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Electron microscopy and histochemistry of oncospheral hook formation by the cestode Catenotaenia pusilla
International Journal for Parasitology, 1973, Vol. 3. pp. 27-33. Pergamon Press. Printed in Great Britaik
ELECTRON MICROSCOPY AND HISTOCHEMISTRY OF ONCOSPHERAL HOOK FORMATION BY THE CESTODE CATENOTAENIA PUSILLA ZDZISLAW Department
of Comparative
SWIDERSKI
Anatomy and Physiology, University of Geneva, 1211 Geneva 4, Switzerland (Received 4 May 1972) Abstract
SW~DERSKI Z. 1973. Electron microscopy and histochemistry of oncospheral hook formation by the cestode Catenotaenia p&la. International Journal for Parasitology 3: 27-33. Ultrastructure and histochemistry of oncospheral hook development in the cestode C. pusilla are described. The six anlagen of embryonal hooks appear in six specialized hook forming cells (oncoblasts) in the advanced phase of the preoncosphere. Electron microscopy shows a close connection of hook primordium with an abundance of free ribosomes, extended Golgi regions, and mitochondrial aggregations, evidently engaged in the hook morphogenesis. The shank completion occurs simultaneously with the progressive degeneration of hook forming cells, which completely disappears in the last phase of hook development. The mature oncospheral hook is a heterogeneous, bipartite structure composed of a dense outer sheath or cortex and a less dense inner core. Histochemistry shows evident changes in the reactivity for -SH, and -S-Sgroups through the consecutive stages of hook development. The early hook anlage shows strongly positive reaction for sulfhydryl (-SH) groups (unconsolidated prekeratin), which remains in the subsequent stages mainly in the zone of keratinization, undergoing continuous displacement toward the base during hook maturation. The sulfhydryl groups of prokeratin through the oxidation process form bisulfite (-SS-) links of mature hook keratin; reactivity for -SH groups completely disappears. The difference in electron density between the outer and inner part of the hook corresponds to a different reactivity for -S--Slinks; the outer sheath shows evidently stronger reaction than the inner core. INDEX KEY WORDS: Catenotaeniapusilla; cellular differentiation; cestode hook; cestodes; circular septate desmosome; connective tissue; Golgi; histochemistry; hook formation; keratin differentiation; keratinization; mitochondria; muscle attachment; oncoblasts; oncospheres; oncospheral hook development; prekeratin; ribo- and polyribosomes; sulfhydryl groups; ultrastructure. INTRODUCTION THE DEVELOPMENT of oncospheral hooks in cestode embryos represents a particularly interesting example of cellular differentiation and specialization towards an intracellular mode of formation of dense structures. As observed on the ultrastructural level, it represents also a good example of specialization of certain cytoplasmic regions and cellular organelles of hook forming cells, namely : mitochondria, Golgi complexes and ribo- or polyribosomes, which, in the light of the present study, seem to be actively engaged in this process. The ultrastructural aspect of oncospheral hook formation is until now virtually unknown. The present investigation is the first attempt of a detailed approach and fine structural analysis of all consecutive stages of hook development. Due to the small size of cells of the tapeworm embryos and their very weak afhnity for dyes, previous light microscopical studies on four cyclophyllidean cestodes by Ogren (1955-1961) provided very scanty and uncertain information concerning the cytological aspect of this process. It refers also to the observations on the development of oncospheral 27
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hooks included in the light microscope studies on cestode embryogenesis (Rybicka, 1966; Swiderski, 1966/67; Mokhtar, 1970). These technical difficulties and the small dimensions of cells make light microscopic interpretation very puzzling and may explain much misleading information in the four above mentioned papers, including a statement on the extracellular origin of embryonal hooks in Hymenolepis nana (Ogren, 1955) revised later on by the same author (Ogren, 1961). MATERIAL
AND
METHODS
Adult specimens of the cestode, Catenotaeniapusilla (Goeze, 1782), were obtained from naturally infected laboratory white mice. Electron microscopy
After removal from their hosts, and a brief rinse in Krebs-Ringer-bicarbonate medium, posterior segments of the strobila were immediately immersed in chilled fixative. The mature and gravid proglottides were cut into fragments smaller than 1 mm and fixed for 15 h either in 2 % osmium tetroxide or Karnovsky’s fixative, containing 2 % paraformaldehyde and 3 % glutaraldehyde in 0.067 M cacodylate buffer at pH 7.4 at 4°C (Karnovsky, 1965). After ethanol dehydration the tissue was infiltrated overnight in an equal mixture of Epon 812 and propylene oxide, embedded in Epon (Luft, 1961) and polymerized for 48 h at 60°C. Sections cut with glass knives on a LKB Ultrotome were collected on formvar-carbon membranes, stained with lead citrate (Reynolds, 1963) or lead hydroxide (Karnovsky, 1961), and examined in an Ehniskop I Siemens, AEI EM 6B, Hitachi HS-7S and Philips EM 300 at 60-80 kV. Histochemistry
The mature and gravid proglottids, cut into small pieces, were fixed in 10 per cent cold neutral formalin, Carnoy’s or Bouin’s fixatives. Standard techniques were employed for embedding in paraihn. For paraffin sections, the following histochemical tests were used: (1) DDD reaction (Barnett & Seligman, 1952; in Pearse, 1961) for detecting the sulfhydryl groups (-SH); (2) The ferric ferricyanide method for -SH groups (Chevremont & Frederic, 1943; in Pearse, 1961). The control sections were treated before staining with saturated phenyl mercuric chloride in butanol for 2-3 days. (3) Performic Acid-S&X (PFAS) method (Pearse, 1961) for -S-Slinks. (4) Performic Acid-Alcian Blue (PFAAB) method for -S-Slinks (Adams & Sloper, 1956; in Pearse, 1961). Control slides for this method were stained with Schiff reagent without oxidation in performic acid. RESULTS
The ultrastructural characteristic of oncospheral hook development in the tapeworm Catenotaenia pusilla shows certain similarities with that observed in the previously studied davaineid cestode Inermicapsfer madagascariensis (Swiderski, 1970). Morphological differentiation of specialized cells, ‘oncoblasts’ (Ogren, 1955), which are determined for the formation of these dense structures, is already visible in the preoncospheral phase of embryogenesis (Fig. la). The first sign of their differentiation consists in the polar displacement of their nuclei (Fig. 2). In the opposite, cytoplasmic pole of the
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HOOKFORMAnON
IN
Catenotaenia pusilla
29
oncoblast the ‘hook forming center’ then appears. Further specialization of this region of the cytoplasm leads mainly to a tight accumulation of numerous mitochondria, free riboand polyribosomes and extended Golgi complexes (Fig. 3). The primordium or anlage of the hook appears morphologically as an electron dense material of more or less spherical form, localized within the large, central vesicle of the Golgi complex (Fig. 3). As the anlage enlarges, it elongates into a hooklet and becomes progressively a heterogeneous, bipartite structure composed of an outer cortex of a very high electron density and an inner core of moderate density. Such a hooklet represents the beginning of blade formation (Fig. lc). Later stages of hook development show a simultaneous increase and elongation of electron dense material. Transformed into a more or less completed hook blade, it remains, however, within the oncoblast (Fig. lc) and is surrounded by its cellular membrane until the beginning of the shank formation. During further elongation of the shank or hook handle, the blade protrudes continuously outside the oncoblast (Fig. Id and Fig. 4). The protruding blade, as seen on Fig. Id., is surrounded by a cytoplasmic collar, forming a circular, septate desmosome, visible in this region in later stages (Figs. 5 and 6). On both sides of the desmosome which is extended into the basal lamina of the oncospheral surface, appear two rings encircling the blade of the hook, both of which are composed of a dense matrix (Fig. If and Fig. 6). The part between the blade and completed shank of the hook increases in width, forming the thickly enlarged guard or collar of the mature hook. The mature oncospheral hook of C. pus&z is a heterogeneous, bipartite structure. Its two components: the outer sheath (or cortex) and the inner core show a marked difference in both the electron density and in the histochemical reactivity for keratin (Figs. 7 and 8; Table 1). TABLE
I.-HLSTOCHEMICAL
CHANGES
OBSERVED
DURING
ONCOSPHERAL
HOOK
Histochemical
for (-SH)
MORPHOGENESIS
tests:
groups
+ + + - + -
IN
C.pusilla
Strongly positive Positive Questionable Negative
for (-S-S-)
links
Consecutive stages of hook development
(i) Early hook anlage (Fig. lb) (ii) Blade completion (Fig. lc) (iii) Shank formation (blade completed protruding outside oncoblast) (Fig. Id) (iv) Shank completed (base keratization: degeneration of oncoblast) (Fig. le) (v) Mature hook (Fig. If) 1. 2. 3. 4.
(BL) (SH) (B) (cx) (cr)
1
2
3
t-f
++
-
-I+
++
+
+
++
+
+
+
-+
-+
++ +
+++
:+ -
++ + -+ +i-
-
4
+ r: +
DDD reaction (Barrnett and Seligman, 1952; in Pearse, 1961). Ferric Ferricyanide method for -SH groups (Chkvremont and FrBderic, 1943; in Pearse, 1961). Performic Acid-Schiff method (PFAS) according to Pearse, 1961. Performic Acid-Aclain Blue method for -S--Sgroups (PFAAB) according to Adams and Sloper, 1956, in Pearse, 1961.
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SVVJDERSKI
I.J.P. VOL. 3. 1973
FIG. la. The general topography of the embryo of C. pudla in the early stage of hook formation (preoncospheral phase of embryogenesis). Note two hook-forming cells (HFC) with characteristic displa~ment of their nuclei into one pole of the cell and spherical, electron dense hook-primordia. FIGS. lb-f. The five consecutive stages of hook development: (b) early oncoblast with hook anlage formation, (c) early oncoblast with intracellular outline of blade, (d) late oncoblast with protruding outside blade and early shank formation, (e) late, degenerating oncoblast with outline of shank completed. (f) mature oncospheral hook completion. 3
BL
c
CE CP
CT cr cx D
Em G HFC HM Hi%4
Key to abbreviations base of the hook blade of the hook collar or guard of the hook cytoplasmic sheath surrounding the shank of the hook capsule (external envelope of the embryo) connective tissue inner core of the hook cortex or outer sheath of the hook circular septate desmosome embryophore Golgi complex hook forming cells or oncoblasts hook muscles hook muscle attachments
of Figs. t-8. HP hook primordi~ or hook anlage HRM hook region membrane IE inner envelope mitochondria ii nucleus nucleus of outer envelope nuclei of inner envelope OE outer envelope pene~ation gland PO r ribo- or polyribosomes RO degenerating oncoblast ‘somatic cells’ representing perinuclear SC areas of muscle system shank or handle of the hook SH somatic muscles of the oncosphere sit4
$
FIG. 2. Part of the embryo of C. pusillu in the preoncospheral phase of development. Two oncoblasts (HFC) showing characteristic displacement of their nuclei (N) into one pole of the cell, considered as the first sign of their differentiation. Stained with lead hydroxide. ( x 16,000).
[I.J.P.
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FIG. 3. Two oncoblasts (HFC) showing further specialization of the cytoplasmic pole of the cells transformed into a ‘hook forming center’ containing a tight accumulation of numerous mitochondria (m), free ribo- and polyribosomes (v) and extended Golgi complexes (C). The oncoblast at the upper right side of the electron micrograph shows an electron dense material (HP) concentrated within the large central vesicle of the Golgi complex (C). Stained with lead hydroxide. (X 24,000).
FIG. 4. Oncoblast at the beginning of shank (SIT) formation showing the completely formed blade, (BL) protruding outside the cell. Stained with lead citrate. ( x 23,000). FIG. 5. Hook in the stage of shank completion (SH). Note the residual part of a degenerating oncoblast (RO), two bands of hook muscles (HM) oriented toward the opposite sides of the hook collar (C), and circular septate desmosome (D), surrounding protruding blade (BL) of the hook. Hook region membrane (HRM) draped over the blade (BL). Stained with lead citrate. (x 23,000).
FIG. 6. Mature oncospheral hook with hook muscles (XM) attached to its collar C) and distal extremity of the shank, forming enlarged base (B). Note the layer of connective tissue (CT) between the base (B) and hook muscle attachment (NM); thin layer of the same tissue also surrounds the shank surface. Also, the shank is surrounded from the base to its collar by a thick sheath of cytoplasm (CL?). Hook muscles (HI@) show direct contact (incurved arrows) with the cytoplasm of so-called ‘somatic cells’ (SC). Stained with lead hydroxide. (x38,00@. FIG. 7. The cross-section of the hook through its shank, showing two layers of different density: the inner core (CY)having a much lower density than the surrounding outer sheath or cortex (cx). Stained with lead citrate. (x 90,000). FIG. 8. Detail of a longitudinal section through the shank. Differentiation of keratin as above. Stained with lead citrate. ( x 90,000).
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31
Histochemical results (Table 1) show that the electron dense material of a hook anlage and that of the early blade is unconsolidated prekeratin rather than keratin. Only methods for -SH groups are positive in this stage, while those for -S-Slinks give a negative result. The test for -S-Slinks becomes positive with the stage of blade completion, when oxydated -SH groups form bisulfite links of the hardening keratin. At this stage, particularly intense reaction was observed in the outer layer of the completed blade, representing a zone of active keratinization. In later stages, the keratinization zone undergoes a continuous displacement towards the future basal part of the hook. The fully hardened keratin of the mature oncospheral hook remains positive for the bisulfite links test, but, the reactivity for sulfhydryl (-SH) groups completely disappears. One of the principal chemical changes which takes place during oncospheral hook morphogenesis is therefore the oxydation of -SH groups in the zone of keratinization of the developing hook for form -S-Sgroups of mature hook keratin.
DISCUSSION
Until now, the formation of embryonal hooks has only been studied by means of light microscopy in the four following species: Oochoristica symmtrica (Ogren, 1957), Dilepis undula (Ogren, 1958), Hymenolepis nana (Ogren, 1955) and Hymeno~epis diminuta (Ogren, 1961; Moczori, 1971). The present electron microscopic study of embryonal hook formation in the tapeworm C. pusiIla principally confirms Ogren’s statement on the intracellular origin of the embryonal hooks of cestode. However, many details in his description (Ogren, 1957, 1958, 1961) cannot receive any support from the results obtained. In particular it refers to the following: (1) the assertion that ‘even mature hooks are closely associated with the oncoblast nucleus’ and ’ are confined within a cell membrane’; (2) the statement that ‘the myoblast attachments are made to the oncoblast surface and not to the hook itself’; and (3) the suggestion that ‘the ergastoplasm of oncoblasts and other embryonic cells in the oncosphere may have an appearance similar to the above (i.e. composed of cytomembranes and minute granular ribosomes), when high resolution electron photo-micrographs become available’ (Ogren, 1961). Mature hooks of the oncosphere of C. pusilla are never associated with the nucleus or with the cell membrane of the oncoblast (Fig. If and Fig. 6), since the former has already disappeared during the shank completion. Hook muscles are attached directly to the hook itself (Fig. 6). ~yoblast attachment to the surface of the oncoblast was never observed either in mature or in immature hooks. Ogren’s suggestion predicting the extensively developed granular endoplasmic reticulum cannot be confirmed by observations on the species examined. Despite the large concentration of free ribosomes in the cytoplasm of the oncoblasts, no profiles of the endoplasmic reticulum were ever detected. This is in agreement with the present knowledge on the ultrastructural characteristic of protein synthesi~ng cells. It has been shown that protein synthesis does not necessarily require an extensive development of cytoplasmic membranes, but, can take place directly on free ribosomes. In fact, rapidly proliferating and growing cells may often have a high concentration of cytoplasmic ribosomes and do not show any evidence of endoplasmic reticulum. As it was summarized by Fawcett (1966), ‘the close
32
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association of ribosomes with the membranes of the reticulum appears to be necessary only when the product of protein synthesis is to be exported from the cell as a secretion’. In spite of a complete lack of endoplasmic reticulum in the oncoblasts of C. pusillu, the Golgi complex, which plays an important role in the concentration of secretory products, is extensively developed, especially in the stage of blade formation. The presence of the Golgi complex in the oncoblasts of H. diminuta and three other species examined seems to be overlooked by Ogren (1955, 1957, 19.58, 1961). If they were observed at all, they were perhaps noted as a ‘bunch of straw and granules’, ‘granules of a looped appearance’ or ‘active coils’ (Ogren, 1961). The characteristic shape of the hooks produced by oncoblasts is a very fascinating and at the same time a rather enigmatic question. According to Ogren (1961) ‘as the blade elongates it approaches the cell membrane and is curved to the correct shape; the protein is soft at this time and would tend to follow the nearly circular shape of the cell’. Such an explanation cannot, however, be accepted for C. pusilla, because in this species the early blade of the embryonal hook (Fig. lc) does not follow the oval shape of the oncoblast, but touches its membrane only at one point. The ultrastructure of the mature hooks of C. pusilla and their musculature generally resembles that of Hymenolepis citelli (Collin, 1968), Taenia taeniaeformis (Nieland, 1968) and Hymenolepis diminuta (Pence, 1970). The most decisive difference between the oncospheral hooks of C. pusilla and three above mentioned species is the number of zones of hook material observed on the cross and longitudinal sections. In C. pusilla as in Dipylidium caninum (Pence, 1967) there are only two zones of different electron density in the mature hook of the oncosphere: dense outer cortex and less dense inner core. Similar organization of hook material was observed also in the embryonal hooks of Inermicapsifer madagascariensis (Swiderski, 1970). In the embryonal hooks of T. taeniaeformis (Nieland, 1968), H. citelli (Collin, 1968) and H. diminuta (Pence, 1970) there are three layers of different density: outer granular zone: middle fibrous layer, and central core. Central cores of all examined species are analogous, however, in C. pusilla it does not appear to be laminated as in H. citelli (Collin, 1968) or crystalloid as in T. taeniueformis (Nieland, 1968). The outer granular zone was never observed in the oncospheral hooks of C. pusillu, in which the outer cortex corresponds generally with the middle fibrous layer described in other species. The keratinaceous character of embryonal hooks (particularly their outer cortex or middle fibrous layer) have been reported in all species examined so far. According to Pence (1970) only the middle fibrous layer of the oncospheral hooks of H. diminuta elicits a positive reaction for keratin; histochemical tests on the inner core in this species are positive for phospholipids, which are not extractable with pyridine and do not react with Sudan black B. The oncospheral hooks of C. pusilla show a positive reaction for keratin both in their core and cortex; however, there is a marked difference in reactivity between the moderately stained core and intensely stained outer sheath or cortex. It may indicate that the outer sheath is probably richer in disulfide bonds than the inner core (Dvorak, 1969). The chemical changes observed in the consecutive stages of oncospheral hook morphogenesis in C. pusilla follow those observed usually during the keratinization process (Mercer, 1961). The early hook anlage shows a strongly positive reaction for -SH groups (unconsolidated prekeratin), which, through the oxydation process form -S-Slinks of mature hook keratin (Table 1). Hook maturation, which begins with the stage of shank completion,
X.J.P. VOL. 3. 1973
HOOK FORMATiON IN Catenotaenia pusiib
33
is accompanied therefore by an increase in the reactivity for -S-Slinks (cystine) and a decrease in the reactivity for sulfhydryl groups (-SH), which, in the mature hook, completely disappears. SimultaneousIy with hook development, the zone of active kerat~nization undergoes continuous displacement from the hook blade towards its base. As observed generally in the keratinization process (Mercer, 1961), the zone of keratinization may be subdivided into three regions: unconsolidated prekeratin, progressive hardening zone, and fully hardened zone, which correspond respectively to the three following phases: anagen, catagen and telogen, distinguished in the keratinization process. ft seems quite probable that the above mentioned sub-zones and phases may occur also during oncospheral hook morphogenesis. Further studies based on birefringence measurements, X-ray diffraction and electron microscope autoradiography (administration of 35S-labelled radiocystine) would seem to be particularly promising in finding an answer for this question. REFERENCES COLLIN W. K. 1968. Electron microscope studies of the muscle and hook systems of hatched oncospheres of Hymenoiepis citelli McLeod. 1933 (Cestoda: Cvcloohvllidea). Journal ofParasitoloav 54: 74-88. DVORAKJ. A. 1969. Hymenolepis micr&toma: Interfer&e microscopy of embryonic-lateral hooks--II. Structure and reaction to 2-mercaptoethanol. Experimental Parasitology 26: 101-l 10. FAWCEIT D. W. 1966. An Atlas of Fine Structure. The Cell Its Organelles and Inclusions. W. B. Saunders. Philadelphia 1966. KARNOVSKY M. J. 1961. Simple methods for ‘staining with lead’ at high pH in electron microscopy. Journal of Biophysicai and Biochemical Cvtoloav 11: 729-732. KAR-NOVSKY M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal of Cell Biology 27: 137 A. LUFT J. H. 1961. Improvements in epoxy resin embedding methods. Journal of BiophysicuZ and Biochemical Cytology 9: W-414. MERCERE. H. 1961. Keratin and Keratinization. Pergamon Press, Oxford, 1961. Moczofi T. 1971. Hist~hemi~l study of the development of embryonic hooks in Hymenolepis diminuta (Cestoda). Acta Parasito~ogica Polonica 19: 269-274. MOKHTARF. 1970. Recherches sur l’embryologie des Cestodes Tetraphyllides. Thesis. University of Montpellier. NIELAND M. L. 1968. Electron microscope observations on the egg of Taenia taeniueformis. Journal of Parasitologv 54: 957-969. OGREN R. Eyi955. Development and morphology of glandular regions in oncospheres of Hymenolepis nana. Proceedktgs of the Pennsylvania Academy of Sciences 29: 258-264. OGREN R. E. 19571 Morpholo~ and development bf oncospheres of the cestode Oochoristicu symmetrica Baylis. Journal of Parasitology 43: 505520. OGREN R. E. 1958. The hexacanth embryo of a dilepidid tapeworm-I. The development of hooks and contractile parenchyma. Journal of Parasitology 44: 477-483. OGRENR. E. 1961. Observations on hook development in the oncoblasts of hexacanth embryo from Hymenolepis diminuta, a tapeworm of mammals (Cestoda: Cyclophyllidea). Proceedings of the Pennsylvania Academy of Sciences 35: 23-31. PEARCEA. Cl. E. 1961. Hisfochemistry ~eoreticaZ and Applied. Little, Brown, Boston. PENCED. B. 1967, The fine structure and hist~hemist~ of infective eggs of ~~y~idiurn eaniuum. Journal of Parasitology 53: 1041-1054. PENCE D. B. 1970. Electron microscope and histochemical studies on the eggs of HymenoIepis diminuta. Journal of Parasitology 56: 84-97. REYNOLDSE. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 11: 208-212. RYBKCKAR. 1966. Embryogenesis in Cestodes. Advances in Parasitology 4: 107-186. SW~DER~KIZ. 1966167. Emb~onic development of the cestode ~repanidotaenia ~~ceo~ata (Bloch, 1782). Acta Parasito~ogica Polonica 14: 409418. SWIDERSKIZ. 1970. Electron microscopy of embryonal hook formation by the cestode Znermicapsijr madagascariensis (Cyclophyllidea, Davaineidae). Proceedings of the Second International Congress of Parasitology, pp. 337-338, Washington, D.C. 1970. SWWERSKIZ. 1972. La structure fine de l’oncosphere du Cestode CatenotaeniapusiZIa(Goeze, 1782) (Cyclophyllidea, Catenotaeniidae). La Cellule 69: 205-237.
ELECTRON MICROSCOPY AND HISTOCHEMISTRY OF ONCOSPHERAL HOOK FORMATION BY THE CESTODE CATENOTAENIA PUSILLA ZDZISLAW Department
of Comparative
SWIDERSKI
Anatomy and Physiology, University of Geneva, 1211 Geneva 4, Switzerland (Received 4 May 1972) Abstract
SW~DERSKI Z. 1973. Electron microscopy and histochemistry of oncospheral hook formation by the cestode Catenotaenia p&la. International Journal for Parasitology 3: 27-33. Ultrastructure and histochemistry of oncospheral hook development in the cestode C. pusilla are described. The six anlagen of embryonal hooks appear in six specialized hook forming cells (oncoblasts) in the advanced phase of the preoncosphere. Electron microscopy shows a close connection of hook primordium with an abundance of free ribosomes, extended Golgi regions, and mitochondrial aggregations, evidently engaged in the hook morphogenesis. The shank completion occurs simultaneously with the progressive degeneration of hook forming cells, which completely disappears in the last phase of hook development. The mature oncospheral hook is a heterogeneous, bipartite structure composed of a dense outer sheath or cortex and a less dense inner core. Histochemistry shows evident changes in the reactivity for -SH, and -S-Sgroups through the consecutive stages of hook development. The early hook anlage shows strongly positive reaction for sulfhydryl (-SH) groups (unconsolidated prekeratin), which remains in the subsequent stages mainly in the zone of keratinization, undergoing continuous displacement toward the base during hook maturation. The sulfhydryl groups of prokeratin through the oxidation process form bisulfite (-SS-) links of mature hook keratin; reactivity for -SH groups completely disappears. The difference in electron density between the outer and inner part of the hook corresponds to a different reactivity for -S--Slinks; the outer sheath shows evidently stronger reaction than the inner core. INDEX KEY WORDS: Catenotaeniapusilla; cellular differentiation; cestode hook; cestodes; circular septate desmosome; connective tissue; Golgi; histochemistry; hook formation; keratin differentiation; keratinization; mitochondria; muscle attachment; oncoblasts; oncospheres; oncospheral hook development; prekeratin; ribo- and polyribosomes; sulfhydryl groups; ultrastructure. INTRODUCTION THE DEVELOPMENT of oncospheral hooks in cestode embryos represents a particularly interesting example of cellular differentiation and specialization towards an intracellular mode of formation of dense structures. As observed on the ultrastructural level, it represents also a good example of specialization of certain cytoplasmic regions and cellular organelles of hook forming cells, namely : mitochondria, Golgi complexes and ribo- or polyribosomes, which, in the light of the present study, seem to be actively engaged in this process. The ultrastructural aspect of oncospheral hook formation is until now virtually unknown. The present investigation is the first attempt of a detailed approach and fine structural analysis of all consecutive stages of hook development. Due to the small size of cells of the tapeworm embryos and their very weak afhnity for dyes, previous light microscopical studies on four cyclophyllidean cestodes by Ogren (1955-1961) provided very scanty and uncertain information concerning the cytological aspect of this process. It refers also to the observations on the development of oncospheral 27
28
ZDZISLAWSWIDERSKI
I.J.P. VOL.
3. 1973
hooks included in the light microscope studies on cestode embryogenesis (Rybicka, 1966; Swiderski, 1966/67; Mokhtar, 1970). These technical difficulties and the small dimensions of cells make light microscopic interpretation very puzzling and may explain much misleading information in the four above mentioned papers, including a statement on the extracellular origin of embryonal hooks in Hymenolepis nana (Ogren, 1955) revised later on by the same author (Ogren, 1961). MATERIAL
AND
METHODS
Adult specimens of the cestode, Catenotaeniapusilla (Goeze, 1782), were obtained from naturally infected laboratory white mice. Electron microscopy
After removal from their hosts, and a brief rinse in Krebs-Ringer-bicarbonate medium, posterior segments of the strobila were immediately immersed in chilled fixative. The mature and gravid proglottides were cut into fragments smaller than 1 mm and fixed for 15 h either in 2 % osmium tetroxide or Karnovsky’s fixative, containing 2 % paraformaldehyde and 3 % glutaraldehyde in 0.067 M cacodylate buffer at pH 7.4 at 4°C (Karnovsky, 1965). After ethanol dehydration the tissue was infiltrated overnight in an equal mixture of Epon 812 and propylene oxide, embedded in Epon (Luft, 1961) and polymerized for 48 h at 60°C. Sections cut with glass knives on a LKB Ultrotome were collected on formvar-carbon membranes, stained with lead citrate (Reynolds, 1963) or lead hydroxide (Karnovsky, 1961), and examined in an Ehniskop I Siemens, AEI EM 6B, Hitachi HS-7S and Philips EM 300 at 60-80 kV. Histochemistry
The mature and gravid proglottids, cut into small pieces, were fixed in 10 per cent cold neutral formalin, Carnoy’s or Bouin’s fixatives. Standard techniques were employed for embedding in paraihn. For paraffin sections, the following histochemical tests were used: (1) DDD reaction (Barnett & Seligman, 1952; in Pearse, 1961) for detecting the sulfhydryl groups (-SH); (2) The ferric ferricyanide method for -SH groups (Chevremont & Frederic, 1943; in Pearse, 1961). The control sections were treated before staining with saturated phenyl mercuric chloride in butanol for 2-3 days. (3) Performic Acid-S&X (PFAS) method (Pearse, 1961) for -S-Slinks. (4) Performic Acid-Alcian Blue (PFAAB) method for -S-Slinks (Adams & Sloper, 1956; in Pearse, 1961). Control slides for this method were stained with Schiff reagent without oxidation in performic acid. RESULTS
The ultrastructural characteristic of oncospheral hook development in the tapeworm Catenotaenia pusilla shows certain similarities with that observed in the previously studied davaineid cestode Inermicapsfer madagascariensis (Swiderski, 1970). Morphological differentiation of specialized cells, ‘oncoblasts’ (Ogren, 1955), which are determined for the formation of these dense structures, is already visible in the preoncospheral phase of embryogenesis (Fig. la). The first sign of their differentiation consists in the polar displacement of their nuclei (Fig. 2). In the opposite, cytoplasmic pole of the
I.J.P. VOL.
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HOOKFORMAnON
IN
Catenotaenia pusilla
29
oncoblast the ‘hook forming center’ then appears. Further specialization of this region of the cytoplasm leads mainly to a tight accumulation of numerous mitochondria, free riboand polyribosomes and extended Golgi complexes (Fig. 3). The primordium or anlage of the hook appears morphologically as an electron dense material of more or less spherical form, localized within the large, central vesicle of the Golgi complex (Fig. 3). As the anlage enlarges, it elongates into a hooklet and becomes progressively a heterogeneous, bipartite structure composed of an outer cortex of a very high electron density and an inner core of moderate density. Such a hooklet represents the beginning of blade formation (Fig. lc). Later stages of hook development show a simultaneous increase and elongation of electron dense material. Transformed into a more or less completed hook blade, it remains, however, within the oncoblast (Fig. lc) and is surrounded by its cellular membrane until the beginning of the shank formation. During further elongation of the shank or hook handle, the blade protrudes continuously outside the oncoblast (Fig. Id and Fig. 4). The protruding blade, as seen on Fig. Id., is surrounded by a cytoplasmic collar, forming a circular, septate desmosome, visible in this region in later stages (Figs. 5 and 6). On both sides of the desmosome which is extended into the basal lamina of the oncospheral surface, appear two rings encircling the blade of the hook, both of which are composed of a dense matrix (Fig. If and Fig. 6). The part between the blade and completed shank of the hook increases in width, forming the thickly enlarged guard or collar of the mature hook. The mature oncospheral hook of C. pus&z is a heterogeneous, bipartite structure. Its two components: the outer sheath (or cortex) and the inner core show a marked difference in both the electron density and in the histochemical reactivity for keratin (Figs. 7 and 8; Table 1). TABLE
I.-HLSTOCHEMICAL
CHANGES
OBSERVED
DURING
ONCOSPHERAL
HOOK
Histochemical
for (-SH)
MORPHOGENESIS
tests:
groups
+ + + - + -
IN
C.pusilla
Strongly positive Positive Questionable Negative
for (-S-S-)
links
Consecutive stages of hook development
(i) Early hook anlage (Fig. lb) (ii) Blade completion (Fig. lc) (iii) Shank formation (blade completed protruding outside oncoblast) (Fig. Id) (iv) Shank completed (base keratization: degeneration of oncoblast) (Fig. le) (v) Mature hook (Fig. If) 1. 2. 3. 4.
(BL) (SH) (B) (cx) (cr)
1
2
3
t-f
++
-
-I+
++
+
+
++
+
+
+
-+
-+
++ +
+++
:+ -
++ + -+ +i-
-
4
+ r: +
DDD reaction (Barrnett and Seligman, 1952; in Pearse, 1961). Ferric Ferricyanide method for -SH groups (Chkvremont and FrBderic, 1943; in Pearse, 1961). Performic Acid-Schiff method (PFAS) according to Pearse, 1961. Performic Acid-Aclain Blue method for -S--Sgroups (PFAAB) according to Adams and Sloper, 1956, in Pearse, 1961.
30
ZDZISLAW
SVVJDERSKI
I.J.P. VOL. 3. 1973
FIG. la. The general topography of the embryo of C. pudla in the early stage of hook formation (preoncospheral phase of embryogenesis). Note two hook-forming cells (HFC) with characteristic displa~ment of their nuclei into one pole of the cell and spherical, electron dense hook-primordia. FIGS. lb-f. The five consecutive stages of hook development: (b) early oncoblast with hook anlage formation, (c) early oncoblast with intracellular outline of blade, (d) late oncoblast with protruding outside blade and early shank formation, (e) late, degenerating oncoblast with outline of shank completed. (f) mature oncospheral hook completion. 3
BL
c
CE CP
CT cr cx D
Em G HFC HM Hi%4
Key to abbreviations base of the hook blade of the hook collar or guard of the hook cytoplasmic sheath surrounding the shank of the hook capsule (external envelope of the embryo) connective tissue inner core of the hook cortex or outer sheath of the hook circular septate desmosome embryophore Golgi complex hook forming cells or oncoblasts hook muscles hook muscle attachments
of Figs. t-8. HP hook primordi~ or hook anlage HRM hook region membrane IE inner envelope mitochondria ii nucleus nucleus of outer envelope nuclei of inner envelope OE outer envelope pene~ation gland PO r ribo- or polyribosomes RO degenerating oncoblast ‘somatic cells’ representing perinuclear SC areas of muscle system shank or handle of the hook SH somatic muscles of the oncosphere sit4
$
FIG. 2. Part of the embryo of C. pusillu in the preoncospheral phase of development. Two oncoblasts (HFC) showing characteristic displacement of their nuclei (N) into one pole of the cell, considered as the first sign of their differentiation. Stained with lead hydroxide. ( x 16,000).
[I.J.P.
5.p.301
FIG. 3. Two oncoblasts (HFC) showing further specialization of the cytoplasmic pole of the cells transformed into a ‘hook forming center’ containing a tight accumulation of numerous mitochondria (m), free ribo- and polyribosomes (v) and extended Golgi complexes (C). The oncoblast at the upper right side of the electron micrograph shows an electron dense material (HP) concentrated within the large central vesicle of the Golgi complex (C). Stained with lead hydroxide. (X 24,000).
FIG. 4. Oncoblast at the beginning of shank (SIT) formation showing the completely formed blade, (BL) protruding outside the cell. Stained with lead citrate. ( x 23,000). FIG. 5. Hook in the stage of shank completion (SH). Note the residual part of a degenerating oncoblast (RO), two bands of hook muscles (HM) oriented toward the opposite sides of the hook collar (C), and circular septate desmosome (D), surrounding protruding blade (BL) of the hook. Hook region membrane (HRM) draped over the blade (BL). Stained with lead citrate. (x 23,000).
FIG. 6. Mature oncospheral hook with hook muscles (XM) attached to its collar C) and distal extremity of the shank, forming enlarged base (B). Note the layer of connective tissue (CT) between the base (B) and hook muscle attachment (NM); thin layer of the same tissue also surrounds the shank surface. Also, the shank is surrounded from the base to its collar by a thick sheath of cytoplasm (CL?). Hook muscles (HI@) show direct contact (incurved arrows) with the cytoplasm of so-called ‘somatic cells’ (SC). Stained with lead hydroxide. (x38,00@. FIG. 7. The cross-section of the hook through its shank, showing two layers of different density: the inner core (CY)having a much lower density than the surrounding outer sheath or cortex (cx). Stained with lead citrate. (x 90,000). FIG. 8. Detail of a longitudinal section through the shank. Differentiation of keratin as above. Stained with lead citrate. ( x 90,000).
I.J.P.
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HOOK FORMATION
IN
Catenotaenia prrsilla
31
Histochemical results (Table 1) show that the electron dense material of a hook anlage and that of the early blade is unconsolidated prekeratin rather than keratin. Only methods for -SH groups are positive in this stage, while those for -S-Slinks give a negative result. The test for -S-Slinks becomes positive with the stage of blade completion, when oxydated -SH groups form bisulfite links of the hardening keratin. At this stage, particularly intense reaction was observed in the outer layer of the completed blade, representing a zone of active keratinization. In later stages, the keratinization zone undergoes a continuous displacement towards the future basal part of the hook. The fully hardened keratin of the mature oncospheral hook remains positive for the bisulfite links test, but, the reactivity for sulfhydryl (-SH) groups completely disappears. One of the principal chemical changes which takes place during oncospheral hook morphogenesis is therefore the oxydation of -SH groups in the zone of keratinization of the developing hook for form -S-Sgroups of mature hook keratin.
DISCUSSION
Until now, the formation of embryonal hooks has only been studied by means of light microscopy in the four following species: Oochoristica symmtrica (Ogren, 1957), Dilepis undula (Ogren, 1958), Hymenolepis nana (Ogren, 1955) and Hymeno~epis diminuta (Ogren, 1961; Moczori, 1971). The present electron microscopic study of embryonal hook formation in the tapeworm C. pusiIla principally confirms Ogren’s statement on the intracellular origin of the embryonal hooks of cestode. However, many details in his description (Ogren, 1957, 1958, 1961) cannot receive any support from the results obtained. In particular it refers to the following: (1) the assertion that ‘even mature hooks are closely associated with the oncoblast nucleus’ and ’ are confined within a cell membrane’; (2) the statement that ‘the myoblast attachments are made to the oncoblast surface and not to the hook itself’; and (3) the suggestion that ‘the ergastoplasm of oncoblasts and other embryonic cells in the oncosphere may have an appearance similar to the above (i.e. composed of cytomembranes and minute granular ribosomes), when high resolution electron photo-micrographs become available’ (Ogren, 1961). Mature hooks of the oncosphere of C. pusilla are never associated with the nucleus or with the cell membrane of the oncoblast (Fig. If and Fig. 6), since the former has already disappeared during the shank completion. Hook muscles are attached directly to the hook itself (Fig. 6). ~yoblast attachment to the surface of the oncoblast was never observed either in mature or in immature hooks. Ogren’s suggestion predicting the extensively developed granular endoplasmic reticulum cannot be confirmed by observations on the species examined. Despite the large concentration of free ribosomes in the cytoplasm of the oncoblasts, no profiles of the endoplasmic reticulum were ever detected. This is in agreement with the present knowledge on the ultrastructural characteristic of protein synthesi~ng cells. It has been shown that protein synthesis does not necessarily require an extensive development of cytoplasmic membranes, but, can take place directly on free ribosomes. In fact, rapidly proliferating and growing cells may often have a high concentration of cytoplasmic ribosomes and do not show any evidence of endoplasmic reticulum. As it was summarized by Fawcett (1966), ‘the close
32
ZDZISLAW SWIDERSKJ
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association of ribosomes with the membranes of the reticulum appears to be necessary only when the product of protein synthesis is to be exported from the cell as a secretion’. In spite of a complete lack of endoplasmic reticulum in the oncoblasts of C. pusillu, the Golgi complex, which plays an important role in the concentration of secretory products, is extensively developed, especially in the stage of blade formation. The presence of the Golgi complex in the oncoblasts of H. diminuta and three other species examined seems to be overlooked by Ogren (1955, 1957, 19.58, 1961). If they were observed at all, they were perhaps noted as a ‘bunch of straw and granules’, ‘granules of a looped appearance’ or ‘active coils’ (Ogren, 1961). The characteristic shape of the hooks produced by oncoblasts is a very fascinating and at the same time a rather enigmatic question. According to Ogren (1961) ‘as the blade elongates it approaches the cell membrane and is curved to the correct shape; the protein is soft at this time and would tend to follow the nearly circular shape of the cell’. Such an explanation cannot, however, be accepted for C. pusilla, because in this species the early blade of the embryonal hook (Fig. lc) does not follow the oval shape of the oncoblast, but touches its membrane only at one point. The ultrastructure of the mature hooks of C. pusilla and their musculature generally resembles that of Hymenolepis citelli (Collin, 1968), Taenia taeniaeformis (Nieland, 1968) and Hymenolepis diminuta (Pence, 1970). The most decisive difference between the oncospheral hooks of C. pusilla and three above mentioned species is the number of zones of hook material observed on the cross and longitudinal sections. In C. pusilla as in Dipylidium caninum (Pence, 1967) there are only two zones of different electron density in the mature hook of the oncosphere: dense outer cortex and less dense inner core. Similar organization of hook material was observed also in the embryonal hooks of Inermicapsifer madagascariensis (Swiderski, 1970). In the embryonal hooks of T. taeniaeformis (Nieland, 1968), H. citelli (Collin, 1968) and H. diminuta (Pence, 1970) there are three layers of different density: outer granular zone: middle fibrous layer, and central core. Central cores of all examined species are analogous, however, in C. pusilla it does not appear to be laminated as in H. citelli (Collin, 1968) or crystalloid as in T. taeniueformis (Nieland, 1968). The outer granular zone was never observed in the oncospheral hooks of C. pusillu, in which the outer cortex corresponds generally with the middle fibrous layer described in other species. The keratinaceous character of embryonal hooks (particularly their outer cortex or middle fibrous layer) have been reported in all species examined so far. According to Pence (1970) only the middle fibrous layer of the oncospheral hooks of H. diminuta elicits a positive reaction for keratin; histochemical tests on the inner core in this species are positive for phospholipids, which are not extractable with pyridine and do not react with Sudan black B. The oncospheral hooks of C. pusilla show a positive reaction for keratin both in their core and cortex; however, there is a marked difference in reactivity between the moderately stained core and intensely stained outer sheath or cortex. It may indicate that the outer sheath is probably richer in disulfide bonds than the inner core (Dvorak, 1969). The chemical changes observed in the consecutive stages of oncospheral hook morphogenesis in C. pusilla follow those observed usually during the keratinization process (Mercer, 1961). The early hook anlage shows a strongly positive reaction for -SH groups (unconsolidated prekeratin), which, through the oxydation process form -S-Slinks of mature hook keratin (Table 1). Hook maturation, which begins with the stage of shank completion,
X.J.P. VOL. 3. 1973
HOOK FORMATiON IN Catenotaenia pusiib
33
is accompanied therefore by an increase in the reactivity for -S-Slinks (cystine) and a decrease in the reactivity for sulfhydryl groups (-SH), which, in the mature hook, completely disappears. SimultaneousIy with hook development, the zone of active kerat~nization undergoes continuous displacement from the hook blade towards its base. As observed generally in the keratinization process (Mercer, 1961), the zone of keratinization may be subdivided into three regions: unconsolidated prekeratin, progressive hardening zone, and fully hardened zone, which correspond respectively to the three following phases: anagen, catagen and telogen, distinguished in the keratinization process. ft seems quite probable that the above mentioned sub-zones and phases may occur also during oncospheral hook morphogenesis. Further studies based on birefringence measurements, X-ray diffraction and electron microscope autoradiography (administration of 35S-labelled radiocystine) would seem to be particularly promising in finding an answer for this question. REFERENCES COLLIN W. K. 1968. Electron microscope studies of the muscle and hook systems of hatched oncospheres of Hymenoiepis citelli McLeod. 1933 (Cestoda: Cvcloohvllidea). Journal ofParasitoloav 54: 74-88. DVORAKJ. A. 1969. Hymenolepis micr&toma: Interfer&e microscopy of embryonic-lateral hooks--II. Structure and reaction to 2-mercaptoethanol. Experimental Parasitology 26: 101-l 10. FAWCEIT D. W. 1966. An Atlas of Fine Structure. The Cell Its Organelles and Inclusions. W. B. Saunders. Philadelphia 1966. KARNOVSKY M. J. 1961. Simple methods for ‘staining with lead’ at high pH in electron microscopy. Journal of Biophysicai and Biochemical Cvtoloav 11: 729-732. KAR-NOVSKY M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. Journal of Cell Biology 27: 137 A. LUFT J. H. 1961. Improvements in epoxy resin embedding methods. Journal of BiophysicuZ and Biochemical Cytology 9: W-414. MERCERE. H. 1961. Keratin and Keratinization. Pergamon Press, Oxford, 1961. Moczofi T. 1971. Hist~hemi~l study of the development of embryonic hooks in Hymenolepis diminuta (Cestoda). Acta Parasito~ogica Polonica 19: 269-274. MOKHTARF. 1970. Recherches sur l’embryologie des Cestodes Tetraphyllides. Thesis. University of Montpellier. NIELAND M. L. 1968. Electron microscope observations on the egg of Taenia taeniueformis. Journal of Parasitologv 54: 957-969. OGREN R. Eyi955. Development and morphology of glandular regions in oncospheres of Hymenolepis nana. Proceedktgs of the Pennsylvania Academy of Sciences 29: 258-264. OGREN R. E. 19571 Morpholo~ and development bf oncospheres of the cestode Oochoristicu symmetrica Baylis. Journal of Parasitology 43: 505520. OGREN R. E. 1958. The hexacanth embryo of a dilepidid tapeworm-I. The development of hooks and contractile parenchyma. Journal of Parasitology 44: 477-483. OGRENR. E. 1961. Observations on hook development in the oncoblasts of hexacanth embryo from Hymenolepis diminuta, a tapeworm of mammals (Cestoda: Cyclophyllidea). Proceedings of the Pennsylvania Academy of Sciences 35: 23-31. PEARCEA. Cl. E. 1961. Hisfochemistry ~eoreticaZ and Applied. Little, Brown, Boston. PENCED. B. 1967, The fine structure and hist~hemist~ of infective eggs of ~~y~idiurn eaniuum. Journal of Parasitology 53: 1041-1054. PENCE D. B. 1970. Electron microscope and histochemical studies on the eggs of HymenoIepis diminuta. Journal of Parasitology 56: 84-97. REYNOLDSE. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 11: 208-212. RYBKCKAR. 1966. Embryogenesis in Cestodes. Advances in Parasitology 4: 107-186. SW~DER~KIZ. 1966167. Emb~onic development of the cestode ~repanidotaenia ~~ceo~ata (Bloch, 1782). Acta Parasito~ogica Polonica 14: 409418. SWIDERSKIZ. 1970. Electron microscopy of embryonal hook formation by the cestode Znermicapsijr madagascariensis (Cyclophyllidea, Davaineidae). Proceedings of the Second International Congress of Parasitology, pp. 337-338, Washington, D.C. 1970. SWWERSKIZ. 1972. La structure fine de l’oncosphere du Cestode CatenotaeniapusiZIa(Goeze, 1782) (Cyclophyllidea, Catenotaeniidae). La Cellule 69: 205-237.