help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1383
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/7/3426    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, Y.
Right arrow Articles by Leung, F. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, Y.
Right arrow Articles by Leung, F. C.
Endocrinology Vol. 148, No. 7 3426-3440
Copyright © 2007 by The Endocrine Society

Epidermal Growth Factor (EGF) Receptor Ligands in the Chicken Ovary: I. Evidence for Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Potential Oocyte-Derived Signal to Control Granulosa Cell Proliferation and HB-EGF and Kit Ligand Expression

Yajun Wang, Juan Li, Crystal Ying Wang, Amy Ho Yan Kwok and Frederick C. Leung

Department of Zoology, The University of Hong Kong, Hong Kong, China

Address all correspondence and requests for reprints to: Dr. Frederick C. Leung, Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, China. E-mail: fcleung{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is increasing evidence that epidermal growth factor (EGF) receptor (EGFR) ligand and Kit ligand (KL) play critical roles in controlling follicular development in mammals. Because little is known about their expressions in the ovary of nonmammalian vertebrate, our study aimed to examine the expression, hormonal regulation, and interaction of HB-EGF and KL in the chicken ovary. Using semiquantitative RT-PCR, we demonstrated that ovarian HB-EGF expression increased dramatically with the posthatching ovarian growth. In line with this finding, HB-EGF was shown to be produced primarily by the growing oocytes and capable of stimulating the proliferation of granulosa cells in prehierarchal (3 mm) and preovulatory follicles (F5 and F1). Although HB-EGF expression is mainly restricted to the oocytes, its expression in cultured granulosa cells could be transiently yet strongly induced by HB-EGF and other EGFR ligands including EGF and TGF-{alpha}. And the inducing effect of HB-EGF was completely abolished by AG1478 (10 µM) or PD98059 (100 µM), indicating that the action of HB-EGF is mediated by EGFR and intracellular MAPK/ERK signaling pathway. Unlike mammals, only KL-1, not the other three isoforms identified (KL-2, -3, and -4), was detected to be predominantly expressed in the chicken ovary. Interestingly, KL expression in undifferentiated and differentiated granulosa cells could be transiently down-regulated by HB-EGF, implying an intrafollicular communication between growing oocyte and surrounding granulosa cells through the interplay of EGFR ligand and KL. Collectively, our data suggest that HB-EGF is likely a paracrine signal from the oocyte to regulate granulosa cell proliferation and HB-EGF and KL expression during ovarian follicular development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS GENERALLY believed that pituitary gonadotropins, including FSH and LH, play pivotal roles in the regulation of ovarian development and functions in vertebrates (1, 2). However, increasing evidence suggests that locally produced peptide growth factors could modify or mediate the actions of gonadotropins (1, 2, 3). The orchestrated actions of these paracrine/autocrine factors, together with the cyclic control of gonadotropins, ensure the release of mature oocyte for subsequent fertilization (1, 2, 4). Among these peptide factors, epidermal growth factor (EGF) receptor (EGFR) ligand and Kit ligand (KL) have attracted much attention in this regard (1, 5, 6, 7, 8).

EGFR ligands are made up of seven members including EGF, TGF-{alpha}, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), epiregulin (ER), betacellulin (BTC), and epigen (9). In mammals, both EGFR and its ligands including EGF, TGF-{alpha}, AR, ER, and BTC have been shown to be expressed in granulosa cells or thecal cells (10, 11, 12, 13). Interestingly, TGF-{alpha} and/or EGF could also be detected in pig, cow, and human oocytes (14, 15, 16). Consistent with their expressions, multiple biological actions of EGFR ligand have been reported in mammalian ovary. Exogenous EGF is able to stimulate proliferation and prevent apoptosis of granulosa cells (17, 18). It also modulates ovarian steroidogenesis and granulosa cell differentiation including stimulating progesterone production and inhibiting FSH-induced biosynthesis of estradiol and receptors of FSH and LH in cultured granulosa cells (19, 20, 21, 22). Moreover, a number of recent studies demonstrated that EGFR ligands including AR, ER, and BTC could mimic LH-surge actions including stimulating cumulus expansion and oocyte maturation in ovulatory follicles (10, 11, 12, 23, 24, 25), indicating that EGFR ligands also act as the endogenous mediators of LH actions during ovulatory process (6, 10, 12, 24).

KL (also called stem cell factor, SCF; mast cell growth factor, MGF; steel factor, SF) is another peptide growth factor extensively studied in mammalian ovary (5, 8, 26). At the embryonic stage, KL has been shown to be involved in the migration, proliferation, and survival of the primordial germ cell (27, 28, 29). During folliculogenesis, KL is detected to be expressed abundantly in the granulosa cells and demonstrated to be involved in the primordial to primary follicle transition, thecal cell recruitment and proliferation, as well as ovarian steroidogenesis (5, 7, 8, 30, 31). Moreover, KL is involved in controlling oocyte growth, survival and meiotic arrest (7, 8, 32, 33, 34). As a granulosa cell-derived factor, the expression of KL could be regulated by the paracrine factors from oocyte and thecal cell, also outlining a central role of KL in the intrafollicular communication between granulosa cell and both its neighbors, oocyte and thecal cell (35, 36, 37, 38, 39, 40, 41, 42).

In contrast to the extensive studies on EGFR ligand and KL in mammalian ovary, there is little or no information on the expression, hormonal regulation, and roles of EGFR ligand and KL in the ovary of nonmammalian vertebrate species, raising an important question on how the concept or hypothesis developed in mammals can be applied to other vertebrates. Despite the fact that exogenous TGF-{alpha} and EGF have been demonstrated to be capable of stimulating proliferation, preventing differentiation and apoptosis (43, 44, 45, 46, 47, 48, 49, 50, 51), and suppressing basal and gonadotropin-induced progesterone secretion of granulosa cells in chickens (43, 45, 46, 49, 52), nevertheless, where and when EGFR ligands are produced, as well as how their expressions are regulated in chicken ovary, remains largely unknown (47). The pioneering studies hypothesized that the germinal disc, a region occupying less than 1% of the oocyte volume but containing the nucleus and 99% of the organelles of oocyte within the preovulatory follicle, might serve as a growth center (53, 54, 55), which could control the proliferation, differentiation, and apoptosis of granulosa cell by synthesizing EGF (44, 49, 50, 54, 55). However, due to the multiplicity of EGFR ligand, further extensive studies on each EGFR ligand are required to advance this hypothesis (44, 50). Recently, all EGFR ligands except HB-EGF have been cloned in our laboratory. And our preliminary studies have shown that chicken ovary expresses all 7 EGFR ligands, among which HB-EGF appears to be expressed abundantly (56, 57, 58, 59). Because the information on ovarian HB-EGF is limited and controversial in mammals (60, 61); therefore, the prior aim of this study was to examine the spatiotemporal expression, regulation, and role of HB-EGF in chicken ovary. Due to the lack of information on KL expression in the ovary of chicken, the expression of KL and its interaction with ovarian HB-EGF were also investigated and discussed in the present study.

Chicken ovary is an ideal model for the study of the mechanisms involved in follicular development (62). It contains follicles at distinct developmental stages including thousands of cortical follicles (<1 mm in diameter), hundreds of white follicles (1–5 mm in diameter), five or six small yellow follicles (5–8 mm in diameter), and five or six preovulatory follicles (10–40 mm in diameter) arranged in hierarchy, whereby the largest preoulvatory follicle (F1) is destined to be ovulated first, followed by the second largest preovulatory follicle (F2) 24–26 h later (45). As in mammals, the follicle atresia involving the apoptosis of granulosa cell occurs frequently in the prehierarchal follicles (1–8 mm in diameter) (62, 63). And there also exists a follicle selection mechanism by which a single follicle from a cohort of small yellow follicles is selected each day into the preovulatory hierarchy under the influence of FSH and locally produced factors including EGFR ligands (45, 47, 48, 51). All these features make the hen an attractive model for deciphering the mechanisms regarding ovarian follicular growth, atresia, selection, and maturation. Given the unique evolutionary position of chicken possessed in vertebrates, the findings on ovarian EGFR ligand and KL from this model system would contribute significantly to our better understanding of the conserved roles of EGFR ligand and KL in controlling ovarian follicular development and functions in vertebrates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and hormones
All chemicals were bought from Sigma-Aldrich (St. Louis, MO), and restriction enzymes were obtained from Amersham Biosciences (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) unless stated otherwise. Mouse EGF (mEGF) was obtained from Invitrogen (Carlsbad, CA). Recombinant human (rh) TGF-{alpha}, rhHB-EGF, PD98059, Tyrphostin AG1478, and phorbol 12-myristate 13-acetate (PMA) were purchased from Calbiochem (Calbiochem, La Jolla, CA). They were first dissolved in appropriate solution according to manufacturers’ instructions and then diluted to the desired concentrations with medium before use.

Animals
Sexually immature (from 3–16 wk) and mature chickens and chicken embryos are of a local chicken strain (Shek-ki) in Hong Kong, which were kindly provided by Kadoorie Agriculture Research Centre (Hong Kong). All experiments were performed under license from Government of the Hong Kong Special Administrative Region and endorsed by the Animal Experimentation Ethics Committee of the University of Hong Kong.

Total RNA extraction
Adult chickens were killed and different tissues including brain, heart, small intestine, kidney, liver, lung, muscle, pituitary, spleen, testis, and ovary (the five or six preovulatory follicles were removed because the large amount of yellow yolk released from these follicles would greatly affect the RNA extraction, RNA quality, and RT reaction) were collected. Total RNA was extracted from chicken tissues with Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions and dissolved in DEPC-treated H2O.

Sampling of ovaries (or follicles) from embryos, sexually immature, and mature chickens
To examine the changes in expression of ovarian HB-EGF and EGFR at the organ level, ovaries from two consecutive periods, embryogenesis (from d 11–20) and sexual maturation (from 3 wk of age to adult), were collected. To avoid contamination from other tissues, the embryonic ovaries were isolated under stereoscope and the attached extraovarian tissues were carefully removed with needles. To ensure samples of the right stages collected, the left ovaries from the same batch of embryos and small part of ovary at posthatching stage were fixed in Bouin Solution and subjected to histological sectioning and microscopic observation. After confirmation of their stages, total RNA was extracted from embryonic and sexually immature and mature ovaries (the five or six preovulatory follicles removed).

To examine the expression of HB-EGF and EGFR in growing follicles at prehierarchal stage, follicles of various sizes were removed from adult chicken ovaries. In this experiment, according to their diameter, the follicles were divided into three groups: 1 mm (0.5–1.5 mm in diameter), 3 mm (2.5–3.5 mm in diameter), and 6 mm (5.5–6.5 mm in diameter) follicles. Then, total RNA was extracted and subjected to semiquantitative RT-PCR assay.

Preparation of total RNA from oocyte and granulosa cell layer of 3-mm follicles
According to our method previously established in zebrafish ovary (64), we developed a modified protocol to extract total RNA from oocyte and granulosa cell layer of 3-mm follicles. Briefly, healthy adult ovaries were removed from four to six chickens and 3-mm follicles were collected in 1x PBS solution at 4 C. To extract total RNA from oocytes, eight to ten 3-mm white follicles were cut open by a pair of small scissors to collect ooplasm (including the white yolk) in a 1.5-ml tube (placed on ice in advance). Then, total RNA was extracted from ooplasm with Tri Reagent. To prepare total RNA from granulosa cell layer, the broken 3-mm follicles were washed three times with 1x PBS (4 C) to ensure minimal amount of ooplasm enclosed. Then, granulosa cell layer was carefully separated from the opened follicles and washed three times with 1x PBS by centrifugation (at 3000 rpm, 2 min) to minimize ooplasm contamination. Finally, total RNA was extracted with Tri Reagent (Molecular Research Center).

To examine the purity of total RNA extracted from the two compartments, the expression of chicken bone morphogenetic protein 15 gene (BMP-15) (GenBank accession no. AY725199) in oocyte and granulosa cell layer was examined by semiquantitative RT-PCR assay. As expected, BMP-15 was detected to be expressed in the oocyte nearly exclusively but not in the granulosa cell layer, indicating the high purity of total RNA isolated from two compartments (see Fig. 2BGo). Then, the same RT samples were used to evaluate the relative mRNA levels of HB-EGF and EGFR in the two compartments.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		FIG. 2. A, Abundant expressions of HB-EGF and EGFR in 1-, 3-, and 6-mm ovarian follicles. B–D, Relative expression levels of BMP-15 (B), HB-EGF (C), and EGFR (D) in oocyte and granulosa cell layer of 3-mm follicles. Each value represents the mean ± SEM of four independent RT-PCRs. ***, P < 0.001 vs. oocyte.

 
Cloning and sequencing of cDNAs coding for chicken KL
To identify chicken KL isoforms, based on the full-length cDNA sequence cloned by others (GenBank accession no.: D13516) (65), four pairs of gene-specific primers were designed to amplify the full-length or partial cDNAs coding for KL. Using RT products from cultured granulosa cells (F1 follicle) as template, PCR was performed with Expand High FidelityPLUS PCR System (Roche Diagnostics, Basel, Switzerland) using a cycle profile (35 cycles) of 30 sec at 94 C, 30 sec at 56 C, and 90 sec at 72 C, followed by 10 min extension. The PCR products were first cloned into pBluescript SK(+/–) (Stratagene, La Jolla, CA) and sequenced by ABI3100 (PerkinElmer, Foster City, CA). The cDNA sequence of each KL isoform was finally determined by sequencing at least two independent clones.

Granulosa cell culture and RNA extraction
Whole ovaries were removed from four to six egg-laying chickens. According to the method described by Gilbert et al. (66), granulosa cell layers were isolated from 3 mm, 6 mm, F5 (the fifth largest preovulatory follicle), F4 + 5 (the fourth and fifth largest preovulatory follicles), and F1 (the largest preovulatory follicle) follicles and washed with Medium 199 (Invitrogen). To disperse cells, granulosa cell layer was digested by 0.3% collagenase for 10–15 min at 37 C (Invitrogen). After washing the dispersed cells three times with Medium 199 by centrifugation (1200 rpm, 3 min), the granulosa cells were re-suspended in Medium 199 supplemented with 10% fetal bovine serum (vol/vol), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen) and incubated for 18–24 h at 37 C with 5% CO2 in a 24-well plate (1.5 x 105 cells/well) before treatment. In each experiment, the cell viability before culture was determined to be more than 90% by trypan blue exclusion analysis and all treatments were carried out under serum-free condition.

To extract total RNA from cultured cells, the medium was first discarded from 24-well plate. Then, 300 µl of Tri Reagent (Molecular Research Center, Inc.) were added to each well, and the plate was shaken for 10 min at 600 rpm on the Thermomixer Comfort (Eppendorf, Hamburg, Germany). The extract from each well was then transferred to a microtube containing 75 µl chloroform, vortexed, and spun for 30 min at 4 C. The aqueous phase from each sample was transferred to a new tube containing isopropanol, vortexed for 30 sec, left at –20 C for 30 min, and centrifuged for 30 min at 4 C. After washing with 75% ethanol and brief air drying, the RNA pellet was dissolved in 10 µl DEPC-H2O and stored at –80 C.

RT-PCR
RT was performed at 42 C for 2 h in a total volume of 10 µl consisting of 3 µl total RNA (0.5–2 µg), 1x Single Strand Buffer (Invitrogen), 10 mM dithiothreitol, 0.5 mM each deoxynucleotide triphosphate, 0.5 µg oligo-deoxythymidine, and 100 U Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). All negative controls were carried out under the same condition without reverse transcriptase added in the 10 µl of reaction mix.

PCR was carried out in a total volume of 15 µl consisting of 1x PCR buffer, 0.2 mM each deoxynucleoitide triphosphate, 2.0 mM MgCl2, 0.2 µM each primer, and 0.3 U Taq DNA polymerase (Invitrogen) on the PTC-225 Peltier Thermal Cycler (MJ Research Inc., Waltham, MA). To detect the expression of ErbB4s (ErbB4a and ErbB4b) and KL in the granulosa cells, ovaries, and extraovarian tissues, 35 cycles of reaction were performed with profiles of 30 sec at 95 C, 30 sec at 56 C, and 60 sec at 72 C. Primers used in this experiment are listed in Table 1Go.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Primers used for RT-PCR and semiquantitative RT-PCR assays

 
Validation of semiquantitative RT-PCR assay for HB-EGF, EGFR, KL, BMP-15, and ß-actin
The optimal cycle number was first determined for semiquantitative PCR analysis according to our previously established method (67, 68, 69). In brief, the RT reaction (0.6 µl) from cultured granulosa cells or whole ovaries was used as template for PCR amplification. The primers used for HB-EGF, EGFR, BMP-15, KL, and ß-actin are listed in Table 1Go. PCR was carried out in a volume of 15 µl consisting of 1x PCR buffer, 0.2 mM each deoxynucleotide triphosphate, 2.5 mM MgCl2, 0.2 µM each primer, and 0.3 U of Taq polymerase on the PTC-225 Peltier Thermal Cycler (MJ Research Inc.) for various cycles with the profile of 30 sec at 94 C, 30 sec at 58 C (or 56 C for EGFR), and 60 sec at 72 C. The PCR products from different cycles of amplification were visualized on a UV-transilluminator after electrophoresis on 2% agarose gel containing ethidium bromide, and the signal intensity was quantified with Quantity One software (Bio-Rad, Hercules, CA). The cycle numbers that generate half-maximal amplification were used for subsequent semiquantitative analysis of gene expression, and they are 29 cycles for HB-EGF, 26 cycles for EGFR and BMP-15, 30 cycles for KL, and 23 cycles for ß-actin. The specificity of PCR amplification was confirmed by sequencing PCR products directly or cloning the PCR products into pBluescript SK (+/–) (Stratagene) followed by sequencing. To validate the feasibility of the semiquantitative RT-PCR assays, PCR amplification (15 µl) was performed on 4 µl of serially diluted PCR products (HB-EGF, EGFR, BMP-15, and ß-actin) or plasmids (containing KL fragment) to evaluate the correlation between input of template and output of PCR amplification. As expected, there is a clear linear relationship between the amount of template inputs and PCR amplification for target genes, indicating the workability of the RT-PCR assay for quantitating their mRNA levels (67, 68, 69).

Cell proliferation assay
Freshly isolated granulosa cells from 3 mm, F5, and F1 follicles were cultured in Medium 199 supplemented with 10% fetal bovine serum (vol/vol), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen) and incubated at 37 C with 5% CO2 in a 96-well plate (~1 x 104 cells/well). Twelve hours later, the medium was replaced by serum-free Medium 199 containing 10 ng/ml HB-EGF (cells cultured in Medium 199 without HB-EGF were used as controls) and granulosa cells were incubated for additional 36 h. The effect of HB-EGF on cell proliferation was quantified by CellTiter 96 AQueous One Solution (Promega) according to the manufacturer’s instructions (the mean value from four wells with Medium 199 only was used to subtract the background absorbance).

Data analysis
The mRNA level of each gene in each sample was first calculated as the ratio to that of ß-actin, which was amplified as the internal control and then expressed as the percentage of the control group. The data were analyzed by Student’s t test or one-way ANOVA followed by Dunnett test using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). We performed each experiment at least twice using different batches of animals to confirm the results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Developmental expression profiles of HB-EGF and EGFR in the chicken ovary
To elucidate the potential roles of HB-EGF in the chicken ovary, we first examined the expression profiles of ovarian HB-EGF and EGFR during embryogenesis (from d 11–20) and sexual maturation. At the embryonic stage, the primordial germ cell undergoes rapid proliferation and finally forms numerous oocytes devoid of surrounding granulosa cells in the ovarian cortex of left ovary by the time of hatching (d 20) (Fig. 1BGo). During this period, the expression level of HB-EGF was relatively low (Fig. 1AGo). After hatching, the oocyte is gradually surrounded by one layer of granulosa cells and starts to grow within the first 3 wk (Fig. 1BGo). In concurrence with this process, the expression level of HB-EGF increased dramatically in 3-wk ovaries and was further elevated with the advancement of ovarian development (Fig. 1AGo). HB-EGF expression reached the maximum in 10-wk ovaries and was sustained in 16-wk (appearance of small white follicles, ≤ 3 mm in diameter) and adult ovaries (Fig. 1AGo). In contrast, EGFR was detected to be abundantly expressed in both embryonic and posthatching ovaries at a more or less constant level (Fig. 1AGo).


Figure 1
View larger version (84K):
[in this window]
[in a new window]

  		FIG. 1. A, Temporal expression profiles of chicken ovarian HB-EGF and EGFR during embryogenesis and sexual maturation. The expression levels were normalized to that of ß-actin and expressed as the percentage of the level of e11. e11, e15, and e20 represent chicken embryos from d 11, 15, and 20 of incubation respectively; w3, w6, w10, w13, and w16 represent ovaries from 3-, 6-, 10-, 13-, and 16-wk-old sexually immature chickens, respectively. N, Number of chicken or embryo examined. Each value represents the mean ± SEM. *, P < 0.001 vs. e11. B, Histological sections of the ovaries at different developmental stages (a, e20 embryo; b, 3-wk-old chicken; c, 6-wk-old chicken). Arrow in panel a indicates the numerous denude oocytes in the ovarian cortex. Arrows in panels b and c show the growing oocytes surrounded by single layer of granulosa cell (scale bar, 50 µm).

 
To examine the expression of HB-EGF in growing follicles, follicles of diameter ranging from 1–6 mm were used. As shown in Fig. 2AGo, high expression level of HB-EGF could be easily detected in all follicles of different developmental stages (Fig. 2AGo), suggesting that the sudden increase in HB-EGF expression is likely to be caused by the emergence of numerous growing follicles in developing ovaries (Fig. 1BGo).

Evidence for HB-EGF being an oocyte-derived factor of 3-mm follicle
The dramatic increase in the expression of HB-EGF in posthatching ovary, instead of embryonic ovary, implies that growing oocyte may contribute significantly to the elevated expression of HB-EGF. To test this possibility, total RNA from oocyte and granulosa cell layer of 3-mm follicles was prepared and subjected to semiquantitative RT-PCR assay. Interestingly, HB-EGF was detected to be expressed predominantly in the oocyte but only weakly in granulosa cells (Fig. 2CGo), a spatial expression pattern similar to that of oocyte-derived BMP-15 examined (Fig. 2BGo). In contrast, EGFR was abundantly expressed in the surrounding granulosa cells but weakly in the oocyte. Although the oocyte may express EGFR as demonstrated in mammals (14), minor contamination of RNA from the granulosa cells may also contribute to the weak signal detected (Fig. 2DGo).

Effect of HB-EGF on the proliferation of granulosa cells in prehierarchal and preovulatory follicles
Because the growing oocyte is the major source of HB-EGF, it suggests that oocyte-derived HB-EGF may stimulate granulosa cell proliferation. To test this hypothesis, undifferentiated granulosa cells from prehierarchal follicles (3 mm) were treated with rhHB-EGF (10 ng/ml). As expected, rhHB-EGF stimulated the proliferation of undifferentiated granulosa cells, leading to the formation of a sheet of tightly organized granulosa cell layer within a short incubation period (36 h) (Fig. 3BGo). In contrast, granulosa cells without HB-EGF treatment grew slowly and only formed small patches of aggregated cells. Furthermore, HB-EGF could increase the cell number of differentiated granulosa cells from preovulatory follicles (F5 and F1) (Fig. 3BGo). Using CellTiter 96 AQueous One Solution Cell Proliferation Assay kit, the effect of HB-EGF on cell proliferation was easily monitored (Fig. 3AGo).


Figure 3
View larger version (76K):
[in this window]
[in a new window]

  		FIG. 3. Effects of recombinant human HB-EGF (rhHB-EGF, 10 ng/ml, 36 h) on the proliferation of cultured granulosa cells from 3-mm, F5, and F1 follicles. A, The stimulatory effect of rhHB-EGF on cell proliferation was quantified by CellTiter 96 AQueous One Solution. Each value represents mean ± SEM, n = 4. **, P < 0.001 vs. respective control (without HB-EGF treatment, 36 h). B, Pictures showing granulosa cells from 3-mm (a and b), F5 (c and d), and F1 (e and f) follicles cultured in the absence (on left) and presence (on right) of 10 ng/ml rhHB-EGF.

 
Regulation of HB-EGF expression by HB-EGF in cultured ovarian granulosa cell
The lower expression level of HB-EGF in granulosa cell layer leads us to speculate that oocyte-derived HB-EGF may have a negative impact on its expression. Contradictory to our expectation, the expression of HB-EGF was strongly induced by rhHB-EGF (200 ng/ml) in cultured granulosa cells from F4 + 5 follicles. The expression of HB-EGF reached a peak level at 4 h of the treatment and declined afterwards (Fig. 4AGo). However, the significant stimulatory effect could still be observed at 24 h after treatment (Fig. 4AGo). To investigate the dose-dependent effect of rhHB-EGF, 4 h of the treatment was used in the following experiments. As shown in Fig. 5Go, HB-EGF strongly induced HB-EGF expression in cultured granulosa cells from 3-mm, 6-mm, F4 + 5, and F1 follicles in a clear dose-dependent manner. The stimulatory effect of rhHB-EGF was evident at 2 ng/ml and reached the maximal level at 20 ng/ml (Fig. 5Go). Interestingly, the inducing effect of rhHB-EGF appears to be much stronger in differentiated granulosa cells (from F4 + 5 and F1 follicles) than in undifferentiated cells (from 3- and 6-mm follicles) (Fig. 5Go), although the underlying mechanism remains to be determined.


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 4. Effects of HB-EGF (200 ng/ml) (A), TGF-{alpha} (10 nM) (B), and EGF (150 nM) (C) on the expression of chicken HB-EGF in cultured granulosa cells from F4 + 5 ovarian follicles (0–24 h). The expression levels were normalized by ß-actin and expressed as the percentage of respective control (0 h). Each value represents mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of each graph. *, P < 0.05; **, P < 0.001 vs. control.

 

Figure 5
View larger version (39K):
[in this window]
[in a new window]

  		FIG. 5. Dose-dependent effect of HB-EGF (0–200 ng/ml, 4 h) on the expression of chicken HB-EGF in cultured granulosa cells from 3-mm (A), 6-mm (B), F4 + 5 (C), and F1 (D) follicles. Each value represents mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of each graph. *, P < 0.05; **, P < 0.001 vs. control.

 
Regulation of HB-EGF expression by EGF and TGF-{alpha} in cultured granulosa cells
The induction of HB-EGF expression by rhHB-EGF prompts us to examine whether other EGFR ligand could stimulate HB-EGF expression in cultured granulosa cells. As expected, both recombinant human TGF-{alpha} (rhTGF-{alpha}) and mEGF strongly induced HB-EGF expression in a clear time- and dose-dependent manner (Figs. 4Go and 6Go). However, rhTGF-{alpha} is far more potent than mEGF, as 1 nM of TGF-{alpha} could induce the maximal expression of HB-EGF in cultured granulosa cells from F1 and F4 + 5 follicles, whereas 150 nM of mEGF was required to induce a higher expression of HB-EGF under the same condition (Fig. 6Go).


Figure 6
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 6. Effects of EGF (0–150 nM, 4 h) and TGF-{alpha} (0–10 nM, 4 h) on chicken HB-EGF expression in cultured granulosa cells from F4 + 5 (A and B) and F1 follicles (C and D). Each value represents mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of each graph. *, P < 0.05; **, P < 0.001 vs. control.

 
Involvement of EGFR and MAPK signaling pathway in mediating the stimulatory effect of HB-EGF on HB-EGF expression in cultured granulosa cells
To demonstrate that the effect of rhHB-EGF on HB-EGF expression is mediated by EGFR and intracellular MAPK/ERK signaling pathway, two pharmaceutical drugs were used in this study. One is Tyrphostin AG1478, a specific inhibitor of EGFR tyrosine kinase; and the other is PD98059, a potent and selective inhibitor of MAPK kinase. In the first experiment, administration of AG1478 (10 µM) completely abolished the rhHB-EGF-induced expression of HB-EGF in cultured granulosa cells, suggesting that the stimulatory effect is mediated by EGFR (Fig. 7AGo). In the second experiment, PD98059 (100 µM) treatment not only abolished the inducing effect of rhHB-EGF, but also effectively suppressed the basal expression level of HB-EGF (Fig. 7BGo), indicating that the activation of intracellular MAPK signaling pathway is required for both basal and HB-EGF-induced expression of HB-EGF in granulosa cells.


Figure 7
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 7. A, Effects of HB-EGF (HB; 200 ng/ml, 4 h) on the chicken HB-EGF expression in cultured granulosa cells from F4 + 5 follicles in the presence or absence of Tyrphostin AG1478 (AG; 10 µM). B, Effects of HB-EGF (HB; 200 ng/ml, 4 h) on the chicken HB-EGF expression in cultured granulosa cells from F4 + 5 follicles in the presence or absence of PD98059 (PD; 100 µM). In experiments A and B, AG1478 and PD98059 were added 1 h before HB-EGF treatment (C, control). C and D, Time- and dose-dependent effects of PMA on the chicken HB-EGF expression in cultured granulosa cells from F4 + 5 follicles. Each value represents mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of each graph. *, P < 0.05; **, P < 0.001 vs. control; ##, P < 0.001 vs. HB.

 
EGFR ligand is reported to activate protein kinase C (PKC) signaling pathway (70). Thus, to determine whether the activation of PKC can induce HB-EGF expression, PMA (a potent activator of PKC) was used in this experiment. Interestingly, PMA strongly stimulated HB-EGF expression in clear time- and dose-dependent manners, suggesting that PKC or PKC-activated signaling pathways (such as MAPK signaling pathway) may be involved in controlling HB-EGF expression in granulosa cells (Fig. 7Go, C and D). However, whether PKC can directly mediate the effect of HB-EGF on HB-EGF expression in granulosa cells is still under investigation.

Differential expression of two ErbB4 isoforms in ovarian and extraovarian tissues
It has been reported that ErbB4, another receptor of HB-EGF, could mediate the actions of HB-EGF on target cells (71). Because two ErbB4 isoforms (ErbB4a and ErbB4b), with or without signal peptide, have been identified by us (ErbB4a, GenBank accession no. DQ069275; ErbB4b, DQ069276; Wang, Y., J. Li, and F. C. Leung, unpublished data), thus, to examine their expressions in the ovarian and extraovarian tissues, three pairs of primers were used (Table 1Go). Interestingly, using primers to amplify the common region of both isoforms, ErbB4 was detected to be expressed in all of the tissues except intestine (Fig. 8Go); however, using specific primers, ErbB4b was detected to be preferentially expressed in the ovary and testis, whereas ErbB4a was expressed in most of the tissues examined including brain, kidney, muscle, testis, and ovary (Fig. 8Go).


Figure 8
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 8. Differential expression of ErbB4a and ErbB4b in adult chicken tissues. Numbers in brackets indicate the PCR cycles used. +, RT with reverse transcriptase; –, RT without reverse transcriptase.

 
Down-regulation of KL expression by HB-EGF in cultured granulosa cells
It is well documented that granulosa cell-derived KL can directly act on oocyte to promote its growth, whereas oocyte-derived growth differentiation factor 9 (GDF-9) and BMP-15 are able to regulate KL expression in granulosa cell, indicating the presence of regulatory feedback loops between oocyte and granulosa cells in mammals (8, 36, 37, 40). To extend this concept, we also attempted to find a link between HB-EGF and KL in this study.

Experiment 1. Identification of KL-1, KL-2, KL-3, and KL-4 from cultured ovarian granulosa cells.
There are two types of KL mRNA transcripts (KL-1 and KL-2) identified in mammals, whereas only one KL transcript (KL-1) has been reported in chickens (65). To examine the expression of KL-1 and other KL isoforms in chickens, we first cloned the alternatively spliced KL transcripts from cultured granulosa cells (F1). In addition to KL-1 (GenBank accession no. DQ870920), the other three KL transcripts (containing either complete ORF or partial ORF), named KL-2 (GenBank accession no. DQ870921), KL-3 (GenBank accession no. DQ870922), and KL-4 (GenBank accession no. DQ870923) respectively, were identified (Fig. 9Go). Comparison of KL cDNA sequences with the chicken genome database (http://www.ensembl.org/Gallus_gallus) revealed that, as in mammals, the difference between KL-1 and KL-2 depends on the presence or absence of exon 6 (34 amino acids), which encodes a stretch of 34 amino acids containing the major proteolytic cleavage site (Fig. 9Go, A and B) (72). In contrast, KL-3 lacks exon 4 (60 amino acids), and KL-4 lacks exons 4 and 6 (94 amino acids) (Fig. 9Go).


Figure 9
View larger version (51K):
[in this window]
[in a new window]

  		FIG. 9. A, Alignment of deduced amino acid sequences of 4 chicken KL isoforms (cKL-1, cKL-2, cKL-3, and cKL-4) with those of mouse KL-1 (mKL-1) and KL-2 (mKL-2). The deletion of 60 amino acid residues (encoded by exon 4) or 34-amino acid residues (encoded by exon 6) in KL isoforms is shaded and underlined. Transmembrane domain (TMD) is shaded in (A). Arrowhead in panel A indicates the major cleavage site for release of soluble KL peptide. Dots indicate the amino acid residues identical with those of chicken KL-1 (cKL-1). B, Genomic organization of chicken KL gene. Exons of KL gene are numbered. Deletions of exon 6 or exon 4, or both, result in the formation of three putative isoforms, cKL-2, cKL-3, and cKL-4, respectively. Dotted line in panel B indicates the deleted exon(s). The incomplete N termini of cKL-2 and cKL-4 are underlined in panel A or shaded in panel B.

 
Experiment 2. KL-1 is expressed predominantly in the ovarian and extraovarian tissues.
To evaluate the relative mRNA abundance of KL isoforms in the ovarian and extraovarian tissues, RT-PCR assay was used. In freshly isolated granulosa cells, KL-1 is the predominant isoform expressed, whereas other transcripts with deletion of exon 6 or exon 4, or both, were detected to be expressed at an extremely low level (Fig. 10AGo). Similarly, KL-1 is also expressed in cultured granulosa cells predominantly (Fig. 10BGo). Interestingly, under culture condition, the proportion of KL-3 transcript (without exon 4) seemed to increase significantly, whereas the other two isoforms (KL-2 and KL-4) were hardly to be detected (Fig. 10BGo).


Figure 10
View larger version (70K):
[in this window]
[in a new window]

  		FIG. 10. Detection of KL isoforms in freshly isolated granulosa cells (A), cultured granulosa cells (B), ovarian (C), and extraovarian tissues (D). In A and B, follicles of different stages (3 mm, 6 mm, F4 + 5, and F1) were used. Panel a shows the PCR products using primers detecting the presence (upper strong band) and absence of exon 6 (lower weak band denoted as {Delta}E6). Similarly, in Panel b, primers recognize the presence (upper strong band) and absence of exon 4 (lower weak band denoted as {Delta}E4). Panel c shows the amplification of full-length KL isoforms. In A, C, and D, KL-1 is the predominant isoform detected. In B, only two bands were visible and they were identified as KL-1 and KL-3, respectively, the other two isoforms (KL-2 and KL-4) were hardly to be detected due to their extremely low levels of expression. e8, e12, e16, and e20 represent ovaries from d-8, -12, -16, and -20 chicken embryos respectively; w3, w6, w10, w16, and Ad represent ovaries from 3-, 6-, 10-, and 16-wk, and adult chickens, respectively. PCR cycle numbers used for KL and ß-actin genes were 35 and 23, respectively. All negative controls are not shown. B, Brain; H, heart; I, small intestine, K, kidney; Li, liver; Lu, lung; M, muscle; P, pituitary; S, spleen; T, testis.

 
Furthermore, KL-1 was detected to be predominantly expressed in both embryonic (from d 8–20) and posthatching ovaries (Fig. 10CGo).

The transcripts without exon 6 or exon 4 were detected in extraovarian tissues; however, KL-1 was shown to be expressed predominantly (Fig. 10DGo).

Experiment 3. Down-regulation of KL expression by HB-EGF in cultured granulosa cells.
Because both KL-2 and KL-4 were expressed at an extremely low level in cultured granulosa cells (Fig. 10BGo) and undetectable in the semiquantitative RT-PCR assay, thus only two bands, denoted as KL-1 (large band) and KL-3 (minor band) respectively, were detected in the following experiments. As shown in Fig. 11Go, HB-EGF could significantly down-regulate the steady-state mRNA level of KL-1 in cultured granulosa cells from F4 + 5 follicles in a time-dependent manner. The inhibitory effect was maximal at 4 h of the treatment but gradually diminished after lengthened duration of treatment (12–24 h) (Fig. 11Go). Using the 4 h as duration of treatment, rhHB-EGF inhibited KL-1 expression in undifferentiated (3 and 6 mm) and differentiated (F4 + 5 and F1) granulosa cells in a dose-dependent manner (Fig. 12Go). Interestingly, in all experiments performed, the maximal inhibitory effect of rhHB-EGF was found at 20 ng/ml, a dosage used for inducing the maximal expression of HB-EGF in granulosa cells (Figs. 5Go and 12Go).


Figure 11
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 11. Time course of HB-EGF (200 ng/ml) effect on chicken KL-1 expression in cultured granulosa cells from F4 + 5 follicles (0–24 h). Each value represents mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of the graph. Because KL-1 and KL-3 (without exon 4) were detectable in semiquantitative RT-PCR assay, whereas KL-2 and KL-4 were undetectable due to their extremely low levels of expression. Therefore, two bands detected are denoted as KL-1 (bright band) and KL-3 (faint band), respectively, in Figs. 11–13GoGo. *, P < 0.01 vs. control.

 

Figure 12
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 12. Dose-dependent effects of HB-EGF (0–200 ng/ml) on chicken KL-1 and KL-3 expression in cultured granulosa cells from 3-mm (A), 6-mm (B), F4 + 5 (C), and F1 (D) follicles. Each value represents mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of each graph. Arrows indicate the faint band corresponding to KL-3. * and #, P < 0.01 vs. respective control.

 
Because KL-3 was barely detectable in semiquantitative RT-PCR assay; thus, the quantitative analysis of KL-3 was included only in part of the experiments. Interestingly, similar to KL-1, the declining trend in KL-3 expression was also observed in most experiments regardless of its low expression level (Fig. 12Go).

Like HB-EGF, both TGF-{alpha} and EGF could down-regulate KL-1 expression in cultured granulosa cells (data not shown).

Experiment 4. Involvement of EGFR in mediating the inhibitory action of HB-EGF on KL expression in cultured granulosa cells.
To examine whether EGFR could mediate the inhibitory action of HB-EGF, Tyrphostin AG1478 was used. Once again, AG1478 completely blocked the suppressive action of HB-EGF on KL-1 (and KL-3) expression in cultured granulosa cells from F4 + 5 follicles, indicating that the inhibitory effect is mediated by EGFR (Fig. 13Go).


Figure 13
View larger version (35K):
[in this window]
[in a new window]

  		FIG. 13. Effects of HB-EGF (HB; 200 ng/ml, 4 h) on the chicken KL-1 and KL-3 expression in cultured granulosa cells from F4 + 5 follicle in the absence or presence of Tyrophostin AG1478 (AG; 10 µM). AG1478 was added 1 h before HB-EGF treatment. Each value represents mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of the graph. Arrow indicates the faint band corresponding to KL-3. * and #, P < 0.01 vs. respective control (C); a and b, P < 0.01 vs. HB.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we clearly demonstrated that HB-EGF is primarily synthesized by the growing oocyte and is able to stimulate granulosa cell proliferation and HB-EGF expression in chickens. Moreover, the soluble KL (KL-1), but not the other three KL forms (KL-2, KL-3, and KL-4), was detected to be predominantly expressed in developing ovaries and its expression in undifferentiated and differentiated granulosa cells could be down-regulated by HB-EGF. These data, for the first time, depict a potential intrafollicular communication network between growing oocyte and surrounding somatic cells, which involves an interaction of oocyte-derived EGFR ligand and granulosa cell-derived KL in the ovarian follicles of chickens.

HB-EGF was detected to be expressed in the embryonic, immature, and mature ovaries of chickens, and its expression was stage dependent in this study. In contrast, the expression of HB-EGF in ovarian follicles is controversial in mammals. In humans, no immunoreactive HB-EGF protein was detected in preantral, antral, and preovulatory follicles, or oocyte throughout follicular growth (61). In rats, HB-EGF was expressed in most granulosa cells of all developing follicles but not in those of preovulatory follicles (60). The difference in temporal expression profile of ovarian HB-EGF among species is likely to be due to the species difference. Despite the lack of information on HB-EGF expression in embryonic ovaries of mammals, our study demonstrated that the relatively low expression of HB-EGF and abundant expression of EGFR could be detected in embryonic ovaries (from d 11–20), suggesting a role of HB-EGF in the proliferation and survival of primordial germ cells in chickens. Interestingly, the expression of HB-EGF increased dramatically in the ovary at posthatching stage. This temporal change is synchronous to the first cohort of oocytes entering the growth phase, indicating a close association of ovarian HB-EGF expression with the early stage of follicular development (Fig. 1Go).

The dramatic increase in ovarian HB-EGF expression raises the question concerning the origin of HB-EGF. In this study, HB-EGF was detected to be predominantly expressed in the growing oocyte but not in granulosa cell (3-mm follicle) (Fig. 2Go), whereas EGFR is abundantly expressed in the granulosa cell. This interesting information provides important clues to the potential roles of HB-EGF in oocyte-somatic cell communication and leads us to hypothesize that oocyte-derived EGFR ligand may act as a paracrine (or juxtacrine) factor to signal the surrounding somatic cells during follicular development. Despite the fact that the expression of HB-EGF contrasts the findings reported in human and rat ovaries (60), there are lines of evidence supporting that oocyte is a potential source of EGFR ligand in both vertebrates and invertebrates. In humans, both TGF-{alpha} and EGF have been detected in oocytes of primordial and preantral follicles by immunohistochemical staining (15). A similar finding has been reported in porcine ovarian follicles. The strongest signals for EGF mRNA and protein were detected in oocyte, whereas the strongest EGFR signal was detected in cumulus, granulosa, and thecal cells in pigs (14). In chickens, EGF has been localized to the germinal disc of the oocyte right under the oolemma by immunohistochemistry. Further evidence demonstrated that oocyte could secrete EGF to stimulate the proliferation and inhibit the differentiation of overlying granulosa cells in a paracrine manner (49, 50). In zebrafish, similar spatial expression pattern of EGF and EGFR in fully grown ovarian follicle was reported by us in a previous study (73). Interestingly, during the oogenesis of Drosophila, oocyte could produce Gurken, a ligand resembling vertebrate TGF-{alpha}, to control the dorsal-ventral patterning and follicle cell fate via activation of Drosophila EGFR, which is expressed exclusively in the surrounding follicle epithelial cells (74). Considering the close evolutionary origin of EGFR ligands, the conserved spatial distribution of EGFR ligand and EGFR in oocyte and follicle cells among different species, from invertebrates to vertebrates, strongly suggests that EGFR ligand-EGFR interaction may be one of the most primitive mechanisms of oocyte-somatic cell communication (Fig. 14AGo), although the list of oocyte-derived EGFR ligand, as well as the time window of their actions, remains largely unknown (or overlooked) in most vertebrate species.


Figure 14
View larger version (32K):
[in this window]
[in a new window]

  		FIG. 14. A, Conserved spatial expression patterns of EGFR ligand and EGFR in oocyte and surrounding follicle cells (or granulosa cells) of Drosophila (74 ), zebrafish (73 ), and chicken (49 50 ). In Drosophila, Grk, resembling vertebrate TGF-{alpha}, could activate Drosophila EGFR expressed in follicle cells. In zebrafish and chickens, EGFR ligand, such as EGF and HB-EGF, could activate EGFR abundantly expressed in the follicle cells (or granulosa cells in chickens). B, Hypothetical model for the paracrine/juxtacrine actions of oocyte (or germinal disc)-derived HB-EGF on granulosa cell layer and thecal cell layer. In this model, granulosa cell-derived HB-EGF and KL function as two important mediators of oocyte-derived HB-EGF actions in growing ovarian follicles. However, both the actions of KL on oocyte and thecal cell layer of developing follicle and actions of granulosa cell-produced HB-EGF on thecal cell layer are yet unclear. In addition, BMP-15 is expressed in the growing oocyte predominantly; however, its interrelationship with granulosa cell-derived KL and EGFR ligand remains to be clarified.

 
Chicken oocyte growth requires rapid proliferation of surrounding granulosa cells (Fig. 1BGo); thus, we speculate that one major role of oocyte-derived HB-EGF is to stimulate proliferation of granulosa cells. Administration of recombinant human HB-EGF could strongly stimulate the proliferation of undifferentiated granulosa cells in 3-mm follicle (Fig. 3Go, A and B), substantiating that HB-EGF is a powerful mitogen for granulosa cell of growing follicles at early developmental stage. Interestingly, the mitogenic effect of HB-EGF could also be observed in differentiated granulosa cells from preovulatory follicles (Fig. 3BGo). This concurs with the previous studies that other EGFR ligands such as TGF-{alpha} and EGF could stimulate the proliferation of differentiated granulosa cells (43, 49, 50). Although the site of HB-EGF production in the preovulatory follicles has not yet been determined, this finding is still of potential physiological relevance. In view of the fact that the dramatic increase in oocyte diameter during the final rapid-growth phase requires active proliferation of surrounding granulosa cells (53, 75), only the high proliferative potency of granulosa cell in response to mitogen such as HB-EGF could meet the requirement (Fig. 3BGo) (43).

The production of HB-EGF by growing oocyte also encourages us to investigate the potential mediators which could amplify or exert its impacts on other follicular compartments, i.e. granulosa cell layer and thecal cell layer. Granulosa cell-derived HB-EGF appears to be a suitable candidate. The expression of HB-EGF could be strongly induced by HB-EGF in cultured granulosa cells from both prehierarchal (3 and 6 mm) and preovulatory follicles (F4 + 5 and F1). This induction suggests a profound effect of oocyte-derived HB-EGF on granulosa cell and thecal cell of growing follicles. On one hand, granulosa cell-derived HB-EGF could amplify (or exert) the actions of oocyte-derived HB-EGF on neighboring granulosa cells in a paracrine/juxtacrine manner (Fig. 14BGo); or it may act as part of a closed-loop positive feedback mechanism to amplify the actions of oocyte-derived HB-EGF in an autocrine manner if it works on the same granulosa cell (Fig. 14BGo). And presumably, with the regional distribution of the germinal disc in large yolky follicles, the mediatory role of granulosa cell-derived HB-EGF would become more evident (54, 55). On the other hand, granulosa cell-derived HB-EGF could regulate thecal cell proliferation, differentiation and function in a paracrine/juxtacrine manner via activation of EGFR in thecal cells (Fig. 14BGo), in which EGFR is abundantly expressed (76).

Like HB-EGF, other EGFR ligand, such as EGF and TGF-{alpha}, could strongly induce HB-EGF expression in ovarian granulosa cells. This induction also suggests that granulosa cell-derived HB-EGF may function as a mediator of EGFR ligands from oocyte, as well as from other compartments, of ovarian follicle (47).

Although EGFR ligand is a potent inducer of HB-EGF expression in granulosa cells, the relatively low expression of HB-EGF in granulosa cell layer has been noted in vivo (Fig. 2Go), clearly indicating that pituitary hormones and other local factors could modulate HB-EGF expression in granulosa cells.

It is generally believed that HB-EGF could activate EGFR and downstream MAPK/ERK signaling pathway in target cells (77). And this is demonstrated to be the case in cultured chicken granulosa cell in this study. Specific inhibition of EGFR tyrosine kinase activity by Tyrphostin AG1478 completely abolished the stimulatory effect of HB-EGF on HB-EGF expression, suggesting a critical mediatory role played by EGFR (Fig. 7AGo).

In contrast, the role of ErbB4, another receptor of HB-EGF (71, 77), in mediating ovarian HB-EGF action remains elusive. In this study, two isoforms of ErbB4, ErbB4a and ErbB4b, are coexpressed in chicken ovary. ErbB4a contains a typical signal peptide and is predicted to function as a membrane-spanning receptor (GenBank accession no. DQ069275), whereas ErbB4b lacks a signal peptide, supporting that ErbB4b is likely to be a cytosolic component (GenBank accession no. DQ069276; Wang, Y., J. Li, and F. C. Leung, unpublished data). Because ErbB4 differs from other ErbB family members in its capacity to function as a STAT5A nuclear chaperone to enhance gene transcription (78, 79); thus, the mediatory role of ErbB4, particularly ErbB4a, remains to be determined. Although ErbB4a mRNA could be detected in the ovary, its expression level appears to be low because the PCR signal was detectable only after 35 cycles of amplification. In agreement with our finding, the low mRNA level of ErbB4 was detected in human luteinized granulosa cells by RT-PCR, and most intriguingly, the immunoreactivity of ErbB4 was predominantly localized in the nuclei (80). In rats, no ErbB4 mRNA has been detected in the ovary using RT-PCR assay (60). All the evidence tends to support that ErbB4 is unlikely to be a principal mediator of HB-EGF actions in the ovaries of chicken and mammalian species.

As a well-known granulosa cell-derived factor, KL appears to play a critical role in oocyte growth of mammals (8, 34, 35). The production of HB-EGF by growing oocyte also leads us to speculate a cross talk between growing oocyte and granulosa cells, partially achieved by the interaction of oocyte-derived HB-EGF and granulosa cell-derived KL. Due to the lack of information on ovarian KL isoforms in chickens, the expression of KL isoforms was investigated in our initial study. Strikingly, unlike mammals, there are four types of KL mRNA transcripts identified (KL-1, KL-2, KL-3, and KL-4) (36, 37, 42, 81), among which KL-3 and KL-4 have not been reported in any other vertebrate species. Because KL-3 and KL-4 involve the deletion of exon 4 encoding 60 amino acids (Fig. 9Go), which contain domains critical for the formation of noncovalent KL homodimer and receptor activation (Kit) (82, 83); thus, whether these KL isoforms could interfere the dimer formation and receptor activation is unclear. Despite the fact that the multiple isoforms of KL are coexpressed, KL-1 but not the other three isoforms is predominantly expressed in vivo (Fig. 10Go), strongly suggesting a dominant role of KL-1 in the growing ovary. Because the major proteolytic cleavage site of KL-1 precursor is fully conserved between chicken and mammals (Fig. 9AGo) (81), it is conceivable that the soluble KL (KL-1), instead of membrane bound KL, plays a critical physiological role in ovarian and extraovarian tissues (Fig. 10Go). This finding contrasts the study in mice that generation of solely soluble KL in vivo causes a deficiency in primordial germ cells and female infertility (8, 72). Despite this difference between chicken and mammals, high expression level of KL-1 could be easily detected in freshly isolated or cultured granulosa cells, and its expression could be suppressed by HB-EGF (Figs. 11–13GoGoGo). These findings support our speculation on a potential intrafollicular communication network between growing oocyte and surrounding somatic cells, which may be achieved by EGFR ligand and KL. In light of the fact that the receptor of KL (c-Kit) is abundantly expressed in oocyte and thecal cells in all species examined (7, 8, 84), it is therefore tempting to speculate that chicken KL may function as another mediator of oocyte-derived HB-EGF actions, not only in modifying thecal cell proliferation and functions, but also in controlling oocyte survival and growth in a potential regulatory feedback loop (Fig. 14BGo).

The regulation of granulosa cell-derived KL expression by oocyte-specific factors, including GDF-9 and BMP-15, appears to be a common feature of oocyte-somatic cell communication during follicular development in mammals (8, 35, 36, 37, 40). Meanwhile, KL also stimulates oocyte growth and inhibits oocyte-derived BMP-15 expression in a feedback manner (40). All the evidence points out that KL is likely a key component of the oocyte-somatic cell communication network (7, 8). Similar to GDF-9 and BMP-15, HB-EGF suppresses KL expression in both undifferentiated and differentiated granulosa cells. This action, together with the stimulatory effect on granulosa cell proliferation and HB-EGF expression, strongly suggests that oocyte-derived EGFR ligands, such as HB-EGF in chickens, may play an important role in oocyte-somatic cell communication, which is essential for normal ovarian follicular development and functions (8) (Fig. 14Go, A and B).

Although the present study only represents the first step toward uncovering the oocyte-somatic cell communication in chicken ovary, our findings, together with pioneering studies on EGF expressed at the germinal disc (44, 49, 50), strongly support the notion that oocyte at the early developmental stage, or the germinal disc region of oocyte within the large yolky follicle, plays an active role in controlling granulosa cell proliferation, differentiation and apoptosis, at least in part, by producing EGFR ligands (Fig. 14Go) (49, 50).


   Acknowledgments
 
This work was supported by Research Grant Council of the Hong Kong Government, HKU7345/03M.


   Footnotes
 
The cDNA sequences encoding chicken KL-1, KL-2, KL-3, and KL-4 have been submitted to the DDBJ/EMBL/GenBank databases under accession nos. DQ870920, DQ870921, DQ870922, and DQ870923, respectively.

Disclosure Statement: Y.W., J.L., C.Y.W., A.H.Y.K., and F.C.L. have nothing to disclose.

First Published Online March 29, 2007

Abbreviations: AR, Amphiregulin; BMP-15, bone morphogenetic protein 15; BTC, betacellulin; EGF, epidermal growth factor; ER, epiregulin; KL, Kit ligand; EGFR, EGF receptor; GDF-9, growth differentiation factor 9; HB-EGF, heparin-binding EGF-like growth factor; mEGF, mouse EGF; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; rh, recombinant human.

Received October 11, 2006.

Accepted for publication March 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ge W 2005 Intrafollicular paracrine communication in the zebrafish ovary: the state of the art of an emerging model for the study of vertebrate folliculogenesis. Mol Cell Endocrinol 237:1–10[CrossRef][Medline]
  2. McGee EA, Hsueh AJ 2000 Initial and cyclic recruitment of ovarian follicles. Endocr Rev 21:200–214[Abstract/Free Full Text]
  3. Hsueh AJ, McGee EA, Hayashi M, Hsu SY 2000 Hormonal regulation of early follicle development in the rat ovary. Mol Cell Endocrinol 163:95–100[CrossRef][Medline]
  4. Eppig JJ, Wigglesworth K, Pendola FL 2002 The mammalian oocyte orchestrates the rate of ovarian follicular development. Proc Natl Acad Sci USA 99:2890–2894[Abstract/Free Full Text]
  5. Hutt KJ, McLaughlin EA, Holland MK 2006 Kit ligand and c-Kit have diverse roles during mammalian oogenesis and folliculogenesis. Mol Hum Reprod 12:61–69[Abstract/Free Full Text]
  6. Conti M, Hsieh M, Park JY, Su YQ 2006 Role of the epidermal growth factor network in ovarian follicles. Mol Endocrinol 20:715–723[Abstract/Free Full Text]
  7. Skinner MK 2005 Regulation of primordial follicle assembly and development. Hum Reprod Update 11:461–471[Abstract/Free Full Text]
  8. Thomas FH, Vanderhyden BC 2006 Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth. Reprod Biol Endocrinol 4:19[CrossRef][Medline]
  9. Harris RC, Chung E, Coffey RJ 2003 EGF receptor ligands. Exp Cell Res 284:2–13[CrossRef][Medline]
  10. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M 2004 EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303:682–684[Abstract/Free Full Text]
  11. Sekiguchi T, Mizutani T, Yamada K, Kajitani T, Yazawa T, Yoshino M, Miyamoto K 2004 Expression of epiregulin and amphiregulin in the rat ovary. J Mol Endocrinol 33:281–291[Abstract]
  12. Shimada M, Hernandez-Gonzalez I, Gonzalez-Robayna I, Richards JS 2006 Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor. Mol Endocrinol 20:1352–1365[Abstract/Free Full Text]
  13. Singh B, Armstrong DT 1995 Transforming growth factor {alpha} gene expression and peptide localization in porcine ovarian follicles. Biol Reprod 53:1429–1435[Abstract]
  14. Singh B, Rutledge JM, Armstrong DT 1995 Epidermal growth factor and its receptor gene expression and peptide localization in porcine ovarian follicles. Mol Reprod Dev 40:391–399[CrossRef][Medline]
  15. Reeka N, Berg FD, Brucker C 1998 Presence of transforming growth factor {alpha} and epidermal growth factor in human ovarian tissue and follicular fluid. Hum Reprod 13:2199–2205[Abstract/Free Full Text]
  16. Glister C, Groome NP, Knight PG 2003 Oocyte-mediated suppression of follicle-stimulating hormone- and insulin-like growth factor-induced secretion of steroids and inhibin-related proteins by bovine granulosa cells in vitro: possible role of transforming growth factor {alpha}. Biol Reprod 68:758–765[Abstract/Free Full Text]
  17. Tilly JL, Billig H, Kowalski KI, Hsueh AJ 1992 Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 6:1942–1950[Abstract]
  18. Hammond JM, English HF 1987 Regulation of deoxyribonucleic acid synthesis in cultured porcine granulosa cells by growth factors and hormones. Endocrinology 120:1039–1046[Abstract]
  19. Mondschein JS, Schomberg DW 1981 Growth factors modulate gonadotropin receptor induction in granulosa cell cultures. Science 211:1179–1180[Abstract/Free Full Text]
  20. Knecht M, Catt KJ 1983 Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J Biol Chem 258:2789–2794[Free Full Text]