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

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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 Univers i ty 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, Univers i ty of B r i t i s h Columbia We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1990 © Stephen Sean Campbell, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ANATOMY  The University of British Columbia Vancouver, Canada Date OCTOBER 3, 1990 DE-6 (2/88) i i ABSTRACT All developing embryos contain an extracellular matrix (ECM) consisting of proteins, glycoproteins, and proteoglycans. These components are important for morphogenetic processes such as cell migration, cell differentiation and cell death. The ECM of the starfish, Pi saster ochraceus. consists of three major components: A hyaline layer which coats the external surface of the embryo; a basal lamina which lines the basal surfaces of the epithelia; and a blastocoelic component which f i l l s the embryonic cavity or blastocoel. Observations of chemically fixed asteroid embryos have revealed the hyaline layer to contain five sub-layers of fibrous strands encrusted with amorphous material. Strands of a similar nature form a meshwork within the f l u i d - f i l l e d blastocoel. Recent studies of the living embryo, however, have suggested that the ECM within the blastocoel of echinoderms, including the asteroid, is a gel-like substance and not a f l u i d with extracellular fibres. Since artefacts imposed by chemicals such as aldehydes and osmium are well documented, a method of preservation, which does not involve the use of these chemicals, may resolve the apparent conflict over the nature of the ECM of the asteroid embryo. Freeze substitution, an expensive cryofixation technique which has proven successful in fixing vertebrate tissue, does not require the use of aldehydes and osmium. The i n i t i a l objective of this study was to devise an inexpensive, easily employable freeze substitution technique which would allow good preservation of cellular and extracellular elements of the embryonic starfish, Pi saster ochraceus. A plunge freezing apparatus was constructed which consisted of a Dewer flask f i l l e d with liquid nitrogen, a small cup was f i l l e d with cryogen and inserted into the nitrogen, and a motor which constantly stirred the cryogen. Embryos were isolated on copper freeze-fracture grids and plunged into the cryogen. After considering four different cryogens and four separate cryoprotectants, cryoprotecting asteroid embryos with propylene glycol and plunging them into supercooled propane was found to provide optimal preservation. Frozen embryos were freeze substituted in anhydrous ethanol at -90 °C, osmicated, and embedded for ultrastructural and histochemical analysis. Following freeze substitution, the blastocoel appears to contain a gel-like substance, rich in sulfated GAG's, with extracellular fibres and not a fl u i d with fibres. In addition, the hyaline layer was found to consist of at least six sub-layers of greater thickness than was seen in chemically fixed embryos. Histochemical studies demonstrated that both sulfated and unsulfated GAG's were present in these layers. The morphological differences among the sub-layers suggest that some sub-layers may have unique functions while others may have functions shared by other sub-layers. Freeze substitution also revealed the presence of microvillus associated bodies, structures which may represent major attachment points of the hyaline layer to the epithelium. Although the fixation of asteroid embryos by freeze substitution is a lengthy process, taking four to five days, the resulting preservation, particular!ly of the ECM components, j u s t i f i e s i t s use over chemical fixations. Material preserved by freeze substitution can be used for histochemical studies and, since aldehydes and heavy metals are not necessary for successful preservation, may also prove useful for immunocytochemical studies. i v TABLE OF CONTENTS PAGE Abstract i i L i s t of Tables v L i s t of Figures vi L i s t of Abbreviations v i i i Acknowledgements xi 1. INTRODUCTION 1 2. MATERIALS AND METHODS 11 2.1 Rearing of Asteroid Embryos 11 2.2 Cryof ix ing of Embryos 13 2.3 Chemical F ixat ion and Embedding 18 2.4 Microscopy: Morphology 18 2.5 Histochemistry 19 3. 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 ixat ion 57 3.6 Histochemistry 62 4. DISCUSSION 68 4.1 Freeze Subst i tu t ion 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 vials needed for freeze substitution 16 3 LM cross-section of an embryo frozen in Freon 12 24 4 LM cross-section of an embryo frozen in LN2 slush 24 5 TEM of the ectoderm and ECM of an embryo frozen in LN2 slush 26 6 TEM of the blastocoel of an embryo frozen in 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 in propane 34 b TEM of the blastocoel of the embryo in Fig. 10a 34 11a TEM of the ectoderm and HL of an embryo treated with 10% glycerol prior to freezing in propane 36 b TEM of the blastocoel and BL of the embryo in Fig. 11a 36 12 TEM of an embryo treated with 15% glycerol prior to in propane 36 13a TEM of the ectoderm and HL of an embryo treated 10% ethylene glycol prior to freezing in propane. 38 b TEM of the blastocoel of the embryo in Fig. 13a 38 14a TEM of the HL of an embryo treated with 15% ethylene glycol prior to freezing in propane 40 b TEM of the ectoderm of the embryo in Fig. 14a 40 15 LM mid-sagittal section of an embryo treated with 15% propylene glycol prior to freezing in propane 44 v i i FIGURE PAGE 16 TEM of the BL and blastocoel of an embryo prepared like that in Fig. 15 46 17 TEM of the esophageal fibres in an embryo prepared like that in Fig. 15 48 18 TEM of the dorsal web of an embryo prepared like that in that in Fig. 15 48 19 TEM of the HL of an embryo prepared like that in Fig. 15 and an inset LM of the HL a living embryo 50 20 TEM of the six sub-layers of the HL of an embryo prepared like that in Fig. 15 52 21 Stereo pair of TEM's of the HL of an embryo prepared like that in Fig. 15 54 22 TEM of the HL and the microvillus associated body of an embryo prepared like that in Fig. 15 54 23a Higher magnification TEM of the microvillus associated body in Fig. 22 56 b TEM cross section of the microvillus associated body 56 c High magnification TEM oblique section of the microvillus associated body 56 d TEM sagittal section of microvillus filaments 56 24a TEM of the HL of a chemically fixed embryo 59 b TEM of the BL of the blastocoel of a chemically fixed embryo 59 25 TEM of the esophageal fibres of a chemically fixed embryo 61 26 TEM of the dorsal web of a chemically fixed embryo 61 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 v i i i LIST OF ABBREVIATIONS AB alcian blue aq aqueous BL basal lamina(e) b blastocoel B boundary sub-layer BM basement membrane bpt boiling point temperature C coelom c specific heat CEC Cri t i c a l Electrolyte Concentration cf. compare °C degrees Centigrade DMSO Dimethylsulfoxide dH20 d i s t i l l e d water Ec ectoderm ECM extracellular 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 ix HI hyaline 1 H2, hyaline 2 H3 hyaline 3 In India ink particles IV intervillus sub-layer J Joule(s) k thermal conductivity K Kelvin kV kilovolts LD lamina densa LL lamina lucida LM light microscope (microscopy) LN2 liquid nitrogen m metre(s) M mesenchyme mg mi 11igram(s) mJ mi 11iJoules ml m i l l i l i t r e ( 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) X OM coarse outer meshwork 0s0 4 osmium tetroxide P plasma membrane PAS periodic acid - Schiff reagent s second(s) AT difference between bpt and mpt TEM transmission electron microscope Th Thermos V glass vial xi ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Bruce Crawford who showed me what science is a l l about and how to approach i t . He was very supportive of my goals in l i f e and spent countless hours assisting 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 for 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. INTRODUCTION Extracellular matrix (ECM) is present in a l l developing embryos. It is secreted by the cells into the extracellular spaces and serves numerous functions including separating populations of cells to prevent their interaction, serving as a substrate upon which cells may migrate and inducing the differentiation of certain cell types (Gilbert, 1988). The ECM of vertebrate embryos includes glycoproteins such as collagen (von der Mark, 1980; Wartiovarra et a l . , 1980; Hayashi, 1988), laminin (Hakamori, 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 and heparan sulfate (Kvist and Finnegan, 1970a,b; Pratt et a l . , 1975; Solursh and Morriss, 1977; Weston et a l . , 1978; Toole, 1981; Tucker and Erickson, 1986). Elements of the ECM appear to be essential mediators of many developmental processes (Grobstein, 1967; Wessels, 1977; Hay, 1981, 1984; Bernfield et a l . , 1984). Some examples of these processes include the successful branching of the mouse salivary gland (Grobstein and Cohen, 1965; Wessels and Cohen, 1968), the induction of chick vertebral cartilage 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 embryos also depend upon an ECM for successful development. For example, the sea urchin or echinoid ECM consists of three major components: A hyaline layer that coats the external surface of the embryo; a basal lamina that lines the basal surfaces of the ectoderm and endoderm; and a blastocoelic component that f i l l s the embryonic cavity or blastocoel. - 2 -The blastocoel contains the largest amount of ECM in the developing echinoid. The fibrous ECM components found in the blastocoel are synthesized during gastrulation. They form a network which is confined by the basal laminae of the walls of the embryonic cavity (Endo and Noda, 1977; Katow and Solursh, 1979; Galileo and Morrill, 1985; Morrill and Santos, 1985). It is thought that a fluid occupies the spaces between fibers (Pointer, 1978). As in vertebrate embryos, biochemical studies have indicated the presence of several macromolecules in the echinoid ECM including GAG's (Karp and Solursh, 1974; Solursh and Katow, 1982), proteoglycans (Oguri and Yamagata, 1978), collagen (Pucci-Minafra et a l . , 1972; Golob et a l . , 1974; Crise-Benson and Benson, 1979; Wessel and McClay, 1987; Benson et a l . , 1990), fibronectin and laminin (Spiegel et a l . , 1980, 1983; Katow et a l . , 1982; Wessel et a l . , 1984; McCarthy and Burger, 1987). Studies have suggested a correlation between the presence of GAG's in the blastocoel and the migration of mesenchyme cells (Katow and Solursh, 1981; Lane and Solursh, 1988). In addition, fibronectin has been shown to be important for mesenchymal cell migration in vitro (Katow, 1987; Solursh and Lane, 1988). Collagen is also necessary for important morphogenetic processes during echinoid gastrulation (Katow, 1986; Wessel and McClay, 1987) such as archenteron formation and gut differentiation (Mizoguchi et a l . , 1983; Mizoguchi and Yasumasu, 1983a,b; Mizoguchi et a l . , 1989). In embryos of the starfish Pi saster ochraceus. the ECM of the blastocoel appears to be secreted by cells of ectodermal and endodermal origin during blastulation and f i l l s the entire body cavity just prior to gastrulation (Abed and Crawford, 1986b). Previous chemical fixation methods suggested that starfish ECM, like that of the echinoid, is also fibrous in nature and surrounded by a flu i d (Crawford and Chia, 1981; Abed and Crawford, 1986b; Crawford, 1989). Crawford (1990) demonstrated - 3 -distinct extracellular fibers in the regions of the esophagus and the inner aspect of the dorsal ectoderm. He suggested that these fibers may be involved in the migration of the presumptive esophageal smooth muscle cells and in maintaining the constriction at the middle of the embryo. Summers et a l . (1987) have shown ultrastructurally that the fibers of the echinoid blastocoel are much more anastomosing and mesh-like than was shown in the asteroid (Crawford and Chia, 1981; Abed and Crawford, 1986b; Crawford, 1990). Strathmann (1989), however, indicates that the blastocoels of embryonic echinoderms are f i l l e d with a gelatinous substance and not a flu i d containing a fibrous meshwork. It is apparent, therefore, that a method of fixation is needed that would preserve the ECM within the blastocoel of echinoderms in a manner which is closer to that found in vivo than is currently possible. It may then be possible to determine the arrangement of the ECM within the blastocoel and the significance of this arrangement with respect to normal morphogenesis. The basal lamina (BL), the second ECM component, separates parenchymal cells from the connective tissue (Leblond and Inoue, 1989). In the gastrulating echinoid, the parenchyma includes the wall of the blastocoel and the basal surfaces of the presumptive alimentary canal, the archenteron, and the coelomic pouches (Endo and Uno, 1960; Wolpert and Mercer, 1963; Okazaki and Niijima, 1964; Gibbons et a l . , 1969). This ECM is a natural substrate upon which most cells grow, and i t may act as a semi-permeable barrier to macromolecules (Farquhar, 1981). At the ultrastructural level, the basal lamina is seen to consist of two layers, a lamina lucida and a lamina densa, similar to that of vertebrate basal laminae. Biochemical and histochemical studies of the echinoid basal lamina have revealed collagen types III and IV, fibronectin, laminin and heparan sulfate proteoglycan (Davidson, 1974; Spiegel et a l . , 1980; Wessel - 4 -et a l . , 1984; Wessel and McClay, 1987). The basal lamina of asteroid embryo has been shown to play an intricate role in mouth formation during morphogenesis (Crawford and Abed, 1983; Abed and Crawford, 1986a,b). Histochemical and cytochemical studies have shown that this BL contains structures suggestive of proteoglycans and collagen f i b r i l s (Crawford, 1989). However, different fixation methods result in variations in the morphology of the BL (Crawford, 1989). It is perhaps possible that the true morphology of the asteroid BL is yet to be determined. The outer ECM (Spiegel and Spiegel, 1979), the hyaline layer (HL), forms at f e r t i l i z a t i o n upon fusion of the cortical granules with the plasmalemma of the egg and the subsequent release of their contents into the extracellular space (Kane and Hersh, 1959; Endo, 1961; Runnstrom, 1966; Anderson, 1968; Holland, 1979, 1980; Hylander and Summers, 1982). The egg is then firmly attached to the HL by microvilli (Dan, 1960; Wolpert and Mercer, 1963; Burgess and Schroeder, 1977; Begg and Rebhun, 1978; Katow and Solursh, 1980). Numerous functions have been suggested for the HL including a substrate for cells (Herbst, 1900; Dan, 1960; Chambers, 1940; Vacquier and Mazia, 1968; Citkowitz, 1971, 1972; Kane, 1973), a source of cellular attachment during morphogenesis (Dan, 1960; Gustafson and Wolpert, 1967; Wolpert and Mercer, 1967; Adelson and Humphreys, 1988; Spiegel et a l . , 1989), a f i l t e r for organic molecules (Spiegel et a l . , 1989), lubrication (Crawford and Abed, 1986) and protection from mechanical and bacterial perturbation (Lundgren, 1973). Spiegel et a l . (1989) have composed a general schematic diagram of the HL based on ultrastructural studies of several marine invertebrate embryos. These authors divided the HL into two major zones - an inner zone and an outer zone. The inner zone consists of a dense meshwork of thin fibers while the outer zone is made up of more loosely arranged fibers. A dense border of compacted fibers and - 5 -granules separates the two zones. The HL maintains i t s attachment to the epithelium via microvilli which have ECM specializations or microvillus associated bodies (MVAB's) at their tips which are continuous with the outer zone. However, after treatment with the dye Ruthenium Red and ultrastructural analysis, Morrill et a l . (1987) suggested that the echinoid HL contains four distinct regions of varying thickness. Evidence for this model, however, is s t i l l unpublished. Biochemical studies of the echinoid HL have demonstrated the presence of several high molecular weight proteins. Hyalin, a major component of the HL (Yazaki, 1968; Kane and Stephens, 1969; Hylander and Summers, 1982) had been shown to reside primarily in the outer regions of the HL (McClay and Fink, 1982). Results from immunocytochemical studies, using a monoclonal antibody for hyalin, have suggested that hyalin is a major constituent of the MVAB's and this cell-HL adhesion molecule may be essential for normal morphogenesis (Adelson and Humphreys, 1988). Echinonectin, a putative substrate adhesion molecule, is another macromolecule isolated from the HL (Alliegro et a l . , 1988; Veno et a l . , 1990). Other large molecules have been isolated but their precise functions have not yet been determined (Hall and Vacquier, 1982; McCarthy and Spiegel, 1983). The HL of the starfish, Pisaster ochraceus. has not been studied as extensively as that of the sea urchin. The ultrastructure of the asteroid HL, after chemical fixation in the presence of anionic dyes, has been described in detail by Crawford and Abed (1986). They showed that the HL consists of four major zones - a coarse outer meshwork, a supporting layer, a boundary layer and an intervillus layer. The supporting layer was further subdivided into two minor zones - HI and H2. Lectin histochemical studies of the asteroid HL have revealed an unequal distribution of glycoconjugates throughout these zones (Reimer and 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 and asteroid morphological studies, chemical fixatives were used to prepare the embryos for microscopy. However, i t seems that for every fixation technique employed, variations in ultrastructure were observed. This suggests that much of our current knowledge of developmental processes, such as cell migration and c e l l - c e l l or cell-ECM interactions, could be based upon results which may be artefactual. Chemical fixation, as reviewed by Hayat (1981), usually involves treating a specimen with glutaraldehyde, paraformaldehyde and/or osmium tetroxide. Chemicals such as these are thought to aid in stabilizing molecules that may otherwise be extracted or relocated during dehydration of the tissue. This stabilization is achieved by intermolecular and intramolecular cross-linking, thereby creating a lattice which will withstand the effects of dehydration. These actions, however, may reduce the antigenicity of many compounds, denature proteins and rearrange cellular and extracellular components (Hayat, 1981). If these alterations or artefacts could be reduced or eliminated, the analysis of biological tissue based on morphological studies may prove more reliable. Rapidly freezing or cryofixing tissues is a method by which such artefacts could be significantly reduced. Cellular and extracellular components may be suspended in their l i f e - l i k e states, after rapid freezing, in the absence of chemicals such as aldehydes and heavy metals. It has long been suggested that v i t r i f i c a t i o n , the process of rapid freezing in the absence of ice crystal formation (Pryde and Jones, 1952), is the ideal method for fixing biological tissues (Luyet, 1937). Franks (1977) has taken this idea further by stating that the vitreous state is equivalent to the solidified i n vivo state. However, in order to v i t r i f y - 7 -uncryoprotected tissue at normal atmospheric pressure by plunge freezing, 4 cooling rates must exceed 10 Kelvin/second (K/s) (Moor, 1964, 1973; Riehle, 1968; Riehle and Hochli, 1973) and the specimen must be very small, usually less than 300 nm thick (Bruggler and Mayer, 1980; Dubochet et a l . , 1982). If the specimen exceeds this size, then ice crystal formation is i nevi table. Ice crystal formation is described adequately by the classical nucleation theory (Angel 1 , 1982; Franks, 1982). According to this theory, there are two types of ice crystal nucleation: heterogeneous or extrinsic nucleation, and homogeneous or intrinsic nucleation. Heterogeneous nucleation occurs when a foreign particle, in pure water, provides a site upon which ice crystals become seeded. Homogeneous nucleation, on the other hand, occurs in supercooled water when a few water molecules assume a configuration resembling that of ice, thereby acting as a template upon which crystalization can occur. Gilkey and Staehlin (1986) have described the thermodynamic properties of ice crystal nucleation but, because the physics of ice are beyond the scope of this thesis, they will not be discussed here other than to say that at the c r i t i c a l cooling rate for v i t r i f i c a t i o n of any given hydrated biological specimen, heterogeneous nucleation is inhibited and only homogeneous nucleation can occur. Since so many sites of homogeneous nucleation arise simultaneously, the system 'jams up' and so l i d i f i e s without crystal growth, hence vi t r i f y i n g the ti ssue. When v i t r i f i c a t i o n is unachievable in multi-component systems and ice crystals do form, phase separations occur (Robards and Sleytr, 1985). That i s , as the ice crystals grow, solutes such as proteins, electrolytes and sugars are extruded leaving two separate phases - the pure crystalline water or solvent phase and the aqueous solution or eutectic phase which - 8 -resides between the growing crystals (Plattner and Bachmann, 1982). The extrusion of solute continues as the temperature decreases and/or as ice crystal growth propagates until the remaining eutectic simultaneously freezes. The temperature at which this phenomenon occurs is the eutectic temperature and is unique to that solution (Rey, 1960). At this point, the entire system is in the solid state. Phase separation, which does not occur during v i t r i f i c a t i o n , results in numerous physiological alterations including pH changes (Franks, 1977), salting out (Gordon, 1975), and changes in immunogenic behavior (Birkeland, 1976). Obviously, these are deleterious to the i n vivo state of the specimen so these artefacts imposed by phase separation must be eliminated as much as possible in order to ju s t i f y the use of cryofixation techniques over chemical fixation. Many of these problems can be overcome by the use of compounds, known as cryoprotectants, which impede the growth of ice crystals. The effects of such chemicals, however, are not clearly understood and they may be just as detrimental to the tissue as is chemical fixation. Once v i t r i f i c a t i o n has been achieved, the tissue must be brought into a medium which is suitable for subsequent procedures. In the case of histochemical or immunocytochemical studies, this may involve the use of a resin. In order to successfully transfer the tissue from the frozen state to a resin, the v i t r i f i e d water must be substituted by non-aqueous solvents such as ethanol or acetone at temperatures below the crystallization temperature of ice. This process, called freeze substitution, does not require the use of chemical fixatives and, in principle, may give better preservation than chemical fixation. Currently, there are devices on the market in excess of $20,000 which fix tissues by freeze substitution. With such equipment, a specimen is rapidly frozen in a liquified gas and then stored in liquid nitrogen. The frozen specimen then undergoes substitution - 9 -over a period of several days and is subsequently embedded into a medium which is convenient for the investigator. The only foreign chemicals to which the specimen need be exposed prior to embedding are the cryogen, the nitrogen, and the solvent, although osmium tetroxide and aldehydes have been added by some investigators during the later stages of the substitution (Barlow and Sleigh, 1978; Meissner and Schwarz, 1990). Freeze substitution helps to maintain the antigenicity of the tissue and has been shown to give more detailed ultrastructure than chemical fixation in tissues rich in ECM such as cartilage (Arsenault et a l . , 1988). The disadvantages of freeze substitution include the cost, the time involved from freezing to embedding (4-6 days), the hazard of using some explosive cryogens and, with most biological specimens, the use of cryoprotective chemi cals. Since commercial freezing equipment was beyond the financial capabilities of our laboratory, the i n i t i a l portion of this study was to devise inexpensive and easily employable techniques for rapidly freezing and freeze substituting embryos of the starfish, Pi saster ochraceus. It was hoped that such methods would lead to the better preservation of the embryos, in a manner closer to their living form than is possible using conventional chemical fixatives. Once this aspect of the study was completed, these techniques were used to preserve each of the different elements of the asteroid ECM for both histochemical and ultrastructural examination. The results of these studies were then analysed in detail in an attempt to a) determine the nature of the material within the blastocoel, including the basal lamina and, b) determine the structure of the hyaline layer. Finally, the ECM of the freeze substituted embryos were compared to that of embryos prepared by chemical fixation in an attempt to - 10 -gain a better understanding of the ECM components and their arrangements, and to perhaps gain some insight into their roles in morphogenesis. - 11 -2. MATERIALS AND METHODS 2.1 Rearing o f A s t e r o i d Embryos Obtaining adult starfish and seawater Ripe adult starfish, Pisaster ochraceus. were collected from the intertidal zones at Vancouver and Victoria B.C. between April and June, 1988. The starfish were maintained at 9-12 °C in aquaria supplied with circulating seawater in the Department of Zoology. Light was kept at a constant level so as to avoid undesired spawning. Seawater for the culturing of embryos was obtained from the coastal waters of Victoria and from the Department of Fisheries in West Vancouver. Water retrieved from the latter source came from seventy feet below the surface where the salinity was comparable to that of the former site (unpublished data). Prior to i t s use in culturing, seawater was filtered through a #1 Whatman f i l t e r and aerated with a bubbling stone in a cold room maintained at 10-11 °C. Glass and plastic ware Glass and plastic wares used exclusively for culturing embryos were labelled to avoid accidental contamination, and were rinsed with only seawater or tapwater but never with detergents. After a culture had been completed, a l l containers were wiped clean, rinsed with tapwater and then with seawater so that they could be utilized in subsequent cultures. Isolation of gametes Arms of ripe starfish were removed, thereby exposing the gonads. After visualization with the naked eye, ovaries were dissected and removed, placed on a clean petri dish, washed with filtered seawater, and treated - 12 -with 10 ml of 0.1 mg/ml of 1-methyl adenine for ninety minutes in order to induce oocyte maturation. Testes were l e f t 'dry' in a separate clean petri dish until the eggs were ready to be f e r t i l i z e d at which time 1-2 drops of concentrated semen were placed in 25 ml of seawater producing a milky solution. Samples of this solution were examined under a light microscope to ensure that at least 10-20% of the sperm were motile, and that the pattern of motility was normal. Ferti1ization When the eggs had matured (as indicated by the disappearance of the germinal vesicles), they were placed in a 1 l i t r e plastic beaker containing 300-400 ml of fresh seawater at 10-11 °C. Several culturing beakers were f i l l e d with approximately 300 ml of seawater, and eggs were added until roughly 50% of the bottom of each beaker was covered. The cultures were f e r t i l i z e d by adding ten to fifteen drops of sperm-suspension to each beaker. 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 plastic conical centrifuge tubes. The tubes were then placed in ice and the embryos were allowed to settle. 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 for 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 flasks, one with an insert cup, were f i l l e d with liquid nitrogen (LN2). The flask with the insert cup was used for the actual freezing process, while the other two flasks stored a reserve supply of LN2 and temporarily housed the frozen embryos in 1.5 ml freezer vials on canes. A large ring stand with the freezing flask upon its base was placed inside a fume cabinet. An eddy current motor, with a stirring rod connected to i t , was clamped above the flask. The stirring rod extended half way into the insert cup, which was immersed as far into the LN2 as possible without allowing i t to overflow into the cup (Fig. 1). While the rod was stirr i n g , the container of liquid cryogen was inverted and sprayed into the cup. It was important to maintain the stirring to avoid freezing of the cryogen, and to keep the LN2 level as high as possible to reduce evaporation of the cryogen. The embryos were prepared for freezing as follows: following pretreatment with a cryoprotectant, i f appropriate, 1 u l of the embryo suspension was placed on a copper freeze fracture grid so as to form a monolayer; the grid was then plunged into the stirring liquid cryogen and kept moving for about five seconds. Subsequently, the grid was plunged into the adjacent LN2 and then transferred to a freezer vial to be stored temporarily in one of the other Dewer flasks. Once four grids had accumulated in a v i a l , the vial was capped and transferred to a LN2 refrigerator for an indefinite storage period. After the entire freezing process was completed, the cryogen was allowed to evaporate in the fume cabinet by l i f t i n g the cup out of the LN2. - 14 -b) Liquid nitrogen slush LN2 was poured into a small glass Thermos which was, in turn, placed inside of an insulated vacuum chamber. The chamber was evacuated, causing the LN2 to boil vigourously, until the LN2 began to solidify forming a slush. At this time, the chamber was quickly opened and the grid with embryos was plunged into the slush, kept in motion for 5 seconds, and then dropped into a storage vial as above. Crvoprotection In order to protect the embryos from the damage of ice crystal formation, four different intracellular cryoprotective solutions were tested. Solutions of 10% DMSO, 10% and 15% glycerol, 10% and 15% ethylene glycol, and 15% propylene glycol were prepared in 100% seawater. Excess water was removed from the tubes of packed embryos and they were resuspended for 20-30 minutes in 15 ml of a cryoprotective solution over ice. Subsequently, the tubes were centrifuged at 120xg for 30 seconds, the excess solution was removed, and the pellet of embryos in the small amount of remaining liquid was frozen in propane as described above. Freeze substitution To prepare for freeze substitution, 150 g of dry ice was placed in the bottom of a wide mouth Thermos. Acetone was slowly added to the thermos until i t was half f u l l after which LN2 was added until the temperature of the slurry reached -90° to -95 °C as monitered by a Cole-Parmer Digi-Sense thermocouple. Up to five 10 ml glass vials were f i l l e d with 100% ethanol, saturated with alcian blue i f desired to stain ECM (Behnke and Zelander, 1970), capped, and immersed into the slurry (Fig. 2). When - 15 -Fig. 1: Photograph of the apparatus used for plunge freezing asteroid embryos. A stirring rod powered by an eddy current motor (Mo) ensures continuous movement of the cryogen within the insert cup (arrow) which sits inside the liquid nitrogen-filled Dewer flask (F). Embryos on EM grids are frozen by plunging them into the cryogen and then into the surrounding liquid nitrogen before being stored in liquid nitrogen. Fig. 2: Photograph of the Thermos (Th) and glass vials (V) used for freeze substitution. The Thermos is f i l l e d with an acetone/dry ice/liquid nitrogen slurry (-90° to -95 °C) and the glass vials are f i l l e d with anhydrous ethanol and placed into the slurry. The frozen specimens are then dropped into the pre-cooled vials and inserted into the Thermos which is capped and placed in a -60 °C refrigerator for 4 to 5 days. - 17 -the temperature of the ethanol reached -90 °C, one sample of frozen embryos was placed in each glass v i a l . The Thermos was then put in a -60 °C freezer for four days. At least once per day, however, LN2 was added to the Thermos in order to maintain the temperature below -85 °C to avoid ice crystal formation. After four days of freeze substitution, the vials were removed from the Thermos and placed in a -20 °C freezer for one hour. They were then placed in a 4 °C refrigerator for one hour and f i n a l l y brought to room temperature over one hour. Embedding cryofixed material Cryofixed embryos were embedded in either Epon (Luft, 1961) or JB4 (Polysciences, Inc.). For Epon embedding, embryos were washed in reagent grade acetone two or three times during the transition 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 acetone/Epon. The next morning, the embryos were pipetted into plastic trays f i l l e d 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 inf i l t r a t e d for three hours at room temperature with catalyzed Solution A (100 ml Solution A + 0.9 g of peroxide catalyst) and transferred to fresh JB4 (25 ml catalyzed Solution A + 1 ml Solution B). The JB4 was then allowed to polymerize at room temperature overnight in aluminum f o i l plates covered with parafilm to reduce oxygen exposure. - 18 -2.3 Chemical Fixation and Embedding Glutaraldehyde fixation Packed embryos were immersed in 1% glutaraldehyde in 80% seawater (pH 7.0) saturated with alcian blue (Behnke and Zelander, 1970) at room temperature for four hours. Following a rinse in 1.25% NaHC03 buffer (pH 7.2), the embryos were post-fixed in 2% OsO^ in the same buffer (pH 7.4) for one hour, washed with the buffer, and stained en bloc for 45 minutes with 2% uranyl acetate (aqueous). 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 fixation Packed embryos were fixed for paraffin embedding by immersion in 8% formalin in seawater (pH 8.0) for four hours. The embryos were dehydrated through an increasing series of ethanol concentrations (30%, 50%, 70%, 95%, and 100%) and cleared with two successive washes in xylene. They were then immersed in 1:1 xylene/paraffin at 60 °C for 30 minutes followed by three 30 minute immersions in paraffin at 60 °C. Finally, the embryos were embedded in paraffin blocks for sagittal sectioning. 2.4 Microscopy: Morphology Epon embedded embryos were mounted on aluminum stubs for transverse or sagittal sectioning and sectioned on a Porter-Blum MT-1 ultramicrotome. For light microscopy, 1 um thick sections were cut with a glass knife and stained with Richardson's stain (0.5% methylene blue, 0.5% Azure II and 0.5% Borax in d i s t i l l e d water, Richardson et a l . , 1960). Sections were viewed with a Zeiss Photo-microscope III and photographed with Kodak FX 5060 Panatomic X film. The film was developed in D-76 f u l l strength for - 19 -seven minutes, washed with tapwater and fixed for ten minutes with Kodak rapid f i x . For transmission electron microscopy (TEM), Epon embedded embryos were sectioned with a Dupont diamond knife. Silver to grey coloured sections were picked up with carbon coated, 100 mesh copper or nickel grids. If the sections were to be used for stereo photographs, thicker sections which were violet to gold in colour were cut and retrieved with uncoated 150 mesh grids. Sections were stained by floating the grids for 10 minutes each on drops of 2% uranyl acetate (aq) and lead citrate. Grids with thick sections for stereo imaging were completely immersed in the stains in order to achieve maximum staining. Sections were viewed with a Phillips 301 TEM at 60 kV and photographed with Kodak Eastman fine grain 5302 35 mm film. Stereo pairs were photographed 12° apart. Film was developed for five minutes with Kodak D-19 f u l l strength, rinsed with tapwater and fixed for ten minutes with Kodak rapid f i x . 2.5 Histochemistry JB4 embedded embryos were mounted and sections 2-3 um thick were cut with a 'dry' knife. Sections were picked up with an eyelash and floated on drops of water on clean glass slides. The slides were placed on a 60° C hotplate for 3 minutes in order to adhere the sections to the slides. Slides were then stained with PAS, Alcian blue or Toluidine blue (Appendix #1) and covered with glass coverslips. Sections were viewed with a Zeiss Photo-microscope III and photographed as above. Paraffin embedded embryos were sectioned at 7 um with a steel knife on an American Optical Corporation Spencer '820' microtome. Ribbons of sections were floated on warm water and picked up on albumin coated glass slides. The slides were placed in a 60 °C oven for at least one hour prior to staining (Appendix #1) in order to adhere the sections to the slides. Sections were viewed and photographed as described for 0B4 sections. - 21 -3. RESULTS 3.1 Cryogens Embryos frozen in supercooled Freon 12 (dichlorodifluoromethane, mpt=l15 K) lacked the dumb-bell shape seen in vivo (Crawford and Chi a, 1978) but, instead, demonstrated shape distortion and cellular destruction (Fig. 3). Large irregular spaces were present within the epithelia, no distinction could be made between the nuclei and the cytoplasm, and a continuous basement membrane was not present. Thick, irregularily distributed strands of ECM made up the hyaline layer (HL) and f i l l e d the blastocoel. The pattern of ECM in the blastocoel resembled that of a honeycomb in some areas, but this arrangement was not consistent within different regions of the same embryo nor in sections of different embryos. The ECM of the HL was reduced to a series of strands, lacking the structural organization described by Crawford and Abed (1986). When liquid nitrogen (LN2) slush (mpt=63 K) was used as a cryogen, the embryonic shape was altered (Fig. 4) although not as severely as i t was after freezing with Freon 12 (cf. Figs. 3 and 4). Cellular preservation appeared adequate at the LM level. The ECM of the blastocoel had a strand-like morphology similar to that seen after freezing in Freon but in contrast to the honeycomb pattern seen after Freon fixation, strands of ECM after freezing in LN2 slush seemed to be oriented in parallel lines of random origin. A basement membrane was diffucult to resolve and the HL appeared to consist of fibrous strands (Fig. 4). At the ultrastructural level, the rough endoplasmic reticulum and nuclei appeared to be well preserved but the membranes of intracellular granules were often damaged or destroyed (Fig. 5). The ECM consisted of thick, amorphous strands both within the blastocoel (Fig. 6) and comprising the HL (Fig. 5). In addition to fibrous strands in the HL, a 0.5 nm thick mat of ECM was also present - 22 -at the apical surface of the ectoderm, possibly as a result of HL components collapsing (Fig. 5). The basal lamina adhered tightly to the epithelium and i t had strands of blastocoelic ECM extending from i t to the center of the embryo (Fig. 5 and 6). When frozen in liquid ethane (mpt=90 K), the overall shape of the embryos was maintained. The cells appeared to be free of ice crystal damage at the LM level (Fig. 7). The ECM of the blastocoel, however, had a very regular, fibrous appearance, similar to that described in embryos frozen in Freon 12 and LN2 slush, but exhibited more parallel strands per unit area than after freezing with the latter two cryogens. The HL appeared as a fibrous, tri-layered structure. The outermost sub-layer of the HL appeared as a network of fibres arranged in a honeycomb pattern which rested upon a slightly thicker mat of ECM. An area with l i t t l e fibrous material, the third sub-layer, existed between the mat of ECM and the epithelium. The basement membrane appeared continuous with the ECM of the blastocoel and was diffucult to resolve at the LM level. Finally, embryos frozen in liquid propane (mpt=84 K) demonstrated l i t t l e shape distortion at the LM level (Fig. 8). The ECM was diffucult to resolve at the LM level, suggesting that i t was more dispersed than was seen in embryos frozen in Freon 12, LN2 slush and ethane. At the ultrastructural level, intercellular spaces were present in many areas of the embryo (Fig. 9a). The HL was poorly preserved relative to previous chemical fixations (Crawford and Abed, 1986; Spiegel et a l . , 1989) showing some evidence of zonular organization but no distinct, identifiable sub-layers (Fig. 9a). The blastocoel (Fig. 9b) contained thinner fibres (40 nm) than those seen after cryofixation in LN2 slush (cf. Figs. 6 and 9b). The fibres of the propane fixed embryos were arranged in a parallel fashion but were sparsely distributed, which probably accounted for their - 23 -F i g . 3: 1.0 um cross-section through the esophageal region of an embryo frozen in Freon 12, embedded in Epon, and stained with Richardson's stain. The shape of the embryo is distorted and cells of ectodermal (Ec), endodermal (En), and mesenchymal (M) origin f a i l to demonstrate cellular detail and show vacuolation, indicating ice crystal damage. The ECM of the blastocoel is arranged in random strands with some areas resembling a honeycomb pattern (arrows). The hyaline layer (HL) has been reduced to a series of radiating fibres. x800. F i g . 4: 1.0 y.m cross-section through the coelomic region of an embryo frozen in liquid nitrogen slush, embedded in Epon and stained with Richardson's stain. The shape of the embryo has been distorted but the cells appear well preserved at the LM level. The ECM of the hyaline layer and blastocoel appears to be distributed as random strands (arrows). Coelom (C), Esophagus (Es), Ectoderm (Ec). x900. 24 - 25 -Fig. 5: TEM of an area of the ectoderm of an embryo frozen in liquid nitrogen slush, embedded in Epon, and stained with uranyl acetate and lead citrate. Intracellular membranes are often poorly distinguishable (arrows) but in general, the cellular preservation is good. The hyaline layer (HL) appears as a thick mat of ECM upon the apex of the epithelium with some radiating strands. The basal lamina is tightly adhered to the epithelium (arrowheads). xl9700. Fig. 6: TEM of the blastocoel of an embryo frozen in liquid nitrogen slush, embedded in Epon, and stained with uranyl acetate and lead citrate. Interconnecting strands of ECM (arrows) occupy the blastocoel. The basal lamina (arrowheads) appears tightly adhered to the ectoderm (Ec). x7500 26 - 27 -Fig. 7: 1.0 \m section of the posterior region of an embryo frozen in liquid ethane, embedded in Epon, and stained with Richardson's stain. The ectodermal (Ec) and endodermal (En) cells appear well preserved at this magnification. The ECM of the blastocoel consists of regular strands continuous with the basement membrane (arrows). The hyaline layer (HL) is fibrous in appearance and seems to consist of three distinct zones. x2200. Fig. 8: 1.0 \m cross-section through the region of the esophagus (Es) of an embryo frozen in liquid propane, embedded in Epon, and stained with Richardson's stain. The shape of the embryo has been preserved and the ina b i l i t y to resolve the ECM suggests that i t is much more dispersed than was seen in the previous cryofixed embryos. x670. - 29 -Fig. 9a: TEM of a region of the ectoderm of an embryo frozen in liquid propane, embedded in Epon, and stained with uranyl acetate and lead citrate. The hyaline layer (HL) is fibrous in appearance with some evidence of zonular organization. The cells are well preserved but numerous intercellular spaces (*) suggest that shrinkage has occurred. x9800. Fig. 9b: TEM of a region of the blastocoel adjacent the endoderm (En) of an embryo frozen in liquid propane, embedded in Epon, and stained with uranyl acetate and lead citrate. The ECM appears very fibrous (arrows) and these fibres are often continuous with the basal lamina (arrowheads). x20400. 30 - 31 -poor resolution at the LM level (Fig. 8). The BL was thin, relative to the blastocoelic fibers, and adhered close to the epithelium (Fig. 9b). 3.2 Cryoprotection Embryos cryoprotected with 10% DMSO and frozen in propane demonstrated widespread ice crystal damage at the ultrastructural level (Figs. 10a and b). Cellular organelles were indistinguishable and the ECM of the blastocoel and the HL were irregular and fibrous, not unlike those seen after freezing in Freon 12, LN2 slush and ethane. Embryos cryoprotected by pretreatment with 10% and 15% glycerol prior to freezing in propane differed ultrastructurally. Those treated with 10% glycerol exhibited ice damage which varied quantitatively throughout the embryo. In some areas, the organelles 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 intercellular and basal cytoplasmic processes suggested that some cell shrinkage had occurred (Fig. l i b ) . The ECM of the blastocoel was strand-like in appearance throughout the entire embryo (Fig. l i b ) . The HL, consisting of three fibrous sub-layers (Fig. 11a), included an outer network of thick and thin fibres which was connected to a much thicker band or supporting layer. Beneath this and extending to the epithelium was a region with relatively few fibrous elements interconnecting the supporting layer with the plasmalemma of the epithelium. There were also some microvilli present in this region. Embryos treated with 15% glycerol, on the other hand, appeared to demonstrate excellent preservation of cellular organelles (Fig. 12). Cells were free of ice crystal damage, with electron dense cytoplasms, intact organelles and well defined nuclei. Most cell s , however, appeared to have undergone tremendous shrinkage as indicated by the many cytoplasmic - 32 -processes and the separation of the epithelial cells from the BL and HL as well as from each other. The ECM of the blastocoel was evenly dispersed, showing no signs of collapse or disruption, and the HL appeared multi-layered and well preserved relative to chemically fixed hyaline layers (Crawford and Abed, 1986). Embryos treated with 10% ethylene glycol prior to freezing in propane had some cellular ice crystal damage which could be resolved at the ultrastructural level. Intracellular granules and many organelles, including nuclei, endoplasmic reticulum, and ribosomes could be distinguished but the nuclei and cytoplasm contained small vacuoles, a characteristic of ice crystal damage (Fig. 13a). Cells of the ectoderm and endoderm showed no differences in quality of preservation. The ECM of the blastocoel, however, exhibited coarse irregular strands adjacent to the endoderm but was much more evenly dispersed towards the ectoderm (Fig. 13b). This pattern, suggestive of a gradient, indicated that freezing and/or cryoprotection of ECM elements was effective through the ectoderm but only partially into the blastocoel. As the freezing rate decreased towards the center of the embryo, thereby increasing the likelihood of ice crystal formation (Van Harreveld and Crowe11, 1964), there was an insufficient quantity of ethylene glycol to inhibit the growth of ice crystals. Embryos treated with 15% ethylene glycol demonstrated excellent ultrastructural preservation of the cells and ECM, especially the HL. However, only a few random regions of the embryo, probably those areas f i r s t exposed to the cryogen (Fig. 14a), were well preserved. Elsewhere, in the same embryo, cellular ice crystal damage and ECM strands were present (Fig. 14b) as seen in previous preparations (cf. 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 in Epon, and staining with uranyl acetate and lead citrate, showing extensive cellular ice crystal damage and a collapsed hyaline layer (HL). X9900. F i g . 10b: TEM of a region of the blastocoel of an embryo prepared as described in Fig. 10a. The fibrous ECM (arrows), the damaged ectoderm (Ec) and endoderm (En) are shown. X12300. - 35 -F i g . 11a: TEM of the ectoderm of an embryo treated with 107. glycerol prior to freezing in propane, embedding in Epon, and staining with uranyl acetate and lead citrate. The hyaline layer (HL) is shown to consist of three distinct zones of fibers. A well preserved cell is shown (arrow). xl2700. F i g . l i b : TEM of the blastocoel of the embryo described in Fig. 11a. The ECM is very fibrous and is continuous with the basal lamina (arrows) of the endoderm (En). Extending from the ectoderm (Ec) are numerous cytoplasmic processes (arrowheads) which obscure its basal lamina, xl1000. F i g . 12: TEM of an embryo treated with 15% glycerol prior to freezing in liquid propane, embedding in Epon, and staining with uranyl acetate and lead citrate. The ECM of the blastocoel (b) is homogeneously distributed and a basal lamina is clearly present lining the ectoderm (arrows). Although cellular preservation is excellent, the numerous cytoplasmic processes and the separation of the ectodermal (Ec) and endodermal (En) cells from the ECM (*) indicate shrinkage has occurred. x4400. 36 - 37 -Fig. 13a: TEM of the ectoderm of an embryo treated with 10% ethylene glycol prior to freezing in propane, embedding in Epon, and staining with uranyl acetate and lead citrate. Areas of cellular ice crystal damage are present (arrows) and the hyaline layer (HL) appears damaged, consisting of irregularly distributed strands of ECM. There appears to be some zonular organization in the HL. xl3500. Fig. 13b: TEM of the blastocoel of an embryo prepared as described in Fig. 13a. The ECM adjacent to the ectoderm (Ec) is more homogeneous than the fibrous components (arrows) near the endoderm (En) suggesting the presence of a gradient of freezing despite good preservation of the cells of the endoderm themselves. x7200. - 39 -Fig. 14a: TEM of the hyaline layer of an embryo treated with 15% ethylene glycol prior to freezing in propane, embedding in Epon, and staining with uranyl acetate and lead citrate. Multiple sub-layers can be seen. x24900. Fig. 14b: TEM of a region of the ectoderm of an embryo prepared as described in Fig. 14a. Ice crystal damage is clearly visible in the nuclei (N), and the hyaline layer (HL) lacks the detail seen in Fig. 14a. x9500. 40 - 41 -Embryos cryoprotected with 15% propylene glycol prior to freezing in propane appeared to be preserved better than any of the those preserved using different cryogens and cryoprotectants. At the LM level, the shape of the embryo was maintained, the cells were well preserved, and the blastocoel appeared to contain a homogeneously, lightly stained material (Fig. 15). Cell ultrastructure was well preserved, showing no evidence of ice crystal damage (Figs. 16, 18 and 19). The membranes of intracellular granules had a punctate appearance, possibly indicating damage during fixation (Fig. 16), and the rough endoplasmic reticulum was frequently distended (Fig. 19). The nuclei lacked vacuolation, demonstrating a heterochromatic periphery and one central nucleolus (Fig. 16). The ECM was preserved in a way so as to reveal the interesting morphological relationships with the cells as described below. 3.3 ECM of the Blastocoel and Basal Lamina An ECM consisting of short extracellular fibres and amorphous material of intermediate density was distributed homogeneously throughout the embryonic cavity. The TEM showed these small twig-like ECM components evenly scattered throughout a l l planes of section, with no set pattern or orientation (Figs. 16, 17 and 18). In at least two locations - radiating from the esophagus interconnecting i t di s t a l l y to the ectoderm (Fig. 17) and in the dorsoposterior region of the embryo termed the dorsal web (Fig. 18) - distinct fibres existed which travelled in somewhat of a sinusoidal path. These fibres measured approximately 50 nm in diameter and appeared either alone or frequently in bundles of three or more. The basal lamina appeared thicker (200 nm) than after chemical fixation (Crawford, 1989) and two distinct zones, the lamina densa and lamina lucida, could be identified (Fig. 16). The morphology of the basal - 42 -lamina did vary, usually in thickness, in different 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 thick, which compared well with measurements taken in vivo (Fig. 19). This outer ECM did not follow each ridge or trough of the epithelium but seemed to have a shape independent of i t . The HL consisted of a multi-layered ECM whose density varied amongst each sub-layer and appeared to consist of six sub-layers - the intervillus layer, hyaline 1 (HI), H2, H3, the boundary layer, and the coarse outer meshwork (Fig. 20). The coarse outer meshwork, the outermost sub-layer, consisted of an ECM which was homogeneously distributed in a pattern characteristic of the ECM of the blastocoel. However, the ECM of this sub-layer became more diffuse towards its outer limit (Fig. 19). The boundary layer, shown as a stereo pair (Fig. 21), appeared to be a 250 nm thick array of hairpin-like loops of matrix strands. This sub-layer was the major site of termination for m i c r o v i l l i , although some extended into the outer meshwork (Fig. 22). Within the boundary layer, the tips of the microvilli formed microvillus associated bodies (Spiegel et a l . , 1989) consisting of electron dense material lining the inner and outer surfaces of the plasma membranes (Figs. 23a-d). The extramembranous portion of this material appeared to be continuous with the loops of the boundary layer. In cross section, this cap of material appeared to radially project thin filamentous structures from the external densities to the loops of the boundary layer (Figs. 23b). In addition, filaments could be seen running the length of the microvilli and i t is possible that these filaments were attached to the inner dense region of the microvillus associated body (Fig. 23d). - 43 -Figs. 15 through 23 are sections of embryos treated with 15% propylene glycol prior to freezing in liquid propane, embedding in Epon, and staining with uranyl acetate and lead citrate. Fig. 15: 1.0 um, mid-sagittal section of an embryo prepared as described above and stained with Richardson's stain. The ECM of the blastocoel (b) and the hyaline layer (arrows) do not appear to contain the thick, fibrous structures that were seen after cryofixation by other methods. Esophagus (Es). x470.. 4 4 - 45 -F i g . 16: TEM of the basal lamina of the ectoderm (Ec) adjacent to the blastocoel (b). The basal lamina consists of a patchy lamina lucida (LL) and a thick lamina densa (LD) which appears to contain tiny f i b r i l s and granules. The ECM of the blastocoel consists predominately of twig-like structures (arrowheads) and amorphous material. x27600 4 6 - 47 -F i g . 17: TEM of extracellular fibres (arrows) radiating from the esophagus (Es). x!4700. F i g . 18: TEM of the dorsal web showing extracellular fibres (arrows) within the meshwork of 'twig-like' ECM. Endoderm (En), Ectoderm (Ec). x31200. - 49 -Fig. 19: A comparison of the thickness of hyaline layers (HL) seen in a living embryo treated with India ink particles (insert) and an embryo prepared by freeze substitution. Both hyaline layers are approximately 4.0 \im thick. Ectoderm (Ec), Blastocoel (b), India ink particles (In). x22800 (inset: x990). 5 0 - 51 -Fig. 20: TEM of the hyaline layer demonstrating its convoluted morphology and showing i t s six sub-layers: The intervillus sub-layer (IV); the supporting layer, consisting of HI, H2 and H3; the boundary sub-layer (B); and the outer meshwork (OM). X41500. 52 - 53 -F i g . 21: Stereo pair (a & b) of TEM's demonstrating the looping arrangement of the boundary sub-layer (B). Intervillus sub-layer (IV), supporting layer (HI, H2 and H3), outer meshwork (OM). X57500. F i g . 22: TEM showing the relationship between microvilli and the hyaline layer. A microvillus associated body connecting a microvillus to the boundary sub-layer (B) is indicated (arrowhead). Intervillus sub-layer (IV), supporting layer (HI, H2 and H3), outer meshwork (OM). X57000. - 55 -Figs. 23a-d are high magnification TEM's of the microvillus associated body. Fig. 23a: Higher magnification TEM of Fig. 22 showing the dense material lining the inner and outer surfaces of the plasma membrane (P) of the tip of the microvillus and the continuity of the outer dense region with the fibres of the boundary sub-layer (B). x83300. Fig. 23b: Cross-section through the tip of a microvillus (arrowhead) showing the continuity of the outer dense material with the boundary sub-layer (B). X58400. Fig. 23c: Oblique section showing the inner and outer densities of the microvillus tip and the radiating strands of ECM which are continuous with the boundary sub-layer (B). Plasma membrane (P). xl50000. Fig. 23d: Sagittal section of a microvillus demonstrating filaments running i t s length (arrows). X48000. 56 - 57 -The HI sub-layer was an electron dense mat of ECM upon which the boundary layer originated (Figs. 20, 21). Below HI was the sub-layer, H2, which consisted of a 125 nm thick, amorphous belt of ECM (Figs. 20, 21) under which was H3 (Figs. 20, 21), a distinct transition layer between H2 and the intervillus layer, the sub-layer adjacent the epithelia. H3 appeared to be an intermittant, electron dense material embedded in the base of the H2 sub-layer and interfacing with the i n t e r v i l l u s layer. H3 could be differentiated from the components of the i n t e r v i l l u s layer based on i t s greater electron density. Mi c r o v i l l i extended through the in t e r v i l l u s sub-layer into the outer zones of the HL (Fig. 22). Since the shape of the HL did not perfectly reflect that of the epithelium, i t was the thickness of the intervillus sub-layer that varied in order to account for the convoluting characteristics of the boundary and supporting sub-layers of the HL (Fig. 20). In addition to m i c r o v i l l i , an ECM sli g h t l y more fibrous in appearance than that of the OM existed throughout the in t e r v i l l u s layer. 3.5 Chemical F i x a t i o n The morphology of Pi saster ochraceus embryos fixed with glutaraldehyde and osmium tetroxide has been previously described (Crawford and Chi a, 1978, 1982; Crawford and Abed, 1983, 1986; Abed and Crawford, 1986a,b; Crawford, 1989, 1990). The embryos were prepared in this study for comparative purposes. The HL after this preparation consisted of five distinct sub-layers - the coarse outer meshwork, the boundary layer, HI and H2, and the intervillus layer - in total appoximately 2 um thick (Fig. 24a). The coarse outer meshwork, which varied in thickness from 0.7 um to 1.2 um, consisted of random clumps of ECM of consistent density throughout; the 150 nm thick boundary layer - 58 -Fig. 24a: TEM of the hyaline layer of an embryo fixed with glutaraldehyde and alcian blue, embedded in Epon, and stained with uranyl acetate and lead citrate showing the five sub-layers: The intervillus sub-layer (IV); the supporting layer (HI and H2); the boundary sub-layer (B); and coarse outer meshwork (OM). x35800. (photograph courtesy of Dr. B. Crawford). Fig. 24b: TEM of the basal lamina of an embryo prepared as described in Fig. 24a showing the lamina lucida (LL) and the lamina densa (LD). The fibrous components of the blastocoel are indicated (arrowheads). x33200. (photograph courtesy of Dr. B. Crawford). 5 9 - 60 -Fig. 25: TEM of an embryo prepared as described in Fig. 24a showing fibres (arrows) radiating from the esophagus (Es). Mesenchyme cell (M). X26500. (photograph courtesy of Dr. B. Crawford). Fig. 26: TEM of an embryo fixed as described in Fig. 24a showing the dorsal web (arrows). Ectoderm (Ec); Endoderm (En). xl0400. (photograph courtesy of Dr. B. Crawford). 61 - 62 -appeared to contain looping structures which were similar to that seen after cryofixation but somewhat condensed; HI was 50 nm thick and very electron dense; H2 was amorphous and lacked the homogeneity seen after cryofixation; and the intervillus layer was relatively electron lucent, lacking the abundant extracellular material seen after cryofixation. A distinct H3 sub-layer could not be seen after this fixation. The BL, in general, was thinner (10 nm) than the cryofixed BL and could also be separated into a lamina densa and a lamina lucida as previously described by Crawford (1989). The adjacent blastocoel contained many 30 nm thick, dense fibres (Fig. 24b) but as with cryofixed embryos, these fibres were found in greatest numbers radiating from the esophagus (Fig. 25) and in the dorsal web (Fig. 26). There was no dispersed gel-like ECM in the blastocoel after this type of fixation. 3.6 H i s t o c h e m i s t r y (Table I ) PAS Cryofixed embryos embedded in JB4 had numerous large intracellular granules which stained intensely PAS positive and a HL which stained very li g h t l y PAS positive (Fig. 27). Formalin fixed embryos embedded in paraffin had weakly positive PAS staining areas in the hyaline layer and the basement membrane and well defined positive staining of cellular granules (Fig. 28). Alcian blue At pH 3.2 and 2.5, the hyaline layer, basement membrane, and ECM of the blastocoels of the cryofixed and formalin fixed embryos were intensely alcian blue positive (Fig. 29, 30). A few small intracellular granules - 63 -were alcian blue positive in the cryofixed embryos but such granules could not be resolved in the formalin fixed material. At pH 1.0, both the cryofixed and the formalin fixed embryos showed intensely positive alcian blue staining of the hyaline layer and the basement membrane. Formalin fixed embryos also revealed intensely positive alcian blue staining of the hyaline layer and the blastocoel, but only moderately positive staining of the basement membrane. The blastocoel of the cryofixed embryos stained moderately alcian blue positive. Alcian blue positive cellular granules could not be resolved in either preparation. When stained at pH 0.5 and 0.2, there was no alcian blue staining in the cryofixed embryos. However, intensely positive alcian blue staining was present in the hyaline layer, basement membrane and the blastocoel of the formalin fixed embryos at pH 0.5 and moderate staining of these components was present at pH 0.2. PAS/Alcian blue (DH 2.5) Embryos prepared by cryofixation and formalin fixation showed similar staining patterns to that described above: only the large, intracellular granules were PAS positive; a few small granules that could only be resolved in the JB4 embedded embryos were alcian blue positive; the hyaline layer, the blastocoel, and the basement membrane were intensely alcian blue positive. Toluidine blue Neither the cryofixed nor the formalin fixed embryos demonstrated metachromasia. This came as no surpise as the cryofixed embryos, embedded in JB4, could not be hydrated due to the hydrophobic properties of the plastic resin. The embryos fixed in formalin and embedded in paraffin, on - 64 -the other hand, were so shrunken and damaged that even i f metachromasia was present, i t could not be clearly resolved. Embryos fixed by both methods showed basophilia when stained with toluidine blue. The hyaline layer of cryofixed embryos was intensely basophilic while the basement membrane demonstrated moderate basophilia. The hyaline layer of the formalin fixed embryos exhibited moderate basophilia. The ECM of the blastocoel did not demonstrate basophilia in either preparation. - 65 -Table I : The Res u l t s o f Histochemical S t a i n i n g o f C r y o f i x e d  and Formalin Fixed A s t e r o i d EmbryosJ Histochemical stain Cryofixed embryos Formalin fixed embryos Component2 PAS ++ ++ G +/- HL — — b - +/- BM Alcian blue pH 3.2 +/- _ G ++ ++ HL ++ ++ b ++ ++ BM Alcian blue pH 2.5 +/- _ G ++ ++ HL ++ ++ b ++ ++ BM Alcian blue pH 1.0 _ _ G ++ ++ HL +/- ++ b ++ +/- BM Alcian blue pH 0.5 _ G - ++ HL - ++ b - ++ BM Alcian blue pH 0.2 _ G — +/- HL - +/- b - +/- BM PAS and Alcian PAS++:AB+/- PAS++:AB- G blue pH 2.5 PAS -:AB++ PAS-:AB+/- HL PAS -:AB++ PAS-:AB++ b PAS -:AB++ PAS-:AB+/- BM Toluidine blue _ G ++ +/- HL — - b +/- — BM '++, intensely positive staining; +/-, moderately positive staining; -, no staining visualized. 2G, granules; HL, hyaline layer; b, blastocoel; BM, basement membrane. - 66 -Figs. 27, 29 and 30 are 2.0 um sections of embryos cryoprotected with 15% propylene glycol prior to freezing in propane, embedding in JB4, and stained with either PAS or alcian blue. Fig. 27: Cryofixed embryo, stained with PAS, showing intensely positive staining in the intracellular granules (arrowheads) and moderately positive staining in the HL. x580. Fig. 28: 7 um section of a formalin fixed embryo, stained with PAS, showing intensely positive staining in the intracellular granules (arrowheads) and moderately positive staining in the HL. The moderately positive staining BM cannot be clearly resolved in this section. x460. Fig. 29: Cryofixed embryo, stained with alcian blue at pH 3.2, showing intensely positive staining in the HL and basement membrane (arrowheads). The homogeneous staining of the blastocoel is present but cannot be seen in this picture. x390. Fig. 30: Cryofixed embryo, stained with alcian blue at pH 2.5, showing a similar distribution of alcian blue positive material as seen in Fig. 29. x380. - 68 -4. DISCUSSION In conformity with the objectives of this study, the Discussion is divided into two sections. 4.1 Freeze Substitution of Asteroid Embryos Conditions for adequate plunge freezing When plunge freezing an uncryoprotected tissue, adequate freezing will occur only i f the optimal conditions outlined by Elder et a l . (1982) and Robards and Sleytr (1985) are met. These conditions include using the smallest possible tissue block, keeping the cryogen in motion, having a sufficient depth of cryogen, having a high entry velocity into the cryogen and choosing a cryogen with a melting point less than 140 K, good thermal conductivity, high specific heat, and a boiling point far removed from its 3 melting point. Tissue sizes of less than 1mm are necessary though not always sufficiently small to be thoroughly frozen but because the embryos used in this study never exceeded this size (approx. 500um in length), only the number of embryos frozen at one time required attention. Freezing rates are dependent upon the rate of heat transfer through the tissue which decreases exponentially as the distance from the surface of the tissue increases (Van Harreveld and Crowe11, 1964). Since asteroid embryos in the larval stage have blastocoels f i l l e d with a hydrated extracellular matrix that has a very poor thermal conductivity, i t became even more c r i t i c a l to obtain maximum possible freezing rates. Previously successful v i t r i f i c a t i o n studies involved freezing extremely thin layers of solutions in liquid cryogens (Bruggeler and Mayer, 1980; Dubochet et a l . , 1982). This concept was applied to the current system by placing a monolayer or sheet of embryos on a copper freeze fracture EM grid, removing - 69 -as much water from the grid as possible and immediately plunging the grid into the cryogen with fine-tipped stainless steel forceps. In addition to the low mass and high thermal conductivity, these grids allowed those specimens in the windows of the grids to be exposed to the cryogen on both sides simultaneously, unlike solid supports which allow only one side of the specimen to come in direct contact with the cryogen. Freezing tissue on EM grids is not entirely new. Adrian et a l . (1984) cryofixed viruses using copper mesh grids while Porter and Anderson (1982) froze cultured kidney cells on gold grids. To my knowledge, whole eukaryotic organisms have never been cryofixed successfully prior to the present study using this method. It is generally accepted that cryogen s t i r r i n g is essential for optimal freezing although few authors actually indicate whether or not their cryogen was st i r r i n g at the time the tissue was plunged into i t . The st i r r i n g of cryogen in the present study was in a horizontal direction. Murray et a l . (1984) have devised a countercurrent plunge freezing system in which the cryogen circulates vertically. They maintain that during horizontal s t i r r i n g , circumferential velocity differences and variations in the depth of the cryogen due to a stir r i n g vortex create a large vertical temperature gradient which is detrimental to the speed of freezing. But, because successful cryofixation was eventually achieved with horizontal s t i r r i n g in the present study, the st 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 was somewhat controversial (Stephenson, 1956) but there is a now consensus in the literature that a high plunging velocity correlates well with the depth of freezing into the tissue (Costello and Corless, 1978; Elder et a l . , 1982; Robards and Sleytr, 1985). Numerous mechanical plunging devices ranging from spring activated - 70 -triggers (Robards and Crosby, 1983) to costly solenoid operated electromagnetic plungers (Escraig, 1982) have been constructed in order to standardize and maximize plunging velocities. Steinbrecht (1982), however, has shown that plunging velocity is proportional only to the yield of well frozen specimens and not to the overall quality of freezing. Because the embryos in this study were plunged quickly (>1.0 m/s) by hand into the cryogen in manner similar to that described by Allenspach and Childress (1986), i t was never determined i f a plunging velocity:freezing efficiency relationship existed in this system. As a rule of thumb, however, the grids were plunged into the cryogen as rapidly as possible to avoid pre-cooling induced by the evaporating nitrogen and cryogen and to expose the entire grid surface to the cryogen as synchronously as possible. Cryogens Other conditions required for optimal cryoquenching revolve around the properties of the cryogen. From a thermodynamic viewpoint, the melting point (mpt), boiling point (bpt), specific heat, and thermal conductivity are the most important parameters (Table II). The mpt of the cryogen should be low (<140 K) and the bpt should be high (>175 K), such that the difference between them is as wide as possible (Robards and Sleytr, 1985). If the mpt and bpt are not far removed, the Leidenfrost phenomenon may result (Robards and Sleytr, 1985). The Leidenfrost phenomenon is the formation of a vapour jacket around the specimen due to the relatively high temperature of the specimen causing the cryogen immediately surrounding i t to b o i l . This slows the freezing rate dramatically, thereby allowing widespread ice crystal formation. The specific heat of the cryogen, which is a measure of the storage capacity of heat per unit mass, should be high. This ensures temperature s t a b i l i t y during specimen plunging because - 71 -the cryogen is able to absorb the temperature of the specimen without effecting i t s own temperature. Finally, the thermal conductivity of the cryogen, the a b i l i t y to carry away heat, should also be high in order to disperse the heat throughout i t s volume and to maintain the temperature at its melting point. On the basis of these c r i t e r i a , four cryogens were chosen for this study; Freon 12, liquid nitrogen slush, ethane and propane. Each had several qualities that theoretically made them sufficiently good cryogen candidates to study frozen asteroid embryos at the ultrastructural level. Since halocarbons have been commonly used as cryogens (Rebhun, 1972; Rosenkranz, 1975; Somlyo et a l . , 1977; Nagele et a l . , 1985;), and were readily available in the lab at the beginning of the present study, Freon 12 was the f i r s t tested. The results clearly demonstrate the poor freezing properties of Freon 12 for this system. The mpt of Freon 12 is 115 K, and the bpt 243 K (Table II), thus satisfying the above c r i t e r i a for low mpt and a far removed bpt (128 K). The specific heat and thermal conductivity values are also relatively low, 0.854 J/g/K and 138 mJ/m/s/K, respectively. Since the latter two values are low, i t is possible that Freon 12 did not maintain i t s mpt temperature upon introduction of the specimen thus resulting in conditions which encouraged ice crystal formation. Liquid nitrogen has been previously used as a cryogen (Rosenkranz, 1975). Because LN2 is boiling at room temperature, i t s use in cryogenic procedures would result in the Leidenfrost effect. Since LN2 is inexpensive and easily obtainable, in the past i t was used more as a coolant for other "cryogens than as a cryogen i t s e l f . If reduced to its mpt, however, the Leidenfrost effect may be suppressed because some of its thermodynamic properties improve to levels acceptable for the present - 72 -TABLE II: The Thermodynamic Properties of the Crvoaens U s e d J k Cryogen mpt bpt AT C 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 in Kelvin bpt = Boiling point in Kelvin AT = Difference between the mpt and bpt c = Specific heat in joules/gram/Kelvin near the mpt. k = Thermal conductivity in millijoules/metre/second/Kelvin near the mpt. ref. Weast, R. (1982). - 73 -study. The specific heat of LN2 slush is higher than that of propane at 2.06 J/g/K and the mpt is extremely low at 63 K (Table II) suggesting that these features, at least in theory, should make LN2 a good cryogen. Tests on asteroid embryos, however, revealed cellular ice crystal damage and extensive phase separation within the blastocoel. This was probably due to the Leidenfrost effect. Because only a 14 K gradient exists between the mpt and the bpt, and the thermal conductivity is poor, being only slightly higher than Freon at 153 mJ/m/s/K (Table II), i t is possible that a vapour jacket, which would dramatically slow the freezing process, could easily form resulting in a poor structural preservation of the embryos. Bald (1984) has shown that LN2 slush would make an excellent cryogen, in theory, at 33.5 atmospheres but these conditions are beyond the a b i l i t y of this laboratory so this theory remains to be tested. It has been previously stated that liquid ethane is an excellent cryogen for plunge freezing (Silvester et a l . , 1982; Dubochet et a l . , 1985). Ethane has the highest specific heat (2.27 J/g/K) and thermal conductivity (240 mJ/m/s/K) values of the four cryogens used in this study and i t s mpt (90 K) is comparable with that of propane (84 K) (Table II). These outstanding thermodynamic properties should have allowed for optimal freezing of asteroid embryos. However, the bpt of ethane is low (184 K) leaving a 94 K difference between i t and the mpt which is a much lower difference than is present in Freon 12 and propane (Table II). It is possible that the Leidenfrost phenomenon played a role in the overwhelming phase separation in the blastocoel of the asteroid embryos resulting in the widespread strand-like ECM structures seen after freezing with this cryogen. The pattern of ECM seen after ethane fixation relative to the larger but fewer strands seen after liquid nitrogen fixation correlates with a higher mpt/bpt differential in ethane than in the latter. This - 74 -suggests that due to the higher ethane d i f f e r e n t i a l , the good specific heat and thermal conductivity, ice crystals did not have as much time to grow as in prior fixations, but there was s t i l l enough ice crystal formation to significantly disrupt the ECM. Propane has been used as a cryogen by numerous researchers (Burnstein and Maurice, 1978; Elder et a l . , 1982; Porter and Anderson, 1982; Steinbrecht, 1982; Meissner and Schwarz, 1990). The temperature difference between the bpt (231 K) and mpt (84 K) is 147 K, the largest differential of the four cryogens tested in this study. Its specific heat is 1.92 J/g/K which ranks just behind ethane and i t s thermal conductivity is 219 mJ/m/s/K (Table II). The 'useful heat-sink capacity', a term coined by Plattner and Bachmann (1982), is the energy difference between the specimen stabilization temperature (approx. 173 K) and the mpt of the cryogen, in this case 84 K. Propane, according to the above authors, has the highest 'useful heat-sink capacity' of the cryogens they reviewed (120 0/ml), which included the four used in this study, suggesting that propane is the optimal cryogen of those tested for ultrastructural studies. 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 for plunge freezing and the results from this study support this. Cellular ice crystal damage was present after propane freezing but the phase separation in the asteroid blastocoel was not as distinct as in the previous cryofixations. Such damage could easily be distinguished at the EM level, however. Since propane is one of the most effective cryogens known, these results probably reflect the thickness of the embryos and the accompanying water on the grid and suggest that optimal freezing for ECM preservation could not be achieved by cryoquenching in propane alone. It therefore became necessary - 75 -to incorporate cryoprotectants into the embryos in order to intervene in the ice crystal growth process. Cryoprotectants Cryoprotective chemicals essentially impede ice crystalization within tissues by lowering the freezing point, increasing the supercooling capacity, decreasing the c r i t i c a l cooling rate, lowering the eutectic point, removing or decreasing the quantity of 'unbound' water and removing nuclei for crystallization (Robards and Sleytr, 1985) thus enhancing the likelihood of v i t r i f i c a t i o n . The four cryoprotectants used in this study, DMSO, glycerol, ethylene glycol and propylene glycol do some or a l l of these with different efficiencies which are reflected in the cryoprotected embryos. The success of a perspective cryoprotectant seems, to depend upon its molecular structure. Polyols have been used extensively in the past as cryoprotectants. Glycerol, for instance, is non-destructive to proteins and non-toxic to most living systems (Fink, 1986). The three hydroxyl groups ^enable multiple hydrogen bonds to form per molecule which readily interfere with the mobilization of ice crystals by occupying water molecules. Glycerol is a very viscous liquid which in i t s e l f may substantially retard the growth of crystals (Skaer, 1982). When 10% glycerol was used to cryoprotect the asteroid embryos, ice damage and phase separation were present in a l l regions of the embryo. Since the time of exposure to a l l cryoprotectants was held constant, i t is unlikely that incomplete penetration was the cause of poor cryoprotection. This suggests that there was an insufficient concentration of glycerol, therefore the concentration was increased to 15%. These results showed superior ultrastructural preservation with no evidence of ice damage but the osmotic - 76 -effects of glycerol were quite evident. Glycerol is used by many authors to cryoprotect tissues, so the artefacts induced by i t are well known. Shrinkage of the cells is one such artefact (Skaer, 1982). The ECM was preserved in a gel-like pattern, which indicates the presence of amorphous or perhaps vitreous ice. This is probably reflected by the a b i l i t y of glycerol to interact with unbound water which normally would have formed ice crystals, pushing the ECM to the periphery as large strands. However, even though the ECM pattern correlates with the gel-filled-blastocoel hypothesis proposed by Strathmann (1989), the vast amount of shrinkage and the i n a b i l i t y to offset this by decreasing the concentration while s t i l l retaining thorough preservation without ice crystal formation eliminates glycerol as a good candidate for cryoprotecting asteroid embryos under the described conditions. Dimethylsulfoxide (DMSO) is a well established cryoprotectant. Since i t is used almost exclusively for preserving cell v i a b i l i t y (Robards and Sleytr, 1985), l i t t l e work has been done to show how DMSO preserves cell u l a r ultrastructure. DMSO is a polar molecule that can form hydrogen bonds only by accepting protons. It therefore does not have as much interacting potential as the polyols. If used in high enough concentrations, this lack of hydrogen bonding may be compensated for. However, when used at concentrations greater than 15%, it s effects become toxic (Robards and Sleytr, 1985). When applied to asteroid embryos at concentrations of 5% and 10%, DMSO failed to protect the embryos from ice crystal formation suggesting that i t inadequately interacts with 'unbound' water. Ethylene glycol has rarely been used with success as a cryoprotectant (Richter, 1968a). Its hydrogen bonding capacity exceeds that of DMSO but is lower than glycerol due to one less hydroxyl group. It can interact - 77 -with 'unbound' water molecules to impede ice crystal formation but may also interact with i t s e l f thereby blocking i t s own water binding characteristics. The results of the present study demonstrated that freezing was inconsistent throughout starfish embryos treated with ethylene glycol. When used at a concentration of 10%, a freezing gradient was present in the blastocoel. The quality of freezing was good at the surface of the embryo but this quality diminished towards the center. This suggests that at the surface of the embryo, which contacted the cryogen f i r s t , and for a small distance into the blastocoel the concentration of ethylene glycol and the rate of freezing was adequate to inhibit resolvable phase separation. However, further into the embryo, the concentration of ethylene glycol, though s t i l l the same, does not appear to be sufficient to compensate for the f a l l in the freezing rate brought about by the poor thermal conductivity of the blastocoel. This allowed for the growth of ice crystals resulting in separation of water and ECM. When the ethylene glycol is increased to 15%, some areas of the embryo were very well preserved. However, many areas, largely in the center of the embryo, revealed ice crystal damage. It is possible that ethylene glycol may be a good cryoprotectant for monolayers of c e l l s , since there would be no blastocoel from which heat must be transferred. But, for large, hydrated organisms such as the embryonic asteroid, ethylene glycol does not adequately interfere with ice crystal growth in the center of the embryo unless very high concentrations are used. Such concentrations would probably induce large amounts of osmotic shrinkage rendering the material unsuitable for ultrastructural study. When ethylene glycol is methylated, the resulting molecule is propylene glycol. Fahy et a l . (1987) discussed in detail the effects of methylation upon polyols and 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 with a terminal unsaturated methyl group. It is possible that terminal methyl groups produce an enthalpically favourable reorganization of water. In addition, these terminal methyls may inhibit 'self-linking' of the molecule, though usually only a problem with amides, thereby allowing the molecule to f u l f i l l i t s complete water binding potential. Propylene glycol was determined to be the best of the four cryoprotectants studied here, and Boutron and Kaufmann (1979) showed this to be also true in binary systems (cryoprotectant plus water). Its a b i l i t y to interfere with ice crystal growth on a consistent basis and not induce excessive shrinkage in asteroid embryos has allowed for this type of fixation to reveal the more detailed ultrastructure discussed below. Currently, propylene glycol is also being studied for i t s a b i l i t y to cryopreserve viable blood cells (Takahashi et a l . , 1986) and corneas (Rich and Armitage, 1990). Not a lot of rapid freezing studies have involved the use of propylene glycol so the present study certainly demonstrates its u t i l i t y as a cryoprotectant for plunge freezing marine invertebrate embryos. Since propylene glycol has one free methyl group, and methyl groups enhance v i t r i f i c a t i o n , i t would seem logical to assume that the addition of a methyl group to the other terminus, creating 2,3-butanediol, would produce a better cryoprotectant than propylene glycol (Boutron et a l . , 1986; Mehl and Boutron, 1987). This compound, however, was not utilized in this study but might prove useful in future work. Freeze substitution As discussed in the Introduction, the aim of freeze substituting asteroid embryos was to preserve them in manner which induced minimal - 79 -artefact, including the denaturation of proteins caused by the harsh chemical treatments used in conventional fixation. A wide-mouth Thermos, placed in a -60 °C freezer, was used as a chamber for freeze substitution because of the lack of a -90 °C freezer which would provide the temperature necessary for the substitution to occur. Anhydrous ethanol was used as the substituting medium because i t was readily available, inexpensive, and had been successfully used by other authors (Simpson, 1941; Fernandez-Moran, 1960). The substitution was a four day duration to ensure completion as diffusion is very slow at such low temperatures, thus reducing the chances of ice crystal formation during the substitution. Acetone (mpt=177 K) was f i r s t used as a substituting medium in this study but i t s i n f e r i o r i t y was quickly realized due to i t s relatively poor solu b i l i t y in ice and poor water transporting a b i l i t i e s . Ethanol (mpt=158 K), therefore, served as a more efficient substituting f l u i d and would do so at lower temperatures than acetone i f necessary. Recently, methanol, sometimes used in conjunction with aldehydes (Zalokar, 1966; Meissner and Schwarz, 1990), has become widely accepted as an excellent substituting medium for biological material (Glauert, 1974; Barlow and Sleigh, 1979; Steinbrecht, 1982; Maitland and Arsenault, 1989) but i t was not utilized in this study. Since the temperature of the substitution chamber ranged from 190 K to 170 K, depending on how recently the LN2 was added, methanol (mpt=175 K) could have frozen which would, in effect, have terminated the substitution. By using ethanol, such accidental freezing was much less l i k e l y . Methanol has been well documented as having the fastest substitution time below 183 K (Barlow and Sleigh, 1979; Humbel and Muller, 1984) whereas acetone has one of the slowest substitution times (Van Harreveld et a l . , 1965; Zalokar, 1966; Muller et a l . , 1980). Unfortunately, l i t t l e work has been done in determining the substituting - 80 -properties of ethanol other than being shown to have better substituting capabilities 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 resulting in the relocation or removal of such compounds. As suggested by Harvey (1982), i t would come as no surprise, then, i f autoradiographic or cytochemical analysis revealed low concentrations or inappropriate distributions of certain ions, for example, in freeze substituted asteroid embryos. But since the present study was primarily morphological and histochemical in nature, these drawbacks will not be discussed any further other than to acknowledge their probable existence for future reference. 4.2 Morphology and Histochemistry Blastocoel The ECM of the blastocoel of cryofixed, JB4 embedded embryos stained alcian blue positive at pH 3.2, 2.5, and 1.0. When fixed in formalin and embedded in paraffin, however, additional positive staining was seen in the blastocoel at pH 0.5. This suggests that both sulfated and unsulfated glycosaminoglycans (GAG's) are present within the larval blastocoel. Macromolecules containing sulfated components have been shown to be necessary for mesenchyme cell migration in echinoids (Karp and Solursh, 1974; Katow and Solursh, 1981; Lane and Solursh, 1988). Solursh and Katow (1982) have further characterized some of these components to be GAG molecules such as heparan sulfate, chondroitin 6-sulfate and dermatan sulfate. Since histochemical staining in the developing asteroid suggests the presence of such components, and similar histochemical results have been obtained from the echinoid (Sugiyama, 1972; Katow and Amemiya, 1986), i t can be postulated that proteoglycans with sulfated and unsulfated acidic - 81 -GAG's as well as glycoproteins are major constituents of the ECM of the blastocoel in the larval stage. Histochemical staining with alcian blue using the c r i t i c a l electrolyte concentration (CEC) method (Scott and Dorling, 1965) may allow for a more accurate assessment of the presence of specific sulfated GAG's. The lack of positive PAS staining within the blastocoel suggests that neutral GAG's such as hyaluronic acid are not a major constituent of the blastocoel at this stage of morphogenesis. Ultrastructural analysis of the blastocoel of freeze substituted five-day-old Pi saster ochraceus embryos reveals a twig-like meshwork of short fibres of varying thickness as well as a distinct population of fibers in the esophageal and dorsoposterior regions of the embryos. The meshwork pattern is similar to that seen in a r t i f i c i a l gels of ECM following rapid fixation (MacKenzie and Luyet, 1962, 1967; Allenspach and Kraemer, 1989). The fibres lack the amorphous coat of alcianophilic material seen in the chemically fixed embryos described previously (Crawford and Chi a, 1982; Abed and Crawford, 1986b). In the past, i t has been argued as to whether the blastocoel of the asteroid embryo is f l u i d - f i l l e d with extracellular fibres or g e l - f i l l e d (Crawford and Chi a, 1982; Abed and Crawford, 1986b; Crawford, 1990). Observations of chemically fixed embryos favour the former. Recent experiments by Strathmann (1989), however, have demonstrated that small particles will not penetrate the opened blastocoels of living echinoderm embryos and that they also maintain their shape after being cut open. If f l u i d - f i l l e d , one would expect the embryos to collapse after such a dissection due to the sudden drop in hydrostatic pressure. This suggests that a gel occupies the primary body cavity and the fibres seen after chemical fixation may represent elements of the collapsed gel. Recent ultrastructural work on echinoids fixed by freeze substitution also - 82 -suggests that the blastocoel may be f i l l e d with a gel (Ruppert and Balser, 1986; Summers et a l . , 1987). The histochemical staining of the asteroid embryo with alcian blue demonstrated a homogeneously stained ECM within the blastocoel thus providing further evidence for the presence of a gel. These histochemical and ultrastructural findings along with the above mentioned studies support Strathmann's findings in vivo and strongly suggest that the blastocoel is f i l l e d with a gel and not a f l u i d with fibres. The advantages of a g e l - f i l l e d blastocoel over a fibrous, f l u i d - f i l l e d cavity, as discussed by Strathmann (1989), are as follows: A gel permits the embryo to assume numerous shapes which can f a c i l i t a t e feeding by positioning bands of c i l i a in optimal locations for food retrieval; a gel can maintain the integrity of tentacles or lobes better than a f l u i d can; because of the viscosity of a gel, large larvae may develop with minimal cell u l a r material such as a simple, outer epithelium, and; the visco-elastic properties of a gel would make opposing muscles unnecessary. This latter point especially pertains to the asteroid embryo. During morphogenesis, mesenchyme cells migrate to the presumptive esophagus and differentiate into smooth muscle c e l l s . These muscle cells wrap themselves around the esophagus, interdigitating with each other to form a continuous muscular tube in order to constrict the lumen (Reimer and Crawford, manuscript in preparation). Upon constriction of the esophagus, i t is possible that the attached radial esophageal fibres in turn draw in the ectoderm. During relaxation, since opposing esophageal muscles do not exist, the gel allows for rebounding of the ectoderm which, via the esophageal fibres, pulls at the esophageal musculature thereby dilating the lumen. It is plausible, therefore, that opposition to the esophageal musculature could exist but i t would be in the form of extracellular fibres and a gel or ground substance-like connective tissue. - 83 -Strathmann (1989) has further hypothesized that epithelial or mesenchymal cells may possess the a b i l i t y to dissolve and reform the gel thereby permitting greater freedom for distributing cells and creating shapes. If true, the 'contact-guidance' theory, which hypothesizes that cells migrate along networks of fibres or guide wires (Gustafson and Wolpert,1962, 1963), will need to be modified. In the asteroid embryo, then, mesenchymal cells use fibrous ECM elements as guide-wires to traverse the blastocoel. Such fibres are shown to exist in two major regions in the asteroid embryo - radiating from the esophagus, as discussed in the previous paragraph, and forming the dorsal web. Crawford and Chi a (1982) have shown by time-lapse cinemicrography that mesenchyme cells in living embryos actively traverse the region occupied by the esophageal fibres. These c e l l s , which are not muscle forming c e l l s , may be synthesizing or modifying the fibres (Abed and Crawford, 1986b). However, i t is possible that in order for the cells to migrate along a fibre, they must simultaneously dissolve the surrounding gel. In addition, the viscosity of a gel may provide better support for mesenchyme cells than would a f l u i d . It is also possible that such fibres maintain the shape of the embryo. The esophageal fibres described above, in addition to their role in muscle opposition, may stabilize the position of the ectoderm opposite the esophagus so that areas rostral and caudal to i t can continue to expand during morphogenesis. This is analogous to blowing up a balloon while maintaining a constriction around the middle of i t so that only areas above and below the constriction can expand. In the asteroid embryo then, a central constriction or waist forms during gastrulation and the oral hood and caudal regions of the embryo expand at a greater rate than the mid-section. The migrating population of mesenchyme cells may, then, - 84 -maintain the integrity of these fibres in order to retain the cylindrical or dumb-bell shape of the embryo seen in late gastrula. The role of the fibres situated on the dorsal web is not obvious at the larval stage of development. Mesenchyme cells are rarely present in the dorsal web and structural support does not appear to be particularily necessary at this site. Perhaps, the web is forming in order to serve a purpose at a later stage of morphogenesis. Basal lamina The BL, like the HL, stained alcian blue positive under acidic conditions and also l i g h t l y PAS positive. Together these observations suggest the presence of sulfated and neutral GAG's. These observations are in agreement with Davidson's findings (1974) in the echinoid BL in which uronic acid-containing sulfated GAG's and an intensely PAS positive glycoprotein were recognized. More recent studies have revealed other macromolecules in the echinoid BL including collagen types III and IV, fibronectin, laminin and heparan sulfate proteoglycan (Spiegel et a l . , 1980, 1983; Wessel et a l . , 1984; Wessel and McClay, 1987) A much more detailed immunocytochemical study is required, however, in order to accurately determine the composition of the asteroid basal lamina. In the present study, at the ultrastructural level, the basal lamina appears 'bushier' after cryofixation than after chemical fixation. Distinct f i b r i l s and granules, which may be collagen and proteoglycan respectively, seem to make up a large component of this ECM structure. This suggests that the BL may be a much more loosely-knit network of f i b r i l s and ground substance instead of a compact mat or feltwork as previously described (Crawford, 1989). Such a loose network may be able to better f a c i l i t a t e the diffusion of molecules between the epithelia and the - 85 -blastocoel than would a tightly packed mat of ECM. This latter morphology may be a direct result of the collapsing effects of chemical fixation. Neither the tubule-like structures nor the membrane puckerings between the BL and the epithelia, as observed by Crawford (1989), were seen in freeze substituted embryos. Perhaps these structures, which may be involved in transmitting mechanical forces between the blastocoel and the epithelia, are also artefacts of chemical fixation. Hyaline Layer ^ The perceived structure of the echinoid HL has evolved with better fixation techniques. It was once thought to be a single layer of extracellular material enveloping the developing embryo (Herbst, 1900). Since then, however, a two tiered structure (Wolpert and Mercer, 1963; Spiegel et a l . , 1989), and now a three to four layered structure is implicated (Lundgren, 1973; Morrill et a l . , 1987). Numerous functions attributed to the HL include a scaffold for holding cells together (Herbst, 1900; Chambers, 1940; Dan, 1960; Gustafson and Wolpert, 1967; Wolpert and Mercer, 1967; Vacquier and Mazia, 1968; Citkowitz, 1971, 1972; Kane, 1973; Adelson and Humphreys, 1988), a f i l t e r for nutrient collection (Spiegel et a l . , 1989), a barrier against mechanical and bacterial perturbation (Lundgren, 1973), and a source of lubrication while swimming (Crawford and Abed, 1986). Since i t s morphology is very complex, i t is unlikely that there is only one function to the HL. The HL of the Pi saster ochraceus embryo is similar in structure, function and probably composition to the echinoid HL. Observations in the present study, as well as those reported earlier (Crawford and Abed, 1986) following chemical fixation, have shown that the HL consists of five sub-layers: The intervillus layer; the supporting layer, consisting of HI - 86 -and H2; the boundary layer; and the coarse outer meshwork (Crawford and Abed, 1986). Reimer and Crawford (1990), u t i l i z i n g lectin histochemistry, revealed an unequal distribution of glycoconjugates throughout the HL (Reimer and Crawford, 1990), thus further supporting the idea that the HL has multiple functions. The morphology of the asteroid HL following freeze substitution is quite different than was described by Crawford and Abed (1986). The coarse outer meshwork is a homogeneously distributed ECM, like that in the blastocoel following freeze substitution. Since the latter is now thought to be gelatinous (Strathmann, 1989), this outer ECM is probably also a gel. Like the capsule of a bacterium, this outer gel may f a c i l i t a t e movement of the animal through the water as suggested by Crawford and Abed (1986) and may protect the entire embryo (Lundgren, 1973), particularly the - inner layers of the HL from perturbation. The boundary sub-layer is a repeating series of ECM loops. This looping pattern has never been previously described so i t s significance, at this stage, is purely speculative. Its continuity with the outer meshwork suggests that i t anchors this gel to the rest of the HL. Since i t appears to be the major attachment to and perhaps a source of the substance of the outer meshwork, this looping arrangement may be such to provide an increased surface area for these two functions. If the boundary layer is the source of the gel, this would suggest that some sub-layers of the HL are precursors to the next sub-layer as opposed to each sub-layer being synthesized and secreted separately by the epithelium. Pulse-chase experiments would l i k e l y resolve this conflict. In addition, microvilli terminated in the boundary layer. The ECM structures at the tip of each microvillus resembled the microvillus associated bodies visualized in other embryos by Spiegel et a l . (1989), and the dense matrix at the tips of microvilli in chicken intestinal microvilli - 87 -(Mooseker and Tilney, 1975). Filaments ran the length of the asteroid microvilli and appeared to terminate in the dense area inside the plasmalemma of each microvillus t i p . ECM fibres then radiated from the external dense region, and became continuous with the boundary layer. This external component of the microvillus associated body may contain a hyalin-like protein. Adelson and Humphreys (1988) localized hyalin to the microvillus associated bodies of echinoids and, based upon perturbation studies, suggested that hyalin is a major cell-HL adhesion molecule necessary for morphogenesis of echinoderms. Further immunocytochemical studies may prove useful in determining i f hyalin has adhesive functions in the asteroid embryo. The continuity which exists from within the epithelium to the attachment point of the HL, the microvillus associated body, is a direct physical link between the intracellular components and the HL; i t may provide a mode of communication between the cells and the HL. It is possible that the cells may be capable of directly controlling the characteristics of the HL. For example, when asteroid embryos are treated with a monoclonal antibody raised to their HL, the embryos retard development, shed the 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 site or foundation for the looping fibres of the boundary layer. The density and degree of compaction of HI suggest that this sub-layer also provides a great deal of structural support for the upper sub-layers. Sub-layer H2, the homogeneous belt of ECM underlying HI, may represent a gel-like substance which is less hydrated than the blastocoelic matrix or coarse outer meshwork. Its molecular arrangement and anhydrous characteristics may be such that phase separations do not occur during freezing, thus, perhaps accounting for i t s homogeneity. This - 88 -sub-layer may act as a f i l t e r (Spiegel et a l . , 1989) or a barrier, and provide an indirect source of attachment to the epithelium due to the microvilli passing through i t s gel on their way to the boundary layer. Sub-layer H3 has never been visualized previously. This sub-layer, underlying' H2, may be a transition or line of demarcation between the supporting layer and the intervillus layer. A monoclonal antibody to H3 suggests that this sub-layer is of different composition than the int e r v i l l u s layer (Crawford, personal communication). The sub-layer nearest the epithelium, the in t e r v i l l u s layer, contains ECM components which appear to be much more heterogeneously arranged than other areas of ECM. This sub-layer may be a part of an extremely hydrated gel which would be much more sensitive to ice crystal formation during fixation. This gel may provide hydrostatic pressure to maintain the remaining sub-layers above the epithelium. The ECM components of the in t e r v i l l u s layer may represent the apical lamina described in the echinoid (Hall and Vacquier, 1982). The results of the histochemical analysis of the HL suggest that a large amount of acidic GAG's and a small amount of neutral GAG's such as hyaluronic acid are present throughout the six sub-layers. The fact that the supporting layer stained more alcian blue positive than the rest of the HL is l i k e l y due to i t s more dense and compact organization and not a difference in composition. Positive PAS results were distinguishable in chemically fixed embryos but were not as clear in cryofixed material. The fact that the cryofixed embryos were embedded in JB4, a resin which does not incorporate stains as easily as paraffin, may be the source of this c o n f l i c t . If a thorough histochemical analysis of the asteroid ECM is to be carried out in the future, this variable will need to be eliminated. Alcian blue staining suggests that sulfated GAG's are a major constituent - 89 -of the asteroid HL. The results from previous studies in the echinoid (Solursh and Katow, 1982) suggest that these molecules could be heparan sulfate, chondroitin 6-sulfate and/or dermatan sulfate. Thus, proteoglycans containing these GAG's could represent a large component of the HL. As in the blastocoel, staining with alcian blue using the c r i t i c a l electrolyte concentration method (Scott and Dorling, 1965) may allow for a more accurate assessment of the presence of specific sulfated GAG's. 4.3 Summary The i n i t i a l goal of this study was to devise an inexpensive, easily employable freeze substitution technique which would allow good preservation of cellular and extracellular elements of the embryonic starfish, Pi saster ochraceus. A plunge freezing apparatus was constructed which consisted of a Dewer flask f i l l e d with liquid nitrogen, a small cup which was f i l l e d with with cryogen and inserted into the nitrogen, and a motor which constantly stirred the cryogen. Embryos were isolated on copper freeze-fracture grids and plunged into the cryogen. After considering four separate cryogens and four different cryoprotectants, cryoprotecting asteroid embryos with propylene glycol and plunging them into supercooled propane appeared to provide optimal preservation. Frozen embryos were freeze substituted in anhydrous ethanol at -90 °C, osmicated, and embedded for ultrastructural and histochemical analysis. Results from embryos preserved using the freeze substitution technique described above revealed that the blastocoel appears to contain a gel-like substance with extracellular fibres and not a f l u i d with extracelluar fibres, as previously thought. In addition, the hyaline layer, an ECM surrounding the outside of the embryo which was previously thought to contain three or four sub-layers, was found to consist of at least six - 90 -sub-layers. Histochemical studies demonstrated that both sulfated and unsulfated GAG's were present in these layers. The morphological differences among the sub-layers suggest that some sub-layers may have unique functions while others may have functions shared by other sub-layers. Studies of material preserved in this manner also demonstrate the presence of microvillus associated bodies. These are located at the tips of the microvilli and may represent sites of attachment between the microvilli and the hyaline layer. Although fixing asteroid embryos by freeze substitution is a lengthy process, taking four to five days, the resulting preservation, pa r t i c u l a r i l y of the ECM components, j u s t i f i e s i t s use over chemical fixations. Material preserved by freeze substitution can be used for histochemical studies and, in addition, since aldehydes and heavy metals are not necessary for successful preservation, may also prove useful for immunocytochemical studies. 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PAS stain for carbohydrates (modified after McManus, 1948) Schiff 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 intermittently for 2-3 hours or until the solution turns brownish or straw-coloured. Add 1 g of decolourizing charcoal, shake and f i l t e r . Repeat until the solution clears. Schiff reagent can be stored in the refrigerator for 2-3 months or until i t recolourizes. Sulfite wash Potassium metabisulfite (anhydrous)..0.9 g IN HCl 7.5 ml d i s t i l l e d water 150 ml Make immediatly prior to use. Periodic Acid (0.5% ag.) 1. Immerse slide in periodic acid for five minutes. 2. Wash with dH20 3. Immerse in Schiff reagent for 30 minutes (JB4) or 15 minutes (paraffin). 4. Wash with s u l f i t e wash for 5 minutes. 5. Wash with running tapwater for 10 minutes. 6. Wash with dH20. 7. Counterstain with hematoxylin by placing drops of the stain on the slides for four minutes on a 60° C hotplate. 8. Wash with dH20. 9. Cover with a glass coverslip - JB4. Dehydrate, clear, mount in DPX (BDH Co.) and cover - paraffin 2. pH dependent Alcian Blue test for acid GAG's (modified after Steedman, 1950; Mowry, 1956; Cook, 1982) Alcian blue 8GX 1% in 10% sulfuric acid (pH 0.2) 1% in 0.2N HCl (pH 0.5) 1% in 0.1N HCl (pH 1.0) 1% in 3% acetic acid (pH 2.5) 1% in 0.5% acetic acid (pH 3.2) 1. De-wax and hydrate paraffin sections. 2. Immerse slides in unstained solvent of the desired pH stain at room temperature. 3. Stain in alcian blue of desired pH for 5 hours at 60° C (JB4) or for 5 minutes at room temperature (paraffin) 4. Rinse with dH20 - 104 -5. Counterstain with 1% Neutral Red for 30 seconds (paraffin) or 1% phenylene-diamene in 1:1 methanol/propanol for 90 seconds (JB4), both at room temperature. 6. Rinse with dH20 7. Rinse JB4 sections with 100% ethanol for 1 minute or until the excess phenylene-diamene is washed away. 8. Dehydrate, clear (paraffin) and mount in DPX. 9. Cover with a glass coverslip. Alcian blue-PAS test for neutral and acid GAG's (modified from Cook, 1982) 1. Stain with Alcian blue (pH 2.5) as per Appendix 1.2 omitting counterstain. 2. Rinse with dH20. 3. Stain with PAS omitting counterstain. 4. Dehydrate, clear (paraffin) and mount in DPX. 5. Cover with a glass coverslip. Toluidine Blue for basophilia and metachromasia (modified after L i l l i e , 1965) Acetate buffer pH 4.0 0.1N sodium acetate (8.2g/l) 36 ml 0.1N acetic acid (6 ml/1) 164 ml Stain 49 ml acetate buffer 1 ml IX Toluidine blue (aq) , 1. De-wax and hydrate sections (paraffin) 2. Immerse in stain for 1 hour at room temperature. 3. Rinse with dH20. 4. Dehydrate, clear (paraffin) and mount in DPX (for basophi1ia)...or 5. Dehydrate for 1 minute in acetone. 6. Immerse in 1:1 acetone/xylene for 1 minute, then clear with xylene and mount in DPX (for metachromasia). 7. Cover with glass coverslip. 

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