EXPERIMENTAL PARASITOLOGY 20, 177-185 (1967)

Embryogenesis in Hymenolepis diminuta

III. Distribution of Ribonucleic Acid1

Krystyna Rybicka2

Department of Biology, Vanderbilt University, Nashville, Tennessee

(Submitted for publication, 8 December 1966)

RYBICKA, KRYSTYNA. 1967. Embryogenesis in Hymenolepis diminuta. III. Distri- bution of ribonucleic acid. Experimental Parasitology 20, 177-185. Distribution of RNA in gonads and embryos of Hymenolapis diminuta was studied by the use of Azure B and RNase control. RNA accumulated in testes during spermatogenesis; the sperm was deprived of this acid. High accumulation of RNA occurred in the ovary. Oocytes contained fine granules of RNA dispersed in the cytoplasm and in large vitelline granules. The nucleolus of the oocyte was rich in RNA. During cleavage most of embryonic RNA was accumulated in the macromeres destined to form the outer envelope, and in mesomeres. In the early preoncosphere phase most of the embryonic RNA occurred in the cells forming the oncospheral organelles (hook- forming cells and penetration glands). At the end of development most of the embryonic RNA disappeared what was presumably connected with the degeneration of embryonic cells. The accumulation of RNA increased in the germinative cells. The increase of RNA occurred in the embryonic envelope during the preoncosphere phase in spite of the lack of cellular divisions in the envelope.

The role of RNA in embryonic develop- ment of most animal groups seems to be well established. However, few publications exist that deal with this question in cestode embryonic development. First observations in this field date back to the study of Vogel (1929), who was the first to use pyronin for staining cestode embryos. The cyto- plasm of some cells stained very deeply, and he referred to these cells as “plastin” cells. Although an histochemical interpreta- tion could not be given at that time, it has appeared later that the “plastin” or “germi- native” cells contained almost the entire

’ This investigation received financial support from the World Health Organization.

* Present address: Department of Parasito- logy, Polish Academy of Sciences, Pasteura 3, Warsaw, Poland.

amount of oncospheral RNA, and that these cells are responsible for the further development of the larva-( see Rybicka, 1966a).

Some fragmentary observations on the occurrence of RNA during cestode em- bryogenesis have been given by Silverman (1954), Ogren, (1956, 1957a,b, 1959, 1961, 1962a), Rybicka (1961a.b. 1962a,b, 1964a,b), Bogitsh (1963), Coil (1963), and Cheng and Jacknick (1964).

The present study is based on morpho- genesis in Hymenolepis dinzinuta as de- scribed by Rybicka (1966b), and deals with observations of the changes in RNA distribution in the embryo.

Azure B was used as stain for RNA. It appears much more useful than pyronin which, according to the author’s experi-

177

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ence, does not give good results in cyclo- phyllidean cestodes.

MATERIAL AND METHODS

Adult cestodes were obtained from a rat 17 days after infection with ten larvae. The worms were fixed in Carnoy’s (3 : 1)) em- bedded in paraffin, and sectioned longitudi- nally at 5 p. The details of the preparation of material were described previously (Ry- bicka, 1966b).

The slides were stained with Azure B, 0.25 mg/ml at pH 4.0, for 2 hours at 40°C (Flax and Himes, 1952). Control sections were incubated in RNase for 1 hour at 37°C in a solution of enzyme, 1 mg/ml of glass distilled water (Pearse, 1961).

The results showed a metachromatic staining of RNA and green staining of DNA.

RESULTS

Gonads. RNA was present in the testes during all phases of spermatogenesis. The most intense, almost homogenous stain oc- curred in the cytoplasm and nucleoli of early spermatogonia (Fig. 1) . During growth of spermatocytes, the stain became less intense, and the RNA occurred in the form of fine granules dispersed in the cyto- plasm (Fig. 1) . Mature sperm did not pos- sess RNA (Fig. 1) . The residual cyto- plasm remaining in the testes after sperm release stained dark blue, which was labile to RNase.

The young oogonia resembled young spermatogonia in their staining reaction. Their cytoplasm and nucleoli stained prom- inently. During oogenesis, RNA became dispersed in the form of fine granules in the ooplasm. In growing oocytes (Fig. 2) the vitelline granules were formed, and these were distinguishable due to their ho- mogenous, light blue-violet stain. In addi- tion these vitelline granules appeared to be surrounded by aggregations of fine cyto- plasmic granules of RNA. The staining re-

action of the dispersed granules and of the vitelline granules disappeared after RNase digestion. The nucleolus of the oocyte en- larged and showed a heavy deposit of RNA.

The cytoplasm of the vitelline cells con- tained some RNA (Fig. 2), but this was much less prominent than that found in the oocytes. The cytoplasm of the vitelline cells became vacuolated and the individual vacuoles were divided by a delicate net- work of RNA granules. In the nucleus of the vitelline cell, a structure resembling the nucleolus was observed. However, the dark stain of a similar structure remained after digestion, and it was difficult to judge whether there was a nucleolus closely asso- ciated with an agglomeration of chromatin, or whether the dark stain was due to the presence of the chromatin only.

Maturation divisions occurred in the oocyte immediately after it entered the uterus. At this time, the sperm nucleus grew in the oocyte and the mature ovum contained two pronuclei (male and fe- male) (Fig. 3). The content of RNA in the oocyte slightly decreased during maturation in comparison with that present in the ov- ary. The fine granules appeared less dense, but the homogenous stain of vitelline gran- ules remained unchanged. A very marked decrease of RNA occurred in the nucleolus during oocyte maturation (Fig. 3).

The structure of the vitelline cell changed after entering the uterus. The net- work of RNA granules observed in the cells of the vitelline gland changed into a more compact thin layer surrounding the nucleus of the cell (Fig. 3).

Cleavage. During early cleavage the structure of one large blastomere remained similar to that of the ovum (see Rybicka, 1966b). The vitelline granules were easily distinguishable in this macromere. After several unequal divisions of the macro- mere, the equal division occurred and led to the formation of two daughter macro-

EMBRYOGENESIS IN Hymenolepis diminuta. III 179

FIG. 1. Testis. R, Residual cytoplasm; S, spermatogonia; SP, sperm; SP,, primary spermatocytes in division of maturation.

FIG. 2. Ovary (upper) and vitelline gland (bottom). Vitelline granules surrounded by fine granules of RNA are visible in the ooplasm.

meres. The vitelline material was divided romere contained finely dispersed droplets unequally among them and the cytoplasm of RNA. As cleavage progressed 5 macro- of macromere which obtained most of this meres were formed. Three macromeres of material stained intensely with Azure B the inner envelope contained clearly less (Fig. 4). The cytoplasm of the other mac- RNA than the two macromeres of the outer

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envelope. On the other hand, nucleolar RNA increased in the former cells at the time of detachment of the macromeres from the embryo (Fig. 5).

The cytoplasm and the nucleoli of the mesomeres formed during cleavage con- tained large amounts of RNA.

Preoncosphere. The differences in the cytoplasmic RNA of cells forming the em- bryonic envelope disappeared in the preon- cosphere phase, when all 5 macromeres formed one syncytial layer surrounding the embryo (Fig. 6). However, the differences described above in nucleolar RNA re- mained also in the preoncosphere phase.

Generally the early embryonic envelope stained less intensely than the embryo it- self. In the embryo, cell differentiation and RNA distribution appeared to be interre- lated. In the early preoncosphere the great- est accumulation of RNA appeared in the hook-forming cells, where it was dispersed in the form of fine granules (Fig. 6). The presence of small hook rudiments was ob- served at this time by the use of phase- contrast microscopy. Two cells at the oppo- site hemisphere of the embryo showed also a relatively large amount of RNA. Similar cells were found also in the somewhat older preoncosphere (Fig. 7), and these were considered to form the penetration glands. Two large unstained areas were seen around these cells (Fig. 7). These areas presumably represent some material that had accumulated in the gland and did not stain with Azure B. Other embryonic cells localized near these glands were positive for the presence of RNA.

Comparison of Fig. 6 with Fig. 7 shows that the distribution of RNA in the em- bryo remained generally unchanged. In the hook region, RNA was dispersed in the form of fine droplets. In the central part of the embryo a new type of cell appeared, having a large nucleus with a dense layer of RNA surrounding it.

As the embryonic envelope proceeded through its differentiation, the shell was formed on its surface. The participation of the two nuclei in shell formation and their subsequent degeneration was described elsewhere (Rybicka, 196613). In the early phase of the shell formation this hardening layer stained light blue with Azure B, and this stain was not abolished by RNase treatment. Later, the shell did not react with the stain and remained colorless.

On the inner side of the envelope the embryophore formed. It was colorless and visible only by phase-contrast microscopy. The nuclei of the inner envelope showed large nucleoli rich in RNA (Fig. 7). The cytoplasmic layer between the shell and the embryophore, defined as the inner en- velope, contained RNA which decreased as development proceeded.

In the late phase of the preoncosphere, the amount and location of RNA observed inside the embryo changed (Fig. 8). Par- ticularly characteristic of this phase was the decrease of RNA in the hook region. Si- multaneously, more RNA was accumulated in the hemisphere opposite to the hooks. A densely stained layer surrounded several nuclei of germinative cells which accumu- lated RNA at the end of embryonic de- velopment.

The cytoplasmic layer of the inner enve- lope contained RNA.

Oncosphere. Most of the oncospheral RNA was accumulated in the cytoplasm and nucleoli of germinative cells (Fig. 9). The hook region and the penetration glands were almost depleted of RNA.

Almost all RNA disappeared from the embryonic envelope. Some traces were found beneath the shell.

DISCUSSION

The accumulation of RNA in spermato- gonia has been mentioned by Cheng and Jacknick (1964). These authors have ob-

EMBRYOGENESIS IN Hymenolepis diminuta. III 181

FIG. 3. Oocytes in uterus. Note a weak staining of nucleolus. FIG. 4. Early cleavage. MI, Macromeres of the inner envelope; MO, macromeres of the outer

envelope. FIG. 5. Late cleavage. MI, Macromeres of the inner envelope; MO, macromeres of the outer

envelope. FIG. 6. Early preoncosphere. GL, Gland-forming cell; H, hook-forming cells.

FIG. 7. Older preoncosphere. GL, Gland; H, hook-forming cells. FIG. 8. Late preoncosphere. Note a decrease in RNA in the hook region.

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FIG. 9. Oncosphere. G, Germinative cells; GL, glands; H, hook region.

served the presence of RNA in mature sperm of H. diminuta. This observation does not agree with the present results. As known to this author, RNA has never been observed in the sperm of other cestoda, or for that matter, in the sperm of other ani- mals. Brachet (1960)) discussing this ques- tion, has concluded that protein and nucleic acid anabolism in sperm is exceed- ingly low if it exists at all. It seems that the results obtained by Cheng and Jacknick (1964) were rather due to the use of pyro- nin, which hardly gives a specific reaction for RNA in cyclophyllideans. In older testes, a prominent stain is observed in the residual cytoplasm extruded from sperma- tozoa, but this is presumably not active metabolically as it has been found for other animals (Brachet, 1960).

The large amount of RNA in oogonia is typical for most animals. Brachet (1960) has pointed out that during the course of oogenesis, while the amount of RNA seems often to decrease, it is in fact rather a mere dilution of RNA in the oocyte which, in turn, has increased tremendously in vol-

ume. This same observation seems to be true for oogenesis in H. diminuta.

In the developed oocyte, RNA is dis- persed in the form of fine granules through- out the cytoplasm. Bjorkman and Thorsell ( 1964) have shown dense ribosomal bodies in the oocyte of Fasciola hepatica by electron microscopy. These bodies are iden- tical with basophilic bodies found in the light microscope which are labile to RNase. Although it is difficult to compare the struc- ture of F. hepatica with the structure of H. diminuta, it seems quite likely that the fine RNA granules observed in the oocyte of the latter might represent the presence of ribosomal bodies. An electron microscope study would be of value in solving this question.

In addition to the fine granules of RNA in the ooplasm, the large ooplasmic bodies or vitelline granules stain intensely in the oocyte. These granules represent a charac- teristic feature of the oocyte of H. dimi- nuta, and have been observed earlier when stained with hematoxylin (Rybicka, 1966b). The term vitelline granules has

EMBRYOGENESIS IN Hymenolepis diminuta. III 183

been used very loosely. In fact, this mate- rial has never been chemically defined. Some speculations can be made by com- parison with the oocyte of a trematode, Gorgoderina attenuata (Koulish, 1965). This author has described the “nucleolus- like” cytoplasmic body which seems to be similar to the vitelline granules in H. dimi- nuta. It should be pointed out that the dis- persion of this material into large granules is typical for some cyclophyllidean species as described by Saint-Remy (1900) in Anoplocephala mumillana, by Bona (1957) in Paricterotaenia porosa, by Og- ren (1955 and 1956b) in Hymenolepis nana and Mesocestoides corti, and by Ry- bicka (1964a) in Moniezia expansa; while in the other species like Taenia serrata de- scribed by Janicki ( 1907)) Multiceps smythi by Johri (1957), and Dilepis un- dula by Ogren (1962b), one large accumu- lation was observed similar to that found by Koulish (1965) in the trematode. The ultratructural study of the latter author in- dicates the resemblance of this body to that of the nucleolus. Although the present ob- servations by light microscopy are not suffi- cient for comparison with the results ob- tained by the electron microscope, it seems worthy to note that the homogenous stain with Azure B occurs both in the vitelline granules and in the nucleoli of the oocyte of H. diminuta (the latter stain being much heavier). It would be also of interest to point out some speculations of Brachet (1960) concerning the presence of phos- phoproteins in the nucleolus and in the yolk and their possible relationship.

After entering the uterus the oocyte un- dergoes maturation, and at this time the amount of its RNA decreases. This change is particularly prominent in the nucleolus, which is only slightly stained and resembles the vitelline granules.

In review of the general difference in RNA content among the blastomeres dur-

ing cleavage, it should be pointed out that the large macromeres of the inner enve- lope, which do not possess vitelline mate- rial, stain the weakest, whereas the greatest amount of RNA is accumulated in macro- meres of the outer envelope containing the vitelline material.

The accumulation of RNA in the meso- meres is to be expected as these are the true embryonic cells which, by further mul- tiplication, form the oncosphere.

During the preoncosphere phase, RNA accumulates inside the embryo and appears first in the cells forming the embryonic or- ganelles (e.g., the hooks). Observations on the presence of RNA in hook-forming cells confirm the detailed study of Ogren ( 1961) . This author has also observed the early accumulation of RNA at the time when the hooks began to develop and the subsequent decrease in RNA as the hooks grew.

Simultaneously with hook formation, RNA accumulates in two cells of the op- posite pole. These cells presumably par- ticipate in the formation of the penetration glands.

In the late phase of development, the germinative cells are formed, and these ac- cumulate RNA. The localization of germ- inative cells in the vicinity of the penetra- tion glands does not, however, prove, by any means, that the former can originate from the glands as has been suggested by Ogren (1962a).

In general, a lot of RNA was observed in the multiplying embryonic cells during the preoncosphere phase. At the end of de- velopment, most of the embryonic RNA disappears except in the germinative cells where RNA accumulates. The disappear- ance of RNA is clearly connected with degeneration of most of the embryonic cells. This degeneration has been observed for the first time in Diorchis ransomi (Ry- bicka, 1961a,b) and has been confirmed

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by Rybicka (1964a,b) in other cyclophyl- lidean species such as Dipylidium caninum and Moniezia expansa. Review of the lit- erature dealing with embryogenesis in ces- todes (Rybicka, 1966a) indicates that this degeneration is typical for all the cyclophyl- lidea. It is noteworthy that such an event does not represent any peculiarity con- nected with development of parasites. Saunders ( 1966)) analyzing the degenera- tion of cells in embryogenesis of different organisms, has stated that “death of cells is the usual accompaniment of embryonic growth and differentiation.”

A separate problem represents the ac- cumulation of RNA in the embryonic en- velope. The macromeres, which detach from the embryo to form the envelopes, do not divide further during embryogenesis. It seems somewhat surprising that the cyto- plasm and nucleoli of these cells contain large accumulations of RNA when, on the other hand, this accumulation is known to be connected with cellular divisions (Brachet, 1959). However, the embryonic envelope greatly increases in size during the preoncosphere phase and, presumably, protein synthesis occurs in this layer. Be- sides, the recent study (Rybicka, in prep- aration) indicates that the embryonic en- velope is not an inactive layer serving to protect the embryo. On the contrary, it seems to have a high metabolic activity and thus may represent an important function for the embryo. This can explain also the high accumulation of RNA in the envelopes.

When the embryo is ready to hatch, the active function of the embryonic envelopes decreases. Some rudiments of RNA are us- ually found between the shell and the em- bryophore. Coil (1963) has studied the dis- tribution of RNA in the embryonic enve- lopes of two cyclophyllideans. He has found some differences in RNA distribution in the two species and has concluded that these can be of importance in taxonomy

higher than the generic level. It seems, how- ever, that the rudiments of RNA remain- ing in the envelopes of mature oncospheres cannot be more active metabolically. Some analogy can be made with the cytoplasmic RNA remaining in the residual cytoplasm in the testes after sperm is released. Thus there are some doubts whether the differ- ences in RNA distribution in such residual layers, which do not play a further role in the development, can be considered of such high taxonomic importance.

ACKNOWLEDGMENTS

The author is greatly indebted to Dr. Burton J. Bogitsh for the laboratory facilities and for kind revision of the manuscript.

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