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Endocrinology, doi:10.1210/en.2007-0195
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Endocrinology Vol. 148, No. 12 6164-6175
Copyright © 2007 by The Endocrine Society

Hormone-Induced Expression of Tumor Necrosis Factor {alpha}-Converting Enzyme/A Disintegrin and Metalloprotease-17 Impacts Porcine Cumulus Cell Oocyte Complex Expansion and Meiotic Maturation via Ligand Activation of the Epidermal Growth Factor Receptor

Yasuhisa Yamashita, Ikkou Kawashima, Yoshiari Yanai, Masahide Nishibori, JoAnne S. Richards and Masayuki Shimada

Department of Applied Animal Science (Y.Yam., I.K., Y.Yan., M.N., M.S.), Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; and Department of Molecular and Cellular Biology (J.S.R.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Masayuki Shimada, Ph.D., Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, 739-8528, Japan. E-mail: mashimad{at}hiroshima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNF{alpha}-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNF{alpha}-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LH INDUCES NUMEROUS morphological and physiological changes in preovulatory follicles during the ovulation process. In the cumulus-oocyte complex (COC), the oocyte resumes meiotic maturation to metaphase II, cumulus cells secrete a hyaluronan-rich matrix, and the expanded, matrix-rich COC is extruded from the follicle (1). COC expansion requires LH-induced expression of specific genes encoding hyaluronan synthase 2 (Has2) (2, 3), TNF{alpha}-induced protein 6 (Tnfaip6) (4, 5), pentraxin 3 (Ptx3) (6, 7), and other factors that stabilize the matrix (1). TNF{alpha}IP6 binds to and delivers the heavy chain of the serum-derived factor inter-{alpha}-trypsin inhibitor (I{alpha}I) to the hyaluronan backbone to stabilize matrix formation (5, 8). Pentraxin 3 is also secreted by cumulus cells and binds selectively to TNF{alpha}IP6 thereby providing an additional layer to the scaffolding network of the matrix (6, 7). In Ptx3-null mice, the number of ovulated COCs is reduced to about half, and the ovulated oocytes exhibit impaired developmental competence, suggesting that matrix factors play important roles in the ovulation process and oocyte function.

Because cumulus cells of mouse and rat possess few if any LH receptors, the molecular and biological mechanisms by which LH impacts cumulus expansion appear to be indirect. Park et al. (9) provided the first evidence in the mouse that LH stimulated the expression of genes encoding members of the epidermal growth factor (EGF)-like factor family, amphiregulin (Areg), ß-cellulin (Btc), and epiregulin (Ereg) and that these factors secreted from granulosa cells act on cumulus cells via a paracrine mechanism. Recent microarray, RT-PCR, and Western blot analyses of samples derived from murine COCs before and during ovulation show that these EGF-like factors are also expressed in cumulus cells (10). Although the initial induction of these genes occurs in response to the paracrine effects of prostaglandin E2 (PGE2) and EGF-like factors secreted from LH-stimulated granulosa cells, COCs eventually maintain expression of Areg and Ereg by autocrine mechanisms also involving PGE2 (10). Specifically, when COCs or follicle-enclosed oocytes were cultured with AREG, the expressions of Ptgs2, Has2, and Tnfaip6 mRNAs were induced in cumulus cells, COC expansion occurred, and oocytes resumed meiotic maturation (9, 10).

AREG and EREG are expressed as transmembrane precursors that are cleaved at one or more sites in the extracellular domain to release a soluble EGF domain (11, 12). Therefore, metalloprotease activity is critical for the release of the EGF domain to activate EGF receptor (EGFR) on the target cells (13, 14). Sahin et al. (12) showed that TNF{alpha}-converting enzyme (TACE) is the main sheddase of AREG and EREG in mouse embryonic cells. TACE, also known as ADAM17 is a member of a disintegrin and metalloprotease (ADAM) family of proteases. The expression of TACE/ADAM17 has been observed in many tissues, including heart, brain, lung, liver, muscle, kidney, and testis (15). Tace/Adam17-null mice die soon after birth (16), thereby precluding analyses of fertility. However, transcription of the Tace/Adam17 gene is regulated by SP1/SP3 sites within its proximal promoter region (15), a feature common to many genes induced by LH during the ovulatory process (17, 18, 19). Additionally, our microarray analyses of mouse cumulus cells show that Tace/Adam17 is expressed during the ovulatory process (20). Thus, we hypothesized that Tace/Adam17 is expressed in granulosa cells and cumulus cells and that TACE protease activity contributes to release EGF-like factors from the cells during ovulation process. The data showing that a broad metalloprotease inhibitor, galardin, suppressed LH-induced cumulus expansion and oocyte meiotic resumption of rat follicle-enclosed COCs or porcine COCs, whereas the negative effects were completely reversed by the addition of EREG (21, 22), supported this theory. However, precise information about the expression and function of TACE/ADAM17 in COC expansion remained lacking.

To analyze the physiological role of TACE/ADAM17 in more detail, COCs were obtained from porcine ovaries and used in a well-characterized in vitro culture model in which we have shown previously that the metalloprotease inhibitor galardin significantly suppressed cumulus expansion and oocyte meiotic maturation (21). The induction of Tace/Adam17, Areg, and Ereg mRNA was analyzed in cumulus cells of porcine COCs cultured with FSH and LH for specific time intervals. TACE/ADAM17 enzymatic activity was examined by in vitro assay using a specific fluorescent substrate. Additionally, to examine the role of endogenous TACE/ADAM17, granulosa cells from small antral follicles obtained from porcine ovaries or from rat preovulatory follicles, respectively, were cultured with gonadotropins and/or TNF{alpha}-processing inhibitor-2 (TAPI-2). Some rat granulosa cells were transfected with Tace/Adam17 small interfering RNA (siRNA) and then treated with LH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) were purchased from Asuka Seiyaku (Tokyo, Japan). Highly purified porcine FSH and porcine LH were a gift from the National Hormone and Pituitary Program (Rockville, MD). Forskolin, phorbol 12-myristate 13-acetate (PMA), and galardin were purchased from Sigma Chemical Co. (St. Louis, MO). AG1478, PD98059, and TAPI-2 were purchased from Calbiochem (San Diego, CA). Forskolin, PMA, galardin, and TAPI-2 were dissolved in ethanol, and AG1478 and PD98059 were dissolved in dimethylsulfoxide. DMEM/F12 medium and penicillin-streptomycin were from Invitrogen (Carlsbad, CA). Fetal bovine serum was obtained from Life Technologies Inc. (Grand Island, NY). Oligonucleotide poly-(dT) was purchased from Amersham Pharmacia Biotech (Newark, NJ), and avian myeloblastosis virus reverse transcriptase was from Promega (Madison, WI). Routine chemicals and reagents were obtained from Nakarai Chemical Co. (Osaka, Japan) or Sigma.

In vitro culture of porcine COCs and granulosa cells
Isolation of COCs was described previously (23). Briefly, porcine ovaries were collected from 5- to 7-month-old prepubertal gilts at a local slaughterhouse. COCs were collected from intact healthy antral follicles measuring from 3–5 mm in diameter. Oocytes having evenly granulated cytoplasm with at least four layers of unexpanded cumulus cells were selected and were washed three times with maturation medium [modified NCSU37 (21) supplemented with 10% (vol/vol) fetal bovine serum and 7 mM taurine (Sigma)]. The COCs were cultured (20 per well) for up to 40 h in 300 µl of the maturation medium supplemented with both 20 ng/ml FSH and 500 ng/ml porcine LH at 39 C in a humidified incubator (95% air, 5% CO2). Under these conditions, cumulus expansion was induced in porcine COCs (21). Some COCs were cultured with 50 µM TAPI-2 (24), 10 µM galardin (21), 1 µM AG1478 (9), or 10 µM PD98059 (20). Inhibitor-free medium supplemented only with ethanol or dimethylsulfoxide served as controls. At selected time intervals after culture, COCs were collected and then separated by 0.1% hyaluronidase (Sigma) treatment into cumulus cells and oocytes for RNA, protein isolation, and determination of oocyte nuclear status.

Assessment of cumulus expansion was based on the diameter of COCs measured with an eyepiece micrometer using phase-contrast microscopy (Olympus IMT2; Olympus, Tokyo, Japan) and a x10 objective as described previously (21). The diameter selected for measurement was defined as the greatest distance across the COC expanded matrix.

The oocytes were fixed with acetic acids/ethanol (1:3) for 48 h and stained with aceto-lacmoid before examination under a phase-contrast microscope (x400) for evaluation of their chromatin configuration.

Isolation and culture of granulosa cells were described previously (25) with some modification. Briefly, granulosa cells were isolated from intact healthy antral follicles measuring from 3–5 mm in diameter. The cells were treated with trypsin (Calbiochem) for 1 min and DNase (Sigma), and 2 x 106 cells per well were cultured in 12-well culture plates in 1% serum containing DMEM/F12 medium containing penicillin and streptomycin. After the granulosa cells were cultured for 8 h, the cells were washed and then treated with forskolin (10 µM) to mimic LH stimulation of cAMP production and PMA (20 nM) to activate diacylglycerol-mediated signaling or 100 ng/ml FSH in serum-free medium.

Rat granulosa cell culture
Immature female Wister rat were obtained from Clea Japan (Tokyo, Japan). On d 23 of age, female rats were injected ip with 20 IU eCG to stimulate follicular growth and to induce LH receptor (Lhcgr) in granulosa cells. Animals were housed under a 14-h light, 10-h dark schedule in the Experimental Animal Center at Hiroshima University and provided food and water ad libitum. Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Hiroshima University.

Granulosa cells were harvested by needle puncture from ovaries of eCG-primed immature rats as described previously (19). Briefly, the isolated cells were cultured for 8 h in 12-well culture plates in 1% serum-containing medium (DMEM/F12 containing penicillin and streptomycin), and then cells were washed. The granulosa cells were cultured for 4 h in fresh, serum-free medium containing both 10 µM forskolin and 20 nM PMA or 1 µg/ml LH and harvested for protein and RNA analysis.

siRNA treatment procedure in cultured rat granulosa cells
Tace/Adam17 Silencer Predesigned siRNA was purchased from Ambion (Austin, TX). The sequences were sense GGAAAGCGAGUACAGUGUGtt and antisense CACACUGUACUCGCUUUCCtc. Scrambled siRNA duplex (Ambion) was used as a negative control. Rat granulosa cells (2 x 106 per well) were plated in 12-well culture plates before transfection. Transfection of siRNA (1 or 10 nM) was accomplished with the HVJ envelope vector kit GenomONE neo (Ishihara Sangyo, Tokyo, Japan) according to the manufacturer’s instructions. Cells were incubated at 37 C in a CO2 incubator. Three hours after transfection, the medium was changed and granulosa cells were cultured with both 10 µM forskolin and 20 nM PMA or with 1 µg/ml LH for up to 4 h.

Western blot analysis
Cumulus cells or granulosa cells were lysed in Laemmli sample buffer and protein extracts stored at –80 C until use. Protein concentrations were determined by a Lowry assay using a protein assay kit (Bio-Rad, Hercules, CA) according to manufacturer’s procedure. After denaturing by boiling for 5 min, 10 µl of each protein sample (10 µg) was separated by SDS-PAGE on 12.5% polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane (Amersham Biosciences, Uppsala, Sweden). The membrane was blocked with 5% (wt/vol) nonfat dry milk (Amersham) in 0.1% (vol/vol) Tween 20 (Sigma)/PBS (T-PBS). Primary antibodies were added in 2.5% (wt/vol) nonfat dry milk in T-PBS, and incubated overnight at 4 C. A rabbit polyclonal antibody against the human ADAM17 (Sigma) was used at a dilution of 1:2000. Anti-phospho-ERK1/2, total ERK1/2, and phospho-p38MAP kinase antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA) and diluted at 1:2000, 1:1000, and 1:1000, respectively. Anti-ß-actin mouse monoclonal antibody (Sigma) was diluted at 1:10,000. After four washes in T-PBS, the membranes were incubated for 1 h with a 1:2000 dilution of goat antirabbit IgG or antimouse IgG horseradish-peroxidase-labeled antibody (Cell Signaling) in 2.5% (wt/vol) nonfat dry milk in T-PBS at room temperature. After five washes of 10 min each with T-PBS, peroxidase activity was visualized using the ECL Western blotting detection system (Amersham), according to the manufacturer’s instructions. After exposure to x-ray film, the intensity of specific bands was quantified by densitometric scanning using a Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD).

RNA isolation
After cumulus cells were separated from COCs or after granulosa cells were cultured, they were washed three times in PBS. Total RNA was extracted from the cells using the SV Total RNA Isolation System (Promega, Madison, WI) according to the instruction manual and dissolved in nuclease-free water (26). The final RNA concentrations were determined by absorbance using a spectrophotometer.

Real-time RT-PCR analyses
Total RNA was reverse transcribed using 500 ng poly-dT (Amersham Pharmacia Biotech, Newark, NJ) and 0.25 U avian myeloblastosis virus-reverse transcriptase (Promega) at 42 C for 75 min and 95 C for 5 min. cDNA and primers were added to 15 µl total reaction volume provided in the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). PCR were then performed using the iCycler thermocycler (Bio-Rad). Conditions were set to the following parameters: 10 min at 95 C followed by 45 cycles each of 15 sec at 95 C and 1 min at the annealing temperature. Each annealing temperature and specific primers pairs were selected and analyzed as indicated in Table 1Go.


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  		TABLE 1. List of primers used for RT-PCR and the expected size

 
Determination of ADAM17 activity
Cumulus cells were lysed in 25 mM Tris buffer (pH 7.4) containing 1% (vol/vol) Triton X-100, 1 mM phenylmethylsulfonyl fluoride (Sigma), 10 µg/ml leupeptin (Sigma). and 3 mM aprotinin (Sigma). The lysed samples were centrifuged, and the supernatants were stored at –80 C until analyzed. Protein concentration was determined by Lowry assay, and 20 µg protein extract was used to determine the protease activity, using 4 mM fluorogenic peptide III (R&D Systems, Minneapolis, MN) as a substrate. ADAM17 cleaves this peptide sequence between Ala and Val, separating the fluorochrome from the quencher, thus allowing the detection of the fluorescence. The reaction was performed at 37 C, and the fluorescence intensity was determined with a spectrofluorometer (Shimazu, Kyoto, Japan) using an excitation wavelength at 320 nm and emission at 405 nm every 15 min for 2 h. Some protein samples were incubated with either anti-ADAM17 rabbit polyclonal antibody diluted at 1:50 that was also used for Western blotting or IgG purified from normal rabbit serum (Sigma) at 4 C overnight. Then the immune complex was precipitated by protein A-Sepharose beads (Sigma), and this precipitate was used for protease assay as described above.

Statistical analysis
Statistical analyses of data from three separate culture experiments were carried out by one-way ANOVA followed by Duncan’s multiple ranges test using STATVIEW (Abacus Concepts, Inc., Berkeley, CA). All percentage data were subjected to arc-sine transformation before statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Areg, Ereg, and Tace/Adam17 mRNAs are induced in cumulus cells of porcine COCs cultured with FSH and LH
To analyze the induction of the EGF-like ligands Areg and Ereg as well as Tace/Adam17 in cumulus cells, porcine COCs were isolated from ovaries (0 h) and then cultured with FSH and LH for 5, 10, 20, 30, and 40 h (Fig. 1Go, A, C, and E). After culture, cumulus cells were isolated by hyaluronidase treatment, total RNA was prepared, and real time-RT-PCR analyses were performed. As shown, a dramatic induction of each gene was observed in cumulus cells of COCs cultured with FSH and LH for 5 h and was maintained at 10 h. Expression levels of Areg and Ereg declined progressively thereafter, whereas high levels of Tace/Adam17 mRNA persisted until 20 h but declined significantly thereafter. When COCs were cultured for 10 h in the absence of agonist, expression of each gene was significantly lower as compared with those in COCs cultured with FSH and LH (Fig. 1Go, B, D, and F).


Figure 1
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  		FIG. 1. The induction of Areg, Ereg, or Tace/Adam17 mRNA expression in cumulus cells of porcine COCs is dependent on FSH and LH. A, C, and E, Temporal changes of Areg (A), Ereg (C), or Tace/Adam17 (E) mRNA expression in cumulus cells of COCs cultured with FSH and LH for 0, 5, 10, 20, 30, or 40 h. For reference, the 0-h COC value was set as 1 and the data presented as the fold increase. Values are mean ± SEM of three independent culture experiments. *, Significant differences observed as compared with that in COCs before culture (0 h) (P < 0.05). B, D, and F, Effect of FSH and LH on expression of Areg (B), Ereg (D), or Tace/Adam17 (F) mRNA in cumulus cells of COCs cultured for 10 h. 0 h, COCs isolated just after collection; Free, COCs cultured without FSH and LH for 10 h; FSH+LH, COCs cultured with FSH and LH for 10 h.

 
Levels of TACE/ADAM17 protein and activity are increased by FSH and LH in COCs
To analyze TACE/ADAM17 protein levels, cumulus cells were prepared from COCs cultured as in Fig. 1Go and protein samples subjected to Western blot analyses using a specific anti-ADAM17 antibody (Fig. 2AGo). An immunoreactive band at about 80 kDa corresponds to TACE/ADAM17. This band was present in low amounts in COCs before culture but increased dramatically at 10 h, and the high level was maintained up to 40 h, indicating that TACE/ADAM17 protein was induced but more stable than Tace/Adam17 mRNA, which had declined significantly in cumulus cells of COCs by 30 h.


Figure 2
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  		FIG. 2. TACE/ADAM17 protein levels and activity in cumulus cells of COCs. A, TACE/ADAM17 protein in COCs that were cultured with FSH and LH for 0, 5, 10, 20, 30, or 40 h was analyzed by Western blotting. ß-Actin was used as an internal control for loading and transfer. For reference, the 0-h COC value was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent culture experiments. *, Significant differences observed as compared with that in COCs before culture (0 h) (P < 0.05). B, Luminescence of fluorogenic peptide III incubated up to 120 min with protein extracts from cumulus cells of COCs before culture (0 h) or cultured with FSH and LH for 40 h. The level of fluorescence of 0-h COCs before incubation with substrate at 37 C was set as 1 and the data presented as fold increase. C, Luminescence of fluorogenic peptide III incubated at 37 C for 105 min with either the supernatant or the anti-ADAM17 antibody precipitate. Total protein extract, supernatant (S), or precipitate (P) was incubated with fluorescent peptide substrate. For reference, the value of 0-h COC after incubation with fluorescent substrate at 37 C for 105 min was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent culture experiments. D, The temporal changes of TACE/ADAM17 protease activity in cumulus cells of COCs cultured with FSH and LH up to 40 h. TACE/ADAM17 activity in cumulus cells of COCs cultured with FSH and LH for 0, 5, 10, 20, 30, or 40 h was analyzed. For reference, the value of 0-h COC after incubation with fluorescent substrate at 37 C for 105 min was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent culture experiments. *, Significant differences observed as compared with that in COCs before culture (0 h) (P < 0.05). FL, COCs cultured with FSH and LH for 40 h; free, COCs cultured without hormones for 40 h.

 
To determine TACE/ADAM17 protease activity in cumulus cells, the fluorogenic peptide III was used as a specific synthetic substrate. In this assay, the fluorescence emitted from the cleaved substrate was measured and defined as TACE/ADAM17 protease activity. In the first experiments, the optimal reaction time was determined using 20 µg protein extracts prepared from cumulus cells of COCs before and at selected time intervals after culture with FSH and LH. Protein samples were incubated at 37 C up to 120 min with the fluorescence measured every 15 min (Fig. 2BGo). In cumulus cells of COCs before culture, a low level of fluorescence was maintained during incubation time; however, the intensity was dramatically increased in the protein samples from cumulus cells of COCs cultured with FSH and LH for 40 h. Because the maximal level of activity was reached at 105 min, this incubation period was used for subsequent analyses. When TACE/ADAM17 was precipitated with antihuman ADAM17 antibody, a high level of activity was detected in the precipitate, whereas the level in the supernatant was reduced to the 0-h control level (Fig. 2CGo). These results indicated that the cleavage of the synthetic peptide was induced via a TACE/ADAM17-dependent manner. Because ADAM10 can also cleave this substrate and is expressed in porcine cumulus cells (data not shown) in a constitutive manner, the 0-h (basal) level of fluorescence is most likely due to the activity of this other endogenous protease.

Using this assay system, the temporal changes of TACE/ADAM17 activity in cumulus cells of cultured COCs was analyzed (Fig. 2DGo). TACE/ADAM17 activity was significantly increased at 5 h of culture with FSH and LH as compared with that in COCs before culture (0 h). The high activity was maintained up to 40 h, a pattern similar to that observed for protein levels. TACE/ADAM17 activity in COCs cultured for 40 h in the absence of hormones remained low (Fig. 2DGo).

Effects of TACE/ADAM17 inhibitor TAPI-2 on cumulus cell function
In previous studies, we have shown that the rapid FSH-mediated phosphorylation of p38 MAPK is independent of AREG. In contrast, FSH-mediated phosphorylation of ERK1/2 is regulated in part by FSH induction and activation of AREG (10) and activation of sarcoma oncogene (Src) tyrosine kinase and SRC-related family tyrosine kinases (SFKs) (26). In porcine COCs, the expression of Areg and Ereg mRNAs was induced by FSH and LH. The gonadotropins also significantly increased both the expression levels and activity of TACE/ADAM17. To determine whether TACE/ADAM17 might modulate the activity of AREG and EREG in porcine COCs via its ability to cause EGF-like factor shedding, the effects of the TACE/ADAM17-selective inhibitor TAPI-2 or the broad metalloprotease inhibitor galardin on ERK1/2 phosphorylation in cumulus cells of COCs were analyzed. As shown in Fig. 3AGo, phospho-ERK1/2 was detected in cumulus cells of COCs cultured with FSH and LH for 10 h but not at 0 h. The intensity of the phospho-ERK signal decreased in COCs cultured with TAPI-2 or galardin, whereas the addition of EGF to the inhibitor-containing medium reversed the negative effect of the TACE/ADAM17 inhibitors. Additionally, the FSH+LH-induced phosphorylation of ERK1/2 in cumulus cells of cultured COCs was suppressed by either the EGFR tyrosine kinase inhibitor (AG1478) or the MEK1 inhibitor (PD98059) (Fig. 3BGo). Thus, the release of EGF-like factors from the surface of cumulus cells by TACE/ADAM17 is required for the activation of EGFR signaling pathway.


Figure 3
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  		FIG. 3. FSH+LH-induced phosphorylation of ERK1/2 in cumulus cells of porcine COCs was dependent on EGF-like factor release and EGFR activation. A, Effect of TAPI-2 or galardin (Gal) on the phosphorylation of ERK1/2 in cumulus cells of COCs cultured with FSH and LH for 10 h. Results in each panel are representative of two separate experiments. C (control), COCs cultured with FSH and LH for 10 h; TAPI, COCs cultured with FSH, LH, and TAPI-2 for 10 h; Gal, COCs cultured with FSH, LH, and galardin for 10 h; +EGF, EGF added to FSH+LH and each inhibitor-containing medium. B, Phosphorylation of ERK1/2 in cumulus cells of COCs was suppressed by EGFR tyrosine kinase inhibitor (AG1478) or the MEK1 inhibitor (PD98059), respectively. free, COCs cultured without any hormones for 10 h. Results in each panel are representative of two separate experiments.

 
It has been shown that EGFR inhibitor AG1478 suppresses the expression of Ptgs2, Has2, and Tnfaip6 mRNAs in cumulus cells of mice (9, 10). Therefore, we investigated whether the treatment with TAPI-2 affected the expression of these specific genes via the release of EGF-like factors. COCs were cultured with or without TAPI-2 or galardin in the presence of FSH and LH for 20 or 30 h. Additional COCs were cultured with EGF added to the inhibitor-containing medium. Levels of Ptgs2, Has2, and Tnfaip6 mRNAs increased in cumulus cells of COCs cultured for 20 and 30 h with hormones compared with COCs cultured in medium alone (Fig. 4Go, A, B, D, E, G, and H). The induced expression of these genes was not affected by treatment with TAPI-2 or galardin at the 20-h time interval (Fig. 4Go, B, E, and H) but were significantly repressed by each inhibitor at 30 h of culture (Fig. 4Go, C, F, and I). The addition of EGF to media containing TAPI-2 or galardin reversed the inhibitor effects of these compounds (Fig. 4Go, C, F, and I).


Figure 4
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  		FIG. 4. Effect of TAPI-2 or galardin on the expression of Ptgs2, Has2, or Tnfaip6 mRNAs in cumulus cells of COCs cultured with FSH and LH for 20 or 30 h. A, D, and G, The temporal changes of Ptgs2 (A), Has2 (D), and Tnfaip6 (G) mRNA levels in cumulus cells of COCs cultured with FSH and LH. For reference, the 0-h COC value was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent culture experiments. Significant differences were observed as compared with that in COCs before culture (0 h): *, P < 0.05; **, P < 0.01. B, E, and H, The levels of Ptgs2 (B), Has2 (E), and Tnfaip6 (H) mRNAs in cumulus cells of COCs cultured with FSH and LH (C, control); FSH, LH, and TAPI-2 (TAPI); or FSH, LH, and galardin (Gal) for 20 h. free, COCs cultured without hormones for 20 h. C, F, and I, The levels of Ptgs2 (C) Has2 (F), and Tnfaip6 (I) mRNAs in cumulus cells of COCs cultured with FSH and LH (C, control); FSH, LH, and TAPI-2 (TAPI); or FSH, LH, and galardin (Gal) for 30 h. Significant differences are indicated: *, P < 0.05; **, P < 0.01.

 
In the next experiment, we examined temporal effects of TAPI-2 and galardin on cumulus expansion of porcine COCs. The diameter of COCs cultured in the presence of FSH and LH with or without TAPI-2 for 20 h was similar to that of COCs before culture or cultured without agonists for 20 h (Fig. 5AGo). However, after 40 h of culture, cumulus expansion was increased notably by FSH and LH and was suppressed in COCs cultured with inhibitors (Fig. 5BGo). The reduced expansion of COCs was completely reversed by addition of EGF to the inhibitor-containing medium (Fig. 5BGo). In addition, when COCs were cultured with FSH and LH for 40 h, meiotic maturation of the oocytes had reached metaphase II, whereas fewer oocytes had matured in COCs cultured in the presence of inhibitors (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effect of TAPI-2 and galardin on diameter of COCs cultured with FSH and LH for 20 or 40 h. A, COCs were cultured for 20 h with FSH and LH (C, control); FSH, LH, and TAPI-2 (TAPI); or FSH, LH, and galardin (Gal). Values are mean ± SEM of three independent culture experiments. B, COCs were cultured for 40 h with FSH and LH (C, control); FSH, LH, and TAPI-2 (TAPI); or FSH, LH, and galardin (Gal). Significant differences are indicated: *, P < 0.05; **, P < 0.01. C, The number of oocytes reaching meiotic metaphase II (MII) was analyzed after 40 h culture with FSH and LH. *, Meiotic progression to the MII stage was significantly suppressed by either TAPI-2 or galardin (P < 0.05).

 
When COCs were cultured with FSH and LH in the presence of either AG1478 or PD98059, the expression of Ptgs2, Has2, and Tnfaip6 mRNAs in cumulus cells was significantly suppressed at 20 h (Fig. 6AGo) and cumulus expansion was reduced at 40 h (Fig. 6BGo), indicating that these events occur in a sequential and defined temporal pattern in response to activation of the EGFR pathway


Figure 6
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  		FIG. 6. Effects of AG1478 or PD98059 on cumulus cell functions during in vitro culture of COCs with FSH and LH. A, The levels of Ptgs2, Has2, and Tnfaip6 mRNAs in cumulus cells of COCs cultured with AG1478 (AG) or PD98059 (PD) in the presence of FSH and LH for 20 h. The free COC value was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent culture experiments. *, Expression of each gene was significantly increased by 20 h of culture with FSH and LH (P < 0.01) as compared with that observed in COCs cultured without any agonists (free); # and **, mRNA levels were significantly suppressed by AG1478 (#) or PD98059 (**) as compared with those in COCs cultured without any inhibitor (P < 0.05). B, The cumulus expansion of porcine COCs was suppressed by AG1478 or PD98059. COCs were cultured with FSH, LH, and AG1478 (AG) or with FSH, LH, and PD98059 (PD) for 40 h.

 
TAPI-2 impacts gonadotropin-mediated phosphorylation of ERK1/2 in porcine granulosa cells
The effect of TAPI-2 on the gonadotropin-induced phosphorylation of ERK1/2 in granulosa cells was also determined by using a porcine granulosa cell culture system. Because porcine granulosa cells recovered from small antral follicles do not express LH receptor (data not shown), the cells from these follicles were cultured with FSH alone or forskolin+PMA to mimic FSH/LH stimulation. Tace/Adam17 mRNA was induced significantly within 1 h and maintained for 4 h in granulosa cells cultured with either FSH alone or forskolin+PMA (Fig. 7AGo). In these same cells, Areg mRNA increased significantly with forskolin+PMA and FSH at 1 and 4 h, respectively, compared with cells without agonists (Fig. 7BGo). When the porcine granulosa cells were cultured with FSH, ERK1/2 phosphorylation also increased dramatically at 4 h of culture but not before (Fig. 4CGo), and this response to FSH was reduced by the addition of TAPI-2 (Fig. 7CGo). In contrast, forskolin+PMA rapidly induced ERK1/2 phosphorylation within 1 h, but the addition of TAPI-2 caused no effect (Fig. 7CGo).


Figure 7
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  		FIG. 7. The effects of TAPI-2 on the function of EGF-like factor in porcine granulosa cells. A and B, Kinetic changes of Tace/Adam17 (A) and Areg (B) mRNA levels in porcine granulosa cells cultured with FSH or forskolin and PMA (For+PMA). For reference, the 0-h granulosa cells value was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent culture experiments. Significantly higher levels of Tace/Adam17 and Areg mRNA were observed in granulosa cells cultured with FSH or For+PMA as compared with that in granulosa cells cultured without any agonists: *, P < 0.05; **, P < 0.01. C, The phosphorylation of ERK1/2 in porcine granulosa cells cultured with TAPI-2 in the presence of FSH or For+PMA. Results in each panel are representative of two separate experiments.

 
TACE/ADAM17 and EGF-like factors impact ERK1/2 phosphorylation in rat granulosa cells
To determine whether TACE/ADAM17 modulation of EGF-like factor shedding and activation of the EGFR pathway occurred in a rodent model, in vitro culture of rat granulosa cells was done. Because the rat granulosa cells isolated from eCG-primed ovaries respond to LH, the cells were cultured with either LH or forskolin+PMA. LH alone as well as forksolin+PMA induced expression of Tace/Adam17 and Areg mRNAs (Fig. 8Go, A and B) and increased phosphorylation of ERK1/2 (Fig. 8CGo) within 1 h; maximal levels were observed at 2 and 4 h of culture. The addition of TAPI-2 to the LH-containing medium did not alter the phosphorylation of ERK1/2 at 1 h but did reduce ERK1/2 phosphorylation in cells cultured with LH and TAPI-2 for 2 and 4 h (Fig. 8CGo). In contrast, forskolin+PMA-mediated ERK1/2 phosphorylation was not suppressed by TAPI-2 at any time interval examined (Fig. 8CGo), a response similar to that in porcine granulosa cells (Fig. 7CGo).


Figure 8
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  		FIG. 8. The role of TACE/ADAM17 and EGF-like factors in the phosphorylation of ERK1/2 in rat granulosa cells. A and B, The levels of Tace/Adam17 (A) or Areg (B) mRNAs in rat granulosa cells. Rat granulosa cells were cultured with 500 ng/ml LH or forskolin and PMA (For+PMA). For reference, the 0-h granulosa cells value was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent culture experiments. Culture with LH or For+PMA significantly increased the levels of Tace/Adam17 and Areg mRNAs as compared with those in granulosa cells cultured without any agonists: *, P < 0.05; **, P < 0.01. C, Phosphorylation of ERK1/2 in rat granulosa cells cultured with TAPI-2 in the presence of LH or For+PMA. Results in each panel are representative of two separate experiments. D, TACE/ADAM17 protein levels were suppressed by Tace/Adam17 siRNA. Granulosa cells were transfected with Tace/Adam17 siRNA (siRNA) or scrambled siRNA duplex as a negative control (NC) for 5 h and then treated with LH for 4 h. TACE/ADAM17 protein levels were analyzed by Western blotting. ß-Actin was used as an internal control. Results in each panel are representative of two separate experiments. E, The effects of Tace/Adam17 siRNA on the phosphorylation of p38 MAPK and ERK1/2 were analyzed by Western blotting. Granulosa cells transfected with 10 nM Tace/Adam17 siRNA (siRNA) or scrambled siRNA duplex as a negative control (NC) for 5 h and treated with LH for up to 4 h. F, Expression of Tnfaip6 gene expression in granulosa cells was suppressed by Tace/Adam17 siRNA. Granulosa cells were transfected with Tace/Adam17 siRNA (siRNA) or scrambled siRNA duplex as a negative control (NC) for 5 h and treated with LH for 4 h. For reference, the 0-h granulosa cells value was set as 1 and the data presented as fold increase. Values are mean ± SEM of three independent experiments.

 
To determine the selectivity of TAPI-2 function on TACE/ADAM17 protease activity, rat granulosa cells were transfected with Tace/Adam17 siRNA. The levels of TACE/ADAM 17 protein were suppressed by the transfection with Tace/Adam17 siRNA but not by the control siRNA (negative control) (Fig. 8DGo). LH-mediated phosphorylation of ERK1/2 was not affected by the Tace/Adam17 siRNA at 1 h but was reduced at 2 and 4 h of culture (Fig. 8EGo) in a manner similar to that observed in cells cultured with LH and TAPI-2 (Fig. 8CGo). Tace/Adam17 siRNA transfection did not affect the phosphorylation of p38 MAPK or the total amount of ERK1/2 in rat granulosa cells (Fig. 8EGo). Expression of the EGFR target gene Tnfaip6 was significantly increased in the granulosa cells cultured with LH at 4 h but was significantly reduced when granulosa cells were also transfected with Tace/Adam17 siRNA (Fig. 8FGo). The addition of EGF increased the level of Tnfaip6 mRNA in granulosa cells transfected with Tace/Adam17 siRNA (Fig. 8FGo), indicating the possibility that activation/shedding of the EGFR ligands is regulated by TACE/ADAM17 activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The process of COC expansion is one hallmark of ovulation that is triggered in preovulatory follicles by the LH surge. COCs isolated from preovulatory follicles can also be induced to expand if cultured in defined medium containing FSH, FSH and LH, or EGF (27, 28). The first critical link between the gonadotropins and growth factor activity was documented recently by studies of Park et al. (9) who showed that hCG-induced cumulus expansion was dramatically suppressed by an EGFR tyrosine kinase inhibitor. Moreover, LH has been shown to induce the expression of the EGF-like factors Areg, Btc, and Ereg in mural granulosa cells during ovulation process (9, 29, 30), leading to the concept that an EGFR network is an essential mediator of LH action in granulosa cells during ovulation. This concept was expanded by the observation that cumulus cells as well as granulosa cells are induced to express Areg, Btc, and Ereg mRNAs and that this involves paracrine as well as autocrine regulatory loops. Using a well-defined porcine COC expansion model, the present study documents that the EGFR network is also activated and hormonally regulated in porcine COCs. Additionally the network may require the induction and the activation of TACE/ADAM17. Moreover, because the time interval for gonadotropin-mediated expansion of porcine COCs extends over a longer time interval than that of the mouse or rat, this model reveals that the responses of COCs to the EGF-like factors (ERK1/2 phosphorylation) are dependent on precise temporal changes in cumulus cell function.

The function of EGF-like factors is controlled at many levels. They are produced as inactive precursors spanning the cell membrane, and activation of these factors requires proteolytic shedding of the ligand domain to activate the EGFR on target cells (13, 14, 31). The broad metalloprotease inhibitor galardin suppresses LH-induced cumulus expansion and meiotic oocyte maturation of rat follicle-enclosed COC or porcine COCs (21, 22), suggesting that induction and/or activation of a metalloprotease might be involved in the processing of EGF-like factors during the ovulation process. Herein, we document that levels of TACE/ADAM17 mRNA and protein are induced rapidly (within 5 h) in cumulus cells of porcine COCs cultured with FSH and LH. Importantly, TACE/ADAM17 enzyme activity was increased by FSH and LH in a similar temporal pattern and maintained during the 40 h of culture. When endogenous protease activity was reduced by the treatment with TACE/ADAM17 inhibitor TAPI-2, multiple downstream targets of EGFR activation were suppressed, including Ptgs2, Has2, and Tnfaip6 mRNA levels in cumulus cells, expansion of COCs, meiotic maturation of oocytes to the metaphase II, and phosphorylation of ERK1/2. Moreover, the negative effects of TAPI-2 on these downstream targets of EGFR activation were each reversed at selected time intervals by the simultaneous addition of EGF. These results support those of Sahin et al. (12) who showed that TACE/ADAM17 can act as a sheddase of AREG and EREG in mouse embryonic cells.

The specific roles of activated/released AREG and EREG appear to depend on precise temporal and hormone-induced changes in cumulus cell function. For example, although Areg, Ereg, and Tace/Adam17 mRNA were induced and TACE/ADAM17 enzyme activity was increased in cumulus cells of porcine COCs within 5 h of culture with FSH and LH, the inhibitory effects of TAPI-2 on COC expansion and expression of Ptgs2, Has2, and Tnfaip6 mRNA in cumulus cells were not observed until 30 h of culture. The reasons for the delay in TAPI-2 sensitivity (AREG and EREG activation) are not entirely clear. However, it is possible that the cumulus cells need to acquire sufficient levels of EGFR or a specific downstream component of the EGFR pathway to mediate the response. Alternatively, cumulus cells may need to acquire specific responses to FSH and LH. Prochazka et al. (32) reported that EGF alone does not promote expansion of porcine COCs recovered from small antral follicles, whereas FSH strongly induced an EGF response within 3 h. These results indicate that the function of the EGFR pathway in cumulus cells of porcine COCs is FSH dependent. We also show that EGF alone does not induce cumulus expansion (data not shown), whereas at 10 h culture, TAPI-2 repressed the FSH-induced phosphorylation of ERK1/2, and the addition of EGF reversed the negative effects of the inhibitor. Thus, cumulus cells of porcine COCs collected from small antral follicles 3–5 mm in diameter acquire the competence to respond to EGF ligands within at least 10 h culture with FSH. Because porcine COCs do not exhibit expansion at 10 or 20 h of culture, additional changes in cumulus cell function occur in response to FSH and LH from 20–40 h. These changes include the appearance of the LH receptor (LHCGR) on cumulus cells that reaches maximal levels at 20 h (33). The addition of LH to FSH-containing medium at this time increases cAMP levels in the cumulus cells (34). After an additional 20-h culture period with LH, the level of Lhcgr mRNA in cumulus cells is markedly reduced (35), whereas high levels of cAMP are maintained in COCs until 40 h (36). The maintenance of cAMP at 40 h is most likely due to the increased production and action of PGE2 (37, 38). Because the induction of Ptgs2 mRNA in cumulus cells of mouse COCs is induced by the stimulation of EGF-like factors (10), it is likely that EGF-like factors induce expression of this gene in porcine cumulus cells after 30 h. From these results, we propose that during the later (20–40 h) culture period, the TACE/ADAM17-mediated release of AREG and EREG leads to the production of PGE2 that maintains cAMP production required for COC expansion during this time period. It is also likely that other factors controlling matrix formation and stabilization are also induced during the later time interval.

Specific temporal responses to FSH, LH, and TAPI-2 were also observed in cultured porcine and rat granulosa cells. Interestingly, LH-mediated phosphorylation of ERK1/2 in rat granulosa cells was not affected after 1 h exposure to TAPI-2 or after transfection with Tace/Adam17 siRNA, whereas the level of ERK1/2 phosphorylation was reduced after 2 or 4 h culture with either antagonist. In contrast, when rat or porcine granulosa cells were cultured with forskolin and PMA, no effect of the TAPI-2 was observed on ERK1/2 phosphorylation at any time interval, indicating that the mechanisms by which forskolin+PMA activate ERK1/2 differ from those of LH and do not require ligand activation of the EGFR. However, because the EGFR tyrosine kinase inhibitor AG1478 significantly suppressed ERK1/2 phosphorylation in rat granulosa cells cultured with forskolin+PMA (10), ligand-independent EGFR activation appears to be involved. One explanation for the delayed effects of TAPI-2 may be that the effects of LH (39) as well as FSH (25, 40) are mediated by an intermediate pathway. Because FSH potently and rapidly activates Rous sarcoma oncogene (26) and the EGFR by a mechanism that is blocked by a SFK inhibitor, a member of the SRC family or a SRC-related kinase may be involved. If similar mechanisms are operative in porcine granulosa cells and cumulus cells, the FSH+LH-stimulated phosphorylation of ERK1/2 may be mediated at early time intervals by FSH+LH activation of SFKs, whereas the maintenance of ERK1/2 phosphorylation in these cells may be dependent the extracellular EGFR ligands that are induced, proteolytically modified, and secreted by the cell itself. However, additional studies will be needed to determine whether a SFK or SRC-related tyrosine kinase or other kinase mediates gonadotropin activation of ERK1/2 in immature porcine granulosa cells.

In summary, the present study documents that the protease/sheddase TACE/ADAM17 is induced and activated in cumulus cells of porcine COCs concomitantly with the expression of the EGF-like factors Areg and Ereg. TACE activity was required for the activation of EGFR downstream signaling and phosphorylation of ERK1/2, which might be induced by the shedding of EGF-like factors. However, the effects of the activated EGFR ligands are dependent on the stage of cumulus cell differentiation and the acquisition of mechanisms permitting matrix formation, expansion, and meiotic maturation, including but not restricted to expression of Ptgs2, Has2, and Tnfaip6 mRNA. Thus, the gonadotropins and TACE/ADAM17-mediated EGF-like factor activation work coordinately to achieve full COC expansion and meiotic maturation.


   Acknowledgments
 
Porcine FSH and LH were kindly provided by Dr. A. F. Parlow, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Disease. We thank Mr. T. Okazaki, N. Noma, and K. Hayakawa for technical assistance and the staff of the Meat Inspection Office in Hiroshima City for supplying the porcine ovaries.


   Footnotes
 
This work was supported in part by Grant-in-Aid for Scientific Research (M.S., No. 18688016; M.N., No. 18658109) and Research Fellowship for Young Scientist (Y.Y., No. 08254) from the Japan Society for the Promotion of Science, and NIH-HD-16229 and HD-07495 (Project III, Specialized Cooperative Centers Program in Reproduction and Infertility Research (J.S.R.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 27, 2007.

Abbreviations: ADAM, A disintegrin and metalloprotease; AREG, amphiregulin; COC, cumulus-oocyte complex; eCG, equine chorionic gonadotropin; EGF, epidermal growth factor; EGFR, EGF receptor; EREG, epiregulin; hCG, human chorionic gonadotropin; PGE2, prostaglandin E2; PMA, phorbol 12-myristate 13-acetate; SFK, SRC-related family tyrosine kinase; siRNA, small interfering RNA; SRC, steroid receptor coactivator; TACE, TNF{alpha}-converting enzyme; TAPI-2, TNF{alpha}-processing inhibitor-2; TNF{alpha}IP6, TNF{alpha}-induced protein 6; T-PBS, 0.1% Tween 20/PBS.

Received February 9, 2007.

Accepted for publication September 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals