UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Morphology and histochemistry of the extracellular matrix of embryos following freeze substitution of… Cambell, Stephen Sean 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-UBC_1990_A6_7 C36.pdf [ 14.66MB ]
JSON: 831-1.0098110.json
JSON-LD: 831-1.0098110-ld.json
RDF/XML (Pretty): 831-1.0098110-rdf.xml
RDF/JSON: 831-1.0098110-rdf.json
Turtle: 831-1.0098110-turtle.txt
N-Triples: 831-1.0098110-rdf-ntriples.txt
Original Record: 831-1.0098110-source.json
Full Text

Full Text

MORPHOLOGY AND HISTOCHEMISTRY OF THE EXTRACELLULAR MATRIX OF EMBRYOS FOLLOWING FREEZE SUBSTITUTION OF THE STARFISH PISASTER OCHRACEUS By STEPHEN SEAN CAMPBELL B . S c , The U n i v e r s i t y of B r i t i s h Columbia,  1989  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES Department of Anatomy, U n i v e r s i t y of B r i t i s h Columbia  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September ©  1990  Stephen Sean Campbell,  1990  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  publication of  by  his  or  her  DE-6  (2/88)  an advanced  Library shall make  it  It  is  granted  by the  understood  that  head of copying  my or  this thesis for financial gain shall not be allowed without my written  ANATOMY  The University of British Columbia Vancouver, Canada  Date  representatives.  for  agree that permission for extensive  scholarly purposes may be  permission.  Department of  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  OCTOBER 3 , 1990  ii ABSTRACT All  developing  embryos  contain  an  consisting of proteins, glycoproteins, and are  important  for morphogenetic  differentiation  and  cell  processes  death.  The  extracellular  proteoglycans. such ECM  of  ochraceus. consists of three major components: the  external  surfaces  of  surface the  of the  epithelia;  embryo; a basal and  a  as  (ECM)  These components  cell  the  matrix  migration,  starfish,  cell  Pi saster  A hyaline layer which  coats  lamina which lines the basal  b l a s t o c o e l i c component  which  fills  the  revealed  the  embryonic cavity or blastocoel. Observations of chemically  fixed  asteroid embryos have  hyaline layer to contain f i v e sub-layers amorphous material. fluid-filled  of fibrous strands  encrusted  with  Strands of a s i m i l a r nature form a meshwork within  blastocoel.  Recent  studies  of  the  living  embryo,  the  however,  have suggested that the ECM within the blastocoel of echinoderms, including the  asteroid, i s a g e l - l i k e  fibres. are  substance and  not  a f l u i d with e x t r a c e l l u l a r  Since artefacts imposed by chemicals such as aldehydes and osmium  well  documented, a method of preservation, which does not  use of these chemicals,  may  resolve the apparent c o n f l i c t  involve the  over the  nature  of the ECM of the asteroid embryo. Freeze  substitution, an  expensive  cryofixation technique  which  has  proven successful in f i x i n g vertebrate t i s s u e , does not require the use of aldehydes and osmium. inexpensive, allow  good  embryonic  easily  The i n i t i a l employable  preservation starfish,  of  objective of this study was  freeze  cellular  Pi saster  substitution technique and  ochraceus.  cup  was  filled  with  cryogen and  of  the  A plunge freezing apparatus  was  with  liquid  inserted into the  motor which constantly s t i r r e d the cryogen.  an  which would  e x t r a c e l l u l a r elements  constructed which consisted of a Dewer flask f i l l e d small  to devise  nitrogen, a  nitrogen,  and  a  Embryos were isolated on copper  freeze-fracture grids and plunged into the cryogen. different  cryogens  asteroid  embryos with  propane was found freeze  and  four  cryoprotectants,  propylene glycol and plunging  to provide  substituted  separate  After considering four  in  optimal  anhydrous  them into  preservation.  ethanol  at  cryoprotecting  Frozen  -90  °C,  supercooled  embryos were  osmicated,  and  embedded f o r u l t r a s t r u c t u r a l and histochemical analysis. Following  freeze  substitution, the blastocoel  g e l - l i k e substance, rich i n sulfated GAG's, with not  a fluid  with  fibres.  In addition,  consist of at least six sub-layers chemically sulfated  fixed and  morphological  embryos.  unsulfated  GAG's  were  studies  differences among the sub-layers  layer  thickness  present  associated  Freeze bodies,  was  demonstrated  in  found to  than was seen in  these  that  layers.  both The  suggest that some sub-layers  may have unique functions while others may have functions sub-layers.  to contain a  e x t r a c e l l u l a r fibres and  the hyaline  of greater  Histochemical  appears  substitution also revealed  shared  by other  the presence of microvillus  structures which may represent  major attachment  points  of the hyaline layer to the epithelium. Although the f i x a t i o n of asteroid embryos by freeze substitution i s a lengthy  process,  particular!ly fixations.  four  of the ECM Material  histochemical necessary  taking  studies  for  components,  preserved and,  successful  immunocytochemical studies.  to f i v e  by  since  days,  the r e s u l t i n g preservation,  justifies  freeze  substitution  aldehydes  preservation,  i t s use over  and  may  heavy  also  chemical  can be  used for  metals  are not  prove  useful  for  iv  TABLE OF CONTENTS  PAGE Abstract  ii  L i s t of Tables  v  L i s t of Figures  vi  L i s t of Abbreviations  viii  Acknowledgements  xi  1.  INTRODUCTION  1  2.  MATERIALS AND METHODS  11  2.1  Rearing of Asteroid Embryos  11  2.2  C r y o f i x i n g of Embryos  13  2.3  Chemical F i x a t i o n and Embedding  18  2.4  Microscopy:  18  2.5  Histochemistry  3.  4.  Morphology  19  RESULTS  21  3.1  Cryogens  21  3.2  Cryoprotection  31  3.3  ECM of the Blastocoel and Basal Lamina  41  3.4  Hyaline Layer  42  3.5  Chemical F i x a t i o n  57  3.6  Histochemistry  62  DISCUSSION  68  4.1  Freeze S u b s t i t u t i o n of Asteroid Embryos  68  4.2  Morphology and Histochemistry  80  4.3  Summary  89  5.  REFERENCES  91  6.  APPENDIX  103  LIST OF TABLES TABLE I  The Results of Histochemical Staining of Cryofixed and Formalin Fixed Asteroid Embryos  II  The Thermodynamic Properties of the Cryogens Used  vi  LIST OF FIGURES FIGURE  PAGE  1  Photograph of the apparatus used to plunge freeze embryos  16  2  Photograph of the Thermos and glass v i a l s needed f o r freeze substitution  16  3  LM cross-section of an embryo frozen in Freon 12  24  4  LM cross-section of an embryo frozen i n LN2 slush  24  5  TEM of the ectoderm and ECM of an embryo frozen i n LN2 slush  26  6  TEM of the blastocoel of an embryo frozen i n LN2 slush  26  7  LM of an embryo frozen in ethane  28  8  LM cross-section of an embryo frozen in propane  28  9a  TEM of the ectoderm and HL of an embryo frozen in propane  30  b  TEM of the blastocoel and BL of an embryo frozen in propane  30  10a  TEM of the ectoderm and HL of a DMSO treated embryo frozen  b 11a  b 12 13a  b 14a  b 15  in propane  34  TEM of the blastocoel of the embryo in F i g . 10a  34  TEM of the ectoderm and HL of an embryo treated with 10% glycerol p r i o r to freezing i n propane  36  TEM of the blastocoel and BL of the embryo i n F i g . 11a  36  TEM of an embryo treated with 15% glycerol p r i o r to in propane TEM of the ectoderm and HL of an embryo treated 10% ethylene  36  glycol p r i o r to freezing in propane.  38  TEM of the blastocoel of the embryo in F i g . 13a  38  TEM of the HL of an embryo treated with 15% ethylene glycol p r i o r to freezing in propane  40  TEM of the ectoderm of the embryo in F i g . 14a  40  LM mid-sagittal section of an embryo treated with 15% propylene glycol prior to freezing in propane  44  vii  FIGURE 16 17 18 19 20 21 22 23a  PAGE  TEM of the BL and blastocoel of an embryo prepared l i k e that in Fig. 15  46  TEM of the esophageal fibres i n an embryo prepared l i k e that i n Fig. 15  48  TEM of the dorsal web of an embryo prepared l i k e that in that in Fig. 15  48  TEM of the HL of an embryo prepared l i k e that i n Fig. 15 and an inset LM of the HL a l i v i n g embryo  50  TEM of the six sub-layers l i k e that in Fig. 15  52  of the HL of an embryo prepared  Stereo pair of TEM's of the HL of an embryo prepared l i k e that in Fig. 15  54  TEM of the HL and the microvillus associated body of an embryo prepared l i k e that in Fig. 15  54  Higher magnification  TEM of the microvillus associated  body i n Fig. 22  56  b  TEM cross section of the microvillus associated  c  High magnification associated  d 24a b  body  TEM oblique section of the m i c r o v i l l u s  body  TEM sagittal  56  56  section of microvillus filaments  TEM of the HL of a chemically  56  fixed embryo  59  TEM of the BL of the blastocoel of a chemically  fixed embryo  fixed embryo  59  25  TEM of the esophageal f i b r e s of a chemically  26  TEM of the dorsal web of a chemically  27  LM of a cryofixed embryo stained with PAS  67  28  LM of a formalin fixed embryo stained with PAS  67  29  LM of a cryofixed embryo stained with alcian blue at pH 3.2  67  30  LM of a cryofixed embryo stained with alcian blue at pH 2.5  67  fixed embryo  61 61  viii  LIST OF ABBREVIATIONS AB  alcian blue  aq  aqueous  BL  basal  b  blastocoel  B  boundary sub-layer  BM  basement membrane  bpt  b o i l i n g point temperature  C  coelom  c  s p e c i f i c heat  CEC  Critical  cf.  compare  °C  degrees Centigrade  DMSO  Dimethylsulfoxide  dH 0  d i s t i l l e d water  Ec  ectoderm  ECM  e x t r a c e l l u l a r matrix  En  endoderm  Es  esophagus  F  Dewer Flask  g  gram(s)  xg  times the acceleration of gravity  G  cytoplasmic granule(s)  GAG  glycosaminoglycan  HCl  hydrochloric acid  HL  hyaline layer  2  lamina(e)  E l e c t r o l y t e Concentration  ix HI  hyaline 1  H2,  hyaline 2  H3  hyaline 3  In  India ink p a r t i c l e s  IV  intervillus  J  Joule(s)  k  thermal conductivity  K  Kelvin  kV  kilovolts  LD  lamina densa  LL  lamina lucida  LM  l i g h t microscope (microscopy)  LN2  liquid  m  metre(s)  M  mesenchyme  mg  mi 11igram(s)  mJ  mi 11iJoules  ml  millilitre(s)  mm  mi 11imetre(s)  jam  micrometre(s)  Mo  eddy current motor  mpt  melting point temperature  MVAB  microvillus associated body  N  nucleus  N  Normality  nm  nanometre(s)  sub-layer  nitrogen  X OM 0s0  coarse outer meshwork 4  osmium tetroxide  P  plasma membrane  PAS  periodic acid - S c h i f f reagent  s  second(s)  AT  difference between bpt and  TEM  transmission  Th  Thermos  V  glass v i a l  mpt  electron microscope  xi  ACKNOWLEDGEMENTS I would l i k e to express my sincere gratitude to Dr. Bruce Crawford who showed me what science i s a l l about and how to approach i t . He was very supportive of my goals i n l i f e and spent countless hours a s s i s t i n g me in achieving these goals. I am thankful to my wife, Jackie, who was very patient with me through my studies and was always there f o r me when times were d i f f i c u l t . I am also very grateful to Dr. Ravindra Shah who frequently took the time to discuss my research and writing. This work was supported by Dr. Crawford's operating grant from The Natural Science and Engineering Research Council of Canada. Summer scholarships were provided by The Medical Research Council of Canada and NSERC and a postgraduate scholarship was provided by NSERC.  - 1 -  1.  E x t r a c e l l u l a r matrix is secreted functions  by the c e l l s including  interaction, inducing ECM  serving  INTRODUCTION  (ECM) i s present  in a l l developing  embryos. It  into the e x t r a c e l l u l a r spaces and serves numerous  separating as a  populations  substrate  the d i f f e r e n t i a t i o n  of c e l l s  upon  which  of certain c e l l  cells  types  of vertebrate embryos includes glycoproteins  may  their  migrate and  (Gilbert,  1988).  The  such as collagen (von der  Mark,  1980; Wartiovarra  1984;  Schuger et a l . , 1990) and fibronectin (Newgreen  and Thiery, 1980;  Mayer et a l . , 1981; Hay, 1981, 1984) and proteoglycans  which are made up  largely of glycosaminoglycans (GAG's) such as hyaluronic  acid, chondroitin  sulfate 1975; and  and heparan  Solursh  et a l . , 1980; Hayashi,  to prevent  and Morriss, 1977; Weston et a l . , 1978; Toole,  Erickson, 1986).  1981; Tucker  Elements of the ECM appear to be essential mediators (Grobstein,  Bernfield et a l . , 1984).  the successful branching 1965;  (Hakamori,  sulfate (Kvist and Finnegan, 1970a,b; Pratt et a l . ,  of many developmental processes 1984;  1988), laminin  1967; Wessels, 1977; Hay, 1981,  Some examples of these processes  of the mouse salivary gland  include  (Grobstein and Cohen,  Wessels and Cohen, 1968), the induction of chick vertebral c a r t i l a g e  synthesis  by notochord  and neural  tube  (Hay, 1981),  the alignment of  feather germs (Stuart et a l . , 1972) and the induction of corneal  epithelium  (Toole, 1981). Invertebrate development.  embryos  also  depend  upon  ECM  f o r successful  For example, the sea urchin or echinoid ECM consists of three  major components: embryo; a basal  A hyaline  lamina that  layer that coats lines  the basal  the external  surfaces  endoderm; and a blastocoelic component that f i l l s blastocoel.  an  surface of the  of the ectoderm and  the embryonic cavity or  - 2 The blastocoel echinoid.  The  contains  fibrous  the largest  ECM  amount of ECM  components  found  in  in the developing  the  blastocoel  are  synthesized during gastrulation.  They form a network which i s confined by  the  of the embryonic  basal  1977;  laminae of  Katow  Santos,  and  the walls  Solursh,  1985).  I t i s thought  fibers (Pointer, 1978). indicated  the  including  GAG's  (Karp  (Oguri  Galileo that  a  and fluid  Morrill,  of  several  and  macromolecules  Solursh,  1974;  Studies  have  blastocoel Lane  and  et a l . ,  1982),  (Pucci-Minafra et a l . ,  1979; Wessel  and McClay,  et a l . ,  1980,  1984; McCarthy and Burger, 1987).  and the migration of mesenchyme c e l l s  1988).  1987)  ECM  suggested a correlation between the presence of GAG's in the  Solursh,  processes  Katow,  1990), fibronectin and laminin (Spiegel 1982; Wessel  and  studies have echinoid  and  1988).  In addition,  (Katow and Solursh,  fibronectin  has  been  important for mesenchymal c e l l migration i n v i t r o (Katow, Lane,  Morrill  in the  Solursh  1972; Golob et a l . , 1974; Crise-Benson and Benson,  1983; Katow et a l . ,  1985;  occupies the spaces between  and Yamagata, 1978), collagen  1987; Benson et a l . ,  (Endo and Noda,  As in vertebrate embryos, biochemical  presence  proteoglycans  1979;  cavity  Collagen  during  is  echinoid  also  necessary  gastrulation  such as archenteron formation  for  (Katow,  shown to be  1987; Solursh and  important  1986;  1981;  morphogenetic  Wessel  and gut d i f f e r e n t i a t i o n  and  McClay,  (Mizoguchi et  a l . , 1983; Mizoguchi and Yasumasu, 1983a,b; Mizoguchi et a l . , 1989). In  embryos  blastocoel origin  the  starfish  appears to be secreted  during  gastrulation methods  of  blastulation (Abed  suggested  and that  and  Pi saster by  fills  Crawford, starfish  cells  ochraceus. of  ectodermal  the  ECM  and  of  the  endodermal  the entire body cavity just prior to 1986b).  ECM,  like  Previous  chemical  that of the echinoid,  fixation i s also  fibrous in nature and surrounded by a f l u i d  (Crawford and Chia, 1981; Abed  and  Crawford  Crawford,  1986b;  Crawford,  1989).  (1990)  demonstrated  - 3 -  distinct aspect  e x t r a c e l l u l a r fibers in the regions of the esophagus and the inner  of  the  dorsal  ectoderm.  involved in the migration and  in maintaining  He  suggested  that  these  fibers  may  be  of the presumptive esophageal smooth muscle c e l l s  the c o n s t r i c t i o n at the middle of the  embryo.  Summers  et a l . (1987) have shown u l t r a s t r u c t u r a l l y that the fibers of the echinoid blastocoel  are much more anastomosing and  asteroid  (Crawford  1990).  Strathmann  embryonic fluid  and  method  (1989),  echinoderms  containing of  Chia,  1981;  Abed  however,  are  filled  is  needed  with  that  and  Crawford,  indicates a  a fibrous meshwork.  fixation  mesh-like than was  It  that  gelatinous  blastocoels  substance  preserve  the  1986b; Crawford,  the  i s apparent,  would  shown in  and  therefore,  the  ECM  of  not  a  that  a  within  the  blastocoel of echinoderms in a manner which is closer to that found i n vivo than  is  currently possible.  arrangement of the ECM  It may  then  be  possible  within the blastocoel and  the  to  determine  the  significance of  this  arrangement with respect to normal morphogenesis. The basal lamina (BL), the second ECM cells  from  the  connective  tissue  component, separates  (Leblond  gastrulating echinoid, the parenchyma includes and  the  basal  archenteron, Mercer, is  surfaces  and  1963;  a natural  the  coelomic  Okazaki  and  substrate  semi-permeable  of  the  pouches  Niijima, 1964;  (Endo  to  Inoue,  the  presumptive and  wall  1989).  of  macromolecules  alimentary  canal,  the  Uno,  Wolpert  and  1960;  grow, and  i t may  (Farquhar,  1981).  l e v e l , the basal lamina i s seen to consist of two  lamina  and  laminae.  a  Biochemical  lamina have revealed  lamina and  densa,  histochemical  collagen  heparan sulfate proteoglycan  similar to  types  studies  III and  (Davidson, 1974;  IV,  the  the blastocoel  ultrastructural lucida  In  Gibbons et a l . , 1969).  upon which most c e l l s  barrier  and  parenchymal  that of  of the  This act  as  At  a the  layers, a  vertebrate  basal  echinoid  basal  f i b r o n e c t i n , laminin  Spiegel  ECM  et a l . , 1980;  and  Wessel  - 4 et  a l . , 1984;  Wessel  embryo has been morphogenesis  1989).  McClay,  and  and  Abed,  cytochemical  suggestive  of  The  1983;  basal  studies  Abed  have  lamina  fixation  of  Crawford,  that  collagen  methods  of the BL (Crawford, 1989).  and  shown  proteoglycans and  However, d i f f e r e n t  morphology  1987).  asteroid  shown to play an i n t r i c a t e role in mouth formation during  (Crawford  Histochemical structures  and  result  this  1986a,b).  BL contains  fibrils  (Crawford,  in variations  It i s perhaps  possible  in the  that  the  true morphology of the asteroid BL i s yet to be determined. The outer forms  at  ECM  (Spiegel  fertilization  upon  plasmalemma of the egg and the  and Spiegel, fusion  1979), the  of  the  hyaline  cortical  layer  (HL),  granules with  the subsequent release of t h e i r  contents into  e x t r a c e l l u l a r space (Kane and Hersh, 1959; Endo, 1961; Runnstrom,  Anderson, 1968; Holland, 1979, 1980; Hylander and Summers, 1982). is  then firmly  attached  to the HL by m i c r o v i l l i  (Dan,  the  1960;  1966;  The egg  Wolpert and  Mercer, 1963; Burgess and Schroeder, 1977; Begg and Rebhun, 1978; Katow and Solursh,  1980).  Numerous  functions  including a substrate for c e l l s Vacquier and Mazia, 1968;  have  (Herbst,  Wolpert  al.,  1989),  lubrication bacterial composed  and a  Mercer,  filter  a  for  1967;  general  studies of several  Dan,  1960;  for  the  Chambers,  HL  1940;  (Dan, 1960; Gustafson and Wolpert,  Adelson and Humphreys, 1988;  organic  molecules  (Crawford and Abed, 1986) perturbation  1900;  suggested  Citkowitz, 1971, 1972; Kane, 1973), a source of  c e l l u l a r attachment during morphogenesis 1967;  been  (Lundgren,  and protection  1973).  schematic diagram  (Spiegel  Spiegel  et  et  a l . , 1989),  from mechanical and et  a l . (1989)  of the HL based on  marine invertebrate embryos.  Spiegel  ultrastructural  These authors divided  HL into two major zones - an inner zone and an outer zone.  have  the  The inner zone  consists of a dense meshwork of thin fibers while the outer zone i s made up of  more loosely  arranged f i b e r s .  A dense  border of compacted  fibers and  - 5 granules  separates  the  two  epithelium  via microvilli  associated  bodies  outer  zone.  HL  contains  after  have  proteins.  Hyalin,  Stephens,  1969;  a  from  Hylander  immunocytochemical  have suggested cell-HL  treatment  that  adhesion  (Adelson and  with  of  the  may  be  a  had  Veno  their  precise  1982;  McCarthy and Spiegel, 1983). The  HL of the  Other  have not  starfish,  been  after  described  chemical in detail  fixation  shown  to  antibody  boundary  layer  further subdivided studies  of  the  an  into two asteroid  for hyalin,  for  normal  this  morphogenesis  a putative substrate adhesion HL  (Alliegro  et a l . ,  large molecules have been isolated but yet  been determined  the  The  (Hall  not  and  Vacquier,  been studied  of  layer.  minor zones - HI have  revealed  as  ultrastructure of the asteroid  presence  intervillus  HL  reside  of the MVAB's and  anionic  by Crawford and Abed (1986).  and  and  Results  dyes,  has  The  and an  glycoconjugates throughout these zones (Reimer and  been  They showed that the  consists of four major zones - a coarse outer meshwork, a supporting a  weight  Kane  monoclonal  Pisaster ochraceus. has  in  1968;  Fink, 1982).  essential  extensively as that of the sea urchin. HL,  echinoid  (McClay and  Echinonectin,  1988;  functions  and  molecular  (Yazaki,  hyalin i s a major constituent  a l . , 1990).  Red  Evidence for this  high  molecule, i s another macromolecule isolated from the et  the  studies of the  HL  1982)  of the HL  Humphreys, 1988).  with  Ruthenium  thickness.  several  of  studies, using  molecule  continuous  dye  Biochemical  Summers,  regions  are  the  of varying  component  and  which  microvillus  et a l . (1987) suggested that the echinoid  presence  major  s p e c i a l i z a t i o n s or  tips  unpublished.  the  primarily in the outer  HL maintains i t s attachment to the ECM  their  four d i s t i n c t regions  demonstrated  The  have  analysis, M o r r i l l  model, however, i s s t i l l HL  which  (MVAB's) at  However,  ultrastructural  zones.  H2.  supporting Lectin  unequal  HL  layer,  layer  was  histochemical  d i s t r i b u t i o n of  Crawford, 1990).  It i s ,  - 6 -  therefore,  possible  that  i f the HL  has  several  functions,  then  each  sub-layer may have i t s own unique function. In most of the echinoid fixatives seems  were  that  used  for  ultrastructure  to prepare  every  were  cell-ECM  observed.  involves osmium  Chemical  treating  stabilizing  of  employed,  suggests  be  fixation,  that  based  variations  much  such  as  of our current  results  by  these  in  migration and c e l l - c e l l  upon  as reviewed  However, i t  Hayat  which  may  (1981),  are  thought  be  usually  with glutaraldehyde, paraformaldehyde  Chemicals  molecules  dehydration  This  could  a specimen  tetroxide.  technique  processes, such as c e l l  interactions,  artefactual.  morphological studies, chemical  the embryos f o r microscopy.  fixation  knowledge of developmental or  and asteroid  to  and/or aid in  that may otherwise be extracted or relocated during  the  tissue.  This  stabilization  is  achieved  by  intermolecular and intramolecular cross-linking, thereby creating a l a t t i c e which w i l l may  withstand the effects  reduce  rearrange  the a n t i g e n i c i t y  cellular  alterations  of dehydration.  of many  and e x t r a c e l l u l a r  or artefacts  could  These  compounds,  components  be reduced  actions,  denature  (Hayat,  however,  proteins and  1981).  or eliminated,  I f these  the analysis of  biological tissue based on morphological studies may prove more r e l i a b l e . Rapidly artefacts  freezing  could  components  may  be be  or cryofixing  tissues  significantly  reduced.  suspended  their  in  i s a method Cellular  life-like  states,  freezing, i n the absence of chemicals such as aldehydes It  has  long  been  suggested  that  vitrification,  freezing in the absence of i c e crystal is  the ideal  method f o r f i x i n g  equivalent  to the s o l i d i f i e d  tissues  by stating  i n vivo state.  that  such  extracellular after  rapid  and heavy metals.  the process  of rapid  formation (Pryde and Jones,  biological  (1977) has taken this idea further  and  by which  (Luyet, 1937). the vitreous  1952), Franks  state i s  However, i n order to v i t r i f y  - 7 uncryoprotected  tissue at normal  atmospheric pressure  by  plunge freezing,  4 cooling  rates  Riehle, 1968;  must  exceed  Riehle and  usually less than 300 1982).  I f the  nm  10  Kelvin/second  Hochli, 1973)  (K/s)  (Moor,  1964,  1973;  and the specimen must be very small,  thick (Bruggler  specimen exceeds this  and  Mayer, 1980;  size,  then  Dubochet et a l . ,  ice crystal  formation  is  i nevi table. Ice  crystal  nucleation  theory  there are two nucleation, nucleation upon  (Angel 1 , 1982;  and  homogeneous  nucleation:  or  ice  c r y s t a l s become  resembling  of  discussed  ice  are  beyond  here other  vitrification nucleation  'jams  the  According  classical  to this  nucleation.  theory,  extrinsic  Heterogeneous  i c e , thereby  of  any  up'  and  of  ice  the  given  i s i n h i b i t e d and  sites  Gilkey and  of  than to  of  that  hydrated  at  acting  as  nucleation  on  the  template  but,  they w i l l  the  critical  cooling  specimen,  growth,  can  hence  the  not  rate  be for  heterogeneous occur.  arise simultaneously,  crystal  upon  because  thesis,  homogeneous nucleation  without  a  this  biological  homogeneous nucleation  solidifies  nucleation,  Staehlin (1986) have described  crystal  scope  say  only  Homogeneous  a site  water when a few water molecules assume a  that of  thermodynamic properties  many  by  heterogeneous or  intrinsic  seeded.  c r y s t a l i z a t i o n can occur.  physics  adequately  Franks, 1982).  hand, occurs in supercooled  which  so  described  types of ice crystal  configuration  the  is  occurs when a foreign p a r t i c l e , in pure water, provides  which  other  formation  the  Since system  vitrifying  the  i s unachievable in multi-component systems and  ice  t i ssue. When v i t r i f i c a t i o n  crystals do form, phase separations is,  as the  sugars  are  water or  occur (Robards and S l e y t r , 1985).  ice c r y s t a l s grow, solutes extruded  solvent  leaving  phase and  two the  such as  separate  proteins,  phases  -  aqueous solution or  the  That  e l e c t r o l y t e s and pure  eutectic  crystalline phase which  - 8 resides  between  extrusion  the growing crystals  of solute  crystal  growth  freezes.  The  (Plattner  and Bachmann,  propagates  until  the  remaining  eutectic  including changes  i s in the  solid  vitrification,  pH  state.  results  Phase  (Franks,  1977),  in immunogenic  behavior  (Birkeland,  by phase  to the i n vivo  separation  At this point, the  separation,  which does not  in numerous physiological  changes  deleterious  simultaneously  temperature at which this phenomenon occurs i s the eutectic  system  occur during  salting  out  1976).  alterations  (Gordon,  1975),  Obviously,  problems  state of the specimen so these artefacts imposed  must be eliminated  cryoprotectants,  can  be  which  and  these are  as much as  possible  in order to  j u s t i f y the use of cryofixation techniques over chemical f i x a t i o n . these  The  continues as the temperature decreases and/or as ice  temperature and i s unique to that solution (Rey, 1960). entire  1982).  overcome  by  the  use  of  compounds,  impede the growth of i c e c r y s t a l s .  Many of  known  as  The effects of  such chemicals, however, are not c l e a r l y understood and they may  be just as  detrimental to the tissue as i s chemical f i x a t i o n . Once v i t r i f i c a t i o n a  medium  which  histochemical resin.  has been achieved, the tissue must be brought into  i s suitable  for subsequent  or immunocytochemical  studies,  procedures. this may  involve  the case of the use of a  In order to successfully transfer the tissue from the frozen  to a r e s i n , the v i t r i f i e d water must be substituted such  In  as  ethanol  or  temperature of i c e . require  acetone This  temperatures  process,  the use of chemical  preservation  at  called  by non-aqueous  below  freeze  solvents  the c r y s t a l l i z a t i o n  substitution,  does  f i x a t i v e s and, in p r i n c i p l e , may give  than chemical f i x a t i o n .  Currently,  state  not  better  there are devices on the  market in excess of $20,000 which f i x tissues by freeze substitution. such equipment, a specimen i s rapidly  frozen  stored  specimen then undergoes substitution  in l i q u i d  nitrogen.  The frozen  in a l i q u i f i e d  With  gas and then  - 9 over a period which  of several  days and  i s subsequently embedded into a medium  i s convenient for the investigator.  which the specimen  need be exposed  nitrogen,  solvent,  been  and  added  the by  some  The  only foreign  chemicals to  prior to embedding are the cryogen, the  although  osmium  investigators  tetroxide  during  substitution (Barlow and Sleigh, 1978;  the  and  aldehydes  later  stages  have  of  Meissner and Schwarz, 1990).  the  Freeze  substitution helps to maintain the a n t i g e n i c i t y of the tissue and has been shown  to  tissues  give rich  more in  detailed  ECM  such  disadvantages  of freeze  from  to embedding  freezing  cryogens  and,  with  ultrastructure  as  cartilage  substitution  most  than  chemical  (Arsenault et  include  the cost,  fixation  a l . , 1988). the  time  in The  involved  (4-6 days), the hazard of using some explosive  biological  specimens,  the  use  of  cryoprotective  chemi c a l s . Since  commercial  capabilities devise  of our  freezing  laboratory,  inexpensive and  easily  the i n i t i a l  employable  and  freeze  was  hoped that such methods would  embryos,  substituting  equipment  completed, elements  chemical these  of the  examination. an  attempt  blastocoel,  to their  fixatives.  techniques asteroid  ECM  lead  used  for both  the  of this  techniques  financial  study was  for rapidly  to  freezing  Pi saster ochraceus.  It  to the better preservation of the living  Once  were  beyond  portion  embryos of the s t a r f i s h ,  in a manner closer  conventional  was  this  form  than  aspect  i s possible using of  to preserve each histochemical and  the  study  of the  was  different  ultrastructural  The results of these studies were then analysed in detail in to  a)  determine  including  the hyaline layer.  the  the basal  nature  of  the  material  lamina and, b) determine  within  the  the structure of  F i n a l l y , the ECM of the freeze substituted embryos were  compared to that of embryos prepared by chemical f i x a t i o n  in an attempt to  - 10 -  gain  a better understanding  of the ECM  components and t h e i r arrangements,  and to perhaps gain some insight into t h e i r roles in morphogenesis.  - 11 -  2. 2.1  MATERIALS AND METHODS  R e a r i n g o f A s t e r o i d Embryos  Obtaining adult s t a r f i s h and seawater Ripe  adult  starfish,  intertidal  zones  at Vancouver  1988.  starfish  The  circulating  Pisaster and  ochraceus. were Victoria  were maintained at 9-12  seawater  in the Department  B.C.  collected  from  between April  °C in aquaria  of Zoology.  Light  and  the June,  supplied was  with  kept at a  constant level so as to avoid undesired spawning. Seawater  for the culturing  of embryos was  waters of V i c t o r i a and from the Department Water  retrieved  surface  where  (unpublished  from the  salinity  data).  through a #1  the l a t t e r  Prior  was  of Fisheries in West Vancouver.  source came from seventy feet comparable  to  that  of  the  below  former  to i t s use in culturing, seawater was  Whatman f i l t e r  room maintained at 10-11  obtained from the coastal  and  aerated  the site  filtered  with a bubbling stone in a cold  °C.  Glass and p l a s t i c ware Glass labelled  and  to  plastic  avoid  wares used  accidental  exclusively  for culturing  contamination, and  seawater or tapwater but never with detergents. completed, a l l containers were wiped  were  After  embryos were  rinsed  with  only  had  been  a culture  clean, rinsed with tapwater and then  with seawater so that they could be u t i l i z e d in subsequent cultures.  Isolation of gametes Arms  of  ripe  starfish  were  removed,  thereby exposing the  gonads.  After v i s u a l i z a t i o n with the naked eye, ovaries were dissected and removed, placed  on a clean  petri  dish, washed with f i l t e r e d  seawater, and treated  - 12 -  with 10 ml of 0.1 mg/ml of 1-methyl induce oocyte maturation. dish until  Testes were l e f t  'dry' i n a separate clean petri  the eggs were ready to be f e r t i l i z e d  concentrated solution.  adenine f o r ninety minutes i n order to  semen were placed  at which time 1-2 drops of  i n 25 ml of seawater  Samples of this solution were examined  to ensure that  at least  10-20% of the sperm  producing  a milky  under a l i g h t microscope  were motile,  and that the  pattern of m o t i l i t y was normal.  Ferti1ization When the eggs had matured  (as indicated  by the disappearance of the  germinal v e s i c l e s ) , they were placed i n a 1 l i t r e p l a s t i c beaker containing 300-400 ml of fresh seawater at 10-11 °C. filled  with  beaker.  culturing  beakers were  approximately 300 ml of seawater, and eggs were added  roughly 50% of the bottom fertilized  Several  by adding  of each beaker was covered.  ten to f i f t e e n  drops  until  The cultures were  of sperm-suspension  to each  Seventy two hours after f e r t i l i z a t i o n , hatched, swimming blastulae  were poured into clean beakers of seawater and the remaining unsuccessfully developed embryos, which were s t i l l  clustered on the bottom of the beaker,  were discarded.  Harvesting the embryos When the embryos reached 5 to 5 1/2 days of development,  they were  concentrated by pouring the cultures into 50 ml p l a s t i c conical  centrifuge  tubes. settle.  The tubes were then placed i n i c e and the embryos were allowed to After  settling,  the concentrated embryos were transferred with a  clean glass pipette to three or four 15 ml conical  glass centrifuge tubes.  The tubes were centrifuged at 120xg f o r 1-3 minutes to further concentrate the embryos.  - 13 -  2.2  Cryofixation of Embryos  Freezing a) Liquid Freon 12, Ethane and Propane For freezing in Freon  12, ethane and propane, three Dewer f l a s k s ,  with an insert cup, were f i l l e d with l i q u i d nitrogen (LN2). the insert cup was two  used  for the actual  freezing  one  The flask with  process, while  the other  flasks stored a reserve supply of LN2 and temporarily housed the frozen  embryos  in 1.5  freezing  flask  ml  freezer v i a l s  upon i t s base was  current motor, with a s t i r r i n g flask.  The  stirring  into the cup  ( F i g . 1).  cryogen  inverted  LN2  placed  A  large ring  inside  half way  sprayed  stand  with  a fume cabinet.  to i t , was  clamped  An  the eddy  above the  into the insert cup, which  as possible without  While the rod was and  maintain the s t i r r i n g  canes.  rod connected  rod extended  immersed as f a r into the  was  on  was  allowing i t to overflow  s t i r r i n g , the container of liquid  into  the  cup.  It  was  important  to  to avoid freezing of the cryogen, and to keep the LN2  level as high as possible to reduce evaporation of the cryogen. The  embryos  pretreatment suspension  with was  were a  prepared  for  placed  on  a copper  freeze fracture  then plunged  kept  five  for about  into the adjacent LN2 temporarily  in  accumulated  in a  refrigerator process  was  one  and of  vial,  for an  as  follows:  cryoprotectant, i f appropriate, 1 u l  monolayer; the grid was moving  freezing  seconds.  stirring  Subsequently,  of  the the  other vial  Dewer was  flasks.  capped  storage period.  the cryogen  cabinet by l i f t i n g the cup out of the  was  allowed  LN2.  and  the  so as  liquid  Once  embryo  to form a cryogen  the grid  then transferred to a freezer v i a l  indefinite  completed,  into the  grid  following  was  and  plunged  to be stored  four  transferred  grids  had  to  LN2  a  After the entire freezing to evaporate  in the  fume  - 14 -  b) Liquid nitrogen slush LN2  was  poured into a small glass Thermos which was,  inside of an insulated vacuum chamber. the  LN2  slush.  to boil At this  embryos was  vigourously,  until  The chamber was  the LN2  time, the chamber was  in turn, placed  evacuated, causing  began to s o l i d i f y  quickly  opened  and  the  forming a grid  with  plunged into the slush, kept in motion f o r 5 seconds, and then  dropped into a storage vial as above.  Crvoprotection In  order  formation, tested. glycol, water  four  protect  and  resuspended  different  embryos  from  intracellular  15% propylene glycol removed  from  the  f o r 20-30 minutes  the  damage of  cryoprotective  ice crystal  solutions  were  were prepared in 100%  tubes  of  in 15 ml  packed  seawater.  embryos  and  of a cryoprotective  Excess  they  were  solution  over  Subsequently, the tubes were centrifuged at 120xg for 30 seconds, the  excess solution was of  the  Solutions of 10% DMSO, 10% and 15% g l y c e r o l , 10% and 15% ethylene  was  ice.  to  removed, and the p e l l e t of embryos in the small amount  remaining l i q u i d was frozen in propane as described above.  Freeze substitution To prepare for freeze substitution, 150 g of dry ice was bottom until the  of a wide mouth Thermos. i t was  slurry  half f u l l reached  ethanol,  was  after which LN2 was -90°  Digi-Sense thermocouple. 100%  Acetone  saturated  to Up  -95  to f i v e  with alcian  °C  as  10 ml  slowly added  added  until  monitered glass  vials  blue i f desired  placed in the to the thermos  the temperature of by  a  were  Cole-Parmer filled  to stain ECM  and Zelander, 1970), capped, and immersed into the s l u r r y (Fig.  2).  with  (Behnke When  - 15 -  Fig.  1:  Photograph embryos. ensures cup  of the apparatus  into  for plunge  freezing  asteroid  A s t i r r i n g rod powered by an eddy current motor continuous movement of the  (arrow) which  flask  used  (F).  cryogen  s i t s inside the l i q u i d  Embryos on  EM  grids  are  within  the  (Mo)  insert  n i t r o g e n - f i l l e d Dewer  frozen  by  plunging them  the cryogen and then into the surrounding l i q u i d  nitrogen  before being stored in l i q u i d nitrogen.  Fig.  2:  Photograph  of the Thermos (Th) and  freeze substitution. ice/liquid  nitrogen  vials  filled  are  slurry. pre-cooled  The  vials  (V)  used for  The Thermos i s f i l l e d with an acetone/dry slurry  with  frozen  glass  (-90°  anhydrous specimens  to  -95  ethanol are  then  °C) and  and  the  glass  placed into the  dropped  v i a l s and inserted into the Thermos which  and placed in a -60 °C r e f r i g e r a t o r for 4 to 5 days.  into  the  i s capped  - 17 -  the  temperature  embryos was °C freezer to  of  the  ethanol  reached  placed in each glass v i a l . f o r four days.  At  least  -90  °C,  one  sample  The Thermos was  of  frozen  then put in a  once per day, however, LN2 was  -60  added  the Thermos in order to maintain the temperature below -85 °C to avoid  ice  crystal formation. After four days of freeze  the  Thermos and  placed  placed  substitution,  the v i a l s  were removed from  in a -20 °C freezer for one hour.  in a 4 °C r e f r i g e r a t o r  f o r one  hour and  finally  They were then brought  to room  temperature over one hour.  Embedding cryofixed material Cryofixed  embryos were embedded  in either  Epon (Luft,  1961)  or  JB4  acetone  two  (Polysciences, Inc.). For or  Epon embedding, embryos were washed in reagent grade  three times during the t r a n s i t i o n from 4 °C to room temperature before  immersing  in  2%  OsO^  in  acetone  for  two  hours  at  room  temperature.  Following osmication, the embryos were once again washed with acetone and left  overnight in 1:1  pipetted  acetone/Epon.  into p l a s t i c trays f i l l e d  The  next  morning,  the  embryos were  with fresh 100% Epon 812 and cured in a  60 °C oven for 24 hours. For  JB4  embedding, embryos were washed with fresh 100% ethanol after  warming to room temperature  as above.  The  embryos  were  infiltrated  for  three hours at room temperature with catalyzed Solution A (100 ml Solution A +  0.9  g of  catalyzed polymerize  peroxide catalyst)  Solution  A  +  1 ml  at room temperature  and  Solution  transferred B).  The  to  fresh  JB4 was  overnight in aluminum f o i l  with parafilm to reduce oxygen exposure.  JB4  (25  ml  then allowed to plates  covered  - 18 -  2.3  Chemical  Fixation and Embedding  Glutaraldehyde f i x a t i o n Packed embryos were immersed in 1% glutaraldehyde in 80% seawater (pH 7.0)  saturated  temperature 7.2), for with  with  alcian  for four hours.  blue  in 2% OsO^  hour, washed with the buffer, and  2%  uranyl  and  Zelander,  Following a rinse in 1.25%  the embryos were post-fixed  one  (Behnke  acetate (aqueous).  1970)  NaHC0  3  at  room  buffer (pH  in the same buffer (pH  stained en  7.4)  bloc for 45 minutes  After a wash with d i s t i l l e d  water, the  embryos were dehydrated with increasing concentrations of ethanol, immersed twice in 100% propylene oxide, and embedded in Epon 812 (Luft, 1961).  Formalin f i x a t i o n Packed embryos were fixed formalin in seawater (pH 8.0)  for paraffin  embedding by  for four hours.  The  immersion  embryos were dehydrated  through an increasing series of ethanol concentrations (30%, 50%, and  100%)  and  cleared with two successive washes in xylene.  immersed in 1:1 30  minute  in paraffin  at  60  embedded in paraffin blocks for sagittal  2.4  Microscopy:  70%,  °C.  Finally,  95%,  They were then  xylene/paraffin at 60 °C for 30 minutes followed by  immersions  in 8%  the  three  embryos were  sectioning.  Morphology  Epon embedded embryos were mounted on aluminum stubs for transverse or sagittal For  light  stained 0.5%  sectioning  and  microscopy,  with  Borax  viewed with  sectioned on  1 um  Richardson's in d i s t i l l e d  a  Porter-Blum  ultramicrotome.  thick sections were cut with a glass knife and stain  water,  (0.5% methylene Richardson  The  film  was  blue,  0.5%  Azure  et a l . , 1960).  a Zeiss Photo-microscope III and  5060 Panatomic X f i l m .  MT-1  developed  photographed in D-76  II  and  Sections were with  full  Kodak  FX  strength for  - 19 -  seven  minutes,  washed with  tapwater  and fixed  f o r ten minutes with Kodak  rapid f i x . For transmission electron microscopy sectioned  with  a Dupont diamond knife.  were picked up with carbon sections  were to be used  (TEM), Epon embedded embryos were Silver  to grey  coloured sections  coated, 100 mesh copper or nickel grids. f o r stereo photographs,  thicker  sections which  were v i o l e t to gold in colour were cut and retrieved with uncoated grids. drops  I f the  150 mesh  Sections were stained by f l o a t i n g the grids f o r 10 minutes each on of  2%  uranyl  acetate  (aq) and  lead  citrate.  Grids  with  thick  sections f o r stereo imaging were completely immersed in the stains in order to achieve maximum staining.  Sections were viewed with a P h i l l i p s 301 TEM  at 60 kV and photographed with Kodak Eastman fine  grain  Stereo  was  pairs  were  photographed  minutes with Kodak D-19 f u l l  12° apart.  Film  strength, rinsed with  5302 35 mm  developed  tapwater  film.  f o r five  and fixed for  ten minutes with Kodak rapid f i x .  2.5  Histochemistry JB4 embedded  embryos were mounted and sections 2-3 um  with a 'dry' knife.  Sections were picked up with an eyelash and floated on  drops of water on clean glass s l i d e s . hotplate  thick were cut  f o r 3 minutes  in order  The slides  to adhere  were placed on a 60° C  the sections to the s l i d e s .  Slides were then stained with PAS, Alcian blue or Toluidine blue #1) and covered  with  glass coverslips.  (Appendix  Sections were viewed with a Zeiss  Photo-microscope III and photographed as above. Paraffin on  an American  embedded Optical  sections  were floated  slides.  The slides  prior  to staining  embryos were sectioned at 7 um with a steel Corporation  Spencer  '820' microtome.  on warm water and picked up on albumin  were placed (Appendix  #1)  i n a 60 °C oven  knife  Ribbons of coated glass  f o r at least  one hour  in order to adhere the sections to the  slides. sections.  Sections  were  viewed  and  photographed  as  described  for  0B4  - 21 -  3. 3.1  RESULTS  Cryogens Embryos  mpt=l15  K)  1978)  but,  (Fig.  3).  frozen  lacked  in  the  supercooled  dumb-bell  Freon  shape  seen  12  (dichlorodifluoromethane,  in  vivo  (Crawford  instead, demonstrated shape d i s t o r t i o n and Large  i r r e g u l a r spaces  were present  distinction  could  be  the  continuous  basement  distributed  strands  blastocoel.  The  made between membrane  of ECM  was  made up  pattern  of  honeycomb in some areas,  but  ECM this  nuclei  not the  in  cellular  within and  the  present.  hyaline  the  epithelia,  cytoplasm,  layer (HL)  not  and  no a  irregularily and  filled  resembled  was  Chi a,  destruction  Thick,  blastocoel  arrangement  the  and  that  the of  consistent  a  within  d i f f e r e n t regions  of the same embryo nor  in sections of d i f f e r e n t embryos.  The  HL  series  ECM  of  the  was  reduced  to  a  of  strands,  lacking  the  structural organization described by Crawford and Abed (1986). When l i q u i d nitrogen (LN2) embryonic  shape was  altered  a f t e r freezing with appeared  adequate  Freon at  the  slush (mpt=63 K) was  ( F i g . 4)  12  although  not  ( c f . Figs. 3 and  4).  LM  level.  The  ECM  used as a cryogen, the as  severely  Cellular  of  the  as  it  was  preservation  blastocoel  had  a  strand-like morphology s i m i l a r to that seen a f t e r freezing in Freon but in contrast to the honeycomb pattern after random  freezing in LN2 origin.  appeared level,  to the  preserved  A  slush  seemed to  basement membrane was  consist of fibrous rough  seen a f t e r Freon f i x a t i o n , strands of  endoplasmic  strands reticulum  be  oriented  in  diffucult ( F i g . 4). and  parallel  to resolve At  nuclei  ECM  lines  and  the  of HL  the u l t r a s t r u c t u r a l appeared  to  be  well  but the membranes of i n t r a c e l l u l a r granules were often damaged or  destroyed  ( F i g . 5).  The  ECM  consisted  of thick, amorphous strands  within the blastocoel ( F i g . 6) and comprising to fibrous  strands  in the  HL,  a 0.5  nm  the HL  thick mat  ( F i g . 5). of ECM  was  both  In addition also  present  - 22 at  the  apical  surface  of  the  ectoderm,  components collapsing ( F i g . 5). epithelium and  i t had  The  possibly  basal  as  a  lamina adhered  strands of blastocoelic ECM  result tightly  extending  of to  HL the  from i t to the  center of the embryo ( F i g . 5 and 6). When frozen embryos  was  in  maintained.  damage at the LM very  regular,  frozen unit  appeared the  HL  than  as  ethane  The  (mpt=90 K),  cells  ( F i g . 7).  LN2  after  freezing  rested  as  upon  slightly  fibrous material, the third the epithelium.  The  be  similar to  that  with  the  latter  structure.  seen  at  fibres  in  LM  embryos  ultrastructural the  frozen  some  of  frozen  in  liquid  level,  evidence  (40 nm)  suggesting in  Freon  intercellular The  HL  of  zonular  ( F i g . 9a).  The  fibres  in  ECM.  propane  was  that 12,  i t was  LN2  a An  in  strands  organization  The  area mat  ECM  and  blastocoel  no  ( F i g . 9b)  was  of  of  little ECM  the ECM  and of  demonstrated diffucult  was  At  the  in many areas of to  et a l . , 1989) distinct,  contained slush  to  than  ethane.  relative  Spiegel  HL  pattern  with  more dispersed  preserved  but  The  per  level.  spaces were present poorly  embryos  honeycomb  (mpt=84 K)  slush  than those seen a f t e r cryofixation in LN2  The  the  crystal  cryogens.  existed between the  f i x a t i o n s (Crawford and Abed, 1986;  sub-layers  9b).  level,  embryo ( F i g . 9a).  chemical  ice  outermost sub-layer  arranged  thicker mat  sub-layer,  two  d i f f u c u l t to resolve at the LM  embryos  the  of  described  The  l i t t l e shape d i s t o r t i o n at the LM level ( F i g . 8). resolve  free  shape of  basement membrane appeared continuous with  the blastocoel and was Finally,  to  overall  slush, but exhibited more p a r a l l e l  a network of a  the  The ECM of the blastocoel, however, had a  appearance,  12 and  appeared  a fibrous, t r i - l a y e r e d  appeared  which  level  fibrous  in Freon area  liquid  previous showing  identifiable thinner  fibres  ( c f . Figs. 6  and  of the propane fixed embryos were arranged in a parallel  fashion but were sparsely d i s t r i b u t e d , which probably accounted for their  - 23 -  F i g . 3:  1.0  um  cross-section  embryo frozen Richardson's  through  in Freon 12, stain.  The  the  esophageal  embedded  shape of  in Epon, and the  (Ec), endodermal (En), and  origin  demonstrate  to  vacuolation,  indicating  blastocoel  is  resembling  a  honeycomb  cellular  crystal  damage.  in  random  strands  pattern  stained  1.0  y.m  frozen with  cross-section in l i q u i d  Richardson's  distorted but The  ECM  of  distributed  through  (arrows).  nitrogen stain.  the  slush, The  coelomic embedded  shape of  the c e l l s appear well the  as  hyaline  layer  random strands  (Es), Ectoderm (Ec).  x900.  and  The with  The  ECM  and  region  blastocoel Coelom  show the  some  areas  hyaline  layer  x800.  of an embryo  in Epon and the  (M)  of  embryo  stained has  preserved at the LM  (arrows).  with  and  (HL) has been reduced to a series of radiating f i b r e s .  F i g . 4:  an  mesenchymal  detail  ice  arranged  of  embryo i s distorted  c e l l s of ectodermal fail  region  appears (C),  been level. to  be  Esophagus  24  - 25 -  Fig. 5:  TEM  of an  nitrogen acetate poorly  area slush,  and  strands.  i s good.  TEM  of  slush, lead the  Epon,  (arrows) but The  The  the  basal  embryo frozen  and  stained  Intracellular in  with  membranes general,  in l i q u i d uranyl  are  the  often  cellular  hyaline layer (HL) appears as a thick  upon the apex of the  (arrowheads).  Fig. 6:  in  citrate.  distinguishable  of ECM  ectoderm of an  embedded  lead  preservation mat  of the  epithelium  with  some radiating  lamina i s t i g h t l y adhered to the  epithelium  xl9700.  blastocoel of an  embedded  citrate. blastocoel.  in  embryo  Epon, and  Interconnecting The  basal  stained strands  lamina  adhered to the ectoderm (Ec).  frozen  x7500  with of  in  liquid  uranyl ECM  nitrogen  acetate  and  (arrows) occupy  (arrowheads) appears  tightly  26  - 27 -  Fig. 7:  1.0  \m  section  liquid  of  the posterior region of an embryo frozen in  ethane, embedded in Epon, and  stain.  The  well  ectodermal  preserved  blastocoel  at  (Ec)  this  consists  of  and  endodermal  magnification. regular  strands  basement membrane (arrows).  The  in  consist  appearance  and  seems  stained  to  hyaline of  with  Richardson's  (En)  cells  The  ECM  continuous layer (HL) three  appear of  the  with  the  i s fibrous  distinct  zones.  x2200.  Fig. 8:  1.0 of  \m an  cross-section embryo frozen  stained  with  and  the  in l i q u i d  Richardson's  been preserved that  through  the  cryofixed embryos.  inability  x670.  of the esophagus  propane, embedded  stain.  i t i s much more dispersed  region  The to  in Epon,  (Es) and  shape of the embryo has resolve  than was  the  seen  ECM  suggests  in the previous  - 29 -  Fig. 9a:  TEM of a region of the ectoderm of an embryo frozen in l i q u i d propane, embedded lead  citrate.  in Epon, and stained with  uranyl  The hyaline layer (HL) i s fibrous in appearance  with some evidence of zonular organization.  Fig. 9b:  preserved  but numerous  shrinkage  has occurred.  intercellular  embryo  stained very  frozen  with  fibrous  uranyl  in  The c e l l s are well  spaces  (*)  suggest  that  x9800.  TEM of a region of the blastocoel adjacent an  acetate and  liquid  propane,  the endoderm (En) of  embedded  acetate and lead c i t r a t e .  (arrows) and  these  with the basal lamina (arrowheads).  fibres  in Epon, and The ECM appears  are often  x20400.  continuous  30  - 31 -  poor resolution at the LM level (Fig. 8). The BL was t h i n , r e l a t i v e to the blastocoelic f i b e r s , and adhered close to the epithelium  3.2  ( F i g . 9b).  Cryoprotection Embryos cryoprotected  widespread b).  in propane demonstrated  i c e crystal damage at the u l t r a s t r u c t u r a l level  Cellular  blastocoel  with 10% DMSO and frozen  organelles  were  indistinguishable  and  and the HL were i r r e g u l a r and fibrous,  (Figs. 10a and  the  ECM  not unlike  of the  those seen  a f t e r freezing in Freon 12, LN2 slush and ethane. Embryos cryoprotected  by pretreatment with 10% and 15% glycerol prior  to freezing i n propane d i f f e r e d u l t r a s t r u c t u r a l l y . glycerol  exhibited  i c e damage which varied  embryo.  In some areas, the organelles  Those treated  quantitatively  with 10%  throughout the  were intact and the cytoplasm was  very electron dense (Fig. 11a) while in other areas, evidence of ice damage was present.  In addition, the presence of numerous i n t e r c e l l u l a r and basal  cytoplasmic processes suggested that some c e l l lib). the  The ECM of the blastocoel entire  embryo  was strand-like  (Fig. l i b ) .  sub-layers ( F i g . 11a), included  shrinkage had occurred (Fig.  The  HL,  i n appearance throughout  consisting  of  an outer network of thick  three  and thin fibres  which was connected  to a much thicker  band or supporting layer.  this  to the epithelium  was a region  and extending  fibrous of  elements  interconnecting  the epithelium.  region.  There  were  with  Beneath  relatively  few  the supporting layer with the plasmalemma also  some  microvilli  present  in this  Embryos treated with 15% g l y c e r o l , on the other hand, appeared to  demonstrate excellent preservation were  fibrous  free  of i c e crystal  organelles  and well  defined  undergone  tremendous  of c e l l u l a r organelles  damage, with nuclei.  shrinkage  as  electron  Most c e l l s , indicated  (Fig. 12). Cells  dense cytoplasms, intact however, appeared to have by  the many  cytoplasmic  - 32 processes well  and the separation of the e p i t h e l i a l  as from each other.  showing  no  signs  multi-layered  c e l l s from the BL and HL as  The ECM of the blastocoel was evenly dispersed,  of  collapse  or  disruption,  and well  preserved  relative  to  and  the  chemically  HL  appeared  fixed  hyaline  layers (Crawford and Abed, 1986). Embryos treated with 10% ethylene glycol had  some  cellular  ultrastructural including  i c e crystal  level.  nuclei,  distinguished  damage  which  Intracellular  endoplasmic  but the nuclei  p r i o r to freezing in propane  granules  reticulum,  be  and  and  and cytoplasm  c h a r a c t e r i s t i c of i c e crystal  could  resolved many  small  endoderm 13b).  however,  exhibited coarse  but was much  This  pattern,  more evenly suggestive  irregular  dispersed  strands  towards  of a gradient,  only  partially  into  be  vacuoles,  a  the blastocoel.  The ECM of the adjacent  to the  the ectoderm ( F i g .  indicated  and/or cryoprotection of ECM elements was e f f e c t i v e but  could  damage ( F i g . 13a). C e l l s of the ectoderm and  endoderm showed no differences in quality of preservation. blastocoel,  organelles,  ribosomes  contained  at the  that  through  As the freezing  freezing  the ectoderm rate  decreased  towards the center of the embryo, thereby increasing the likelihood of ice crystal  formation  insufficient  (Van  Harreveld  quantity of ethylene  and  Crowe11,  glycol  1964),  to i n h i b i t  there  was  the growth  an  of ice  crystals. Embryos ultrastructural  treated  with  preservation  However, only a few random first in  exposed  15% ethylene of the c e l l s regions  to the cryogen  the same embryo,  cellular  glycol and  demonstrated ECM,  of the embryo,  especially probably  damage  and  ECM  the HL.  those  ( F i g . 14a), were well preserved. i c e crystal  excellent  areas  Elsewhere,  strands  were  present ( F i g . 14b) as seen in previous preparations ( c f . Figs. 14b and 13a).  - 33 -  F i g . 10a:  TEM of a region of ectoderm in an embryo treated with 10% DMSO prior  to freezing in propane, embedding i n Epon, and staining  with  uranyl  cellular (HL).  F i g . 10b:  acetate  i c e crystal  and damage  lead and  citrate, a  showing  collapsed  extensive  hyaline  layer  X9900.  TEM of a region described  of the blastocoel  in F i g . 10a.  The fibrous  of an  embryo  ECM (arrows),  ectoderm (Ec) and endoderm (En) are shown.  X12300.  prepared  as  the damaged  - 35 -  F i g . 11a:  TEM  of the  prior  ectoderm of an  TEM  of  The  ECM  lamina  lead c i t r a t e .  The  the  i s shown (arrow).  blastocoel of  i s very (arrows)  the  fibrous and of  the  hyaline layer  embryo described is  continuous  endoderm  which obscure i t s basal lamina,  TEM  staining  of an embryo treated with  (HL)  A well  xl2700.  (En).  ectoderm (Ec) are numerous cytoplasmic  F i g . 12:  107. glycerol  shown to consist of three d i s t i n c t zones of f i b e r s .  preserved c e l l  Fig. l i b :  treated with  to freezing in propane, embedding in Epon, and  with uranyl acetate and is  embryo  in Fig.  with  the  Extending  processes  11a. basal  from  the  (arrowheads)  xl1000.  15% glycerol  prior  to  freezing  in l i q u i d propane, embedding in Epon, and staining with uranyl acetate and  lead c i t r a t e .  The  ECM  distributed  and  a  homogeneously present  lining  preservation and  the  ectoderm  i s excellent, the  basal  (arrows).  blastocoel (b) i s lamina  is  clearly  Although  cellular  numerous cytoplasmic  processes  the separation of the ectodermal  c e l l s from the ECM  of the  (Ec) and  endodermal  (*) indicate shrinkage has occurred.  (En)  x4400.  36  - 37 -  F i g . 13a:  TEM of the ectoderm glycol  prior  of an embryo  to freezing  staining  with  uranyl  cellular  ice crystal  treated  i n propane,  acetate and damage  with 10% ethylene  embedding i n Epon, and  lead  citrate.  are present  Areas  (arrows)  of  and the  hyaline layer (HL) appears damaged, consisting of i r r e g u l a r l y distributed  strands of ECM.  organization in the HL.  Fig. 13b:  TEM of the blastocoel Fig.  13a.  The  homogeneous  ECM  than  endoderm  (En)  freezing  despite  xl3500.  of an embryo prepared as described in  adjacent to the ectoderm  the  fibrous  suggesting good  endoderm themselves.  There appears to be some zonular  the  components presence  preservation  x7200.  of  (Ec) i s more  (arrows) of the  a  near the  gradient  cells  of  of the  - 39 -  F i g . 14a:  TEM  of  the  ethylene Epon,  hyaline  glycol  and  layer  of  prior to freezing  staining  with  uranyl  Multiple sub-layers can be seen.  Fig. 14b:  TEM  of a region  described in  the  detail  embryo  treated  with  15%  i n propane, embedding in acetate  and  lead  citrate.  x24900.  of the ectoderm  in Fig. 14a. nuclei  an  of an embryo prepared as  Ice crystal damage i s c l e a r l y v i s i b l e  (N), and  seen in Fig. 14a.  the hyaline x9500.  layer  (HL) lacks  the  40  - 41 -  Embryos cryoprotected propane  appeared  to  be  with  preserved  using d i f f e r e n t cryogens and of  the  embryo  blastocoel (Fig.  was  Cell  ice crystal  damage (Figs. 16,  had  fixation  ( F i g . 16),  a  punctate  (Fig.  heterochromatic preserved  to contain  in  the a  cells  and  19).  rough nuclei  The  the  way  so  as  to  LM  the  preserved, stained  the  material  showing no evidence of  reticulum  vacuolation,  the  shape  and  intracellular  i n d i c a t i n g damage  endoplasmic  reveal  preserved  level,  membranes of  periphery and one central nucleolus a  those  lightly  possibly  lacked  to freezing in  the  were well  19).  appearance,  The  At  well preserved,  18 and  the  of  homogeneously,  ultrastructure was  granules  distended  better than any  cryoprotectants.  maintained,  appeared  15).  15% propylene glycol p r i o r  was  during  frequently  demonstrating  ( F i g . 16).  interesting  The  ECM  a was  morphological  relationships with the c e l l s as described below.  3.3  ECM of the Blastocoel and Basal Lamina An  of  ECM  intermediate  embryonic evenly  density  cavity.  The  was TEM  distributed showed  these  scattered throughout a l l planes  orientation from the and  consisting of short e x t r a c e l l u l a r fibres and amorphous material  (Figs. 16,  17 and  18).  esophagus interconnecting  homogeneously small  twig-like  of section, with  In at least two i t distally  throughout  no  ECM  the  components  set pattern  or  locations - radiating  to the  ectoderm  (Fig.  17)  in the dorsoposterior region of the embryo termed the dorsal web (Fig.  18) - d i s t i n c t path.  fibres existed which travelled  These fibres  in somewhat of a sinusoidal  measured approximately 50 nm  in diameter and  appeared  either alone or frequently in bundles of three or more. The fixation  basal  lamina  (Crawford,  1989)  appeared and  two  lamina l u c i d a , could be i d e n t i f i e d  thicker  (200  nm)  distinct  zones,  ( F i g . 16).  The  than the  after  lamina  chemical densa  and  morphology of the basal  - 42 -  lamina did vary,  usually in thickness, in d i f f e r e n t regions  of the embryo  as described by Reimer and Crawford (in preparation).  3.4  Hyaline Layer The  HL  appeared  to  be  a  convoluted  ECM  approximately  4  um  which compared well with measurements taken i n vivo ( F i g . 19). ECM  did  not  follow each ridge or  have a shape independent of  it.  trough of The  HL  and  sub-layers  - the  layer, and  the coarse outer meshwork ( F i g . 20).  the  outermost  distributed  sub-layer,  in  a  However, the ECM limit  pattern of this  ( F i g . 19).  appeared strands. although  to  be  The a  250  of  sub-layer  thick  some extended tips  into  H3,  which  the  was  of the  the  boundary  ECM  of  the  blastocoel.  became more d i f f u s e towards i t s outer  array  of  a stereo  hairpin-like  pair ( F i g . 21), loops  outer  meshwork  microvilli  (Fig.  formed  of  matrix  for m i c r o v i l l i ,  22).  Within  consisting of electron dense material  the  of the  and  outer  surfaces  plasma membranes  lining  (Figs. 23a-d).  extramembranous portion of this material appeared to be continuous with loops  of  the  boundary  layer.  In  cross  section,  appeared to r a d i a l l y project thin filamentous densities  to  filaments  could  possible  that  the be  loops  of the  these filaments  this  cap  the  length  were attached  the m i c r o v i l l u s associated body ( F i g . 23d).  The the  of  material  structures from the  external  boundary layer (Figs. 23b).  seen running  the  microvillus associated  bodies (Spiegel et a l . , 1989) inner  ECM  homogeneously  the major s i t e of termination the  seemed to  The coarse outer meshwork,  boundary layer, shown as nm  but  outer  appeared to consist of six  ECM of  This  of a multi-layered  1 (HI), H2,  an  characteristic  This sub-layer was  boundary layer, the  layer, hyaline  consisted  epithelium  consisted  whose density varied amongst each sub-layer intervillus  the  thick,  of  the  to the  microvilli inner  In addition, and  i t is  dense region of  - 43 Figs. glycol  15 through 23 are sections  of embryos  treated  with  15% propylene  p r i o r to freezing in l i q u i d propane, embedding i n Epon, and staining  with uranyl acetate and lead c i t r a t e .  F i g . 15:  1.0  um,  described of  mid-sagittal  section  of  an  embryo  prepared  above and stained with Richardson's s t a i n .  the blastocoel  appear to contain  (b) and the hyaline  layer  as  The ECM  (arrows) do not  the thick, fibrous structures that were seen  a f t e r cryofixation by other methods.  Esophagus (Es).  x470..  4 4  - 45 -  F i g . 16:  TEM of the basal blastocoel lucida contain  lamina of the ectoderm  (b). The basal  (LL) and a thick tiny f i b r i l s  (Ec) adjacent to the  lamina consists lamina  densa  and granules.  of a patchy  (LD) which  x27600  appears  to  The ECM of the blastocoel  consists predominately of twig-like structures amorphous material.  lamina  (arrowheads) and  46  - 47 -  F i g . 17:  TEM  of  extracellular  esophagus  F i g . 18:  TEM  of  within  fibres  (arrows)  radiating  showing  extracellular  from  the  (Es). x!4700.  the dorsal the  web  meshwork  Ectoderm (Ec). x31200.  of  'twig-like'  ECM.  fibres  (arrows)  Endoderm  (En),  - 49 -  Fig.  19:  A comparison of the thickness  of hyaline layers (HL) seen in a  l i v i n g embryo treated with India ink p a r t i c l e s ( i n s e r t ) and an embryo are  prepared by freeze  approximately 4.0  substitution.  \im thick.  Both  Ectoderm  hyaline  layers  (Ec), Blastocoel  (b), India ink p a r t i c l e s (In). x22800 ( i n s e t : x990).  50  - 51 -  Fig. 20:  TEM  of  the  morphology and  hyaline showing  layer  i t s six sub-layers:  sub-layer (IV); the supporting H3; the boundary sub-layer  X41500.  demonstrating  its The  convoluted intervillus  layer, consisting of HI, H2 and  (B); and the outer  meshwork  (OM).  52  - 53 -  F i g . 21:  Stereo  pair  arrangement sub-layer  (a of  &  b)  the  of  TEM's  boundary  ( I V ) , supporting  demonstrating  sub-layer  layer  the  (B).  (HI, H2  looping  Intervillus  and  H3),  outer  meshwork (OM). X57500.  F i g . 22:  TEM  showing  hyaline  the  layer.  microvillus (arrowhead).  relationship A  to  microvillus  the  boundary  Intervillus  between  microvilli  associated sub-layer  sub-layer  (HI, H2 and H3), outer meshwork (OM).  body (B)  and  connecting is  a  indicated  ( I V ) , supporting X57000.  the  layer  - 55 Figs. 23a-d are high magnification TEM's of the m i c r o v i l l u s associated body.  F i g . 23a:  Higher  magnification  material  lining  TEM  of  F i g . 22  the inner and outer  showing  surfaces  the  dense  of the plasma  membrane (P) of the t i p of the m i c r o v i l l u s and the continuity of  the outer  sub-layer (B).  F i g . 23b:  Cross-section  dense  region  with  the f i b r e s  x83300.  through  the t i p o f a m i c r o v i l l u s (arrowhead)  showing the continuity of the outer boundary sub-layer (B).  F i g . 23c:  Oblique  continuous  F i g . 23d:  dense material  with the  X58400.  section showing the inner and outer densities of the  microvillus  (P).  of the boundary  t i p and the r a d i a t i n g strands with  the boundary sub-layer  o f ECM which are  (B). Plasma membrane  xl50000.  Sagittal  section  of a  m i c r o v i l l u s demonstrating  running i t s length (arrows).  X48000.  filaments  56  - 57 The  HI  sub-layer  was  an  electron  dense mat  of  Below HI  was  boundary layer originated (Figs. 20, 21). which  consisted  of  under which was and  the  a  H3  125  nm  thick,  (Figs. 20,  intervillus  21),  layer,  the  appeared  to  be  an  base of  the  H2  sub-layer and  a distinct  its  greater  electron  the  HL  the thickness for  the  did not  electron  dense material  i n t e r f a c i n g with the  density.  sub-layers  of  the  HL  the i n t e r v i l l u s  Chemical The  perfectly reflect  (Fig.  and Chi a,  Pi saster  osmium  tetroxide  Crawford,  1986a,b; Crawford, for  consisted  of  boundary  layer,  of ECM  in  In  of  and  varied  20).  the  embedded  extended HL  the  layer.  H3  layer based through  to  the  Since  epithelium,  the  it  was  in order to account  boundary  addition  H3  in  ( F i g . 22).  that of the  of  epithelia.  H2  and  supporting  microvilli,  an  ECM  existed throughout  Fixation  (Crawford  appoximately  21)  intervillus  in appearance than that of the OM  morphology  study  (Figs. 20,  layer.  glutaraldehyde  this  H2,  intervillus  Microvilli  characteristics  s l i g h t l y more fibrous  3.5  the  of the i n t e r v i l l u s sub-layer that varied  convoluting  sub-layer,  the  adjacent  i n t e r v i l l u s sub-layer into the outer zones of the shape of  the  t r a n s i t i o n layer between  could be d i f f e r e n t i a t e d from the components of the on  upon which  amorphous belt of ECM  sub-layer  intermittant,  ECM  1982;  distinct HI  um  thickness  and  thick from  sub-layers -  (Fig. 0.7  has  previously  and  um  to  1983,  HL  the  coarse  after  coarse  outer  consisted  Abed  prepared  this  layer  with  described  1986;  outer  intervillus  um,  fixed  embryos were  The  The 1.2  Abed,  The  the  24a).  been  and  1990).  purposes.  H2,  embryos  Crawford  1989,  comparative  five  2  1978,  ochraceus  and in  preparation  meshwork, -  in  the  total  meshwork, which of  random clumps  of consistent density throughout; the 150 nm thick boundary layer  - 58 -  F i g . 24a:  TEM  of  the  hyaline  glutaraldehyde stained five  with  and  and  uranyl  coarse  outer  TEM  of the  in  F i g . 24a  densa  (LD).  indicated Dr. B.  basal  and  and  embedded lead  intervillus H2);  embryo  the  fixed in  citrate sub-layer  Epon,  and  showing  the  (IV);  the  boundary sub-layer  (OM).  with  (B);  x35800.  (photograph  of an embryo prepared  as described  Crawford).  lamina  fibrous  (arrowheads).  Crawford).  an  blue,  meshwork  showing the The  of  acetate The  layer (HI  courtesy of Dr. B.  F i g . 24b:  alcian  sub-layers:  supporting  layer  lamina  lucida  (LL)  components of the x33200.  and  the  lamina  blastocoel are  (photograph  courtesy  of  59  - 60 -  F i g . 25:  TEM  of an  embryo  prepared  f i b r e s (arrows) radiating cell  F i g . 26:  (M).  X26500.  web (arrows).  i n F i g . 24a  from the esophagus (Es).  showing  Mesenchyme  (photograph courtesy of Dr. B. Crawford).  TEM of an embryo fixed dorsal  as described  as described  Ectoderm  i n F i g . 24a showing the  (Ec); Endoderm (En).  (photograph courtesy of Dr. B. Crawford).  xl0400.  61  - 62 -  appeared after  to  contain  looping  cryofixation  electron  dense; H2  cryofixation; lacking  the  and  structures  which  were  but  somewhat condensed; HI  was  amorphous and  the  intervillus  lacked  layer  similar  was  50  the  was  nm  to  that  seen  and  very  thick  homogeneity  seen  r e l a t i v e l y electron  abundant e x t r a c e l l u l a r material  seen  after  after lucent,  cryofixation.  A  d i s t i n c t H3 sub-layer could not be seen a f t e r t h i s f i x a t i o n . The could  BL,  in general, was  also  be  previously  separated  described  contained many 30  into  by  nm  thinner a  (Fig.  dispersed  3.6  nm)  than  densa  (1989).  the  and  a  The  and  g e l - l i k e ECM  in  in greatest  the  dorsal  in the blastocoel  web  cryofixed lamina  adjacent  thick, dense f i b r e s ( F i g . 24b)  25)  Histochemistry  lamina  Crawford  embryos, these f i b r e s were found esophagus  (10  but  and  lucida  as  blastocoel  as with cryofixed  numbers radiating (Fig.  BL  26).  from  There  was  the no  a f t e r t h i s type of f i x a t i o n .  (Table I)  PAS Cryofixed  embryos embedded  granules which stained  intensely  lightly  (Fig.  PAS  paraffin the  positive  had  basement  weakly p o s i t i v e membrane  and  in JB4 PAS  27). PAS well  had  numerous  p o s i t i v e and Formalin  large i n t r a c e l l u l a r  a HL  fixed  embryos  staining areas in the defined  positive  which stained embedded  hyaline  staining  layer  of  very in and  cellular  granules ( F i g . 28).  A l c i a n blue At pH the  3.2  blastocoels  alcian  and  2.5,  of the  blue p o s i t i v e  the  hyaline  cryofixed ( F i g . 29,  and 30).  layer, basement membrane, and formalin A  few  ECM  of  fixed embryos were intensely small  i n t r a c e l l u l a r granules  - 63 were a l c i a n blue p o s i t i v e i n the cryofixed not be resolved At  i n the formalin  pH 1.0, both  intensely  positive  alcian  blue  blue  and the formalin  staining  of  the  fixed  of the hyaline  layer  and  cryofixed  embryos stained  When stained the  cryofixed  was  present  the  formalin  and the  intensely  positive but only  The blastocoel  moderately a l c i a n blue p o s i t i v e .  p o s i t i v e c e l l u l a r granules could not be resolved  showed  layer  the blastocoel,  moderately p o s i t i v e staining of the basement membrane. the  embryos  hyaline  Formalin fixed embryos also revealed  staining  could  material.  the cryofixed alcian  basement membrane.  fixed  embryos but such granules  i n either  of  Alcian blue  preparation.  at pH 0.5 and 0.2, there was no a l c i a n blue staining in  embryos.  However,  i n the hyaline fixed  intensely  positive  alcian  blue  staining  layer, basement membrane and the blastocoel  embryos  at  pH  0.5  and  moderate  staining  of  of  these  components was present at pH 0.2.  PAS/Alcian blue (DH 2.5) Embryos prepared by cryofixation and formalin staining  patterns  to that  granules  were  resolved  i n the JB4 embedded  PAS  described  positive;  a  above:  few  small  f i x a t i o n showed  only the large, granules  that  similar  intracellular  could  only  be  embryos were a l c i a n blue p o s i t i v e ; the hyaline  layer, the blastocoel, and the basement membrane were intensely a l c i a n blue positive.  Toluidine  blue  Neither metachromasia. in  JB4, could  plastic  resin.  the cryofixed This  nor the formalin  came as no surpise  not be hydrated  due  fixed  embryos  as the cryofixed  to the hydrophobic  The embryos fixed i n formalin  demonstrated  embryos,  embedded  properties  of the  and embedded  i n p a r a f f i n , on  - 64 -  the other hand, were so shrunken and damaged that even i f metachromasia was present, i t could  not be c l e a r l y resolved.  showed  when stained  basophilia  cryofixed  embryos  demonstrated  was  with  intensely  moderate basophilia.  embryos exhibited  t o l u i d i n e blue.  basophilic The hyaline  moderate basophilia.  demonstrate basophilia i n either  Embryos  while  fixed  by both methods  The hyaline  layer of  the basement  membrane  layer of the formalin  The ECM of the blastocoel  preparation.  fixed  did  not  - 65 -  T a b l e I : The R e s u l t s o f H i s t o c h e m i c a l S t a i n i n g o f C r y o f i x e d and F o r m a l i n F i x e d A s t e r o i d  Histochemical stain  Cryofixed embryos  PAS  A l c i a n blue pH 3.2  A l c i a n blue pH 2.5  A l c i a n blue pH 1.0  A l c i a n blue pH 0.5  EmbryosJ  Formalin fixed embryos  ++  ++ +/-  —  —  -  +/-  +/++ ++ ++  _  ++ ++ ++  +/++ ++ ++  ++ ++ ++  _  _  ++ +/++  ++ ++ +/-  _  _  -  ++ ++ ++ _  A l c i a n blue pH 0.2 —  PAS and A l c i a n blue pH 2.5  G HL b BM G HL b BM G HL b BM  G HL b BM  +/-  -  —  +/-  2  G HL b BM  PAS++:ABPAS-:AB+/PAS-:AB++ PAS-:AB+/_  staining;  G HL b BM  G HL b BM  Toluidine blue  '++, intensely p o s i t i v e no staining v i s u a l i z e d .  2  +/+/+/-  PAS++:AB+/PAS -:AB++ PAS -:AB++ PAS -:AB++ ++  Component  —  +/-, moderately  positive  G HL b BM staining; -,  G, granules; HL, hyaline layer; b, blastocoel; BM, basement membrane.  - 66 Figs. 27,  29  and  propylene  glycol  30  are  2.0  um  prior  to  freezing  stained with either PAS  F i g . 27:  sections  of embryos cryoprotected  in  propane,  embedding  Cryofixed  embryo, stained  staining  in  7 um  section  showing  the  with PAS,  intracellular  of  a formalin  intensely  The  resolved  F i g . 29:  moderately  granules  Cryofixed  embryo, stained  intensely  positive  Cryofixed a  similar  and  The  embryo, in  staining  seen in F i g . 29.  x380.  and  the  BM  with  PAS,  intracellular  staining  cannot  in  the  be  clearly  3.2,  showing  x460.  with a l c i a n blue at pH in the  HL  homogeneous staining  d i s t r i b u t i o n of  stained  moderately p o s i t i v e  staining  embryo, stained  (arrowheads)  x580.  and of  present but cannot be seen in this picture.  F i g . 30:  JB4,  showing intensely positive  staining  positive  in t h i s section.  (arrowheads).  fixed  positive  granules (arrowheads) and HL.  15%  or a l c i a n blue.  moderately p o s i t i v e staining in the HL.  F i g . 28:  in  with  basement the  blue  blastocoel  is  x390.  with a l c i a n blue at pH alcian  membrane  positive  2.5,  showing  material  as  - 68 -  4.  In conformity into two  4.1  DISCUSSION  with the objectives of this  study, the Discussion  sections.  Freeze Substitution of Asteroid Embryos  Conditions  f o r adequate plunge freezing  When plunge freezing an uncryoprotected occur only Robards  i f the  and  smallest  optimal  Sleytr  possible  conditions  (1985) are tissue  outlined  met.  block,  t i s s u e , adequate freezing w i l l  These  keeping  choosing  a cryogen with a melting  conductivity, high  melting  point.  s p e c i f i c heat, and  Tissue  always s u f f i c i e n t l y used  in  this  sizes of  small  study  point  this  only the number of embryos frozen at one Freezing  rates  are  dependent  (Van  embryos  stage  in  the  extracellular  larval  matrix  even more c r i t i c a l successful  This  has  to obtain  vitrification  solutions in l i q u i d 1982).  that  but  the  good thermal  and  involved  to  necessary though because the  not  embryos length),  time required attention. rate of the  heat transfer through  distance  from the  Crowe11, 1964).  maximum possible  applied  K,  (approx. 500um in  filled  a very poor thermal  studies  was  are  blastocoels  cryogens (Bruggeler  concept  1mm  size  upon the  Harreveld have  using  in motion, having a  less than 140  frozen  the t i s s u e which decreases exponentially as of the t i s s u e increases  cryogen  include  a b o i l i n g point f a r removed from i t s 3  less than  exceeded  Elder et a l . (1982) and  entry v e l o c i t y into the cryogen  to be thoroughly  never  by  conditions  the  s u f f i c i e n t depth of cryogen, having a high and  i s divided  surface  Since asteroid  with  a  hydrated  conductivity, i t became  freezing rates.  Previously  freezing extremely thin layers of  and  Mayer, 1980;  the  current  Dubochet et a l . ,  system  monolayer or sheet of embryos on a copper freeze fracture EM  by  placing  a  g r i d , removing  - 69 -  as much water from the grid as possible into the cryogen with fine-tipped the  low mass  and high  thermal  and immediately  plunging  s t a i n l e s s steel forceps. conductivity,  these  the grid  In addition to  grids  allowed  those  specimens i n the windows o f the grids to be exposed to the cryogen on both sides the on  simultaneously,  unlike  solid  supports which allow only  specimen to come i n d i r e c t contact EM grids  using  i s not e n t i r e l y new.  copper  kidney c e l l s have  never  mesh  grids  on gold been  while  grids.  cryofixed  with the cryogen.  Adrian  Porter  one side of  Freezing  et a l . (1984) cryofixed  and Anderson  (1982) froze  To my knowledge, whole eukaryotic successfully  p r i o r to the present  tissue viruses cultured  organisms  study  using  t h i s method. It  i s generally  optimal  freezing  accepted  although  t h e i r cryogen was s t i r r i n g stirring  of cryogen  that  cryogen  few authors  stirring  actually  indicate  i s essential f o r whether  or not  at the time the tissue was plunged into i t . The  i n the present  study was i n a horizontal  direction.  Murray et a l . (1984) have devised a countercurrent plunge freezing system in  which  the cryogen  horizontal the  stirring,  circulates  vertically.  circumferential  They  maintain  v e l o c i t y differences  depth of the cryogen due to a s t i r r i n g vortex create  temperature because stirring  gradient  successful  which  i s detrimental  cryofixation  was  that  during  and variations in a large  vertical  to the speed of freezing.  eventually  achieved  with  But,  horizontal  in the present study, the s t i r r i n g apparatus was not altered, and  i t was, therefore, not necessary to test these findings. The  importance  of the plunging  velocity  (Stephenson, 1956) but there i s a now consensus high plunging v e l o c i t y correlates well  was somewhat controversial i n the l i t e r a t u r e that a  with the depth of freezing  into the  tissue ( C o s t e l l o and Corless, 1978; Elder et a l . , 1982; Robards and S l e y t r , 1985).  Numerous mechanical plunging devices ranging from spring  activated  - 70 -  triggers  (Robards  and  Crosby,  1983)  electromagnetic  plungers (Escraig, 1982)  standardize  maximize plunging  and  has shown that plunging frozen  specimens and  embryos  in t h i s  cryogen  in  (1986), i t was relationship grids  costly  Steinbrecht  v e l o c i t y i s proportional  not  quickly  that  were  plunged  pre-cooling  induced  in  As  a  the  cryogen  as  rapidly  evaporating  to  Because the  hand  into  and  the  Childress  velocity:freezing efficiency  system.  the  by  Allenspach  this  into by  m/s)  by  never determined i f a plunging  in order  only to the y i e l d of well  (>1.0  described  operated  (1982), however,  to the o v e r a l l q u a l i t y of f r e e z i n g .  s i m i l a r to  existed  solenoid  have been constructed  velocities.  study were plunged  manner  to  rule  nitrogen  of  thumb, however,  as  and  possible  cryogen and  to  the  avoid  to expose  the entire grid surface to the cryogen as synchronously as possible.  Cryogens Other conditions properties point are  of  (mpt),  the  should  cryogen.  boiling  most be  the  required  low  point  important (<140  for optimal  From  a  thermodynamic  (bpt), s p e c i f i c  parameters  K) and  the bpt  cryoquenching revolve around the  (Table should  viewpoint,  heat, and II).  The  be high  the  melting  thermal  conductivity  mpt  the  (>175  of  cryogen  K), such that  the  difference between them i s as wide as possible (Robards and  S l e y t r , 1985).  If  phenomenon  the  result  mpt  and  bpt  are  (Robards  and  Sleytr,  formation  boil.  a  high.  1985).  This  specimen causing  slows  widespread ice crystal is  f a r removed, the The  Leidenfrost  Leidenfrost  of a vapour jacket around the specimen due  temperature of the to  not  measure  of  the  the  storage  is  the  to the r e l a t i v e l y high  the cryogen immediately surrounding i t  freezing  formation.  phenomenon  may  rate  The  capacity  dramatically,  specific of  heat  This ensures temperature s t a b i l i t y during  thereby  allowing  heat of the cryogen, which per  unit mass, should  specimen plunging  be  because  - 71 -  the  cryogen  effecting  i s able  i t s own  to absorb  the  temperature.  temperature  Finally,  of  the  the thermal  specimen  without  conductivity  cryogen, the a b i l i t y to carry away heat, should also  of the  be high in order to  disperse the heat throughout i t s volume and to maintain the temperature at i t s melting point. On study;  the basis of these c r i t e r i a , Freon  several  12,  liquid  qualities  that  nitrogen  four cryogens were chosen  slush,  theoretically  ethane  and  f o r this  propane.  made them s u f f i c i e n t l y  Each  good  candidates to study frozen asteroid embryos at the u l t r a s t r u c t u r a l Since  cryogen  level.  halocarbons have been commonly used as cryogens (Rebhun,  Rosenkranz, readily  had  1975;  Somlyo et a l . , 1977;  available  Nagele  et  a l . , 1985;),  1972;  and  were  in the lab at the beginning of the present study, Freon  12 was the f i r s t tested.  The results c l e a r l y demonstrate  properties of Freon 12 f o r this system. the bpt 243 K (Table I I ) , thus  The mpt  satisfying  the poor freezing  of Freon 12 i s 115 K, and  the above c r i t e r i a  f o r low  mpt  and a f a r removed bpt (128 K).  The s p e c i f i c heat and thermal conductivity  values  low,  are  also  respectively. Freon  Since the  12 did not  specimen  relatively  thus  latter  maintain  resulting  two  i t s mpt in  0.854  J/g/K  and  values are low, temperature  conditions  138  mJ/m/s/K,  i t i s possible  upon  introduction  which  encouraged  ice  used  as a cryogen  that  of the crystal  formation. Liquid 1975).  nitrogen  Because  LN2  procedures  would  inexpensive  and  coolant mpt,  has  been  is boiling result  easily  in  previously  at room temperature, the  obtainable,  f o r other "cryogens  Leidenfrost in the  past  than as a cryogen  however, the Leidenfrost e f f e c t may  i t s use  effect. i t was  itself.  (Rosenkranz, in cryogenic  Since used  more  I f reduced  be suppressed because  LN2 as  is a  to i t s  some of i t s  thermodynamic properties improve to levels acceptable f o r the present  - 72 -  TABLE I I :  The Thermodynamic Properties o f the Crvoaens U s e d J  Cryogen  mpt  bpt  AT  C  k  Freon 12  115  243  128  0.854  138  Nitrogen  63  77  14  2.06  153  Ethane  90  184  94  2.27  240  Propane  84  231  147  1.92  219  mpt = Melting point i n Kelvin bpt = B o i l i n g point i n Kelvin AT  = Difference between the mpt and bpt  c = S p e c i f i c heat i n joules/gram/Kelvin  near the mpt.  k = Thermal conductivity i n millijoules/metre/second/Kelvin  r e f . Weast, R. (1982).  near the mpt.  - 73 -  study. 2.06  The  J/g/K  specific and  heat of LN2  the mpt  slush i s higher  asteroid  embryos,  however,  should make LN2  revealed  cellular  the  Leidenfrost e f f e c t .  mpt  and the bpt, and  Because only  the thermal  higher than Freon at 153  form  resulting  in  a 14  crystal  This was  K gradient  that Tests  damage  probably  and  due  to  exists between the  conductivity i s poor, being only  slightly  mJ/m/s/K (Table I I ) , i t i s possible that a vapour  dramatically slow the a  a good cryogen. ice  extensive phase separation within the b l a s t o c o e l .  jacket, which would  that of propane at  i s extremely low at 63 K (Table II) suggesting  these features, at least in theory, on  than  poor  structural  freezing process,  preservation  of  the  could  easily  embryos.  Bald  (1984) has shown that LN2  slush would make an excellent cryogen, in theory,  at  these  33.5  atmospheres  but  conditions  are  beyond  the  ability  of  this  laboratory so t h i s theory remains to be tested. It  has  been  previously  plunge  freezing  cryogen  for  1985).  Ethane  has  the  stated  that  (Silvester  highest  liquid  et  ethane  a l . , 1982;  specific  heat  (2.27  i s an  excellent  Dubochet  et a l . ,  J/g/K) and  thermal  conductivity (240 mJ/m/s/K) values of the four cryogens used in this and  i t s mpt  (90  These outstanding freezing leaving  K)  i s comparable with  a 94  difference  that of propane (84 K) (Table I I ) .  thermodynamic properties should  of asteroid embryos.  However, the  K difference between i t and  than  i s present  in  Freon  study  12  bpt  have allowed of  the mpt and  ethane i s low which  propane  possible that the Leidenfrost phenomenon played  for optimal  is a  (Table  (184  much II).  K)  lower It i s  a role in the overwhelming  phase separation in the blastocoel of the asteroid embryos resulting in the widespread cryogen. larger with  a  strand-like The  but  pattern  fewer  higher  ECM of  strands  mpt/bpt  structures  ECM seen  seen  seen a f t e r after  differential  freezing  ethane f i x a t i o n  liquid in  after  nitrogen  ethane  than  with  relative  fixation in the  this to  the  correlates  latter.  This  - 74 -  suggests that due to the higher ethane d i f f e r e n t i a l , the good s p e c i f i c heat and thermal conductivity, ice c r y s t a l s did not have as much time to grow as in  prior  fixations,  but  there was  s i g n i f i c a n t l y disrupt the  still  enough i c e crystal  formation to  ECM.  Propane has been used as a cryogen by numerous researchers (Burnstein and  Maurice,  1978;  Steinbrecht, 1982;  Elder  et  a l . , 1982;  Porter  Meissner and Schwarz, 1990).  between the bpt (231  K) and mpt  Bachmann  'useful  (1982),  stabilization  'useful  difference  differential J/g/K  conductivity i s 219 mJ/m/s/K  heat-sink capacity', a term coined by Plattner and  is  the  temperature  this case 84 K.  The temperature  1982;  Its s p e c i f i c heat i s 1.92  which ranks j u s t behind ethane and i t s thermal The  Anderson,  (84 K) i s 147 K, the largest  of the four cryogens tested i n this study.  (Table I I ) .  and  energy  (approx.  difference  173  K)  and  between  the mpt  the  specimen  of the cryogen, in  Propane, according to the above authors, has  the highest  heat-sink capacity' of the cryogens they reviewed (120 0/ml), which  included optimal  the  four  used  in  this  study,  suggesting  cryogen of those tested f o r u l t r a s t r u c t u r a l  that  propane  studies.  is  the  In addition,  according to the c r i t e r i a suggested by Elder et a l . (1982) and Robards and Sleytr  (1985),  propane should be an excellent cryogen f o r plunge  and the r e s u l t s from this study support t h i s . was  freezing  C e l l u l a r ice crystal damage  present a f t e r propane freezing but the phase separation in the asteroid  blastocoel damage  was  could  not  as  easily  distinct  be  as  in the  distinguished  at  previous the  EM  cryofixations.  level,  however.  Such Since  propane i s one of the most e f f e c t i v e cryogens known, these results probably r e f l e c t the thickness of the embryos and the accompanying water on the grid and  suggest  achieved  that  optimal  by cryoquenching  freezing  for  ECM  in propane alone.  preservation  could  not  be  It therefore became necessary  - 75 -  to  incorporate  cryoprotectants  into the embryos i n order  to intervene i n  the i c e crystal growth process.  Cryoprotectants Cryoprotective tissues  by  capacity,  chemicals  lowering  the  decreasing  freezing  for crystallization  these with  ethylene  increasing  cooling  rate,  the  supercooling  lowering  the eutectic  the quantity of 'unbound' water and removing  (Robards and S l e y t r ,  l i k e l i h o o d of v i t r i f i c a t i o n . DMSO, g l y c e r o l ,  point,  the c r i t i c a l  point, removing or decreasing nuclei  e s s e n t i a l l y impede i c e c r y s t a l i z a t i o n within  1985) thus  The four cryoprotectants  glycol  and propylene  used i n this  glycol  d i f f e r e n t e f f i c i e n c i e s which are r e f l e c t e d  enhancing the study,  do some or a l l of i n the cryoprotected  embryos. The  success  molecular  of a perspective cryoprotectant  structure.  cryoprotectants. and  non-toxic  Polyols  have  been  used  G l y c e r o l , f o r instance,  to most  living  systems  seems, to depend  upon i t s  extensively i n the past as  i s non-destructive  (Fink,  1986).  to proteins  The three  hydroxyl  groups ^enable multiple hydrogen bonds to form per molecule which interfere  with  molecules.  the  mobilization  Glycerol  substantially  is a  retard  very  the growth  of  ice crystals  viscous  liquid  of c r y s t a l s  by  occupying  which  (Skaer,  readily  in  itself  1982).  water may  When 10%  glycerol was used to cryoprotect the asteroid embryos, i c e damage and phase separation exposure  i n a l l regions  to a l l cryoprotectants  incomplete that  were present  there  was  of the embryo.  held  constant,  Since the time of  i t i s u n l i k e l y that  penetration was the cause of poor cryoprotection. was  concentration  an i n s u f f i c i e n t was  increased  concentration to  15%.  of g l y c e r o l ,  These  results  This suggests therefore the  showed  superior  u l t r a s t r u c t u r a l preservation with no evidence of i c e damage but the osmotic  - 76 -  e f f e c t s o f glycerol were quite evident. to  cryoprotect  t i s s u e s , so the artefacts induced  Shrinkage o f the c e l l s preserved or  Glycerol i s used  by many  by i t are well  i s one such a r t e f a c t (Skaer,  1982).  glycerol  The ECM was  to i n t e r a c t  This  with  i s probably  reflected  unbound water which  by the a b i l i t y of  normally  would  have formed  ice c r y s t a l s , pushing the ECM to the periphery as large strands. though  hypothesis the  the ECM pattern  retaining  correlates  with  However,  the g e l - f i l l e d - b l a s t o c o e l  proposed by Strathmann (1989), the vast amount of shrinkage and  inability  glycerol  known.  i n a g e l - l i k e pattern, which indicates the presence o f amorphous  perhaps vitreous i c e .  even  authors  to o f f s e t  thorough  this  by decreasing  preservation  as a good candidate  without  the concentration  i c e crystal  while  formation  still  eliminates  f o r cryoprotecting asteroid embryos under the  described conditions. Dimethylsulfoxide it  i s used  Sleytr,  almost  1985),  (DMSO) i s a well established cryoprotectant.  e x c l u s i v e l y for preserving  little  work  cellular ultrastructure. bonds  only  potential  concentrations,  this  done  to show  protons. as  lack  the  I t therefore polyols.  o f hydrogen  If  bonding  However, when used at concentrations  greater  toxic  When applied  (Robards  concentrations crystal  and S l e y t r ,  1985).  o f 5% and 10%, DMSO f a i l e d  formation  viability  suggesting  than  (Robards and  how DMSO  DMSO i s a polar molecule that  by accepting  interacting  has been  cell  Since  preserves  can form hydrogen  does  not have  used  in  high  as much enough  may be compensated  for.  15%, i t s effects become to asteroid  embryos at  to protect the embryos from ice  that i t inadequately  i n t e r a c t s with  'unbound'  water. Ethylene  glycol  (Richter, 1968a). is  lower than  has r a r e l y been used with  success  as a cryoprotectant  Its hydrogen bonding capacity exceeds that o f DMSO but  glycerol due to one less  hydroxyl  group.  I t can interact  - 77 with  'unbound' water molecules  interact  with  itself  characteristics. freezing was glycol.  The  to impede ice crystal formation but may also  thereby results  blocking  of  the  inconsistent throughout  When  used  at  a  its  present  starfish  concentration  of  own  study  10%,  a  of  quality  suggests first,  but  this  that at the  and  diminished  surface of the  f o r a small  the  However, further into  ethylene g l y c o l , though s t i l l compensate thermal  f o r the  fall  i n the  the  embryo,  glycol would large,  may be  revealed be  no  freezing  This allowed  induce  i c e crystal  organisms  damage.  which  such  as  i n t e r f e r e with  heat the  ethylene glycol.  glycol  It  the poor  f o r the growth of ice  some areas of the embryo largely  in the center of  i s possible that  must  be  ice crystal  ethylene  since there  transferred.  But,  for  growth in the center of the Such concentrations would  shrinkage  rendering  the material  study. is  methylated,  the  Fahy et a l . (1987) discussed  upon polyols and  cryogen  embryonic asteroid, ethylene glycol  large amounts of osmotic  unsuitable f o r u l t r a s t r u c t u r a l  methylation  the  This  ECM.  embryo unless very high concentrations are used.  propylene  contacted  rate brought about by  However, many areas,  blastocoel from  does not adequately  When  center.  a good cryoprotectant f o r monolayers of c e l l s ,  hydrated  probably  the  embryo, the concentration of  When the ethylene glycol i s increased to 15%,  the  was  adequate to i n h i b i t resolvable  c r y s t a l s r e s u l t i n g in separation of water and  preserved.  gradient  the same, does not appear to be s u f f i c i e n t to  conductivity of the blastocoel.  were very well  ethylene  blastocoel the concentration of  ethylene glycol and the rate of freezing was phase separation.  that  good at the surface  towards  embryo, which  distance into  demonstrated  freezing  The q u a l i t y of freezing was  embryo  binding  embryos treated with  present i n the blastocoel. the  water  resulting in d e t a i l  molecule  is  the effects of  amides with respect to v i t r i f i c a t i o n .  Methyl  - 78 -  groups appear to allow v i t r i f i c a t i o n to occur at much lower concentrations than  the  same molecule  possible  that  with  a terminal unsaturated methyl  terminal methyl  groups  produce  of water.  'self-linking'  of the molecule, though usually only a problem  allowing  the  molecule  to  these  enthalpically  reorganization  thereby  In addition,  an  group.  It i s  favourable  terminal methyls may  fulfill  its  complete  inhibit  with amides,  water  binding  potential. Propylene  glycol  was  determined  cryoprotectants studied here, and  to  Boutron  be  and  the  best  of  i n t e r f e r e with ice crystal  excessive  shrinkage  fixation  to  Currently,  in  reveal  the  propylene  more  glycol  and Armitage, use  1990).  of propylene  has  detailed  is  cryopreserve viable blood c e l l s  embryos  also  allowed  for  ultrastructure  being  (Takahashi  studied  not  this  et a l . , 1986)  induce  type  discussed  for  this  Its a b i l i t y  growth on a consistent basis and  asteroid  four  Kaufmann (1979) showed  to be also true in binary systems (cryoprotectant plus water). to  the  below.  its ability  and  of  to  corneas (Rich  Not a l o t of rapid freezing studies have involved the  glycol  so the  present  study  certainly  demonstrates  its  u t i l i t y as a cryoprotectant f o r plunge freezing marine invertebrate embryos. Since propylene  glycol  has  one  free methyl  enhance v i t r i f i c a t i o n , i t would seem logical a  methyl  produce  group a  to  better  the  other  terminus,  cryoprotectant than  1986; Mehl and Boutron, 1987).  group,  and  methyl  groups  to assume that the addition of  creating  propylene  2,3-butanediol,  glycol  This compound, however, was  (Boutron  would  et a l . ,  not u t i l i z e d in  this study but might prove useful in future work.  Freeze substitution As asteroid  discussed embryos  in the was  to  Introduction, the preserve  them  aim  of  freeze  in manner which  substituting  induced  minimal  - 79 -  artefact,  including  chemical placed  the  denaturation of  treatments used  of  the  lack  caused  in conventional f i x a t i o n .  i n a -60 °C freezer, was  because  proteins  of  a  used as a chamber f o r freeze  -90  °C  freezer  as  the  substituting  inexpensive, 1941;  and  had  been  Fernandez-Moran,  medium  successfully  1960).  which  Acetone but  the  chances  of  (mpt=177 K) was  its inferiority  solubility  used  first  formation  at  quickly  realized  than acetone  sometimes used in conjunction Schwarz,  1990),  has  readily  available,  other authors  (Simpson,  a four day duration to  during  due  to  the  substitution.  i t s relatively  substituting  with aldehydes  material  (Glauert,  1974;  an  and  would  do  Recently, methanol,  (Zalokar, as  poor  Ethanol (mpt=158  fluid  i f necessary.  become widely accepted  medium f o r b i o l o g i c a l  the  Anhydrous ethanol was  i n i c e and poor water transporting a b i l i t i e s .  lower temperatures  provide  used as a substituting medium in this study  K), therefore, served as a more e f f i c i e n t so  substitution  would  was  by  harsh  i s very slow at such low temperatures, thus  i c e crystal  was  it  The substitution was  ensure completion as d i f f u s i o n reducing  because  the  A wide-mouth Thermos,  temperature necessary f o r the substitution to occur. used  by  1966;  Meissner and  excellent  Barlow  and  substituting Sleigh,  1979;  Steinbrecht, 1982; Maitland and Arsenault, 1989) but i t was not u t i l i z e d in this 190  study. K  to  Since the temperature of the substitution chamber ranged from  170  K,  depending  on  how  recently  (mpt=175 K) could have frozen which would, substitution. likely.  By  Methanol  using has  ethanol, been  such  well  the LN2  was  in e f f e c t ,  accidental  documented  freezing as  whereas  acetone  has  et  al.,  1965;  Unfortunately,  little  Harreveld  one  work has  of  the  Zalokar,  slowest 1966;  methanol  have terminated the was  having  substitution time below 183 K (Barlow and Sleigh, 1979; 1984)  added,  the  less  fastest  Humbel and Muller,  substitution Muller  much  et  been done in determining the  times al.,  (Van 1980).  substituting  - 80 -  properties  of  ethanol  other  than  being  shown to have better substituting  c a p a b i l i t i e s than acetone but not better than methanol (Steinbrecht, 1982). As  with  any  substituting f l u i d ,  certain  cellular  components  are  soluble in ethanol, thereby  r e s u l t i n g i n the r e l o c a t i o n or removal of such  compounds.  Harvey (1982), i t would  then,  As  if  suggested  by  autoradiographic  or  cytochemical  come as  analysis  no surprise, revealed  low  concentrations or inappropriate d i s t r i b u t i o n s of c e r t a i n ions, for example, in  freeze  substituted asteroid embryos.  p r i m a r i l y morphological  and  not  further  be  discussed  any  But  histochemical other  since the  in nature,  than  to  present  these  study  drawbacks  acknowledge  their  was will  probable  existence f o r future reference.  4.2  Morphology and  Histochemistry  Blastocoel The alcian  ECM  blue  of the blastocoel of cryofixed, JB4 p o s i t i v e at pH 3.2,  2.5,  and  1.0.  embedded  When fixed  embryos stained in formalin  embedded in p a r a f f i n , however, additional p o s i t i v e s t a i n i n g was blastocoel  at  pH  0.5.  glycosaminoglycans Macromolecules necessary 1974;  sulfate. the  f o r mesenchyme  have  molecules  can  are  cell  as  of  such  both  the  components in  Lane and  sulfate,  have  echinoids  Solursh, 1988).  some  of  these  chondroitin  and  similar  from the echinoid (Sugiyama, 1972;  be postulated that proteoglycans  larval  unsulfated blastocoel.  been  shown  (Karp  and  to  be  Solursh,  Solursh and Katow  components 6-sulfate  staining in the developing  components,  seen in the  sulfated and  within  migration  characterized heparan  that  present  sulfated  Since histochemical  presence  suggests  Solursh, 1981;  further  such  been obtained it  (GAG's)  containing  Katow and  (1982)  This  and  to  and  be  GAG  dermatan  asteroid suggests  histochemical  results  have  Katow and Amemiya, 1986),  with sulfated and unsulfated acidic  - 81  GAG's  as  well  blastocoel using  as  glycoproteins  in the  the  critical  Dorling, 1965) specific  larval  may  blastocoel  stage.  major constituents  Histochemical  electrolyte  allow  sulfated  are  -  GAG's.  suggests that  The  lack  neutral  of  the  staining with  concentration  f o r a more accurate  of  (CEC)  method  ECM  of  the  alcian  blue  (Scott  and  assessment of the presence of  positive  GAG's such as  PAS  s t a i n i n g within  hyaluronic  acid are  the  not  a  major constituent of the blastocoel at this stage of morphogenesis. Ultrastructural five-day-old short  Pi saster  f i b r e s of  fibers  in the  pattern  varying  following  rapid  Kraemer,  1989).  is  in  the  asteroid  past,  the  is  and  Chi a,  (Crawford  Observations experiments particles  of by  will  embryos and fluid-filled, dissection that  a  chemical  gel  as  to  that  lack  a  in  Luyet,  the  twig-like distinct  regions  seen  of  the  artificial 1962,  amorphous  fixed  1982;  would  to the  fixation  may  work  substituted  1967;  coat  embryos  meshwork  of  population  of  embryos.  The  gels  of  ECM  Allenspach  and  of a l c i a n o p h i l i c  described  previously  been argued as to whether the blastocoel of with  Abed  Crawford,  embryos  (1989), the  e x t r a c e l l u l a r f i b r e s or  and  fixed  penetrate  occupies  ultrastructural  well  a  freeze  favour  however, opened  1986b;  have  former.  Recent  demonstrated of  expect  the  embryos  to  living  collapse  sudden drop in hydrostatic pressure. the  primary  represent on  body  cavity  elements  echinoids  fixed  of by  and  the  the  collapsed  freeze  gel-filled 1990).  the  blastocoels  the  Crawford,  they also maintain t h e i r shape a f t e r being  one  due  as  dorsoposterior  fluid-filled  Strathmann  that  reveals  of  Abed and Crawford, 1986b).  chemically  not  embryos  chemically  i t has  embryo  blastocoel  (MacKenzie and  fibres  (Crawford and Chi a, 1982; In  and  fixation  seen  the  thickness  similar  The  of  ochraceus  esophageal  meshwork  material  analysis  that  small  echinoderm  cut open. after This  fibres  If  such  a  suggests  seen  gel.  substitution  after Recent also  - 82 -  suggests that the blastocoel 1986;  may  Summers et a l . , 1987).  be f i l l e d The  with a gel (Ruppert and  histochemical staining  of the  embryo with a l c i a n blue demonstrated a homogeneously stained blastocoel These  thus  providing  histochemical and  mentioned  studies  suggest  that  further  evidence  ultrastructural  support  Strathmann's  the blastocoel  is filled  f o r the  along  findings  in  with  a  gel  ECM  and  of  with  vivo  asteroid  within the  presence  findings  Balser,  a gel.  the  and  not  a  above  strongly  fluid  with  fibres. The advantages of a g e l - f i l l e d cavity, the  as discussed by Strathmann  embryo  to  positioning  assume  numerous  bands of c i l i a  blastocoel  over a f i b r o u s ,  (1989), are as follows:  shapes  which  can  A gel permits  facilitate  of the v i s c o s i t y  cellular  material  visco-elastic This  latter  of a g e l , large  such  as  a  simple,  point  especially  mesenchyme  pertains  cells  the esophagus, tube  manuscript possible  exist,  the  It  order  to  the  can;  develop with minimal  epithelium,  asteroid  and;  the  unnecessary.  embryo.  to the presumptive These muscle  constrict Upon  the attached radial  gel  by  During  esophagus and  c e l l s wrap themselves  i n t e r d i g i t a t i n g with each other to form a continuous  preparation).  During  esophageal lumen.  in  in  that  ectoderm.  outer  to  migrate  d i f f e r e n t i a t e into smooth muscle c e l l s .  muscular  larvae may  a fluid  properties of a gel would make opposing muscles  morphogenesis,  around  feeding  in optimal locations f o r food r e t r i e v a l ; a gel  can maintain the i n t e g r i t y of tentacles or lobes better than because  fluid-filled  relaxation,  allows  for  the  lumen  constriction esophageal  since  fibres  opposing  rebounding  of  of  the  (Reimer the  and  Crawford,  esophagus,  in turn  esophageal ectoderm  draw  muscles which,  i t is in the do  not  via  the  f i b r e s , p u l l s at the esophageal musculature thereby d i l a t i n g the is  plausible,  therefore,  that  opposition  to  the  esophageal  musculature could e x i s t but i t would be in the form of e x t r a c e l l u l a r fibres and a gel or ground substance-like connective t i s s u e .  - 83 -  Strathmann mesenchymal thereby  cells  may  has  I f true, migrate  the  along  Wolpert,1962,  further  possess  permitting greater  shapes. cells  (1989)  the a b i l i t y  freedom  for  will  of  need  guide  be modified.  then, mesenchymal c e l l s use fibrous ECM  elements  or  reform the gel  cells  theory, which or  epithelial  and  distributing  fibres  to  that  to dissolve  'contact-guidance'  networks  1963),  hypothesized  and  creating  hypothesizes  wires  that  (Gustafson  In the asteroid  and  embryo,  as guide-wires to traverse  the blastocoel.  Such f i b r e s are shown to e x i s t in two major regions in the  asteroid  embryo  -  previous  paragraph,  have shown by embryos  and  forming  traverse the  the  esophagus,  the dorsal web. that  the f i b r e s  (Abed  that  order  the  for  and cells  Crawford, to  by  the may  1986b).  in  the  and Chi a (1982) in  esophageal be  living fibres.  synthesizing or  However, i t i s possible  along  simultaneously dissolve the surrounding g e l .  discussed  Crawford  cells,  migrate  as  mesenchyme c e l l s  region occupied  which are not muscle forming  modifying in  from  time-lapse cinemicrography  actively  These c e l l s ,  radiating  a  fibre,  they  must  In addition, the v i s c o s i t y of  a gel may provide better support f o r mesenchyme c e l l s than would a f l u i d . It embryo. in  is The  also  possible  esophageal  that  such  fibres  maintain  the  f i b r e s described above, in addition  muscle opposition, may  stabilize  shape of to t h e i r  the position of the ectoderm  the role  opposite  the esophagus so that areas r o s t r a l and caudal to i t can continue to expand during  morphogenesis.  This  i s analogous  to  blowing  up  a  balloon while  maintaining a c o n s t r i c t i o n around the middle of i t so that only areas above and  below  central and  the  constriction  can  expand.  In  the  asteroid  c o n s t r i c t i o n or waist forms during gastrulation  caudal  mid-section.  regions The  of  the  migrating  embryo  expand  population  of  at  a  and  greater  mesenchyme  embryo then, a the oral rate  cells  than  may,  hood the then,  - 84 -  maintain the i n t e g r i t y of these f i b r e s in order to retain or dumb-bell The the  cylindrical  shape of the embryo seen in late gastrula.  role of the f i b r e s  larval  situated on the dorsal  stage of development.  the dorsal web necessary  the  and  structural  at t h i s  site.  web  Mesenchyme c e l l s  support does not  Perhaps,  the web  i s not obvious  are r a r e l y  appear to be  at  present in  particularily  i s forming in order to serve a  purpose at a l a t e r stage of morphogenesis.  Basal  lamina The  BL,  conditions  like  and  the  also  HL,  stained  lightly  PAS  alcian  blue  positive.  positive  Together  suggest the presence of sulfated and neutral GAG's. in  agreement with  uronic  acid-containing  glycoprotein  were  macromolecules fibronectin, 1980,  Davidson's  1983;  detailed  findings  sulfated  recognized.  in the  echinoid BL  laminin and Wessel  et  heparan  immunocytochemical  and  study  an  recent  observations  is  intensely have  PAS  and  McClay,  required,  in which positive  revealed  collagen types  proteoglycan  Wessel  acidic  These observations are  studies  including sulfate  a l . , 1984;  these  (1974) in the echinoid BL  GAG's  More  under  other  III and  (Spiegel 1987)  IV,  et a l . ,  A much more  however,  in  order  to  accurately determine the composition of the asteroid basal lamina. In  the present study, at the u l t r a s t r u c t u r a l  appears  'bushier'  Distinct  fibrils  respectively, This  suggests  fibrils  and  after  and  seem that ground  cryofixation  granules,  to make up the  BL  may  substance  which a be  instead  previously described (Crawford, 1989). better f a c i l i t a t e  may  large a  than be  after  of  more a  the basal  chemical  collagen  component  much  level,  and  of this  compact  mat  or  Such a loose network may  the d i f f u s i o n of molecules  fixation.  proteoglycan  ECM  loosely-knit  lamina  structure. network  of  feltwork as be able to  between the e p i t h e l i a and  the  - 85 -  blastocoel may  be  than would a t i g h t l y packed  a direct  Neither BL and  result  of  the  mat  collapsing  This l a t t e r morphology  effects  of  chemical  fixation.  the tubule-like structures nor the membrane puckerings between the the e p i t h e l i a , as observed  substituted  embryos.  transmitting  by Crawford  Perhaps these  mechanical  techniques.  extracellular then,  Spiegel  et  implicated  and  involved in  the  epithelia,  ^  perceived structure  fixation  blastocoel  be  in freeze  fixation.  Hyaline Layer The  (1989), were seen  structures, which may  forces between the  are also artefacts of chemical  Since  of ECM.  It  material however,  of the echinoid HL  was  once  enveloping  thought  the  has  to  evolved  be  developing  a  two  tiered  structure  (Wolpert  a l . , 1989),  and  now  three  four  (Lundgren,  1973;  a  Morrill  et  to  single  embryo  a  better  layer  (Herbst,  and  of  1900).  Mercer,  layered  a l . , 1987).  with  1963;  structure  is  Numerous functions  attributed to the HL include a s c a f f o l d f o r holding c e l l s together (Herbst, 1900;  Chambers, 1940;  Mercer,  1967;  Dan,  1960;  Vacquier and Mazia,  Gustafson and Wolpert,  1967;  Wolpert  1968;  1972;  Kane,  Adelson and Humphreys, 1988), a f i l t e r al.,  1989),  (Lundgren, Abed,  a  barrier  against  Citkowitz, 1971,  f o r nutrient c o l l e c t i o n  mechanical  and  bacterial  1973;  (Spiegel et perturbation  1973), and a source of l u b r i c a t i o n while swimming (Crawford  1986).  and  Since i t s morphology i s very complex, i t i s u n l i k e l y  and that  there i s only one function to the HL. The  HL  of  the  Pi saster  ochraceus  embryo  i s similar  function and probably composition to the echinoid HL. present study, as well following  chemical  sub-layers: The  as those reported e a r l i e r  fixation,  intervillus  have  layer;  shown the  that  in  structure,  Observations in the  (Crawford and Abed, the  HL  supporting layer,  consists  of  consisting  1986) five of HI  - 86 -  and  H2; the boundary  Abed, 1986). revealed  an  layer; and the coarse  unequal  distribution  has multiple functions.  (1986).  meshwork  Reimer and Crawford (1990), u t i l i z i n g  (Reimer and Crawford,  substitution  outer  i s quite  (Crawford  lectin  of glycoconjugates  1990), thus further supporting  histochemistry,  throughout  The morphology of the asteroid HL following freeze different  than  was described  by Crawford  that i n the blastocoel following freeze s u b s t i t u t i o n .  Like the capsule of a bacterium,  and Abed ECM, l i k e  Since the l a t t e r i s  now thought to be gelatinous (Strathmann, 1989), this outer  movement o f the animal  the HL  the idea that the HL  The coarse outer meshwork i s a homogeneously d i s t r i b u t e d  also a g e l .  and  ECM i s probably  t h i s outer gel may f a c i l i t a t e  through the water as suggested by Crawford and Abed  (1986) and may protect the e n t i r e embryo (Lundgren, 1973), p a r t i c u l a r l y the - inner layers o f the HL from perturbation. The  boundary  sub-layer  i s a repeating  series o f ECM  looping pattern has never been previously described this  stage,  i s purely speculative.  I t s continuity with  to be the major attachment to and perhaps a source meshwork,  increased  surface  the source are  this  arrangement  f o r these  of the g e l , this would  precursors  synthesized  area  looping  to the next  and  secreted  may  the outer meshwork  suggest that  sub-layer separately  the  such  to  provide  an  I f the boundary layer i s some sub-layers  as opposed by  Since i t appears  of the substance of the  be  two functions.  This  so i t s s i g n i f i c a n c e , at  suggests that i t anchors this gel to the rest of the HL.  outer  loops.  to each  of the HL  sub-layer  epithelium.  being  Pulse-chase  experiments would l i k e l y resolve this c o n f l i c t . In addition, m i c r o v i l l i structures  at  the  t i p of  terminated each  i n the boundary  microvillus  resembled  associated bodies v i s u a l i z e d i n other embryos by Spiegel the dense matrix  at the t i p s of m i c r o v i l l i  layer.  The ECM  the m i c r o v i l l u s  et a l . (1989), and  i n chicken i n t e s t i n a l  microvilli  - 87 -  (Mooseker  and  microvilli  Tilney,  and  plasmalemma  of  1975).  appeared each  to  Filaments terminate  microvillus  ran in  tip.  ECM  the the  length dense  fibres  of the area  asteroid  inside  then radiated  from the  external dense region, and became continuous with the boundary layer. external  component  of  h y a l i n - l i k e protein. microvillus studies,  microvillus  associated  body  may  Adelson and Humphreys (1988) l o c a l i z e d  associated  suggested  necessary  the  bodies of  that  hyalin  f o r morphogenesis  of  echinoids is  a  and,  based  the  This  contain  a  hyalin to the  upon  perturbation  major  cell-HL  adhesion  molecule  echinoderms.  Further  immunocytochemical  studies may prove useful in determining i f hyalin has adhesive functions in the  asteroid  epithelium body,  embryo.  the  continuity  i s a direct  physical  link  between  characteristics with  development,  of  the HL.  a monoclonal shed  the  exists  from  within  the m i c r o v i l l u s  the i n t r a c e l l u l a r  provide a mode of communication  I t i s possible that the c e l l s may  treated  which  to the attachment point of the HL,  the HL; i t may HL.  The  between  associated  components  the c e l l s  be capable of d i r e c t l y  For example,  when  asteroid  the  and  and the  controlling embryos are  antibody raised to t h e i r HL, the embryos retard  disrupted  HL,  and  synthesize  a  new  one  before  resuming normal morphogenesis (Crawford, personal communication). Sub-layers HI, H2 and H3  constitute  the supporting  layer of the HL.  HI appears to be the anchorage s i t e or foundation f o r the looping fibres of the boundary that  this  layer.  The  sub-layer also  the upper sub-layers. HI,  may  represent  blastocoelic and  anhydrous  matrix  density provides  and degree of compaction a great  deal  of HI  of structural  support for  Sub-layer H2, the homogeneous belt of ECM  a gel-like  substance which  or coarse outer  characteristics  may  meshwork. be  such that  i s less  phase  underlying  hydrated  Its molecular  suggest  than the  arrangement  separations  occur during freezing, thus, perhaps accounting f o r i t s homogeneity.  do not This  - 88 sub-layer provide  may an  act as  indirect  microvilli  passing  Sub-layer  H3  supporting  source  through  has  underlying' H2,  suggests  a filter  be  layer and that  a  The ECM  on  sub-layer  is  a  epithelium to  of  the  This  demarcation  A monoclonal  different  much more  fixation. remaining  be  This  gel  sub-layers  i n t e r v i l l u s layer may (Hall and Vacquier, The large  may  results  above  difference  that  due  in  the  incorporate  of  the  GAG's and  to  composition.  than  the  layer, contains arranged  The  lamina  ECM  hydrated  formation  pressure  to  during  maintain  components  described  than  the  of  the  i n the echinoid  analysis of the  HL  suggest that  throughout the  six sub-layers.  The  fact  a  that  and  compact  organization  and  not  P o s i t i v e PAS  results  were distinguishable in  embryos but were not as c l e a r in cryofixed material.  cryofixed embryos were embedded stains as  be  out  blue  H3  a small amount of neutral GAG's such as  i t s more dense  I f a thorough  Alcian  epithelium.  histochemical  conflict. carried  to  layer stained more alcian blue p o s i t i v e than the rest of the  chemically fixed fact  the  1982).  hyaluronic acid are present  is likely  layer.  between  composition  i c e crystal  hydrostatic  represent the apical  amount of a c i d i c  the supporting  the  the  sub-layer,  be a part of an extremely  s e n s i t i v e to  provide  and  to  antibody  the epithelium, the i n t e r v i l l u s  gel  would  due  boundary  components which appear to be much more heterogeneously  which  barrier,  communication).  This sub-layer may  not  or  previously.  layer.  other areas of ECM.  HL  way  line  of  personal  the  their  or  intervillus  sub-layer nearest  to  visualized  transition  i n t e r v i l l u s layer (Crawford,  et a l . , 1989)  attachment  been  the  this  of  i t s gel  never  may  (Spiegel  in the  easily  as  paraffin,  histochemical  future, this  staining suggests  that  i n JB4, may  be  need  The  a resin which does the  source  of  analysis of the asteroid ECM  variable w i l l  a  to be  this i s to  eliminated.  sulfated GAG's are a major constituent  - 89 -  of  the  asteroid  (Solursh  and  sulfate,  HL.  The  Katow, 1982)  proteoglycans As  previous  suggest that  chondroitin  the HL.  results from  6-sulfate  these  molecules  and/or  containing these GAG's could  studies  could  dermatan represent  a  in the  echinoid  be  heparan  sulfate.  Thus,  large component  of  in the blastocoel, staining with a l c i a n blue using the c r i t i c a l  e l e c t r o l y t e concentration  method (Scott and  Dorling, 1965)  may  allow for a  more accurate assessment of the presence of s p e c i f i c sulfated GAG's.  4.3  Summary The  initial  employable  goal  freeze  preservation starfish,  of  of  this  study was  substitution  cellular  and  Pi saster ochraceus.  to devise  technique  motor  filled  which  copper  with  with  constantly  considering  four  cryoprotecting  asteroid  into supercooled embryos  were  with  cryogen and  grids  separate  the  liquid  and  allow  of  the  and  into  four  embryonic  a small  in  were  the  cup  glycol  optimal  anhydrous  isolated  cryogen.  different  propylene  propane appeared to provide substituted  good  constructed  nitrogen,  Embryos  plunged  with  easily  inserted into the nitrogen, and  cryogen.  cryogens  embryos  freeze  would  A plunge freezing apparatus was  stirred  freeze-fracture  which  inexpensive,  e x t r a c e l l u l a r elements  which consisted of a Dewer f l a s k f i l l e d which was  an  a on  After  cryoprotectants, and  plunging  preservation. ethanol  at  them Frozen  -90  °C,  osmicated, and embedded for u l t r a s t r u c t u r a l and histochemical analysis. Results described  above revealed  substance fibres,  as  surrounding contain  from embryos preserved  with  that the blastocoel appears to contain a g e l - l i k e  extracellular fibres  previously the  three  using the freeze substitution technique  or  thought.  outside four  of  the  sub-layers,  In  and  not  a  fluid  addition,  the  embryo which  was  was  found  to  with  hyaline  extracelluar  layer,  previously  consist  of  at  an  thought least  ECM to six  - 90 -  sub-layers.  Histochemical  unsulfated  GAG's  differences  among  unique  present  demonstrated in  these  the sub-layers  suggest  while  may  functions  sub-layers. the  were  studies  Studies  others  of material  t i p s of the m i c r o v i l l i  layers.  that  have  preserved  presence o f m i c r o v i l l u s associated  that  some  sulfated and  The  morphological  sub-layers  functions  may  shared  by  have other  i n this manner also demonstrate  bodies.  and may represent  both  These are located  sites  o f attachment  at the  between the  m i c r o v i l l i and the hyaline layer. Although f i x i n g asteroid embryos by freeze process,  taking  particularily fixations. histochemical are  four  o f the ECM Material  five  studies.  technique  substituted  the  resulting  justifies  i t s use  freeze  substitution  could  preservation,  With very  cultured  encouraging (M. E l l i o t , personal  some  likely  tissue. rat  over  chemical  can be  used f o r  modification,  In f a c t ,  preliminary  epithelial  communication).  metals  may also prove useful f o r this  be applied to other  kidney  lengthy  preservation,  and, i n addition, since aldehydes and heavy  embryos and perhaps vertebrate freeze  by  f o r successful  immunocytochemical  days,  components,  preserved  studies  not necessary  substitution  to  substitution i s a  freeze  invertebrate results from  cells  are  very  - 91 5.  REFERENCES  Abed, M. and B.J. Crawford (1986a) U l t r a s t r u c t u r a l aspects of mouth formation i n the s t a r f i s h Pisaster ochraceus. J . Morphol. 188:239-250. Abed, M. and B.J. Crawford (1986b) Changes i n structure and location of e x t r a c e l l u l a r matrix of the blastocoel during development of the s t a r f i s h Pisaster ochraceus. Can. J . Zool. 64:1436-1443. Adelson, D.L. and T. Humphreys (1988) Sea urchin morphogenesis and cell-hyalin adhesion are perturbed by a monoclonal antibody hyalin. Development 104:391-402.  for  Adrian, M., J . Dubochet, J . Lepault and A.W. McDowall (1984) Cryo-electron microscopy of viruses. Nature 308:32-36. Allenspach, A.L. and C P . Childress (1986) Morphology of the chick embryonic axis a f t e r quick-freezing. J . Cell B i o l . 103:245a. Allenspach, A.L. and T.G. Kraemer (1989) Ice crystal patterns i n artificial gels of extracellular matrix macromolecules quick-freezing and freeze-substitution. Cryobiology 26:170-179.  after  A l l i e g r o , M.C., C A . Ettensohn, C A . Burdsal, H.P. Erickson and D.R. McClay (1988) Echinonectin: A new embryonic substrate adhesion protein. J . C e l l B i o l . 107:2319-2327. Anderson, E. (1968) Oocyte d i f f e r e n t i a t i o n in the sea urchin, Arabacia punctulata. with p a r t i c u l a r reference to the o r i g i n of the c o r t i c a l granules and t h e i r p a r t i c i p a t i o n in the c o r t i c a l reaction. J . Cell Biol. 37:514-539. Angell, C A . (1982) Supercooled water. In: Water - A comprehensive treatise. V o l . 7. Water and Aqueous Solutions at Subzero Temperatures. F. Franks, ed. Plenum, New York, pp 1-81. Arsenault, A.L., F.P. Ottensmeyer and I.B. Heath (1988) An electron microscopic study of murine epiphyseal c a r t i l a g e : Analysis of fine structure and matrix v e s i c l e s preserved by slam freezing and freeze s u b s t i t u t i o n . J . U l t r a s t r u c t . Res. 98:32-47. Bald, W.B. (1984) The r e l a t i v e e f f i c i e n c y of cryogenic f l u i d s used i n the rapid quench cooling of b i o l o g i c a l samples. J . Microsc. 134:261-270. Barlow, D.I. and M.A. Sleigh (1979) Freeze substitution f o r preservation of c i l i a t e d surfaces f o r scanning electron microscopy. J . Microsc. 115:81-95. Begg, D.A. and L.I. Rebhun (1978) pH induced changes i n actin associated with the sea urchin egg cortex. J . Cell B i o l . 79:274a. Behnke, 0. and T. Zelander (1970) Preservation of i n t r a c e l l u l a r substances by the c a t i o n i c dye alcian blue i n preparative for electron microscopy. J . U l t r a s t r u c t . Res. 31:424-438.  procedures  - 92 -  Benson, S., L. Smith, F., Wilt, and R. Shaw (1990) secretion of collagen by cultured sea urchin Res. 188:141-146.  The synthesis and micromeres. Exp.  Cell.  B e r n f i e l d , M., S.D. Banerjee, J.E. Koda and A.C. Rapraeger (1984) Remodeling of the basement membrane as a mechanism of morphogenetic tissue i n t e r a c t i o n . In: The Role of the E x t r a c e l l u l a r Matrix in Development. R.L. T r e l s t a d , ed. A.R. L i s s , New York, pp. 545-572. Birkeland, S.A. (1976) The immunological capacity of peripheral lymphocytes in a blast-transformation system using frozen-stored c e l l s . Cryobiology 13:433-441. Boutron, P. and A. Kaufmann (1979) S t a b i l i t y of the amorphous state in the system water - 1,2-propanediol. Cryobiology 16:557-568. Boutron, P., P. Mehl, A. Kaufmann and P. Angibaud (1986) Glass-forming tendency and s t a b i l i t y of the amorphous state i n the aqueous solutions of l i n e a r polyalcohols with four carbons. 1. Binary systems water polyalcohol. Cryobiology 23:453-469. Bruggeller, P. and E. Mayer (1980) Complete v i t r i f i c a t i o n i n pure l i q u i d water and d i l u t e aqueous solutions. Nature (London) 288:569-571. Burgess, D.R. and T.E. Schroeder (1977) Polarized bundles of a c t i n filaments within m i c r o v i l l i of f e r t i l i z e d sea urchin eggs. J . Cell Biol. 74:1032-1037. Burstein, N.L. and D.M. Maurice (1978) Cryofixation of tissue surfaces by a propane j e t f o r electron microscopy. Micron 9:191-198. Cook, H. (1982) In: Theory and Practice of H i s t o l o g i c a l Techniques. 2nd ed. J.D. Bancroft and A. Stevens, eds. Churchill Livingston, Edinburgh, p. 616. ^ C o s t e l l o , M.J. and Corless, J.M. (1978) The d i r e c t measurement of temperature changes within freeze fracture specimens during quenching i n l i q u i d coolants. J . Microsc. 112:17-37.  rapid  Crawford, B.J. (1989) U l t r a s t r u c t u r e of the basal lamina and i t s relationship to e x t r a c e l l u l a r matrix of embryos of the s t a r f i s h Pisaster ochraceus revealed by anionic dyes. J . Morphol. 199:349-361. Crawford, B.J. (1990)  Personal communication.  Crawford, B.J. (1990) Changes i n the arrangements of the e x t r a c e l l u l a r matrix, larval shape and mesenchyme c e l l migration during asteroid larval development. J . Morphol. In press. Crawford, B.J. and M. Abed (1983) The role of the basal lamina i n mouth formation i n the embryo of the s t a r f i s h Pi saster ochraceus Morphol. 176:235-246.  J.  - 93 Crawford, B.J. and M. Abed (1986) U l t r a s t r u c t u r a l aspects of the surface coatings of eggs and larvae of the s t a r f i s h , Pisaster ochraceus. as revealed by a l c i a n blue. J . Morphol. 187:23-37. Crawford, B.J. and F.S. Chia (1978) Coelomic pouch formation i n the s t a r f i s h Pi saster ochraceus (Echinodermata:Asteroida). J . Morphol. 157:99-120. Crawford, B.J. and F.S. Chia (1982) Genesis and movement of mesenchyme c e l l s i n embryos of the s t a r f i s h Pi saster ochraceus. In J.M. Lawrence (ed.): International Echinoderms Conference. Tampa Bav. Rotterdam:A.A. Balkema, pp. 505-511. Crise-Benson, N. and S.C. Benson (1979) U l t r a s t r u c t u r e of collagen i n sea urchin embryos. Wilhelm Roux's Arch. Entwicklungsmech. Org. 186:65-70. Dan, K. (1960) Cyto-embryology of echinoderms and amphibians. Int. Rev. C y t o l . 9:321-368. Davidson, J.M. (1974) On the r o l e of the e p i t h e l i a l basal lamina i n echinoid morphogenesis. Ph.D. dissertation, Stanford University, Stanford CA., 211 pp. Dempsey, G.P. and S. B u l l i v a n t (1976) A copper block method f o r freezing non-cryoprotected tissue to produce i c e - c r y s t a l - f r e e regions for electron microscopy. 1. Evaluation using freeze-substitution. J. Microsc. 106:251-260. Dubochet, J . , J . Lepault, R. Freeman, J.A. Berriman and J.-C. Homo (1982) Electron microscopy of frozen water and aqueous solutions. J. Microsc. 128:219-237. Dubochet, J . , M. Adrian, J . Lepault and A.W. McDowall (1985) Cryo-electron microscopy of v i t r i f i e d b i o l o g i c a l specimens. Trends Biochem. S c i . 10:143-146. Elder, H.Y., C C . Gray, A.G. Jardine, J.N. Chapman and W.H. Biddlecombe (1982) Optimum conditions f o r cryoquenching of small tissue blocks in l i q u i d coolants. J . Microsc. 126:45-61. E l l i o t , M. (1990)  Personal communication.  Endo, Y. (1961) Changes i n the c o r t i c a l layer of sea urchin eggs at f e r t i l i z a t i o n as studied with the electron microscope. 1. Clvpeaster japonicas. Exp. C e l l . Res. 25:383-397. Endo, Y. and Y.D. Noda (1977) Ultrastructure of blastocoel of sea urchin embryos. Zool. Mag. 86:309. Endo, Y. and N. Uno (1960) Zool. Mag. 69:8.  I n t e r c e l l u l a r bridges i n sea urchin blastula.  Escraig, J . (1982) New instruments which f a c i l i t a t e rapid freezing at 83K and 6K. J . Microsc. 126:221-229.  - 94 Fahy, G.M., D.I. Levy and S.E. A l i (1987) Some emerging p r i n c i p l e s underlying the physical properties, b i o l o g i c a l actions, and u t i l i t y of v i t r i f i c a t i o n solutions. Cyrobiology 24:196-213. Farquhar, M.G. (1981) The glomerular basement membrane, a s e l e c t i v e macromolecular f i l t e r . In: Cell Biology of the E x t r a c e l l u l a r Matrix. E.D. Hay, ed. Plenum, New York, pp. 335-378. Farrant, J . , C A . Walter, H. Lee, G.J. Morris and K.J. Clarke (1977) Structural and functional aspects of b i o l o g i c a l freezing techniques. J . Microsc. 111:17-34. Fernandez-Moran, H. (1960) Low temperature preparation techniques f o r electron microscopy of b i o l o g i c a l specimens based on rapid freezing with l i q u i d helium I I . Ann. N.Y. S c i . 85:689-713. Fink, A.L. (1986) Effects of cryoprotectants on enzyme structure. Cryobiology 23:28-37. Franks, F. (1977) Ill:3-16.  Biological freezing and c r y o f i x a t i o n .  J . Microsc.  Franks, F. (1982) The properties of aqueous solutions at subzero temperatures. In: Water - A Comprehensive T r e a t i s e . V o l . 7. Water and Aqueous Solutions at Subzero Temperatures. F. Franks, ed. Plenum, New York, pp. 215-338. G a l i l e o , D.S. and J.B. M o r r i l l (1985) Patterns of c e l l s and e x t r a c e l l u l a r material of the sea urchin Lvtechinus variegatus (Echinodermata; Echinoidea) embryo, from hatched blastula to late gastrula. J. Morphol. 185:387-402. Gibbons, J.R., L.G. Tilney and K.R. Porter (1969) Microtubules i n the formation and development of the primary mesenchyme i n Arbacia punctulata 1. The d i s t r i b u t i o n of microtubules. J . Cell Biol. 41:201-227. G i l b e r t , S.F. (1988) Developmental Biology. U.S.A. pp. 539-547.  Sinauer Associates, Inc.  Gilkey, J.C. and L.A. Staehlin (1986) Advances i n u l t r a r a p i d freezing f o r the preservation of c e l l u l a r u l t r a s t r u c t u r e . J . Electron Microsc. Tech. 3:177-210. Glauert, A.M. (1974) Fixation, dehydration and embedding of b i o l o g i c a l specimens. In: Practical Methods i n Electron Microscopy. V o l . 3. A.M. Glauert, ed. North-Holland, Amsterdam. Golob, R., C J . Chetsanga and P. Doty (1974) The onset of collagen synthesis i n sea urchin embryos. Biochim. Biophys. Acta 349:135-141. Gordon, J.E. (1975) In: The Organic Chemistry of E l e c t r o l y t e Solutions Wiley, New York, pp. 10-49.  - 95 Grobstein, C. (1967) Mechanisms of organogenetic tissue i n t e r a c t i o n . Cancer Inst. Monogr. 26:279-299.  Natl.  Grobstein, C. and 0. Cohen (1965) Collagenase: E f f e c t on the morphogenesis of embryonic s a l i v a r y epithelium i n v i t r o . Science 150:626-628. Gustafson, T., and L. Wolpert (1962) C e l l u l a r mechanisms i n the morphogenesis of the sea urchin larva. Change in shape sheets. Exp. C e l l . Res. 27:260-279.  of  cell  Gustafson, T., and L. Wolpert (1963) The c e l l u l a r basis of morphogenesis and sea urchin development. Int. Rev. C y t o l . 15:139-214. H a l l , H.G. and V.D. Vacquier (1982) The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer. Dev. B i o l . 89:168-178. Harvey, D.M.R. (1982)  Freeze-substitution.  0. Microsc. 127:209-221.  Hay, E.D. (1981) Collagen and Embryonic Development. In: C e l l Biology of E x t r a c e l l u l a r Matrix. E.D. Hay, ed. Plenum, New York, pp. 379-409. Hay, E.D. (1984) Cell-matrix interactions in the embryo: C e l l shape, c e l l surface, c e l l skeletons, and t h e i r r o l e in d i f f e r e n t i a t i o n . In: The Role of E x t r a c e l l u l a r Matrix in Development. E.D. Hay, ed. Alan R. L i s s , Inc., New York, pp. 1-31. Hayashi, M., Y. Ninomiya, K. Hayashi, T.F. Linsenmayer, B.R. Olsen and R.L. Trelstad (1988) Secretion of collagen types I and II by e p i t h e l i a l and endothelial c e l l s i n the developing chick cornea demonstrated by i n s i t u hybridization and immunohistochemistry. Development 103:27-36. Hayat, M.A. (1981) Fixation f o r Electron Microscopy. Academic Press, New York, pp. 1-182. Herbst, C. (1900) Uber das auseinamdergehen von Forschungsund Gewebezellen imkalkfreiem Medium. Wilhelm Roux' Arch. Entwicklungsmech. Organ 9:424-463. Holland, N.D. (1979) Electron microscope study of the c o r t i c a l the ophiuroid echinoderm. Tissue Cell 11:445-455.  reaction of  Holland, N.D. (1980) Electron microscope study of the c o r t i c a l reaction in eggs of the s t a r f i s h (Pateria miniata). Cell T i s s . Res. 205:67-76. Humbel, B. and M. Muller (1984) Freeze-substitution and low temperature embedding. In: Proc. 8th Eur. Reg. Conf. Electron Microscopy. Budapest. A. Csanady, P. Rolich and D. Szabo, eds. 3:1789. Hylander, B.L. and R.G. Summers (1982) An u l t r a s t r u c t u r a l immunocytochemical l o c a l i z a t i o n of hyalin in the sea urchin egg. B i o l . 93:368-380.  Dev.  - 96 Kane, R.E. and R.T. Hersh (1959) The i s o l a t i o n and preliminary characterization of a major soluble protein of the sea urchin Exp. C e l l . Res. 16:59-69.  egg.  Kane, R.E. and R.E. Stephens (1969) A comparitive study of the i s o l a t i o n of the cortex and the role of the calcium-insoluble protein i n several species of sea urchin eggs. J . Cell B i o l . 41:133-144. Karp, G.C. and M. Solursh (1974) Acid mucopolysaccharide metabolism, the c e l l surface and primary mesenchyme c e l l a c t i v i t y i n the sea urchin embryo. Dev. B i o l . 41:110-123. Katow, H. (1986) Behavior of sea urchin primary mesenchyme c e l l s i n a r t i f i c i a l e x t r a c e l l u l a r matrices. Exp. C e l l . Res. 162:401-410. Katow, H. (1987) I n h i b i t i o n of c e l l surface binding of f i b r o n e c t i n and fibronectin-promoted c e l l migration by synthetic peptides i n sea urchin primary mesenchyme c e l l s In v i t r o . Dev. Growth and D i f f e r . 29:579-589. Katow, H. and M. Solursh (1979) U l t r a s t r u c t u r e of blastocoel material in blastulae and gastrulae of the sea urchin Lvtechinus p i c t u s . J . Exp. Zool. 210:561-567. Katow, H. and M. Solursh (1980) U l t r a s t r u c t u r e of primary mesenchyme c e l l ingression i n the sea urchin Lvtechinus pictus. J . Exp. Zool. 213:231-246. Katow, H., and M. Solursh (1981) U l t r a s t r u c t u r a l and time-lapse studies of primary mesenchyme c e l l behavior i n normal and s u l f a t e deprived sea urchin embryos. Exp. C e l l . Res. 136:233-245. Katow, H., K.M. Yamada and M. Solursh (1982) Occurence of f i b r o n e c t i n on the primary mesenchyme c e l l surface during migration i n the sea urchin embryo. D i f f e r e n t i a t i o n 22:120-124. K v i s t , T.N. and C V . Finnegan (1970a) The d i s t r i b u t i o n o f glycosaminoglycans i n the axial region of the developing I. Histochemical a n a l y s i s . J . Exp. Zool. 175:220-240.  chick  embryo.  K v i s t , T.N. and C V . Finnegan (1970b) The d i s t r i b u t i o n of glycosaminoglycans i n the axial region of the developing II. Biochemical a n a l y s i s . J . Exp. Zool. 175:241-258.  chick  embryo.  Lane, M.C and M. Solursh (1988) Dependence of sea urchin primary mesenchyme c e l l migration on Xyloside- and S u l f a t e - s e n s i t i v e surface-associated components. Dev. B i o l . 127:78-87.  cell  Leblond, C P . and S. Inoue (1989) Structure, composition, basement membrane. Am. J . Anat. 185:367-390.  and assembly of  L i l l i e , R.D. (1965) Histopathologic Technic and P r a c t i c a l 3rd ed. McGraw-Hill, New York, pp. 493-524.  Histochemistry.  Luft, J.H. (1961) Improvements i n Epoxy resin embedding methods. Biophys. Biochem. C y t o l . 9:409-414.  J.  - 97 Lundgren, B. (1973) Surface coatings of the sea urchin larva as revealed by ruthenium red. 3. Submicr. Cytol. 5:61-70. Luyet, B.O. (1937) The v i t r i f i c a t i o n of organic c o l l o i d s and protoplasm. Biodynamica 1:1-14. Maitland, M.E. and A.L. Arsenault (1989) Freeze-substitution staining of rat growth plate c a r t i l a g e with a l c i a n blue f o r electron microscopic study of proteglycans. 3. Histochem. Cytochem. 37:383-387. Mayer, B.W., E.D. Hay and R.O. Hynes (1981) Immunocytochemical l o c a l i z a t i o n of f i b r o n e c t i n i n embryonic chick trunk and area vasculosa. Dev. Biol. 82:267-286. McCarthy, R.A. and M.M. Burger (1987) In vivo embryonic expression of laminin and i t s involvement i n c e l l shape change i n the sea urchin Sphaerechinus g r a n u l a n s . Development 101:659-671. McCarthy, R.A. and M. Spiegel (1983) Protein composition of the hyaline layer of the sea urchin embryos and reaggregating c e l l s . Cell Differ. 13:93-102. McClay, D.R. and R.D. Fink (1982) Sea urchin hyalin: function i n development. Dev. B i o l . 92:285-293.  Appearance and  McManus, J.F.A. (1948) H i s t o l o g i c a l and histochemical uses of periodic a c i d . Stain Technol. 23:99-108. Mehl, P. and P. Boutron (1987) Glass-forming tendency and s t a b i l i t y of the amorphous state i n the aqueous solutions of l i n e a r polyalcohols with four carbons. Cryobiology 24:355-367. Meissner, D.H. and H. Schwarz (1990) Improved cryoprotection and freeze-substitution of embryonic quail retina: A TEM study on u l t r a s t r u c t u r a l preservation. 3. Electron Microsc. Tech. 14:348-356. Mizoguchi, H., A. Fujiwara and I. Yasumasu (1983) Degeneration of archenteron in sea urchin embryos caused by a.a'-dipyridyl. Differentiation 25:106-112. Mizoguchi, H. A. Fujiwara and I. Yasumasu (1989) Synthesis of collagen-like proteins i n embryonic organs of the sea urchin, Hemicentrotus pulcherrimus. Develop. Growth and D i f f e r . 31:189-196. Mizoguchi, H. and I. Yasumasu (1983a) I n h i b i t i o n of archenteron formation by the i n h i b i t o r s of prolyl hydroxylase i n sea urchin embryos. Cell Differ. 12:225-231. Mizoguchi, H. and I. Yasumasu (1983b) Effect of a.a'-dipyridyl on exogut formation on vegetalized embryos of the sea urchin. Growth and D i f f e r . 25:57-64.  Develop.  Monne, L. and D.B. Slautterback (1950) D i f f e r e n t i a l staining of various polysaccharides i n sea urchin eggs. Exp. C e l l . Res. 1:477-491.  - 98 Moor, H. (1964) Die G e f r i e r f i x a t i o n lebender Zellen und ihre Anwendung in der Elektronenmik.rosk.opie. Z. Z e l l f o r s c h . 62:546-580. M o r r i l l , J.B. and L.L. Santos (1985) A scanning electron microscopical overview of c e l l u l a r and e x t r a c e l l u l a r patterns during b l a s t u l a t i o n and gastrulation i n the sea urchin, Lytechinus vari egatus. In: JM C e l l u l a r and Molecular Biology of Invertebrate Development. R.H. Sawyer and R.M. Showman, eds. University of South Carolina Press. M o r r i l l , J.B., R.G. Summers and C. Nislow (1987) Correlative SEM and TEM analysis of ectodermal hyaline layer i n the sea urchin, Lvtechinus variegatus. J. Cell Biol. 105:86a. Mowry, R.W. (1956) Observations on the use of the sulpheric ether f o r the sulphation of hydroxyl groups i n tissue sections. J . Histochem. Cytochem. 4:407. Muller, M., T.H. Marti and S. Kriz (1980) Improved structural preservation by f r e e z e - s u b s t i t u t i o n . In: Proc. 7th Eur. Conf. Electron Microscopy. The Hague. P Brederoo and W. de Proester, eds. 2:270. Murray, P. W. Le R., A.W. Robards and P.R. Waites (1989) Countercurrent plunge cooling: a new approach to increase r e p r o d u c i b i l i t y in the quick freezing of b i o l o g i c a l t i s s u e . J . Microsc. 156:173-182. Nagele, R.G., M.C. Kosciuk, S.M. Wang, D.A. Spero and H. Lee (1985) A method f o r preparing quick-frozen, freeze-substituted cells for transmission electron microscopy and immunocytochemistry. J . Microsc. 139:291-301. Newgreen, D. and J.P. Thiery (1980) Fibronectin i n early avian embryos: synthesis and d i s t r i b u t i o n along the migratory pathways of neural crest c e l l s . Cell Tissue Res. 211:269-291. Oguri, K. and T. Yamagata (1978) Appearance of a proteoglycan i n developing sea urchin embryos. Biochim. Biophys. Acta 341:385-393. Okazaki, K. and L. Niijima (1964) "Basement membrane" i n sea urchin larvae. Embryologia 8:89-100. Plattner, H. and L. Bachmann (1982) Cryofixation: A tool i n biologicaal u l t r a s t r u c t u r a l research. Int. Rev. C y t o l . 79:237-304. Pointer, D.A. (1978) Blastocoelic f l u i d of the sea urchin embryo: Its components and t h e i r s i g n i f i c a n c e i n development. Ph.D Dissertation, University of C a l i f o r n i a , Berkeley. 139 pp. Porter, K.R. and K.L. Anderson (1982) The structure of the cytoplasmic matrix preserved by freeze-drying and freeze-substitution. Eur. J . Cell B i o l . 29:83-96. Pratt, R.M., M.A. Larsen and M.C. Johnston (1975) Migration of cranial neural crest c e l l s i n a c e l l free hyaluronate r i c h matrix. Dev. B i o l . 44:289-305.  - 99 -  Pryde, J.A and G.O. Jones (1952) (London) 170:685-688.  Properties of vitreous water.  Nature  Pucci-Minafra, I., C. Casano and C. LaRosa (1972) Collagen synthesis and spicule formation i n sea urchin embryos. Cell D i f f e r . Dev. 1:157-165. Reimer, C.L. and B.J. Crawford (1990) Lectin histochemistry of the hyaline layer i n the asteroid, Pisaster ochraceus J . Morphol. 203:361-375. Reimer, C.L. and B.J. Crawford (1990) Formation o f the esophageal musculature i n the s t a r f i s h embryo, Pi saster ochraceus. Manuscript i n preparation. Rey, L.R. (1960) Thermal analysis of eutectics i n freezing solutions. Ann. N.Y. Acad. S c i . 85:510-534. Rich, S.J. and W.J. Armitage (1990) component of a v i t r i f i c a t i o n 27:42-54.  Propane-1,2-diol as a potential solution f o r corneas. Cryobiology  Richardson, K.C., L. J a r r e t t and E.H. Finke (1960) Embedding i n Epoxy resins f o r u l t r a thin sectioning i n electron microscopy. Stain Tech. 53:313-323. Richter, H. (1968a) Die Reaktion hochpermeabler Pflanzenzellen auf die Gefrierschutzstoffe (Glyzerin, Athylenglykol, Dimethylsulfoxid). Protoplasma 65:155-166. Riehle, U. (1968b) Fast freezing o f organic specimens f o r electron microscopy. Chem. Ing. Tech. 40:213-218. Riehle, U. and Hoechli (1973) The theory and technique of high pressure freezing, In: Freeze-etching. Techniques and Applications. E.L. Benedetti and P. Favard, eds. (Soc. Francaise Microscopie Electronique, P a r i s ) , pp. 31-61. Robards, A.W. and P. Crosby (1983) Optimization of plunge freezing: l i n e a r r e l a t i o n s h i p between cooling rate and entry v e l o c i t y into propane. Cryo-Letters 4:23-32. >~  liquid  Robards, A.W. and U.B. Sleytr (1985) Low temperature methods i n b i o l o g i c a l electron microscopy. In: Practical Methods i n Electron Microscopy. Vol. 10. A.M. Glauert, ed. E l s e v i e r , Amsterdam. Rosenkranz, J . (1975) Course of temperature v a r i a t i o n i n an object during freeze-etching procedures. Arzneim Forsch. 25:454-455. Runnstrom, R. (1966) The v i t e l l i n e membrane and c o r t i c a l p a r t i c l e s i n sea urchin eggs and t h e i r functions i n maturation and f e r t i l i z a t i o n . Adv. Morphol. 5:221-225. Ruppert, E.E. and E.J. Balser (1986) Nephridia i n the larvae of hemichordates and echinoderms. B i o l . B u l l . 171:188-196.  - 100 Ryan, K.P., D.H. Purse, S.G. Robinson and J.W. Wood (1987) The r e l a t i v e e f f i c i e n c y of cryogens used f o r plunge-cooling b i o l o g i c a l specimens. J . Microsc. 145:89-96. Schuger, L., S. O'Shea, J . Rheinheimer and J . Varani (1990) Laminin i n lung development: Effect of anti-laminin antibody i n murine lung morphogenesis. Dev. B i o l . 137:26-32. Scott, J.E. and J . Dorling (1965) D i f f e r e n t i a l glycosaminoglycans (mucopolysaccharides) solutions. Histchemi. 5:221-233.  s t a i n i n g of acid by alcian blue  in  salt  S i l v e s t e r , N.R., S. Marchese-Ragona and D.N. Johnston (1982) The r e l a t i v e e f f i c i e n c y of various f l u i d s i n the rapid freezing of protozoa. J. Microsc. 128:175-186. Simpson, W.L. (1941) freezing drying.  An experimental analysis of the Altmann technique of Anat. Rec. 80:173-189.  Skaer, H. (182) Chemical cryoprotection f o r u l t r a s t r u c t u r a l Microsc. 125:137-147.  studies.  J.  Solursh, M. and H. Katow (1982) I n i t i a l characterizations of sulfated macromolecules in the blastocoels of mesenchyme blastulae of Strong!yocentrotus purpuratus and Lvtechinus pictus. Dev. Biol. 94:325-336. Solursh, M. and M.C. Lane (1988) E x t r a c e l l u l a r matrix t r i g g e r s a directed c e l l migratory response i n sea urchin primary mesenchyme c e l l s . Dev. Biol. 130:397-401. Solursh, M. and G.M. Morriss (1977) Glycosaminoglycan synthesis in rat embryos during the formation of primary mesenchyme and neural f o l d s . Dev. B i o l . 57:75-86. Somlyo, A.V. and J . Si 1 cox (1979) Cryoultramicrotomy f o r electron probe analysis. In: Microbeam Analysis i n Biology. C P . Lechene and R.R. Warner eds., Academic Press, New York, pp. 535-555. Spiegel, E. and M. Spiegel (1979) The hyaline layer i s a collagen-containing e x t r a c e l l u l a r matrix i n sea urchin reaggregating c e l l s . Exp. Cell Res. 123:434-441. Spiegel, E., M. Burger and M. Spiegel (1980) sea urchin. J . Cell B i o l . 87:309-313.  embryos  and  Fibronectin i n the developing  Spiegel, E., M. Burger and M. Spiegel (1983) Fibronectin and laminin in the e x t r a c e l l u l a r matrix and basement membrane of sea urchin embryos. Exp. C e l l . Res. 144:47-55. Spiegel, E., L. Howard and M. Spiegel (1989) E x t r a c e l l u l a r matrix of sea urchin and other marine invertebrate embryos. J . Morphol. 199:71-92. Steedman, H.F. (1950) A l c i a n blue 8GS: Micr. S c i . 91:477-479.  A new stain f o r mucin.  Quart. J .  - 101 Steinbrecht, R.A. substitution 125:187-192.  -  (1982) Experiments on freezing damage with freeze using moth antennae as test objects. J. Microsc.  Stephenson, J.L. (1956) Ice crystal growth during the rapid freezing of t i s s u e s . J . Biophys. Biochem. Cytol. 2:45-53. Strathmann, R.R. (1989) Existence and functions of a gel f i l l e d primary body c a v i t y i n development of echinoderms and hemichordates. Biol. B u l l . 176:25-31. Stuart, E.S., B. Garber and A. Moscona (1972) An analysis of feather germ formation in normal development and in skin treated with hydrocortisone. J . Exp. Zool. 179:97-110. Sugiyama, K. (1972) Occurrence of mucopolysaccharides in early development of the sea urchin embryo and i t s r o l e in gastrulation. Develop. Growth & D i f f e r . 14:63-73. Summers, R.G., J.B. M o r r i l l , C. Nislow, A. Yudin and G. Cherr (1987) Optimal preservation of sea urchin blastocoelic matrix freeze-substitution f i x a t i o n . J . Cell B i o l . 105:85a.  by  Takahashi, T., A Hirsh, E.F. Erbe, J.B. Bross, R.L. Steere and R.J. Williams (1986) Polymers protect monocytes from freezing injury through high temperature e x t r a c e l l u l a r glass formation. Cryobiology 23:556. Toole, B.P. (1981) Glycosaminoglycans i n morphogenesis. In: Cell Biology of the E x t r a c e l l u l a r Matrix. E.D. Hay, ed. Plenum, New York, pp. 259-294. Tucker, R.P. and C A . Erickson (1986) Pigment c e l l pattern formation in Taricha torosa: The role of the e x t r a c e l l u l a r matrix in c o n t r o l l i n g pigment c e l l migration and d i f f e r e n t i a t i o n . Dev. B i o l . 118:268-285. Van Harreveld, A., and J . Crowell (1964) Electron microscopy a f t e r rapid freezing on a metal surface and substitution f i x a t i o n . Anat. Rec. 149:381-386. Veno, P.A., M.A. Strumski and W.H. Kinsey (1990) P u r i f i c a t i o n and characterization of echinonectin, a carbohydrate-binding protein from sea urchin eggs. Develop. Growth and D i f f e r . 32:315-319. von der Mark, K. (1980) Immunological studies on collagen type t r a n s i t i o n in chondrogenesis. In: Immunological Approaches to Development and D i f f e r e n t i a t i o n . Part I I . M. Frielander, ed. Academic Press, Nre York, pp. 199-225. Wartiovarra, J . , I. Leivo and A. Vaheri (1980) Matrix glycoproteins in early mouse development and in d i f f e r e n t i a t i o n of teratocarcinoma cells. In: The Cell Surface: Mediator^ of Developmental Processes. S. Subtelny and N.K. Wessels, eds. Academic Press, New YOrk, pp. 305-324.  -  102 -  Weast, R., ed. (1982) CRC Handbook of Chemistry and Physics. Rubber Company, Ohio.  Chemical  Wessel, G.M., R.M. Marchase and D.R. McClay (1984) Ontogeny of the basal lamina i n the sea urchin embryo. Dev. B i o l . 103:235-245. Wessel, G.M. and D.R. McClay (1987) Gastrulation i n the sea urchin embryo requires the deposition of crosslinked collagen within the e x t r a c e l l u l a r matrix. Dev. B i o l . 121:149-165. Wessells, N.K. (1977) Menlo Park, CA.  Tissue Interactions and Development.  W.A. Benjamin,  Wessells, N.K. (1968) Effects of collagenase on developing e i p t h e l i a i n vitro: Lung, u r e t e r i c bud and pancreas. Dev. B i o l . 18:294-309. Weston, O.A., M.A. Derby and O.E. Pinter (1978) Changes i n the extracellular environment of neural crest cells during migration. Zoon 6:103-113.  early  Wolpert, L. and E.H. Mercer (1963) An electronmicroscope study of the development of the b l a s t u l a of the sea urchin embryo and i t s radial polarity. Exp. C e l l . Res. 30:280-300. Yazaki, I. (1968) Immunological analysis of the calcium p r e c i p i t a b l e protein of sea urchin eggs. I. Hyaline layer substance. Embryologia 10:131-141. Zalokar, M. (1966) A simple freeze-substitution method f o r electron microscopy. J . U l t r a s t r u c t . Res. 15:469-479.  - 103 6.  Appendix - Histochemical Staining  Techniques  1.  PAS stain f o r carbohydrates (modified a f t e r McManus, 1948) S c h i f f reagent ( L i l l i e , 1965) Basic fuchsin 1.0 g Potassium metabisulfite (anhydrous)..1.9 g IN HCl 15 ml d i s t i l l e d water 85 ml Combine the above, shaking i n t e r m i t t e n t l y f o r 2-3 hours or u n t i l the solution turns brownish or straw-coloured. Add 1 g o f decolourizing charcoal, shake and f i l t e r . Repeat u n t i l the solution c l e a r s . S c h i f f reagent can be stored i n the r e f r i g e r a t o r f o r 2-3 months or u n t i l i t recolourizes. S u l f i t e wash Potassium metabisulfite IN HCl d i s t i l l e d water  (anhydrous)..0.9 g 7.5 ml 150 ml  Make immediatly p r i o r to use. Periodic Acid 1. 2. 3. 4. 5. 6. 7. 8. 9.  2.  (0.5% ag.)  Immerse s l i d e i n periodic acid f o r f i v e minutes. Wash with dH20 Immerse i n S c h i f f reagent f o r 30 minutes (JB4) or 15 minutes (paraffin). Wash with s u l f i t e wash f o r 5 minutes. Wash with running tapwater f o r 10 minutes. Wash with dH20. Counterstain with hematoxylin by placing drops of the stain on the s l i d e s f o r four minutes on a 60° C hotplate. Wash with dH20. Cover with a glass coverslip - JB4. Dehydrate, c l e a r , mount i n DPX (BDH Co.) and cover - p a r a f f i n  pH dependent Alcian Blue test f o r acid GAG's (modified a f t e r Steedman, 1950; Mowry, 1956; Cook, 1982) Alcian blue 8GX 1% i n 10% s u l f u r i c acid (pH 0.2) 1% i n 0.2N HCl (pH 0.5) 1% i n 0.1N HCl (pH 1.0) 1% i n 3% acetic acid (pH 2.5) 1% i n 0.5% acetic acid (pH 3.2) 1. 2. 3. 4.  De-wax and hydrate p a r a f f i n sections. Immerse s l i d e s i n unstained solvent of the desired pH stain at room temperature. Stain i n a l c i a n blue of desired pH f o r 5 hours at 60° C (JB4) or for 5 minutes at room temperature (paraffin) Rinse with dH20  - 104 5.  6. 7. 8. 9.  Counterstain with 1% Neutral Red f o r 30 seconds (paraffin) or 1% phenylene-diamene i n 1:1 methanol/propanol f o r 90 seconds (JB4), both at room temperature. Rinse with dH20 Rinse JB4 sections with 100% ethanol f o r 1 minute or u n t i l the excess phenylene-diamene i s washed away. Dehydrate, c l e a r ( p a r a f f i n ) and mount i n DPX. Cover with a glass c o v e r s l i p .  A l c i a n blue-PAS test f o r neutral and acid GAG's (modified from Cook, 1982) 1. 2. 3. 4. 5.  Stain with A l c i a n blue (pH 2.5) as per Appendix 1.2 omitting counterstain. Rinse with dH20. Stain with PAS omitting counterstain. Dehydrate, clear ( p a r a f f i n ) and mount i n DPX. Cover with a glass c o v e r s l i p .  Toluidine Blue f o r basophilia and metachromasia (modified a f t e r L i l l i e , 1965) Acetate buffer pH 4.0 0.1N sodium acetate (8.2g/l) 0.1N a c e t i c acid (6 ml/1)  36 ml 164 ml  Stain 49 ml acetate buffer 1 ml IX Toluidine blue (aq) , 1. De-wax and hydrate sections (paraffin) 2. Immerse i n stain f o r 1 hour at room temperature. 3. Rinse with dH20. 4. Dehydrate, clear ( p a r a f f i n ) and mount i n DPX ( f o r basophi1ia)...or 5. Dehydrate f o r 1 minute i n acetone. 6. Immerse i n 1:1 acetone/xylene f o r 1 minute, then clear with xylene and mount i n DPX ( f o r metachromasia). 7. Cover with glass c o v e r s l i p .  


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items