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Biology of Reproduction 65, 777-783 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Second Messengers on Gap Junctional Intercellular Communication of Ovine Luteal Cells Throughout the Estrous Cycle1

Anna T. Grazul-Bilska2,a,b, Lawrence P. Reynoldsa,b, Jerzy J. Bilskia,b, and Dale A. Redmera

a Department of Animal and Range Sciences b Cell Biology Center, North Dakota State University, Fargo, North Dakota 58105

ABSTRACT

Corpora lutea (CL) from Days 5, 10, and 15 after superovulation were enzymatically dispersed, and a portion of the cells were elutriated to obtain fractions enriched with small or large luteal cells. Mixed, small, and large luteal cell fractions were incubated with no treatment or with agonists or antagonists of cAMP (dbcAMP or Rp-cAMPS), protein kinase C (PKC; TPA or H-7), or calcium (A23187, EGTA, or A23187 + EGTA). The rate of contact-dependent gap junctional intercellular communication (GJIC) was evaluated by laser cytometry. Media were collected for progesterone (P4) radioimmunoassay, and luteal cells cultured with no treatment were fixed for immunocytochemistry or frozen for Western blot analysis. Luteal cells from each stage of the estrous cycle exhibited GJIC. The dbcAMP increased (P < 0.05) GJIC for all cell types across the estrous cycle. The Rp-cAMPS decreased (P < 0.05) GJIC for small luteal cells on Day 5 and for all cell types on Days 10 and 15. The TPA inhibited (P < 0.01), but H-7 did not affect, GJIC for all cell types across the estrous cycle. The A23187 decreased (P < 0.05) GJIC for large luteal cells touching only small or only large luteal cells, whereas A23187 + EGTA decreased (P < 0.05) GJIC for all cell types across the estrous cycle. For the mixed and large luteal cell fractions, dbcAMP increased (P < 0.05), but TPA and A23187 + EGTA decreased (P < 0.05), P4 secretion. The A23187 alone decreased (P < 0.05) P4 secretion by large, but not by mixed, luteal cells. For all days and cell types, the rate of GJIC and P4 secretion were correlated (r = 0.113–0.249; P < 0.01). Connexin 43 was detected in cultured luteal cells by immunofluorescence and Western immunoblotting. Thus, intracellular regulators like cAMP, PKC, or calcium appear to regulate GJIC, which probably is an important mechanism for coordinating function of the ovine CL.

corpus luteum function, ovary, signal transduction, progesterone

INTRODUCTION

The corpus luteum (CL) exhibits regular periods of growth, differentiation, and regression during each estrous cycle and/or pregnancy [1–3]. Successful development and function of the CL requires proliferation, differentiation, and interaction between steroidogenic and nonsteroidogenic luteal cells [2–8]. Rapid tissue growth and differentiation, such as that exhibited by the ovine CL, must be highly regulated and coordinated [2, 7, 8].

Growth and cellular interactions may be mediated by several mechanisms, including humoral (e.g., endocrine or paracrine) as well as contact-dependent (e.g., gap junctional) pathways [6–8]. Gap junctional intercellular communication (GJIC) probably is critical for coordination of cellular metabolism and function during growth and differentiation of organs and tissues [9, 10]. Cellular interactions mediated by contact-dependent and contact-independent mechanisms have been demonstrated for the CL of several species [6–8, 11, 12]. Several small-molecular-size regulators of luteal function may be transported through gap junctions from cell to cell [13].

The growth, differentiation, and regression of the CL are regulated by several factors, including luteinizing hormone (LH), a major luteotropin, and prostaglandin F2{alpha} (PGF), which is recognized as a major luteolytic factor [3, 4]. Both LH and PGF may activate second messengers, including cAMP, protein kinase C (PKC), or calcium [4–6]. Several studies have evaluated the effects of LH, PGF, and agonists and antagonists of cAMP, PKC, or calcium on ovine luteal morphology, progesterone (P4) production, or intracellular signaling [4, 14–23]. However, to our knowledge, the effects of second messengers on contact-dependent cellular interactions within ovine luteal tissues have not yet been evaluated.

The function and structure of gap junctions are regulated by numerous factors, including stage of development, hormones, growth factors, and intracellular regulators [7–10, 24, 25]. Structural and functional gap junctions and/or gap junctional proteins have been shown in the luteal tissues of several species. Structural gap junctions have been demonstrated by electron microscopy and by immunohistochemical detection of specific gap junctional proteins, such as connexin (Cx), in luteal tissues of several species [7, 8]. Connexin 43 seems to be one of the major gap junctional proteins in luteal tissues [7, 8]. Functional gap junctions have been demonstrated for ovine and bovine luteal cells in vitro and in situ using dye-transfer techniques [7, 8]. We have shown previously that GJIC among luteal cells is affected by several regulators of luteal function, including LH and PGF [7, 8].

The aim of this study was to evaluate the in vitro effects of agonists or antagonists of cAMP, PKC, or calcium on GJIC and P4 secretion of ovine luteal cells; the relationship between GJIC and P4 secretion; and the expression of Cx43 in cultured luteal cell types throughout the estrous cycle.

MATERIALS AND METHODS

Materials

For cell cultures, Dulbecco modified Eagle medium (DMEM), Ham F-10 medium, Ca2+- and Mg2+-free Hank balanced salt solution, fetal bovine serum (FBS), calf serum (CS), crystalline bovine insulin, trypan blue stain (0.4% [w/v]), and penicillin-streptomycin were purchased from Gibco (Grand Island, NY). Bovine serum albumin (fraction V), dimethylsulfoxide, transferrin, hydrocortisone, etiocholan-3-ol-17-one, nitroblue tetrazolium, NAD+, N6,2'-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP), o-tetradecanoylphorbol 13-acetate (TPA), calcium ionophore (A23187), and ethylene glycol-bis(ß-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) were purchased from Sigma (St. Louis, MO). 5-Carboxy-fluorescein diacetate acetoxymethyl ester (CFDA-AM) was purchased from Molecular Probes (Eugene, OR). Cyclic AMP antagonist (Rp-cAMPS) was purchased from BioLog, Life Science Institute (La Jolla, CA). Protein kinase C inhibitor (H-7) was purchased from Toronto Research Chemicals (North York, ON, Canada).

For localization of Cx43 by immunocytochemistry, Cx43 antibody was obtained from Dr. E.M. Hendrix (Southern Missouri State University, Springfield, MO) and Dr. W.J. Larsen (University of Cincinnati, OH). Fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit IgG) was purchased from Boehringer-Mannheim (Indianapolis, IN), Triton X-100 from Malinckrodt (Paris, KY), and normal goat serum from Vector Labs (Burlingame, CA). For detection of Cx43 by Western immunoblot, antibody against Cx43 was purchased from Zymed (San Francisco, CA), Immobilon-P membrane from Millipore (Bedford, MA), and peroxidase-labeled anti-rabbit antibody and Western blotting detection enhanced chemiluminescence (ECL) reagents from Amersham (Little Chalfont, UK). For the P4 radioimmunoassay (RIA), P4 standard was purchased from Sigma and tritiated P4 from DuPont/New England Nuclear Products (Boston, MA). The P4 antibody (GDN-337) was provided by Dr. G.D. Niswender (Colorado State University, Fort Collins, CO). For induction of superovulation, follicle-stimulating hormone (FSH; Super-Ov) was purchased from Ausa International (Tyler, TX).

Tissue Collection

Ovaries were collected on Days 5, 10, and 15 (n = 6 ewes/day) of the estrous cycle from superovulated ewes that had previously exhibited at least one estrous cycle of normal duration (15–18 days). To induce superovulation, FSH was injected i.m. twice daily on Days 13–15 of the estrous cycle as described previously [26, 27]. Day 0 of the estrous cycle (i.e., standing estrus) was determined using vasectomized rams. At slaughter, CL were dissected from the ovaries, weighed, and then dissociated with collagenase followed by elutriation as described below. In addition, one CL from each ewe was frozen in liquid nitrogen for determination of P4 concentrations by RIA [1]. Protocols for this study and animal care were approved by the Institutional Animal Care and Use Committee.

Dissociation and Culture of Luteal Cells

Luteal tissues were dissociated using collagenase as described previously [25, 26, 28]. A portion of the dispersed (i.e., mixed) population of luteal cells was used to obtain fractions enriched with small or large luteal cells through an elutriation procedure as described by Grazul-Bilska et al. [26]. The mixed (i.e., nonelutriated) population of luteal cells (not including nonsteroidogenic cells) contained 94.1% ± 0.4% (mean ± SEM) small and 5.9% ± 0.4% large luteal cells on Day 5 and 60.9% ± 9.1% small and 39.1% ± 9.1% large luteal cells on Days 10 and 15 of the estrous cycle. The small luteal cell fraction was not contaminated by large luteal cells, whereas the large luteal cell fraction contained 17.5% ± 3.0% (4–29%) small luteal cells across the estrous cycle. Viability of luteal cells was 87.2% ± 3.2% before elutriation. After elutriation across the estrous cycle, viability was 84.9% ± 2.2% for small and 79.9% ± 3.9% for large luteal cell fractions.

Luteal cells were resuspended in plating medium (DMEM containing 1% [v/v] FBS, 1% [v/v] CS, and antibiotics [100 U of penicillin and 100 µg/ml of streptomycin]) and preincubated for 24 h in 35-mm Petri dishes at 37°C in a humidified atmosphere (5% CO2 and 95% air). Due to low serum concentrations, the rate of attachment on nonsteroidogenic cells to the dish was low, and the proportion of nonsteroidigenic cells after 24 h of incubation was negligible [26]. For evaluation of GJIC or immunocytochemistry, the mixed luteal cell or small luteal cell fractions were plated at a concentration of 2.5 x 104 per cloning cylinder/dish, and large luteal cells were plated at a concentration of 0.5 x 104 per cloning cylinder/dish. Cloning cylinders were used to obtain subconfluent cultures within a small area (50 mm2) of the Petri dish. For Western immunoblot, cells were plated at a concentration of 3 x 105 cells per 35-mm Petri dish.

After preincubation, plating medium was changed to serum-free medium [25], and luteal cells were incubated in this serum-free medium with no treatment, dbcAMP (2 mM), Rp-cAMPS (300 µM), TPA (100 ng/ml), H-7 (100 µM), A23187 (1 µM), or EGTA (100 µM) for 2 h. The dbcAMP is an agonist of cAMP, whereas Rp-cAMPS is an antagonist of cAMP [29]. The TPA is an activator of PKC [30], and H-7 is an inhibitor of PKC [31]. The A23187 is a calcium ionophore, and EGTA is a calcium chelator [32]. The dose of each factor and the time of incubation were established based on the results of previous as well as preliminary experiments [7, 8, 25, 28, 29].

For each ewe, two dishes with the mixed or large luteal cell fractions were assigned to each treatment for evaluation of GJIC. After incubation with treatments, media were collected for determination of P4 concentrations, and GJIC was measured by evaluation of fluorescence recovery after photobleaching (FRAP) as described below. Moreover, two dishes with the mixed, small, or large luteal cells cultured without treatments were fixed in ethanol:glacial acetic acid (5.7:1 [v/v]) for 20 min and used for immunofluorescent detection of Cx43. An additional three dishes of each type of cells were scraped with a rubber policeman, and cells were stored at -70°C until used for detection of Cx43 by Western immunoblot analysis. Two other dishes of each type of luteal cell culture were fixed in formalin for 10 min and then used for detection of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) [26, 29].

Analysis of Contact-Dependent GJIC by FRAP

Gap junctional intercellular communication between luteal cells in culture was monitored by dye coupling with a Meridian ACAS 570 workstation (Meridian Instruments, Okemos, MI) using the FRAP technique [25, 26, 28, 29]. Briefly, after incubation with treatments, medium was removed from each dish, and fresh serum-free medium containing the fluorescent probe (CFDA-AM, 15 µM) was added. After a 10-min incubation (22°C), dishes were rinsed three times with serum-free medium to remove excess CFDA-AM. Dishes were then placed onto the interactive laser cytometer, and three fields (180 x 180 µm per field) on each dish were identified for scanning. For each field, 6–12 cells were selected and analyzed for initial fluorescence intensities. Immediately after measurement of initial fluorescence, the fluorescent probe was photobleached in four to eight selected cells in each field. To determine the rate of FRAP, the fluorescence intensity of all selected cells was quantified every 4 min for 8 min after photobleaching. As reported previously, only the linear portion of the fluorescence recovery curve (i.e., the first 4 min after photobleaching) was used. Treatments did not affect the initial fluorescence intensities of the luteal cells.

Identification of Luteal Cell Types

As we have described previously [25, 26, 28], the initial identification of steroidogenic luteal cells in culture was based on the characteristic granular appearance of the cytoplasm. Morphological classification was confirmed for two dishes from each culture by staining for 3ß-HSD as described previously [26, 28]. The criteria used for identification of specific steroidogenic luteal cell types have been described in detail elsewhere [25, 26, 28]. The majority (90–99%) of cultured luteal cells were steroidogenic cells as determined based on 3ß-HSD staining (data not shown). For all cultures, the rate of FRAP was determined for small luteal cells in contact only with small luteal cells (S-S), for large luteal cells in contact only with small luteal cells (L-S), and for large luteal cells in contact only with large luteal cells (L-L).

Immunocytochemistry

The presence of Cx43 in cultured luteal cells was visualized using immunofluorescence as described previously [33–35]. Fixed luteal cells were treated for 20 min with blocking buffer consisting of PBS (0.01 M phosphate and 0.14 M NaCl [pH 7.3]) containing 0.3% (v/v) Triton X-100 and 1% (v/v) normal goat serum, followed by incubation with a rabbit polyclonal antibody against Cx43 overnight at 4°C. Detection of the primary antibody was accomplished using an fluorescein isothiocyanate-conjugated secondary antibody (i.e., goat anti-rabbit IgG). Control staining consisted of replacing the primary antibody with the same dilution of rabbit serum.

Image Analysis

For the mixed fraction of luteal cells, the percentage of the total area that exhibited immunofluorescent staining for Cx43 was evaluated quantitatively with an image-analysis system (VIDAS version 2.5; Roche Image Analysis Systems, Los Altos, CA) as described previously [33]. For each luteal cell culture, seven randomly chosen fields that exhibited a monolayer of cells (0.025 mm2 per field) were evaluated in each culture plate (n = 14 measurements/culture). Background fluorescence was minimal and adjusted to the same level for each section by the image-analysis system. The data are reported as the percentage (mean ± SEM) of the total area within each field that exhibited positive Cx43 staining.

Western Immunoblot Analysis

Western immunoblot analysis was performed using a previously validated procedure [33–35]. Briefly, the mixed, small, or large luteal cell fraction was collected after culture without treatments from additional dishes, homogenized in buffer (1% [w/v] cholic acid and 0.1% SDS [w/v] in PBS), and sonicated with an ultrasonic processor (Sonics & Materials, Danbury, CT). Due to an insufficient number of cells as well as the large number of samples, we performed Western immunoblot only for those cells cultured without treatments. Samples of protein from cultured luteal cell types (100 µg; n = 3–5 ewes/day per cell type) and rat heart (50 µg; used as a control tissue) were added to loading buffer, boiled for 2 min, and then applied to a 12% polyacrylamide gel with a 3% stacking gel. After electrophoresis, separated proteins were electroblotted onto an Immobilon-P membrane and then immunoblotted with a rabbit polyclonal antibody against Cx43. Next, membranes were incubated with a peroxidase-labeled anti-rabbit antibody, followed by detection with ECL reagents. Densitometry was performed with a densitometer (Model PDSI; Molecular Dynamics, Sunnyvale, CA). After scanning the autoradiograph, a grid was drawn so that each sample was contained within rectangles of equal size, and the densitometric volume contained within each rectangle was then measured.

Progesterone RIA

Progesterone concentrations in benzene-hexane extracts of luteal homogenates and unextracted luteal conditioned media were measured by RIA [1, 25, 26]. The sensitivity of the assay was 12.5 pg/tube, and the intra- and interassay coefficients of variation were 4.5% and 8.9%, respectively. The recovery of 3H-P4 added to samples before extraction was 72.8% ± 1.2%, and concentrations of P4 in extracts were adjusted for recovery.

Statistical Analysis

Data for CL weights, luteal P4 concentrations, effects of treatments on the rates of GJIC and P4 secretion by luteal cells, area of positive staining for Cx43 in cultured luteal cells, and densitometry of Western immunoblots were analyzed using general linear model analysis of variance [36]. Correlations between the rates of GJIC and P4 were evaluated using PROC CORR of SAS [36]. When an F-test was significant (P < 0.05), differences between specific means were evaluated with the Bonferroni multiple comparison procedure [37].

RESULTS

Mean weights of CL were 209 ± 6, 363 ± 23, and 274 ± 14 mg on Days 5, 10, and 15, respectively, and were greatest (P < 0.05) on Day 10, less (P < 0.05) on Day 15, and least (P < 0.05) on Day 5 of the estrous cycle. Mean P4 concentrations in luteal tissues were 11.4 ± 0.7, 18.9 ± 1.9, and 5.1 ± 2.2 µg/g CL on Days 5, 10, and 15, respectively, and were greatest (P < 0.05) on Day 10, less (P < 0.05) on Day 5, and least (P < 0.05) on Day 15 of the estrous cycle.

Across all treatments in both experiments, the rate of GJIC of isolated (i.e., not touching any other cells), photobleached large and small cells (n = 123) was 0.1% ± 0.01% per minute over 4 min, which demonstrates that cells not in direct contact with other cells did not regain fluorescence after photobleaching; that is, FRAP was contact-dependent for luteal cells, as we have previously reported [25, 26]. The fluorescence value for nonphotobleached cells (n = 1693), whether in contact with other cells or not, was 99.5% ± 1% of the initial fluorescence values at the end of 4-min period, which shows that nonspecific photobleaching, leakage of a fluorescent probe from the cells, and a shift in the equilibrium of the fluorescent dye within the cells also were negligible. Luteal cells communicated with each other, and the basal rates of GJIC (i.e., no treatment) were similar throughout the estrous cycle within each cell type (Table 1).


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  		TABLE 1. Effects of agonists and antagonists second messengers on GJIC of ovine luteal cell types throughout the estrous cycle.a

The rate of GJIC was affected by agonists or antagonists of the various second messengers (Table 1). Across the estrous cycle, dbcAMP (cAMP agonist) increased (P < 0.05) the rate of GJIC for all cell types. On Day 5, Rp-cAMPS (cAMP antagonist) decreased (P < 0.01) the rate of GJIC for L-L cells but not for S-S or L-S cells. In contrast, Rp-cAMPS decreased (P < 0.05) the rate of GJIC for all cell types from Days 10 to 15 of the estrous cycle. Throughout the estrous cycle, TPA (PKC activator) completely inhibited (P < 0.01) the rate of GJIC for all cell types, but H-7 (PKC inhibitor) did not affect the rate of GJIC. Throughout the estrous cycle, A23187 (calcium ionophore) alone decreased (P < 0.01) the rate of GJIC for L-S and L-L cells, but it did not affect the rate of GJIC for S-S cells. However, in the presence of EGTA (calcium chelator), A23187 inhibited (P < 0.01) GJIC for S-S cells at all stages of the estrous cycle, but EGTA alone did not affect GJIC of luteal cell types throughout the estrous cycle. This demonstrates synergistic effects of A23187 and EGTA on GJIC of S-S, but not that of L-S or L-L, cells.

Basal secretion of P4 by the mixed luteal cell fraction was greater (P < 0.05) on Day 10 than on Days 5 and 15 of the estrous cycle, which were similar (8.7 ± 0.5 vs. 4.4 ± 0.5 and 3.8 ± 1.2 ng/ml, respectively). Due to the large variation, basal secretion of P4 by large luteal cells only tended to be greater (P < 0.15) on Day 10 than on Days 5 and 15 of the estrous cycle (5.8 ± 1.6 vs. 3.3 ± 0.5 and 1.8 ± 0.9 ng/ml, respectively). The effects of the second-messenger agonists and antagonists were similar for Days 5, 10, and 15 of the estrous cycle; therefore, data are combined for all days (Fig. 1). In the mixed luteal cell cultures, secretion of P4 was increased (P < 0.05) by dbcAMP and decreased (P < 0.05) by TPA and A23187 + EGTA, but it was not affected by Rp-cAMPS, H-7, A23187, or EGTA alone (Fig. 1). In cultures of large luteal cells, secretion of P4 was increased (P < 0.05) by dbcAMP; decreased (P < 0.05) by TPA, A23187, and A23187 + EGTA; and not affected by Rp-cAMPS, H-7, or EGTA alone (Fig. 1).



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  		FIG. 1. Effects of agonists and antagonists of second messengers on P4 secretion by mixed and large luteal cells throughout the estrous cycle. Data are expressed as a percentage of the no treatment (control) value. For values of basal P4 production, see Results. *P < 0.05 vs. no treatment (control)

Table 2 shows coefficients of correlation between the rates of GJIC and P4 secretion in cultures of mixed and large luteal cell fractions on Days 5, 10, and 15 of the estrous cycle. Across all days and luteal cell types, the coefficient of correlation was significant (P < 0.01) but low (range, 0.113–0.249).


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  		TABLE 2. Correlation coefficients between rate of GJIC (% FRAP/min) and P4 concentration (ng/ml) in cultured media from ovine luteal cells cultures throughout the estrous cycle.a

Immunofluorescent staining showed that Cx43 was present in mixed, small, and large luteal cell cultures throughout the estrous cycle (Fig. 2). Punctate staining was located at the periphery of the cells at sites of cell-cell contact (Fig. 2). In mixed luteal cell cultures, image analysis demonstrated that the area of positive staining for Cx43 was similar (P > 0.05) on Days 5, 10, and 15 of the estrous cycle (0.108% ± 0.005%, 0.128% ± 0.012%, and 0.081% ± 0.013%, respectively).



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  		FIG. 2. Immunofluorescent localization of Cx43 in cultured A) mixed, B) small, and C) large luteal cells from Day 10 of the estrous cycle, as well as control staining (D). Note that staining for Cx43 exhibits brightly fluorescent, punctate areas in regions of cell contact. The pattern of staining was similar for Days 5, 10, and 15 of the estrous cycle; therefore, a representative culture from Day 10 of the estrous cycle is shown. S, Small luteal cells; L, large luteal cell. Bar = 20 µm

Western immunoblot analysis revealed bands of 43–45 kDa for cultured mixed, small, and large luteal cell fractions from all stages of the estrous cycle (Fig. 3). Densitometry demonstrated that expression of Cx43 was similar (P > 0.05) across the estrous cycle for mixed luteal cells (5330 ± 572, 6180 ± 960, and 4892 ± 315 optical density [OD] units on Days 5, 10, and 15 of the estrous cycle, respectively; n = 5 ewes/day). The Cx43 expression was also similar (P > 0.05) for large and small luteal cell fractions (9116 ± 938 and 6832 ± 638 OD units, respectively; n = 3 ewes/day per cell type) across the estrous cycle (Fig. 3).



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  		FIG. 3. Representative Western immunoblot of Cx43 in cultured ovine luteal cells from Days 5, 10, and 15 of the estrous cycle. Lanes 1–3 represent mixed luteal cells from Days 5, 10, and 15, respectively. Lane 4 represents rat heart, which was used as a positive control. Lanes 5–10 represent small (lanes 5, 7, and 9) or large (lanes 6, 8, and 10) luteal cells from Days 5 (lanes 5 and 6), 10 (lanes 7 and 8) and 15 (lanes 9 and 10) of the estrous cycle. The Mr value is indicated (x 103)

DISCUSSION

The present data demonstrate that ovine luteal cell types communicated with each other via contact-dependent mechanisms, and that the rate of GJIC was affected by intracellular regulators. Cyclic AMP appears to be a potent stimulator of GJIC, whereas activation of PKC or high intracellular calcium levels appear to be potent inhibitors of GJIC of ovine luteal cells. In addition, the gap junctional protein Cx43 was expressed in cultured ovine luteal cells across the estrous cycle.

In luteal tissues, several second messengers may be involved in intracellular signaling during growth, differentiation, and regression of the CL. Cyclic AMP, PKC, and/or calcium are thought to mediate cellular responses to the major regulators of luteal function, including LH and PGF [3, 4, 14–16].

In the present experiment, the cAMP agonist stimulated, whereas the cAMP antagonist inhibited, GJIC of luteal cells. Similar effects have been observed for bovine luteal cell types [28]. In numerous cell-culture systems, cAMP has been shown to be an important regulator of gap junction function [7, 8]. It also has been demonstrated that cAMP may affect the rate of transcription of Cx43 mRNA, stability of mRNA, permeability or conductance of gap junctions, expression of gap junctional proteins and mRNA encoding these proteins, and thereby may affect the number and function of gap junctions [10, 24, 29, 38]. However, at present, the specific mechanism by which cAMP-dependent protein kinase might affect GJIC of ovine luteal cells is not clear.

In ewes, small and large luteal cells contain a cAMP-dependent protein kinase system, and adenyl cyclase activation increases intracellular concentrations of cAMP [17]. However, cAMP appears to increase P4 secretion in small, but not in large, luteal cells [14, 17, 26, 39]. In the present experiment, we observed more than a twofold increase in P4 secretion by the mixed luteal cells in response to dbcAMP. We also found a slight increase in P4 secretion by large luteal cell fractions in response to dbcAMP, but this was likely due to contamination by small luteal cells. However, cAMP antagonist did not affect P4 secretion by luteal cells. Nevertheless, in the present study, activation of cAMP-dependent protein kinase had stimulatory effects on GJIC and P4 secretion by luteal cells. Inhibition of cAMP-dependent protein kinase had moderate inhibitory effects on GJIC but not on P4 secretion. Perhaps stronger inhibition of cAMP-dependent protein kinase is required to completely inhibit GJIC and P4 secretion. The present data indicate that cAMP-dependent protein kinase is involved in regulation of GJIC in both small and large ovine luteal cells.

In the present experiment, activation of PKC strongly inhibited GJIC, but an inhibitor of PKC did not affect GJIC of ovine luteal cells. Similar effects of PKC activators or inhibitors on GJIC have been reported for bovine luteal cells as well as numerous other cell types [7, 8, 28, 38, 40]. The effects of TPA on intercellular communication may include closure of gap junction channels, reduction of gap junction numbers, and a decrease of gap junctional protein expression [38]. In luteal tissues, TPA may inhibit GJIC by acting through any, or all, of these mechanisms.

Protein kinase C mediates the intracellular action of a number of hormones and neurotransmitters [41]. Protein kinase C activity has been detected in ovine luteal tissues [21, 22, 42], and PKC has been demonstrated to be involved in regulation of ovine luteal function by affecting P4 secretion, activity of the cholesterol side-chain cleavage enzyme, and cell viability [18, 21, 22]. In the present experiment, TPA decreased P4 secretion by mixed and large luteal cells. Inhibitory effects of TPA on ovine mixed and large luteal cell function also have been demonstrated by others [15, 18, 21, 22]. Because TPA inhibited both GJIC and P4 secretion by ovine luteal cells in the present study, we suggest a possible functional relationship between these processes.

Calcium has been demonstrated to be involved in regulation of gap junction function in numerous tissues, including CL [7, 8, 10, 38, 43, 44]. In the present experiment, the calcium ionophore decreased GJIC between large and small luteal cells and between large luteal cells, but it did not affect GJIC between small luteal cells. However, in the presence of EGTA, the calcium ionophore inhibited GJIC among all ovine luteal cell types. Similarly, increased intracellular calcium resulting from calcium ionophore treatment or microinjection of calcium ions inhibited GJIC in several cell types [10, 38, 43, 44]. The inhibitory effects of high calcium concentrations on gap junction function may be indirect through abolishing the generation of cAMP [43] or by activation of the PKC pathway [15]. Gap junctions are permeable to physiological concentrations of calcium ions, and physiological concentrations of intracellular calcium do not inhibit gap junction function [10]. Therefore, it has been hypothesized that calcium does not affect gap junction function at physiological concentrations [38]. However, under pathological and/or toxicological conditions, calcium likely plays an important role as a safekeeper of normal communication within cell communities [45].

Numerous studies have demonstrated that second messengers are important regulators of gap junction function in many tissues, including ovine luteal tissues [7, 8, 10, 38]. Second messengers are also involved in regulation of luteal function [3, 14, 15]. In addition, gap junctions are important for transfer of second messengers within tissues [9, 45]. A reciprocal relationship exists between gap junctions and second messengers: gap junctions are dynamically regulated by second-messenger pathways, and the extent by which second messengers are spread from one cell to another depends on the rate of conductance of gap junctions [10].

Data from our study demonstrated an interrelationship between GJIC and P4 secretion. In the presence of dbcAMP, which enhanced GJIC, secretion of P4 by luteal cells also was increased. Similarly, in the presence of TPA or A23187, which inhibited GJIC, P4 secretion was decreased. We previously have reported that both GJIC and P4 secretion were enhanced in bovine or ovine luteal cell cultures in the presence of LH, dbcAMP, or forskolin [25, 26, 28, 29]. In addition, we and others have demonstrated that suppression of Cx43 production by oligonucleotides decreased LH or ACTH-induced steroid secretion by bovine luteal cells [46] or adrenal cells [47], respectively. These data suggest that gap junctions indeed may be involved in the regulation of luteal P4 secretion. On the other hand, the possibility that P4 affects gap junction function cannot be excluded. This subject requires further study.

The present experiment also demonstrated presence of the gap junctional protein Cx43 in cultured luteal cells throughout the estrous cycle. In addition, the expression of Cx43 in luteal cells in vitro was similar throughout the estrous cycle. This lack of differences in Cx43 levels may explain our inability to demonstrate differences in the rate of GJIC across stages of the estrous cycle for ovine luteal cells in vitro during the present study as well as previously [26]. In contrast, in vivo expression of Cx43 is greater during the early and mid luteal phases than during the late luteal phase for bovine, ovine, and baboon CL [33, 34, 48]. The differences in Cx43 expression in vivo versus in vitro may be explained by the possibility that luteal cells from the late luteal phase of the estrous cycle may restore production of Cx43 during culture and, thereby, establish functional gap junctional channels.

Connexin 43 likely is the major gap junctional protein in luteal tissues, because it is localized on the cellular borders and is expressed at relatively high levels throughout the estrous cycle [7, 8, 33, 34, 48]. In several other cell or tissue types, Cx43 also is recognized as the major gap junctional protein [49, 50]. However, Cx43 is not the only gap junctional protein expressed in ovine CL. Other connexins, such as Cx26 and Cx32, have been immunolocalized in ovine luteal tissues in vivo and/or in vitro ([34]; unpublished results). In vivo, Cx26 and Cx32 are associated with connective tissues and blood vessels rather than with parenchyma, but in vitro, Cx32 has been detected in the cytoplasm of some parenchymal cells ([34]; unpublished results). Closer characterization of the presence and role of Cx26 and Cx32 in luteal tissues is currently under study.

In conclusion, activation of cAMP-dependent protein kinase increases GJIC and P4 secretion, but inhibition of cAMP-dependent protein kinase decreases GJIC but not P4 secretion. In addition, activation of PKC and increased intracellular calcium concentrations inhibit GJIC and P4 secretion by luteal cells. This indicates a relationship between the rate of GJIC and P4 secretion. Therefore, GJIC may be important for signal transduction within luteal tissues during growth, differentiation, and regression, and second messengers may be critical in the regulation of GJIC throughout the estrous cycle.

ACKNOWLEDGMENTS

We thank Mr. J.D. Kirsch and Mr. K. Kraft for expert technical assistance and Ms. J. Berg for typing the manuscript.

FOOTNOTES

First decision: 18 January 2001.

1 Supported by NIH grant R29 HD30348 to A.T.G.-B. Back

2 Correspondence. FAX: 701 231 7590; anna_grazul-bilska{at}ndsu.nodak.edu Back

Accepted: April 16, 2001.

Received: December 10, 2000.

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