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

0013-7227/03/$15.00/0 Printed in U.S.A. Endocrinology 144(3):1008 –1019 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-220435 Decreased Expression of Tumor Necrosis Factor-␣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, JOANNE S. RICHARDS AND 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 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␣-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 C UMULUS-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 serumderived factor inter-␣-trypsin inhibitor (I␣I), which by covalent cross-linking of its heavy chains to HA functions to stabilize the cumulus cell matrix (5). When assembly of I␣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 Abbreviations: C, Chondroitinase; COC, cumulus oocyte complex; COX, cyclooxygenase; H, hyaluronidase; HA, hyaluronan; HAS-2, HA synthase-2; hCG, human chorionic gonadotropin; I␣I, inter-␣-trypsin inhibitor; PG, prostaglandin; PMSG, pregnant mare serum gonadotropin; SDS, sodium dodecyl sulfate; SDS/2-ME, SDS and 6% ␤-mercaptoethanol; TSG-6, TNF␣-stimulated gene 6. 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␣-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. (Endocrinology 144: 1008 –1019, 2003) 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-␣-stimulated gene 6 (TSG-6) (9) initially characterized as a TNF-␣ 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 ratelimiting 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 (17–19). COX-2 is the critical 1008 Ochsner et al. • Regulation of TSG-6 mRNA in Cumulus Cells 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-1␤ will rescue infertility in these mice and restore ovulation rates. Additionally, mice null for one of the PGE2 receptors, EP2, are also infertile (23–25) 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 in COX-2 null mice as well as TSG-6 and HAS-2 expression in EP2 null mice. Materials and Methods Materials Pregnant mare serum gonadotropin (PMSG) (Gestyl) was purchased from Professional Compounding Center of America (Houston, TX). hCG (Pregnyl) was purchased from (...truncated)