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-Stimulated Gene 6 Blocks Cumulus Cell-Oocyte Complex Expansion
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 37232-2372; and Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology (R.H.G.), Rice University, Houston, Texas 77005-1892
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.
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-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 E2 (PGE2) 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, PGE2, EP2 receptor, and selected protein kinase inhibitors on TSG-6 production as well as specific antibodies to the TSG-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 PGE2 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. |
Introduction |
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trypsin inhibitor (II) (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 II 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 II 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 II) 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 II do not become covalently transferred to hyaluronan (25). It is possible therefore that the approximately 120-kDa complexes formed between TSG-6 and II 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 II 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/II 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.
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Materials and Methods |
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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), PGE2 (500 ng/ml), or forskolin (10 µM) in 50–500 µl reactions at 37 C in a humidified incubator (95% air, 5% CO2). After 24 h of incubation, COCs were collected for RNA and protein isolation or visualized using an Axiovert S100 (Zeiss) inverted scope and x10 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 x10 objective was placed inside an incubator with 95% air, 5% CO2. An external power supply was used to drive a 6-V illumination lamp with a blue filter at 3 V. The NIH image program (http://rsb.info.nih.gov/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 µg/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 µM forskolin for 24 h. The concentrations of inhibitors based on previous studies in our laboratory (28) were as follows: PD98059 (20 µM), H89 (10 µM), SB203580 (20 µM), and LY294002 (25 µM).
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% ß-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 II polyclonal antibody (Dako Corp., Carpinteria, CA) was used at a dilution of 1:5000 in PBS reagents. Membranes were then washed 3 x 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 1x Thermocycle buffer, 500 ng oligo-deoxythymidine primer, 1 mM deoxynucleotide triphosphates, 4 mM MgCl2, 20 U RNAsin, and 2.5 U avian myeloblastosis virus-reverse transcriptase in a volume of 20 µl 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), 2.5 U Taq polymerase in 1x Thermocycle buffer, and 2.5 mM MgCl2 in a volume of 100 µl. 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 http://www.genome.wi.mit.edu/genome 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)].
Analysis of TSG-6/II complex formation
TSG-6/II complexes were formed in vitro from human recombinant TSG-6 and II, 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 µg/ml final concentration; 2.7 µM based on a mass of 30 kDa) was incubated with II (320 µg/ml final concentration; 1.8 µM based on a mass of 180 kDa) in the absence or presence of Q75 or A38 (1 or 10 µg; 0.27 or 2.7 µM, respectively), in 20 mM HEPES-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2 (total volume 25 µl) 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 µl) were analyzed by Western blotting with a rabbit antihuman polyclonal serum against TSG-6 as described by Nentwich et al. (29).
TSG-6/II complexes were also formed in vitro using forskolin (10 µM) to stimulate mouse COC expansion during culture in the absence or presence of 5 µg/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/II complexes formed by this method.
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). 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 PGE2) 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 http://endo.endojournals.org).
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, 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 PGE2, 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 PGE2 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 II 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 II 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 SB203580 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 SB203580 then stimulated with forskolin for 24 h. Inhibition of p38 MAPK with SB203580 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.
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, FSH stimulated COC expansion of both the heterozygote and null complexes whereas PGE2 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 PGE2 cell signaling in EP2 null COCs. Figure 3C displays the incorporation of II 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 II. The approximately 80-kDa heavy chains of II 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.
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). 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 http://endo.endojournals.org). 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 http://endo. 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.
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).
To determine if the A38 antibody blocked the formation of TSG-6/II complexes, two approaches were taken. When purified human TSG-6 was incubated with purified human II, for 5 or 120 min, an approximately 120-kDa species was detectable by Western blotting with an antihuman TSG-6 polyclonal antiserum (29) (Fig. 5A). The formation of this approximately 120-kDa TSG-6/II complex could be inhibited at both time points by the inclusion of 10 µg 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 µg). 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/II 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 formation of the TSG-6/II 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 II not only in a biochemical assay but also in the context of a physiologically relevant model.
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Discussion |
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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 II 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/II 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 II provided in the culture media (1% serum), free TSG-6 is still present. Therefore, some unbound TSG-6 may be unavailable to II. 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 expansion, 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 II 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 cross-linking 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 II 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 II 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 II and other matrix molecules such as hyaluronan. These results have implications for understanding unresolved cases of infertility and for improving IVF procedures.
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Acknowledgments |
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I and Dr. Jayne Lesley for providing the A38 and Q75 rat monoclonal antibodies. |
Footnotes |
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See the supplemental data published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.
Abbreviations: COC, Cell-oocyte complex; COX-2, cyclooxygenase 2; EP2, PGE2 receptor subtype; hCG, human chorionic gonadotropin; II, inter- trypsin inhibitor; PMSG, pregnant mare serum gonadotropin; PGE2, prostaglandin E2; SDS, sodium dodecyl sulfate; SDS/2-ME, 2% SDS and 6% ß-mercaptoethanol; TSG-6, TNF--stimulated gene 6.
Received April 17, 2003.
Accepted for publication July 7, 2003.
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 F. J. Diaz, K. Wigglesworth, and J. J. Eppig Oocytes determine cumulus cell lineage in mouse ovarian follicles J. Cell Sci., April 15, 2007; 120(8): 1330 - 1340. [Abstract] [Full Text] [PDF]
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M. Hsieh, D. Lee, S. Panigone, K. Horner, R. Chen, A. Theologis, D. C. Lee, D. W. Threadgill, and M. Conti Luteinizing Hormone-Dependent Activation of the Epidermal Growth Factor Network Is Essential for Ovulation Mol. Cell. Biol., March 1, 2007; 27(5): 1914 - 1924. [Abstract] [Full Text] [PDF]
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 R. Forteza, S. M. Casalino-Matsuda, M. E. Monzon, E. Fries, M. S. Rugg, C. M. Milner, and A. J. Day TSG-6 Potentiates the Antitissue Kallikrein Activity of Inter-{alpha}-inhibitor through Bikunin Release Am. J. Respir. Cell Mol. Biol., January 1, 2007; 36(1): 20 - 31. [Abstract] [Full Text] [PDF]
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M. Shimada, I. Hernandez-Gonzalez, I. Gonzalez-Robanya, and J. S. Richards Induced Expression of Pattern Recognition Receptors in Cumulus Oocyte Complexes: Novel Evidence for Innate Immune-Like Functions during Ovulation Mol. Endocrinol., December 1, 2006; 20(12): 3228 - 3239. [Abstract] [Full Text] [PDF]
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T. Takahashi, J. D. Morrow, H. Wang, and S. K. Dey Cyclooxygenase-2-derived Prostaglandin E2 Directs Oocyte Maturation by Differentially Influencing Multiple Signaling Pathways J. Biol. Chem., December 1, 2006; 281(48): 37117 - 37129. [Abstract] [Full Text] [PDF]
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Y.-Q. Su, M. Nyegaard, M. T. Overgaard, J. Qiao, and L. C. Giudice Participation of Mitogen-Activated Protein Kinase in Luteinizing Hormone-Induced Differential Regulation of Steroidogenesis and Steroidogenic Gene Expression in Mural and Cumulus Granulosa Cells of Mouse Preovulatory Follicles Biol Reprod, December 1, 2006; 75(6): 859 - 867. [Abstract] [Full Text] [PDF]
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I. Hernandez-Gonzalez, I. Gonzalez-Robayna, M. Shimada, C. M. Wayne, S. A. Ochsner, L. White, and J. S. Richards Gene Expression Profiles of Cumulus Cell Oocyte Complexes during Ovulation Reveal Cumulus Cells Express Neuronal and Immune-Related Genes: Does this Expand Their Role in the Ovulation Process? Mol. Endocrinol., June 1, 2006; 20(6): 1300 - 1321. [Abstract] [Full Text] [PDF]
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M. Shimada, I. Hernandez-Gonzalez, I. Gonzalez-Robayna, and J. S. Richards Paracrine and Autocrine Regulation of Epidermal Growth Factor-Like Factors in Cumulus Oocyte Complexes and Granulosa Cells: Key Roles for Prostaglandin Synthase 2 and Progesterone Receptor Mol. Endocrinol., June 1, 2006; 20(6): 1352 - 1365. [Abstract] [Full Text] [PDF]
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 W. Selbi, A. J. Day, M. S. Rugg, C. Fulop, C. A. de la Motte, T. Bowen, V. C. Hascall, and A. O. Phillips Overexpression of Hyaluronan Synthase 2 Alters Hyaluronan Distribution and Function in Proximal Tubular Epithelial Cells J. Am. Soc. Nephrol., June 1, 2006; 17(6): 1553 - 1567. [Abstract] [Full Text] [PDF]
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V. Sriraman, M. D. Rudd, S. M. Lohmann, S. M. Mulders, and J. S. Richards Cyclic Guanosine 5'-Monophosphate-Dependent Protein Kinase II Is Induced by Luteinizing Hormone and Progesterone Receptor-Dependent Mechanisms in Granulosa Cells and Cumulus Oocyte Complexes of Ovulating Follicles Mol. Endocrinol., February 1, 2006; 20(2): 348 - 361. [Abstract] [Full Text] [PDF]
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T. Pakarainen, F.-P. Zhang, L. Nurmi, M. Poutanen, and I. Huhtaniemi Knockout of Luteinizing Hormone Receptor Abolishes the Effects of Follicle-Stimulating Hormone on Preovulatory Maturation and Ovulation of Mouse Graafian Follicles Mol. Endocrinol., October 1, 2005; 19(10): 2591 - 2602. [Abstract] [Full Text] [PDF]
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S. A. Kuznetsova, A. J. Day, D. J. Mahoney, M. S. Rugg, D. F. Mosher, and D. D. Roberts The N-terminal Module of Thrombospondin-1 Interacts with the Link Domain of TSG-6 and Enhances Its Covalent Association with the Heavy Chains of Inter-{alpha}-trypsin Inhibitor J. Biol. Chem., September 2, 2005; 280(35): 30899 - 30908. [Abstract] [Full Text] [PDF]
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D. J. Mahoney, B. Mulloy, M. J. Forster, C. D. Blundell, E. Fries, C. M Milner, and A. J. Day Characterization of the Interaction between Tumor Necrosis Factor-stimulated Gene-6 and Heparin: IMPLICATIONS FOR THE INHIBITION OF PLASMIN IN EXTRACELLULAR MATRIX MICROENVIRONMENTS J. Biol. Chem., July 22, 2005; 280(29): 27044 - 27055. [Abstract] [Full Text] [PDF]
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M. S. Rugg, A. C. Willis, D. Mukhopadhyay, V. C. Hascall, E. Fries, C. Fulop, C. M. Milner, and A. J. Day Characterization of Complexes Formed between TSG-6 and Inter-{alpha}-inhibitor That Act as Intermediates in the Covalent Transfer of Heavy Chains onto Hyaluronan J. Biol. Chem., July 8, 2005; 280(27): 25674 - 25686. [Abstract] [Full Text] [PDF]
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J. M. A. Emmen, J. F. Couse, S. A. Elmore, M. M. Yates, G. E. Kissling, and K. S. Korach In Vitro Growth and Ovulation of Follicles from Ovaries of Estrogen Receptor (ER){alpha} and ER{beta} Null Mice Indicate a Role for ER{beta} in Follicular Maturation Endocrinology, June 1, 2005; 146(6): 2817 - 2826. [Abstract] [Full Text] [PDF]
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C. D. Blundell, A. Almond, D. J. Mahoney, P. L. DeAngelis, I. D. Campbell, and A. J. Day Towards a Structure for a TSG-6{middle dot}Hyaluronan Complex by Modeling and NMR Spectroscopy: INSIGHTS INTO OTHER MEMBERS OF THE LINK MODULE SUPERFAMILY J. Biol. Chem., May 6, 2005; 280(18): 18189 - 18201. [Abstract] [Full Text] [PDF]
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J. S. Richards, I. Hernandez-Gonzalez, I. Gonzalez-Robayna, E. Teuling, Y. Lo, D. Boerboom, A. E. Falender, K. H. Doyle, R. G. LeBaron, V. Thompson, et al. Regulated Expression of ADAMTS Family Members in Follicles and Cumulus Oocyte Complexes: Evidence for Specific and Redundant Patterns During Ovulation Biol Reprod, May 1, 2005; 72(5): 1241 - 1255. [Abstract] [Full Text] [PDF]
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K. W. Sanggaard, H. Karring, Z. Valnickova, I. B. Thogersen, and J. J. Enghild The TSG-6 and I{alpha}I Interaction Promotes a Transesterification Cleaving the Protein-Glycosaminoglycan-Protein (PGP) Cross-link J. Biol. Chem., March 25, 2005; 280(12): 11936 - 11942. [Abstract] [Full Text] [PDF]
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M. Shimada, M. Nishibori, Y. Yamashita, J. Ito, T. Mori, and J. S. Richards 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 Endocrinology, October 1, 2004; 145(10): 4603 - 4614. [Abstract] [Full Text] [PDF]
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 Y.-C. Hu, P.-H. Wang, S. Yeh, R.-S. Wang, C. Xie, Q. Xu, X. Zhou, H.-T. Chao, M.-Y. Tsai, and C. Chang Subfertility and defective folliculogenesis in female mice lacking androgen receptor PNAS, August 3, 2004; 101(31): 11209 - 11214. [Abstract] [Full Text] [PDF]
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D. Mukhopadhyay, A. Asari, M. S. Rugg, A. J. Day, and C. Fulop Specificity of the Tumor Necrosis Factor-induced Protein 6-mediated Heavy Chain Transfer from Inter-{alpha}-trypsin Inhibitor to Hyaluronan: IMPLICATIONS FOR THE ASSEMBLY OF THE CUMULUS EXTRACELLULAR MATRIX J. Biol. Chem., March 19, 2004; 279(12): 11119 - 11128. [Abstract] [Full Text] [PDF]
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