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The Fabrication and Collagenous Substructure of the Eggshell Membrane in the Isthmus of the Hen Oviduct
Matrix Vol. 1111991, pp. 313-320 © 1991 by Gustav Fischer Verlag, Stuttgart
The Fabrication and Collagenous Substructure of the Eggshell Membrane in the Isthmus of the Hen Oviduct J. L. ARIAS 1 , M. S. FERNANDEZ 1 , J. E. DENNIS and A. I. CAPLAN Skeletal Research Center, Department of Biology, Case Western Reserve University, Cleveland, OH 44 106, USA. 1 Visiting Scientists from University of Chile, Santiago.
Abstract The eggshell of the chicken consists of a bi-Iayered shell membrane overlaid with a thick, calcified shell matrix. The shell membranes and matrix are deposited onto the egg as it passes through the oviduct. To assess the temporal and spatial aspects of the fabrication of type X collagen within the eggshell extracellular matrix, the immunohistochemical localization of type X collagen was studied in three regions of the hen oviduct (magnum, isthmus and uterus), in the membranes of uncalcified eggshells obtained from the oviduct prior to mineral deposition and in eggshell membrane and calcified eggshell matrix. Additionally, immunohistochemicallocalization of type I and III collagens was done in order to determine possible co-localization of collagen types or to define tissue compartments. None of the collagen epitopes assayed was found in the shell matrix. Type X collagen epitope was immunohistochemically localized only to the epithelial cell layer lining the isthmus region of the oviduct and in the shell membranes of both uncalcified and calcified eggshells. Antitype III collagen monoclonal antibody delineated the inter-tubular gland connective tissue of the oviduct and was negative in the shell layers under conditions which gave strong connective tissue reactivity. Type I collagen epitope was exposed after pepsin treatment of the tissue and co-localizes with the distribution of type III collagen. Type I collagen co-localized with type X collagen in the shell membranes of uncalcified shells. The type I collagen epitope was reactive in the shell membrane of the uncalcified shells, but could only be detected in calcified shells following pepsin digestion. These observations suggest that the type I collagen epitope is masked during the process of calcification of the shell. Type X collagen is localized to regions adjacent to mineral deposits in the eggshell but is never colocalized with mineral deposits. We hypothesize that extracellular matrices containing typeX collagen can function as a mineralization barrier. Key words: eggshell, hen, oviduct, shell membranes, type I collagen, type III collagen, type X collagen.
Introduction The avian oviduct is a complex tubular organ which is responsible for the transport of the egg and the secretion of a diverse mixture of extracellular matrix components which surround the egg white. The extracellular matrix components assemble into complex layers the outer regions of which provide the scaffolding for calcium carbonate
deposition of the eggshell proper. The eggshell consists of a number of extracellular matrix layers which are sequentially fabricated during the passage of the egg through the oviduct. The first layers covering the egg white are the fibrillar inner and outer shell membranes (Fig. 1). The most external portion of the outer membrane consists of discrete aggregations of organic material, called mammillae, forming the mammillary layer. These mammillae are suggested
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to be the sites where the organic matrix of the shell is attached and where the initiation of calcium carbonate crystal formation takes place (Creger et aI., 1976; Stemberger et aI., 1977; Baumgartner et aI., 1978). The hard shell proper, the palisade or calcite layer, is organized upon this shell matrix with the most external layer referred to as the cuticle. The oviduct is organized into five regions on the basis of differences in morphological features and function (Coste, 1847). These regions are, from proximal to distal, the infundibulum which receives the ovum, the magnum which secretes albumen, the isthmus which secretes precursors of the shell membranes, the uterus or shell gland in which deposition of calcium carbonate takes place and the vagina. In addition to its principal role of depositing the shell membrane (Coste, 1847; Pearl and Curtis, 1912), the isthmus supplies water, copper, iron and calcium to the developing egg (Schraer and Schraer, 1965; Robinson et aI., 1966). The microstructure and ultrastructure of the avian isthmus have been studied (Richardson, 1935; Turchini and Broussy, 1938; Hoffer, 1971), but neither the histological nor the biochemical analysis of the isthmus have clarified the nature of the process by which the amorphous secretory products of its tubular glands are transformed into the double-layered fibrous shell membrane. Also, the molecular composition of the secretory granules of the isthmus cells and of the shell membranes and shell matrix themselves have not been completely elucidated (Hoffer, 1971). However the collagenous nature of the shell membrane proteins has been suspected based indirectly on the fact that factors affecting collagen synthesis, such as copper and ascorbate deficiency and lathyrogens, also affect formation of the shell membranes (Thornton and Moreng, 1958; 1959; Baumgartner et aI., 1978). Co-localization of type I and III collagen has been described in the intercellular loose connective tissue of the quail oviduct (Perche et aI., 1990).
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Fig. 1. A drawing of a complete radial cross-section of the chicken eggshell (modified from Arias et ai., 1991 a).
Collagen types I and V with polyclonal antibodies (Wong et aI., 1984) and collagen type X with monoclonal antibodies (Arias et aI., 1991 a) have been detected and localized immunohistochemically within the shell membranes. However, with monoclonal antibody IIBe, to type I collagen, positive reaction is only detected after pretreatment of the shell membranes with pepsin (Arias et aI., 1991 a). These collagen fibers are coated with a proteoglycan-rich material that binds the fibers into a network (Wong et aI., 1984). The proteoglycans have been partially characterized as being rich in keratan and dermatan sulfate (Arias et aI., 1991 b). The composition of the shell matrix is less well known and only some y-carboxyglutamic rich proteins (Abatangelo et aI., 1978; Krampitz et aI., 1980) and proteoglycans (Arias et aI., 1991 b) have been described. In addition, some precursors of the shell matrix proteins have been described to be synthesized in the liver (Eckert et aI., 1986; Schade, 1987). To examine the temporal and spatial aspects of eggshell extracellular matrix deposition, the secretion products of the oviduct which comprise the shell membranes were studied by immunohistochemistry. Localization of types I and X collagen was studied in three regions within the oviduct, in the shell membranes of complete calcified eggshells, and in the newly deposited shell membranes located in the oviduct prior to eggshell calcification.
Material and Methods
Entire oviducts from two-year-old White Leghorn laying hens were dissected. In one oviduct, an egg was located 5 cm away from the uterus. Small pieces of the magnum, isthmus, uterus and the shell membranes covering the uncalcified egg were isolated and immersed directly in 15% sucrose in phosphate-buffered saline (PBS) for 30 min, transferred to Tissue-Tek O. C. T. compound (Miles Inc.,
Type X Collagen in the Hen Oviduct Elkhart, IN) and frozen in liquid nitrogen and 8-[A,m sections were cut with a Minotome cryostat. Additionally, pieces of eggshell from a completely formed egg were fixed in 2 % paraformaldehyde, 0.1 % glutaraldehyde in PBS for 12 h and then decalcified in 20% EDTA for 24 h and prepared for cryosectioning as above. Sections were placed on albumin-coated microscope slides and the slides immersed in PBS (pH 7.3) for 5 min. Some sections were digested with 0.5% pepsin in 0.05% acetic acid for 30min at 3rc. All sections were blocked for 5 min with 3 % bovine serum albumin (BSA) in PBS (BSA/PBS). Sections were exposed to the following monoclonal antibodies: anti-type X collagen (AC9/DEI0; Schmid and Linsenmayer, 1985), anti-type I collagen (IIB6; Linsenmayer et aI., 1979) and anti-type III collagen (3B2; provided by Dr. R. Mayne). The antitype III collagen antibody, which is a specific marker for the loose connective tissue was used to delineate the inter-tubular space. Additionally, since type III and type X collagens have never been co-localized in any other tisssue, the distinct distribution of these antibodies confirms the specificity of these probes. Sections were incubated in primary antibody for 1 h at room temperature, rinsed with 0.1 % BSA/PBS, washed with PBS for 5 min and blocked again with 3 % BSA/PBS for 5 min. The sections were then incubated in fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Cappel Lab., Cochranville, PAl for 1 h at room temperature; secondary antibody was used at a 1 :200 dilution in 1 % BSA/PBS. After this incubation, the sections were washed with PBS, mounted with coverslips in Fluoromount B (Fisher Biotech., Orangeburg, NY) and examined with an Olympus BH-2 microscope. As a control for non-specific staining by the secondary antibody, PBS or non-specific IgG was used in place of the primary antibody. Identical photographic exposures were taken for both control and experimental specimens. For histological identification serial sections were stained with Mallory-Heidenhain (Humason, 1979) and examined with bright field optics for comparison with immunostained sections.
Results
Oviduct The wall of the oviduct consists of a mucosa, a submucosa, a tunica muscularis and a serosa. The magnum and its Fig. 2. Immunostaining of the magnum wall with 3B2, a monoclonal antibody to type III collagen. (A) Cross-section of magnum mucosa and submucosa stained with Mallory-Heidenhain. An intense red stain is observed in the cells of the tubular glands (arrow) (X 220). (B) Immunopositive fluorescence is located along blood vessels walls (v) and around the smooth muscle layers (m) (X 250). (C) The fluorescence indicative of type III collagen is located in the connective tissue between the tubular glands of the magnum (X 250). g = glands; e = epithelium.
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tubular glands are covered by a simple columnar epithelium, with ciliated and goblet cells lining the magnum lumen and acinar cells forming the tubular glands (Fig. 2A). The cytoplasm of the gland cells contains secretory granules
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J. L. Arias et ai. tions of the surface epithelium form short ducts through which the tubular glands communicate with the lumen. The gland epithelium is formed by only a single cell type referred to as principal cells. These cells contain numerous large secretory granules which appear blue when stained with Mallory-Heidenhain. TypeIII collagen is located in the loose connective tissue surrounding the tubular glands (Fig. 4 B). After pepsin digestion, a positive reaction with anti-type I collagen antibody appears in the loose connective tissue colocalized with type III collagen as in the magnum. A strong reactivity with antibody to type X collagen is observed in the tubular gland cells (Fig.4 C), on the apical surface of the mucosal epithelial cells and in the glandular and isthmallumen (Fig. 4 D). The distribution of the reactive epitope of type X collagen does not overlap with that of epitope of type III collagen. The uterine mucosa is covered with a columnar epithelium and also possesses subepithelial glands. However, there is a greater proportion of loose connective tissue in this region than in the more proximal parts of the oviduct (Fig. 5 A). The epithelial cells consist of two cell types: ciliated and non-ciliated cells. The uterine gland cells, unlike the magnum and isthmal gland cells, do not show any evidence of secretory activity when stained sections are observed with the light microscope. The strong, widespread reactivity against type III collagen confirms that a large proportion of the uterine glands consists of loose connective tissue (Fig. 5 B). There was no reactivity against type X collagen in this region and for type I collagen it was again, as in the magnum and isthmus, a positive reaction in the loose connective tissue only after pepsin digestion. In addition, reactivity against type III collagen in the egg layers was never observed.
Fig. 3. (A) 1mmunostaining of the magnum wall treated with pepsin to anti-type I collagen monoclonal antibody 11 8 6 . 1mmunopositive fluorescence is located in the connective tissue between the tubular glands (g). (X 250). (B) The same antibody in non-pepsintreated samples shows no reactivity. (X 250). characteristic of egg white proteins which stain intense red with Mallory-Heidenhain; the same intense red material is detected in the gland lumen. Type III collagen delineates the loose connective tissue between tubular glands and surrounds the smooth muscle cells of the muscular layer (Fig.2B). The largest proportion of the magnum wall is occupied by the glandular cells surrounded by the loose connective tissue (Fig. 2 C). Immunostaining with type X collagen antibody was not observed in this region. Immunostaining for type I collagen was observed in the connective tissue only after digestion with pepsin (Fig. 3 A). Its distribution appears similar to that of type III collagen which did not require pepsin digestion to be visualized (see Fig.2C). The isthmal mucosa is covered by an epithelium made up of ciliated and mucus-secreting cells (Fig. 4 A). Invagina-
Shell membranes The shell membranes obtained before they reach the uterus, stain intensely for types X (Fig. 6 A) and I (Fig. 6 B) collagen. In decalcified eggshells (Fig. 6 C), type X collagen is localized exclusively to the shell membranes, and not to the shell matrix (Fig. 6 D). Type I collagen antibody reactivity is negative in the shell membranes, mammillary knobs and shell matrix under conditions where the antibody to type X collagen is reactive in the shell membranes. However, as we have shown elsewhere (Arias et ai., 1991 a), treatment of shell membranes from calcified eggshells with pepsin exposes the type I collagen epitope so that reactivity with the antibody to type I collagen is detected. The shell membranes in uterus do not require pretreatment with pepsin to give a positive reaction to type I collagen.
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Fig. 4. Immunostaining of the isthmus wall with monoclonal antibodies 3B2 (anti-type III collagen) and AC9IDEIO (anti-type X collagen). (A) Cross-section of the isthmal mucosa stained with Mallory-Heidenhain. The secretory granules in the cells of the tubular glands appear blue (arrow) (X 244). (B) An intense immunofluorescence (3B2 antibody) is observed in the loose connective tissue between the tubular glands (X 250). (C) Immunofluorescent staining by anti-type X collagen antibody is localized to the cells of the tubular glands (X 220) and (D) also localized at the luminal surface of the isthmal epithelial cells (X 220). g = gland; e = epithelium; L = lumen.
Discussion Despite the findings that proteoglycans and collagens contribute significantly to the composition of the organic matrix of the eggshell (Wong et ai., 1984; Arias et ai., 1991 a, b), little is known about the function of these matrix components in the calcification process within the oviduct. The presence of type X collagen associated with the glandular cells of the shell membrane-secreting region (isthmus) of the hen's oviduct is reported here. Type I and X collagens were located in the shell membranes of the uncalcified egg, but only type X collagen could be observed in the shell membranes of the mineralized egg. As demonstrated previously, type I collagen was immunohistologically co-
localized in these shell membranes with type X collagen only after pepsin treatment. Additionally, both collagens were found only in the shell membranes (non-mineralized) and not in the shell matrix proper (mineralized). Type III collagen was found in the loose connective tissue of the magnum, isthmus and uterus; while type I collagen was found co-localized with type III collagen only after pepsinization of the tissue. The three secretory regions of the hen oviduct are responsible for the elaboration of a multiple array of materials which comprise the layers surrounding the egg yolk. However, only the isthmus and uterus are responsible for the formation of the eggshell. The magnum region synthesizes the egg white or albumen, consisting of glycoproteins with
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Fig. 5. Immunostaining of the uterine wall with monoclonal antibody 3B2 (anti-type III collagen). (A) Cross-section of the uterus stained with Mallory-Heidenhain. Secretory granules in the cytoplasm of the glandular cells were not detected with this staining (X 200). (B) An intense fluorescence with 3B2 antibody is observed in the abundant loose connective tissue (X 220). g = gland; e = epithelium; L = lumen. complex N-linked oligosaccharides (Brockhausen et ai., 1988; 1989). The growth and cytodifferentiation of the magnum as well as the synthesis of egg white proteins are regulated by hormones, especially estrogen and progesterone (Kohler et ai., 1968; Oka and Schimke, 1969; Perche et ai., 1989; Pageaux et ai., 1989). The isthmus or shell membrane-secreting region of the oviduct has been characterized histochemically and ultrastructurally (Richardson, 1935; Hoffer, 1971). Three main types of cells have been described lining the epithelium of the isthmal mucosa and the tubular glands (Hoffer, 1971). These are the ciliated columnar epithelial cells, the mucous cells and the principal cells. It has also been established that the secretory granules of the principal cells contain a non-
sulfated protein-polysaccharide which does not contain detectable carboxylic groups, but possesses sulfhydryl and disulfide groups (Hoffer, 1971). The synthesis of sulfated glycoproteins as components of eggshell membranes has been suggested to occur in both the magnum and the isthmus (Picard et ai., 1973; Paul-Gardais et ai., 1974). The presence of type X collagen epitope in the principal cells of the isthmus and its simultaneous occurrence in the shell membranes is consistent with the suggested role of this region in the assembly of the shell membranes. It is of interest to consider that anti-type X collagen reactivity is found only in the isthmus, indicating a regional specialization of the oviduct for the synthesis of this collagen type. The occurrence of type X collagen in a system of mineralization other than hypertrophic chondrocytes may help in the understanding of the mechanisms underlying biological mineralization. Type X collagen is found in the shell membrane-secreting region of the oviduct and in the eggshell membranes, which do not mineralize. Similarly, immunolocalization of type X collagen has not been observed to be concentrated in the focal calcification sites nor in association with matrix vesicles suggesting an absence of a direct role for type X collagen in nucleation and matrix vesicle function during endochondral ossification (Poole and Pidoux, 1989; Poole et ai., 1989). From these observations we hypothesize that type X collagen functions to inhibit mineralization and thereby establish boundaries or regions which are protected from mineral deposition. In fact, it appears that type X collagen, when secreted from the isthmus tubular gland cells, is assembled in the oviductal lumen, at some distance from the cells, shaping the shell membranes which surround the egg white. These shell membranes do not mineralize but act as substrate for the deposition of the mammillary knobs and shell matrix which direct nucleation and growth of the crystals forming the hard shell proper. The easy detection of type I collagen in the shell membranes in uterus revealed by the antibody used here and the necessity of pepsin treatment of the shell membranes from calcified eggshells to exhibit its epitope (Arias et aI., 1991 a) is not easily interpreted. One possible explanation is that early in the passage of the shell membranes through the uterus, the epitope to type I collagen is accessable but, later during passage it becomes masked in such a way that it can only be exposed after pepsin digestion. As we suggested elsewhere (Arias et aI., 1991 a), type X collagen could be masking the type I collagen epitope, although a similar effect by other molecules (e. g. proteoglycans) cannot be excluded. Cross-links may also be involved in the masking of the type I collagen epitope. It is known that the reaction with I1B6 antibody is highly dependent on the collagen conformation and also that it has less sensitivity than polyclonal antibodies against type I collagen raised in rabbits (Linsenmayer et ai., 1979). Although different from elastin
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Fig. 6. Immunostaining of the shell membranes and/or shell matrix produced by; (A) anti-type X collagen antibody and (B) anti-type I collagen antibody, on shell membranes located in the oviduct before calcification occurs (X 280). (C) Cross-section of the shell membrane (sm) and shell matrix (mx) of a decalcified eggshell stained with Mallory-Heidenhain. The shell membranes stain purple and the matrix blue (X 280). (D) Cross-section of decalcified eggshell exposed to anti-type X antibody showing the positive immunoreaction to the shell membranes (sm) but not to the shell matrix (mx) (X 280).
(Starcher and King, 1980; Crombie et aI., 1981; Leach et aI., 1981), the proteins of the eggshell membranes are highly crosslinked by disulfide bonds (Leach, 1982) and by desmosine and isodesmosine groups (Candlish and Scougall, 1969; Balch and Cooke, 1970; Starcher and King, 1980; Leach et aI., 1981; Crombie et aI., 1981), due to a strong lysyloxidase activity in the isthmus (Harris et aI., 1980). Further research is required to elucidate the contribution of cross-linking to the masking of the epitope of this monoclonal antibody. The origin of the type I collagen of the shell membranes is still unknown. With the methodology used here, we were unable to detect type I collagen in any secretory cell of the different regions of the oviduct.
Acknowledgements We thank Dr. T.F. Linsenmayer (Tufts University, Boston, MA) and Dr. R. Mayne (University of Alabama, Birmingham, AL) for their generous gifts of the monoclonal antibodies and Dr. Dave Carrino (Case Western Reserve University, Cleveland, OH) for critical reading of the manuscript. This work was supported by the University of Chile (Santiago), by NIH and by D.O.E. through Battelle Pacific Northwest Laboratories.
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gens of the chicken eggshell membranes. Connect. Tiss. Res. 26: 37~45, 1991a. Arias, ].L., Fernandez, M.S., Dennis, J.E., Carrino, D.A. and Caplan, A. I.: Immunolocalization of proteoglycans in the avian eggshell. 1991 b (in preparation). Balch, D. and Cooke, R.A.: A study of the composition of hen's eggshell membranes. Am. Bioi. Anim. Biochem. Biophys. 10: 13~25, 1970. Baumgartner, S., Brown, D.J., Salevsky, E. and Leach, R. M., Jr.: Copper deficiency in the laying hen.]. Nutr. 108: 804~811, 1978. Brockhausen, I., Carver, ]. P. and Schachter, H.: Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC study of the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes. Biochem. Cell. Bioi. 66: 1134~1151, 1988. Brockhausen, I., Hull, E., Hindsgaul, 0., Schachter, H., Shah, R. N., Michnick, S. W. and Carver, J. P.: Control of glycoprotein synthesis.]. Bioi. Chem. 265: 11211 ~ 11221,1989. Candlish, ].K. and Scougall, R.K.: L-5-hydroxylysine as a constituent of the membranes of the hen's egg. Int.]. Prot. Chem. 1: 299~302, 1969. Coste, M.: Histoire genera Ie et particuliere du developpement des corps organises, publiee sous les auspices de M. Villemain. V. Masson, Paris, 1847, pp. 1847-1859. Creger, C. R., Phillips, H. and Scott,].].: Formation of an eggshell. Poultry Sci. 55: 1717-1723, 1976. Crombie, G., Snider, R., Faris, C. and Franzblau, c.: Lysinederived-cross-links in the egg shell membrane. Biochim. Biophys. Acta 640: 365-367, 1981. Eckert, ]., Glock, H., Schade, R., Krampitz, G., Enbergs, H. and Petersen, J.: Synthesis of a precursor polypeptide of egg matrix in the liver of laying hen. J. Anim. Physiol. Anim. Nutr. 56: 258-265,1986. Harris, E. D., Blount, J. E. and Leach, R. M., Jr.: Localization of lysyloxidase in hen oviduct: implications in egg shell membrane formation and composition. Science 208: 55 -56,1980. Hoffer, A. P.: The ultrastructure and cytochemistry of the shell membrane-secreting region of the Japanese quail oviduct. Am. ]. Anat. 131: 253-288, 1971. Humanson, G. L.: Animal Tissue Techniques. W. H. Freeman and Co., San Francisco, 1979, pp. 661. Kohler, P. O. and O'Malley, B. W.: Estrogen-induced morphologic changes in monolayer cultures of immature chick oviduct. Endocrinology 81: 1422-1427,1967. Krampitz, G., Meisel, H. and Witt-Krause, W.: Identification of carboxy-glutamic acid in ovocalcin. Naturwissenschaften 67: 38~39, 1980. Linsenmayer, T. F., Hendrix, M.]. R. and Little, C. D.: Production and characterization of a monoclonal antibody to chicken type I collagen. Proc. Natl. Acad. Sci. USA 76: 3703-3707, 1979. Leach, R.M., Jr., Rucker, R.B. and Van Dyke, G.P.: Egg shell membrane protein: a non elastin desmosine/isodesmosine-containing protein. Arch. Biochem. Biophys. 207: 353-359,1981. Leach, R.M., Jr.: Biochemistry of the organic matrix of the eggshell. Poultry Sci. 61: 2040-2047, 1982. Oka, T. and Schimke, R. T.: Interaction of estrogen and progesterone in chick oviduct development. I. Antagonist effect of progesterone on estrogen-induced proliferation of tubular gland cells.]. Cell. Bioi. 41: 8916-831, 1969. Pageaux, ].-F., Dufrene, L., Laugier, c., Perche, O. and Sandoz, D.: Heterogeneity of progesterone receptor expression in
epithelial cells of immature and differentiating quail oviduct. Bioi. Cell. 67: 135-140,1989. Paul-Garda is, A., Picard,]. and Hermelin, B.: Turnover of sulfates in glycopeptides from egg shell membranes and hen oviduct sulfated glycoproteins. Biochim. Biophys. Acta 354: 11-16, 1974. Pearl, R. and Curtis, M. R.: Studies on the physiology of reproduction in the domestic fowl. V. Data regarding the physiology of the oviduct.]. Exp. Zool. 12: 99-132, 1912. Perche, 0., Laine, M.-C., Pageau x, J.-F., Laugier, C. and Sandoz, D.: Modification of cell evagination and cell differentiation in quail hyperstimulation by progesterone. Bioi. Cell. 67: 124-132, 1989. Perche, 0., Hayashi, M., Hayashi, K., Birk, D., Trelstad, R. L. and Sandoz, D.: Origin of type I collagen localized within oviduct epithelium of quail hyperstimulated by progesterone. ]. Cell. Sci. 95: 85-95, 1990. Picard,J., Paul-Gardais, A. and Vedel, M.: Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte. Biochim. Biophys. Acta 320: 427-441, 1973. Poole, A. R. and Pidoux, I.: Immunoelectron microscopic studies of type X collagen in endochondral ossification. ]. Cell. Bioi. 109: 2547-2554, 1989. Poole, A. R., Matsui, Y., Hinek, A. and Lee, E. R.: Cartilage macromolecules and the calcification of cartilage matrix. Anat. Rec. 224: 167-179, 1989. Richardson, K. c.: The secretory phenomena in the oviduct of the fowl, including the process of shell formation examined by the microincineration technique. Phil. Trans. Royal Soc. London Series B 225: 149-195,1935. Robinson, D.S., King, N.R. and Bowen, D.].: Neutral and acidic mucins in the reproductive tract of the laying hen. Proc. Roy. Micro. Soc. 1: 213-214, 1966. Schade, R.: Biochemische und molekulargenetische Aspekte der Eischalenbildung. Arch. Geflugelk. 51: 81-87, 1987. Schmid, T.M. and Linsenmayer, T.F.: Immunohistochemical localization of short chain cartilage collagen (Type-X) in avian tissues.]. Cell. Bioi. 100: 598-605,1985. Schraer, R. and Schraer, H.: Changes in metal distribution of the avian oviduct during the ovulation cycle. Proc. Soc. Exp. Bioi. 119: 937-942, 1965. Starcher, B. C. and King, G. S.: The presence of desmosine and isodesmosine in eggshell membrane protein. Connect. Tiss. Res. 8: 53-55, 1980. Sternberger, B.H., Mueller, W.J. and Leach, R.M., Jr.: Microscopic study of the initial stages of egg shell calcification. Poultry Sci. 56: 537-543, 1977. Turchini, J. and Broussy, J.: Nature histochimique des granulations des glandes de l'oviducte de la poule et formation de l'ovokeratine. Arch. Anat. Micros. 34: 146-152,1938. Thornton, P.A. and Moreng, R.E.: The effect of ascorbic acid on egg quality factors. Poultry Sci. 37: 691 ~698, 1958. Thornton, P.A. and Moreng, R.E.: Further evidence on the value of ascorbic acid maintenance of shell quality in warm environmental temperature. Poultry Sci. 38: 594-598, 1959. Wong, M., Hendrix, M.J. c., von der Mark, K., Little, C. and Sterns, R.: Collagen in the egg shell membranes of the hen. Develop. Bioi. 104: 28-36, 1984. Dr. Arnold I. Caplan, Skeletal Research Center, Department of Biology, Case Western Reserve University, Cleveland, OH 44106, USA.
The Fabrication and Collagenous Substructure of the Eggshell Membrane in the Isthmus of the Hen Oviduct J. L. ARIAS 1 , M. S. FERNANDEZ 1 , J. E. DENNIS and A. I. CAPLAN Skeletal Research Center, Department of Biology, Case Western Reserve University, Cleveland, OH 44 106, USA. 1 Visiting Scientists from University of Chile, Santiago.
Abstract The eggshell of the chicken consists of a bi-Iayered shell membrane overlaid with a thick, calcified shell matrix. The shell membranes and matrix are deposited onto the egg as it passes through the oviduct. To assess the temporal and spatial aspects of the fabrication of type X collagen within the eggshell extracellular matrix, the immunohistochemical localization of type X collagen was studied in three regions of the hen oviduct (magnum, isthmus and uterus), in the membranes of uncalcified eggshells obtained from the oviduct prior to mineral deposition and in eggshell membrane and calcified eggshell matrix. Additionally, immunohistochemicallocalization of type I and III collagens was done in order to determine possible co-localization of collagen types or to define tissue compartments. None of the collagen epitopes assayed was found in the shell matrix. Type X collagen epitope was immunohistochemically localized only to the epithelial cell layer lining the isthmus region of the oviduct and in the shell membranes of both uncalcified and calcified eggshells. Antitype III collagen monoclonal antibody delineated the inter-tubular gland connective tissue of the oviduct and was negative in the shell layers under conditions which gave strong connective tissue reactivity. Type I collagen epitope was exposed after pepsin treatment of the tissue and co-localizes with the distribution of type III collagen. Type I collagen co-localized with type X collagen in the shell membranes of uncalcified shells. The type I collagen epitope was reactive in the shell membrane of the uncalcified shells, but could only be detected in calcified shells following pepsin digestion. These observations suggest that the type I collagen epitope is masked during the process of calcification of the shell. Type X collagen is localized to regions adjacent to mineral deposits in the eggshell but is never colocalized with mineral deposits. We hypothesize that extracellular matrices containing typeX collagen can function as a mineralization barrier. Key words: eggshell, hen, oviduct, shell membranes, type I collagen, type III collagen, type X collagen.
Introduction The avian oviduct is a complex tubular organ which is responsible for the transport of the egg and the secretion of a diverse mixture of extracellular matrix components which surround the egg white. The extracellular matrix components assemble into complex layers the outer regions of which provide the scaffolding for calcium carbonate
deposition of the eggshell proper. The eggshell consists of a number of extracellular matrix layers which are sequentially fabricated during the passage of the egg through the oviduct. The first layers covering the egg white are the fibrillar inner and outer shell membranes (Fig. 1). The most external portion of the outer membrane consists of discrete aggregations of organic material, called mammillae, forming the mammillary layer. These mammillae are suggested
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!MAMMILLARY outer
SHELL MEMBRANES
to be the sites where the organic matrix of the shell is attached and where the initiation of calcium carbonate crystal formation takes place (Creger et aI., 1976; Stemberger et aI., 1977; Baumgartner et aI., 1978). The hard shell proper, the palisade or calcite layer, is organized upon this shell matrix with the most external layer referred to as the cuticle. The oviduct is organized into five regions on the basis of differences in morphological features and function (Coste, 1847). These regions are, from proximal to distal, the infundibulum which receives the ovum, the magnum which secretes albumen, the isthmus which secretes precursors of the shell membranes, the uterus or shell gland in which deposition of calcium carbonate takes place and the vagina. In addition to its principal role of depositing the shell membrane (Coste, 1847; Pearl and Curtis, 1912), the isthmus supplies water, copper, iron and calcium to the developing egg (Schraer and Schraer, 1965; Robinson et aI., 1966). The microstructure and ultrastructure of the avian isthmus have been studied (Richardson, 1935; Turchini and Broussy, 1938; Hoffer, 1971), but neither the histological nor the biochemical analysis of the isthmus have clarified the nature of the process by which the amorphous secretory products of its tubular glands are transformed into the double-layered fibrous shell membrane. Also, the molecular composition of the secretory granules of the isthmus cells and of the shell membranes and shell matrix themselves have not been completely elucidated (Hoffer, 1971). However the collagenous nature of the shell membrane proteins has been suspected based indirectly on the fact that factors affecting collagen synthesis, such as copper and ascorbate deficiency and lathyrogens, also affect formation of the shell membranes (Thornton and Moreng, 1958; 1959; Baumgartner et aI., 1978). Co-localization of type I and III collagen has been described in the intercellular loose connective tissue of the quail oviduct (Perche et aI., 1990).
lnner
Fig. 1. A drawing of a complete radial cross-section of the chicken eggshell (modified from Arias et ai., 1991 a).
Collagen types I and V with polyclonal antibodies (Wong et aI., 1984) and collagen type X with monoclonal antibodies (Arias et aI., 1991 a) have been detected and localized immunohistochemically within the shell membranes. However, with monoclonal antibody IIBe, to type I collagen, positive reaction is only detected after pretreatment of the shell membranes with pepsin (Arias et aI., 1991 a). These collagen fibers are coated with a proteoglycan-rich material that binds the fibers into a network (Wong et aI., 1984). The proteoglycans have been partially characterized as being rich in keratan and dermatan sulfate (Arias et aI., 1991 b). The composition of the shell matrix is less well known and only some y-carboxyglutamic rich proteins (Abatangelo et aI., 1978; Krampitz et aI., 1980) and proteoglycans (Arias et aI., 1991 b) have been described. In addition, some precursors of the shell matrix proteins have been described to be synthesized in the liver (Eckert et aI., 1986; Schade, 1987). To examine the temporal and spatial aspects of eggshell extracellular matrix deposition, the secretion products of the oviduct which comprise the shell membranes were studied by immunohistochemistry. Localization of types I and X collagen was studied in three regions within the oviduct, in the shell membranes of complete calcified eggshells, and in the newly deposited shell membranes located in the oviduct prior to eggshell calcification.
Material and Methods
Entire oviducts from two-year-old White Leghorn laying hens were dissected. In one oviduct, an egg was located 5 cm away from the uterus. Small pieces of the magnum, isthmus, uterus and the shell membranes covering the uncalcified egg were isolated and immersed directly in 15% sucrose in phosphate-buffered saline (PBS) for 30 min, transferred to Tissue-Tek O. C. T. compound (Miles Inc.,
Type X Collagen in the Hen Oviduct Elkhart, IN) and frozen in liquid nitrogen and 8-[A,m sections were cut with a Minotome cryostat. Additionally, pieces of eggshell from a completely formed egg were fixed in 2 % paraformaldehyde, 0.1 % glutaraldehyde in PBS for 12 h and then decalcified in 20% EDTA for 24 h and prepared for cryosectioning as above. Sections were placed on albumin-coated microscope slides and the slides immersed in PBS (pH 7.3) for 5 min. Some sections were digested with 0.5% pepsin in 0.05% acetic acid for 30min at 3rc. All sections were blocked for 5 min with 3 % bovine serum albumin (BSA) in PBS (BSA/PBS). Sections were exposed to the following monoclonal antibodies: anti-type X collagen (AC9/DEI0; Schmid and Linsenmayer, 1985), anti-type I collagen (IIB6; Linsenmayer et aI., 1979) and anti-type III collagen (3B2; provided by Dr. R. Mayne). The antitype III collagen antibody, which is a specific marker for the loose connective tissue was used to delineate the inter-tubular space. Additionally, since type III and type X collagens have never been co-localized in any other tisssue, the distinct distribution of these antibodies confirms the specificity of these probes. Sections were incubated in primary antibody for 1 h at room temperature, rinsed with 0.1 % BSA/PBS, washed with PBS for 5 min and blocked again with 3 % BSA/PBS for 5 min. The sections were then incubated in fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Cappel Lab., Cochranville, PAl for 1 h at room temperature; secondary antibody was used at a 1 :200 dilution in 1 % BSA/PBS. After this incubation, the sections were washed with PBS, mounted with coverslips in Fluoromount B (Fisher Biotech., Orangeburg, NY) and examined with an Olympus BH-2 microscope. As a control for non-specific staining by the secondary antibody, PBS or non-specific IgG was used in place of the primary antibody. Identical photographic exposures were taken for both control and experimental specimens. For histological identification serial sections were stained with Mallory-Heidenhain (Humason, 1979) and examined with bright field optics for comparison with immunostained sections.
Results
Oviduct The wall of the oviduct consists of a mucosa, a submucosa, a tunica muscularis and a serosa. The magnum and its Fig. 2. Immunostaining of the magnum wall with 3B2, a monoclonal antibody to type III collagen. (A) Cross-section of magnum mucosa and submucosa stained with Mallory-Heidenhain. An intense red stain is observed in the cells of the tubular glands (arrow) (X 220). (B) Immunopositive fluorescence is located along blood vessels walls (v) and around the smooth muscle layers (m) (X 250). (C) The fluorescence indicative of type III collagen is located in the connective tissue between the tubular glands of the magnum (X 250). g = glands; e = epithelium.
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tubular glands are covered by a simple columnar epithelium, with ciliated and goblet cells lining the magnum lumen and acinar cells forming the tubular glands (Fig. 2A). The cytoplasm of the gland cells contains secretory granules
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J. L. Arias et ai. tions of the surface epithelium form short ducts through which the tubular glands communicate with the lumen. The gland epithelium is formed by only a single cell type referred to as principal cells. These cells contain numerous large secretory granules which appear blue when stained with Mallory-Heidenhain. TypeIII collagen is located in the loose connective tissue surrounding the tubular glands (Fig. 4 B). After pepsin digestion, a positive reaction with anti-type I collagen antibody appears in the loose connective tissue colocalized with type III collagen as in the magnum. A strong reactivity with antibody to type X collagen is observed in the tubular gland cells (Fig.4 C), on the apical surface of the mucosal epithelial cells and in the glandular and isthmallumen (Fig. 4 D). The distribution of the reactive epitope of type X collagen does not overlap with that of epitope of type III collagen. The uterine mucosa is covered with a columnar epithelium and also possesses subepithelial glands. However, there is a greater proportion of loose connective tissue in this region than in the more proximal parts of the oviduct (Fig. 5 A). The epithelial cells consist of two cell types: ciliated and non-ciliated cells. The uterine gland cells, unlike the magnum and isthmal gland cells, do not show any evidence of secretory activity when stained sections are observed with the light microscope. The strong, widespread reactivity against type III collagen confirms that a large proportion of the uterine glands consists of loose connective tissue (Fig. 5 B). There was no reactivity against type X collagen in this region and for type I collagen it was again, as in the magnum and isthmus, a positive reaction in the loose connective tissue only after pepsin digestion. In addition, reactivity against type III collagen in the egg layers was never observed.
Fig. 3. (A) 1mmunostaining of the magnum wall treated with pepsin to anti-type I collagen monoclonal antibody 11 8 6 . 1mmunopositive fluorescence is located in the connective tissue between the tubular glands (g). (X 250). (B) The same antibody in non-pepsintreated samples shows no reactivity. (X 250). characteristic of egg white proteins which stain intense red with Mallory-Heidenhain; the same intense red material is detected in the gland lumen. Type III collagen delineates the loose connective tissue between tubular glands and surrounds the smooth muscle cells of the muscular layer (Fig.2B). The largest proportion of the magnum wall is occupied by the glandular cells surrounded by the loose connective tissue (Fig. 2 C). Immunostaining with type X collagen antibody was not observed in this region. Immunostaining for type I collagen was observed in the connective tissue only after digestion with pepsin (Fig. 3 A). Its distribution appears similar to that of type III collagen which did not require pepsin digestion to be visualized (see Fig.2C). The isthmal mucosa is covered by an epithelium made up of ciliated and mucus-secreting cells (Fig. 4 A). Invagina-
Shell membranes The shell membranes obtained before they reach the uterus, stain intensely for types X (Fig. 6 A) and I (Fig. 6 B) collagen. In decalcified eggshells (Fig. 6 C), type X collagen is localized exclusively to the shell membranes, and not to the shell matrix (Fig. 6 D). Type I collagen antibody reactivity is negative in the shell membranes, mammillary knobs and shell matrix under conditions where the antibody to type X collagen is reactive in the shell membranes. However, as we have shown elsewhere (Arias et ai., 1991 a), treatment of shell membranes from calcified eggshells with pepsin exposes the type I collagen epitope so that reactivity with the antibody to type I collagen is detected. The shell membranes in uterus do not require pretreatment with pepsin to give a positive reaction to type I collagen.
Type X Collagen in the Hen Oviduct
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Fig. 4. Immunostaining of the isthmus wall with monoclonal antibodies 3B2 (anti-type III collagen) and AC9IDEIO (anti-type X collagen). (A) Cross-section of the isthmal mucosa stained with Mallory-Heidenhain. The secretory granules in the cells of the tubular glands appear blue (arrow) (X 244). (B) An intense immunofluorescence (3B2 antibody) is observed in the loose connective tissue between the tubular glands (X 250). (C) Immunofluorescent staining by anti-type X collagen antibody is localized to the cells of the tubular glands (X 220) and (D) also localized at the luminal surface of the isthmal epithelial cells (X 220). g = gland; e = epithelium; L = lumen.
Discussion Despite the findings that proteoglycans and collagens contribute significantly to the composition of the organic matrix of the eggshell (Wong et ai., 1984; Arias et ai., 1991 a, b), little is known about the function of these matrix components in the calcification process within the oviduct. The presence of type X collagen associated with the glandular cells of the shell membrane-secreting region (isthmus) of the hen's oviduct is reported here. Type I and X collagens were located in the shell membranes of the uncalcified egg, but only type X collagen could be observed in the shell membranes of the mineralized egg. As demonstrated previously, type I collagen was immunohistologically co-
localized in these shell membranes with type X collagen only after pepsin treatment. Additionally, both collagens were found only in the shell membranes (non-mineralized) and not in the shell matrix proper (mineralized). Type III collagen was found in the loose connective tissue of the magnum, isthmus and uterus; while type I collagen was found co-localized with type III collagen only after pepsinization of the tissue. The three secretory regions of the hen oviduct are responsible for the elaboration of a multiple array of materials which comprise the layers surrounding the egg yolk. However, only the isthmus and uterus are responsible for the formation of the eggshell. The magnum region synthesizes the egg white or albumen, consisting of glycoproteins with
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Fig. 5. Immunostaining of the uterine wall with monoclonal antibody 3B2 (anti-type III collagen). (A) Cross-section of the uterus stained with Mallory-Heidenhain. Secretory granules in the cytoplasm of the glandular cells were not detected with this staining (X 200). (B) An intense fluorescence with 3B2 antibody is observed in the abundant loose connective tissue (X 220). g = gland; e = epithelium; L = lumen. complex N-linked oligosaccharides (Brockhausen et ai., 1988; 1989). The growth and cytodifferentiation of the magnum as well as the synthesis of egg white proteins are regulated by hormones, especially estrogen and progesterone (Kohler et ai., 1968; Oka and Schimke, 1969; Perche et ai., 1989; Pageaux et ai., 1989). The isthmus or shell membrane-secreting region of the oviduct has been characterized histochemically and ultrastructurally (Richardson, 1935; Hoffer, 1971). Three main types of cells have been described lining the epithelium of the isthmal mucosa and the tubular glands (Hoffer, 1971). These are the ciliated columnar epithelial cells, the mucous cells and the principal cells. It has also been established that the secretory granules of the principal cells contain a non-
sulfated protein-polysaccharide which does not contain detectable carboxylic groups, but possesses sulfhydryl and disulfide groups (Hoffer, 1971). The synthesis of sulfated glycoproteins as components of eggshell membranes has been suggested to occur in both the magnum and the isthmus (Picard et ai., 1973; Paul-Gardais et ai., 1974). The presence of type X collagen epitope in the principal cells of the isthmus and its simultaneous occurrence in the shell membranes is consistent with the suggested role of this region in the assembly of the shell membranes. It is of interest to consider that anti-type X collagen reactivity is found only in the isthmus, indicating a regional specialization of the oviduct for the synthesis of this collagen type. The occurrence of type X collagen in a system of mineralization other than hypertrophic chondrocytes may help in the understanding of the mechanisms underlying biological mineralization. Type X collagen is found in the shell membrane-secreting region of the oviduct and in the eggshell membranes, which do not mineralize. Similarly, immunolocalization of type X collagen has not been observed to be concentrated in the focal calcification sites nor in association with matrix vesicles suggesting an absence of a direct role for type X collagen in nucleation and matrix vesicle function during endochondral ossification (Poole and Pidoux, 1989; Poole et ai., 1989). From these observations we hypothesize that type X collagen functions to inhibit mineralization and thereby establish boundaries or regions which are protected from mineral deposition. In fact, it appears that type X collagen, when secreted from the isthmus tubular gland cells, is assembled in the oviductal lumen, at some distance from the cells, shaping the shell membranes which surround the egg white. These shell membranes do not mineralize but act as substrate for the deposition of the mammillary knobs and shell matrix which direct nucleation and growth of the crystals forming the hard shell proper. The easy detection of type I collagen in the shell membranes in uterus revealed by the antibody used here and the necessity of pepsin treatment of the shell membranes from calcified eggshells to exhibit its epitope (Arias et aI., 1991 a) is not easily interpreted. One possible explanation is that early in the passage of the shell membranes through the uterus, the epitope to type I collagen is accessable but, later during passage it becomes masked in such a way that it can only be exposed after pepsin digestion. As we suggested elsewhere (Arias et aI., 1991 a), type X collagen could be masking the type I collagen epitope, although a similar effect by other molecules (e. g. proteoglycans) cannot be excluded. Cross-links may also be involved in the masking of the type I collagen epitope. It is known that the reaction with I1B6 antibody is highly dependent on the collagen conformation and also that it has less sensitivity than polyclonal antibodies against type I collagen raised in rabbits (Linsenmayer et ai., 1979). Although different from elastin
Type X Collagen in the Hen Oviduct
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B
Fig. 6. Immunostaining of the shell membranes and/or shell matrix produced by; (A) anti-type X collagen antibody and (B) anti-type I collagen antibody, on shell membranes located in the oviduct before calcification occurs (X 280). (C) Cross-section of the shell membrane (sm) and shell matrix (mx) of a decalcified eggshell stained with Mallory-Heidenhain. The shell membranes stain purple and the matrix blue (X 280). (D) Cross-section of decalcified eggshell exposed to anti-type X antibody showing the positive immunoreaction to the shell membranes (sm) but not to the shell matrix (mx) (X 280).
(Starcher and King, 1980; Crombie et aI., 1981; Leach et aI., 1981), the proteins of the eggshell membranes are highly crosslinked by disulfide bonds (Leach, 1982) and by desmosine and isodesmosine groups (Candlish and Scougall, 1969; Balch and Cooke, 1970; Starcher and King, 1980; Leach et aI., 1981; Crombie et aI., 1981), due to a strong lysyloxidase activity in the isthmus (Harris et aI., 1980). Further research is required to elucidate the contribution of cross-linking to the masking of the epitope of this monoclonal antibody. The origin of the type I collagen of the shell membranes is still unknown. With the methodology used here, we were unable to detect type I collagen in any secretory cell of the different regions of the oviduct.
Acknowledgements We thank Dr. T.F. Linsenmayer (Tufts University, Boston, MA) and Dr. R. Mayne (University of Alabama, Birmingham, AL) for their generous gifts of the monoclonal antibodies and Dr. Dave Carrino (Case Western Reserve University, Cleveland, OH) for critical reading of the manuscript. This work was supported by the University of Chile (Santiago), by NIH and by D.O.E. through Battelle Pacific Northwest Laboratories.
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