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} . Cell Sci. 53, 173-191 (1982) 173Printed in Great Britain © Company of Biologists Limited 198a

COATED VESICLE-MEDIATED TRANSPORT

AND DEPOSITION OF VITELLOGENIC

FERRITIN IN THE RAPID GROWTH

PHASE OF SNAIL OOCYTES

W. BOTTKE, I. SINHA AND I. KEILZoologisches Institut der Universitdt, BadestraQe 9, 4400 Mtinster, FRG

SUMMARY

The main yolk component in oocytes of the pulmonate freshwater snails Planorbariuscornetu L. and Lymnaea stagnalis L. consists of the iron storage protein ferritin and iron-freeapoferritin. Both compounds are deposited in the yolk in the form of large paracrystalloids,tubular structures and randomly dispersed particles. In addition, the plasm contains lysosome-like inclusions with depositions of haemosiderin. Haemosiderin is interpreted as the product ofproteolytic degradation of ferritin. During the rapid growth phase of the oocytes vitellogenicferritin is transported across the basement lamina and taken up by adsorptive endocytosis viacoated pits and vesicles. Formation of yolk bodies occurs by fusion of ferritin-containingvacuoles and empty vesicles that are probably derived from the Golgi apparatus. Uptake offerritin is restricted to the basal region of the oocyte. No involvement of the follicle cells insynthesis and deposition of ferritin could be detected. Secretory cells of the midgut gland arethe most likely site of synthesis of vitellogenic ferritin.

Under conditions of iron overload large masses of ferritin are encountered in the basementlamina of the oocytes. However, no significant increase in the uptake of ferritin could beobserved. With the use of a tannic acid-glutaraldehyde fixation procedure a hitherto unobservedfilamentous or rod-like material was detected inside the lamina and in coated pits. This materialis probably also taken up by the oocytes and integrated into yolk platelets.

Though ferritin is a rather unusual vitellogenic protein, the mode of its uptake and depositionin the oocyte plasm is highly reminiscent of that of typical hormone-induced vitellogenins inother animal groups.

INTRODUCTION

In the development of the yolky eggs of many animal groups certain serum proteins,termed vitellogenins, are the major source of the yolk proteins or vitellins. The vitel-logenins, synthesized outside the oocyte in distant maternal somatic tissues such asthe liver or an analogous organ, are secreted into the bloodstream or haemolymph andtaken from it by the growing oocytes (Wallace & Bergink, 1974; Hagedorn & Kunkel,1979). Synthesis and uptake of the proteins are under stringent hormonal control(Clemens, 1974).

Though the vitellogenins of closely related species are rather similar in amino acidcomposition or carbohydrate content, the uptake is evidently highly selective andinvolves the recognition of subtle differences in structure or charge (Kunkel & Pan,1976). Absorption of vitellogenic proteins is achieved by structural modifications of

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Ferritin transport and deposition in oocytes 175

the oolemma, which include the formation of coated pits and coated vesicles. Recentinvestigations have shown conclusively that coated vesicles function as highly selectivemolecular filters (Goldstein, Anderson & Brown, 1979; Bretscher, Thomson & Pearse,1980).

It is generally assumed that, in addition to the principal component of coatedvesicles, i.e. the protein clathrin (Pearse, 1976), membrane-bound receptors of coatedvesicles are involved in specific recognition and transport of proteins (Roth, Cutting &Atlas, 1976; Silverstein, Steinman & Cohn, 1977; Woods, Woodward & Roth, 1978;Goldstein et al. 1979).

Coated vesicle-mediated uptake of yolk proteins has been observed in a large numberof invertebrate and vertebrate animal groups and is especially well documented ininsects, amphibia and birds (Perry & Gilbert, 1979). In contrast to these observations,the mode of yolk formation and deposition in other groups, e.g. molluscs (with theexception of cephalopods) is enigmatic.

In gastropod snails vitellogenesis is likewise assumed to be controlled by hormones.Evidence for this assumption is indirect and based on countings of vitellogenic oocytesin the gonads in the presence or absence of the presumed hormone (Geraerts & Joosse,

In fact, no vitellogenin or other non-specific exogenous egg protein has been foundin snails (cf. Woods, Paulsen, Engle & Pert, 1958; Wright & Ross, 1963; Figueiredoet al. 1973). Consequently, the target of the gonadotropic hormone is actually unknown.On account of the apparent rarity or lack of endocytotic vesicles at the oocyte surfaceand on account of the lack of biochemical or immunological data, most workers haveassumed that the yolk is formed endogenously. Different cytoplasmic constituentshave been proposed as being involved in the formation of protein yolk (see de Jong-Brink, de Wit, Kraal & Boer, 1976). The yolk of freshwater snails is characterized byits high iron content as shown by cytochemistry. Observations of Heneine, Gazzinelli& Tafuri (1969), dealing with the iron metabolism of planorbid snails, indicate thatiron is transferred from the midgut gland of the animals to the gonad and that ironis bound during the transport to a macromolecular component. These observationsprompted us to isolate and characterize the iron-containing yolk protein. Since this

Fig. 1. Vitellogenetic Planorbarius oocyte of stage 5 at the bottom of a gonadal acinus.Basement lamina (b[), follicle cell (Jc), oocyte (o). x 640.Fig. 2. Detail of a vitellogenetic oocyte and the basement lamina (stage 4). The oocyteis separated from the neighbouring follicle cell by a prominent interfollicular cavity(ifc). Basement lamina (bl), follicle cell (Jc), oocyte (0). x 7200.Fig. 3. Microtubule-containing follicle cell projection in the basement lamina directlyunder the vitellogenetic oocyte. Planorbarius. Follicle cell (Jc), oocyte (0). x 20000.Fig. 4. A desmosome of the zonula adhaerens type connecting a stage 4 oocyte witha follicle cell at the basal region of the follicle. Planorbarius. Follicle cell (Jc), oocyte (0).x 120000.

FIG. 5. Cross-section of a follicle cell projection in the basement lamina exhibiting amultitude of cytoplasmic microtubules. Lymnaea. x 80000.Fig. 6. Fibrillar structure of the Lymnaea basement lamina. Basement lamina (bl),follicle cell (fc). x 80000.


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yolk protein has turned out to be the principal iron-storage protein ferritin (Bottke &Sinha, 1979), we have an intrinsic probe for electron microscopical investigations onthe transport, deposition and site of synthesis. As a result vitellogenic ferritin is clearlyan exogenous protein, which is taken up by the oocytes via the coated vesicle route,thus suggesting a highly specific receptor-mediated uptake for this unusual protein.The way in which ferritin is taken up and deposited is quite comparable to the wayin which typical vitellogenins are internalized by oocytes.

MATERIALS AND METHODS

Materials

Adult snails of Planorbarius corneus L. and Lymnaea stagnate L. were bred in the laboratoryat temperatures of 15-20 °C under long-day conditions. The animals were maintained on astandard diet of commercial Tetra SM 80 food. In some cases iron sulphate was added to thetap water in which the animals lived. Iron is at once oxidized and the animals take up smallamounts of the brownish mud together with their food.

Methods

Isolation of ferritin and X-ray microprobe analysis were done as described previously (Bottke& Sinha, 1979). Ferritin samples were negatively stained with 2 % neutral potassium phospho-tung8tate.

Preparation for electron microscopy involved fixation with 3 % glutaraldehyde, buffered topH 7-7-5, with f-collidine, and osmication in 2 % OsO^. After dehydration the gonads wereembedded in Epon 812 or methacrylate-styrene. Thin sections were cut on a Reichert OmU2ultramicrotome and picked up on Formvar-coated grids. Sections were stained with 5 %uranylacetate followed by lead citrate. They were examined in a Siemens Elmiskop 101 electronmicroscope. In order to outline extracellular spaces we used a tannic acid-glutaraldehydefixative essentially as described by Wagner (1976) and Simionescu & Simionescu (1976). Inorder to detect ferritin particles in the tissue, unstained sections from the same blocks wereinvestigated in parallel. Tannic acid was obtained from Mallinckrodt Inc. (tannic acid AR,code no. 1764). Measurements were done on micrographs at primary magnifications ofx 40000 to 120000. In the case of ferritin, particle measurements were done on focus series.

RESULTS

Structure of the gonad

Oogenesis of P. corneus and L. stagnalis can be divided into 5 stages in analogy toobservations in other snails (de Jong-Brink et al. 1976). In the present report only

Fig. 7. Isolated ferritin molecules from gonads of Planorbarius. Protein shells of themolecules are outlined by negative staining, x 480000.Fig. 8. Unstained preparation of Planorbarius ferritin. Only the inorganic ferritin corescan be recognized, x 280000.Fig. 9. Tubular structure in a Planorbarius yolk platelet made up of hexagonallycrystallized ferritin. x 140000.Fig. 10. Ferritin paracrystalloid in a secretory cell of the midgut gland of Planorbarius.x 200000.

Fig. 11. Aggregation of ferritin molecules in a Planorbarius yolk platelet interpretedas an intermediate stage of haemosiderin formation due to proteolytic degradation offerritin. x 200000.

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stages 4 and 5 sensu de Jong-Brink et al. have been investigated. At these stages theoocyte-follicle cell complex is built up in a polarized manner (Fig. 1). Follicle cellsand the oocyte sit on a rather conspicuous basement lamina whose structure has beendescribed (Starke, 1971) (Fig. 6). Laterally and apically the oocyte is totally sur-rounded by the highly polyploid follicle cells, which show all the characteristics ofprotein-synthesizing cells (Fig. 2) and which contain masses of cytoplasmic micro-tubules (Figs. 3, 5). Between the follicle cells and the oocyte an interfollicular cavityis built starting at stage 4. Only at their basal regions do the follicle cells remain inclose membrane contact with the oocyte by desmosomes (Fig. 4).

Dealing with the structure of the follicle, 2 compartments must be envisaged whereuptake of vitellogenic proteins could occur.(1) Uptake of materials from the interfollicular cavity (Fig. 2). These materials could

be either follicle cell products or haemolymph proteins transported across thebasement lamina into the interfollicular cavity.

(2) Uptake of vitellogenic proteins at the base of the oocyte above the basementlamina. These proteins can be expected to be haemolymph proteins but theinvolvement of secreted proteins from the follicle cells is not excluded.

Our ultrastructural observations combined with autoradiographic results (to bepublished elsewhere) are in favour of the second way.

Structure and deposition of yolk protein

In the electron microscope, animal and plant ferritins exhibit a typical structure andare easily identified (Massover & Cowley, 1975).

Therefore, we isolated the iron-rich protein of snail yolk and compared its structurewith that of horse spleen ferritin. In monolayers of unstained preparations the electron-dense mineral cores of yolk ferritin have diameters of about 6 nm, the individual coresbeing always separated by a distance of at least 11-13 nm (centre to centre) (Fig. 8).The surrounding protein shells become easily visible on negative staining (Fig. 7).Individual whole molecules have a diameter of about 12-12-5 nm and correspond insize to other tissue ferritins. Compared with horse spleen ferritin, which has beeninvestigated most, Planorbarius and Lymnaea ferritins exhibit a rather low contrastin the electron microscope, which is probably due to the low iron content of the cores(Fig. 8). Preliminary estimations of the iron content yielded an average content of

Fig. 12. Yolk platelet of Planorbarius containing paracrystalloids of ferritin (/) andapoferritin (a) and randomly dispersed ferritin particles, x 80000.Fig. 13. X-ray emission spectrum of a ferritin paracrystalloid showing the Fe (Ka)line. Other elements are due to the grid and specimen holder.Fig. 14. Apoferritin paracrystalloid in a yolk platelet of Planorbarius. Apoferritin (a),ferritin (/). x 120000.Fig. 15. Ly8osomal inclusion in a vitellogenetic oocyte of Planorbarius containingmyelin-like membranous structures and haemosiderin depositions (/1). x 80000.Fig. 16. X-ray emission spectrum from a haemosiderin deposition, exhibiting theFe (Ko) and the phosphorus line (P).Fig. 17. High-resolution picture of haemosiderin. The typical structure of denselyaggregated ferritin cores is resolved in certain areas (arrows), x 240000.

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300-600 iron atom9 per molecule as compared with 4500 in horse spleen ferritin(Clegg, Fitton, Harrison & Treffry, 1980). However, as seen in focus series, there isevidently some heterogeneity in iron content between individual molecules of onepreparation. In crude preparations that have not been subjected to ultracentrifugation,profiles of iron-free apoferritin molecules are always encountered together withferritin.

Inside the yolk platelets ferritin molecules occur freely or are deposited in extendedhighly ordered paracrystalloids of variable size (up to 1 /im) (Fig. 12). Various aspectsof the ultrastructure of paracrystalloids have been described already in Planorbarius(Favard & Carasso, 1958) and other snails (de Jong-Brink et al. 1976). Interestingly,apoferritin paracrystalloids are encountered together with ferritin crystalloids insidethe same yolk platelet, both compounds showing no co-crystallizing with one anotherinside the same crystalloid (Figs. 12, 14). In addition, tubular structures whose wallsconsist of monolayers of hexagonally ordered ferritin molecules are frequentlyobserved (Fig. 9). Even when using the tannic acid reagent in order to increase thecontrast of membranes we were unable to detect an association of this ferritin withmembranes. The structures are reminiscent of the 2-dimensional crystalline ferritinmonolayers associated with the surface of lipid droplets as observed by David &Easterbrook (1971) in Phycomyces. Masses of free ferritin molecules are embeddedinto a finely granular undefined matrix (Fig. 12). A dense layer of molecules alwayscovers the inner leaflet of the limiting membrane.

In addition to typical yolk platelets, the oocyte contains deutoplasmic inclusions ofnearly the same size, which are typical on account of their apparently empty appear-ance, i.e. their lack of a matrix and paracrystalloids (Fig. 15). Instead, they containmyelin-like membraneous structures and coarsely granular masses, which on highresolution are resolved into typical ferritin cores (Fig. 17). These are often seen inintimate contact and not separated by a clear space. All intermediate stages betweenirregular aggregations of ferritin molecules (Fig. 11) and masses consisting merely of

Fig. 18. The cortex of a vitellogeneric oocyte of Planorbarius showing large-scaleuptake of ferritin via coated pits. Dense precipitates inside coated pits and at themembrane are due to the tannic acid fixation procedure. Ferritin particles (arrow),basement lamina (Jbt), oocyte (o). x 100000.Fig. 19. Coated pits and basement lamina in Planorbarius heavily contrasted by tannicacid-OsO4. Basement lamina (W), oocyte (0). x 100000.Fig. 20. Ferritin molecules in a coated pit and in the narrow intercellular cleft betweenthe oocyte and a follicle cell projection (arrows). Follicle cell (Jc), oocyte (o). x 80000.Fig. 21. Bristle coat of a coated pit at the basement lamina (arrows). Planorbarius.x 200000.

Fig. 22. Coated pit in the oocyte plasm exhibiting ferritin cores aligned along theouter membrane layer, x 160000.Fig. 23. Coated vesicles with an uncoated tubular projection (arrow). This structureprobably represents an intermediate stage between coated pits and uncoated vesicles.Oocyte (0). x 80000.Fig. 24. A coated vesicle (arrow) shown by tannic acid-glutaraldehyde fixation toencircle, in reality, an extracellular space. The coated vesicles in the vicinity areindeed intracellular vesicles. Planorbarius. Basement lamina (bf). x 120000.


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aggregated cores are seen. We interpret these structures to be the second most abundantiron-storage protein haemosiderin, which is generally assumed to be a product ofproteolytic degradation of ferritin inside lysosomal structures (Fischbach et al. 1971).Our interpretation is supported by microprobe analysis and autoradiography. Intypical ferritin paracrystalloids the Ka-line of the iron spectrum is reliably detected(Fig. 13). No other heavy metal could be observed. In addition, haemosiderin massesalso yield a distinct phosphorus peak (Fig. 16). It is not known whether the phos-phorus signal is due to the enrichment of ferritin cores in haemosiderin, or whetherit indicates the existence of a second phosphorus compound. Interestingly, a distinctphosphorus peak has also been observed in plant ferritin (Sheffield & Bell, 1978).The lysosomal nature of these haemosiderin-containing inclusions is also confirmedby autoradiography (results not shown here). Whereas typical yolk platelets are readilylabelled after injection of tritiated amino acids, using incubation times up to 20 h, noradioactivity was encountered over the lysosome-like structures. Therefore, weinterpret these structures as degradation stages of yolk platelets.

Yolk platelets are often surrounded by profiles of the granular endoplasmic reticulumthat are free from ferritin molecules. No ferritin could be detected inside mitochondria,thus ruling out a direct involvement of these structures in ferritin deposition (seeFavard & Carasso, 1958).

Interestingly, the plasm of the oocytes always contains a small amount of randomlydispersed ferritin molecules. In other cell systems this mode of occurrence is generallytaken as an indication for the autosynthesis of ferritin (Trump & Berezesky, 1977).Consequently, a small-scale autosynthesis in the oocytes cannot be ruled out for Planor-barius and Lymnaea. On the other hand, many of the presumed free particles may inreality be associated with vesicles, which are not easily seen in unstained sections.Some particles may be molecules that have been delocalized from the yolk plateletsduring the process of sectioning.

Fig. 25. Ferritin molecules bound to the membrane of a vitellogenetic Lymnaeaoocyte. Unstained section. Basement lamina (6/). oocyte (o). x 120000.Fig. 26. Ferritin-containing pits of a vitellogenetic Planorbarius oocyte. Unstainedsection. Basement lamina (b[), oocyte (0). x 80000.Fig. 27. Coated pit showing ferritin molecules aligned along the outer membrane layer.Planorbarius. Basement lamina (4/), oocyte (o). x 120000.Fig. 28. Ferritin particles bound at the oolemma of a vitellogenetic Planorbariusoocyte. Unstained section. Basement lamina (bl), oocyte (0). x 240000.Fig. 29. Ferritin-containing vesicle (arrow) in the vicinity of a mature yolk platelet inthe central plasm of a Planorbarius oocyte. Yolk platelet (yp). x 120000.Fig. 30. Ferritin-bearing vesicles and vacuoles in the basal oocyte cortex. Thesestructures are interpreted as precursors of yolk platelets. Planorbarius. x 100 000.Fig. 31. Yolk platelet with crystallized ferritin and surrounding ferritin-bearing (smallarrows) and empty (large arrow) vesicles in the cortex of a Planorbarius oocyte. Yolkplatelet (yp). x 80000.


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Fig. 32. Ferritin depositions in the basement lamina of a vitellogenetic Lymnaeaoocyte under conditions of iron overload due to excessive iron hydroxide consumption.Basement lamina (bl). x 150000.Fig. 33. Ferritin isolated from the Planorbarius haemolymph by ultracentrifugation.x 250000.

Fig. 34. Ferritin particles taken up from the interfollicular cavity of the Lymnaeafollicle under conditions of iron overload. Interfollicular cavity (ifc), oocyte (o), micro-villus of the oocyte (v). x 150000.Fig. 35. Ferritin depositions in the basement lamina directly beneath a coated pit inthe oocyte cortex of Lymnaea. Basement lamina (6/)» oocyte (0). x 150000.

Uptake of ferritin

A careful examination of the cortex of vitellpgenic oocytes reveals large-scale uptakeof ferritin via endocytosis. Characteristic of the oocyte cortex of ferritin-sequesteringoocytes are indentations of the membrane in the form of shallow and deep pits, tubularstructures and profiles of vesicles with a diameter of about 100-150 nm (Fig. 18),


which, on staining with tannic acid, show the typical bristle-like projections character-istic of the polyhedral clathrin lattice of coated pits (Perry & Gilbert, 1979) (Fig. 21).

Due to the generally high contrast, only those ferritin particles that have a highiron content become visible in stained sections (Figs. 18, 20). In unstained sectionsferritin molecules are easily identified and can be allocated to cellular structures, sincethese have a high contrast even in unstained sections due to the tannic acid procedure(Figs. 25-30).

Whereas the basement lamina contains only small amounts of randomly scatteredferritin particles, the oolemma is locally densely studded with ferritin molecules;the individual cores always lying centripetally located some 15-20 nm off the mem-brane (Figs. 27, 28). Assuming a diameter of about 3 nm for the ferritin protein shells,the glycocalyx of the oolemma thus extends up to 17 nm. In favourable sections, i.e.if the trilaminar structure of the membrane is clearly revealed, ferritin particles arenever seen to fill the central cavity of the coated pits, but are, in contrast, alwaysassociated with the membranes (Fig. 27). We investigated whether coated pits existwithout ferritin particles, indicating the presence and uptake of another unidentifiedmaterial. In fact, none were found, the pits always containing considerable amountsof ferritin. Therefore, we conclude that the presence of ferritin at the membrane isa prerequisite for the formation of coated pits.

A considerable heterogeneity in the distribution and frequency of ferritin moleculesis seen at different areas of the same oocyte and between different vitellogenic oocytes;areas that are densely packed with ferritin molecules alternating with those that arenearly empty. We have not been able to establish a correlation between the frequencyof ferritin at the membrane and the occurrence of underlying structures such assinusoids of the circulation system or connective tissue cells. High concentrations offerritin are often seen in the near intercellular space between the oocyte and the under-lying small projections of the follicle cells (Fig. 20).

At a deeper level of the ooplasm no vesicle with a typical coat was seen. Vesiclesthat are partly deprived of their coat, and possibly are transition stages between coatedand uncoated vesicles, are rarely observed (Fig. 23). The uncoated ferritin-bearingvesicles range in size from 150 to 500 nm. In addition to typical vesicles, extendedtubular structures are common. Ferritin-containing vesicles are often seen formingloose aggregations (Figs. 24, 30), suggesting that they are either formed nearly simul-taneously and nearly at the same membrane area and/or that some fusion occurswhereby larger vacuoles are formed. Larger vacuoles with diameters above 500 nm,which already contain typical ferritin crystalloids in addition to dispersed molecules,are suggested to represent transitory stages in the maturation of yolk platelets (Figs.30, 31). These precursors of mature yolk platelets are easily recognized on account oftheir smaller size and the lack of an electron-dense matrix compared with matureyolk platelets. Deeply inside the ooplasm, beyond a distance of 1 /tm from the oolemma,ferritin-containing vesicles are extremely rare (Fig. 29). Other features of the super-ficial cytoplasm include large amounts of uncoated vesicles and vacuoles that are freefrom ferritin, microtubules and cisternae of the rough endoplasmic reticulum. Theempty vesicles and vacuoles are distributed over the whole oocyte plasm, often in close

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vicinity to mature yolk platelets (Fig. 31), suggesting that they may add a furthercomponent, perhaps endogenously formed, to the growing yolk platelets. We interpretthese vesicles as Golgi vesicles since they correspond in size and electron density tothe numerous secretory vesicles of the well-elaborated Golgi apparatus.

If the snails are grown up without addition of iron to the tap water, the uptake offerritin is restricted to the basal region of the oocyte. No ferritin molecules were foundin the interfollicular cavity (Fig. 2). However, under conditions of iron overload, if


the snails are forced to take up large amounts of iron together with their food, dramaticchanges are observed.

(1) The basement lamina now contains abundant ferritin particles, which are randomlydispersed and locally form large aggregates (Figs. 32, 35).

(2) Ferritin molecules now can be found in the interfollicular cavity, from wherethey are removed by coated vesicles as is typical for the basement lamina-oolemmainterface (Fig. 34).

It is an open question whether the striking increase in frequency of ferritin in thebasement lamina reflects an enhanced ferritin synthesis in order to detoxify free iron,or whether those ferritin particles that are not detected under normal conditions dueto their low iron content now become visible. However, in analogy to other tissues orferritins, both events probably take place (Clegg et al. 1980). We have no evidencethat the increase in ferritin under conditions of iron overload results in enhancedendocytotic activity of the oocytes (Fig. 35).

Structure of the basement lamina

The tannic acid procedure yields structures inside the lamina, which are onlypoorly seen after conventional staining procedures. These either consist of shorttubules whose walls are highly tannophilic or, more likely, of tiny filaments or rodswhose contours are outlined by negative staining due to the precipitation of tannicacid inside the lamina. Profiles of these structures are highly reminiscent of cross-sectioned membranes, their diameter roughly corresponding to that of the centralclear layer of membranes. These rods are either scattered randomly in the lamina,their profiles never exceeding lengths of 60 nm, or they are aggregated in dense bundlesthat often show a braided structure probably due to the interweaving of their filamentousor rod-like subunits (Fig. 38). We cannot infer from our pictures whether the bundlesare composed of extended uninterrupted filaments, or whether they consist of theshort 60 nm subunits arranged side-by-side and tandemly. Interestingly, structuresthat, on the basis of their morphology, are identical to those of the lamina are associatedwith the oolemma where they are arranged in a highly geometrical order, always beingorientated perpendicularly to the membrane (Fig. 37). This coat is separated fromthe outer membrane layer by an electron-lucent space of about 5-7-5 nm and extendsup to 60 nm into the basement lamina (Fig. 37). No direct connection between thebundles in the lamina and the coat at the oolemma was observed.

Fig. 36 A-D. A series of sections of a tubular coated pit whose walls are densely studdedwith a filamentous or rod-like coat outlined by the precipitation of tannic acid.Planorbarius. x 120000.

Fig. 37. Oolemma of a Planorbarius oocyte covered with the densely contrasted coatfollowing tannic acid precipitation. Basement lamina (bl), oocyte (o). x 120000.

Fig. 38. Bundles of filaments or rods in the Planorbarius basement lamina. Thismaterial looks identical to that of the coat at the oocyte membrane and that insidecoated pits (see Figs. 36, 39, 40). x 300000.

Figs. 39, 40. Details of the dense layer inside coated pits. Basement lamina (W),oocyte (o). x 200000 and 240000, respectively.

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The oolemma is not homogeneously covered with this coat but areas of coveragealternate with those that are only covered by an amorphous fluffy material. In manyinstances this filamentous layer can be observed inside coated pits (Figs. 36, 39, 40).In all cases analysed by serial sections, the innermost areas of the pits remain free. Itis unclear whether those areas of the oolemma that are covered by the rods also containferritin particles, or whether their occurrence prevents binding of ferritin to the mem-brane, since in stained sections ferritin particles are not reliably identified, and since inunstained sections the coats can be easily confused with obliquely sectioned membranes.

These coats could not be observed at the membranes of the neighbouring somaticcells, i.e. follicle cells, Sertoli cells, or connective tissue cells. The occurrence of thefilamentous coat inside coated pits suggests that this material is taken up by theoocytes together with ferritin. However, we have not been able to trace this materialinside the oocyte, due to the bad penetration of tannic acid into the cells.

We could not detect ferritin molecules in the plasm of the follicle cells. We concludethat follicle cells are not directly involved in synthesis and transport of vitellogenicferritin.

DISCUSSION

On the basis of electron microscopical results it has been generally assumed that theyolk proteins of freshwater snails are formed endogenously inside the oocyte (deJong-Brink et al. 1976). So far, no vitellogenin or other vitellogenic protein has beenidentified in this animal group. In contrast, our present results indicate that the mainyolk protein of the snails is the principal iron-storage protein, ferritin, and that thisprotein is an exogenous protein that is taken up by endocytosis. Our micrographssuggest that the internalization of vitellogenic ferritin occurs via the coated pit-coatedvesicle route as is the case with typical vitellogenins. Recently it has been shown inmany other cell systems that the presence of coated vesicles can be taken as a reliableindication for a highly selective transport across membranes, which probably involvesbinding of the ligands to specific receptors (Roth et al. 1976; Woods et al. 1978;Goldstein et al. 1979; Willingham & Pastan, 1980). Thus our observations favour theidea that the uptake of ferritin occurs via a highly specific receptor-mediated transportsystem. On the other hand, the observations of Anderson & Spielman (1971) inmosquito oocytes clearly demonstrate that other unspecific electron microscopicalmarkers, e.g. heterologous ferritin or peroxidase, are taken up by the oocytes togetherwith vitellogenin during the rapid growth phase.

Moreover, Roth et al. (1976) have presented evidence that maternal yolk proteinsthat bind to specific receptors at the oolemma even stimulate the uptake of theseunspecific markers. In our opinion, the uptake of vitellogenic ferritin in snails isspecific. The presence of large amounts of ferritin at the membrane, compared with therelative rareness of the protein in the basement lamina, provides morphological evidencefor the binding of the protein at the membrane. Ferritin molecules inside coated pitsare likewise membrane-bound and not encountered in the fluid phase of the vesicles.Since ferritin molecules are still bound to membranes deeply inside the oocyte plasm


and even in immature yolk platelets, the binding to the membrane, i.e. probably toa specific membrane-bound receptor, seems to be stable. The fact that no coatedvesicles without ferritin were observed, though extended areas of the oolemma maybe nearly empty of ferritin, also argues against an unspecific uptake. So far, it isdifficult to speculate on the mechanism by which ferritin is recognized by the oocyte.Clearly, recognition cannot be due exclusively to the negative charge of the moleculessince snail ferritins have isoelectric points nearer to the neutral point than the circu-lating extracellular haemoglobins, which are not taken up by the oocytes, as indicatedby electrophoretic results (unpublished results). Inspection of gels stained by thePAS reaction for the presence of carbohydrates did not yield evidence for the occur-rence of sugars in snail ferritins. However, since this method is clearly not sensitiveenough, and since the occurrence of carbohydrates in ferritin is generally a matter ofcontroversy, this point deserves further consideration.

To our knowledge the only other cell system where ferritin has been shown to betaken up in a highly specific manner via coated vesicles, is the erythroid system invertebrates (Fawcett, 1965). Here uptake and storage of ferritin clearly reflect theneed of the cells to store iron for haemoglobin synthesis. Iron storage is also a well-documented phenomenon in oocytes of vertebrates. Here it is stored in the highlyphosphorylated phosvitin (Taborsky, 1980), which is formed by processing of vitel-logenin, but it may also be stored in ferritin in the plasm (Brown & Caston, 1962).However, the biological sense of storing such excessive amounts of iron inside ferritinas the principal yolk component is enigmatic especially in those snails that containonly rather limited amounts of tissue haemoglobins as a possible acceptor for ferritiniron (Lymnaea). Moreover, observations of Morrill, Rubin & Grandi (1976) indicatethat Lymnaeid snails can survive and hatch even if the bulk of deutoplasmic materialis removed early in embryogenesis.

Ferritin iron is mobilized already in the oocytes as indicated by the presence ofhaemosiderin in such structures, which are supposed to derive from yolk platelets.Our electron microscopical observations are confirmed by cytochemical findings ofde Jong-Brink et al. (1976) and zymograms of Morrill et al. (1976), which indicate thepresence of hydrolases in oocytes and early embryos of 2 other freshwater species.However, though degradation of yolk obviously starts early, it proceeds slowly, sincewell-preserved yolk platelets are encountered even in late embryos (Ami, 1974, 1975).

Typical vitellogenins in other animal systems are under direct hormonal controlas far as synthesis and uptake by the oocyte is concerned (Clemens, 1974). In fresh-water snails there are also indications that vitellogenesis is controlled by a gonadotropichormone of the dorsal bodies (Geraerts & Joosse, 1975). In contrast, no ferritin isknown to be under hormonal control. Regarding the conservative structure andfunction of all known ferritins ranging from bacteria upwards to mammals (Clegg etal. 1980), snail ferritin cannot be expected to deviate drastically in function, structureor regulation from those other ferritins. Hence it is difficult to imagine how hormonesinfluence this protein. Ferritin synthesis is clearly induced and maintained by thepresence of iron in the tissue (Clegg et al. 1980). However, one cannot imagine thatvitellogenesis in snails is governed by the presence of iron in the environment of the

190 W. Bottke, I. Sinha and I. Keil

animals. Therefore, we conclude that there is no hormonal influence acting on ferritinmetabolism.

It cannot be ruled out that the oocytes take up small amounts of other proteins,perhaps typical vitellogenins, in addition to ferritin. These proteins could well beregulated by hormones and could also gain access to the oocyte by endocytosis. It isdifficult to speculate as to the site of synthesis of vitellogenic ferritin. We could isolateferritin from the midgut gland and the haemolymph of our animals (Fig. 33). In themidgut gland ferritin occurs inside the basophilic secretory cells (Fig. 10). Consideringobservations of Heneine et al. (1969) dealing with the transport of iron from the midgutgland to the gonad, in addition to our findings, the midgut gland seems to be the mostlikely candidate for yolk synthesis. Obviously, the follicle cells are ruled out as far asferritin synthesis is concerned. Though these cells show all the characteristics of highlyactive cells no ferritin molecules could be detected in their plasm even under conditionsof iron overload. In addition, those areas of the oolemma that face the follicle cells,do not show endocytotic activity. Therefore, we expect that the transport of vitello-genic proteins occurs via the basement lamina exclusively.

Obviously, the basement lamina does not function as a molecular filter that rejectshigh molecular weight proteins, e.g. ferritin. Its highly porous structure is indicatedby the formation of large ferritin aggregates inside the lamina under conditions ofiron overload. Therefore, we expect the clue to the specificity of uptake to reside inthe molecular structure of the ferritin itself and the recognition system of the membrane.

This investigation was supported by the Deutsche Forschungsgemeinschaft and the StiftungVolkswagenwerk. We thank Dr U. Mays for valuable help and discussions.

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{Received 28 May 1981)

coated vesicle-mediated transport and deposition …the main yolk componen in oocytet os f th...

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