OVULATION IS THE process by which a fertilizable

Transcription

1 /03/$15.00/0 Endocrinology 144(10): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /en Disrupted Function of Tumor Necrosis Factor- - Stimulated Gene 6 Blocks Cumulus Cell-Oocyte Complex Expansion SCOTT A. OCHSNER, ANTHONY J. DAY, MARILYN S. RUGG, RICHARD M. BREYER, RICHARD H. GOMER, AND JOANNE S. RICHARDS Department of Molecular and Cellular Biology (S.A.O., J.S.R.), Baylor College of Medicine, Houston, Texas 77030; Medical Research Council Immunochemistry Unit (A.J.D., M.S.R.), Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; Departments of Medicine (Division of Nephrology) and Pharmacology, Vanderbilt University School of Medicine (R.M.B.), Nashville, Tennessee ; and Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology (R.H.G.), Rice University, Houston, Texas During ovulation, the oocyte and surrounding somatic cumulus cells contained within a specialized, mucoid matrix are released from the ovary. One matrix component, TNF- -stimulated gene 6 (TSG-6), is a hyaluronan binding protein induced in cumulus cells of preovulatory follicles by the LH surge and is decreased in cumulus cells of COX-2 and prostaglandin E 2 (PGE 2 ) receptor subtype EP2 null mice that exhibit impaired ovulation and cumulus expansion. To determine if TSG-6 was hormonally induced in cumulus cells in vitro and was functional during the formation of the expanded matrix, we established a cumulus cell-oocyte complex (COC) culture system. This system was used to analyze the effects of FSH, PGE 2, EP2 receptor, and selected protein kinase inhibitors on TSG-6 production as well as specific antibodies to the TSG-6 OVULATION IS THE process by which a fertilizable oocyte and surrounding somatic cumulus cells (the cumulus cell-oocyte complex; COC) are released from the ovary for fertilization. Ovulation is induced by the LH surge which triggers a cascade of events that impact inflammationlike reactions including the induction of cyclooxygenase 2 (COX-2) and the synthesis of prostaglandins (1 5). Importantly, mice null for COX-2 and the prostaglandin E 2 (PGE 2 ) receptor subtype EP2 have fertility defects due to impaired ovulation associated with defective COC expansion (6, 7). Expansion involves the formation of a mucoid extracellular matrix by cumulus cells creating a spherical mass surrounding the oocyte (8 12). The matrix is comprised of components present in other inflammation-like processes. Among them is the glycosaminoglycan hyaluronan (13), which forms the principal structural backbone as well as several hyaluronan binding proteins (14): i.e. the proteoglycan versican (15), the serum-derived factor inter- trypsin inhibitor (I I) (16), and the secreted protein TNF- -stimulated gene 6 (TSG-6) (17). TSG-6 not only binds hyaluronan through its link module domain (18, 19) but also forms stable approximately 120-kDa (probably covalent) complexes with the heavy chains of I I Abbreviations: COC, Cell-oocyte complex; COX-2, cyclooxygenase 2; EP2, PGE 2 receptor subtype; hcg, human chorionic gonadotropin; I I, inter- trypsin inhibitor; PMSG, pregnant mare serum gonadotropin; PGE 2, prostaglandin E 2 ; SDS, sodium dodecyl sulfate; SDS/2-ME, 2% SDS and 6% -mercaptoethanol; TSG-6, TNF- -stimulated gene 6. link module on TSG-6 function. We document that TSG-6 message and protein are induced by camp/protein kinase A/MAPK signaling pathways and that blocking these cascades prevents expansion and the production of TSG-6. FSH but not PGE 2 rescued expansion and production of TSG-6 in the EP2 null COCs, indicating that generation of a camp signal is essential. Furthermore, disruption of the functional interactions between TSG-6, inter- trypsin inhibitor, and hyaluronan with specific antibodies severely altered matrix formation and cumulus expansion, as recorded by time-lapse imaging. Collectively, these results indicate that TSG-6 mrna is induced in cumulus cells in culture by camp and that the secreted TSG-6 protein is a key structural component of the mouse COC matrix. (Endocrinology 144: , 2003) during COC expansion (17, 20). It is well established that these heavy chains become covalently linked to hyaluronan in the cumulus matrix and this is likely to contribute to matrix stability (21). Targeted disruption of I I formation in mice (9, 10) also impairs COC expansion, ovulation, and fertilization providing additional evidence that formation of the COC matrix is of critical importance. In the rodent ovary, TSG-6 mrna is induced rapidly in granulosa cells as well as cumulus cells of preovulatory follicles following an ovulatory dose of human chorionic gonadotropin (hcg) (22, 23). TSG-6 expression persists in cumulus cells but not granulosa cells at the time of ovulation (12 16 h post hcg). Moreover, levels of TSG-6 mrna but not levels of either hyaluronan synthase 2 or versican mrnas are reduced selectively in cumulus cells of preovulatory follicles of the COX-2 null mice and EP2 null mice (15, 24). In addition, the amount of TSG-6 protein incorporated into the COC matrix is reduced significantly in COX-2 null mice and EP2 null mice (24). This selective decrease of TSG-6 in two different animal models with aberrant COC expansion led us to hypothesize that prostaglandin-induced expression of TSG-6 in cumulus cells and its interactions with matrix components (including hyaluronan and I I) are critical for proper formation of the COC matrix. Recent studies with TSG-6 null mice, which do not form a cumulus matrix and show female infertility, has revealed that, in the absence of TSG-6, heavy chains of I I do not become covalently trans- 4376

2 Ochsner et al. TSG-6 Protein Acts in COC Expansion Endocrinology, October 2003, 144(10): ferred to hyaluronan (25). It is possible therefore that the approximately 120-kDa complexes formed between TSG-6 and I I heavy chains act as intermediates in these transfer reactions. Here COCs were isolated from preovulatory follicles of pregnant mare serum gonadotropin (PMSG)-treated immature mice and placed in defined medium with selected hormones, agonists and/or antagonists to determine if TSG-6 protein was induced in cultured COCs and if it complexed with I I as observed in vivo. A rat monoclonal antihuman TSG-6 antibody, which blocks TSG-6 binding to hyaluronan (26), and as determined herein the formation of the approximately 120-kDa TSG-6/I I complex, was used to determine a functional role for TSG-6 in matrix formation and COC expansion. Using time lapse imaging analyses as well as standard photoimaging, we document the dynamics of COC expansion and its disruption by blocking the hyaluronan link module of TSG-6. Materials Materials and Methods Gestyl (PMSG) was purchased from Professional Compounding Center of America (Houston, TX). Ovine FSH-16 was a generous gift from the National Hormone and Pituitary Agency (Rockville, MD). PGE 2 was purchased from Cayman Chemical Co. (Ann Arbor, MI). Forskolin, H89, PD98059, SB203580, and LY were purchased from Calbiochem (La Jolla, CA). 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). EP2 null mice were generated as described (27). Follicular growth was stimulated in 21-d-old female mice by administration of 5 IU PMSG injected ip. Animals were maintained according to the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals. Cumulus COC culture Ovaries were collected from immature mice 48 h after a single injection of 5 IU PMSG and used to collect unexpanded cumulus COCs. Large antral follicles visible on the surface of the ovary were ruptured using a 26-gauge needle allowing the unexpanded COC to flow out into collection medium (MEM with Earle s salts supplemented with 25 mm HEPES, 0.25 mm sodium pyruvate, 3 mm l-glutamine, and 1 mg/ml BSA). Collected COCs were pooled and incubated in expansion medium (collection media plus 1% FBS) with or without FSH (100 ng/ml), PGE 2 (500 ng/ml), or forskolin (10 m) in l reactions at 37 C in a humidified incubator (95% air, 5% CO 2 ). After 24 h of incubation, COCs were collected for RNA and protein isolation or visualized using an Axiovert S100 (Zeiss) inverted scope and 10 objective. In the case of QuickTime movies, a model 4912 charge-coupled device camera (Cohu, San Diego, CA) mounted on a Nikon TMS inverted microscope with a 10 objective was placed inside an incubator with 95% air, 5% CO 2.An external power supply was used to drive a 6-V illumination lamp with a blue filter at 3 V. The NIH image program ( nih-image/) and a Scion (Frederick, MD) framegrabber were used to capture a frame every 250 sec for 400 frames ( 27 h). Rat antihuman TSG-6 link module monoclonal antibodies A38 and Q75 (26) were added at 5 g/ml to the expansion media as indicated in figure legends. In the case of inhibitor coculture, inhibitors were preincubated with unexpanded COCs for 1 h before the addition of 10 m forskolin for 24 h. The concentrations of inhibitors based on previous studies in our laboratory (28) were as follows: PD98059 (20 m), H89 (10 m), SB (20 m), and LY (25 m). Western analysis After isolation, COCs were washed in PBS. Isolated COCs were treated with hyaluronidase (10 U) and chondroitinase (0.04 U) at 37 C for 3 h. In the first experiment, the cumulus cells and oocytes were separated from the digested matrix components by gentle centrifugation. Each fraction was then denatured in a strong denaturing protein loading buffer consisting of 2% sodium dodecyl sulfate (SDS) and 6% -mercaptoethanol (SDS/2-ME). In other experiments, the hyaluronidase and chondroitinase treated COCs were directly denatured 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 extracts were separated in 4 20% gradient acrylamide/sds gels (Bio-Rad Laboratories, Hercules, CA) and transferred to Imobilon-P nylon membranes (Millipore, Bedford, MA). Membranes were blocked with 5% nonfat dry milk in PBS or TBS/T. Primary antibodies were added in 5% nonfat dry milk in PBS or TBS/T and incubated overnight at 4 C. Rabbit antimouse TSG-6 polyclonal antibody (20) was used at a dilution of 1:1000 in PBS reagents. Rabbit antihuman TSG-6 polyclonal antibody was used as described by Nentwich et al. (29). Rat antihuman monoclonal antibodies A38 and Q75 (26) were used as described in the figure legends. Rabbit antimouse PKB polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA) and rat antihuman TSG-6 monoclonal antibody (26) were used at a dilution of 1:1000 in TBS/T reagents. Rabbit antihuman I I polyclonal antibody (Dako Corp., Carpinteria, CA) was used at a dilution of 1:5000 in PBS reagents. Membranes were then washed 3 10 min in PBS or TBS/T followed by incubation for 1 h with a 1:10,000 dilution of donkey antirabbit IgG or a 1:5,000 dilution of goat antirat IgG peroxidase linked antibody (Amersham Life Sciences, Arlington Heights, IL) in 5% nonfat dry milk in PBS or TBS/T. After washing membranes as before, Supersignal chemiluminescent detection reagent (Pierce, Rockford, IL) was used to visualize the specific immunoreactive products by exposure to X-OMAT autoradiographic film. Quantification was performed using a densitometer and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). RNA isolation Cumulus cell RNA was obtained 24 h after COC treatment and culture by homogenization in TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) followed by RNA precipitation in isopropanol. Recovered RNA was then washed in 70% ethanol and dissolved in ribonuclease-free water. RNA was quantified and stored at 80 C until use. RT-PCR analyses Briefly, 300 ng of total RNA was reverse transcribed using 1 Thermocycle buffer, 500 ng oligo-deoxythymidine primer, 1 mm deoxynucleotide triphosphates, 4 mm MgCl 2, 20 U RNAsin, and 2.5 U avian myeloblastosis virus-reverse transcriptase in a volume of 20 l at42cfor 90 min. To these reverse transcriptase reactions was added 500 ng of each primer, 2 Ci [ 32 P]deoxy-CTP (ICN Radiochemicals, Los Angeles, CA), 2.5 U Taq polymerase in 1 Thermocycle buffer, and 2.5 mm MgCl 2 in a volume of 100 l. 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 (30). Ribosomal protein L19 (GenBank accession no. NM_031103), TSG-6 (NM_009398), and COX-2 (NM_01198) were amplified using 24 cycles. Primers for L19 were included as an internal amplification control. PCR products were separated on a 5% polyacrylamide gel and exposed to X-OMAT AR autoradiographic film. Products were quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc.). RT-PCR primers were generated using a web-based prediction program (Rozen, S., and H. Skaletsky, 1998 Primer3; code available at software/other/primer3. html). TSG-6 primers were Sense 5 -ttccatgtctgtgctgctggatgg-3 and Antisense 5 -agcctggatcatgttcaaggtcaaa-3. L19 primers were Sense 5 -ctgaaggtcaaagggaatgtg-3 and Antisense 5 -ggacagagtcttgatgatctc-3. COX-2 primers were Sense 5 -tgtacaagcagtggcaaagg-3 and Antisense 5 -gctgtggatcttgcacattg-3. RT-PCR products were verified by sequencing (TSG-6 and COX-2) or based on previously published work [L19 (31)].

3 4378 Endocrinology, October 2003, 144(10): Ochsner et al. TSG-6 Protein Acts in COC Expansion Analysis of TSG-6/I I complex formation TSG-6/I I complexes were formed in vitro from human recombinant TSG-6 and I I, purified from human serum (kindly provided by Professor Erik Fries, Uppsala, Sweden), as described previously (29) in the absence or presence of the rat antihuman monoclonal antibodies Q75 and A38 (26). Briefly, full-length TSG-6 (at 80 g/ml final concentration; 2.7 m based on a mass of 30 kda) was incubated with I I (320 g/ml final concentration; 1.8 m based on a mass of 180 kda) in the absence or presence of Q75 or A38 (1 or 10 g; 0.27 or 2.7 m, respectively), in 20 mm HEPES-HCl (ph 7.5), 150 mm NaCl, 5 mm MgCl 2 (total volume 25 l) for 5 or 120 min at 37 C. TSG-6 was incubated individually, for 5 or 120 min, under identical conditions as a control. The reaction mixtures (2.5 l) were analyzed by Western blotting with a rabbit antihuman polyclonal serum against TSG-6 as described by Nentwich et al. (29). TSG-6/I I complexes were also formed in vitro using forskolin (10 m) to stimulate mouse COC expansion during culture in the absence or presence of 5 g/ml of either the A38 or Q75 monoclonal antibodies. COCs were collected after 24 h in culture, treated with hyaluronidase (10 U) and chondroitinase (0.04 U) at 37 C for 3 h before denaturation SDS/2-ME. Western analysis of COC extracts with the A38 antibody was used to detect TSG-6/I I complexes formed by this method. Results Induction of COC expansion and TSG-6 synthesis Unexpanded COCs were removed from preovulatory follicles of PMSG-primed mice and cultured in defined medium containing 1% FBS with or without FSH (Fig. 1A). When COCs were cultured in the absence of hormone (control), most of the cumulus cells remained tightly packed around the oocyte with the zona pellucida distinctly visible. In contrast, COCs cultured for 24 h with agonists that stimulate camp production (forskolin, FSH, and PGE 2 ) exhibited a marked structural change. Specifically, the cumulus cells dispersed away from the oocyte into a three dimensional spherical structure with the oocyte at its center. This rearrangement of the cells blurred the image of the zona pellucida and the cell mass appeared dense/fuzzy due to the fact that not all cells were in the plane of vision. Very few cells became flattened on the culture dish with the majority remaining suspended in the expanded complex. The dynamics of FSH-induced COC expansion and matrix formation were clearly observed with time-lapse imaging (published as supplemental movie 1 on The Endocrine Society s Journals Online web site at To determine the capacity of in vitro expanded COCs to produce TSG-6 and the extent to which the secreted TSG-6 protein is incorporated into the de novo hyaluronan matrix components of the COC expansion reactions were separated into three fractions and analyzed. The medium in which the expansion reaction took place was collected separately from the expanded COCs. The COCs were separated into the two remaining fractions via hyaluronidase and chondroitinase digestion under conditions that did not result in cell lysis. Intact cumulus cells and oocytes were separated and collected from the digestion reaction by centrifugation, and the remaining supernatant consisting of the digested extracellular matrix was collected. As seen in Fig. 1B, TSG-6 protein was not detected in any of the three fractions in nonstimulated control (C) COCs. Additionally, TSG-6 protein was not detected in the cumulus cell fraction of COCs cultured with FSH or PGE 2, suggesting that TSG-6 does not become tightly associated with cell membranes and is probably not involved in anchoring the matrix to the cell surface. However, TSG-6 protein was present in the matrix and media fractions of FSH and PGE 2 stimulated COCs (Fig. 1B). Within the matrix fraction two TSG-6 bands were detected. The approximately 120-kDa TSG-6 band (i) represents complexes of TSG-6 and the heavy chains of I I as previously observed in vivo (17). This band accounted for approximately 80% of the TSG-6 detected in this fraction, whereas the remaining 20% was the 36-kDa free form of TSG-6 (ii) (Fig. 1C). This 4:1 ratio of 120-kDa species to 36-kDa monomer was observed in three separate experiments and is similar to results obtained in vivo (17, 24). Analysis of the media fraction also revealed the presence of both the 120-kDa and the 36-kDa forms of TSG-6 representing protein, which was not incorporated into a stable matrix or which detached in the isolation process (Fig. 1B). Thus, TSG-6 and I I form the same stable complex during in vitro stimulated expansion as observed in vivo. COC expansion depends on multiple signaling pathways To further define the signaling pathway(s) downstream of camp stimulated COC expansion, chemical inhibitors of known signaling pathways were used to determine their involvement. As seen in Fig. 2A, forskolin, a direct activator of adenylyl cyclase, was able to stimulate COC expansion. When COCs were pretreated with H89, an inhibitor of the camp-dependent protein kinase A, forskolin-mediated expansion was impaired because cumulus cell movement away from the oocyte was limited (Fig. 2A). Su et al. (32) showed that MAPK activity within cumulus cells was necessary for gonadotropin stimulation of oocyte meiotic resumption and cumulus expansion. The MEK-1 inhibitor PD98059 that blocks the downstream activation of ERK pathways and the p38 MAPK inhibitor SB both completely blocked forskolin-induced COC expansion, indicating that the activity of these kinase cascades is critical for this process (Fig. 2A). In both cases, cumulus cells remained tightly packed around the oocyte with no visible expansion. The fact that both MAPK inhibitors were able to block COC expansion in vitro may indicate that at least two separate, independent pathways are operating downstream of camp production. On the other hand, preincubation with the phosphatidylinositol 3-kinase specific inhibitor LY294002, which blocks FSH/ camp mediation of PKB phosphorylation (28), did not alter COC expansion as greatly as the other inhibitors, indicating this cascade may not be required for camp induced expansion (Fig. 2A). To assess whether these inhibitors were able to affect TSG-6 mrna expression, COCs were pretreated with SB then stimulated with forskolin for 24 h. Inhibition of p38 MAPK with SB resulted in the inhibition of TSG-6 as well as COX-2 mrna in cultured COCs (Fig. 2B). Inhibition of TSG-6 mrna synthesis correlated with the inhibition of COC expansion. Together, the data suggest that in vitro camp-mediated COC expansion depends on the activation of protein kinase A as well as multiple MAPKs independently of phosphatidylinositol 3-kinase and in the case of TSG-6 mrna expression, on p38 MAPK specifically.

4 Ochsner et al. TSG-6 Protein Acts in COC Expansion Endocrinology, October 2003, 144(10): FSH stimulated expansion and TSG-6 production in EP2 null COCs FSH has the ability to induce expansion of COCs from EP2 null mice in vitro despite the abortive expansion of EP2 null COCs in vivo (7, 33). Thus, FSH activation of its receptor and the resulting production of camp are able to compensate for the loss of PGE 2 signaling via the EP2 receptor. To determine if FSH is able to stimulate the production of TSG-6 by EP2 null COCs during in vitro expansion, EP2 heterozygote and null COCs were stimulated with FSH or PGE 2. As shown in Fig. 3A, FSH stimulated COC expansion of both the heterozygote and null complexes whereas PGE 2 stimulated expansion of the EP2 heterozygote complexes but not the EP2 null complexes. The nonexpanded complexes looked similar to those incubated without agonists (as in Fig. 1A, no hormone). Western blot analysis of EP2 heterozygote and null COCs stimulated with FSH demonstrated that TSG-6 protein is synthesized in null COCs in response to this hormone (Fig. 3B). The approximately 120-kDa form of TSG-6 produced in these cultures shows that FSH is able to compensate for the loss of PGE 2 cell signaling in EP2 null COCs. Figure 3C displays the incorporation of I I components into the matrix of EP2 null COCs stimulated with FSH. The blot from Fig. 3B was stripped and reprobed with a polyclonal antibody against I I. The approximately 80-kDa heavy chains of I I were detected as well as the 120-kDa complex of TSG-6 and heavy chain indicating that this critical component of COC expansion is incorporated into the matrix of EP2 null COCs induced to expand in vitro with FSH. TSG-6 is critical for the formation and stabilization of the expanded COC complex Having determined that gonadotropin and prostaglandin stimulation of COC expansion in vitro results in the production of the two known forms of TSG-6 present in the matrix of COCs expanded in vivo, we had a model system in which to analyze the function of TSG-6 in COC expansion. For this, we used two rat monoclonal antihuman TSG-6 antibodies (A38 and Q75) made against the TSG-6 link module (26). The A38 and Q75 monoclonal antibodies both recognize mouse TSG-6 protein on Western blots but only the A38 antibody was able to block hyaluronan binding to recombinant human TSG-6 (26). As in previous experiments, no expansion occurred in the absence of a camp stimulus whereas incubation with forskolin caused COC expansion into a dense appearing, three-dimensional spherical structure surrounding FIG. 1. Stimulation of camp production leads to COC expansion and TSG-6 expression. A, COCs isolated from PMSG primed ovaries of wild-type mice were cultured either with or without FSH (100 ng/ml), forskolin (10 M), or PGE 2 (500 ng/ml) for 24 h. Each treatment consisted of 25 COCs cultured in a 250 l volume in a 96-well culture dish. Panels are bright-field images captured with an inverted microscope at 100 magnification (C, cumulus cells; Z, zona pellucida; O, oocyte). B, Isolated COCs were cultured with 100 ng/ml FSH (F), or 500 ng/ml PGE 2 (P) for 24 h. Control lanes (C) were not cultured with gonadotropin or prostaglandin. Each treatment consisted of 75 COCs cultured in a 2-ml vol in a 24-well culture dish. Representative immunoblot of TSG-6 Western analysis of cultured COC fractions using a rabbit antimouse TSG-6 polyclonal antibody (20). Immunoreactive bands are designated as follows: i, approximately 120-kDa TSG-6/ complex; ii, 36-kDa free TSG-6. C, Graphical representation of TSG-6 immunoreactivity from three independent experiments. Left panel, Relative amounts of TSG-6 protein in the matrix fraction. Right panel, Relative amounts of TSG-6 protein in the media fraction. Results are expressed as the mean SD.

5 4380 Endocrinology, October 2003, 144(10): Ochsner et al. TSG-6 Protein Acts in COC Expansion FIG. 2. Multiple cell signaling pathways impact forskolin induced COC expansion. A, In vitro culture of COCs isolated from PMSG primed ovaries of wild-type mice pretreated for 1 h with either H89 (10 M), PD98059 (20 M), SB (20 M), or LY (25 M) and then stimulated with forskolin (10 M) for 24 h. Each treatment consisted of 50 COCs cultured in a 2-ml vol in a 24-well culture dish. Panels are bright-field images captured with an inverted microscope at 100 magnification. Representative of two separate experiments. B, Semiquantitative RT-PCR analysis of cumulus cell RNA isolated after 24 h culture from one experiment. Similar results were obtained in a separate experiment in which cultured granulosa cells were pretreated with SB and then stimulated with forskolin for 4 h and when the samples were analyzed in the same RT-PCRs (data not shown). As in A, 200 COCs were pretreated with or without SB before the addition of forskolin for 24 h. Left panel, TSG-6 RT-PCR analysis. Right panel, COX-2 RT-PCR analysis. the oocyte (Fig. 4). The simultaneous incubation of COCs with the control Q75 antibody did not alter forskolin stimulated expansion (Fig. 4, Q75 panel; see supplemental movie 2 on The Endocrine Society s Journals Online web site at In sharp contrast, the simultaneous incubation of COCs with forskolin and the A38 antibody severely altered the expansion process (Fig. 4, A38 panel; see supplemental movie 3 on The Endocrine Society s Journals Online web site at endojournals.org). Expansion appeared to begin normally but became disorganized and disrupted (see supplemental movie 3). Specifically, as the cumulus cells moved away from the oocyte, they began to scatter. Individual cumulus cells in the plane of the culture dish were clearly visible and were not attached to a matrix or to each other. Quantitative results of two experiments (n 100 COCs) indicate that expansion occurred in more than 90% of COCs exposed to forskolin in the absence or presence of the Q75 nonblocking antibody. In contrast, expansion was disrupted (i.e. in which cumulus cells detached and fell to the culture plate) in more than 80% of COCs exposed to forskolin in the presence of the A38 antibody (Fig. 4B). To determine if the A38 antibody blocked the formation of TSG-6/I I complexes, two approaches were taken. When purified human TSG-6 was incubated with purified human I I, for 5 or 120 min, an approximately 120-kDa species was

6 Ochsner et al. TSG-6 Protein Acts in COC Expansion Endocrinology, October 2003, 144(10): FIG. 3. FSH but not PGE 2 stimulates COC expansion and TSG-6 production by EP2 null COCs. Isolated COCs from EP2 heterozygous or null mice were cultured with 100 ng/ml FSH or 500 ng/ml PGE 2 for 24 h. A, Panels are bright-field images captured with an inverted microscope at 100 magnification. Each treatment consisted of 25 COCs cultured in a volume of 250 l in a 96-well culture dish. B and C, Representative immunoblots of TSG-6 and I I Western analysis respectively of EP2 heterozygous and null COCs cultured with 100 ng/ml FSH for 24 h. Each lane consists of 80 COCs cultured in vitro in a volume of 2 ml in a 24-well culture dish. The immunoblot in B was first probed with the rat antihuman monoclonal A38 TSG-6 antibody (26) then stripped and reprobed with a polyclonal I I antibody (C). Band designations are as in Fig. 1. vi, Free heavy chains from I I. Representative of three separate experiments. detectable by Western blotting with an antihuman TSG-6 polyclonal antiserum (29) (Fig. 5A). The formation of this approximately 120-kDa TSG-6/I I complex could be inhibited at both time points by the inclusion of 10 g of the A38 antibody (i.e. TSG-6 and A38 at equimolar concentrations), but not when it was present at a sub-stoichiometric amount (i.e. 1 g). The Q75 antibody, however, had no effect on complex formation at either concentration (Fig. 5A). These data reveal that the monoclonal antibody A38 prevents the production of the TSG-6/I I complex in vitro [in addition to inhibiting hyaluronan binding to TSG-6 (26)]. The monoclonal antibody A38 (but not the Q75 antibody) also blocked the FIG. 4. TSG-6 plays a critical role in the formation and stabilization of the expanded COC complex. A, COCs isolated from PMSG primed ovaries of wild-type mice were cultured either with or without forskolin for 24 h. In the case of forskolin stimulation the monoclonal, anti-tsg-6 antibodies A38 and Q75 were added at 5 g/ml as indicated. Each treatment consisted of 25 COCs cultured in a 1-ml vol in a 24-well culture dish. Panels are bright-field images captured with an inverted microscope at 100 magnification. Representative of three separate experiments. B, Graphical representation of COC expansion from two independent experiments. Results are expressed as the mean SD. formation of the TSG-6/I I complex by forskolin-stimulated COC in vitro (Fig. 5B). These data are the first to document that the A38 antibody blocks the interaction of TSG-6 and I I

7 4382 Endocrinology, October 2003, 144(10): Ochsner et al. TSG-6 Protein Acts in COC Expansion not only in a biochemical assay but also in the context of a physiologically relevant model. FIG. 5. A38 antibody blocks the formation of TSG-6/I I complexes in vitro. A, Purified human TSG-6 and I I were incubated together in the presence or absence of the indicated concentrations of either the A38 or the Q75 monoclonal antibodies. Reactions were carried out for 5 or 120 min as indicated. Immunoblots represent TSG-6 Western analysis of in vitro binding assays carried out using the rabbit antihuman TSG-6 polyclonal antiserum (29). Band designations are as in Fig. 1. B, In vitro expansion of COCs isolated from PMSG-treated wild-type mice using 10 M forskolin in the presence or absence of either A38 or Q75 monoclonal antibodies (5 g/ml). Upper panel, Representative TSG-6 Western of COCs 24 h after culture using the A38 monoclonal Discussion Release of a fertilizable oocyte at the time of ovulation requires rupture of the surface epithelium and transport of the expanded COC through the ovulation pore. This latter event is dependent on the synthesis of hyalruonan and its assembly with hyaluronan binding proteins to form the stable, expanded mucoid matrix (8 12). Two factors that have been shown to be necessary for proper matrix formation and cumulus expansion are the enzyme COX-2, which generates prostaglandins (1, 2), and EP2 receptors localized to cumulus cells (7). We have recently shown that synthesis of the hyaluronan binding protein TSG-6 is severely and selectively impaired in cumulus cells (compared with granulosa cells) of both the COX-2 and EP2 null mice (24). These observations led us to hypothesize that this inflammation related factor must be made by and secreted from the cumulus cells where it is critical during the process of matrix formation and expansion. Herein we have documented clearly that TSG-6 message and protein are induced in isolated (granulosa cell free) COCs that expand in vitro in response to various agonists that stimulate camp (including PGE 2, FSH, and forskolin) and that camp-mediated COC expansion and TSG-6 expression require specific kinase cascades, most notably p38 MAPK. Furthermore, the data show that TSG-6 is secreted and is associated with matrix components, most notably, I I, a serum factor that is also obligatory for matrix formation and ovulation (9, 10). Most importantly, we show that a rat antihuman TSG-6 monoclonal antibody (A38) directed to specific target amino acids within the TSG-6 link module blocks the interaction of TSG-6 with the heavy chains of the serum-derived factor I I and prevents proper expansion of the COC. These observations provide direct functional evidence that TSG-6 and its interactions with the heavy chains are essential for the formation and/or stabilization of the matrix. Specifically, the data show that the ratio of free/unbound TSG-6 to the bound TSG-6/I I complex in the COC matrices expanded in vitro was constant in several different experiments and similar to that observed in vivo (17, 24). The 50% loss of TSG-6 protein in COCs isolated from preovulatory follicles of COX-2 and EP2 null mice appears to be sufficient to disrupt this balance and lead to aberrant cumulus expansion and ovulation in these animal models (24). Despite high levels of I I provided in the culture media (1% serum), free TSG-6 is still present. Therefore, some unbound TSG-6 may be unavailable to I I. Alternatively, some unbound TSG-6 may be important for a hyaluronan independent action of TSG-6. Importantly, unbound/free TSG-6 was only observed in COCs collected from follicles in vivo beginning 8 12 h after hcg, a time just preceding ovulation (24). The possible function, if any, of this free pool of TSG-6 is not yet known. To analyze the functional role of the TSG-6 in COC ex- antibody (26). Fifty COCs were loaded per lane. Lower panel, Immunoblot in the upper panel was stripped and reprobed for PKB expression as a loading control. A and B are representative of at least three separate experiments.

8 Ochsner et al. TSG-6 Protein Acts in COC Expansion Endocrinology, October 2003, 144(10): pansion, two specific antibodies (A38 and Q75) generated against a TSG-6/CD44 chimera were used. The A38 antibody but not the Q75 antibody has previously been shown to block the interactions of TSG-6 with hyaluronan (26). Herein we show that the A38 antibody, but not the Q75 antibody, also blocks TSG-6 binding to the heavy chains of I I in both a biochemical assay as well as during a physiological process. This is consistent with the recent findings of Fulop et al. (25), who suggest that thetsg-6/heavy chain complexes are likely to be intermediates in the covalent transfer of heavy chains to hyaluronan. The hyaluronan-binding properties of the TSG-6 link module may also be involved in this transfer reaction. Additionally, an important role for TSG-6 in crosslinking hyaluronan cannot be excluded. The ability of the A38 antibody to disrupt COC expansion most likely occurs via its blocking of TSG-6 function; the epitope of A38 has been localized to Y78 of the TSG-6 link module (26), which forms part of the hyaluronan binding site (34, 35). In addition, TSG-6 has been found to have functions independent of its hyaluronan binding activity. For example, the link module alone was sufficient to block neutrophil migration and potentiate I I anti-plasmin activity, neither of which correlates with the ability of the link module to bind hyaluronan (34). In light of these data, it is possible that the A38 antibody simultaneously not only blocks TSG-6 binding to I I and hyaluronan but may also alter hyaluronan-independent functions of the TSG-6 link module, thereby contributing to the A38 block of COC expansion. Although our data do not resolve which mechanism is most critical, our results demonstrate that the TSG-6 link region is necessary for proper matrix formation and stabilization required for COC expansion. In summary, the microenvironment and selected signaling events within the COC of preovulatory follicles are essential for proper formation of an extracellular matrix critical for ovulation. Induction of COX-2 expression and prostaglandin signaling via EP2 receptors in cumulus cells are emerging as key (inflammation-like) regulators of at least one critical COC component, the hyaluronan binding protein TSG-6. We show for the first time that blocking the activity of the TSG-6 link module disrupts normal COC expansion most likely by preventing its association with I I and other matrix molecules such as hyaluronan. These results have implications for understanding unresolved cases of infertility and for improving IVF procedures. Acknowledgments We thank Professor Erik Fries for providing human I I and Dr. Jayne Lesley for providing the A38 and Q75 rat monoclonal antibodies. Received April 17, Accepted July 7, Address all correspondence and requests for reprints to: J. S. Richards, Baylor College of Medicine, One Baylor Plaza, Houston, Texas joanner@bcm.tmc.edu. This work was supported by the following sources of funding: HD (to J.S.R.), NIH-HD (training grant), Medical Research Council (UK) (to A.J.D.), and GM (to R.M.B.). See the supplemental data published on The Endocrine Society s Journals Online web site at References 1. Sirois J, Simmons DL, Richards JS 1992 Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles. Induction in vivo and in vitro. J Biol Chem 267: Wong WY, Richards JS 1992 Induction of prostaglandin H synthase in rat preovulatory follicles by gonadotropin-releasing hormone. 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