Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis

Joanne E. I. Gittens1,2,3,4, Abdul Amir Mhawi1,2,3,4, Darcy Lidington4,5, Yves Ouellette3,4, and Gerald M. Kidder1,2,3,4

Departments of 1 Physiology and Pharmacology, 2 Obstetrics and Gynaecology, 3 Paediatrics, and 5 Medical Biophysics, University of Western Ontario, London N6A 5C1; and 4 Child Health Research Institute, London, Ontario, Canada N6C 2V5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ovarian granulosa cells are coupled via gap junctions containing connexin43 (Cx43 or alpha -1 connexin). In the absence of Cx43, granulosa cells stop growing in an early preantral stage. However, the fact that granulosa cells of mature follicles express multiple connexins complicated interpretation of this finding. The present experiments were designed to clarify the role of Cx43 vs. these other connexins in the earliest stages of folliculogenesis. Dye injection experiments revealed that granulosa cells from Cx43 knockout follicles are not coupled, and this was confirmed by ionic current injections. Furthermore, electron microscopy revealed that gap junctions are extremely rare in mutant granulosa cells. In contrast, mutant granulosa cells were able to form gap junctions with wild-type granulosa cells in a dye preloading assay. It was concluded that mutant granulosa cells contain a population of connexons, composed of an unidentified connexin, that do not normally contribute to gap junctions. Therefore, although Cx43 is not the only gap junction protein present in granulosa cells of early preantral follicles, it is the only one that makes a significant contribution to intercellular coupling.

intercellular communication; hemichannels; alpha -1 connexin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GAP JUNCTIONS ARE SITES of intercellular membrane channels comprising a large family of subunit proteins, the connexins. Gap junctional intercellular coupling is now well established as an important component of cell signaling pathways involved in development and homeostasis. Its importance has been documented both by mouse knockouts lacking specific connexins and by human diseases associated with connexin gene mutations (reviewed in Ref. 36). However, the developmental or physiological function of gap junctional coupling in any specific situation remains unclear, especially with regard to the molecules that are being transmitted through the channels and the relationship between the different connexins that are often expressed in the same cell.

We have been characterizing a line of connexin43 (Cx43) knockout mice. In addition to dysmorphogenesis of the heart leading to early neonatal death (31, 38), offspring lacking Cx43 exhibit pathophysiological features in a variety of organs. These include slowed cardiac conduction and increased susceptibility to arrhythmias (8, 14, 22), precataractogenic lesions in the lenses (12), delayed ossification and osteoblast dysfunction (21), impaired hematopoiesis (27), reduced germ cell numbers in the fetal gonads (17), and disrupted gametogenesis in both sexes (1, 32). It is the latter consequence of the loss of Cx43 that is the focus of the present report.

Cx43 is already being expressed in mouse ovaries on postnatal day 1, when follicles start to form (17). As follicles grow and reach maturity, the expanding granulosa cell population continues to express Cx43 (19, 35). Given the presence of this connexin from the onset of folliculogenesis, it was not surprising that developing follicles arrest in preantral stages when Cx43 is absent (1). However, interpretation of this finding was complicated by the fact that granulosa cells lacking Cx43 retain the ability to form gap junctions when paired with wild-type granulosa cells in a dye preloading assay (1). Although dye transfer was limited to first-order cells, this suggested the presence of another connexin(s). Indeed, granulosa cells in mature rodent follicles do express other connexins in addition to Cx43, including Cx32 (24, 35), Cx45 (2, 19, 28), Cx37 (37), and possibly Cx57 (the gene encoding Cx57 is transcribed in the mouse ovary, and its porcine homolog, Cx60, has been identified in cumulus granulosa cells; Refs. 15, 25). In interpreting the arrest of folliculogenesis in Cx43-deficient ovaries, therefore, we must consider several possibilities: 1) Cx43 is the only connexin that contributes to granulosa cell gap junctions in the earliest stages of folliculogenesis; 2) Cx43 is coexpressed with other connexins but performs a unique function that cannot be fulfilled by them; or 3) the coexpressed connexins in granulosa cells of early follicles share redundant functions, but all are needed to establish a sufficient level of coupling.

To distinguish between these possibilities, we used dye and current injection as well as electron microscopy to test for the presence of residual gap junctions in granulosa cells from Cx43-deficient juvenile ovaries. As in our previous experiments (1), we used a kidney grafting procedure to circumvent the neonatal lethality caused by the loss of Cx43. In contrast to our earlier results with the preloading assay, we could find no evidence of gap junctional coupling between granulosa cells lacking Cx43. We have interpreted the discordance between these results and those obtained with the preloading assay to indicate the presence of connexons composed of another connexin in granulosa cells from developing follicles.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Mice used as graft recipients were 20- to 22-g Prkdcscid/Prkdcscid females (C.B-17/IcrHsd-scid purchased from Harlan Sprague Dawley, Indianapolis, IN). Cx43-deficient mice were derived from matings of heterozygous (Gja1+/Gja1-) CD1 mice. The ovaries were collected from fetal offspring on day 17.5 of gestation from dams killed by cervical dislocation after CO2 anesthesia. Fetuses were killed by decapitation before removal of ovaries. The genotypes of the ovary donors were determined by PCR as previously described (1).

Grafting of ovaries. The ovaries of either a wild-type or a homozygous mutant donor were transplanted to the right kidney capsule of a mature ovariectomized Prkdcscid/Prkdcscid mouse as previously described (1). In brief, host ovaries were removed through small incisions on both the left and right dorsolateral surfaces, caudal to the last ribs of the mouse. The right kidney was brought to the surface through the right dorsolateral incision, and a small hole was made in its capsule. A pair of wild-type or mutant ovaries was inserted beneath the kidney capsule. The body wall was sutured, and the skin was closed with wound clips.

Follicle isolation. Grafted ovaries were recovered 20-22 days after transplantation and either processed immediately for electron microscopy or transferred to Waymouth MB 752/1 medium (Invitrogen Canada, Burlington, ON, Canada) containing 10% fetal bovine serum (Invitrogen), 0.6% OmniPur HEPES free acid (EM Science, Gibbstown, NJ), 0.5% Fungizone antimycotic (Invitrogen), 0.5% penicillin-streptomycin (Invitrogen), and 1 mg/ml collagenase type 1 (Sigma-Aldrich Canada, Oakville, ON, Canada). Follicles were liberated by repeated aspiration and expulsion with a 1-ml pipettor. Care was taken to ensure that the follicles taken from wild-type ovaries were comparable in size with those from mutant ovaries. Follicles were washed through two dishes of culture medium. Those to be used for the preloading assay were then placed into wells of chamber slides (Nunc Lab-Tec, Naperville, IL) containing 1 ml of medium and cultured for 48 h, during which time the granulosa cells grew over the surface of the wells to form monolayers. Follicles to be used for dye injection or electrical coupling experiments were transferred to 12-mm round coverglasses in 200-µl drops of culture medium. They were incubated in a humidified chamber at 37°C in an atmosphere of 5% CO2 in air for 24 h to allow the follicles to attach to the glass and form granulosa cell monolayers. The coverglasses were transferred to 35 × 10 mm petri dishes containing 3 ml of culture medium and assayed immediately or incubated for an additional 24 h.

Testing for gap junctional coupling by dye microinjection. Ovaries were recovered from graft hosts, and short-term follicle cultures in Waymouth medium were established as described in Follicle isolation. One granulosa cell from each follicle was microinjected with a gap junction-permeant fluorescent dye for 1 min. A variety of fluorescent dyes with varying connexin-dependent gap junction permeabilities were used to assess intercellular coupling. The fluorescent dyes were Lucifer yellow (5% in ddH2O; Molecular Probes, Eugene, OR), 5(and 6)-carboxyfluorescein (0.05% in ddH2O; Molecular Probes), calcein (13 mg/ml in solution containing 100 mM KCl and 20 mM KH2PO4, pH 7.4; Molecular Probes), and 2',7'-dichlorofluorescein (7 mg/ml in the same solution; Sigma-Aldrich). After 5 min, the percentage of microinjected cells that transferred dye to their neighbors was determined.

Testing for gap junctional coupling by current microinjection. Follicles cultured on coverglass were transferred to a 22°C bath chamber containing Waymouth medium. Electrical coupling experiments, conducted at room temperature (~22°C), were completed as previously described (26). Briefly, glass microelectrodes were backfilled with 1 M KCl (tip resistances of 40-80 MOmega ) and connected to electrometers (Intra 767; World Precision Instruments, Sarasota, FL) by chlorinated silver wires. Two adjacent granulosa cells (interelectrode distance ~25 µm) were each impaled with an electrode. On insertion of the microelectrode, a sharp drop in the electrometer reading (identified as the resting membrane potential, Em) was noted. After a stable Em was read in each cell, three to five hyperpolarizing pulses (50 nA, 100-ms duration) of current were injected into one cell (A310 Accupulser; World Precision Instruments), resulting in a change in Em (electrotonic potential change, Delta Em) in the adjacent cell. Because of the limited size and large geometric variability of the granulosa cell monolayers, no significance was attributed to the magnitude of these potential changes. On removal of the electrode from the cell, the measured Em returned to zero: recordings that did not return to within 10% of the baseline were assumed to be ruptured cells, and these recordings were discarded. Membrane and electrotonic potentials were recorded on a Gould RS 3200 two-channel recorder (Gould, Valley View, OH).

Testing for gap junctional coupling by dye preloading. Preantral follicle cultures prepared from ovaries removed directly from a wild-type mouse were preloaded with calcein-AM (a membrane-permeant molecule that gives rise to the membrane-impermeant green fluorescent dye calcein once inside the cell) and 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (diI, a lipophilic, fluorescent dye that stains membranes red) as described by Goldberg et al. (13). Both dyes were purchased from Molecular Probes. The cells were then treated with a 0.25% trypsin-1 mM EDTA solution (Invitrogen) for 2 min at 37°C in an atmosphere of 5% CO2 in air, rinsed, and pipetted onto unlabeled follicles (mutant or wild type) obtained from grafted ovaries. Recipient follicles were cocultured with preloaded granulosa cells for 3 h to allow the preloaded cells to establish gap junctions with the unlabeled granulosa cells. The number of granulosa cells receiving calcein from the preloaded granulosa cell provides an index of the strength of intercellular coupling. In some experiments, the recipient cells were pretreated for 4 h with 200 µM carbenoxolone, a treatment determined to completely and reversibly block dye coupling in cultured wild-type granulosa cells. In other cases, brefeldin A treatment (2 h at 20 µg/ml) was used to block trafficking of nascent connexins to the plasma membrane (11). Both inhibitors were purchased from Sigma-Aldrich.

Electron microscopy. Ovaries recovered from the kidneys of host mice were briefly rinsed in 0.1 M phosphate buffer (pH 7.4) and then cut into small pieces and fixed for 3 h at room temperature in 2.5% glutaraldehyde in the same buffer. After thorough washing in phosphate buffer, the pieces were postfixed in 1% OsO4 in the same buffer for 1 h at room temperature. They were then washed in sodium acetate (2 × 30 min), stained in 1% uranyl acetate overnight at 4°, and washed again in sodium acetate (2 × 30 min) before being dehydrated in an increasing series of ethanol. Infiltration was carried out with a 1:1 mixture of LR White resin (London Resin, Reading, UK) and ethanol for 1 h at room temperature. The pieces were then placed in pure LR White resin on a rotator overnight. After another change of resin (1 h), the pieces were embedded in gelatin capsules (size 00; Polysciences, Warrington, PA) and polymerized for 24 h at 60°C. Thin sections (70-80 nm) were cut and stained with 2.5% aqueous uranyl acetate and Reynolds lead citrate before viewing with a Zeiss EM 902 electron microscope at 80 kV. The proportion of membrane contact length occupied by gap junctions was estimated from measurements on the micrographs.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To examine the state of gap junctional coupling between granulosa cells in the absence of Cx43, wild-type and homozygous mutant ovaries were collected on day 17.5 of gestation and grafted into the kidney capsules of immunocompromised adult female mice, a procedure necessitated by neonatal lethality caused by the Gja1 null mutation (1, 30). Ovaries were recovered 20-22 days after grafting, and cultures of wild-type and mutant follicles were prepared for tests of gap junctional coupling. Granulosa cells were first tested for intercellular dye transfer by microinjection. Microinjection pipettes were filled with one of four gap junction-permeant fluorescent dyes [Lucifer yellow, 5 (and 6)-carboxyfluorescein, calcein, or 2',7'-dichlorofluorescein], and dye was allowed to enter the cell for up to 1 min before the removal of the injection pipette. With wild-type follicles, each of the dyes readily passed from the injected granulosa cell to contacting cells and thence to their neighbors (Fig. 1B). Dye transfer was extensive, with almost all injections resulting in transfer to at least fourth-order cells. In contrast, injected granulosa cells of mutant follicles failed to transfer dye to neighbors despite continuous injection for 1 min (Fig. 1D). Because the lack of dye transfer to neighbors could be a reflection of the cell selected for injection and not characteristic of the entire population of granulosa cells within a single follicle, a few cells within each of several mutant follicles were injected. In all cases, dye was not transferred to even one contacting cell, regardless of which dye was injected (Table 1). These data indicate that granulosa cells from early follicles lacking Cx43 do not contain functional gap junctions.


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Fig. 1.   Dye injection assay for gap junctional coupling between connexin43 (Cx43)-deficient granulosa cells. B: wild-type cells demonstrated extensive coupling (as with Lucifer yellow in this case), with almost all injections resulting in transfer to at least 4th-order cells. D: mutant cells failed to transfer dye to even 1 contacting cell. * Injected cell in the corresponding phase contrast images (A, C). O, oocyte. Scale bar = 50 µm.


                              
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Table 1.   Summary of dye coupling assayed by microinjection

We next performed electrical coupling measurements as a more sensitive means of detecting functional gap junctions between mutant granulosa cells. The resting Em of wild-type and mutant granulosa cells was not different and ranged between 8 and 16 mV. In wild-type granulosa cells, Delta Em were consistently detected during current pulsing. In mutant granulosa cells, however, electrotonic potentials were never detected, indicating that these cells were not electrically coupled. Representative recordings from electrical coupling experiments are depicted in Fig. 2.


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Fig. 2.   Current injection assay for gap junctional coupling between Cx43-deficient granulosa cells. Two adjacent cells of the granulosa cell monolayer were impaled with microelectrodes. Once stable membrane potentials (Em) were acquired, hyperpolarizing pulses of current were injected into the 1st cell (Em1, top panels) and communication of electrotonic potentials was monitored in the 2nd cell (Em2, bottom panels). In wild-type cell pairs (Em1 and Em2 in left panels), electrotonic potentials were noted. In Cx43-deficient cell pairs, however, no electrotonic potentials were communicated to the second cell (Em1 and Em2 in right panels). Figure is representative of 10 recordings from 8-9 granulosa cell monolayers for each genotype.

To confirm that granulosa cells lacking Cx43 are devoid of gap junctions in vivo, we performed electron microscopy on follicles from grafted ovaries (Fig. 3). Regions of closely opposed plasma membranes of any length exhibiting a typical pentalaminar gap junction structure were measured. Numerous gap junctions could be identified between granulosa cells in grafted wild-type follicles, occupying ~7% of the 136.3 µm of opposed membrane length surveyed. In contrast, only one gap junction was identified in homozygous mutant follicles (0.14% of the 178.2 µm of opposed membrane length surveyed) despite extensive regions of close cell apposition. This result indicates that gap junctions are extremely rare in Cx43-deficient granulosa cells.


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Fig. 3.   Cell contact regions of granulosa cells from grafted follicles viewed with an electron microscope. A, B, and C: although numerous regions of close cell apposition (arrows) were evident between granulosa cells in mutant follicles, structures recognizable as gap junctions were extremely rare. Panel C shows the single putative gap junction detected in this analysis (arrow indicates a short segment exhibiting typical pentalaminar structure of a gap junction). D: a typical gap junction (arrows) connects 2 granulosa cells in a wild-type follicle. Scale bars = 400 nm. Inset, gap junction segment between wild-type granulosa cells photographed at twice the magnification.

Our conclusion that mutant granulosa cells lack intercellular coupling is in apparent contradiction with our previous work (1) showing that the loss of Cx43 does not completely eliminate the ability of granulosa cells to form gap junctions. Because the previous experiments had used a preloading assay for dye coupling, we wanted to reassess the situation in our present experiments with the same technique. When calcein- and diI-labeled wild-type granulosa cells were seeded onto a monolayer of mutant granulosa cells, there was indeed evidence of dye transfer from preloaded cells to unlabeled recipients (Fig. 4 and Table 2). As in our earlier study, those recipients did not transfer the dye any further. Thus the preloading assay consistently provides results that are discordant with those of the other tests for gap junctions in mutant follicles. To confirm that dye transfer between wild-type and mutant granulosa cells occurs via gap junction channels, carbenoxolone, a proven gap junction channel blocker, was added to the recipient granulosa cells in advance of the assay. This treatment abolished all dye transfer regardless of the genotype of the recipient cells. Likewise, when mutant granulosa cells were used as dye donors, there was no dye transfer, confirming the requirement for Cx43 in the wild-type donors.


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Fig. 4.   Dye preloading assay for gap junctional coupling with Cx43-deficient granulosa cells. B: dye was transferred from a wild-type preloaded cell (yellow because of merging of the green calcein and red diI signals) to wild-type recipients (green) and thence on to their neighbors. D: recipient mutant cells received dye from a wild-type preloaded cell but did not transfer the dye to any of their neighbors. F: a mutant preloaded cell failed to transfer calcein to any cells in a mutant monolayer. A, C, and E show the corresponding phase contrast images. Scale bar = 50 µm.


                              
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Table 2.   Summary of dye coupling assayed by preloading

Finally, we tested the hypothesis that coupling between wild-type and mutant granulosa cells in the preloading assay results from recruitment of connexons from intracellular stores to the plasma membranes of the mutant cells. Gap junction assembly involves connexon delivery from the Golgi to the plasma membrane in vesicular carriers, a process that is inhibited by brefeldin A (20). Treatment with brefeldin A during the preloading assay at a concentration (20 µg/ml) sufficient to block connexon trafficking (20) failed to block dye transfer, indicating that dye transfer in this assay depends primarily on preformed, undocked connexons in the plasma membranes of the mutant cells. However, this result does not rule out an additional contribution from intracellular connexons that are recruited to the plasma membrane, perhaps in response to contact with a wild-type cell.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The importance of gap junctional intercellular coupling in ovarian folliculogenesis has been demonstrated clearly by knockouts of two different connexin genes, those encoding Cx37 and Cx43. Cx37 contributes to gap junctions linking growing oocytes to surrounding granulosa cells. In the absence of this connexin, coupling between oocytes and granulosa cells is abolished (according to neurobiotin injection experiments) and folliculogenesis is disrupted around the time of antrum formation (5, 33). Cx43, on the other hand, is expressed in granulosa cells, and its absence is associated with follicular arrest in a unilaminar (primary) or early multilaminar (secondary) follicle stage depending on strain background (1).

Although multiple connexins have been identified in mature ovarian follicles (reviewed in Ref. 19), Cx43 is the only gap junction protein that has been clearly shown to be expressed by the granulosa cells of early preantral follicles of the mouse (17, 37). Thus the follicle arrest in Cx43-deficient ovaries may be due to a complete absence of connexins from the granulosa cells. However, when calcein- and diI-labeled wild-type granulosa cells were seeded onto a monolayer of mutant granulosa cells, there was evidence of dye transfer from preloaded cells to unlabeled recipients (Ref. 1 and Fig. 4). The transfer of dye to mutant granulosa cells required that gap junctions be established between the preloaded wild-type cells and the mutant cells, and this, in turn, required that connexons be present in the plasma membranes of the mutant granulosa cells. In contrast, both dye injection and electrical coupling experiments demonstrated that mutant granulosa cells in culture are not coupled with each other. Electron microscopy confirmed that gap junctions are extremely rare in mutant granulosa cells. Collectively, these results suggest that the granulosa cells of preantral follicles express one or more connexins other than Cx43 but that this other connexin does not normally assemble into functional gap junctions in the absence of Cx43. The other connexin(s) could be Cx32, Cx37, Cx45, or Cx57, all of which are reported to be expressed along with Cx43 in granulosa cells of mature mouse follicles (19, 37). Such a rare connexin, coexpressed with Cx43 in granulosa cells of early follicles, might contribute more significantly to gap junction assembly later in folliculogenesis as gap junctions become larger and more numerous. Our finding of only a single gap junction profile in all of the granulosa cells surveyed with the electron microscope indicates that this rare connexin does occasionally form gap junctions, but in such a tiny fraction of the cells that coupling is unlikely to be detected in a mutant cell monolayer.

How can we explain the discrepancy between the results of the preloading experiments and the other coupling assays? We believe the discrepancy arises from the use of wild-type cells as donors, necessitated by the paucity of follicles and the reduced number of granulosa cells per follicle in Cx43-deficient ovaries (1). Wild-type cells could have increased the frequency with which rare hemichannels in the plasma membranes of the mutant cells are able to form intercellular channels. The existence of such hemichannels has been demonstrated in Xenopus oocytes injected with connexin mRNAs, cultured mammalian cell lines transfected with connexin cDNAs, and some primary cells in culture (4, 6, 7, 9, 10, 16, 23, 29, 34). Although it is not possible to visualize hemichannels, we presume that Cx43 hemichannels are present in high numbers in the membranes of dispersed wild-type granulosa cells. As a consequence, the frequency with which hemichannels in mutant plasma membranes find docking partners is much greater when the mutant cells are paired with wild-type cells. In other words, the paucity of hemichannels in Cx43-deficient granulosa cells might restrict their docking, hindering gap junction formation and intercellular coupling within mutant follicles. This concept is illustrated in Fig. 5. Thus, in this situation, the preloading assay gives a false indication of the existence of residual gap junctions between granulosa cells from mutant follicles but does reveal that the mutant cells are competent to engage in gap junctional communication under certain circumstances. In other contexts, it has been shown that the extent of dye transfer observed with a preloading assay correlates well with that seen with microinjection (13).


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Fig. 5.   An hypothesis to explain the ability of mutant cells to form gap junctions with wild-type cells (WT) but not with each other. The use of wild-type cells as dye donors in the preloading assay could increase the efficiency with which rare hemichannels (yellow) in the mutant cells (knockout, KO), either already existing in the plasma membranes or recruited there on cell contact, can form intercellular channels by providing an abundance of docking partners (Cx43 hemichannels, red). Otherwise, preantral granulosa cells lacking Cx43 do not have sufficient hemichannels in their membranes to allow gap junction formation.

Although once considered to represent nothing more than an intermediate step in gap junction assembly (23), gap junction hemichannels are now thought to play a variety of physiological roles in specific contexts (3, 18, 29, 30, 34). The non-Cx43 granulosa cell hemichannels of preantral granulosa cells are apparently too sparse to contribute to gap junctions in most cells; they must therefore serve an independent function, if any. Future experiments will be aimed at discovering what that function might be.

Our results demonstrate the importance of correlating multiple tests for the presence of gap junctional intercellular coupling between connexin-deficient cells. Although the seeding of wild-type cells onto mutant cells may not reflect the true state of gap junctional coupling between mutant cells in vivo, it can reveal the presence of other connexins capable of forming gap junctions under optimal circumstances. In our study, coupling between mutant cells was best measured through the microinjection of gap junction-permeant dyes or ionic current. The lack of dye transfer observed after microinjection of a variety of fluorescent molecules having different molecular weights and ionic charges accurately revealed that the vast majority of mutant cells completely lack communication via gap junctions, a finding confirmed by electrical coupling measurements and consistent with the electron micrographs. Therefore, an accurate evaluation of the level of coupling between cells requires a critical evaluation of the methods used to assess it. Whereas Cx43 may not be the only connexin expressed in granulosa cells during early stages of mouse folliculogenesis, it does appear to be the only one that makes a significant contribution to intercellular coupling and thus plays a distinct role in granulosa cell function.


    ACKNOWLEDGEMENTS

We are grateful to Kevin Barr for technical assistance and conscientious management of the mouse colony, Paul Walton for help with the dye injections, Dale Laird for a critical reading of the manuscript, and Peter Ottensmeyer for providing access to the electron microscope.


    FOOTNOTES

Funding for this work was provided by a grant (to G. M. Kidder) from the Canadian Institutes of Health Research. J. E. I. Gittens is supported by a Doctoral Research Award from the Canadian Institutes of Health Research.

Address for reprint requests and other correspondence: G. M. Kidder, Dept. of Physiology and Pharmacology, Univ. of Western Ontario, London, ON, Canada N6A 5C1 (E-mail: gerald.kidder{at}fmd.uwo.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00277.2002

Received 14 June 2002; accepted in final form 4 December 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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