Calmodulin transit via gap junctions is reduced in the absence of an electric field

Gap junctional transport of Calmodulin (CaM) from epithelial cells to insect oocytes is enhanced by alignment of the molecules via an electric field. It has recently been shown that CaM is needed for uptake of vitellogenins, is produced in the epithelial cells and reaches oocytes via gap junctions. For CaM to transit the gap junctions something must align these elongated molecules with the lumina of the gap junctions. This might be accomplished by the electric field that exists at the membrane of any cell with an Em of >0 mV. Fluorescently labeled CaM was injected into oocytes. At t=0, the epithelial cell/oocyte "fluorescence" ratio showed epithelial cells to be 24%+/-1.5% as bright as the injected oocyte. In follicles which maintained an electric field for one hour the epithelial cell/oocyte fluorescence ratio had risen to 79%+/-1.4%, while for follicles in which the field was cancelled by holding Em at 0 mV the ratio was only 45%+/-1.7%. After termination of the holding current follicles regained their original Em and their original electric field. At the end of a second hour of incubation the ratio had risen to 76%+/-1.2%, very close to what was observed in the untreated control follicles.     

Passage of 17 kDa calmodulin through gap junctions of three vertebrate species

Gap junctions of some vertebrates are capable of passing the elongate molecule, calmodulin, with a molecular weight 8-17 times greater than the previously recognized size limits. Fluorescently labeled calmodulin (FCaM) (17.34 kDa) microinjected into oocytes of ovarian follicles from an amphibian, Xenopus laevis, and from two species of teleost fish, Danio rerio (Zebrafish) and Oryzias latipes (Medaka), is shown to transit their gap junctions and enter the surrounding epithelial cells. Passage of FCaM was terminated when follicles were first treated with 1 mM octanol, a molecule known to down-regulate gap junctions. There was no FCaM detected in the surrounding medium, nor did epithelial cells become fluorescent when follicles were incubated in medium containing dye. Calmodulin is well known to modulate many cytoplasmic reactions; thus, its passage through gap junctions opens possibilities of additional means by which cells may be supplied with this signaling molecule, and by which their supply may be regulated.     

Importance of molecular configuration in gap junctional permeability

Gap junctions between insect oocytes and follicular epithelial cells allow transit of elongate Ca(2+)-binding proteins Calmodulin (CaM, 17kDa) and Troponin-C (Trop-C, 18kDa), but not multi-branched dextran (10kDa) nor the Ca(2+)-binding protein Osteocalcin (Osteo, 6kDa). By microinjection of fluorescently labeled versions of each of these molecules we were able to obtain visual evidence that, despite their lesser molecular weight, molecules with greater cross-sections were unable to transit these gap junctions, while heavier but elongate molecules could. While CaM had previously been shown to pass through gap junctions from oocytes to their surrounding epithelial cells, the ability of CaM and Trop-C to transit the gap junctions between adjacent epithelial cells had not been demonstrated. Evidence shown here demonstrates that the homologous gap junctions among epithelial cells, like the heterologous gap junctions between epithelial cells and the oocyte they surround, allow transit of elongate molecules up to at least 18kDa. Furthermore, the evidence for four different molecules of differing molecular weights and configurations supports the hypothesis that it is molecular configuration, not chemical activity, that primarily determines the observed permeability of gap junctions to molecules 5-6 times larger than the molecular weight limit previously acknowledged.     

Implication of gap junction coupling in amphibian vitellogenin uptake

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.     

Development of gap junctions in normal and mutant ovaries of Drosophila melanogaster

An ovarian follicle of Drosophila consists of an oocyte, 15 nurse cells, and hundreds of follicular epithelial cells. A freeze-fracture analysis of the surfaces between glutaraldehyde-fixed ovarian cells showed that all three cell types were interconnected by gap junctions. This is the first report of gap junctions between adjacent nurse cells, between nurse cells and oocytes, and between follicle cells and oocytes in Drosophila. Since we did not observe intramembranous particle clumping into crystalline patterns and since structurally different gap junctions occurred at different times in development and at different cell-cell interfaces, it is unlikely that fixation artifacts influenced particle distribution in our experiments. A computer-assisted morphometric analysis showed that the extent, size, and morphology of gap junctions varied with development and that these junctions can cover up to 9% of the cell surfaces. To test the role of gap junctions in follicular maturation, we studied ovaries from flies homozygous for the female sterile mutation fs(2)A17, in which follicles develop normally until yolk deposition commences. During the development of mutant follicles, gap junctions became abnormal before any other morphological aspect of the follicle. These studies show that gap junctions are available to play an important role in coordinating intercellular activities between all three cell types in ovarian follicles of Drosophila.     

Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their Vj sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).     

Calmodulin Colocalizes with Connexins and Plays a Direct Role in Gap Junction Channel Gating

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their V_j sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).     

Vitellogenesis in Oncopeltusfasciatus: PLC/IP3, DAG/PK-C pathway triggered by CaM

In Oncopeltus fasciatus, evidence shown here indicates it is calmodulin (CaM) that activates phospholipase-C (PLC), beginning a signalling pathway necessary for endocytic uptake of yolk precursor molecules. Epithelial cell-produced CaM, transported to oocytes via gap junctions, has been shown to be required for receptor-mediated endocytic uptake of vitellogenins (Vgs, the protein precursors of yolk). To determine if CaM was directly or indirectly stimulating the phospholipase-C (PLC) signalling cascade and thus controlling Vg endocytosis we used a series of molecules known to inactivate various elements of the pathway. W-7 prevents CaM from interacting with other molecules. Neomycin isolates PIP₂ from PLC. U-73122 directly inactivates PLC. 2-APB blocks IP₃ receptors which would otherwise cause release of Ca²⁺. Verapamil and CdCl₂ block Ca²⁺ release channels. Staurosporin and calphostin are inhibitors of PK-C. 1-Hexadecyl-2-acetyl glycerol (HAG) binds to diacylglycerol (DAG). Through the use of these antagonists we show here that: (1) the activation of phospholipase-C in this system requires CaM. (2) Stimulated phospholipase-C converts PIP₂ into IP₃ and DAG. (3) IP₃ causes increase in cytosolic Ca²⁺. (4) DAG and Ca²⁺ each stimulate phosphokinase-C, resulting in endocytosis of Vgs.     

Hormonal modulation of gap junctions in rat ovarian follicles

Summary Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24 h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.     

Structural basis for simultaneous binding of two carboxy-terminal peptides of plant glutamate decarboxylase to calmodulin

Activation of glutamate decarboxylase (GAD) by calcium-bound calmodulin (CaM) is required for normal plant growth through regulation of gamma-aminobutyrate and glutamate metabolism. The interaction of CaM with the C-terminal domain of GAD is believed to induce dimerization of the enzyme, an event implicated for Ca(2+)-dependent enzyme activation. Here, we present the solution structure of CaM in complex with a dimer of peptides derived from the C-terminus of Petunia hybrida GAD. The 23 kDa ternary complex is pseudo-symmetrical with each domain of CaM bound to one of the two antiparallel GAD peptides, which form an X-shape with an interhelical angle of 60 degrees. To accommodate the dimeric helical GAD target, the two domains of CaM adopt an orientation markedly different from that seen in other CaM-target complexes. Although the dimeric GAD domain is much larger than previously studied CaM-binding peptides, the two CaM domains appear closer together and make a number of interdomain contacts not observed in earlier complexes. The present structure of a single CaM molecule interacting with two target peptides provides new evidence for the conformational flexibility of CaM as well as a structural basis for the ability of CaM to activate two enzyme molecules simultaneously.     

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.     

Varied effects of 1-octanol on gap junctional communication between ovarian epithelial cells and oocytes of Oncopeltus fasciatus, Hyalophora cecropia, and Drosophila melanogaster

In insect gap junctions, species-specific differences occur in response to the purported gap junction uncoupling agent, 1-octanol. Changes in gap junctional communication between oocytes and their epithelial cells following treatment with 1-octanol were assayed in Oncopeltus fasciatus (the milkweed bug), Hyalophora cecropia (the American silk moth), and Drosophila melanogaster. In all three species, microinjection of untreated control follicles with Lucifer yellow CH revealed extensive dye coupling among epithelial cells and between epithelial cells and their oocytes. Also for all three species, treatment with octanol appeared to completely block dye coupling and increase oocyte input resistance. The effect on electrical coupling varied. In Drosophila, octanol diminished the electrical coupling from 64% (0.64 coupling coefficient) in controls to 53% in treated follicles. In Hyalophora, the coupling ratio remained the same following treatment. In Oncopeltus, octanol actually increased the electrical coupling ratio from 84% in controls to 94% in treated follicles. While 0.5 mM octanol left some Oncopeltus epithelial cells dye coupled to the oocyte, the electrical coupling ratio was increased slightly more by this concentration than by 1 or 5 mM octanol solutions, although the differences were not significant. While input resistance (R(o )) increased in all three following treatment with octanol, there was considerable difference in the magnitude of the response. Average oocyte R(o ) for Oncopeltus increased the least of the three species, rising from 196-240 kOhm. Both Hyalophora, with a nearly fourfold increase from 230-900 kOhm or more, and Drosophila, with a twofold increase from 701 kOhm to over 1.2 MegOhm showed much larger changes. Results shown here indicate that insect gap junctions have more varied responses to this common gap junction antagonist than have been reported for their vertebrate counterparts. Arch.     

Gap junction structure and cell-to-cell coupling regulation: is there a calmodulin involvement?

In most tissues neighboring cells communicate directly with each other by exchanging ions and small metabolites via cell-to-cell channels located at the intermembrane particles of gap junctions. Evidence indicates that the channels close when the [Ca2+]i or [H+]i increases. The channel occlusion (cell-to-cell uncoupling) is mainly a safety device by which cells can isolate themselves from damaged neighboring cells ("healing-over" process). Despite our knowledge of uncoupling agents, the uncoupling mechanism is still poorly understood. Uncoupling treatments have been shown to cause structural changes in gap junctions, characterized by an increase in tightness and regularity (crystallization) of particle packing and a decrease in particle size. Recently these changes have been shown to be induced by Ca2+ or H+ in isolated lens junctions and by Ca2+ in liver junctions, which suggests a close relationship between structural changes and uncoupling, but preliminary studies indicate that the junctional changes may not be synchronous with uncoupling but may lag behind it. However, recent X-ray diffraction data show that the channels of crystalline gap junctions (typical of uncoupled cells) are indeed closed, because they are inaccessible to sucrose (a gap junction permeant). Thus it seems that crystalline junctions are indeed in a non-permeable state, but the occlusion of the channels may precede the crystallization process. In the lens, junction crystallization is inhibited by a calmodulin (CaM) inhibitor, trifluoperazine (TFP). Is CaM involved in the uncoupling mechanism? To test this hypothesis, TFP and calmidazolium (CDZ), the most specific CaM inhibitor, were used on amphibian embryonic cells electrically uncoupled by CO2. Both TFP and CDZ effectively protect the cells from uncoupling, which suggests that CaM participates in the process. As a hypothesis, we propose that channel occlusion follows a CaM-mediated conformational change in the junctional protein. Particle crystallization may follow the conformational changes and result from a modification in electrostatic repulsion among the particles.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Ca signaling regulates ecmB expression,cell differentiation and slug regeneration in Dictyostelium

Ca²⁺ regulates cell differentiation and morphogenesis in a diversity of organisms and dysregulation of Ca²⁺ signal transduction pathways leads to many cellular pathologies. In Dictyostelium Ca²⁺ induces ecmB expression and stalk cell differentiation in vitro. Here we have analyzed the pattern of ecmB expression in intact and bisected slugs and the effect of agents that affect Ca²⁺ levels or antagonize calmodulin (CaM) on this expression pattern. We have shown that Ca²⁺ and CaM regulate ecmB expression and pstAB/pstB cell differentiation in vivo. Agents that increase intracellular Ca²⁺ levels increased ecmB expression and/or pstAB and pstB cell differentiation, while agents that decrease intracellular Ca²⁺ or antagonize CaM decreased it. In isolated slug tips agents that affect Ca²⁺ levels and antagonize CaM had differential effect on ecmB expression and cell differentiation in the anterior versus posterior zones. Agents that increase intracellular Ca²⁺ levels increased the number of ecmB expressing cells in the anterior region of slugs, while agents that decrease intracellular Ca²⁺ levels or antagonize CaM activity increased the number of ecmB expressing cells in the posterior. We have also demonstrated that agents that affect Ca²⁺ levels or antagonize CaM affect cells motility and regeneration of shape in isolated slug tips and backs and regeneration of tips in isolated slug backs. To our knowledge, this is the first study detailing the pattern of ecmB expression in regenerating slugs as well as the role of Ca²⁺ and CaM in the regeneration process and ecmB expression.     

Marker genes identify three somatic cell types in the fetal mouse ovary

The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.     

The gating effect of calmodulin and calcium on the connexin50 hemichannel

Gap junction channels formed by connexin50 (Cx50) are critical for the maintenance of eye lens transparency, which is sensitive to pH and external Ca2+ concentration, but the mechanism is still not clear. In this study we performed dye uptake-leakage assays, patch clamping and confocal co-localization experiments to confirm the function of calmodulin (CaM) and Ca2+ in the Cx50 hemichannel. Below pH 7.4, lucifer yellow (LY)-preloaded Cx50-HeLa cells allow dye to leak out when washed with Ca2+-free solution or incubated in solution containing 50 microg/ml W7 (CaM inhibitor) first, then washed in solution containing 2 mM Ca2+, whereas little or no dye leakage was observed when LY-preloaded Cx50-HeLa cells were incubated in solution containing 2 mM Ca2+. Moreover, in the absence of Ca2+, polarizing pulses applied to Cx50-HeLa activated outward transmembrane currents, which were inhibited by 2 mM external Ca2+. When Cx50-HeLa cells were incubated with 2 mM Ca2+ and 50 microg/ml W7, the transmembrane currents were activated again. This indicates that Ca2+ and CaM play a gating role in Cx50 hemichannels. Either the chelation of Ca2+ or the inhibition of CaM increased the permeability of Cx50 hemichannels. The same phenomena were observed below pH 6.5. Furthermore, CaM could be localized in gap junctions formed by Cx50 below pH 6.5. Our results demonstrate that CaM and Ca2+ can cooperate in the gating control of Cx50 hemichannels.     

Granulosa cells regulate oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms

Mammalian oocytes grow within ovarian follicles in which the oocyte is coupled to surrounding granulosa cells by gap junctions. We report here that growing oocytes isolated from mouse preantral follicles are incapable of recovering from an experimentally induced acidosis, and that oocytes acquire the ability to manage acid loads by activating Na(+)/H(+) exchange during growth. By contrast, granulosa cells from similar preantral follicles possess substantial Na(+)/H(+) exchange capacity, which is attributable to the simultaneous action of two Na(+)/H(+) exchanger isoforms: NHE1 and NHE3. Granulosa cells were also found to possess a V-type H(+)-ATPase that drives partial acidosis recovery when Na(+)/H(+) exchange is inactivated. By monitoring intracellular pH (pH(i)) in small follicle-enclosed oocytes, we found that the oocyte has access to each of these acidosis-correcting activities, such that small follicle-enclosed oocytes readily recover from acidosis in a manner resembling granulosa cells. However, follicle-enclosed oocytes are unable to access these activities if gap-junction communication within the follicle is inhibited. Together, these experiments identify the NHE isoforms involved in regulating oocyte pH(i), indicate that gap junctions allow granulosa cells to exogenously regulate oocyte pH(i) against acidosis until the oocyte has acquired endogenous pH(i) regulation, and reveal that granulosa cells possess multiple mechanisms for carrying out this function.