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Down-Regulated Expression of A Disintegrin and Metalloproteinase with Thrombospondin-Like Repeats-1 by Progesterone Receptor Antagonist Is Associated with Impaired Expansion of Porcine Cumulus-Oocyte Complexes

Department of Applied Animal Science (M.S., M.N., Y.Y., J.I.), Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan; Infertility Center (T.M.), Daigo-Watanabe Clinic, Kyoto 601-1375, 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, Higashi-Hiroshima, Hiroshima 739-8528, Japan. E-mail: mashimad{at}hiroshima-u.ac.jp.

	Abstract

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ADAMTS-1, a member of the A disintegrin and metalloproteinase family of proteases, is expressed in rodent follicles via progesterone receptor (PR)-dependent pathways. However, the functional relationship between ADAMTS-1 expression and PR has not been studied extensively in other species. In the present study, we investigated the time-dependent changes in ADAMTS-1 expression in cumulus cells of porcine cumulus-oocyte complexes (COCs), and the roles of ADAMTS-1 in cumulus expansion during in vitro maturation of oocytes. ADAMTS-1 message was not detected in cumulus cells at the time of collection from the follicles. In response to gonadotropins, ADAMTS-1 mRNA was dramatically up-regulated and reached a maximum at 20 h. The level of mature ADAMTS-1 protein increased in a time-dependent manner with a maximum level at 40 h. The induction of ADAMTS-1 mRNA and protein was significantly decreased by the addition of PR antagonist RU486 to the cultures. However, RU486 did not affect the expression of ADAMTS-4 or factors that had been reported to be required for COC expansion (TSG-6, versican, HA synthase-2). COCs cultured with FSH and LH for 40 h exhibited prominent cumulus expansion. The expansion was reduced significantly by the addition of either RU486 or Galardin, a broad-spectrum matrix metalloproteinase inhibitor. These results suggest that the expression and induction of ADAMTS-1 through receptor-mediated action of progesterone in cumulus cells is one of the essential requirements for gonadotropin-regulated cumulus expansion of porcine COCs.

	Introduction

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THE SURGE OF LH induces marked functional (endocrine, biochemical, and molecular) changes in the preovulatory follicle. Estrogen concentrations decline in follicular fluid as a consequence of the reduced expression of aromatase and 17-hydroxysteroid dehydrogenase in granulosa and theca cells, respectively, whereas progesterone concentrations rise in association with induction of P450 side-chain cleavage enzyme, CYP11A1, in granulosa cells (1, 2). The LH surge also induces ovulation, a process that is inhibited in rats by treatment with either antiprogesterone antiserum (3) or epostane (4), a compound that blocks the synthesis of progesterone, suggesting that the LH-induced increase of progesterone is essential for ovulation. Many of the biological activities of progesterone are mediated by an intracellular receptor, a hormonally regulated DNA-binding protein that belongs to a superfamily of ligand-activated transcription factors (5, 6). Progesterone receptor (PR) mRNA is induced in granulosa cells of rat preovulatory follicles by LH (7, 8). Female mice null for PR (PRKO) fail to ovulate, even in response to exogenous hormones (9). Thus, progesterone- and PR-dependent pathways appear to play important roles in the functional changes that occur in follicular cells during ovulation.

A disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS-1) is a member of the ADAMTS family of metalloproteinases and has been shown to degrade members of the lectican family of proteoglycans (10, 11, 12). ADAMTS-1 has been shown to be induced by LH in granulosa cells via progesterone- and PR-dependent pathways (13, 14, 15). Furthermore, mice null for ADAMTS-1 exhibit impaired fertility (16, 17), suggesting that ADAMTS-1 impacts some aspect of the ovulation process. Recently Russell et al. (18) reported that LH induces in ovulating follicles the expression of versican, one known preferred substrate of ADAMTS-1 (19). Cleavage of versican in ovulated cumulus-oocyte complexes (COCs) has been documented (15), suggesting that ADAMTS-1 or related family members are present and active in the ovulated complexes. Before ovulation, cumulus cells surrounding oocytes synthesize hyaluronan (HA), a glycosoaminoglycan polymer, along with other factors to make a gelatinous matrix, a process that is termed cumulus expansion (20, 21, 22). Versican possesses an N-terminal link-module HA binding domain (23). TNF-stimulated gene 6 (TSG-6) also binds HA and the heavy chains of inter--trypsin inhibitor (IaI) (24, 25, 26). Thus, the expansion of COCs during ovulation involves the organization of the HA-rich matrix that is comprised of HA, IaI heavy chains, TSG-6, and versican. Collectively, these observations suggest that PR and ADAMTS-1 may impact ovulation via the formation or stabilization of the COC complex. However, this hypothesis has not been confirmed.

During in vitro meiotic maturation of COCs, it has been shown in human (27), rat (28), cattle (29), and pig (30, 31, 32) that progesterone is produced by cumulus cells and that the level of progesterone is increased by stimulation with LH and FSH. In our previous studies (33, 34), we showed that the high concentrations of progesterone secreted by porcine COCs accelerated meiotic resumption of oocytes and improved the rate of early embryonic development to the blastocyst stage after in vitro fertilization. We also reported in porcine COCs that the progesterone bound to newly synthesized PR in cumulus cells was associated with reduced proliferative activity of cumulus cells and closed the gap junctional communication within cumulus cells (32, 34). Our previous reports suggest that activation of PR induced the differentiation of cumulus cells. However, little is known about the specific roles of progesterone and PR in either the induction of ADAMTS-1 or cumulus expansion during in vitro maturation of COCs.

In this study, we examined the time-dependent changes in ADAMTS-1 gene expression and its regulation by PR in cumulus cells of porcine COCs. To investigate the roles of ADAMTS-1 in cumulus expansion, COCs were cultured with PR antagonist, RU486, or a broad-spectrum matrix metalloproteinase inhibitor, Galardin. COC expansion and the amount of mRNA and protein were analyzed. In addition, the expression of other genes associated with cumulus expansion [TSG-6, versican, and HA synthase-2 (HAS-2)] was analyzed in control and RU486-treated COCs.

	Materials and Methods

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In vitro culture of porcine COCs
Isolation of porcine COCs was described previously (35). Briefly, porcine ovaries were collected from 5- to 7-month-old prepubertal gilts at a local slaughterhouse. Oocytes were collected from the surfaces of intact healthy antral follicles measuring from 3 to 5 mm in diameter. Oocytes having evenly granulated cytoplasm with at least four layers of unexpanded cumulus cells were selected and washed three times with maturation medium. The COCs were cultured (20/well) for up to 40 h in 300 µl of the maturation medium supplemented with both 20 ng/ml highly purified porcine FSH (National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA) and 500 ng/ml porcine LH (NIDDK) at 39 C in a humidified incubator (95% air, 5% CO₂). The maturation medium was modified NCSU37 (36) supplemented with 10% (vol/vol) fetal calf serum (Life Technologies, Inc., Grand Island, NY), 7 mM Taurine (Sigma, St. Louis, MO) and 4 mM hypoxanthine (Sigma). At selected time intervals, COCs were collected for RNA and protein isolation.

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

In the case of treatment with PR antagonist, RU486 (Sigma), or a broad-spectrum matrix metalloproteinase inhibitor, Galardin (GM 6001, Sigma), COCs were cultured for 0, 20, or 30 h and then further cultured with additional 25 µM RU486 or 1 or 10 µM Galardin for 40, 20, or 10 h, respectively. Some COCs were cultured with 10 µM flutamide (Sigma), a specific androgen receptor (AR) blocking agent, or 10 µM IC182,780 (Tocris, Langford, Bristol, UK), an estrogen receptor (ER) antagonist. The RU486 concentration used herein was the same as that used to test PR function in cumulus cells during meiotic resumption of porcine oocytes (34). Ito et al. (37) reported that 10 µM Galardin significantly decreased the activity of ADAM family in human placenta. The concentration of flutamide (10 µM) suppressed AR-induced expression of prolactin but not the function of PR or ER in human endometrial stromal cells (38). The ER antagonist, IC182,780, specifically inhibited the estrogen-induced proliferative activity of human smooth muscle cells at 10 µM (39). RU486 and flutamide were dissolved in ethanol at 25 or 10 mM and stored at 4 C. Galardin was dissolved in dimethylsulfoxide (DMSO) (Sigma) at 10 mM, and stored at –20 C. IC182,780 was dissolved in ethanol at 10 mM and stored at –20 C. The final concentration of each compound (as described above) was obtained by dilution (1:1000) with the maturation medium. Inhibitor-free medium supplemented only with either 0.1% (vol/vol) ethanol or 0.1% (vol/vol) DMSO to the maturation medium served as a control. The final concentration of vehicles (ethanol or DMSO) were less than 0.1% (vol/vol), which did not affect the function of cumulus cells during meiotic resumption of porcine oocytes (40).

Western blot analysis
COCs were treated with 0.01% (wt/vol) hyaluronidase (Sigma)/PBS for 1 min at 39 C and then mechanically denuded by pipetting with Flame-Draw pipette tips that had inner diameters slightly larger than the oocyte diameter. Intact COCs or cumulus cells were lysed in Laemmli sample buffer and protein extracts stored at –80 C until use. After denaturing by boiling for 5 min, 10 µl of each protein sample was separated by SDS-PAGE on 7.5% polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and then transferred onto polyvinyl difluoride membrane (Amersham Biosciences, Uppsala, Sweden). The membrane was blocked with 5% (wt/vol) nonfat dry milk (Amersham Biosciences) in 0.1% (vol/vol) Tween 20 (Sigma)/PBS (T-PBS). Primary antibodies were added in 5% (wt/vol) nonfat dry milk in T-PBS, and incubated overnight at 4 C. Rabbit antihuman PR polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which allowed recognition of both PR type A (PR-A) and PR type B (PR-B), was used at 1:500 dilution. A rabbit polyclonal antibody against the anti-C terminal of human ADAMTS-1 (Sigma) was used at a dilution of 1:2000. After four washes in T-PBS, the membranes were incubated for 1.5 h with a 1:2000 dilution of goat antirabbit IgG horseradish-peroxidase-labeled antibody (Cell Signaling Technology, Inc., Beverly, MA) in 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 Plus Western blotting detection system (Amersham Biosciences), according to the manufacturer’s instructions. The intensity of the objective bands was quantified by densitometric scanning using a Gel-Pro analyzer (Media Cybernetics, Silver Spring, MD).

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

RT-PCR
RT-PCR was performed according to a coupled one-step procedure using the Access RT-PCR System (Promega) with some modifications (41). Briefly, 10 ng total RNA were reverse transcribed at 48 C for 45 min, denatured at 94 C for 2 min, and amplified for 28, 30, or 33 cycles of denaturation at 94 C for 30 sec, primer annealing at 58 C (PR-B, ADAMTS-4), 56 C (PR-A/B, ß-actin), or 54 C (ADAMTS-1, TSG-6, Versican, HAS-2) for 1 min, and extension at 68 C for 1 min, with a final extension step of 7 min at 68 C. The amplified products were analyzed by electrophoresis on 2% agarose gels. The intensity of the objective bands was quantified by densitometric scanning using a Gel-Pro analyzer. One primer set was directed at the sequence specific for the PR-B (i.e. within the 164 amino acids at the N terminus) and therefore detected only mRNA transcripts encoding PR-B (Table 1; see Fig. 2). The other primer set was directed to the section of the PR common to PR-A and PR-B and therefore detected total PR mRNA (Table 1; also see Fig. 2). The PR primers were based on the human sequence (GenBank accession no. NM00926). The primers used for amplification of ADAMTS-1 or ADAMTS-4 were designed from known cDNA sequence of human ADAMTS-1 (AF207664) or human ADAMTS-4 (NM005099) (Table 1). The TSG-6 primers were based on human sequence (M31165), and versican primers on porcine sequence (AF159384). The application of porcine HAS-2 has been reported by Kimura et al. (42). ß-Actin was used as a control for reaction efficiency and variations in concentrations of mRNA in the original reverse transcription reaction. The ß-actin primers were based on the mouse sequence (GenBank accession no. NM009609). The amplified cDNAs were directly sequenced according to our previous study to verify their authenticity (34).

View larger version (38K): [in this window] [in a new window]   FIG. 2. Time dependent changes in PR mRNA levels and PR protein in cumulus cells of COCs cultured with FSH and LH. A, Schematic diagram indicating positions of the oligonucleotide primers on the putative porcine PR gene. Although unknown, the genomic structure of the porcine PR is depicted in a similar fashion to the human PR gene (DDBJ, accession no. NM 000926). B, RT-PCR analysis of PR-A/B or PR-B mRNA in cumulus cells of COCs. COCs were cultured with FSH and LH for up to 40 h. At selected time intervals, COCs were collected for RNA, and either PR-A/B or PR-B mRNA was detected by RT-PCR. The intensity of the amplified bands was quantified by densitometric scanning using a Gel-Pro analyzer. The respective values of PR-A/B and PR-B were normalized according to those of ß-actin to evaluate arbitrary units of the relative abundance of the targets. Letters (a–f) do not have common designated significance within each product (P < 0.05). Values are mean ± SEM of three replicates. C, The effects of FSH and LH on the expression of PR gene in cumulus cells of COCs cultured for 10 h. free, COCs were cultured for 10 h without FSH and LH; FSH+LH, COCs were cultured for 10 h with FSH and LH. D, Western blot analysis of PR in cumulus cells of COCs. COCs, COCs were cultured with FSH and LH for up to 40 h. At selected time intervals, COCs were collected for protein, and then PR was detected by a rabbit antihuman PR polyclonal antibody. G, Porcine granulosa cells, which were recovered from 3- to 5-mm-diameter follicles, were cultured with 500 ng/ml LH for 10 h. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

	Results

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Time-dependent changes in PR expression in cumulus cells of COCs
To analyze time-dependent changes in PR expression in cumulus cells of COCs, RT-PCR and Western blot analyses were used to determine the induction of PR mRNA and protein levels, respectively. Using this one-step RT-PCR procedure, the intensity for a band of ß-actin mRNA started to increase at 23 cycles and reached a plateau after 33 cycles (Fig. 1). Partial cDNAs were amplified by RT-PCR from porcine cumulus cells using primers that are sequence specific for human PR-B (i.e. within the 164 amino acids at the N terminus) and common to human PR-A and PR-B (Fig. 2A). The similarities of the partial porcine PR-B region and common region sequences were 83.3% to human PR-B and 98.1% to human PR (Shimada, M., Y. Yamashita, J. Ito, T. Okazaki, K. Kawahata, and M. Nishibori, unpublished data). The amount of total PR mRNA (PR-A/B) was increased rapidly (5 h) and reached maximum levels at 20 h and then significantly decreased by additional culture in the presence of FSH and LH for up to 40 h (Fig. 2B). Specific RT-PCR analysis of PR-B showed that the expression of message encoding this isoforms in cumulus cells was also up-regulated by FSH and LH during the first 10 h of culture, whereas the level had significantly decreased between 20 and 40 h (Fig. 2B). The levels of RT-PCR products were not increased when COCs were cultured without FSH and LH for 10 h (Fig. 2C, free). In agreement with RT-PCR analysis, Western blot analysis revealed that PR-A was not detected in COCs before culture but was observed 10 h after culture with FSH and LH (Fig. 2D). PR-A protein showed maximal abundance at 10 h, which subsequently declined in cumulus cells of COCs 40 h after culture with FSH and LH (Fig. 2D). A 120-kDa band (PR-B) was also detected in cumulus cells of COCs that were cultured at 10 h (Fig. 2D) but not at later times of culture (20 or 40 h). This pattern of PR-B abundance closely mimicked the induction of PR-B mRNA. These two isoforms of PR protein were also detected in porcine granulosa cells that were cultured for 10 h with LH (Fig. 2D).

View larger version (27K): [in this window] [in a new window]   FIG. 1. The linear range for detecting ß-actin mRNA was analyzed by increasing PCR cycle numbers. The intensity of the objective bands was quantified by densitometric scanning using a Gel-Pro analyzer.

View larger version (35K): [in this window] [in a new window]   FIG. 3. Time-dependent changes in ADAMTS-1 mRNA level in cumulus cells and protein level in COCs cultured with FSH and LH. A, Comparison of partial cDNA sequences of ADAMTS-1 primers/RT-PCR product with human ADAMTS-1 (accession no. AF207664). Conserved nucleotides are indicated by a dash. B, RT-PCR analysis of ADAMTS-1 mRNA in cumulus cells of COCs. COCs were cultured with FSH and LH for up to 40 h. At selected time intervals, COCs were collected for RNA, and ADAMTS-1 mRNA was detected by RT-PCR. The intensity of the amplified bands was quantified by densitometric scanning. The respective values of ADAMTS-1 were normalized according to those of ß-actin to evaluate arbitrary units of the relative abundance of the targets. Letters (a–d), Ratio with no common letters are significant (P < 0.05). Values are mean ± SEM of three replicates. C, The effects of FSH and LH on the expression of ADAMTS-1 gene in cumulus cells of COCs cultured for 20 h. free, COCs were cultured for 20 h without FSH and LH; FSH+LH, COCs were cultured for 20 h with FSH and LH. D, Western blot analysis of ADAMTS-1 in cumulus cells of COCs. COCs were cultured with FSH and LH for up to 40 h. At selected time intervals, COCs were collected for protein, and then ADAMTS-1 was detected by a rabbit antihuman ADAMTS-1 polyclonal antibody. Pre, The 110-kDa molecular size is ADAMTS-1 precursor; MP, 85-kDa prodomain truncated form of ADAMTS-1. The positions of molecular mass markers (in kilodaltons) are indicated on the left. E, The differences of expression of ADAMTS-1 protein between granulosa cells and cumulus cells of COCs. COCs, COCs were cultured for 10 h with FSH and LH; G, porcine granulosa cells recovered from 3- to 5-mm-diameter follicles were cultured with 500 ng/ml LH for 10 h; pre, 110-kDa molecular size is ADAMTS-1 precursor; MP, 85-kDa prodomain truncated form of ADAMTS-1. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

View larger version (37K): [in this window] [in a new window]   FIG. 3A. Continued

View larger version (31K): [in this window] [in a new window]   FIG. 4. Effects of steroid hormone receptor antagonists on the expression of ADAMTS-1 in cumulus cells of COCs. A, RT-PCR analysis of ADAMTS-1 mRNA in cumulus cells of COCs. Con (control), COCs were cultured for 20 h with FSH, LH, and 0.1% (vol/vol) ethanol; RU, COCs were cultured with FSH, LH, and 25 µM RU486 for 20 h; Flu, COCs were cultured with FSH, LH, and 10 µM flutamide for 20 h; IC, COCs were cultured with FSH, LH, and 10 µM IC182,780 for 20 h. After 20 h culture of COCs, COCs in each treatment group were collected for RNA, and ADAMTS-1 mRNA was detected by RT-PCR. The intensity of the amplified bands was quantified by densitometric scanning. The respective values of ADAMTS-1 were normalized according to those of ß-actin to evaluate arbitrary units of the relative abundance of the targets. The effect of RU486 on the level of ADAMTS-1 mRNA is significant (P < 0.01). Values are mean ± SEM of three replicates. B, The effects of RU486 on the expression of ADAMTS-1 protein in cumulus cells were analyzed by Western blot analysis. COCs were cultured with FSH and LH and/or 25 µM RU486 for 20 or 40 h. Pre, The 110-kDa molecular size is ADAMTS-1 precursor; MP, 85-kDa prodomain truncated form of ADAMTS-1. The positions of molecular mass markers (in kilodaltons) are indicated on the left. C, The effects of flutamide or IC182,780 on the expression of ADAMTS-1 protein in cumulus cells were detected by Western blot analysis. COCs were cultured for 40 h in the presence of FSH and LH supplemented with 10 µM flutamide or 10 µM IC182,780. Con (control), COCs were cultured for 40 h with FSH, LH, and 0.1% (vol/vol) ethanol; Flu, COCs were cultured with FSH, LH, and 10 µM flutamide for 40 h; IC, COCs were cultured with FSH, LH, and 10 µM IC182,780 for 40 h; Pre, 110-kDa molecular size is ADAMTS-1 precursor; MP, 85-kDa prodomain truncated form of ADAMTS-1. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

View larger version (45K): [in this window] [in a new window]   FIG. 5. Effects of RU486 on the expression of TSG-6, versican, HAS-2, or ADAMTS-4 in cumulus cells of COCs cultured with FSH and LH. A, Comparisons of partial cDNA sequences of TSG-6 primers-RT-PCR product with human TSG-6 (accession no. M31165). Conserved nucleotides are indicated by a dash. B, Comparisons of partial cDNA sequences of ADAMTS-4 primer/RT-PCR product with human ADAMTS-4 (accession no. NM005099). Conserved nucleotides are indicated by a dash. C, RT-PCR analysis of TSG-6, versican, HAS-2, or ADAMTS-4 mRNA in cumulus cells of COCs. C (control), COCs were cultured with FSH, LH, and 0.1% (vol/vol) ethanol for 20 h; RU, COCs were cultured with FSH, LH, and 25 µM RU486 for 20 h. After 20 h culture, COCs in each treatment group were collected for RNA, and TSG-6, versican, HAS-2, or ADAMTS-4 mRNA was detected by RT-PCR. The intensity of the amplified bands was quantified by densitometric scanning 1, y-axis shows that the respective values of TSG-6, versican, HAS-2, or ADAMTS-4 were normalized according to those of ß-actin to evaluate arbitrary units of the relative abundance of the targets. Values are mean ± SEM of three replicates.

View larger version (30K): [in this window] [in a new window]   FIG. 5A. Continued

View larger version (76K): [in this window] [in a new window]   FIG. 6. The effects of RU486 or Galardin on cumulus cell expansion of COCs cultured with FSH and LH for 40 h. A, After COCs were cultured with FSH and LH for 40 h in each RU486-treated group, COCs were visualized using phase-contrast microscopy and a x10 objective. Control, COCs were cultured for 40 h with FSH, LH, and 0.1% ethanol. 1, COCs at the collection from the follicles; 2, COCs were cultured with FSH and LH for 0, 20, or 30 h and then further cultured for 40, 20, or 10 h with the addition of 25 µM RU486. More than 30 COCs in each treatment group were observed. Each experiment was repeated three times. B, The diameter of expanded COCs that were cultured for 40 h with RU486 treatment. The diameter was measured with an eyepiece micrometer under phase-contrast microscopy (Olympus IMT2) and a x10 objective and was measured at that distance giving the longest line between two points. Control, COCs were cultured for 40 h with FSH, LH, and 0.1% ethanol; 1, COCs at the collection from the follicles; 2, COCs were cultured with FSH and LH for 0, 20, or 30 h and then further cultured for 40, 20, or 10 h with the addition of 25 µM RU486. *, Forty-hour culture with FSH and LH significantly induced the expansion of COCs as compared with that in COCs at the collection from the follicles (P < 0.01). The treatment with RU486 for 40 h significantly suppressed cumulus expansion of COCs as compared with control (P < 0.01). The treatment with addition of RU486 at 20 h significantly suppressed cumulus expansion of COCs as compared with control (P < 0.05). The diameter of expanded COCs was measured in more than 30 COCs in each treatment group. Each experiment was repeated three times. C, After COCs were cultured for 40 h in each Galardin-treated group, COCs were visualized using phase-contrast microscopy and a x10 objective. Control, COCs were cultured for 40 h with FSH, LH, and 0.1% DMSO; 3, COCs were cultured with FSH and LH for 0, 20, or 30 h and then further cultured for 40, 20, or 10 h with the addition of 1 or 10 µM Galardin. More than 30 COCs in each treatment group were observed. Each experiment was repeated three times. D, The diameter of expanded COCs that were cultured for 40 h with Galardin treatment. The diameter was measured with an eyepiece micrometer using phase-contrast microscopy (Olympus IMT2) and a x10 objective and was measured at that distance giving the longest line between two points. Control, COCs were cultured for 40 h with FSH, LH, and 0.1% DMSO; 1, COCs at the collection from the follicles; 3, COCs were cultured with FSH and LH for 0, 20, or 30 h and then further cultured for 40, 20, or 10 h with the addition of 1 or 10 µM Galardin. *, Forty-hour culture with FSH and LH significantly induced the expansion of COCs as compared with that in COCs at the collection from the follicles (P < 0.01). The treatment with Galardin for 40 h significantly suppressed cumulus expansion of COCs as compared with control (P < 0.01). The diameter of expanded COCs was measured in more than 30 COCs in each treatment group. Each experiment was repeated three times. E, The ADAMTS-1 protein in COCs that were cultured for 40 h with the addition of RU486 at 10, 20, and 30 h. Control, COCs were cultured for 40 h with FSH, LH, and 0.1% ethanol; 4, COCs were cultured with FSH and LH for 0, 10, 20, or 30 h and then further cultured for 40, 30, 20, or 10 h with the addition of 25 µM RU486. After 40 h culture, COCs were collected for protein, and then ADAMTS-1 was detected by a rabbit antihuman ADAMTS-1 polyclonal antibody. Pre, The 110-kDa molecular size is ADAMTS-1 precursor; MP, 85-kDa prodomain truncated form of ADAMTS-1. The positions of molecular mass markers (in kilodaltons) are indicated on the left. 5, The y-axis shows that relative amounts of mature ADAMTS-1 (85 kDa), which are determined using scanning densitometry (data are expressed as fold strength of control as defined 100%). Letters (a–d), Ratio with no common letters are significant (P < 0.05). Values are mean ± SEM of three replicates.

View larger version (20K): [in this window] [in a new window]   FIG. 6A. Continued

	Discussion

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FSH and LH increase production of progesterone (33, 45) and induce the expression of PR (46), especially PR-A (present results) in cumulus cells during in vitro maturation of porcine COCs. These data indicate that activation of ligand bound PR may be involved in gonadotropin-induced cumulus cell differentiation, such as the progesterone production and the close of gap junctional communication (32, 34). ADAMTS-1 is another target of PR action that has identified in epostane-treated rats (13) and in PRKO mice (14). Specifically, expression of ADAMTS-1 message and protein are reduced in the absence of functional PR (13, 14, 15). However, despite the spatiotemporal expression of PR and ADAMTS-1 genes in the equine (47), bovine (48), and primate (49) ovaries, the functional relationship between ADAMTS-1 expression and PR has not been studied extensively. Because PR-A null mice have decreased ovulation (50) and those that lack only the B form are not affected (51), we hypothesized and now show that expression of PR-A in porcine cumulus cells appears to be involved in hormone-mediated differentiation of these cells during COC expansion.

Specifically, the present study documents that PR-A is induced by FSH and LH in a time-dependent manner in cumulus cells of cultured porcine COCs. The appearance of PR-A is temporally and functionally associated with the induction of ADAMTS-1 and proper cumulus cell expansion. In response to gonadotropins, ADAMTS-1 mRNA in cumulus cells is induced and reaches maximal levels at 20 h, whereas the level of mature ADAMTS-1 protein increased in a time-dependent manner with maximum levels at 40 h. Furthermore, we show that the addition of the PR antagonist RU486 selectively suppresses expression of ADAMTS-1 and compromises cumulus expansion of COC. Thus, cumulus expansion appears to be regulated by a progesterone- and PR-dependent pathway in porcine COCs and that the effects of PR are highly specific and dependent in part on ADAMTS-1.

It has been reported that numerous factors expressed in cumulus cells are required for expansion of the COC matrix. For example, induction of the HAS-2 mediates the rapid local production of long HA polymers that form the obligatory backbone of the expanded COC (42, 52). TSG-6 binds to HA and interacts with serum-derived factor IaI heavy chain to stabilize matrix formation (53, 54). Fulop et al. (55) reported that COCs failed to expand in TSG-6-deficient female mice, and Ochsner et al. (26) showed that COCs failed to expand when cultured with a TSG-6-blocking antibody because of the inability of the cumulus cells to assemble their HA-rich extracellular matrix. Versican is also coexpressed with HA at many sites of inflammation, although its function in the COC matrix formation is not entirely clear (18). It has been reported that ADAMTS-4, which is functionally grouped with ADAMTS-1, is also expressed in mouse granulosa cells and cumulus cells (15) as well as in bovine preovulatory follicles (48). However, despite their apparent critical roles in COC expansion, the expression patterns of TSG-6, versican, and HAS-2 were similar in control and RU486-treated COCs. In contrast, induction of ADAMTS-1 message and protein was down-regulated by the PR antagonist but not by the AR or ER antagonist. Moreover, the inhibitory effect of RU486 was time dependent and not observed whether the antagonist was added during the last 10 h of culture. These results suggest that there is a temporal correlation between the induction of ADAMTS-1 in porcine cumulus cells, which is dependent on the progesterone-PR pathway, and cumulus expansion.

Because ADAMTS-1 is a secreted protease (12, 43) that is known to bind extracellular matrix molecules by the presence of thrombospondin type I motifs (56) and to cleave versican, one function of ADAMTS-1 in COC expansion might be related to its functional activity and/or activities. Of note ADAMTS-4 is also expressed in cumulus cells but was not regulated by RU486, suggesting that these two proteases are regulated by different factors. Furthermore, because cumulus expansion was compromised in the RU486-treated cultures despite the continued presence of ADAMTS-4, these two proteases likely control distinct functions. In addition, in PRKO mice in which ADAMTS-1 levels are low, ADAMTS-4 does not appear substitute for ADAMTS-1 (15). What might ADAMTS-1 regulate during expansion? Russell et al. (15) reported that ADAMTS-1 is secreted by ovulated mouse COCs and that the mature protein is detected on cell surfaces as well as in the intercellular matrix. Thus, ADAMTS-1, which is produced in cumulus cells, is secreted from cumulus cells and accumulated within the COC matrix.

ADAMTS-1 contains the zinc-binding catalytic domain sequence and acts as active metalloproteinase (43). That is known to cleave versican (V1 variant) in human aorta in vivo, and this cleavage releases a 70-kDa N-terminal fragment (19). In expanded COCs, a 70-kDa N-terminal V1 fragment appears to be generated by ADAMTS-1 protease-like activity (15). Because the cleaved N-terminal domain of versican binds the HA-rich matrix, it has been thought that the cleaved versican may serve to stabilize cumulus expansion of COC. When COCs were cultured with RU486, outer layers of cumulus cells became adhered to the culture dish with unexpanded cumulus cells remaining suspended in the complex. A similar phenomenon was observed when COCs were cultured with 10 µM Galardin, a metalloproteinase inhibitor, for 40 h. Recently Wu et al. (57) showed that the C-terminal domain of versican can interact with integrins, and this interaction of integrin with the Cterminal of versican activated focal adhesion kinase to promote cell adhesion. Therefore, it is possible that removing the C-terminal domain of versican by ADAMTS-1 protease-like activity is essential for either detachment of COC from mural granulosa layer in vivo or interfering of COC with adhesion to dish in vitro. In the present study, the message of versican in cumulus cells was increased by gonadotropins and the expression was not affected by RU486. However, the precise functions of versican in COC expansion remain to be determined. By analyzing the fragments of versican present in the COC matrix, the dynamics of cumulus expansion of COC and the functions of ADAMTS-1 and versican may be elucidated.

In summary, the present study documents that ADAMTS-1 mRNA in cumulus cells of COCs was dramatically upregulated in response to gonadotropins. The induction of ADAMTS-1 message and protein was down-regulated by the treatment with the PR antagonist, RU486. In the RU486-treated COCs, cumulus cells remained packed around the oocytes with little visible expansion. In contrast, the cumulus cells of COCs treated with RU486 showed patterns of HAS-2, TSG-6, versican, or ADAMTS-4 expression similar to those of COCs without RU486 treatment. These results suggest that progesterone- and PR-dependent functions in cumulus cells are essential for COC expansion in pig, possibly through the action of ADAMTS-1.

	Acknowledgments

 
Porcine FSH and LH were kindly provided by Dr. A. F. Parlow (National Hormone and Pituitary Program, NIDDK, Torrance, CA). We thank T. Okazaki and K. Kawahata for technical assistance and the staff of the Meat Inspection Office (Hiroshima City, Japan) for supplying the porcine ovaries.

	Footnotes

 
This work was supported by the following sources of funding: Grant-in-Aid for Scientific Research (14760179, 15380222, 16780195) (to M.S.), Research Fellowship for Young Scientists (08254) (to Y.Y.) from the Japan Society for the Promotion of Science, and National Institutes of Health Grant NIH-HD-07495 (to J.S.R.).

Current address for J.I.: National Institute for Physiological Sciences, Okazaki, Aichi 444-585, Japan.

Abbreviations: ADAMTS, A disintegrin and metalloproteinase with thrombospondin-like repeats; AR, androgen receptor; COC, cumulus-oocyte complex; DMSO, dimethylsulfoxide; ER, estrogen receptor; HA, hyaluronan; HAS, HA synthase; IaI, inter--trypsin inhibitor; PR, progesterone receptor; PR-A, PR type A; PR-B, PR type B; PRKO, null for PR; T-PBS, Tween 20/PBS; TSG-6, TNF-stimulated gene 6.

Received April 27, 2004.

Accepted for publication June 25, 2004.

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