYO  Minimum Verticle d i s t . from Hindbrain Floor  0  44  44  —  2 75  150  55  77  -  175  250  66  83  350  100  122  -  450  150  110  28  39  650  165  150  38  11  1030  192  165  55  22  1200  220  148  110  38  1500  220  190  120  11  1800  275  202  165  0  2000  330  250  192  0  2200  300  275  250  0  2700  300  230  220  0  4000  330  100  25  0  NOTE:  110  a l lmeasurements are i n microns, obtained by o p t i c a l m e t e r m e a s u r e m e n t s . A c c u r a c y i s +_ 6 m i c r o n s .  55  micro-  - 41  -  F i g u r e 12. S t r u c t u r e o f notochord sheath o f 4 cm. embryo. Inner l a y e r ( I ) , Middle Layer (M), Outer l a y e r o f matrix and mesenchyme ( 0 ) . ABPAS, x 465.  F i g u r e 13. S t r u c t u r e o f notochord o f 4 embryo 150 microns from a n t e r i o r end o f notochord. Azure A pH 4.0, x 690.  cm.  - 43 -  F i g u r e 14. S t r u c t u r e o f notochord sheath 350 microns from a n t e r i o r end of notochord. Azure A pH 4.0, x 580.  F i g u r e 15. S t r u c t u r e o f notochord sheath 1500 microns from a n t e r i o r end o f notochord. Azure A pH 4.0, x 450. Note p r o x i m i t y to f l o o r o f h i n d b r a i n (HB) .  - 44 -  F i g u r e 16. S t r u c t u r e o f notochord sheath 2200 microns from a n t e r i o r end o f notochord. P e r i p h e r a l mesenchyme (M) a t maximum e x t e n t . Notochord adjacent to f l o o r o f h i n d b r a i n (HB). Azure A pH 4.0, x 314.  F i g u r e 16a. Notochord a t 2200 microns from a n t e r i o r end showing metachromacy o f p e r i p h e r a l mesenchyme (M) and extreme orthochromacy o f middle l a y e r o f sheath ( 0 ) . Azure A pH 4.0, x 155.  TABLE V a 4 c m . EMBRYO C O M P O S I T E R E S U L T S OF H E A D , TRUNK, A N D T A I L (No. o f e m b r y o s 3)  TREATMENT  Sheath inner surface  Sheath middle layer  Sheath matrix  Sheath mesench.  Epith. ectoderm  Trunk mesoderm, heart mesoderm  Azure A p H 4.0  0+++  0++++++  M+++  0+-M+++  0+++  0+ M+  Azure A p H 1.5  0+  M++  M+++  - M++  Hyalase A . A p H 4.0  0+  0++-M+  0+  0+-M+  0+  Hyalase A.A p H 1.5  0+ 0+  Methylate A.A p H 4.0 Methylate Saponify A . A p H 4.0 Diastase A . A p H 4.0  0+-0+ 0+  M++  M+-M+  0+-M+  0+  0+ 0+  PAS+++  ABP++++  AB+++  AB+++  ABP++++  AB++ PAS++++  PAS+  AB+++  AB+  AB+-++  AB++  0.6 M N a C l A . A p H 1.5 ABPAS  Diastase ABPAS Saunders -ir Saunders 2 Saunders 3  -++++-  +  +++  +  -++++-  ++  -H+  AB++  ++  NP NP  -  Adjacent about pH  5  4  microns  digestion. moderate  results and at  pH  4  no  orange  present  heparin  was  3).  i s  with  sulphate 1963).  of  azurophilia  pH  1.5,  azurophilia at  pH  hyaluronic very  NaCl  abolished but  the  suggested possible  Acridine  group  to  by  slight  These C-S-A/C,  azurophilia  groups.  a l l staining of  indicating  a  slight  orange  staining  over-sulphated  orange  i s known  sequence  sulphate  presence  as  to  C-S-C  occur  to  1.5.  acid,  Acridine  that  slight  hyaluronidase  that  only  and  at  C-S-C  containing  i n  shark  cartilage  The  presence  of  considerably  hyaluronic  chondroitin  sulphates  i s  supported  Acridine  extreme  layer with that  the  orthochromacy  yolk  around  Azure  appears.  A  at  pH  stage  third  by  the  and  embryos  sheath  of  and  r e a c t i o n of  reflects  has  (Table the  an  may  be  so  and  induced  occurred  i n  by sulphation  I ) .  sheath  area  this  dye-binding  i s overcome  effect  19 61)  layer  4  shift  This  (Pearse,  i n disc The  orthochromatic  metachromatic  oversulphation  cells  to  Jayer  results.  middle  of  at  extreme  s t a i n i n g i s due  was  I t  extreme  uniform  methylation-saponification  heparin  The  dense  similar  restoration of  binding  and  the  presence  most  i s a  following  reduces  M  (Dorfman,  shows  1.5  0.6  extra  acid  and  with  (Saunders  an  The  surface  metachromasia  metachromacy  that  of  be  4  f o l l o w i n g the  Extraction  amount  pH  i n d i c a t e the  indicates  that  at  -  inner  thick which  Diastase  C-S-B.  may  the  (orthochromatic),  metachromasia  a  to  46  of  consists  of  mesenchyme  AMPS a p p e a r i n g  as  a  -  matrix  associated with  surface with there  was  Results  only  neurtral  layer  matrix  anterior  to Azure  which with  present  matrix. of  l e s s e r component.  No  and i tappears form  mesenchyme  that  of the of the  of any  and heart  acid  by  orange  AMPS. contain  a n d C-S-A/C b u t  does a  have  a  l a b i l i t y  B a s e m e n t membrane  and gut areas  polysaccharides  Acridine  m e s o d e r m may  mesoderm  diastase.  acidic  results.  i s demonstrated  dense  i n parts  branchial  than  concentration  of theepithelial  filaments.  to short  and 18).  other  surfaces  b u t they  results  quantity to  of  short  structures do  not  show  characteristics.  l a b i l i t y 17  a  contain  a n d ABPAS  Heart  i n heart  A  of  A  may  quantities of hyaluronic  incubation  noted  cells  trunk  not definite.  AMPS  and v e n t r a l  concentrated  t o i n d i c a t e the presence Both  are  layers  notochord.  according  glycogen  i n 2-3  lateral  i s composed mostly  i n the lateral  Epidermal  are  as  the  and l i t t l e  are present  i s a more  AMPS  dorsal  the matrix acid  On  was  o f mesenchyme  hyaluronic  pre-cartilage  minor  b u t on  polysaccharides  sheath  f a i l s  t h e mesenchyme  matrix  indicate that  C-S-A/C w i t h  the  1  -  t h e mesenchyme.  o f the sheath  considerable  47  This  This  (25 min.) was  t h e 4 cm.  positive material  ectoderm  proved  diastase  not noted stage.  o f PAS  and i n the  t o be  glycogen  digestion  was  lumina by  a  (Figures  i n any developmental  stages  -  48  -  F i g u r e 18. B r a n c h i a l f i l a m e n t s o f 4 cm. e m b r y o f o l l o w i n g s h o r t (25 m i n u t e s ) d i a s t a s e digestion ABPAS, x 160.  -  49  7 cm.  In expanded in  is  t o 6-8  matrix.  are  t h e 7 cm.  I n trunk  p a r t i a l l y  Results in  the notochord  in  skeletal  o f mesenchyme w i t h  and t a i l ,  i n the  areas.  fine  fibrous  in  neural  part  b u t results A n y AMPS  occurs  o f the mantle  particularly Results  neurons  (area  o f bovine  A VII)  cartilage  and  acid i s  some  heart  strongly  and mesonephric  i n mesodermal  structures  membrane,  b u t as  c e l l s . o f some A M P S - l i k e  tube),  material  i n the ventro  surrounding  have  brain  lateral  t h e ependymal  neurons  layer)  (Figure 19).  acid  a n d l e s s e r amounts o f  been  reported  and spinal  cord  present  i n  b y Szabo and  (1962).  comparison shows  matrix  present  resembling  o f which  Roboz-Einstein  (Table  (neural  i n cells  both  tissue  indicate primarily hyaluronic  between  indicate hyaluronic  C-iS-A/C,  i n gut,  i s indication  ectoderm  hyaluronic  n o t as a basement  networks  There  muscle  i n t h e sheath  I n contrast,  mesoderm  stage  arches  t a i l .  L i t t l e  tubule  this  and haemal  matrix  AMPS may b e p r e s e n t  in  increase  C-S-A/C  cartilages.  VI).  like  has  o f C-S-A/C a n d C-S-B  sheath,  i n these  (Table  neural  a  sheath  indicate the presence  acidic  acid  the notochordal  established and considerable  differentiating  indicated  Embryo  embryo  layers  -  o f results  differences between  from  head  i n composition  t h e two r e g i o n s .  and t a i l o f sheath  regions and  C-S-B i s f o u n d  TABLE.VI 7 c m . EMBRYO  AZURE A p H AND ENZYME TREATEMTNS TRUNK (No. o f embryos 5)  REGION  Sheath matrix  Cart. matrix  Trunk* mesoderm struct.  Trunk mesench.  M++++  M++++  M++  M+  0+++-M+  Azure A p H 1.5  M++  M++  0+  -  0+  Hyalase A.A p H 4.0  M+  -**  -**  -**  -**  Hyalase A . A p H 1.5  0+  ~  Methylate A.A p H 4.0  -  Methylate Saponify A.A p H 4.0  -  TREATMENT Azure pH  *  **  Neural ectod.  A  4.0  O  Mesoderm o f g u t tube, (intestine), heart, mesonephric tube a n d t u b u l e s . Cytoplasm i s unstained u n i f o r m 0++.  i nthese  regions  epimyocardium,  and  but a l lnuclei a r e  - 51  -  Ficrure 19. Portion of neural tube of 7 embryo, showing H y a l u r o n i c a c i d (HA) i n layer. A z u r e A pH 4.0, x 128.  cm. mantle  TABLE. V I I 7 c m . EMBRYO  COMPARISON OF HEAD AND T A I L (No. o f embryos 3)  CARTILAGE  REGIONS  HEAD  TAIL TREATMENT  Inner sheath  Sheath matrix  Cart. matrix  Inner sheath  Sheath matrix  Cart. matrix  Azure A pH 4.0  M++++  M++++  M+++  M+  M++++++  M++  Azure A p H 1.5  M++  M++  M++  M+  M+-++  M+-++  Hyalase Azure A pH 4.0  M+  M+  M++  -  M+  Hyalase Azure A pH 1.5  M+  M+  M++  -  M+  Diastase Azure A pH 4.0  M+  M+++  M++++  M++  Methylate Azure A pH 4.0 Methylate Saponify Azure A pH 4.0  M+++  -  in  the  inner  sheath  larger  proportion  matrix  i n  the  cartilage than  i n  the  of  head  matrix  i n head  cartilage  i n  the  arches  which  sheath  but  the  girdle  notochord.  are The  they  indicate  of  i n  the  indicated  i n  the  i n  the  but  i s  head,  not  i n  of  i n  the  the  with  t a i l i n  the  associated  differences  i n  this Skeletal and  notochordal include and  pectoral  with  the  composition  i n  antero-posterior  region  neural  chondrocranium  necessarily  A  sheath  t a i l .  s k e l e t a l cartilages  arches,  head.  Skeletal  developing  structural differences an  t a i l .  indicated  associated  the  may  cartilages  gradient  i n  or,  complexity  c a r t i l a g e .  14  notochord included notochord  cm.  The  area  and  surrounding  epithelial and  the  ectoderm  underlying to  the  posterior  region  were  free  trunk of  i n  the  14  the  cm.  stage  elements:.'  (epidermis), intestinal  bottom  of  (Figures  specimens  swimming.  the  vertebral  c l o s e l y applied  Two  Embryo  examined  was  and  not  C-S-B  indicated  some  but  intensely  consists  are  not  indicate may  more  some  branchial  which  than  regions  haemal  of  i s  region  t a i l  -  t a i l  C-S-B  and  cartilage  elements  the  stains  head  i n  i n  53  examined  However  these  neural  tube,  notochord 21,  22).  had  just  could  the  Sections  epithelium  the 20,  was  which i n  hatched  s t i l l  be  -  54  -  Figure 2 0 . P o r t i o n o f v e r t e b r a l c o l u m n o f 14 cm. e m b r y o , s h o w i n g s k e l e t a l e l e m e n t s , (epidermis ep, i n t e r d o r s a l i , b a s i d o r s a l b, r i b r , b a s i v e n t r a l v, s p i n a l c o r d s) ABPAS, x 34.  - 55  -  Figure 21. Longitudonal s e c t i o n through epidermis and u n d e r l y i n g v e r t e b r a l column o f 14 cm. embryo, ( u n i c e l l u l a r mucous gland u, pigment c e l l s p, muscle and connective t i s s u e m, c a r t i l a g e matrix C) Azure A pH 4.0, x 235.  F i g u r e 22. Cross s e c t i o n through i n t e s t i n a l e p i t h e l i u m and b a s i v e n t r a l c a r t i l a g e o f 14 cm. embryo, (blood v e s s e l b) Azure A pH 4.0, x 235.  considered the  embryos  -  56  because  of  the  skeletal  interdorsal,  basidorsal,  elements  present.  are  surrounded  by  complete  as  yolk  i n  the  neural  notochord  and  centra  of  the  the  notochord,  b a s i v e n t r a l and  i s  completely  are  developing  v e r t e b r a l column  tube is s t i l l  r i b  twice  the  i s  not  diameter  of  centra.  sulphates  the  A/C  cartilage  are  indicated i n high  matrix  intestinal  results  possibly i n very  of  sulphates  (Saunders  sulphates  i n a l l these  following  extraction with  chondroitin  structures  (Saunders  3)  and  with  amounts  heparin, mucins The  diastase  0.6  M  and  at  the  reduced  pH  1.5  M  Azure NaCl  Both  staining at  former latter  i n  the  of  matrix  i n d i c a t e the  chondroitin  4  the  orange  4  epidermal staining  following  presence  cartilage  Azure  pH  presence  and  A  of  matrix  i n cartilage  almost  VIII).  chondroitin  supports  pH  cartilage  s t a i n i n g at  Acridine  staining with  and  A  i n  (Table  presence  i n cartilage  NaCl  the  incubation  azurophilia  A  but  0.3  only.  Azure  extraction  epidermal  areas  glandular  amounts  s t a i n i n g for the  C-S-B  epidermal  structures  supports  sulphates  glandular  of  2)  orange  i n  chondroitin  staining.  slight  glandular  Acridine  that  f o r most  concentration  and  and  i t i s clear  responsible  structures  of  of  around  interventral,  Development  In  The  presence  cartilage  The  cartilage  segmentally.  is  the  intestine. In  the  -  and  matrix at  complete  following hyaluronidase  pH  loss  variable  4  only. following  of  incubation  -  57 -  TABLE 14  c m . EMBRYO  VIII  SKELETAL CARTILAGE AND (No. o f embryos 3)  EPITHELIA  TREATMENT  Cart. matrix  Epid. mucins (glands)  Intest. mucous glands  Azure A pH 4.0  M++++++  M++++  M++++  Azure A p H 1.5  M++  M++++  M++  Hyalase A.A p H 4.0  -  M+  0++++++  0++++  Hyalase A . A 1.5  - 0+  0+-++++  - 0+  Diastase A.A p H 4.0  M++++  M++++  0++++++  ABPAS  AB+-++++  AB++++*  AB++++*  Diastase ABPAS  AB++++**  AB++++**  AB++++**  Methylate A.A p H 4.0 Methylate Saponify A.A p H 4.0  Saunders  1  ++  +++  +  Saunders  2  ++  +++  +  Saunders  3  +-+  +  +-+  0.3M NaCl A.A p H 4.0  0++-M++  0.6M NaCl A.A p H 4.0  *  **  - 0++  M+  S t a i n i n g i suneven, extremely variable. Staining i s uniform.  -  do  not support  structures. than  AMPS  the presence  These  results  hyaluronidase  that  components  present  variable blue and  Azure  C-S-B  o f shark  Staining  is  infer  A  was  of cartilage  i n epidermal  o f cartilage  matrix.  matrix  indicated  that  in  Table  may  be  by  some  decrease areas.  due  C-S-A/C a r e t h e  glands  and i n  became  Alcian uniform  diastase digestion  polysaccharide  materials  the a v a i l a b i l i t y  The  t o such  G-S-B  indicated  o f  presence  an i n t e r f e r e n c e i n  mechanism. Figures  results  which  i n different  i n matrix  Meyer  sulphated  Variable  which  23, 24)  sites  which  Seno and  and, t h a t  mucous  (Figures  i n matrix  that  matrix  preceded  staining  other  following  the major  i f i t was  C-S-B  material  structures  C-S-B.  indicate  amounts i n c a r t i l a g e  present  1.5  be  intense  of  gland  skin.  results  primarily  staining  binding  some  gland  a t pH  more  are  i n intestinal  that  digestion can only  reported  polysaccharide  major  o f C-S-B  material i n epidermal  intensely with  (1963)  -  i s present. The  stains  58  25  o f cartilage VIII.  t o 30 matrix  are included staining  t o show  which  are  some  of the  presented  - 59 -  F i g u r e 24. C a r t i l a g e m a t r i x from 14 cm. embryo, l o n g (2 h o u r s ) d i a s t a s e d i g e s t i o n ,  ABPAS, x  450.  - 60 -  F i g u r e 2 6. C a r t i l a g e o f 14 cm. embryo, w i t h 0.3 M NaCl, Azure A pH 4.0, x 450.  Extracted  F i g u r e 28. Section of epidermis o f 14 cm. embryo, h y a l u r o n i d a s e A z u r e A pH 4.0, x 130.  and cartilage digestion.  F i g u r e 29. S e c t i o n o f epidermis and u n d e r l y i n g t i s s u e s o f 14 cm. embryo, ( u n i c e l l u l a r mucous gland U, pigment c e l l s P, c a r t i l a g e matrix M), Azure A pH 4.0, x 238.  F i g u r e 30. Epidermis and u n d e r l y i n g t i s s u e s o f 14 cm. embryo, e x t r a c t e d w i t h 0.6 M NaCl, Azure A pH 4.0, x 130.  -  63  -  DISCUSSION  Specifity  Specificity pertinent three  component  and  embryos  PAS p o s i t i v e  material  orange  with  developmental  stages  interpreting  in  the f i r s t  both and  sulphate forming  removes only.  Azure  was  by  on  the methyl  esters  and restores  hydroxyl groups  cleavage Material stains  by Acridine and three  d i f f i c u l t y  groups  there  AMPS,  appeared,  some  variation  i s expected  to  This  A l c i a n blue  the  former  staining  staining  alcianophilia  procedure  block  Demethylation  carboxyl  and then  i n  treatments.  but  entirely.  and deacetylation i s designed  and amino only.  no  the latter.  abolished  i n  desulphating  esters  demethylation  acid  stage  Methylation  d i d not block  blue  Saunders'  stages.  the f i r s t  of sulphated  groups  and  the other  of the staining  and carboxyl  the A l c i a n  positive  i n t h e 1 7 - 1 8 mm.  Acetylation  hydroxyl  there  instance  achieved mm.  methyl  subsequent  block  a n d 6-7  Through  studied  problem  Satisfactory  s t a i n s was  A.  responses.  Methylation  A,  as hyaluronic  indication  A l c i a n blue  AMPS;  method.  mm.  i s a  In this  Azure  A l c i a n blue  the results  However,  study.  d i d not react with  was  metachromatic  with  2-4  demonstrated  flourescence  techniques  procedure,  a l l three  through  Methods  t o demonstrate  A c r i d i n e orange  agreement between  was  used  o f t h e ABPAS  flourescent  that  of staining  t o any histochemical  s t a i n s were  stage  o f Staining  to  to restore  i s applicable i n  -  i d e n t i f i c a t i o n However,  i t was  abolished or  found  A while  l i t t l e  in  o f acidic  place  o r Azure  treatment.  new  A when  sulphated  s t a i n i n g method  with  A l c i a n  and thus  observed  (1964)  i t was  found  sequence was u s e f u l  A was  treatment.  ABPAS  the stain results  and neutral  A  are substituted with  are subjected  t o  results  developed  by  has  A l c i a n this  using  staining.  specific  used were  A  Scott,  overcome  s t a i n i n g  the  methylation-  i n assessing  not consistent.  only  when  t h e degree  groups r e s p e c t i v e l y ,  i n conjunction  used  with  treatment.  was  this  Acetylation  to locate  s t a i n i n g with  s t a i n i n g however,  by the deacetylation b y Yamada  that  polysaccharide  A l c i a n blue  suggested  treatment  not stain  and sulphate  d e a c e t y l a t i o n was u s e f u l  affected  groups  apparently  more  blue  staining.  similar  f o rA l c i a n blue  s t a i n i n g due t o c a r b o x y l  staining.  AMPS  treatment  A l c i a n  o f the  and A l c i a n blue-PAS  and allows  general  demethylation  glycogen  AMPS w i t h  w i l l  AMPS  o f this  blue.  In  when A z u r e  part  hydroxyl  sulphated  and Dellovo  d i f f i c u l t i e s  are  the latter  i s that  (1964)  AMPS  such  and  polysaccharides.  on sulphated  groups  Yamada  Quintarelli  of  that  o r no a f f e c t  explanation  known  and acid  the acetylation part  possible  blue  -  a l l s t a i n i n g o f sulphated  Azure  had  o f neutral  64  ABPAS  frequently  I t may b e , a s  (19 64)  that  strongly acidic  groups  affected by the a l k a l i  used  i n the deacetylation  treatment. In  t h e 1 4 cm. e m b r y o ,  there  was c o n f l i c t  between  -  Acridine heparin  orange  and Azure  i n epidermal  65  A  -  r e s u l t s .  unicellular  The former  mucous  glands,  matrix  and i n intestinal  epithelial  method  indicated heparin  i n cartilage matrix  only the  basic Azure  orange  difference A  solution  with  acetic  between  acid.  difference  i s probably  hyaluronic  a c i d were  would  suggest  interfered ference  that  with  that  and uniform  as  (1961) b u l l  stated  testis  specific studies with  nasal  was  o f  sections  diastase  interference  inter-  and  treatment.  with  Walker  Azure  A  acid,  that  o f  t h e enzyme  satisfactory.  and  o f those  1962; Mancini,  o f a p a r t i c u l a r AMPS was b a s e d  several 'Hyalase',  materials  Vilar,  the specifity  However,  Curran  was  preparations,  i n the extraction  study  and  commercial  C-S-A a n d C-S-C,  commercial  In this  c r i t i c i z e d  (1961)  o f the action showed  used  has been  196 3 ) .  1963; Zugibe,  1960).  type  septum c a r t i l a g e .  analyses  success  and S i r l i n ,  hyaluronidase  following  (Szirmai,  o f AMPS h a v e  This  staining o f cartilage  use o f hyaluronidase  that  and/or  material  This  i n control  protein  f o rh y a l u r o n i c  F i o r i n i ,  zation  uneven  hyaluronidase  apparent  (Kato and  noted  non-specific  sulphates  other  dense  The  f o rthe s t a i n i n g  o r some  became  o f horse  i s that  protein  i nAlcian blue  The  HCl and Acridine  chondroitin  extraction.  the latter  only.  and staining.  was v e r y  staining  while  present  which  (1963)  i n cartilage  s t i l l  matrix  Szirmai  with  The e x p l a n a t i o n  the NaCl  was noted  glands  t h e two methods  i s acidified  indicated  Stein  o f  the characteri-  on several  c r i t e r i a  -  i n a d d i t i o n to h y a l u r o n i d a s e  66 -  lability.  Relative acidity,  r e s i s t a n c e t o s a l t e x t r a c t i o n a t d i f f e r e n t m o l a r i t i e s and b l o c k i n g and u n b l o c k i n g  o f s p e c i f i c a c i d groups were  n e c e s s a r y f a c t o r s i n the i d e n t i f i c a t i o n o f a s p e c i f i c AMPS. I t became apparent t h a t there a r e l i m i t s to the degree o f i d e n t i f i c a t i o n p o s s i b l e .  With the techniques  employed i d e n t i f i c a t i o n o f glycogen, n e u t r a l  polysaccharides  (probably o l i g o s a c c h a r i d e s ) , h y a l u r o n i c a c i d , c h o n d r o i t i n sulphate A/C and c h o n d r o i t i n sulphate  B was a c h i e v e d .  I t must be noted t h a t such i d e n t i f i c a t i o n s were the l e a s t difficult  i n the youngest developmental s t a g e s .  As  development proceeds, AMPS c o n t a i n i n g areas become more complex w i t h  the a d d i t i o n o f other m a t e r i a l s and p o s i t i v e  i d e n t i f i c a t i o n o f AMPS becomes  difficult.  Embryonic Development o f AMPS  An  examination o f the r e s u l t s i n the d i f f e r e n t  embryonic stages  c l e a r l y shows a p r o g r e s s i o n  appearance o f AMPS (Table  IX).  Neutral polysaccharides polysaccharides  i n the  and p o s s i b l y some a c i d  were the main c o n s t i t u e n t o f c l e a v i n g  cells.  Some h y a l u r o n i c a c i d was suggested b u t there was n o t s u f f i c i e n t q u a n t i t y t o permit p o s i t i v e i d e n t i f i c a t i o n . The  d e f i n i t e l o c a l i z a t i o n o f the p o l y s a c c h a r i d e s  can be  TABLE GENERAL  SUMMARY  OF  RESULTS  Neutral Polysaccharides  STAGE Cleavage  cleavage furrows ,general cytoplasm.  2-4  ectoderm notochord endoderm mesoderm  mm.  6 - 7 mm.  17-18  4  cm.  7  cm.  14  mm.  cm.  ce c ce ce  l e l l  ls, lls, ls, ls.  FROM  TABLES  Hyaluronic  IX  I - V I I I LOCATION  acid  OF  I D E N T I F I A B L E AMPS  C-S-A/C-S-C  C-S-B  ECM, perinotoch. sheath.  endoderm c e l l s , mesoderm cells, neural ecto.cells,  ECM, mesoderm cells, neural ecto.cells,  perinotoch. sheath.  notoch.cells, notoch. sheath.  notoch. sheath, mesod. basement membranes, mesenchyme, t r u n k mesod.  notoch.sheath.  inner sheath, h e a r t mesod., branchial f i l aments , epidermis.  p r e c a r t . o f parachordals , peripheral shth. mesenchyme, t r u n k mesod., h e a r t mesod., mesoneph.tube mesoderm.  precart. o f parachordals, middle sheath, periph.sheath mesenchyme.  middle sheath.  gut,heart mesod., mesoneph.tube  sheath,mid., periph.matrix,  sheath mid.  mesod, neural  skeletal cart., neural tube.  tube.  cart.matrix.  cart.matrix,  epidermis  Heparin  - 68 -  attributed glycogen are of  to a  fixation  sometimes  large  seen  ( 0 . 3 1 mm.  X  polysaccharides  precipitation  thin  with  neural layer  identified were  ECM.  The  area  with  layers.  material  structure  of migrating  mesencyme  Gustafson  and Wblpert  Steinberg  proposed  that  of ruptured  by  t h e a c t i o n o f enzymes  be  true  are  i n systems  several  proposal whole  reasons  with  skate  ECM  respect  embryo.  cells  ECM  'pseudopod' by  development. produced  from  i n dissociated tissues  against  t o the appearance First,  like  tissue  described  f o r dissociation.  argue  labelled  to the  i s artificially  poly-  described  membrane  to adjacent  of dissociated c e l l s . which  positively  of the  i t was  i n sea urchin  used  very  thin  Material'  similar  cells  mm.  polysaccharides  some  (1963),  extensions  (1963)  with  extracellular  i s a basement  are very  chromosomes  this  possessed  and Steinberg  extracellular  i n t h e 2-4  a c i d was  but neutral  Because  s t r u c t u r e o f ECM  f i r s t  and as a uniform,  The e x t e n s i o n s  the  during  to the overlying  of the 'Extracellular  (1960)  cells  occurs  associated  Hyaluronic  a n d AMPS m a t e r i a l  Moscona  identified  cells  ectoderm  component.  characteristics by  o f mesoderm  i n this  saccharide  of  and the p o s i t i o n  the displacment  extracellularly  the notochord.  the main  Cleaving  and yolky  that  a c i d was  and epidermal around  0 . 1 3 mm.)  c e l l s .  to that  C-P-B-formalin.  I t appeared  extensions  i n l i v e r  infers  Hyaluronic embryo.  a r t e f a c t comparable  appears  This  However,  may  there  Steinberg;' s o f ECM only  i n the  i n specific  -  developmental embryonic  locations  artefact. mesoderm does  stages.  and cannot mm.  and endoderm,  between  f o r example  The appearance  and  mm.  a  stage  developmental  form  o f ECM  suggest may of  be  t h e ECM  with  c e l l  i n t h e 2-4  around  the notochord.  have  form  adhesion sense. ECM  would  be  form  or to keep The h i g h l y  tissues  mm.  times  form  a c t as a  may  but  which  layer  layer  ECM  where  procedures the  form.  i n a  tissue  form  most  that  to function  the  acutal  Possible  i n tissue chemical  structure  diffusion  Most  between  areas  suggests  chemical  i n the  i n association  embryo  separated  hydrated  easily  be  suggest  procedures.  appears  t h e smooth could  embryo  stages which  b u t i n some  This  mm.  are differences  I n t h e 6-7  appears.  and splanchnic  location  as t h e smooth  form  fixation  between  i n t h e 2-4  separated i n histological  of this  could  appear  specific  and epidermis but  a t different  stage  to a  somatic  developmental  mm.  layer  been  appearance  functions  in  does  i n t h e smooth  'pseudopod' ECM  mesoderm  There  i n  appears  e x t e n s i o n s o r i n 'pseudopod'  b u t some  tissues  ECM  to histochemical  layers  is  ascribed  between  functions due  appears  and i t s specific  a t different  artefacts  be  o f ECM  function.  different  ECM  embryo  mesoderms. t h e 6-7  -  Secondly,  I n t h e 6-7  n o t appear  69  barrier  o f AMPS to  large  molecules.  in gut  Hyaluronic  acid  i s found  b a s e m e n t membrane  like  structures  tube  and mesonephros.  T h i s was  i n t h e 1 7 - 1 8 mm. around  embryo  the developing  not included  as  ECM  -  because  i t appeared  specific manner  organ  stages.  not  with  i n the  finely  persists  Such  a  the  f i r s t  C-S-A/C w e r e of  of  u n t i l  the  of  4  i n the  layer  of  the  In  the  4  in  the  middle  layer  14  cm.  embryo  i n unicellular  preceded  the  notochord  (fibroblast) is  to  packed  be  of  cm.  the  by  appear  6-7  matric  a  mm.  mucous  In of  or  i n a  formed.  AMPS  fibroblast  cells  as  a  i n a  uniform  C-S-A/C  glands  stage,  precartilage  elements,  i n  sheath,  and  peripheral  was  sheath  of  of  skeletal  the  to  identified  and  i n the  condensation  appeared  acid  other  that  mesenchyme  place where then  i n  the  C-S-B  cartilage of  or  embryo.  fibroblasts  embryo,  condensation  sheath, cells  or  appears  were  notochord  notochord  production of  i t  mesonephric  matrix.  stage.  the  and  hyaluronic  parachordal chondrocranium  i n mesenchyme  was  but  demonstrated  cm.  like  mesenchyme  to  the  not  extracellularly  The  between  a  development,  duct  development,  sheath  cartilaginous  notochord.  stages  s u l p h a t e d AMPS  identified the  throughout  cartilage  sulphates were  areas  cm.  later  i n  developmental  Wolffian  processes In  function  later  i s found  7  with  basement membrane  i n any  and  notochord  Chondroitin  material  cm.,  component  The  embryonic  4  concerned  i t may  epimyocardium,  c e l l s .  as  directly  found  branching  fibroblast  middle  ECM.  Hyaluronic acid  tubules  around  to  were  associated  as  be  -  development but  similar  structures  to  70  i n  the 4  the epidermis.  cm.  embryo  cells  around  mesenchyme cartilage  between  the  closely  matrix.  No  neutral  -  polysaccharides these  cells  AMPS w e r e  were  either  not identified  the i n t e r s t i t i a l  in  the matrix  fibroblasts  AMPS m a t r i x the  study  showed  only  both  i n intercellular  are  present  outside  weight  physiological C-S-A/C  embryo of  not.  were  spinal  more  advanced  identified and were  Szabo  and Roboz-Einstein acid  may  which  was  positive  indicate  and are  that  precursors  polymerized  i n locations suggestive function.  i n one area n o t found  Hyaluronic  i nboveine  i n nerve  (1961)  intercellular  c o n t a i n i n g AMPS cells  with  AMPS.  appeared  identified  agreed  sulphate  and  This  sulphate  stages.  surrounding  f o rAMPS w e r e  matrix.  c a l l y  hyaluronic  tests  o r biochemical  cord,  This  that  fibroblasts  t o form  Some A M P S  staining  demonstrable  Stein and F i o r i n i  w i t h i n fibroblast  the c e l l  was no  fibroblasts  were  Vilar,  b u t histochemical  molecular  and  which  o f Mancini,  low  a  There  i n cultures o f fibroblasts  matrix  b u t only  The d e n s i t y o f AMPS  d i f f e r e n c e between  into  o f AMPS.  t o depend on t h e p r o x i m i t y o f  and those  incorporated  the formation  w i t h i n the. f i b r o b l a s t s  matrix.  seemed  i n association with  o r after  one t o another.  morphological  -  demonstrated  before  in  71  white  (196 2 ) . tissue  acid  o f t h e 7 cm.  acid  matter  might  Hyaluronic  i n spinal  They  o f  cord  has been  biochemi-  (spinal  cord)  suggested  that  function i n myelin  cons t r u e t i o n . The portion  morphological  o f the notochord  by  structure o f the anterior  and associated  parachordal  -  precursors adjacent inverse  i s of  tissue  and  the  of  floor  notochord appears  exists  the the  of  distance between  and  to  This  idea  Madden (St.  maximum  spinal  chondrogenesis contain  these The  regions in  of  showed  somites  7  cm.  than  the  caudal  the  14  results  can  size of  the  study  of  the  extent of also  of  chick  presence  be  enzymes  hind  on  one  on  to  hand  the  other.  and  somites  notochord  necessary  alone  brain  precarti-  Lash  embryo  to  related  Glick,  of  the  the  the  notochord  or  f o r  would  such  an  of  not  not  be  by  C-S-B,  lack  However,  of  epidermal Azure  stained  and  t a i l  with  characteristics  neutral polysaccharides,  gradient of  The  other  noted.  glands  Acridine  considered positive.  no  heparin  unicellular A  and  differences  there were  identification  both  head  advanced  antero-posterior  and  from  revealed slight  showed more  proposed  embryo  cartilage  region cartilage  cartilage.  matrix  cm.  embryo  and  greater content  cartilage  of  an  notochord  hind brain  cultured  of  Cranial  e.g.  The  and  that  i n the  comparison  the  of  The  that  extent of  the  floor  of  enzymes.  intensity  examples  the  contain the  but  composition.  greater  by  shows  maximum  the  notochord  cultured  cord would  The  view  maximum  p a r a c h o r d a l s may  i s supported  16-17)  Va  distance from  dimensions  (1964) w h i c h  the  notochord.  the  point of  Table  o n l y when  the  the  the  the  between  hind brain.  touch  materials  from  v e r t i c a l  i s reached  to  -  interactions.  relation  notochord  lage  interest  72  i n i n  orange extractive  -  techniques more  used  l i k e l y  i n this  that  matrix  heparin.  Mathews  cartilages  were  not extracted  (1962)  and bovine  cartilages  suspect  the indicated presence identified In  Raja in  cleavage  in  and f i n a l l y  more  advanced  content  o f these  6-7 mm.  stages  relates  t o morphogenetic  the  f i r s t  production  ECM,  may  tissue  polysaccharides  i n neural  sulphated  tube  AMPS  development.  are intracellular  the 4  cm.  c e l l s .  a c t i v i t i e s  stage.  to the high  and  Their yolk  I n t h e 2 - 4 mm.  o f neutral  rates  i t has  and  polysaccharides  o f different  and might  tissues  possibly indicate f o r AMPS  (Figure 30).  neural  extension  AMPS  o f oligosaccharide precursors  Hyaluronic with  acidic  formed  t o  i s that  neutral  relates  the gradient  a t different  presence  from  u n t i l  reason  i n the development o f  o f cartilage  areas  shark  date.  to strongly acidic  i n cleaving cells  occurring  t o  o f  12% protein  o f heparin  appears  to weakly  a  stained as  study  Another  polysaccharides  i n certain  presence  there  stages  Neutral persist  cartilage.  a progression  stages  and thus  noted  i n cartilage  review,  binoculata  stages  shark  and i t i s  by protein i n  i n a biochemical  i n adult  been  are suspect  AMPS b o u n d  content  not  -  procedure  sulphated  cartilage  73  acid,  polysaccharides  o f major  organ  the weakly i n stages  forming  f u n c t i o n as an agent  movement,  acidic  occurring  o f neurulation and  areas  o f tissue  o r as a medium  AMPS  i n the form adhesion,  governing  some  o f o f  chemical  -  74  aspect,  o f the extracellular  system  development hyaluronic  associated with to  specific  ECM b u t w i t h o u t  Hyaluronic stages  acid  Their in  function  some  sulphate and  matrix  embryo.  differences  glands also  advanced  and  adult  i n adult  o f cartilage may  participate  development. layer  matrix.  Chondroitin  o f the  notochord  o f the epidermis  be a component  o f  i nt h e cartilage  embryo.  i s no o b v i o u s  i n function  component.  participate  and persist  as a s t r u c t u r a l  I t may  i n the very  and C  and they  tissue  similar  matrix.  component  o f neural  B appears  There  AMPS  formation  AMPS  i n a form  development  A  o f organ  t o be  polysaccharide  sulphates  i n u n i c e l l u l a r mucous  advanced  systems  through  i s structural  aspects  appears  o f cartilage  i n cartilage  as t h e major  I n stages  acid  organ  persists  Chondroitin  stages  space.  the neutral  as a component  primarily  -  explanation  f o r the  and l o c a l i z a t i o n o f the various  discussed. The  previous  results  biochemical  cartilages  cited  obtained analyses  elsewhere.  i n this  study  agree  o f shark  skins  and  with  TABLE METABOLIC  STEPS  IN  THE  X  BIOSYNTHESIS  OF  CHONDROITIN  SULPHATES  Glucose  r  G l u c o s e - l - P -*  I-  glucose-6P  ->-  N-acetylglucosamine  DPN  t  ATP  UDP-Glucuronic  acid  -**Hyaluronic  acid  N - a c ee tt y l g l u c o s a m i n e - 6 - P  1-«  UTP  N - a c ee tt y l g l u c o s a m i n e - l - P  I  I  UDP-N-acetylgalactosamine  Oligosaccharide PAPS :(3 p h o s p h o a d e n o s i n e phosphosulphate) 1  Chondroitin  V  Glucosamine-6-P •Acetyl-CoA  - UTP  UDP-Glucose  j-<  Glutamine  • ATP  sulphate  -« 5  UTP  1  N-acetylgalactosamine-l-P *  ATP  N-acetylgalactosamine  from  Kent,  P. W.  Some B i o c h e m i c a l a s p e c t s o f S u l p h a t e d M u c o p o l y s a c c h a r i d e s , B i o c h e m i c a l S o c i e t y Symp. No. 20, Cambr. U n i v . P r e s s , 1 9 6 1 .  -  76  -  SUMMARY  Embryonic  stages o f the elasmobranch,  r a n g i n g from cleavage to immediate  Raja b i o n c u l a t a ,  p r e h a t c h i n g were  examined by h i s t o c h e m i c a l techniques to f o l l o w a c i d mucopolysaccharide A p r o g r e s s i o n was  (AMPS)  development.  observed from  (1)  intracellular  n e u t r a l p o l y s a c c h a r i d e s i n c l e a v i n g stage t o ,  (2)  a combination o f e x t r a c e l l u l a r n e u t r a l p o l y s a c c h a r i d e s and weakly a c i d i c a c i d mucopolysaccharides  (hyaluronic  a c i d ) a s s o c i a t e d w i t h c e l l processes i n n e u r u l a t i n g embryos to,. (3)  extracellular strongly acidic  s u l p h a t e d a c i d mucopolysaccharides  (chondroitin  sulphates) i n l a t e r stages, p a r t i c u l a r l y i n areas of  cartilage  development.  H y a l u r o n i c a c i d f i r s t appeared i n the e a r l y n e u r u l a i n an e x t r a c e l l u l a r form a s s o c i a t e d w i t h c e l l processes and, as a smooth l a y e r around the notochord and on the v e n t r a l s u r f a c e o f n e u r a l and epidermal  ectoderm.  N e u t r a l p o l y s a c c h a r i d e s were a s s o c i a t e d w i t h t h i s extracellular material  (ECM) which o c c u r r e d between  some a d j a c e n t t i s s u e l a y e r s b u t not between somatic and a p l a n c h n i c mesoderm.  In post neurulae, h y a l u r o n i c  a c i d appeared i n a basement membrane l i k e a s s o c i a t e d w i t h the development  form  o f mesonephric  and  gut tubes and as a t h i n l a y e r around the notochord. In  older, embryos h y a l u r o n i c a c i d appeared i n  mesenchyme  associated with  development, matrix  to  i n fibroblast  and i n cartilage  Sulphated  AMPS  immediate  appeared  development.  notochord  sheath,  (fibroblasts)  of  the notochord  epidermal Both  tube  like  embryonic becomes  The  adult  area  i d e n t i f i e d  i nt h e  mesenchyme and i n skeletal layer  i d e n t i f i e d i n o f the neural  embryo.  In later  obtained  the histochemical  i s least  d i f f i c u l t  and  stages, i t  AMPS s p e c i f i c a l l y ,  matrix.  previously reported skins  i n early  developmental  i n immediate  prehatching  biochemical  cartilages.  mm.  o f  i n the middle  a n d C-S-A/C w e r e  i n cartilage  shark  i n areas  sheath matrix  (17-18  and i n the u n i c e l l u l a r  d i f f i c u l t to i d e n t i f y  agreed with of  C-S-A/C w e r e  o f AMPS u s i n g  stages.  results  stages  p a r t i c u l a r l y  i n the mantle  described  particularly  precartilage  i n later  C-S-B a p p e a r e d  acid  c e l l s  Identification  tubule  glands.  o f t h e 7 cm.  techniques  only  i n associated  sheath  mucous  hyaluronic  neuron  produced  and precartilage  matrix.  and mesonephric  matrix.  prehatching)  cartilage  cartilage  heart  stages analyses  - 78 -  LITERATURE CITED  Anno, K., Seno, N., Kawaguchi, M. 19 62. A comparison o f Glucosamine and Galactosamine content o f c a r t i l a g e from v a r i o u s s o u r c e s . Biochim. Biophys. A c t a . , 58: 87. Bloom, W. and Fawcett, D. W. 1962. A Text Book o f H i s t o l o g y . Saunders Co., London. Chen, C. M. C. and Chang, H. L. 1963. Methyl-green Pyronin as a s t a i n f o r Mast c e l l s i n p a r a f f i n sections. S t a i n Tech., 3_8: 133. C l a r k , R. S. 1919-1922. Rays and Skates No. 1. Egg Capsules and Young. J . Mar. B i o l . A s s o c . U.K.N.S., 12.: 577. Clemens and Wilbee. 1946. F i s h e s o f the P a c i f i c c o a s t o f Canada. F i s h e r i e s Res. Board o f Can., B u l l . 68.. C u l l i n g , C. F. A. 1963. Handbook o f H i s t o p a t h o l o g i c a l Techniques. Butterworths, London. Curran, R. C. 1961. The h i s t o l o g i c a l demonstration o f Connective t i s s u e Mucopolysaccharides. Biochem. Soc. Symp. No. 20., Cambridge. Davidson, E . and Small, W. 1963. Metabolism i n Vivo o f c o n n e c t i v e t i s s u e P o l y s a c c h a r i d e s . Biochim. Biophys. A c t a . 69.: 445. Dorfman, A. 19 63. Polysaccharides of connective t i s s u e . J . Histochem. Cytochem., 1.1: 2. G l i c k , M. C., Lash, J . W. and Madden, J..W. 1964. Enzymic a c t i v i t i e s a s s o c i a t e d w i t h the i n d u c t i o n o f chondrogenesis i n v i t r o . Biochim. Biophys. A c t a . , 83: 84. Gustafson, T. 1963. C e l l u l a r mechanisms i n the morphogenesis o f the sea u r c h i n embryo. E x p t l . C e l l . Res., 32: 570. Gustafson, T. and Wolpert L. 1963. S t u d i e s on the c e l l u l a r b a s i s o f morphogenesis i n the sea u r c h i n embryo. E x p t l . C e l l Res., 29.: 561.  -  Kato,  K.  79  -  I . and S i r l i n , J . L. 1963. Aspects o f mucopolysaccharide production i n larval insect salivary c e l l s . J . Histochem. Cytochem., 11: 163.  Kelly,  J . W., Bloom, Quaternary histochemis Cytochem.,  Libby,  E . L . a n d G i l b e r t , P . W. 1960. Reproduction i n the clear-nosed skate, Raja ecrlanteria., Anat. R e c , 138: 3 6 5 .  Lhotka,  G. D. a n d S c o t t , J . E . 1963. Ammonium compounds i n c o n n e c t i v e tissue try. Selective unblocking. J . Histochem. ,11: 7 9 9 .  J . F. 1964. Histochemical l o c a l i z a t i o n s p o l y s a c c h a r i d e s i n t h e d e v e l o p i n g human Nature, 202: 1124.  M c C o n n a c h i e , P . R. a n d F o r d , P . the elasmobranch yolk 43: 128. Mancini,  Muir,  Histochemistry of Can. J . o f Biochem.,  R. E . , V i l a r , 0 . , S t e i n , E . a n d F i o r i n i , E . 1961. A histochemical aid radioautographic study o f the participation of fibroblasts i n the production of mucopolysaccharides i n connective tissue. J . H i s t o c h e m . C y t o c h e m . , 9_: 2 7 8 .  Materazzi,  Mathews,  1964. stalk.  of aorta.  G. 1963. A c e t y l a t i o n blockade o f the chromotropy and b a s o p h i l i a o f v a r i o u s h i s t o l o g i c s u b s t r a t e s . J. Histochem. Cytochem., JL1: 59.  M. B . 1962. Sodium c h o n d r o i t i n s u l p h a t e - p r o t e i n complexes o f c a r t i l a g e . Preparation from shark. B i o c h i m . B i o p h y s . A c t a . , 58,: 9 2 .  H.  1961. Chondroitin sulphates and mucopolysaccharides of connective S o c . Symp. No. 2 0 , C a m b r i d g e .  sulphated tissue. Biochem.  Ozello,  L . a n d B e m b r y , J . Y. 1964. E f f e c t s o f 17B o e s t r a d i o l on t h e p r o d u c t i o n o f a c i d m u c o p o l y s a c c h a r i d e s by c e l l c u l t u r e s o f human f i b r o b l a s t s . Nature, 203: 80.  Pearse,  A.  Prockop,  G. E . 1961. Histochemistry Theoretical and Applied. J . A. C h u r c h i l l L t d . , London.  D. J . , P e t t e n g i l l , 0 . a n d H o l t z e r , H . 1964. Incorporation of sulfate and the synthesis of collagen by cultures o f embryonic chondrocytes. B i o c h i m . B i o p h y s . A c t a . , 83_: 1 8 9 .  -  Rogers,  H.  80  -  J. 1961. The s t r u c t u r e and f u n c t i o n of hyaluronate. Biochem. Soc. Symp. No. 20, Cambridge.  Saunders,  A . M. 1964. Histochemical identification mucopolysaccharides with Acridine orange. H i s t o c h e m . C y t o c h e m . , 12.: 164.  Schiller,  S. 19 6 3 . Annals of  Scott,  E., Q u i n t a r e l l i , G. a n d D e l l o v a , M. C. 1964. The c h e m i c a l and h i s t o c h e m i c a l p r o p e r t i e s of Alcian blue. H i s t o c h e m i e , 4.: 73.  Seno,  J.  N.  Mucopolysaccharides t h e New York Academy  of of  of J.  acid  normal mast c e l l s . Science, 103: 199.  a n d M e y e r , K. 1963. Comparative biochemistry skin. The m u c o p o l y s a c c h a r i d e s o f s h a r k s k i n . Biochim. Biophys. Acta., 78.: 258.  of  Shackleford, J . M. a n d B e n t l e y , H . P. 1964. Carbohydrate h i s t o c h e m i s t r y o f the s a l i v a r y g l a n d s and pancreas in cystic fibrosis. J . Histochem. Cytochem., 12: 512. Spicer,  S.  S. 1960. Histochemistry of rodent mucopolysaccharides. J . H i s t o c h e m . C y t o c h e m . , 8_: 18.  Spicer,  S.  S. 1963. Histochemical properties of m u c o p o l y s a c c h a r i d e s and b a s i c p r o t e i n s i n c e l l s . Ann. N.Y. A c a d . S c i . , 103: 322.  S.  Spicer,  mast  S. a n d L i l l i e , R. D. 1959. S a p o n i f i c a t i o n as a means o f s e l e c t i v e l y r e v e r s i n g t h e methylation blockade of tissue basophilia. J. Histochem. C y t o c h e m . , 7_: 123.  Steinberg,  M. S. 1963. "ECM": Its nature, origin function i n c e l l aggregation. Exptl. Cell 30: 257.  Szabo,  M. and R o b o z - E i n s t e i n , E. 1962. Acidic polysaccharides i n the c e n t r a l nervous system. Arch. B i o c h e m . B i o p h y s . , 98.: 406.  M.  Szirmai,  TeWinkel,  J . A. 1963. Quantitative approaches i n histochemistry of mucopolysaccharides. Cytochem., L l : 24.  and Res.,  the J. Histochem.  L. E. 1950. N o t e s on o v u l a t i o n , ova and early d e v e l o p m e n t i n the Smooth D o g f i s h , M u s t e l i s c a n i s , d u r i n g the f i r s t three months of gestation. B i o l . B u l l . , 99.: 474.  -  Walker,  P.  81  -  G. 1 9 6 1 . The e n z y m a t i c d e g r a d a t i o n o f mucopolysaccharides. B i o c h e m . S o c . Symp. 20, Cambridge.  No.  Williams,  G. a n d J a c k s o n , D. S . 1956. for acid mucopolysaccharides. 189.  Yamada,  K.  1963a. A contribution t o the histochemistryof acid mucin i n the g a l l bladder epithelium o f the of the toad, Bufo f u l g a r i s japonicus. Acta. Histochem., 15: 50.  Yamada,  K. by  1963b. means  Staining of Alcian  Two o r g a n i c f i x a t i v e s S t a i n Tech., 31:  of sulphated polysaccharides blue. Nature, 198; 799.  Yamada,  K.  1964. The r e a c t i o n s o f s u l p h a t e d p o l y s a c c h a r i d e s to several h i s t o c h e m i c a l tests. J . Histochem. C y t o c h e m . , 12.: 3 2 7 .  Zugibe,  F.  T. 1 9 6 2 . The d e m o n s t r a t i o n s o f t h e i n d i v i d u a l a c i d m u c o p o l y s a c c h a r i d e s i n human a o r t a s , coronary arteries and cerebral a r t e r i e s . Methods. I d e n t i f i c a t i o n and significance with aging. J . H i s t o c h e m . C y t o c h e m . , TO: 4 4 1 .  Zugibe,  F.  T. 1963. Mucopolysaccharides wall. J . Histochem. Cytochem.,  of the a r t e r i a l 1_1: 3 5 .  -  ADDITIONAL  Bennet,  H.  82  -  REFERENCES  S. 1 9 6 3 . Morphological aspects o f e x t r a c e l l u l a r polysaccharides. J . Histochem. Cytochem., 1 1 : 1 4 .  G. S . a n d D a l f e r e s , E . R. 1 9 6 3 . Identification of a c i d mucopolysaccharides by glass paper chromatography. Biochim. Biophys. Acta., 5 8 :  Berenson,  3 4 .  Bollet,  A.  J . 1 9 6 3 . The p r e s e n c e o f h y a l u r o n i d a s e i n v a r i o u s mammalian t i s s u e s . J . Biol. Chem., 2 3 8 :  B. 1 9 6 3 . sulphates and s k i n .  Clausen,  Fisher,  Kao  I n f l u e n c e o f age on c h o n d r o i t i n a n d c o l l a g e n o f human a o r t a , myocardium Lab. Invest., 12.: 5 3 8 .  E . R. a n d L i l l i e , R. D. lation on basophilia. 2.:  Gore,  3 5 2 2 .  1 9 5 4 . The e f f e c t o f methyJ . Histochem. Cytochem.,  8 1 .  I . , T a n a k a , Y. a n d W h i t e , H. A . 1 9 6 4 . a c i d mucopolysaccharide changes by Arch, o f Path., 78.: 1 8 1 .  A r t e r i a l protamine.  Tang,  K . Y . , H i t t , W. E . , D a w s o n , R. L . a n d M c G a v a c k , T . 1962. Connective tissue VII. Changes i n p r o t e i n and hexosamine c o n t e n t o f bone a n d c a r t i l a g e o f rats a t d i f f e r e n t ages. Proc. Soc. Exptl. B i o l , and Med., 1 1 0 : 5 3 8 . H . a n d W i n d r u m , G. M . 1 9 5 4 . Sulphation techniques in histochemistry with special reference to metachromasia. J . H i s t o c h e m . C y t o c h e m . , 2_: 1 9 6 .  Kramer,  Lison,  Love,  L.  R.  Mathews,  1 9 5 4 . A l c i a n blue 8 G with Chlorantine Fast Red 5 B . A t e c h n i q u e f o rselective s t a i n i n g o f mucopolysaccharides. S t a i n Tech., 29.: 1 3 1 . a n d W a l s h , R. J . 1 9 6 3 . Studies o f the cytochemistry o f nucleoprotein. Improved s t a i n i n g m e t h o d s w i t h T o l u i d i n e b l u e a n d Ammonium Molybdate. J . H i s t o c h e m . C y t o c h e m . , 1_1: 1 8 8 . M. B . a n d H i n d s , L. 1 9 6 2 . Acid mucopolysaccharides and f r o g metamorphosis. Fed. Proc., 2jL. 1 6 7 . :  Nanto,  V.  1 9 6 3 . On t h e e l e c t r o p h o r e t i c s e p a r a t i o n o f a c i d mucopolysaccharides on c e l l u l o s e acetate sheets. A c t a . Chem. S c a n c . , 1 7 . : 8 5 7 .  H.  -  83  -  Revetto,  C. 1964. A l c i a n blue, A l c i a n yellow. A new method f o r the i d e n t i f i c a t i o n o f d i f f e r e n t acid groups. J . H i s t o c h e m . C y t o c h e m . , 12.: 4 4 .  Shaw,  E. a n d M a r t i n , B. F. 1962. H i s t o l o g i c a l and h i s t o c h e m i c a l s t u d i e s on mammalian knee joint tissues. J . A n a t . , 9j6: 3 5 9 .  N.  Stidworthy, G. H . , E l l i s , P . a n d G o t t s c h a l k , R. 1963. Separation o f sulphated u r i n a r y acid mucopolysaccharides u t i l i z i n g cetyltrimethylammoniumbromide and e t h y l a l c o h o l . Proc. Soc. Exp. B i o l , a n d Med., 1 1 2 : 8 6 1 . Takeuchi,  J . 1961. Staining of sulphated mucopolysaccharides on f i l t e r p a p e r b y means o f A c r i f l a v i n e . Stain T e c h . , 36.: 1 5 9 .  Terner,  J . Y. 1964. Histochemical alkylation: A methyl iodide and the e f f e c t on tissues. H i s t o c h e m . C y t o c h e m . , 12.: 5 0 4 .  study J .  of  Terner,  J . Y . a n d L e v . R. 1963. Lactone formation i n the histochemical evaluation of acid polysaccharides and mucin. J . H i s t o c h e m . C y t o c h e m . , JLL: 8 0 4 .  -  84  APPENDIX  Structure  Hyaluronic  1:3  sulphate  Polymer 1:3  position,  testicular  A  Polymer 1:3  hyaluronidase  CO.H  NHAc  B  labile.  and  N-acetylgalactosamine, group  a t C  4  labile.  HO.S-0  OH  CH.-OH  NHAc  (C-S-B)  o f iduronic  glycosidic  hyaluronidase  (C-S-A)  H0.S-0 CH.-OH  sulphate  N-acetylglucosamine,  l i n k a g e , one sulphate  OH  Chondroitin  and  linkage, testicular  o f glucuronic acid  glycosidic  CO,H  beta  Mucopolysaccharides  o f glucuronic acid  glycosidic  Chondroitin  beta  A  acid Polymer  beta  o f Acid  -  acid  and  N-acetylgalactosamine,  l i n k a g e , one sulphate  group  a t C  4  - 85 -  position,  not l a b i l e  Chondroitin  _H°»S-<>  CH..OH  sulphate  C  Polymer beta  1:3  hyaluronidase.  7 ' , H>  HO.S-0  0H  (C-S-C)  o f glucuronic  glycosidic  position,  t o t e s t i c u l a r  linkage,  acid  and  N-acetylgalactosamine,  one sulphate  t e s t i c u l a r hyaluronidase  group  a t C6  l a b i l e .  CO.H  NHAc  QH  NHAc  Heparin Polymer sulphate  groups  additional possibly  o f glucosamine  a t C 2 and C 4  sulphate,  alpha  glycosidic  CH.OH 1  H  H OH H >4_ o H  positions,  1:4, n o t l a b i l e  COOH 1  H  SO,H  Glucuronide  3 ^  alpha  H ./I—o. H > H y  1  H  NH  1  OH  o  SO,H  H  NH  SO,H SO,H ,  Glucosaminide  3  i  4  ^  Clucuronide  ^  an  hyaluronidase,  CH.OH  1  two  1:3 o r  a  \ O H H y ^ _-o — -.  o  1  COOH 1  a  H "°\H  1  linkage  acid,  may h a v e  t o t e s t i c u l a r  1  OH  and glucuronic  Glucosaminide  -  86  -  APPENDIX  Histochemical  A.  Fixation  (Williams  Fix chloride  small  (Bromide  Methods  and  at  water  dehydration.  tissue  temperature.  i n 0.5%  cetylpyridinium-  i n 4%  aqueous  Wash w e l l  (Alternate  hexadecyltrimethyl-ammonium  B.  1956)  satisfactory)  formaldehyde before  room  Employed  Jackson,  pieces of  proved  B  chloride  i n running  nomenclature or  bromide,  i s  CPC  or  CPB.)  Staining 1.  Azure  A  (Szirmai,  Stain (Fisher  to  d i s t i l l e d in  f o r 30  Scientific)  adjusted  4.0  or  water,  minutes  i n 0.2%  i n d i s t i l l e d 1.5  with  dehydrate  water  HCl.  Azure with  Rinse  A pH  i n  i n a i r , clear,  mount  permount.  2.  Alcian  blue  PAS  Stain blue  (G.  running acid  T.  Gurr)  water  Schiff  f o r 10  orange  Treat  3  treat  with  acid, follow  rinse,  Alcian  rinse with  i n Periodic  alcohol  permount.  (Saunders,  parallel  f o r 5 minutes,  minutes,  minutes,  i n  i n 1.0%  acetic  treatment, water  3.  '  1960)  minutes  i n 3%  mount  Acridine  (Spicer,  f o r 30  dehydration,  CPB  1963)  wash  1964)  slides  i n 1.0%  i n running water  ribonuclease  1 mg/ml  CPC f o r i n  or 10 glass  -  d i s t i l l e d  at  Treat  slide  wash in  orange  M  NaCl  differentiate acid  f o r 10  not  come  due  Enzyme  i n 0.01  to  again, stain  orange  (G.  wash  f o r T.  i n  3 Gurr)  f o r 5 minutes  i n  M  (pH  acetic  f o r 10  acetic  3  as  M  acid  slide  NaCl  acid  minutes,  M  slide  be  2,  i n 0.01  up  out  necessary to to  of  i n  0.1% 3.2),  differentiate  f o r 10  minutes.  but M  acetic  30  prolong  minutes,  the  u n t i l  sections.  i n running water, (E.  interpret,  to hyaluronic  chondroitin  C.  2  i n Flourmount To  3,  slide  may  washed w e l l  mounted  hours.  minutes.  differentiation  is  Acridine  i n 0.6  It  does  i n CPB  minutes,  i n 0.01  Treat  are  1  i n running water  0.3  f o r two  water. Stain  Acridine  45°C  f o r 10  i n 0.1%  d i s t i l l e d  -  water  running water minutes  87  sulphates  A l l slides a i r dried  and  Gurr).  i n slide  acid, and  stain  1  red  i n slide heparin  flourescence  2,  and  to i n  slide  heparin.  treatments 1.  Hyaluronidase Incubate  in  (Pearse.  1961;  sections  f o r 3 hours  1 mg/ml h y a l u r o n i d a s e i n 0.85%  well  i n d i s t i l l e d  water.  Culling,  Control  at  saline, sections  1963) 3 7°C wash are  -  incubated  -  i n 0.85% s a l i n e . AMPS  in  88  enzyme  staining i n control  treated  slides  C-S-A/C o r a m i x t u r e testicular 2.  at a l l is are  37°C  D.  Salt  t o remove  glycogen.  a  extractions  portion  (Kelly,  f o r 30  and  i f incubation Control  water  b u t this  o f the glycogen  Bloom  malt  minutes  Oligosaccharides  3 t o 24 h o u r s .  i n d i s t i l l e d  large  water  may b e r e m o v e d  from  (NBC  i n 0.1% commercial  d i s t i l l e d  incubated  compounds.  1963)  i n glass  prolonged  acid,  specified.)  sections  carbohydrates  remove  o f these  (Culling,  Digest diastase  i shyaluronic  hyaluronidase  Diastase  slides b u tnot  and Scott,  slides may  present.  19 6 3 ;  Saunders,  1964) 1.  Treat  necessary), at  room  stain of  sections  f o r 10 m i n u t e s  i n 0.3 M  temperature,  as required.  chondroitin  2.  Treat  in  0.01 M  NaCl  sulphates  sections acetic  i n 0.01 M  rinse AMPS  well  acetic  i n running  staining w i l l and/or  as before  acid.  (longer i f  AMPS  acid water,  consist  heparin.  b u t i n 0.6 M  NaCl  staining w i l l  be  heparin. For is  a required  this  treatment  fixative.  (1 a n d 2)  CPB/C-formalin  -  E.  Selective blocking 1.  89  and unblocking  treatment  Methylation-demethylation 1954;  Spicer, Treat  hours with  a t 60°C HCl.  I960:  w e l l  and  t r e a t i n 1 % KOH minutes,  forms  blocking removes groups  i n ethanol,  rinse w e l l  methyl a l l acid  thus  hydrolyzes  esters  from  0.1  N  and  stain.  celloidonize,  hydrate  sulphage  o n AMPS.  f o r 1-4  a t 25°C f o r  on carboxyl  groups  the methyl  sections,  i n ethanol,  esters  made  hydrate  i n 70% ethanol  Methylation and  sections  methanol  parallel  L i l l i e ,  1964)  dehydrated  i n preheated  Rinse  (Fisher and  Yamada,  ethanol  Methylate  30  -  and  stain.  groups  groups  thus  Demethylation  the  carboxyl  restoring s t a i n i n g to carboxyl  groups  only. 2.  A c e t y l a t i o n - d e a c e t y l a t i o n (Pearse, Treat  acetic hours  uncoated  anhydride a t 25°C  dehydrated  i n anhydrous  o r f o r one h a l f  19 61)  sections  pyridine  i n 40%  f o r 1-24  t o s i xh o u r s  a t  60°C. To acetylated and  saponify  sections  t r e a t i n 0.1  minutes  a t room  N  w e l l KOH  thus  esters  blocking  i n ethanol,  i n 70% ethanol  rinse celloidonize, f o r 45  temperature.  Acetylation acetyl  or deacetylate,  a n d may PAS  blocks  hydroxyl  hydrolyze  staining.  alpha  groups amino  Deacetylation  with groups  removes  -  the  esters  staining 3.  -  and restores  Sulphation  (Kramer  concentrated  sulphuric  Rinse  dehydrated  sections  acid  and  30  10  ml.  i n  of  ml. of  f o r 10-20 m i n u t e s  a t  w e l l  hydrate  i n ethanol,  room  stain. Sulphation  on  PAS  1954;  top) containing  acetic acid  temperature.  and Windrum,  uncoated  c o p l i n j a r (screw  and  group  1960)  Treat  glacial  hydroxyl  only.  Spicer,  a  90  hydroxyl  groups  induces  carried  on  sulphate  esters  polysaccharides.  

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