Visualization of the real microarchitecture of glycoprotein matrices with ...

Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________

Visualization of the real microarchitecture of glycoprotein matrices with scanning electron microscopy G. Familiari*, M. Relucenti, A. Familiari, E. Battaglione, G. Franchitto, and R. Heyn Laboratory of Electron Microscopy “Pietro M. Motta”, Department of Anatomy, University of Rome La Sapienza, Rome, Italy. Preparation of biological samples for scanning electron microscopy considers fixation, dehydration and drying, such procedures strongly stress the delicate filamentous structure of glycoprotein matrices, due to their extremely hydration state. Therefore, in order to allow the observation of the real microarchitecture of these matrices, a specific technique was developed by us. Mucus covering the jejunum surface and the zona pellucida surrounding the mature oocyte were examined. The technique consists in the use of ruthenium red (RR), saponin, osmium and thiocarbohydrazide. RR prevents the dissolution and/or the alteration of glycoproteins and polyanionic carbohydrates induced by aqueous fixatives, whereas saponin is a detergent of soluble proteins. Osmium-thiocarbohydrazide preserves matrix filaments from the mechanical stress induced by dehydration and critical point drying, thus minimizing filaments packing and shrinkage. Considering the role of glycoprotein matrices in many biological functions, this technique can be usefully applied to further investigate other similar structures in various physiological and pathological conditions.

Keywords scanning electron microscopy, glycoproteins, matrix, zona pellucida, intestinal mucus.

Introduction Extracellular matrices are an intricate network of macromolecules that bind the cells together but also influence their survival, development, shape, polarity, and behaviour. Extracellular matrices are made up of various protein fibers interwoven in a hydrated gel composed of a network of glycosaminoglycan (GAG) chains [1]. GAGs are a heterogeneous group of negatively charged polysaccharide chains that are covalently linked to protein to form proteoglycan molecules (except for hyaluronan). They occupy a large volume and form hydrated gels in the extracellular space. Proteoglycans are also found on the surface of cells, where they function as co-receptors to help cells respond to secreted signal proteins [1]. The zona pellucida (ZP) and gastrointestinal mucus are to be considered as particular extracellular matrices, covering the oocyte and the gastrointestinal surface epithelium, respectively. Mammalian ZP is the extracellular matrix that surrounds the oocyte during folliculogenesis up to blastocyst hatching. ZP plays also an important role in fertilization [2-4]. The ZP is made up of three species-specific glycoproteins: mZP1, mZP2, mZP3 [5] arranged in a delicate filamentous matrix. Long interconnected filaments, which are polymers of mZP2 and mZP3 [6] compose the ZP. An mZP2–mZP3 dimer is located every 140 Å or so along the filaments, thus imposing a structural periodicity that can be seen in electron micrographs of dissolved ZP. Filaments, in turn, are cross-linked by mZP1 to create a 3-D scaffold in the matrix. Thus, each of the ZP glycoproteins plays a structural role during ZP assembly. A protective mucus gel composed predominantly of mucin glycoproteins that are synthesized and secreted by goblet cells covers the gastrointestinal epithelium [7]. The mucus gel layer is an integral structural component of the gastrointestinal tract, contributing with protection, lubrication, and transport between the luminal contents and the epithelial lining [8]. The major gel-forming glycoprotein *

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Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________

components called mucins supply the viscoelastic, polymer-like properties of the mucus [8]. Mucins consist of a peptide backbone containing alternating glycosylated and nonglycosylated domains, with Olinked glycosylated regions comprising 70–80% of the polymer. The four primary mucin oligosaccharides are N-acetylglucosamine, N-acetylgalactosamine, fucose, and galactose [8]. Mucin oligosaccharide chains are often terminated with sialic acid or sulfate groups, which account for the polyanionic nature of mucins at a neutral pH [8]. It is very difficult to preserve the structure of glycoprotein matrices, such as the ZP or the gastrointestinal mucus, during procedures for electron microscopy, because they are extremely hydrated. Solutions of ethanol or glutaraldehyde used for electron microscopy may cause shrinkage of the glycoprotein structure and may produce artifacts, thus explaining why only fragments of the mucus layer are observed on fixed sections [9]. Moreover freezing and drying procedures may stress the filaments, causing their alignment in the direction of shear or flow, influencing in that way the apparent organization of molecules [10]. For all the above reasons, the development of high resolution scanning electron microscopy (SEM) technique is required to really assesses the three dimensional structure that glycoprotein filaments assume in the matrices. An analysis of the three dimensional microarchitecture of the ZP and the intestinal mucus is here proposed.

Conventional SEM analysis In case of the standard method [11, 12], specimens were fixed in a 3.0% glutaraldehyde solution in 0.1 M cacodylate buffer at pH 7.4 for two to five days. The specimens were carefully washed twice, each for ten minutes, without stirring, in 0.1 M cacodylate buffer at pH 7.4. Postfixation was made with a solution of 1.0% osmium tetroxide in 0.1 M cacodylate buffer at pH 7.4. Samples were rinsed gently in distilled water twice, 10 min each, to remove excess of osmium tetroxide, dehydrated in series of ascending concentrations of acetone/ethanol (30%-50%-70%-95%-100%-100%-100%, 20 minutes each) and critical point-dried in a CPD020 Balzers device (Balzers, Liechtenstein). Dehydrated specimens were mounted on aluminium stubs with silver paint and sputtered with platinum at 10-15 mA for 1 min (obtaining a 3 nm-thick film) in an EMITECH K550 sputtering device (EMITECH ltd. Ashford, Kent, England). Observations were made with a Hitachi S-4000 field-emission scanning electron microscope operating at 5-10 kV using secondary electrons and with a working distance of 5-9 mm.

High resolution SEM technique for glycoproteins We have developed and applied the following eight-step schedule [11, 12]: 1) The extraction-stabilization procedure was made with 0.02% saponin and 1.0% RR in cacodylate buffer 0.1 M at pH 7.4 for 15-45 min; 2) fixation was obtained by immersion in 3.0% glutaraldehyde plus 0.02% saponin and 1.0% RR in cacodylate buffer overnight; 3) four washes, 25 min each, using a solution of 0.02% saponin and 1.0% RR in cacodylate buffer 0.1 M at pH 7.4 for 1 h; 4) postfixation was performed with a solution of 1.0% osmium tetroxide containing 0.02% saponin and 0.75% RR in cacodylate buffer for 2 h; 5) impregnation with two solutions, 1% thiocarbohydrazide in distilled water (T), 1.0% osmium tetroxide (O) in distilled water. The solutions were used alternatively in four steps (T/O/T/O for 20 /60 /20 /60 min); 6) samples were washed four times 10 min each in distilled water and then dehydrated in a series of ascending concentrations of acetone/ethanol (30%-50%-70%-95%-100%-100%-100%, 20 min each);

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7) the drying procedure was made with a critical point dryer device CPD020 Balzers (Balzers, Liechtenstein) in liquid carbon dioxide; samples were then mounted with silver paint on aluminium stubs. 8) Specimens were finally observed in a Hitachi S-4000 field emission scanning electron microscope operating at 5-10 kV using secondary electrons, with a working distance of 5-9 mm. Note that no sputtering coating was performed.

Zona Pellucida visualized by high resolution SEM After standard SEM preparation the outer surface of the ZP was usually characterized by a spongy appearance, due to the presence of numerous branches surrounding fenestrations (Fig. la). In control coated specimens, at high magnification, the surface of the branches as well as fenestrations of the outer ZP displayed a granular appearance (Fig. 1b). Specimens treated with the RR-saponin-T/O/ T/O method showed the outer zona surface consisting of fine filaments arranged in a regular alternance of tight and large meshed networks. These filaments were 22-28 nm in thickness and showed a globule-bearing structure (Fig. 1c). The tight meshed arrangement of filaments was in correspondence of branches whereas large meshed network of filaments was within the fenestrations. Fig. 1 Mouse oocyte. a) Standard SEM with platinum coating, note the spongy appearance of the ZP (2000X). b) Greater magnification of the square shown in a, note the granular appearance of the outer aspect of the ZP (50000X). c) Saponin, RR, T/O/T/O treatment. At high magnification note the presence of filaments arranged in complex networks on the outer aspect of the ZP (50000X). (From ref. [11] with permission).

a)

b)

c)

Mucus covering the jejunum surface visualized by high resolution SEM After standard SEM preparation, jejunum samples evidenced large areas of the villous surface lacking the mucus layer (Fig. 2a). In the same specimens, at high magnification, the residual mucus appeared as small patches made up of a granular material (Fig. 2b). Specimens treated with the RR-saponin-T/O/ T/O method evidenced the mucus forming a continuous layer covering almost the entire surface of the jejunal villi (Fig. 2c). When seen at high magnification the

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mucus was characterized by filaments, measuring 30-50 nm in thickness, arranged in regular meshed networks (Fig. 2d).

a)

b)

c) d) Fig. 2 Mouse jejunum. a) Standard SEM with platinum coating. The mucus forms patches of amorphous material on the villous surface (110X). b) Standard SEM with platinum coating. Higher magnification of the squared area shown in a, note the granular appearance of the mucus (10000X). c) Saponin, RR, T/O/T/O treatment. The mucus layer forms a continuous sheet covering the entire surface of the villi (200X). d) Saponin, RR, T/O/T/O treatment. Higher magnification of the squared area shown in c. At high resolution, the mucus displays filaments that are arranged in a regular meshwork, revealing a globule-bearing structure (15000X). (From ref. [11] with permission)

Concluding remarks Several ultrastructural methods are available to study the glycoprotein matrix for both transmission [6] and scanning electron microscopy [10] observation. All the techniques characterized the glycoprotein matrix as a filamentous structure. In particular, methods comprising rapid freeze-drying or freeze substitution [10] showed fibrils ranging from 50 to 500 nm in diameter, arranged to form interconnected layers; therefore, the relevant diameter of filaments observed using these methods was likely related to packing of thin filaments, not properly stabilized. On the contrary, the RR-saponin-T/O/T/O method used in our study offers several advantages, in terms of stabilization and preservation of the structure and, consequently, of the morphology of thin filaments. The role of RR is to stabilize glycoproteins and polyanionic carbohydrates, avoiding their dissolution and/or alteration induced by aqueous fixatives. The addition of RR was reported as causing the collapse of polyanionic chains, making them appearing as condensed granules [13]. However, in specimens processed for the high resolution SEM technique, thin filamentous structures devoid of condensed

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granules were observed, this was due to the use of saponin. In fact, this kind of detergent of soluble proteins prevents the formation of globular artifacts. The treatment with osmium-thiocarbohydrazide increases molecular weight, yielding a very fine structure under the electron beam, but also hardens and protect the glycoprotein matrix filaments from the mechanical stress induced by dehydration and critical point drying, thus decreasing filaments shrinkage and packing. Our technique introduces particular improvements that made possible to show that: 1) the filaments’ network is arranged in a very regular way; 2) the thickness of mucus filaments is smaller and more precise than that obtained with other preparation methods [10], and 3) the thickness of ZP filaments is homogenous, according with transmission electron microscopy data obtained by dissolution of the ZP matrix and observation by means of negative staining [6]. In conclusion, performing the high resolution SEM technique developed in our laboratory for the study of glycoprotein matrices (ZP or mucus) it is possible to obtain a more detailed view of the structural organization of these special structures. The glycoprotein matrices play a fundamental role in many important biological functions, so this technique can be usefully applied to further investigate the morphodynamic changes of these and other similar structures in various physiological and pathological conditions. Acknowledgements: This work was supported by Italian M.U.R. and University of Rome “La Sapienza” grants (1988-2006).

References [1] B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts and P. Walter, in: Molecular Biology of the Cell, 4th ed. Garland Science Publ., New York, pp. 1065-1126 (2002). [2] G. Familiari, R. Heyn, M. Relucenti, S.A. Nottola and A.H. Sathananthan, International Review of Cytology, 249, 53 (2006). [3] P.M. Motta, S.A. Nottola, G. Familiari, S. Makabe, T. Stallone and G. Macchiarelli, International Review of Cytology, 223, 177 (2003). [4] H.M. Florman and T. Ducibella, in: Knobil and Neill’s Physiology of Reproduction, editor JD Neill. Elsevier Academic Press (2006), pp. 55-112 [5] E.S. Litscher and P.M. Wassarman, Histology and Histopathology, 22, 337 (2007). [6] J..M. Greve and P.M. Wassarman, Journal of Molecular Biology, 181, 253 (1985). [7] B. Deplancke and H.R. Gaskins, The American Journal of Clinical Nutrition, 73 (suppl), 1131S (2001). [8] J.F. Forstner, M.G. Oliver, F.A. Sylvester, in: M.J. Blaser, P.D. Smith, J.I. Ravdin, H.B. Greenberg, R.L. Guerrant eds., Infections of the gastrointestinal tract, Raven Press, New York (1995), pp.71-88. [9] A. Allen and A. Leonard, Gastroenterologie Clinique et Biologique, 9, 9 (1985). [10] J.M. Sturgess, in P.C. Braga and L. Allegra, Methods in Bronchial Mucology, eds. Raven Press, New York (1988), pp. 245-253. [11] G. Familiari, S.A. Nottola, G. Macchiarelli, A. Familiari, P.M. Motta, Microscopy Research and Technique, 23, 225 (1992). [12] G. Familiari, M. Relucenti, R. Heyn, G. Micara, S. Correr, Microscopy Research and Technique, 69, 415 (2006). [13] E.B. Hunziker and R.K. Schenk, The Journal of Cell Biology, 98, 277 (1984).

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