Epidermal Growth Factor Family Members: Endogenous Mediators of the Ovulatory Response
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内分泌学杂志 2005年第1期
The Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation (H.A., X.C., S.M., M.P., A.T.), Weizmann Institute of Science, Rehovot 76100, Israel; and Division of Reproductive Biology (M.C.), Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317
Address all correspondence and requests for reprints to: Alex Tsafriri, The Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: alex.tsafriri@weizmann.ac.il.
Abstract
Previous studies showed that epidermal growth factor (EGF) and TGF mimic the action of LH on the resumption of oocyte maturation. We tested whether EGF-like agents, such as amphiregulin (AR), epiregulin (ER), and betacellulin (BTC), also mediate the LH stimulation of the ovulatory response in the rat. LH induced transient follicular expression of AR, ER, and BTC mRNA, reaching a maximum after 3-h incubation. Furthermore, the addition of ER, AR, and BTC to the culture medium could mimic some of LH actions. AR and ER fully simulated LH-induced resumption of meiosis in vitro, whereas BTC was less effective. To study the putative involvement of EGF-like factors in mediation of LH signal, the effect of the EGF receptor kinase inhibitor AG1478 was tested. When added with LH, AG1478, but not its inactive analog AG43, reduced EGF receptor phosphorylation and oocyte maturation compared with follicles treated with LH only. In addition to the inhibition of resumption of meiosis, AG1478 administration into the bursa (3 μg/bursa) resulted in 51% (P < 0.0005) inhibition of ovulation in the treated ovaries, compared with the untreated contralateral ones, as well as to the vehicle-treated ovaries (P < 0.02). LH, as well as ER, induced the expression of genes associated with the ovulatory response like rat hyaluronan synthase-2, cycloxygenase-2, and TNF-stimulated gene 6 mRNA, whereas AG1478 inhibited this effect of LH. Release of EGF-like factors from the membrane is dependent on activated metalloproteases. Indeed, Galardin, a broad-spectrum metalloprotease inhibitor, but not a specific matrix metalloprotease 2 and 9 inhibitor, suppressed meiotic maturation induced by LH. Conversely, meiotic maturation induced by ER was not affected by Galardin, thus, supporting the notion that LH releases follicular membrane-bound EGF-like agents. In summary, EGF-like factors such as ER, AR, and BTC seem to mediate, at least partially, the LH stimulation of oocyte maturation, ovulatory enzyme expression, and ovulation.
Introduction
IN MOST MAMMALS, the surge of LH stimulates the ovulatory response in Graafian follicles. It includes the resumption of meiotic maturation; differentiation of mural granulosa cells, usually referred to as luteinization, a reprogramming of their protein and steroidogenic activity; expansion or maturation of the cumulus cells surrounding the oocytes; and, finally, with rupture of the follicle wall and release of a fertilizable ovum (1). The physiological role of gonadotropins is well established: activation of G protein-coupled receptors (GPCRs) mediated by the adenyl cyclase-cAMP system in the ovary and the preovulatory follicle (2). Therefore, the ability of several other paracrine factors, using different transduction pathways to mimic the ovulatory actions of LH was quite puzzling. Thus, it has been shown that epidermal growth factor (EGF) (3) or TGF (4) induce the maturation of follicle-enclosed oocytes (FEOs) in the rat, EGF stimulates oocyte maturation and cumulus expansion in vitro in the pig (5) and in the human (6), and that GnRH stimulates ovulatory changes in vitro (7) and in vivo (8).
Amphiregulin (AR), epiregulin (ER), and betacellulin (BTC) are members of the family of EGF receptor (EGFR) ligands. Members of this family are structurally and functionally related integral membrane proteins that can be proteolytically processed and released from cell surface (9). Recently, Park et al., (10) have presented evidence that LH stimulates the expression of EGF-family members AR, ER, and BTC in mouse preovulatory follicles and that these EGF-like factors trigger meiosis and cumulus expansion in such follicles in culture. It was suggested, therefore, that ovarian EGF-factors serve as paracrine mediators of LH in the induction of ovulation.
In this study, we extended these observations using rat FEOs in culture. We have confirmed that LH/human chorionic gonadotropin (hCG) stimulated follicular expression of AR, ER, and BTC mRNA; that addition of these EGF-like factors to cultured follicles stimulated the resumption of meiotic maturation and the expression of several genes clearly associated with cumulus expansion and ovulation; and the EGFR-specific kinase inhibitor AG1478, but not its inactive analog AG43, blocked LH stimulation of follicular EGFR phosphorylation, resumption of meiosis, and expression of ovulation-associated genes in vitro.
In addition, we showed that an EGFR kinase inhibitor, AG1478, attenuated LH/hCG-stimulated ovulation in vivo. Furthermore, it has been shown that EGF-like factors are bound to the plasma membrane and released by the activation of metalloproteases (11). Accordingly, we have demonstrated that Galardin, a broad-spectrum metalloprotease inhibitor, suppressed resumption of meiosis in FEOs only when triggered by LH, but not ER, supporting the notion that LH stimulation of ovulation is mediated, at least partially, by the release of follicular cell membrane-bound EGF-like factors.
Materials and Methods
Animals
Rats of our Wistar-derived colony were provided with water and rat chow ad libitum and housed in air-conditioned rooms illuminated 14 h/d. The experiments were carried out in accordance with the principles and guidelines for the use of laboratory animals and approved by the Weizmann Institute of Sciences (Rehovot, Israel) research animal committee.
Immature female Wistar rats (23–24 d old) were injected subcutaneously with 12 IU pregnant mare serum gonadotropins (PMSGs) (Sanofi SNA, Libourne, France) to enhance preovulatory follicular development. For explanting FEOs for culture, the animals were killed 48–50 h after PMSG by using CO2 and subsequent cervical dislocation.
Intrabursal injections and the ovulatory response in vivo
Two days after PMSG treatment, the bursa of rats was injected with the indicated doses of AG1478 (Calbiochem, San Diego, CA) or vehicle (12) between 1230 and 1330 h. For intrabursal injection, animals were anesthetized by a cocktail of ketamine (40–60 mg/kg) and diazepam (2–3 mg/kg), and one of the ovaries was exteriorized via a small lumbosacral incision. A 29-gauge needle was threaded into the ovarian bursa via adjoining fat pad. The location of the injection was confirmed by observation of the swelling of the bursa. After injection of the inhibitor or vehicle (50 μl/bursa), the ovary was replaced into the abdominal cavity, and the skin was sutured. The contralateral ovary served as control. After 30 min, animals were treated with 5 IU hCG. Vehicle treated-animals were used as an additional control. Ovulated cumulus oocyte complexes collected 18 h after hCG treatment were isolated from the oviduct and counted under a dissecting microscope.
Culture of preovulatory follicles
The preovulatory follicles were isolated 48 h after PMSG injection as previously described (13). FEOs (10–15/dish) were cultured in Leibovitz’s L-15 medium (Life Technologies, Inc., Grand Island, NY) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) (Life Technologies, Inc.), and 5% fetal calf serum (Sera-Lab, Crawley Down, UK). FEOs were cultured at 37 C in a controlled atmosphere of 50% O2, 1.3% CO2, and 48.7% N2. After 60 min of preequilibration, ovine LH (10–100 ng/ml) (generously provided by Dr. A. F. Parlow and the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health), AG1478 (10 μM) and AG43 (10 μM) (Calbiochem), AR (10–1000 nM) (R&D Systems, Minneapolis, MN), ER (1–250 nM) (R&D Systems), or BTC (10–100 nM) (R&D Systems) were added. Galardin (20 μM) (Biomol, Plymouth Meeting, PA) or (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid (100–500 μM) (Calbiochem) was added to the culture 30 min before LH or ER stimulation. The same vehicle solution was included in control culture media. At the end of the culture period, the follicles were punctured to release and collect the cumulus oocyte complexes under a dissecting microscope. Oocyte maturation was assessed by scoring groups of 10–15 released oocytes using Nomarski interference microscopy. The mean ± SEM values of the groups of oocytes for each treatment are given.
Semiquantitative RT-PCR analysis
The expression of rat (r) hyaluronan synthase-2 (rHAS-2), cycloxygenase-2 (rCOX-2), TNF-stimulated gene 6 (rTSG-6), rAR, rER, and rBTC was examined by relative semiquantitative RT-PCR. Total RNA was extracted from ovaries and preovulatory follicles at the indicated time intervals, using Tri-reagent (Sigma, St. Louis, MO). For each sample, 250 ng RNA from ovaries or 60–80 ng of RNA from preovulatory follicles was reverse transcribed using oligo dT primer (Promega, Madison, WI) followed by PCR amplification. Specific primers were used to amplify the following cDNAs: HAS-2 (forward, 5'-gcttgaccctgcctcatctgtgg-3'; reverse, 5'-ctggttcagccatctcagatatt-3'), COX-2 (forward, 5'-ctgcttttcaaccagcagttcc-3'; reverse, 5'-tctgcagccatttctttctctc-3'), TSG-6 (forward, 5'-cgaagcgaatctttaaatcccc-3'; reverse, 5'-ctaaaccgtccagctaagaac-3'), AR (forward, 5'-ccacaggggactatgactac-3'; reverse, 5'-ttacggcggagacaaagac-3'), ER (forward, 5'-ccaccttctacaagcagtatc-3'; reverse, 5'-tcactctctcgtattcttccc-3'), BTC (forward, 5'-ggtcttgtgattctccagtg-3'; reverse, 5'-cttccttcttctttttgcgatg-3'), which amplified 403-, 376-, 394-, 408-, 407-, and 400-bp products, respectively. A fragment of S-16 cDNA, which served as an internal standard, was amplified in parallel using the following primers: forward, 5'-cgttcaccttgatgagcccatt-3'; and reverse, 5'-tccaagggtccgctgcagtc-3', which amplified a 100-bp product.
Each band was scanned and quantified by Fluor-STM Multimager (Bio-Rad, Hercules, CA). Briefly, the same size rectangle was used to surround each band, and its intensity was determined using the Quantity one version 4.2.1 software (Bio-Rad) adjusted for ethidium bromide gel. The background in the blank region of the gel was subtracted from the total area of the screened samples.
Immunoblotting
Samples (75 μg whole cell lysates per each lane) were separated by SDS-7.5% PAGE and electrophoretically transferred to a nitrocellulose membrane. The samples were immunoblotted with polyclonal anti-EGFR (Cell Signaling Technology, Inc., Beverly, MA) or polyclonal anti-PY1068 EGFR (Cell Signaling Technology, Inc.) as indicated in the legends. The polyclonal antibodies for immunoblots were detected with horseradish peroxidase-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) antibodies. Immunoblotting was performed according to manufactures instructions. Specific signals were visualized on x-ray by enhanced chemiluminescence detection system (Pierce, Rockford, IL) and scanned and quantified by Fluor-STM Multimager (Bio-Rad) adjusted for x-ray film as described above.
Histology
Ovaries after superovulation were excised and fixed in Bouin’s solution overnight, then washed with 70% ethanol and embedded in paraffin wax. Sections, 5 μm thick, were mounted on Super-frost/PLUS Micro slides (Fisher Scientific, Pittsburgh, PA), then rehydrated and stained with hematoxylin and eosin for 10 min, and then washed.
Statistical analysis
Statistical analysis by ANOVA, student’s t test, and 2 test were performed whenever appropriate. Data were expressed as mean ± SEM of pooled results obtained from at least three replicate cultures and two separate experiments. Otherwise, the results of two replicates were given. For the analysis of oocyte maturation in vivo (see Fig. 6), SE values were calculated according to the method described (14). Values of P < 0.05 were considered to be significant.
FIG. 6. Inhibition of meiotic maturation and ovulation by AG1478 administration in vivo. Unruptured large antral follicles were counted and the meiotic status of entrapped oocytes defined in serial sections of AG1478-treated and their control, untreated contralateral ovaries. The data are presented as the percentage of large antral follicles with oocytes and the percentage of mature oocytes entrapped in them. Numbers on columns indicate the numbers of counted follicles and oocytes.
Results
Stimulation of follicular EGF-like factors mRNA by LH
To test the involvement of EGF-like factors in the LH pathway, FEO cultures were initially treated with 100 ng/ml LH for 3 and 9 h. At the end of culture, levels of rER, rAR, and rBTC mRNA were determined by semiquantitative RT-PCR. LH brought about transient expression of rAR, rER, and rBTC mRNA with a maximum at 3 h followed by a marked decrease at 9 h of culture. Although LH further stimulated the basal BTC expression, it induced both AR and ER, which were undetected in unstimulated follicles (Fig. 1).
FIG. 1. LH-induced expression of rER, rAR, and rBTC mRNAs in preovulatory follicles in vitro. Preovulatory follicles (13 14 ) were explanted and cultured for the indicated period with and without LH (100 ng) in three independent experiments. Upper panel, Gel showing expression of the three genes examined and S16 as standard; lower panel, summary of the results. Mean ± SEM values are shown. LH-treated follicles differ significantly from untreated ones cultured for the same time (b, P < 0.0001; c, P < 0.002 for rER and rBTC and P < 0.03 for rAR) as revealed by one-way ANOVA analysis.
Effect of EGF-like factors on meiosis
In view of the above findings, we tested whether the addition of EGF-like factors, such as ER, AR, and BTC in vitro reproduces, some actions of LH. Complete (with AR and ER) or partial (BTC) stimulation of the resumption of meiosis in FEO was obtained (Fig. 2A). ER was more potent in causing maximal maturation (88% germinal vesicle breakdown, GVB) at the concentration of 100 nM. Ten-fold higher dose of AR was required to reach a similar maturation rate.
FIG. 2. EGF-like growth factors and the resumption of meiosis in rat FEOs. A, Induction of meiosis by EGF-like ligands. B, Inhibition of LH-induced maturation by AG1478. Preovulatory follicles were cultured for 24 h with EGF-like factors, LH, or LH+AG1478 (10 μM) as indicated. The number of examined oocytes is indicated on columns. The bars represent the percentage of GVB (mean ± SEM) of at least three groups of oocytes in two independent experiments (excluding 10 nM BTC with only two replicates, not included in statistical analysis). b, Versus control, P < 0.03; c, vs. control, P < 0.0002; d, vs. control, P < 0.0001; e, vs. LH, P < 0.0001.
EGF signal transduction and LH action
In vitro. To examine the role of EGF signal transduction in LH stimulation of meiosis, we have tested the effect of the EGFR kinase inhibitor, AG1478, on LH-stimulated follicles. For this purpose, follicles were cultured with LH and AG1478. AG1478, but not AG43 its inactive analog, blocked the LH-induced EGFR phosphorylation (Fig 3, A and B) and oocyte maturation (Figs. 2B and 3C) (1% GVB with AG1478 vs. >88% with LH or LH + AG 43) compared with control follicles (4% GVB).
FIG. 3. EGFR phosphorylation and induction of resumption of meiosis by LH. A and B, EGF receptor phosphorylation. Follicles were either untreated or incubated with LH (1 μg/ml), LH + AG43 (10 μM), and LH + AG1478 (10 μM) (lanes 1–4, respectively, in A) for 6 h followed by immunoblotting with the EGFR-phophospecific PY1068 antibody and, after stripping by Ponceau Red for 2 h, with the EGFR-specific antibody. In two separate experiments, each including eight rats, 39 follicles were cultured for each treatment variable. Total protein extracted was applied to each lane. A, representative blot. B, summary of two repeat experiments. The phosphorylated EGFR immunoblotting for each lane was standardized by EGFR. The bars show the ratios for each variable as related to the mean of AG43, which is determined as 100%. The dots represent the values of two independent experiments. C, Resumption of meiosis in rat FEOs. Preovulatory follicles were cultured for 6 h with indicated additives (LH, 1 μg/ml; AGs, 10 μM). The number of oocytes examined is indicated on the columns. The bars represent the percentage of mature oocytes ± SEM.
In vivo. To examine the inhibitory effects of AG1478 in vivo, it was injected into the ovarian bursa before hCG administration (Fig. 4). Local administration of 3 μg/bursa into the periovarian sac resulted in 51% (14.5 ± 2.6 ova/ovary) inhibition of ovulation in treated ovary, compared with untreated contralateral ovary (28.5 ± 2.3 ova/ovary) (P < 0.0005) or with vehicle-treated ovaries (21.3 ± 2.3 ova/ovary) (P < 0.02). Intrabursal administration of lower or higher concentrations of AG1478 revealed no significant differences in ovulation rate between treated ovaries and untreated ones or vehicle-treated ovaries.
FIG. 4. Effect of AG1478 on ovulation. The vehicle (control) or the drug was injected unilaterally to the periovarian sac (bursa). Number of rats on the columns; bars indicate mean ± SEM. *, P < 0.02 AG1478-treated vs. vehicle-treated control and P < 0.0005 vs. untreated contralateral ovary as obtained by Student’s t test analysis.
Histological appearance
The histological appearance of the AG1478-treated ovaries with the effective dose of 3 μg/bursa was examined and compared with the untreated contralateral ovaries (Fig. 5). Most of the large antral follicles of control ovaries underwent follicular rupture during the ovulation processes and the mature ovum was released (Fig. 5A). Confirming the counts of ovulated oocytes, the histological examination of AG1478-treated ovaries revealed oocytes entrapped in the majority of the large antral follicles (Fig. 5B). These included oocytes with intact germinal vesicle (GV) with immature unexpanded cumuli (Fig. 5C), GVB oocytes at the first metaphase/anaphase (Fig. 5D), and oocytes that completed the first meiotic division as evidenced by the presence of the polar body (Fig. 5E). In control ovaries, entrapped oocytes were also found in large antral follicles at different morphological stages (Fig. 6). Therefore, we have analyzed quantitatively the number of large antral follicles with entrapped oocytes, as well as their meiotic status. The serial ovarian sections were scored for oocytes entrapped and for the ratio of immature oocytes in large antral follicles. The control and AG1478-treated ovaries contained 35 and 62% large follicles with entrapped oocytes, respectively (2 test P < 0.0005) (Fig. 6). Moreover, the ratio of entrapped immature oocytes (GV) in the inhibitor-treated ovaries was 5-fold higher compared with the contralateral untreated ovaries (2 test P < 0.0005).
FIG. 5. Histological appearance of control (A) and AG1478-treated (B–E) ovaries. B, Follicles with entrapped oocytes, one at metaphase (black arrow) and the second with GV (white arrow). C, Immature oocyte with intact GV. D, Mature oocyte at metaphase II. E, Mature oocyte with first polar body. Each bar, 100 μm.
Expression of rHAS-2, rCOX-2, and rTSG-6 mRNA in follicles in culture
Several genes, such as COX-2, HAS-2, and TSG-6, have been previously shown to be essential for the ovulatory response, including cumulus expansion. Therefore, we have tested their mRNA expression after LH and ER stimulation of follicles in culture. We found that both LH and ER induced the expression of rCOX-2, rHAS-2, and rTSG-6 mRNA (Fig. 7, upper panel), but ER was less effective than LH. To test whether LH action on some of the key genes associated with ovulation is mediated by the EGF signal transduction network, we have examined the effect of AG1478. Therefore, the expression of rHAS-2, rCOX-2, and rTSG-6 mRNA in FEOs treated by LH or LH plus AG1478 after 6 h incubation was compared. Very low expression of the tested enzymes was detected in control group; however, LH stimulated mRNA expression of rHAS-2, rCOX-2, and TSG-6 with 3.8-, 27-, and 7.3-fold increase, respectively. By contrast, AG1478 suppressed the LH-stimulated expression of these enzymes (Fig. 8). The expression of these enzymes was tested also in ovaries treated by AG1478 administration into the periovarian sac. The relative semiquantitative RT-PCR revealed no significant differences in the ovarian expression of rHAS-2, rCOX-2, and rTSG-6 mRNAs between AG1478-treated and untreated-contralateral ovary in vivo (Fig. 9). Nevertheless, the expression of respective mRNAs was lower when compared with vehicle-treated control levels (P < 0.025, <0.025, and <0.05, respectively).
FIG. 7. LH and ER stimulation of rat HAS-2, COX-2, and TSG-6 mRNA expression in preovulatory follicles in vitro. Preovulatory follicles were explanted and cultured for different periods as described. A representative gel is shown (upper panel) and the cumulative results of three independent experiments (lower panel).
FIG. 8. Inhibition of LH-induced expression of rHAS-2, rCOX-2, and rTSG-6 mRNAs by AG1478. Preovulatory follicles were explanted and cultured for 6 h. Other details as in Fig. 7.
FIG. 9. Ovarian expression in vivo of rHAS-2, rCOX-2, and rTSG-6 mRNAs in AG1478- or vehicle-injected rats. The inhibitor or vehicle was injected unilaterally into the ovarian bursa (50 μl; gray columns), and hCG was administered ip 30 min afterward. The contralateral ovaries served as controls (black columns). The rats were killed 6 h after hCG stimulation of ovulation, and the ovaries were processed for semiquantitative RT-PCR. Treatments with different letters differ significantly as revealed by one-way ANOVA analysis (AG1478 injected vs. vehicle injected).
Effects of Galardin on the LH- and ER-induced maturation of FEOs
To test whether the action of LH on resumption of meiosis is dependent upon proteolytic activity, suggested to be required for the release of cell membrane-bound EGF-like factors (11), we used a broad spectrum metalloprotease inhibitor, Galardin, and a potent matrix metalloprotease (MMP)-2/MMP-9 inhibitor [(2R)-2-[(4biphenylsulfonyl)amino]-3-phenylpropionic acid]. Only Galardin (20 μM) prevented the LH-induced resumption of meiosis in FEO in culture but not that of exogenous ER (Fig. 10). By contrast, the specific MMP-2/MMP-9 inhibitor (100–500 μM) did not affect resumption of meiosis (>80% GVB).
FIG. 10. The effect of Galardin on LH- or ER-induced maturation of rat FEOs. The broad-spectrum MMP inhibitor Galardin (20 μM) was added 30 min before ER or LH. Treatments with different letters differ significantly (P < 0.001) as revealed by one-way ANOVA analysis.
Discussion
Interactions, or cross-talk, between diverse signaling networks is now a widely established concept in cell biology, development, and carcinogenesis. Such interactions have now been recognized to include also triple membrane passing signal mechanisms that involve the activity of metalloproteases in the processing and release of transmembrane precursors of EGF family growth factors. Thus, the seven known EGFR ligands and four receptor tyrosine kinases (RTKs or ErbBs) (15, 16) were activated also by ligands stimulating GPCRs (17, 18). Now, the ovulatory response, recurring each reproductive cycle, is identified to include, in addition to GPCR, EGFR ligands released by a metalloprotease.
After the demonstration of the stimulation of the resumption of meiosis by EGF and TGF in rat preovulatory follicles (3, 4), the ability of EGF to induce meiosis was shown in many additional species including mouse (19), human (20), and several ruminant species (for review, see Ref. 21). In addition, EGFR mRNA and protein were identified in follicle cells, including mural granulosa cells, cumulus cells, and the oocyte (see Ref. 21). Finally, evidence for follicular synthesis of EGF-like factors was presented. Thus, LH stimulation of ER mRNA expression was detected by subtractive hybridization in the rat (22) and the cow (23). Gonadotropic stimulation protocols in in vitro fertilization patients stimulated the expression of AR and ER in granulosa cells (24).
Using the well-characterized model of rat preovulatory follicles in culture, we have confirmed and extended the recent report (10) based on mouse follicles and oocytes implicating locally produced EGF-like agents in the mediation of ovulation triggered by LH/hCG. Thus, we have confirmed that LH/hCG stimulated follicular expression of three EGF-like factor (AR, ER, and BTC) mRNA; that addition of ER, AR, and BTC to cultured follicles stimulated the resumption of meiotic maturation and the expression of several genes clearly associated with cumulus expansion and ovulation; and the EGFR-specific kinase inhibitor AG1478, but not its inactive analog AG43, blocked LH stimulation of EGFR phosphorylation, resumption of meiosis, and expression of ovulation-associated genes.
Injection of AG1478 into the ovarian bursa provided further support for the suggested mediation of LH-stimulated ovulation by paracrine EGF ligands. Thus, the drug significantly inhibited the ovulation from the treated ovaries, compared with untreated contralateral ones or vehicle-treated ovaries. This effect was biphasic; a dose lower than 3 μg/bursa was ineffective, as were higher doses. This latter finding may be related to additional cooperative interactions of the inhibitor with extrafollicular or extraovarian systems conducive with ovulation.
Histological scrutiny revealed that in the ovaries treated with the effective dose of the inhibitor, the large preovulatory follicles with entrapped oocytes had a markedly higher proportion of immature, GV oocytes, compared with the oocytes entrapped in control ovaries. In other large follicles with entrapped oocytes, the meiotic process has been resumed, and in some, the first meiotic division was even completed as evidenced by the first polar body. Such separation between ovarian responses has been already observed in response to gonadotropic stimulation, resumption of meiosis having a lower threshold than completion of the first meiotic division and follicle rupture requiring even a stronger stimulus (25, 26). Thus, our in vivo studies strongly support the physiological mediation of LH action on ovulation by ovarian EGF-like ligands.
Cumulus maturation or expansion involves the formation of a mucoid extracellular matrix by cumulus cells. The matrix is comprised mostly of the glycosaminoglycan hyaluronan (HA) forming a structural backbone as well as several HA binding proteins: the proteoglycan versican, the serum-derived factor inter trypsin inhibitor, and the secreted protein TSG-6 (27). Cumulus expansion and HA synthesis are required for the release of fertilizable ova at ovulation (28). Likewise, TSG-6 is also essential for ovulation because TSG-6 null female mice have a markedly lower ovulation rate than normal (29).
The LH surge also triggers a cascade of inflammation-related responses, including the induction of COX-2 and the synthesis of prostaglandins. Pharmacological inhibition of COX-2 in several mammalian species suppresses ovulation (1), and mice with null mutation for either COX-2 or the prostaglandin E2 receptor subtype EP2 have impaired ovulation associated with defective cumulus expansion (30, 31, 32). Expression of COX-2 and the resulting prostaglandins seem to be involved at two steps required for the ovulation of a fertilizable ovum: cumulus expansion (33) and follicle rupture, most probably through activation of collagenolytic enzymes (1). These two responses could be separated by the inhibition of hyaluronic acid synthesis, which prevented cumulus expansion but not rupture of the follicle wall, and the mature oocytes remained entrapped within the ruptured follicles (28). The precise interactions of prostaglandins, cumulus expansion, and follicle rupture remain to be elucidated. The examination of the follicular expression of the three genes HAS-2, TSG-6, and COX-2 essential for these ovulatory changes provided molecular markers for stimulation of ovulation by LH/hCG and the role of paracrine EGF family members in mediating this response. This is especially valuable in cultured rat follicles that do not undergo rupture. LH/hCG and ER stimulated follicular mRNA of HAS-2, TSG-6, and COX-2 in vitro. Furthermore, AG1478 inhibited the expression of these mRNA species in response to LH/hCG in cultured follicles and also in vivo in the whole ovary. This latter result is of special interest. The data in Fig. 9 show that both ovaries, the one injected with the inhibitor and the untreated contralateral one, showed similar reduction in the expression of these three marker RNAs when compared with the control animals injected with the vehicle only. Yet, there was a significant reduction in ovulation rate of the AG1478-treated ovaries compared with the untreated ones or vehicle-treated controls. These results indicate that the drug probably reached the untreated ovary too. In this case (see Fig. 4), the reduced ovulation from the inhibitor-treated ovary is somewhat puzzling. It is probably related to the preferential uptake of the drug into the follicular compartment of the treated ovaries or differential sensitivity of various ovarian cells to the drug. These possibilities need further examination in the future.
The broad-spectrum metalloprotease inhibitor Galardin, but not a specific MMP-2/MMP-9 inhibitor, prevented the stimulation of the resumption of meiosis in rat follicles by LH. Thus, we demonstrate in a physiological unit comprised of several cell types the use of triple membrane passing signal mechanisms and transactivation of RTK by the stimulation of GPCR by LH. The failure of a specific MMP-2/MMP-9 inhibitor to block this activity of LH is of interest because they were implicated in the transactivation of the EGFR by GnRH in mouse gonadotropes cell line (11). The determination of the follicular metalloprotease involved in the processing of EGF-like factors remains to be established.
In conclusion, our results provide an explanation to the previously enigmatic LH-mimicking actions of EGF and TGF- in the preovulatory follicle. Furthermore, they support the physiological role of follicular EGF-like factors in mediating the ovulatory response to LH in terms of resumption of meiosis, activation of genes involved in cumulus expansion, and follicle rupture in vitro and the release of the mature ovum in vivo. In view of the available evidence for EGFR by the somatic cells of the follicle as well as by the oocyte the precise cellular site(s) of EGF-like factors on ovulatory responses like the resumption of meiosis and cumulus maturation remain to be carefully dissected. These studies may impact the development of new approaches for fertility treatments and contraception.
Acknowledgments
We thank Dr. R. Seger and Dr. N. Dekel for helpful discussions and support throughout this study, Mrs. A. Tsafriri for her excellent technical help, and Dr. A. F. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases’ National Hormone and Pituitary Program, National Institute of Child Health and Human Development for the gonadotropins.
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Address all correspondence and requests for reprints to: Alex Tsafriri, The Bernhard Zondek Hormone Research Laboratory, Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: alex.tsafriri@weizmann.ac.il.
Abstract
Previous studies showed that epidermal growth factor (EGF) and TGF mimic the action of LH on the resumption of oocyte maturation. We tested whether EGF-like agents, such as amphiregulin (AR), epiregulin (ER), and betacellulin (BTC), also mediate the LH stimulation of the ovulatory response in the rat. LH induced transient follicular expression of AR, ER, and BTC mRNA, reaching a maximum after 3-h incubation. Furthermore, the addition of ER, AR, and BTC to the culture medium could mimic some of LH actions. AR and ER fully simulated LH-induced resumption of meiosis in vitro, whereas BTC was less effective. To study the putative involvement of EGF-like factors in mediation of LH signal, the effect of the EGF receptor kinase inhibitor AG1478 was tested. When added with LH, AG1478, but not its inactive analog AG43, reduced EGF receptor phosphorylation and oocyte maturation compared with follicles treated with LH only. In addition to the inhibition of resumption of meiosis, AG1478 administration into the bursa (3 μg/bursa) resulted in 51% (P < 0.0005) inhibition of ovulation in the treated ovaries, compared with the untreated contralateral ones, as well as to the vehicle-treated ovaries (P < 0.02). LH, as well as ER, induced the expression of genes associated with the ovulatory response like rat hyaluronan synthase-2, cycloxygenase-2, and TNF-stimulated gene 6 mRNA, whereas AG1478 inhibited this effect of LH. Release of EGF-like factors from the membrane is dependent on activated metalloproteases. Indeed, Galardin, a broad-spectrum metalloprotease inhibitor, but not a specific matrix metalloprotease 2 and 9 inhibitor, suppressed meiotic maturation induced by LH. Conversely, meiotic maturation induced by ER was not affected by Galardin, thus, supporting the notion that LH releases follicular membrane-bound EGF-like agents. In summary, EGF-like factors such as ER, AR, and BTC seem to mediate, at least partially, the LH stimulation of oocyte maturation, ovulatory enzyme expression, and ovulation.
Introduction
IN MOST MAMMALS, the surge of LH stimulates the ovulatory response in Graafian follicles. It includes the resumption of meiotic maturation; differentiation of mural granulosa cells, usually referred to as luteinization, a reprogramming of their protein and steroidogenic activity; expansion or maturation of the cumulus cells surrounding the oocytes; and, finally, with rupture of the follicle wall and release of a fertilizable ovum (1). The physiological role of gonadotropins is well established: activation of G protein-coupled receptors (GPCRs) mediated by the adenyl cyclase-cAMP system in the ovary and the preovulatory follicle (2). Therefore, the ability of several other paracrine factors, using different transduction pathways to mimic the ovulatory actions of LH was quite puzzling. Thus, it has been shown that epidermal growth factor (EGF) (3) or TGF (4) induce the maturation of follicle-enclosed oocytes (FEOs) in the rat, EGF stimulates oocyte maturation and cumulus expansion in vitro in the pig (5) and in the human (6), and that GnRH stimulates ovulatory changes in vitro (7) and in vivo (8).
Amphiregulin (AR), epiregulin (ER), and betacellulin (BTC) are members of the family of EGF receptor (EGFR) ligands. Members of this family are structurally and functionally related integral membrane proteins that can be proteolytically processed and released from cell surface (9). Recently, Park et al., (10) have presented evidence that LH stimulates the expression of EGF-family members AR, ER, and BTC in mouse preovulatory follicles and that these EGF-like factors trigger meiosis and cumulus expansion in such follicles in culture. It was suggested, therefore, that ovarian EGF-factors serve as paracrine mediators of LH in the induction of ovulation.
In this study, we extended these observations using rat FEOs in culture. We have confirmed that LH/human chorionic gonadotropin (hCG) stimulated follicular expression of AR, ER, and BTC mRNA; that addition of these EGF-like factors to cultured follicles stimulated the resumption of meiotic maturation and the expression of several genes clearly associated with cumulus expansion and ovulation; and the EGFR-specific kinase inhibitor AG1478, but not its inactive analog AG43, blocked LH stimulation of follicular EGFR phosphorylation, resumption of meiosis, and expression of ovulation-associated genes in vitro.
In addition, we showed that an EGFR kinase inhibitor, AG1478, attenuated LH/hCG-stimulated ovulation in vivo. Furthermore, it has been shown that EGF-like factors are bound to the plasma membrane and released by the activation of metalloproteases (11). Accordingly, we have demonstrated that Galardin, a broad-spectrum metalloprotease inhibitor, suppressed resumption of meiosis in FEOs only when triggered by LH, but not ER, supporting the notion that LH stimulation of ovulation is mediated, at least partially, by the release of follicular cell membrane-bound EGF-like factors.
Materials and Methods
Animals
Rats of our Wistar-derived colony were provided with water and rat chow ad libitum and housed in air-conditioned rooms illuminated 14 h/d. The experiments were carried out in accordance with the principles and guidelines for the use of laboratory animals and approved by the Weizmann Institute of Sciences (Rehovot, Israel) research animal committee.
Immature female Wistar rats (23–24 d old) were injected subcutaneously with 12 IU pregnant mare serum gonadotropins (PMSGs) (Sanofi SNA, Libourne, France) to enhance preovulatory follicular development. For explanting FEOs for culture, the animals were killed 48–50 h after PMSG by using CO2 and subsequent cervical dislocation.
Intrabursal injections and the ovulatory response in vivo
Two days after PMSG treatment, the bursa of rats was injected with the indicated doses of AG1478 (Calbiochem, San Diego, CA) or vehicle (12) between 1230 and 1330 h. For intrabursal injection, animals were anesthetized by a cocktail of ketamine (40–60 mg/kg) and diazepam (2–3 mg/kg), and one of the ovaries was exteriorized via a small lumbosacral incision. A 29-gauge needle was threaded into the ovarian bursa via adjoining fat pad. The location of the injection was confirmed by observation of the swelling of the bursa. After injection of the inhibitor or vehicle (50 μl/bursa), the ovary was replaced into the abdominal cavity, and the skin was sutured. The contralateral ovary served as control. After 30 min, animals were treated with 5 IU hCG. Vehicle treated-animals were used as an additional control. Ovulated cumulus oocyte complexes collected 18 h after hCG treatment were isolated from the oviduct and counted under a dissecting microscope.
Culture of preovulatory follicles
The preovulatory follicles were isolated 48 h after PMSG injection as previously described (13). FEOs (10–15/dish) were cultured in Leibovitz’s L-15 medium (Life Technologies, Inc., Grand Island, NY) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) (Life Technologies, Inc.), and 5% fetal calf serum (Sera-Lab, Crawley Down, UK). FEOs were cultured at 37 C in a controlled atmosphere of 50% O2, 1.3% CO2, and 48.7% N2. After 60 min of preequilibration, ovine LH (10–100 ng/ml) (generously provided by Dr. A. F. Parlow and the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health), AG1478 (10 μM) and AG43 (10 μM) (Calbiochem), AR (10–1000 nM) (R&D Systems, Minneapolis, MN), ER (1–250 nM) (R&D Systems), or BTC (10–100 nM) (R&D Systems) were added. Galardin (20 μM) (Biomol, Plymouth Meeting, PA) or (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid (100–500 μM) (Calbiochem) was added to the culture 30 min before LH or ER stimulation. The same vehicle solution was included in control culture media. At the end of the culture period, the follicles were punctured to release and collect the cumulus oocyte complexes under a dissecting microscope. Oocyte maturation was assessed by scoring groups of 10–15 released oocytes using Nomarski interference microscopy. The mean ± SEM values of the groups of oocytes for each treatment are given.
Semiquantitative RT-PCR analysis
The expression of rat (r) hyaluronan synthase-2 (rHAS-2), cycloxygenase-2 (rCOX-2), TNF-stimulated gene 6 (rTSG-6), rAR, rER, and rBTC was examined by relative semiquantitative RT-PCR. Total RNA was extracted from ovaries and preovulatory follicles at the indicated time intervals, using Tri-reagent (Sigma, St. Louis, MO). For each sample, 250 ng RNA from ovaries or 60–80 ng of RNA from preovulatory follicles was reverse transcribed using oligo dT primer (Promega, Madison, WI) followed by PCR amplification. Specific primers were used to amplify the following cDNAs: HAS-2 (forward, 5'-gcttgaccctgcctcatctgtgg-3'; reverse, 5'-ctggttcagccatctcagatatt-3'), COX-2 (forward, 5'-ctgcttttcaaccagcagttcc-3'; reverse, 5'-tctgcagccatttctttctctc-3'), TSG-6 (forward, 5'-cgaagcgaatctttaaatcccc-3'; reverse, 5'-ctaaaccgtccagctaagaac-3'), AR (forward, 5'-ccacaggggactatgactac-3'; reverse, 5'-ttacggcggagacaaagac-3'), ER (forward, 5'-ccaccttctacaagcagtatc-3'; reverse, 5'-tcactctctcgtattcttccc-3'), BTC (forward, 5'-ggtcttgtgattctccagtg-3'; reverse, 5'-cttccttcttctttttgcgatg-3'), which amplified 403-, 376-, 394-, 408-, 407-, and 400-bp products, respectively. A fragment of S-16 cDNA, which served as an internal standard, was amplified in parallel using the following primers: forward, 5'-cgttcaccttgatgagcccatt-3'; and reverse, 5'-tccaagggtccgctgcagtc-3', which amplified a 100-bp product.
Each band was scanned and quantified by Fluor-STM Multimager (Bio-Rad, Hercules, CA). Briefly, the same size rectangle was used to surround each band, and its intensity was determined using the Quantity one version 4.2.1 software (Bio-Rad) adjusted for ethidium bromide gel. The background in the blank region of the gel was subtracted from the total area of the screened samples.
Immunoblotting
Samples (75 μg whole cell lysates per each lane) were separated by SDS-7.5% PAGE and electrophoretically transferred to a nitrocellulose membrane. The samples were immunoblotted with polyclonal anti-EGFR (Cell Signaling Technology, Inc., Beverly, MA) or polyclonal anti-PY1068 EGFR (Cell Signaling Technology, Inc.) as indicated in the legends. The polyclonal antibodies for immunoblots were detected with horseradish peroxidase-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) antibodies. Immunoblotting was performed according to manufactures instructions. Specific signals were visualized on x-ray by enhanced chemiluminescence detection system (Pierce, Rockford, IL) and scanned and quantified by Fluor-STM Multimager (Bio-Rad) adjusted for x-ray film as described above.
Histology
Ovaries after superovulation were excised and fixed in Bouin’s solution overnight, then washed with 70% ethanol and embedded in paraffin wax. Sections, 5 μm thick, were mounted on Super-frost/PLUS Micro slides (Fisher Scientific, Pittsburgh, PA), then rehydrated and stained with hematoxylin and eosin for 10 min, and then washed.
Statistical analysis
Statistical analysis by ANOVA, student’s t test, and 2 test were performed whenever appropriate. Data were expressed as mean ± SEM of pooled results obtained from at least three replicate cultures and two separate experiments. Otherwise, the results of two replicates were given. For the analysis of oocyte maturation in vivo (see Fig. 6), SE values were calculated according to the method described (14). Values of P < 0.05 were considered to be significant.
FIG. 6. Inhibition of meiotic maturation and ovulation by AG1478 administration in vivo. Unruptured large antral follicles were counted and the meiotic status of entrapped oocytes defined in serial sections of AG1478-treated and their control, untreated contralateral ovaries. The data are presented as the percentage of large antral follicles with oocytes and the percentage of mature oocytes entrapped in them. Numbers on columns indicate the numbers of counted follicles and oocytes.
Results
Stimulation of follicular EGF-like factors mRNA by LH
To test the involvement of EGF-like factors in the LH pathway, FEO cultures were initially treated with 100 ng/ml LH for 3 and 9 h. At the end of culture, levels of rER, rAR, and rBTC mRNA were determined by semiquantitative RT-PCR. LH brought about transient expression of rAR, rER, and rBTC mRNA with a maximum at 3 h followed by a marked decrease at 9 h of culture. Although LH further stimulated the basal BTC expression, it induced both AR and ER, which were undetected in unstimulated follicles (Fig. 1).
FIG. 1. LH-induced expression of rER, rAR, and rBTC mRNAs in preovulatory follicles in vitro. Preovulatory follicles (13 14 ) were explanted and cultured for the indicated period with and without LH (100 ng) in three independent experiments. Upper panel, Gel showing expression of the three genes examined and S16 as standard; lower panel, summary of the results. Mean ± SEM values are shown. LH-treated follicles differ significantly from untreated ones cultured for the same time (b, P < 0.0001; c, P < 0.002 for rER and rBTC and P < 0.03 for rAR) as revealed by one-way ANOVA analysis.
Effect of EGF-like factors on meiosis
In view of the above findings, we tested whether the addition of EGF-like factors, such as ER, AR, and BTC in vitro reproduces, some actions of LH. Complete (with AR and ER) or partial (BTC) stimulation of the resumption of meiosis in FEO was obtained (Fig. 2A). ER was more potent in causing maximal maturation (88% germinal vesicle breakdown, GVB) at the concentration of 100 nM. Ten-fold higher dose of AR was required to reach a similar maturation rate.
FIG. 2. EGF-like growth factors and the resumption of meiosis in rat FEOs. A, Induction of meiosis by EGF-like ligands. B, Inhibition of LH-induced maturation by AG1478. Preovulatory follicles were cultured for 24 h with EGF-like factors, LH, or LH+AG1478 (10 μM) as indicated. The number of examined oocytes is indicated on columns. The bars represent the percentage of GVB (mean ± SEM) of at least three groups of oocytes in two independent experiments (excluding 10 nM BTC with only two replicates, not included in statistical analysis). b, Versus control, P < 0.03; c, vs. control, P < 0.0002; d, vs. control, P < 0.0001; e, vs. LH, P < 0.0001.
EGF signal transduction and LH action
In vitro. To examine the role of EGF signal transduction in LH stimulation of meiosis, we have tested the effect of the EGFR kinase inhibitor, AG1478, on LH-stimulated follicles. For this purpose, follicles were cultured with LH and AG1478. AG1478, but not AG43 its inactive analog, blocked the LH-induced EGFR phosphorylation (Fig 3, A and B) and oocyte maturation (Figs. 2B and 3C) (1% GVB with AG1478 vs. >88% with LH or LH + AG 43) compared with control follicles (4% GVB).
FIG. 3. EGFR phosphorylation and induction of resumption of meiosis by LH. A and B, EGF receptor phosphorylation. Follicles were either untreated or incubated with LH (1 μg/ml), LH + AG43 (10 μM), and LH + AG1478 (10 μM) (lanes 1–4, respectively, in A) for 6 h followed by immunoblotting with the EGFR-phophospecific PY1068 antibody and, after stripping by Ponceau Red for 2 h, with the EGFR-specific antibody. In two separate experiments, each including eight rats, 39 follicles were cultured for each treatment variable. Total protein extracted was applied to each lane. A, representative blot. B, summary of two repeat experiments. The phosphorylated EGFR immunoblotting for each lane was standardized by EGFR. The bars show the ratios for each variable as related to the mean of AG43, which is determined as 100%. The dots represent the values of two independent experiments. C, Resumption of meiosis in rat FEOs. Preovulatory follicles were cultured for 6 h with indicated additives (LH, 1 μg/ml; AGs, 10 μM). The number of oocytes examined is indicated on the columns. The bars represent the percentage of mature oocytes ± SEM.
In vivo. To examine the inhibitory effects of AG1478 in vivo, it was injected into the ovarian bursa before hCG administration (Fig. 4). Local administration of 3 μg/bursa into the periovarian sac resulted in 51% (14.5 ± 2.6 ova/ovary) inhibition of ovulation in treated ovary, compared with untreated contralateral ovary (28.5 ± 2.3 ova/ovary) (P < 0.0005) or with vehicle-treated ovaries (21.3 ± 2.3 ova/ovary) (P < 0.02). Intrabursal administration of lower or higher concentrations of AG1478 revealed no significant differences in ovulation rate between treated ovaries and untreated ones or vehicle-treated ovaries.
FIG. 4. Effect of AG1478 on ovulation. The vehicle (control) or the drug was injected unilaterally to the periovarian sac (bursa). Number of rats on the columns; bars indicate mean ± SEM. *, P < 0.02 AG1478-treated vs. vehicle-treated control and P < 0.0005 vs. untreated contralateral ovary as obtained by Student’s t test analysis.
Histological appearance
The histological appearance of the AG1478-treated ovaries with the effective dose of 3 μg/bursa was examined and compared with the untreated contralateral ovaries (Fig. 5). Most of the large antral follicles of control ovaries underwent follicular rupture during the ovulation processes and the mature ovum was released (Fig. 5A). Confirming the counts of ovulated oocytes, the histological examination of AG1478-treated ovaries revealed oocytes entrapped in the majority of the large antral follicles (Fig. 5B). These included oocytes with intact germinal vesicle (GV) with immature unexpanded cumuli (Fig. 5C), GVB oocytes at the first metaphase/anaphase (Fig. 5D), and oocytes that completed the first meiotic division as evidenced by the presence of the polar body (Fig. 5E). In control ovaries, entrapped oocytes were also found in large antral follicles at different morphological stages (Fig. 6). Therefore, we have analyzed quantitatively the number of large antral follicles with entrapped oocytes, as well as their meiotic status. The serial ovarian sections were scored for oocytes entrapped and for the ratio of immature oocytes in large antral follicles. The control and AG1478-treated ovaries contained 35 and 62% large follicles with entrapped oocytes, respectively (2 test P < 0.0005) (Fig. 6). Moreover, the ratio of entrapped immature oocytes (GV) in the inhibitor-treated ovaries was 5-fold higher compared with the contralateral untreated ovaries (2 test P < 0.0005).
FIG. 5. Histological appearance of control (A) and AG1478-treated (B–E) ovaries. B, Follicles with entrapped oocytes, one at metaphase (black arrow) and the second with GV (white arrow). C, Immature oocyte with intact GV. D, Mature oocyte at metaphase II. E, Mature oocyte with first polar body. Each bar, 100 μm.
Expression of rHAS-2, rCOX-2, and rTSG-6 mRNA in follicles in culture
Several genes, such as COX-2, HAS-2, and TSG-6, have been previously shown to be essential for the ovulatory response, including cumulus expansion. Therefore, we have tested their mRNA expression after LH and ER stimulation of follicles in culture. We found that both LH and ER induced the expression of rCOX-2, rHAS-2, and rTSG-6 mRNA (Fig. 7, upper panel), but ER was less effective than LH. To test whether LH action on some of the key genes associated with ovulation is mediated by the EGF signal transduction network, we have examined the effect of AG1478. Therefore, the expression of rHAS-2, rCOX-2, and rTSG-6 mRNA in FEOs treated by LH or LH plus AG1478 after 6 h incubation was compared. Very low expression of the tested enzymes was detected in control group; however, LH stimulated mRNA expression of rHAS-2, rCOX-2, and TSG-6 with 3.8-, 27-, and 7.3-fold increase, respectively. By contrast, AG1478 suppressed the LH-stimulated expression of these enzymes (Fig. 8). The expression of these enzymes was tested also in ovaries treated by AG1478 administration into the periovarian sac. The relative semiquantitative RT-PCR revealed no significant differences in the ovarian expression of rHAS-2, rCOX-2, and rTSG-6 mRNAs between AG1478-treated and untreated-contralateral ovary in vivo (Fig. 9). Nevertheless, the expression of respective mRNAs was lower when compared with vehicle-treated control levels (P < 0.025, <0.025, and <0.05, respectively).
FIG. 7. LH and ER stimulation of rat HAS-2, COX-2, and TSG-6 mRNA expression in preovulatory follicles in vitro. Preovulatory follicles were explanted and cultured for different periods as described. A representative gel is shown (upper panel) and the cumulative results of three independent experiments (lower panel).
FIG. 8. Inhibition of LH-induced expression of rHAS-2, rCOX-2, and rTSG-6 mRNAs by AG1478. Preovulatory follicles were explanted and cultured for 6 h. Other details as in Fig. 7.
FIG. 9. Ovarian expression in vivo of rHAS-2, rCOX-2, and rTSG-6 mRNAs in AG1478- or vehicle-injected rats. The inhibitor or vehicle was injected unilaterally into the ovarian bursa (50 μl; gray columns), and hCG was administered ip 30 min afterward. The contralateral ovaries served as controls (black columns). The rats were killed 6 h after hCG stimulation of ovulation, and the ovaries were processed for semiquantitative RT-PCR. Treatments with different letters differ significantly as revealed by one-way ANOVA analysis (AG1478 injected vs. vehicle injected).
Effects of Galardin on the LH- and ER-induced maturation of FEOs
To test whether the action of LH on resumption of meiosis is dependent upon proteolytic activity, suggested to be required for the release of cell membrane-bound EGF-like factors (11), we used a broad spectrum metalloprotease inhibitor, Galardin, and a potent matrix metalloprotease (MMP)-2/MMP-9 inhibitor [(2R)-2-[(4biphenylsulfonyl)amino]-3-phenylpropionic acid]. Only Galardin (20 μM) prevented the LH-induced resumption of meiosis in FEO in culture but not that of exogenous ER (Fig. 10). By contrast, the specific MMP-2/MMP-9 inhibitor (100–500 μM) did not affect resumption of meiosis (>80% GVB).
FIG. 10. The effect of Galardin on LH- or ER-induced maturation of rat FEOs. The broad-spectrum MMP inhibitor Galardin (20 μM) was added 30 min before ER or LH. Treatments with different letters differ significantly (P < 0.001) as revealed by one-way ANOVA analysis.
Discussion
Interactions, or cross-talk, between diverse signaling networks is now a widely established concept in cell biology, development, and carcinogenesis. Such interactions have now been recognized to include also triple membrane passing signal mechanisms that involve the activity of metalloproteases in the processing and release of transmembrane precursors of EGF family growth factors. Thus, the seven known EGFR ligands and four receptor tyrosine kinases (RTKs or ErbBs) (15, 16) were activated also by ligands stimulating GPCRs (17, 18). Now, the ovulatory response, recurring each reproductive cycle, is identified to include, in addition to GPCR, EGFR ligands released by a metalloprotease.
After the demonstration of the stimulation of the resumption of meiosis by EGF and TGF in rat preovulatory follicles (3, 4), the ability of EGF to induce meiosis was shown in many additional species including mouse (19), human (20), and several ruminant species (for review, see Ref. 21). In addition, EGFR mRNA and protein were identified in follicle cells, including mural granulosa cells, cumulus cells, and the oocyte (see Ref. 21). Finally, evidence for follicular synthesis of EGF-like factors was presented. Thus, LH stimulation of ER mRNA expression was detected by subtractive hybridization in the rat (22) and the cow (23). Gonadotropic stimulation protocols in in vitro fertilization patients stimulated the expression of AR and ER in granulosa cells (24).
Using the well-characterized model of rat preovulatory follicles in culture, we have confirmed and extended the recent report (10) based on mouse follicles and oocytes implicating locally produced EGF-like agents in the mediation of ovulation triggered by LH/hCG. Thus, we have confirmed that LH/hCG stimulated follicular expression of three EGF-like factor (AR, ER, and BTC) mRNA; that addition of ER, AR, and BTC to cultured follicles stimulated the resumption of meiotic maturation and the expression of several genes clearly associated with cumulus expansion and ovulation; and the EGFR-specific kinase inhibitor AG1478, but not its inactive analog AG43, blocked LH stimulation of EGFR phosphorylation, resumption of meiosis, and expression of ovulation-associated genes.
Injection of AG1478 into the ovarian bursa provided further support for the suggested mediation of LH-stimulated ovulation by paracrine EGF ligands. Thus, the drug significantly inhibited the ovulation from the treated ovaries, compared with untreated contralateral ones or vehicle-treated ovaries. This effect was biphasic; a dose lower than 3 μg/bursa was ineffective, as were higher doses. This latter finding may be related to additional cooperative interactions of the inhibitor with extrafollicular or extraovarian systems conducive with ovulation.
Histological scrutiny revealed that in the ovaries treated with the effective dose of the inhibitor, the large preovulatory follicles with entrapped oocytes had a markedly higher proportion of immature, GV oocytes, compared with the oocytes entrapped in control ovaries. In other large follicles with entrapped oocytes, the meiotic process has been resumed, and in some, the first meiotic division was even completed as evidenced by the first polar body. Such separation between ovarian responses has been already observed in response to gonadotropic stimulation, resumption of meiosis having a lower threshold than completion of the first meiotic division and follicle rupture requiring even a stronger stimulus (25, 26). Thus, our in vivo studies strongly support the physiological mediation of LH action on ovulation by ovarian EGF-like ligands.
Cumulus maturation or expansion involves the formation of a mucoid extracellular matrix by cumulus cells. The matrix is comprised mostly of the glycosaminoglycan hyaluronan (HA) forming a structural backbone as well as several HA binding proteins: the proteoglycan versican, the serum-derived factor inter trypsin inhibitor, and the secreted protein TSG-6 (27). Cumulus expansion and HA synthesis are required for the release of fertilizable ova at ovulation (28). Likewise, TSG-6 is also essential for ovulation because TSG-6 null female mice have a markedly lower ovulation rate than normal (29).
The LH surge also triggers a cascade of inflammation-related responses, including the induction of COX-2 and the synthesis of prostaglandins. Pharmacological inhibition of COX-2 in several mammalian species suppresses ovulation (1), and mice with null mutation for either COX-2 or the prostaglandin E2 receptor subtype EP2 have impaired ovulation associated with defective cumulus expansion (30, 31, 32). Expression of COX-2 and the resulting prostaglandins seem to be involved at two steps required for the ovulation of a fertilizable ovum: cumulus expansion (33) and follicle rupture, most probably through activation of collagenolytic enzymes (1). These two responses could be separated by the inhibition of hyaluronic acid synthesis, which prevented cumulus expansion but not rupture of the follicle wall, and the mature oocytes remained entrapped within the ruptured follicles (28). The precise interactions of prostaglandins, cumulus expansion, and follicle rupture remain to be elucidated. The examination of the follicular expression of the three genes HAS-2, TSG-6, and COX-2 essential for these ovulatory changes provided molecular markers for stimulation of ovulation by LH/hCG and the role of paracrine EGF family members in mediating this response. This is especially valuable in cultured rat follicles that do not undergo rupture. LH/hCG and ER stimulated follicular mRNA of HAS-2, TSG-6, and COX-2 in vitro. Furthermore, AG1478 inhibited the expression of these mRNA species in response to LH/hCG in cultured follicles and also in vivo in the whole ovary. This latter result is of special interest. The data in Fig. 9 show that both ovaries, the one injected with the inhibitor and the untreated contralateral one, showed similar reduction in the expression of these three marker RNAs when compared with the control animals injected with the vehicle only. Yet, there was a significant reduction in ovulation rate of the AG1478-treated ovaries compared with the untreated ones or vehicle-treated controls. These results indicate that the drug probably reached the untreated ovary too. In this case (see Fig. 4), the reduced ovulation from the inhibitor-treated ovary is somewhat puzzling. It is probably related to the preferential uptake of the drug into the follicular compartment of the treated ovaries or differential sensitivity of various ovarian cells to the drug. These possibilities need further examination in the future.
The broad-spectrum metalloprotease inhibitor Galardin, but not a specific MMP-2/MMP-9 inhibitor, prevented the stimulation of the resumption of meiosis in rat follicles by LH. Thus, we demonstrate in a physiological unit comprised of several cell types the use of triple membrane passing signal mechanisms and transactivation of RTK by the stimulation of GPCR by LH. The failure of a specific MMP-2/MMP-9 inhibitor to block this activity of LH is of interest because they were implicated in the transactivation of the EGFR by GnRH in mouse gonadotropes cell line (11). The determination of the follicular metalloprotease involved in the processing of EGF-like factors remains to be established.
In conclusion, our results provide an explanation to the previously enigmatic LH-mimicking actions of EGF and TGF- in the preovulatory follicle. Furthermore, they support the physiological role of follicular EGF-like factors in mediating the ovulatory response to LH in terms of resumption of meiosis, activation of genes involved in cumulus expansion, and follicle rupture in vitro and the release of the mature ovum in vivo. In view of the available evidence for EGFR by the somatic cells of the follicle as well as by the oocyte the precise cellular site(s) of EGF-like factors on ovulatory responses like the resumption of meiosis and cumulus maturation remain to be carefully dissected. These studies may impact the development of new approaches for fertility treatments and contraception.
Acknowledgments
We thank Dr. R. Seger and Dr. N. Dekel for helpful discussions and support throughout this study, Mrs. A. Tsafriri for her excellent technical help, and Dr. A. F. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases’ National Hormone and Pituitary Program, National Institute of Child Health and Human Development for the gonadotropins.
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