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Biology of Reproduction 64, 625-633 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Dynamics of Connexin 43 Levels and Distribution in the Mink (Mustela vison) Anterior Pituitary Are Associated with Seasonal Changes in Anterior Pituitary Prolactin Content1

María L. Vitale2,a, Julie Cardina, Norton B. Gilulab, M. Eloísa Carbajala, and R.-Marc Pelletiera

a Département de Pathologie et biologie cellulaire, Faculté de Médecine, Université of Montréal, Montréal, Québec, Canada H3T 1J4 b Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037-1037

ABSTRACT

Because in mammals the anterior pituitary lacks innervation, we investigated whether gap junctions established between selected cells within the gland are part of an intrapituitary mechanism to ensure physiological synchronization of cells involved in the control of hormone secretion. We report here the dynamics of anterior pituitary connexin 43 (Cx43)-gap junctions throughout the mink (Mustela vison) annual reproductive cycle and its relationship with the anterior pituitary prolactin (PRL) content that parallels variations in serum PRL levels documented in the literature. We found that PRL anterior pituitary levels were maximal in spring and during lactation and that they were minimal in autumn and winter. Anterior pituitary Cx43 levels were maximal during periods of high PRL secretion. During these periods, Cx43-positive gap junctions localized to stellate-shaped cells occupying the center of anterior pituitary follicles and to the rounded cells occupying the remaining follicles. Connexin 43-positive gap junctions were also observed between adjacent follicles. During periods of low PRL pituitary content, Cx43-positive gap junctions localized to the stellate cells but not to the cells of the remaining follicles. Moreover, Cx43 labeling was undetected between adjacent follicles. To assess between which cells within the mink anterior pituitary the Cx43 gap junctions were established, the different anterior pituitary cell populations were separated by a discontinuous Percoll gradient, and Western blot analyses of each cell population using Cx43 antibodies were performed. The immunoblots showed a Cx43 immunoreactive band associated with the cell layer enriched in S-100-positive, stellate-shaped cells. The result was confirmed by fluorescence microscopy studies that showed that Cx43-mediated gap junctions were established preferentially between the cultured S-100-positive, elongated cells. The results show that in mink stellate cells, the junctional machinery associated with the Cx43 protein varies in synchrony with the anterior pituitary PRL content throughout the mink annual reproductive cycle. It is suggested that the Cx43 gap junctions on the stellate cells play an important role in the synchronization of cellular activity within selected follicles of the anterior pituitary, thus contributing to the control of PRL secretion during the annual reproductive cycle.

anterior pituitary, lactation, pituitary, pituitary hormones, prolactin, seasonal reproduction

INTRODUCTION

Anatomical evidence shows that anterior pituitary cells are arranged to allow intercellular communication among different cell types. The pituitary gland consists of more or less complete follicles that contain hormone-secreting (granular) cells and folliculo-stellate (FS; agranular) cells [1]. The function of FS cells remains unclear, although experimental evidence suggests that they play a role in the paracrine regulation of anterior pituitary hormone secretion by allowing interfollicle connection [2–6]. Intercellular communication mainly occurs via gap junctions. Gap junctions enable exchange of small molecules and thus facilitate metabolic cell-to-cell coordination and synchronization of cellular responses. Connexins (Cx) are the structural proteins of gap junctions. Connexin 43 [7, 8] and Cx26 [7] are expressed in rat anterior pituitary. In the rat, gap junctions were reported to join endocrine cells [9], FS cells [10–16], FS cells and gonadotroph cells [8], and FS cells and lactotroph cells [17, 18]. Because in mammals the anterior pituitary lacks direct innervation, gap junction-mediated cell-to-cell communication within the gland must be indispensable for the adequate cell-to-cell coordination and synchronization required to ensure appropriate and timed hormone secretion.

In the present work, we measured gap junction protein Cx43 levels and determined the changes in the localization of the protein in mink anterior pituitary in relation to the annual cyclic reproductive cycle. In the mink, a short-day seasonal breeder, blood prolactin (PRL) concentration has been reported highest in spring and the lactation period and lowest in autumn and winter [19–22]. Gonadotropin serum levels are low in spring and high in winter [23–25]. Our results indicate that mink anterior pituitary expresses Cx43 and that both the levels of the protein and the number of Cx43-positive gap junctions increase during periods of increased anterior pituitary PRL content. Immunohistochemical studies revealed that gap junction protein Cx43 labeling between anterior pituitary cells varied during periods of high and low PRL anterior pituitary content. Furthermore, our results indicate that Cx43 gap junctions localize preferentially to S-100-positive, stellate-shaped cells in the mink anterior pituitary.

MATERIALS AND METHODS

Animals

Adult male and female mink were purchased from Visonnière St. Damase, St. Damase, Québec, Canada and RBR Fur Farm, St. Marys, Ontario, Canada. Animals were kept under natural lighting conditions and were allowed food and water ad libitum. Mink were killed in the morning, at different intervals of the annual reproductive cycle specifically, in autumn and winter when PRL serum levels are low and gonadotropin serum levels are high, and in spring when PRL serum levels are high and gonadotropin serum levels are low [20–22, 26]. In addition, female mink were killed during the lactation period, when PRL secretion is high [21]. Animals were anesthetized by an i.p. injection of Somnotol (0.2 ml/kg body weight). After decapitation, the anterior lobe of the pituitary gland was dissected free from the posterior lobe and processed according to the corresponding protocol. The posterior lobe of the pituitary and the brain were dissected in male mink. The experimental protocol was approved by the Université de Montréal Animal Care Committee (Protocol no. 99-114).

Preparation of Mink Anterior Pituitary Cell Cultures and Separation of Different Anterior Pituitary Cell Populations

Mink anterior pituitaries were dissected from females and males killed during spring. Two to three anterior pituitaries were diced in small pieces and dispersed into a single cell population by incubation with Mg2+- and Ca2+-free Lockes solution (154 mM NaCl, 2.6 mM KCl, 1.25 mM K2HPO4, 0.50 mM KH2PO4, 10 mM Hepes, 10 mM glucose; pH 7.2) containing 0.10% trypsin, 0.2% collagenase D, and 0.3% BSA for 2–3 h at 37°C. Digestion was stopped by addition of a volume of Dulbeccos modified Eagles medium (DMEM) containing 0.2% of soybean trypsin inhibitor. Four to five anterior pituitaries were used for the preparation of anterior pituitary-enriched cultures. Preparation of enriched cultures was performed by using a discontinuous Percoll gradient as described before [27, 28]. Briefly, after enzymatic dispersion, anterior pituitary cells were recovered by centrifugation, rinsed with DMEM, and resuspended in DMEM. Two million cells were placed at the top of a discontinuous Percoll gradient (70, 60, 50, 35, 25%). After centrifugation, each interface was recovered and the same interfaces within the same experiment were pooled. In this way, a total of five cell fractions were obtained. Each cell fraction was divided in two halves. The cells of the first half were put in culture, and after 2 days in culture, the percentage of each type of anterior pituitary cell in the fraction was calculated by fluorescence microscopy (see below). The remaining half was used to detect Cx43 by immunoblotting. Heterogeneous anterior pituitary cells and enriched populations of anterior pituitary cells were rinsed with DMEM, resuspended in culture medium (DMEM supplemented with 10% fetal calf serum, antibiotics, and fungicide), and plated on poly-L-lysine-coated glass. Cells were cultured at 37°C in a 95% air-5% CO2 atmosphere.

Electrophoresis and Immunoblotting

Anterior pituitaries harvested throughout the annual reproductive cycle (eight or nine animals per experimental condition) were homogenized by sonication (Fisher Sonic dismembranator, model 300; Fisher, Farmington, NY) in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) containing a cocktail of protease inhibitors. Total proteins in the homogenates (15 µg) were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were saturated with Blotto and tested for the presence of Cx43 with a specific affinity-purified polyclonal antibody (1:5000 dilution). This polyclonal antibody was raised against {alpha}1-connexin and has been extensively characterized [29, 30]. When the presence of Cx43 was studied in the different fractions of the Percoll gradients, cells in the remaining half of the fractions were treated with cold acetone overnight, the preparation was next centrifuged, and the pellet resuspended in electrophoresis buffer to a final protein concentration of 3 mg/ml. Thirty micrograms of proteins per fraction were loaded on the gels and subjected to the same procedure as the anterior pituitary homogenates. Antigen-antibody complexes were revealed by enhanced chemiluminescence detection.

Determination of Relative PRL, GH, and FSH Anterior Pituitary Content

Twenty micrograms of anterior pituitary homogenates were subjected to 15% SDS-PAGE, and proteins were transferred onto nitrocellulose membranes. Membranes were incubated with either anti-hPRL (NIDDK-anti-hPRL-IC-5; 1:2000 dilution), anti-rGH (NIDDK-anti-rGH-IC-1; 1:50 000), or anti-hFSH (NIDDK-anti-ßFSH-IC-3; 1:750). In immunoblottings, anti-hPRL recognized one band of molecular mass of 21.5 kDa in rat anterior pituitary homogenates and several bands in mink anterior pituitary homogenates. The two strongest bands had a molecular mass of 13 kDa and 24 kDa. Both bands showed the same variations throughout the annual reproductive cycle. The 24-kDa band was scanned to calculate PRL content in the mink anterior pituitary. Anti-rGH recognized a 22.5-kDa band in both rat and mink anterior pituitary homogenates. Anti-hßFSH detected a 25.5-kDa band in rat anterior pituitary homogenates and a band of 26 kDa in mink anterior pituitary homogenates. After detection of antigen-antibody complexes by enhanced chemiluminescence, films were scanned and the optical density of the bands was calculated with the aid of the PC-compatible Scion Imaging Software (Scion Corporation, Frederick, MD). Eight to nine animals were used per experimental condition.

Immunoperoxidase Labeling

Freshly dissected anterior pituitaries were quickly immersion-fixed in Bouins solution. Tissues were dehydrated in ethanol and cleared in xylene before paraffinization. Five-micrometer-thick coronal sections were mounted on glass slides coated with 3-aminopropyltriethoxysilane, deparaffinized, and rehydrated in xylene and ethanol. To inhibit potential endogenous peroxidase activity, tissue sections were exposed to 0.6% H2O2 in Tris-buffered saline (TBS: 140 mM NaCl, 50 mM Tris-HCl, pH 7.4) for 10 min. They were then washed for 5 min in TBS containing 0.1% Tween-20 (TBST) [31]. Next, tissue sections were incubated for 60 min at 37°C with 1% nonfat milk in TBST to block unspecific labeling and incubated overnight at room temperature (RT) with anti-Cx43 antibody (1:2000 dilution) and next with biotinylated anti-rabbit IgG (1:1000 dilution) for 60 min followed by horseradish-peroxidase (HRP)-conjugated streptavidin (1:300 dilution). Slides were washed in TBST and incubated for 10 min at RT in 0.01% H2O2, 0.05% diaminobenzidine tetrachloride, and 10 mM imidazole in TBS (pH 7.7) [32]. The sections were counterstained with methylene blue and mounted with Permount. Negative controls included the omission of the first or second antibodies. Mink neurohypophysis was used as a positive control. Pictures were taken with Kodak Technical Pan films and a Carl Zeiss Axiophot. Quantitative analyses of Cx43 immunolabeling were carried out on microphotographs obtained from eight different animals per experimental condition. Four sections were analyzed per animal. The Cx43-immunopositive dots at the cell membranes in a 100-µm x 100-µm area were counted without knowing from which experimental group the photographs were taken.

Fluorescence Microscopy

Heterogenous anterior pituitary cell cultures or enriched cell cultures were allowed to grow for 2 to 3 days and processed for fluorescence microscopy. Dishes were removed from the incubator, rinsed with Lockes solution containing 1.2 mM MgCl2 and 2.2 mM CaCl2, and immediately fixed with 3.7% formaldehyde, permeabilized with acetone, and processed for fluorescence microscopy as previously described [33]. Coverslips were thoroughly rinsed with PBS and incubated for 1 h at RT with 3% nonfat milk in PBS. All antibody dilutions were prepared in 1% nonfat milk in PBS. To calculate the percentage of each type of anterior pituitary cell in either heterogeneous cell cultures or enriched cell cultures, cells were incubated with antibodies directed against each anterior pituitary hormone or against the S-100 protein, a marker for FS cells. Next, cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody together with rhodamine-phalloidin (1:1500). Rhodamine-phalloidin labels actin filaments in all cells thus allowing the calculation of the total number of cells per coverslip. One hundred rhodamine-positive cells per coverlip were counted and those that were doubled labeled with rhodamine-phalloidin and with an antibody to a particular anterior pituitary hormone were recorded to calculate the percentage of that hormone-secreting cell in that fraction of the gradient. Four coverslips per fraction of the gradient and per anterior pituitary hormone were analyzed. The experiment was repeated four times. To study gap junction localization, anterior pituitary cell cultures were double labeled for S-100 and Cx43. After blocking, cells were incubated with S-100 antibody (1:500 dilution) followed by Cy3-Fab anti-rabbit IgG (1:400 dilution). Next, cells were incubated with affinity-purified polyclonal Cx43 antibody (1:200 dilution) followed by incubation with FITC anti-rabbit IgG (1:400 dilution). Cells were observed with a Carl Zeiss Axiophot fluorescence microscope equipped with filters for fluorescein and rhodamine. Photographs were taken with T-MAX Kodak films.

Statistical Analysis

Data were evaluated by one-way analysis of variance, and differences between means were analyzed by the Keuls multiple-range test or the Student t-test according to the number of groups [34].

Protein Measurement

Proteins in the samples were measured by the Bradford dye binding assay [35].

Sources of Chemicals and Antibodies

Enzymes for anterior pituitary cell dispersion, protease cocktail and chemiluminescence kits (Lumi-lightplus) were purchased from Boehringer-Mannheim (Laval, QC, Canada). Antibiotics, soybean trypsin inhibitor, poly-L-lysine, 3-aminopropyltriethoxysilane, S-100 antibody, and FITC-conjugated secondary antibodies were purchased from Sigma Chemical Co. (St. Louis, MO). The Cy3 Fab anti-rabbit IgG and biotinylated anti-rabbit IgG were from Jackson ImmunoResearch Lab (BioCan Scientific, Mississauga, ON, Canada). Percoll was from Pharmacia (St. Anne de Bellevue, PQ, Canada). Antibodies against anterior pituitary hormones were generously provided by Dr. A.F. Parlow, National Hormone and Pituitary Program. Sommotol (sodium pentobarbital) was purchased from MTC Pharmaceuticals (Cambridge, ON, Canada). Rhodamine-phalloidin was purchased from Molecular Probes (Eugene, OR). The Bradford dye binding assay was from BioRad (Mississauga, ON, Canada).

RESULTS

To evaluate the activity of the mink anterior pituitary, the PRL, FSH, and GH content of mink pituitaries was measured by densitometric analysis of immunoreactive bands in Western blots and compared to published serum levels (Fig. 1). The relative PRL content of anterior pituitaries harvested during spring and the lactation period was higher than the PRL content of anterior pituitaries harvested during autumn and winter. Growth hormone relative anterior pituitary content was similar during autumn and winter and spring but decreased in lactating mink, whereas FSH content of anterior pituitaries harvested during autumn and winter was higher than anterior pituitaries harvested during spring and the lactation period.



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  		FIG. 1. Anterior pituitary PRL, GH, and FSH content at different moments of the mink reproductive cycle. Mink anterior pituitaries were dissected at different moments of the annual reproductive cycle. Tissues were homogenized by sonication, and total proteins were separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and subsequently incubated with either anti-hPRL or anti-rGH or anti-hFSH followed by incubation with an HRP-labeled secondary antibody. Antigen-antibody complexes were revealed by chemiluminescence detection. Relative anterior pituitary hormone content was estimated by scanning the immunoreactive bands followed by measurement of the density of the bands with the Scion Imaging Software. The lowest reading for each hormone was taken as 1. Values shown are the mean ± SEM; eight to nine animals were tested per experimental condition. *P < 0.01, GH, lactation period versus spring and autumn and winter; *P < 0.01, PRL, spring and lactation period versus autumn and winter; ·P < 0.02, PRL, lactation period versus spring; **P < 0.05, FSH, autumn and winter versus spring and lactation period

Expression of Cx43 in the mink anterior pituitary throughout the annual reproductive cycle was determined by Western blot analysis. Mink anterior pituitary lysates were subjected to electrophoresis followed by immunoblotting using an antibody against Cx43 (Fig. 2A). The antibody, raised against rat Cx43, recognized a broad band of a molecular weight of 42–46 kDa in mink brain and anterior pituitary (Fig. 2A). To explore whether anterior pituitary Cx43 levels were affected during a change in the endocrine status of the animals, Western blots were carried out on anterior pituitaries harvested during different periods of the annual reproductive cycle. Figure 2B shows that Cx43 levels of mink anterior pituitaries harvested during winter and autumn (adult females killed at the beginning of the month of March [Mf] and adult males killed at the end of the month of November [Nm]) were lower than those observed in anterior pituitaries harvested from females during the first week of lactation (Lf) and from females killed during spring (adult female mink at the beginning of the month of April [Af]). The results suggest a temporal correlation between the increase in anterior pituitary Cx43 levels and the increase in anterior pituitary PRL content.



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  		FIG. 2. Western blot analyses of Cx43 levels in the anterior pituitary during different periods of the mink annual reproductive cycle. A) The anterior pituitary and the brain were removed from male mink. Tissues were sonicated, and 30 µg (anterior hypophysis) and 10 µg (brain) total protein samples were loaded per well. After electrophoretic migration, proteins were electrotransferred onto nitrocellulose membranes. Membranes were sequentially incubated with Cx43 antibodies and with HRP-conjugated secondary antibody. Antigen-antibody complexes were revealed by chemiluminescence. A broad Cx43 immunoreactive band with a molecular weight of 42–44 kDa was observed in brain (Br) as well as in anterior pituitary (AP) homogenates. B) Representative blots of anterior pituitaries harvested during periods of low PRL anterior pituitary levels, i.e., winter and autumn (females killed at the beginning of March [Mf] and male in November [Nm]), and during periods of high anterior pituitary PRL levels (lactating females during the first week of lactation (Lf) and in spring (nonlactating females killed at the beginning of April [Af]). The Cx43 immunoreactive bands were stronger in lactating females (Lf) and females killed in spring (Af) than in mink killed in winter and autumn (Mf and Nm). C) The same as B but with a longer exposure to show the presence of Cx43 in anterior pituitaries harvested in winter and autumn

The possibility that changes in the secretory activity of anterior pituitary were correlated to changes in Cx43-mediated gap junctions was further analyzed by assessing the localization of the Cx43-positive gap junction within the gland at different moments of the annual reproductive cycle. Immunoperoxidase labeling on anterior pituitary sections was performed with the same antibody used in the biochemical studies. Sections were weakly counterstained to avoid masking Cx43 staining while allowing visualization of the cellular architecture of the gland. Moreover, to ascertain whether the preparation of the tissues for immunohistochemistry did not affect the labeling intensity, the neurohypophysis, a tissue known to express high amounts of Cx43-positive gap junctions [8], was immunolabeled following the same procedure as the one used for anterior pituitary sections. The mink neurohypophysis was heavily labeled with the Cx43 antibody and the labeling corresponded to intercellular junctions (Fig. 3, NH, arrowheads). The pars intermedia showed a less intense labeling (Fig. 3, PI, arrowhead). Mink anterior pituitaries were dissected out at different periods of the annual seasonal reproductive cycle and during the lactation period. The mink anterior pituitary is organized in follicles (Fig. 4, dashed lines). Immunolabeling of Cx43 in anterior pituitary sections appeared as minute dots associated with plasma membranes (Fig. 4, A–C). The tissue sections shown in Figure 4, A and B, are representative micrographs of adult mink anterior pituitaries harvested during the season of high anterior pituitary PRL content (A, lactating mink; B, nonlactating female mink killed in April). In the anterior pituitary of these animals, Cx43 gap junctions were detected near the center (Fig. 4, A and B, closed arrowheads) and close to the periphery of anterior pituitary follicles, joining, respectively, cells within the same follicle (Fig. 4, A and B, open arrowheads) and cells from adjacent follicles (Fig. 4, A and B, open arrows). The micrograph in Figure 4C is representative of the Cx43 labeling obtained in anterior pituitaries harvested during periods of low PRL pituitary content (female mink killed at the beginning of March). Here, the Cx43-positive gap junctions occupied the center of the follicles (Fig. 4C, closed arrowheads), and Cx43-positive gap junctions between neighboring follicles were no longer observed. Quantitative analyses of the number of Cx43-positive gap junctions revealed that there was a twofold increase in the number Cx43-positive junctions in mink anterior pituitary during the period of high anterior pituitary PRL content when compared to the period of low anterior pituitary PRL (Table 1).



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  		FIG. 3. Immunohistochemical localization of Cx43 in the mink posterior pituitary. Abundant Cx43 labeling (arrowheads) is observed in the neurohypophysis (NH), whereas the pars intermedia (PI) shows a less intense immunolabeling for Cx43 (arrowheads). x690



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  		FIG. 4. Immunohistochemical localization of Cx43 in the mink anterior hypophysis during periods of high and low PRL secretion. Anterior pituitaries were harvested during periods of high PRL anterior pituitary content (A, lactating female and B, nonlactating female killed at the beginning of April) and during periods of low PRL anterior pituitary content (C, female killed at the beginning of March). The Cx43 immunolabeling showed a punctate pattern typical of gap junctions. To facilitate the identification of pituitary follicles some of them are encompassed within a dashed line. In A and B, the micrographs show gap junction Cx43 labeling localized to cells near the center of follicles (closed arrowheads); thin cytoplasmic projections of these cells surrounding the more peripherally located cells also presented Cx43 labeling (open arrowheads). In addition, cells located near the periphery of the follicles and adjoining adjacent follicles were Cx43 positive (open arrows). During periods of low anterior pituitary PRL content (C), Cx43 immunolabeling (closed arrowheads) was diminished and concentrated near the center of the follicles and the follicular cavity. x690


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  		TABLE 1. Quantitative analysis of Cx43-positive gap junctions in mink anterior pituitary during periods of high and low PRL secretion.a

The localization of Cx43-positive gap junctions to a particular or several cell types in the mink anterior pituitary was next investigated. Mink anterior pituitaries were dissected during a period of high anterior pituitary PRL content and were dispersed by enzymatic digestion to a single cell suspension. The cell suspension thus obtained was loaded on top of a discontinuous Percoll gradient to separate the different cell populations present in the gland. After centrifugation, five interfaces or fractions were obtained, and each was divided into two halves. Cells in the first half of each fraction were cultured for 2–3 days. The remaining half was used in Western blot analyses to study the association of Cx43 with the cells in the different fractions. Cultured cells were processed for fluorescence microscopy using antibodies directed to each anterior pituitary hormone. The preparations were viewed under the fluorescence microscope and the percentage of each cell type in each fraction of the gradient was calculated. Figure 5 summarizes the results of these experiments. The open bars indicate the percentage of each type of cell in the whole anterior pituitary cell culture and the dashed bars indicate the concentration of each type of cell after the gradient. The proportion of lactotroph cells in the whole anterior pituitary cell culture was 39 ± 8%, somatotrophs accounted for 20 ± 7%, and S-100 positive, stellate-shaped cells represented 8 ± 3% of the cells in the cultures (Fig. 5A). The LH-positive cells were 9 ± 3%, TSH-positive cells accounted for 5 ± 2%, and ACTH-positive cells were 3 ± 1% of total anterior pituitary cells in the cultures (not shown). Therefore, lactotroph and somatotroph cells were the most abundant cells within the mink anterior pituitary cell cultures. After the gradient, the third fraction was enriched in lactotrophs (Fig. 5A, fraction 3). Fluorescence microscopy studies of this fraction showed lactotroph cells with a rounded shape, some of them containing easily identifiable clusters of cytoplasmic PRL-containing granules (Fig. 5B, top micrograph). The fourth fraction of the gradient was enriched in somatotrophs (Fig. 5A, fraction 4). Most somatotroph cells in the fourth fraction were rounded, but some of them were flattened and polygonal (Fig. 5B, middle micrograph). Lastly, S-100-positive, stellate-shaped cells were recovered mostly in the first layer of the gradient (Fig. 5A, fraction 1). After 2 or 3 days in culture, most S-100-positive cells in the first fraction were in clusters of two or more cells; only a few were single (Fig. 5B, bottom micrograph). Western blot analyses on the presence of Cx43 in the different cell fractions of the gradient showed that Cx43 immunoreactivity was concentrated in the first fraction (Fig. 5C) where most S-100-positive, stellate-shaped cells were recovered. The results suggest that Cx43 gap junctions may be associated with S-100-positive, stellate-shaped cells in the mink anterior hypophysis. To confirm this possibility, anterior pituitary cells were cultured for 3 days, and we double stained the cells for S-100 and Cx43.



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  		FIG. 5. Association of Cx43 gap junctions to specific cell populations of the mink anterior pituitary. Mink anterior pituitaries were harvested during the period of high PRL anterior pituitary content. Tissues were submitted to mild enzymatic digestion, and anterior pituitary cells in the suspension were plated and cultured for 2 or 3 days. To separate the different populations of anterior pituitary cells the cell suspension was applied to the top of a discontinuous Percoll gradient. After centrifugation five cell layers or fractions were obtained and each fraction was divided into two halves. The cells in the first half of each cell fraction were cultured for 2 or 3 days to evaluate the percentage of each type of anterior pituitary cell in that fraction. The remaining half of the fractions was used in Western blot analyses to assess the presence of Cx43. A) The percentage of lactotrophs, somatotrophs, and S-100-positive, stellate-shaped cells in mink anterior pituitary cell cultures (open bars) and their distribution in the different cell fractions of the gradient (dashed bars). Micrographs in B illustrate the appearance of cell layers enriched in lactotrophs, somatotrophs, and S-100-positive, stellate-shaped cells after being processed for fluorescence microscopy using specific antibodies. x582. C) Western blot analyses of the presence of Cx43 in each layer of the gradient. A Cx43 immunoreactive band is seen in the first fraction of the gradient

The S-100-positive, elongated cells were observed in the cultures (Fig. 6A, close arrows). The same cells, shown in Figure 6B, are Cx43 positive. The Cx43 gap junctions were preferentially established between S-100-positive, stellate cells (Fig. 6B, open arrows).



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  		FIG. 6. Fluorescence microscopy studies of Cx43 gap junctions between cultured folliculo-stellate cells. Anterior pituitary cells cultured for 3 days were processed for fluorescence microscopy. Cells were double labeled with antibodies against S-100 protein (A) and Cx43 (B). The Cx43 gap junctions (B, open arrows) were predominant between S-100-positive, elongated folliculo-stellate cells (closed arrows). x640

DISCUSSION

The present work shows the dynamics of the Cx43 expression and localization in the anterior pituitary throughout the mink annual reproductive cycle and the relationship of these two parameters with the anterior pituitary hormone content. Increased levels of Cx43 protein and increased number of Cx43-positive gap junctions in the mink anterior pituitary coincided with two different periods of the annual reproductive cycle characterized by a high anterior pituitary PRL content, i.e., spring and the lactation period. This observation indicates the existence of a link between the Cx43-mediated gap junctions and the cellular elements responsible for the control of PRL secretion at the anterior pituitary level and agrees with published reports showing that modifications in the expression of the Cx43 protein and/or in the number, size, and localization of Cx43-mediated gap junctions correlate to changes in the physiology of the tissue bearing the junctions [36]. The number of gap junctions between myometrial cells increases during pregnancy to coordinate uterine contractions [29]. In the testis, the localization of the Cx43-positive gap junction is modified in accordance with the changes in the seasonal spermatogenic activity [37].

The antibody raised against rat Cx43 used in the present work recognized a broad band in mink brain and anterior pituitary immunoblottings. In immunohistochemical experiments performed on pituitary tissue sections, the same antibody recognized a protein that localized to gap junctions. The increased anterior pituitary Cx43 levels shown by immunoblotting analyses during periods of high anterior pituitary PRL concentration correlated with an increased number of Cx43-positive gap junctions and with a wider distribution of Cx43-positive gap junctions. On the contrary, during periods of low anterior pituitary PRL content, the diminished Cx43 levels coincided with a reduction of the number of Cx43-positive gap junctions and with a distribution of Cx43-positive gap junctions that was confined near the stellate cell body that occupied the center of the follicle. Moreover, Cx43 labeling was detected neither between adjacent follicles nor on the cytoplasmic projections of stellate-shaped cells as was the case during periods of increased PRL pituitary content. The larger number of Cx43 gap junctions and their wider distribution suggest that during periods of the annual reproductive cycle characterized by increased PRL anterior pituitary content (the present work) and increased PRL levels [19–22, 26], the anterior pituitary can adapt to seasonal requirements of intercellular communication to ensure proper hormone secretion. In this sense, the present results agree with findings in the guinea pig anterior pituitary where groups of gap junction-coupled cells were suggested to synchronize and coordinate GH secretion in vitro [38].

Although many reports have demonstrated the presence of gap junctions in the anterior pituitary gland, there is still debate over the type of cells coupled by the junctions. Fletcher et al. [9] initially suggested that gap junctions localize to endocrine cells, while Soji and Herbert [10] reported the presence of gap junctions only between FS cells. Later, gap junctions were reported between FS cells and also within gonadotrophs [8]. Morand et al. [18] demonstrated that functional Cx43 gap junctions form between FS cells and between FS cells and endocrine cells, particularly lactotrophs, in rat anterior pituitary cell cultures. The present biochemical experiments show that Cx43 immunoreactivity was associated with a cell fraction enriched in S-100-positive stellate-shaped cells (FS cells) of the mink anterior hypophysis. Mink FS cells are morphologically heterogeneous [39]. Particularly, S-100-positive, stellate-shaped cells are more abundant during periods of high than during periods of low anterior pituitary PRL content [39]. The distribution of Cx43-positive gap junctions shown in the present work overlaps with the distribution of S-100-positive, stellate-shaped cells reported in the mink anterior pituitary [39]. Moreover, there is a positive correlation between the increased number of Cx43-positive gap junctions and their wider distribution during periods of high anterior pituitary PRL content and the presence of a larger number of S-100-positive, stellate-shaped cells in the mink pituitary during the same periods of the annual reproductive cycle. To further characterize the association of Cx43 with FS cells or other cells in the mink anterior pituitary, cell culture studies were carried out. In these studies, most Cx43-positive gap junctions were detected between flat S-100-positive cells. However, few Cx43-positive gap junctions were observed between S-100-positive and S-100-negative cells, indicating that not all Cx43-positive gap junctions localize exclusively between FS cells. The S-100-negative cells could be lactotropes, gonadotropes, or somatotropes as described by other authors, although we were unable to label those cells with antibodies to anterior pituitary hormones. The fact that most Cx43-positive gap junctions did not involve an endocrine cell but rather two FS cells, at least in culture, does not rule out the possibility that the endocrine cells' activity may not be regulated by a higher level of intercellular communication among FS cells. The FS cells mainly exert a paracrine control on anterior pituitary activity by releasing regulatory factors such as S-100, follistatin, activin, and interleukin-6 [40–44]. A better synchronization between FS cells may ensure a better coordination of the endocrine activity within the anterior pituitary.

The changes in Cx43 expression and localization recorded in the present study take place in the hypophysis in situ, that is to say, in the hypophysis of animals that responded normally to endogenous signals to increase or decrease the secretion of the hormone. Therefore, increased levels of Cx43 protein together with a large number and wider distribution of Cx43-positive gap junctions detected during periods of increased PRL content may reflect the establishment of a high degree of cell synchronization within the anterior pituitary. A network formed by gap junction-bearing FS cells could create a syncytium that could favor interaction between cells within individual follicles and between adjacent follicles to establish and maintain the coordination between selected cellular players involved in the control of PRL secretion at the level of the anterior pituitary to ensure adequate and timely release of the hormone.

NOTE ADDED IN PROOF

It is with great sadness that we learned of the passing away of Dr. Norton B. Gilula.

ACKNOWLEDGMENTS

The authors thank Dr. Parlow and the National Hormone and Pituitary Program of the NIDDKD for the gift of anterior pituitary hormone antibodies. The authors also thank the Canada Foundation for Innovation for grants to purchase the fluorescence and inverted microscopes.

FOOTNOTES

First decision: 6 July 2000.

1 This work was funded by the Natural Sciences and Engineering Research Council of Canada grant OGP0194652 to M.L.V. M.L.V. is supported by a scholarship from Fonds de la recherche en santé du Québec. Back

2 Correspondence: María L. Vitale, Département de Pathologie et biologie cellulaire, Faculté de Médecine, University of Montréal, 2900 Edouard-Montpetit, Montréal, PQ, Canada H3T 1J4. FAX: 514 485 4482; maria.leiza.vitale{at}umontreal.ca Back

Accepted: September 27, 2000.

Received: June 2, 2000.

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