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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ovarian granulosa cells are
coupled via gap junctions containing connexin43 (Cx43 or -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; -1 connexin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 M) 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,
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
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, 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.
|
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.
|
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.
|
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ackert, CL,
Gittens JEI,
O'Brien MJ,
Eppig JJ,
and
Kidder GM.
Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse.
Dev Biol
233:
258-270,
2001[ISI][Medline].
2.
Alcoléa, S,
Théveniau-Ruissy M,
Jarry-Guichard T,
Marics I,
Tzouanacou E,
Chauvin JP,
Briand JP,
Moorman AFM,
Lamers WH,
and
Gros D.
Downregulation of connexin45 gene products during mouse heart development.
Circ Res
84:
1365-1379,
1999
3.
Bruzzone, S,
Guida L,
Zocchi E,
Franco L,
and
De Flora A.
Connexin43 hemichannels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells.
FASEB J
15:
10-12,
2001
4.
Bukauskas, FF,
Elfgang C,
Willecke K,
and
Weingart R.
Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human cells.
Biophys J
68:
2289-2298,
1995[Abstract].
5.
Carabatsos, MJ,
Sellitto C,
Goodenough DA,
and
Albertini DF.
Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence.
Dev Biol
226:
167-179,
2000[ISI][Medline].
6.
Castro, C,
Gómez-Hernandez JM,
Silander K,
and
Barrio LC.
Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations.
J Neurosci
19:
3752-3760,
1999
7.
Contreras, JE,
Sánchez HA,
Eugenin EA,
Speidel D,
Theis M,
Willecke K,
Bukauskas FF,
Bennett MVL,
and
Sáez JC.
Metabolic inhibition induces opening of unopposed connexin43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture.
Proc Natl Acad Sci USA
99:
495-500,
2002
8.
Eloff, BC,
Lerner DL,
Yamada KA,
Schuessler RB,
Saffitz JE,
and
Rosenbaum DS.
High resolution optical mapping reveals conduction slowing in connexin43 deficient mice.
Cardiovasc Res
51:
681-690,
2001[ISI][Medline].
9.
Eskandari, S,
Zampighi GA,
Leung DW,
Wright EM,
and
Loo DDF
Inhibition of gap junction hemichannels by chloride channel blockers.
J Membr Biol
185:
93-102,
2002[ISI][Medline].
10.
Francis, D,
Stergiopoulos K,
Ek-Vitorín JF,
Cao FL,
Taffet SM,
and
Delmar M.
Connexin diversity and gap junction regulation by pHi.
Dev Genet
24:
123-136,
1999[ISI][Medline].
11.
Fujiwara, T,
Oda K,
Yokota S,
Takatsuki A,
and
Ikehara Y.
Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum.
J Biol Chem
263:
18545-18552,
1988
12.
Gao, Y,
and
Spray DC.
Structural changes in lenses of mice lacking the gap junction protein connexin43.
Invest Ophthalmol Vis Sci
39:
1198-1209,
1998[Abstract].
13.
Goldberg, GS,
Bechberger JF,
and
Naus CCG
A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer.
Biotechniques
18:
490-497,
1995[ISI][Medline].
14.
Guerrero, PA,
Schuessler RB,
Davis LM,
Beyer EC,
Johnson CM,
Yamada KA,
and
Saffitz JE.
Slow ventricular contraction in mice heterozygous for a connexin43 null mutation.
J Clin Invest
99:
1991-1998,
1997
15.
Itahana, K,
Tanaka T,
Morikazu Y,
Komatu S,
Ishida N,
and
Takeya T.
Isolation and characterization of a novel connexin gene, Cx-60, in porcine ovarian follicles.
Endocrinology
139:
320-329,
1998
16.
John, SA,
Kondo R,
Wang SY,
Goldhaber JI,
and
Weiss JN.
Connexin43 hemichannels opened by metabolic inhibition.
J Biol Chem
274:
236-240,
1999
17.
Juneja, SC,
Barr KJ,
Enders GC,
and
Kidder GM.
Defects in the germ line and gonads of mice lacking connexin43.
Biol Reprod
60:
1263-1270,
1999
18.
Kamermans, M,
Fahrenfort I,
Schultz K,
Janssen-Bienhold U,
Sjoerdsma T,
and
Weiler R.
Hemichannel-mediated inhibition in the outer retina.
Science
292:
1178-1180,
2001
19.
Kidder, GM,
and
Mhawi AA.
Gap junctions and ovarian folliculogenesis.
Reproduction
123:
613-620,
2002
20.
Lauf, U,
Giepmans BNG,
Lopez P,
Braconnot S,
Chen SC,
and
Falk MM.
Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells.
Proc Natl Acad Sci USA
99:
10446-10451,
2002
21.
Lecanda, F,
Warlow PM,
Sheikh S,
Furlan F,
Steinberg TH,
and
Civitelli R.
Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction.
J Cell Biol
151:
931-943,
2000
22.
Lerner, DL,
Yamada KA,
Schuessler RB,
and
Saffitz JE.
Accelerated onset and increased incidence of ventricular arrhythmias induced by ischemia in Cx43-deficient mice.
Circulation
101:
547-552,
2000
23.
Li, H,
Liu TF,
Lazrak A,
Peracchia C,
Goldberg GS,
Lampe PD,
and
Johnson RG.
Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells.
J Cell Biol
134:
1019-1030,
1996[Abstract].
24.
Li, R,
and
Mather JP.
Lindane, an inhibitor of gap junction formation, abolishes oocyte directed follicle organizing activity in vitro.
Endocrinology
138:
4477-4480,
1997
25.
Manthey, D,
Bukauskas F,
Lee CG,
Kozak CA,
and
Willecke K.
Molecular cloning and functional expression of the mouse gap junction gene connexin-57 in human HeLa cells.
J Biol Chem
274:
14716-14723,
1999
26.
Mao, AJ,
Bechberger J,
Lidington D,
Galipeau J,
Laird DW,
and
Naus CCG
Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43.
J Biol Chem
275:
34407-34414,
2000
27.
Montecino-Rodriguez, E,
and
Dorshkind K.
Regulation of hematopoiesis by gap junction-mediated intercellular communication.
J Leukoc Biol
70:
341-347,
2001
28.
Okuma, A,
Kuraoka A,
Iida H,
Inai T,
Wasano K,
and
Shibata Y.
Colocalization of connexin 43 and connexin 45 but absence of connexin 40 in granulosa cell gap junctions of rat ovary.
J Reprod Fertil
107:
255-264,
1996[Abstract].
29.
Plotkin, LI,
Manolagas SC,
and
Bellido T.
Transduction of cell survival signals by connexin-43 hemichannels.
J Biol Chem
277:
8648-8657,
2002
30.
Quist, AP,
Rhee SK,
Lin H,
and
Lal R.
Physiological role of gap-junctional hemichannels: extracellular calcium-dependent isosmotic volume regulation.
J Cell Biol
148:
1063-1074,
2000
31.
Reaume, AG,
De Sousa PA,
Kulkarni S,
Langille BL,
Zhu D,
Davies TC,
Juneja SC,
Kidder GM,
and
Rossant J.
Cardiac malformation in neonatal mice lacking connexin43.
Science
267:
1831-1834,
1995[ISI][Medline].
32.
Roscoe, WA,
Barr KJ,
Mhawi AA,
Pomerantz DK,
and
Kidder GM.
Failure of spermatogenesis in mice lacking connexin43.
Biol Reprod
65:
829-838,
2001
33.
Simon, AM,
Goodenough DA,
Li E,
and
Paul DL.
Female infertility in mice lacking connexin37.
Nature
385:
525-529,
1997[ISI][Medline].
34.
Stout, CE,
Costantin JL,
Naus CCG,
and
Charles AC.
Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.
J Biol Chem
277:
10482-10488,
2002
35.
Valdimarsson, G,
De Sousa PA,
and
Kidder GM.
Co-expression of gap junction proteins in the cumulus-oocyte complex.
Mol Reprod Dev
36:
7-15,
1993[ISI][Medline].
36.
White, TW,
and
Paul DL.
Genetic diseases and gene knockouts reveal diverse connexin functions.
Annu Rev Physiol
61:
283-310,
1999[ISI][Medline].
37.
Wright, CS,
Becker DL,
Lin JS,
Warner AE,
and
Hardy K.
Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation.
Reproduction
121:
77-88,
2001
38.
Ya, J,
Erdtsieck-Ernste EBHW,
de Boer PAJ,
van Kempen MJA,
Jongsma H,
Gros D,
Moorman AFM,
and
Lamers WH.
Heart defects in connexin43-deficient mice.
Circ Res
82:
360-366,
1998