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Ion physiology of vitellogenic follicles
Journal of Insect Physiology 48 (2002) 915–923 www.elsevier.com/locate/jinsphys
Review
Ion physiology of vitellogenic follicles William H. Telfer a,∗, Richard I. Woodruff b a
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA Department of Biology, West Chester University, West Chester, PA 19383-2130, USA
b
Received 30 April 2002; received in revised form 20 June 2002; accepted 20 June 2002
Abstract The ion physiology of vitellogenic follicles from a lepidopteran (Hyalophora cecropia) and a hemipteran (Rhodnius prolixus) are compared. Similarities that can be expected to occur in vitellogenic follicles of many other insects include: (1) gap junctions, which unite the cells of a follicle into an integrated electrical system, (2) transmembrane K+ and H+ gradients that account for over 60% of follicular membrane potentials, (3) absence of a Cl⫺ potential, (but the opening of channels to this anion when vitellogenesis terminates in H. cecropia), (4) an electrogenic proton pump that supplements follicular membrane potentials, (5) Ca2+ action potentials evoked by injecting depolarizing currents into oocytes, and (6) the use of osmotic pressure to control epithelial patency. Differences include: a Na+/K+-ATPase that accounts for about 20% of the follicular resting potential in R. prolixus but is absent from H. cecropia, and an intrafollicular Ca2+ current that moves from oocyte to nurse cells through cytoplasmic bridges in H. cecropia. Evidence is also summarized for two promising mechanisms that require further substantiation: (1) transmission via gap junctions of a follicle cell product that promotes endocytosis in the oocyte; and (2) transport of the proton pump back and forth between cell surface and endosomes as the membrane that carries it recycles through successive rounds of vitellogenin uptake. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Gap junction; Ion gradients; Osmotic pressure; Proton pump; Resting potential
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916
2.
Cell-to-cell coupling through gap junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917
3.
Follicular membrane potentials: the fraction due to ion gradients . . . . . . . . . . . . . . . . . . 917
4.
Follicular membrane potentials: the fraction due to a proton pump
5.
Proton pumps and the processing of endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918
6.
Ca2+ action potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919
7.
A Ca2+ pump in the trophic cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919
8.
Osmotic pressure and volume changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 920
9.
Future questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921
Corresponding author. Tel.: +1-215-898-7150; fax: +1-215-898-8780. E-mail addresses: [email protected] (W.H. Telfer); rwoodruff@ wcupa.edu (R.I. Woodruff). ∗
0022-1910/02/$ - see front matter. 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 2 ) 0 0 1 5 2 - X
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1. Introduction Insect follicles whose oocytes are depositing yolk have been characterized in depth over the last several decades by morphological and molecular methods (reviewed by Raikhel and Dhadialla, 1992; Raikhel and Snigirevskaya, 1998). Of concern here is a perspective that supplements this background with the findings of microelectrode technology (Fig. 1). While available information from other insects is also included, we focus on the ion physiology of follicles from the two insects that have been most intensively studied in this regard, Hyalophora cecropia and Rhodnius prolixus. These species are also of interest because they have evolved divergent adaptations. Nurse cells, whose products in both cases reach the vitellogenic oocyte through open cytoplasmic connectives, are present within each follicle in H. cecropia while being clustered in a distant, apical tropharium in R. prolixus. Ionic functions that support nurse cell activity are thus intrafollicular in one case and extrafollicular in the other. Perhaps more importantly, inorganic ion compositions of the hemolymph of the two species differ strikingly and this leads to fundamental differences in membrane physiology. Follicles are defined here as vitellogenic when their
Fig. 1. Photomicrograph of an early vitellogenic follicle of Hyalophora cecropia impaled by voltage-measuring capillary microelectrodes in the oocyte (OOC) and a nurse cell (NC), and one currentcarrying microelectrode, also in the oocyte. Opacity of the oocyte is due to accumulated yolk. The epithelium of follicle cells is columnar over the oocyte (oce) and squamous over the nurse cells (nce). The oocyte nucleus (the germinal vesicle, GV) appears as a clear, spherical area embedded in yolk. Scale bar=200 micrometers. (This figure is from Woodruff et al, 1992.)
oocytes are endocytosing and storing vitellogenin, a female-specific hemolymph protein that is synthesized by the fat body and reaches the oocyte through spaces between the follicle cells. Immunoelectron microscopy has confirmed in a lepidopteran that vitellogenin uptake initiates and terminates in synchrony with the opening and closing of these spaces (Zimowska et al., 1994, 1995). There are also convenient markers of vitellogenesis in living follicles. Initiation is correlated in H. cecropia with the onset of electrical coupling and membrane hyperpolarization (Woodruff and Telfer, 1990). And termination can be identified by the loss of ability to stain with vital dyes such as trypan blue that fill the intercellular spaces of vitellogenic follicles and are endocytosed by the oocyte (Telfer and Anderson, 1968; Anderson and Telfer, 1970) (Fig. 2). Between onset and termination, the number of vitellogenic follicles in an ovariole varies
Fig. 2. A chain of 24 vitellogenic follicles and 7 post-vitellogenic follicles dissected from one of the 8 ovarioles of Hyalophora cecropia. The chain had been stained for 2 minutes in 1% trypan blue and then washed in a dissecting solution. The dye is retained only by follicles with open intercellular spaces. Scale: the largest staining follicle is 2.0 mm long.
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from only one in R. prolixus to several dozen or more in many lepidopterans.
2. Cell-to-cell coupling through gap junctions Microelectrodes provide information about ion physiology in the follicle as a whole, but heterologous gap junctions are so numerous that they obscure the electrogenic contributions of individual cells (Wollberg et al., 1975; Woodruff, 1979). Coupling coefficients1 between oocytes and follicle cells are as high as 0.89 in Oncopeltus fasciatus, 0.94 in H. cecropia and 0.65 in Drosophila melanogaster (Adler and Woodruff, 2000). Coupling extends to adjacent follicles, with coefficients between oocytes in neighboring follicles being as high or higher than 0.40 (Woodruff, 1979). That coupling is mediated by gap junctions has been confirmed by electron microscopy of lanthanum-impregnated and freeze-fractured follicles in R. prolixus (Huebner, 1981). In H. cecropia the oocyte and follicle cells are separated during vitellogenesis by a several micrometer thick vitelline envelope, but fingers of ooplasm penetrate this layer, and terminate on the surface of adjacent follicle cells (Woodruff, 1979; Telfer et al., 1982). Inner leaflet to inner leaflet thickness of the apposed membranes is 10–20 nanometers, the correct dimension for gap junctions. An estimated several dozen fingers of ooplasm terminate on every follicle cell. Since the vitelline envelope does not extend to the cap of nurse cells, broader areas of contact can occur between these cells and the follicle cells and lanthanum permeation reveals extensive fields of gap junctions in this region (Woodruff et al., 1986). Coupling can also be monitored by cytoplasm to cytoplasm transfer of fluorescent dyes. When fluorescein (molecular weight, 376) was iontophoresed into vitellogenic oocytes in H. cecropia fluorescence appeared in the surrounding epithelial cells within 5 minutes, and within 30 minutes had reached the oocytes in adjacent follicles (Woodruff, 1979). In O. fasciatus both dye and electrical coupling were lost when follicles were exposed to 0.5 millimolar ethyl methanesulfonate, or a pH 5 incubation medium (Anderson and Woodruff, 2001). But dye and electrical coupling are not always equivalent, for in 1 millimolar octanol, a third traditional uncoupling agent,
1
Coupling coefficients were obtained by injecting a current pulse into an oocyte and simultaneously recording the voltage responses of the oocyte (⌬V1) and a follicle cell (⌬V2). The coupling coefficient is then ⌬V2/⌬V1. Coupling coefficients are affected by input resistances of the conjoined cells, as well as by gap junctional patency. They are used here as qualitative indices to the presence of gap junctions that are open to inorganic ions.
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only dye transfer was lost (Adler and Woodruff, 2000). The gap junctions of O. fasciatus follicles apparently remain partially open in octanol, so that inorganic ions can still travel from cell to cell. The possibility of semi-open gap junctions becomes especially important when coupling is studied at the onset of vitellognesis. In H. cecropia, coupling is restricted to vitellogenic follicles, previtellogenic follicles exhibiting neither electrical nor dye coupling (Woodruff and Telfer, 1990). In the largest previtellogenic follicles of O. fasciatus, fluorescent dyes injected into the oocyte were unable to diffuse into the follicle cells (Anderson and Woodruff, 2001). But these cells were nevertheless electrically coupled and gap junctions were therefore present and partially open. In Locusta migratoria also, electrical coupling was detected between previtellogenic follicles (Wollberg et al., 1975), but whether these follicles had also developed dye coupling has not been reported. The dye-coupling level of gap junctional patency is necessary for endocytosis in the oocyte in O. fasciatus (Anderson and Woodruff, 2001)—uncoupling agents, including octanol, blocked endocytosis in the oocyte. Inhibition appeared not to be a pharmacological effect on endocytosis per se, because injecting a soluble fraction of lysed epithelial cells into uncoupled oocytes promoted the resumption of endocytosis. Uncoupling also inhibited endocytosis in insects from five other orders, including Actias luna, D. melanogaster, Acheta domestica, Tenebrio molitor, and Xylocopa virginica (Waksmonski and Woodruff, 2002). The finding is consistent with an established literature on the transmission of physiological and developmental regulators across heterologous gap junctions (reviewed in Lo, 1999).
3. Follicular membrane potentials: the fraction due to ion gradients The resting potential (Em) of vitellogenic follicles is generated by trans-membrane ion gradients, supplemented by metabolically powered electrogenic pumps. In H. cecropia, ion gradients account for 65% of Em between oocyte and medium when the follicle is incubated in a standard medium adopted for this study (Woodruff et al., 1992). A similar figure, 69%, was obtained for vitellogenic follicles of R. prolixus (O’Donnell and Sharda, 1994). In both species K+ and H+ potentials were evident, but ion substitutions failed to detect potentials due to Ca2+, Mg2+ or, in most follicles, Cl⫺. Absence of a Cl⫺ conductance is of developmental interest because, as described below, increased permeation of this anion is required for the onset of an osmotically driven, post-vitellogenic growth phase in H. cecropia. K+ potentials also dominate Em in vitellogenic follicles
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of the cyclorrhaphan dipterans Sarcopohaga bullata (Verachtert et al., 1989) and D. melanogaster (Sun and Wyman, 1993). In the former, neither Ca2+, Mg2+, nor Na+ substitution affected Em, but replacement of Cl⫺ led to temporary depolarization, suggesting greater permeation by this anion than was detected in H. cecropia and R. prolixus.
and Wieczorek, 1997) that energizes co-transport of amino acids across membranes (Martin and Harvey, 1994). By contrast, in R. prolixus, which has a more conventional high Na+/low K+ hemolymph, about half of the energy-dependent component of follicular Em can be inhibited by ouabain (O’Donnell and Sharda, 1994).
5. Proton pumps and the processing of endosomes 4. Follicular membrane potentials: the fraction due to a proton pump The electrogenic fraction of Em in R. prolixus includes contributions from both Na+/K+- and H+-ATPases (O’Donnell and Sharda, 1994), while in H. cecropia only an H+-ATPase contribution has been detected. That a proton pump is involved in the latter was first seen in follicles that were initiating electrical coupling and, coincidentally, starting to produce yolk (Woodruff and Telfer, 1990). Em averaged ⫺21 millivolts in previtellogenic follicles, and was not affected by respiratory inhibition with 10 millimolar azide. Cytoplasmic pH was relatively unregulated, averaging close to the pH 6.5 of the incubation medium. But with the onset of coupling, cytoplasmic pH rose to 7.4 and Em began to rise to the 35–45 millivolt steady state levels characteristic of vitellogenesis. Azide reversed these effects, causing both Em and cytoplasmic pH to return to their previtellogenic levels. These indications were confirmed by studies on follicles at more advanced stages of vitellogenesis. Here also depolarization with 10 millimolar azide was tracked by a synchronous fall in cytoplasmic pH (Stynen et al., 1988). Furthermore, the active component was abolished by 150 nanomolar bafilomycin (Wang and Telfer, 1998), an inhibitor of vesicular-type H+-ATPase. By contrast, ouabain, an inhibitor of the Na+/K+ ATPase that energizes membranes in most animal cells did not affect Em. Two biological factors underlie the dependence of Em on a proton pump in H. cecropia. First, the follicles develop in situ in an unusual ionic environment (Hausman, 1970), with the high K+ and Mg2+, and low pH and Na+ characteristic of hemolymph in leaf-eating lepidopterans (Florkin and Jeuniaux, 1974). With hemolymph Na+ concentrations often around 1 millimolar or less, transmembrane gradients of this ion do not drive the co-transport mechanisms that carry hydrophilic nutrients and metabolites across cell membranes. Ouabain-resistant electrogenesis is well known in other lepidopteran tissues, including the midgut of Manduca sexta (Harvey and Wieczorek, 1997) and intersegmental muscle fibers in caterpillars of Chilo partellus (Dawson et al., 1989). In the midgut, membranes are energized by a vesicular type H+-ATPase located in the goblet cells, and protons are used to generate a K+ gradient (reviewed by Harvey
A second factor derives from the high rate of endocytosis in vitellogenic oocytes. In theory, therefore, it applies to vitellogenic follicles of all insects, without regard to the Na+/K+ composition of their hemolymph. A general model for selective endocytosis of proteins in animal cells includes the binding of ligands to cell surface receptors at the pH of blood, dissociation of binding after internalization, when coated vesicles are acidified, and the possibility that pumps, receptors and other membrane components required for endocytosis can be reused in subsequent rounds of ligand uptake (reviewed by Goldstein et al., 1985). Acidification is achieved by an inwardly directed, vesicular type H+-ATPase in the membrane of the coated vesicle. Several features of insect vitellogenesis are consistent with this model. Vitellogenin binding to receptors is inhibited by mildly acidic conditions in both biochemical assays (Koenig and Lanzrein, 1985; Rohrkasten and Ferenz, 1985; Osir and Law, 1986) and in whole follicle incubations (DiMario and Mahowald, 1986). In the oocyte cortex, electron micrographs reveal configurations consistent with receptor/ligand dissociation: interspersed among coated vesicles are endosomes containing a homogeneous matrix of yolk precursors instead of a lining of adsorbed ligands. Endosomes, rather than coated vesicles, are seen in membrane fusion configurations that allow the matrix material to be formed into yolk bodies (e.g., Roth and Porter, 1964; Stay, 1965; Anderson, 1969; reviewed by Raikhel and Dhadialla, 1992). Weak bases inhibit vitellogenesis in D. melanogaster (DiMario and Mahowald, 1986) and it was suggested that this is because they can neutralize protons as the latter are pumped into the coated vesicles. Vesicular acidification was seen more directly in H. cecropia when sucrose density gradient centrifugation was used to monitor the uptake of labeled vitellogenin (Stynen et al., 1988). Endocytosis transferred label within 10 minutes from the incubation medium to a population of intracellular vesicles (density 1.15), and in pulse/chase experiments this label was transferred within 30 minutes to heavier particles (density 1.20) that were presumed to be nascent yolk spheres. Nigericin and monensin, ionophores that exchange intravesicular protons for cytoplasmic K+, prevented the second transfer. Endocytosis was not inhibited, because label accumulated in density
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1.15 particles; only subsequent transfer to the heavier particles was blocked. Relevant to its potential secondary function as an electrogenic component of Em is the probability that H+ATPase cycles back and forth between endosomes and plasma membrane. Calculations based on surface/volume relations indicate that only 1% of endosomal membrane in H. cecropia is required to cover the yolk spheres (Telfer et al., 1982). In several insects immunoelectron microscopy has identified a reservoir of excess membrane that is potentially available for return to the oocyte surface (Van Antwerpen et al., 1993; Raikhel and Snigirevskaya, 1998). Assuming that H+ATPase is included in the recycling reservoir, then the question becomes whether it is reinserted into preformed coated vesicles, or into plasma membrane, along with vitellogenin receptors and clathrin. The latter route would explain the 20% of Em that is due to a proton pump in R. prolixus follicles (O’Donnell and Sharda, 1994). Although this blood-feeding hemipteran has a conventional high Na+/low K+ hemolymph, only half of the electrogenic component of follicular Em is sensitive to ouabain. The electrogenic proton pump was interpreted in this case in the context of pH regulation in the oocyte, but the possibility was pointed out that it cycles back and forth between endosomes and plasma membrane. A monoclonal antibody against the 20 kDa subunit of Manduca sexta H+-ATPase localized the proton pump at two sites (Janssen et al., 1995)—the surface of the oocyte, where endosomes are being acidified, and the outer (or basal) surface of the follicle cells. Both sites could, in principle, contribute to the active component of Em. In the case of R. prolixus with its two membrane ATPases, one wonders whether the proton pump will prove to be restricted to the vitellogenic surface of the oocyte. The Na+/K+ ATPase is known to be located in follicle cells, because ouabain binds to isolated follicle cell membranes (Ilenchuk and Davey, 1987). And it is also active in the oocyte, for extracellular ion currents measured with a vibrating probe identified Na+ currents exiting both apical and basal ends of vitellogenic follicles (Diehl-Jones and Huebner, 1992, 1993). These currents were inhibited by ouabain and were accordingly interpreted as generated by a Na+/K+ ATPase. They are produced by the follicle itself, because they persisted in late vitellogenesis, when the oocyte has separated from the trophic cord that connects it to the nurse cells. And they are produced at least in part by the oocyte, for they also persisted after experimental removal of follicle cells. 6. Ca2+ action potentials As noted, Ca2+ does not contribute measureably to follicular resting potentials in Sarcophaga bullata
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(Verachtert et al., 1989), H. cecropia (Woodruff et al., 1992), or R. prolixus (O’Donnell and Sharda, 1994). On the other hand, there are strong electrochemical gradients favoring seepage of Ca2+ into the cytoplasm—in the case of H. cecropia, an external 5,000 micromolar Ca2+ concentration as opposed to a measured 0.6 micromolar ooplasmic activity, and an Em of around ⫺40 millivolts. This gradient comes into play when a Ca2+ action potential is induced by a depolarizing current injected into the oocyte (O’Donnell, 1985; O’Donnell, 1988). Action potentials were demonstrated in vitellogenic follicles of H. cecropia and columbia, L. migratoria, Periplaneta americana, Tenebrio molitor, and R. prolixus, but could not be demonstrated in D. melanogaster. They were visible only when channel blockers were used to suppress the K+ potential that dominates the electrical properties of these follicles. They lasted several seconds longer than the 1 second pulse used to evoke them, and during this period Em rose to values of +10 to +20 millivolts. The action potential was inhibited by Ca2+ channel blockers, but not by Na+-free media and was thus attributed to voltage-gated Ca2+ channels. A function during vitellogenesis is not apparent but it was speculated that, as in many other animal eggs, controlled Ca2+ entry may at a later time be an activating step associated with fertilization.
7. A Ca2+ pump in the trophic cap Ca2+ extrusion can be inferred from the electrical properties of the cytoplasmic bridges that connect the oocyte of H. cecropia to its intrafollicular cap of seven nurse cells. A focused, 2–5 millivolt steady state potential is maintained across these connections (Woodruff and Telfer, 1973, 1974). The transbridge potential arises at the onset of vitellogenesis when the hyperpolarization characteristic of that transition affects the nurse cells more strongly than the oocyte (Woodruff and Telfer, 1990). It persists for over four days, until the bridges and nurse cells disintegrate one day prior to the end of vitellogenesis. Ion selective microelectrodes showed that Ca2+ activity is three times higher in the less electronegatively charged oocyte than in the nurse cells (0.65 versus 0.18 micromolar). By contrast, cytoplasmic activities of K+, Mg2+ and Cl⫺ differed between oocyte and nurse cells by less than 5%, with the two cations having slightly higher activities in the more negative nurse cells while the anion was higher in the oocyte (Woodruff and Telfer, 1994). Cytoplasmic pH did not detectably differ across the bridges. That the transbridge potential is powered by Ca2+ extrusion from the nurse cells was confirmed when follicles were incubated in 10 millimolar azide. The transbridge potential then disappeared and Ca2+ activities
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equalized, rising in both oocyte and nurse cells to about 1.5 micromolar levels. The extracellular return pathway of the Ca2+ current is, in principle, guided by the hemisphere of follicle cells that cover the nurse cell cap (Fig. 1, nce). Unlike the follicle cells surrounding the oocyte, these cells form an electrically tight epithelium (Woodruff et al., 1986), and the escape route of any ions pumped out of the nurse cells would necessarily be beneath the epithelium and toward the oocyte. This model was supported by measurements with a vibrating probe, which detected a strong exit current from the nurse cells wherever patches of the epithelial barrier had been experimentally removed. Completion of the circuit would require leakage of Ca2+ through plasma membranes into the oocyte and/or its follicle cells. The gradient between oocyte and nurse cells is tightly focused in the 20–30 micrometer long cytoplasmic bridges, and is of sufficient magnitude over that short distance to affect the diffusion of proteins between the two cells (Woodruff and Telfer, 1974, 1980). Fluorescein-labeled proteins with a net positive charge crossed the channels into the nurse cells after injection into the oocyte, but did not as readily move in the reverse direction. The same protein, modified by methylcarboxylatin to yield a net negative charge, behaved in the opposite manner. Differences in charge-based mobility should thus generate differences in the cytoplasmic protein composition of the oocyte and its nurse cells. This has been confirmed with isoelectric focusing of soluble proteins in A. luna (Cole and Woodruff, 1997) and D. melanogaster (Cole and Woodruff, 2000). Differences in microinjected protein movements based on net electrical charge have also been seen in extrafollicular nurse tissue, the tropharium, of R. prolixus (Huebner, 1984) and O. fasciatus (Woodruff and Anderson, 1984). In both insects, negatively charged fluorescent proteins injected into the tropharium diffused throughout its syncytial core and into the beginnings of the cytoplasmic chords that attach it to each oocyte. Basic proteins injected at the same site migrated instead peripherally into nucleated pockets of cytoplasm that radiate outward from the core. In O. fasciatus the most peripheral trophocytes were found to be 8 millivolts electronegataive to the core. For R. prolixus differences in electrical potential between the outpocketings and the core have not been reported, but the contrasting directions of acidic and basic protein migration are consistent with the nucleated pockets being electronegative to the core here also. Whatever the mechanism of retention, the oocyte, as in species with intrafollicular nurse tissues, must have suppressed levels of soluble basic proteins. Whether the nurse cells are intrafollicular or extrafollicular, transcriptional activity in the oocyte nucleus, as revealed by the incorporation of tritiated uridine, is generally not detectable during vitellogenesis (reviewed in
Telfer, 1975). In H. cecropia, suppression begins with the onset of vitellogenesis, when the transbridge potential is first detected (Woodruff and Telfer, 1990). When the intercellular bridges were experimentally depolarized by abolishing the Ca2+ gradient, uridine incorporation resumed in the oocyte. Three different treatments had this effect—a calcium buffer, a calcium ionophore, and vanadate ions, which inhibit calcium extrusion by the nurse cells (Woodruff et al., 1998).
8. Osmotic pressure and volume changes K+ channels in H. cecropia follicles are osmotically gated. They are maximally open at the osmotic pressure of hemolymph but they close in more dilute solutions, and Em becomes unresponsive to changes in external K+ concentrations (Woodruff et al., 1992). Another example of sensitivity to osmotic pressure was seen in D. melanogaster, in which hyperosmotic media caused a 3–4 mV hyperpolarization of the oocyte, without affecting the nurse cells (Singleton and Woodruff, 1994). When predicting in situ properties from the ion physiology of follicles incubated in vitro, therefore, it is important to work at the osmotic pressure of the hemolymph in which they form yolk. Two cases have been analyzed in which vitellogenesis is affected by changes in follicle cell volume. In R. prolixus the promotion of vitellogenesis by juvenile hormone is mediated by the ability of this hormone to control epithelial patency. Patency is lost when vitellogenic follicles are incubated in the absence of juvenile hormone, but is maintained during incubation in the presence of this hormone. The response to juvenile hormone entails a 50% reduction in follicle cell volume (AbuHakima and Davey, 1977), is inhibited by ouabain (AbuHakima and Davey, 1979), and is preceded by a juvenile hormone-dependent increase in ouabain binding by follicle cell membranes in late previtellogenic follicles (Ilenchuk and Davey, 1987). The juvenile hormone effect is mediated by calcium-dependent protein kinase (PKC), which, in the presence of juvenile hormone, phosphorylates a polypeptide resembling in size the 100 kDa subunit of Na+/K+-ATPase (Sevala and Davey, 1993). A second case involves cyclic AMP-dependent protein kinase (PKA) rather than PKC, the promotion of follicle cell swelling rather than shrinking, and the termination of vitellogenesis rather than its maintenance. When lepidopteran follicles reach a species-specific length—e.g., 2 mm in H. cecropia (Telfer and Anderson, 1968) and 1.8 mm in M. sexta (Nijhout and Riddiford, 1974)—vitellogenesis ends and a period of water uptake ensues. H. cecropia follicles increase their volume during this period by 50%, and assume the slightly flattened ellipsoid shape of a mature egg. Isotonicity of isolated
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yolk spheres changes from 0.6 to 0.9 osmolar, clear evidence that swelling and shape change are driven by the turgor pressure of solute uptake. Ion substitution studies indicated that Cl⫺ and K+ are required as osmolytes (Wang and Telfer, 1998). The epithelial phase of osmotic swelling can be triggered by pharmcological activation of PKA (Wang and Telfer, 1996, 1997). Among the strategies that can be used to increase PKA activity, the one employed in the osmotic swelling study was incubation in Sp-cAMPS, an analog of cyclic AMP that is cell-permeant and resistant to hydrolysis by phosphodiesterase (Dostmann et al., 1990); in a concentration of 1 mM the follicle cells began to swell within 30 minutes. Also within 30 minutes two changes in ion physiology were detected—onset of a Cl⫺ conductance and a transitory 30% hyperpolarization (Wang and Telfer, 1998). Opening Cl⫺ channels should have a depolarizing effect on follicle membranes (Woodruff et al., 1992), so hyperpolarization was due to other factors. A drop in cytoplasmic pH from 7.26 to 7.06 suggested that an increase in substrate availability for the electrogenic H+-ATPase contributed to the change in Em (Wang and Telfer, 1998). A Cl⫺ channel blocker, anthracene-9-carboxylic acid, prevented the increase in conductance and also prevented osmotic swelling and hyperpolarization (Wang and Telfer, 1998). The follicle cells contain 80% of total follicular PKA (Wang and Telfer, 2000), and it is therefore likely that they are the primary site of a cascade of events leading from PKA activation through Cl⫺ channel opening and finally to epithelial swelling.
9. Future questions The power of comparative studies is made evident in this review by the similarities and differences between a lepidopteran and a heteropteran. Other orders need to be investigated, as well as families with other life styles within the two orders represented here. H. cecropia is among the lepidopterans that make yolk as pharate adults and may therefore have fundamental differences in ion physiology from species in which vitellogenesis is triggered by juvenile hormone in post-eclosion, nectar-feeding moths and butterflies. And R. prolixus, with its hemolymph rich in Na+ derived from mammalian blood meals, may well differ fundamentally from heteropterans with vegetarian diets that are poor in Na+. Examples of questions raised here that might yield to a comparative approach include why selective concentrating of vitellogenin in yolk seems to be associated with a low Cl⫺ conductance, with plasma membrane energization by a protein pump, and with gap junctions sufficiently patent to transmit intermediate-sized solutes from follicle cells to the oocyte.
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We can also hope that developmental studies will add to an understanding of follicular physiology by expanding the list of gains and losses in membrane functions at the onset and termination of vitellogenesis. And in the longer run, how do internal, programmatic cues (Swevers and Iatrou, 1992), or hormonal stimulation (Ilenchuk and Davey, 1987) evoke these gains and losses? References Abu-Hakima, R., Davey, K.G., 1977. The action of juvenile hormone on the follicle cells of Rhodnius prolixus: the importance of volume changes. Journal of Experimental Biology 69, 33–44. Abu-Hakima, R., 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 Biochemistry 9, 195–198. Adler, E.L., Woodruff, R.I., 2000. Varied effects of 1-octanol on gap junctional communication between ovarian epithelial cells and oocytes in Oncopeltus fasciatus and Drosophila melanogaster. Archives of Insect Biochemistry and Physiology 43, 22–32. Anderson, E., 1969. Oogenesis in the cockroach, Periplaneta americana, with special reference to the specializations of the oolemma and the fate of coated vesicles. Journal de Microscopie 8, 721–738. Anderson, K., Woodruff, R.I., 2001. A gap junctionally transmitted epithelial cell signal regulates endocytotic yolk uptake in Oncopeltus faciatus. Developmental Biology 239, 68–78. Anderson, L.M., Telfer, W.H., 1970. Trypan blue inhibition of yolk deposition—a clue to follicle cell function in the cecropia moth. Journal of Embryology and Experimental Morphology 23, 35–52. Cole, R.W., Woodruff, R.I., 1997. Charge dependent distribution of endogenous proteins within ovarian follicles of Actias luna. Journal of Insect Physiology 43, 275–287. Cole, R.W., Woodruff, R.I., 2000. Vitellogenic ovarian follicles of Drosophila exhibit a charge-dependent distribution of endogenous soluble proteins. Journal of Insect Physiology 46, 1239–1248. Dawson, J., Djamgoz, M.B.A., Hardie, J., Irving, S.N., 1989. Components of resting membrane electrogenesis in lepidopteran skeletal muscle. Journal of Insect Physiology 35, 659–666. Diehl-Jones, W., Huebner, E., 1992. Spacial and temporal transcellular current patterns during oogenesis. Developmental Biology 153, 302–311. Diehl-Jones, W., Huebner, E., 1993. Ionic basis of bioelectric currents during oogenesis in an insect. Developmental Biology 158, 301– 316. DiMario, P.J., Mahowald, A.P., 1986. The effects of pH and weak bases on the in vitro endocytosis of vitellogenin by oocytes of Drosophila melanogaster. Tissue and Cell Research 246, 103–108. Dostmann, W.R., Taylor, S.S., Genieser, H.-G., Jastorff, B., Doskeland, S.O., Ogreid, D., 1990. Probing the cyclic nucleotide binding sites of cAMP-dependent protein kinases I and II with analogs of adenosine 3⬘:, 5⬙-cyclic phosphorothioates. Journal of Biological Chemistry 265, 10484–10491. Florkin, M., Jeuniaux, C., 1974. Hemolymph: Composition. In: Rockstein, M. (Ed.), Insect Physiology. Academic Press, New York, London, pp. 256–305. Goldstein, J.L., Brown, M.S., Anderson, R.G.W., Russell, D.W., Schneider, W.J., 1985. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annual Review of Cell Biology 1, 1–39. Harvey, W.R., Wieczorek, H., 1997. Animal plasma membrane energization by chemiosmotic H+ V-ATPase. Journal of Experimental Biology 200, 203–216.
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Hausman, S.J. (1970). Ionic Homeostasis in the saturniid moth, Hyalophora cecropia (L). Masters Thesis in Zoology, University of Pennsylvania, Philadelphia, PA. Huebner, E., 1981. Oocyte–follicle cell interaction during normal oogenesis and atresia in an insect. Journal of Ultrastructure Research 74, 95–104. Huebner, E., 1984. Developmental cell interactions in female reproductive organs. Advances in Invertebrate Reproduction 3, 97–105. Ilenchuk, T.T., Davey, K.G., 1987. The development of responsiveness to juvenile hormone in the follicle cells of Rhodnius prolixus. Insect Biochemistry 17, 525–529. Janssen, I., Hendrickx, K., Klein, U., DeLoof, A., 1995. Immunolocalization of a proton V-ATPase in ovarian follicles of the tobacco hornworm Manduca sexta. Archives of Insect Biochemistry and Physiology 28, 131–141. Koenig, K.R., Lanzrein, B., 1985. Binding of vitellogenin receptors in oocyte membrane preparations of the ovoviviparous cockroach, Naupheta cinerea. Insect Biochemistry 15, 735–747. Lo, W.C., 1999. Genes, gene knockouts, and mutations in the analysis of gap junctions. Developmental genetics 24, 1–4. Martin, F.G., Harvey, W.R., 1994. Ionic circuit analysis of K+/H+ antiport and amino acid/K+ symport energized by as proton motive force in Manduca sexta larval midgut vesicles. Journal of Experimental Biology 196, 77–92. Nijhout, M.M., Riddiford, L.M., 1974. The control of egg maturation by juvenile hormone in the tobacco hornworm moth, Manduca sexta. Biological Bulletin 146, 377–392. O’Donnell, M.J., 1985. Calcium action potentials in developing oocytes of an insect, Rhodnius prolixus. Journal of Experimental Biology 119, 287–300. O’Donnell, M.J., 1988. Potassium channel blockers unmask electrical excitability in insect follicles. Journal of Experimental Zoology 245, 137–143. O’Donnell, M.J., Sharda, R.C., 1994. Membrane potential and pH regulation in vitellogenic oocytes of an insect, Rhodnius prolixus. Physiological Zoology 67, 7–28. Osir, E.O., Law, J.H., 1986. Specific binding and uptake of 125 Ilabelled vitellogenin by follicles of the tobacco hornworm, Manduca sexta. Archives of Insect Biochemistry and Physiology 3, 513–528. Raikhel, A.S., Dhadialla, T.S., 1992. Accumulation of yolk proteins in insect oocytes. Annual Review of Entomology 37, 217–251. Raikhel, A.S., Snigirevskaya, E.S. (Eds.), 1998. Vitellogenesis. Insecta, Microscopic Anatomy of Invertebrates, 11C. Wiley-Liss, pp. 933–955. Rohrkasten, A., Ferenz, H.-J., 1985. In vitro study of selective endocytosis of vitellogenin by locust oocytes. Roux’s Archives of Developmental Biology 194, 411–416. Roth, T.F., Porter, K.R., 1964. Yolk protein uptake in the oocyte of the mosquito Aedes aegypti. Journal of Cell Biology 20, 313–332. Sevala, V.L., Davey, K.G., 1993. Juvenile hormone dependent phosphorylation of a 100 kDa polypeptide is mediated by protein kinase C in the follicle cells of Rhodnius prolixus. Invertebrate Reproduction and Development 23, 1898–1903. Singleton, K., Woodruff, R.I., 1994. The osmolarity of adult Drosophila hemolymph and its effect on oocyte-nurse cell electrical polarity. Developmental Biology 161, 279–288. Stay, B., 1965. Protein uptake in the oocytes of the cecropia moth. Journal of Cell Biology 26, 49–62. Swevers, L., Iatrou, K., 1992. Early establishment of autonomous implementation of a developmental program controlling silkmoth chorion gene expression. Developmental Biology 150, 12–22. Stynen, D., Woodruff, R.I., Telfer, W.H., 1988. Effects of ionophores on vitellogenin uptake by Hyalophora oocytes. Archives of Insect Biochemistry and Physiology 8, 261–276. Sun, Y.A., Wyman, R.J., 1993. Lack of an oocyte to nurse cell voltage difference in Drosophila. Developmental Biology 155, 206–215.
Telfer, W.H., 1975. Development and physiology of the oocyte-nurse cell syncytium. Advances in Insect Physiology 11, 223–319. Telfer, W.H., Anderson, L.M., 1968. Functional transformations accompanying the inition of a terminal growth phase in the cecropia moth oocyte. Developmental Biology 17, 512–535. Telfer, W.H., Huebner, E., Smith, D.S., 1982. The cell biology of vitellogenic follicles in Hyalophora and Rhodnius. In: King, R.C., Akai, H. (Eds.), Insect Ultrastructure. Plenum, New York, pp. 118–149. Van Antwerpen, R., Conway, R., Law, J.H., 1993. Protein and lipoprotein uptake by ceveloping oocytes of the hawkmoth Manduca sexta. An ultrastructural and immunocytochemical study. Tissue and Cell 25, 205–218. Verachtert, B., Amelinckx, M., De Loof, A., 1989. Potassium and chloride dependence of the membrane potential of vitellogenic follicles of Sarcophaga bullata (Dioptera). Journal of Insect Physiology 35, 143–148. Waksmonski, S.L. and Woodruff, R.I., 2002. For uptake of yolk precursor, epithelial cell–oocyte gap junctional communication is required by insects representing six different orders. Journal of Insect Physiology, in press. Wang, Y., Telfer, W.H., 1996. Cyclic nucleotide-induced termination of vitellogenin uptake by Hyalophora cecropia follicles. Insect Biochemistry and Molecular Biology 26, 85–94. Wang, Y., Telfer, W.H., 1997. cAMP-stimulated termination of vitellogenesis in Hyalophora cecropia: formation of a diffusion barrier and the loss of patency. Journal of Insect Physiology 43, 675–684. Wang, Y., Telfer, W.H., 1998. cAMP-induced water uptake in a moth ovary: inhibition by bafilomycin and anthracene-9-carboxylic acid. Journal of Experimental Biology 201, 1627–1635. Wang, Y., Telfer, W.H., 2000. Cyclic nucleotide-dependent protein phosphorylation in vitellogenic follicles of Hyalophora cecropia. Insect Biochemistry and Molecular Biology 30, 29–34. Wollberg, Z., Cohen, E., Kalina, M., 1975. Electrical properties of developing oocytes of the migratory locust, Locusta migratoria. Journal of Cellular Physiology 88, 145–158. Woodruff, R.I., 1979. Electrotonic junctions in cecropia moth ovaries. Developmental Biology 69, 281–295. Woodruff, R.I., Anderson, K.L., 1984. Nutritive cord connection and dye-coupling of the follicular epithelium to the growing oocytes in the telotrophic ovarioles in Oncopeltus fasciatus, the milkweed bug. Roux’s Archives of Developmental Biology 193, 158–163. Woodruff, R.I., Telfer, W.H., 1973. Polarized intercellular bridges in ovarian follicles of the cecropia moth. Journal of Cell Biology 58, 172–188. Woodruff, R.I., Telfer, W.H., 1974. Electrical properties of ovarian cells linked by intercellular bridges. Annals of the New York Academy of Science 238, 408–419. Woodruff, R.I., Telfer, W.H., 1980. Electrophoresis of proteins in intercellular bridges. Nature (London) 286, 84–86. Woodruff, R.I., Telfer, W.H., 1990. Activation of a new physiological state at the onset of vitellogenesis in Hyalophora follicles. Developmental Biology 138, 410–420. Woodruff, R.I., Telfer, W.H., 1994. Steady-state gradient in calcium ion activity across the intercellular bridges connecting oocytes and nurse cells in Hyalophora cecropia. Archives of Insect Biochemistry and Physiology 25, 9–20. Woodruff, R., Huebner, E., Telfer, W., 1986. Ion currents in Hyalophora ovaries: the role of the epithelium and the intercellular spaces of the trophic cap. Developmental Biology 117, 405–416. Woodruff, R.I., Munz, A., Telfer, W.H., 1992. Steady-state potentials in ovarian follicles of a moth, Hyalophora cecropia. Journal of Insect Physiology 38, 49–60. Woodruff, R.I., Dittmann, F., Telfer, W.H., 1998. Ca2+ current from oocyte to nurse cells and suppression of uridine incorporation in the germinal vesicle of Hyalophora cecropia. Invertebrate Reproduction and Development 34, 157–164. Zimowska, G., Shirk, P.D., Silhacek, D.L., Shaaya, E., 1994. Yolk
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sphere formation is initiated in oocytes before development of patency in follicles of the moth Plodia interpunctella. Wilhelm Roux’s Archives of Developmental Biology 203, 215–226. Zimowska, G., Shirk, P.D., Silhacek, D.L., Shaaya, E., 1995. Termin-
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ation of vitellogenesis in follicles of the moth, Plodia interpunctella: changes in oocyte and follicular epithelial cell activities. Archives of Insect Biochemisry and Physiology 29, 357–380.
Review
Ion physiology of vitellogenic follicles William H. Telfer a,∗, Richard I. Woodruff b a
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA Department of Biology, West Chester University, West Chester, PA 19383-2130, USA
b
Received 30 April 2002; received in revised form 20 June 2002; accepted 20 June 2002
Abstract The ion physiology of vitellogenic follicles from a lepidopteran (Hyalophora cecropia) and a hemipteran (Rhodnius prolixus) are compared. Similarities that can be expected to occur in vitellogenic follicles of many other insects include: (1) gap junctions, which unite the cells of a follicle into an integrated electrical system, (2) transmembrane K+ and H+ gradients that account for over 60% of follicular membrane potentials, (3) absence of a Cl⫺ potential, (but the opening of channels to this anion when vitellogenesis terminates in H. cecropia), (4) an electrogenic proton pump that supplements follicular membrane potentials, (5) Ca2+ action potentials evoked by injecting depolarizing currents into oocytes, and (6) the use of osmotic pressure to control epithelial patency. Differences include: a Na+/K+-ATPase that accounts for about 20% of the follicular resting potential in R. prolixus but is absent from H. cecropia, and an intrafollicular Ca2+ current that moves from oocyte to nurse cells through cytoplasmic bridges in H. cecropia. Evidence is also summarized for two promising mechanisms that require further substantiation: (1) transmission via gap junctions of a follicle cell product that promotes endocytosis in the oocyte; and (2) transport of the proton pump back and forth between cell surface and endosomes as the membrane that carries it recycles through successive rounds of vitellogenin uptake. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Gap junction; Ion gradients; Osmotic pressure; Proton pump; Resting potential
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916
2.
Cell-to-cell coupling through gap junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917
3.
Follicular membrane potentials: the fraction due to ion gradients . . . . . . . . . . . . . . . . . . 917
4.
Follicular membrane potentials: the fraction due to a proton pump
5.
Proton pumps and the processing of endosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918
6.
Ca2+ action potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919
7.
A Ca2+ pump in the trophic cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919
8.
Osmotic pressure and volume changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 920
9.
Future questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921
Corresponding author. Tel.: +1-215-898-7150; fax: +1-215-898-8780. E-mail addresses: [email protected] (W.H. Telfer); rwoodruff@ wcupa.edu (R.I. Woodruff). ∗
0022-1910/02/$ - see front matter. 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 2 ) 0 0 1 5 2 - X
. . . . . . . . . . . . . . . . 918
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1. Introduction Insect follicles whose oocytes are depositing yolk have been characterized in depth over the last several decades by morphological and molecular methods (reviewed by Raikhel and Dhadialla, 1992; Raikhel and Snigirevskaya, 1998). Of concern here is a perspective that supplements this background with the findings of microelectrode technology (Fig. 1). While available information from other insects is also included, we focus on the ion physiology of follicles from the two insects that have been most intensively studied in this regard, Hyalophora cecropia and Rhodnius prolixus. These species are also of interest because they have evolved divergent adaptations. Nurse cells, whose products in both cases reach the vitellogenic oocyte through open cytoplasmic connectives, are present within each follicle in H. cecropia while being clustered in a distant, apical tropharium in R. prolixus. Ionic functions that support nurse cell activity are thus intrafollicular in one case and extrafollicular in the other. Perhaps more importantly, inorganic ion compositions of the hemolymph of the two species differ strikingly and this leads to fundamental differences in membrane physiology. Follicles are defined here as vitellogenic when their
Fig. 1. Photomicrograph of an early vitellogenic follicle of Hyalophora cecropia impaled by voltage-measuring capillary microelectrodes in the oocyte (OOC) and a nurse cell (NC), and one currentcarrying microelectrode, also in the oocyte. Opacity of the oocyte is due to accumulated yolk. The epithelium of follicle cells is columnar over the oocyte (oce) and squamous over the nurse cells (nce). The oocyte nucleus (the germinal vesicle, GV) appears as a clear, spherical area embedded in yolk. Scale bar=200 micrometers. (This figure is from Woodruff et al, 1992.)
oocytes are endocytosing and storing vitellogenin, a female-specific hemolymph protein that is synthesized by the fat body and reaches the oocyte through spaces between the follicle cells. Immunoelectron microscopy has confirmed in a lepidopteran that vitellogenin uptake initiates and terminates in synchrony with the opening and closing of these spaces (Zimowska et al., 1994, 1995). There are also convenient markers of vitellogenesis in living follicles. Initiation is correlated in H. cecropia with the onset of electrical coupling and membrane hyperpolarization (Woodruff and Telfer, 1990). And termination can be identified by the loss of ability to stain with vital dyes such as trypan blue that fill the intercellular spaces of vitellogenic follicles and are endocytosed by the oocyte (Telfer and Anderson, 1968; Anderson and Telfer, 1970) (Fig. 2). Between onset and termination, the number of vitellogenic follicles in an ovariole varies
Fig. 2. A chain of 24 vitellogenic follicles and 7 post-vitellogenic follicles dissected from one of the 8 ovarioles of Hyalophora cecropia. The chain had been stained for 2 minutes in 1% trypan blue and then washed in a dissecting solution. The dye is retained only by follicles with open intercellular spaces. Scale: the largest staining follicle is 2.0 mm long.
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from only one in R. prolixus to several dozen or more in many lepidopterans.
2. Cell-to-cell coupling through gap junctions Microelectrodes provide information about ion physiology in the follicle as a whole, but heterologous gap junctions are so numerous that they obscure the electrogenic contributions of individual cells (Wollberg et al., 1975; Woodruff, 1979). Coupling coefficients1 between oocytes and follicle cells are as high as 0.89 in Oncopeltus fasciatus, 0.94 in H. cecropia and 0.65 in Drosophila melanogaster (Adler and Woodruff, 2000). Coupling extends to adjacent follicles, with coefficients between oocytes in neighboring follicles being as high or higher than 0.40 (Woodruff, 1979). That coupling is mediated by gap junctions has been confirmed by electron microscopy of lanthanum-impregnated and freeze-fractured follicles in R. prolixus (Huebner, 1981). In H. cecropia the oocyte and follicle cells are separated during vitellogenesis by a several micrometer thick vitelline envelope, but fingers of ooplasm penetrate this layer, and terminate on the surface of adjacent follicle cells (Woodruff, 1979; Telfer et al., 1982). Inner leaflet to inner leaflet thickness of the apposed membranes is 10–20 nanometers, the correct dimension for gap junctions. An estimated several dozen fingers of ooplasm terminate on every follicle cell. Since the vitelline envelope does not extend to the cap of nurse cells, broader areas of contact can occur between these cells and the follicle cells and lanthanum permeation reveals extensive fields of gap junctions in this region (Woodruff et al., 1986). Coupling can also be monitored by cytoplasm to cytoplasm transfer of fluorescent dyes. When fluorescein (molecular weight, 376) was iontophoresed into vitellogenic oocytes in H. cecropia fluorescence appeared in the surrounding epithelial cells within 5 minutes, and within 30 minutes had reached the oocytes in adjacent follicles (Woodruff, 1979). In O. fasciatus both dye and electrical coupling were lost when follicles were exposed to 0.5 millimolar ethyl methanesulfonate, or a pH 5 incubation medium (Anderson and Woodruff, 2001). But dye and electrical coupling are not always equivalent, for in 1 millimolar octanol, a third traditional uncoupling agent,
1
Coupling coefficients were obtained by injecting a current pulse into an oocyte and simultaneously recording the voltage responses of the oocyte (⌬V1) and a follicle cell (⌬V2). The coupling coefficient is then ⌬V2/⌬V1. Coupling coefficients are affected by input resistances of the conjoined cells, as well as by gap junctional patency. They are used here as qualitative indices to the presence of gap junctions that are open to inorganic ions.
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only dye transfer was lost (Adler and Woodruff, 2000). The gap junctions of O. fasciatus follicles apparently remain partially open in octanol, so that inorganic ions can still travel from cell to cell. The possibility of semi-open gap junctions becomes especially important when coupling is studied at the onset of vitellognesis. In H. cecropia, coupling is restricted to vitellogenic follicles, previtellogenic follicles exhibiting neither electrical nor dye coupling (Woodruff and Telfer, 1990). In the largest previtellogenic follicles of O. fasciatus, fluorescent dyes injected into the oocyte were unable to diffuse into the follicle cells (Anderson and Woodruff, 2001). But these cells were nevertheless electrically coupled and gap junctions were therefore present and partially open. In Locusta migratoria also, electrical coupling was detected between previtellogenic follicles (Wollberg et al., 1975), but whether these follicles had also developed dye coupling has not been reported. The dye-coupling level of gap junctional patency is necessary for endocytosis in the oocyte in O. fasciatus (Anderson and Woodruff, 2001)—uncoupling agents, including octanol, blocked endocytosis in the oocyte. Inhibition appeared not to be a pharmacological effect on endocytosis per se, because injecting a soluble fraction of lysed epithelial cells into uncoupled oocytes promoted the resumption of endocytosis. Uncoupling also inhibited endocytosis in insects from five other orders, including Actias luna, D. melanogaster, Acheta domestica, Tenebrio molitor, and Xylocopa virginica (Waksmonski and Woodruff, 2002). The finding is consistent with an established literature on the transmission of physiological and developmental regulators across heterologous gap junctions (reviewed in Lo, 1999).
3. Follicular membrane potentials: the fraction due to ion gradients The resting potential (Em) of vitellogenic follicles is generated by trans-membrane ion gradients, supplemented by metabolically powered electrogenic pumps. In H. cecropia, ion gradients account for 65% of Em between oocyte and medium when the follicle is incubated in a standard medium adopted for this study (Woodruff et al., 1992). A similar figure, 69%, was obtained for vitellogenic follicles of R. prolixus (O’Donnell and Sharda, 1994). In both species K+ and H+ potentials were evident, but ion substitutions failed to detect potentials due to Ca2+, Mg2+ or, in most follicles, Cl⫺. Absence of a Cl⫺ conductance is of developmental interest because, as described below, increased permeation of this anion is required for the onset of an osmotically driven, post-vitellogenic growth phase in H. cecropia. K+ potentials also dominate Em in vitellogenic follicles
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of the cyclorrhaphan dipterans Sarcopohaga bullata (Verachtert et al., 1989) and D. melanogaster (Sun and Wyman, 1993). In the former, neither Ca2+, Mg2+, nor Na+ substitution affected Em, but replacement of Cl⫺ led to temporary depolarization, suggesting greater permeation by this anion than was detected in H. cecropia and R. prolixus.
and Wieczorek, 1997) that energizes co-transport of amino acids across membranes (Martin and Harvey, 1994). By contrast, in R. prolixus, which has a more conventional high Na+/low K+ hemolymph, about half of the energy-dependent component of follicular Em can be inhibited by ouabain (O’Donnell and Sharda, 1994).
5. Proton pumps and the processing of endosomes 4. Follicular membrane potentials: the fraction due to a proton pump The electrogenic fraction of Em in R. prolixus includes contributions from both Na+/K+- and H+-ATPases (O’Donnell and Sharda, 1994), while in H. cecropia only an H+-ATPase contribution has been detected. That a proton pump is involved in the latter was first seen in follicles that were initiating electrical coupling and, coincidentally, starting to produce yolk (Woodruff and Telfer, 1990). Em averaged ⫺21 millivolts in previtellogenic follicles, and was not affected by respiratory inhibition with 10 millimolar azide. Cytoplasmic pH was relatively unregulated, averaging close to the pH 6.5 of the incubation medium. But with the onset of coupling, cytoplasmic pH rose to 7.4 and Em began to rise to the 35–45 millivolt steady state levels characteristic of vitellogenesis. Azide reversed these effects, causing both Em and cytoplasmic pH to return to their previtellogenic levels. These indications were confirmed by studies on follicles at more advanced stages of vitellogenesis. Here also depolarization with 10 millimolar azide was tracked by a synchronous fall in cytoplasmic pH (Stynen et al., 1988). Furthermore, the active component was abolished by 150 nanomolar bafilomycin (Wang and Telfer, 1998), an inhibitor of vesicular-type H+-ATPase. By contrast, ouabain, an inhibitor of the Na+/K+ ATPase that energizes membranes in most animal cells did not affect Em. Two biological factors underlie the dependence of Em on a proton pump in H. cecropia. First, the follicles develop in situ in an unusual ionic environment (Hausman, 1970), with the high K+ and Mg2+, and low pH and Na+ characteristic of hemolymph in leaf-eating lepidopterans (Florkin and Jeuniaux, 1974). With hemolymph Na+ concentrations often around 1 millimolar or less, transmembrane gradients of this ion do not drive the co-transport mechanisms that carry hydrophilic nutrients and metabolites across cell membranes. Ouabain-resistant electrogenesis is well known in other lepidopteran tissues, including the midgut of Manduca sexta (Harvey and Wieczorek, 1997) and intersegmental muscle fibers in caterpillars of Chilo partellus (Dawson et al., 1989). In the midgut, membranes are energized by a vesicular type H+-ATPase located in the goblet cells, and protons are used to generate a K+ gradient (reviewed by Harvey
A second factor derives from the high rate of endocytosis in vitellogenic oocytes. In theory, therefore, it applies to vitellogenic follicles of all insects, without regard to the Na+/K+ composition of their hemolymph. A general model for selective endocytosis of proteins in animal cells includes the binding of ligands to cell surface receptors at the pH of blood, dissociation of binding after internalization, when coated vesicles are acidified, and the possibility that pumps, receptors and other membrane components required for endocytosis can be reused in subsequent rounds of ligand uptake (reviewed by Goldstein et al., 1985). Acidification is achieved by an inwardly directed, vesicular type H+-ATPase in the membrane of the coated vesicle. Several features of insect vitellogenesis are consistent with this model. Vitellogenin binding to receptors is inhibited by mildly acidic conditions in both biochemical assays (Koenig and Lanzrein, 1985; Rohrkasten and Ferenz, 1985; Osir and Law, 1986) and in whole follicle incubations (DiMario and Mahowald, 1986). In the oocyte cortex, electron micrographs reveal configurations consistent with receptor/ligand dissociation: interspersed among coated vesicles are endosomes containing a homogeneous matrix of yolk precursors instead of a lining of adsorbed ligands. Endosomes, rather than coated vesicles, are seen in membrane fusion configurations that allow the matrix material to be formed into yolk bodies (e.g., Roth and Porter, 1964; Stay, 1965; Anderson, 1969; reviewed by Raikhel and Dhadialla, 1992). Weak bases inhibit vitellogenesis in D. melanogaster (DiMario and Mahowald, 1986) and it was suggested that this is because they can neutralize protons as the latter are pumped into the coated vesicles. Vesicular acidification was seen more directly in H. cecropia when sucrose density gradient centrifugation was used to monitor the uptake of labeled vitellogenin (Stynen et al., 1988). Endocytosis transferred label within 10 minutes from the incubation medium to a population of intracellular vesicles (density 1.15), and in pulse/chase experiments this label was transferred within 30 minutes to heavier particles (density 1.20) that were presumed to be nascent yolk spheres. Nigericin and monensin, ionophores that exchange intravesicular protons for cytoplasmic K+, prevented the second transfer. Endocytosis was not inhibited, because label accumulated in density
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1.15 particles; only subsequent transfer to the heavier particles was blocked. Relevant to its potential secondary function as an electrogenic component of Em is the probability that H+ATPase cycles back and forth between endosomes and plasma membrane. Calculations based on surface/volume relations indicate that only 1% of endosomal membrane in H. cecropia is required to cover the yolk spheres (Telfer et al., 1982). In several insects immunoelectron microscopy has identified a reservoir of excess membrane that is potentially available for return to the oocyte surface (Van Antwerpen et al., 1993; Raikhel and Snigirevskaya, 1998). Assuming that H+ATPase is included in the recycling reservoir, then the question becomes whether it is reinserted into preformed coated vesicles, or into plasma membrane, along with vitellogenin receptors and clathrin. The latter route would explain the 20% of Em that is due to a proton pump in R. prolixus follicles (O’Donnell and Sharda, 1994). Although this blood-feeding hemipteran has a conventional high Na+/low K+ hemolymph, only half of the electrogenic component of follicular Em is sensitive to ouabain. The electrogenic proton pump was interpreted in this case in the context of pH regulation in the oocyte, but the possibility was pointed out that it cycles back and forth between endosomes and plasma membrane. A monoclonal antibody against the 20 kDa subunit of Manduca sexta H+-ATPase localized the proton pump at two sites (Janssen et al., 1995)—the surface of the oocyte, where endosomes are being acidified, and the outer (or basal) surface of the follicle cells. Both sites could, in principle, contribute to the active component of Em. In the case of R. prolixus with its two membrane ATPases, one wonders whether the proton pump will prove to be restricted to the vitellogenic surface of the oocyte. The Na+/K+ ATPase is known to be located in follicle cells, because ouabain binds to isolated follicle cell membranes (Ilenchuk and Davey, 1987). And it is also active in the oocyte, for extracellular ion currents measured with a vibrating probe identified Na+ currents exiting both apical and basal ends of vitellogenic follicles (Diehl-Jones and Huebner, 1992, 1993). These currents were inhibited by ouabain and were accordingly interpreted as generated by a Na+/K+ ATPase. They are produced by the follicle itself, because they persisted in late vitellogenesis, when the oocyte has separated from the trophic cord that connects it to the nurse cells. And they are produced at least in part by the oocyte, for they also persisted after experimental removal of follicle cells. 6. Ca2+ action potentials As noted, Ca2+ does not contribute measureably to follicular resting potentials in Sarcophaga bullata
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(Verachtert et al., 1989), H. cecropia (Woodruff et al., 1992), or R. prolixus (O’Donnell and Sharda, 1994). On the other hand, there are strong electrochemical gradients favoring seepage of Ca2+ into the cytoplasm—in the case of H. cecropia, an external 5,000 micromolar Ca2+ concentration as opposed to a measured 0.6 micromolar ooplasmic activity, and an Em of around ⫺40 millivolts. This gradient comes into play when a Ca2+ action potential is induced by a depolarizing current injected into the oocyte (O’Donnell, 1985; O’Donnell, 1988). Action potentials were demonstrated in vitellogenic follicles of H. cecropia and columbia, L. migratoria, Periplaneta americana, Tenebrio molitor, and R. prolixus, but could not be demonstrated in D. melanogaster. They were visible only when channel blockers were used to suppress the K+ potential that dominates the electrical properties of these follicles. They lasted several seconds longer than the 1 second pulse used to evoke them, and during this period Em rose to values of +10 to +20 millivolts. The action potential was inhibited by Ca2+ channel blockers, but not by Na+-free media and was thus attributed to voltage-gated Ca2+ channels. A function during vitellogenesis is not apparent but it was speculated that, as in many other animal eggs, controlled Ca2+ entry may at a later time be an activating step associated with fertilization.
7. A Ca2+ pump in the trophic cap Ca2+ extrusion can be inferred from the electrical properties of the cytoplasmic bridges that connect the oocyte of H. cecropia to its intrafollicular cap of seven nurse cells. A focused, 2–5 millivolt steady state potential is maintained across these connections (Woodruff and Telfer, 1973, 1974). The transbridge potential arises at the onset of vitellogenesis when the hyperpolarization characteristic of that transition affects the nurse cells more strongly than the oocyte (Woodruff and Telfer, 1990). It persists for over four days, until the bridges and nurse cells disintegrate one day prior to the end of vitellogenesis. Ion selective microelectrodes showed that Ca2+ activity is three times higher in the less electronegatively charged oocyte than in the nurse cells (0.65 versus 0.18 micromolar). By contrast, cytoplasmic activities of K+, Mg2+ and Cl⫺ differed between oocyte and nurse cells by less than 5%, with the two cations having slightly higher activities in the more negative nurse cells while the anion was higher in the oocyte (Woodruff and Telfer, 1994). Cytoplasmic pH did not detectably differ across the bridges. That the transbridge potential is powered by Ca2+ extrusion from the nurse cells was confirmed when follicles were incubated in 10 millimolar azide. The transbridge potential then disappeared and Ca2+ activities
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equalized, rising in both oocyte and nurse cells to about 1.5 micromolar levels. The extracellular return pathway of the Ca2+ current is, in principle, guided by the hemisphere of follicle cells that cover the nurse cell cap (Fig. 1, nce). Unlike the follicle cells surrounding the oocyte, these cells form an electrically tight epithelium (Woodruff et al., 1986), and the escape route of any ions pumped out of the nurse cells would necessarily be beneath the epithelium and toward the oocyte. This model was supported by measurements with a vibrating probe, which detected a strong exit current from the nurse cells wherever patches of the epithelial barrier had been experimentally removed. Completion of the circuit would require leakage of Ca2+ through plasma membranes into the oocyte and/or its follicle cells. The gradient between oocyte and nurse cells is tightly focused in the 20–30 micrometer long cytoplasmic bridges, and is of sufficient magnitude over that short distance to affect the diffusion of proteins between the two cells (Woodruff and Telfer, 1974, 1980). Fluorescein-labeled proteins with a net positive charge crossed the channels into the nurse cells after injection into the oocyte, but did not as readily move in the reverse direction. The same protein, modified by methylcarboxylatin to yield a net negative charge, behaved in the opposite manner. Differences in charge-based mobility should thus generate differences in the cytoplasmic protein composition of the oocyte and its nurse cells. This has been confirmed with isoelectric focusing of soluble proteins in A. luna (Cole and Woodruff, 1997) and D. melanogaster (Cole and Woodruff, 2000). Differences in microinjected protein movements based on net electrical charge have also been seen in extrafollicular nurse tissue, the tropharium, of R. prolixus (Huebner, 1984) and O. fasciatus (Woodruff and Anderson, 1984). In both insects, negatively charged fluorescent proteins injected into the tropharium diffused throughout its syncytial core and into the beginnings of the cytoplasmic chords that attach it to each oocyte. Basic proteins injected at the same site migrated instead peripherally into nucleated pockets of cytoplasm that radiate outward from the core. In O. fasciatus the most peripheral trophocytes were found to be 8 millivolts electronegataive to the core. For R. prolixus differences in electrical potential between the outpocketings and the core have not been reported, but the contrasting directions of acidic and basic protein migration are consistent with the nucleated pockets being electronegative to the core here also. Whatever the mechanism of retention, the oocyte, as in species with intrafollicular nurse tissues, must have suppressed levels of soluble basic proteins. Whether the nurse cells are intrafollicular or extrafollicular, transcriptional activity in the oocyte nucleus, as revealed by the incorporation of tritiated uridine, is generally not detectable during vitellogenesis (reviewed in
Telfer, 1975). In H. cecropia, suppression begins with the onset of vitellogenesis, when the transbridge potential is first detected (Woodruff and Telfer, 1990). When the intercellular bridges were experimentally depolarized by abolishing the Ca2+ gradient, uridine incorporation resumed in the oocyte. Three different treatments had this effect—a calcium buffer, a calcium ionophore, and vanadate ions, which inhibit calcium extrusion by the nurse cells (Woodruff et al., 1998).
8. Osmotic pressure and volume changes K+ channels in H. cecropia follicles are osmotically gated. They are maximally open at the osmotic pressure of hemolymph but they close in more dilute solutions, and Em becomes unresponsive to changes in external K+ concentrations (Woodruff et al., 1992). Another example of sensitivity to osmotic pressure was seen in D. melanogaster, in which hyperosmotic media caused a 3–4 mV hyperpolarization of the oocyte, without affecting the nurse cells (Singleton and Woodruff, 1994). When predicting in situ properties from the ion physiology of follicles incubated in vitro, therefore, it is important to work at the osmotic pressure of the hemolymph in which they form yolk. Two cases have been analyzed in which vitellogenesis is affected by changes in follicle cell volume. In R. prolixus the promotion of vitellogenesis by juvenile hormone is mediated by the ability of this hormone to control epithelial patency. Patency is lost when vitellogenic follicles are incubated in the absence of juvenile hormone, but is maintained during incubation in the presence of this hormone. The response to juvenile hormone entails a 50% reduction in follicle cell volume (AbuHakima and Davey, 1977), is inhibited by ouabain (AbuHakima and Davey, 1979), and is preceded by a juvenile hormone-dependent increase in ouabain binding by follicle cell membranes in late previtellogenic follicles (Ilenchuk and Davey, 1987). The juvenile hormone effect is mediated by calcium-dependent protein kinase (PKC), which, in the presence of juvenile hormone, phosphorylates a polypeptide resembling in size the 100 kDa subunit of Na+/K+-ATPase (Sevala and Davey, 1993). A second case involves cyclic AMP-dependent protein kinase (PKA) rather than PKC, the promotion of follicle cell swelling rather than shrinking, and the termination of vitellogenesis rather than its maintenance. When lepidopteran follicles reach a species-specific length—e.g., 2 mm in H. cecropia (Telfer and Anderson, 1968) and 1.8 mm in M. sexta (Nijhout and Riddiford, 1974)—vitellogenesis ends and a period of water uptake ensues. H. cecropia follicles increase their volume during this period by 50%, and assume the slightly flattened ellipsoid shape of a mature egg. Isotonicity of isolated
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yolk spheres changes from 0.6 to 0.9 osmolar, clear evidence that swelling and shape change are driven by the turgor pressure of solute uptake. Ion substitution studies indicated that Cl⫺ and K+ are required as osmolytes (Wang and Telfer, 1998). The epithelial phase of osmotic swelling can be triggered by pharmcological activation of PKA (Wang and Telfer, 1996, 1997). Among the strategies that can be used to increase PKA activity, the one employed in the osmotic swelling study was incubation in Sp-cAMPS, an analog of cyclic AMP that is cell-permeant and resistant to hydrolysis by phosphodiesterase (Dostmann et al., 1990); in a concentration of 1 mM the follicle cells began to swell within 30 minutes. Also within 30 minutes two changes in ion physiology were detected—onset of a Cl⫺ conductance and a transitory 30% hyperpolarization (Wang and Telfer, 1998). Opening Cl⫺ channels should have a depolarizing effect on follicle membranes (Woodruff et al., 1992), so hyperpolarization was due to other factors. A drop in cytoplasmic pH from 7.26 to 7.06 suggested that an increase in substrate availability for the electrogenic H+-ATPase contributed to the change in Em (Wang and Telfer, 1998). A Cl⫺ channel blocker, anthracene-9-carboxylic acid, prevented the increase in conductance and also prevented osmotic swelling and hyperpolarization (Wang and Telfer, 1998). The follicle cells contain 80% of total follicular PKA (Wang and Telfer, 2000), and it is therefore likely that they are the primary site of a cascade of events leading from PKA activation through Cl⫺ channel opening and finally to epithelial swelling.
9. Future questions The power of comparative studies is made evident in this review by the similarities and differences between a lepidopteran and a heteropteran. Other orders need to be investigated, as well as families with other life styles within the two orders represented here. H. cecropia is among the lepidopterans that make yolk as pharate adults and may therefore have fundamental differences in ion physiology from species in which vitellogenesis is triggered by juvenile hormone in post-eclosion, nectar-feeding moths and butterflies. And R. prolixus, with its hemolymph rich in Na+ derived from mammalian blood meals, may well differ fundamentally from heteropterans with vegetarian diets that are poor in Na+. Examples of questions raised here that might yield to a comparative approach include why selective concentrating of vitellogenin in yolk seems to be associated with a low Cl⫺ conductance, with plasma membrane energization by a protein pump, and with gap junctions sufficiently patent to transmit intermediate-sized solutes from follicle cells to the oocyte.
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We can also hope that developmental studies will add to an understanding of follicular physiology by expanding the list of gains and losses in membrane functions at the onset and termination of vitellogenesis. And in the longer run, how do internal, programmatic cues (Swevers and Iatrou, 1992), or hormonal stimulation (Ilenchuk and Davey, 1987) evoke these gains and losses? References Abu-Hakima, R., Davey, K.G., 1977. The action of juvenile hormone on the follicle cells of Rhodnius prolixus: the importance of volume changes. Journal of Experimental Biology 69, 33–44. Abu-Hakima, R., 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 Biochemistry 9, 195–198. Adler, E.L., Woodruff, R.I., 2000. Varied effects of 1-octanol on gap junctional communication between ovarian epithelial cells and oocytes in Oncopeltus fasciatus and Drosophila melanogaster. Archives of Insect Biochemistry and Physiology 43, 22–32. Anderson, E., 1969. Oogenesis in the cockroach, Periplaneta americana, with special reference to the specializations of the oolemma and the fate of coated vesicles. Journal de Microscopie 8, 721–738. Anderson, K., Woodruff, R.I., 2001. A gap junctionally transmitted epithelial cell signal regulates endocytotic yolk uptake in Oncopeltus faciatus. Developmental Biology 239, 68–78. Anderson, L.M., Telfer, W.H., 1970. Trypan blue inhibition of yolk deposition—a clue to follicle cell function in the cecropia moth. Journal of Embryology and Experimental Morphology 23, 35–52. Cole, R.W., Woodruff, R.I., 1997. Charge dependent distribution of endogenous proteins within ovarian follicles of Actias luna. Journal of Insect Physiology 43, 275–287. Cole, R.W., Woodruff, R.I., 2000. Vitellogenic ovarian follicles of Drosophila exhibit a charge-dependent distribution of endogenous soluble proteins. Journal of Insect Physiology 46, 1239–1248. Dawson, J., Djamgoz, M.B.A., Hardie, J., Irving, S.N., 1989. Components of resting membrane electrogenesis in lepidopteran skeletal muscle. Journal of Insect Physiology 35, 659–666. Diehl-Jones, W., Huebner, E., 1992. Spacial and temporal transcellular current patterns during oogenesis. Developmental Biology 153, 302–311. Diehl-Jones, W., Huebner, E., 1993. Ionic basis of bioelectric currents during oogenesis in an insect. Developmental Biology 158, 301– 316. DiMario, P.J., Mahowald, A.P., 1986. The effects of pH and weak bases on the in vitro endocytosis of vitellogenin by oocytes of Drosophila melanogaster. Tissue and Cell Research 246, 103–108. Dostmann, W.R., Taylor, S.S., Genieser, H.-G., Jastorff, B., Doskeland, S.O., Ogreid, D., 1990. Probing the cyclic nucleotide binding sites of cAMP-dependent protein kinases I and II with analogs of adenosine 3⬘:, 5⬙-cyclic phosphorothioates. Journal of Biological Chemistry 265, 10484–10491. Florkin, M., Jeuniaux, C., 1974. Hemolymph: Composition. In: Rockstein, M. (Ed.), Insect Physiology. Academic Press, New York, London, pp. 256–305. Goldstein, J.L., Brown, M.S., Anderson, R.G.W., Russell, D.W., Schneider, W.J., 1985. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annual Review of Cell Biology 1, 1–39. Harvey, W.R., Wieczorek, H., 1997. Animal plasma membrane energization by chemiosmotic H+ V-ATPase. Journal of Experimental Biology 200, 203–216.
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Telfer, W.H., 1975. Development and physiology of the oocyte-nurse cell syncytium. Advances in Insect Physiology 11, 223–319. Telfer, W.H., Anderson, L.M., 1968. Functional transformations accompanying the inition of a terminal growth phase in the cecropia moth oocyte. Developmental Biology 17, 512–535. Telfer, W.H., Huebner, E., Smith, D.S., 1982. The cell biology of vitellogenic follicles in Hyalophora and Rhodnius. In: King, R.C., Akai, H. (Eds.), Insect Ultrastructure. Plenum, New York, pp. 118–149. Van Antwerpen, R., Conway, R., Law, J.H., 1993. Protein and lipoprotein uptake by ceveloping oocytes of the hawkmoth Manduca sexta. An ultrastructural and immunocytochemical study. Tissue and Cell 25, 205–218. Verachtert, B., Amelinckx, M., De Loof, A., 1989. Potassium and chloride dependence of the membrane potential of vitellogenic follicles of Sarcophaga bullata (Dioptera). Journal of Insect Physiology 35, 143–148. Waksmonski, S.L. and Woodruff, R.I., 2002. For uptake of yolk precursor, epithelial cell–oocyte gap junctional communication is required by insects representing six different orders. Journal of Insect Physiology, in press. Wang, Y., Telfer, W.H., 1996. Cyclic nucleotide-induced termination of vitellogenin uptake by Hyalophora cecropia follicles. Insect Biochemistry and Molecular Biology 26, 85–94. Wang, Y., Telfer, W.H., 1997. cAMP-stimulated termination of vitellogenesis in Hyalophora cecropia: formation of a diffusion barrier and the loss of patency. Journal of Insect Physiology 43, 675–684. Wang, Y., Telfer, W.H., 1998. cAMP-induced water uptake in a moth ovary: inhibition by bafilomycin and anthracene-9-carboxylic acid. Journal of Experimental Biology 201, 1627–1635. Wang, Y., Telfer, W.H., 2000. Cyclic nucleotide-dependent protein phosphorylation in vitellogenic follicles of Hyalophora cecropia. Insect Biochemistry and Molecular Biology 30, 29–34. Wollberg, Z., Cohen, E., Kalina, M., 1975. Electrical properties of developing oocytes of the migratory locust, Locusta migratoria. Journal of Cellular Physiology 88, 145–158. Woodruff, R.I., 1979. Electrotonic junctions in cecropia moth ovaries. Developmental Biology 69, 281–295. Woodruff, R.I., Anderson, K.L., 1984. Nutritive cord connection and dye-coupling of the follicular epithelium to the growing oocytes in the telotrophic ovarioles in Oncopeltus fasciatus, the milkweed bug. Roux’s Archives of Developmental Biology 193, 158–163. Woodruff, R.I., Telfer, W.H., 1973. Polarized intercellular bridges in ovarian follicles of the cecropia moth. Journal of Cell Biology 58, 172–188. Woodruff, R.I., Telfer, W.H., 1974. Electrical properties of ovarian cells linked by intercellular bridges. Annals of the New York Academy of Science 238, 408–419. Woodruff, R.I., Telfer, W.H., 1980. Electrophoresis of proteins in intercellular bridges. Nature (London) 286, 84–86. Woodruff, R.I., Telfer, W.H., 1990. Activation of a new physiological state at the onset of vitellogenesis in Hyalophora follicles. Developmental Biology 138, 410–420. Woodruff, R.I., Telfer, W.H., 1994. Steady-state gradient in calcium ion activity across the intercellular bridges connecting oocytes and nurse cells in Hyalophora cecropia. Archives of Insect Biochemistry and Physiology 25, 9–20. Woodruff, R., Huebner, E., Telfer, W., 1986. Ion currents in Hyalophora ovaries: the role of the epithelium and the intercellular spaces of the trophic cap. Developmental Biology 117, 405–416. Woodruff, R.I., Munz, A., Telfer, W.H., 1992. Steady-state potentials in ovarian follicles of a moth, Hyalophora cecropia. Journal of Insect Physiology 38, 49–60. Woodruff, R.I., Dittmann, F., Telfer, W.H., 1998. Ca2+ current from oocyte to nurse cells and suppression of uridine incorporation in the germinal vesicle of Hyalophora cecropia. Invertebrate Reproduction and Development 34, 157–164. Zimowska, G., Shirk, P.D., Silhacek, D.L., Shaaya, E., 1994. Yolk
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sphere formation is initiated in oocytes before development of patency in follicles of the moth Plodia interpunctella. Wilhelm Roux’s Archives of Developmental Biology 203, 215–226. Zimowska, G., Shirk, P.D., Silhacek, D.L., Shaaya, E., 1995. Termin-
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ation of vitellogenesis in follicles of the moth, Plodia interpunctella: changes in oocyte and follicular epithelial cell activities. Archives of Insect Biochemisry and Physiology 29, 357–380.