A procedure has been developed for separating the oocytes and follicular epithelium-nurse cell complexes making up the vitellogenic ovarian follicle of the Cecropia moth. Both components remained viable during short-term in vitro incubation in female blood. Isolated epithelial cells were found by autoradiography to incorporate tritiated amino acids and to secrete a fixable, non-dialysable labelled material. Isolated oocytes incubated in a blood medium containing this tritiated, dialysed follicle cell product incorporated it in small cortical yolk bodies, presumably by pinocytosis. Quantitative perchloric acid-precipitation and scintillation counting indicated that the amount of labelled material incorporated by the oocytes increased with time. These results provide direct confirmation of a follicle contribution to the yolk.

Isolated oocytes were also tested for their ability to incorporate labelled amino acids. Fixable label was observed autoradiographically throughout the oocyte cytoplasm, with the greatest concentration in the cortex, but little appeared in the yolk spheres. The amount of perchloric acid-precipitable amino acid in oocytes incubated in female blood increased with time for up to 2 h and then remained constant or decreased slightly. In medium that had been previously conditioned by follicle cells and dialysed, however, incorporation of labelled amino acid continued for at least 4 h. A possible interpretation of this result is that stimulation of pinocytosis by the epithelial cell products causes increased turnover of cell membrane and demands continued synthesis of new proteins.

Labelled female blood proteins were not incorporated into yolk to an appreciable extent by isolated oocytes, even in the presence of follicle cell product. Perhaps extracellular pre-concentration, as occurs in the intact follicle, is necessary for effective accrual of blood proteins. The female blood proteins did become associated with the oocyte cortex, however, and exhibited a higher affinity for the oocyte than male blood proteins. Thus preferential adsorption to the oocyte surface may be a component of the selection process in vitellogenesis.

Proteins enter the vitellogenic oocyte of the Cecropia moth from 3 sources: large amounts of haemolymph protein, incorporated by pinocytosis, are stored in yolk spheres as a major nutritive provision for the embryo (Telfer, 1965); the yolk spheres also receive a secretory product, believed to be at least partly protein, from the follicular epithelium surrounding the oocyte (Anderson & Telfer, 1969); and part of the cytoplasm of the oocyte, including protein, is derived from the nurse cells (Melius, 1966).

The simultaneous occurrence of these protein movements and the possibility of additional protein synthesis within the oocyte itself have hampered definitive study of individual protein formation and transport events. Recently, however, with development of methods for in vitro incubation and labelling, it has been possible to distinguish unambiguously between proteins synthesized within the follicle and those formed elsewhere within the animal (Anderson & Telfer, 1969; Melius & Telfer, 1969; Hausman, Anderson & Telfer, 1971). This procedure for short-term in vitro experimentation has now been extended to oocytes and epithelia isolated from each other and in recombination, in order to permit sorting out of the intrafollicular activities related to protein formation and accumulation.

Both isolated oocytes and follicle (epithelial) cell-nurse cell complexes have been found to survive for at least 4 h during incubation in female blood and to carry on some of their normal functions. The success of the isolation and incubation procedures renders feasible, for the first time, direct analysis of the contributions and capabilities of each cell type. The initial studies, reported here, were focused on the vitellogenic functions of the oocyte, since its activities have been particularly obscured by contributions from the associated cells.

The investigation, utilizing autoradiographic and quantitative determination of isotopic labelling, dealt with 2 questions: whether the oocyte itself synthesizes yolk proteins, in addition to those received from extracellular sources; and whether the isolated oocyte is able to accomplish the selective uptake and concentration of extra-cellular materials normally entailed in yolk sphere genesis. The results indicated that the proteins synthesized by the oocyte remain localized primarily in the cytoplasm of the cortex, with little observed in the yolk spheres. The isolated oocyte is also capable of pinocytosis, incorporating labelled extracellular, non-dialysable follicle cell products at a rate comparable to that observed in the intact follicle. Labelled female blood proteins, however, were not taken up in detectable amounts, even though they became adsorbed to the oocyte surface. The surface interaction was apparently selective, since labelled male blood proteins were adsorbed to a lesser extent. Thus the oocyte may participate in the selection of blood-borne proteins for vitellogenesis, but apparently requires the presence of an intact follicular epithelium to amass these proteins as yolk.

Isolation of oocytes and epithelial cell-nurse cell complexes

The removal of the epithelial and nurse cells from vitellogenic oocytes involved 3 steps: (1) extraction of extracellular materials, including concentrated blood protein (Anderson & Telfer, 1970b) by soaking the follicles in Cecropia saline (Anderson & Telfer, 1969), modified to contain a low concentration of K+; (2) digestion of the basement lamina and remaining extra-cellular materials with pronase; and (3) separation of the cells by gentle pipetting in low K+ saline containing o-i M sucrose to induce osmotic shrinkage.

Female pupae on day 18–21 of adult development were dissected in low K+ saline (1 mM KC1, 15 mM MgCl2 4 mM CaCl2, 180 mM tris-succinate buffer, pH 6*3) and the ovariole sheath removed from the vitellogenic follicles approximately 1·3–1·8 mm long. In experiments where labelled material was to be extracted and measured quantitatively, identical segments containing 6–12 follicles were removed from each of the 8 ovarioles and processed individually; each sample then consisted of 1 or 2 segments. For autoradiographic experiments the vitellogenic follicles from each ovary were processed together and 6–10 oocytes selected at random for each experimental treatment.

After 1-5 h of soaking the follicles were treated in a depression slide for 5 min with a solution of pronase (1 mg/ml; Calbiochem, Grade B) in low K+ saline. The basement lamina became thinner during this treatment and disappeared during subsequent washing. After pronase digestion the oocytes ruptured if allowed to contact the surface of the medium, so that all subsequent transfers were carried out by means of a fire-polished, broad-mouthed pipette.

The pronase was rinsed off with cold low K+ sucrose solution, either by dilution or by transferring the follicles through a series of baths. The latter method of washing yielded the greatest percentage of intact oocytes. The follicles were transferred to a Syracuse watch glass containing cold low K+ sucrose solution and gently pulled in and out of the pipette. In most cases the epithelium came off the oocytes in sheets with the nurse cells attached, leaving intact, denuded oocytes. Occasionally a small percentage of the oocytes began to disintegrate shortly thereafter, and were discarded. The remainder of the isolated oocytes remained intact for at least 30 min in the low K+-sucrose saline.

The epithelium-nurse cell complex was always removed easily and viable oocytes obtained when the above procedure was followed exactly. However, the cells tended to resist separation if the pronase was not fresh, if the follicles were not soaked for at least 1 h prior to pronase treatment, or if the female from which the follicles were taken was younger than 18 days of adult development. Higher concentrations of pronase or longer treatment with the enzyme increased the incidence of oocyte lysis. The technique was in general not successful with follicles shorter than 13 mm or longer than 2-0 mm.

Incubation and viability tests

The denuded oocytes were transferred to 0·1–0·2 ml of various experimental media contained in depression slides, the depression was covered with a coverslip, and the slide was incubated in a humidified Petri dish with shaking. This incubation procedure has been shown to be adequate for the survival and vitellogenic functioning of intact follicles (Hausman et al. 1971). At the end of most incubations, the viability of the oocytes was tested prior to preparation for autoradiography or extraction by addition of about 0–1 ml of a 1 % suspension of trypan blue (Anderson & Telfer, 1970a) to the medium. After 10 min the dye was washed off by dilution and the oocytes examined under the dissecting microscope. In cases of cell death the deep blue of the cytoplasm, due to penetration of the colloidal dye, was readily apparent. When female blood was used as the incubation medium (see Hausman et al. 1971, for details of preparation),90 % or better of the oocytes survived a 3–4 h incubation in the majority of the experiments. In a few experiments 30–50 % of the oocytes showed large patches of blue cytoplasm in the trypan blue test after incubation. These were excluded from the recorded results. Survival of the denuded oocytes in Cecropia saline was poor; about 50% disintegrated and less than 10% excluded trypan blue after 3 h of incubation. Male blood was much better than saline but not as good as female blood, permitting survival of 40–60 % of the oocytes.

Isotopic labelling

Isolated oocytes or epithelia were labelled in female blood to which had been added 0·1 mCi/ml [3H]histidine (L-histidine-2,5-T, Amereham Searle, 1 mCi/ml, 40 Ci/mmol), [3H]-leucine (L-leucine-4,5-T, New England Nuclear, 1 mCi/ml, 5 Ci/mmol or 38-5 Ci/mmol; the latter preparation was used only in Expts. 2 and 3, Table 2), or tritiated blood proteins, obtained by injecting developing pupae with [3H]leucine as described previously (Hausman et al. 1971).

Table 1.

Incorporation of [3H]histidine-labelled material from dialysed, epithelial cell-conditioned medium

Incorporation of [3H]histidine-labelled material from dialysed, epithelial cell-conditioned medium
Incorporation of [3H]histidine-labelled material from dialysed, epithelial cell-conditioned medium
Table 2.

Effect of follicle cell-conditioned medium on [3H]leucine incorporation by isolated oocytes

Effect of follicle cell-conditioned medium on [3H]leucine incorporation by isolated oocytes
Effect of follicle cell-conditioned medium on [3H]leucine incorporation by isolated oocytes

For autoradiography, the cells were fixed in Bouin’s solution, embedded in paraffin, sectioned at 2 or 10 μm, deparaffinized, hydrated, and coated with NTB-2 liquid emulsion (Kodak). The slides were developed with Kodak D-n after 7-8 weeks. The amount of labelling varied somewhat from cell to cell and from one region of a given oocyte to another, as might be expected following so drastic a treatment. Regions which appeared normal morphologically usually showed the maximum amount of label, and so were assumed to be the healthiest and most representative for photographing.

For quantitative extraction of incorporated label, the oocytes in each sample were rinsed with Cecropia saline, transferred to 0·2–0·4 ml saline, frozen and thawed, and homogenized with a capillary pipette. Aliquots of 0·1 ml of each sample were dried on glass-fibre filter disks and subjected to perchloric acid (PCA) precipitation and ethanol-ether washing by the method of Mans & Novelli (1961). The disks were layered with 3 ml of scintillation fluid (0·5% PPO (2,5-diphenyloxazole) and 0·03% POPOP (1,4-bis-2-(5-phenyloxazolyl)-benzene) in toluene) and counted for 5·10 min in a Packard Tricarb scintillation counter. The degree of quenching was found to be similar for all samples within an experiment. The cpm value for each sample was corrected, for background radioactivity and used to calculate the total cpm per oocyte or per group of oocytes.

Duplicate samples were run in most experiments because of the variability in labelling observed autoradiographically. Results from the duplicates were usually in good agreement, indicating that the variations in incorporation between oocytes averaged out from sample to sample. All experiments were repeated at least 3 times.

Incorporation of tritiated amino acids by isolated oocytes

Autoradiographs of denuded oocytes incubated in female blood containing 0·1 mCi/ml [3pH]leucine or [3pH]histidine for 3 h revealed that both precursors were incorporated in fixable form by the oocytes (Figs. 2, 3). Silver grains were evident over the entire oocyte cytoplasm, with the greatest concentration over the cortical region.

Fig. 1.

Matched samples of isolated oocytes were incubated in female blood containing 0·1 mCi/ml [3PH]histidine and assayed for PCA-precipitable radioactivity at the times specified. Dots represent duplicate samples at each time point. Two other experiments gave similar results; see also Table 2.

Fig. 1.

Matched samples of isolated oocytes were incubated in female blood containing 0·1 mCi/ml [3PH]histidine and assayed for PCA-precipitable radioactivity at the times specified. Dots represent duplicate samples at each time point. Two other experiments gave similar results; see also Table 2.

Figs. 2, 3.

Autoradiographs of peripheral portions of isolated oocytes incubated for 3 h in female blood containing 0·1 mCi/ml [3H]leucine (Fig. 2) or [3H]hi8tidine (Fig. 3). 10-μm sections, ×880.

Figs. 2, 3.

Autoradiographs of peripheral portions of isolated oocytes incubated for 3 h in female blood containing 0·1 mCi/ml [3H]leucine (Fig. 2) or [3H]hi8tidine (Fig. 3). 10-μm sections, ×880.

That the observed labelling was due to protein synthesis rather than adsorption of free amino acid was indicated by the marked inhibitory effect of 1 mM cyclo-heximide (compare Fig. 4 with Fig. 2). Further confirmation was sought by investigating whether labelling increased with time, as would be expected in the case of protein synthesis. The amount of perchloric acid-precipitable radioactivity per oocyte in matched samples incubated in female blood with either [3pH]leucine or [3pH]histidine increased with time from 30 min to 2 h (Fig. 1). The rate of incorporation decreased slightly during this period, and after 2 h the amount of label incorporated remained constant or decreased somewhat. However, incorporation continued for at least 4 h if the blood used had been conditioned by isolated epithelial cells, as is demonstrated in the following paragraphs.

Fig. 4.

Oocyte incubated for 3 h in blood containing 0·1 mCi/ml [3pH]leucine as in Fig. 2 but in the presence of 1 mM cycloheximide. (Irregular inclusions deep in the oocyte are histological artifacts.) 10-μm section, ×880.

Fig. 4.

Oocyte incubated for 3 h in blood containing 0·1 mCi/ml [3pH]leucine as in Fig. 2 but in the presence of 1 mM cycloheximide. (Irregular inclusions deep in the oocyte are histological artifacts.) 10-μm section, ×880.

In autoradiographs of 2-μm sections, in which there is little overlap of the larger yolk spheres with cytoplasm, the silver grains appeared over the cytoplasm rather than over the yolk spheres (Fig. 5), although a low degree of labelling of small yolk spheres in addition to the cytoplasm cannot be ruled out. The pattern and degree of cytoplasmic labelling in the isolated oocytes was similar to that observed in oocytes of whole follicles incubated in the same manner. The oocyte can therefore synthesize much of its own cytoplasmic protein and may not rely on the epithelium or nurse cells for significant amounts of these components.

Fig. 5.

Oocyte labelled with [3H]leucine as in Fig. 2. 2-μm section, ×880.

Fig. 5.

Oocyte labelled with [3H]leucine as in Fig. 2. 2-μm section, ×880.

The absence of amino acid incorporation in the peripheral yolk spheres of the isolated oocytes, on the other hand, is in contrast to the marked labelling of these organelles in whole follicles incubated in the presence of tritiated amino acid. This labelling has been attributed to pinocytosed secretion products of the follicular epithelium (Anderson & Telfer, 1969), a conclusion which is supported by the absence of detectable label in the yolk spheres of isolated oocytes in the present experiments. Either the synthetic products of the oocyte are not added to the yolk spheres, or such contributions as normally do occur are withheld under the experimental conditions employed.

Behaviour of isolated epithelial cells in vitro

The follicular epithelium-nurse cell complexes isolated from the oocytes could be maintained in large sheets by gentle handling during the separation or dispersed into small clumps by repeated pipetting. Most of the cells remained alive during removal and subsequent incubation for 3–4 h in female blood, since only 10-20% were deeply stained by the trypan blue viability test. An occasional clump or sheet of follicle cells became firmly reattached to an isolated oocyte when the two were incubated together (Fig. 6).

Fig. 6.

Phase-contrast micrograph of a sheet of follicle cells partially reattached to the surface of an isolated oocyte. 10-μm section, x 340.

Fig. 6.

Phase-contrast micrograph of a sheet of follicle cells partially reattached to the surface of an isolated oocyte. 10-μm section, x 340.

The ability of the isolated epithelial and nurse cells to incorporate amino acids and secrete proteins was tested using a procedure similar to that employed for the denuded oocytes. The separated complexes were washed several times with Cecropia saline by gentle centrifugation and resuspended; most sheets were broken into small clumps by this treatment. The washed cells from 50-80 follicles were incubated for 3·4 h in 0·1 ml of female blood containing 0·1 mCi/ml [3pH] histidine, fixed in Bouin′s, and processed for autoradiography.

Both epithelial cells and nurse cells incorporated [3pH]histidine in fixable form in their cytoplasm and nuclei (Fig. 7) and PCA-precipitable label appeared in the incubation medium, as indicated by the filter disk method. A source of the labelled material in the medium was suggested when clumps of follicle cells became folded over to form a pocket or cyst: the resultant cavity often contained a fixable, flocculent material that was moderately labelled in autoradiographs (Fig. 7). In some instances the label in the follicle cells was greatest at the surface adjacent to the cavity, as in Fig. 7, suggesting that the radioactive extracellular material tended to adhere to the surface from which it was secreted. The possibility that at least some of it was produced by the nurse cells also present cannot of course be ruled out, though under in vivo circumstances the nurse cells transfer their products to the oocyte through cytoplasmic connectives, rather than secreting them into the extracellular spaces.

Fig. 7.

Autoradiograph of a sheet of isolated follicle cells incubated in female blood containing 0·1 mCi/ml [3H]histidine for 3 h. The sheet is folded over to form a pocket (p), which is filled with a flocculent, labelled material. The surfaces of the follicle cells adjacent to the pocket (s) are intensely labelled. The extracellular material is pulled away from the follicle cell surfaces in some regions, presumably as a result of shrinkage in fixation. 10-μm section, ×340.

Fig. 7.

Autoradiograph of a sheet of isolated follicle cells incubated in female blood containing 0·1 mCi/ml [3H]histidine for 3 h. The sheet is folded over to form a pocket (p), which is filled with a flocculent, labelled material. The surfaces of the follicle cells adjacent to the pocket (s) are intensely labelled. The extracellular material is pulled away from the follicle cell surfaces in some regions, presumably as a result of shrinkage in fixation. 10-μm section, ×340.

The extracellular substance released by the isolated epithelial cell-nurse cell complexes might well include the histidine-labelled product secreted by the epithelium of the intact follicle for pinocytosis by the oocyte. It therefore seemed a suitable material with which to test the pinocytotic ability of the isolated oocytes.

Incorporation of labelled follicle cell product by oocytes

In an experiment to discover whether isolated oocytes would take up released follicle cell product, oocytes were incubated for 3 h in 0·1 ml of female blood containing 0·1 mCi/ml [3pH]histidine and the isolated follicle and nurse cells from 50 to 80 follicles. In autoradiographs, fixable label was observed in dense concentrations at the oocyte surface and in small cortical yolk spheres, as well as in the cytoplasm (Fig. 8). The quantity of fixable [3pH]histidine label in the cortex was an order of magnitude greater in the presence of the epithelial cells than in their absence (compare Figs. 3 and 8), though the concentration of label was the same in the medium in both cases.

Fig. 8.

Autoradiograph of isolated oocyte incubated in female blood with 0·1 mCi/ml fHJhistidine as in Fig. 3 but in the presence of the isolated follicle cells from about 80 follicles. Silver grains are densely concentrated over small bodies in the cortex (arrows). 10-μm section, ×880.

Fig. 8.

Autoradiograph of isolated oocyte incubated in female blood with 0·1 mCi/ml fHJhistidine as in Fig. 3 but in the presence of the isolated follicle cells from about 80 follicles. Silver grains are densely concentrated over small bodies in the cortex (arrows). 10-μm section, ×880.

To determine whether the labelled content of the cortical spheres was in fact produced by the follicle or nurse cells, rather than by the oocyte under some undetected stimulus from them, saline-washed epithelium-nurse cell complexes from about 80 follicles were incubated in 0·2 ml of female blood containing 0·1 mCi/ml [3H]histidine for 3–4 h. The cells were removed by centrifugation, and the medium was dialysed against Cecropia saline for 3 h. As a control identical samples of blood containing labelled histidine but without epithelial cells were incubated, centrifuged, and dialysed in parallel. Isolated oocytes were then incubated in mixtures consisting of 70% dialysed media and 30% whole female blood to provide any needed dialysable nutrients. Unlabelled histidine (1 HIM) was included to reduce incorporation of any remaining free [3pH]histidine.

Autoradiography demonstrated (Fig. 9) that the follicular epithelium-nurse cell mixtures had in fact synthesized and secreted a non-dialysable material which appeared to be fixed at the surface of the oocyte and formed into small bodies up to 5 /tm in diameter in the cortex. Some more diffuse labelling occurred in the cortex, but this could well have been due to pinosomes, which in this system are smaller than the limit of resolution of the light microscope (Stay, 1965; Telfer & Smith, 1970) and which had not been organized into cortical bodies. Quantitative extractions showed that the amount of labelled material taken up by the oocytes increased with time for at least 4h (Table 1), and that little labelling of the cells occurred in the control medium. Inclusion of 1 mM dinitrophenol in the medium greatly reduced the amount of labelled product incorporated by the oocytes (Table 1, Expt. 2), indicating an active process rather than passive absorption.

Fig. 9.

Autoradiograph of an isolated oocyte incubated in dialysed female blood which had been pre-conditioned by follicle cells in the presence of [3H]histidine. Again label was concentrated over numerous cortical bodies (arrows). 10-μm section, ×880.

Fig. 9.

Autoradiograph of an isolated oocyte incubated in dialysed female blood which had been pre-conditioned by follicle cells in the presence of [3H]histidine. Again label was concentrated over numerous cortical bodies (arrows). 10-μm section, ×880.

These results constitute direct confirmation of the proposal that the follicle cells contribute a flxable, histidine-labelling material to the yolk (Anderson & Telfer, 1969). In addition they indicate that the isolated epithelial cells continue to produce and release this material during incubation in vitro, and that isolated oocytes remained capable of sequestering the material, presumably by pinocytosis. The intensity with which the cortical bodies labelled with tritiated follicle cell product, and, as noted above, the absence of obvious labelling in these bodies in isolated oocytes incubated by themselves in [3H]histidine, confirm the conclusions from experiments with intact follicles that endogenously synthesized yolk proteins come primarily from the epithelium.

Stimulation of protein synthesis in oocytes by conditioned medium

While intact follicles labelled in vitro by the methods employed here will continue to incorporate amino acids for at least 6 h (Anderson & Telfer, 1969), isolated oocytes, as indicated in the first section of this report, cease to do so rather abruptly within about 2 h (Fig. 1, p. 739). An unanticipated discovery was that the presence of follicle cell product at least doubled the duration of this period of protein synthesis.

Unlabelled conditioned medium was obtained by incubating follicular epithelium-nurse cell complexes in female blood, but with omission of the radioactive amino acid. Matched samples of isolated oocytes were then incubated in this medium after centrifugation and in untreated blood, each containing 0·1 mCi/ml [3H]leucine. After 2h of incubation, the same amount of PCA-precipitable label had been incorporated by both samples (Table 2). After 4 h, however, the oocytes in conditioned medium had accumulated almost twice as much fixable label as at 2 h, while the label in the control oocytes, incubated in blood alone, had remained constant. Dialysed conditioned medium mixed with 30% whole female blood also was effective in promoting continued protein synthesis, indicating that a high-molecular weight material was responsible.

As shown in the previous section, a non-dialysable component of the conditioned medium is taken up by the isolated oocytes, presumably by pinocytosis, suggesting a possible interpretation of the simultaneous stimulatory effect on amino acid incorporation: the proteins synthesized after 2 h of incubation were constituents of the cell membrane whose formation is suppressed when no membrane turnover occurs and is triggered when membrane is removed from the cell surface by pinocytosis. According to this interpretation, pinocytosis does not occur in isolated oocytes at a rate rapid enough to require continued protein synthesis, but is induced by the follicle cell secretion products with consequent increase in membrane turnover.

Uptake of labelled female and male blood proteins

The small, intensely labelled cortical bodies containing the follicle cell products in the isolated oocytes resembled those generated by whole follicles labelled in saline in the absence of blood proteins, rather than the larger, more moderately labelled yolk spheres normally laid down during incubation in female blood. The pattern suggested that blood proteins, though present in abundance in the medium, were not incorporated by oocytes as readily in isolation as in intact follicles, even though epithelial products are readily accumulated in both cases.

This question was investigated by including dialysed labelled female blood proteins in the incubation medium. Previous experiments have shown that such proteins are capable of supporting normal yolk formation in whole follicles and of effecting readily detectable labelling of newly-formed yolk spheres (Hausman et al. 1971). Comparable labelling of the peripheral yolk spheres of isolated oocytes was never observed following exposure to labelled female blood proteins mixed with female blood, with follicle cell-conditioned blood, or with blood containing living follicle cells. The isolated oocytes seemed then incapable of carrying on normal blood protein uptake, in spite of their apparent viability and ability both to synthesize proteins and to pinocytose follicle cell products.

In all of the incubation media tried, the labelled female blood proteins became associated with the oocyte surface and were seen in occasional small cortical yolk spheres (Fig. 10). This interaction of the female proteins with the oocyte cortex was of interest, since it has been suggested that vitellogenin, the sex-limited protein used in preference to all others for yolk formation, is selectively adsorbed by the oocyte prior to pinocytosis (Telfer, 1961). This possibility was tested by incubating isolated oocytes in 2 media similar with respect to protein composition and content of low-molecular weight substances, but with a tritium label borne by female blood proteins in one case and by male blood proteins in another.

Figs. 10, 11.

Autoradiographs of isolated oocytes incubated in a mixture of labelled female blood proteins and male blood (Fig. 10) or labelled male blood proteins plus female blood (Fig. 11). Silver grains are seen mainly at the surface of the oocytes and over occasional peripheral yolk spheres (arrows). 10-μm sections, ×1800.

Figs. 10, 11.

Autoradiographs of isolated oocytes incubated in a mixture of labelled female blood proteins and male blood (Fig. 10) or labelled male blood proteins plus female blood (Fig. 11). Silver grains are seen mainly at the surface of the oocytes and over occasional peripheral yolk spheres (arrows). 10-μm sections, ×1800.

The first medium was obtained by mixing equal parts of dialysed labelled female blood proteins and unlabelled developing male blood. For the second, unlabelled developing female blood, which is similar in composition to male blood except for the presence of vitellogenin, was mixed with equal parts of dialysed male blood proteins with approximately the same amount of PCA-precipitable label as the female proteins. The media were then closely matched in protein composition and radioactivity, differing only in the location of the label. Unlabelled leucine (1 mM) was included to reduce incorporation of any remaining labelled amino acid. The female blood protein labelling procedure used has been shown to result in 50-70% of total blood protein label being in the specific yolk precursor, vitellogenin (Pan, 1971).

In autoradiographs of oocytes incubated in these media for 2–3 h, labelled material was observed associated with the oocyte surface and cortex, in distinctly higher concentrations when the label was borne by the female proteins (Fig. 10) than when the male proteins were labelled (Fig. 11). Quantitative extractions of matched samples consisting of 8–12 oocytes incubated in the two media confirmed this conclusion (Table 3), indicating 1·5 to 2·5 times more label associated with the oocytes incubated in the medium with labelled female proteins.

Table 3.

Association of labelled blood proteins with isolated oocytes

Association of labelled blood proteins with isolated oocytes
Association of labelled blood proteins with isolated oocytes

Such a difference could arise if the oocyte exhibited a selective preference for female proteins, or if the female proteins associated with the oocyte contained more labelled amino acids per molecule than the male proteins. Although the latter possibility cannot be ruled out in these experiments, the results may suggest that specific adsorption of proteins to the oocyte surface represents one component of the selection process in vitellogenesis.

The use of isolated oocytes in vitro has made it possible to resolve several questions that could not be readily attacked with whole-follicle preparations. It is now apparent, for example, that, after incubation in a medium containing tritiated amino acid, the pattern of labelled-protein distribution in the oocyte cytoplasm, particularly its high concentration in the cortex, is not due exclusively to proteins imported from the epithelium or from the nurse cells, for the same distribution is seen in oocytes labelled after separation from these cells. The high intensity of amino acid incorporation in the cortex of the oocyte might have been predicted from the presence of more RNA there than in most other regions of the oocyte (Pollack & Telfer, 1969) and presumably reflects protein synthesis associated with the high rate of membrane turnover due to pinocytosis. This suggestion receives support from the observation that protein synthesis continued longer than 2 h only if pinocytosis was occurring under stimulation by the follicle cell products.

Little if any label was transferred from the cortical cytoplasm to the yolk spheres of isolated oocytes. Because of the failure of the isolated oocytes to generate yolk spheres from blood protein, however, the possibility remains that some transfer may occur under more normal conditions when new yolk spheres are being generated.

Autoradiographic studies of whole Cecropia follicles (Anderson & Telfer, 1969), as well as earlier cytological investigations of the follicles of a variety of insects (see Nath, 1968), led to the proposal that histologically fixable secretion products of the follicular epithelium are pinocytosed by the oocyte along with blood proteins during yolk deposition. This conclusion is decisively confirmed here by the observation that a non-dialysable histidine-labelled material, released from isolated follicle cell-nurse cell complexes, is incorporated into cortical bodies by isolated oocytes. The fact that follicle cells continue to release this material after separation from the oocyte now opens the way for its biochemical isolation and characterization.

Vitellogenin and female blood proteins have been shown to support vitellogenesis in intact follicles in vitro (Hausman et al. 1971). The failure of the isolated oocytes to incorporate blood proteins must therefore be ascribed either to the absence of an intact epithelium or to a functional lesion in the cortex caused by the process of epithelium removal. The latter explanation is weakened by the demonstration that isolated oocytes retain their ability to incorporate non-dialysable secretory products of the epithelium into cortical bodies, thus seeming to be pinocytotically competent. While the question remains unanswered, a likely possibility is that the absence of the pre-concentration mechanism associated with the epithelium is responsible. In the intact follicle many blood proteins become concentrated in the extracellular spaces, and vitellogenin is selectively withdrawn or released from this concentrate for pinocytotic incorporation by the oocyte (Anderson & Telfer, 1970ft). Perhaps this extra-cellular preconcentration is necessary for effective filling of the pinocytotic vesicles, so that those formed by isolated oocytes receive only small amounts of blood protein.

A preferential association of female blood proteins, compared to male blood proteins, might indicate that the adsorption of vitellogenin to the oocyte surface is the final step in the selectivity of its uptake. Though specificity in protein incorporation has been described for other cells, including amphibian oocytes (Wallace & Jared, 1969), and a variety of mammalian cells (Kiipffer cells: Gans, Subramanian & Tan, 1968; sarcoma cells: Ryser & Hancock, 1965; and leucocytes: Viti & Bocci, 1970), information as to how selectivity is accomplished has been lacking. Adsorption to the cell surface is the first step in the pinocytotic uptake of proteins by amoeba (Brandt & Pappas, 1960) and in phagocytosis of bacteria and other material by macrophages (Allen & Cook, 1970). The results described here present direct evidence that selectivity could reside in this adsorption process.

This work was supported by Grant 4463 from the National Science Foundation to Dr W. H. Telfer. The author is grateful to Dr Telfer for his support and advice and for critical reading of the manuscript.

Allen
,
J. W.
&
Cook
,
G. M. W.
(
1970
).
A study of the attachment phase of phagocytosis by murine macrophages
.
Expl Cell Res
.
59
,
105
116
.
Anderson
,
L. M.
&
Telfer
,
W. H.
(
1969
).
A follicle cell contribution to the yolk spheres of moth oocytes
.
Tissue fef Cell
1
,
633
644
.
Anderson
,
L. M.
&
Telfer
,
W. H.
(
1970a
).
Trypan blue inhibition of yolk deposition—a clue to follicle cell function in the Cecropia moth
.
J. Embryol. exp Morph
.
23
,
35
52
.
Anderson
,
L. M.
&
Telfer
,
W. H.
(
1970b
).
Extracellular concentrating of proteins in the Cecropia moth follicle. J
.
cell. Physiol
.
76
,
37
53
.
Brandt
,
P. W.
&
Pappas
,
G. D.
(
1960
).
An electron microscope study of pinocytosis in ameba. I. The surface attachment phase
.
J. biophys. biochem. Cytol
.
8
,
675
687
.
Gans
,
H.
,
Subramanian
,
V.
&
Tan
,
B. H.
(
1968
).
Selective phagocytosis: a new concept in protein catabolism
.
Science, N.Y
.
159
,
107
110
.
Hausman
,
S. J.
,
Anderson
,
L. M.
&
Telfer
,
W. H.
(
1971
).
The dependence of Cecropia yolk formation in vitro on specific blood proteins
.
J. CellBiol
. (in the Press).
Mans
,
R. J.
&
Novelli
,
G. D.
(
1961
).
Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disc method
.
Archs Biochem. Biophys
.
94
,
48
53
.
Melius
,
M. E.
(
1966
).
An Autoradiographic Analysis of Blood Protein Uptake and Protein Yolk Sphere Formation by Cecropia Moth Oocytes
.
Doctoral dissertation, University of Pennsylvania
.
Melius
,
M. B.
&
Telfer
,
W. H.
(
1969
).
An autoradiographic analysis of yolk deposition in the Cecropia moth oocyte
.
J. Morph
.
129
,
1
16
.
Nath
,
V.
(
1968
).
Animal Gametes (Female)
.
Bombay, London and New York
:
Asia Publishing House
.
Pan
,
M. L.
(
1971
).
The synthesis of vitellogenin in the Cecropia silkworm
.
J. Insect Physiol
.
17
,
677
689
.
Pollack
,
S. B.
&
Telfer
,
W. H.
(
1969
).
RNA in Cecropia moth ovaries: sites of synthesis, transport and storage
.
J. exp. Zool
.
170
,
1
23
.
Ryser
,
H. J.
&
Hancock
,
R.
(
1965
).
Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture
.
Science, N.Y
.
150
,
501
503
.
Stay
,
B.
(
1965
).
Protein uptake in the oocytes of the Cecropia moth
.
J. Cell Biol
.
26
,
49
62
.
Telfer
,
W. H.
(
1961
).
The route of entry and localization of blood proteins in the oocyte of saturniid moths
.
J. biophys. biochem. Cytol
.
9
,
747
759
.
Telfer
,
W. H.
(
1965
).
The mechanism and control of yolk formation
.
A. Rev. Ent
.
10
,
161
184
.
Telfer
,
W. H.
&
Smith
,
D. S.
(
1970
).
Aspects of egg formation
.
In Insect Ultrastructure, Symp. R. ent. Soc. Lond. No. 5
(ed.
C.
Neville
), pp.
117
134
.
Oxford and Edinburgh
:
Blackwell Scientific Publications
.
Viti
,
A.
&
Bocci
,
V.
(
1970
).
Minimal catabolism of native I-y-globulin by polymorphonuclear leucocytes
.
Expl Cell Res
.
61
,
206
207
.
Wallace
,
R. A.
&
Jarhd
,
D. W.
(
1969
).
Protein incorporation in vitro by oocytes of Xenopus laevis
.
J. Cell Biol
.
43
,
153
A.