2006 Bagchi Edn2 PGR Ovulation Mol Endo

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A Novel Pathway Involving Progesterone Receptor,Endothelin-2, and Endothelin Receptor B ControlsOvulation 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 ofMolecular 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 piv-

otal 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 per-formed gene expression profiling using ovaries

from mice subjected to gonadotropin-induced su-

perovulation in the presence and in the absence of

CDB-2914, a synthetic PGR antagonist. Prominent

among the genes that were down-regulated in re-

sponse to CDB-2914 was endothelin (ET)-2, a po-

tent vasoactive molecule. ET-2 mRNA was tran-

siently induced in mural granulosa cells of the

preovulatory follicles immediately preceding ovu-

lation. This induction was absent in the ovaries of

PGR null mice, indicating a critical role of this re-

ceptor in ET-2 expression. To investigate the func-

tional 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 cumulusgranulosa 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-regu-

lated 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 auto-

crine manner on multiple cell types within the pre-

ovulatory follicle to control the final events leading

to its rupture. (Molecular Endocrinology 20:

27842795, 2006)

OVULATION IN MAMMALS is a unique biologicalprocess in which a mature follicle within the ovaryruptures 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 pellu-

cida, 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 leadsto follicular rupture remains largely unknown (24). It is

widely thought, however, that a preovulatory surge ofthe LH initiates the expression of critical gene path-ways that underlie the morphological and biochemicalalterations that occur before ovulation.

Several lines of evidence suggest that progesterone(P) receptor (PGR) is a key regulatory molecule in theovary, acting downstream of LH during ovulation. LHrapidly and selectively induces PGR expression in mu-ral granulosa cells of the preovulatory follicles (57).Earlier studies in rats indicated that ovulation is inhib-ited 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 asRU486 and Org-31710, also inhibited ovulation, sug-gesting that P acting via its receptor controls ovulation(10, 11). Creation of a Pgr knockout (PRKO) mousemodel by Lydon et al. (12) and Conneely et al. (13)provided an unequivocal demonstration of the criticalrole played by this receptor in the ovulatory process.The PRKO mice failed to ovulate even when stimulatedwith exogenous gonadotropins. In these mutant mice,follicles developed normally to the preovulatory stage,and the characteristic expansion of the cumulus-oo-cyte complex (COC) appeared to occur. The follicles,however, failed to rupture, resulting in trapped oocyteswithin the ovarian tissue (12).

First Published Online August 3, 2006* Y.-P.C. and J.K. contributed equally to the manuscript.

Abbreviations: BW, Body weight; CG, chorionic gonado-tropin; cGK II, cGMP-dependent protein kinase II; COC, cu-mulus-oocyte complex; DIG, Digoxygenin; eNOS, endothelialNO synthase; ET, endothelin; ETR, ET receptor; NO, nitricoxide; P, progesterone; PGR, progesterone receptor; PMA,phorbol 12-myristate 13 acetate; PMSG, pregnant mare se-rum gonadotropin; PRKO, Pgrknockout; VIC, vasoactive in-testinal contractor; WT, wild type.

Molecular Endocrinology is published monthly by TheEndocrine Society (http://www.endo-society.org), theforemost professional society serving the endocrinecommunity.

0888-8809/06/$15.00/0 Molecular Endocrinology 20(11):27842795Printed in U.S.A. Copyright 2006 by The Endocrine Society

doi: 10.1210/me.2006-0093

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PGR is a well-known ligand-inducible transcriptionfactor (14, 15). It is likely that hormone-occupied PGRtriggers the expression of specific gene networks inthe granulosa cells of the ovarian follicles, and theproducts of these genes mediate the hormonal effectsduring ovulation. To understand the molecular basis ofthe ovulatory program, it is critical to identify the PGR-regulated molecules that are induced or suppressed atthe time of ovulation and analyze their functional rolesduring this complex physiological process.

In this study, we employed CDB-2914, a novel an-tiprogestin, as a tool to uncover the PGR-regulatedpathways during ovulation in mice. This compoundbinds to PGR with high affinity and impairs its gene-regulatory function (1619). Our results indicated thatadministration of CDB-2914 strongly blocked LH-in-duced ovulation in mice. Using oligonucleotide mi-croarrays, we identified several genes the expressionof which is markedly repressed in response to CDB-

2914 in mouse ovary at the time of ovulation. One ofthese genes encoded the vasoactive intestinal con-tractor (VIC), the mouse ortholog of human endothelin(ET)-2. ET-2 is a vasoactive molecule that mediatesvarious physiological functions such as vasodilation,vasoconstriction, and smooth muscle contraction inother tissues (2022). Here we present evidence that aunique signaling pathway in the preovulatory follicle,involving LH, PGR, ET-2, and its downstream targetmolecules, plays a critical role during ovulation.

RESULTS

Administration of CDB-2914 Blocks Ovulation

in Mice

A previous study reported an antiovulatory activity ofCDB-2914 in rats (16). To investigate its effects onovulation in mice, gonadotropin-induced superovula-tion was performed in the absence and in the presenceof this drug. Mice were treated with pregnant mareserum gonadotropin (PMSG) for 48 h followed by

treatment with human (h) chorionic gonadotropin (CG)to induce the ovulatory response. Vehicle or CDB-2914 was administered ip 1 h before the hCG injection.The number of released oocytes was counted 18 hafter hCG treatment. As shown in Fig. 1A, an averageof 3040 oocytes were released from the ovaries ofeach mouse subjected to the superovulation protocol.Histological analysis of the ovarian sections of vehicle-treated mice at 18 h after hCG injection exhibitednumerous corpora lutea, indicating efficient ovulation(Fig. 1B, panel a). Administration of increasing dosesof CDB-2914 led to a progressive decline in the num-ber of released oocytes when compared with micethat received vehicle alone (Fig. 1A). Ovarian sectionsof CDB-2914-treated mice showed the presence ofmany unruptured follicles at 18 h after hCG treatment,confirming impaired ovulation (Fig. 1B, panel b). Thefailure of follicular rupture upon CDB-2914 treatmentclosely resembled the ovarian phenotype of the PRKO

mice and is consistent with the previously reportedantiprogestational activity of this drug. Collectively,our results indicated that CDB-2914 exerts its inhibi-tory effects on ovulation in mice by blocking the func-tion 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 thatare regulated by PGR during ovulation. Mice weresubjected to superovulation in the absence and in thepresence of CDB-2914 as described above, and ova-

ries were collected 12 h after hCG treatment. mRNAwas isolated from vehicle or CDB2914-treated miceand hybridized to mouse oligonucleotide microarrays(Affymetrix, Santa Clara, CA) representing approxi-mately 11,000 genes as described previously (23). Ourstudies identified several mRNAs the expression ofwhich in the ovary altered at least 2-fold in response toCDB-2914 (Cheon, Y.C., and I.C. Bagchi, unpublishedresults). One of the mRNAs that was markedly down-regulated by CDB-2914 encoded the vasoactive intes-

Fig. 1. Administration of CDB-2914 to Mice Inhibits OvulationA, 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 werecollected 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 humanET-2 (2729). VIC differs from the human ET-2 in onlyone of 21 amino acid residues and mimics its biolog-ical function (2729). Therefore, in this paper, we willrefer to VIC as mouse ET-2.

To verify the results of our microarray analysis, weexamined, by Northern blotting, ovarian RNAs ob-tained from mice subjected to superovulation in theabsence or in the presence of CDB-2914. Treatmentwith CDB-2914 did not alter the level of ovarian PgrmRNA induced by hCG (data not shown). As shown inFig. 2A, a strong signal corresponding to ET-2 mRNAwas observed at 12 h after hCG administration (lane 1).This signal was undetectable in the ovaries of CDB-2914-treated mice (lane 2), confirming the results ofour 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 ex-pression during gonadotropin-induced superovula-tion. Total ovarian RNA was collected at 0, 4, 8, 12,and 16 h after hCG injection and subjected to Northernblot analysis. As shown in Fig. 2B, upper panel, ET-2transcript was undetectable at 08 h post hCG admin-istration. A dramatic rise in the level of this transcriptwas observed at 12 h, which declined to an undetect-able level by 16 h post hCG treatment.

In mice subjected to gonadotropin-induced super-ovulation, follicular rupture typically occurs at 1112 hafter hCG administration. To further narrow down thewindow of expression of ET-2 gene during superovu-lation, we performed Northern blot analysis usingovarian RNA collected at 10, 11, 12, and 13 h afterhCG treatment (Fig. 2C). We found that the level of

ET-2 mRNA rose sharply at 11 h, increased further at12 h, and then declined abruptly by 13 h. A transientsurge of ET-2 mRNA expression, therefore, occurs inthe ovary between 1112 h after hCG stimulation, andit 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 ex-pression of ET-2 mRNA, we analyzed the expressionof this gene in the ovaries of PRKO mice. Wild-type(WT) and PRKO females were subjected to gonado-

tropin-induced superovulation. Ovarian RNA was iso-lated at 0, 4, 8, 12, and 16 h after hCG injection andanalyzed by Northern blotting. As shown in Fig. 3A,upper panel, a transient but robust expression of ET-2mRNA was observed in the ovarian tissues collectedfrom WT mice at 12 h post hCG. In contrast, theexpression of ET-2 mRNA was undetectable in PRKOmice, establishing that PGR is essential for inductionof ET-2 expression in the ovary during ovulation.

We next examined the spatial expression of ET-2mRNA in the ovary at the time of ovulation by employ-ing in situ hybridization. As shown in Fig. 3B, using anantisense probe specific for ET-2, we observed a

strong hybridization signal in the mural granulosa cellsof the preovulatory follicles at 12 h after hCG injection(Fig. 3B, panel a). No ET-2 mRNA was detected in theprimary or secondary follicles. Ovarian sections hy-bridized with the corresponding sense RNA probe ofequal length did not exhibit any significant signal (Fig.3B, panel b). We also failed to detect any signal cor-responding to ET-2 mRNA in the ovarian sections ofPRKO mice at 12 h after hCG injection (Fig. 3B, panelc). These results demonstrated that the expression ofET-2 is induced exclusively in the preovulatory folliclesduring ovulation, and this induction is dependent onPGR.

We also analyzed the PGR regulation of ET-2 ex-

pression in primary cultures of granulosa cells. Previ-

Fig. 2. ET-2 mRNA Expression in the OvaryA, Mice (n 4) were treated with PMSG and 48 h later with

hCG. One hour before hCG administration, mice were in-jected with vehicle or CDB-2914 (40 mg/kg BW). The ovarieswere collected 12 h after hCG treatment, and mRNA (1 g)was analyzed by Northern blotting followed by hybridizationwith 32P-labeled cDNA probes corresponding to ET-2 and36B4, 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) ob-tained from ovaries of superovulated mice at 0, 4, 8, 12, and16 h after hCG administration (n 2 at each time point) wassubjected to Northern blot analysis. Top panel representssignals obtained after hybridization with a 32P-labeled ET-2cDNA probe. The bottom panel shows the same blot afterhybridization 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 timepoint) and subjected to Northern blot analysis. The intensitiesof the ET-2 mRNA signals were quantitated by densitometricscanning and normalized with respect to the 36B4 signals inthe same blot. The relative intensities representing ET-2mRNA levels at different times during superovulation werethen plotted. The normalized value of the 12 h samplewas setat 100%.

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ous studies showed that treatment of cultures ofPMSG-primed granulosa cells with forskolin and phor-bol 12-myristate 13 acetate (PMA), which activate ad-

enylyl cyclase and protein kinase C pathways, respec-tively, mimics LH signaling that, in turn, stimulates Pgrgene expression in these cells (31). Consistent withthese reports, we observed a marked induction of PgrmRNA within 4 h of addition of forskolin and PMA togranulosa cells (Fig. 4A). At 12 h, the level of ET-2mRNA rose steeply in these cells (Fig. 4, B and C). It isof interest to note that the temporal expression patternof Pgr and ET-2 mRNAs in granulosa cells in vitro isremarkably similar to that seen for these genes inresponse to LH in vivo. Addition of CDB-2914, whichblocks PGR function, inhibited the induction of ET-2mRNA in a dose-dependent manner (Fig. 4B). In

agreement with this finding, when granulosa cells iso-lated from ovaries of PMSG-primed PRKO mice weretreated with forskolin and PMA, we failed to detect anyinduction of ET-2 mRNA (Fig. 4C). Collectively, theseresults strengthened our view that ET-2 is a down-stream target of PGR-dependent mechanisms in thepreovulatory follicles.

Expression of ET Receptors (ETRs) in the Ovary

The cellular functions of ET-2 are mediated via the ETRisoforms, ETR-A and ETR-B (2022). We, therefore,used Northern blotting to monitor the expression ofETR-A and ETR-B mRNAs in the mouse ovary at var-

ious times after hCG administration. As shown in Fig.

5A, signals corresponding to ETR-A and ETR-BmRNAs 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. Using32P-labeled ETR-A and ETR-B cDNA probes of iden-

tical specific activity, we observed a relatively strongersignal of ETR-B compared with ETR-A in these tis-sues, suggesting that ETR-B is likely the predominant

ET receptor in the ovary during ovulation. We alsoexamined the expression of these receptors in theovaries of PRKO mice and found that the expressionlevels of ETR-A and ETR-B in these mice are compa-

rable to those seen in the WT animals (data notshown).

We next localized the site of expression of ET-2receptors during ovulation. Mice were subjected to

superovulation, ovaries were collected 12 h after hCGadministration, and immunohistochemistry was per-formed using ETR-A- and ETR-B-specific antibodies(Fig. 5B). Ovarian sections stained with the ETR-A

antibody showed weak immunostaining in the muraland cumulus granulosa cells (Fig. 5B, panel b). Incontrast, the ETR-B antibody exhibited relativelystrong immunostaining in the mural cells and particu-

larly intense staining was seen in the cumulus cells(Fig. 5B, panel c). Most interestingly, closer examina-tion of higher magnification micrographs revealed dis-tinct 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).

Fig. 3. PGR Regulates Ovarian Expression of ET-2 mRNAA, 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-labeledET-2 and 36B4 cDNA probes. The results are representative of two independent experiments. B, WT (129 Sv) and PRKO micewere 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 fromWT mice hybridized with antisense and sense ET-2 probes, respectively. Panel c is a section from PRKO mice hybridized withthe ET-2 antisense probe.



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Although ETR-B staining was observed in follicles of

all stages, it was most prominent in the preovulatoryfollicles, particularly in the COC. These results indi-cated that ETR-B is the likely mediator of the func-tional effects of ET-2 during the final events leading tothe rupture of the preovulatory follicles. Because it isexpressed in multiple cell types within the tissue, itpresents multiple potential sites of action of ET-2 dur-ing the rupture process.

Administration of ETR Antagonists Impairs

Ovulation in the Mouse

To address the role of ET-2 in ovulation, we employedselective 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 hafter hCG priming. The impact of these drugs on ovu-

lation was assessed by counting the number of re-

leased oocytes at 18 h after hCG injection (Fig. 6A).

Whereas administration of JKC-301 led to a statisti-

cally significant reduction (25%; P 0.05) in the

number of released oocytes compared with the vehi-

cle-treated control group, similar treatment with BQ-

788 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 adminis-

tration of the antagonist BQ-788 has any effect on the

Fig. 4. PGR Regulates Expression of ET-2 mRNA in Primary Cultures of Granulosa CellsA, 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 withP (10 nM), PMA (20 nM), and forskolin (Fo, 10 M) for 0 and 4 h. Total RNA was isolated from these cells and analyzed byquantitative RT-PCR (Q-PCR) using gene-specific primers for PGR and 36B4. Expression of PgrmRNA was normalized against36B4 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 RNAwas isolated from these cells and subjected to Q-PCR using ET-2 and 36B4 primers. Expression of ET-2mRNA was normalized against 36B4 mRNA expression. Fold changes were computed relative to the expression level of ET-2 incells 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 and36B4. Expression of ET-2 mRNA was normalized against 36B4 mRNA expression. Fold changes were computed relative to theexpression level of ET-2 in cells collected from WT mice at 0 h.



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magnitude of inhibition of ovulation. The drug wasadministered at 4, 6, or 8 h after hCG injection, and thenumber of released oocytes was counted at 18 h. Asshown in Fig. 6B, administration of BQ-788 at timesthat are increasingly closer to the time of ovulationresulted in a progressive decline in the number ofreleased oocytes. We observed greater than 85% in-hibition in oocyte release when the drug was givenonly 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 sec-tions of vehicle-treated control mice showed numer-ous corpora lutea, indicating successful ovulation. Incontrast, ovarian sections from BQ-788-treated miceshowed many unruptured follicles and only a few cor-pora lutea (Fig. 6C, right panel). Taken together, theseresults indicated that the blockade of ETR-B by BQ-788 effectively suppressed ovulation by inhibiting fol-licular rupture.

Because ET-2 is a downstream target of PGR reg-

ulation during the ovulatory period, we considered the

Fig. 5. Expression Profiles of ETR-A and ETR-B in Mouse Ovary during OvulationA, 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 analysisusing preimmune serum (a), ETR-A antibody (b), and ETR-B antibody (c). Panels d and e show higher magnification ( 20) of anovarian 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 immu-nostaining.



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possibility that it may mediate some of the effects ofthis receptor during ovulation. We, therefore, investi-gated whether certain of the known PGR-regulatedgenes in granulosa cells are also regulated by ET-2. Totest this possibility, we examined the expression of thegene encoding cGMP-dependent protein kinase II

(cGK II, Prkg2), a molecule recently reported to beregulated by PGR-induced mechanisms (34). Amarked induction of cGK II mRNA was seen in culturedgranulosa cells 12 h after administration of forskolinand PMA (Fig. 7A). As expected, this induction wassuppressed when PGR function was inhibited byCDB-2914. We next examined the effects of the block-ade of the ET-2 pathway on cGK II mRNA expressionin granulosa cells. As shown in Fig. 7B, addition of theETR-B antagonist BQ-788 to cultured granulosa cellsdid not significantly alter the level of ET-2 mRNA. Wenoted, however, a marked reduction in the level of cGKII mRNA in response to BQ-788, indicating that theexpression 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 antago-nist, to identify the gene networks that are regulated

by this receptor during ovulation. Compared with

RU486, a well-known and widely used PGR antago-

nist, CDB-2914 displays improved specificity and ef-

ficacy 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-de-

pendent antiovulatory activity when administered on

the day of proestrus (16). In the present study, we

found that administration of CDB-2914 to mice under-

going gonadotropin-induced superovulation effec-

tively blocked the release of oocytes from the ovary,

presumably by inhibiting PGR-dependent pathways.

Fig. 6. Mice Treated with an ETR-B Antagonist Exhibit Impaired Follicular RuptureA, 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 indicatedtimes after hCG treatment. Control represents mice treated with vehicle alone at 6 h after hCG administration. The number ofreleased oocytes was counted 18 h after hCG treatment. C, Hematoxylin and eosin-stained ovarian sections of mice treated withvehicle (left panel) or BQ-788 (right panel) at 8 h after hCG injection. The ovarian sections were collected at 18 h after hCGadministration. CL, Corpus luteum; UF, unruptured follicles.



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To obtain a comprehensive understanding of the genenetworks underlying PGR function during ovulation,we performed gene expression profiling in the ovariantissue under conditions in which the activity of thisreceptor is inhibited by CDB-2914. Our study demon-strated that ET-2 expression, which occurs immedi-ately preceding ovulation, is blocked when PGR isoccupied by CDB-2914. Therefore, we identified ET-2as a downstream target of PGR regulation during theovulatory process.

The lack of ET-2 expression in the ovaries of PGRnull mice upon gonadotropin stimulation confirmed

the role of this receptor in ET-2 induction during ovu-

lation. This conclusion was further strengthened byour in vitro studies using primary cultures of granulosacells. Previous studies showed that addition of fors-kolin and PMA to granulosa cells isolated from PMSG-primed mice mimics LH action and induces Pgr ex-pression (31). In our experiments, treatment of primarycultures of granulosa cells with these reagents led to aremarkable induction of ET-2 mRNA in these cells. NoET-2 mRNA was detected when granulosa cells ob-tained from PRKO mice were treated similarly, indicat-ing that PGR is essential for ET-2 induction. In situhybridization analysis revealed that ET-2 mRNA ex-

pression occurred only in the mural granulosa cells of

Fig. 7. ET-2 Regulates Expression of cGK II mRNA in Primary Culture of Granulosa CellsA, 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 and36B4. Expression of cGK II mRNA was normalized against 36B4 mRNA expression. Fold changes were computed relative to theexpression level of cGK II in cells collected from mice at 0 h. B and C, The cells were treated with or without indicatedconcentrations of P, PMA, forskolin, and ETR-B antagonist, BQ788, for 0 h (column 1) and 12 h (columns 2 and 3), and analyzedfor the expression of ET-2 (panel B) and cGK II (panel C). Expression of ET-2 and cGK II mRNA was normalized against 36B4expression. Fold changes were computed relative to the expression level of ET-2 or cGK II in cells collected from mice at 0 h.



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the preovulatory follicles. These cells are also knownto be the exclusive sites of Pgr expression in thepreovulatory follicles during ovulation (57). Therefore,PGR and ET-2 expression are spatially linked. Ourresults, however, did not reveal whether PGR inducesET-2 expression directly or indirectly. During super-ovulation of mice, PGR expression peaks at 24 h afterhCG stimulation. The peak expression of ET-2, on theother hand, occurs only at 1112 h after hCG treat-ment. Given this difference in their temporal expres-sion patterns, it is possible that PGR regulates ET-2through indirect mechanisms. Direct regulation of atarget gene by PGR involves interaction of the recep-tor with a P response element motif in its regulatoryregion or tethering to a promoter-bound transcriptionfactor (14, 15). An in silico analysis of the 5-regulatoryregion of ET-2 did not reveal any consensus P re-sponse element motif, although interaction via tether-ing to another transcription factor remains a possibil-

ity. It is also conceivable that PGR induces a mediatormolecule, which in turn regulates the expression ofET-2. Further work would be needed to decipher themechanisms by which PGR regulates ET-2expression.

The ET family of ligands is composed of ET-1, ET-2,and ET-3 (2022). Interestingly, of these three struc-turally related isoforms, only ET-2 is robustly induceddownstream of LH and PGR during the ovulatory pro-cess in mice. In Northern blotting experiments, wefailed to detect any mRNA signal corresponding toET-1 or ET-3 in the ovary under the superovulationconditions (data not shown). There is an extensive

literature of the biological roles of ETs in diverse tis-sues (2022). The physiological effects of ETs are me-diated by two distinct seven transmembrane G pro-tein-coupled receptor subtypes, ETR-A and ETR-B(20). Germline mutation of either ETR leads to perinatalor juvenile lethality in the mouse (35, 36). Mice defi-cient in ETR-A exhibited defects in craniofacial andcardiac neural crest development (35). In contrast,mice deficient in ETR-B suffer from severe melanocyteand enteric neuron defects and die as juveniles (36).

To determine the consequences of loss of ET-2signaling through each receptor during ovulation, weused two selective peptide inhibitors: JKC-301 for

ETR-A and BQ-788 for ETR-B (32, 33, 36). Thesepeptides structurally resemble ETs and act as com-petitive inhibitors. Systemic administration of BQ-788,the ET-B antagonist, before ovulation led to as muchas 85% reduction in the number of released ova.These results are highly consistent with the recentfindings of Ko et al. (37), who showed that a directintraovarian injection of a small amount of tezosentan,a pan-antagonist of ETR, immediately before ovulationefficiently blocked follicular rupture in the rat. Theseresults strongly implied that the blockade of ovulationby the ETR antagonists is a consequence of disruptionof ET function within the ovary itself rather than asecondary 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 todissect the individual roles of these receptor subtypesduring ovulation. Our results, indicating that BQ-788 ismore effective than JKC-301 in blocking ovulation,suggest that ETR-B plays a more critical role thanETR-A in mediating the effects of ET-2 during thisprocess. A likely explanation of this finding is the ob-served difference in the expression levels of ETR-Aand ETR-B in the ovary. By the criteria of Northernblotting and immunohistochemical analyses, the ex-pression level of ETR-B appears to be substantiallyhigher than that of ETR-A.

In contrast to the expression of its ligand ET-2,which is seen only transiently in the preovulatory fol-licles preceding ovulation, ETR-B expression was de-tected in follicles of all stages of development in thePMSG-treated ovary. Its expression increased onlymodestly upon hCG stimulation. Immunohistochemi-cal localization of the ETR-B provided interesting clues

regarding the site(s) of action and function of ET-2 inthe preovulatory follicle. We found that ETR-B is ex-pressed in three different cell types within the ovary:mural granulosa cells, cumulus cells, and the endo-thelial cells of the wreath of capillaries bordering thetheca interna. Interestingly, the expression of ETR-Bwas more intense in the COC compared with the muralgranulosa cells. We postulate that ET-2 produced bythe mural granulosa cells of the preovulatory folliclesacts in an autocrine or paracrine manner at one ormore sites of ETR-B expression within the ovary tocontrol follicular rupture.

The existence of ETR-B in the endothelial cells of the

large capillaries in the theca interna indicates the pos-sibility that ET-2 could regulate the ovulatory processby ETR-mediated vasodilation and increased vascularpermeability. It is believed that certain aspects of theovulatory process resemble an acute inflammatory re-action involving marked changes in permeability of thefollicle-blood barriers, leading to accumulation of fol-licle fluid and increase of intrafollicular pressure (1, 38).Earlier studies showed that in rats subjected to super-ovulation, the ovarian blood volume increases signifi-cantly starting at 4 h after hCG treatment and reachesa peak that is markedly higher than the control level at10 h post hCG near the time of follicular rupture (39).

This increased blood flow is known to be important forovulation, contributing to the hyperemia and edemathat occur before follicular rupture (40). ETs are knownto signal through a nitric oxide (NO)-mediated path-way. They stimulate endothelial nitric oxide (NO) syn-thase (eNOS) under various physiological conditions,which in turn catalyzes the production of NO fromL-arginine (41, 42). The NO diffuses across the endo-thelium into neighboring smooth muscle and inducesvasodilation. Interestingly, previous studies haveshown that the expression of eNOS required for NOproduction reaches a peak at 12 h after hCG injectionand plays a critical role in ovulation (4346). It is tempt-ing 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 sig-naling and in this manner plays a role in the inductionof local vasodilation (Fig. 8).

Expression of ETR-B in the mural and cumulus gran-ulosa cells presents additional possible mechanismsof ET-2 action during ovulation (Fig. 8). It is conceiv-able that ET-2 secreted by the mural granulosa cells ofthe preovulatory follicles acts back on these samecells in an autocrine manner. The intense expressionof ETR-B in the COC is also intriguing, suggesting aparacrine role of ET-2 at this site. An important findingof our study is the identification of cGK II as a down-stream target of ET-2 in the preovulatory follicle. Re-cent work by Richards and co-workers (34) indicatedthat PGR-dependent pathways are responsible for theinduction of the cGK II gene in the granulosa cells andCOC at the time of ovulation. We propose that ET-2synthesized in response to PGR in the granulosa cellsacts on ETR-B in mural cells in an autocrine manner.

ET-2 can also act on cumulus cells of the COC in aparacrine manner to induce synthesis of cGK II inthese cells. The functional significance of cGK II ex-pression in mural granulosa cells or COC is presentlyunclear. It is of interest, however, to note that manycomponents of the cGMP pathway are present in thegranulosa cells and activated during LH-induced ovu-lation. Both membrane-bound and soluble guanylatecyclases, which produce cGMP, are expressed inthese cells (30, 47). As mentioned above, NO signal-

ing, which activates soluble guanylate cyclases, is alsoinduced in the ovarian tissue during ovulation (43). Thefuture challenge is to determine the precise nature ofthe molecular circuitry involving PGR, ET-2, NOS/NO,cGMP-producing enzymes, and cGK II signaling path-way that operates downstream of LH signaling to im-pact on the function of mural granulosa cells and theCOC 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 SigmaChemical Co. (St. Louis, MO). The ETR inhibitors BQ-788 andJKC-301 were purchased from EMD Biosciences, Inc. (SanDiego, CA) and Alexis Biochemicals (San Diego, CA),

respectively.

Animals and Tissue Collection

All experiments involving animals were conducted in accor-dance with the National Institutes of Health (NIH) standardsfor the use and care of animals and were approved by theInstitutional Animal Care and Use Committee at the Univer-sity of Illinois at Urbana-Champaign.

To induce superovulation, the immature CD-1, 129Sv, orPRKO mice (2428 d old) were injected ip with 5 IU of PMSGand 48 h later by 5 IU of hCG. The animals were killed atvarious times after hCG injection, and ovaries were collectedfor Northern blot analysis. PRKO were bred and homozy-gotes were confirmed by genotyping as described previously(12, 23).

To assess the effect of CDB-2914 on superovulation inmice, the compound was dissolved in sesame oil and admin-istered ip at 20 or 40 mg/kg body weight (BW) 1 h before hCGinjection. The oviducts were collected at 18 h after hCGinjection, and oocytes were counted. For histological analy-sis, ovaries were collected and fixed in 4% paraformaldehydeat 4 C. After paraffin embedding, ovarian sections were sub-

jected to hematoxylin and eosin staining (Sigma ChemicalCo.).

To confirm Pgr regulation of ovarian gene expression,CDB-2914 was administered ip to mice subjected to super-ovulation 1 h before hCG injection. Ovaries were collectedfrom these mice at 12 h after hCG, and mRNA was extractedand analyzed.

GeneChip Analysis

For microarray analysis, RNA samples were processed andanalyzed using mouse Affymetrix GeneChips following the

Affymetrix protocol as described previously (23).

RNA Analysis

Northern blot analysis was performed as described previ-ously (23).

The in situ hybridization was performed as described pre-viously (24). Briefly, tissues were fixed in 4% paraformalde-hyde at 4 C. Cryostat sections were cut at 8 m and attachedto 3-aminopropyl triethylsilane (Sigma)-coated slides.Digoxygenin (DIG)-labeled sense or antisense RNA probecomplementary to nucleotides 240 to 643 of mouse ET-2cDNA was used. DIG-labeled RNA probes were synthesized

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 themural granulosa cells of the preovulatory follicles. Signalingvia LH receptor in these cells induces PGR, which in turnleads to the expression of ET-2. ET-2 acts in an autocrine orparacrine manner via ETR-B on mural and/or cumulus gran-ulosa cells (GC) and endothelial cells of large capillaries toinduce downstream signaling molecules, which include cGKII. LH signaling in the ovary is also known to induce nitricoxide synthases, eNOS and iNOS, which are thought to beinvolved in cGMP production. cGMP activates cGK II, whichphosphorylates as yet unknown downstream molecules thatare likely to participate in pathways that control follicularrupture. iNOS, Inducible nitric oxide synthase.



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from the cDNAs using T3 or T7 RNA polymerase and DIG-labeled nucleotides, according to the manufacturers speci-fications (Roche Applied Science, Indianapolis, IN).

Immunohistochemistry

Polyclonal antibodies against ETR-A and ETR-B receptorswere purchased from Alexis Chemical (Carlsbad, CA) andCalbiochem (La Jolla, CA), respectively, and diluted 1:500 forimmunohistochemistry. The antibodies were raised againstC-terminal peptide of rat ET-A and ET-B receptors and re-acted with rat, mouse, and human antigens. Paraffin-embed-ded ovarian tissues were sectioned at 4 m and mounted onslides. Sections were washed in PBS for 20 min and thenincubated in a blocking solution containing 10% normal rab-bit serum for 10 min before incubation in primary antibodyovernight at 4 C. Immunostaining was performed using Avi-din-Biotin kit for rabbit primary antibody (Vector Laboratories,Burlingame, CA) and the diaminobenzidine chromogen. Sec-tions were counterstained with hematoxylin, mounted, andexamined under bright field. Red deposits indicate the sitesof immunostaining.

Inhibitor Studies

The ETR inhibitors JKC-301 (10 mg/kg BW) and BQ-788 (10mg/kg BW) were administered ip to mice subjected to super-ovulation at 4, 6, and 8 h after hCG injection. The oviductswere collected at 18 h after hCG injection, and oocytes werecounted. The ovaries from mice treated with BQ-788 werealso collected for histological analysis.

Statistical Analysis

Thedata were rank transformed, and ANOVA (25) followed byDunnetts one-sided test against the control were performed(26). Data are reported as mean SD. P 0.05 was consid-

ered as statistically significant.

Acknowledgments

The PRKO mice were kindly provided by F. J. DeMayo ofBaylor 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, Illinois61802. E-mail: [email protected].

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 con-ducted in a facility constructed with support from ResearchFacilities Improvement Program Grant C06 RR16515-01 fromthe National Center for Research Resources, NIH (to I.C.B.).

Disclosure Statement: The authors have nothing todeclare.

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