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

0013-7227/04/$15.00/0 Printed in U.S.A. Endocrinology 145(10):4603– 4614 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2004-0542 Down-Regulated Expression of A Disintegrin and Metalloproteinase with Thrombospondin-Like Repeats-1 by Progesterone Receptor Antagonist Is Associated with Impaired Expansion of Porcine CumulusOocyte Complexes MASAYUKI SHIMADA, MASAHIDE NISHIBORI, YASUHISA YAMASHITA, JUNYA ITO, TAKAHIDE MORI, AND JOANNE S. RICHARDS Department of Applied Animal Science (M.S., M.N., Y.Y., J.I.), Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan; Infertility Center (T.M.), Daigo-Watanabe Clinic, Kyoto 601-1375, Japan; and Department of Molecular and Cellular Biology (J.S.R.), Baylor College of Medicine, Houston, Texas 77030 ADAMTS-1, a member of the A disintegrin and metalloproteinase family of proteases, is expressed in rodent follicles via progesterone receptor (PR)-dependent pathways. However, the functional relationship between ADAMTS-1 expression and PR has not been studied extensively in other species. In the present study, we investigated the time-dependent changes in ADAMTS-1 expression in cumulus cells of porcine cumulus-oocyte complexes (COCs), and the roles of ADAMTS-1 in cumulus expansion during in vitro maturation of oocytes. ADAMTS-1 message was not detected in cumulus cells at the time of collection from the follicles. In response to gonadotropins, ADAMTS-1 mRNA was dramatically up-regulated and reached a maximum at 20 h. The level of mature ADAMTS-1 protein increased in a time-dependent manner with a maxi- T HE SURGE OF LH induces marked functional (endocrine, biochemical, and molecular) changes in the preovulatory follicle. Estrogen concentrations decline in follicular fluid as a consequence of the reduced expression of aromatase and 17␣-hydroxysteroid dehydrogenase in granulosa and theca cells, respectively, whereas progesterone concentrations rise in association with induction of P450 side-chain cleavage enzyme, CYP11A1, in granulosa cells (1, 2). The LH surge also induces ovulation, a process that is inhibited in rats by treatment with either antiprogesterone antiserum (3) or epostane (4), a compound that blocks the synthesis of progesterone, suggesting that the LH-induced increase of progesterone is essential for ovulation. Many of the biological activities of progesterone are mediated by an intracellular receptor, a hormonally regulated DNA-binding protein that belongs to a superfamily of ligand-activated transcription factors (5, 6). Progesterone receptor (PR) Abbreviations: ADAMTS, A disintegrin and metalloproteinase with thrombospondin-like repeats; AR, androgen receptor; COC, cumulusoocyte complex; DMSO, dimethylsulfoxide; ER, estrogen receptor; HA, hyaluronan; HAS, HA synthase; IaI, inter-␣-trypsin inhibitor; PR, progesterone receptor; PR-A, PR type A; PR-B, PR type B; PRKO, null for PR; T-PBS, Tween 20/PBS; TSG-6, TNF␣-stimulated gene 6. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. mum level at 40 h. The induction of ADAMTS-1 mRNA and protein was significantly decreased by the addition of PR antagonist RU486 to the cultures. However, RU486 did not affect the expression of ADAMTS-4 or factors that had been reported to be required for COC expansion (TSG-6, versican, HA synthase-2). COCs cultured with FSH and LH for 40 h exhibited prominent cumulus expansion. The expansion was reduced significantly by the addition of either RU486 or Galardin, a broad-spectrum matrix metalloproteinase inhibitor. These results suggest that the expression and induction of ADAMTS-1 through receptor-mediated action of progesterone in cumulus cells is one of the essential requirements for gonadotropin-regulated cumulus expansion of porcine COCs. (Endocrinology 145: 4603– 4614, 2004) mRNA is induced in granulosa cells of rat preovulatory follicles by LH (7, 8). Female mice null for PR (PRKO) fail to ovulate, even in response to exogenous hormones (9). Thus, progesterone- and PR-dependent pathways appear to play important roles in the functional changes that occur in follicular cells during ovulation. A disintegrin and metalloproteinase with thrombospondinlike repeats (ADAMTS-1) is a member of the ADAMTS family of metalloproteinases and has been shown to degrade members of the lectican family of proteoglycans (10 –12). ADAMTS-1 has been shown to be induced by LH in granulosa cells via progesterone- and PR-dependent pathways (13–15). Furthermore, mice null for ADAMTS-1 exhibit impaired fertility (16, 17), suggesting that ADAMTS-1 impacts some aspect of the ovulation process. Recently Russell et al. (18) reported that LH induces in ovulating follicles the expression of versican, one known preferred substrate of ADAMTS-1 (19). Cleavage of versican in ovulated cumulus-oocyte complexes (COCs) has been documented (15), suggesting that ADAMTS-1 or related family members are present and active in the ovulated complexes. Before ovulation, cumulus cells surrounding oocytes synthesize hyaluronan (HA), a glycosoaminoglycan polymer, along with other factors to make a gelatinous matrix, a process that is termed cumulus expansion (20 –22). Versican possesses an N-terminal link-module HA binding domain (23). TNF␣- 4603 4604 Endocrinology, October 2004, 145(10):4603– 4614 Shimada et al. • PR Pathway Acts in COC Expansion stimulated gene 6 (TSG-6) also binds HA and the heavy chains of inter-␣-trypsin inhibitor (IaI) (24 –26). Thus, the expansion of COCs during ovulation involves the organization of the HArich matrix that is comprised of HA, IaI heavy chains, TSG-6, and versican. Collectively, these observations suggest that PR and ADAMTS-1 may impact ovulation via the formation or stabilization of the COC complex. However, this hypothesis has not been confirmed. During in vitro meiotic maturation of COCs, it has been shown in human (27), rat (28), cattle (29), and pig (30 –32) that progesterone is produced by cumulus cells and that the level of progesterone is increased by stimulation with LH and FSH. In our previous studies (33, 34), we showed that the high concentrations of progesterone secreted by porcine COCs accelerated meiotic resumption of oocytes and improved the rate of early embryonic development to the blastocyst stage after in vitro fertilization. We also reported in porcine COCs that the progesterone bound to newly synthesized PR in cumulus cells was associated with reduced proliferative activity of cumulus cells and closed the gap junctional communication within cumulus cells (32, 34). Our previous reports suggest that activation of PR induced the differentiation of cumulus cells. However, little is known about the specific roles of progesterone and PR in either the induction of ADAMTS-1 or cumulus expansion during in vitro maturation of COCs. In this study, we examined the time-dependent chang (...truncated)