EXPERIMENTAL
PARASITOLOGY
Capillaria
44286297
(1976)
hepafica:
Fine
Structure
of Egg Shell
GEORGEJ. GRIGONIS, JR., AND GENE B. SOLOMON 1 Department School
of Animal of Veterinary Philadelphia,
(Accepted
Biology and Department Medicine, University Pennsylvania 19174,
for publication
of Pathobiology,
of Pennsylvania, U.S.A.
12 December 1975)
GRIGONIS, G. J., JR.,AND SOLOMON, G. B. 1976. Capillaria hepatica: Fine structure of egg shell. Experimental Parasitology 40, 286-297. The structure of the Capillaria hepatica egg shell was studied with the electron microscope and correlated with light microscope histochemical observations. The shellis composed of fibrous and nonfibrous components, both of which stain for protein. The fibrous component, the major portion of the shell, consists of submicroscopic fibers. The nonfibrous component is located in the outer region of the shell but is not always visible; when present it has a reticulated appearance in electron micrographs. The fibrous component is divided into outer and inner regions. The outer region is composed of radially arranged pillars which are connected at their outer surface by a beam-like network and are anchored at the base to a compact inner region. The inner region consists of a series of concentrically arranged lamellae above which is located a nonlaminated region where the pillar bases originate. At each polar end of the shell is a single opening plugged with a material which contains acid mucopolysaccharide. The fine structure of the body of the plug is unresolvable with the electron microscope; its outer surface is impregnated with electron dense particles. Externally the shell is covered by a 250 p1 thick continuous membrane which is in close opposition to the surrounding host tissue. INDEX DESCRIPTORS: Capillaria hepatica; Helminth egg; Egg shell structure; Electron microscopy; Fine structure; Submicroscopic anatomy; Histochemistry; Mice.
Experimental granuloma formation to Capillaria hepatica eggs is composed of both cellular and humoral components (Solomon and Soulsby 1973; Raybourne et al. 1974; Raybourne and Solomon 1975). Although egg antigens used in the above studies were prepared (Solomon et al. 1974), neither the site of origin nor the source of egg-derived antigens has as yet been clarified. It seemed clear that further knowledge of the structure and function of the C. hepatica egg shell was required for ‘a fuller understanding of the source and role of antigens involved in experimental egg granulom’a formation. The current series of 1 Author to whom reprint requests should be sent.
papers is directed toward this goal. The first paper details the fine structure of the C. hepatica egg shell in situ following freeze-dry fixation of infected liver. This information will serve as a basis for studies concerned with changes in the C. hepatica egg following isolation from infected liver and with the release of antigens contributing to the immunological response. Subsequent publications will deal with the origin and characterization of egg antigens and with the response of cells directly involved in the cellular reaction of experimental egg granulomas. MATERLALSANDMETHODS
The strain of C. hepatica used and the methods for maintenance of stock infections
286 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved
CUpikiU
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FIG. la. Capillaria hepatica egg in situ. Photomicrograph of section, stained with toluidine blue showing egg shell structure in relation to surrounding tissue. The embryo has fallen out in sectioning and hence is not seen. The egg shell consists of two regions: (a) inner, 2.5-3.0 pm thick, (b) outer pillared region, 2.5 pm thick. At the polar ends two microscopic plugs are located; only one (c) is seen in this photograph, because of the plane of section; host tissue (d). The clear spot just below the plug is caused by a film defect. X1080. FIG. lb. C. hepatica egg in liver tissue, freeze dried paraffin embedded section stained for protein with toluidine blue at pH 5.0. Inner shell (a) and the pillars of the outer shell (b) stain with a blue-green metachromasia. The polar plugs (c) stain pale blue and the shell matrix, when present, stains with a pink metachromasia. No staining of the shell or plug occurred at pH 2.0; host tissue (d); embryo (e). X1080. FIG. lc. C. hepatica egg fixed and embedded as in Fig. lb, stained with Alcian blue (pH 0.5). Staining at this pH is indicative of the presence of sulphated acid mucopolysaccharides. The plug (c) stains most intensely at the base and to a lesser degree in the outer region. The inner membrane (between the inner shell and the embryo) also stains and is marked by the arrow. A similar staining reaction occurred at pH 1.0, but no staining was observed at pH 2.5. X 1080.
were identical to those reported by Solomon and Soulsby ( 1973). Electron Microscopy Liver specimens from freshly killed 35day stock-infected mice were cut in a moist box to a thickness of 0.1-0.2 mm. In order to confirm the presence of C. hepatica eggs, press preparations were examined with the light microscope. Hepatic tissue without eggs was discarded. When eggs were observed, adjacent tissue regions were placed on thin aluminum foil and frozen ultrarapidly (at a rate of freezing greater than -5000 C per second) in Freon 13, cooled to -180 C with liquid nitrogen, and swirled vigorously to prevent significant ice crystal formation (Gersh 1973). Specimens were vacuum dried at a temperature below -40 C and subsequently
postfixed in vacua with the protein crosslinking agent heptafluorodimethyloctanedione ( HEFDOD ) . Postfixation with HEFDOD vapors in vacua reduces the possibility of extraction of soluble components (Gersh 1973). After postfrxation, the tissue was infiltrated with 95% ethanol and subsequently with water-soluble Durcupan, which was slowly polymerized. Sections were cut with a Huxley ultramicrotome using a DuPont diamond knife, ‘and mounted directly on uncoated 400-mesh copper grids. Sections 600-800 A thick were used for low magnification electronmicrographs; other sections about 400 A thick were used for high magnification, high resolution electronmicrographs. Some sections were stained with uranyl acetate while others were left unstained. All sections were carbon coated in #avacuum evaporator and then photo-
288
GRIGONIS
AND
SOLOMON
Alcian blue (Alcianblau 8GS, Roboz Surgical Instument Company, Washington, D.C.) according to the method of Lev and Spicer
(1964).
A 0.1%
toluidine
blue
solu-
tion, in water, adjusted to pH 2.0 and pH 5.0 with HCI was employed to stain for protein (Singer 1954). Sections were photographed using a Zeiss Photomicroscope with a planapochromatic oil immersion objective (100X ). OBSERVATIONS
FIG. 2. Diagrammatic representation of the major structural features of the Capillaria hepatica egg shell as reconstructed from observations of sections with the light microscope and electron micrographs. (A) Section through the C. hepatica egg shell as seen in electron photomicrographs and corresponding to the plane of section in ( B ). The shell matrix which fills the shell space in a certain percentage of eggs is not shown. (B) Overhead view of the C. hepatica egg shell surface illustrating the beam-like network in relation to the pillars. The precise surface geometry of the network and beams is not known; however, preliminary observations indicate that the pillars are arranged pentagonally.
graphed with an Hitachi HU 11-A electron microscope. Histochemistry For histochemical studies, slabs of liver 2-3 mm thick from stock-infected animals were freeze-dried and embedded in paraffin and sectioned with a Spencer sliding microtome. Sections were mounted dry on albuminized slides. To demonstrate the presence of acid mucopolysaccharide within the egg shell, paraffin sections of stock-infected tissue were stained with
In the interest of brevity, detailed descriptions are given in the figure legends. The C. hepatica egg shell is divided into inner and outer regions (Fig. la). Polar portions of the shell have openings which are plugged with a stainable material. Histological staining of the egg shell with toluidine blue takes place at pH 5.0, but not at pH 2.0. This indicates that the shell contains protein (Fi,. 0 lb). The polar plugs stain similarly with toluidine blue (Fig. lb) and thus contain protein also. In addition,
the
plugs
staiu
positively
for
acid
mucopolysaccharide with Alcian blue. This reaction extends beyond the base of the plug as a fine layer surrounding the embryo, which corresponds to the inner membrane (Fig. lc). With the electron microscope the shell is resolved into fibrous and nonfibrous components. The fibrous components comprise the inner and outer regions as seen with the light microscope. The structure of the fibrous component is shown in diagrammatic form in Fig. 2. This is used as a basis for the detailed terminology and subsequent descriptions of the various parts of the egg shell. The fibrous component consists of laminated and nonlaminated portions of the inner shell (Figs. 2, 6) and pillars and connecting beam-like structures of the outer shell (Figs. 2, 3). In the space between the pillars and beneath the beams of the outer shell there is a protein network, referred to as the nonfibrous component, or shell matrix (Figs. 3, 6). The overall construc-
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EGG SHELL FINE STRUCTURE
289
FIG. 3. Segment of a longitudinal section of the Capillaria hepatica egg shell illustrating some of the details of shell construction. The outer membrane (OM) is continuous over the outer portion of the egg shell adjacent to host tissue (HT). The outer shell of the egg consists of a series of radially arranged pillars (PIL) connectedby a beam-likenetwork ( BM ). Located between the pillars is a shell matrix (SM). Pillars originate from the nonlaminated (NONLAM) portion of the inner shell. X20,000.
tion of the C. hepatica egg shell and especially the outer region is similar to the “meshwork” in the physical gill, or plastron, as seen in the integument and egg shells of certain insects (Hinton 1969). Within the fibrous component, individual fiber dimensions and spacings are approximately uniform (Fig. 12), but differ in orientation. The laminated portion (Figs, 6, 7) is characterized by six to seven concentric lamellae. The fibers in each lamella are parallel to the surface of the egg. In the interlamellar spaces the fibers turn out of the lamellar plane resulting in a concentric C pattern that is strikingly similar to that of the arthropod endocuticle (Locke 1961, 1974). In the center of the interlamellar spaces, cross-sections of the indi-
vidual fibers are seen (Fig. 7). The fibers turning out of the lamellar plane of the outermost lamellae ‘are packed in bundles that make up the nonlaminated portion (Figs. 6, 12). Here the bundle arrangements are complex and no definite pattern can be resolved. A somewhat recognizable order is resumed in the vicinity of the pillar bases,where fiber bundles aggegate and are directed upward, normal to the egg surface, initiating pillar formation (Fig. 9)., From a study of longitudinal (Fig. 10) and cross (Fig. 11) sections of pillars, one can see that most of the fibers are oriented in the direction of the pillar axis. It is possible that some fibers may be arranged circularly or spirally on the outside of the pillars. Beneath the outer membrane the pillars
290
GIUGONIS
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SOLOMON
FIG. 4. Longitudinal section through the polar region of the egg shell showing the microscopic opening closed by a plug. It is about 4.0 pm at the constriction (CON) and about 6.0 Frn at the opening of the mouth (MT). The neck (NK) arises from the nonlaminated portion of the inner shell (NON-LAM) in a manner similar to the pillars. The shell matrix (SM) is visible. The major portion of the plug (PL) is a homogeneous matrix. At the border line of the outer portion of the plug there is a layer, about 0.55-0.65 pm thick, of dense granular material (GM). The outer membrane ( OM) lies over the plug in close association with the granular material. X11,000.
branch, forming an interconnecting beam- brane is in contact with the shell matrix like structure (Figs. 2, S), parallel to the or shell space (Fig. 3). At the poles it is egg surface. attached to the plug material (Figs. 4, 6). The nonfibrous protein network (shell The total thickness of the membrane is matrix) is present in 10-X% of the eggs. about 250 A, with a less dense center 100 These consisted chiefly of solitary eggs and A wide bordered by two 75 A thick layers eggs located at the outside edge of egg (Fig. 8). At the polar regions, however, the clusters. The matrix is formed by the com- membrane appears as a single layer about municating walls of submicroscopic vacu- 250 A without subdivisions (Fig. 5). oles (Fig. 6). The vacuoles are not empty The polar openings of the egg shell are but contain a stainable material whose den- plugged with a predominantly homogenesity is appreciably greater than the embed- ous matrix, the submicroscopic structure ding medium (Fig. 6). of which is not resolvable (Fig. 4). At the Covering the shell externally is a con- outer border of the plug is a dense layer tinuous membrane attached to and sup- of granular material, consisting of stacks of ported by the beam-like structure (Fig. 8). small doughnut-shaped particles ( Fig. 5). In areas of the egg shell surface not cov- The shape of these particles can be seen ered by the beam-like structures the mem- only at the lower edge of the outer border
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hepatica:
EGG
since in the remainder of the granular zone the stacks are too densely packed to recognize any substructure. In unstained sections the contrast of the granular material with respect to the plug matrix is similar to that in the stained preparation. The outer membrane is usually close to the granular material and, in many cases, in direct contact with it (Fig. 5). DISCUSSION
These observations confirm the findings of Inatomi (1960) that the egg shell of C. hepatica consists of an inner ‘and outer layer with two pol’ar openings plugged with stainable material. They differ from his descriptions in that no submicroscopic pores are present in frozen-dried specimens
FIG. 5. Enlargement of outer border background, representing protein and order to increase the contrast of the granular material (GM). The granules 500 A outer diameter and 250 A inner (PL). x54,000.
SHELL
FINE
STRUCTURE
291
in the outer shell; in fact, the entire shell is covered by a continuous outer membrane supported by a beam-like network. Furthermore, Inatomi’s interpretations of the outer shell structures are Iapparently incorrect, since the dense septa and holes described by him are, in reality, the pillars and shell space, respectively, as observed in our frozen-dried specimens. The observations reported here on the laminated and nonlaminated structure of the inner shell and its interconnection with the pillars of the outer shell also confirm those of Inatomi ( 1960). But the precise relations of the inner shell to the pillars and the beam-like structures were not noted by him. The present study has, in addition, shown that the inner shell and a portion of the outer shell
line of plug in Fig. 4 to illustrate the granules. The polysaccharide has been printed with less contrast in granules. The outer membrane (OM) is close to the are made up of small doughnut-shaped particles about diameter (Inset X500,000). Homogeneous plug matrix
292
GRIGONIS
AND
h,ave ‘a fibrous fine structure. Iats dimensiorral uniformity throughout demonstrates the continuity between each portion of the fibrous shell and is further suggestive of the existence of a basic structural polymer. The nature of the fibrous component of the egg shell is not clear. There are two chief possibilities at present. (1) Fiber dimensions and spacings observed in the fibrous shell are remarkably similar to those of the chitin-protein complex of insect cuticles (Rudall 1963). The presence or absence of chitin in the C. hepaticu egg shell has not been adequately proven (Monne and H&rig 1954; Mariano 1967) since exis-
SOLOMON
tent histochemical tests for chitin are nonspecific and the methods depend largely on the amount of free uncomplexed chitin (Hackman 1974). Therefore, it seems plausible that a structural polysaccharide such as ‘chitin may be present though undetectable by present histochemical methods. (2) Keratin, another structural polymer widely present in the animal kingdom has ‘also been suggested to occur in the C. hepatica egg shell ( Monne and Hiinig 1954). This was based on certain solubility characteristics and the histochemical demonstration of sulfur containing proteins. Although the shell did not manifest all the solubility
FIG. 6. Longitudinal section of inner shell showing concentric lamellae. The electron dense lamellae (LM) are about 0.28-0.125 pm thick, the thinner ones are centrally located, i.e., toward the embryo. The interlamellar spaces (IN LM) are less electron dense and vary in thickness from about 0.125 to 0.37 pm with the thinner ones again being centrally located. External to the laminated portion is the nonlaminated (NON-LAM) portion of the inner shell. It is from 0.64-1.40 ym thick and its electron density is variable. From this region originate the base of the pillars and the neck of the polar openings; X20,000. The shell matrix (SM) is characterized by the presence of submicroscopic vacuoles (SUB VAC) (Inset). Their walls (WAL) are of uneven density. The contents of the vacuoles are also denser than the density of the plastic embedding medium ( “); X54,000.
Cupillaria
hepatica:
EGG
SHELL
FINE
STRUCTURE
293
FIG. 7. Longitudinal section of laminated portion of the inner shell. Fibers of similar dimension and spacing to the pillar fibers (Figs. 9-11) are in the lamellae and the interlamellar spaces. The fibers are oriented chiefly lengthwise in the plane of the lamella (Box 1). They curve in the interlamellar regions (Box 2) and run perpendicular to the plane of section in the center of the interlamellar spaces (Box 3). Abbreviations as given previously. X135,000.
characteristics of keratin it was noted that quinone-tanned keratin-like proteins do display the solubilities observed (Monn6 and Hiinig 1954). The fiber dimensions in the fibrous shell do not correspond to #anyfiber dimensions of mammalian keratin (Fraser and MacRae 1973), but they are similar to FIG. 8. A pillar in the outer region of the egg shell. The pillar base, not shown in the figure, originates from the nonlaminated portion of the inner shell region. The pillar is about 2.5 pm long and about 0.7 pm in diameter, extending the full thickness of the outer shell region. Near the surface of the outer shell the pillar expands forming a beam (BM) extending to an adjacent pillar. The beams, in a structural sense, act to support the outer membrane ( OM ) . In the center of the pillar near the surface, there is a small irregular depression (PIL DEP) which is bridged by the outer membrane. Shell matrix (SM). ~37,800.
294
GRIGONIS
AND
SOLOMON
FIG. 9. Longitudinal section through the pillar base (circles) of the nonlaminated segment and the aggregation form the pillars (between the arrows). X 135,000.
region illustrating of these bundles
the fiber bundles turning upward to
those of avian feather kcratin (Filshic and Rogers 1962; Fraser and MacRae 1973). No definitive conclusion can thus be made at this time concerning the structural polymer of the outer shell. Further studies on this topic will be reported in a later article of this series. The pillar and beam-like network of the outer shell closely resemble the plastron or physical gill of a number of insect egg shells as well as the integument of certain aquatic insects (Hinton 1969). Hinton showed that such a system makes it possible for the insect or egg to breathe while under water Fro. 10. Longitudinal section through a pillar. The structural basis of the pillars is fibrillar. The fibers in some areas run in the same direction as the long axis of the column which is indicated by the arrows (Box 1). Other fibers tend to curve away from the long axis (Box 2). Fiber cross sections are also seen (Box 3 ) X 108,000.
Capillaria
hepaticu:
EGG
due to the formation ‘of ,a gas film of constant volume having an extensive air-water interface. We suggest that the C. heputicu egg shell may function similarly during egg development since ‘a number of physical requirements necessary for plastron function ‘are similar to those needed for C. heputicu egg development ( Luttermoser I938 ) . The existence ‘of the shell matrix has not been reported elsewhere. In the specimens prepared by freezing and drying, matrix was ‘observed in N-15% of the specimens and reacted positively for protein. Mariano (1967) described a similar reaction, but as he w’as not ,aware of the existence of matrix, he could not attribute this staining reaction to any specific part of the shell. The plugs that fill the polar openings have a peculiar layer in close association with the continuous outer membrane. These
FIG. 11. Cross section of pillar through its chiefly in the center of the pillar ( Box 1) . At as either short, straight segments (Box 2), may be interpreted as bundles of fibers which
SHELL
FINE
STRUCTLJRE
295
granules were also descrbied by Inatomi ( 1960). They do not show a selectivity for any of the stains used in this study. In fact, the upper half of the plug had a somewhat lesser affinity for the Alcian blue than the remainder of the plug. The nature and function of the granules is uncertain. It is possible that they may serve as a foundation for the deposition of the plug matrix during egg shell formation. Histochemical staining used in this study has indicated the presence of protein in all parts of the egg shell. The polar plugs contain acid mucopolysaccharide and protein. This suggests that the plugs may resemble in function the protein-polysaccharide complex commonly seen in connective tissues of vertebrates ( Catchpole 1973). This suggested function will be elaborated on in the second report of this series.
center. Fibrous material is seen in cross section the outer border of the pillar the fibers are seen or curved segments (Box 3). Both of the latter encircle the periphery of the pillar. X135,000.
GRIGONIS AND SOLOMON
296
FIG. 12. High magnification electron micrograph of a tangential section of the nonlaminated part of the inner shell. Fibrillar material in cross section has a circular profile about 30 A in diameter (single arrow). In well organized regions the fibers have a center to center spacing of about 60 A (between the arrows a). Circled areas illustrate the fiber bundles present in the nonlaminated segment. X300,000.
ACKNOWLEDGMENTS The authors wish to express their sincere appreciation to Professor Isidore Gersh for his interest, advice, and assistance in this study. Dr. K. A. Wright of the University of Toronto kindly provided, from his own collection, several electron micrographs of the C. hepatica egg. These were used for study purposes only. Mrs. Rosalina Espiritu is gratefully acknowledged for her technical assistance. Supported in part by United States Public Health Service Research Grant AI-10898, USPHS Research Training Grant AI-00302, and USPHS Biomedical Science Support Grant RR 0’7083-09 Sub 21.
REFERENCES CATCHWLE, H. R. 1973. Capillary permeability. III. Connective tissue. In “The Inflammatory (B. W. Zweifach, L. Grant, and R. T. Process” McCluskey, eds. ), 2nd ed., Vol. 2. Academic Press, New York.
FILSHIE, B. K., AND ROGERS, G. E. 1962. An electron microscope study of the fine structure of feather keratin. Journal of Cell Biology 13, 1-12. FRASER, R. D. B., AND MACHAE, T. P. 1973. “Conformation in Fibrous Proteins and Related Synthetic Polypeptides.” Academic Press, New York. GERSH, I. 1973. “Submicroscopic Cytochemistry, Vol. I. Proteins and Nucleic Acids.” Academic Press, New York. HACKMAN, R. H. 1974. Chemistry of the insect cuticle. In “The Physiology of Insecta” (M. Rockstein, ed.), 2nd ed., Vol. 6. Academic Press, New York. HINTON, H. E. 1969. Respiratory systems of insect shells. Annual Review of EntomoZogy 14, 343368. INATOMI, S. 1960. Submicroscopic structure of the egg shell of helminth. III. A study on CapiZZuria hepatica. Acta Medica Okayama 14, 261-264. LEV, R., AND SPICER, S. S. 1964. Specific staining of sulphate groups with alcian blue at low pH. Journal of Histochemistry and Cytochemistry 12, 309.
CU$hiU
he@iCU:
EGG
LOCKE, M. 1961. Pore canals and related structures in insect cuticle. Journal of Biophysical and Biochemical Cytology 10, 589-618. LOCKE, M. 1974. The structure and formation of the integument in insects. In “The Physiology of Insecta” (M. Rockstein, ed.), 2nd ed., Vol. 6. Academic Press, New York. LUTTERMOSER, G. W. 1938. Factors influencing the development and viability of the eggs of Capilluria hepatica. American Journal of Hygiene 27, 275-289. MARIANO, M. 1967. Histochemical investigation on egg shell polysaccharides in CapiZlaria hepatica ( Bancroft 1918, Capillaridiae, Trichuroidea) . Acta Histochemica 26, 144-150. MONN~, L., AND H~NIC, G. 1954. On the properties of the egg envelopes of the parasitic nematodes Trichuris and Capillariu. Arkives fiir Zoologie 6, 559-562. RAYBOURNE, R., AND SOLOMON, G. B. 1975. Cap% laria hepatica: Granuloma formation to eggs. III. Anti-immunoglobulin augmentation and
SHELL
FINE
reagin activity ogy 38,87-95. RAYBOLJRNE, E. J. L. formation responses. 252.
297
STRUCTURE
in mice.
Experimental
Parasitol-
R. B., SOLOMON,G. B., AND SOULSBY, 1974. Capillariu hepatica: Granuloma to eggs. II. Peripheral immunological Experimental Parasitology 36, 244-
RUDALL, K. M. 1963. The chitin/protein of insect cuticles. Advances in Insect 1,257-313.
complexes Physiology
SINGER, M. 1954. The staining of basophilic ponents. Journal of Histochemistry and chemistry 2, 322-333. SOLOMON, G. B., RAYBOURNE, E. J. L. 1974. Serological infected with Capillaria Parasitology 60, 732-734.
comCyto-
R. B., AND SOULSBY, studies on rodents hepatica. Journal of
SOLOMON, G. B., AND SOULSBY, E. J. L. 1973. Granuloma formation to Capillaria hepatica eggs. I. Descriptive definition, Experimental Parasitology 33,458-467.
PARASITOLOGY
Capillaria
44286297
(1976)
hepafica:
Fine
Structure
of Egg Shell
GEORGEJ. GRIGONIS, JR., AND GENE B. SOLOMON 1 Department School
of Animal of Veterinary Philadelphia,
(Accepted
Biology and Department Medicine, University Pennsylvania 19174,
for publication
of Pathobiology,
of Pennsylvania, U.S.A.
12 December 1975)
GRIGONIS, G. J., JR.,AND SOLOMON, G. B. 1976. Capillaria hepatica: Fine structure of egg shell. Experimental Parasitology 40, 286-297. The structure of the Capillaria hepatica egg shell was studied with the electron microscope and correlated with light microscope histochemical observations. The shellis composed of fibrous and nonfibrous components, both of which stain for protein. The fibrous component, the major portion of the shell, consists of submicroscopic fibers. The nonfibrous component is located in the outer region of the shell but is not always visible; when present it has a reticulated appearance in electron micrographs. The fibrous component is divided into outer and inner regions. The outer region is composed of radially arranged pillars which are connected at their outer surface by a beam-like network and are anchored at the base to a compact inner region. The inner region consists of a series of concentrically arranged lamellae above which is located a nonlaminated region where the pillar bases originate. At each polar end of the shell is a single opening plugged with a material which contains acid mucopolysaccharide. The fine structure of the body of the plug is unresolvable with the electron microscope; its outer surface is impregnated with electron dense particles. Externally the shell is covered by a 250 p1 thick continuous membrane which is in close opposition to the surrounding host tissue. INDEX DESCRIPTORS: Capillaria hepatica; Helminth egg; Egg shell structure; Electron microscopy; Fine structure; Submicroscopic anatomy; Histochemistry; Mice.
Experimental granuloma formation to Capillaria hepatica eggs is composed of both cellular and humoral components (Solomon and Soulsby 1973; Raybourne et al. 1974; Raybourne and Solomon 1975). Although egg antigens used in the above studies were prepared (Solomon et al. 1974), neither the site of origin nor the source of egg-derived antigens has as yet been clarified. It seemed clear that further knowledge of the structure and function of the C. hepatica egg shell was required for ‘a fuller understanding of the source and role of antigens involved in experimental egg granulom’a formation. The current series of 1 Author to whom reprint requests should be sent.
papers is directed toward this goal. The first paper details the fine structure of the C. hepatica egg shell in situ following freeze-dry fixation of infected liver. This information will serve as a basis for studies concerned with changes in the C. hepatica egg following isolation from infected liver and with the release of antigens contributing to the immunological response. Subsequent publications will deal with the origin and characterization of egg antigens and with the response of cells directly involved in the cellular reaction of experimental egg granulomas. MATERLALSANDMETHODS
The strain of C. hepatica used and the methods for maintenance of stock infections
286 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved
CUpikiU
hepUth2:
EGG SHELL FINE STRUCTURE
287
FIG. la. Capillaria hepatica egg in situ. Photomicrograph of section, stained with toluidine blue showing egg shell structure in relation to surrounding tissue. The embryo has fallen out in sectioning and hence is not seen. The egg shell consists of two regions: (a) inner, 2.5-3.0 pm thick, (b) outer pillared region, 2.5 pm thick. At the polar ends two microscopic plugs are located; only one (c) is seen in this photograph, because of the plane of section; host tissue (d). The clear spot just below the plug is caused by a film defect. X1080. FIG. lb. C. hepatica egg in liver tissue, freeze dried paraffin embedded section stained for protein with toluidine blue at pH 5.0. Inner shell (a) and the pillars of the outer shell (b) stain with a blue-green metachromasia. The polar plugs (c) stain pale blue and the shell matrix, when present, stains with a pink metachromasia. No staining of the shell or plug occurred at pH 2.0; host tissue (d); embryo (e). X1080. FIG. lc. C. hepatica egg fixed and embedded as in Fig. lb, stained with Alcian blue (pH 0.5). Staining at this pH is indicative of the presence of sulphated acid mucopolysaccharides. The plug (c) stains most intensely at the base and to a lesser degree in the outer region. The inner membrane (between the inner shell and the embryo) also stains and is marked by the arrow. A similar staining reaction occurred at pH 1.0, but no staining was observed at pH 2.5. X 1080.
were identical to those reported by Solomon and Soulsby ( 1973). Electron Microscopy Liver specimens from freshly killed 35day stock-infected mice were cut in a moist box to a thickness of 0.1-0.2 mm. In order to confirm the presence of C. hepatica eggs, press preparations were examined with the light microscope. Hepatic tissue without eggs was discarded. When eggs were observed, adjacent tissue regions were placed on thin aluminum foil and frozen ultrarapidly (at a rate of freezing greater than -5000 C per second) in Freon 13, cooled to -180 C with liquid nitrogen, and swirled vigorously to prevent significant ice crystal formation (Gersh 1973). Specimens were vacuum dried at a temperature below -40 C and subsequently
postfixed in vacua with the protein crosslinking agent heptafluorodimethyloctanedione ( HEFDOD ) . Postfixation with HEFDOD vapors in vacua reduces the possibility of extraction of soluble components (Gersh 1973). After postfrxation, the tissue was infiltrated with 95% ethanol and subsequently with water-soluble Durcupan, which was slowly polymerized. Sections were cut with a Huxley ultramicrotome using a DuPont diamond knife, ‘and mounted directly on uncoated 400-mesh copper grids. Sections 600-800 A thick were used for low magnification electronmicrographs; other sections about 400 A thick were used for high magnification, high resolution electronmicrographs. Some sections were stained with uranyl acetate while others were left unstained. All sections were carbon coated in #avacuum evaporator and then photo-
288
GRIGONIS
AND
SOLOMON
Alcian blue (Alcianblau 8GS, Roboz Surgical Instument Company, Washington, D.C.) according to the method of Lev and Spicer
(1964).
A 0.1%
toluidine
blue
solu-
tion, in water, adjusted to pH 2.0 and pH 5.0 with HCI was employed to stain for protein (Singer 1954). Sections were photographed using a Zeiss Photomicroscope with a planapochromatic oil immersion objective (100X ). OBSERVATIONS
FIG. 2. Diagrammatic representation of the major structural features of the Capillaria hepatica egg shell as reconstructed from observations of sections with the light microscope and electron micrographs. (A) Section through the C. hepatica egg shell as seen in electron photomicrographs and corresponding to the plane of section in ( B ). The shell matrix which fills the shell space in a certain percentage of eggs is not shown. (B) Overhead view of the C. hepatica egg shell surface illustrating the beam-like network in relation to the pillars. The precise surface geometry of the network and beams is not known; however, preliminary observations indicate that the pillars are arranged pentagonally.
graphed with an Hitachi HU 11-A electron microscope. Histochemistry For histochemical studies, slabs of liver 2-3 mm thick from stock-infected animals were freeze-dried and embedded in paraffin and sectioned with a Spencer sliding microtome. Sections were mounted dry on albuminized slides. To demonstrate the presence of acid mucopolysaccharide within the egg shell, paraffin sections of stock-infected tissue were stained with
In the interest of brevity, detailed descriptions are given in the figure legends. The C. hepatica egg shell is divided into inner and outer regions (Fig. la). Polar portions of the shell have openings which are plugged with a stainable material. Histological staining of the egg shell with toluidine blue takes place at pH 5.0, but not at pH 2.0. This indicates that the shell contains protein (Fi,. 0 lb). The polar plugs stain similarly with toluidine blue (Fig. lb) and thus contain protein also. In addition,
the
plugs
staiu
positively
for
acid
mucopolysaccharide with Alcian blue. This reaction extends beyond the base of the plug as a fine layer surrounding the embryo, which corresponds to the inner membrane (Fig. lc). With the electron microscope the shell is resolved into fibrous and nonfibrous components. The fibrous components comprise the inner and outer regions as seen with the light microscope. The structure of the fibrous component is shown in diagrammatic form in Fig. 2. This is used as a basis for the detailed terminology and subsequent descriptions of the various parts of the egg shell. The fibrous component consists of laminated and nonlaminated portions of the inner shell (Figs. 2, 6) and pillars and connecting beam-like structures of the outer shell (Figs. 2, 3). In the space between the pillars and beneath the beams of the outer shell there is a protein network, referred to as the nonfibrous component, or shell matrix (Figs. 3, 6). The overall construc-
cUpihZ&2
hpUtiCU:
EGG SHELL FINE STRUCTURE
289
FIG. 3. Segment of a longitudinal section of the Capillaria hepatica egg shell illustrating some of the details of shell construction. The outer membrane (OM) is continuous over the outer portion of the egg shell adjacent to host tissue (HT). The outer shell of the egg consists of a series of radially arranged pillars (PIL) connectedby a beam-likenetwork ( BM ). Located between the pillars is a shell matrix (SM). Pillars originate from the nonlaminated (NONLAM) portion of the inner shell. X20,000.
tion of the C. hepatica egg shell and especially the outer region is similar to the “meshwork” in the physical gill, or plastron, as seen in the integument and egg shells of certain insects (Hinton 1969). Within the fibrous component, individual fiber dimensions and spacings are approximately uniform (Fig. 12), but differ in orientation. The laminated portion (Figs, 6, 7) is characterized by six to seven concentric lamellae. The fibers in each lamella are parallel to the surface of the egg. In the interlamellar spaces the fibers turn out of the lamellar plane resulting in a concentric C pattern that is strikingly similar to that of the arthropod endocuticle (Locke 1961, 1974). In the center of the interlamellar spaces, cross-sections of the indi-
vidual fibers are seen (Fig. 7). The fibers turning out of the lamellar plane of the outermost lamellae ‘are packed in bundles that make up the nonlaminated portion (Figs. 6, 12). Here the bundle arrangements are complex and no definite pattern can be resolved. A somewhat recognizable order is resumed in the vicinity of the pillar bases,where fiber bundles aggegate and are directed upward, normal to the egg surface, initiating pillar formation (Fig. 9)., From a study of longitudinal (Fig. 10) and cross (Fig. 11) sections of pillars, one can see that most of the fibers are oriented in the direction of the pillar axis. It is possible that some fibers may be arranged circularly or spirally on the outside of the pillars. Beneath the outer membrane the pillars
290
GIUGONIS
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FIG. 4. Longitudinal section through the polar region of the egg shell showing the microscopic opening closed by a plug. It is about 4.0 pm at the constriction (CON) and about 6.0 Frn at the opening of the mouth (MT). The neck (NK) arises from the nonlaminated portion of the inner shell (NON-LAM) in a manner similar to the pillars. The shell matrix (SM) is visible. The major portion of the plug (PL) is a homogeneous matrix. At the border line of the outer portion of the plug there is a layer, about 0.55-0.65 pm thick, of dense granular material (GM). The outer membrane ( OM) lies over the plug in close association with the granular material. X11,000.
branch, forming an interconnecting beam- brane is in contact with the shell matrix like structure (Figs. 2, S), parallel to the or shell space (Fig. 3). At the poles it is egg surface. attached to the plug material (Figs. 4, 6). The nonfibrous protein network (shell The total thickness of the membrane is matrix) is present in 10-X% of the eggs. about 250 A, with a less dense center 100 These consisted chiefly of solitary eggs and A wide bordered by two 75 A thick layers eggs located at the outside edge of egg (Fig. 8). At the polar regions, however, the clusters. The matrix is formed by the com- membrane appears as a single layer about municating walls of submicroscopic vacu- 250 A without subdivisions (Fig. 5). oles (Fig. 6). The vacuoles are not empty The polar openings of the egg shell are but contain a stainable material whose den- plugged with a predominantly homogenesity is appreciably greater than the embed- ous matrix, the submicroscopic structure ding medium (Fig. 6). of which is not resolvable (Fig. 4). At the Covering the shell externally is a con- outer border of the plug is a dense layer tinuous membrane attached to and sup- of granular material, consisting of stacks of ported by the beam-like structure (Fig. 8). small doughnut-shaped particles ( Fig. 5). In areas of the egg shell surface not cov- The shape of these particles can be seen ered by the beam-like structures the mem- only at the lower edge of the outer border
CUpi&&
hepatica:
EGG
since in the remainder of the granular zone the stacks are too densely packed to recognize any substructure. In unstained sections the contrast of the granular material with respect to the plug matrix is similar to that in the stained preparation. The outer membrane is usually close to the granular material and, in many cases, in direct contact with it (Fig. 5). DISCUSSION
These observations confirm the findings of Inatomi (1960) that the egg shell of C. hepatica consists of an inner ‘and outer layer with two pol’ar openings plugged with stainable material. They differ from his descriptions in that no submicroscopic pores are present in frozen-dried specimens
FIG. 5. Enlargement of outer border background, representing protein and order to increase the contrast of the granular material (GM). The granules 500 A outer diameter and 250 A inner (PL). x54,000.
SHELL
FINE
STRUCTURE
291
in the outer shell; in fact, the entire shell is covered by a continuous outer membrane supported by a beam-like network. Furthermore, Inatomi’s interpretations of the outer shell structures are Iapparently incorrect, since the dense septa and holes described by him are, in reality, the pillars and shell space, respectively, as observed in our frozen-dried specimens. The observations reported here on the laminated and nonlaminated structure of the inner shell and its interconnection with the pillars of the outer shell also confirm those of Inatomi ( 1960). But the precise relations of the inner shell to the pillars and the beam-like structures were not noted by him. The present study has, in addition, shown that the inner shell and a portion of the outer shell
line of plug in Fig. 4 to illustrate the granules. The polysaccharide has been printed with less contrast in granules. The outer membrane (OM) is close to the are made up of small doughnut-shaped particles about diameter (Inset X500,000). Homogeneous plug matrix
292
GRIGONIS
AND
h,ave ‘a fibrous fine structure. Iats dimensiorral uniformity throughout demonstrates the continuity between each portion of the fibrous shell and is further suggestive of the existence of a basic structural polymer. The nature of the fibrous component of the egg shell is not clear. There are two chief possibilities at present. (1) Fiber dimensions and spacings observed in the fibrous shell are remarkably similar to those of the chitin-protein complex of insect cuticles (Rudall 1963). The presence or absence of chitin in the C. hepaticu egg shell has not been adequately proven (Monne and H&rig 1954; Mariano 1967) since exis-
SOLOMON
tent histochemical tests for chitin are nonspecific and the methods depend largely on the amount of free uncomplexed chitin (Hackman 1974). Therefore, it seems plausible that a structural polysaccharide such as ‘chitin may be present though undetectable by present histochemical methods. (2) Keratin, another structural polymer widely present in the animal kingdom has ‘also been suggested to occur in the C. hepatica egg shell ( Monne and Hiinig 1954). This was based on certain solubility characteristics and the histochemical demonstration of sulfur containing proteins. Although the shell did not manifest all the solubility
FIG. 6. Longitudinal section of inner shell showing concentric lamellae. The electron dense lamellae (LM) are about 0.28-0.125 pm thick, the thinner ones are centrally located, i.e., toward the embryo. The interlamellar spaces (IN LM) are less electron dense and vary in thickness from about 0.125 to 0.37 pm with the thinner ones again being centrally located. External to the laminated portion is the nonlaminated (NON-LAM) portion of the inner shell. It is from 0.64-1.40 ym thick and its electron density is variable. From this region originate the base of the pillars and the neck of the polar openings; X20,000. The shell matrix (SM) is characterized by the presence of submicroscopic vacuoles (SUB VAC) (Inset). Their walls (WAL) are of uneven density. The contents of the vacuoles are also denser than the density of the plastic embedding medium ( “); X54,000.
Cupillaria
hepatica:
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STRUCTURE
293
FIG. 7. Longitudinal section of laminated portion of the inner shell. Fibers of similar dimension and spacing to the pillar fibers (Figs. 9-11) are in the lamellae and the interlamellar spaces. The fibers are oriented chiefly lengthwise in the plane of the lamella (Box 1). They curve in the interlamellar regions (Box 2) and run perpendicular to the plane of section in the center of the interlamellar spaces (Box 3). Abbreviations as given previously. X135,000.
characteristics of keratin it was noted that quinone-tanned keratin-like proteins do display the solubilities observed (Monn6 and Hiinig 1954). The fiber dimensions in the fibrous shell do not correspond to #anyfiber dimensions of mammalian keratin (Fraser and MacRae 1973), but they are similar to FIG. 8. A pillar in the outer region of the egg shell. The pillar base, not shown in the figure, originates from the nonlaminated portion of the inner shell region. The pillar is about 2.5 pm long and about 0.7 pm in diameter, extending the full thickness of the outer shell region. Near the surface of the outer shell the pillar expands forming a beam (BM) extending to an adjacent pillar. The beams, in a structural sense, act to support the outer membrane ( OM ) . In the center of the pillar near the surface, there is a small irregular depression (PIL DEP) which is bridged by the outer membrane. Shell matrix (SM). ~37,800.
294
GRIGONIS
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SOLOMON
FIG. 9. Longitudinal section through the pillar base (circles) of the nonlaminated segment and the aggregation form the pillars (between the arrows). X 135,000.
region illustrating of these bundles
the fiber bundles turning upward to
those of avian feather kcratin (Filshic and Rogers 1962; Fraser and MacRae 1973). No definitive conclusion can thus be made at this time concerning the structural polymer of the outer shell. Further studies on this topic will be reported in a later article of this series. The pillar and beam-like network of the outer shell closely resemble the plastron or physical gill of a number of insect egg shells as well as the integument of certain aquatic insects (Hinton 1969). Hinton showed that such a system makes it possible for the insect or egg to breathe while under water Fro. 10. Longitudinal section through a pillar. The structural basis of the pillars is fibrillar. The fibers in some areas run in the same direction as the long axis of the column which is indicated by the arrows (Box 1). Other fibers tend to curve away from the long axis (Box 2). Fiber cross sections are also seen (Box 3 ) X 108,000.
Capillaria
hepaticu:
EGG
due to the formation ‘of ,a gas film of constant volume having an extensive air-water interface. We suggest that the C. heputicu egg shell may function similarly during egg development since ‘a number of physical requirements necessary for plastron function ‘are similar to those needed for C. heputicu egg development ( Luttermoser I938 ) . The existence ‘of the shell matrix has not been reported elsewhere. In the specimens prepared by freezing and drying, matrix was ‘observed in N-15% of the specimens and reacted positively for protein. Mariano (1967) described a similar reaction, but as he w’as not ,aware of the existence of matrix, he could not attribute this staining reaction to any specific part of the shell. The plugs that fill the polar openings have a peculiar layer in close association with the continuous outer membrane. These
FIG. 11. Cross section of pillar through its chiefly in the center of the pillar ( Box 1) . At as either short, straight segments (Box 2), may be interpreted as bundles of fibers which
SHELL
FINE
STRUCTLJRE
295
granules were also descrbied by Inatomi ( 1960). They do not show a selectivity for any of the stains used in this study. In fact, the upper half of the plug had a somewhat lesser affinity for the Alcian blue than the remainder of the plug. The nature and function of the granules is uncertain. It is possible that they may serve as a foundation for the deposition of the plug matrix during egg shell formation. Histochemical staining used in this study has indicated the presence of protein in all parts of the egg shell. The polar plugs contain acid mucopolysaccharide and protein. This suggests that the plugs may resemble in function the protein-polysaccharide complex commonly seen in connective tissues of vertebrates ( Catchpole 1973). This suggested function will be elaborated on in the second report of this series.
center. Fibrous material is seen in cross section the outer border of the pillar the fibers are seen or curved segments (Box 3). Both of the latter encircle the periphery of the pillar. X135,000.
GRIGONIS AND SOLOMON
296
FIG. 12. High magnification electron micrograph of a tangential section of the nonlaminated part of the inner shell. Fibrillar material in cross section has a circular profile about 30 A in diameter (single arrow). In well organized regions the fibers have a center to center spacing of about 60 A (between the arrows a). Circled areas illustrate the fiber bundles present in the nonlaminated segment. X300,000.
ACKNOWLEDGMENTS The authors wish to express their sincere appreciation to Professor Isidore Gersh for his interest, advice, and assistance in this study. Dr. K. A. Wright of the University of Toronto kindly provided, from his own collection, several electron micrographs of the C. hepatica egg. These were used for study purposes only. Mrs. Rosalina Espiritu is gratefully acknowledged for her technical assistance. Supported in part by United States Public Health Service Research Grant AI-10898, USPHS Research Training Grant AI-00302, and USPHS Biomedical Science Support Grant RR 0’7083-09 Sub 21.
REFERENCES CATCHWLE, H. R. 1973. Capillary permeability. III. Connective tissue. In “The Inflammatory (B. W. Zweifach, L. Grant, and R. T. Process” McCluskey, eds. ), 2nd ed., Vol. 2. Academic Press, New York.
FILSHIE, B. K., AND ROGERS, G. E. 1962. An electron microscope study of the fine structure of feather keratin. Journal of Cell Biology 13, 1-12. FRASER, R. D. B., AND MACHAE, T. P. 1973. “Conformation in Fibrous Proteins and Related Synthetic Polypeptides.” Academic Press, New York. GERSH, I. 1973. “Submicroscopic Cytochemistry, Vol. I. Proteins and Nucleic Acids.” Academic Press, New York. HACKMAN, R. H. 1974. Chemistry of the insect cuticle. In “The Physiology of Insecta” (M. Rockstein, ed.), 2nd ed., Vol. 6. Academic Press, New York. HINTON, H. E. 1969. Respiratory systems of insect shells. Annual Review of EntomoZogy 14, 343368. INATOMI, S. 1960. Submicroscopic structure of the egg shell of helminth. III. A study on CapiZZuria hepatica. Acta Medica Okayama 14, 261-264. LEV, R., AND SPICER, S. S. 1964. Specific staining of sulphate groups with alcian blue at low pH. Journal of Histochemistry and Cytochemistry 12, 309.
CU$hiU
he@iCU:
EGG
LOCKE, M. 1961. Pore canals and related structures in insect cuticle. Journal of Biophysical and Biochemical Cytology 10, 589-618. LOCKE, M. 1974. The structure and formation of the integument in insects. In “The Physiology of Insecta” (M. Rockstein, ed.), 2nd ed., Vol. 6. Academic Press, New York. LUTTERMOSER, G. W. 1938. Factors influencing the development and viability of the eggs of Capilluria hepatica. American Journal of Hygiene 27, 275-289. MARIANO, M. 1967. Histochemical investigation on egg shell polysaccharides in CapiZlaria hepatica ( Bancroft 1918, Capillaridiae, Trichuroidea) . Acta Histochemica 26, 144-150. MONN~, L., AND H~NIC, G. 1954. On the properties of the egg envelopes of the parasitic nematodes Trichuris and Capillariu. Arkives fiir Zoologie 6, 559-562. RAYBOURNE, R., AND SOLOMON, G. B. 1975. Cap% laria hepatica: Granuloma formation to eggs. III. Anti-immunoglobulin augmentation and
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reagin activity ogy 38,87-95. RAYBOLJRNE, E. J. L. formation responses. 252.
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in mice.
Experimental
Parasitol-
R. B., SOLOMON,G. B., AND SOULSBY, 1974. Capillariu hepatica: Granuloma to eggs. II. Peripheral immunological Experimental Parasitology 36, 244-
RUDALL, K. M. 1963. The chitin/protein of insect cuticles. Advances in Insect 1,257-313.
complexes Physiology
SINGER, M. 1954. The staining of basophilic ponents. Journal of Histochemistry and chemistry 2, 322-333. SOLOMON, G. B., RAYBOURNE, E. J. L. 1974. Serological infected with Capillaria Parasitology 60, 732-734.
comCyto-
R. B., AND SOULSBY, studies on rodents hepatica. Journal of
SOLOMON, G. B., AND SOULSBY, E. J. L. 1973. Granuloma formation to Capillaria hepatica eggs. I. Descriptive definition, Experimental Parasitology 33,458-467.
