Gap Junctional Communication and Connexin43 Expression in Relation to Apoptotic Cell Death and Survival of Granulosa Cells
Department of Human Anatomy, Embryology, Histology and Medical Physics (DVK,SM,KD), and Department of Physiology and Pathophysiology (LL), Ghent University, Ghent, Belgium
Correspondence to: Dmitri V. Krysko, Dept. of Human Anatomy, Embryology, Histology and Medical Physics, Faculty of Medicine, Ghent University, Godshuizenlaan 4, B-9000 Ghent, Belgium. E-mail: dmitri.krysko{at}UGent.be
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Summary |
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Key Words: granulosa cells apoptosis gap junctions connexin43 FRAP
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Introduction |
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Most studies on apoptosis regulation, including ours, are focusing predominantly on intracellular signaling pathways (D'Herde et al. 2000; Krysko et al. 2001
), while tissue function obviously involves cell-to-cell communication. Granulosa cells can communicate either through local production of intraovarian factors such as cytokines (Adashi 1992
) and growth factors (Adashi et al. 1991
), which act as paracrine and/or autocrine modulators, or through gap junctions. Gap junctions are channels formed by two hemichannels, each formed by six connexin subunits contributed by each cell. All known connexins are transmembrane proteins with four transmembrane domains, two extracellular loops, one intracellular loop, and cytoplasmic amino and carboxyl termini (Bruzzone et al. 1996
; Munari-Silem and Rousset 1996
; Trosko and Ruch 1998
). Cx43 is the major gap junctional protein expressed in granulosa cells that makes a significant contribution to intercellular coupling (Dekel 1987
; Grazul-Bilska et al. 1997
; Farioli-Vecchioli et al. 2000
; Sommersberg et al. 2000
; Rosenfeld et al. 2001
; Gittens et al. 2003
). It has been shown that gap junctions play an important role in granulosa cell development, differentiation, and luteinization (Kidder and Mhawi 2002
). Classically, it is believed that gap junctions provide a way for cell-to-cell diffusion of hydrophilic molecules with a molecular mass of less than 1 kD, such as cAMP, Ca2+, IP3, and ATP (Kumar and Gilula 1996
). More recently, however, several studies demonstrated that Cx43 could affect signaling pathways independently of its contribution to gap junction channels (Moorby and Patel 2001
; Plotkin and Bellido 2001
). Despite the increased interest of research in this field, little is known regarding the involvement of gap junctional intercellular communication and Cx43 expression during apoptosis. In a previous study, we observed by electron microscopy (EM) in 24-hr serum-free cultured granulosa cell explants, in which apoptosis is elicited by gonadotropin withdrawal, an increased number of huge gap junctions in comparison with uncultured granulosa cells (D'Herde and Leybaert 1997
). This observation prompted us to investigate the relation between connexin expression, cell coupling, apoptosis, and survival of the granulosa cell explants.
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Materials and Methods |
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Quantification of Apoptosis by DAPI Staining
Granulosa cell explants were rinsed in 10 mM PBS, pH 7.4, and fixed in 4% buffered formaldehyde. Thereafter, granulosa cell explants were rinsed again in PBS and stained with a 1:1000 solution of DAPI (2',6'-diamidino-2-phenylindole; Sigma-Aldrich) in PBS. Mounted granulosa cell explants were examined in a Leica DM IRB/E inverted microscope and apoptotic cells were identified by their characteristic fragmented and condensed chromatin masses. Small groups of apoptotic bodies were counted as remnants of one apoptotic cell. Apoptotic indexes (AIs) are expressed as the number of apoptotic nuclei per number of total nuclei counted in the same microscopic field. This AI was averaged for 10 fields, giving a total number of 1500 cells counted per experiment.
Antibodies
A mouse monoclonal antibody (IgG1) raised against the peptide sequence corresponding to the amino acid residues 252270 of the native rat Cx43 was used for immunocytochemistry (ICC) and Western blotting and was purchased from ProBio (Poole, UK) and BD Biosciences (Erembodegem, Belgium), respectively. These monoclonal antibodies were reported to crossreact with avian Cx43 (Beyer 1990; Musil et al. 1990
). Affinity-purified secondary goat anti-mouse (IgG) antibody conjugated to horseradish peroxidase (HRP) were obtained from Cell Signaling Technology (Westburg, Leusden, The Netherlands) and secondary rabbitFITC anti-mouse (IgG) antibody was purchased from DAKO (Glostrup, Denmark).
Immunocytochemistry of Connexin43
After rinsing of granulosa cell explants with PBS, they were fixed for 20 min in 3% PBS-buffered paraformaldehyde. Next, explants were incubated for 20 min in 3% BSA and then for 10 min in 1% glycine. After 45-min incubation in 10% normal rabbit serum, the explants were incubated, directly and without a rinsing step, overnight at 4C with mouse monoclonal anti-Cx43 antibody (1:400 dilution of 1 mg/ml stock; or for the controls the corresponding IgG1 at the same concentration; DAKO). Then the granulosa explants were incubated for 45 min with rabbitFITC anti-mouse IgG (1:40). For double staining, the granulosa explants were stained with DAPI as described above, rinsed with 0.9% NaCl, and mounted with fluorescent mounting medium. Unless otherwise described, explants were rinsed thoroughly with PBS between all steps.
Quantification of Cx43 Immunofluorescence
Quantitative measurements of Cx43 immunofluorescence (Laing et al. 1997) were performed by determining the average fluorescence intensity in images obtained by confocal laser scanning microscopy (CLSM) of granulosa cell sheets or explants immunostained as described above. Images were acquired with a custom-made video-rate CLSM (Sanderson and Parker 2003
) built around a Nikon Eclipse TE300 (Analis; Ghent, Belgium) equipped with a x40 oil immersion objective (CFI Plan Fluor; Nikon) and with 488-nm excitation from an argon laser.
Protein Extraction and Western Blotting Analysis of Cx43 Expression
Granulosa cell explants were lysed in 50 µl of ice-cold modified RIPA buffer (50 mM NaCl, 0.5% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 250 mM -glycerophosphate, 25 mM Tris, pH 8.2) containing a standard phosphatase inhibitor cocktail 2 (20 µl/ml; Sigma-Aldrich) and complete EDTA-free protease inhibitor tablet (20 µl/ml; Roche Diagnostics, Brussels, Belgium) for 10 min and sonicated on ice three times for 10 sec using a Vibracell VC-130 sonicator. The protein concentration of each sample was measured according to Bradford (1976)
using the BioRad protein assay kit (BioRad Laboratories; Nazareth/Eke, Belgium) and a microtiter plate reader model 550 (Bio-Rad Laboratories). Protein samples were solubilized in Laemlli sample buffer and were heated at 100C for 10 min and centrifuged at 13,000 x g for 7 min to remove cell debris. Rat brain lysate was obtained from BD Biosciences (Erembodegem, Belgium) and was used as a positive control for Cx43 protein expression on Western blots. Approximately 55 µg of total protein from each sample was separated on 12% Tris-HCl precast gels (BioRad Laboratories). After separation, proteins were transferred to a nitrocellulose membrane (VWR; Zaventem, Belgium), blocked for 1 hr in TBS containing 5% non-fat dried milk and 0.1% Tween (blocking buffer), and incubated overnight at 4C with monoclonal antibodies to Cx43 diluted 1:250 in blocking buffer. After extensive washes in blocking buffer, membranes were probed for 1 hr with HRP-conjugated secondary goat anti-mouse IgG antibody diluted in blocking buffer (1:2000) and developed by enhanced chemiluminescence (Cell Signaling Technology; Westburg) according to the manufacturer's instructions. The time of exposure (typically 10 min) to X-ray films (Kodak Biomax Light-1; Sigma-Aldrich) was identical for all experimental conditions. The intensity of the bands after Western blotting was determined by scanning of the X-ray films followed by quantitative analysis using Total-lab software (Phoretix).
Transmission Electron Microscopy
The granulosa explants were fixed by immersion for 2 hr in 2% glutaraldehyde buffered with 0.1 M Na-cacodylate containing 1 mM CaCl2 (pH 7.4). After a rinse in 0.1 M Na-cacodylate containing 7.5% sucrose, the explants were osmicated in 2% OsO4 in 0.1 M Na-cacodylate and embedded in LX medium (Ladd; Burlington, VT). Ultrathin sections were stained with uranyl acetate and lead citrate and were analyzed on a Jeol 1200 EXII electron microscope.
Fluorescence Recovery After Photobleaching (FRAP)
Gap junctional coupling was investigated in granulosa cell sheets or explants using the FRAP technique (Braet et al. 2003). For this purpose, the sheets or explants were loaded with the gap junction-permeable fluorescent tracer 5-carboxyfluorescein diacetate (CFDA; Molecular Probes, Leiden, The Netherlands) by incubation in Hank's buffered salt solution (HBSS-HEPES) containing 50 µM CFDA for 60 min at room temperature and under continuous shaking. The preparations were then rinsed three times with HBSS-HEPES and transferred to the microscope stage of the CLSM described above. The loading and rinsing solutions contained in addition 1 mM probenecid (Sigma) to prevent rapid dye extrusion from the cells (Di Virgilio et al. 1988
; D'Herde and Leybaert 1997
). Fluorescence within a single cell was photobleached by spot exposure to the 488-nm line of an argon laser over a period of 10 sec. Spot exposure was done by reducing the scan amplitude of the horizontal and vertical scanning mirrors. CFDA fluorescence before and after photobleaching was recorded in confocal imaging mode with normal scanning mirror settings. Recovery of fluorescence because of dye influx from neighboring cells was quantified at 4 min after the start of photobleaching. A correction was made for the baseline bleaching effect associated with repeated exposure to the 488-nm imaging light. This was done by recording the fluorescence profile in cells not exposed to the spot bleach and correcting the recovery signal in proportion to the baseline fluorescence decay.
Gap Junction Blocking by 18 -Glycyrrhetinic Acid
Eighteen -glycyrrhetinic acid (3
-hydroxy-11-oxo-18
, 20
-olean-12-en-29-oic acid, AGA; Sigma) was dissolved in dimethylsulfoxide (DMSO; UCB, Leuven, Belgium). To study the effect of gap junction blocking on cell survival, granulosa cell explants were incubated for 24 hr with AGA doses ranging from 25 µM to 250 µM and AIs were calculated on DAPI- stained granulosa cell explants as described above. Cell coupling after treatment with AGA (250 µM, 24 hr) was investigated using FRAP.
Statistical Analysis
The data are expressed as mean ± SEM, with n denoting the number of experiments. Statistical significance was tested using a t-test for unpaired observations and using a p value of less than 0.05. Multiple groups were compared using variance analysis, followed by the Dunnett test for multiple comparisons to a control group or the Student-Newman-Keuls test for comparison of all groups among each other. Curve fittings for doseresponse relations were performed with non-linear least-square procedures available in the program Inplot.
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Results |
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Electron Microscopy
Electron microscopic analysis of granulosa cells demonstrated rare gap junctions in freshly isolated granulosa cells despite the positive Cx43 immunoreactivity described above, and showed huge gap junctions in 24h-SF and 24h-SF-H conditions (Figure 4) . In 24h-SF-Ca conditions, many short gap junctions were observed in accordance with the ICC punctate pattern (Figure 4). Internalization of gap junctions did not occur, as evidenced by the absence of annular junctions.
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Discussion |
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Granulosa cell explants provide a model system closely mimicking the apoptotic process that occurs in vivo during spontaneous follicular atresia (D'Herde and Leybaert 1997). Contributing factors to explain the relatively low apoptotic rates in 24h-SF granulosa cell explants might be the presence of native extracellular matrix, i.e., the vitelline and basement membranes, which are still present in the used model system and which are known to promote survival (Amsterdam et al. 1989
; Boudreau et al. 1995
). Second, as reviewed by Johnson (2000)
, death-suppressing members of the Bcl-2 family (Bcl-xLong, Bcl-2) are expressed in granulosa cells from preovulatory follicles. Another contributing factor is the expression of IAP-1, an inhibitor of apoptosis protein found in preovulatory hen granulosa cells (Johnson et al. 1998
).
The data of the present study demonstrated that, in freshly isolated granulosa cells, Cx43 immunolabeling was observed all around the cells, even in those regions of the plasma membrane where visible contact with any apposing plasma membrane of neighboring cells was missing. In the absence of any apposing cells, immunolabeling in the non-junctional plasma membrane regions is assumed to represent hemichannels of Cx43 (Musil and Goodenough 1991; Zampighi et al. 1999
; Quist et al. 2000
). Quantification of the Cx43 immunoreactivity (as revealed by ICC and Western blotting) in freshly isolated granulosa cell sheets showed a higher amount of Cx43 protein compared with 24h-SF or 24h-SF-H supplemented cultures in which at EM level long gap junctions between neighboring cells are easily detected. Despite the detected Cx43 immunolabeling and coupling of the cells as assessed by FRAP, extremely rare gap junctions were detected by EM analysis in these freshly isolated granulosa cells. This discrepancy between ultrastructural detection of gap junctions and ICC detection of their major component Cx43 might be explained by the fact that immunoreactivity is not confined only to gap junctions but also reveals Cx43 present in the cytoplasm or organized as connexin hemichannels. Moreover, one should consider the fact that clustering of gap junctions into plaques is the primary basis for their ultrastructural identification (Goodenough and Revel 1970
; McNutt and Weinstein 1970
).
In 24h-SF cultured granulosa cell explants, the apoptosis-inducing condition, well-organized gap junctions were observed (as revealed by ICC and EM), providing a better coupling of the granulosa cells than in freshly isolated granulosa cells as measured by FRAP analysis. However, a lower immunoreactive signal was detected in these 24h-SF granulosa cells (as revealed by quantitative analysis of both the confocal images and immunoblots) compared with freshly isolated granulosa cells. These apparently inconsistent findings underscore the fact that the abundance of Cx43 does not necessarily correlate with the amount of intracellular communication (Lau et al. 1992; Kurata and Lau 1994
; Laing et al. 1997
; Sia et al. 1999
).
In agreement with the data from the present study, Wilson et al. (2000) found an average increase of gap junctional coupling during initiation of apoptosis in serum-deprived (24-hr) cultures of a normal diploid epithelial cell line (WB-F344), assessed by the scrape load/dye transfer method. In the same study, individual cells were analyzed by the FRAP assay to compare nuclear morphology to differences in dye transfer during serum deprivation. The authors demonstrated that most apoptotic bodies did not communicate with neighboring cells. This is in line with the present ICC findings revealing that granulosa cells with clear apoptotic nuclear morphology do not show Cx43 immunolabeling at their plasma membrane. Kalvelyte et al. (2003)
reported that expression of Cx43 in HeLa-transfected cells accelerates the transition of cells from an early phase of apoptosis to the late phase.
To verify whether cell coupling is indeed a means of propagating a cell death message, experiments using the gap junctional intercellular communication inhibitor AGA were carried out. This compound was shown to block cell coupling (Davidson et al. 1986), presumably by disassembling gap junctional channels and affecting their dephosphorylation (Guan et al. 1996
). In the present model system, gap junctional coupling was significantly inhibited after long-term AGA exposure. Moreover, it was demonstrated that, by blocking gap junctional coupling with AGA, AIs were dose-dependently decreased (Figure 6). In agreement with the present data, Krutovskikh et al. (2002)
showed that in BC31 cells (a rat bladder carcinoma cell line) cell coupling and apoptosis were significantly inhibited by treatment with AGA. In addition, Lin et al. (1998)
reported that gap junctions achieved by transfection of Cx43 could mediate the propagation of a death signal between dying and healthy glial cells in a co-culture system.
A further argument in support of a role of cell coupling in the propagation of a death signal among granulosa cells comes from DAPI-stained granulosa explants in the 24h-SF condition (Figure 2J), which demonstrates that granulosa cells did not die randomly but, rather, in tracks of closely associated cells. This finding supports the observation described by Cotrina et al. (1998) and Krutovskikh et al. (2002)
that communicating cells die by clusters. Although apoptosis in granulosa cell explants is associated with increased cell coupling (as discussed above), it is important to note that increased cell coupling does not necessarily induce apoptosis per se, as illustrated in the Ca2+-rescue protocol. Therefore, our findings and those of others (Lin et al. 1998
; Sai et al. 2001
; Krutovskikh et al. 2002
; Kalvelyte et al. 2003
) are consistent with the hypothesis that gap junctional intercellular communication is indeed needed for cellcell transmission of a death signal and thus may contribute to the regulation of the apoptotic process in diverse model systems.
It is known that gap junctions are permeable to several second messengers, such as inositol-1,4,5-triphosphate, ATP, cAMP, and calcium.
In a rat bladder carcinoma line, it has been demonstrated that a reduction of gap junction permeability with oleamide, which appears not to affect the passage of Ca2+ ions, did not abrogate coordinated cell death by clusters, suggesting that Ca2+ ions are involved as the signal propagating cell death (Krutovskikh et al. 2002).
Interestingly, Cx43 immunoreactivity (as studied by ICC and immunoblotting) was inversely correlated with apoptosis (Figure 3C), suggesting that Cx43 expression plays a role in the survival process. Sasson and Amsterdam (2002) reported that apoptosis induction by LH and forskolin was accompanied by increased expression of Cx43 in human luteinized granulosa cells, a discrepancy that might be related to the use of different cell models and apoptosis-inducing stimuli. Several reports point to the fact that Cx43 plays a role in promoting survival in response to extracellular cues independently of gap junctional intercellular communication (Plotkin and Bellido 2001
; Plotkin et al. 2002
). Huang et al. (2001)
demonstrated that regulation of apoptosis by Cx43 in human glioblastoma cells is mediated by downregulation of the apoptosis inhibitor protein Bcl-2 and hence is not linked to gap junction channel function. Recently, Lin et al. (2003)
demonstrated that forced expression of Cx43 as well as two other members of the connexin family (Cx32 and Cx40) increased the resistance of astrocytes to injury and that the anti-death activity of connexin proteins was independent of the functional status of gap junctions.
In conclusion, the present study suggests that gap junctions mediate the propagation of a cell death message, while independently Cx43 expression is needed for survival. Future studies will focus on the experimental modulation of Cx43 expression and its effects on the major pro- and anti-apoptotic signaling pathways.
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Acknowledgments |
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We are very grateful to Barbara De Bondt, Dominique Jacobus, and Hubert Stevens for excellent technical assistance.
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Footnotes |
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Received for publication December 16, 2003; accepted April 18, 2004
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adashi EY (1992) The potential relevance of cytokines to ovarian physiology. J Steroid Biochem Mol Biol 43:439444[CrossRef][Medline]
Adashi EY, Resnick CE, Hurwitz A, Ricciarelli E, Hernandez ER, Roberts CT, Leroith D, et al. (1991) Insulin-like growth factors: the ovarian connection. Hum Reprod 6:12131219[Abstract]
Amsterdam A, Rotmensch S, Furman A, Venter EA, Vlodavsky I (1989) Synergistic effect of human chorionic gonadotropin and extracellular matrix on in vitro differentiation of human granulosa cells: progesterone production and gap junction formation. Endocrinology 124:19561964[Abstract]
Beyer EC (1990) Molecular cloning and developmental expression of two chick embryo gap junction proteins. J Biol Chem 265:1443914443
Boudreau N, Sympson CJ, Werb Z, Bissell MJ (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267:891893[Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
Braet K, Vandamme W, Martin PE, Evans WH, Leybaert L (2003) Photoliberating inositol-1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap 26. Cell Calcium 33:3748[CrossRef][Medline]
Bruzzone R, White TW, Goodenough DA (1996) The cellular internet: on-line with connexins. Bioessays 18:709718[Medline]
Cotrina ML, Kang J, Lin JHC, Bueno E, Hansen TW, He L, Liu Y, et al. (1998) Astrocytic gap junctions remain open during ischemic conditions. J Neurosci 18:25202537
Davidson JS, Baumgarten IM, Harley EH (1986) Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochem Biophys Res Commun 134:2936[Medline]
Dekel N (1987) Interaction between the oocyte and the granulosa cells in the preovulatory follcile. In Armstrong DT, Freisen HG, Leung PCK, Moger W, Ruf KB, eds. Endocrinology and Physiology of Reproduction. New York, Plenum Publishing, 197209
D'Herde K, De Prest B, Mussche S, Schotte P, Beyaert R, Coster RV, Roels F (2000) Ultrastructural localization of cytochrome c in apoptosis demonstrates mitochondrial heterogeneity. Cell Death Differ 7:331337[CrossRef][Medline]
D'Herde K, Leybaert L (1997) Intracellular free calcium related to apoptotic cell death in quail granulosa cell sheets kept in serum-free culture. Cell Death Differ 4:5965[CrossRef]
Di Virgilio F, Steinberg TH, Swanson JA, Silverstein SC (1988) Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions. J Immunol 140:915920
Farioli-Vecchioli S, Raes S, Espeel M, Roels F, D'Herde K (2000) Inverse expression of peroxisomes and connexin-43 in the granulosa cells of the quail follicle. J Histochem Cytochem 48:167178
Gilbert AB, Evans AJ, Perry MM, Davidson MH (1977) A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus). J Reprod Fertil 50:179181[Medline]
Gittens JE, Mhawi AA, Lidington D, Ouellette Y, Kidder GM (2003) Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis. Am J Physiol 284:C880887
Goodenough DA, Revel JP (1970) A fine structural analysis of intercellular junctions in the mouse liver. J Cell Biol 45:272290
Grazul-Bilska AT, Reynolds LP, Redmer DA (1997) Gap junctions in the ovaries. Biol Reprod 57:947957[Medline]
Guan X, Wilson S, Schlender KK, Ruch RJ (1996) Gap-junction disassembly and connexin 43 dephosphorylation induced by 18 beta-glycyrrhetinic acid. Mol Carcinog 16:157164[CrossRef][Medline]
Huang RP, Hossain MZ, Huang R, Gano J, Fan Y, Boynton AL (2001) Connexin 43 (cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells. Int J Cancer 92:130138[CrossRef][Medline]
Jiang JY, Cheung CK, Wang Y, Tsang BK (2003) Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia. Front Biosci 8:d222237[Medline]
Johnson AL (2000) Granulosa cell apoptosis: conservation of cell signaling in an avian ovarian model system. Biol Signals Recept 9:96101[CrossRef][Medline]
Johnson AL, Bridgham JT, Digby MR, Lowenthal JW (1998) Expression of the inhibitor of T-cell apoptosis (ita) gene in hen ovarian follicles during development. Biol Reprod. 58:414420[Abstract]
Kalvelyte A, Imbrasaite A, Bukauskiene A, Verselis VK, Bukauskas FF (2003) Connexins and apoptotic transformation. Biochem Pharmacol 66:16611672[CrossRef][Medline]
Kidder GM, Mhawi AA (2002) Gap junctions and ovarian folliculogenesis. Reproduction 123:613620[CrossRef][Medline]
Krutovskikh VA, Piccoli C, Yamasaki H, Yamasali H (2002) Gap junction intercellular communication propagates cell death in cancerous cells. Oncogene 21:19891999[CrossRef][Medline]
Krysko DV, Roels F, Leybaert L, D'Herde K (2001) Mitochondrial transmembrane potential changes support the concept of mitochondrial heterogeneity during apoptosis. J Histochem Cytochem 49:12771284
Kumar NM, Gilula NB (1996) The gap junction communication channel. Cell 84:381388[Medline]
Kurata WE, Lau AF (1994) p130gag-fps disrupts gap junctional communication and induces phosphorylation of connexin43 in a manner similar to that of pp60v-src. Oncogene 9:329335[Medline]
Laing JC, Tadros PN, Westphale EM, Beyer EC (1997) Degradation of connexin43 gap junctions involves both the proteasome and the lysosome. Exp Cell Res 236:482492[CrossRef][Medline]
Lau AF, Kanemitsu MY, Kurata WE, Danesh S, Boynton AL (1992) Epidermal growth factor disrupts gap-junctional communication and induces phosphorylation of connexin43 on serine. Mol Biol Cell 3:865874[Abstract]
Lin JH, Weigel H, Cotrina ML, Liu S, Bueno E, Hansen AJ, Hansen TW, et al. (1998) Gap-junction-mediated propagation and amplification of cell injury. Nature Neurosci 1:494500[CrossRef][Medline]
Lin JHC, Yang J, Liu S, Takano T, Wang X, Gao Q, Willecke K, et al. (2003) Connexin mediates gap junction-independent resistance to cellular injury. J Neurosci 23:430441
McNutt NS, Weinstein RS (1970) The ultrastructure of the nexus. A correlated thin-section and freeze-cleave study. J Cell Biol 47:666688
Moorby C, Patel M (2001) Dual functions for connexins: Cx43 regulates growth independently of gap junction formation. Exp Cell Res 271:238248[CrossRef][Medline]
Munari-Silem Y, Rousset B (1996) Gap junction-mediated cell-to-cell communication in endocrine glands-molecular and functional aspects: a review. Eur J Endocrinol 135:251264[Medline]
Musil LS, Beyer EC, Goodenough DA (1990) Expression of the gap junctional protein connexin-43 in embryonic chick lens: molecular cloning, ultrastructural localization and post-translational phosphorylation. J Membr Biol 116:163175[Medline]
Musil LS, Goodenough DA (1991) Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol 115:13571374[Abstract]
Mussche S, Leybaert L, D'Herde K (2000) First and second messenger role of calcium. Survival versus apoptosis in serum-free cultured granulosa explants. Ann NY Acad Sci 926:101115
Onagbesan OM, Peddie MJ (1988) Induction of ovulation and oviposition in female quail with luteinizing hormone, luteinizing hormone releasing hormone, or progesterone. Gen Comp Endocrinol 71:124131[Medline]
Plotkin LI, Bellido T (2001) Bisphosphonate-induced, hemichannel-mediated, anti-apoptosis through the Src/ERK pathway: a gap junction-independent action of connexin43. Cell Commun Adhes 8:377382[Medline]
Plotkin LI, Manolagas SC, Bellido T (2002) Transduction of cell survival signals by connexin-43 hemichannels. J Biol Chem 277:86488657
Quist AP, Rhee SK, Lin H, Lal R (2000) Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol 148:10631074
Rosenfeld CS, Wagner JS, Roberts RM, Lubahn DB (2001) Intraovarian actions of oestrogen. Reproduction 122:215226
Sai K, Kang KS, Hirose A, Hasegawa R, Trosko JE, Inoue T (2001) Inhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cell: relation to down-regulation of gap junctional intercellular communication. Cancer Lett 173:163174[CrossRef][Medline]
Sanderson MJ, Parker I (2003) Video-rate confocal microscopy. In Marriott G, Parker I, eds. Biophotonics. Methods in Enzymology. Vol 360. San Diego, Academic Press, 447481
Sasson R, Amsterdam A (2002) Stimulation of apoptosis in human granulosa cells from in vitro fertilization patients and its prevention by dexamethasone: involvement of cell contact and bcl-2 expression. J Clin Endocrinol Metab 87:34413451
Sia MA, Woodward TL, Turner JD, Laird DW (1999) Quiescent mammary epithelial cells have reduced connexin43 maintain a high level of gap junction intercellular communication. Dev Genet 24:111122[CrossRef][Medline]
Sommersberg B, Bulling A, Salzer U, Frohlich U, Garfield RE, Amsterdam A, Mayerhofer A (2000) Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone. Biol Reprod 63:16611668
Tilly JL (1997) Apoptosis and the ovary: a fashionable trend or food for thought? Fertil Steril 67:226228[CrossRef][Medline]
Trosko JE, Ruch RJ (1998) Cell-cell communication in carcinogenesis. Front Biosci 3:D208236[Medline]
Wilson MR, Close TW, Trosko JE (2000) Cell population dynamics (apoptosis, mitosis, and cell-cell communication) during disruption of homeostasis. Exp Cell Res 254:257268[CrossRef][Medline]
Zampighi GA, Loo DD, Kreman M, Eskandari S, Wright EM (1999) Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes. J Gen Physiol 113:507524