Epidermal Growth Factor in the Germinal Disc and Its Potential Role in Follicular Development in the Chicken¹

^a Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

	ABSTRACT

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The germinal disc region (GDR; germinal disc + overlying granulosa cells) of the hen's ovarian follicle secretes one or more factors that stimulate proliferation of, and decrease progesterone (P₄) production by, granulosa cells. Destruction of the GDR results in apoptosis and atresia of the follicle. These data suggested that the GDR produces a growth factor(s) to sustain the development of the follicle. These findings prompted us to investigate two questions: 1) Is epidermal growth factor (EGF) or transforming growth factor (TGF), which binds to the EGF receptor, present in the GDR? 2) Does EGF regulate granulosa cell functions in the hen? Immunocytochemistry revealed that EGF, but not TGF, was present in the germinal disc of the four largest preovulatory follicles of the hen. TGF was found only in the theca interna. To determine whether EGF regulates granulosa cell functions, granulosa layer explants (13 mm in diameter) from the second-largest preovulatory follicle were cultured for 36 h with 0, 0.017, or 0.17 µM EGF. Proliferation, apoptosis, and P₄ production of granulosa layer explants were then measured by using a colorimetric method for determining viable cell number, gel electrophoresis, and RIA, respectively. EGF regulates several functions of granulosa layer explants by stimulating proliferation, inhibiting apoptosis, and decreasing basal P₄ production. These data indicate that EGF is present in the germinal disc and may be one of the factors that regulate follicular development in the hen.

	INTRODUCTION

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At ovulation, the hen oocyte is 30 mm in diameter and consists of a large volume of yolk and the germinal disc (Fig. 1). The germinal disc contains most of the oocyte's organelles and the nucleus of the oocyte and is the avian counterpart to the mammalian oocyte. The oocyte is surrounded by a single layer of granulosa cells. Granulosa cells near the germinal disc are proliferative as determined by [³H]thymidine incorporation and morphology, whereas those distal to the germinal disc are differentiated based on morphology and progesterone (P₄) production [1, 2]. GDR (germinal disc + overlying granulosa cells)-conditioned medium (GDR-CM) was found to contain a peptide/protein factor(s) that stimulates proliferation of, and inhibits P₄ production by, granulosa cells [1]. Destruction of the GDR results in atresia, apoptosis, and ovulation failure of the treated follicle [3, 4]. These data indicate that the GDR plays a vital role in regulating follicular growth and may be a source of growth factors.

View larger version (47K): [in this window] [in a new window]   FIG. 1. Diagram of a cross section of the hen preovulatory follicle. The oocyte consists of a large amount of yolk and the germinal disc, which appears as a white plaque 3 mm in diameter on the surface of the oocyte, and is surrounded by the follicular wall. The germinal disc and overlying granulosa cells are associated tightly and form a unit called the GDR. (Modified from [1].)

Although the oocyte was once considered a passive participant in follicular growth, studies have revealed its importance in granulosa cell steroidogenesis [5], ovulation [3], and cumulus expansion [6]. Studies have shown that oocytes from other species produce growth factors to regulate follicular development [7–9]. One of the growth factors the mammalian oocyte produces is epidermal growth factor (EGF). In the human, EGF and its receptor were present only in oocytes of preantral follicles. Furthermore, immunostaining for EGF increased as follicles matured to the preovulatory stage, at which time EGF and its receptor were localized to the granulosa and theca interna layers as well [8]. These data suggest that EGF may play a role in the development of the oocyte and the follicle in the human and other species.

Our objective was to elucidate further the role of the GDR in regulating follicular growth. The observation that EGF in the mammal causes physiological responses similar to those induced by GDR-CM, and that transforming growth factor (TGF) binds to the EGF receptor, led us to focus on EGF and TGF as potential growth factors in the GDR. We asked two questions: 1) Is EGF or TGF present in the GDR? 2) Does EGF regulate granulosa cell functions in the hen? To answer question 1, immunocytochemistry (ICC) was used to determine whether EGF and TGF are present in the four largest preovulatory follicles of the hen (F1—largest preovulatory follicle, F2—second largest, etc.). To answer question 2, a proliferation assay, gel electrophoresis, and RIA were used to measure the effects of EGF on proliferation and apoptosis of, and P₄ production by, granulosa layer explants.

	MATERIALS AND METHODS

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Animals

Mature single-comb white Leghorn hens were individually caged and exposed to 17L:7D. Feed and water were provided ad libitum. Oviposition was recorded every 30 min from 0700 to 1200 h and again at 1700 h. Hens with regular sequences of 4–12 eggs were used.

Experiment 1: Is EGF or TGF Present in the GDR?

F1–F4 follicles were removed from hens 1 h after oviposition and were placed in 10% neutral buffered formalin (pH 7.4) for 24 h. Follicular walls from different parts of the follicle were obtained and prepared for ICC as previously described [10]. Slides were incubated with 10% normal goat serum to block nonspecific binding. The primary antibody for EGF was a polyclonal rabbit anti-human EGF antibody (Oncogene Research Products, Cambridge, MA), and the secondary antibody was a biotinylated anti-rabbit IgG (HistoMark Streptavidin-HRP Systems; Kirkegaard & Perry Laboratories, Gaithersburg, MD). Streptavidin-peroxidase and then Histomark Orange were used to develop staining. To localize TGF, the primary antibody was a monoclonal mouse anti-recombinant human TGF antibody (Oncogene Research Products), and the secondary antibody was a phosphatase-labeled goat anti-mouse IgG. BCIP/NBT phosphatase substrate (Kirkegaard & Perry Laboratories) was used to develop staining. All slides were counterstained with methyl green. Negative controls, omitting the first antibody or incubating with increasing amounts of corresponding growth factors, were performed to demonstrate specificity of antibodies. Chicken intestine, a known source of EGF, was used as a positive control in this study to confirm the specificity of the antibody. Photomicrographs were taken using Sony (Tokyo, Japan) digital photo camera DKC-5000 and processed with Adobe (Mountain View, CA) Photoshop 4.0.

Experiment 2: Does EGF Regulate Granulosa Cell Functions in the Hen?

TGF was not tested in this experiment because it was not present in the GDR. The F2 follicle was chosen as a representative follicle because EGF immunostaining was identical in F1–F4 follicles. The F2 follicle was removed from the hen 1 h after oviposition to allow ovulation to occur. The granulosa layer was isolated, and three 13-mm diameter circular "punches" equidistant from the GDR were collected. The three punches from the same F2 follicle were cultured with one of three doses of human EGF (0, 0.1, or 1.0 µg/ml) in 500 µl medium. The punches were cultured for 36 h at 39°C in a humidified 5% CO₂:95% air incubator in tissue culture-treated 24-well cell culture clusters (Costar, Cambridge, MA). Culture medium contained 25 mM Hepes, 3.7 g/L sodium bicarbonate, 10 g/L Dulbecco's Modified Eagle's medium, 100 U/ml penicillin, and 100 mg/ml streptomycin. The medium was filter sterilized and adjusted to pH 7.4. This experiment was repeated six times.

Measurement of Proliferation

CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) is a colorimetric method for determining viable cell number. After 36-h culture, 100 µl One Solution Reagent was added to each well of the 24-well plates, and incubation was carried out for 1 h. Then 100 µl was removed from each well and absorbance read at 490 nm. The One Solution Reagent contains MTS tetrazolium (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium), which is bioreduced by cells into a colored formazen product soluble in culture medium. Absorbance is directly proportional to the number of cells. This experiment was repeated six times.

Determination of Apoptosis

After 36-h culture, all granulosa punches were analyzed for apoptotic DNA fragmentation. The DNA extraction procedure was adapted from Tilly and Hsueh [11] with minor modifications. Briefly, tissues were homogenized with a Polytron (Kinematica, Luzern, Switzerland) at setting 1 in 0.3 ml of homogenization buffer (0.1 M sodium chloride, 0.01 M EDTA, 0.2 M sucrose, and 0.3 M Tris-HCl, pH 8). Homogenates were then processed as previously described [11]. After extraction and quantification of DNA, 10–15 µg of DNA was loaded onto a 2% agarose (Sigma Chemical Co., St. Louis, MO) gel with ethidium bromide (0.5 µg/ml) and separated by electrophoresis for 3.5–4 h at 50 volts using single-strength TAE (40 mM Tris-acetate, 1 mM EDTA) as running buffer. Gels were destained overnight in double-distilled water and visualized and photographed on a UV transilluminator. This experiment was repeated six times.

Measurement of P₄ Production

P₄ concentration of the spent medium was determined by RIA according to a previous study [12]. This experiment was repeated six times.

Statistics

ANOVA was used to compare the differences between treatment groups in the second experiment, which measured proliferation and P₄ production. Experiment 2 was repeated at least six times. Differences were considered significant when p < 0.05.

	RESULTS

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Experiment 1: Is EGF or TGF present in the GDR?

F1–F4 follicles (n = 4) were examined for the presence of EGF (Fig. 2) and TGF (Fig. 3). A representative F2 follicle is shown because all follicles had identical immunostaining for both EGF and TGF. EGF immunostaining (Fig. 2, A and B, arrowheads) was present immediately below the oolemma (Fig. 2B, asterisks) of the germinal disc. Staining for EGF was absent from the follicular wall immediately adjacent to the germinal disc (Fig. 2, C and D) and follicular walls from other parts of the follicle (not shown). No staining for TGF was found in the germinal disc. However, TGF was uniformly present in the theca interna of the follicular wall (Fig. 3, A and C, and Fig. 3, B and D, arrowheads). Negative controls omitting the first antibody were performed, and no staining was detected for either growth factor. Incubation with increasing amounts of human EGF partially diminished immunostaining for EGF. Incubation with increasing amounts of human TGF abolished immunostaining for TGF (data not shown).

View larger version (79K): [in this window] [in a new window]   FIG. 2. Immunolocalization of EGF in the F2 follicle of the hen. A) x200 and B) x1000 magnification of the germinal disc and its overlying follicular wall. C) x200 and D) x1000 magnification of follicular walls immediately adjacent to the germinal disc. Arrowheads indicate the immunostaining for EGF (orange-brownish color). Bars = 50 and 10 µm for x200 and x1000 magnifications, respectively. GD, Germinal disc; Gr, granulosa layer; Th, theca layer; asterisks, oolemma.

View larger version (86K): [in this window] [in a new window]   FIG. 3. Immunolocalization of TGF in the F2 follicle of the hen. A) x200 and B) x1000 magnification of the germinal disc and its overlying follicular wall. C) x200 and D) x1000 magnification of follicular walls, which do not contain the germinal disc. Arrowheads indicate the immunostaining for TGF (dark purple). Bars = 50 and 10 µm for x200 and x1000 magnifications, respectively. GD, Germinal disc; Gr, granulosa layer; TI, theca interna; TE, theca externa; asterisks, oolemma.

Experiment 2: Does EGF Regulate Granulosa Cell Functions in the Hen?

EGF significantly stimulated proliferation of F2 granulosa cells at the highest dose (Fig. 4A). Furthermore, apoptotic DNA fragmentation in granulosa layer explants decreased with increasing concentrations of EGF in the culture medium (Fig. 4B). In granulosa explants cultured without EGF, four light bands of DNA fragments were apparent (Fig. 4B, 4 arrowheads). In granulosa explants cultured with 0.017 µM EGF, only two light bands of DNA were apparent (Fig. 4B, arrowheads). Finally, granulosa explants cultured with 0.17 µM EGF demonstrated no DNA fragmentation (no band present). This experiment was repeated six times and the results were consistent. P₄ production by F2 granulosa layer explants was significantly decreased when culture was performed with EGF (Fig. 4C).

View larger version (27K): [in this window] [in a new window]   FIG. 4. Effects of increasing amounts of EGF on granulosa layer explants after 36-h culture. A) Proliferation of granulosa layer explants measured by the CellTiter 96 AQueous One Solution Cell Proliferation Assay. B) Apoptotic DNA fragmentation of granulosa layer explants (arrowheads) as determined by gel electrophoresis of total DNA (10–15 µg). C) P4 production by granulosa layer explants measured by RIA. Asterisk indicates statistical significance (p < 0.05). Each bar represents the mean (SEM of six experiments).

	DISCUSSION

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Our novel finding is the identification of EGF in the germinal disc. EGF was located in the germinal disc immediately below the oolemma. Staining for EGF was absent from the remainder of the oocyte, the granulosa cells contiguous to the germinal disc, and the follicular wall. Thus, the immunostaining for EGF was specific for the germinal disc. In contrast, TGF was present uniformly in the theca interna of the follicular wall, but not in the oocyte. Addition of EGF to granulosa layer explants stimulated proliferation of, inhibited apoptosis of, and decreased basal production of P₄ by, granulosa layer explants from the F2 follicle.

However, the source of EGF in the germinal disc is unknown. One possibility is that EGF is imported into the oocyte with yolk proteins. As the oocyte grows, it imports yolk proteins from the blood—first white yolk and then yellow yolk during the rapid growth phase. Interestingly, the oolemma overlying the germinal disc is unique in that yellow yolk proteins do not enter the oocyte at that site. As a result, the germinal disc is not covered with yolk and displaced to the middle of the oocyte. Rather, the germinal disc remains attached to the oolemma. EGF was found only in the germinal disc, suggesting that EGF may be imported with white yolk proteins. However, if this occurred, the entire germinal disc should have shown immunostaining for EGF because the germinal disc is filled with white yolk. Furthermore, EGF is not imported with yellow yolk proteins, because the yellow yolk portion of the oocyte had no immunostaining for EGF. Thus, the presence of EGF in the germinal disc is apparently not due to protein uptake by the oocyte during yolk deposition. A second possible source of EGF in the germinal disc is the overlying granulosa layer. The absence of EGF staining in the granulosa cells suggests that they are not the source of EGF. A third possibility is that the germinal disc produces EGF. It has been shown that synthesis and accumulation of RNA and proteins are major activities of growing oocytes [13]. The accumulation of EGF immediately below the oolemma suggests that EGF may be ready for release. Studies are in progress to determine whether the germinal disc synthesizes EGF.

The presence of EGF in the hen follicle has been examined previously. In an ICC study by Onagbesan et al. [14], EGF was ubiquitous, present in the granulosa, theca interna, and theca externa layers. The discrepancy between our results and those of Onagbesan et al. may be due to the use of different antibodies and tissue preparation (paraffin-embedded versus frozen sections). Furthermore, we used the chicken intestine, a known source of EGF, as a positive control in this study (not shown) to confirm the specificity of our antibody.

In the second experiment we found that the addition of EGF to granulosa layer explants had effects on granulosa layer functions. EGF stimulated proliferation and inhibited apoptosis of granulosa layer explants from the F2 follicle. This finding confirms the earlier finding by Yoshimura and Tamura [15] that EGF stimulated granulosa cell proliferation. The same result has been reported for the mammal [16]. The inhibition of apoptosis correlates with earlier studies [3, 4] in which destruction of the GDR resulted in apoptosis of the follicle. Apoptosis may be due to the loss of EGF present in the germinal disc. Lack of EGF causes apoptosis of rat granulosa cells as well [17]. EGF also decreased basal P₄ production by granulosa layer explants from the F2 follicle. This is the same effect as described for GDR-CM [1]. Our results also confirm the finding of Pulley and Marrone [18] that EGF inhibits P₄ biosynthesis in cultured chicken granulosa cells. On the basis of these findings we hypothesize that the germinal disc synthesizes EGF, which is stored below the oolemma. The EGF is released in response to a certain signal and binds to receptors on the granulosa cells. This then leads to activation of EGF tyrosine kinase receptor that ultimately results in the alteration of granulosa cell functions such as steroid biosynthesis and growth.

In summary, EGF was present in the germinal disc of the hen's oocyte and was found to regulate several functions of granulosa layer explants by stimulating proliferation, inhibiting apoptosis, and decreasing basal P₄ production. These findings explain results of previous studies in which destruction of the GDR of the hen's preovulatory follicle resulted in death of the follicle via apoptosis. The loss of EGF after GDR destruction may be a contributing factor to apoptosis in the aftermath of that treatment. The observation that the addition of EGF to granulosa culture caused physiological responses similar to those of GDR-CM (stimulating proliferation and decreasing production of P₄ by granulosa cells) suggests that EGF may be the factor secreted by the germinal disc. Studies are underway to test this hypothesis.

Investigations of oocyte-follicle interactions are difficult due to the variety of cell types involved, the changes occurring as the oocyte/follicle grows and matures, the constantly changing hormonal milieu, and the large number of growth factors that may be involved. However, in this study we have begun to shed light on at least one factor (EGF) that the oocyte uses to control its destiny.

	ACKNOWLEDGMENTS

 
The authors are grateful to Dr. Rex Hess (University of Illinois at Urbana-Champaign, IL) for his microscopic equipment and support on image processing.

	FOOTNOTES

 
¹ Supported by National Science Foundation (NSF IBN-92–07535 and IBN-96–30957) and the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture (#35–324). This work was presented at the SSR 30th annual meeting, Portland, Oregon, 1997, Abstract 173. K.K.V. and H.-C.Y. contributed equal amount of work on this project.

² Correspondence: Janice M. Bahr, ASL 326, 1207 West Gregory Drive, Urbana, IL 61801. FAX: (217) 333–8286; j-bahr{at}uiuc.edu

Accepted: April 21, 1998.

Received: January 5, 1998.

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