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Regular Article |
a Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
ABSTRACT
The germinal disc (GD) of the chicken oocyte produces factors that influence proliferation and differentiation of granulosa cells. Granulosa cells proximal to the GD are more proliferative, whereas granulosa cells distal to the GD are more differentiated. Previously, we had found epidermal growth factor (EGF) was present in the GD. In this study, we tested the hypothesis that EGF is the GD-derived paracrine factor that stimulates proliferation of granulosa cells. Northern analysis, reverse transcription-polymerase chain reaction, and radioimmunoassay indicated that the GD and granulosa cells but not theca cells are the sources of EGF in chicken preovulatory follicles. However, only the conditioned medium from the GD region (GDR = GD + overlying granulosa cells) but not the granulosa cell-conditioned medium stimulated proliferation of granulosa cells. Pretreatment of conditioned media with EGF antibody abolished the proliferation-stimulating effect of the GDR-conditioned medium. We conclude that EGF is one of the paracrine factors produced by the GD to stimulate proliferation of granulosa cells. Granulosa cells proximal to the GD express a proliferative phenotype possibly because they are exposed to a greater amount of EGF derived from the GD.
follicle, granulosa cells, growth factors, ovary, ovum
INTRODUCTION
Development of a healthy follicle relies on not only a tightly regulated hypothalamic-pituitary-gonadal axis but also a coordinated interaction between the oocyte and its surrounding cells. The oocyte receives information and nutrients from granulosa cells while releasing signals that influence the growth and differentiation of granulosa cells. In the rabbit, removal of the oocyte induced spontaneous luteinization of granulosa cells in the antral follicle [1]. Co-culturing of rat granulosa cells with oocytes resulted in inhibition of spontaneous luteinization of cultured rat granulosa cells, suggesting that oocytes secrete factors that prevent spontaneous luteinization [2]. In the rodent and other species, factors secreted by oocytes regulate granulosa steroidogenesis [3–5], stimulate granulosa proliferation [6], inhibit plasminogen activator production by granulosa cells [7], and stimulate cumulus expansion in response to FSH [8–12]. Murine oocytes also suppressed expression of LH receptor mRNA in cultured granulosa cells [13]. Furthermore, murine follicles that did not express the oocyte-specific factor, growth differentiation factor-9 (GDF-9), failed to develop beyond the primary stage [14]. These observations clearly indicate that mammalian oocytes are actively involved in regulating growth and differentiation of follicles.
The avian oocyte also plays a role in follicular development. The oocyte, which can reach a size of 40 mm in diameter before ovulation, consists of a large amount of yolk and a structure called the germinal disc (GD). The GD is a white plaque about 3–4 mm in diameter on the surface of the oocyte. The GD contains the nucleus and 99% of the organelles of the oocyte even though the GD occupies less than 1% of the oocyte volume. The GD structurally and functionally resembles the mammalian oocyte. The GD and its overlying granulosa cells are associated through gap junctions and interdigitations [15] and form a unit called the GD region (GDR). The GDR is considered the growth center of the chicken follicle [16] because granulosa cells proximal to the GDR are highly proliferative, whereas granulosa cells distal to the GDR are more differentiated and produce more progesterone in response to LH [17–19]. In addition, destruction of the GDR of preovulatory follicles 24 h before ovulation resulted in atresia and apoptosis of follicles and failure to ovulate [20–22].
The ability of GDR-conditioned medium to stimulate 3H-thymidine incorporation and decrease progesterone production by granulosa cells indicated that the GD produces paracrine factors that influence functions of granulosa cells. Sensitivity to heat and proteinase suggested that factors in GDR-conditioned medium were peptides or proteins [23]. We found by immunocytochemistry that epidermal growth factor (EGF) was present in the GD. Moreover, EGF in vitro mimicked the effect of GDR-conditioned medium to stimulate proliferation of and to decrease progesterone production by granulosa cells [24]. However, there is no direct evidence to indicate that EGF is the paracrine factor produced by the GD to regulate functions of granulosa cells. The active transportation of proteins such as low-density lipoproteins into the chicken oocyte raises the possibility that EGF in the GD may be synthesized by other follicular compartments and deposited into the GD along with yolk proteins. To demonstrate that EGF is one of the GD-derived paracrine factors responsible for stimulating proliferation of granulosa cells, we conducted experiments to answer the following questions: 1) What are the sources of EGF in the chicken preovulatory follicle? 2) Does the GD synthesize EGF mRNA and its protein? and 3) Does EGF derived from the GD stimulate proliferation of granulosa cells?
MATERIALS AND METHODS
Animals
Single-comb white Leghorn hens in their first year of reproductive age and with laying clutches of at least five eggs were used in all the studies described. Birds were maintained individually and provided with feed and water ad libitum. The lighting schedule was 17L:7D, with lights-on at 0400 h and lights-off at 2100 h. The times of oviposition were monitored daily at 1-h intervals between 0700 h and 1200 h and once at 1700 h for late oviposition.
Tissue Collection
Hens were killed by cervical dislocation 1–2 h after oviposition. Preovulatory follicles (F1–F3) were removed and immediately placed in ice cold PBS. Granulosa and theca layers were separated as previously described [25].
Reverse Transcriptase-Polymerase Chain Reaction
Chicken-specific EGF primer sets were designed according to the cDNA sequence from GenBank and published data [26]. The sense 5'-primer (5'-CAC AAC GGC GGC CAG TGC TA-3') and the antisense 3'-primer (5'-ATG AAG AGC AGC AGC AGC ACC AGC-3') generated a 170 base-pair (bp) cDNA fragment corresponding to chicken EGF nucleotides 1577 to 1746. The W. M. Keck Center for Functional and Comparative Genomics at the University of Illinois at Urbana-Champaign synthesized the primer sets. RETROscript First-Strand Synthesis Kit (Ambion Inc., Austin, TX) was used to generate single-stranded cDNA from isolated total RNA (2 µg). The PCR was performed using reagents from TaqBead Hot Start Polymerase (Promega, Madison, WI), and then the general PCR protocol was followed (29 cycles at 94°C for 2 min, 55°C for 1.5 min, and 72°C for 1.5 min followed by a 5-min extension at 72°C).
Synthesis of Biotinylated Riboprobes
We used an RNA Biotin Labeling Kit with Streptavidin-AP (NEN Life Science Products, Boston, MA) to generate riboprobes from cloned plasmids. Cloned plasmids were first linearized with either NotI (for PCR products with 5'-3' orientation) or EcoRI (for PCR products with 3'-5' orientation) restriction enzyme. Complete linearization was confirmed by gel electrophoresis of the digested plasmid. The linear plasmid (2 µl, 0.5 µg/µl) was transcribed into RNA in vitro by mixing with 5 µl Biotin Nucleotide Mix (4x), 11 µl diethylpyrocarbonate-treated water, and 2 µl of RNA polymerase (T7 for 5'-3' orientation and T3 for 3'-5' orientation). The mixture was incubated for 2 h at 37°C in a water bath followed by addition of 1 µl DNase for 15 min at 37°C. The RNA probes were aliquoted and stored at -80°C. The yield of RNA probes was quantified by a spectrophotometer.
Northern Analysis
Total RNA from ovarian tissues was isolated using TRIzol Reagent (Life Technologies, Gaithersburg, MD). Ten micrograms of total RNA were separated on a 1.5% denaturing agarose gel. RNA was then transferred from the gel to a Magnagraph nylon membrane (MSI, Westborough, MA) overnight by standard capillary transfer. After transfer, the membrane was checked for the presence of ethidium bromide-stained RNA, rinsed briefly in 0.1x SSC, and air dried for 15 min. The RNA was then ultraviolet (UV) cross-linked to a membrane with a Stratagene UV Crosslinker (Stratagene, La Jolla, CA). Prehybridization was performed by incubating the membrane in 10 ml of prehybridization buffer (50% formamide and 0.5% SDS in 5x Denhardts solution and 6x SSPE [3 M sodium chloride, 0.2 M sodium phosphate, 0.02 M EDTA, pH 7.4]) at 55°C for 3 h. The prehybridization buffer was then removed and 10 ml of hybridization buffer with biotinylated RNA probes (20–50 ng/ml) were added. Hybridization was performed overnight at 55°C in the hybridization oven. The membrane was washed twice with 2x SSPE and 0.5% SDS for 30 min at room temperature followed by two more washes with 0.1x SSPE and 0.5% SDS for 30 min at 65°C. The membrane was then washed with 1x SSPE and 0.1% SDS for 5 min at room temperature followed by incubating in block solution (1x SSPE, 0.5% SDS, 0.5% Hammerstein Casein) for 30 min. The membrane was incubated in the streptavidin conjugate 1x solution for 30 min and then washed three times in blocking buffer for 5 min each. The membrane was finally washed three times for 5 min each with 1x SSPE and 0.1% SDS followed by two 2-min washes in assay buffer (10 mM Tris-HCl pH 9.5, 10 mM NaCl, 1 mM MgCl2). Two milliliters of CDP-Star (NEN, Boston, MA) substrate were added onto the membrane and a piece of plastic wrap was placed over the membrane to evenly distribute the substrate. Then the membrane was blot dried, wrapped in Saran wrap (Borden, North Andover, MA), placed in a cassette, and exposed to X-OMAT Blue XB-1 film (Kodak, Rochester, NY) for 5 min to 30 min at room temperature. The density of the bands was analyzed densitometrically with Collage Image Analysis Software (Fotodyne Inc., Hartland, WI). The relative density of EGF was normalized with density of correspondent 28S rRNA bands.
Epidermal Growth Factor Radioimmunoassay
Epidermal growth factor radioimmunoassay (RIA) was carried out as described previously with modification [27]. Briefly, 1 mCi Na125I was neutralized with 10 µl of 0.2 M phosphate buffer (pH 7.4) and added to 10 µg of EGF in 10 µl of 0.2 M phosphate buffer. Eight-hundred nanograms of chloramine-T in 10 µl of water were then added and incubated for 30 sec at room temperature. The reaction was stopped by adding 200 µl of phosphate buffer with 2.5% BSA. The free 125I and labeled EGF were then separated by eluting the reaction mixture through a 0.7 cm x 30 cm Econocolumn (Bio-Rad, Hercules, CA) packed with Sephadex G-50 in 0.05 M Tris-base buffer. To measure EGF in the conditioned medium, 100 µl of standards (0.5–64 ng/ml of mouse EGF) or conditioned medium (450 µl) were incubated with the primary antibody (12.5 µl; 1:4000 dilution in normal rabbit serum) and 10 000 cpm 125I-EGF for 2 h at 37°C or 18–20 h at room temperature. The final volume of the reaction was brought to 500 µl with PBS with 1% BSA. Then, 100 µl EDTA (0.1 M) and 100 µl of 2% normal rabbit serum in PBS were added to the reaction followed by addition of the secondary antibody (200 µl; 1:10 dilution) and 500 µl of 6% polyethylene glycol for 15 min at room temperature. The reaction tubes were centrifuged for 15 min at 1800 x g at 4°C. The supernatant was aspirated and the pellet counted for 1 min in a gamma counter. Nonspecific binding was determined by replacing the primary antibody with normal mouse serum. An internal control of 5 ng/ml mouse EGF was run in every assay to determine the intraassay and interassay variations. Hormone concentrations were calculated using the RIAEIA Parallelism Program with Hot Recovery in SAS written by Dr. Ming-Che J. Wu. Intraassay and interassay coefficients of variation were 6.3% and 10.5%, respectively.
Conditioned Medium for Explant Culture and RIA
Conditioned media (CM) were prepared by culturing either four GDR or four proximal granulosa punches (4 mm in diameter) without the GD in 0.5 ml of serum-free Dubeccos modified Eagles medium (DMEM) for 12 h at 39°C in a tissue culture-treated 24-well cell culture cluster (Costar, Cambridge, MA) in a humidified 5% CO2:95% air incubator. Either a rabbit anti-mouse EGF antibody (1:4000 dilution, Sigma) or normal rabbit serum was added to the medium before culture. For explant culture, a granulosa explant (13 mm in diameter) from the F1 follicle was added to the conditioned medium at the end of 12 h and cultured for another 24 h. For RIA, 450 µl of conditioned medium was used to measure EGF concentration.
Measurement of Cell Proliferation
CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was used to determine viable cell number. At the end of the culture, 100 µl of One Solution Reagent was added to each well followed by 1 h of incubation. Then, 100 µl of medium was removed from each well and absorbency was read at 490 nM. The One Solution Reagent contains MTS tetrazolium (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium), which is bio-reduced by cells into a colored formazen product, which is soluble in culture medium. Absorbency is directly proportional to the number of cells.
Statistics
ANOVA was used to compare the differences between different tissue sources. The experiments were repeated five times for Northern analysis, conditioned medium studies, and RIA. Differences were considered significant when P < 0.05.
RESULTS
What Are the Sources of EGF in the Chicken Preovulatory Follicle?
We used RT-PCR to detect the presence of EGF mRNA in GDR, granulosa, and theca layers of preovulatory follicles. The 170-bp fragment for EGF was present in GDR and granulosa samples, whereas no product was detected in theca sample or when cDNA was replaced with water (Fig. 1). The 170-bp fragments from chicken tissues were cloned and sequenced. The 170-bp fragment sequence was identical to the chicken EGF cDNA sequence from GenBank. Northern blot analysis was performed to determine the presence and size of EGF mRNA from ovarian tissues. The 170-bp DNA product from RT-PCR was used as a template to generate a biotinylated riboprobe for chicken EGF. A 2-kilobase (kb) mRNA transcript was detected in granulosa layers but not theca layers of the three largest preovulatory follicles (F1, F2, and F3; Fig. 2).
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Does the GD Synthesize mRNA and Protein for EGF?
Due to technical difficulties in isolating the GD, we compared the difference between the GD region (GDR = GD + proximal granulosa cells) and proximal granulosa cells without the GD (Fig. 3). The difference between these two samples should indicate the contribution of the GD. The GDRs or proximal granulosa cells of F1, F2, or F3 follicles were pooled from five to six birds according to the size of the follicles. There was no difference in EGF mRNA abundance among follicles and the result for the F1 follicle was shown as a representation. Northern blot analysis indicated the presence of a 2-kb transcript for EGF in both GDR and proximal granulosa cells. To differentiate the abundance of EGF mRNA in the GDR and granulosa cells, densitometric readings of bands for EGF transcript and 28S rRNA were obtained. The relative abundance of EGF transcript was normalized with 28S rRNA and the abundance of EGF transcript in GDR was set as 100%. The EGF transcripts in the GDR were 30% more abundant than those in proximal granulosa cells (Fig. 3, n = five birds, P < 0.01). The ethidium bromide-stained 28 and 18S rRNA demonstrated relatively equal loading of total RNA. No signal was detected using the sense riboprobe (data not shown). The presence of EGF in conditioned media of the GDR and proximal granulosa cells (Fig. 4) was measured by EGF RIA. The concentration of EGF in the GDR-conditioned medium (mean ± SEM = 45.30 ± 3.99 pM, n = five birds) was 45% higher than that in the proximal granulosa conditioned medium (mean ± SEM = 25.56 ± 5.96 pM, n = five birds, P < 0.05).
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Does EGF Derived from the GD Stimulate Granulosa Proliferation?
To examine the biologic activity of EGF in conditioned media, a rabbit anti-mouse EGF antibody was added to conditioned media to neutralize the EGF in the conditioned medium. Without the addition of anti-EGF antibody, the GDR-conditioned medium significantly increased the number of granulosa cells compared with proximal granulosa-conditioned medium or control unconditioned medium alone (Fig. 5, n = five birds, P < 0.05). Addition of antibody to the conditioned media abolished the stimulatory effect of GDR-conditioned medium on proliferation of granulosa cells. Addition of normal rabbit serum to the conditioned medium that served as the control for the antibody did not have any effect on granulosa cell proliferation. After addition of anti-EGF antibody, EGF in the conditioned medium was undetectable (data not shown).
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DISCUSSION
We found that 1) both GD and its overlying granulosa cells synthesize EGF mRNA and protein, whereas the theca layer of preovulatory follicles is not a source of EGF; and 2) EGF is one of the paracrine factors from the GD that stimulates proliferation of granulosa cells.
The source of EGF in the avian follicle has been controversial. Onagbesan et al. [28] used a polyclonal antibody against recombinant human EGF (whole protein; Biogenesis, Bournemouth, UK) in frozen sections of large white and preovulatory chicken follicles. They found that EGF was ubiquitously present in granulosa, theca externa, and theca interna layers of the follicles. However, Volentine et al. [24] used a similar polyclonal anti-human EGF antibody (Oncogene Research Products, Cambridge, MA) on paraffin sections obtained from formalin-fixed follicles and found immunostaining for EGF was only present in the GD of preovulatory follicles. The discrepancy of these two reports may result from sources of antibodies, methods of tissue processing, and sensitivity of the assay. Localization of EGF mRNA should resolve these problems and identify the cell types that produce EGF in the follicle. Our results from RT-PCR and Northern blot analysis clearly demonstrated that the GD and granulosa cells but not the theca layer produce EGF mRNA. These results were further verified by using an EGF RIA to detect EGF in the conditioned media from the GDR and granulosa cells without the GD. However, we have to emphasize that the conclusion that the GD produces EGF is inferential. Because of technical difficulties in isolating the GD, we compared the difference between granulosa cells with the GD (GDR) and granulosa cells without the GD. The difference between these two samples should indicate the contribution of the GD. Quantitative results from Northern analysis and RIA for EGF showed that the GDR has 30% and 45% more mRNA and protein, respectively, than granulosa cells without the GD. These data indicate that the GD of the chicken oocyte is able to synthesize EGF mRNA and protein.
The presence of EGF in the oocyte has been reported in many other species beside chickens. In Japanese quails, a relative of the chicken, EGF was found in oocytes of developing follicles [29]. The cytoplasm of hamster oocytes at all stages of follicular development showed noticeable immunostaining for EGF [30]. The mRNA and protein for EGF were detected in porcine oocytes at all follicular stages [31]. In humans, immunostaining for EGF was observed in the oocyte and the staining intensity increased as the oocyte reached the preovulatory stage [32]. Although numerous studies indicated that EGF is a potent mitogen for chicken, rodent, porcine, and human granulosa cells in vitro [33, 34], we have very limited knowledge of the physiological role of EGF produced by the oocyte.
To study the function of EGF derived from the GD, we examined the effect of GDR-conditioned medium on proliferation of granulosa cells before and after neutralization of EGF in the medium with an anti-EGF antibody. The ability of the GDR-conditioned medium to stimulate proliferation of granulosa cells is consistent with the previous study [23]. Neutralization of EGF in the GDR-conditioned medium abolished the stimulatory effect of the conditioned medium on proliferation of granulosa cells, indicating that EGF is the mitogenic factor derived from the GDR. It is very interesting that even though both GDR and granulosa-conditioned media contained EGF, only the GDR-conditioned medium was able to elicit a stimulatory effect on proliferation of granulosa cells. Our explanation is that there may be a threshold for the action of EGF on granulosa cells. In our culture system, the concentration of EGF in the conditioned media is at the picomole or fentomole level. Granulosa cells in the follicle should be exposed to a higher level of EGF produced by themselves, the GD, or both, depending on their location because they are in a confined environment instead of in a 0.5-ml culture medium. The constant presence of EGF from granulosa cells may define the basal responsiveness of granulosa cells to EGF. As a result, granulosa cells proliferate in response to EGF only when they are exposed to a higher level of EGF such as that in the GDR-conditioned medium.
The ability of GD-derived EGF to stimulate proliferation of granulosa cells provides an explanation to why granulosa cells in the GDR are more proliferative than granulosa cells distal to the GDR. It has been known for years that granulosa cells in the GDR have a higher mitotic rate [16], have more cells in the S and G2/M phases [17], and incorporated more bromodeoxyuridine [18] in vivo than the peripheral granulosa cells. Granulosa cells in the GDR also incorporated more tritiated thymidine than their peripheral counterparts in culture [19]. The proliferative phenotype of granulosa cells in the GDR may be attributed to their exposure to extra EGF derived from the GD (Fig. 6).
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This research is the first to demonstrate that EGF produced by the chicken oocyte has the paracrine function to stimulate proliferation of granulosa cells. Our findings provide a comparative model to illustrate the universal role of the oocyte in determining the fate of the follicle.
FOOTNOTES
1 Supported by National Science Foundation grants IBN-92-07535 and IBN-96-30957. This work was presented at the 32nd annual meeting for the Society for the Study of Reproduction at Pullman, Washington, abstract 1. 
2 Correspondence: Janice M. Bahr, ASL 326, 1207 West Gregory Drive, Urbana, IL 61801. FAX: 217 333 8286; j-bahr{at}uiuc.edu
3 Current address: Box 3709 Nanaline Duke, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710.
Accepted: September 5, 2000.
Received: June 5, 2000.
REFERENCES
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Y. Wang and W. Ge Cloning of Epidermal Growth Factor (EGF) and EGF Receptor from the Zebrafish Ovary: Evidence for EGF as a Potential Paracrine Factor from the Oocyte to Regulate Activin/Follistatin System in the Follicle Cells Biol Reprod, September 1, 2004; 71(3): 749 - 760. [Abstract] [Full Text] [PDF]
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