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Endocrinology Vol. 144, No. 3 1008-1019
Copyright © 2003 by The Endocrine Society

ARTICLE

Decreased Expression of Tumor Necrosis Factor-{alpha}-Stimulated Gene 6 in Cumulus Cells of the Cyclooxygenase-2 and EP2 Null Mice

Scott A. Ochsner, Darryl L. Russell, Anthony J. Day, Richard M. Breyer and Joanne S. Richards

Department of Molecular and Cellular Biology (S.A.O., D.L.R., J.S.R.), Baylor College of Medicine, Houston, Texas 77030; Medical Research Council Immunochemistry Unit (A.J.D.), Department of Biochemistry, University of Oxford, OX13QU Oxford, United Kingdom; and Departments of Medicine (Division of Nephrology) and Pharmacology (R.M.B.), Vanderbilt University School of Medicine, S3223 Medical Center North, Nashville, Tennessee 37232-2372

Address all correspondence and requests for reprints to: J. S. Richards, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovulation, the release of fertilizable oocytes from mature follicles, involves tissue remodeling and increased prostaglandin (PG) signaling. Cyclooxygenase (COX)-2 is the rate-limiting enzyme during PG synthesis. Female mice null for either COX-2 or the PGE2 receptor EP2 are infertile, show decreased ovulation, and exhibit abnormal cumulus expansion. Cumulus expansion is the production of a complex extracellular matrix surrounding the cumulus-oocyte complex (COC). Matrix components consist of hyaluronan, proteoglycans, and proteins with hyaluronan binding domains. One such hyaluronan binding protein is TNF{alpha}-stimulated gene 6 (TSG-6). By various methods, we show induction of TSG-6 and hyaluronan synthase-2 mRNA in ovaries of mice treated with pregnant mare serum gonadotropin and human chorionic gonadotropin. By in situ hybridization, we show that both genes are expressed in periantral mural granulosa cells and cumulus cells of the mouse ovary. Notably, RT-PCR and in situ hybridization show that TSG-6 mRNA but not hyaluronan synthase-2 mRNA expression is selectively reduced in cumulus cells of COX-2 and EP2 null mice. Western analysis further confirms that TSG-6 protein is reduced in isolated COCs but remains covalently associated with inter{alpha}-trypsin inhibitor in COX-2 null mice. These observations identify TSG-6 as a target of PG action and show that its production in ovulatory follicles is associated with proper formation of the cumulus-derived extracellular matrix.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CUMULUS-OOCYTE COMPLEX (COC) expansion is the formation of a highly complex extracellular matrix by cumulus cells and mural granulosa cells surrounding the antrum. This process is initiated in preovulatory follicles by the LH surge. The main structural component of this matrix is the glycosaminoglycan hyaluronan (HA) (1). HA, a polymer of glucuronic acid and N-acetylglucosamine, is extremely long and can exceed 5000 kDa in size (2). The enzyme responsible for HA production by cumulus cells is hyaluronan synthase-2 (HAS-2). HAS-2 cDNA has been isolated from COCs induced to expand in vivo (3). Chemical inhibition of glucosamine synthesis, the building block of HA, blocks cumulus expansion and ovulation showing HA to be a critical component of the cumulus-derived matrix (4).

Another critical component of this matrix is the serum-derived factor inter-{alpha}-trypsin inhibitor (I{alpha}I), which by covalent cross-linking of its heavy chains to HA functions to stabilize the cumulus cell matrix (5). When assembly of I{alpha}I (a compound of two heavy chains and the light chain, bikunin) is prevented by targeted disruption of the bikunin gene, proper delivery of the heavy chain component is impaired, cross-linking does not occur appropriately, and the HA matrix is not formed resulting in a block of ovulation (6, 7).

The other components of the cumulus-derived matrix are proteoglycans and HA binding proteins (8). The importance of these factors is less well characterized. An example is the secreted HA binding protein TNF-{alpha}-stimulated gene 6 (TSG-6) (9) initially characterized as a TNF-{alpha} and IL-1 inducible gene in fibroblasts (10). TSG-6 has also been isolated as a gene expressed in expanding mouse COCs and as a gonadotropin induced gene in the rat ovary where it was shown to be rapidly induced in preovulatory follicles in response to an ovulatory dose of human chorionic gonadotropin (hCG) (11, 12). TSG-6 was shown to be highly expressed in periantral mural granulosa cells and cumulus cells between 4 and 8 h after hCG administration. In addition, the protein localizes to extracellular matrix surrounding cumulus and mural granulosa cells from ovulatory follicles (13). Interestingly, the hCG induction of TSG-6 mRNA could be reduced with indomethacin pretreatment, indicating this gene might be a potential target of prostaglandin (PG) regulation (12).

PGs throughout the body are common mediators of inflammatory responses including ovulation, which exhibits many features of inflammation (14, 15). PGs are synthesized from arachidonic acid by cyclooxygenase (COX), the rate-limiting enzyme in this conversion process (16). Of the two isoforms of COX, COX-2 is induced by LH/hCG in mural granulosa and cumulus cells, whereas COX-1 is constitutively expressed in the theca (17, 18, 19). COX-2 is the critical producer of PG signaling in the ovary as its knockout results in female infertility (20). These mice exhibit impaired cumulus expansion and have severely reduced ovulation rates (21, 22). This phenotype is reversible, as PG E2 (PGE2) or IL-1ß will rescue infertility in these mice and restore ovulation rates. Additionally, mice null for one of the PGE2 receptors, EP2, are also infertile (23, 24, 25) and exhibit impaired cumulus expansion and have reduced ovulation rates (22, 23). Taken together, these observations indicate that PG signaling within the follicle plays a role in cumulus-oocyte expansion, matrix formation, and ovulation. It is therefore reasonable to expect components of the cumulus-oocyte matrix to be disrupted in mice with aberrant PG signaling.

The objective of this study was to examine the expression of genes encoding components of the COC to determine whether their expression patterns were altered in COX-2/EP2 null mice, thereby identifying potential targets for PG action that impact cumulus cell function and extracellular matrix formation. To this end, we have analyzed the expression of HAS-2, TSG-6, and levels of I{alpha}I in COX-2 null mice as well as TSG-6 and HAS-2 expression in EP2 null mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Pregnant mare serum gonadotropin (PMSG) (Gestyl) was purchased from Professional Compounding Center of America (Houston, TX). hCG (Pregnyl) was purchased from Organon Special Chemicals (West Orange, NJ). All RT-PCR reagents were obtained from Promega Corp. (Madison, WI). Genosys (Houston, TX) synthesized all oligonucleotides. Routine chemicals and reagents were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma (St. Louis, MO).

Animals and hormone treatments
Wild-type C57BL/6 mice were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). COX-2 null mice were obtained from The Jackson Laboratory (Bar Harbor, ME) (20). EP2 null mice were generated as in (24) as well as kindly provided by Stephen Tilley (25). Follicular growth and ovulation were stimulated in mice at 21 d of age by the following hormonal regimen: 5 IU PMSG was injected ip followed 48 h later by 5 IU hCG injected ip. Whole ovaries, COCs, or granulosa cells were isolated from mice 48 h after PMSG treatment as well as at selected time intervals after hCG treatment as indicated in results and figure legends. Animals were maintained according to the NIH Guide for Care and Use of Laboratory Animals.

RNA isolation
Whole ovary mouse RNA was obtained by tissue homogenization in TRIzol Reagent (Life Technologies, Inc., Gaithersburg, MD) followed by RNA precipitation in isopropanol. Recovered RNA was then washed in 70% ethanol and dissolved in ribonuclease-free water. COCs or granulosa cells were isolated from COX-2 and EP2 heterozygous and null mice treated with PMSG for 48 h followed by hCG for 4 or 12 h as described by Joyce et al. (19) or Zeleznik et al. (26), respectively. RNA was isolated using TRIzol Reagent as above. RNA was quantified and stored at -80 C until use.

RT-PCR analysis
RT-PCR primers were generated by a web-based prediction program (27). TSG-6 primer pairs were 5'-ttccatgtctgtgctgctggatgg-3' and 5'-agcctggatcatgttcaaggtcaaa-3' based on mouse TSG-6 sequence (accession no. U83893). LH receptor primer pairs were 5'-atcccagccactgagttcattc-3' and 5'-cttatccataaccaccataccag-3' based on mouse LHR sequence (accession no. M81310). Ribosomal S16 primer pairs were 5'-tccaagggtccgctgcagtc-3' and 5'-cgttcaccttgatgagcccatt-3' based on mouse rbS16 sequence (accession no. M11408). Ribosomal L19 primer pairs were 5'-ctgaaggtcaaagggaatgtg-3' and 5'-ggacagagtcttgatgatctc-3' based on rat rbL19 sequence (accession no. J02650). HAS-2 primer pairs were as published (28). Briefly, 150–300 ng of total RNA was reverse transcribed using 1x Thermocycle buffer, 500 ng oligo-deoxythymidine primer, 1 mM deoxy-NTPs, 4 mM MgCl2, 20 U RNAsin, and 2.5 U avian myeloblastosis virus-reverse transcriptase at 42 C for 90 min. To these reverse transcriptase reactions was added 500 ng of each primer, 2 µCi [32P]deoxy-CTP (ICN Radiochemicals, Los Angeles, CA), and 2.5 U Taq polymerase in 1x Thermocycle buffer and 2.5 mM MgCl2. PCR conditions were 94 C for 2 min followed by multiple cycles of 95 C for 30 sec, 60 C for 45 sec, and 72 C for 60 sec with a 72-C extension for 10 min (29). L19 and TSG-6 were amplified using 23 cycles, LHR, and S16 using 25 cycles. Has-2 was amplified using a 55 C annealing temperature and 25 cycles. Primers for rbS16 and rbL19 were included as internal amplification controls. PCR products were separated on a 5% polyacrylamide gel and exposed to autoradiographic film (Kodak, Rochester, NY). Products were quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

In situ hybridization
In situ hybridization was done as described by Wilkensen (30). Briefly, [35S]-uridine triphosphatase (Amersham Pharmacia Biotech, Piscataway, NJ)-labeled antisense and sense probes from rat TSG-6 and mouse HAS-2 were made using the Riboprobe In-Vitro Transcription Systems kit (Promega Corp.). Dr. Larry Espey (Trinity University, San Antonio, TX) generously provided the rat TSG-6 cDNA template. Dr. Martin Matzuk (Baylor College of Medicine, Houston, TX) generously provided the mouse HAS-2 cDNA template. Ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 7 µm onto silane-coated slides. After deparaffinization and rehydration, sections were pretreated with 20 µg/ml proteinase K and 0.1 M triethanolamine/acetic anhydride before coating with labeled probe and incubated overnight at 55 C. Slides were washed with increasing stringency using 5x SSC followed by 50% formamide/2x SSC/100 mM ß-mercaptoethanol for 30 min each at 65 C. Slides were then treated with 20 µg/ml ribonuclease A for 30 min at 37 C followed by final washes of 2x SSC and 0.1x SSC for 15 min at 65 C and dehydrated. To visualize the [35S]-uridine triphosphate label, slides were dipped in photographic NTB-2 emulsion (Kodak, Rochester, NY). D-19 developer and fixer (Kodak) were used to develop the slides 72 h later followed by hematoxylin counterstain.

Western analysis
Solubilized whole ovary cell extracts were prepared as described previously (31). Briefly, whole mouse ovaries were homogenized in PE buffer (10 mM potassium phosphate, pH 6.8; and 1 mM EDTA) with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (10 mM), sonicated, and microcentrifuged to remove large insoluble material. Supernatant was collected as the detergent solubilized fraction.

After isolation, COCs were washed in 1x PBS. Isolated COCs were directly solubilized with a strong denaturing protein loading buffer consisting of 2% sodium dodecyl sulfate (SDS) and 6% ß-mercaptoethanol (SDS/2-ME) or treated with hyaluronidase (10 U) and/or chondroitinase (C) (0.04 U) at 37 C for 3 h before denaturation in SDS/2-ME. In the case of urea extraction, COC samples were extracted at 4 C for 1 h in urea extraction buffer [6 M urea, 0.05 M sodium acetate (pH 6.0), 0.1% Triton, and protease inhibitors] and microcentrifuged to pellet insoluble material. The supernatant was collected as the urea solubilized fraction and the remaining pellet was solubilized in SDS/2-ME.

All protein extracts were stored at -80 C until use. Samples were boiled at 100 C for 5 min before gel loading. Protein extract was separated in 10% or 4–15% gradient acrylamide/SDS gels (Bio-Rad Laboratories, Inc., Hercules, CA) and transferred to Imobilon-P nylon membranes (Millipore Corp., Bedford, MA).

Membranes were blocked with 5% nonfat dry milk in 1x PBS. Primary antibodies were added in 5% nonfat dry milk in 1x PBS at room temperature for 1 h or overnight at 4 C in the case of TSG-6. For COX-2 immunoblots rabbit antimurine COX-2 polyclonal antibody (Caymam Chemical, Ann Arbor, MI) was used at a dilution of 1:1000. For I{alpha}I immunoblots, rabbit antihuman I{alpha}I polyclonal antibody (DAKO Corp., Carpinteria, CA) was used at a dilution of 1:5000. For TSG-6 immunoblots, rabbit antimouse TSG-6 polyclonal antibody (32) was used at a dilution of 1:1000. Membranes were then washed 3 x 10 min in 1x PBS followed by incubation with 1:10,000 dilution of donkey antirabbit IgG peroxidase-linked antibody (Amersham Life Sciences, Arlington Heights, IL) in 5% nonfat dry milk in 1x PBS. After washing membranes as before, specific signal was detected using Supersignal chemiluminescent detection reagents (Pierce Chemical Co., Rockford, IL). Treated membranes were exposed to autoradiographic film (Kodak) to visualize any bands. Quantification was performed using a densitometer and ImageQuant software (Molecular Dynamics, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HAS-2 is expressed in wild-type and COX-2 null mice
To analyze HAS-2 expression in mutant mouse models, semiquantitative RT-PCR analysis as well as in situ hybridization were used to determine the induction and localization of HAS-2 mRNA. In wild-type mouse ovaries, HAS-2 mRNA was rapidly induced 4 h after an ovulatory dose of hCG and was maintained above levels seen in PMSG treated mice up to 24 h after hCG (Fig. 1AGo). In situ hybridization showed that mRNA induction was evident as early as 2 h after hCG administration and that the peak of expression occurred at 4 h (Fig. 1BGo). HAS-2 expression was localized to cumulus cells surrounding the oocyte and to adjacent mural granulosa cells of large preovulatory follicles. Little HAS-2 mRNA localized to mural granulosa cells not located near the COC in the antral cavity. By 12 h after hCG administration, HAS-2 mRNA expression was restricted to cumulus cells and absent from mural granulosa cells.



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  		Figure 1. hCG induces expression of HAS-2 mRNA. A, Message levels of HAS-2 and S16 (internal control) were analyzed by semiquantitative RT-PCR analysis using specific primer sets. Whole ovary RNA was isolated from wild-type animals treated with PMSG (48 h) followed by hCG (4–24 h). Top panel, Representative autoradiograph of HAS-2 RT-PCR in mouse whole ovary. Bottom panel, Combined mean ± SD of three experiments. B, In situ localization of HAS-2 mRNA in ovarian sections using a 35S-labeled antisense probe. Upper panels, Bright-field images; lower panels, dark-field images. Sense probe was included as a negative control. Mice were treated with PMSG (48 h) followed by hCG (2–12 h).

 
To assess HAS-2 mRNA expression in COX-2 null mice, in situ hybridization was again used to examine COX-2 heterozygous and null mice. Four hours after hCG stimulation, when HAS-2 mRNA expression is high in cumulus and mural granulosa cells, the COX-2 heterozygous and null mice showed patterns of HAS-2 mRNA expression similar to that of wild-type mice (Fig. 2AGo). Both periantral mural granulosa cells and cumulus cells surrounding the oocyte expressed HAS-2 mRNA. In addition, RT-PCR analysis of whole ovary RNA from COX-2 null and heterozygous mice did not reveal a difference in HAS-2 expression levels at a similar time interval (Fig. 2BGo, part 1). In agreement with this, RT-PCR analysis of COCs isolated from follicles of COX-2 null and heterozygous mice 12 h after hCG administration from three separate experiments did not reveal a significant difference in HAS-2 message levels despite high variability between experiments (Fig. 2BGo, part 2). Thus, the ovaries of COX-2 null mice maintain the capacity to produce HAS-2 mRNA in response to an ovulatory dose of hCG.



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  		Figure 2. Cumulus cell expression of HAS-2 is not disrupted in the COX-2 null mouse. A, In situ localization of HAS-2 mRNA in COX-2 null and heterozygous ovarian sections using a 35S-labeled antisense probe. Upper panels are bright-field images, and lower panels are dark-field images. Mice were treated with PMSG (48 h) followed by hCG (4 h). B, Semiquantitative RT-PCR analysis of HAS-2 message using specific primer sets. Part 1, Top panel, Representative autoradiograph of HAS-2 RT-PCR of whole ovary RNA isolated from COX-2 null and heterozygous mice treated with PMSG 48 h followed by hCG 4 h. Bottom panel, The combined mean ± SD of two experiments. Part 2, Top panel, Representative autoradiograph of HAS-2 RT-PCR of RNA isolated from COCs of COX-2 null and heterozygous mice treated with PMSG (48 h) followed by hCG (12 h). Brackets indicate samples from three separate experiments. Bottom panel, The combined mean ± SD of three experiments.

 
TSG-6 mRNA expression is altered in COX-2 null mice
We next analyzed expression of the HA binding protein, TSG-6 because its expression increased with hCG administration. Semiquantitative RT-PCR analysis was initially used to analyze the dynamics of TSG-6 mRNA induction in wild-type mice (Fig. 3AGo). As seen in previous studies using a PMSG-hCG rat model (12), TSG-6 mRNA was rapidly induced 4 h after hCG treatment and persisted as long as 12 h after hCG injection. In a pattern similar to that of HAS-2 expression, TSG-6 mRNA was localized to mural granulosa cells lining the antrum as well as cumulus cells surrounding the oocyte in wild-type mice (Fig. 3BGo). Expression was most intense in cumulus cells and mural granulosa cells adjacent to the cumulus complex and decreased in mural granulosa cells farther away from the antral cavity. By 12 h after hCG treatment, expression was mainly limited to cumulus cells and low in mural granulosa cells. Follicles from mice treated with PMSG alone showed no detectable TSG-6 mRNA by this method.



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  		Figure 3. hCG induces expression of TSG-6 mRNA. A, Analysis of TSG-6 and S16 (internal control) message by semiquantitative RT-PCR using specific primer sets. Whole ovary RNA was isolated from wild-type mice treated with PMSG (48 h) followed by hCG (4–24 h). Top panel, Representative autoradiograph of TSG-6 RT-PCR in mouse whole ovary. Bottom panel, The combined mean ± SD of four experiments. B, In situ localization of TSG-6 mRNA in ovarian sections using a 35S-labeled antisense probe. Upper panels are brightfield images, and lower panels are darkfield images. Mice were treated with PMSG (48 h) followed by hCG (4–12 h).

 
When whole ovarian RNA from COX-2 null and heterozygous mice was examined by RT-PCR for TSG-6 mRNA expression 4 h after hCG administration, no difference was observed between samples (Fig. 4AGo, part 1). In situ hybridization analysis of COX-2 null and heterozygous mice, however, revealed aberrant TSG-6 mRNA localization. Specifically, TSG-6 mRNA expression in cumulus cells appeared to be absent in COX-2 null mice (Fig. 4BGo). This was evident at 4, 6, 8, and 12 h after hCG administration. This loss was specific for cumulus cells, as mural granulosa cell expression of TSG-6 in COX-2 null mice appeared normal. To quantify the loss of TSG-6 mRNA in isolated COCs, additional RT-PCR analyses were done (Fig. 4AGo, part 2). Although the levels of TSG-6 mRNA varied across three separate experiments, overall TSG-6 mRNA levels were 50% less (P < 0.05) in COCs of null mice than the amount observed in COCs of heterozygotes. Thus, it seems likely that the regulated expression of TSG-6 mRNA in cumulus cells is dependent on COX-2 expression and the action of PGs.



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  		Figure 4. Cumulus cell expression of TSG-6 is disrupted in COX-2 null mice. A, Semiquantitative RT-PCR analysis of TSG-6 message using specific primer sets. Part 1, Top panel, Representative autoradiograph of TSG-6 RT-PCR of whole ovary RNA isolated from COX-2 null and heterozygous mice treated with PMSG (48 h) followed by hCG (4 h). Bottom panel, Combined mean ± SD of two experiments. Part 2, Top panel, Representative autoradiograph of TSG-6 RT-PCR of RNA isolated from COCs of COX-2 null and heterozygous mice treated with PMSG (48 h) followed by hCG (12 h). Brackets indicate samples from three separate experiments. Bottom panel, The combined mean ± SD of three experiments. *, Significant difference as determined by a Student’s t test where P < 0.05 was considered significant. B, In situ localization of TSG-6 mRNA in COX-2 null and heterozygous ovarian sections using a 35S-antisense probe. Upper panels are bright-field images, and lower panels are dark-field images. Mice were treated with PMSG (48 h) followed by hCG (4–12 h). Arrows point to COCs in preovulatory follicles.

 
TSG-6 mRNA expression is altered in EP2 null mice
Having determined TSG-6 mRNA expression in cumulus cells is mediated (at least in part) by PGs, it was necessary to determine which PG pathway might be responsible. The EP2 null mice, with a similar infertility phenotype to that of the COX-2 null mice, proved useful for this purpose. In Fig. 5AGo, in situ hybridization was used to analyze TSG-6 mRNA localization in ovaries of EP2 heterozygous and null mice. Expression of TSG-6 in the EP2 heterozygous mouse 4 h after hCG administration was similar to that observed in wild-type and COX-2 heterozygous mouse ovaries. Namely, TSG-6 mRNA was localized to the mural granulosa cells lining the antrum as well as cumulus cells surrounding the oocyte. Twelve hours after hCG administration, TSG-6 message was only detectable in cumulus cells as previously seen in wild-type and COX-2 heterozygous mice. However, in mice lacking the EP2 receptor, expression of TSG-6 mRNA in cumulus cells was not detectable 4 or 12 h after hCG, whereas TSG-6 mRNA expression in mural granulosa cells persisted as it did in the COX-2 null mice (Fig. 5AGo).



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  		Figure 5. Cumulus cell expression of TSG-6 is disrupted in EP2 null mice. A, In situ localization of TSG-6 mRNA in EP2 null and heterozygous ovarian sections using a 35S-antisense probe. Upper panels are dark-field images, and lower panels are bright-field images. Mice were treated with PMSG (48 h) followed by hCG (4 or 12 h). Arrows point to COCs in preovulatory follicles. B, COCs and granulosa cells (GC) were isolated from ovaries of EP2 null and heterozygous mice treated with PMSG (48 h) followed by hCG (4 h). Top panel, Semiquantitative RT-PCR analysis using specific primer sets for TSG-6, HAS-2, LHR (down-regulated in cumulus cells after hCG administration), and S16 (internal control). Each lane is a pool of RNA isolated from three animals and analyzed in one experiment. Lower panel, Densitometric analysis of the TSG-6 and HAS-2 RT-PCR.

 
To further confirm that the pattern of TSG-6 expression in EP2 null mice was identical to that observed in COX-2 null mice, semiquantitative RT-PCR analyses were done using RNA prepared from pools of COCs and granulosa cells from EP2 null mice. TSG-6 mRNA was lower in COCs of EP2 null mice compared with heterozygous mice whereas HAS-2 mRNA did not change (Fig. 5BGo). Furthermore, the decrease of TSG-6 mRNA levels in COCs of EP2 null mice was of a similar magnitude to that seen in the COX-2 null COCs, approximately 50%. As a control for cumulus cell isolation, LH receptor mRNA was also analyzed by RT-PCR. The LH receptor is down-regulated specifically in cumulus cells by factors from the oocyte (33). In both the EP2 null and heterozygous mice, the LH receptor was lower in COCs than in isolated granulosa cells indicating the absence of granulosa cells in the COC pools (Fig. 5BGo). Taken together, TSG-6 mRNA induction in cumulus cells may be partially mediated by PGE2 acting through the EP2 PG receptor.

Western analysis of TSG-6 and ITI protein expression patterns in COCs
Having established that TSG-6 mRNA was significantly reduced in cumulus cells of COX-2 and EP2 null mice, we next analyzed the expression of TSG-6 protein. For this, COCs were isolated from mice treated with PMSG or PMSG followed by hCG for 4, 6, 8, and 12 h (Fig. 6Go). Samples were either directly denatured in SDS/2-ME loading buffer (designated untreated, U), digested with hyaluronidase (H), C, or both enzymes (H + C) followed by denaturation in SDS/2-ME buffer. Proteins were resolved by SDS-PAGE and probed with an antibody specific for TSG-6. As shown in Fig. 6AGo, TSG-6 immunoreactivity was observed in COCs isolated 12 h after hCG but not in whole ovarian extracts indicating that TSG-6 protein was concentrated in COCs. Two immunoreactive TSG-6 bands were detected in COC samples collected from the ovary 12 h or from the oviduct 16 h after hCG administration. The 36-kDa TSG-6 band (designated as ii in Fig. 6, A and BGo) coincides with the native protein and was only detected in COCs collected 12 h and 16 h after hCG administration (Fig. 6, A and BGo). A second immunoreactive band at approximately 120 kDa (designated as i in Fig. 6, A and BGo) was first observed at 6 h (commensurate with the expression of TSG-6 mRNA) and remained present at 8, 12, and 16 h. It is important to note that this 120-kDa species was resistant to H and C digestion alone or in combination, suggesting that this complex containing TSG-6 is not covalently linked to HA or to chondroitin sulfate moieties.



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  		Figure 6. TSG-6 and I{alpha}I protein expression in whole ovary and isolated COCs. Western analysis of whole ovary (WO) and COC extracts from wild-type animals treated with PMSG (48 h) followed by hCG (4–16 h). WO extracts and COCs were digested without (U, untreated) or with H or with C alone or in combination (H + C). Thirty COCs were loaded per lane. Representative autoradiographs from at least three experiments. A, TSG-6 and COX-2 Western analyses of WO and isolated COCs. B, TSG-6 Western analysis of isolated COCs. C, I{alpha}I Western analysis of isolated COCs. (i, 120-kDa TSG-6 band; ii, 36-kDa TSG-6 band; iii, 120-kDa I{alpha}I band; iv, 86-kDa heavy chain of I{alpha}I; {star}, 120-kDa I{alpha}I band).

 
Previously, two groups have characterized a 120-kDa TSG-6 band that was shown to be linked to the heavy chain of I{alpha}I (13, 34). Therefore, the blot from 6B was analyzed for the temporal appearance of I{alpha}I in isolated COCs. Negligible amounts of I{alpha}I were observed in untreated COCs directly denatured in loading buffer (i.e. no H + C digestions) with the exception of the 12-h sample when multiple immunoreactive I{alpha}I bands were detected in low amounts including one at 120 kDa (Fig. 6CGo, iii). In contrast, pretreatment of the COCs with H + C before denaturation in SDS/2-ME buffer greatly facilitated the detection of immunoreactive I{alpha}I. The major band at 86 kDa (Fig. 6CGo, iv), present 6 h after hCG, represents the heavy chain of I{alpha}I and increases in concentration at 8 and 12 h coincident with the increased movement of serum proteins into the follicular fluid and the increased cross-linking of I{alpha}I to HA (and possibly other components) of the matrix (35). The identity of other higher molecular weight bands is not known. A 120-kDa I{alpha}I band was detectable (Fig. 6CGo, iii, star) in the 8- and 12-h (H + C) samples. This species corresponds to the 120-kDa I{alpha}I band observed in the 12-h untreated sample.

Because in situ hybridization and RT-PCR analyses revealed a specific loss of TSG-6 mRNA in cumulus cells of preovulatory follicles, we next analyzed TSG-6 protein levels in COCs isolated from COX-2 and EP2 null and heterozygous mice. In Fig. 7AGo, untreated COCs from COX-2 null and heterozygous mice were denatured in SDS/2-ME buffer and resolved by SDS-PAGE. COX-2 null mouse COCs contained 50% less (P = 0.037) TSG-6 36-kDa protein than COX-2 heterozygous mouse COCs. Similar results were obtained from solubilized COC fractions of EP2 null and heterozygous mice using a urea detergent (Fig. 8Go). When the corresponding insoluble pellet was extracted in SDS/2-ME, results showed that not all the TSG-6 was extracted from the matrix by urea and that the 36-kDa species was more readily extracted from the null samples than the heterozygote samples (Fig. 8Go). Reasons for this apparent difference are not fully understood but support different matrix compositions between null and heterozygous samples. The magnitude of this protein loss matches the loss of TSG-6 mRNA in COCs after hCG administration in COX-2 and EP2 null mice (Fig. 4AGo, part 2; and Fig. 5BGo). A longer exposure of the blot in Fig. 7AGo revealed the presence of a 120-kDa species and showed that the amount of this immunoreactive band did not differ significantly (P = 0.621) between the null and heterozygous samples (Fig. 7BGo). Stripping the same blot and probing with an antibody for COX-2 confirmed the genotypes of the mice (Fig. 7CGo). To analyze the levels of I{alpha}I, the blot used in Fig. 7AGo was stripped and analyzed for I{alpha}I immunoreactivity. Because these samples were not digested with H + C, most of the immunoreactive I{alpha}I remained in a large complex at the top of the gel; no discernible bands were detected between 200 and 28 kDa (Fig. 7DGo). However, when the top portion of the blot above 186 kDa was removed and the remaining portion was again probed for I{alpha}I, immunoreactive bands were observed at approximately 120 kDa and 130 kDa in the samples from the COX-2 null and heterozygous mice (Fig. 7EGo). There was no significant difference in the 120-kDa band between the COX-2 null and heterozygous isolated COCs (P = 0.133).



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  		Figure 7. Protein expression of TSG-6 is disrupted in COX-2 null animals. Western analysis of COC extracts from COX-2 null and heterozygous animals treated with PMSG (48 h) followed by hCG (12 h). Thirty untreated, solubilized (SDS/2-ME) COCs were loaded per lane. A, TSG-6 Western analysis. Top panel, Short exposure of TSG-6 Western. Bottom panel, Densitometric analysis of the 36-kDa TSG-6 band (ii). B, TSG-6 Western analysis. Top panel, Long exposure of TSG-6 Western from A. Bottom panel, Densitometric analysis of the 120-kDa TSG-6 band (i). C, COX-2 Western analysis of immunoblot used in A, B, D, and E. D, Immunoblot from panel A was stripped and reprobed for I{alpha}I, revealing high molecular weight complexes (v). E, The top portion of the immunoblot (above 186 kDa) in panel D was removed. The remaining portion, less than 186 kDa, was probed again for I{alpha}I showing the 120-kDa complex (iii). Densitometric analyses performed in A and B represent the combined mean ± SD of three experiments. *, Significant difference as determined by a Student’s t test where P < 0.05 was considered significant (i, ii, iii, and iv are as in Fig. 6Go; v = I{alpha}I immunoreactivity without H + C digestion).

 


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  		Figure 8. Protein expression of TSG-6 is disrupted in EP2 null animals. Western analysis of COC extracts from EP2 null and heterozygous animals treated with PMSG (48 h) followed by hCG (12 h). Ten micrograms of solubilized (urea buffer) COC extract and the remaining pellet (SDS/2-ME) were analyzed for levels of the 36-kDa TSG-6 species. The heterozygous level was set to 100%, and the null level was expressed as a percentage of the heterozygous level. Top panel, Representative autoradiograph of a 36-kDa TSG-6 band. Bottom panel, Densitometric analysis of the 36-kDa TSG-6 band from two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Successful completion of ovulation requires that a fertilizable oocyte encased in an expanded cumulus matrix be released from the rupture site of the ovulating follicle. Evidence that formation of the cumulus matrix is essential for extrusion of the oocyte is increasing, as ovulation is impaired if the synthesis of HA and/or its interactions with the heavy chain of I{alpha}I are blocked (6, 7, 36, 37, 38). Furthermore, PGs appear to play a key intrafollicular role in regulating genes involved in matrix formation and cumulus expansion. Specifically, targeted disruption of two genes either COX-2 or the PGE2 receptor EP2 results in lower ovulation rates and impaired cumulus expansion (21, 22, 23). Although the mechanisms by which PGs mediate cumulus expansion and subsequent ovulation are not entirely understood, our data provide insight into one target of PG action, namely the HA binding protein TSG-6.

Specifically, our results show that induction of HAS-2 mRNA is probably not a PG-mediated event because HAS-2 induction by hCG was similar in COX-2 heterozygous and null mice. However, because COX-2 and EP2 are required for optimal ovulation and expansion, PGs may impact the incorporation of HA into a stable cumulus-derived matrix. One component critical for stable matrix formation is the serum factor I{alpha}I. Its entry into the follicle is dependent on LH-mediated dissolution of the basement membrane. Once within the preovulatory follicle, a granulosa cell factor catalyzes the covalent linkage of the heavy chain of I{alpha}I to HA and a majority of I{alpha}I is covalently linked to HA in ovulated COCs (35). Thus, PGs may induce and or activate this granulosa cell factor that facilitates the incorporation of I{alpha}I into the matrix. Clear evidence for this is not yet known. Our data, however, suggest that at least in the COX-2 null animals I{alpha}I is able to form a covalent interaction with HA in a similar manner to that observed in COX-2 heterozygous animals in response to hCG administration. Because the COX-2 null animals have impaired cumulus expansion despite the ability of I{alpha}I to covalently link to HA, other HA cross-linking species not present in serum must either be activated or produced in part by PG signaling.

In addition, and most importantly, our results provide the first evidence that the expression of TSG-6 in cumulus cells is a target of PG action. It will be important to determine whether PGE2 activation of the EP2 receptor is sufficient by itself to induce TSG-6 mRNA. Future experiments are planned to address whether PGE2 can rescue cumulus cell expression of TSG-6 in COX-2 and EP2 null mice. Previously, Fujimoto et al. (39) showed that in cervical smooth muscle cells PGE2 was able to induce TSG-6 lending further evidence to the pathway described herein. As such, these results also provide the first evidence that TSG-6 may play an essential role in the formation or function of the matrix of the COC. The functions of TSG-6 in the ovary or other tissues remain unclear but several are implicated. For example, TSG-6 has been reported to impact leukocyte migration, possibly by modifying extracellular matrix remodeling (40). TSG-6 can prevent degradation of aggrecan in inflamed synovial tissue by blocking the activity of the protease ADAMTS-4 (41). TSG-6 has been reported to potentiate the I{alpha}I-mediated inhibition of plasmin protease activity (40). In addition, TSG-6 may compete with the cell surface CD44 receptor for binding of HA (41). Any of these functions could modify events occurring at the site of inflammation. These functions may depend on the factors with which TSG-6 interacts. TSG-6 has been shown to interact with HA via the link module (42, 43), I{alpha}I by mechanisms that are not entirely clear and may be tissue specific (13, 34), and chondroitin-4-sulfate containing glycosaminoglycans (44).

Although the interactions of TSG-6 with I{alpha}I have received the most attention with respect to inflammation in synovial tissue and the ovary, these interactions appear to be tissue specific. For example, in synovial tissue or in serum, TSG-6 interacts with I{alpha}I via chondroitin sulfate bonds that also form the link between the I{alpha}I heavy chain and bikunin (34). However, our data and that of Mukhopadhyay et al. (13) show that in COCs the 120-kDa TSG-6 band is not sensitive to C. Thus, if the heavy chain of I{alpha}I is linked to TSG-6 a different bonding mechanism is involved in the ovary.

Of note, unbound intact TSG-6 is observed only in COCs isolated at 12 and 16 h after hCG (Fig. 6Go, A and B). Thus, TSG-6 appears to be linked rapidly to some factor following its synthesis and release into the antrum from 4–8 h after hCG. The appearance of the 36-kDa TSG-6 band at 12 and 16 h may reflect a decrease in a coupling reaction, increased degradation of previously bound TSG-6 or changes in the linking of TSG-6 to matrix factors. Each of these suggests that the regulation and localization of TSG-6 may differ markedly before and after ovulation. At these later time points, TSG-6 may preferentially interact with versican which is induced markedly between 8 and 12 h after hCG and is localized to the matrix within the antrum (45). In this manner, TSG-6 may impact the function of ADAMTS-1, a protease that is also induced at 12 h after hCG and has been shown to cleave versican, at least in vitro (46). Thus, TSG-6 may play more than one role by modifying pro or antiinflammatory events in the follicle and COC during ovulation.

Selective induction of TSG-6 by PGs and EP2 receptor activation is indicated by the consistent decrease of TSG-6 mRNA and protein in cumulus cells compared with mural granulosa cells in both the COX-2 and EP2 null mice. This supports two possible mechanisms by which LH regulates this molecule. In mural granulosa cells, LH may act directly to induce TSG-6 expression through cAMP production. In cumulus cells, LH may act indirectly to increase cAMP via activation of GDF-9. In support of this indirect pathway are data that GDF-9 stimulates the expression of COX-2, production of PGE2, and the expression of EP2 (47).

The obligatory roles for PGs, PGE2, cAMP, and I{alpha}I in vivo can be partially reproduced in vitro. For many years, FSH and serum have been used in vitro to induce stable cumulus expansion. This regimen, however, may bypass the need for local production of PGE2. Specifically, a recent study by Matsumoto et al. (22) has indicated that COC expansion may not be impaired in the COX-2 and EP2 null mice. The authors report that cumulus complexes from null mice expand in vitro when incubated with FSH and serum and that the COCs were visibly similar in heterozygous and null mice. This experimental paradigm may not, however, reconstitute the milieu of the intact follicle. The pharmacological dose (1 µg) of FSH used does not resemble intrafollicular levels of FSH after the LH surge. However, their results, in addition to those of Hizaki et al. (23), show that expansion in vitro is mediated by a cAMP signaling mechanism whether initiated by large doses of FSH or cAMP itself. In vivo, a critical cAMP signaling event within cumulus cells is initiated by PGE2 binding to the EP2 receptor, an activator of adenylate cyclase and protein kinase A (48). Our results, as well as those of Davis et al. (21), show that at least in vivo when the PGE2 signaling pathway is disrupted, cumulus expansion is impaired indicating that the pharmacological paradigm of expansion in vitro does not necessarily mimic the physiological induction of expansion in vivo.

In either case, cumulus cell specific expression of TSG-6 is downstream of a PG-EP2-mediated event in preovulatory follicles. Our studies show that the lack of PG-regulated expression of TSG-6 in cumulus cells of the COX-2 and EP2 null mice may contribute to impaired cumulus cell expansion and lower ovulation rates seen in these animals.


   Footnotes
 
This work was supported by the following sources of funding: NIH Grant HD-16229 (to J.S.R.), NIH Grant HD-07165 (training grant), Medical Research Council (UK) (to A.J.D.), and NIH Grant GM-15431 (to R.M.B.).

Abbreviations: C, Chondroitinase; COC, cumulus oocyte complex; COX, cyclooxygenase; H, hyaluronidase; HA, hyaluronan; HAS-2, HA synthase-2; hCG, human chorionic gonadotropin; I{alpha}I, inter-{alpha}-trypsin inhibitor; PG, prostaglandin; PMSG, pregnant mare serum gonadotropin; SDS, sodium dodecyl sulfate; SDS/2-ME, SDS and 6% ß-mercaptoethanol; TSG-6, TNF{alpha}-stimulated gene 6.

Received April 23, 2002.

Accepted for publication November 8, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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