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. 2019 Aug 29;18(20):2742–2756. doi: 10.1080/15384101.2019.1656952

EGFR promotes the proliferation of quail follicular granulosa cells through the MAPK/extracellular signal-regulated kinase (ERK) signaling pathway

Yan Wu a,b,✉, Hongwei Xiao a,#, Jinsong Pi a, Hao Zhang a, Ailuan Pan a, Yuejin Pu a, Zhenhua Liang a, Jie Shen a, Jinping Du a
PMCID: PMC6773241  PMID: 31465245

ABSTRACT

Follicles develop into preovulatory follicles during folliculogenesis and the majority of small yellow follicles become atretic and gets reabsorbed. In this study, based the RNA-seq results of duck ovary, epidermal growth factor receptor (EGFR) was selected as a candidate gene in follicular development and the role was explored. The results demonstrated that EGFR-P8 was the quail EGFR core promoter. It had an E2F4 binding site within EGFR core promoter. E2F4 overexpression significantly increased EGFR expression in quail granulosa cells (GCs). However, the effect was abolished when the GCs were treated with corynoxeine, an inhibitor of the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) signaling pathway. Moreover, luciferase reporter assay and chromatin immunoprecipitation experiments showed that E2F4 upregulated the expression of EGFR expression, which increased E2 and P4 production. In addition, EGFR regulated GCs proliferation and affected follicular development. Taken together, our findings suggested that EGFR, which was regulated by E2F4, enhanced the expression of MAPK/ERK pathway components and follicular development. These results provided an important basis for an improved understanding of the MAPK/ERK pathway and new insight into the development of quail follicles.

KEYWORDS: Quail, follicular granulosa cells, follicle development, EGFR, E2F4, MAPK/ERK

Introduction

The developmental status of avian follicles determined the reproductive performance of avians. In the reproductive period, only 5% of follicles can develop into preovulatory follicles, and most of small yellow follicles (<8 mm in diameter) go into atretic and reabsorbed [1,2]. Usually, during peak laying periods, only one small yellow follicle is selected one day to join the hierarchy of preovulatory follicles and begins rapid growth and final differentiation in preparation for ovulation [3–5]. Therefore, elucidate the follicular development process and related mechanisms could help us to improve egg production. Unfortunately, the precise mechanisms underlying the selection of a specific follicle for ovulation are unknown. For investigating neuroendocrine components of reproduction, Japanese quail is an advantageous model.

It has been reported the EGF ligand/receptor system plays important physiological roles in cell survival, proliferation, adhesion, invasion, motility and angiogenesis [6–8]. Epidermal growth factor receptor (EGFR) is a member of transmembrane receptor tyrosine kinases ErbB family. In gonadotropin induced oocyte maturation, the activation of EGFR plays a physiological role [9]. Our previous results of RNA-seq showed that EGFR gene was differential expression in the duck ovaries between laying peak and before laying. Therefore, we selected EGFR as a candidate gene that potentially regulates follicular development and selection in quails. In tumors, EGFR overexpression activates numerous signaling pathways that induce cell proliferation, survival, and invasion, such as the Ras/mitogen-activated protein kinase (MAPK), Src kinase, Janus kinase signal transducer and activator of transcription (JAK/STAT), and phosphatidylinositol 39-kinase (PI3K)-Akt pathways [10]. Nevertheless, a mechanism coordinating the EGFR-mediated regulation of quail follicular granulosa cells proliferation and differentiation through the MAPK signaling pathway remains to be elucidated.

Thus, in the present study, we examined the expression, localization, core promoter region, transcription factor bind sites and role of EGFR in quail follicular granulosa cells (GCs) and tested the hypothesis that the stimulation of EGFR expression by the transcription factor E2F4 elicits the transactivation of EGFR and MAPK/ERK pathway in follicular GCs. The findings of this study lay the foundation for further elucidation of the molecular mechanisms underlying follicular development.

Materials and methods

Ethics statement

All animal experiments were carried out according the guidelines established by the Administration of Affairs Concerning Experimental Animal (Ministry of Science and Technology, China, 2004). Tissues were collected from 20-week-old egg-laying quails. The quails were raised with ad libitum and slaughtered in the laboratory humanely.

Cell culture, cell transfection and dual-luciferase reporter assays

The small yellow follicles were separated from egg laying quails and putted them into ice-cold phosphate-buffered saline (PBS). We removed the yolks from the follicles carefully with ophthalmic scissors. Granulosa cells were separated from small yellow follicles and then treated with 0.2% collagenase II (Gibco, Gaithersburg, MD, USA) for 30 min at 37°C, then oscillated in a vortex pan every five minutes. After centrifugation, the GCs were resuspended in culture media (the M199, 10% fetal bovine serum and 1% penicillin and streptomycin) and then seeded in 6 or 24-well culture plates with the density of 1 × 106 or 2 × 105 cells/well, respectively. Trypan blue was used to detect the number of cells. The cells were cultured at 37°C in 5% CO2. When cells achieved 80–90% confluence, transfection assays were conducted.

GCs and CHO (Chinese hamster ovary) cells (which were gotten from China Center for Type Culture Collection, Shanghai, China) were seeded in 24-well plates for transient transfection. After 16 to 24 h, the cells were transfected with an overexpression vector or siRNA using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, California, USA). The experiments were carried with triplicate for each construct. The luciferase activity in the transfected cells was measured on the basis of the instructions of manufacturer with a PerkinElmer 2030 Multilabel Reader (PerkinElmer).

Plasmid construction

Nine EGFR promoter deletion fragments were obtained from quail genomic DNA using the primers P1-P9 (shown in supplementary table S1). The purified PCR products were digested with KpnI and XhoI (NEB, Ipswich, MA, USA) and ligated into the pGL3-Basic vector (Promega). Quail EGFR (XM_015854890) and E2F4 (XM_015873747) were amplified, and then digested with KpnI and XhoI (NEB, Ipswich, MA, USA) and cloned into the pcDNA3.1 (+) vector (Promega). The amplified product of EGFR was also digested with EcoRI and XhoI (NEB, Ipswich, MA, USA) and cloned into pCMV-HA-N vector (Clontech). The mutant of the binding site was produced by the TaKaRa MutanBEST Kit (Dalian, China) and mutation primers (shown in supplementary table S1).

RNA interference

The short interfering RNAs (siRNAs) targeted EGFR and E2F4 were synthesized and designed by Guangzhou RiboBio (China). The oligonucleotides are showed in supplementary table S1. siRNAs were transfected into quail follicular GCs and Chinese hamster ovary (CHO) cells with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) by the instructions of manufacturer.

Chromatin immunoprecipitation (chIP)

As E2F4-specific antibodies for immunoprecipitation were not available, the pCMV-HA-N-E2F4 were constructed and then this vector was transfected into GCs. The ChIP assays were carried out using the EZ-ChIP™ Kit (Millipore, Boston, MA, USA) to evaluate the binding of endogenic E2F4 to EGFR promoter in GCs. We used an AVCX130 system (Sonics & Materials, Newtown, CT, USA) for cell sonication. Anti-HA (Abcam, ab9110) and normal mouse-IgG (Millipore) antibodies were used for the overnight immunoprecipitation reactions at 4°C. The purified DNA which comes from the samples and the input controls was amplified by PCR. The sequences of primers were listed in the supplementary table 1.

Quantitative real-time PCR

Using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), total RNA was extracted from cells. And it was measured using the NanoQuant Plate (TECAN, Infinite M200PRO). After transfection for 24 h, the cellular RNA was extracted. Reverse transcription was performed using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). All of the primers used in the QPCR experiment are listed in supplementary table S1. QPCR was carried out by LightCycle® 480 II (Roche) with THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). The 2−ΔΔCt method [11] was used to standardize the expression levels of genes to expression of β-actin using.

Western blotting analysis

The RIPA lysis buffer (Beyotime, Beijing, China) were used to generate Cell protein lysates. After transfection for 48 h, the cellular protein was extracted and separated using SDS-PAGE and transferred to PVDF membranes, which blocked with skim milk. Then, antibodies specific for EGFR (1:2000, Abcam, Cambridge, MA, USA), E2F4 (1:1000, Abcam, Cambridge, MA, USA) and β-actin (1:1000, Abcam, Cambridge, MA, USA) were used for immunoblotting. We have quantified the protein expression levels compared to β-actin expression using ImageJ 1.42q (Wayne Rasband, National Institutes of Health, USA).

Enzyme-linked immunosorbent assay (ELISA)

E2 and P4 levels in the GCs culture media were tested by the Chicken Estradiol (E2) and Progesterone (P4) ELISA Kit (Hefei Laier Biotechnology Co., Ltd., Anhui, China) at 0 h, 24 h, 48 h and 72 h after transfection with the EGFR-pc vector according to the instructions of manufacturer. Normalized E2 and P4 values were determined by extrapolation from a standard curve.

Cell counting kit-8 assay

The proliferation of GCs was detected using a Cell Counting Kit-8 assay (Dojindo, Kumamoto Japan). The cells were seeded in a 96-well plate and transfected at 70% confluence with the EGFR-pcDNA3.1 plasmid or siRNA specific for EGFR. After transfection 0, 24, 48, and 72 h, 10 μL of CCK-8 per 100 μL medium were added into every well. Subsequently, the plates were incubated at 37°C for 1 h. The absorbance was evaluated at 450 nm by the PerkinElmer 2030 Multilabel Reader.

5-ethynyl-2ʹ-deoxyuridine (EdU) assay

After GCs were transfected for 24 h, the GCs were incubation with 50 μM EdU (RiboBio, China) for 2 h. Next, the cells were fixed in 4% paraformaldehyde for 30 min and neutralized using 2 mg/mL glycine solution. And then, permeabilized using 0.5% Triton X-100. Subsequently, 1 × Apollo reaction cocktail (RiboBio, China) was added to the GCs, and then incubation for 30 min. The nuclear stain Hoechst 33,342 was then added for DNA content analysis and incubation for 30 min, too. A fluorescence microscope (IX35, Olympus, Japan) was used to capture six randomly selected fields to visualize the number of EdU-stained cells.

Apoptosis and cell cycle analysis

EGFR-pcDNA3.1(+), pcDNA3.1(+), siRNA-EGFR or siRNA-NC were transfected into quail GCs cells. After transfecting for 24 h, the cells were harvested. The apoptosis rates and cell cycle profiles were measured and determined by Fluorescence-activated cell sorting (FACS). Aapoptosis and the cell cycle were analyzed using the Annexin V-FITC Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China) according to the instructions of manufacturer.

Statistical analysis

The mean ± S.D were used to present all the data. Each treatment has three replicates. Differences between different groups were analyzed by a two-tailed t-test. An independent-samples t-test was used to evaluate the significant differences. A statistically significant difference was showed when P < 0.05.

Results

EGFR is differentially expressed between small yellow follicles and white follicles

In order to investigate the ovulation mechanism, we investigated if the EGFR gene was differentially expressed in different grade follicles (small yellow follicles and white follicles). Q-PCR analyzes showed that the EGFR was expressed at significantly higher transcript levels in tissues and granulosa cells of white follicles than those of small yellow follicles (P < 0.05) (Figure 1(a,c)). The western blotting analyzes results were in accordance with the Q-PCR analyzes (Figure 1(b,d)).

Figure 1.

Figure 1.

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EGFR gene expression in tissue and granulosa cells from different follicles. (a,c) The relative mRNA expression results of the tissue and granulosa cells from different follicles; (b,d) The relative protein expression results of the tissue and granulosa cells from different follicles. Note: WF means white follicle; SWF means small yellow follicle.

EGFR promotes the proliferation and differentiation of GCs and CHO cells

To verify the effect of EGFR on the proliferation and differentiation of GCs and CHO cells, we constructed an EGFR overexpression vector and designed EGFR-specific siRNA. The Q-PCR and Western Blotting results showed that EGFR expression levels in GCs were significantly increased or suppressed upon the transfection with EGFR-pcDNA3.1 (EGFR-pc) or siRNA-EGFR (EGFR-si), respectively (Supplementary Fig. S1(a–d)). Flow cytometry showed that EGFR overexpression induced G0/G1 phase arrest in GCs and CHO cells (Figure 2(a,b)); EGFR knockdown induced S phase arrest in the GCs and CHO cells (Figure S2(a,b)). The Q-PCR results showed that EGFR overexpression induced a very significantly increase in the expression of BCL2 (B-cell lymphoma-2, an anti-apoptotic marker gene [12,13]) (p < 0.01) and a significant decrease in the expression of c-Myc (an apoptosis marker gene [14,15]) (p < 0.01) (Figure2(c,d)); EGFR knockdown had induced the opposite results (Figure S2(c,d)). The CCK-8 results showed that the cell viability for overexpression EGFR was increased from 0 h to 96 h and there was significant difference between EGFR-pc and pcDNA3.1 (Figure 2(e)). Moreover, the result of EdU strain assay showed that there were significantly more cells in the EGFR overexpression group than in the control group (Figure 2(f)). These results showed that EGFR promoted cell proliferation.

Figure 2.

Figure 2.

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Effect of EGFR on the proliferation and differentiation of GCs and CHO cells. (a-d) Cell cycle arrest profiles upon EGFR overexpression and knockdown. (e-h) The expression of genes related to proliferation and apoptosis upon EGFR overexpression or knockdown. (i) Cell proliferation analyzed by CCK-8 assay. (j) ELISA analysis of E2 and P4 production in quail GCs transfected with EGFR-pc or pcDNA3.1 after 0 h, 24 h, 48 h and 72 h. (k-n) Apoptosis in each group detected by annexin V-FITC/PI double staining.

Figure 2.

Figure 2.

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(Continued.)

Estrogen and progesterone (P4) have important roles in the regulation of ovulation [16]. Therefore, it was essential to determine whether EGFR affects estradiol (E2) and P4 secretion in GCs. As shown in Figure 2(g), EGFR upregulated E2 and P4 production. These results showed that EGFR affects ovulation by regulating EGFR expression levels and E2 and P4 production in GCs.

To further confirm that EGFR promotes the proliferation of GCs and CHO cells, we used the FITC-Annexin V/PI double-staining method to examine apoptosis rates by flow cytometry. EGFR overexpression significantly reduced the cells number in early and late apoptosis in comparison with the empty plasmid control group (P < 0.05) (Figure 2(h,i)); downregulation of EGFR showed the opposite results (Figure S2(e,f)). Overall, the above results revealed that EGFR promotes the proliferation and differentiation of GCs and CHO cells.

Identification and characterization of the quail EGFR promoter

In order to identify the promoter region and regulatory factors of the quail EGFR gene, a series deletion in potential promoter region were predicted by online neural network promoter prediction online software and detected by luciferase analysis. Luciferase activity of GCs and CHO cells revealed that EGFR-P8 (- 425 bp/-222 bp) was the potential core promoter and was important for EGFR (Figure 3(a)).

Figure 3.

Figure 3.

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Identification of the E2F4-binding site in EGFR promoter region. (a) Luciferase assays show the activity of a series of deletion constructs in both quail GCs and CHO cells. (b) The JASPAR database was used to determine the potential transcription factor binding sites with a core match and matrix match of at least 0.8. (c) EGFR-P8 reporter constructs were cotransfected with E2F4-pc into growing GCs and CHO cells. (d) Point mutations in the E2F4-binding site in the EGFR promoter were analyzed using luciferase assays. Luciferase activity was analyzed 24 h after transfection.

In order to further find the transcription factors which bind the core promoter of EGFR using the transcription factor prediction database JASPAR, an E2F4 transcription factor binding site was distinguished in EGFR-P8 domain (Fig. S3). To examine if the activity of the quail EGFR promoter was influenced by E2F4, an E2F4 overexpression plasmid (E2F4-pc) was generated and cotransfected with the EGFR-P8 plasmid into growing GCs and CHO cells. After transfection for 24 h, luciferase assays results showed EGFR-P8 promoter activity was significantly increased by E2F4-pc (Figure 3(b)).

To confirm the essentiality of the E2F4 binding site at −399 to −389, this site was mutated using the wild-type EGFR-P8 plasmid as the template. Luciferase assays in GCs and CHO cells revealed that compared with the wild-type construct, the activity of mutant construct (E2F4-mut) was significantly decreased (Figure 3(c)).

E2F4 binding site detected in the EGFR promoter

To further confirm if the transcription factor-E2F4 was bind to the core promoter of quail EGFR in vivo, chromatin immunoprecipitation (ChIP) experiments were performed with growing GC cells. The pCMV-HA-N-E2F4 was constructed and then transfected into GCs. A 189-bp DNA fragment was amplified from material precipitated by the anti-HA antibody from GCs but, not from precipitated with the anti-IgG antibody (Figure 4). All the results demonstrated that E2F4 was specifically binded to the quail EGFR promoter.

Figure 4.

Figure 4.

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Binding of E2F4 to the EGFR promoter region as analyzed by chromatin immunoprecipitation (ChIP).

E2F4 regulates EGFR expression in gcs and CHO cells

In order to further verification that E2F4 was regulated EGFR expression, the E2F4-pcDNA3.1 vector or E2F4-siRNA was transfected into GCs or CHO cells, respectively. E2F4 overexpression significantly increased EGFR and BCL2 expression and decreased c-Myc expression, as shown by QPCR (Figure 5(a–c)) and Western-blotting analysis (Figure 5(d–f)) (P < 0.05). E2F4 knockdown significantly suppressed EGFR mRNA (Fig. S4(a–c)) and protein levels (Fig. S4(d–f)) (P < 0.01). All the results indicated that E2F4 was binding to core promoter of EGFR, upregulates EGFR expression and promotes the proliferation of GCs and CHO cells.

Figure 5.

Figure 5.

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E2F4 regulated EGFR expression in GCs and CHO cells. (a-c) E2F4-pcDNA3.1 or pcDNA3.1 was transfected into GCs and CHO cells, and EGFR, BCL2 and c-Myc mRNA expression levels were measured by qRT-PCR. (d-f) E2F4-pcDNA3.1 or pcDNA3.1 was transfected into GCs and CHO cells, and EGFR, BCL2 and c-Myc protein expression levels were measured by Western blot analysis. (g-i) E2F4-si or siRNA NC was transfected into GCs and CHO cells, and EGFR, BCL2 and c-Myc mRNA expression levels were measured by qRT-PCR. (j-l) E2F4-si or siRNA NC was transfected into GCs and CHO cells, and EGFR, BCL2 and c-Myc protein expression levels were measured by Western blot analysis.

E2F4 regulates the gene expression of MAPK/ERK pathway components via the EGFR gene

In this study, we supposed that E2F4 might play a role in the MAPK/ERK signaling pathway by regulating EGFR gene expression. First, the EGFR-pc was transfected into GC cells. EGFR overexpression was significantly promoted EGFR expression, and this was consistent with the results of QPCR (Figure 6(a)) and Western-blot analysis (Figure 6(b)) (P < 0.01), demonstrating the EGFR-pc plasmid was successful expressed. Furthermore, EGFR overexpression increased mRNA and protein expression level of HRas, Raf1, MEK2 or c-Myc, which were key genes or downstream targets in the MAPK/ERK pathway (Figure 6(c–j)). E2F4 overexpression increased the mRNA expression of HRas, Raf1, MEK2 and c-Myc compared with the control (Figure 6(k–r)). These results suggested that E2F4 promotes MAPK/ERK signaling via the EGFR gene.

Figure 6.

Figure 6.

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Overexpression of EGFR and E2F4 promoted the expression of key genes or downstream target genes in the MAPK/ERK signaling pathway. (a) Transfection of the EGFR-pc vector into GCs resulted in increased EGFR mRNA and (b) protein expression levels. (c-j) The expression levels of key genes or downstream target genes (HRas, Raf1, MEK2 and c-Myc) were upregulated when cells were transfected with the EGFR-pc or E2F4-pc vector (K-R).

Figure 6.

Figure 6.

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(Continued.)

To further verify that E2F4 promotes MAPK/ERK signaling via the EGFR gene, growing cells were treated with corynoxeine (an inhibitor of the MAPK/ERK signaling pathway that targets MEK and ERK) before transfection of the E2F4 and EGFR vectors. The optimum treatment time and concentration in this study were 2 h and 30 µM/L, respectively. After treatment with the inhibitor and transfection of the E2F4 or EGFR overexpression vectors, the expression levels of EGFR, E2F4 and an upstream gene in the MAPK/ERK signaling pathway (HRas) were significantly increased (P < 0.01) (Figure 7(a–f)), whereas the expression of MEK2 and a downstream target gene in the MAPK/ERK pathway (c-Myc) disappeared. These results further suggested that E2F4 promotes MAPK/ERK signaling via the EGFR gene and promotes cell proliferation.

Figure 7.

Figure 7.

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After treatment with an inhibitor, EGFR and E2F4 overexpression promoted the expression of EGFR, E2F4 and HRas, an upstream gene in the MAPK/ERK signaling pathway. (a), (c) and (e) show the mRNA expression of EGFR, E2F4 and HRas, respectively, in cells transfected with EGFR-pc and/or E2F4-pc after treatment with an inhibitor of the MAPK/ERK signaling pathway. The protein expression of these genes is shown in (b), (d) and (f).

Discussion

Granulosa cells (GCs) of normal proliferation and differentiation were critical for the ovarian follicles development [17]. However, the regulatory mechanism of follicular development was still unclear.

Many previous studies had indicated that in the process of oocyte maturation, cumulus expansion and ovulation, the activity of EGFR was essential; in cumulus cells, EGFR autocrine stimulation of activated epidermal growth factor-like peptide, to amplify the signal, and mediates transmission and mammals from outer periphery of LH stimulated oocytes [18–21]. However, how EGFR regulated follicular development and ovulation in poultry, which differ from mammals, had not been reported. In this study, EGFR was detected and displayed differential expression in small yellow follicles, white follicles, and the granulosa cells from small yellow follicles and white follicles of quail. These findings suggested that EGFR may mediate follicular development or ovulation. In order to further understand the molecular mechanism of EGFR was regulated follicular development and ovulation, the cell cycle profile, cell proliferation and apoptosis rates, and the expression of genes related to cell proliferation and apoptosis were determined in GCs and CHO cells (Figure 2). These results showed the EGFR promotes proliferation and differentiation of GCs and CHO cells.

A variety of intracellular and extracellular signals was responded by transcriptional regulatory a cell or organism and adapt accordingly [22]. The E2F was a family of transcription factor proteins and had a variety of functions; all E2Fs play important roles in cell cycle regulation [23,24]. The regulatory functions of E2F family members can affect cell cycle, cell differentiation, DNA damage response and cell death [25,26]. Most members of the E2F protein family contain several evolutionarily conserved domains, comprising a DNA-binding domain and a dimeric domain, for interacting with differentiation-regulating transcription factors [27]. For instance, previous studies results showed E2F4 can bind to promoter region of ACSL1 to drive its transcription [28], to the promoter region of SIRT5 to regulation of SIRT5 expression in bovine adipocytes [29], or to the EZH2 promoter to repress EZH2 transcription [30]. In this study, the 5ʹ flanking region of the quail EGFR gene were isolated and the promoter activity and the regulatory elements were analyzed. The EGFR-P8 (−425 bp/-222 bp) region had the maximal promoter activity, as detected by luciferase reporter analysis (Figure 3(a)). We found that E2F4 regulated EGFR transcription through binding to EGFR core promoter (EGFR-P8) through site directed mutagenesis, ChIP and overexpression E2F4 experiments. These results showed that E2F4 positively regulates EGFR gene expression.

EGFR can be activated by EGF-like peptide and promotes several signaling pathways, including the JAK/STAT, PI3K and MAPK pathways [31–33]. The basic role of ERK1/2 (also named as MAPK3/1) in effecting ovulatory stimulus in periovulatory follicle had been widely recorded [34,35]. The EGFR and ERK1/2 dependent gene transcription activation was finally upregulates EGF-like peptides production in cumulus cells and GCs. Activating this signaling and maintains the EGF network throughout the follicle to regulate cumulus expansion, ovulation and GC luteinization [36–38]. The activity of EGFR-ERK1/2 was important for fertility because it was regulated all kinds of events related with ovulation. This suggested that EGFR may regulate GC proliferation and differentiation via the MAPK/ERK pathway. In this study, we found that the overexpression of E2F4 or EGFR promoted the expression of the key genes, such as HRas, Raf1, and MEK2 and downstream genes, such as c-Myc, of the MAPK/ERK signaling pathway. Furthermore, corynoxeine, an inhibitor of the MAPK/ERK pathway that targeted MEK and ERK, was used to verify that E2F4 promoteed MAPK/ERK signaling via the EGFR gene. All the results indicated that E2F4 promotes EGFR expression and GCs proliferation via the MAPK/ERK pathway.

In this paper, our results provided direct proof that EGFR was participated in quail ovulation and was regulating by E2 and P4 production. Potential mechanism of EGFR participation in the suppression of E2 and P4 production was preliminarily illustrated. In addition, EGFR expression was upregulated by E2F4 (Figure 8). We also showed that EGFR regulates GC proliferation and follicular atresia through the MAPK/ERK signaling pathway. These findings strongly suggested that, during follicular development, genes in the MAPK/ERK signaling pathway were regulated by E2F4 via EGFR.

Figure 8.

Figure 8.

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A graphical abstract showing the main findings of this study. The EGFR gene is regulated by the transcription factor E2F4, and this regulation probably contributes to follicular development by promoting the EGFR/ERK signaling pathway.

Funding Statement

This work was supported by the National Natural Science Foundation of China (31601937).

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

The supplementary data for this article can be accessed here.

Supplemental Material

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