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Biology of Reproduction 67, 154-160 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Epidermal Growth Factor Enhances Expression of Connexin 43 Protein in Cultured Porcine Preantral Follicles1

Digbo Bolambaa, Anthony A. Floyda, John J. McGloneb, and Vaughan H. Leea,b

a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 b Pork Industry Institute, Department of Animal Science and Food Technology, Texas Tech University,Lubbock, Texas 79409-2141


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.

follicle, follicular development, granulosa cells, growth factors, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Throughout ovarian folliculogenesis, communication among and between granulosa cells and oocytes is required for successful follicular development, oocyte maturation, and ovulation [1–4]. This cell-to-cell communication in ovarian follicles is directed by mechanisms that require intact gap junctions [3, 4]. Gap junctions are thought to play a crucial role in regulating cell growth and development in various tissues, including ovaries [5–7]. Structurally, gap junctions are aggregates of intercellular membrane channels that allow the direct exchange of nutrients, ions, metabolites, and other potential signaling molecules smaller than 1200 Da between adjacent cells [6, 8–10]. Each intercellular channel is formed by two connexons (hemichannels), one contributed by each of the adjacent cells [5, 7]. Each connexon is composed of a hexamer of integral membrane proteins termed connexins, which constitute a large family of at least 20 related proteins that are distinguished by their molecular weights [6].

In ovaries, the major connexin found in granulosa cell-granulosa cell gap junctional communication is connexin 43 (Cx43) [11–15]. This connexin appears to play a critical role in early follicular growth; the absence of the Cx43 gene disrupted progression of follicles beyond primary stages in transgenic mouse ovaries [4]. In pig ovaries, the Cx43 gene transcript and protein have been identified and localized to growing follicles [12–14]. Recently, Melton et al. [12] reported that substantial amounts of Cx43 are initially detected in granulosa cells following activation of follicular development, suggesting an association between the enhancement of intercellular gap junctional communication and onset of follicular growth.

Several factors such as GnRH analogues [16], gonadotropins [14, 16, 17], steroids [18], and kinases [16, 19] regulate the expression and/or phosphorylation of Cx43 protein in the ovarian follicle cells. The involvement of mitogenic growth factors, such as epidermal growth factor (EGF), in this system has not been reported. Numerous in vitro studies have revealed that EGF is involved in different cellular processes such as proliferation, differentiation, and follicle growth. The potential relevance of EGF to ovarian physiology, especially to regulation of ovarian cell proliferation and differentiation, has been reviewed by May and Schomberg [20]. EGF alone or with serum and/or insulin stimulates in vitro proliferation of porcine granulosa cells obtained from preantral [21] and immature antral [21–23] follicles and stimulates FSH receptor binding in these cells [24]. EGF stimulates growth of preantral follicles in cows [25, 26], hamsters [27], mice [28], and humans [29] in vitro. EGF in combination with serum also stimulates proliferation of cultured porcine theca cells [30]. EGF can also stimulate nuclear maturation of porcine oocytes in vitro through intracellular protein kinase A (PKA) by increasing the content of both catalytic subunits of approximately 52 and 40 kDa [31]. Additionally, EGF regulates Cx43 in cells from a variety of tissues. EGF increases the cellular level of Cx43 protein in parallel with enhancing communication in human kidney epithelial cells [32, 33]. These findings suggest that EGF may influence the early follicle growth by enhancing Cx43 gap junctional intercellular coupling in porcine preantral follicles.

In the present study we tested the hypothesis that EGF upregulates the expression of Cx43 protein in porcine granulosa cells during early stages of follicle growth in vitro. Two culture systems were used to test this hypothesis: 1) the culture of ovarian explants [34] and 2) the culture of isolated preantral follicles [35]. In these immature follicles collected from prepubertal pigs, EGF regulation of expression of Cx43 was evaluated using Western blotting.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian Explant Cultures

Ovaries from fourteen 32- to 40-day-old gilts (3 or 4 gilts per replicate, 4 independent replicates), which contain a relatively high population of primary and secondary follicles [36], were surgically removed and placed on ice in fresh Waymouth MB 752/1 medium (WAY medium; Life Technologies, Grand Island, NY) supplemented with 1% antibiotics (10 000 IU/ml penicillin and 10 000 µg/ml streptomycin) (Cellgro; Fisher Scientific, Fair Lawn, NJ). Ovaries were dissected into small pieces (~2 mm), rinsed twice in WAY medium, and cultured using the method described by Wandji et al. [34] with some modifications. These explants were cultured on PET track-etched membrane Cell Culture Insert (3.0 µm pore size, Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) in 24-well cluster dishes (Cell Wells; Corning Glass Works, Corning, NY) at 37°C in a humidified atmosphere of 5% CO2 and 95% air for 6 days. The culture medium was WAY medium containing 0.23 mM pyruvic acid (Sigma Chemical Company, St. Louis, MO), 1% ITS+ (6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenium, 0.3 mg/ml BSA, 1.25 mg/ml linoleic acid; Collaborative Biomedical Products, Bedford, MA). Ovarian explants were incubated in the presence of 0, 50, or 500 ng/ml recombinant mouse EGF (Upstate Biotechnology, Lake Placid, NY). Three hundred microliters of medium was placed in the compartment below membranes, and every second day 200 µl of medium was replaced. At the end of culture, ovarian explants from each treatment group were collected, snap-frozen in liquid nitrogen, and stored at -80°C. Each treatment was applied in 3 or 4 wells in each of 4 independent replicates.

Preantral Follicle Cultures

To determine the optimal age of gilts yielding large numbers of early preantral follicles, ovaries from 35- to 114-day-old gilts (2–4 gilts/replicate; 12 independent replicates) were surgically removed and processed as described for explant cultures. The ovarian pieces were incubated in 2.5 ml WAY medium containing 500 U/ml collagenase type V and 40 U/ml DNase I, type IV (Sigma) per g tissue for 1 h at 39°C with shaking. After enzymatic digestion, the suspension was filtered through a Cellector tissue sieve (Fisher) to remove large debris, and 25 ml of collection medium was added to stop enzyme activity. The collection medium was WAY medium containing 1 mg/ml BSA, Fraction V (Sigma), 0.23 mM pyruvic acid (Sigma), and 5 mg/ml Hepes (Sigma), with pH adjusted between 7.2 and 7.5 with sterile 1.0 N HCl or NaOH (Sigma). The diluted suspension was centrifuged for 10 min at 1000 rpm and 20°C, and the supernatant was discarded. The pellet was resuspended in 5 ml of collection medium, and free-floating follicles were collected manually with a glass pipette mounted on an Accropet pipettor (Bel-Art Products, Pequannock, NJ). Follicles remaining in the tissue were removed mechanically using two 25-ga 5/8 needles (Fisher) attached to a 1-ml syringe (Fisher) and collected. Follicles were rinsed, and intact preantral follicles were selected, counted, and measured with a calibrated ocular micrometer at 20x magnification on a dissecting microscope.

To determine the effect of EGF on expression of Cx43 protein in cultured isolated follicles, 3 of 12 independent replicates were used. A total of 1470 healthy preantral follicles (with 2 or more layers of granulosa cells) collected from 70- to 86-day-old gilts (optimal age of gilts yielding large numbers of early preantral follicles) were cultured for 8 days using the method described by Hirao et al. [35] with some modifications. Follicles were washed 3 times in culture medium consisting of WAY medium (pH 7.2–7.5) with 0.3 mg/ml BSA, Fraction V (Sigma), 0.23 mM pyruvic acid (Sigma), ITS (5 µg /ml insulin, 5 µg /ml transferrin, 5 ng/ml selenium; Sigma), and 1 mg/ml Fetuin (Sigma). Collagen solution (300 µl/well; Collaborative Biomedical Products, Bedford, MA) was placed in 24-well culture dishes (Corning), and 25–30 follicles/well with a small volume of culture medium were put into the well and overlaid with an additional 300 µl of collagen solution. After gelatinization (60 min, 39°C, 5% CO2 and 95% air), 1 ml of culture medium was added to each well. The collagen solution was prepared according to the protocol provided by the manufacturer with the following modifications: 0.05 N NaOH (Sigma) containing 22 mg/ml NaHCO3 (Sigma) and 47.7 mg/ml Hepes (Sigma) was used instead of 1 N NaOH as suggested, and the pH was adjusted to 7.2–7.5. Follicles were incubated in the presence of 0, 50, or 500 ng/ml recombinant mouse EGF (Upstate Biotechnology) at 39°C in a humidified atmosphere of 5% CO2 and 95% air for 8 days. Five hundred microliters of medium was replaced on Days 2 and 5 of culture. Conditioned medium from Day 5 to Day 8 of culture was collected and stored at -20°C for subsequent progesterone and estradiol assays. Collagenase type V (200 µl, 500 U/ml; Sigma) was added to wells and incubated for 60 min (39°C, 5% CO2 and 95% air), and follicles were collected manually with a glass pipette and placed in wells containing WAY medium with 2% fetal bovine serum (Hyclone Laboratories, Logan, UT) to stop collagenase reaction. Follicles were washed two more times in this medium, transferred to Eppendorf tubes, and stored at -80°C until protein solubilization.

Western Blot Analysis

Proteins from cultured ovarian explants and cultured preantral follicles were solubilized for one-dimensional PAGE according to the method of Lee and Dunbar [37]. Solubilized proteins (30 µg) were separated by 12.5% PAGE, transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA), and probed with anti-rat Cx43 antibodies (Zymed, South San Francisco, CA) [12]. Specificity of anti-rat Cx43 was verified by adsorbing antibody with rat Cx43 peptide (Zymed) according to protocol provided by the manufacturer. Phosphorylation of Cx43 in porcine ovarian samples was evaluated using alkaline phosphatase agarose beads (Sigma). One hundred micrograms of solubilized protein was incubated with 2.5 units of phosphatase overnight at 37°C. Samples were reduced with 4% 2-mercaptoethanol, and 30 µg of sample was separated and transferred for analysis. Phosphatase enzyme was omitted from mock treatment samples, which served as negative controls. Membranes were probed with rabbit anti-rat Cx43 antibodies at 0.05 µg/ml in Tris-buffered saline containing 0.05% Tween 20, and labeling was visualized with the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). Membranes were exposed to x-ray film and developed through an X-OMAT AR x-ray film processor. The optical density (OD) of Cx43 bands was measured by densitometric analysis with AlphaEase software (Alpha Innotech Corp., San Leandro, CA). OD values from treated samples were compared with those of controls based on equal amounts of total protein loaded in each lane.

Steroid Assays

Progesterone and estradiol concentrations were each determined by RIA (Diagnostics Products, Los Angeles, CA) in 100-µl aliquots of unextracted medium samples, as described previously [38]. The sensitivities of the assays were 0.1 ng/ml and 5 pg/ml per tube, and the intraassay coefficients of variation were 4.2% and 3.9% for progesterone and estradiol, respectively.

Statistical Analysis

Cx43 proteins were quantified using 3 independent replicates of ovarian explant cultures and 3 independent replicates of isolated preantral follicle cultures. The effect of EGF on the level of proteins was analyzed with log-transformed OD data [39] and one-way ANOVAs. If significant differences were found, treatment differences were ascertained with a Fisher protected least significant differences (LSD) test. The effect of EGF on the amount of steroid secreted by cultured isolated preantral follicles was also analyzed using one-way ANOVAs followed by the Fisher protected LSD test if the effect was significant. The influence of age of gilts on the number of healthy secondary follicles collected, expressed as a percentage of total healthy secondary follicles over all age groups, was analyzed with a chi-square test. All analyses were performed using StatView software for Macintosh (Abacus Concepts, Calabasas, CA). For all comparisons, a probability of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation State of Cx43 Protein in Immature Porcine Ovaries

Western blot analysis using rabbit anti-rat Cx43 immunoglobin demonstrated a characteristic doublet of 2 immunoreactive protein species (43 and 45 kDa; Fig. 1, lane 1). When samples were treated with alkaline phosphatase, the doublet was reduced to a single band (Fig. 1, lane 2) that migrated at 43 kDa, indicating that the 45-kDa band is the phosphorylated form of Cx43. Mock treatment of a similar sample without phosphatase did not affect the appearance of the 43- and 45-kDa bands (Fig. 1, lane 1). Preincubation of anti-rat Cx43 with the rat Cx43 peptide abolished binding of antibody to Cx43 proteins in pig ovarian protein samples (Fig. 1, lane 3). Subsequent incubation of this membrane with anti-Cx43 not adsorbed with Cx43 peptide verified presence of Cx43 in this control sample (Fig. 1, lane 4).



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  		FIG. 1. Western blot analysis of Cx43 protein in porcine ovaries. Cx43 proteins expressed in porcine ovarian samples appear as a doublet migrating at 43 and 45 kDa. Lane 1: mock-treated control; lane 2: treated with alkaline phosphatase and probed with anti-Cx43; lane 3: probed with anti-Cx43 adsorbed with Cx43 peptide; lane 4: blot from lane 3 reprobed with nonadsorbed anti-Cx43

Effect of EGF on the Expression of Cx43 in Porcine Ovarian Explant Cultures

Western blot analysis to detect Cx43 protein in the homogenates of the cultured porcine ovarian explants revealed the presence of 2 immunoreactive bands of 43 and 45 kDa after treatment with 0, 50, or 500 ng/ml of mouse EGF (Fig. 2A). The intensity of the bands increased with rising doses of EGF. This increase was not due to a difference in the amount of protein samples loaded, as demonstrated by a coomassie-stained gel that showed consistent loading of samples (Fig. 2B). This experiment was repeated 3 times, and average relative OD was graphed (Fig. 3). The amount of total Cx43 protein (phosphorylated plus nonphosphorylated) increased markedly after treatment with 50 ng/ml (P < 0.05) and 500 ng/ml (P < 0.001) of EGF (Fig. 3). The amounts of phosphorylated Cx43 proteins did not differ from that of nonphosphorylated Cx43 proteins within each EGF treatment group (Table 1).



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  		FIG. 2. Analysis of Cx43 proteins in porcine ovarian explant culture samples. A) Western blot of proteins (30 µg) isolated from ovarian explant culture samples and probed for Cx43. C, Control; E50, 50 ng/ml of recombinant mouse EGF; E500, 500 ng/ml of recombinant mouse EGF. The intensity of Cx43 bands (phosphorylated and nonphosphorylated forms) increased with rising doses of EGF. B) Coommassie-stained gel confirmed consistent loading of samples with respect to amount of total protein



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  		FIG. 3. EGF-stimulated expression of Cx43 protein in cultured porcine ovarian explants. Mean (SEM) of the integrated OD for total (phosphorylated plus nonphosphorylated) Cx43 bands was calculated from 3 independent replicates. Asterisk above bars denotes significant differences between treatments and control *P < 0.05; **P < 0.001


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  		TABLE 1. Mean percentages of phosphorylated and nonphosphorylated Cx43 proteins

Influence of Age of Gilts on the Percentage of Preantral Follicles Collected

This procedure yielded healthy preantral follicles consisting of a normal centrally located oocyte surrounded by one or more layers of granulosa cells, as determined with a dissecting microscope. The basal lamina that normally surrounds this complex was partially degraded by the enzyme treatment. Age did affect the number of healthy secondary follicles collected per gilt after enzymatic digestion (Table 2). Ovaries from the youngest pigs (35–38 days old) yielded fewer (P < 0.01) follicles than did those from the 2 groups of older pigs. The ovaries from gilts 60–63 days of age yielded fewer (P < 0.01) follicles than did those from gilts 69–70 days of age, but the percentage of follicles collected from ovaries of gilts 60–63 and 86–114 days of age did not differ. Ovaries from the 2 groups of older pigs yielded similar percentages of follicles.


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  		TABLE 2. Number of preantral follicles collected from different age gilts

Effect of EGF on the Expression of Cx43 in Porcine Preantral Follicle Cultures

Mean size of 27 collected follicles at Day 0 (i.e., day of collection of follicles and the beginning of culture) was 288.9 µm. Follicles remained intact throughout the 8-day culture period (Fig. 4). Western blot analysis to detect Cx43 protein in the homogenates of the cultured porcine ovarian preantral follicles revealed the presence of 2 immunoreactive bands of 43 and 45 kDa (Fig. 5A). The intensity of the bands increased with rising doses of EGF. This increase in intensity was not due to a difference in the amount of protein sample loaded, as demonstrated by a coommassie-stained gel that showed consistent loading of samples (Fig. 5B). These experiments were repeated 3 times, and average relative OD was graphed (Fig. 6). The amount of total Cx43 protein (phosphorylated plus nonphosphorylated) increased markedly after treatment with 50 ng/ml (P < 0.01) and 500 ng/ml (P < 0.001) of EGF (Fig. 6). The amounts of phosphorylated Cx43 proteins did not differ from that of nonphosphorylated Cx43 proteins within each EGF treatment (Table 1). Follicles from control cultures contained more nonphosphorylated than phosphorylated Cx43.



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  		FIG. 4. Morphology of cultured porcine preantral porcine follicles. A) Day 0 of culture. B) Day 8 of culture. Bars = 215 µm



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  		FIG. 5. Analysis of Cx43 proteins in cultured porcine preantral follicles. A) Western blot of proteins (30 µg) isolated from cultured preantral follicle samples and probed for Cx43. C, Control; E50, 50 ng/ml of recombinant mouse EGF; E500, 500 ng/ml of recombinant mouse EGF. The intensity of Cx43 bands (phosphorylated and nonphosphorylated forms) increased with rising doses of EGF. B) Coommassie-stained gel confirmed consistent loading of samples with respect to amount of total protein.



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  		FIG. 6. EGF-stimulated expression of Cx43 protein in cultured porcine ovarian preantral follicles. Mean (SEM) of the integrated OD for total (phosphorylated plus nonphosphorylated) Cx43 bands were calculated from 3 independent replicates. Asterisk above bars denotes significant differences between treatments and controls *P < 0.01; **P < 0.001

Treatment with EGF did not significantly affect the amounts of progesterone secreted by porcine preantral follicles in culture (Table 3). Although the differences were not significant, EGF tended (P < 0.07) to diminish progesterone levels by 47.1% and 41.2% with 50 ng/ml and 500 ng/ml of EGF, respectively. Cultured follicles did not secrete detectable levels of estradiol in culture (Table 3).


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  		TABLE 3. Progesterone and estradiol secretion by porcine preantral follicle cultures between Day 5 and Day 8 of culture.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In a previous study of Cx43 in prepubertal pig ovaries, Cx43 expression in granulosa cells was associated with initiation of early follicular development [12]. In the present study, we investigated the effect of the growth factor EGF on the expression of Cx43 protein in cultured ovarian explants and isolated preantral follicles of pigs and obtained new information concerning the potential role of EGF in the expression of Cx43 gap junctions during early ovarian follicular development in pigs. This is the first report addressing the regulation of porcine Cx43 gap junction protein by EGF using 2 different preantral follicle culture systems.

The anti-rat Cx43 antibody used in this study revealed 2 molecular species of 43 kDa and 45 kDa representing the nonphosphorylated and the phosphorylated forms of the Cx43 protein, respectively. The existence of phosphorylated forms of Cx43 found in ovarian follicles in this study has been previously reported for pigs [13, 14], rats [16, 17, 40], and cows [41]. Similarly, phosphorylated and nonphosphorylated forms of Cx43 proteins are found in nonreproductive tissues such as brain [42], heart [43], and lens [44]. The phosphorylation of Cx43 can influence the biological activity of this protein and may be essential for the assembly of Cx43 into gap junctions [45].

The two different culture systems used in the present study have been used for the initiation of growth of primordial follicles in ovarian explants of cows and baboons [34, 46] and for growth in collagen matrices of isolated preantral follicles from pigs and cows [35, 47]. The use of these culture systems for preantral follicles (ovarian explants and isolated follicles) allowed evaluation of the effects of EGF on expression of Cx43 protein during early stages of follicular development. The explant model supports follicle growth in a more natural cellular environment compared with the culture of isolated follicles. Although EGF-stimulated Cx43 in explant cultures likely reflects expression in growing follicles, increased expression in the interstitial cells cannot be ruled out in this model. The experiments with isolated follicles were used to further address that issue and revealed that EGF directly enhances Cx43 expression in cultured preantral follicles.

The results of Western blot analysis in this study showed that the expression of both phosphorylated and nonphosphorylated Cx43 increased in a dose-dependent manner after treatment of cultures with EGF. This is the first report demonstrating an increase of both phosphorylated and nonphosphorylated forms of Cx43 protein levels by EGF in culture of ovarian preantral follicles. Lenhart et al. [14] found that the level of total Cx43 protein expression in ovaries did not change during the cycle or after treatment of prepubertal pigs with eCG, whereas phosphorylation of the protein increased significantly with follicle size. After an LH surge or hCG injection, expression of Cx43 decreased dramatically [14]. Similar findings have been reported in other species. Incubation of rat antral follicles with FSH or eCG alone increased the amount of Cx43 protein [40, 48], which was associated with a dramatic elevation of its phosphorylated forms [17, 40]. In addition, treatment of immature rats with estradiol alone or in combination with progesterone [18, 49] resulted in increased Cx43 protein levels in granulosa cells of preantral and antral follicles. The increase in Cx43 protein levels associated with natural or induced follicular growth has been found in cows [41, 50, 51] and sheep [52, 53]. The present study suggests that growth factors such as EGF can stimulate Cx43 expression in preantral follicles, possibly as part of controlling early stages of follicular development prior to FSH dependent events.

Granot and Dekel [16] found that short exposure of rat antral follicles to LH stimulated phosphorylation of Cx43 followed by immediate dephosphorylation, but longer incubation with this hormone resulted in disappearance of the protein because of attenuation of its gene expression. Incubation of follicles with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced phosphorylation of Cx43 that was completely blocked by the PKC inhibitor staurosporine [16]. Granot and Dekel [16] suggested that PKA- and PKC-dependent pathways might mediate the phosphorylation of ovarian Cx43. Regulation of the amount and/or phosphorylation state of Cx43 protein by PKA and PKC has been reported also for pigs. Injection of PKA inhibitor, PKC or PKC activators (TPA or OAG [1-oleoyl-2-acetyl-sn-glycerol]) into porcine primary ovarian granulosa cells resulted in a reduction or cessation of cell-to-cell transfer of fluorescent dye, but communication resumed with the injection of the PKA catalytic subunit or after exposure of cells to FSH [19]. With longer exposures to either PKC activator, communication could not be similarly restored, possibly because of the lack of aggregates of Cx43 in the membrane [19]. These results indicate that cell-to-cell communication is reversibly regulated by protein phosphorylation mediated by PKA and PKC and that the amplitude of communication is a reflection of interactions between these 2 enzymes. Because EGF receptor is a tyrosine protein kinase, EGF is thought to mediate its biological actions through a complex signal transduction pathway involving the activation of mitogen-activated protein kinases (MAPK) [54–58]. MAPK also interact with PKA and PKC signal transduction pathways, which could explain how multiple factors influence expression of Cx43 [59, 60].

Results of the present study indicate that EGF may influence preantral follicle development by enhancing the expression of Cx43 protein gap junctions. The presence of both phosphorylated and nonphosphorylated forms of Cx43 could be a mechanism used by growth factors and/or hormones to regulate follicular growth during early folliculogenesis. Further research is required to determine whether this increase in gap junction expression helps control early follicular development or is just a consequence of follicular growth. EGF and its receptor are synthesized locally in the porcine ovary [61] and have been implicated in regulation of development of preantral follicles [27, 62]. It remains to be determined whether endogenous EGF influences Cx43 expression in preantral follicles and what effect this has on early follicular development.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Sam Prien, Mary Catherine Hastert, Tim Borsos, Deirdre Anderson, Stanley Harris, Naomi Reiter, and Edward Carrasco for technical assistance and Dr. Jorge Flores for critical reading of the manuscript.


   FOOTNOTES
 
First decision: 1 October 2001.

1 This work was supported by Texas Higher Education Coordinating Board ATP grant 6182-90-2962 to V.H.L. and J.J.M. Back

2 Correspondence: Vaughan H. Lee, Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430. FAX: 806 743 2990;vaughan.lee{at}ttmc.ttuhsc.edu Back

Accepted: January 30, 2002.

Received: September 6, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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R. Marchal, M. Caillaud, A. Martoriati, N. Gerard, P. Mermillod, and G. Goudet
Effect of Growth Hormone (GH) on In Vitro Nuclear and Cytoplasmic Oocyte Maturation, Cumulus Expansion, Hyaluronan Synthases, and Connexins 32 and 43 Expression, and GH Receptor Messenger RNA Expression in Equine and Porcine Species
Biol Reprod, September 1, 2003; 69(3): 1013 - 1022.
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