ABSTRACT
The yolk of insect eggs consists largely of proteins derived pinocytotically from the maternal blood (Telfer, 1965). Vitellogenic oocytes can also sequester the acidic colloid trypan blue: when injected into the blood, the dye, like the blood proteins, is deposited in yolk spheres in the cortex of the oocyte. This behavior of trypan blue was first described in the scorpian fly by Ramamurty (1964), and has since been confirmed in a cricket (Sander & Vollmar, 1967) and in the cecropia moth (Telfer & Anderson, 1968).
Ovarian follicles of the cecropia moth have also been exposed to trypan blue in vitro-, under these conditions the dye is not only incorporated into cortical yolk spheres but is in addition bound at the oocyte surface and in the intercellular spaces which serve as passageways for blood proteins across the follicular epithelium. The occurrence of extracellular trypan blue binding, particularly along the route of blood protein entry, suggested that the dye may be interacting with a normal component of the mechanism for vitellogenic blood protein accumulation. This concept is supported by the finding that trypan blue can be an effective inhibitor as well as a participant in yolk formation (Ramamurty, 1964; Korfsmeier, 1966; Telfer & Anderson, 1968).
The follicular structures and passageways which have proved relevant to blood protein accumulation and trypan blue effects in cecropia were examined with the electron microscope by Stay (1965) and by King & Aggarwal (1965). To enter a follicle blood proteins must first cross the porous wall of the ovariole, and then an acellular basement lamina. They next pass between the singlelayered, columnar follicle cells (Telfer, 1961), and finally penetrate the vitelline membrane, which is too thin to be readily visualized by light microscopy, to reach the folded oocyte surface, the brush border, where pinocytosis occurs. Pinocytotic vesicles in the oocyte cortex then fuse to yield layer upon layer of 15–20 μ wide, membrane-bound yolk spheres. The structural relationships of the basement lamina, the follicle cells, and the oocyte with its contained yolk spheres may be observed in Figs. 1–4 and 7–10.
Trypan blue incorporation into yolk spheres in vivo 24 h after injection. Follicle cells are to the left. Bouin’s fixation, 10μ. sections, × 250.
Injection on day 17. All yolk spheres formed since the time of dye injection contain the dye. FC = follicle cell; BL = basement lamina; BB = brush border of oocyte.
Fig. 2. Injection on day 20. The blue yolk spheres are smaller and form a shallower layer than those in Fig. 1.
Fig. 3. Injection on day 20, followed by injection of dialysed male pupal blood proteins 12 h later. Note the marked recovery of yolk sphere genesis, compared to Fig. 2.
Fig. 4. Injection on day 20, followed by injection of cecropia saline 12 h later. Little recovery of yolk formation, compared to Figs. 1 and 3, is evident.
Trypan blue incorporation into yolk spheres in vivo 24 h after injection. Follicle cells are to the left. Bouin’s fixation, 10μ. sections, × 250.
Injection on day 17. All yolk spheres formed since the time of dye injection contain the dye. FC = follicle cell; BL = basement lamina; BB = brush border of oocyte.
Fig. 2. Injection on day 20. The blue yolk spheres are smaller and form a shallower layer than those in Fig. 1.
Fig. 3. Injection on day 20, followed by injection of dialysed male pupal blood proteins 12 h later. Note the marked recovery of yolk sphere genesis, compared to Fig. 2.
Fig. 4. Injection on day 20, followed by injection of cecropia saline 12 h later. Little recovery of yolk formation, compared to Figs. 1 and 3, is evident.
Incorporation of the blood proteins into the yolk involves both the concentration and the selection of proteins: a sex-limited female protein (Telfer, 1954), termed here cecropia vitellogenin, is about 20 times more concentrated in the yolk than in the blood, while other cecropia proteins are taken up with distinctly less avidity, and proffered vertebrate and crustacean proteins, injected into the hemolymph, are largely excluded (Telfer, 1960). It is clear from these findings that the blood proteins are somehow selectively manipulated as they follow the extracellular pathway from blood to yolk.
The experiments described below were initiated to determine whether the reactions of trypan blue with the moth follicle could be used to clarify the mechanism of blood protein uptake. The results to be presented suggest that the active accumulation of trypan blue into yolk and its inhibitory effect can be traced to its affinity for a material which is secreted by the follicle cells and may be required for the induction of pinocytosis at the oocyte surface.
MATERIALS AND METHODS
Trypan blue (Harleco) was prepared by dialysis against distilled water for at least 24 h. After drying in dishes on a hot plate at 100 °C the dye was autoclaved for sterilization and removal of volatile contaminants. The optical density of dilute solutions of the purified dye in distilled water was determined at 590 mμ and compared with similar dilutions of a dye standard (Harleco Parstains). The dye preparation was found to be at least 95% pure. The optical density of the dye in solution was linear with concentration from 2 to 10 μ g/ml, the range within which the experimental determinations fell. The absorption coefficient was 0·086 O.D. units/μ M.
For experimental use a 1% solution of purified trypan blue was prepared in a medium containing 0·020 M-KCI, 10−4 M-MgCl2, 10−4 M-CaCl2, and 0·25 M-Tris-succinate buffer, pH 6·2. Except for the divalent cations, which precipitate the hydrophobic colloid at higher concentrations, the composition of the medium was patterned after the buffered physiological saline described below.
The two ovaries of Hyalophora cecropia L. consist of a total of eight equivalent ovarioles, each containing, at the stages studied here, about 30 vitellogenic follicles in linear sequence. Follicles, 1·3–1·8 mm long, from females in the last week of the pupal–adult transformation were exposed to the dye either in vivo or in vitro. For the in vivo experiments, a volume of 1% trypan blue solution equal in ml to one-twentieth the fresh weight of the animal in grams was injected into the hemocoele of females ranging in age from the 15th to the 21 st day of adult development. Twenty-four hours later the animals were bled and dissected under a buffered saline consisting of 0·040 M-KC1, 0·015 M-MgCl2, 0·004 M-CaCl2, and 0·11 M-Tris-succinate buffer, pH 6·2–6·3 (Anderson & Telfer, in preparation) and hereafter referred to as cecropia saline. The concentration of dye in the blood of the animal at the time of dissection was estimated by determining the optical density at 590 mμ, of 1:200 dilutions of the blood in the trypan blue suspension medium after an ether extraction for clarification of the solution. The concentrations of vitellogenin and the major carotenoid protein of the blood were determined by the immunochemical method of Oudin (Telfer & Williams, 1953; Preer & Telfer, 1957), with 17-day female blood as the standard. Tests were performed on 1:10 and 1:100 dilutions of the blood and the two results averaged.
To obtain follicles for in vitro studies without exposure to saline the abdomen was opened by an incision in the dorsal mid-line and follicles removed two or three at a time by grasping the interfollicular connective with forceps. Whole ovarioles could often be removed intact by this method from day 19 and older females. The follicles were placed in approximately 0·1 ml of 1% trypan blue in a sterile spot plate depression, which was then covered with a coverslip. The volume of the medium was just sufficient to immerse the follicles. The cultures were incubated at 26 °C on a mechanical shaker at shakes/s with a stroke length of 5 cm. Under these conditions vitellogenic follicles usually remained viable for at least several hours, as indicated by exclusion of the dye from the cytoplasm (Parker, 1961). Death of some or all of the cells of the follicles ensued, however, if unpurified dye was used, if the volume of solution was greater than 0·2 ml, if the culture was not adequately aerated by shaking, if the culture plates were insufficiently clean, or if the follicles were handled roughly during dissection.
Dye-treated follicles were fixed in Bouin’s solution, embedded in paraffin wax, and sectioned at 5 or 10μ. While the follicle cells and intercellular spaces may have been somewhat distorted by these procedures, Bouin’s solution is the best fixative thus far found for the visualization of trypan blue in the system. The sections were examined directly after removal of the paraffin wax with xylene or after counterstaining with paracarmine. For autoradiography, de-paraffinized sections were hydrated prior to coating with NTB-2 emulsion (Kodak). Autoradiograms were developed after a 3-week exposure.
When extractions of dye from follicles were necessary, the follicles were frozen and thawed in cecropia saline and homogenized. Aqueous sodium lauryl sulfate was added to a final concentration of 1% and the solution homogenized again. Brief shaking with 4 ml ether removed lipid and denatured beclouding proteins, which collected at the interface when the mixture was centrifuged. The aqueous layer was then centrifuged at 10000g for 15 min, yielding 2 ml of an optically clear solution containing the trypan blue. The extract was allowed to stand for 24 h for evaporation of residual ether and the optical density measured at 590 mμ..
RESULTS
Trypan blue incorporation by follicles in vivo
Twenty-four hours after injection of trypan blue into the blood of cecropia females in the last week of adult development, the dye was found to be localized in the outer layers of yolk spheres, formed since the time of dye injection, and nowhere else in the follicle (Telfer & Anderson, 1968). Further investigation has now revealed that the dye was concentrated in the yolk relative to the blood to a degree similar to that observed for vitellogenin. This intensive uptake of trypan blue was demonstrated by measuring the amount of dye accumulated during the 24 h exposure period, and the volume of yolk which it occupied, in the follicles of ten injected females. In each case all of the vitellogenic follicles were deeply stained. Follicles of a given size from each of the eight ovarioles of the animal were pooled: one follicle of each size class was fixed in Bouin’s solution for histological processing in order to measure the volume of yolk occupied by the dye (Figs. 1–4), while the other seven follicles were frozen in cecropia saline for quantitative extraction of the dye (Table 1). Four to nine samples were processed in this way for each animal.
The total volume of the trypan blue-containing yolk was calculated by subtracting the volume of the oocyte at the time of injection (i.e. the volume of the unstained yolk lying within the blue layer) from its volume at the time of dissection. Since the oocyte approximates a prolate ellipsoid, the two volumes could be calculated from the longitudinal and transverse diameters measured in median longitudinal section. Corrections were made for shrinkage during histological processing by measuring oocyte diameters prior to fixation as well as in section.
The concentration of dye in the yolk was calculated by dividing the volume of dyed yolk in a given sample of follicles into the total amount of dye extracted from them. This value was then compared with the concentration in the blood to obtain a concentration factor for the sequestration of the dye (Table 1). (The concentration of dye in the blood was somewhat variable, presumably because of differences in blood volume or in rate of removal of the dye. These variations did not appear to affect the results in any consistent way.) The data presented demonstrate that trypan blue was concentrated by a factor of about 7–19 in the process of being taken up into yolk. This ability to imitate vitellogenin indicates that trypan blue has a stronger affinity for the protein accumulating mechanism than any other foreign colloid to which the system has thus far been exposed (Telfer, 1960). The fact that, like vitellogenin, the dye appears to climb a concentration gradient in entering the yolk can best be explained by assuming that it combines physically with the selective uptake mechanism, or perhaps with vitellogenin itself.
This interpretation is confirmed by the finding that trypan blue, as it participates in yolk formation, also acts as an inhibitor of the process (Ramamurty, 1964; Korfsmeier, 1966; Telfer & Anderson, 1968). The inhibition was manifested in cecropia by a reduction both in the thickness of the stratum of yolk produced in 24 h and in the diameters of the dyed yolk spheres composing the stratum. Autoradiographic studies indicate that vitellogenic oocytes ordinarily produce a 100 μ. deep stratum of yolk in 24 h (Melius & Telfer, 1969). The dyed strata generated in the follicles of trypan-blue injected animals on the other hand, varied from 85 μ. (Fig. 1) to as thin as 20–25μ. (Fig. 2), and the thinner the stratum, the smaller the yolk spheres were. The inhibitory effect was due to the dye, rather than to dilution of the blood by the injected fluid, since injection of trypan blue suspension medium alone into day-20 animals did not result in the formation of smaller yolk spheres.
The inhibition seemed to be greatest when the action of the concentration mechanism on the dye was most effective. When the data from the ten animals are considered together and plotted graphically (Fig. 5), it is evident that the reduction in the depth of dyed yolk spheres varied, from animal to animal, with the factor by which the dye was concentrated in moving from blood to yolk. The more avidly the dye was accumulated, the more pronounced its inhibitory effect.
Relation of the degree of inhibition of yolk formation by trypan blue to the concentration of the dye in the yolk. Inhibition was less (greater depth of dyed yolk) when the concentration factor was low.
The wide variation in the concentration factor for trypan blue and in its inhibitory effects in follicles from different animals apparently resulted from differences in the concentration of vitellogenic proteins in the blood. This relationship was initially suggested by the fact that dye concentration was greater and inhibition was more severe in advanced stages of adult development (compare Figs. 1 and 2), when the rapid growth of the oocytes has caused a 50–80% reduction in the concentration of vitellogenin in the blood (Telfer & Rutberg, 1960). Measurements of vitellogenin and carotenoid protein concentration in the blood of the animals used in these experiments confirmed that the concentration factor for trypan blue accumulation as well as reduction in depth of dyed yolk was inversely correlated with blood protein concentration (Table 1, Fig. 6).
Relation of the concentration of trypan blue in yolk (closed circles) and the depth of stained yolk (open circles) to the concentration of vitellogenin in the blood. The latter values were determined by the Oudin method and are expressed as percentage of the concentration of vitellogenin in the blood sample used as the standard for the test. With greater concentrations of vitellogenin the dye was less concentrated and a deeper layer of dyed yolk was formed.
Relation of the concentration of trypan blue in yolk (closed circles) and the depth of stained yolk (open circles) to the concentration of vitellogenin in the blood. The latter values were determined by the Oudin method and are expressed as percentage of the concentration of vitellogenin in the blood sample used as the standard for the test. With greater concentrations of vitellogenin the dye was less concentrated and a deeper layer of dyed yolk was formed.
In order to obtain more direct evidence that depletion of blood proteins, rather than some other aspect of the ageing of the animal, was the element determining the degree of concentration and inhibition, the capacity of added blood proteins to ameliorate the severe inhibitory effects of trypan blue in day-20 animals was tested. The blood proteins from a diapausing male pupa, which are qualitatively the same as those in the blood of the female except for lack of vitellogenin (Telfer, 1954), were dialysed against cecropia saline, concentrated to twice their initial titer by pervaporation, and dialysed again. A female on the 20th day of adult development was injected with trypan blue and then after 12 h with the solution of male blood proteins (volume in ml equal to one-fifteenth the weight of the animal in grams). Twelve hours later the animal was dissected. The blood proteins had, as predicted, visibly reduced both the concentration of the dye in the yolk and the inhibition of yolk formation (Fig. 3). Compared with animals of the same age injected with trypan blue alone (Fig. 2), the follicles of this female displayed a deeper stratum of less intensely dyed yolk and larger yolk spheres. It is of interest that the tiny yolk spheres which must have formed in the first 12 h after dye injection (as in Fig. 2) disappeared after the protein injection, presumably fusing with the newly-made, larger yolk spheres to form the inner layer of darker spheres in Fig. 3.
Female proteins, derived from either pupal blood or from yolk, also partially reversed dye inhibition. Since injection of a comparable volume of cecropia saline into day 20 females instead of dialysed blood proteins alleviated the inhibition only slightly (Fig. 4), the effect seemed primarily due to the added proteins.
The means by which the blood proteins suppress trypan blue uptake is uncertain at present; presumably these molecules either compete with trypan blue for binding sites or themselves adsorb the dye, thus reducing its effective concentration. Either case confirms the idea that trypan blue and the blood proteins are actively accumulated by a common mechanism.
The in vivo analysis thus revealed three important aspects of the interaction of trypan blue with follicles in the process of protein yolk deposition. The dye is concentrated by the pinocytotic mechanism in the same manner that this mechanism concentrates vitellogenin from the blood. Secondly, in the process of being concentrated, the dye inhibits yolk deposition, resulting in the formation of a thinner stratum of smaller yolk spheres. Finally, the inhibition is severe only late in adult development when yolk precursors are at a low level in the blood, and can be partially prevented by injecting dialysed blood proteins into the blood. These observations raised the possibility that the dye’s inhibitory action is exerted directly on the functioning of the protein uptake mechanism. Identification of the site and mode of the dye’s influence might then lead also to an elucidation of this mechanism. Additional information regarding the basis for the dye’s effect on yolk formation was therefore sought in an analysis of dye uptake in vitro.
Trypan blue incorporation by follicles in vitro
Treatment of follicles with 1% trypan blue in vitro led to a more direct and extreme expression of the effects seen in vivo and provided in addition new evidence regarding the mechanism of action of the dye. Follicles incubated in trypan blue in vitro, like those in vivo, readily incorporated dye into yolk spheres (Telfer & Anderson, 1968). However, yolk sphere generation, which was inhibited to various degrees by trypan blue in vivo, soon stopped altogether in the presence of the dye in vitro. During the first 30 min of incubation in trypan blue solution, pale blue 5–10 μ. wide spheres similar to those formed in vivo predominated in the oocyte cortex (Fig. 7). After 1 h 1–5 μ., intensely-staining spheres were scattered in the cortex (Fig. 8). With longer incubation periods little if any increase in the number or size of dyed yolk spheres was evident (Figs. 9, 10), although the cells of the follicles usually remained alive for 4h or more, as evidenced by their ability to exclude the dye from the cytoplasm.
Localization of trypan blue in follicles incubated in 1% trypan blue for 30 min (Fig. 7), 1 h (Fig. 8), 2 h (Fig. 9), and 3 h (Fig. 10). After dye treatment the follicles were rinsed for 15 min in cecropia saline modified to contain 0 035 M-Mg2+. The dye is bound in the basement lamina, between the follicle cells and close to the surface of the oocyte. Pale blue spheres formed initially (Fig. 7) give way to small, darker spheres, with no subsequent increase in blue yolk formed (Figs. 8–10). Bouin’s fixation, 10 μ. sections, stained with paracarmine, × 650.
Localization of trypan blue in follicles incubated in 1% trypan blue for 30 min (Fig. 7), 1 h (Fig. 8), 2 h (Fig. 9), and 3 h (Fig. 10). After dye treatment the follicles were rinsed for 15 min in cecropia saline modified to contain 0 035 M-Mg2+. The dye is bound in the basement lamina, between the follicle cells and close to the surface of the oocyte. Pale blue spheres formed initially (Fig. 7) give way to small, darker spheres, with no subsequent increase in blue yolk formed (Figs. 8–10). Bouin’s fixation, 10 μ. sections, stained with paracarmine, × 650.
In order to obtain quantitative confirmation of the cessation of yolk formation, measured samples of follicles were incubated in dye solution for increasing intervals of time, rinsed, and treated with a low-Mg2+ solution (see below) in order to release the extracellularly bound dye. The dye contained in the yolk of the oocytes plus the small amount remaining in the basement lamina and brush border were extracted by the detergent-ether method. The quantitative results of the experiment illustrated in Figs. 7–10 are presented in Table 2 and confirm the conclusion drawn from the sectioned follicles, that the total amount of yolk-bound dye did not increase after half an hour, in spite of the initial speed with which it was incorporated and the continued viability of the cells.
Thus in vitro the visible concentration of trypan blue and the concomitant reduction in yolk sphere size and rate of yolk formation were seen in extreme form. At the same time a new feature of the reactivity of the dye appeared: in follicles incubated for as little as 5 min in 1% trypan blue the dye was conspicuously bound in the basement lamina, the spaces between the follicle cells, and the brush border of the oocyte (Figs. 7–10 and Telfer & Anderson, 1968).
Dye was held in the spaces during washing for at least 30-60 min in cecropia saline, in amounts proportional to the concentration of Mg2+ in the wash solution. Dye retention could therefore be promoted by elevating the concentration of Mg 2+ to 0·035–0·050 M, and dye release could be effected by transferring the follicles to saline containing only 10−4 M-Mg2+. After 1 h in the latter solution trypan blue was retained in the cortical yolk spheres and in greatly reduced amounts in the basement lamina and the brush border, but was no longer detectable in the spaces between the follicle cells (see Fig. 13). Removal of retained dye by this method proved to be convenient in studies on the reversibility of trypan blue inhibition of yolk deposition, described below.
Absence of intense extracellular dye binding in vivo was attributed to the lower concentration of trypan blue in the blood—approximately 0·05% instead of 1%—and to an inhibitory effect of blood proteins on extracellular dye retention (Anderson & Telfer, in preparation).
During the course of follicular maturation, the capacity for extracellular dye binding is lost simultaneously with the cessation of blood protein uptake (Telfer & Anderson, 1968), so that dye binding appears to entail materials involved in the formation of yolk. The fact that pronounced extracellular dye accumulation occurred in vitro under the same conditions as complete inhibition of dye uptake into yolk confirms this relationship, and suggests further that the dye may suppress yolk formation by immobilizing an esssential precursor in the extracellular spaces. Though other interpretations are possible, that particular possibility is supported by the following experiments which demonstrate that, along with inhibiting vitellogenesis, trypan blue causes a follicle cell product which would normally be transferred to the yolk to accumulate instead between the follicle cells and the oocyte.
Trypan blue immobilization of follicle cell product
Along with inhibiting yolk formation, in vitro exposure to 1% trypan blue interfered with the transmission to the yolk of a follicle cell secretion product which can be labelled with tritiated histidine. This finding provided a clue, perhaps even the key, to an understanding of how the dye inhibits yolk formation. It has been demonstrated that a histidine-labelled product arising in the follicle cells occupies the intercellular spaces and is incorporated into the yolk spheres as they are formed at the periphery of the oocyte (Anderson & Telfer, in preparation). The extent and degree of histidine labelling in the epithelium and nascent yolk spheres during incubation of follicles in blood containing [3H]histidine is similar to that observed in follicles after injection of the amino acid into the intact animal (Telfer & Anderson, 1968; Melius & Telfer, 1969). Follicles thus incubated show uniform labelling of the cells and spaces of the epithelium; after 2 h a layer of medium-sized yolk spheres in the oocyte cortex is labelled with an intensity comparable to the follicle cells.
A different pattern of labelling was seen when follicles were treated with trypan blue prior to incubation in blood with tritiated histidine. After a 30 min dye treatment the follicles were rinsed briefly in cecropia saline and transferred to a solution containing 50% female blood in cecropia saline. An aqueous solution of tritiated histidine (L-histidine-2, 5-T, approx. 12000 mCi/ml, Nuclear of Chicago) was added to this mixture to yield a final radioactivity of 0·1 mCi/ ml. Though the blood proteins caused release of some of the extracellular dye (Anderson & Telfer, in preparation), histological preparations indicated that a considerable amount remained in the brush border between the follicle cells and the oocyte cortex.
Autoradiographic analysis of follicles incubated in this manner for 2 h revealed label in the follicle cells and in a fixable material that had accumulated in high concentrations between the follicle cells and the oocyte (Fig. 12). (Control follicles, incubated in 50% blood plus [3H]histidine, formed labelled cortical yolk spheres in the normal manner (Fig. 11).) While therewas a scattering of silver grains over the cytoplasm of the oocyte between the yolk spheres in the dye-treated follicles, few of the cortical yolk spheres were labelled. Thus, label which, in the absence of previous dye treatment, would have been incorporated into yolk spheres appeared instead with greater intensity in the dye-filled zone between the oocyte and the follicle cells. The fact that the extracellular zone retaining the greatest concentration of the dye was also the zone which the labelled product was unable to traverse, suggests that trypan blue had prevented the usual movement of label from the follicle cells to the oocyte by binding the follicle cell product in an insoluble, extracellular complex.
Bouin’s fixation, 10 μ. sections. Figs. 13 and 14 are of sections stained with para-carmine, × 650.
Trypan blue inhibition of follicle cell product transfer. The follicles shown are representative of the 4–6 follicles tested from each of five animals. Fig. 11. Incorporation of [3H]histidine in the follicle cells and peripheral yolk spheres during a 3 h incubation in 50% female blood. Most of the silver grains overlying the follicle cells are out of focus. Follicles thus treated serve as controls for the follicle shown in Fig. 12.
Incorporation of [3H]histidine in a follicle incubated as in Fig. 11 (as part of the same experiment), but after a 30 min treatment with 1% trypan blue in vitro. Labelled material is concentrated at the inner surface of the follicle cells, instead of in the yolk spheres. (Direct microscopic examination confirms that the black band observed in the photograph represents a dense accumulation of silver grains.)
Fig. 13–15. Recovery of yolk formation after trypan blue treatment. Fig. 13. Follicle incubated in 1% trypan blue for 2 h and then treated with low Mg2+ solution for release of extracellular trypan blue. Dyed yolk spheres are seen just below the oocyte surface; little dye is present extracellularly.
Fig. 14, 15. Follicles treated as in Fig. 13, then incubated in dialysed female blood proteins containing [3H]histidine for 2 h. The dilution and deeper penetration of the dye in the yolk (Fig. 14) and the appearance of histidine-labelled material in peripheral yolk spheres (Fig. 15) indicate a recovery of yolk formation. Similar results were obtained with follicles from two additional animals.
Bouin’s fixation, 10 μ. sections. Figs. 13 and 14 are of sections stained with para-carmine, × 650.
Trypan blue inhibition of follicle cell product transfer. The follicles shown are representative of the 4–6 follicles tested from each of five animals. Fig. 11. Incorporation of [3H]histidine in the follicle cells and peripheral yolk spheres during a 3 h incubation in 50% female blood. Most of the silver grains overlying the follicle cells are out of focus. Follicles thus treated serve as controls for the follicle shown in Fig. 12.
Incorporation of [3H]histidine in a follicle incubated as in Fig. 11 (as part of the same experiment), but after a 30 min treatment with 1% trypan blue in vitro. Labelled material is concentrated at the inner surface of the follicle cells, instead of in the yolk spheres. (Direct microscopic examination confirms that the black band observed in the photograph represents a dense accumulation of silver grains.)
Fig. 13–15. Recovery of yolk formation after trypan blue treatment. Fig. 13. Follicle incubated in 1% trypan blue for 2 h and then treated with low Mg2+ solution for release of extracellular trypan blue. Dyed yolk spheres are seen just below the oocyte surface; little dye is present extracellularly.
Fig. 14, 15. Follicles treated as in Fig. 13, then incubated in dialysed female blood proteins containing [3H]histidine for 2 h. The dilution and deeper penetration of the dye in the yolk (Fig. 14) and the appearance of histidine-labelled material in peripheral yolk spheres (Fig. 15) indicate a recovery of yolk formation. Similar results were obtained with follicles from two additional animals.
When dye treatment was followed by labelling in whole blood rather than in blood diluted with saline, the amount of dye and of label remaining in the interzone was reduced and greater transfer of label to the cortical yolk spheres occurred. Thus follicle cell product piled up following trypan blue treatment only if dye remained in the intercellular region.
Reversibility of in vitro inhibition by trypan blue
If the short duration of yolk sphere formation by the oocyte in 1% trypan blue is in fact caused by the blockage of transfer of the follicle cell product, rather than by some unrelated phenomenon, such as depletion of nutrients or a sublethal injury, then the inhibition should be reversible even in the absence of the dialyzable fraction of the blood: removal of the dye should allow normal follicle cell product transfer and yolk sphere formation to be resumed in the presence of dialysed blood proteins. An experiment was therefore designed to test the ability of the follicle to recover its vitellogenic activity after severe inhibition by trypan blue.
For this purpose follicles were incubated in 1% trypan blue for 2 h and then transferred to low magnesium saline for 1 h to release extracellularly bound dye (Fig. 13). Sample follicles fixed at this point in the experiment revealed that trypan blue was retained only in the cortical yolk spheres and the basement lamina. While a separation had developed between the follicle cells and the basement lamina, the follicles appeared otherwise intact. In order to test the ability of such follicles to function, the remaining ones were further incubated in female blood proteins dialysed against trypan blue suspension medium and containing 0·1 mCi/ml [3H]histidine. After an additional 3 h of culture in this medium the follicles were fixed for demonstration of trypan blue (Fig. 14) and for autoradiography (Fig. 15).
Not surprisingly, the experimental regimen led to morphological distortion, including vacuole formation in the oocyte cortex, as well as the basement lamina separation noted earlier in the procedure. Despite these conditions, there were two signs that yolk deposition had been resumed. Trypan blue-stained yolk spheres had been displaced from the cortex and many were now larger and paler, as though they had fused with unstained yolk spheres. The cortex was now occupied by yolk spheres which were both unstained (Fig. 14) and heavily labelled (Fig. 15), and had therefore been produced during the labelling period after the removal of extracellular trypan blue. Extracellular accumulation of label was not extensive. Removal of trypan blue from the intercellular spaces therefore had permitted the follicles to recover their ability to form protein yolk spheres and to transfer labelled product from follicle cells to yolk. The inhibitory effect of the dye on yolk formation is therefore dependent on its continued presence in the intercellular spaces and may well be due to its binding and immobilization of the follicle cell product.
DISCUSSION
Trypan blue has been used extensively as an experimental tool in the study of teratogenesis (e.g. Beck & Lloyd, 1963). The suggestion that the teratogenic effects of the dye result from inhibition of certain enzymes acting on acidic compounds (Beck, Lloyd & Griffiths, 1967) parallels the proposition made here, that the dye interferes with the reactions of an extracellular binder with acidic blood proteins.
The observed activities of trypan blue in the cecropia follicle are in keeping with its chemical nature. Trypan blue is a disazo dye bearing free amino and sulfonic acid groups; it has a net negative charge. Though having a molecular weight of 960, in aqueous solution it forms a hydrophobic colloid with a molecular weight of approximately 10000, by comparison to a related dye, Congo red (Fieser & Fieser, 1957). Its negative charge and colloidal nature render it physically similar to the acidic blood proteins, and this resemblance presumably accounts for its ability to participate in blood protein accumulation by the oocyte. On the other hand, the relatively small size of the aggregates, and their hydrophobic nature, in combination with a high charge density, would be expected to result in greater chelating action than is exercised by the large, hydrophilic proteins. Thus both the dye’s similarity to blood proteins in reacting with the proposed extracellular binder and gaining entry into the yolk, and its deviation from normal protein behaviour in imparting excessive stability to the extracellular complex, find an explanation in its chemical nature.
Trypan blue inhibits concurrently both yolk formation and transfer of the follicle cell product. A reasonable interpretation of this situation is that the follicle cell product plays a critical role in the process of blood protein uptake by the oocyte, so that its arrest in the extracellular spaces by trypan blue results in the cessation of protein deposition. While other possibilities for the mechanism of inhibition by trypan blue cannot be ruled out, alternative explanations do not find convincing support in the available experimental results. For instance, a direct internal or external effect of trypan blue on the pinocytotic process seems unlikely. Since oocytes can recover their normal capacity for protein uptake after removal of extracellular dye, in spite of large amounts of dye in the yolk, trypan blue apparently does not act as an internal poison. Furthermore, the dye failed to inhibit pinocytosis externally during the first 30 min of in vitro incubation but was sequestered in substantial amounts in the cortical yolk spheres.
The subsequent cessation of dye uptake in vitro, on the other hand, accords well with the hypothesis that inhibition of pinocytosis results from arrest of an essential component in the intercellular spaces. Presumably the combination of trypan blue with follicle cell product already present in the brush border of the oocyte at the beginning of the incubation accounted for early dye accumulation. Product more remotely situated in the spaces, stabilized there by the dye, was not free to replenish that which was incorporated from the oocyte surface by pinocytosis.
An obvious possibility for the proposed essential function of the follicle cell product is that it reacts with the acidic blood proteins and facilitates their uptake by the oocyte, in the same manner that basic proteins have been found to promote pinocytosis in other systems (Ryser & Hancock, 1965; Chapman-Andresen, 1965). The observation that the follicle cell product is taken up into small cortical bodies in the absence of blood protein (Anderson & Telfer, in preparation) suggests that it may in fact be capable of activating pinocytosis.
Furthermore, the retention of trypan blue throughout the extracellular spaces during in vitro treatment indicates that follicle cell product is present in these regions as well as the oocyte surface, and is thus available for interaction with blood proteins as they cross the basement lamina. Histidine-labelled product is observed in all parts of the spaces (Anderson & Telfer, in preparation). The possibility of involvement of the blood proteins in an extracellular complex extending throughout the spaces receives support from the demonstration, to be reported later, that blood proteins are more concentrated in the spaces than in the blood. In contrast to trypan blue, however, blood proteins are readily released from the spaces during soaking in cecropia saline. The dye apparently stabilizes the normally fluid complex in the space, presumably because its chelating power is greater than that of the blood protein, and so hinders passage of the follicle cell product and associated protein from the spaces to the oocyte surface.
The available evidence can be summarized best in the context of a model of the sequence of events leading to deposition of blood proteins in yolk. Blood protein combination with the follicle cell product in the extracellular spaces creates a labile complex, which passes across the vitelline membrane to the oocyte surface, and there the follicle cell product stimulates pinocytotic uptake of the blood proteins. Trypan blue interferes with this process by transforming the fluid complex into an immobile precipitate.
While some features of this model are clearly speculative, it provides a starting-point for further experimentation and emphasizes the importance of careful attention to extracellular phenomena in the study of pinocytosis of proteins.
SUMMARY
The colloidal acidic dye, trypan blue, injected into the blood of developing cecropia females, is concentrated in the yolk spheres of ovarian follicles to a degree similar to that observed for vitellogenic blood proteins. At the same time, the dye inhibits yolk formation, as evidenced by a reduction both in yolk sphere size and in depth of the layer of newly-formed spheres.
The concentration of the dye and the degree of inhibition are reduced by high concentrations of blood proteins; these relationships support the idea that the dye is incorporated into the oocyte by the same specific mechanism as the blood proteins.
In follicles treated with 1% trypan blue in vitro, the dye was bound in large amounts in the extracellular spaces and entered the yolk in concentrated form, but for a short time only. This severe suppression, which was interpreted as an extreme form of the inhibition observed in vivo, could be reversed by removing extracellular bound dye.
The dye’s inhibition of yolk formation in vitro was accompanied by stabilization of a [3H]histidine-labelled secretion product of the follicle cells in the intercellular spaces, thus preventing its transfer to the yolk. The follicle cell product is suggested to be a necessary agent in the pinocytotic uptake of blood proteins.
RÉSUMÉ
Etude de la fonction des cellules folliculeuses chez cecropia: inhibition de la vitellogènese par le bleu trypan
Le bleu trypan, colorant acide colloidal, injecté dans le sang de cecropia femelles en développement, est concentré dans les granules vitellins des follicules ovariens à un degré comparable à celui que l’on observe pour les protéines vitellines du sang. Le colorant inhibe en même temps la formation de vitellus, comme le montre la réduction de la taille des granules vitellins et de l’épaisseur de la couche des granules néoformés.
La concentration du colorant et le degré d’inhibition sont diminués par des concentrations élevées de protéines du sang; ces relations de cause à effet suggèrent que le colorant est incorporé dans l’ovocyte par le même mécanisme que les protéines du sang.
Dans des follicules traités in vitro par du bleu trypan à 1%, le colorant est en grande partie limité aux espaces extra-cellulaires et pénètre dans le vitellus sous forme concentrée, mais pour un bref laps de temps seulement. Cette répression sévère, qui est interprétée comme une forme extrême de l’inhibition observée in vivo, est réversible: si le colorant extra-cellulaire est retiré, elle ne s’observe pas.
L’inhibition in vitro de la vitellogenèse par le colorant s’accompagne de la stabilisation d’un produit marqué à l’histidine tritiée, sécrété dans les espaces inter-cellulaires par les cellules folliculeuses; le transport du produit dans le vitellus est ainsi empêché. On peut supposer que ce produit des cellules folli-culeuses est un facteur nécessaire à l’absorption par pinocytose des protéines du sang.
ACKNOWLEDGEMENTS
This work was supported by the National Science Foundation (grant number 4463 and a Predoctoral Fellowship to L. M. A.).