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Molecular Endocrinology 20(11):27842795 Copyright 2006 by The Endocrine Society doi: 10.1210/me.2006-0093

A Novel Pathway Involving Progesterone Receptor, Endothelin-2, and Endothelin Receptor B Controls Ovulation in Mice
Gopinath S. Palanisamy, Yong-Pil Cheon,* Jaeyeon Kim,* Athilakshmi Kannan, Quanxi Li, Marcey Sato, Srinivasa R. Mantena, Regine L. Sitruk-Ware, Milan K. Bagchi, and Indrani C. Bagchi Department of Veterinary Biosciences (G.S.P., Y.-P.C., A.K., Q.L., S.R.M., I.C.B.) and Department of Molecular and Integrative Physiology (J.K., M.S., M.K.B.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61802; and Population Council (R.L.S.-W.), New York, New York 10021
The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture. (Molecular Endocrinology 20: 27842795, 2006)

VULATION IN MAMMALS is a unique biological process in which a mature follicle within the ovary ruptures and releases a fertilizable oocyte. In most mammals, the follicle protrudes markedly from the ovarian surface at the time of ovulation, and in many instances a thin translucent stigma, the macula pellucida, forms at the apex of the follicle as a sign of impending rupture (1). It has been suggested that the rupture of the follicle is a culmination of a profound inflammatory response (2). Although morphological features of the ovulatory process are well described, the molecular cascade of events that ultimately leads to follicular rupture remains largely unknown (24). It is
First Published Online August 3, 2006 * Y.-P.C. and J.K. contributed equally to the manuscript. Abbreviations: BW, Body weight; CG, chorionic gonadotropin; cGK II, cGMP-dependent protein kinase II; COC, cumulus-oocyte complex; DIG, Digoxygenin; eNOS, endothelial NO synthase; ET, endothelin; ETR, ET receptor; NO, nitric oxide; P, progesterone; PGR, progesterone receptor; PMA, phorbol 12-myristate 13 acetate; PMSG, pregnant mare serum gonadotropin; PRKO, Pgr knockout; VIC, vasoactive intestinal contractor; WT, wild type. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

widely thought, however, that a preovulatory surge of the LH initiates the expression of critical gene pathways that underlie the morphological and biochemical alterations that occur before ovulation. Several lines of evidence suggest that progesterone (P) receptor (PGR) is a key regulatory molecule in the ovary, acting downstream of LH during ovulation. LH rapidly and selectively induces PGR expression in mural granulosa cells of the preovulatory follicles (57). Earlier studies in rats indicated that ovulation is inhibited by treatment with either an anti-P antiserum, or epostane, a compound that blocks the synthesis of P (8, 9). Administration of PGR antagonists, such as RU486 and Org-31710, also inhibited ovulation, suggesting that P acting via its receptor controls ovulation (10, 11). Creation of a Pgr knockout (PRKO) mouse model by Lydon et al. (12) and Conneely et al. (13) provided an unequivocal demonstration of the critical role played by this receptor in the ovulatory process. The PRKO mice failed to ovulate even when stimulated with exogenous gonadotropins. In these mutant mice, follicles developed normally to the preovulatory stage, and the characteristic expansion of the cumulus-oocyte complex (COC) appeared to occur. The follicles, however, failed to rupture, resulting in trapped oocytes within the ovarian tissue (12).
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PGR is a well-known ligand-inducible transcription factor (14, 15). It is likely that hormone-occupied PGR triggers the expression of specific gene networks in the granulosa cells of the ovarian follicles, and the products of these genes mediate the hormonal effects during ovulation. To understand the molecular basis of the ovulatory program, it is critical to identify the PGRregulated molecules that are induced or suppressed at the time of ovulation and analyze their functional roles during this complex physiological process. In this study, we employed CDB-2914, a novel antiprogestin, as a tool to uncover the PGR-regulated pathways during ovulation in mice. This compound binds to PGR with high affinity and impairs its generegulatory function (1619). Our results indicated that administration of CDB-2914 strongly blocked LH-induced ovulation in mice. Using oligonucleotide microarrays, we identified several genes the expression of which is markedly repressed in response to CDB2914 in mouse ovary at the time of ovulation. One of these genes encoded the vasoactive intestinal contractor (VIC), the mouse ortholog of human endothelin (ET)-2. ET-2 is a vasoactive molecule that mediates various physiological functions such as vasodilation, vasoconstriction, and smooth muscle contraction in other tissues (2022). Here we present evidence that a unique signaling pathway in the preovulatory follicle, involving LH, PGR, ET-2, and its downstream target molecules, plays a critical role during ovulation.

treatment with human (h) chorionic gonadotropin (CG) to induce the ovulatory response. Vehicle or CDB2914 was administered ip 1 h before the hCG injection. The number of released oocytes was counted 18 h after hCG treatment. As shown in Fig. 1A, an average of 3040 oocytes were released from the ovaries of each mouse subjected to the superovulation protocol. Histological analysis of the ovarian sections of vehicletreated mice at 18 h after hCG injection exhibited numerous corpora lutea, indicating efficient ovulation (Fig. 1B, panel a). Administration of increasing doses of CDB-2914 led to a progressive decline in the number of released oocytes when compared with mice that received vehicle alone (Fig. 1A). Ovarian sections of CDB-2914-treated mice showed the presence of many unruptured follicles at 18 h after hCG treatment, confirming impaired ovulation (Fig. 1B, panel b). The failure of follicular rupture upon CDB-2914 treatment closely resembled the ovarian phenotype of the PRKO mice and is consistent with the previously reported antiprogestational activity of this drug. Collectively, our results indicated that CDB-2914 exerts its inhibitory effects on ovulation in mice by blocking the function of PGR. Identification of ET-2 as a Downstream Target of Regulation by CDB-2914 in the Ovary We next employed CDB-2914 to identify genes that are regulated by PGR during ovulation. Mice were subjected to superovulation in the absence and in the presence of CDB-2914 as described above, and ovaries were collected 12 h after hCG treatment. mRNA was isolated from vehicle or CDB2914-treated mice and hybridized to mouse oligonucleotide microarrays (Affymetrix, Santa Clara, CA) representing approximately 11,000 genes as described previously (23). Our studies identified several mRNAs the expression of which in the ovary altered at least 2-fold in response to CDB-2914 (Cheon, Y.C., and I.C. Bagchi, unpublished results). One of the mRNAs that was markedly downregulated by CDB-2914 encoded the vasoactive intes-

RESULTS Administration of CDB-2914 Blocks Ovulation in Mice A previous study reported an antiovulatory activity of CDB-2914 in rats (16). To investigate its effects on ovulation in mice, gonadotropin-induced superovulation was performed in the absence and in the presence of this drug. Mice were treated with pregnant mare serum gonadotropin (PMSG) for 48 h followed by

Fig. 1. Administration of CDB-2914 to Mice Inhibits Ovulation A, Mice were treated with PMSG and 48 h later with hCG. One hour before hCG administration, animals (n 18) were treated with vehicle (n 6), CDB-2914 at 20 mg/kg BW (n 6), and CDB-2914 at 40 mg/kg BW (n 6), respectively. Oviducts were collected 18 h after the hCG injection, and the number of released oocytes was counted. The data are represented as means SEM. B, Hematoxylin and eosin staining of ovarian sections of vehicle (panel a) or CDB-2914-treated (40 mg/kg BW; panel b) mice. The results are representative of three independent experiments. CL, Corpus luteum, UF, unruptured follicles.

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tinal contractor (VIC), the mouse ortholog of human ET-2 (2729). VIC differs from the human ET-2 in only one of 21 amino acid residues and mimics its biological function (2729). Therefore, in this paper, we will refer to VIC as mouse ET-2. To verify the results of our microarray analysis, we examined, by Northern blotting, ovarian RNAs obtained from mice subjected to superovulation in the absence or in the presence of CDB-2914. Treatment with CDB-2914 did not alter the level of ovarian Pgr mRNA induced by hCG (data not shown). As shown in Fig. 2A, a strong signal corresponding to ET-2 mRNA was observed at 12 h after hCG administration (lane 1). This signal was undetectable in the ovaries of CDB2914-treated mice (lane 2), confirming the results of our microarray analysis.

ET-2 mRNA Expression Is Transiently Induced in the Ovary at the Time of Ovulation We next determined the temporal profile of ET-2 expression during gonadotropin-induced superovulation. Total ovarian RNA was collected at 0, 4, 8, 12, and 16 h after hCG injection and subjected to Northern blot analysis. As shown in Fig. 2B, upper panel, ET-2 transcript was undetectable at 08 h post hCG administration. A dramatic rise in the level of this transcript was observed at 12 h, which declined to an undetectable level by 16 h post hCG treatment. In mice subjected to gonadotropin-induced superovulation, follicular rupture typically occurs at 1112 h after hCG administration. To further narrow down the window of expression of ET-2 gene during superovulation, we performed Northern blot analysis using ovarian RNA collected at 10, 11, 12, and 13 h after hCG treatment (Fig. 2C). We found that the level of ET-2 mRNA rose sharply at 11 h, increased further at 12 h, and then declined abruptly by 13 h. A transient surge of ET-2 mRNA expression, therefore, occurs in the ovary between 1112 h after hCG stimulation, and it precisely overlaps with the time of follicular rupture. PGR Regulates the Expression of ET-2 mRNA in Granulosa Cells of Preovulatory Follicles To investigate whether PGR controls the ovarian expression of ET-2 mRNA, we analyzed the expression of this gene in the ovaries of PRKO mice. Wild-type (WT) and PRKO females were subjected to gonadotropin-induced superovulation. Ovarian RNA was isolated at 0, 4, 8, 12, and 16 h after hCG injection and analyzed by Northern blotting. As shown in Fig. 3A, upper panel, a transient but robust expression of ET-2 mRNA was observed in the ovarian tissues collected from WT mice at 12 h post hCG. In contrast, the expression of ET-2 mRNA was undetectable in PRKO mice, establishing that PGR is essential for induction of ET-2 expression in the ovary during ovulation. We next examined the spatial expression of ET-2 mRNA in the ovary at the time of ovulation by employing in situ hybridization. As shown in Fig. 3B, using an antisense probe specific for ET-2, we observed a strong hybridization signal in the mural granulosa cells of the preovulatory follicles at 12 h after hCG injection (Fig. 3B, panel a). No ET-2 mRNA was detected in the primary or secondary follicles. Ovarian sections hybridized with the corresponding sense RNA probe of equal length did not exhibit any significant signal (Fig. 3B, panel b). We also failed to detect any signal corresponding to ET-2 mRNA in the ovarian sections of PRKO mice at 12 h after hCG injection (Fig. 3B, panel c). These results demonstrated that the expression of ET-2 is induced exclusively in the preovulatory follicles during ovulation, and this induction is dependent on PGR. We also analyzed the PGR regulation of ET-2 expression in primary cultures of granulosa cells. Previ-

Fig. 2. ET-2 mRNA Expression in the Ovary A, Mice (n 4) were treated with PMSG and 48 h later with hCG. One hour before hCG administration, mice were injected with vehicle or CDB-2914 (40 mg/kg BW). The ovaries were collected 12 h after hCG treatment, and mRNA (1 g) was analyzed by Northern blotting followed by hybridization with 32P-labeled cDNA probes corresponding to ET-2 and 36B4, a housekeeping gene encoding a ribosomal protein. Lanes 1 and 2 represent mRNAs from mice treated with vehicle and CDB-2914, respectively. B, mRNA (1 g) obtained from ovaries of superovulated mice at 0, 4, 8, 12, and 16 h after hCG administration (n 2 at each time point) was subjected to Northern blot analysis. Top panel represents signals obtained after hybridization with a 32P-labeled ET-2 cDNA probe. The bottom panel shows the same blot after hybridization with a 32P-labeled 36B4 probe. C, mRNA (1 g) was isolated from ovaries of superovulated mice at 10, 11, 12, and 13 h after hCG administration (n 3 at each time point) and subjected to Northern blot analysis. The intensities of the ET-2 mRNA signals were quantitated by densitometric scanning and normalized with respect to the 36B4 signals in the same blot. The relative intensities representing ET-2 mRNA levels at different times during superovulation were then plotted. The normalized value of the 12 h sample was set at 100%.

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Fig. 3. PGR Regulates Ovarian Expression of ET-2 mRNA A, Female WT and PRKO mice of same genetic background (129 Sv) were subjected to superovulation. mRNA (1 g) was obtained from ovaries of mice at 0, 4, 8, 12, and 16 h after hCG administration and analyzed by Northern blotting using 32P-labeled ET-2 and 36B4 cDNA probes. The results are representative of two independent experiments. B, WT (129 Sv) and PRKO mice were subjected to superovulation, and ovaries were collected 12 h after hCG administration. Ovarian sections were analyzed by in situ hybridization employing a DIG-labeled antisense RNA probe specific for ET-2 gene. Panels a and b represent sections from WT mice hybridized with antisense and sense ET-2 probes, respectively. Panel c is a section from PRKO mice hybridized with the ET-2 antisense probe.

ous studies showed that treatment of cultures of PMSG-primed granulosa cells with forskolin and phorbol 12-myristate 13 acetate (PMA), which activate adenylyl cyclase and protein kinase C pathways, respectively, mimics LH signaling that, in turn, stimulates Pgr gene expression in these cells (31). Consistent with these reports, we observed a marked induction of Pgr mRNA within 4 h of addition of forskolin and PMA to granulosa cells (Fig. 4A). At 12 h, the level of ET-2 mRNA rose steeply in these cells (Fig. 4, B and C). It is of interest to note that the temporal expression pattern of Pgr and ET-2 mRNAs in granulosa cells in vitro is remarkably similar to that seen for these genes in response to LH in vivo. Addition of CDB-2914, which blocks PGR function, inhibited the induction of ET-2 mRNA in a dose-dependent manner (Fig. 4B). In agreement with this finding, when granulosa cells isolated from ovaries of PMSG-primed PRKO mice were treated with forskolin and PMA, we failed to detect any induction of ET-2 mRNA (Fig. 4C). Collectively, these results strengthened our view that ET-2 is a downstream target of PGR-dependent mechanisms in the preovulatory follicles. Expression of ET Receptors (ETRs) in the Ovary The cellular functions of ET-2 are mediated via the ETR isoforms, ETR-A and ETR-B (2022). We, therefore, used Northern blotting to monitor the expression of ETR-A and ETR-B mRNAs in the mouse ovary at various times after hCG administration. As shown in Fig.

5A, signals corresponding to ETR-A and ETR-B mRNAs were observed in the ovaries at 0, 4, 8, or 12 h post hCG treatment, indicating constitutive expression of the receptors during the ovulatory period. Using 32 P-labeled ETR-A and ETR-B cDNA probes of identical specific activity, we observed a relatively stronger signal of ETR-B compared with ETR-A in these tissues, suggesting that ETR-B is likely the predominant ET receptor in the ovary during ovulation. We also examined the expression of these receptors in the ovaries of PRKO mice and found that the expression levels of ETR-A and ETR-B in these mice are comparable to those seen in the WT animals (data not shown). We next localized the site of expression of ET-2 receptors during ovulation. Mice were subjected to superovulation, ovaries were collected 12 h after hCG administration, and immunohistochemistry was performed using ETR-A- and ETR-B-specific antibodies (Fig. 5B). Ovarian sections stained with the ETR-A antibody showed weak immunostaining in the mural and cumulus granulosa cells (Fig. 5B, panel b). In contrast, the ETR-B antibody exhibited relatively strong immunostaining in the mural cells and particularly intense staining was seen in the cumulus cells (Fig. 5B, panel c). Most interestingly, closer examination of higher magnification micrographs revealed distinct ETR-B immunostaining in the endothelial cells of large capillaries that lie along the inner border of the theca interna (Fig. 5B, panel e, indicated by arrows).

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Fig. 4. PGR Regulates Expression of ET-2 mRNA in Primary Cultures of Granulosa Cells A, Mice were treated with PMSG and ovaries were collected at 48 h. Granulosa cells were obtained from preovulatory follicles by needle puncture and cultured overnight in DMEM-F12 medium containing 1% fetal bovine serum. The cells were treated with P (10 nM), PMA (20 nM), and forskolin (Fo, 10 M) for 0 and 4 h. Total RNA was isolated from these cells and analyzed by quantitative RT-PCR (Q-PCR) using gene-specific primers for PGR and 36B4. Expression of Pgr mRNA was normalized against 36B4 mRNA expression. Fold changes were computed relative to the expression level of PGR in cells collected from mice at 0 h. B, The cells were treated with or without indicated concentrations of P, PMA, forskolin, and CDB-2914 for 0 h (column 1) and 12 h (columns 25). Total RNA was isolated from these cells and subjected to Q-PCR using ET-2 and 36B4 primers. Expression of ET-2 mRNA was normalized against 36B4 mRNA expression. Fold changes were computed relative to the expression level of ET-2 in cells collected from mice at 0 h. C, Granulosa cells from WT or PRKO mice were treated with P (10 nM), PMA (20 nM), and forskolin (10 M) for 0, 4, 8, 12, and 16 h. Total RNA was isolated from these cells and subjected to Q-PCR using primers for ET-2 and 36B4. Expression of ET-2 mRNA was normalized against 36B4 mRNA expression. Fold changes were computed relative to the expression level of ET-2 in cells collected from WT mice at 0 h.

Although ETR-B staining was observed in follicles of all stages, it was most prominent in the preovulatory follicles, particularly in the COC. These results indicated that ETR-B is the likely mediator of the functional effects of ET-2 during the final events leading to the rupture of the preovulatory follicles. Because it is expressed in multiple cell types within the tissue, it presents multiple potential sites of action of ET-2 during the rupture process. Administration of ETR Antagonists Impairs Ovulation in the Mouse To address the role of ET-2 in ovulation, we employed selective antagonists of ETR-A and ETR-B. We used JKC-301, a specific inhibitor of ETR-A, and BQ-788, a

specific inhibitor of ETR-B (32, 33). In superovulation experiments, each inhibitor was administered ip at 6 h after hCG priming. The impact of these drugs on ovulation was assessed by counting the number of released oocytes at 18 h after hCG injection (Fig. 6A). Whereas administration of JKC-301 led to a statistically significant reduction ( 25%; P 0.05) in the number of released oocytes compared with the vehicle-treated control group, similar treatment with BQ788 resulted in a more striking, approximately 75%, decline in the rate of ovulation. BQ-788, therefore, appeared to be a more effective blocker of ovulation than JKC-301. We next examined whether the timing of administration of the antagonist BQ-788 has any effect on the

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Fig. 5. Expression Profiles of ETR-A and ETR-B in Mouse Ovary during Ovulation A, mRNA (1 g) was isolated from ovaries of superovulated mice at 0, 4, 8, and 12 h after hCG administration and subjected to Northern blot analysis employing 32P-labeled ETR-A (top panel), ETR-B (middle panel), and 36B4 (bottom panel) cDNA probes. B, Ovaries from superovulated mice were collected at 12 h after hCG treatment and subjected to immunohistochemical analysis using preimmune serum (a), ETR-A antibody (b), and ETR-B antibody (c). Panels d and e show higher magnification ( 20) of an ovarian section obtained from a superovulated mouse at 12 h after hCG treatment. Panel d, Hematoxylin and eosin staining. The red spots indicate blood vessels. Panel e, Immunostaing using ETR-B antibody. Reddish brown stains in the mural and cumulus granulosa layers indicate specific ETR-B immunostaining. The arrows indicate large capillaries that also exhibit ETR-B immunostaining.

magnitude of inhibition of ovulation. The drug was administered at 4, 6, or 8 h after hCG injection, and the number of released oocytes was counted at 18 h. As shown in Fig. 6B, administration of BQ-788 at times that are increasingly closer to the time of ovulation resulted in a progressive decline in the number of released oocytes. We observed greater than 85% inhibition in oocyte release when the drug was given only 4 h before the time of ovulation. We also performed a histological analysis of the ovaries of mice treated with BQ-788 at 8 h after hCG

injection. As shown in Fig. 6C, left panel, ovarian sections of vehicle-treated control mice showed numerous corpora lutea, indicating successful ovulation. In contrast, ovarian sections from BQ-788-treated mice showed many unruptured follicles and only a few corpora lutea (Fig. 6C, right panel). Taken together, these results indicated that the blockade of ETR-B by BQ788 effectively suppressed ovulation by inhibiting follicular rupture. Because ET-2 is a downstream target of PGR regulation during the ovulatory period, we considered the

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Fig. 6. Mice Treated with an ETR-B Antagonist Exhibit Impaired Follicular Rupture A, Mice (n 12) were subjected to superovulation and divided into three groups of four animals each. Each group was then injected with vehicle or JKC-301 (10 mg/kg BW) or BQ-788 (10 mg/kg BW). The injections (ip) were given at 6 h after hCG treatment. The number of released oocytes was counted 18 h after hCG administration. The data are represented as means SEM. B, Mice (n 4 at each time point) were subjected to superovulation and treated with BQ-788 (10 mg/kg BW) at indicated times after hCG treatment. Control represents mice treated with vehicle alone at 6 h after hCG administration. The number of released oocytes was counted 18 h after hCG treatment. C, Hematoxylin and eosin-stained ovarian sections of mice treated with vehicle (left panel) or BQ-788 (right panel) at 8 h after hCG injection. The ovarian sections were collected at 18 h after hCG administration. CL, Corpus luteum; UF, unruptured follicles.

possibility that it may mediate some of the effects of this receptor during ovulation. We, therefore, investigated whether certain of the known PGR-regulated genes in granulosa cells are also regulated by ET-2. To test this possibility, we examined the expression of the gene encoding cGMP-dependent protein kinase II (cGK II, Prkg2), a molecule recently reported to be regulated by PGR-induced mechanisms (34). A marked induction of cGK II mRNA was seen in cultured granulosa cells 12 h after administration of forskolin and PMA (Fig. 7A). As expected, this induction was suppressed when PGR function was inhibited by CDB-2914. We next examined the effects of the blockade of the ET-2 pathway on cGK II mRNA expression in granulosa cells. As shown in Fig. 7B, addition of the ETR-B antagonist BQ-788 to cultured granulosa cells did not significantly alter the level of ET-2 mRNA. We noted, however, a marked reduction in the level of cGK II mRNA in response to BQ-788, indicating that the expression of this gene is regulated by signaling downstream of ET-2 in granulosa cells (Fig. 7C).

DISCUSSION Steroidal antagonists that bind PGR and block its gene-regulatory activity are powerful tools to identify the molecular pathways that mediate the function of this receptor in an adult target tissue (23). In this study, we used CDB-2914, a novel PGR-selective antagonist, to identify the gene networks that are regulated by this receptor during ovulation. Compared with RU486, a well-known and widely used PGR antagonist, CDB-2914 displays improved specificity and efficacy due to its lower antiglucocorticoid activity and better binding affinity to PGR (17, 18). Previous studies in the rat showed that CDB-2914 displays dose-dependent antiovulatory activity when administered on the day of proestrus (16). In the present study, we found that administration of CDB-2914 to mice undergoing gonadotropin-induced superovulation effectively blocked the release of oocytes from the ovary, presumably by inhibiting PGR-dependent pathways.

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Fig. 7. ET-2 Regulates Expression of cGK II mRNA in Primary Culture of Granulosa Cells A, Granulosa cells were treated with P, PMA, forskolin (Fo), and indicated concentrations of CDB-2914 for 0 h (column 1) and 12 h (columns 25). Total RNA was isolated from these cells and analyzed by Q-PCR using gene-specific primers for cGK II and 36B4. Expression of cGK II mRNA was normalized against 36B4 mRNA expression. Fold changes were computed relative to the expression level of cGK II in cells collected from mice at 0 h. B and C, The cells were treated with or without indicated concentrations of P, PMA, forskolin, and ETR-B antagonist, BQ788, for 0 h (column 1) and 12 h (columns 2 and 3), and analyzed for the expression of ET-2 (panel B) and cGK II (panel C). Expression of ET-2 and cGK II mRNA was normalized against 36B4 expression. Fold changes were computed relative to the expression level of ET-2 or cGK II in cells collected from mice at 0 h.

To obtain a comprehensive understanding of the gene networks underlying PGR function during ovulation, we performed gene expression profiling in the ovarian tissue under conditions in which the activity of this receptor is inhibited by CDB-2914. Our study demonstrated that ET-2 expression, which occurs immediately preceding ovulation, is blocked when PGR is occupied by CDB-2914. Therefore, we identified ET-2 as a downstream target of PGR regulation during the ovulatory process. The lack of ET-2 expression in the ovaries of PGR null mice upon gonadotropin stimulation confirmed the role of this receptor in ET-2 induction during ovu-

lation. This conclusion was further strengthened by our in vitro studies using primary cultures of granulosa cells. Previous studies showed that addition of forskolin and PMA to granulosa cells isolated from PMSGprimed mice mimics LH action and induces Pgr expression (31). In our experiments, treatment of primary cultures of granulosa cells with these reagents led to a remarkable induction of ET-2 mRNA in these cells. No ET-2 mRNA was detected when granulosa cells obtained from PRKO mice were treated similarly, indicating that PGR is essential for ET-2 induction. In situ hybridization analysis revealed that ET-2 mRNA expression occurred only in the mural granulosa cells of

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the preovulatory follicles. These cells are also known to be the exclusive sites of Pgr expression in the preovulatory follicles during ovulation (57). Therefore, PGR and ET-2 expression are spatially linked. Our results, however, did not reveal whether PGR induces ET-2 expression directly or indirectly. During superovulation of mice, PGR expression peaks at 24 h after hCG stimulation. The peak expression of ET-2, on the other hand, occurs only at 1112 h after hCG treatment. Given this difference in their temporal expression patterns, it is possible that PGR regulates ET-2 through indirect mechanisms. Direct regulation of a target gene by PGR involves interaction of the receptor with a P response element motif in its regulatory region or tethering to a promoter-bound transcription factor (14, 15). An in silico analysis of the 5 -regulatory region of ET-2 did not reveal any consensus P response element motif, although interaction via tethering to another transcription factor remains a possibility. It is also conceivable that PGR induces a mediator molecule, which in turn regulates the expression of ET-2. Further work would be needed to decipher the mechanisms by which PGR regulates ET-2 expression. The ET family of ligands is composed of ET-1, ET-2, and ET-3 (2022). Interestingly, of these three structurally related isoforms, only ET-2 is robustly induced downstream of LH and PGR during the ovulatory process in mice. In Northern blotting experiments, we failed to detect any mRNA signal corresponding to ET-1 or ET-3 in the ovary under the superovulation conditions (data not shown). There is an extensive literature of the biological roles of ETs in diverse tissues (2022). The physiological effects of ETs are mediated by two distinct seven transmembrane G protein-coupled receptor subtypes, ETR-A and ETR-B (20). Germline mutation of either ETR leads to perinatal or juvenile lethality in the mouse (35, 36). Mice deficient in ETR-A exhibited defects in craniofacial and cardiac neural crest development (35). In contrast, mice deficient in ETR-B suffer from severe melanocyte and enteric neuron defects and die as juveniles (36). To determine the consequences of loss of ET-2 signaling through each receptor during ovulation, we used two selective peptide inhibitors: JKC-301 for ETR-A and BQ-788 for ETR-B (32, 33, 36). These peptides structurally resemble ETs and act as competitive inhibitors. Systemic administration of BQ-788, the ET-B antagonist, before ovulation led to as much as 85% reduction in the number of released ova. These results are highly consistent with the recent findings of Ko et al. (37), who showed that a direct intraovarian injection of a small amount of tezosentan, a pan-antagonist of ETR, immediately before ovulation efficiently blocked follicular rupture in the rat. These results strongly implied that the blockade of ovulation by the ETR antagonists is a consequence of disruption of ET function within the ovary itself rather than a secondary effect due to the inhibitory action of this drug in another tissue. The use of ETR-A- and ETR-

B-specific antagonists in our studies allowed us to dissect the individual roles of these receptor subtypes during ovulation. Our results, indicating that BQ-788 is more effective than JKC-301 in blocking ovulation, suggest that ETR-B plays a more critical role than ETR-A in mediating the effects of ET-2 during this process. A likely explanation of this finding is the observed difference in the expression levels of ETR-A and ETR-B in the ovary. By the criteria of Northern blotting and immunohistochemical analyses, the expression level of ETR-B appears to be substantially higher than that of ETR-A. In contrast to the expression of its ligand ET-2, which is seen only transiently in the preovulatory follicles preceding ovulation, ETR-B expression was detected in follicles of all stages of development in the PMSG-treated ovary. Its expression increased only modestly upon hCG stimulation. Immunohistochemical localization of the ETR-B provided interesting clues regarding the site(s) of action and function of ET-2 in the preovulatory follicle. We found that ETR-B is expressed in three different cell types within the ovary: mural granulosa cells, cumulus cells, and the endothelial cells of the wreath of capillaries bordering the theca interna. Interestingly, the expression of ETR-B was more intense in the COC compared with the mural granulosa cells. We postulate that ET-2 produced by the mural granulosa cells of the preovulatory follicles acts in an autocrine or paracrine manner at one or more sites of ETR-B expression within the ovary to control follicular rupture. The existence of ETR-B in the endothelial cells of the large capillaries in the theca interna indicates the possibility that ET-2 could regulate the ovulatory process by ETR-mediated vasodilation and increased vascular permeability. It is believed that certain aspects of the ovulatory process resemble an acute inflammatory reaction involving marked changes in permeability of the follicle-blood barriers, leading to accumulation of follicle fluid and increase of intrafollicular pressure (1, 38). Earlier studies showed that in rats subjected to superovulation, the ovarian blood volume increases significantly starting at 4 h after hCG treatment and reaches a peak that is markedly higher than the control level at 10 h post hCG near the time of follicular rupture (39). This increased blood flow is known to be important for ovulation, contributing to the hyperemia and edema that occur before follicular rupture (40). ETs are known to signal through a nitric oxide (NO)-mediated pathway. They stimulate endothelial nitric oxide (NO) synthase (eNOS) under various physiological conditions, which in turn catalyzes the production of NO from L-arginine (41, 42). The NO diffuses across the endothelium into neighboring smooth muscle and induces vasodilation. Interestingly, previous studies have shown that the expression of eNOS required for NO production reaches a peak at 12 h after hCG injection and plays a critical role in ovulation (4346). It is tempting to speculate that ET-2 produced by the mural granulosa cells acts on the endothelial cells of the

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blood vessels in the theca interna to induce NO signaling and in this manner plays a role in the induction of local vasodilation (Fig. 8). Expression of ETR-B in the mural and cumulus granulosa cells presents additional possible mechanisms of ET-2 action during ovulation (Fig. 8). It is conceivable that ET-2 secreted by the mural granulosa cells of the preovulatory follicles acts back on these same cells in an autocrine manner. The intense expression of ETR-B in the COC is also intriguing, suggesting a paracrine role of ET-2 at this site. An important finding of our study is the identification of cGK II as a downstream target of ET-2 in the preovulatory follicle. Recent work by Richards and co-workers (34) indicated that PGR-dependent pathways are responsible for the induction of the cGK II gene in the granulosa cells and COC at the time of ovulation. We propose that ET-2 synthesized in response to PGR in the granulosa cells acts on ETR-B in mural cells in an autocrine manner. ET-2 can also act on cumulus cells of the COC in a paracrine manner to induce synthesis of cGK II in these cells. The functional significance of cGK II expression in mural granulosa cells or COC is presently unclear. It is of interest, however, to note that many components of the cGMP pathway are present in the granulosa cells and activated during LH-induced ovulation. Both membrane-bound and soluble guanylate cyclases, which produce cGMP, are expressed in these cells (30, 47). As mentioned above, NO signal-

ing, which activates soluble guanylate cyclases, is also induced in the ovarian tissue during ovulation (43). The future challenge is to determine the precise nature of the molecular circuitry involving PGR, ET-2, NOS/NO, cGMP-producing enzymes, and cGK II signaling pathway that operates downstream of LH signaling to impact on the function of mural granulosa cells and the COC during the ovulatory process.

MATERIALS AND METHODS

Reagents CDB-2914 was a generous gift from the Population Council (New York, NY). PMSG and hCG were purchased from Sigma Chemical Co. (St. Louis, MO). The ETR inhibitors BQ-788 and JKC-301 were purchased from EMD Biosciences, Inc. (San Diego, CA) and Alexis Biochemicals (San Diego, CA), respectively. Animals and Tissue Collection All experiments involving animals were conducted in accordance with the National Institutes of Health (NIH) standards for the use and care of animals and were approved by the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign. To induce superovulation, the immature CD-1, 129Sv, or PRKO mice (2428 d old) were injected ip with 5 IU of PMSG and 48 h later by 5 IU of hCG. The animals were killed at various times after hCG injection, and ovaries were collected for Northern blot analysis. PRKO were bred and homozygotes were confirmed by genotyping as described previously (12, 23). To assess the effect of CDB-2914 on superovulation in mice, the compound was dissolved in sesame oil and administered ip at 20 or 40 mg/kg body weight (BW) 1 h before hCG injection. The oviducts were collected at 18 h after hCG injection, and oocytes were counted. For histological analysis, ovaries were collected and fixed in 4% paraformaldehyde at 4 C. After paraffin embedding, ovarian sections were subjected to hematoxylin and eosin staining (Sigma Chemical Co.). To confirm Pgr regulation of ovarian gene expression, CDB-2914 was administered ip to mice subjected to superovulation 1 h before hCG injection. Ovaries were collected from these mice at 12 h after hCG, and mRNA was extracted and analyzed. GeneChip Analysis

Fig. 8. A Hypothetical Signaling Network Involving LH/CG, PGR, ET-2, and ETR-B Controls Ovulation The pituitary surge of LH/CG before ovulation acts on the mural granulosa cells of the preovulatory follicles. Signaling via LH receptor in these cells induces PGR, which in turn leads to the expression of ET-2. ET-2 acts in an autocrine or paracrine manner via ETR-B on mural and/or cumulus granulosa cells (GC) and endothelial cells of large capillaries to induce downstream signaling molecules, which include cGK II. LH signaling in the ovary is also known to induce nitric oxide synthases, eNOS and iNOS, which are thought to be involved in cGMP production. cGMP activates cGK II, which phosphorylates as yet unknown downstream molecules that are likely to participate in pathways that control follicular rupture. iNOS, Inducible nitric oxide synthase.

For microarray analysis, RNA samples were processed and analyzed using mouse Affymetrix GeneChips following the Affymetrix protocol as described previously (23). RNA Analysis Northern blot analysis was performed as described previously (23). The in situ hybridization was performed as described previously (24). Briefly, tissues were fixed in 4% paraformaldehyde at 4 C. Cryostat sections were cut at 8 m and attached to 3-aminopropyl triethylsilane (Sigma)-coated slides. Digoxygenin (DIG)-labeled sense or antisense RNA probe complementary to nucleotides 240 to 643 of mouse ET-2 cDNA was used. DIG-labeled RNA probes were synthesized

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from the cDNAs using T3 or T7 RNA polymerase and DIGlabeled nucleotides, according to the manufacturers specifications (Roche Applied Science, Indianapolis, IN). Immunohistochemistry Polyclonal antibodies against ETR-A and ETR-B receptors were purchased from Alexis Chemical (Carlsbad, CA) and Calbiochem (La Jolla, CA), respectively, and diluted 1:500 for immunohistochemistry. The antibodies were raised against C-terminal peptide of rat ET-A and ET-B receptors and reacted with rat, mouse, and human antigens. Paraffin-embedded ovarian tissues were sectioned at 4 m and mounted on slides. Sections were washed in PBS for 20 min and then incubated in a blocking solution containing 10% normal rabbit serum for 10 min before incubation in primary antibody overnight at 4 C. Immunostaining was performed using Avidin-Biotin kit for rabbit primary antibody (Vector Laboratories, Burlingame, CA) and the diaminobenzidine chromogen. Sections were counterstained with hematoxylin, mounted, and examined under bright field. Red deposits indicate the sites of immunostaining. Inhibitor Studies The ETR inhibitors JKC-301 (10 mg/kg BW) and BQ-788 (10 mg/kg BW) were administered ip to mice subjected to superovulation at 4, 6, and 8 h after hCG injection. The oviducts were collected at 18 h after hCG injection, and oocytes were counted. The ovaries from mice treated with BQ-788 were also collected for histological analysis. Statistical Analysis The data were rank transformed, and ANOVA (25) followed by Dunnetts one-sided test against the control were performed (26). Data are reported as mean SD. P 0.05 was considered as statistically significant.

Acknowledgments
The PRKO mice were kindly provided by F. J. DeMayo of Baylor College of Medicine (Houston, TX).

Received February 22, 2006. Accepted July 24, 2006. Address all correspondence and requests for reprints to: Indrani C. Bagchi, Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802. E-mail: ibagchi@uiuc.edu. This work was supported by National Institutes of Health (NIH) Grants U54 HD 299901-12 (to I.C.B. and R.L.S.-W.), and R01-HD-044611 (to M.K.B.). This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR16515-01 from the National Center for Research Resources, NIH (to I.C.B.). Disclosure Statement: The authors have nothing to declare.

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