Embryogenesis in Hymenolepis diminuta IV. Distribution of succinic dehydrogenase, reduced form of nicotinamide-adenine dinucleotide oxido-reductase, and cytochrome oxidase

Embryogenesis in Hymenolepis diminuta IV. Distribution of succinic dehydrogenase, reduced form of nicotinamide-adenine dinucleotide oxido-reductase, and cytochrome oxidase

EXPERIMENTAL PARASITOLOGY 20, 255-262 Embryogenesis IV. Distribution (1967) in Hymenolepis of Succinic of Nicotinamide-Adenine and Dehydrogen...

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EXPERIMENTAL

PARASITOLOGY

20, 255-262

Embryogenesis IV. Distribution

(1967)

in

Hymenolepis

of Succinic

of Nicotinamide-Adenine and

Dehydrogenase, Dinucleotide

Cytochrome Krystyna

Department

of Biology,

Submitted

diminuta

Reduced

Form

Oxido-Reductase,

Oxidasel Rybicka”

Vanderbilt

University,

for publication,

Nashville,

12 January

Tennessee

37203

1967

RYBICKA, KRYSTYNA. 1967. Embryogenesis in Hymenolepis diminuta IV. Distribution of succinic dehydrogenase, reduced form of nicotinamide-adenine dinucleotide oxido-reductase, and cytochrome oxidase. Experimental Parasitology 20, 255262. High activity of succinic dehydrogenase and NADH-oxido-reductase occurred in gonads and embryos of Hymenolepis diminuta. In embryos the highest enzymatic activity occurred in the inner envelope during the preoncosphere phase. The active layer seems to be connected with the formation of the shell and the embryophore. Toward the end of development, enzymatic activity in the envelope decreased. Succinic dehydrogenase and NADH-oxido-reductase activity demonstrable inside the embryos showed lower ac:ivity than was observed in the envelope. Cytochrome oxidase was not demonstrated in the course of embryogenesis, but some trace; of this enzyme appeared in the mature oncosphere with varied reaction. The study raises the problem of terminal oxidation processes in cestode embryos.

Although the presence of tricarboxylic acid cycle in cestodes or its possible modification is not fully clear as yet (Smyth, 1962; Read and Simmons, 1963; von Brand, 1966), some biochemical and histochemical investigations have indicated the presence of mitochondrial enzymes oxidizing the intermediates of this cycle. The presence of succinic dehydrogenase (SDH) in some species of the genus Hymenolepis has been shown by quantitative data of Read (1952), Goldberg and Nolf (1954)) and Bogitsh and Nunnally (1966). Read (1952) has reported the presence of cyto’ This investigation received financial support from the World Health Organization. ’ Present address: Department of Parasitology, Polish Academy of Sciences, Pasteura 3, Warsaw, Poland.

chrome oxidase in Hymenolepis diminuta. Histochemical evidence of SDH in the genus Hymenolepis is mainly concerned with the cuticule and parenchyma (Hedrick, 1956; Rothman and Lee, 1963; Waitz and Schardein, 1964; Bogitsh and Nunnally, 1966). The localization of NADHoxido-reductase in the cuticle of four cestodes (including three Hymenolepis spp.) has been found by Rothman and Lee (1963). Cytochrome oxidase has been reported in the cuticle of H. diminuta and H. nana (Waitz and Schardein, 1964). There are, however, few reports dealing with the distribution of the enzymes in the reproductive system and developing embryos of H. diminuta. Hedrick (1956) has noted high activity of SDH in testes, ovary, and occasional activity in the eggs (local-

25.5

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RYBICKA

ized beneath the shell) of H. diminuta. Similar observations have been published by Bogitsh and Nunnally (1966) in H. microstoma. Waitz and Schardein (1964) mentioned the presence of cytochrome oxidase in the oncospheres of H. diminuta. The aim of the present study is to analyze the distribution of SDH, NADHand cytochrome oxidase oxido-reductase, during embryogenesis in H. diminuta. MATERIALS

AND METHODS

Adult tapeworms were taken from laboratory rats approximately 3 weeks post infection. They were divided into fragments about 2 cm long and frozen with liquid nitrogen. After freezing, the fragments were put into optimal cutting temperature (OCT) compound and frozen at -30°C. The arrangement of subsequent fragments of worms in blocks permitted one to define the sequence of developmental phases in the embryos. Sections 4 p, 6 p, and 8 p thick were cut in an Ames Lab-Tek cryostat. The nitro blue tetrazolium (NBT) technique as outlined by Nachlas et al. (1957) was used for the study of SDH. Control sections were handled in one of two ways. One group was placed in a medium from which the substrate, sodium succinate, was omitted. Another group of control sections was placed in a complete medium to which sodium malonate was added in two concentrations: (1) a molar concentration of malonate/succinate, l/l, and (2) a molar concentration of malonate/succinate, 2/ 1. incubation times in media ranged from 5 to 10 minutes at 37°C. NADH-oxido-reductase was studied by the use of tetranitroblue tetrazolium according to the method of Novikoff (1963). Sections were incubated 30 minutes at 37°C. In control sections, NADH was omitted from the medium. Two methods were used for the study of

cytochrome oxidase. One method utilized diphenylamine with the addition of 8(Buramino-1,2,3,4-tetrahydroxyquinoline stone, 1962). Incubation time ranged from 1 to 2 hours at room temperature. Control sections were incubated in the medium containing 0.001 M KCN. The second method was the modified osmication method (Seligman, 1965), using N-benzylphenylene diamine, 0.1 M phosphate buffer at pH 7.4, catalase, I-naphtol, and cytochrome c, incubated at room temperature for 10 minutes. Osmication, suggested in this method, was omitted. In all cases, cardiac muscle of the rat was used as positive control. IPEsULTs Succinic

Dehydrogenase

The reproductive glands (testes and ovary) showed very high enzymatic activity. There were some slight differences in the intensity of stain observed in particular phases of spermatogenesis (Fig. 1) . Spermatogonia localized at the periphery of testis showed very heavy accumulations of formazan. Sometimes the heavy stain was observed in groups of cells located in the central part; in the other instance, the stain was more dispersed. Frozen sections did not permit definition of subsequent phases of spermatogenesis. Sperm did not show any succinic dehydrogenase activity. The same lack of formazan formation was observed in sperm ducts filled with sperm. A high accumulation of succinic dehydrogenase was found in the ovary, and somewhat less accumulation occurred in the vitelline gland (Fig. 2). Oocytes entering the uterus showed a high activity of SDH, as did cleaving embryos (Fig. 3). At the end of cleavage, highest enzymatic activity was observed at the embryo periphery, in the region of the macromeres (Rybicka, 1966) (Fig. 4). A

EMBRYOGENESIS

IN

Hymenolepis

diminuta

IV.

FIG. 1. Section through the testis (SDH). Sperm (arrow) unstained. FIG. 2. Section through the ovary (upper) and the vitelline gland (bottom) (SDH).

clear . unstained space was usually seen near the macromeres, presumably connectc:d with the vitelline cell which is at this time filled with glycogen, and did not displ ,ay any other cytoplasmic structures (Ryl bicka, 1966, 1967a). EI nzyme activity occurred in the embry9

onic envelope immediately after its formation (Fig. 5). Inside the enclosed e mbryo, droplets of formazan could also bc: seen. High enzymatic activity remained in the embryonic envelope through the enti .re preoncosphere phase. The shell began t .o form at the surface of the enzymatically active

FIG. 3. Two cleaving embryos (SDH). FIG. 4. The end of cleavage (SDH). FIG. 5. Early preoncosphere (SDH). FIGS. 6, 7, 8. Subsequent phases of preoncosphere development. High activity of succinic dehydrogenase in the inner envelope: E, embryophore; S, shell. FIG. 9. Late preoncosphere. Succinic dehydrogenase accumulated beneath the membrane of inner envelope. FIG. 10. Oncosphere. Decrease in the activity of succinic dehydrogenase in the inner envelope.

EMBRYOGENESIS

IN

Hymenolepis

layer (Fig. 5). As shell formation proceded (Fig. 6), it detached somewhat from the active cytoplasmic layer which surrounded the embryo, and on the inner surface of the active layer the embryophore began to form (Fig. 7). Following embryophore formation, the highest enzymatic activity occurred at the periphery of the inner envelope (Fig. 8). Thus the active layer was somewhat separated from the embryophore, and seemed to move toward the shell. Figure 9 shows, however, that the layer of highest activity was not directly connected with the shell. Succinic dehydrogenase activity was concentrated at the periphery of the inner envelope beneath its membrane. The concentration of the enzyme in the inner envelope decreased toward the end of development. In the mature oncosphere (Fig. lo), when the rough layer was already formed on the shell surface, traces of succinic dehydrogenase activity were seen lining the inner surface of the inner envelope. Generally, SDH activity was present inside the embryo during the course of development. However, the embryonic cells were much less active than the cytoplasmic layer surrounding the embryo. In the oncosphere, the region containing germinative cells showed higher activity than the other parts of the embryo, although some formazan deposit was usually observed in the hook region. Review of sections from the different parts of the strobila indicated that the greatest enzymatic activity occurred in the gonads and in the embryonic envelope during the preoncosphere phase. In general, the reproductive glands, the embryos, and the tegument showed much higher accumulation of formazan than did the parenchyma. Control sections showed some formazan formation. The greatest difference in stain intensity between normal and control sec-

diminuta

IV.

259

tions occurred in the reproductive system, while that in parenchyma was less prominent. The addition of malonate to the medium (malonate/succinate: l/ 1) , partially diminished the reaction. The second medium (malonate/succinate: 2/ 1 ), greatly inhibited the reaction. The intensity of formazan formation in these sections was similar to that observed in the medium deprived of succinate. NADH-Oxido-Reductase

The distribution of NADH-reductase in the tapeworm was generally similar to that of SDH. Highest activity occurred in the gonads, the embryonic envelope during the preoncosphere phase, and in the tegument. Formazan formation in these sites was much stronger than in the parenchyma. Detailed observations of the embryos indicated some slight differences in the distribution of NADH-oxido-reductase when compared with that of SDH. The former showed relatively higher activity inside the embryos and relatively lower within the envelope. High accumulation of NADHreductase occurred in the fully formed oncospheres. The control sections incubated in the medium deprived of NADH did not show any reaction. Cytochrome

Oxidase

Cytochrome oxidase results were generally poor. No reactions occurred with Seligman’s ( 1965) method. Some very slight stain appeared after the use of p-aminodiphenylamine. In this case, the clearly positive reaction was observed in the tegument and in the parenchyma under the tegument. Early embryos did not show any cytochrome oxidase activity. A slight stain occurred occasionally in the embryos toward the end of the development. Contrary to the results obtained with SDH and NADHreductase, this slight stain occurred inside the embryos and not in the embryonic envelope.

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The irregularity in its occurrence, and the very slight intensity, raise some doubts as to whether the reaction can be regarded as positive. KCN completely inhibited the reactions. The cardiac muscle of the rat gave positive results, indicating that the medium was reactive. DISCUSSION

The high accumulation of succinic dehydrogenase and NADH-oxido-reductase in gonads of H. diminuta confirms the observations of Hedrick (1956) and Bogitsh and Nunnally (1966). These investigators have found a similar accumulation of the former enzyme in gonads of H. diminuta and H. microstoma, respectively. The variation observed in the distribution of succinic dehydrogenase inside the testes can be explained speculatively in that the more disperse activity is perhaps connected with the growth of spermatocytes, and results in a loose distribution of mitochondria in these cells. It is also possible that the high accumulation of the enzyme observed at times in the central part of testis results from the residual cytoplasm after the sperm are formed. No succinic dehydrogenase activity is observed in sperm. These observations agree with the electron microscope findings of Rosario (1964), who found large mitochondria in the residual cytoplasm of H. diminuta and none in the sperm. The cellular origin of the embryonic envelope which is formed by the detachment of five macromeres from the embryo (Rybicka, 1966) explains the localization of succinic dehydrogenase and NADH-oxidoreductase in this layer. During the differentiation of the embryonic envelope into shell and embryophore, a cytoplasmic layer (the inner envelope) remains between them. It is covered by a thin membrane which has been shown by Voge and Berntzen (1961). The enzymatic activity occurs in the inner

envelope beneath this membrane. It was surprising to find that the enzymatic activity of the envelope is much higher than that of the embryo. This observation leads to the hypothesis that the envelope contains a particularly high accumulation of mitochondria, and that it represents, perhaps, a basic metabolic layer in the embryogenesis, supplying both the energy and the nutritive material for the developing embryo. Localization of the most active enzymatic layer of the envelope near the shell at the time when the latter starts to form, and near the embryophore at the beginning of its formation, suggests also that this active layer may participate in the synthesis of material forming both the shell and the embryophore. Most enzymatic activity in the envelope is observed during the preoncosphere phase when the embryonic structures and protective layers are formed. Hedrick (1956) and Bogitsh and Nunnally (1966) have mentioned the high accumulation of succinic dehydrogenase in the area under the shell of mature eggs. A review of the photographs presented by the latter authors indicates, however, that this accumulation occurs also in the preoncosphere, before the embryo matures. Thus, these results agree with the present observations. Toward the end of the development, enzymatic activity decreases in the inner envelope. The function of the embryonic envelopes seems to be restricted to the protection of the embryo (supplied by the shell and the embryophore) . Bogitsh and Nunnally ( 1966) have analyzed the distribution of succinic dehydrogenase. The distribution of the enzyme seems to coincide with the regional distribution of glycogen. However, it should be pointed out that in the detailed examination of organs this coincidence does not always occur. For example, testes and ovary of H. diminuta are glycogen-free (Ry-

EMBRYOGENESIS

IN

Hymenolepis

bicka, 1967a), but they show a high activity of succinic dehydrogenase. On the other hand, glycogen occurs in sperm (Rybicka, 1967a) which do not show any enzymatic activity. A close parallelism in localization of glycogen (Rybicka, 1967a) and activity of mitochondrial enzymes occurs in the embryonic envelope. It seems quite probable that this accumulation of glycogen represents a nutritive material which is quickly metabolized in this layer and is used by the developing embryo or for the formation of its shell and embryophore. The distribution of succinic dehydrogenase and NADH-oxido-reductase seem to coincide, to a great extent, with the distribution of RNA (Rybicka, 1967b). This correlation seems quite obvious, as the metabolism of the developing embryo is mainly connected with protein synthesis. The positive reaction for cytochrome oxidase found in the tegument and in the parenchyma confirms the findings of Read (1952) and Waitz and Schardein (1964). Very confusing, however, is the lack of the enzyme in the embryos and particularly in their envelopes, since these structures show a high metabolic activity. This observation illustrates, once more, the unclear question of terminal oxidation in cestodes. Von Brand (1966) has quoted the opinion of Bueding that, in H. diminuta, the anerobic dehydrogenation of NADH leading to the reduction of fumarate to succinate may provide the energy in the form of ATP, as has been suggested for Ascaris (Bueding, 1962). It is, however, also possible that this generally similar oxidation may differ in details as it has been found in an acanthocephalan Moniliformis dubius (Bryant and Nicholas, 1965). The problem in H. diminuta cannot be solved by the use of cytochemical methods only. The present observations, however, seem to support the suggestion of Read and

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Simmons (1963) concerning the possibility of two different pathways in cestode metabolism. In the tegument, which is exposed to a low oxygen tension, terminal oxidation involving cytochrome c may occur, while an alternative pathway may exist in embryonic metabolism. Keeping in mind the great activity of mitochondrial enzymes observed in embryos, it seems quite probable that this alternative pathway is of a great importance in the general metabolism of cestode. Waitz and Schardein (1964) have found cytochrome oxidase in oncospheres of H. diminuta. The present results dealing with the oncospheres are quite doubtful. Assuming, however, that some cytochrome oxidase activity may occur in the oncospheres, the event may be explained as a preparation of the embryo to aerobic conditions connected with its passage to the intermediate host. ACKNOWLEDGMENT

The author is grateful to Dr. Burton J. Bogitsh for the laboratory facilities, for his kind help in the preparation of photographs, and for the revision of the manuscript. REFERENCES B. J., AND NUNNALLY, D. A. 1966. Histochemistry of Hymenolepis microstoma (Cestoda: Hymenolepididae) II. Regional distribution of succinic dehydrogenase. Parasitology 56, 55-61. BRAND VON, T. 1966. “Biochemistry of Parasites.” Academic Press, New York. BRYANT, C., AND NICHOLAS, W. L. 1966. Studies on the oxidative metabolism of Moniliformis dubius (Acanthocephala). Comparative Biochemistry and PhysioZugy 17, 825-840. BUEDING, E. 1962. Comparative aspects of carbohydrate metabolism. Federation Proceedings 21, 1039-1046. BURSTONE, M. S. 1962. “Enzyme Histochemistry and Its Application in the Study of Neoplasm.” Academic Press, New York. GOLDBERG, E., AND NOLF, L. 0. 1954. Succinic dehydrogenase activity in the cestode HymenolBOGITSH,

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epis

nana.

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Parasitology

3, 275-

284. HEDRICK, R. M. 1956. The distribution of succinic dehydrogenase activity in Hymenolepis diminuta and Raillietina cesticillus. Journal of Parasitology 43, (Sec. 2, Suppl.), 34. NACHLAS, M. M., Tsou, K. C., SOUZA, E., CHENG, C. S., AND SELIGMAN, A. M. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitro-phenyl substituted ditetrazole. Journal of Histochemisiry

and

Cytochemistry

5, 420436.

NOVIKOFF, A. B. 1963. Electron zymes. In Histochemical and Proceedings

of

1st

International

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en-

Cytochemical Congress,

Paris, 1960 (Wegmann, P., ed.) Macmillan. READ, C. P. 1952. Contributions to cestode enzymology. I. The cytochrome system and succinic dehydrogenase in Hymenolepis diminuta. Experimental Parasitology 1, 353-362. READ, C. P., AND SIMMONS, J. E., JR. 1963. Biochemistry and physiology of tapeworms. Physiological

Reviews

43, 263-305.

ROSARIO, B. 1964. An electron microscope study of spermatogenesis in cestodes. Journal of Ultrastructure Research 11, 412-427. ROTHMAN, A. H., AND LEE, D. L. 1963. Histochemical demonstration of dehydrogenase

activity in the cuticule of cestodes. Experimental Parasitology 14, 333-336. RYBICKA, K. 1966. Embryogenesis in Hymenolepis diminuta. I. Morphogenesis. Experimental Parasitology 19, 366-379. RYBICKA, K. 1967a. Embryogenesis in Hymenolepis diminuta. II. Glycogen distribution in the embryos. Experimental Parasitology 20, 9% 105. RYBICKA, K. 1967b. Embryogenesis in Hymenolepis diminuta. III. Distribution of ribonucleic acid. Experimental Parasitology 20, 177185. SELIGMAN, A. M. 1965. Some recent trends and advances in enzyme histochemistry. The Sinai Hospital Journal 12, 73-83. SMYTH, J. D. 1962. “Introduction to Animal Parasitology.” The English Universities Press, London. VOGE, M., AND BERNTZEN, A. K. 1961. In vitro hatching of oncospheres of Hymenolepis diminuta (Cestoda: Cyclophyllidea). Journal of Parasitology 47, 813-818. WAITZ, J. A., AND SCHARDEIN, J. L. 1964. Histochemical studies of four cyclophyllidean cestodes. Journal of Parasitology 50, 271277.