The role of an epithelial occlusion zone in the termination of vitellogenesis in Hyalophora cecropia ovarian follicles

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BIOLOGY

71,

115-127 (I9791

The Role of an Epithelial Occlusion Zone in the Termination Vitellogenesis in Hyalophora cecropia Ovarian Follicles’ ELAINE Biology

Department,

University

C.

of

RUBENSTEIN”

of Pennsylvania,

Philadelphia,

Pennsylvania

Received June 1.2, 1978; accepted in revised form January

19174

29, 1979

At the end of vitellogenesis, the follicular epithelium of Hyalophora cecropia follicles forms an occlusion zone that can halt the access of horseradish peroxidase to the oocyte surface in living follicles, and of lanthanum nitrate in fixed preparations. It is proposed that this barrier is responsible for terminating the uptake of blood proteins by the oocyte. Although three types of interfollicle cell junctions were observed, only tight junctions appeared to be responsible for the observed impermeability. Sodium dodecyl sulfate-acrylamide gel electrophoresis of [“Hlleucinelabeled proteins revealed no change in the protein synthetic pattern during the transformation of follicles from vitellogenesis to the subsequent terminal growth period; in addition, pinocytotic figures continued to be formed in the postvitellogenic oocyte. These findings suggest that the epithelial secretion which the oocyte is known to deposit in yolk during vitellogenesis continues to be sequestered in the absence of blood proteins after occlusion zone formation. The proposal explains the origin of a layer of membrane-limited bodies which occupy the cortex of the oocyte in mature silkworm eggs, and which differ markedly in appearance from the protein yolk spheres assembled earlier. INTRODUCTION

specific mix of protein yolk precursors by (Telfer, 1960; Roth and Vitellogenic oocytes of Hyalophora cec- micropinocytosis ropia form their yolk from a mixture of Porter, 1964; Stay, 1965). During the last few days of metamorphoproteins, at least one of which is synthesized sis, one follicle in each ovariole terminates by the fat body, and one by the follicular blood protein uptake every 4 to 5 hr, even epithelium. The fat body secretes the major yolk precursor, vitellogenin (Pan et al., though the composition of the blood re1969; Pan, 1971), which reaches the ovary mains fully adequate for the support of yolk via the blood, and then permeates the in- deposition in younger follicles (Telfer and Rutberg, 1960). This paper is concerned tercellular spaces of the follicular epithewith the cellular changes that cause the lium (Telfer, 1961). Other blood proteins also permeate the intercellular spaces of follicle to stop accumulating blood proteins the follicle, and the epithelium adds to this in an apparently optimal environment. In view of what is already known about yolk mixture by secreting a noncirculating vitellogenic protein of its own (Anderson and deposition in Hyalophora, the essential questions are as follows: Does the oocyte Telfer, 1969; Bast and Telfer, 1976). The lose its capacity to undergo pinocytosis, as oocyte is thus bathed in a heterogeneous Anderson (1969) proposed for Periplaneta? complex of extracellular macromolecules Or does the epithelium enforce termination from which it adsorbs and incorporates a by becoming impermeable to blood pro’ Supported by NIH Training Grant PHS 5TOl GM teins? Or, finally, is the vitellogenic secre00849 and by NSF Grant BMS 73-01461 to William H. tion of the epithelium itself no longer proTelfer. duced, and is this protein an essential pro‘Present address: Department of Biology, Skidmore College, Saratoga Springs, N.Y. 12866. moter of oocyte pinocytosis? 115 0012-1606/79/070115-13$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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The results described here indicate that the termination of blood protein uptake is enforced by the formation of an occlusion zone which makes the epithelium impermeable to macromolecules. They further indicate that the vitellogenic follicle cell product continues to be synthesized by the epithelium during the terminal growth phase, and that the oocyte continues to engage in pinocytosis. This finding confirms an earlier observation that the follicle cell product can be accumulated by the oocyte in the absence of blood proteins (Anderson and Telfer, 1969) and also explains the origin of a new kind of yolk body that appears in the oocyte cortex during the terminal growth phase. Thus, of the three parameters studied here, the occlusion zone alone enforces the end of blood protein uptake in Hyalophora, while the endogenous secretory and pinocytotic functions that had characterized vitellogenesis appear to play a continuing role in the development of the follicle. MATERIALS

AND

METHODS

Electron Microscopy Females on the 18th or 19th day of the pupal-adult molt were opened by a midline dorsal incision of the abdomen. The abdominal walls were pinned aside in the absence of dissecting solution, and the entire animal was covered with 3% glutaraldehyde in 0.06 M sodium cacodylate buffer at pH 7.4. Intact ovarioles were dissected out and transferred immediately to vials containing the glutaraldehyde fixative. In several preliminary experiments the ovarioles were immersed for 5 min just prior to fixation in 1% (w/v) trypan blue in 70 mJ4 KCl, 0.1 n-&f MgCL, 0.1 n&f CaCk, 0.25 M Tris-succinate buffer, pH 6.2. Trypan blue has been shown to stain only those follicles engaged in blood protein uptake (Telfer and Anderson, 1968). After destaining for several minutes in buffered Cecropia saline (BCS: 40 mM KCl, 15 mM MgCh, 5 mM CaC12,0.11 M Tris-succinate buffer, pH 6.2) the vitellogenic follicles were identified, and the in-

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dividual follicles, their sequence noted, fixed in glutaraldehyde. However, in most experiments, ovarioles were not stained with trypan blue; they were processed as one unit, and the individual follicles were not separated until the time of embedding. After 4 hr of fixation at room temperature, the tissue was gradually cooled to 4OC! and then washed overnight in 0.06 it4 sodium cacodylate buffer. It was postfixed in 1% osmium tetroxide in 0.06 M sodium cacodylate for 2 hr at 4”C, dehydrated, and then separated into individual follicles which were embedded in Araldite 502. In some cases, the tissue was stained en bloc, prior to dehydration, with 0.5% aqueous uranyl acetate (pH 4.5) for 1 hr in the dark at room temperature. Silver and gray sections cut on a Porter-Blum MT-l ultramicrotome were collected on 75-mesh copper grids which had been coated with Parlodion and lightly carboned. Unless otherwise stated, the sections were stained with uranyl acetate and lead citrate before examination with a Phillips 200 electron microscope. Horseradish peroxidase experiments were carried out according to the method of Graham and Karnovsky (1966). Day 18 to 19 females were injected with 0.3 ml of 1% (w/v) horseradish peroxidase (Sigma, Type IV) in sterile BCS. After an incubation of variable length the animal was dissected in the glutaraldehyde fixative. Prior to postfixation, the follicles were incubated in DAB reaction mixture [O.l% (w/v), 3,3’diaminobenzidine (DAB), 0.9% (v/v) Hz02 in 0.5 M Tris-HCl buffer at pH 7.61 for 1.5 hr at room temperature. Postfixation, dehydration, and embedding were according to the standard procedure. Lanthanum nitrate treatment was according to the method of Revel and Karnovsky (1967). A 4% (w/v) stock solution of lanthanum nitrate was brought to pH 7.8 by the gradual addition of 0.1 N NaOH. This stock was added to the glutaraldehyde, wash, and osmium solutions to yield a final lanthanum concentration of 1%.

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Protein Synthesis Culture Day 18 to 19 females were dissected in BCS. Chains of 5-12 follicles were dissected out of the ovariole sheath and transferred to a depression slide containing 100 ~1 of blood from an l&day female, 20 ~1 of [3H]leucine (1 mCi/ml distilled water, New England Nuclear), and a few crystals of twice-recrystallized phenylthiourea (PTU), an inhibitor of melanin formation. After 3 to 4 hr of incubation at 25”C, the follicles were soaked for 2-5 min in BCS to remove unincorporated surface-bound precursor. In order to stage the follicles with regard to vitellogenic activity, they were stained in trypan blue, and each follicle was then placed individually in a prechilled test tube and frozen at -20°C.

Gel Electrophoresis The discontinuous sodium dodecyl sulfate (SDS)-acrylamide gel system of Laemmli (1970) was employed to separate protein samples into their component polypeptides. Electrophoresis was carried out in 1.5-mm-thick slabs. Individual follicles were homogenized and dissolved in a volume of sample buffer (5% 2-mercaptoethanol, 2% SDS, 62 mM Tris-HCl, pH 6.8) sufficient to insure a 3:l ratio by weight of SDS to protein. To protect against proteolytic enzyme degradation, the samples were made 1 mJ4 in phenylmethylsulfonyl fluoride (PMSF) by the addition of 0.1 M PMSF dissolved in 2-propanol and boiled for 10 min. Solubilized protein samples were assayed for protein content (Bramhall et al., 1969) and incorporated radioactivity (Mans and Novelli, 1961) by methods which assay cold TCA-precipitable protein only. Separating gels of 15% acrylamide, 0.39% bisacrylamide and stacking gels of 5% acrylamide were used. Electrophoresis was carried out at 30 mA until the tracking dye, pyronin Y, had migrated to about 1 cm from the bottom of the separating gel (3.54 hr). Radioactivity was analyzed in slab gels

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by the autoradiographic method of Bonner and Laskey (1974). To make detection of tritium possible, the gels were impregnated with PPO (2,5-diphenyloxazole) before drying onto a sheet of filter paper. The dried gel was covered with a sheet of Kodak SB-54 X-ray film, and exposed for 2 weeks at -70°C. RESULTS

Follicular

Ultrastructure

In order to identify the ultrastructural changes accompanying the end of blood protein uptake, chains of follicles were fixed for electron microscopy after trypan blue staging. Shown in Fig. 1 are three of the several consecutive follicles examined from one particular ovariole. The first two follicles, (A) and (B), had bound the dye extracellularly, and thus were vitellogenic. The next follicle (C) did not bind trypan blue, but did not yet show chorion deposition, and thus was in the terminal growth stage. In this particular sequence, there were also two older terminal growth follicles which showed the same fine structure as the follicle in (C). The most conspicuous and consistent ultrastructural changes occurring at the transition from vitellogenesis to terminal growth are in the vitelline membrane. In the second follicle before the cessation of trypan blue uptake (Fig. lA), the vitelline membrane had already thickened from about 0.2 pm as in earlier vitellogenic follicles (Stay, 1965) to 0.3-0.4 pm. In the next older follicle (Fig. lB), the last to stain with trypan blue, the vitelline membrane had thickened further, to approximately 1.0 pm. Within the follicle cells, just basal to the microvilli, were membrane-bound vesicles of about 0.3 pm diameter which contained an amorphous, electron-dense material. In the vitelline membrane, just adjacent to the follicle cell microvilli, were discrete clumps of a material similar in appearance to that contained in the follicle cell vesicles. In the first follicle to exclude trypan blue

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ELAINE

C. RUBENSTEIN

Occlusion

(Fig. 1C) the vitelline membrane had thickened to 1.3-1.6 pm, due largely to the further addition of an electron-dense stratum of material resembling that contained in the follicle cell vesicles of the previous follicle. It seems probable, therefore, that the dense stratum is a follicle cell product. The new layer now formed a continuous sheet 0.2-0.5 pm thick, and although having greater electron density than the layer laid down during vitellogenesis, it had a similar granular appearance. An additional feature of the new vitelline membrane was an outer covering of overlapping, plate-like structures, in cross section 18 nm wide and l-l.3 pm long. Once this layer had been deposited, the general appearance and thickness of the vitelline membrane layer remained constant throughout terminal growth. Despite its thickness and electron density, the vitelline layer is sufficiently porous and extensible to allow the uptake of water and water-soluble metabolites and the increase in oocyte volume that continue during the terminal growth stage (Telfer and Anderson, 1968). As will be seen below, there are also grounds for believing that it continues to be permeable to the vitellogenie protein secreted by the epithelium. Experiments to be reported elsewhere show that a hydrophobic permeability barrier between the epithelium and the oocyte does not appear until the endochorion is deposited at the end of terminal growth. The last follicle to stain with trypan blue possessed a somewhat different type of vesicle in the cortical ooplasm. In younger

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follicles, up to the stage shown in Fig. lA, the peripheral yolk spheres contained an amorphous, uniformly distributed material and an abundance of pinocytotic vesicles (Fig. 1D). Within the cortical ooplasm of the largest staining follicle, and those that followed, one sees instead membranebound vesicles containing a discrete clump of electron-dense material surrounded by an electron-lucent zone (Fig. 1E). These are presumably the cortical refractile bodies described by Telfer and Anderson (1968) in light micrographs of terminal growth follicles. Pinocytotic vesicles and coated pits were present in terminal growth oocytes, although less abundant than in vitellogenic oocytes (Fig. 1E). In the following experiments on the origin of barriers to macromolecular penetration, the youngest terminal growth follicle was identified as the first in the chain of follicles to possess the outer dense layer of the vitelline membrane and the plate-like structures. The end of vitellogenesis could in this way be identified without exposing the follicle to trypan blue, thus avoiding the possibility of ultrastructural artifacts induced by this strongly anionic dye. Horseradish

Peroxidase

If a permeability barrier to vitellogenin is formed at the initiation of terminal growth, it could occur at any of several loci: the basement lamina, the follicular epithelium, or the augmented vitelline membrane with its plate-like structures. To examine these possibilities, horseradish peroxidase (mo-

FIG. 1. Three successive follicles stained with trypan blue before fixation. Ultrastructural changes coincident with the termination of vitellogenesis are most conspicuous in the vitelline membrane (vm). (A) The second to last vitellogenic follicle. The vitelline membrane, separating follicle cells (fc) from oocyte (oo), has increased in size from younger vitellogenic follicles. (B) The last vitellogenic follicle. There is a further increase in vitelline membrane size. Also note the presence of membrane-bound vesicles (v) containing an electron-dense material in the follicle cells and what appears to be the contents of these vesicles dumped at the vitelline membrane (arrowheads). (C) The fist terminal growth follicle. An additional electron-dense layer (vm”) has been added to the preexisting vitelline membrane (vm’). Also present are the overlapping plate-like structures. (D) In a vitellogenic follicle the cortical ooplasm contains numerous pinocytotic vesicles (pv) as well as nascent yolk spheres (ys). (E) In contrast, terminal growth follicles possess cortical refractile bodies (crb), membrane-bound vesicles consisting of an electron-dense core surrounded by an electron-lucent zone, and a reduced number of pinocytotic vesicles. (A)-(C), x 20,930; (D) and (E), x 26,390.

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lecular weight 40,000; particle size 4-6 nm), which is considerably smaller than vitellogenin (molecular weight 500,000; Pan and Wallace, 1974), was employed. At the outset controls were performed that showed no detectable effects of horseradish peroxidase on the rate of yolk sequestration during in uiuo incubations of up to 24 hr. Autoradiograms of follicles labeled in uiuo with tritiated amino acids have shown that a lOOpm stratum of yolk spheres is normally deposited every 24 hr (Melius and Telfer, 1969); light microscopy of thick sections of follicles taken from animals that had been injected with horseradish peroxidase indicated that yolk spheres containing the enzyme had been deposited at a similar rate. Follicles from animals which had not been injected with the enzyme showed no deposition of electron-dense reaction product, indicating that there was no demonstrable ovarian peroxidase activity. Thus the injected enzyme was a valid test of the distribution of exogenous protein in normally developing follicles. Further studies, involving the omission of the different components of the horseradish peroxidase-DABH202 reaction mixture, coupled with comparison of sections with and without heavy metal staining, confirmed that the horseradish peroxidase reaction product can be accurately distinguished from the heavy metal stain used to enhance tissue structure, or from any structures or deposits with natural electron density. These controls also demonstrated an apparently un-

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avoidable cytoplasmic leaching effect of the reaction mixture. Vitellogenic follicles exposed to horseradish peroxidase for 3.5 hr in vitro and reacted with the DAB reaction mixture (Fig. 2A) had a homogeneous, electron-dense precipitate within the follicular intercellular spaces, throughout the vitelline membrane, the spaces between the follicle cell microvilli, within the long crevices of the oocyte brush border, in pinocytotic vesicles, and in newly formed yolk spheres. This pattern of extracellular horseradish peroxidase distribution was present in all vitellogenic follicles. By contrast, in follicles which had deposited the thickened vitelline layer and which were therefore no longer stainable with trypan blue, a dramatic change had occurred in the pattern of horseradish peroxidase distribution. The enzyme continued to traverse the basement lamina and to enter the basal end of the intercellular spaces of the epithelium, but it had failed to penetrate beyond a point about 3 pm from the vitelline membrane (Fig. 2B). The intercellular spaces apical to this point, the vitelline membrane, and the crevices of the oocyte brush border were free of electron-dense deposits. The same distribution was seen in all terminal growth and chorionating follicles that were examined. At the point of occlusion, areas of extremely close apposition of adjacent follicle cell membranes were prominent. The apposition was so close that at some points

FIG. 2. (A) A vitellogenic follicle after horseradish peroxidase treatment. Horseradish peroxidase reaction product is seen in the intercellular spaces (is) between the follicle cells (fc), in the vitelline membrane (vm), within the long crevices of the oocyte brush border, within the oocyte (00) in pinocytotic vesicles, and in newly formed yolk spheres. (B) A terminal growth follicle after horseradish peroxidase treatment. In contrast, the penetration of horseradish peroxidase in a terminal growth follicle is halted before reaching the vitelline membrane. The reaction product is only found basal to an occlusion zone (oz) about 3 pm from the vitelhne membrane. (A) and (B), x 20,930. FIG. 3. (A) Tight junction in a horseradish-peroxidase-treated follicle. At the endpoint of horseradish peroxidase penetration a tight junction can be distinguished by regions of extremely close apposition of adjacent follicle cell membranes (arrowheads). (B) A desmosome (d) near the follicle cell microvilh. There is an increased density of the intercellular space, and microtubules (mt) are found in the adjacent cytoplasm. (C) Septate junction in a vitellogenic follicle. A variable number of septae are seen traversing the interfollicle cell space. (A), X 112,770; (B) and (C), x 61,870.

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the opposing outer leaflets appeared fused, as in tight junctions. In Fig. 3A it can be seen that horseradish peroxidase had not penetrated such an area of close membrane apposition. Lanthanum Nitrate To examine further the nature of the occlusion zone formed at the initiation of terminal growth, colloidal lanthanum nitrate was presented to follicles during fixation. Because of some difficulty in visualizing the lanthanum deposits in material which had been stained with heavy metals, unstained sections were routinely examined. Lanthanum nitrate, when presented to vitellogenic follicles during fixation, permeated all of the intercellular spaces of the epithelium, traversed the vitelline membrane, and filled the crevices and the coated pits of the oocyte surface (Fig. 4A). In contrast, the first terminal growth follicle in any given developmental sequence (Fig. 4B) exhibited an epithelial occlusion zone at approximately the same site as that observed in horseradish-peroxidase-treated follicles. Lanthanum nitrate penetrated, and fairly densely packed, the now-narrowed intercellular spaces of the epithelium; however, it was never found closer than about 2 pm from the oocyte surface. From terminal growth through chorion formation, the oocyte surface is no longer accessible to colloidal lanthanum nitrate. In summary, the observations made of

FIG. 4. (A) A vitellogenic follicle treated with lanthanum nitrate during fixation. Lanthanum nitrate deposits (la) are found between the follicle cells (fc) throughout the intercellular spaces (is), the vitelline membrane (vm), and the crevices and coated pits of the oocyte (00) surface. (B) A terminal growth follicle treated with lanthanum nitrate during fixation. Here, the penetration of the tracer is halted by an occlusion zone (02) and never reaches the vitelhne membrane. These sections were not stained with heavy metals. (A), x 6ooo; (B), x 16,300.

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5

abcdefgh

ch FIG. 5. An autoradiogram of a 15% acrylamide-SDS slab gel on which consecutive [“Hlleucine-labeled follicles were electrophoresed. The vitellogenic follicle cell product (arrowhead), synthesized during vitellogenesis (a), continues to be synthesized during at least part of the terminal growth phase (b-0. The protcm synthetic pattern of follicles does not change dramatically until chorion synthesis begins (g-i). vg, Vitellogenic; tg, terminal growth; ch, chorionating

horseradish peroxidase and colloidal lanthanum nitrate penetration in developing Hyalophora follicles demonstrate that a zone of occlusion is formed coincident with the end of trypan blue staining and blood protein uptake. The exclusion of horseradish peroxidase from the apical ends of terminal growth follicles suggests that a molecule such as vitellogenin, which is considerably larger than horseradish peroxidase, must similarly be denied access to the oolemma. Because lanthanum nitrate penetration is halted in approximately the same area and at the same developmental stage as horseradish peroxidase, it is further suggested that the same occlusion zone is being detected by both labels.

Interfollicle

Cell Junctions

Because tight junctions have not yet been clearly established as a common feature of invertebrate tissue, the other junctional structures found between follicle cells were examined. Although desmosomes and septate junctions were also observed, there was no evidence to indicate that they, rather than the tight junctions, were responsible for the observed impermeabilities. Desmosomes (Fig. 3B) were first described in vitellogenic Hyalophora follicles by Stay (1965). They are a common feature of vitellogenic and older follicles. Their role in inter-follicle cell adhesion was suggested

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by Stay (1965) with the observation that even after the cell shrinkage which accompanied osmium fixation, desmosomes were found at the ends of finger-like projections connecting adjacent follicle cells. A uniform distance of 14-17 nm between membranes is maintained throughout the junctional region, and, as in other insect tissue, Hyalophora’s interfollicle cell desmosomes seem to be associated with microtubules rather than microfilaments or tonofilaments (Overton, 1974). In cross section, the microtubules appear to run parallel to the desmosomes in the adjacent cytoplasm. There is some increased electron density of the intercellular space, with no obvious order, unless perhaps a transverse fibrillar one. No median line or microfilaments adjacent to the junction were apparent, although these are often described as common desmosome features. There was no reason to suspect any role other than an adhesive one for these desmosomes, in that their appearance was constant throughout development, and they were never associated with the occlusion of the tracer molecules. In insects, septate junctions have been suggested as both transepithelial permeability barriers and areas where electrical coupling occurs between cells (Satir and Gilula, 1973). Septate junctions (Fig. 3C) were found in vitellogenic as well as terminal growth and older follicles. They consisted of parallel membranes 12-18 nm apart, traversed by a variable number of electron-dense septa. When sections of lanthanum-treated follicles were examined without heavy metal staining, the septa were negatively outlined. A tangential plane through a junction revealed the pleated structure of the septa thus showing Hyalophora’s septate junctions to be of the comb type first described by Locke (1965) and later suggested by Danilova et al. (1969) to be the standard form in insect tissue. As was the case with desmosomes, septate junctions were found in all developmental stages and were found traversed

by the tracer molecules. Thus it seems improbable that septate junctions are responsible for the observed impermeabilities. Horseradish Peroxidase Distribution the End of Vitellogenesis

after

In two other insects lack of oocyte pinocytotic activity was suggested as the immediate cause of the termination of vitellogenesis (Anderson, 1969; Anderson and Spielman, 1971). Evidence presented here indicates that in Hyalophora, however, pinocytosis may continue after blood protein uptake has ceased. The validity of this difference is confirmed by a striking difference between Hyalophora and Periplaneta in the termination of horseradish peroxidase uptake. Anderson (1969) found that Periplaneta oocytes which no longer contained pinocytotic configurations and had not taken up injected enzyme from the blood were nevertheless directly bathed by horseradish peroxidase, which permeated all of the intercellular spaces of the follicle. A very different result was obtained in Hyalophora. Horseradish peroxidase was never observed apical to the occlusion zone in follicles which, according to their vitelline membrane structure, had initiated terminal growth. With 4-5 hr of developmental time separating neighboring follicles, well over half of the ovarioles dissected after 3.5 hr of incubation should have contained one follicle that had formed its occlusion zone in the presence of the enzyme. The invariable absence of enzyme between the epithelium and the oocyte therefore implies that blood proteins trapped in this region by the occluding zone are subsequently removed, presumably by pinocytosis, and in fact, coated pinocytotic pits and vesicles were seen in the cortical ooplasm of terminal growth follicles. Protein Synthesis Patterns With the discovery of occlusion zone formation at the outset of terminal growth, determining when the synthesis of the vi-

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Occlusion

tellogenic follicle cell product terminates became of special interest, in that the possibility arose that secretion and pinocytosis of this protein continued in the absence of blood proteins. To determine this, ovarioles were labeled in vitro with [3H]leucine, and consecutive follicles spanning the developmental stages from late vitellogenesis to early chorion formation were analyzed individually by SDS-acrylamide gel electrophoresis. Figure 5 is an autoradiogram of such a series after electrophoresis. Trypan blue staining had indicated that follicle (a) (and several before it, not included here) were vitellogenic. Chorion synthesis had begun in follicles (g)-(i), as is shown by the heavy labeling of small-molecular-weight polypeptides (Paul et al., 1972; Bast and Telfer, 1976). The five intermediate follicles, (b)-(f), were therefore in terminal growth, and their labeling patterns proved to be indistinguishable from those of the vitellogenic follicles. The vitellogenic epithelial product was previously identified as the most heavily labeled component in tube gels of follicles which had been labeled with [3H]leucine during yolk formation. The band which satisfied these criteria under our conditions is identified by an arrow in Fig. 5. Along with all other components labeling in follicles (a)-(f), it continued to be labeled throughout the terminal growth period. In other experiments the number of terminal growth follicles varied from 2 to 5, but the result was always the same. There was no detectable change in the synthetic pattern of the follicle during late vitellogenesis and terminal growth, while the transformation to chorion formation was, by contrast, accompanied by a sweeping reorganization. In combination with the indication of pinocytotic activity in terminal growth oocytes, the continued synthesis of the epithelial product implies that the endogenous aspects of yolk deposition continue after the occlusion zone has blocked blood protein access. This in turn can ex-

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plain the origin of the new population of cortical bodies appearing in the oocyte during terminal growth. DISCUSSION The results establish that of those characteristics of vitellogenic follicles studiedepithelial permeability, protein synthetic profiles, and oocyte pinocytosis-only the first is altered at the end of blood protein uptake. The occlusion zone responsible for this transformation possesses several characteristics of the zonula occludens, or tight junction, which was first described by Farquhar and Palade (1963). It is impermeable to horseradish peroxidase in living follicles and to lanthanum nitrate in fixed material; in thin sections it appears as a region of punctate, pentalaminar fusions. While it has been suggested that conventional tight junctions, as defined by freeze-fracture criteria, may not occur in invertebrate tissue (Satir and Gilula, 1973)) a functional permeability barrier clearly exists in this case, with an appearance and behavior in sectioned materials that would be difficult to distinguish from vertebrate tight junctions. For several reasons, septate junctions appear not to be the structures responsible for the observed impermeabilities. They exist in vitellogenic follicles when the intercellular spaces are fully permeable, and at this stage are indistinguishable ultrastructurally from those in terminal growth follicles. In addition, the septate junctions of terminal growth follicles were never seen in the regions where tracer penetration ceased, and in fact were found traversed by the tracers. By contrast both the location and the temporal correlation between the onset of impermeability and the first appearance of tight junctions makes them the more logical candidates for occlusion in this case. In two insects where the permeability of the follicular epithelium to tracer molecules was previously studied, tight junctions were not believed to be responsible for the ces-

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sation of blood protein uptake (Anderson, 1969; Anderson and Spielman, 1971). In Hyalophora however it is now clear that occlusion zone formation is a key event. Inaccessible to blood proteins, but still supplied with epithelial secretion, the oolemma continues pinocytosis, but instead of the normal vitellogenin-containing yolk spheres, a special cortical structure is formed which presumably contains only the follicle cell product. These bodies have a characteristic ultrastructure (Telfer and Smith, 1970) which has also been seen in the egg cortex of Ephestia kuhniella (Cruickshank, 1972) and of Bombyx mori (Takesue et al., 1976) and thus they appear to occur widely among Lepidoptera. They have not been described in other insect orders, however, and this suggests that epithelial secretion and occluding zone formation may be special lepidopteran characteristics. The loss of trypan blue binding activity, which has been a convenient index to the transition from vitellogenesis to terminal growth, can now be better explained. During vitellogenesis trypan blue presented to the follicle adheres to all extracellular components, including the basement lamina and the proteins in the channels between the epithelial cells. It is especially heavily bound to the vitelline membrane layer and matrix that occupy the zone between the epithelium and the oocyte. In terminal growth follicles the dye is no longer detectably bound in the latter zone, while the reduction in basal binding is somewhat more gradual (Fig. 17 in Telfer and Anderson, 1968). It can now be proposed that trypan blue, with a molecular weight of 960, cannot pass the occlusion zone, and that the more gradual reduction in basal binding is due to a slower disassembly of the blood protein-rich intercellular matrix characterizing the vitellogenic follicles. A striking characteristic of ovarioles stained with trypan blue for 5-15 min in vitro is that the largest heavily staining

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follicle is usually followed immediately by an older neighbor in which no dye has reached the apical side of the epithelium. Intermediate patterns of staining are rarely seen, and this implies a high degree of coordination within the epithelium. The first and last cells in the epithelium to form effective barriers to the dye do so with a time difference that is well under the 4- to 5-hr developmental lag between successive follicles. Compared to the l-week time scale of vitellogenesis, occlusion zone formation is therefore a rapid and closely coordinated epithelial transformation. Finally, it should be emphasized that occlusion zone formation is not an isolated developmental change. The results described here revealed an acceleration of vitelline membrane deposition and a change in character of the secretory product which yields the more densely staining outer vitelline membrane layer, all beginning several hours before occlusion zone formation. Peroxidase labeling showed that these changes do not in themselves block the uptake of blood proteins, however, and it is not until the epithelium forms its occlusion zone that the termination of vitellogenesis finally takes place. I am extremely grateful to Dr. William Telfer for his valuable suggestions during all phases of this work and for his critical reading of the manuscript. REFERENCES ANDERSON, E. (1969). Oogenesis in the cockroach Periplaneta americana, with special reference to the specialization of the oolemma and the fate of coated vesicles. J. Microsc. 8, 721-738. ANDERSON, L. M., and TELFER, W. H. (1969). A follicle cell contribution to the yolk spheres of moth oocytes. Tissue Cell 1, 633-644. ANDERSON, W. S. and SPIELMAN, A. (1971). Permeability of the ovarian follicle of Aedes aegypt mosquitoes. J. Cell Biol. 50, 201-221. BAST, R. E., and TELFER, W. H. (1976). Follicle cell protein synthesis and its contribution to the yolk of the Cecropia moth oocyte. Develop. Biol. 52,83-97. BONNER, W. M., AND LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biothem. 46,83-88.

ELAINE C. RUBENSTEIN

Occlu sion Zone in H. cecropia Follicles

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