Aota Path. Jap. 27(1): 26- 39, 1977
FINE STRUCTURAL STUDY O F ANNULATE LAMELLAE COMPLEXES I N HUMAN TUMORS Iwao NAKAYAMA, Akira MORIUCHI,Yoshitaugi TAIRA, Osamu TAKAHARA, and Nobuo TSUDA* Takashi ITOGA
*
Department of Central Diqmstic Laboratmy. Nagasaki University Haapital, Nagaaaki Department of Pdhlogy. Atomic Dieeaae Inetitute Nagasaki University School of Medicine, Nagaaaki (Received on April 12, 1976)
Specific intracytoplasmic organelles, annulate lamellae and radial cisternae, have been studied In several human tumors. Annulate lamellae are observed in all cases of leiomyoma, lelomyosarcoma, rhabdomyosarcoma and malignant melanoma, whereas radial cisternae are only found in a case of leiomyosarcoma. Annulate lamellae are characterized by stacks of parallel arrayed long cisternae showing alternative arrangement of annuli and sacs. Some of these cisternae are connected directly with rough-surfaced endoplasmic reticulum and there is continuity with the lumen and membrane. Radial cisternae are mainly composed of two structures: numerous short clsternae, which are a variant of annulate lamellae, and numerous spherical particles derived from the cisternae. The cisternae are arranged parallel or radially around particles measuring up to 1100 A in diameter. These particles consisting !of an amorphous high electron dense materlal without distinct limiting membrane are organized in groups and vary in number. There is no evidence of a direct relationship between these structures and viral infection. ACTA PATH JAP. 27: 25-39,
1977.
Iratroduction In 1956 SWIFT^^ was the first to describe annulate lamellae, a specific intracytoplasmic organelle which is composed of stacks of parallel lamellae. These annulate lamellae are characterized by periodically arranged annuli resembling those of the nuclear envelope. KESSEL~~ compared annulate lamellae with annuli of the nuclear envelope in tunicate oocytes; he published an excellent paper on the morphogenesis of both intranuclear and intractyoplasmic annulate lamellae. Although annulate lamellae are commonly observed in the cytoplasm of both invertebrate and vertebrate germ ~ells2,4,7,11,14,1~,19,29,2~,29,92, they have been also observed in the cytoplasm of somatic cellsg?6P,experimental tumors %'%27,a1 as well as human tumors.1q8 The function of annulate lamellae has remained obscure, although SWIFT*^ suggested that annulate lamellae may function in nuclear-cytoplasmic transfer. Several reports1612'J#22have appeared on the origin of annulate lamellae and the relationship between annulate lamellae and virus infection in viral infected cells and virus induced tumor.
26
AN'NULATH LAMELLAE 00bfPLEXES
Acta Path. Jap.
In recent years PATRIZI and MIDDELKAMP** reported annulate lamellae and radial cisternae associated with two types of spherical particles in rubella virus infected RK13 cells and suggested that these intracytoplasmic organelles may be caused by virus infection, because the structures were absent from non-infected cells. These intracytoplasmic organelles, annulate lamellae and radial cisternae were referred to as annulate lamellae complexes>* because of demonstration of intermediate form between the two organelles. It is of interest that the appearance of annulate lamellae complexes may or may not be directly connected to viral infection as an etiological agent for the development of the neoplasm. In this paper annulate lamellae in several human benign and malignant tumors were studied by electron microscopy and, based upon their fine structure, a hypothetical relationship between development of annulate lamellae complexes and virus infection is discussed.
Materials and Me&ds The histological diagnosis, age and sex of the patients and original site of the tumor for each oase are given in Table 1.
For light microscopic studiea, a portion of each tumor was fixed in 10% neutral formalin, embedded in paraffin and stained with hematoxylin-eoein, periodic acid Schiff (PAS), reticulin, phosphotungstic acid hematoxylin (PTAH) and melanin stains. For electron microscopic study, tissuea obtained in the operating Mom were quiokly transferred into 1.6% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). During fixation the tissuea were separated into numerous small pieces and p l d in fresh fixative for an additionel 4 hours. After washing overnight in phosphate buffer, tissues were post-fixed in 2% osmium-tetraxide for 2 houre, dehydrated in a graded seriee of ethanol followed by propylene-oxide, and embedded in Epon 812. Semithin sections of the entire area were cut and stained with buffered toluidine blue to identify the tumor tissue. Thin sections from selected portions of the blocks were out with grasn knives on a JEOL JUM-7 ultramiomtome end were double stained with a saturated aqueous solution of uranyl aoetate followed by lead citrate'.. Micrographs were taken with a JEM lOOB electron micromope with side entry type. Annulate lamellae and radial oisternae were observed from various directions using rotation and tilting of the specimens to obtain a t h dimensional structure. Table 1. &a&iah Case
No. Age
1
66
a s
46
4
51
46
and R d t a
Sex
Histological Dix.
Site of origin
F F F F
Leiomyoearcoma Rhabdomyoearcoma Malignant melanoma Leiomyoma
Mesocolon Retroperitoneum Nasal cavity Small intestine
AL
RC
+ + + + + -
AL: Annulate lamellae RC: Redial oisternae
Results Annulate LameUae
Annulate lamellae were observed in all cases examined in this study. Since the Figs. 1 and 2. Annulate lamellee are composed of a considerably variable number of cisternae. Each cisterna is bounded by smooth membrane having periodic narrowing. Both ends of cieternae in Fig. 2 demonstrate formation of vesicles (V). There are short profiles of rough endoplasmio reticulum with numerous ribosomes around annulate lamellee. Fig. 1 x 43, OOO and Fig. 2. x 36,000.
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28
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Fig. 6. Annular regions are palisaded in a line on the stacks of the cisternee (arrow). Each cisterna shows an alternating arrangement of annuli and sac%. Amorphous material with high electron density are observed around the annuli. x 46,000.
fine structural characteristics of annulate lamellae are essentially similar from one case to the other, the common morphological characteristics of annulate lamellae encountered in all materials will be described here. Annulate lamellae were composed of stacks of cisternae, bounded by smooth membrane. The number of the cisternae in a single stack was considerably different from one case to the other and even in the same case, and varied from two to as many as more than one dozen (Figs. 1-2). Each cisterna had periodic narrowing8 of its lumen, which were referred to as annuli, lumina or pore with resemblance to that of the nuclear envelope. In Bsomeof the annuli, two membrane structures appeared to fuse into a single membrane having more electron density and the other still had a double membrane with more electron density (Fig. 3 arrow). With the tilting of the same annulate lamellae the fine structure of the well developed annular region was not altered to double membrane but showed a single electron dense membrane (Fig. 4). Figs. 3 and 4. Photographs of thew annulate lamellae were taken from the same area a t a different angle. The photograph shown in Fig. 3 was taken while keeping the specimen on a horizontal plane and in Fig. 4 the specimen was tilted to an angle of 26'. After tilting, the h e structure of the annular region is not altered to double membrane but shows a more distinct single membrane with high electron density (arrow). Figs. 3 and 4 x 60,000.
30
Acta Path. Jap.
Fig. 6. Photograph shows annular regiona cut in almoet a tangential plane through the annuli of annulate lamellee. Spherical particlea arranged in a line consist of high electron dense materials which are embedded in lees electron dense materials but do not have distinct limiting membrane. The left upper porition (arrow) of the photograph shows a remnant of annulate lamellae. x 60,000.
These annuli appeared to be palisaded in a line on stacks of the cisternae. Amorphous material with high electron density was associated with each annuli (Fig. 6). Each sac located between annuli of a cisterna varied considerably, ranging from 714 A to more than 1900 A in length and the mean length of annuli measured 750 A. The distance between two cisternae was about 890 A. If the plane of section passed through the annuli, spherical particles surrounded by less electron dense materials appeared and were arranged in a line (Fig. 6). These particles measuring approximately 1100 A in diameter consisted of high electron dense material formed by an aggregation of small and high electron dense dots and had no distinct limiting membrane between two different electron dense materials (Fig. 6). Depending on the plane of section an intermediate form between typical annuli and these particles was observed (Fig. 7). Both edges of the annuli were connected with sacs which contained electron lucid material. Fig. 7. Annulate lamellae of an intermediate form between Fig. 6 and 6. There are many rodlike structure with electron lucid materials around annulate Ismellee. x 43,000. Fig. 8. Annulate lamellae show direct connection with rough endoplaemic reticulum with luminal and membranoue continuity. x 46,000.
-;
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31
32
ANNULATE LAMELLAE OOWLEXES
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Fig. 11. Portion of radial cisternae. The ckternae bounded by smooth membrene are densely packed together and radially arranged around the dusted spherical particles but do not demonstrate annuli. A single stack of the cisternee exhibits characteristic double zonal structure of high (HD) and low electron density (LD). The particles are embedded in e less electron dense matrix without limiting membrane. Newly forming particles (arrow), located in the periphery of the clueter, appear to be produced by coalescence of several cisternae. x 54,Ooo.
Some cisternae of annulate lamellae were directly connected with rough-surfaced endoplasmic reticulum showing luminal and membrane continuity (Figs. 2, 8)) whereas other annulate lamellae demonstrated formation of a moderate number of vesicles at the end of the cisternae (Fig. 2). There were numerous polysomes and rod-like structure containing electron lucid material located around annulate lamellae (Figs. 2, 9).
Radial cisternae observed only in a case of leiomyosarcoma were composed of bundles of short cisternae, bounded by smooth membrane, and of spherical particles with high electron density. A single bundle of cisternae was in parallel and oriented in a radial arrangment around the centrally aggregated particles (Fig. 10). Since these Fig. 9. Aggregeted ribosomes ere occesione~~y BBBn eround annulate Iemel~ae. x 36,000. Fig. 10. Redial cisternae consist of stacks of cisternae end numeroua spherical particles. The spherical particles ere clustered on both ends of the ckternse, have- high electron density and are embedded in a less electron dense matrix. x 37,000.
34
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Fig. 14. Annulate lamellae. and two elements of radial cisternae, bundle of short cisternae and spherical particles are closely associated. X 31,000.
cisternae were densely packed together, the cytoplasmic matrix between these cisternae was not visible in a large part of a single bundle (Fig. 11). Each cisterna was wavy and significantly shorter in length than that of annulate lamellae. Some of these cisternae were connected directly with rough-surfaced endoplasmic reticulum with luminal and membrane continuity (Figs. 12, 13). Although the periodical arranged annuli observed in the cisternae of annulate lamellae were not present in the well developed cisternae of radial cisternae (Figs. 12, 13), an intermediate form between two typical cisternae was also present (Fig. 14). Each bundle of cisternae showed a characteristic appearance with double zones of different electron density arranged alternately (Figs. 10-13). One of them composed of amorphous high electron dense material transversed straightly on a bundle of the cisternae and measured 930 A in width, and the other consisted of electron lucid part of the cisternae which varied considerably in length. Figs. 12 and 13. Photograph shown in Fig. 12 was taken from the specimen maintained on a horizontal plane and in Fig. 13 the specimen was tilted to an angle of 45'. Right side of the clustered spherical particles in Fig. 12 have no direct connection with cisbrnae (double arrow). With tilting short profiles of the cistarnee appear in the same area of Fig. 13 (double arrow). Some of the cisternae demonatrate direct connection with rough endoplasmic reticulum (arrow). Depending upon the plane of section through the cieternae, aggregated small vesicles (SV) are found near the annulate lamehe complexes. Figs. 12 and 13
x 31,Ooo.
30
ANNWLATn
aOMPLEXES
A& Pdh. Jap.
On the plane of section the bundle of cisternae appeared as membrane bounded vesicles measuring up to 690 A in diameter located in one area of annulate lamellae complexes (Figs. 12, 13). These vesicles were distinctly different from the particles, which appeared in annulate lamellae depending upon the plane of section through the annular region, having single limiting membrane and definite size. Spherical particles which were one of the peculiar elements of annulate lamellae complexes were located on one side or both sides of cisternae (Figs. 11-14). These particles were composed of high electron dense material without distinct limiting membrane but were embedded in a substance of moderate electron density (Figs. 11-14). They measured up to 1200 A in diameter, were clustered in a group, and their number varied considerably, ranging from 10 to as many as 60. At the periphery of aggregated spherical particles, immature forms of these structures were occasionally observed and appeared to be produced by coalescence of several cisternae at the zone of high electron density into irregular bodies and by separation from cisternae at the electron opaque zone (Fig. 11). Therefore the remaining membrane structure of the cisternae was still visible in some of these bodies. Some particles located near the periphery of the cluster appeared to have no direct connection with the cisternae on electron micrographs taken from a horizontal plane of section, however, they had direct connections with the cisternae, when the specimen was tilted to various angles (Figs. 12, 13).
Dascwr& Annulate lamellae and radial cisternae have been studied in both benign and malignant human tumors. Annulate lamellae were observed in all cases, whereas radial cisternae were found only in a case of leiomyosarcoma. The fine structural characteristics of annulate lamellae are identical to those observed in the germ cells of both invertebrate and somatic ~]ls@?6N as well 88 experimental t u m o r ~ ~ ~ ~ ~ ~ 7 ~ ~ 1 . Annulate lamellae are characterized by stacks of parallel arrayed cisternae bounded by smooth membrane and each cisterna is composed of alternately arranged annuli and sacs. This arrangment of both elements resembles nuclear pores in some aspects. K E S S E Lhas ~ ~reported an excellent paper on the morphogenesis of both intranuclear and intracytoplasmic annulate lamellae. He concluded that the blebbing of the outer nuclear envelope contributes to the vesicular and rough endoplasmic reticulum and perhaps to the differentiation of cytoplasmic annulate lamellae. Our studies support his idea on the morphogenesis of annulate lamellae because annulate lamellae observed in this study were frequently directly connected with rough endoplasmic reticulum with luminal and membrane continuity, although they were located in the cytoplasm quite some distance from the nucleus and showed no direct connections with the nuclear membrane. With regard to similarity of h e structure of the nuclear envelope and annulate lamellae, the ultrastructure of nuclear pore has been compared with annular region of
27(1): 1977
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the annulate lamellae. Several investigators6tlltl490 described that the nuclear pores were bridged by a membrane structure which was interpreted as a diaphragm or a portion of the nuclear membrane. In contrast to nuclear pores there are two different hypotheses concerning the presence of membrane across the annular region of the annulate lamellae. R o s s ~ ~and , BALINSKand DOVIS~ have shown the annular sugregion in annulate lamellae to be transversed by a membrane, whereas KESSEL~~ gested that the annular region in at least some annulate lamellaeisnot bridged b y a membrane. In our study the annular region of annulate lamellae showed a single electron dense membrane but exhibited no double membrane either in the horizontal plane or after tilting of the same annulate lamellae to various angles. We failed to observe the presence of a hole without any crossing membrane in the annular region. However, cross sections through the annular region showed electron dense spherical particles without any distinct membrane structure, which were definitely different from the particles of radial cisternae with absence of cisternae associated with the particles. This result suggests that some part of the annular region might not be bridged by membrane structure and therefore the particles without membrane structure were produced at the annular region. The function of annulate lamellae has remained obscure, although several hypotheses concerning this subject have appeared. In our studies the annulate lamellae do not have ribosomes associated with their membrane structure but have amorphous materials with either high or low electron density which was suggested by M E R R I A N ~ ~ to be ribonucleoprotein in nonparticulate form. However, the annulate lamellae have direct connection with rough-surfaced endoplasmic reticulum. The connection of two types of cisternae might suggest that annulate lamellae represent a specific form of the rough endoplasmic reticulum and the fine structural difference of these two cisternae may support the view of functional differences. Some annulate lamellae in our study have developed to radial cisternae having more complicated structure, which are never associated with rough-surfaced endoplasmic reticulum. Furthermore KANE~O showed that following the extraction of one-half of the cellular protein in the Arbacian egg, the annulate lamellae were unaffected, while the endoplasmic reticulum was much more markedly affected. PASTEELS et aL21 noted that most of the vesicular, rough-surfaced endoplasmic reticulum was present in zone 2 while annulate lamellae were observed in zone 3 in the space between platelates in the centrifuged fertilized egg of Paracentrotus livkdus. These fine structural characteristics and several previous o b s e r v a t i o n s ~ 0 ~ ~ ~ ~ ~ ~ ~ 2 ~ ~ 2 ~ suggest that the annulate lamellae are a structure of considerable importance in physical and chemical activities of the cell. Radial cisternae described by PATRIZI and MIDDELKAMP~~ were also observed in this study. It was considered that radial cisternae were a more developed form of annulate lamellae, because the intermediate types of cisternae between annulate lamellae and radial cisternae are present, although the fine structure of completely developed radial cisternae was somewhat different from that of annulate lamellae.
38
AXNULATE L-LLAE
OO?KPWXNS
Acta P d h . Jap.
Radial cisternae were characterized by two elements: short, parallel arrayed cisternae arranged in a group and numerous spherical particles. Each group of cisternae occasionally showed radial arrangment around the spherical particles. These particles consisting of high electron dense material without distinct limiting membrane appeared to be produced by cisternae after a coalescence of two or three cisternae at the end and separation of them a t the electron lucid part. These radial cisterane observed in this study are identical to those described by PATRIZI and MIDDELKAMP~~ in rabbit kidney cells (RK-13 cells) a t 7-8 days after rubella virus infection, although type two particles described by them were not found in this study. Several investigators~2~lS~I6.eP described annulate lamellae or its complexes in the cytoplasm of the cells infected by several viruses, virus induced experimental tumor and even in the human Burkitt’s lymphoma1 considered to be produced by the EV virus. It seems to us that the appearance of the annulate lamellae and its complexes would be an active response of the host cell to virus infection, however, the formation of the annulate lamellae and radial cisternae is not always limited to viral infection but appeared as a result of nonspecific active cell reaction against various physical and pathological stimuli including virus infection, because we failed to observe any viral particles in any of our cases. Furthermore an identical structure has been observed in as well as human virus infected cell and in several 0ocytes~JOJ1JS~*~, somatic cells@I26J@ tumors8 which are not considered to be caused by virus. Acknozulegement: The authors are grateful to Mr. Masanobu ANAMI, Miss Sachie HAYASHIand M k Hitomi HAMADA for their skilled technical assistance.
Referenoes 1. ACHONQ, B.G., and EPSTEIN,M.A.: Fine structure of the Burkitt’s Tumor. J. Nat. Cancer Inst. 36: 877-897, 1986. 2. AFZELIUS, B.A.: Electron microscopy on the besophilic structure of the sea urchin egg. Z. Zellfarsch 45: 660475, 1957. 3. BALINSRY,B.I., and Dov~s,R.J.: Origin and differentiation of cytoplaamic structurea in the oocytea of xenopus leavie. Acta Embryo1 Morphol. Exptl. 6 : 55-108, 1963. S., and MEEK, G.A.: The origin and fate of nuclear membrane in 4. BARER,R., JOSEPH, meiosis. Proc. Roy. SOC.B. 152: 353-368, 1960. 5. BARNES,B.G., and DAVIS,J.M.:
The structure of nuclear pores in mammalian tissue. J. Ultrastruct. Res. 3 : 131-146, 1959. 6. BINQQELI,M.F. : Abnormal intranuclear and cytoplasmic formations associated with chemically induced, transplatable chicken sarcoma. J. Biophys. Biochem. Cytol. 5: 143-152, 1952.
7.
GROSS, P.R., PJIILPOTT, D.E., and NASS,S.: Electron microscopy of the centrifuged sea urchin egg, with a note on the structure of the ground cytoplasm. J. Biophys. Biochem. Cytol. 7: 135-142, 1960.
F., MIN, K.W., KRISKO, I., and GYORKPIY, P.: The usefullnees of electron micros8. GYORKEY, copy in the diagnosis of human tumors. Human Pathology 6 : 421-441, 1975. 9. HAEBISON,G.: The presence of annulate lamellee in the sea gull adrenal. Am. Soc. Cell Biol. Second Ann. Meeting (Abstr), 1982. 10. KANE,R.E.: The effect of partial protein extraction on the structure of the egg of the Be8 urchin. Arbacia punctulata. J. Biophys. Biochem. cytol. 7: 21-26, 1960. R.G.: Intranuclear and cytoplasmic annulate lamellee in tunicate oocytea. J. 11. KESSEL,
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39
Cell Biol. 24: 171-187, 1965. E.S.: Electron microscopy of monkey kidney cell culturea 12. KIM, K.S.W., and BOATMAN, infected with rubella virus. J. Viral 1: 205-214, 1987. 13. KOESTNER,A,, KASZA, L., and KIND IN^, 0.: Electron microscopy of tiseue cultures infected with porcine polioencephalomyelitis virus. Am. J. Path. 48: 129-147, 1966. 14. MAEOWALD,A.P.: Fine structure of pole cells and polar granules in D m p h i l a Melanogaster. J. Exptl. Zool. 151: 201-216, 1962. N.G.: The nuclear envelope of plant cells. J. Ultrastruct. Rss. 3: 328-333, 1960. 15. MARINOS, , PARDO, M., and RAPOZA, N.P.: Pathologenesis of oncogenic 16. MERKOW,L.P., S L ~ U NM., simian a d e n o v i m M.The origii of annulate lamellae in LLC-MK 2 cell infected with SV 30. J. Ultrastruct. Ree. 30: 344-353, 1970. 17. MERRIAN,R.W.: The origin and fate of annulate lamellee in maturing sand dollar eggs. J. Biophys. Biochem. Cytol. 5: 117-122, 1959. E., and WADDINGITON, C.H.: The submicroscopic structure of the Drosophila egg. 18. OEADA, J. Embryol. Exptl. Morphol. 7: 583-697, 1959. G.E.: Studies on the endoplasmic reticulum 11. Simple disposition in cell in situ. 19. PALADE, J. Biophys. Biochem. Cytol. 1 : 567481, 1956. Studies on the pathognesis of M.,and R ~ z AN.P.: , 20. MERKOW. L., SLIFEIN, M., PARDO, simian adenovirus induced tumors 111. The histopathology and ultrestructure of intracranial neoplasm induced by SV 20. J. Nat. Cancer Inst. 41 : 1051-1070, 1968. J.J.. CASTIAIJX, P., and VANDERMWRSSCHE, G.: Cytochemical localizations and 21. PASTEELS, ultrastructure in the fertilized unsegmented egg of Paracentrotus lividus. J. Biophys. Biochem. Cytol. 4: 575678, 1958. G., and MIDDELKAMP,J.N.: Development and changes of annulate lamellae 22. PATRIZI, complexes in Rubella virus-infected RK-13 cells. J. Ultrastruct. Ree. 31 : 407423, 1970. L.I.: Electron microscopy of besophilic structure of some invertebrate oocytes. J. 23. REBHITN, Biophys. Biochem. Cytol. 2 : 93-104, 1956. E.S.: The use of lead citrate a t high pH es an electron opaque stain in electron 24. REYNOLDS, microscopy. J. Cell Biol. 17: 208-212. 1963. 25. ROSS,M.H.: Annulate lamellee in the adrenal cortex of the fetal rat. J. Ultrastruct. Res. 7: 373383, 1962. A.: Besophilic lamellar system in the crayfish spermatocyte. J. Biochys. 26. RUTEIKANN, Biochem. Cytol. 4: 267-274, 1968. 27. SOWLZ, Z. : Elektronermikcroskopische Unbrsuchungen eines Mammakarzinomas der Ratte. Oncologia 10: 317-329, 1957. G., BAUTUS,E., HANOCO-QUERTIER, J., and BRACHET,J.: Ultrastructure of 28. STEINBRT, xenopua laevia oocytes after injection of an extract from progesterone-treated oocytee. J. Ultrastruct. Ree. 49: 188-210, 1974. 29. SWIBT, H.: The fine structure of annulate lamellee. Biophys. Biochem. Cytol. 2 : 415425, 1956. 30. WATSON,M.L.: Further observations on the nuclear envelope of the animal cell. J. Biophys. Biochem. Cytol. 6 : 147-156, 1969. W. : Vergleichende electronen-mikroskopiache Untersuchung 31. WESSEL,W., and BERNHARD. von Ehrlich and Yoshida Ascitesturnonellen. Z. Krebsforsch 62: 1W162, 1957. 32. WISOHNITZER, S.J.: Observation on the annulate lamellee of immature amphibian oocytea. J. Biophys. Biochem. Cytol. 8: 558683, 1960.
FINE STRUCTURAL STUDY O F ANNULATE LAMELLAE COMPLEXES I N HUMAN TUMORS Iwao NAKAYAMA, Akira MORIUCHI,Yoshitaugi TAIRA, Osamu TAKAHARA, and Nobuo TSUDA* Takashi ITOGA
*
Department of Central Diqmstic Laboratmy. Nagasaki University Haapital, Nagaaaki Department of Pdhlogy. Atomic Dieeaae Inetitute Nagasaki University School of Medicine, Nagaaaki (Received on April 12, 1976)
Specific intracytoplasmic organelles, annulate lamellae and radial cisternae, have been studied In several human tumors. Annulate lamellae are observed in all cases of leiomyoma, lelomyosarcoma, rhabdomyosarcoma and malignant melanoma, whereas radial cisternae are only found in a case of leiomyosarcoma. Annulate lamellae are characterized by stacks of parallel arrayed long cisternae showing alternative arrangement of annuli and sacs. Some of these cisternae are connected directly with rough-surfaced endoplasmic reticulum and there is continuity with the lumen and membrane. Radial cisternae are mainly composed of two structures: numerous short clsternae, which are a variant of annulate lamellae, and numerous spherical particles derived from the cisternae. The cisternae are arranged parallel or radially around particles measuring up to 1100 A in diameter. These particles consisting !of an amorphous high electron dense materlal without distinct limiting membrane are organized in groups and vary in number. There is no evidence of a direct relationship between these structures and viral infection. ACTA PATH JAP. 27: 25-39,
1977.
Iratroduction In 1956 SWIFT^^ was the first to describe annulate lamellae, a specific intracytoplasmic organelle which is composed of stacks of parallel lamellae. These annulate lamellae are characterized by periodically arranged annuli resembling those of the nuclear envelope. KESSEL~~ compared annulate lamellae with annuli of the nuclear envelope in tunicate oocytes; he published an excellent paper on the morphogenesis of both intranuclear and intractyoplasmic annulate lamellae. Although annulate lamellae are commonly observed in the cytoplasm of both invertebrate and vertebrate germ ~ells2,4,7,11,14,1~,19,29,2~,29,92, they have been also observed in the cytoplasm of somatic cellsg?6P,experimental tumors %'%27,a1 as well as human tumors.1q8 The function of annulate lamellae has remained obscure, although SWIFT*^ suggested that annulate lamellae may function in nuclear-cytoplasmic transfer. Several reports1612'J#22have appeared on the origin of annulate lamellae and the relationship between annulate lamellae and virus infection in viral infected cells and virus induced tumor.
26
AN'NULATH LAMELLAE 00bfPLEXES
Acta Path. Jap.
In recent years PATRIZI and MIDDELKAMP** reported annulate lamellae and radial cisternae associated with two types of spherical particles in rubella virus infected RK13 cells and suggested that these intracytoplasmic organelles may be caused by virus infection, because the structures were absent from non-infected cells. These intracytoplasmic organelles, annulate lamellae and radial cisternae were referred to as annulate lamellae complexes>* because of demonstration of intermediate form between the two organelles. It is of interest that the appearance of annulate lamellae complexes may or may not be directly connected to viral infection as an etiological agent for the development of the neoplasm. In this paper annulate lamellae in several human benign and malignant tumors were studied by electron microscopy and, based upon their fine structure, a hypothetical relationship between development of annulate lamellae complexes and virus infection is discussed.
Materials and Me&ds The histological diagnosis, age and sex of the patients and original site of the tumor for each oase are given in Table 1.
For light microscopic studiea, a portion of each tumor was fixed in 10% neutral formalin, embedded in paraffin and stained with hematoxylin-eoein, periodic acid Schiff (PAS), reticulin, phosphotungstic acid hematoxylin (PTAH) and melanin stains. For electron microscopic study, tissuea obtained in the operating Mom were quiokly transferred into 1.6% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). During fixation the tissuea were separated into numerous small pieces and p l d in fresh fixative for an additionel 4 hours. After washing overnight in phosphate buffer, tissues were post-fixed in 2% osmium-tetraxide for 2 houre, dehydrated in a graded seriee of ethanol followed by propylene-oxide, and embedded in Epon 812. Semithin sections of the entire area were cut and stained with buffered toluidine blue to identify the tumor tissue. Thin sections from selected portions of the blocks were out with grasn knives on a JEOL JUM-7 ultramiomtome end were double stained with a saturated aqueous solution of uranyl aoetate followed by lead citrate'.. Micrographs were taken with a JEM lOOB electron micromope with side entry type. Annulate lamellae and radial oisternae were observed from various directions using rotation and tilting of the specimens to obtain a t h dimensional structure. Table 1. &a&iah Case
No. Age
1
66
a s
46
4
51
46
and R d t a
Sex
Histological Dix.
Site of origin
F F F F
Leiomyoearcoma Rhabdomyoearcoma Malignant melanoma Leiomyoma
Mesocolon Retroperitoneum Nasal cavity Small intestine
AL
RC
+ + + + + -
AL: Annulate lamellae RC: Redial oisternae
Results Annulate LameUae
Annulate lamellae were observed in all cases examined in this study. Since the Figs. 1 and 2. Annulate lamellee are composed of a considerably variable number of cisternae. Each cisterna is bounded by smooth membrane having periodic narrowing. Both ends of cieternae in Fig. 2 demonstrate formation of vesicles (V). There are short profiles of rough endoplasmio reticulum with numerous ribosomes around annulate lamellee. Fig. 1 x 43, OOO and Fig. 2. x 36,000.
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Fig. 6. Annular regions are palisaded in a line on the stacks of the cisternee (arrow). Each cisterna shows an alternating arrangement of annuli and sac%. Amorphous material with high electron density are observed around the annuli. x 46,000.
fine structural characteristics of annulate lamellae are essentially similar from one case to the other, the common morphological characteristics of annulate lamellae encountered in all materials will be described here. Annulate lamellae were composed of stacks of cisternae, bounded by smooth membrane. The number of the cisternae in a single stack was considerably different from one case to the other and even in the same case, and varied from two to as many as more than one dozen (Figs. 1-2). Each cisterna had periodic narrowing8 of its lumen, which were referred to as annuli, lumina or pore with resemblance to that of the nuclear envelope. In Bsomeof the annuli, two membrane structures appeared to fuse into a single membrane having more electron density and the other still had a double membrane with more electron density (Fig. 3 arrow). With the tilting of the same annulate lamellae the fine structure of the well developed annular region was not altered to double membrane but showed a single electron dense membrane (Fig. 4). Figs. 3 and 4. Photographs of thew annulate lamellae were taken from the same area a t a different angle. The photograph shown in Fig. 3 was taken while keeping the specimen on a horizontal plane and in Fig. 4 the specimen was tilted to an angle of 26'. After tilting, the h e structure of the annular region is not altered to double membrane but shows a more distinct single membrane with high electron density (arrow). Figs. 3 and 4 x 60,000.
30
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Fig. 6. Photograph shows annular regiona cut in almoet a tangential plane through the annuli of annulate lamellee. Spherical particlea arranged in a line consist of high electron dense materials which are embedded in lees electron dense materials but do not have distinct limiting membrane. The left upper porition (arrow) of the photograph shows a remnant of annulate lamellae. x 60,000.
These annuli appeared to be palisaded in a line on stacks of the cisternae. Amorphous material with high electron density was associated with each annuli (Fig. 6). Each sac located between annuli of a cisterna varied considerably, ranging from 714 A to more than 1900 A in length and the mean length of annuli measured 750 A. The distance between two cisternae was about 890 A. If the plane of section passed through the annuli, spherical particles surrounded by less electron dense materials appeared and were arranged in a line (Fig. 6). These particles measuring approximately 1100 A in diameter consisted of high electron dense material formed by an aggregation of small and high electron dense dots and had no distinct limiting membrane between two different electron dense materials (Fig. 6). Depending on the plane of section an intermediate form between typical annuli and these particles was observed (Fig. 7). Both edges of the annuli were connected with sacs which contained electron lucid material. Fig. 7. Annulate lamellae of an intermediate form between Fig. 6 and 6. There are many rodlike structure with electron lucid materials around annulate Ismellee. x 43,000. Fig. 8. Annulate lamellae show direct connection with rough endoplaemic reticulum with luminal and membranoue continuity. x 46,000.
-;
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ANNULATE LAMELLAE OOWLEXES
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Fig. 11. Portion of radial cisternae. The ckternae bounded by smooth membrene are densely packed together and radially arranged around the dusted spherical particles but do not demonstrate annuli. A single stack of the cisternee exhibits characteristic double zonal structure of high (HD) and low electron density (LD). The particles are embedded in e less electron dense matrix without limiting membrane. Newly forming particles (arrow), located in the periphery of the clueter, appear to be produced by coalescence of several cisternae. x 54,Ooo.
Some cisternae of annulate lamellae were directly connected with rough-surfaced endoplasmic reticulum showing luminal and membrane continuity (Figs. 2, 8)) whereas other annulate lamellae demonstrated formation of a moderate number of vesicles at the end of the cisternae (Fig. 2). There were numerous polysomes and rod-like structure containing electron lucid material located around annulate lamellae (Figs. 2, 9).
Radial cisternae observed only in a case of leiomyosarcoma were composed of bundles of short cisternae, bounded by smooth membrane, and of spherical particles with high electron density. A single bundle of cisternae was in parallel and oriented in a radial arrangment around the centrally aggregated particles (Fig. 10). Since these Fig. 9. Aggregeted ribosomes ere occesione~~y BBBn eround annulate Iemel~ae. x 36,000. Fig. 10. Redial cisternae consist of stacks of cisternae end numeroua spherical particles. The spherical particles ere clustered on both ends of the ckternse, have- high electron density and are embedded in a less electron dense matrix. x 37,000.
34
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Fig. 14. Annulate lamellae. and two elements of radial cisternae, bundle of short cisternae and spherical particles are closely associated. X 31,000.
cisternae were densely packed together, the cytoplasmic matrix between these cisternae was not visible in a large part of a single bundle (Fig. 11). Each cisterna was wavy and significantly shorter in length than that of annulate lamellae. Some of these cisternae were connected directly with rough-surfaced endoplasmic reticulum with luminal and membrane continuity (Figs. 12, 13). Although the periodical arranged annuli observed in the cisternae of annulate lamellae were not present in the well developed cisternae of radial cisternae (Figs. 12, 13), an intermediate form between two typical cisternae was also present (Fig. 14). Each bundle of cisternae showed a characteristic appearance with double zones of different electron density arranged alternately (Figs. 10-13). One of them composed of amorphous high electron dense material transversed straightly on a bundle of the cisternae and measured 930 A in width, and the other consisted of electron lucid part of the cisternae which varied considerably in length. Figs. 12 and 13. Photograph shown in Fig. 12 was taken from the specimen maintained on a horizontal plane and in Fig. 13 the specimen was tilted to an angle of 45'. Right side of the clustered spherical particles in Fig. 12 have no direct connection with cisbrnae (double arrow). With tilting short profiles of the cistarnee appear in the same area of Fig. 13 (double arrow). Some of the cisternae demonatrate direct connection with rough endoplasmic reticulum (arrow). Depending upon the plane of section through the cieternae, aggregated small vesicles (SV) are found near the annulate lamehe complexes. Figs. 12 and 13
x 31,Ooo.
30
ANNWLATn
aOMPLEXES
A& Pdh. Jap.
On the plane of section the bundle of cisternae appeared as membrane bounded vesicles measuring up to 690 A in diameter located in one area of annulate lamellae complexes (Figs. 12, 13). These vesicles were distinctly different from the particles, which appeared in annulate lamellae depending upon the plane of section through the annular region, having single limiting membrane and definite size. Spherical particles which were one of the peculiar elements of annulate lamellae complexes were located on one side or both sides of cisternae (Figs. 11-14). These particles were composed of high electron dense material without distinct limiting membrane but were embedded in a substance of moderate electron density (Figs. 11-14). They measured up to 1200 A in diameter, were clustered in a group, and their number varied considerably, ranging from 10 to as many as 60. At the periphery of aggregated spherical particles, immature forms of these structures were occasionally observed and appeared to be produced by coalescence of several cisternae at the zone of high electron density into irregular bodies and by separation from cisternae at the electron opaque zone (Fig. 11). Therefore the remaining membrane structure of the cisternae was still visible in some of these bodies. Some particles located near the periphery of the cluster appeared to have no direct connection with the cisternae on electron micrographs taken from a horizontal plane of section, however, they had direct connections with the cisternae, when the specimen was tilted to various angles (Figs. 12, 13).
Dascwr& Annulate lamellae and radial cisternae have been studied in both benign and malignant human tumors. Annulate lamellae were observed in all cases, whereas radial cisternae were found only in a case of leiomyosarcoma. The fine structural characteristics of annulate lamellae are identical to those observed in the germ cells of both invertebrate and somatic ~]ls@?6N as well 88 experimental t u m o r ~ ~ ~ ~ ~ ~ 7 ~ ~ 1 . Annulate lamellae are characterized by stacks of parallel arrayed cisternae bounded by smooth membrane and each cisterna is composed of alternately arranged annuli and sacs. This arrangment of both elements resembles nuclear pores in some aspects. K E S S E Lhas ~ ~reported an excellent paper on the morphogenesis of both intranuclear and intracytoplasmic annulate lamellae. He concluded that the blebbing of the outer nuclear envelope contributes to the vesicular and rough endoplasmic reticulum and perhaps to the differentiation of cytoplasmic annulate lamellae. Our studies support his idea on the morphogenesis of annulate lamellae because annulate lamellae observed in this study were frequently directly connected with rough endoplasmic reticulum with luminal and membrane continuity, although they were located in the cytoplasm quite some distance from the nucleus and showed no direct connections with the nuclear membrane. With regard to similarity of h e structure of the nuclear envelope and annulate lamellae, the ultrastructure of nuclear pore has been compared with annular region of
27(1): 1977
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the annulate lamellae. Several investigators6tlltl490 described that the nuclear pores were bridged by a membrane structure which was interpreted as a diaphragm or a portion of the nuclear membrane. In contrast to nuclear pores there are two different hypotheses concerning the presence of membrane across the annular region of the annulate lamellae. R o s s ~ ~and , BALINSKand DOVIS~ have shown the annular sugregion in annulate lamellae to be transversed by a membrane, whereas KESSEL~~ gested that the annular region in at least some annulate lamellaeisnot bridged b y a membrane. In our study the annular region of annulate lamellae showed a single electron dense membrane but exhibited no double membrane either in the horizontal plane or after tilting of the same annulate lamellae to various angles. We failed to observe the presence of a hole without any crossing membrane in the annular region. However, cross sections through the annular region showed electron dense spherical particles without any distinct membrane structure, which were definitely different from the particles of radial cisternae with absence of cisternae associated with the particles. This result suggests that some part of the annular region might not be bridged by membrane structure and therefore the particles without membrane structure were produced at the annular region. The function of annulate lamellae has remained obscure, although several hypotheses concerning this subject have appeared. In our studies the annulate lamellae do not have ribosomes associated with their membrane structure but have amorphous materials with either high or low electron density which was suggested by M E R R I A N ~ ~ to be ribonucleoprotein in nonparticulate form. However, the annulate lamellae have direct connection with rough-surfaced endoplasmic reticulum. The connection of two types of cisternae might suggest that annulate lamellae represent a specific form of the rough endoplasmic reticulum and the fine structural difference of these two cisternae may support the view of functional differences. Some annulate lamellae in our study have developed to radial cisternae having more complicated structure, which are never associated with rough-surfaced endoplasmic reticulum. Furthermore KANE~O showed that following the extraction of one-half of the cellular protein in the Arbacian egg, the annulate lamellae were unaffected, while the endoplasmic reticulum was much more markedly affected. PASTEELS et aL21 noted that most of the vesicular, rough-surfaced endoplasmic reticulum was present in zone 2 while annulate lamellae were observed in zone 3 in the space between platelates in the centrifuged fertilized egg of Paracentrotus livkdus. These fine structural characteristics and several previous o b s e r v a t i o n s ~ 0 ~ ~ ~ ~ ~ ~ ~ 2 ~ ~ 2 ~ suggest that the annulate lamellae are a structure of considerable importance in physical and chemical activities of the cell. Radial cisternae described by PATRIZI and MIDDELKAMP~~ were also observed in this study. It was considered that radial cisternae were a more developed form of annulate lamellae, because the intermediate types of cisternae between annulate lamellae and radial cisternae are present, although the fine structure of completely developed radial cisternae was somewhat different from that of annulate lamellae.
38
AXNULATE L-LLAE
OO?KPWXNS
Acta P d h . Jap.
Radial cisternae were characterized by two elements: short, parallel arrayed cisternae arranged in a group and numerous spherical particles. Each group of cisternae occasionally showed radial arrangment around the spherical particles. These particles consisting of high electron dense material without distinct limiting membrane appeared to be produced by cisternae after a coalescence of two or three cisternae at the end and separation of them a t the electron lucid part. These radial cisterane observed in this study are identical to those described by PATRIZI and MIDDELKAMP~~ in rabbit kidney cells (RK-13 cells) a t 7-8 days after rubella virus infection, although type two particles described by them were not found in this study. Several investigators~2~lS~I6.eP described annulate lamellae or its complexes in the cytoplasm of the cells infected by several viruses, virus induced experimental tumor and even in the human Burkitt’s lymphoma1 considered to be produced by the EV virus. It seems to us that the appearance of the annulate lamellae and its complexes would be an active response of the host cell to virus infection, however, the formation of the annulate lamellae and radial cisternae is not always limited to viral infection but appeared as a result of nonspecific active cell reaction against various physical and pathological stimuli including virus infection, because we failed to observe any viral particles in any of our cases. Furthermore an identical structure has been observed in as well as human virus infected cell and in several 0ocytes~JOJ1JS~*~, somatic cells@I26J@ tumors8 which are not considered to be caused by virus. Acknozulegement: The authors are grateful to Mr. Masanobu ANAMI, Miss Sachie HAYASHIand M k Hitomi HAMADA for their skilled technical assistance.
Referenoes 1. ACHONQ, B.G., and EPSTEIN,M.A.: Fine structure of the Burkitt’s Tumor. J. Nat. Cancer Inst. 36: 877-897, 1986. 2. AFZELIUS, B.A.: Electron microscopy on the besophilic structure of the sea urchin egg. Z. Zellfarsch 45: 660475, 1957. 3. BALINSRY,B.I., and Dov~s,R.J.: Origin and differentiation of cytoplaamic structurea in the oocytea of xenopus leavie. Acta Embryo1 Morphol. Exptl. 6 : 55-108, 1963. S., and MEEK, G.A.: The origin and fate of nuclear membrane in 4. BARER,R., JOSEPH, meiosis. Proc. Roy. SOC.B. 152: 353-368, 1960. 5. BARNES,B.G., and DAVIS,J.M.:
The structure of nuclear pores in mammalian tissue. J. Ultrastruct. Res. 3 : 131-146, 1959. 6. BINQQELI,M.F. : Abnormal intranuclear and cytoplasmic formations associated with chemically induced, transplatable chicken sarcoma. J. Biophys. Biochem. Cytol. 5: 143-152, 1952.
7.
GROSS, P.R., PJIILPOTT, D.E., and NASS,S.: Electron microscopy of the centrifuged sea urchin egg, with a note on the structure of the ground cytoplasm. J. Biophys. Biochem. Cytol. 7: 135-142, 1960.
F., MIN, K.W., KRISKO, I., and GYORKPIY, P.: The usefullnees of electron micros8. GYORKEY, copy in the diagnosis of human tumors. Human Pathology 6 : 421-441, 1975. 9. HAEBISON,G.: The presence of annulate lamellee in the sea gull adrenal. Am. Soc. Cell Biol. Second Ann. Meeting (Abstr), 1982. 10. KANE,R.E.: The effect of partial protein extraction on the structure of the egg of the Be8 urchin. Arbacia punctulata. J. Biophys. Biochem. cytol. 7: 21-26, 1960. R.G.: Intranuclear and cytoplasmic annulate lamellee in tunicate oocytea. J. 11. KESSEL,
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Cell Biol. 24: 171-187, 1965. E.S.: Electron microscopy of monkey kidney cell culturea 12. KIM, K.S.W., and BOATMAN, infected with rubella virus. J. Viral 1: 205-214, 1987. 13. KOESTNER,A,, KASZA, L., and KIND IN^, 0.: Electron microscopy of tiseue cultures infected with porcine polioencephalomyelitis virus. Am. J. Path. 48: 129-147, 1966. 14. MAEOWALD,A.P.: Fine structure of pole cells and polar granules in D m p h i l a Melanogaster. J. Exptl. Zool. 151: 201-216, 1962. N.G.: The nuclear envelope of plant cells. J. Ultrastruct. Rss. 3: 328-333, 1960. 15. MARINOS, , PARDO, M., and RAPOZA, N.P.: Pathologenesis of oncogenic 16. MERKOW,L.P., S L ~ U NM., simian a d e n o v i m M.The origii of annulate lamellae in LLC-MK 2 cell infected with SV 30. J. Ultrastruct. Ree. 30: 344-353, 1970. 17. MERRIAN,R.W.: The origin and fate of annulate lamellee in maturing sand dollar eggs. J. Biophys. Biochem. Cytol. 5: 117-122, 1959. E., and WADDINGITON, C.H.: The submicroscopic structure of the Drosophila egg. 18. OEADA, J. Embryol. Exptl. Morphol. 7: 583-697, 1959. G.E.: Studies on the endoplasmic reticulum 11. Simple disposition in cell in situ. 19. PALADE, J. Biophys. Biochem. Cytol. 1 : 567481, 1956. Studies on the pathognesis of M.,and R ~ z AN.P.: , 20. MERKOW. L., SLIFEIN, M., PARDO, simian adenovirus induced tumors 111. The histopathology and ultrestructure of intracranial neoplasm induced by SV 20. J. Nat. Cancer Inst. 41 : 1051-1070, 1968. J.J.. CASTIAIJX, P., and VANDERMWRSSCHE, G.: Cytochemical localizations and 21. PASTEELS, ultrastructure in the fertilized unsegmented egg of Paracentrotus lividus. J. Biophys. Biochem. Cytol. 4: 575678, 1958. G., and MIDDELKAMP,J.N.: Development and changes of annulate lamellae 22. PATRIZI, complexes in Rubella virus-infected RK-13 cells. J. Ultrastruct. Ree. 31 : 407423, 1970. L.I.: Electron microscopy of besophilic structure of some invertebrate oocytes. J. 23. REBHITN, Biophys. Biochem. Cytol. 2 : 93-104, 1956. E.S.: The use of lead citrate a t high pH es an electron opaque stain in electron 24. REYNOLDS, microscopy. J. Cell Biol. 17: 208-212. 1963. 25. ROSS,M.H.: Annulate lamellee in the adrenal cortex of the fetal rat. J. Ultrastruct. Res. 7: 373383, 1962. A.: Besophilic lamellar system in the crayfish spermatocyte. J. Biochys. 26. RUTEIKANN, Biochem. Cytol. 4: 267-274, 1968. 27. SOWLZ, Z. : Elektronermikcroskopische Unbrsuchungen eines Mammakarzinomas der Ratte. Oncologia 10: 317-329, 1957. G., BAUTUS,E., HANOCO-QUERTIER, J., and BRACHET,J.: Ultrastructure of 28. STEINBRT, xenopua laevia oocytes after injection of an extract from progesterone-treated oocytee. J. Ultrastruct. Ree. 49: 188-210, 1974. 29. SWIBT, H.: The fine structure of annulate lamellee. Biophys. Biochem. Cytol. 2 : 415425, 1956. 30. WATSON,M.L.: Further observations on the nuclear envelope of the animal cell. J. Biophys. Biochem. Cytol. 6 : 147-156, 1969. W. : Vergleichende electronen-mikroskopiache Untersuchung 31. WESSEL,W., and BERNHARD. von Ehrlich and Yoshida Ascitesturnonellen. Z. Krebsforsch 62: 1W162, 1957. 32. WISOHNITZER, S.J.: Observation on the annulate lamellee of immature amphibian oocytea. J. Biophys. Biochem. Cytol. 8: 558683, 1960.
