ANNULATE LAMELLAE AND "YOLK NUCLEI"

IN OOCYTES OF

THE DRAGONFLY, LIBELLULA PULCHELLA

INTRODUCTION

So-called "yolk nuclei" or "Balbiani bodies" havebeen depicted on innumerable occasions in oocytes,including those of the insects (cf., 29, 34). Suchstructures consist of one or more localized areas ofthe ooplasm which stain basophilic, are Feulgen-negative, and are frequently characterized as re-sulting from nuclear or nucleolar "emissions (cf.,34) ." During the course of oocyte growth in thedragonfly, as well as in certain other insects, the"yolk nuclei" have been described as increasingin size in the cytoplasm, migrating to the corti-..al ooplasm, and subsequently fragmenting intosmaller, more numerous masses (13) . In some in-

R . G . KESSEL and H. W. BEAMS

From the Department of Zoology, the University of Iowa, Iowa City, Iowa 52240

ABSTRACTAnnulated membranes in the form of single and short lamellae are present adjacent to andparallel to the nuclear envelope in oogonia and early oocyte (synaptene) stages of thedragonfly, Libellula pulchella . These solitary and short annulate lamellae are usually con-tinuous with long, part rough- and part smooth-surfaced cisternae which extend into moredistal areas of the oogonial ooplasm. These particular annulate lamellae then either dis-appear or decrease in number to be replaced by a much more extensive system of annulatelamellae in the cortical ooplasm of previtellogenic oocytes. The differentiation of extensivestacks of annulate lamellae is consistently observed to be restricted to large cytoplasmic areasof considerable electron density . These cytoplasmic regions consist of material which stainsbasophilic and contains RNA but differs structurally from the large number of ribosomeswhich surround the dense masses. The cytoplasmic dense masses, in terms of their formationand staining reactions, are comparable to the "yolk nuclei" or "Balbiani bodies" describedin insect oocytes in earlier studies . The results of the present study thus provide evidencethat the appearance of cortical ooplasmic stacks of annulate lamellae in the dragonfly oocyteis specifically limited to cytoplasmic areas of high electron density which contain RNA butwhich do not have a ribosomal morphology .

sects they were portrayed as then transforminginto yolk material (13), although more recentstudies have shown this conclusion to have beenerroneous (cf., 4, 42) .

Annulate lamellae constitute a class of cyto-membranes which structurally resemble the nu-clear envelope in many respects and are widelydistributed in germ, somatic, and tumor cells(cf., 22 for review) . Certain details in the apparentorigin, structure, and distribution of these cyto-membranes suggest that they may have nuclearmaterials associated with them and thus play an

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important role in cytoplasmic regulation, differ-entiation, and growth (cf. 16-22) .

The present study is concerned with the charac-terization of a heretofore unique relationship be-tween "yolk nuclei" and the differentiation ofannulate lamellae, a relationship which providesmore substantial evidence that the differentiationof these cytomembranes, and perhaps their sub-sequent activity as well, are directly influenced bynuclear materials .

MATERIALS AND METHODS

The organisms used in this investigation, Libellulapulchella, were collected during the months of Maythrough August . The female dragonflies were eitherinjected with the primary fixative or the ovarioleswere excised and placed into the fixative which con-sisted of an ice-cold, 3% solution of glutaraldehyde(38) in 0 .1 M phosphate buffer (pH 7 .2) . Beforefixation, some of the dragonflies were injected with0.1 ml of a 3% solution of horseradish peroxidase(Type II, Sigma Chemical Co., St . Louis, Mo.) ininsect Ringer's . After durations of 15 or 30 min, theovarioles were excised and fixed as indicated above .Following fixation in glutaraldehyde for periods of1-3 hr, the ovarioles were rinsed in several changes ofice-cold, 0 .1 M phosphate buffer (pH 7 .2) for a periodranging in length from several hours to overnight .Individual ovarioles previously exposed to theperoxidase were then incubated with agitation for 20-30 min at 25 °C in 10 ml aliquots of Karnovsky'smedium (10, 15) . Controls consisted of incubation inwhich substrate or H202 was omitted from themedium. The ovarioles were then postfixed for 1-2 hrin an ice-cold, 1 % solution of osmium tetroxide (33)in 0.1 phosphate buffer (pH 7 .2) . The ovarioles wererapidly dehydrated in ascending series of coldethanols, treated with propylene oxide, infiltrated,and then embedded in Epon 812 (28) . The tissueswere sectioned with a Porter-Blum ultramicrotomeequipped with a diamond knife. The sections wereplaced on grids and stained with uranyl acetate (44)and lead citrate (36) . The sections were studied andphotographed in an RCA EMU-3G electron micro-scope .

For light microscopy, sections of Champy's-fixedovaries were stained with Heidenhain's iron hema-toxylin . Sections of Bouin's-fixed ovaries were stainedwith hematoxylin and eosin as well as with mercuricbromphenol blue (30) . Sections of ovaries fixed ineither Carnoy's fluid or 10% neutral buffered forma-lin were stained with the Korson's technique (27) forthe demonstration of nucleic acids . Similarly fixedsections were also stained, some of them after ribo-nuclease digestion, with acidic toludine blue O andIethylene blue (43) also for the demonstration of

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nucleic acids . 1 µ-thick Epon sections of glutaralde-hyde-fixed oocytes weremethylene blue (37) .

RESULTS

stained with azure II and

The dragonfly ovary is composed of numerousovarioles, each of which is characterized as thepanoistic type . Such ovarioles lack special nursecells or trophocytes, and the follicular enveloperepresents the only trophic tissue present (cf., 6) .Previous electron microscope studies have demon-strated that the follicle cells of the dragonflyovariole produce the egg envelopes (4) . For furtherdetails regarding the structure of the dragonflyovariole, the reader is referred to the article byBeams and Kessel (4) .

OogoniaThe oogonia are clustered in the proximal

portion of the ovariole designated as the germar-ium. The cells measure approximately 6-8 µ indiameter and contain large nuclei which occupynearly four-fifths of the total cell volume (Fig . 3) .At this stage, the nucleolar material is in a dis-persed state (Figs. 1-3), a condition which is incontrast to the presence of large and complexnucleoli in growing oocytes . In many cases smallmasses of nucleolar material abut against theinner layer of the nuclear envelope (Figs . 1-3) .The cytoplasm of oogonia contains several smallbut irregularly shaped areas which differ from thesurrounding, more particulate-appearing ooplasm(Fig . 1) . These areas are composed of a homogene-ous but amorphous material which is slightlymore electron opaque than the surrounding ribo-somes (Figs . 7-9) . These dense cytoplasmic massesresemble the dispersed nucleolar components intheir structure and electron density . Such localizedareas of the ooplasm probably represent earlystages in the formation of the "yolk nuclei ."

Annulate lamellae are occasionally observed inoogonia where they are consistently located closeto the nucleus, and are positioned in the ooplasm70-80 mµ away from the outer layer of the nuclearenvelope (Figs. 6, 7, 9) . In some instances, suchannulate lamellae are continuous with nonannu-lated cytoplasmic lamina which extend into moredistal regions of the ooplasm (Fig. 4) and whichare usually devoid of attached granules . The rela-tionship demonstrated in Fig . 4 suggests that anindividual cytoplasmic membranous lamina maybe regionally transformed into an annulate lamella

FIGURE 1 Portion of two oogonia in germarium . Small cytoplasmic mass (CM) probably represents earlystage in formation of "yolk nuclei ." Note structural similarity between the cytoplasmic mass and thedispersed nucleolar elements (NL) . X 19,000 .

FIGURE 2 Fibrous-appearing nucleolus (NL) adjacent to inner layer of nuclear envelope . Note conti-nuity of nucleolar material with nuclear pore (arrows) . Cytoplasm (C) . X 50,000.

FIGURE 3 Cluster of oogonia in germarium of ovariole . Dispersed nucleolar material (NL) in nuclei. Twoof the oogonia are stained black due to accumulation of peroxidase . Continuity between these two oogoniais evident at (C) . Observe, however, that the peroxidase is restricted from the compartments of the Golgicomplex (GA), mitochondria (M), and endoplasmic reticulum (ER) . The unlabeled arrows direct attentionto regions in which the outer layer of the nuclear envelope appears continuous with cytoplasmic laminaeof the endoplasmic reticulum . X 13,600 .

when a portion of the lamina comes in close apposi-tion to the nuclear envelope . Similar characteristicsof structure and density appear to be shared by thematerial associated with the pores of the nuclearenvelope and the annulate lamellae as well as thedispersed nucleolar material and cytoplasmicdense masses (Figs. 2, 6, 9) .

When the ovarioles are exposed to hemolymphcontaining horseradish peroxidase, a number ofoogonia consistently become blackened due to theaccumulation of large amounts of this protein . Insuch preparations, the cytoplasmic connectionsbetween oogonia are clearly observed (Fig . 3) .Such preparations also demonstrate clearly thatmany of the long cytoplasmic laminae previouslydescribed may be continuous with the outer layerof the nuclear envelope (Fig. 3) . The peroxidase is,however, consistently restricted from the internalcompartments of the mitochondria, Golgi com-plexes, and the cytoplasmic laminae (Fig . 3) .On the other hand, the peroxidase can be demon-strated within pinocytotic vesicles of oogonia andoocytes (Figs . 9, 17) .

The annulate lamellae present in the perinuclearooplasm of oogonia and early oocyte stages arecharacteristically single or solitary in occurrence .Thus, extensive stacks of annulate lamellae arenever observed in these early stages ; however, thesolitary and perinuclear annulate lamellae dis-appear or decrease in number during subsequentperiods of oocyte growth since they are rarely ob-served in such oocytes .

The paired centrioles of the oogonium areillustrated in Fig . 5. Here it can also be notedthat they occupy a position in the ooplasm close tothe nuclear envelope . The triplet microtubulescomprising the wall of the centriole are clearlyevident in Fig . 5, and this centriole is surroundedby a halo of a homogeneous-appearing material ofappreciable electron opaqueness .

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Previtellogenie Ooeytes

Growing oocytes, ovoid in shape and 10-12,u attheir maximum width, are characterized by thepresence of one or more densely stained cytoplasmicmass (Fig . 10) . In early oocyte stages, the cyto-plasmic bodies are frequently 1-2 .s in diameter .With the Korson technique (27), the cytoplasmicdense masses stain blue, as do portions of thenucleoli, indicating the presence of ribonucleicacid (Fig . 10) . The presence of RNA within thecytoplasmic bodies is further attested by theirappearance when stained with methylene blue andtoluidine blue O . However, prior treatment of thesections with ribonuclease decreases their stainingintensity. The cytoplasmic masses also stain withmercuric bromphenol blue and they are especiallywell displayed in Champy's-fixed oocytes stainedwith Heidenhain's iron hematoxylin . Thus, on thebasis of both their size and their staining reactions,the cytoplasmic dense masses are comparable tothe "yolk nuclei" described by earlier investigators(6, 29, 34) in light microscope preparations of in-sect oocytes. As the oocytes increase in size, thebasophilic bodies also increase in size and number .They eventually assume a cortical position in theooplasm (Fig . 12) . However, with further growthof the oocyte the basophilic bodies decrease insize, presumably through fragmentation, but re-main predominantly positioned in the corticalooplasm (Fig. 13) .The fine structure of the cytoplasmic dense

masses is illustrated in Fig . 11 which is a stagesimilar to that illustrated in the photomicrographof Figure 10. The cytoplasmic dense mass appearsfinely granular and compact . As a result it ismuch more electron opaque than the more looselyorganized ribosomes located in abundance aroundthe dense masses . The structural distinction be-tween the finely granular nature of the cytoplasmic

FIGURE 4-9 All figures are of oohonia . Dispersed nucleolar material at (NL) in Fig . 4

and 6-9 . Cytoplasmic masses (CM) and nucleolar material in Figs. 7-9 exhibit similarities .Single annulate lamellae (AL) adjacent to the nuclear envelope in Figs . 4, 6, 7, and 9 .Note structural similarity between the material associated with pores of nuclear envelopeand annulate lamellae (arrows) and the material of the nucleolar (NL) and cytoplasmicmasses (CM) . Observe continuity of annulate lamellae (AL) with a non-annulate, mem-branous laminia (L) in Fig. 4 . Paired centrioles (CC) of oogonium adjacent to nucleus (N)in Fig. 5 . Note nature of material constituting halo (H) around one of the centrioles inFig. 5 . Peroxidase (P) is present in intercellular spaces in Figs . 4, 7, and 9 and in vesicles(P) of ooplasm in Figs . 7-9 . Figs . 4, 5, 8, 9, X 50,000 . Fig . 6 X 25,200 ; Fig . 7, X 34,000 .

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dense masses and the ribosomal morphology of thesurrounding ooplasm remains apparent during thecortical migration and fragmentation of the baso-philic bodies (Figs . 14, 15) .

After the fragmentation of the dense, corticalmasses into smaller packets, specialized mem-brane systems which can be recognized as annulatelamellae begin to appear within the cortical masseswhich contain RNA (Figs. 16, 17) . An apparentinitial stage in the differentiation of annulatelamellae within a dense cortical mass is illustratedin Fig. 16 . In this case, portions of three pores orannuli are apparent within that portion of themembrane which is within the dense mass . How-ever, that portion of the membranous laminawhich extends into the adjacent ooplasm is non-annulate . Stages illustrating the progressive fillingof the dense, cortical masses with annulate lamellaeare provided in Figs. 17-25. The dense masseswithin which the annulate lamellae appear exhibita variety of configurations . The progressive in-crease in number of annulate lamellae within thedense masses does not, however, appear to beprecisely regulated . Thus, in ovoid dense masses,annulate lamellae may be present at either end ofthe structure (Fig . 17). In triangular shapedmasses, the annulated membranes may appear in 3different regions (Fig . 22). In other cases, thedistribution of annulate lamellae within the corti-cal dense masses appears even more haphazard(Figs . 18-21) . However, it does appear that thedifferentiation of annulate lamellae in manycases proceeds from the periphery toward theinterior of the dense mass . During the differentia-

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tion of annulate lamellae, short membranoussegments continuous with the annulate lamellaemay extend into the surrounding ooplasm, butthey are never annulate (Figs. 16, 20) . Structureswhich appear to represent single pores or annulican be observed within the dense cortical massesduring early stages in the differentiation of an-nulate lamellae (Figs . 18, 19) .

The cortical dense masses within which annulatelamellae differentiate can be recognized in Eponsections 1 µ thick which have been stained withazure II and methylene blue . The dense massesstain blue as do large whorls of endoplasmicreticulum which are present in the distal cyto-plasm of associated follicle cells . Lines can beobserved to traverse many of the dense massesunder these conditions, and these lines probablyrepresent individual annulate lamellae . With thistreatment, the lamellae generally stain somewhatdarker blue than does the surrounding dense mass .

In many of the cortical masses within whichannulate lamellae are in the process of differ-entiating, the basophilic material may be or-ganized into regions of two different electrondensities . Such a condition is illustrated in Fig . 18in which case the material comprising the twodifferent densities is contiguous. More frequently,however, that portion of the basophilic mass whichpossesses the greater electron density is sphericalin shape and separated from the remainder of thebasophilic mass by a narrow rim of cytoplasm(Figs. 20, 21, 24) ; the latter can be identified bythe loosely packed ribosomes comprising it . Thesignificance of the variation in electron density of

FIGURE 10 Photomicrograph of young oocytes . Dense, cytoplasmic masses (arrows) arepresent which stain similar to portions of the nucleolus (NL) . Carnoy's fixative, Korson'sstain . X 4000 .

FIGURE 11 Electron micrograph of oocyte in a similar stage as that illustrated in Fig . 10.A large, dense mass (DM) is present in the perinuclear ooplasm and is surrounded by nu-merous ribosomes (R) . A portion of the nucleolus (NL) is present in the nucleus (N) . X11,900 .

FIGURE 12 Photomicrograph of oocyte illustrating the large nucleolus (NL) in the nucleusand several large, densely stained masses (DM) in the cortical ooplasm . Champy's fixation,Heidenhain's iron hematoxylin stain . X 3400 .

FIGURE 13 Photomicrograph of section through larger oocyte following the fragmenta-tion of the cortical dense masses . Two small masses in the cortical ooplasm are identi-fied (arrows) in this section. Follicle cell layer (FC) . Champy's fixation, Heidenhain's ironhematoxylin stain. X 3400 .

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FIGURES 14-15 Electron micrographs of cortical masses similar to those present in Fig . 13. Note thefinely granular nature of the dense masses (DM) which differ from the surrounding ribosomes (R) . Mem-branous laminae (L) are present in the ooplasm in Fig . 14 . Peroxidase is present in the perivitelline space(PS) and the intercellular space (IS) between adjacent follicle cells (FC) . Figs . 14 and 15, X 34,000 .

FIGURE 16 Dense, cortical mass penetrated by single membranous lamina (L) . The portion of the mem-brane within the dense mass (DM) is annulate (AL), but is nonannulate as it extends into the surroundingooplasm . Ribosomes (R) . X 50,000 .

the basophilic masses during the differentiationof annulate lamellae within them is not clear .Conceivably, such a condition may representvariations in composition or organization of thebasophilic material. Alternatively, such accumu-lations may represent new material synthesizedby the differentiating basophilic masses . In somecases, smaller areas of increased electron densityalso are associated with the differentiating an-nulate lamellae within the cortical masses (Fig .19) .

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Eventually, the basophilic cortical massesbecome completely filled with annulate lamellae(Figs . 25, 27), a process which is largely com-pleted during the early stages of vitellogenesis .As the number of annulate lamellae reaches amaximum for each stack, the ordered arrangementof the lamellae, characteristic of that observed inother cell types, becomes established (Fig . 27) .At this time, what remains of the cortical basophilicmasses is restricted to the interlamellar space ofthe stack. This region in a stack of annulate

FiGuur; 17 Early stage in the differentiation of annulate lamellae within the cortical basophilic masses(DM) . Annulate lamellae (AL) are present at each end of the dense mass (DM) . Follicle cells (FC) . Peroxi-dase is present in the intercellular space between follicle cells, in the perivitelline space (PS) and withinpinocytotic vesicles (PV) in the cortical ooplasm . Ribosomes (R) . X 34,000 .

lamellae, regardless of cell type, always differsstructurally from the surrounding ooplasm (22) .The differentiated stacks of annulate lamellaepersist throughout vitellogenesis and, while manyof them remain preferentially located in the corti-cal ooplasm, a few are found in deeper areas ofthe ooplasm .

A stack of annulate lamellae in a vitellogenicoocyte is illustrated in Fig . 27 . In this particularinstance the lamellae comprising the stack areclosely aligned, in the sense that the interlamellarspacing is quite uniform . Ooplasmic ribosomes arerestricted from the stack of annulate lamellae .Terminal connections between adjacent lamellaeare established by short segments of smooth-sur-faced membranes. Another condition sometimesencountered is illustrated in Fig . 26 . In this case,those two annulate lamellae at opposite ends ofthe stack are continuous with each other viarough-surfaced endoplasmic reticulum which

forms an extensive loop in the adjacent ooplasm .A similar condition holds with respect to theremaining annulate lamellae in the stack . Thus,there is often an ordered relationship between theannulate lamellae and the endoplasmic reticulumwhich permits a precise interconnection betweenthe two membrane systems following differenti-ation of the annulate lamellae .

The structure of the material comprising thecortical basophilic bodies and the interlamellarmaterial of completely formed stacks of annulatelamellae is illustrated in Figs. 28 and 29 and inthe inset of Fig . 27. From these micrographs, itappears that two small components contributeto this density . One of these components consistsof small, dense granules most of which rangefrom 40-70A in diameter. The second componentis a thin filament which is less than 40A in width .The close packing, and perhaps interconnectionof the two structural components, appears to be

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FIGURE 18 Cortical dense mass (DM) with several randomly arranged annulate lamellae (AL) within it .What appears to be a section of a single annulus or pore is indicated at the unlabeled arrow . A region ofincreased density in the mass is indicated at (D) . Cytoplasmic laminae (L), ribosomes (R), follicle cells(FC) . X 25,000 .

FIGURE 19 Stage in the differentiation of annulate lamellae (AL) within a dense cortical mass (DM) . Aregion of increased density is associated with the annulate lamellae at (D) . Single pores or annuli are indi-cated at (A) . Polysome configurations (arrows) and ribosomes (R) surround the differentiating densemass. X 34,000 .

responsible for the homogeneous appearance of thismaterial in lower magnification electron micro-graphs .

During the origin, migration, and fragmenta-tion of the "yolk nuclei", the ooplasm is char-acterized by numerous ribosomes, mitochondria,packets of microtubules, Golgi complexes, mem-brane-bounded inclusion bodies, and membranouslaminae of variable length ; the last apparentlyrepresent the membranous component of theendoplasmic reticulum (Figs. 14, 18) . The in-dividual membranous laminae are, in fact, widelydistributed throughout the ooplasm and appearsimilar to those membranes which exhibit con-

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tinuity with annulate lamellae differentiatingwithin the cortical basophilic bodies .

Most structural components of the ooplasmare restricted from the dense masses both beforeand during the differentiation of annulate lamellaewithin them, a condition which indicates that thedense masses are tightly compacted . However,at least one microtubule is frequently observed topenetrate the dense masses (Fig . 22). Such acondition may provide a means for the move-ment or the support of the cortical, basophilicmasses. During the differentiation of annulatelamellae, polysome-like configurations are fre-quently present surrounding the dense, corticalmasses (Fig. 19) .

FIGURES 20-21 Stages in the differentiation of annulate lamellae (AL) within the cortical, basophilicmasses . Some of the annulate lamellae are continuous with nonannulate lamellae outside of the corticalmasses (arrows) . Note also that each of the basophilic bodies contains an area of increased electron density(D) which is separated from the dense mass by a narrow region of ribosomes . X 25,000 .

DISCUSSION "yolk nucleus" or "Balbiani body" (6, 29, 34) .The structural nature of the material comprisingthe basophilic dense masses in Libellula appears toconsist of numerous granules and filaments, bothof which are extremely small and closely packedand may be interconnected . As a result, structuraldetail is apparent only at comparatively high

magnification and resolution . It is interesting,

in this connection, that recent studies on otheroocytes have reported the presence of intercon-nected thin filaments and small granules of similarsize in the pores of both the nuclear envelope andannulate lamellae (21, 23) . Those granule-fila-ment complexes described as associated with thepores of annulate lamellae and the nuclearenvelope are in some respects similar to structuralcomponents of nucleoli in the same cells (21) .In several instances, it has been speculated thatthe material comprising the annular material ofnuclear pores may represent a form of "stored

It is well documented that large amounts of RNAaccumulate in the cytoplasm during initialstages of oogenesis (6, 29, 34), and the movementof materials from the nucleus to the cytoplasmhas been described in cytological, cytochemical,and electron-microscopic studies (1, 3, 19, 20,25, 26, 41). In some cases, nucleolar material orportions thereof appears to be transferred fromthe nucleus to the cytoplasm (20, 25, 26) . As inother oocytes, large amounts of RNA accumulatein the cytoplasm during early stages of oogenesisin the dragonfly. However, it is apparent thatin the dragonfly the RNA exists in at least twodistinct morphological forms : (1) a widespread

and general cytoplasmic basophilia produced bynumerous ribosomes and (2) a regionally localizedbasophilia contributed by material of nonribo-

somal morphology previously designated as a

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FIGURES 22-23 Both figures illustrate stages in the differentiation of annulate lamellae (AL) withinthe dense masses (DM) . Note the appearance of the dense masses and the regions in which annulate lamel-lae are located. In Fig. 92, two annulate lamellae appear to be connected with a nonannulated membranewhich forms a loop (L) in the adjacent ooplasm. A microtubule (MT) appears to penetrate the dense massin Fig. 2Q . Fig. 22, X 34,000 ; Fig . 23, X 50,000.

FIGURE 24 Section of a dense mass (DM) to illustrate the "pores" of the differentiating annulate lamellae(AL) in surface view . Two spherical bodies of increased density (D) appear within the dense mass (DM)but are separated from it by a narrow region of ribosomes . X 34,000 .

FIGURE 9,5 Later stage in the differentiation of annulate lamellae within the dense mass which is nowalmost completely filled with the lamellae . Note variations in distance between adjacent lamellae of thestack. X 34,000 .

FIGURE 27 Stack of annulate lamellae in vitellogenic oocyte. Note consistency of material betweenlamellae as compared to surrounding ooplasm . Small granular and filamentous structures in the matrixbetween annulate lamellae are indicated at unlabeled arrows . Terminal connections between some adja-cent lamellae in stack at (C) . Patent pores indicated at (P) . Ribosomes (R) . X 50,000 . The small granules(G) and thin filaments (F) comprising the interlamellar material are illustrated in the insert . X 119,000 .

informational RNA-protein" material (2, 23), aspeculation that could be extended to annulatelamellae on the basis of the structural similaritybetween the pores in the two cases (12) .

Unfortunately, little information is presentlyavailable regarding the chemical nature of theannular material comprising the pores either inthe nuclear envelope or in the annulate lamellae(32) . In addition to the evidence for the presenceof basic proteins (7, 9, 31), there is only sug-gestive evidence for the presence of RNA in as-sociation with the pore complex of the nuclearenvelope (40) . While some studies have indicatedthat DNAse and RNAse have little or no effecton the integrity of the pores in the nuclear en-velope (7, 31), Esser (8) has recently reported ina short communication that both DNAse andpepsin cause a partial breakdown of the nuclearenvelope and the nuclear pores of a diatom .

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It is clear that, during oocyte growth in Libel-lula, stacks of annulate lamellae are found onlywithin the cortical, RNA-containing masses andin no other region of the ooplasm . Such a con-dition in itself is suggestive that the RNA com-prising such areas may store information forthey are subsequently able to exert an influenceon the differentiation of a specific class of porouscytomembranes . If such a conclusion is accepted asa working hypothesis, then the significance of thecontinuity between annulate lamellae and therough-surfaced endoplasmic reticulum in manyother cell types (22) becomes clearer. Thus, theannulate lamellae may provide materials for anddirect the activity of the attached rough-surfacedendoplasmic reticulum which might then functionin the biosynthesis of a specific type of protein or inother substances important at certain times indifferentiation and growth processes . An importantpoint implied in this discussion involves the ques-

FIGURES 28-29 Portion of two basophilic, cortical masses in which annulate lamellae are differentiating .Note the finely granular and compact appearance of the material comprising the masses as compared tothe surrounding ribosomes (R) . The material comprising the masses appears to be composed of small gran-ules (G) and thin filaments (F) . Fig . 28, X 100,000; Fig. 29, X 81,000 .

tion as to why cytoplasmic informational materialsmight be massed together in such significantquantities in oocytes . Thus, in somatic cells, it isgenerally considered that soluble and messengerRNA are not apparent in thin sections with theresolution provided by the electron microscopebecause they are never aggregated in sufficientquantities . However, the oocyte is an unusualcellular system in which many biological ac-tivities are amplified in preparation for eventsunique to this cell type .

In other oocytes, the terms "yolk nucleus" and"Balbiani bodies" have been used to denote avariety of cytoplasmic structures, all of which aregenerally localized in the ooplasm . In somecases, the "yolk nucleus" only refers to a denselystaining cytoplasmic mass while in other instances

the structure has been found to contain arrays of

endoplasmic reticulum, mitochondria, Golgi com-plexes, and a variety of other components . An-nulate lamellae in variable amounts have beendescribed as comprising a component of theseregions in human oocytes (11), hen's egg (5),mollusc oocytes (35), and others.The transformation of cytoplasmic laminae

into annulate lamellae has been suggested forother cell types (12, 22, 24, 39) . Further, in thosestudies concerned with the morphogenesis ofannulate lamellae (11, 12, 14, 16, 17, 22, 24, 39),it has been demonstrated in some cases that thesource of the membranes is the outer layer of thenuclear envelope (16, 17, 22, 24). Such may bethe case also in the dragonfly oocyte . However,the method by which the annulate lamellae form

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within the cortical, basophilic masses in theLibellula oocyte is not clear, but two possibilitiesseem plausible from the morphological observa-tions. One alternative is that the annulate lamellaearise de novo, that is, within and under the in-fluence of the material comprising the densemasses. The only observation that would tend tosupport such a possibility is the presence of whatappeared to be single pores within the densemasses. However, conceivably, such single poresmight represent sections of longer annulatelamellae not included in the section plane . It waspreviously noted that a considerable number ofmembranous lamellae of different lengths wererandomly dispersed throughout the ooplasm dur-ing the differentiation of annulate lamellae,sometimes in close association with the frag-mented "yolk nuclei". Furthermore, annulatelamellae within the dense masses were frequentlyfound to be continuous with nonannulate lamellaein the adjacent ooplasm . Thus, the second alterna-tive should consider the possibility that non-annulate cytoplasmic lamellae penetrate thedense basophilic masses and, as they do, becometransformed into annulated membranes under theinfluence of the material comprising the masses .Included in this suggestion is the possibility thatthe portion of the cytoplasmic lamina not enteringthe dense mass does not become annulate and,therefore, results in the continuity of two morpho-logically distinct types of membranes in some cases .However, in other cell types the nature of thematerial influencing the formation and/or activityof the annulate lamellae is not as morphologicallyapparent as in the dragonfly .

As was previously indicated, solitary annulatelamellae can be found in oogonial stages of de-velopment and their differentiation does notappear to be dependent upon the presence ofdense cytoplasmic masses of material. However,small cytoplasmic masses similar in some respectsto nucleolar materials are usually located in closeproximity to the single annulate lamellae presentin the oogonia . Further, in oogonia and earlyoocytes, the differentiation of solitary annulatelamellae seems to be more dependent upon theproximity of cytomembranous lamina to thenuclear envelope . Thus, the individual annulatelamellae in the oogonia are always closely ap-plied to the outer layer of the nuclear envelope .

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THE JOURNAL OF CELL BIOLOGY • VOLUME 42, 1969

A second feature supporting such a view is thefact that a single, short annulate lamella close tothe nuclear envelope may be continuous with anonannulate membranous lamina which is in allcases more distal to the nucleus than is the an-nulate portion of the lamella. Conceivably, thenucleolar material which is often applied to theinner layer of the nuclear envelope or the ma-terial associated with the nuclear pores, or both,may exert an influence on the differentiation ofsolitary, perinuclearsmall cells .The appearance of annulated cytomembranes

within localized RNA-containing regions of thecytoplasm is a unique observation with respectto the morphogenesis of these membranes. Thefact that stacks of annulate lamellae only appearwithin these granular masses which contain RNAbut do not possess a typical ribosomal morphologyindicates that the material comprising the massescontains information necessary for the formationof these membranes . The material is undoubtedlyimportant also for the subsequent activity ofthe stacked annulate lamellae . The cytoplasmicdense masses do not appear to be required,however, for the differentiation of the solitaryannulate lamellae present in the oogonia . Such avariation may reflect differences in or magnitudeof activity by the annulate lamellae present inthe perinuclear cytoplasm of oogonia as comparedto the more extensive cortical stacks formed laterin the life history of the cell .

It is apparent that the unique features associ-ated with the origin of annulate lamellae in large,regionally localized RNA-containing areas of theooplasm in the Libellula oocyte make this cell afavorable one in which to study the incorporationof various radiochemicals in an attempt to under-stand further details in this unusual relationship .

The authors wish to acknowledge the skillful technicalassistance of Mrs . Robert Decker.This investigation was supported by research

grants (HD-00699, RG-09229, GM-5479, 4706), aResearch Career Development Award (to RGK)from the National Institutes of Health, U . S . PublicHealth Service, and a research grant (G-9879) fromthe National Science Foundation.Received for publication 10 December 1968 and in revisedform 27 February 1969 .

annulate lamellae in these

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R. G. KESSEL AND H. W. BEAMS Annulate Lamellae in Oocytes 201