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The development of responsiveness to juvenile hormone in the follicle cells of Rhodnius prolixus
Insect Biochem. Vol. 17, No. 4, pp. 525-529, 1987
0020-1790/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
THE DEVELOPMENT OF RESPONSIVENESS TO JUVENILE HORMONE IN THE FOLLICLE CELLS OF RHODNIUS PROLIXUS T. T. ILENCHUK and K. G. DAWY Department of Biology, York University, North York, Ontario M3J IP3, Canada (Received 5 May 1986; revised and accepted 23 July 1986)
Abstract--During the previtellogenic phase of the development of the follicle in Rhodnius, the JH-sensitive Na/K ATPase in the membranes of the follicle cells displays a rapid increase in activity, so that the level in vitellogenic follicle cells is about six times that in early previtellogenic cells. The sharp increase in activity during the development of previtellogenic cells is abolished in an allatectomized female. Topical application of JH I early in development restores the level in previtellogenic cells and produces a nearly normal level in vitellogenic cells. Treatment with JH I later in the cycle fails to yield an increase in the activity of the JH-sensitive Na/K ATPase in the previtellogenic cells and leads to only partial restoration of the activity in vitellogenic follicles. These effects of allatectomy and early and late hormone replacement are duplicated by effects on the maximal binding capacity for [3H]JH I of the membranes of follicle cells. These results are interpreted in the light of "activation", the JH-mediated process by which follicles acquire the competence to respond to JH by becoming patent. Key Word Index: Activation, juvenile hormone, binding, Na/K ATPase activity, Rhodnius
INTRODUCTION
In Rhodnius, as in many other insects, juvenile hormone (JH) is required for the full expression of egg maturation. JH is known to govern the production by the fat body of the yolk protein vitellogenin (Coles, 1965). The uptake of vitellogenin from the hemolymph by the developing oocyte is also under the control of JH. The vitellogenin gains access to the oocyte surface by passing between the cells of the follicular epithelium via large spaces which develop between them in response to JH; such a follicle is said to be patent (Pratt and Davey, 1972; Davey and Huebner, 1974). The development of patency is accompanied by a number of changes in the follicle cells, including changes in the gap junctions (Huebner and Injeyan, 1981) and in the cytoskeletal network (Abu-Hakima and Davey, 1977c). It is not yet clear whether all of these changes depend on JH. However, it is clear that the principal action of JH in bringing about patency involves a reduction in volume of the cells, a phenomenon which is reversible and which occurs rapidly in vitro (Abu-Hakima and Davey, 1977a). This action of JH is focussed in the membrane of the follicle cell and involves the enzyme N a / K ATPase (Abu-Hakima and Davey, 1979; Ilenchuk and Davey, 1982). In brief, JH appears to stimulate specific JH-dependent N a / K ATPase sites in the membrane of the vitellogenic follicle cell (Ilenchuk and Davey, 1983). Binding sites for JH have been identified on the membranes of the follicle cells (Ilenchuk and Davey, 1985). These JH-dependent events are all centered on the membrane of the previtellogenic follicle cell and do not involve macromolecular synthesis (AbuHakima and Davey, 1977b). However, it is clear that JH is also required earlier 525
in the development of the follicle cell, before the follicle becomes patent. In their analysis of the possible effects of JH on vitellogenesis, Pratt and Davey (1972) predicted that, in addition to its effects on patency, JH would also affect the follicle immediately before vitellogenesis. This ill-defined, previtellogenic effect was termed "activation". Some additional description of the phenomenon was provided by the discovery that follicle cells in vitellogenic follicles from allatectomized females would not become patent when exposed to JH in vitro (Abu-Hakima and Davey, 1975). The observation that cells from immediately previtellogenic follicles, which do not exhibit patency, nevertheless exhibit some reduction in volume when exposed to JH in vitro (Davey, 1981) confirms that the development of patency depends on more than the ability of the cell to respond to JH by shrinking. That observation also clouds the issue of the timing of the activation and casts some doubt on the validity of the concept of activation as a JH-dependent acquisition of competence to respond to JH. The fact that JH activates a JH-dependent N a / K ATPase offers an opportunity to explore in a more quantitative way the ability of follicle cells at various stages of development to respond to JH. Accordingly, the present paper explores the following questions. Are there differences in the ability of membranes from follicle cells at different stages of development to respond to JH by an increase in the level of ATPase activity? Are there parallel differences in the ability of the membranes to bind JH? Is the acquisition of the ability to respond to JH or to bind the hormone affected by allatectomy? Can any defect in these parameters induced by allatectomy be corrected by JH replacement therapy?
T. T. /LENCHUKand K. G. DAVEY
526
MATERIALS AND METHODS
Juvenile hormone 1 (C18-JH) was a gift from Ayerst Laboratories (Montreal, Quebec, Canada). [3H]ouabain (19.5 Ci/mmol) was purchased from New England Nuclear (Lachine, Quebec, Canada). Tris-ATP was purchased from Sigma (St Louis, Missouri, U.S.A.). ACS and OCS liquid scintillation fluid was purchased from Amersham (Oakville, Ontario, Canada). Membrane filters (HA, pore size 0.45/~m) were purchased from Millipore (Toronto, Ontario, Canada). Tackiwax was purchased from Cenco (Toronto, Ontario, Canada). All other compounds and reagents used were of analytical grade.
Tissue preparation Female Rhodnius prolixus were reared in jars in incubators at a temperature of 28 + I°C under conditions of high relative humidity. The membrane preparations from the follicle cells were prepared according to the methods outlined in an earlier report Olenchuk and Davey, 1982). Briefly, after homogenization in a Tris-HCl (50 mM Tris; pH7.4) buffer, and two centrifugation steps (600g and 13,500g) at 0~°C, the supernatant was centrifuged at 105,000g for 90 min at 0-4°C. The pellet was resuspended in distilled water for subsequent assay.
Allatectomy Allatectomy (CAX) was performed on females within 48 hr of emergence. Briefly, the animals were secured with modelling clay, with their heads positioned in a slightly raised position at the junction of the head and thorax. A sharp scalpel blade was used to remove a section of cuticle to expose the retrocerebral organs. The allatum was removed with fine scissors, and the excised cuticle replaced and secured with Tackiwax. The CAX insects were held in a 10°C incubator for 12-24 hr. The operated insects received one of two regimes of hormone replacement therapy. One group was treated early (EH), and received 100 nl JH I in 5/~1 of acetone on the third day of adult life. These insects were fed on the tenth day of adult life, The second group was treated late (LH), and received the same dose of JH on the second day post feed (i.e. on the twelfth day of adult life). Controls for both groups of insects consisted of CAX females treated at the appropriate time with 5 #1 of acetone. On day 14 post emergence (4 days after feeding), all insects were dissected, and the follicles were removed and grouped according to size. Previtellogenic follicles were less than 400/~m in length and vitellogenic follicles were greater than 400/~m but less than 1600/~m in length.
Juvenile hormone binding assay [3H]JH I was prepared by the procedure of Peter et al. (1979). A more detailed description of the procedures is provided elsewhere (Ilenchuk and Davey, 1985). Ultrafiltration techniques as previously described (Ilenchuk and Davey, 1985) were used to determine the JH binding capacity of membrane preparations from previtellogenic and vitellogenic follicle cells. Aliquots of follicle cell membrane preparations (each containing 0.02 mg protein per ml) were incubated for 2 hr at 37°C in 50mM Tris-HC1 (pH 7.4) in the presence or absence of an excess (1 #M) of unlabelled JH, with [3H]JH I (45.2 Ci/mmol) present in the saturating concentration of 22 nM (0.25/~Ci; see Ilenchuk and Davey, 1985). The [3H]JH I bound to the membranes was separated from the unbound [3H]JH I by filtering 100/~1 samples on membrane filters (0.45/zm pore dia.), which had been soaked overnight in unlabelled JH I. After filtration, the filters were washed four times with 1.0ml of 0.15mM Tris-HC1 (pH7.4). The filters were allowed to dry, placed in 10 ml of OCS scintillation fluid and counted with an efficiency of about 30%. Maximal binding is reported as pmol/mg protein for a number of replicates.
Na/K A TPase assay Na/K ATPase activity present in the membrane preparations was determined by measuring the evolution of inorganic phosphate using the colorimetric methods of Fiske and Subbarow (1925), modified as previously reported (Ilenchuk and Davey, 1982).
[3H]ouabain binding for Na/K A TPase activity Ouabain binds to a single site on the Na/K ATPase complex (Hansen, 1971). The capacity of follicle cell membranes to bind ouabain is, therefore, a reflection of the Na/K ATPase activity resident in those membranes. [3H]Ouabain binding capacity to follicle cell preparations was determined using ultrafiltration techniques (Hansen, 1971), as reported elsewhere (Ilenchuk and Davey, 1983). Briefly, portions of membrane preparation, each containing 0.01mg/ml protein, were added to medium containing 2 mM Tris-ATP, 2 mM MgCI2, I0 mM NaC1, 50 mM Tris-HC1 (pH 7.4), and a saturating concentration, 408 nM (2.0 #Ci), of [3H]ouabain (Ilenchuk and Davey, 1983). The final volume was 250 ~ul. After incubation for 2 hr at 37°C, the samples were filtered through Millipore membrane filters as described above and washed twice with 1.0 ml aliquots of 0.15 mM Tris-HC1 (pH 7.4). The filters were allowed to dry and placed in scintillation vials with 10 ml of ACS scintillation fluid. After 24 hr of dark adaptation the samples were counted at an efficiency of about 30%.
Protein determination Protein concentrations were estimated using the commercially available protein assay kit from Bio-Rad Laboratories (Mississauga, Ontario, Canada) which is based on the methods of Bradford (1976). RESULTS
The development o f JH-sensitive A TPase activity Figure 1 displays the JH-stimulated activity of N a / K ATPase in preparations of membranes from follicles of different sizes as determined by ouabain binding. These data demonstrate that the activity increases by about six times as the follicle grows from a length of 250 to 400 #m, the size at which it enters vitellogenesis. The activity declines sharply after the follicles exceed 1200/~m in length. These events are not simply a function of the great increase in amount of membrane which occurs as the follicle grows, as can be seen from the data on the surface area of the oocyte taken from A b u - H a k i m a and Davey (1977a), and included in Fig. 1.
Effects o f allateetomy and hormone replacement on N a / K A TPase activity The results from these experiments are displayed in Fig. 2. F r o m these results it is clear that allatectomy all but eliminates the JH-stimulated N a / K ATPase activity which is detectable in the previtellogenic follicles of normal females. Membranes prepared from previtellogenic follicle cells from C A X females which were also E H displayed a level of JHstimulated N a / K ATPase significantly greater (P < 0.001) than that from untreated C A X females, and almost identical to that from the membranes of normal cells. Membranes from previtellogenic follicle cells of similar females subjected to LH, however, displayed no increase in level over that in membranes from C A X females receiving no hormone. Allatectomized females do not produce sufficient
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527
contrast, the level in the membranes of vitellogenic follicle cells from LH treated females is less than half as great (P < 0.01) and is only slightly higher than that in the membranes of previtellogenic follicles from normal females. DISCUSSION
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Fig. 1. The relationship among [3H]ouabain binding capacity, follicle length and follicle surface area. [3H]ouabain binding capacity to follicle cell membrane preparations from follicles of different lengths was determined by incubating membrane preparations (0.01 mg protein/ml) for 2hr at 37°C in binding medium containing 2.0#Ci labelled ouabain. The amount of labelled ouabain bound was measured using ultrafiltration techniques as described in Materials and Methods. Data are given as means + SEM for three separate determinations. (The surface area data are from Abu-Hakima and Davey, 1977a.)
The major findings in this paper must be viewed against a background of existing information about the response of the follicle cells to JH and the development of patency. Patency, or the appearance of spaces between the cells of the follicular epithelium, is a JH-dependent event (Pratt and Davey, 1972). Moreover, the acquisition of competence to become patent in response to JH is itself a JHdependent process (Abu-Hakima and Davey, 1975). While patency depends absolutely on the loss of fluid from individual follicle cells (Abu-Hakima and Davey, 1977a), it is clear that the follicle cells acquire the ability to respond to JH by shrinking while they are still in the previtellogenic stage (Davey, 1981). This observation emphasizes the fact that alterations in cell junctions are also involved, although it is not clear whether this is a JH-dependent process (Huebner and Injeyan, 1981).
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vitellogenic cells to permit an assay for the enzyme. However, CAX females which have received EH treatment display a level of JH-stimulated Na/K ATPase activity which is only slightly below that from normal females. However, similar females receiving LH treatment display a level of JH-stimulated activity which is only slightly elevated above the JH-insensitive level, and which is significantly less (P < 0.001) than that in the membranes from CAX females subjected to EH treatment. It is worth noting that the ovaries of these LH females contained many vitellogenic follicles.
Effects of allatectomy and hormone replacement on JH binding capacity Figure 3 displays the results of the determination of maximal binding capacity for [aH]JH I of membrane preparations from follicle cells from CAX and JH-treated females. From these data it is clear that allatectomy reduces the binding capacity of membranes from previtellogenic cells to about half that in normal cells (P < 0.05). Subjecting CAX females to EH all but restores the binding capacity, whereas LH treatment produces a level of [3H]JH I binding which is not significantly different from that in untreated CAX females (P > 0.2). There were insufficient vitellogenic follicles produced in untreated CAX females to permit an assay. However, the membranes from the vitellogenic follicle cells of EH treated females exhibit a level of maximal binding of [3 H]JH I which is not significantly different (P > 0.1) from that of normal females. By
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Fig. 2. Na/K ATPase activity in membrane preparations of cells from previtellogenic and vitellogenic follicles in normal females (NRM) or allatectomized females which had received acetone (CAX), early hormone treatment (EH), or late hormone treatment (LH). EH females received a topical application of JH I (100 nl in 5/~1 acetone) at 48 hr after emergence, while LH females received a topical application of a similar dose of JH I at 48hr after feeding. JHinsensitive Na/K ATPase activity ( - ) was determined using standard assay conditions; while JH-stimulated Na/K ATPase activity (+) was determined using standard assay conditions with the addition of JH I at a final concentration of 4 x 10-7 M. The results are expressed as means + SEM, and the numbers in parentheses represent the number of individual trials.
T. T. ILENCHUKand K. G. DAVEY
528
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Fig. 3. [3H]JH I binding capacity in membrane preparations of cells from previtellogenic, and vitellogenic follicles in normal females (NRM) or allatectomized females which had received acetone (CAX), early hormone treatment (EH), or late hormone treatment (LH). EH females received a topical application of JH I (100 nl JH I in 5/~1 acetone) at 48 hr after emergence, while LH females received a topical application of a similar dose of JH I at 48 hr after feeding. [3H]JH I binding capacity was determined using standard assay conditions. The results are expressed as means + SEM, and the numbers in parentheses represent the number of individual trials.
The reduction in volume of the follicle cells in response to JH involves the activation of a JHdependent Na/K ATPase in the membrane (Ilenchuk and Davey, 1983). The precise mode of action of the hormone in bringing about this effect is not known, but it is clear that the stimulation occurs in isolated membranes, and that JH binds in a specific and saturable way to the membranes (Ilenchuk and Davey, 1985). The present paper explores the appearance of the JH-sensitive Na/K ATPase as well as JH binding sites in the membranes of follicle cells during their development and relates these findings to the concept of activation of the follicle cell as proposed by Pratt and Davey (1972). The first approach involved documenting the appearance of the JH-sensitivite N a / K ATPase during development of the follicle cell. As revealed in Fig. 1, the JH sensitive enzyme first makes its appearance during the period immediately preceding the beginning of viteilogenesis and the appearance of patency. By that time, the JH-sensitive N a / K ATPase has already made its appearance in the membranes of the follicle cells, although that process is relatively rapid. In any case, the fact that previtellogenic follicle cells already possess in their membranes the enzyme which appears to be the primary effector of the action of JH in bringing about the reduction in volume of the follicle cells explains the ability of these cells to shrink in response to the hormone. Is the appearance of the JH-dependent N a / K ATPase itself a JH-dependent event? We have approached this question by examining the activity
of the enzyme in the follicle cells of normal and allatectomized females. For the previtellogenic follicles, it is clear that allatectomy prevents the appearance of the JH-sensitive N a / K ATPase; the N a / K ATPase activity is not increased by the addition of JH to membrane preparations of follicle cells from females allatectomized on the first day of adult life. In dealing with the effects of allatectomy on vitellogenic follicle cells, we were forced to develop a less direct approach. While allatectomized females produce a few vitellogenic follicles, the numbers involved are so small that the collection of enough such follicles for the preparation of membranes is impractical. Allatectomized females which subsequently receive a single topical application of JH I develop many vitellogenic follicles, even when the JH is applied on the second day post feed (i.e. on the twelfth day of adult life). We therefore compared the degree of stimulation of N a / K ATPase activity by JH in membrane preparations from allatectomized females which received a dose of JH on the second day of adult life with those from females treated on the second day post feed. The data were unequivocal: the level of JH-stimulated N a / K ATPase activity in the membranes from females treated early was restored to very near the normal level, while that from females treated late, in spite of having entered vitellogenesis, was very much less. The difference between the stimulated and unstimulated level in the latetreated females was only about 25% of that in the early-treated females. These results on vitellogenic follicle cells are paralleled by those obtained on previtellogenic follicle cells. Those treated early exhibit a level of JHstimulated N a / K ATPase activity which is similar to that found in normal females, while those exposed to JH for only a short time exhibit no activity in terms of JH-sensitive enzyme, as in allatectomized females which have not received JH. A similar approach was used to study the development of the capacity of membranes to bind JH. Previtellogenic follicle cells have only about 50% of the capacity to bind JH when compared to vitellogenic follicle cells. The capacity of membranes from previtellogenic follicle cells to bind JH is sharply reduced in allatectomized females, and this capacity is only partially restored by early treatment with JH and marginally increased by late treatment. In vitellogenic follicles from allatectomized females treated early with JH, the capacity of the membranes to bind JH is markedly enhanced when compared to normal follicles, while that of the membranes from females treated late with JH is not markedly greater than the level found in previtellogenic follicles from normal females. These biochemical observations are in close agreement with observations on the ability of isolated follicle cells from allatectomized females to shrink upon exposure to JH. Follicle cells from both vitellogenic and previtellogenic follicles from allatectomized females do not shrink when exposed in vitro to JH, and the defect is not corrected if the allatectomized females are exposed to JH after feeding (Davey, 1981). However, unpublished data from this laboratory demonstrate that cells from both vitellogenic and previtellogenic follicles are able to
Responsiveness to juvenile hormone in R. prolixus shrink upon exposure to JH if the allatectomized females receive a single dose of JH I on the second day of adult life. All of these data taken together lead to two principal conclusions. First, there are JH-dependent processes at work during the previtellogenic development of the follicle cell that lead to the enhanced appearance in the membrane of at least two structures. On the one hand there is an increased level of JH-sensitive ouabain binding sites, indicating an enhanced level of JH-sensitive Na/K ATPase, and on the other, the ability of the membranes to bind JH is increased. These increases do not occur in the absence of JH and require a prolonged exposure to the hormone in order to develop fully. It is not clear whether these increases represent de novo synthesis of new JH and Na/K ATPase sites, or whether they simply represent a JH-controlled insertion of preexisting elements into the membrane. However, it is worth noting that Koeppe et al. (1981) have demonstrated that JH induces protein synthesis in the follicle cells of Leucophaea. The second principal conclusion concerns the concept of activation. As originally conceived (Pratt and Davey, 1972), the term described an ill-defined set of events which occurred just before the follicles entered vitellogenesis, at a length of 350 to 400/~m; JH was seen as crucial for the entry into vitellogenesis. The present work has provided some description of two JH dependent events, the appearance of Na/K ATPase activity and JH binding sites in the membrane, which are essential for the action of JH on the follicle cells. It is thus tempting to equate these JH-dependent processes with the concept of activation. However, it is important to note that allatectomized females which have been treated with JH exhibit ovaries with many follicles in vitellogenesis, even when the JH is applied after the feed. In one sense it is irrelevant that these follicles are only marginally patent and that the follicle cells themselves, by virtue of the low level of JH-sensitive Na/K ATPase and of JH binding sites, are incapable of responding to JH. There is clearly a JH-dependent process at work which moves the follicles out of the previtellogenic stage, where they tend to accumulate in the absence of JH (Pratt and Davey, 1972), into the vitellogenic phase. While this is a JH-dependent process, it is at the same time distinct from the JH-dependent process by which the follicles acquire the competence to respond to JH by shrinking. If the developmental processes leading to the alteration of the junctional complexes, processes which thus far are not known to be controlled by JH, are added to the obviously JH-dependent processes, a very complex picture emerges. 'Activation' thus describes a complex of at least three processes: the acquisition of competence to respond to JH by shrinking, a process which involves the appearance of JH binding and JH-sensitive Na/K ATPase sites in the membrane; the independent JH-sensitive process by which the follicles poised at the brink of vitellogenesis are moved forward so as to become vitellogenic; and the process, of uncertain relation to JH, by which the pattern of cell junctions is altered. Acknowledgements--We thank Mrs Janet Caverly for
529
skilled technical assistance. Research in this laboratory is supported by grants from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES
Abu-Hakima R. and Davey K. G. (1975) Two actions of juvenile hormone on the folliclecells of Rhodnius prolixus Stal. Can. J. ZooL 53, 1187-1188. Abu-Hakima R. and Davey K. G. (1977a) The action of juvenile hormone on the follicleceils of Rhodnius prolixus: the importance of volume changes. J. exp. Biol. 69, 33-44. Abu-Hakima R. and Davey K. G. (1977b) Effects of hormones and inhibitors of macromolecular synthesis on follicle ceils of Rhodnius..I. Insect Physiol. 23, 913-917. Abu-Hakima R. and Davey K. G. (1977c) The action of juvenile hormone on the folliclecells of Rhodnius prolixus in vitro: the effect of colchicine and cytochalasin B. Gen. comp. Endocr. 32, 360-370. Abu-Hakima R. and Davey K. G. (1979) A possible relationship between ouabain-sensitive (Na+-K+)dependent ATPase and the effect of juvenile hormone on the follicle cells of Rhodnius prolixus. Insect Biochem. 9, 195-198. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Coles G. C. (1965) Studies on the hormonal control of metabolism in Rhodnius prolixus Stal. I. The adult female. J. Insect Physiol. 11, 1325-1330. Davey K. G. (1981) Regulation of cell volume in folliclecells of Rhodnius by JH. In Juvenile Hormone Biochemistry (Edited by Pratt G. E. and Brooks G. T.), pp. 233-240. Elsevier/North Holland Biomedical Press, Oxford. Davey K. G. and Huebner E. (1974) The response of the follicle cells of Rhodnius prolixus to juvenile hormone and antigonadotropin in vitro. Can. J. Zool. 52, 1407-1412. Fiske C. and Subbarow Y. (1925) The colorimetric determination of phosphorus. J. biol. Chem. 66, 375. Hansen O. (1971) The relationship between g-strophanthinbinding capacity and ATPase activity in plasma membrane fragments from ox brain. Biochim. biophys. Acta 233, 122-132. Huebner E. and Injeyan H. (1981) Follicular modulation during oocyte development in an insect: formation and modification of septate and gap junctions. Devl Biol. 83, 101-113. Ilenchuk T. T. and Davey K. G. (1982) Some properties of Na + - K + ATPase in the follicle cells of Rhodnius prolixus. Insect Biochem. 12, 675-679. Ilenchuk T. T. and Davey K. G. (1983) Juvenile hormone increases ouabain-binding capacity of microsomal preparations from vitellogenic follicle cells. Can. J. Biochem. Cell Biol. 61, 826-831. Ilenchuk T. T. and Davey K. G. (1985) The binding of juvenile hormone to membranes of follicle cells in the insect Rhodnius prolixus. Can. J. Biochem. Cell BioL 63, 102-106. Koeppe J. K., Kovalick E. and LaPointe M. C. (1981) Juvenile hormone interactions with ovarian tissue in Leucophaea maderae. In Juvenile Hormone Biochemistry (Edited by Pratt G. E. and Brooks G. T.), pp. 215-231. Elsevier/North-Holland Biomedical Press, Oxford. Peter M. G., Gunawal S. and Emmerich H. (1979) Preparation of optically pure juvenile hormone I labelled in the ester methyl group with tritium at very high specific activity. Experientia 35, 1141-1142. Pratt G. E. and Davey K. G. (1972) The corpus allatum and oogenesis in Rhodnius prolixus (Stal). I. The effects of allatectomy. J. exp. Biol. 56, 201-214.
0020-1790/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
THE DEVELOPMENT OF RESPONSIVENESS TO JUVENILE HORMONE IN THE FOLLICLE CELLS OF RHODNIUS PROLIXUS T. T. ILENCHUK and K. G. DAWY Department of Biology, York University, North York, Ontario M3J IP3, Canada (Received 5 May 1986; revised and accepted 23 July 1986)
Abstract--During the previtellogenic phase of the development of the follicle in Rhodnius, the JH-sensitive Na/K ATPase in the membranes of the follicle cells displays a rapid increase in activity, so that the level in vitellogenic follicle cells is about six times that in early previtellogenic cells. The sharp increase in activity during the development of previtellogenic cells is abolished in an allatectomized female. Topical application of JH I early in development restores the level in previtellogenic cells and produces a nearly normal level in vitellogenic cells. Treatment with JH I later in the cycle fails to yield an increase in the activity of the JH-sensitive Na/K ATPase in the previtellogenic cells and leads to only partial restoration of the activity in vitellogenic follicles. These effects of allatectomy and early and late hormone replacement are duplicated by effects on the maximal binding capacity for [3H]JH I of the membranes of follicle cells. These results are interpreted in the light of "activation", the JH-mediated process by which follicles acquire the competence to respond to JH by becoming patent. Key Word Index: Activation, juvenile hormone, binding, Na/K ATPase activity, Rhodnius
INTRODUCTION
In Rhodnius, as in many other insects, juvenile hormone (JH) is required for the full expression of egg maturation. JH is known to govern the production by the fat body of the yolk protein vitellogenin (Coles, 1965). The uptake of vitellogenin from the hemolymph by the developing oocyte is also under the control of JH. The vitellogenin gains access to the oocyte surface by passing between the cells of the follicular epithelium via large spaces which develop between them in response to JH; such a follicle is said to be patent (Pratt and Davey, 1972; Davey and Huebner, 1974). The development of patency is accompanied by a number of changes in the follicle cells, including changes in the gap junctions (Huebner and Injeyan, 1981) and in the cytoskeletal network (Abu-Hakima and Davey, 1977c). It is not yet clear whether all of these changes depend on JH. However, it is clear that the principal action of JH in bringing about patency involves a reduction in volume of the cells, a phenomenon which is reversible and which occurs rapidly in vitro (Abu-Hakima and Davey, 1977a). This action of JH is focussed in the membrane of the follicle cell and involves the enzyme N a / K ATPase (Abu-Hakima and Davey, 1979; Ilenchuk and Davey, 1982). In brief, JH appears to stimulate specific JH-dependent N a / K ATPase sites in the membrane of the vitellogenic follicle cell (Ilenchuk and Davey, 1983). Binding sites for JH have been identified on the membranes of the follicle cells (Ilenchuk and Davey, 1985). These JH-dependent events are all centered on the membrane of the previtellogenic follicle cell and do not involve macromolecular synthesis (AbuHakima and Davey, 1977b). However, it is clear that JH is also required earlier 525
in the development of the follicle cell, before the follicle becomes patent. In their analysis of the possible effects of JH on vitellogenesis, Pratt and Davey (1972) predicted that, in addition to its effects on patency, JH would also affect the follicle immediately before vitellogenesis. This ill-defined, previtellogenic effect was termed "activation". Some additional description of the phenomenon was provided by the discovery that follicle cells in vitellogenic follicles from allatectomized females would not become patent when exposed to JH in vitro (Abu-Hakima and Davey, 1975). The observation that cells from immediately previtellogenic follicles, which do not exhibit patency, nevertheless exhibit some reduction in volume when exposed to JH in vitro (Davey, 1981) confirms that the development of patency depends on more than the ability of the cell to respond to JH by shrinking. That observation also clouds the issue of the timing of the activation and casts some doubt on the validity of the concept of activation as a JH-dependent acquisition of competence to respond to JH. The fact that JH activates a JH-dependent N a / K ATPase offers an opportunity to explore in a more quantitative way the ability of follicle cells at various stages of development to respond to JH. Accordingly, the present paper explores the following questions. Are there differences in the ability of membranes from follicle cells at different stages of development to respond to JH by an increase in the level of ATPase activity? Are there parallel differences in the ability of the membranes to bind JH? Is the acquisition of the ability to respond to JH or to bind the hormone affected by allatectomy? Can any defect in these parameters induced by allatectomy be corrected by JH replacement therapy?
T. T. /LENCHUKand K. G. DAVEY
526
MATERIALS AND METHODS
Juvenile hormone 1 (C18-JH) was a gift from Ayerst Laboratories (Montreal, Quebec, Canada). [3H]ouabain (19.5 Ci/mmol) was purchased from New England Nuclear (Lachine, Quebec, Canada). Tris-ATP was purchased from Sigma (St Louis, Missouri, U.S.A.). ACS and OCS liquid scintillation fluid was purchased from Amersham (Oakville, Ontario, Canada). Membrane filters (HA, pore size 0.45/~m) were purchased from Millipore (Toronto, Ontario, Canada). Tackiwax was purchased from Cenco (Toronto, Ontario, Canada). All other compounds and reagents used were of analytical grade.
Tissue preparation Female Rhodnius prolixus were reared in jars in incubators at a temperature of 28 + I°C under conditions of high relative humidity. The membrane preparations from the follicle cells were prepared according to the methods outlined in an earlier report Olenchuk and Davey, 1982). Briefly, after homogenization in a Tris-HCl (50 mM Tris; pH7.4) buffer, and two centrifugation steps (600g and 13,500g) at 0~°C, the supernatant was centrifuged at 105,000g for 90 min at 0-4°C. The pellet was resuspended in distilled water for subsequent assay.
Allatectomy Allatectomy (CAX) was performed on females within 48 hr of emergence. Briefly, the animals were secured with modelling clay, with their heads positioned in a slightly raised position at the junction of the head and thorax. A sharp scalpel blade was used to remove a section of cuticle to expose the retrocerebral organs. The allatum was removed with fine scissors, and the excised cuticle replaced and secured with Tackiwax. The CAX insects were held in a 10°C incubator for 12-24 hr. The operated insects received one of two regimes of hormone replacement therapy. One group was treated early (EH), and received 100 nl JH I in 5/~1 of acetone on the third day of adult life. These insects were fed on the tenth day of adult life, The second group was treated late (LH), and received the same dose of JH on the second day post feed (i.e. on the twelfth day of adult life). Controls for both groups of insects consisted of CAX females treated at the appropriate time with 5 #1 of acetone. On day 14 post emergence (4 days after feeding), all insects were dissected, and the follicles were removed and grouped according to size. Previtellogenic follicles were less than 400/~m in length and vitellogenic follicles were greater than 400/~m but less than 1600/~m in length.
Juvenile hormone binding assay [3H]JH I was prepared by the procedure of Peter et al. (1979). A more detailed description of the procedures is provided elsewhere (Ilenchuk and Davey, 1985). Ultrafiltration techniques as previously described (Ilenchuk and Davey, 1985) were used to determine the JH binding capacity of membrane preparations from previtellogenic and vitellogenic follicle cells. Aliquots of follicle cell membrane preparations (each containing 0.02 mg protein per ml) were incubated for 2 hr at 37°C in 50mM Tris-HC1 (pH 7.4) in the presence or absence of an excess (1 #M) of unlabelled JH, with [3H]JH I (45.2 Ci/mmol) present in the saturating concentration of 22 nM (0.25/~Ci; see Ilenchuk and Davey, 1985). The [3H]JH I bound to the membranes was separated from the unbound [3H]JH I by filtering 100/~1 samples on membrane filters (0.45/zm pore dia.), which had been soaked overnight in unlabelled JH I. After filtration, the filters were washed four times with 1.0ml of 0.15mM Tris-HC1 (pH7.4). The filters were allowed to dry, placed in 10 ml of OCS scintillation fluid and counted with an efficiency of about 30%. Maximal binding is reported as pmol/mg protein for a number of replicates.
Na/K A TPase assay Na/K ATPase activity present in the membrane preparations was determined by measuring the evolution of inorganic phosphate using the colorimetric methods of Fiske and Subbarow (1925), modified as previously reported (Ilenchuk and Davey, 1982).
[3H]ouabain binding for Na/K A TPase activity Ouabain binds to a single site on the Na/K ATPase complex (Hansen, 1971). The capacity of follicle cell membranes to bind ouabain is, therefore, a reflection of the Na/K ATPase activity resident in those membranes. [3H]Ouabain binding capacity to follicle cell preparations was determined using ultrafiltration techniques (Hansen, 1971), as reported elsewhere (Ilenchuk and Davey, 1983). Briefly, portions of membrane preparation, each containing 0.01mg/ml protein, were added to medium containing 2 mM Tris-ATP, 2 mM MgCI2, I0 mM NaC1, 50 mM Tris-HC1 (pH 7.4), and a saturating concentration, 408 nM (2.0 #Ci), of [3H]ouabain (Ilenchuk and Davey, 1983). The final volume was 250 ~ul. After incubation for 2 hr at 37°C, the samples were filtered through Millipore membrane filters as described above and washed twice with 1.0 ml aliquots of 0.15 mM Tris-HC1 (pH 7.4). The filters were allowed to dry and placed in scintillation vials with 10 ml of ACS scintillation fluid. After 24 hr of dark adaptation the samples were counted at an efficiency of about 30%.
Protein determination Protein concentrations were estimated using the commercially available protein assay kit from Bio-Rad Laboratories (Mississauga, Ontario, Canada) which is based on the methods of Bradford (1976). RESULTS
The development o f JH-sensitive A TPase activity Figure 1 displays the JH-stimulated activity of N a / K ATPase in preparations of membranes from follicles of different sizes as determined by ouabain binding. These data demonstrate that the activity increases by about six times as the follicle grows from a length of 250 to 400 #m, the size at which it enters vitellogenesis. The activity declines sharply after the follicles exceed 1200/~m in length. These events are not simply a function of the great increase in amount of membrane which occurs as the follicle grows, as can be seen from the data on the surface area of the oocyte taken from A b u - H a k i m a and Davey (1977a), and included in Fig. 1.
Effects o f allateetomy and hormone replacement on N a / K A TPase activity The results from these experiments are displayed in Fig. 2. F r o m these results it is clear that allatectomy all but eliminates the JH-stimulated N a / K ATPase activity which is detectable in the previtellogenic follicles of normal females. Membranes prepared from previtellogenic follicle cells from C A X females which were also E H displayed a level of JHstimulated N a / K ATPase significantly greater (P < 0.001) than that from untreated C A X females, and almost identical to that from the membranes of normal cells. Membranes from previtellogenic follicle cells of similar females subjected to LH, however, displayed no increase in level over that in membranes from C A X females receiving no hormone. Allatectomized females do not produce sufficient
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527
contrast, the level in the membranes of vitellogenic follicle cells from LH treated females is less than half as great (P < 0.01) and is only slightly higher than that in the membranes of previtellogenic follicles from normal females. DISCUSSION
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Fig. 1. The relationship among [3H]ouabain binding capacity, follicle length and follicle surface area. [3H]ouabain binding capacity to follicle cell membrane preparations from follicles of different lengths was determined by incubating membrane preparations (0.01 mg protein/ml) for 2hr at 37°C in binding medium containing 2.0#Ci labelled ouabain. The amount of labelled ouabain bound was measured using ultrafiltration techniques as described in Materials and Methods. Data are given as means + SEM for three separate determinations. (The surface area data are from Abu-Hakima and Davey, 1977a.)
The major findings in this paper must be viewed against a background of existing information about the response of the follicle cells to JH and the development of patency. Patency, or the appearance of spaces between the cells of the follicular epithelium, is a JH-dependent event (Pratt and Davey, 1972). Moreover, the acquisition of competence to become patent in response to JH is itself a JHdependent process (Abu-Hakima and Davey, 1975). While patency depends absolutely on the loss of fluid from individual follicle cells (Abu-Hakima and Davey, 1977a), it is clear that the follicle cells acquire the ability to respond to JH by shrinking while they are still in the previtellogenic stage (Davey, 1981). This observation emphasizes the fact that alterations in cell junctions are also involved, although it is not clear whether this is a JH-dependent process (Huebner and Injeyan, 1981).
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vitellogenic cells to permit an assay for the enzyme. However, CAX females which have received EH treatment display a level of JH-stimulated Na/K ATPase activity which is only slightly below that from normal females. However, similar females receiving LH treatment display a level of JH-stimulated activity which is only slightly elevated above the JH-insensitive level, and which is significantly less (P < 0.001) than that in the membranes from CAX females subjected to EH treatment. It is worth noting that the ovaries of these LH females contained many vitellogenic follicles.
Effects of allatectomy and hormone replacement on JH binding capacity Figure 3 displays the results of the determination of maximal binding capacity for [aH]JH I of membrane preparations from follicle cells from CAX and JH-treated females. From these data it is clear that allatectomy reduces the binding capacity of membranes from previtellogenic cells to about half that in normal cells (P < 0.05). Subjecting CAX females to EH all but restores the binding capacity, whereas LH treatment produces a level of [3H]JH I binding which is not significantly different from that in untreated CAX females (P > 0.2). There were insufficient vitellogenic follicles produced in untreated CAX females to permit an assay. However, the membranes from the vitellogenic follicle cells of EH treated females exhibit a level of maximal binding of [3 H]JH I which is not significantly different (P > 0.1) from that of normal females. By
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Fig. 2. Na/K ATPase activity in membrane preparations of cells from previtellogenic and vitellogenic follicles in normal females (NRM) or allatectomized females which had received acetone (CAX), early hormone treatment (EH), or late hormone treatment (LH). EH females received a topical application of JH I (100 nl in 5/~1 acetone) at 48 hr after emergence, while LH females received a topical application of a similar dose of JH I at 48hr after feeding. JHinsensitive Na/K ATPase activity ( - ) was determined using standard assay conditions; while JH-stimulated Na/K ATPase activity (+) was determined using standard assay conditions with the addition of JH I at a final concentration of 4 x 10-7 M. The results are expressed as means + SEM, and the numbers in parentheses represent the number of individual trials.
T. T. ILENCHUKand K. G. DAVEY
528
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Fig. 3. [3H]JH I binding capacity in membrane preparations of cells from previtellogenic, and vitellogenic follicles in normal females (NRM) or allatectomized females which had received acetone (CAX), early hormone treatment (EH), or late hormone treatment (LH). EH females received a topical application of JH I (100 nl JH I in 5/~1 acetone) at 48 hr after emergence, while LH females received a topical application of a similar dose of JH I at 48 hr after feeding. [3H]JH I binding capacity was determined using standard assay conditions. The results are expressed as means + SEM, and the numbers in parentheses represent the number of individual trials.
The reduction in volume of the follicle cells in response to JH involves the activation of a JHdependent Na/K ATPase in the membrane (Ilenchuk and Davey, 1983). The precise mode of action of the hormone in bringing about this effect is not known, but it is clear that the stimulation occurs in isolated membranes, and that JH binds in a specific and saturable way to the membranes (Ilenchuk and Davey, 1985). The present paper explores the appearance of the JH-sensitive Na/K ATPase as well as JH binding sites in the membranes of follicle cells during their development and relates these findings to the concept of activation of the follicle cell as proposed by Pratt and Davey (1972). The first approach involved documenting the appearance of the JH-sensitivite N a / K ATPase during development of the follicle cell. As revealed in Fig. 1, the JH sensitive enzyme first makes its appearance during the period immediately preceding the beginning of viteilogenesis and the appearance of patency. By that time, the JH-sensitive N a / K ATPase has already made its appearance in the membranes of the follicle cells, although that process is relatively rapid. In any case, the fact that previtellogenic follicle cells already possess in their membranes the enzyme which appears to be the primary effector of the action of JH in bringing about the reduction in volume of the follicle cells explains the ability of these cells to shrink in response to the hormone. Is the appearance of the JH-dependent N a / K ATPase itself a JH-dependent event? We have approached this question by examining the activity
of the enzyme in the follicle cells of normal and allatectomized females. For the previtellogenic follicles, it is clear that allatectomy prevents the appearance of the JH-sensitive N a / K ATPase; the N a / K ATPase activity is not increased by the addition of JH to membrane preparations of follicle cells from females allatectomized on the first day of adult life. In dealing with the effects of allatectomy on vitellogenic follicle cells, we were forced to develop a less direct approach. While allatectomized females produce a few vitellogenic follicles, the numbers involved are so small that the collection of enough such follicles for the preparation of membranes is impractical. Allatectomized females which subsequently receive a single topical application of JH I develop many vitellogenic follicles, even when the JH is applied on the second day post feed (i.e. on the twelfth day of adult life). We therefore compared the degree of stimulation of N a / K ATPase activity by JH in membrane preparations from allatectomized females which received a dose of JH on the second day of adult life with those from females treated on the second day post feed. The data were unequivocal: the level of JH-stimulated N a / K ATPase activity in the membranes from females treated early was restored to very near the normal level, while that from females treated late, in spite of having entered vitellogenesis, was very much less. The difference between the stimulated and unstimulated level in the latetreated females was only about 25% of that in the early-treated females. These results on vitellogenic follicle cells are paralleled by those obtained on previtellogenic follicle cells. Those treated early exhibit a level of JHstimulated N a / K ATPase activity which is similar to that found in normal females, while those exposed to JH for only a short time exhibit no activity in terms of JH-sensitive enzyme, as in allatectomized females which have not received JH. A similar approach was used to study the development of the capacity of membranes to bind JH. Previtellogenic follicle cells have only about 50% of the capacity to bind JH when compared to vitellogenic follicle cells. The capacity of membranes from previtellogenic follicle cells to bind JH is sharply reduced in allatectomized females, and this capacity is only partially restored by early treatment with JH and marginally increased by late treatment. In vitellogenic follicles from allatectomized females treated early with JH, the capacity of the membranes to bind JH is markedly enhanced when compared to normal follicles, while that of the membranes from females treated late with JH is not markedly greater than the level found in previtellogenic follicles from normal females. These biochemical observations are in close agreement with observations on the ability of isolated follicle cells from allatectomized females to shrink upon exposure to JH. Follicle cells from both vitellogenic and previtellogenic follicles from allatectomized females do not shrink when exposed in vitro to JH, and the defect is not corrected if the allatectomized females are exposed to JH after feeding (Davey, 1981). However, unpublished data from this laboratory demonstrate that cells from both vitellogenic and previtellogenic follicles are able to
Responsiveness to juvenile hormone in R. prolixus shrink upon exposure to JH if the allatectomized females receive a single dose of JH I on the second day of adult life. All of these data taken together lead to two principal conclusions. First, there are JH-dependent processes at work during the previtellogenic development of the follicle cell that lead to the enhanced appearance in the membrane of at least two structures. On the one hand there is an increased level of JH-sensitive ouabain binding sites, indicating an enhanced level of JH-sensitive Na/K ATPase, and on the other, the ability of the membranes to bind JH is increased. These increases do not occur in the absence of JH and require a prolonged exposure to the hormone in order to develop fully. It is not clear whether these increases represent de novo synthesis of new JH and Na/K ATPase sites, or whether they simply represent a JH-controlled insertion of preexisting elements into the membrane. However, it is worth noting that Koeppe et al. (1981) have demonstrated that JH induces protein synthesis in the follicle cells of Leucophaea. The second principal conclusion concerns the concept of activation. As originally conceived (Pratt and Davey, 1972), the term described an ill-defined set of events which occurred just before the follicles entered vitellogenesis, at a length of 350 to 400/~m; JH was seen as crucial for the entry into vitellogenesis. The present work has provided some description of two JH dependent events, the appearance of Na/K ATPase activity and JH binding sites in the membrane, which are essential for the action of JH on the follicle cells. It is thus tempting to equate these JH-dependent processes with the concept of activation. However, it is important to note that allatectomized females which have been treated with JH exhibit ovaries with many follicles in vitellogenesis, even when the JH is applied after the feed. In one sense it is irrelevant that these follicles are only marginally patent and that the follicle cells themselves, by virtue of the low level of JH-sensitive Na/K ATPase and of JH binding sites, are incapable of responding to JH. There is clearly a JH-dependent process at work which moves the follicles out of the previtellogenic stage, where they tend to accumulate in the absence of JH (Pratt and Davey, 1972), into the vitellogenic phase. While this is a JH-dependent process, it is at the same time distinct from the JH-dependent process by which the follicles acquire the competence to respond to JH by shrinking. If the developmental processes leading to the alteration of the junctional complexes, processes which thus far are not known to be controlled by JH, are added to the obviously JH-dependent processes, a very complex picture emerges. 'Activation' thus describes a complex of at least three processes: the acquisition of competence to respond to JH by shrinking, a process which involves the appearance of JH binding and JH-sensitive Na/K ATPase sites in the membrane; the independent JH-sensitive process by which the follicles poised at the brink of vitellogenesis are moved forward so as to become vitellogenic; and the process, of uncertain relation to JH, by which the pattern of cell junctions is altered. Acknowledgements--We thank Mrs Janet Caverly for
529
skilled technical assistance. Research in this laboratory is supported by grants from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES
Abu-Hakima R. and Davey K. G. (1975) Two actions of juvenile hormone on the folliclecells of Rhodnius prolixus Stal. Can. J. ZooL 53, 1187-1188. Abu-Hakima R. and Davey K. G. (1977a) The action of juvenile hormone on the follicleceils of Rhodnius prolixus: the importance of volume changes. J. exp. Biol. 69, 33-44. Abu-Hakima R. and Davey K. G. (1977b) Effects of hormones and inhibitors of macromolecular synthesis on follicle ceils of Rhodnius..I. Insect Physiol. 23, 913-917. Abu-Hakima R. and Davey K. G. (1977c) The action of juvenile hormone on the folliclecells of Rhodnius prolixus in vitro: the effect of colchicine and cytochalasin B. Gen. comp. Endocr. 32, 360-370. Abu-Hakima R. and Davey K. G. (1979) A possible relationship between ouabain-sensitive (Na+-K+)dependent ATPase and the effect of juvenile hormone on the follicle cells of Rhodnius prolixus. Insect Biochem. 9, 195-198. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Coles G. C. (1965) Studies on the hormonal control of metabolism in Rhodnius prolixus Stal. I. The adult female. J. Insect Physiol. 11, 1325-1330. Davey K. G. (1981) Regulation of cell volume in folliclecells of Rhodnius by JH. In Juvenile Hormone Biochemistry (Edited by Pratt G. E. and Brooks G. T.), pp. 233-240. Elsevier/North Holland Biomedical Press, Oxford. Davey K. G. and Huebner E. (1974) The response of the follicle cells of Rhodnius prolixus to juvenile hormone and antigonadotropin in vitro. Can. J. Zool. 52, 1407-1412. Fiske C. and Subbarow Y. (1925) The colorimetric determination of phosphorus. J. biol. Chem. 66, 375. Hansen O. (1971) The relationship between g-strophanthinbinding capacity and ATPase activity in plasma membrane fragments from ox brain. Biochim. biophys. Acta 233, 122-132. Huebner E. and Injeyan H. (1981) Follicular modulation during oocyte development in an insect: formation and modification of septate and gap junctions. Devl Biol. 83, 101-113. Ilenchuk T. T. and Davey K. G. (1982) Some properties of Na + - K + ATPase in the follicle cells of Rhodnius prolixus. Insect Biochem. 12, 675-679. Ilenchuk T. T. and Davey K. G. (1983) Juvenile hormone increases ouabain-binding capacity of microsomal preparations from vitellogenic follicle cells. Can. J. Biochem. Cell Biol. 61, 826-831. Ilenchuk T. T. and Davey K. G. (1985) The binding of juvenile hormone to membranes of follicle cells in the insect Rhodnius prolixus. Can. J. Biochem. Cell BioL 63, 102-106. Koeppe J. K., Kovalick E. and LaPointe M. C. (1981) Juvenile hormone interactions with ovarian tissue in Leucophaea maderae. In Juvenile Hormone Biochemistry (Edited by Pratt G. E. and Brooks G. T.), pp. 215-231. Elsevier/North-Holland Biomedical Press, Oxford. Peter M. G., Gunawal S. and Emmerich H. (1979) Preparation of optically pure juvenile hormone I labelled in the ester methyl group with tritium at very high specific activity. Experientia 35, 1141-1142. Pratt G. E. and Davey K. G. (1972) The corpus allatum and oogenesis in Rhodnius prolixus (Stal). I. The effects of allatectomy. J. exp. Biol. 56, 201-214.