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Anatomisches Institut, Universität München (S.F., L.K., N.D., R.G., A.M.), D-80802 Munich, Germany; and Frauenklinik der Klinik am Eichert (C.H.), D-73006 Göppingen, Germany
Address all correspondence and requests for reprints to: Dr. Artur Mayerhofer, Anatomisches Institut, Universität München, Biedersteiner Strasse 29, D-80802 Munich, Germany. E-mail: . mayerhofer{at}lrz.uni- muenchen.de
Abstract
Acetylcholine (ACh) was recently described to be produced by and act on human luteinizing granulosa cells (GCs). Cholinergic agents increase intracellular calcium levels and stimulate GC proliferation via muscarinic receptors. Based on this observation and because endocrine cells of the forming human corpus luteum (CL), which are the in vivo counterparts of GCs, also proliferate in vivo, we hypothesized that ACh may be a factor involved in the regulation of the complex cellular events occurring during ovulation and formation of the CL. We addressed this possibility by investigating ACh/muscarinic receptor-mediated events in GCs. Normally, cultured GCs and their in vivo counterparts are coupled via gap junctions (GJ) consisting of connexin 43. Treatment with carbachol impaired GJ coupling of GCs within seconds, as shown in single cell, whole cell, patch-clamp studies. The cholinergic antagonist atropine and the muscarinic receptor antagonist pirenzepine specifically blocked this effect. Disruption of GJ communication of GCs is probably due to increased phosphorylation of connexin 43 at serine residues, as shown in immunoprecipitation experiments with carbachol-challenged GCs. Ovulation/formation of the CL include reprogramming of luteinizing cells, and in the rat this involves gonadotropin- induced expression of the transcription factor early growth response factor-1 (egr-1). In human GCs we found that carbachol as well as hCG can mimic this effect, as shown by cDNA arrays and semiquantitative RT-PCR. In conclusion, our results obtained in GCs suggest that endogenous, locally produced ACh may contribute to the cellular remodeling of the forming CL via muscarinic receptor/egr-1, thereby affecting proliferation, GJ communication, and regulation of gene expression in luteinizing granulosa cells.
OVULATION IS A complex event that results in the release of the oocyte from the ruptured follicle and the formation of the corpus luteum (CL) (1, 2). During this process, endocrine cells (granulosa and thecal cells) as well as stromal cells migrate, proliferate, and differentiate (1). These different yet interwoven events thus contribute to remodeling in ovarian tissue architecture. How events associated with ovulation and formation of the CL are regulated and orchestrated on the level of endocrine cells is at present not well understood. In the rat ovulation is accompanied by expression of a transcription factor, early growth response factor-1 (egr-1). This factor was shown to be induced by CG in granulosa cells (3), and it was suggested that egr-1 may be a master regulatory factor involved in the reprogramming of (follicular) granulosa cells to form large granulosa-luteal cells of the CL. Which genes are directly regulated as a consequence of egr-1 action in granulosa cells remains to be shown. To our knowledge it is not known whether, for example, genes associated with proliferation or gap junction (GJ) communication are among them.
Proliferation and GJs are of special interest in the context of physiological tissue remodeling in the ovary. Although granulosa cells in the follicle proliferate rapidly, proliferation ceases upon differentiation into large luteal cells in most species (1). However, in some species, including human and pig, evidence was provided that it may also occur later in the CL (1, 2, 4). Follicular granulosa cells and granulosa-luteal cells in the CL are normally coupled by GJs formed between adjacent cells (5, 6). These are membrane-associated protein channels, mainly composed of the GJ protein connexin 43 (Cx43) (7, 8, 9, 10, 11, 12, 13), which forms hexameric units in the membrane. GJs allow passive transfer of small molecules, including second messengers and ions (14). As a consequence, coupled cells respond in a coordinated way to signaling molecules, including hormones (15, 16). How cell proliferation and GJ communication are interrelated in general is not clear, but some hints come from studies of rapidly growing, undifferentiated tumor cells, which usually lack functional GJs (17, 18). Experimental introduction of Cxs and GJs induced differentiation and reduced proliferation rate (19, 20). As a negative correlation between the degree of GJ coupling and proliferation was found in most tumor cells, GJ communication and/or GJ proteins are thought to be suppressors of tumor cell growth and proliferation (18). Whether cellular proliferation and GJ coupling are interrelated in a similar way in nontumor cells, e.g. during physiological tissue remodeling associated with formation of the CL, is not known. Regulatory factors, such as LH and nerve growth factor, were shown to be involved in the regulation of GJ communication in granulosa and thecal cells (7, 21, 22). These factors disrupted GJ communication and increased phosphorylation of Cx43, an effect that may be linked to subsequent proliferation. In contrast, reinforced GJ communication and increased Cx43 gene expression are related to differentiation processes and reduced proliferation rate, as was shown for FSH (11).
We recently provided evidence for a novel intraovarian regulatory system involving ACh, which is produced by rat granulosa cells and human luteinized granulosa cells (GCs) (23). This neurotransmitter can act on ovarian target cells, namely GCs, which possess the muscarinic receptor M1 and M5, and on oocytes, which possess M3 (23). Importantly, stimulation of the muscarinic receptors of human GCs increased intracellular Ca2+ concentrations and resulted in increased proliferation of GCs (24). As proliferation is an important process occurring around the time of ovulation and possibly persists in the human CL, we hypothesized that ACh may be involved in the regulation of CL formation. To explore the possible contribution of ACh/muscarinic receptors to these events, we studied human GCs as a model for the ovulatory follicle/young CL. We investigated whether and how muscarinic receptor activation affects GJ communication and identified a transcription factor linked to ACh/muscarinic receptor action in human GCs. In addition, we examined whether proliferation of endocrine cells occurs in the human CL.
Materials and Methods
The preparation and culture of human GCs, derived from in vitro fertilization patients, have been described previously (24). Paraffin- embedded, fixed human ovaries (n = 3) containing CL were obtained from patients undergoing gynecological surgery. The procedures were approved by the local ethics committees, and patients gave written consent to the use of cells or tissues. Unless otherwise mentioned cells were treated on d 1 or 2 of culture. The following substances were added: carbachol (0.1 µmol/liter; a stable acetylcholine derivate), hCG (10 IU/ml; both from Sigma, Deisenhofen, Germany), and pirenzepine (10 µmol/liter; a selective M1 muscarinic antagonist; Biotrend, Köln, Germany). Concentrations were chosen to match those described previously (25).
Immunoprecipitation and Western blotting as well as RT-PCR were performed as previously described (21, 24, 26). Anti-Cx43 antiserum (1:1000–2000) and antiphosphoserine (1:100) were purchased from Sigma.
Oligonucleotide primers for tubulin and egr-1 used for PCR were synthesized according to the published sequences (tubulin: accession no. K 00558; sense, 5'-CAC CCG TCT TCA GGG CTT CTT GGT TT-3'; antisense, 5'-CAT TTC ACC ATC TGG TTG GCT GGC TC-3'; egr-1: accession no. NM001964; antisense, 5'-ACA AGA AAG CAG ACA AAA GTG-3'; sense, 5'-GGG AAG TGG GCA GAA AGG ATT-3'). The ODs of immunoreactive bands and ethidium bromide-stained cDNAs were analyzed as previously described (26) using ANOVA/Fisher’s protected least significant difference and Scheffé’s post-hoc tests, Student- Newman-Keuls test, or t test, respectively. Verification of cDNAs was achieved by sequencing (26).
A cDNA array technique was used to characterize expression profiles of immediate-early genes in unstimulated control cells and carbachol-stimulated cells. We used the human Pathfinder nonradioactive GEArray (Biomol, Hamburg, Germany) composed of 23 genes. cDNA probes were synthesized from 2 total RNA samples (5 µg each) using SuperArray’s GEAprimer mix for RT. The cDNAs were labeled by integration of biotin-16-dUTP (Roche, Mannheim, Germany). The biotinylated cDNA probes were hybridized to gene-specific cDNA fragments and spotted on the membranes, followed by chemiluminescent detection (CDP-Star, Roche). The relative expression levels of multiple genes involved in a particular pathway were determined. After digitalization (Microtek Scan Maker 4, Kiel, Germany), density analysis (NIH Image 1.40) (26, 27) was performed, and ODs of differently expressed genes were normalized by comparison with the control genes glyceraldehyde-3-phosphate dehydrogenase and actin.
Immunocytochemical methods were described previously (21, 23, 28). Anti-PCNA (1:40) was purchased from Calbiochem (Bad Soden, Germany). Antisteroidogenic acute regulatory protein (anti-StAR) antiserum was a gift from Dr. B. Hales (Chicago, IL) (29). Immunoelectron microscopy for Cx43 was performed using Lowicryl (K4M, Polysciences Europe, Eppenheim, Germany)-embedded GCs, with the same antiserum (1:2,000) as that used for immunoprecipitation and a secondary gold-labeled antiserum (1:20; 10 nm; Aurion, Wageningen, The Netherlands).
Single cell whole cell patch-clamp recordings were performed with cells on d 1–10 of culture as previously described (11, 30, 31). The functional coupling between adjacent cells and the regulation of the resulting cell to cell communication were studied by means of the single cell whole cell patch-clamp technique as described recently (11). Alterations of GJ communication were assessed by measuring the total capacitance (Cpair) of two or more neighboring cells (32, 33, 34). In theory, the Cpair of fully coupled cells should equal the sum of the single cell capacities. A decline in coupling, i.e. an increase in GJ resistance, results in a decrease in Cpair. The Cpair after complete disruption of coupling resembles the capacitance of the single cell connected to the patch-clamp pipette.
We selected pairs or small groups of cells and clamped one of the cells in the whole cell configuration using an EPC-9 patch-clamp amplifier and the software PULSE (HEKA electronic, Lambrecht, Germany). Patch pipettes were pulled from borosilicate glass tubings (GB150–8P, Science Products, Hofheim, Germany) and fire-polished with a DMZ-Universal Puller (Zeitz, Augsburg, Germany). Filled with electrolyte solution, they showed a resistance of 2–4 M
. The extracellular solution contained 140 mmol/liter NaCl, 3 mmol/liter KCl, 1 mmol/liter CaCl2, 10 mmol/liter HEPES, and 10 mmol/liter glucose. The intracellular (pipette) solution contained 130 mmol/liter KCl, 5 mmol/liter NaCl, 1 mmol/liter MgCl2, and 10 mmol/liter HEPES. A free Ca2+ concentration of 100 nmol/ liter was achieved by the addition of 1 mmol/liter CaCl2 and 2 mmol/liter EGTA. The pH of both solutions was adjusted to 7.4 with 10 mol/liter NaOH or 10 mol/liter KOH, respectively. The patched cells were clamped to the actually measured resting membrane potential (-50 to -15 mV). A sinusoidal voltage pulse around -60 mV (110 Hz; peak to peak amplitude, 40 mV) was applied for electrical stimulation. The membrane Cpair could be assessed from the measured admittance using the software lock-in amplifier utility of the EPC-9. A stimulation frequency of 110 Hz differing from that used by Sommersberg et al. (11) for rat GCs was chosen to take into account specific electrical properties of the human GCs (34). Control measurements on single cells (n = 4) were carried out to assure that single cell capacities are constant during any drug application and could not affect the total capacitance in cell pairs or groups. Functional coupling in cell pairs or groups was assessed by application of 5 mmol/liter 1-heptanol (Sigma) at the end of each measurement. Solutions were applied by a fast pressurized perfusion system (MPCU-3, Lorenz Meßgeräte, Lindau, Germany) equipped with a seven-channel perfusion pipette (List Electronic, Darmstadt, Germany) and a ValveLink8 Controller (AutoMate Scientific, Inc., San Francisco, CA). The concentrations of these drugs were similar to levels typically applied in electrophysiological studies, but were different from the concentrations presented in other parts of this study, namely, 100 µmol/liter carbachol, 100 µmol/liter pirenzepine, and 1 µmol/liter atropine (all purchased from Sigma).
Results
Evidence for cell proliferation and Cx43 expression in the CL and cultured human GCs
Using immunocytochemical methods we observed that the proliferation marker proliferating all nuclear antigen (PCNA) is present in the nuclei and cytoplasm of some large granulosa-luteal cells of the human CL (Fig. 1A
). In the CL, luteal cells are functionally coupled with their neighbor cells via GJs, consisting of the GJ protein Cx43 (8, 7, 22, 35) (Fig. 1B). Cultures of human luteinizing GCs, characterized by expression of StAR protein, share this trait (Fig. 2) and are able to proliferate (24). Therefore, they were used to examine ACh/muscarinic receptor-mediated effects.
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Whole cell measurements were performed on 51 independent cell pairs or cell groups gained from 15 different cell preparations. The existence of functional GJs was confirmed by applying 5 mmol/liter 1-heptanol, a decoupling agent, at the end of each measurement. In 92% of these measurements, 1-heptanol induced a fast decrease in Cpair down to single cell values (Fig. 3, B and C). Cholinergic regulation of GJ communication was investigated by applying the ACh agonist carbachol. The addition of 100 µM carbachol reduced electrical coupling in seconds to minutes in 86% of cell pairs or groups (n = 14; Fig. 3A). After application of carbachol over a period of 160 sec, the average change in Cpair was 
Cpair (160 sec) = -14 ± 5%. To examine specificity of carbachol action two different cholinergic antagonists, atropine and pirenzepine, were used. Simultaneous application of carbachol with either 1 µmol/liter atropine (Fig. 3B) or 100 µmol/liter pirenzepine (Fig. 3C) inhibited the carbachol effect in 81% [n = 16 measurements on cell pairs or groups; Cpair (160 sec) = -1.0 ± 0.4%]. The antagonists alone had no significant effect on electrical coupling of adjacent cells [n = 13 measurements; Cpair (160 sec) = -1 ± 1%; Fig. 3, B and C]. The inhibitory effect of the specific muscarinic antagonist pirenzepine in 88% (n = 8; Fig. 3C) clearly demonstrates the involvement of stimulation of muscarinic receptor in GJ regulation. Spontaneous disruption of electrical coupling was observed in 18% of all measurements, exhibiting a run of the curve similar to the carbachol-induced effect. In these experiments the decrease in Cpair started during addition of buffer or even before any drug application. In a few experiments GJ coupling was reduced during the simultaneous application of carbachol and one of the antagonists. These exceptions are probably caused by spontaneous closure of GJs, especially as they occurred with the same incidence (19%) as the unambiguously identified, drug-independent decoupling (18%). This explanation is sustained by the onset of decoupling during application of the antagonist alone in these few cases.
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Using immunoprecipitated Cx43 protein of human GCs treated with carbachol for 5, 10, 15, and 20 min, serine phosphorylation of Cx43 was increased within 5–20 min over values in untreated controls. Increases, as judged from changes in ODs, ranged from 25–400% over values in corresponding untreated cells (not shown; P < 0.05, by t test; n = 7). Samples of three experiments were further analyzed (11). To this end, immunoprecipitated proteins were run in duplicate on gels, one was probed for phosphoserine and the other for Cx43, and served to normalize the results. Increases in three different phosphorylated forms of Cx43 were evaluated and, after normalization to total Cx43, accounted for 10–290% depending on the band (Fig. 4).
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To identify genes induced by stimulation with carbachol in GCs, a cDNA array technique was used that revealed elevated levels of the zinc finger gene egr-1 (Fig. 5A). This observation was verified by semiquantitative RT-PCR experiments using RNA derived from unstimulated GCs and GCs treated with carbachol for different intervals. Results were normalized using tubulin as an internal control. Cells stimulated with hCG served as a positive control for induction of egr-1 (3). Our results show an increase in egr-1 levels compared with unstimulated control levels within 90 min and up to 24 h of treatment (Fig. 5B). The relative levels of increase after 90 min (n = 1), 6 h (n = 2), and 24 h (n = 2), respectively, ranged from 112–188% over the corresponding controls. In addition, we noticed an increase in egr-1 expression after stimulation with hCG (112–136%; n = 2).
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Cell proliferation and differentiation in the ovary are interwoven, as yet poorly understood, processes that are occurring, for example, in the growing follicle (1) and during formation of the CL. The CL forms after the ovulatory rupture of the follicle and involves proliferation and differentiation of thecal cell-derived, small luteal cells and granulosa cell-derived, large luteal cells as well as endothelial cells and other cell types (1). Evidence for cell proliferation of large luteal cells was previously provided by the detection of Ki67 and BrDU incorporation (e.g. in the human or pig CL) (1, 2, 4). This became obvious in the present study, in which PCNA was used to identify proliferating cells in the human CL. Proliferation of luteinizing granulosa cells around or even beyond the periovulatory phase on one side and well documented, intense GJ coupling (7, 22, 36) of these cells on the other side raise the question of how proliferation and GJ communication are linked and regulated in the forming CL. To address this issue we used human luteinized GCs derived from preovulatory follicles, representing in vitro counterparts of large luteal cells of the forming CL. These cells are rather pure in culture and are capable of proliferation, but also express Cx43 and form functional GJ. Importantly, they are also a source of the neurotransmitter ACh and express functional muscarinic receptors (23, 24). As previously shown, ACh represents an intrinsic regulatory factor that can stimulate cell proliferation via para/autocrine pathways (24). In the present study we report details of the consequences of muscarinic receptor activation in GCs. We show that cholinergic stimulation of muscarinic receptors in GCs affected the GJ protein Cx43 and caused disruption of GJ communication. We also report that activation of muscarinic receptor induced expression of the transcription factor egr-1.
To examine how initiation of proliferation by a cholinergic stimulus is related to GJ communication, we employed two approaches, a single cell whole cell patch-clamp technique and immunoprecipitation of Cx43. Our electrophysiological study showed functionally coupled cells that respond to muscarinic receptor agonists with a disruption of GJ communication. Disruption of GJ coupling occurred within seconds to minutes after addition of the cholinergic agonist carbachol. This effect was observed in almost 90% of the cell pairs or groups tested. Coupling was not affected by carbachol in the presence of the muscarinic receptor antagonist pirenzepine or atropine, showing the muscarinic receptor-mediated specificity of the carbachol effect. The single cell patch-clamp technique was previously employed to measure GJ communication in taste receptor cells (31), rat kidney fibroblasts (32), bovine ciliary epithelial cells (33), Rat-1 cells (30), and rat granulosa cells (11). Disruption of GJ communication was reported to be complete after 5–10 min (31, 32, 33), i.e. a period of time similar to this study. Interestingly, carbachol did not alter GJ communication in about 15% of the cell pairs tested. Currently, the reasons for the insensitivity of these cells are not known, but a comparable percentage of the ciliary epithelial cell pairs examined in another study were likewise resistant to carbachol regulation (33).
Gating and thus function of GJ channels are controlled by several mechanisms. Besides Cx turnover, degradation (37), and direct regulation by Ca2+ (38), it is phosphorylation of Cx43 (39) that appears to be of special importance. Cx43 phosphorylation at serine residues is a well established consequence of action of PKA or PKC (5). In human GCs stimulation of M1R/M5R is known to increase intracellular Ca2+ concentrations (24, 40) and most likely activates PKC. It is therefore conceivable that rapid serine phosphorylation of Cx43 may occur. That Cx43 was indeed rapidly, i.e. within minutes, phosphorylated by carbachol in GCs is shown in the present study, thus providing a likely explanation for the disruption of GJ communication observed in our electrophysiological studies.
A recently described mechanism of regulation of Cx43/GJ appears to involve direct phosphorylation of Cx43 by a member of the MAPK family, namely, p42 (41). This was shown in HeLa and mouse cell lines expressing Cx43. Epidermal growth factor increased serine phosphorylation of Cx43 and disrupted GJ communication, and the MEK1 inhibitor PD98059 blocked this effect (41). In the case of human GCs, our preliminary results (not shown) do not indicate involvement of p42/p44 or other MAPKs in the regulation of Cx43 by carbachol. Additional studies will be required to address this point as well as the role of GJ in the process of CL formation, function, and regression.
Ovulation and formation of the CL involve reprogramming of endocrine cells. Recent evidence indicated that the zinc finger transcription factor egr-1 (42, 43, 44) is an important immediate-early gene that acts as a molecular master switch in a variety of normal and pathological states (44). Thus, egr-1 was shown to coordinate regulation of divergent gene families (44). In the rat, egr-1 was recently identified to be induced by CG in ovarian granulosa cells around the time of ovulation (3). We demonstrated that treatment with carbachol as well as with hCG causes an increased expression of egr-1 in human GCs (present report). It is possible that the low levels of egr-1 mRNA detected in untreated controls are due to endogenous ACh produced by GCs (23). Our findings are in line with two previous studies in other cell types showing that egr-1 is among the genes regulated after cholinergic muscarinic receptor activation (42, 43). These novel results therefore not only pinpoint a transcription factor induced by ACh/muscarinic receptor in GCs, but also strongly suggest that the signaling pathway of the intraovarian ACh/muscarinic receptor system and that of the hormones hCG/LH are using the same transcription factor.
In conclusion, the proliferation of endocrine cells is an integral part of physiological tissue rearrangement in the forming CL. Our results obtained in GCs, representing an in vitro model of the forming human CL, identify details of the ACh/muscarinic receptor signaling cascade and lead us to propose that via regulation of GJ and Cx43 as well as via egr-1, the ovarian ACh/muscarinic receptor system is involved in the tissue-remodeling process in this organ.
Acknowledgments
We thank U. Berg and F. D. Berg for providing human GCs; G. Terfloth, M. Rauchfuss, B. Zschiesche, A. Thalhammer, A. Krieger, A. Mauermayer, and G. Prechtner for technical assistance; and A. Bulling for helpful discussions. We are grateful to B. Hales for the gift of StAR protein antiserum.
Footnotes
This work was supported by Deutsche Forschungsgemeinschaft (Ma1080) and Volkswagen-Stiftung.
Abbreviations: ACh, Acetylcholine; CL, corpus luteum; Cpair, total capacitance; Cx43, connexin 43; egr-1, early growth response factor-1; GC, granulosa cell; GJ, gap junction; PCNA, proliferating cell nuclear antigen; StAR, steroidogenic acute regulatory protein.
Received August 21, 2001.
Accepted December 6, 2001.
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