effects of indomethacin, luteinizing hormone (lh), prostaglandin e2 (pge2), trilostane,...

Effects of indomethacin, luteinizing hormone (LH),prostaglandin E2 (PGE2), trilostane, mifepristone,

ethamoxytriphetol (MER-25) on secretion ofprostaglandin E (PGE), prostaglandin F2a (PGF2a)

and progesterone by ovine corpora lutea ofpregnancy or the estrous cycle

L. Kim, Y. S. Weems, P. J. Bridges, B. R. LeaMaster, L. Ching,D. L. Vincent, C. W. Weems*

Dept. of Animal Science, University of Hawaii, Honolulu, HI 96822, USA

Received 20 July 2000; received in revised form 20 September 2000; accepted 19 December 2000

Abstract

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and toexamine autocrine and paracrine roles of progesterone and estradiol-17b on progesterone secretionby the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL ofthe estrous cycle was similar to day-90 CL of pregnancy (P $ 0.05). In experiment 1, secretion ofprogesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P # 0.05)by LH or PGE2. Secretion of progesterone in vitro by CL slices from day-90 pregnant ewes was notaffected by LH (P $ 0.05) while PGE2 increased (P # 0.05) secretion of progesterone. Day 8 ovineCL of the estrous cycle did not secrete (P $ 0.05) detectable quantities of PGF2a or PGE while day-90ovine CL of pregnancy secreted PGE (P # 0.05) but not PGF2a. Secretion of progesterone and PGEin vitro by day-90 CL of pregnancy was decreased (P # 0.05) by indomethacin. The addition of PGE2,but not LH, in combination with indomethacin overcame the decreases in progesterone by indometh-acin (P # 0.05).

In experiment 2, secretion of progesterone in vitro by day-11 CL of the estrous cycle was increasedat 4-h (P # 0.05) in the absence of treatments. Both day-11 CL of the estrous cycle and day-90 CLof pregnancy secreted detectable quantities of PGE and PGF2a (P # 0.05). In experiment 1, PGF2a

* Corresponding author. Tel.:11-808-956-8337; fax:11-808-956-4883.E-mail address:[email protected] (C.W. Weems).

Prostaglandins & other Lipid Mediators 63 (2001) 189–203

0090-6980/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S0090-6980(01)00097-1

secretion by day-8 CL of the estrous cycle and day-90 ovine CL of pregnancy was undetectable, butwas detectable in experiment 2 by day-90 CL. Day 90 ovine CL of pregnancy also secreted more PGEthan day-11 CL of the estrous cycle (P # 0.05), whereas day-8 CL of the estrous cycle did not secretedetectable quantities of PGE (P $ 0.05). Trilostane, mifepristone, or MER-25 did not affect secretionof progesterone, PGE, or PGF2a by day-11 CL of the estrous cycle or day-90 CL of pregnancy (P $0.05). It is concluded that PGE2, not LH, is the luteotropin at day-90 of pregnancy in sheep and thatprogesterone does not modify the response to luteotropins. Thus, we found no evidence for anautocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone,PGE or PGF2a. © 2001 Elsevier Science Inc. All rights reserved.

1. Introduction

Secretion of progesterone is essential for the maintenance of pregnancy in mammalianspecies [1]. In non-pregnant ewes on day-8 of the estrous cycle, secretion of progesterone invitro by CL slices is increased in a dose dependent manner by ovine LH or PGE2 [2]. In88–90-day pregnant ewes secretion of progesterone in vitro is not affected by LH, however,secretion of progesterone is increased by PGE1 or PGE2 [2]. Similar findings are observedin CL slices from non-pregnant Brahman cows on day-14 of the estrous cycle and day-200of pregnancy [3]. This suggests that PGE2, not LH, is regulating luteal secretion of proges-terone by CL from pregnant sheep or cattle.

Progesterone [4] or estradiol-17b [5] may be autocrine or paracrine regulators of CLsecretion of progesterone. Trilostane, a competitive inhibitor of 3b-hydroxysteroid dehydro-genase (3bHSD) [6], increased serum pregnenolone and reduced serum progesterone inrhesus monkeys during the midluteal phase of the menstrual cycle [7]. Trilostane decreasedplacental progesterone secretion in 90-day lutectomized [8] or ovariectomized [9] pregnantewes and increased inferior vena cava PGF2a [9]. A PGF2a metabolite, 13,14-dihydro-15-keto-prostaglandin F2a (PGFM), was increased by trilostane in late pregnant sheep [10].Since 3bHSD also catalyzes the conversion of dehydroepiandrosterone (DHEA) to estradiol-17b, it was expected that estradiol production would be suppressed by trilostane. However,in rhesus monkeys during the mid-luteal phase of the menstrual cycle and in lutectomized [8]or ovariectomized [9] 90-day pregnant ewes, trilostane increased estradiol-17b.

In the pregnant rat, there are conflicting results that mifepristone (RU-486), a progesteronereceptor antagonist [11], both raised and lowered progesterone secretion [12]. In ovariecto-mized ewes at 90 days of pregnancy, mifepristone induced abortion and increased estradiol-17b and PGF2a [13]. In late pregnant ewes, treatment with mifepristone increased basaluterine PGFM and PGE2 and oxytocin-stimulated PGFM release [14].

Estradiol-17b may be involved in regulating CL function, since exogenous estradiol-17bin sheep can prolong the life of the CL [15] or promote luteolysis [16]. In the ovine CL,cytosolic estradiol-17b receptors on large steroidogenic luteal cells are greater than in smallluteal cells [5]. MER-25, a nonsteroidal estrogen antagonist [17], decreased progesterone inpregnant baboons [18]. However, there are no studies with MER-25 on the regulation ofprogesterone secretion by ovine luteal tissue.

The objectives of these experiments were to: 1) determine whether indomethacin, an

190 L. Kim et al. / Prostaglandins & other Lipid Mediators 63 (2001) 189–203

inhibitor of prostaglandin synthesis [19], decreases PGE and progesterone secretion byday-90 ovine CL of pregnancy and whether PGE2, not LH, overcomes indomethacin-induceddecreases in progesterone secretion; and 2) determine whether trilostane, mifepristone, orMER-25 affects secretion of progesterone, PGE, or PGF2a by CL slices from non-pregnantor pregnant ewes.

2. Materials and methods

2.1. General

Mature crossbred Merino ewes were checked twice daily for estrus with vasectomizedbrisket-painted rams. Paint-marked ewes were either removed from the flock and matedtwice each with the same two intact rams of proven fertility or were not bred. The day ofestrus was designated as day 0.

Ewes were fasted for 12 h before surgery and received 1 cc (0.54 mg) of atropine sulfateintramuscularly (i.m.) (Phoenix Pharmaceutical Inc., St. Joseph, MO) 30 min prior toinduction of anesthesia. A polyvinyl catheter (Cole-Parmer Instrument Co., Chicago, IL) of1.02 mm internal diameter was installed in a jugular vein and anesthesia was induced andmaintained with pentobarbital sodium (64.8 mg/ml, Anthony Products Co., Arcadia, CA).Corpora lutea were retrieved by a mid-ventral laparotomy from non-pregnant ewes on day-8post-estrus or day-90 pregnant ewes (experiment 1) and from non-pregnant ewes on day-11post-estrus or from day-90 pregnant ewes (experiment 2). In our previous experiments, bothday-8 or day-11 CL of the estrous cycle have similar weights and secrete similar amounts ofprogesterone per unit mass [2]. All ewes had a single CL present and only one slice per CLwas used for a treatment.

Four slices were taken from each CL, then weighed, diced and incubated at 39°C, pH 7.2under 95% O2/5% CO2 in 3 ml of Medium 199 containing 25 mM Hepes buffer and Earle’ssalts (Gibco, Grand Island, NY), 0.1% bovine serum albumin (BSA) (BSA; Sigma ChemicalCo., St. Louis, MO), 100 IU/ml penicillin (Sigma Chemical Co., St. Louis, MO), 0.2 mg/mlstreptomycin sulfate (Sigma Chemical Co., St. Louis, MO) and 20mg/ml 25-hydroxycho-lesterol (Sigma Chemical Co., St. Louis, MO; 2,3).

Diced CL slices were pre-incubated for 1 h in theabsence of treatments. Each of the fourslices from each CL was randomized to treatments within each experiment. Data forhormones were expressed as pg or ng/ml/100 mg wet weight tissue.

2.2. Experiment 1

Diced CL slices from day-8 post-estrus of non-pregnant ewes (n 5 8) or day-90 pregnantewes (n 5 8) were preincubated for 1 h followed by a 4 hincubation in fresh medium withvehicle, LH (100 ng/ml; LER-1374A), indomethacin (20mM; Sigma Chemical Co., St.Louis, MO), LH1indomethacin, PGE2 (100 ng/ml; Cayman Chemical Co., Ann Arbor, MI),or PGE21indomethacin. Media were decanted from tissue after the 4 h incubation and storedat –20°C until analysis for progesterone [20], PGE [21], and PGF2a [21] by radioimmuno-

191L. Kim et al. / Prostaglandins & other Lipid Mediators 63 (2001) 189–203

assay (RIA). Hormonal data for prostaglandin E (PGE) are expressed as PGE, since theantibody crossreacts equally with PGE1 and PGE2 and cannot be separated chromatograph-ically [21].

2.3. Experiment 2

Diced CL slices from day-11 post-estrus of non-pregnant ewes (n 5 6) and day-90pregnant ewes (n 5 12) were preincubated for 1 h followed by a 4 and 8-h incubation in freshmedium with vehicle, mifepristone (100 ng/ml; Academia Sinica, Beijing, China), trilostane(100 ng/ml; Forrest McKesson, NY), or MER-25 (100 ng/ml; Merrell National Laborato-ries). Media with fresh treatments were replaced at 4 h. Media were decanted from tissueafter the 4 and 8-h incubation and stored at –20°C until analysis for progesterone [20], PGE[21], and PGF2a [21] by RIA.

2.4. Statistical analysis

Data were analyzed for homogeneity of variance by Bartlett’s Box F test [22]. Data on thesecretion of progesterone, PGE, and PGF2a by treated CL from pregnant and non-pregnantewes in vitro were analyzed by a completely randomized design for a factorial ANOVA [22].The sensitivity limit of the assay [21] was used to generate a numerical number for statisticalanalysis since PGE or PGF2a were not detectable in media by RIA by day-8 CL of the estrouscycle. Intra- and interassay coefficients of variation for progesterone, PGE, and PGF2a were4,7; 6,9; and 7 and 10%, respectively.

3. Results

3.1. Experiment 1

Basal secretion of progesterone (ng/ml/100 mg tissue) in vitro by day-8 CL of the estrouscycle or day-90 CL of pregnancy in the absence of treatments was similar (Figs. 1-2;P $0.05). Secretion of progesterone in vitro by day-8 CL slices was increased by LH (Fig. 1;P #0.05) or PGE2 (Fig. 2;P # 0.05). Secretion of progesterone in vitro by day-90 CL slices frompregnant ewes was not affected by LH (Fig. 1;P $ 0.05) but was increased by PGE2 (Fig.2; P # 0.05). Secretion of progesterone by day-90 CL of pregnancy was decreased byindomethacin alone (Figs. 1–2;P # 0.05) or in combination with LH (Fig. 1;P # 0.05).However, PGE2 overcame the inhibitory effects of indomethacin on progesterone secretionby day-90 luteal tissue.

Secretion of PGF2a (pg/ml/100 mg tissue) in vitro by day-8 CL of the estrous cycle orday-90 of pregnancy was undetectable and was not affected by LH (Fig. 3;P $ 0.05),indomethacin alone (Figs. 3–4;P $ 0.05) or in combination with LH (Fig. 3;P $ 0.05). Theaddition of PGE2 increased PGF2a by day-8 CL of the estrous cycle and by day-90 CL ofpregnancy (Fig. 4;P # 0.05).

Secretion of PGE (pg/ml/100 mg tissue) in vitro was undetectable by day-8 CL of the


estrous cycle and did not differ between treatments (Fig. 5;P $ 0.05). PGE was secreted indetectable (P # 0.05) quantities in media of all CL slices from 90-day pregnant ewes (Figs.5–6). Secretion of PGE by CL slices from 90-day pregnant ewes was decreased byindomethacin alone (Fig. 5;P # 0.05) or in combination with LH (Fig. 5;P # 0.05).

3.2. Experiment 2

Basal secretion of progesterone in vitro by day-11 CL of the estrous cycle increased at 4-hin the absence of treatments (Figs. 7-9;P # 0.05). Basal secretion of progesterone in vitroby day-90 CL of pregnancy increases at 4 or 8-h in the absence of treatments (Figs. 7–9;P #0.05) and was similar at 4 and 8 h (Figs. 7–9;P $ 0.05). The addition of trilostane,mifepristone, or MER-25 did not affect secretion of progesterone in vitro by day 11 CL ofthe estrous cycle or day 90 CL of pregnancy and did not differ (P $ 0.05) among treatments(Figs. 7–9;P $ 0.05).

Day 11 CL slices from non-pregnant ewes of the estrous cycle or day 90 CL of pregnancysecreted detectable quantities of PGE (Fig. 10) and PGF2a (Fig, 11) in vitro. Day 90 CL ofpregnancy secreted more PGE than day-11 CL of the estrous cycle (Fig. 10). The additionof trilostane, did not affect secretion of PGE (Fig. 10;P $ 0.05) or PGF2a (Fig. 11; P $0.05) in vitro by day-11 CL of the estrous cycle or day-90 CL of pregnancy nor didmifepristone or MER-25 and did not differ (P $ 0.05) among treatments (data not shown).

Fig. 1. Secretion of progesterone by ovine, corpora luteain vitro from day 8 of the estrous cycle and day 90 ofpregnancy treated with 100 ng/ml ovine lh and 20mm indomethacin. Means6 S.E.M. with different superscriptsare significant atp # 0.05.


4. Discussion

Secretion of progesterone by day-8 CL of the estrous cycle and day-90 CL of pregnancyis similar and supports previous data [2]. Progesterone in peripheral blood increases aftermid-pregnancy in ewes [23] but not in cows [24]. Progesterone secretion increases rapidlyby the ovine placenta around the time the placenta has reached its maximum weight around90-days of gestation [23]. Placental production of progesterone in sheep initially risesbetween day 50 and 70 post coitum and then increases rapidly between days 70 and 100 [26].In day-90 pregnant ewes, half of the circulating progesterone is from the CL and the otherhalf is from the placenta to maintain pregnancy [20]. However, CL are not required tomaintain pregnancy in the ewe after day-55 of gestation [27].

Although LH stimulates the secretion of progesterone by ovine CL of the estrous cyclepresented herein and by others [28–30], LH does not appear to stimulate secretion ofprogesterone by CL slices from 90-day pregnant ewes based on these data and previous data[2]. In addition, the maternal pituitary, the source of LH, is not necessary to maintainpregnancy since hypophysectomy after day-50 of gestation does not cause abortion in sheep[1]. Furthermore, circulating LH and content of LH in the pituitary decreases throughoutgestation in sheep [31].

PGE1 or PGE2, but not LH, appears to be the luteotropin regulating secretion of proges-terone by the ovine CL at day-90 of pregnancy. PGE2 stimulated progesterone secretion invitro by day-8 CL of the estrous cycle and day-90 CL of pregnancy presented herein. Thesecretion of progesterone and PGE by day-90 CL of pregnancy was decreased by treatment

Fig. 2. Secretion of progesterone by ovine corpora luteain vitro from day 8 of the estrous cycle and day 90 ofpregnancy treated with 100 ng/ml PGE2 and 20mm indomethacin. Means6 S.E.M. with different superscripts aresignificant atp # 0.05.


with indomethacin but not by day-8 CL of the estrous cycle. Furthermore, PGE2, not LH, incombination with indomethacin overcame indomethacin-induced decreases in progesteronesecretion by day-90 CL of pregnancy. These data support PGE2, not LH, as the luteotropinregulating progesterone secretion by ovine CL of mid-pregnancy as reported previously [2].PGE was also secreted in detectable quantities by day-11 CL of the estrous cycle but not byday-8 CL of the estrous cycle suggesting that by day-11, CL of the estrous cycle are capableof producing PGE as its own luteotropin. Furthermore, day-90 CL of pregnancy secretedmore PGE than day-11 CL of the estrous cycle. Concentrations of PGE1 or PGE2 in theendometrium and PGE2 in uterine venous plasma increase during early pregnancy [32]. Inaddition, chronic intrauterine infusion of PGE1 or PGE2 adjacent to the ovary with the CLduring the estrous cycle prevents the luteolytic actions of PGF2a [32,33].

PGF2a was secreted in detectable quantities by both day-90 CL of pregnancy and day-11CL of the estrous cycle in experiment 2, however, secretion of PGF2a was undetectable byday-8 CL of the estrous cycle or day-90 CL of pregnancy in experiment 1. The greaterquantity of PGE secreted by day-11 CL of the estrous cycle or 90-day CL of pregnancy inexperiment 2 as well as the amount of PGE secreted may be responsible for the detectableamounts of PGF2a through conversion of PGE to PGF2a by the enzyme PGE2-9-ketoreduc-tase. Prostaglandin E2-9-ketoreductase exists in uterine and luteal tissues of the ewe and theregulation of luteal function in sheep appears to be dependent in part upon relative utero-ovarian concentrations of PGE2 and PGF2a [34].

Trilostane, a 3b-hydroxysteroid dehydrogenase inhibitor, increases serum pregnenoloneand reduces serum progesterone in rhesus monkeys during the midluteal phase of the

Fig. 3. Secretion of PGF2a by ovine corpora luteain vitro from day 8 of the estrous cycle and day 90 of pregnancytreated with 100 ng/ml ovine lh and 20mm indomethacin. Means6 S.E.M. with different superscripts aresignificant atp # 0.05.


Fig. 4. Secretion of PGF2a by ovine corpora luteain vitro from day 8 of the estrous cycle and day 90 of pregnancytreated with 100 ng/ml PGE2 and 20mm indomethacin. Means6 S.E.M. with different superscripts are significantat p # 0.05.

Fig. 5. Secretion of PGE by ovine corpora luteain vitro from day 8 of the estrous cycle and day 90 of pregnancytreated with 100 ng/ml ovine lh and 20mm indomethacin. Means6 S.E.M. with different superscripts aresignificant atp # 0.05.


menstrual cycle [7]. Although trilostane did not affect progesterone secretion in vitro byday-11 CL of the estrous cycle or day-90 CL of pregnancy, trilostane in vivo causes abortionof 90-day lutectomized [8] or ovariectomized [9] pregnant ewes when there is a reduction in

Fig. 6. Secretion of PGE by ovine corpora luteain vitro from day 8 of the estrous cycle and day 90 of pregnancytreated with 100 ng/ml PGE2 and 20mm indomethacin. Means6 S.E.M. with different superscripts are significantat p # 0.05.

Fig. 7. Secretion of progesterone by ovine corpora luteain vitro from day 11 of the estrous cycle and day 90 ofpregnancy treated with 100 ng/ml trilostane. Means6 S.E.M. with different superscripts are significant atp #0.05.


the progesterone:estradiol-17b ratio. The existence of tissue-specific 3bHSD isoforms [35,36] may reflect the ineffectiveness of trilostane on luteal tissue of ovine CL of pregnancy andof the estrous cycle.

Mifepristone, a progesterone receptor antagonist, did not affect secretion of progesterone,

Fig. 8. Secretion of progesterone by ovine corpora luteain vitro from day 11 of the estrous cycle and day 90 ofpregnancy treated with 100 ng/ml mifepristone. Means6 S.E.M. with different superscripts are significant atp #0.05.

Fig. 9. Secretion of progesterone by ovine corpora luteain vitro from day 11 of the estrous cycle and day 90 ofpregnancy treated with 100 ng/ml MER-25. Means6 S.E.M. with different superscripts are significant atp #0.05.


PGE, or PGF2a in vitro by day-11 CL of the estrous cycle or day-90 CL of pregnancy. Theaction of progesterone is mediated by intracellular progesterone receptors which have beenrecently identified in monkey CL [37], however, different isoforms of the receptor inprogesterone target tissues [38,39] may contribute to functional differences in luteal re-sponse. Although it has been suggested that progesterone may play an important role in theregulation of luteal function [4] by either sequestering newly synthesized progesterone or

Fig. 10. Secretion of PGE by ovine corpora luteain vitro from day 11 of the estrous cycle and day 90 of pregnancytreated with 100 ng/ml trilostane. Means6 S.E.M. with different superscripts are significant atp # 0.05.

Fig. 11. Secretion of PGF2a by ovine corpora luteain vitro from day 11 of the estrous cycle and day 90 ofpregnancy treated with 100 ng/ml trilostane. Means6 S.E.M. with different superscripts are significant atp #0.05.


mediating autocrine and/or paracrine actions of progesterone in the CL [4,39], the resultspresented herein with ovine CL does not support this hypothesis.

A study on the proposed auto-regulatory role of progesterone in the human CL during theluteal phase of the menstrual cycle using mifepristone (RU-486) alone in cell culture did notalter progesterone synthesis. However, when mifepristone was added with hCG, mifepris-tone caused a dose-related inhibitory response in progesterone synthesis [40]. This suggeststhat steroidogenic cells may need to be stimulated with a gonadotropin in order to alterprogesterone secretion by the CL with mifepristone.

MER-25, an estrogen receptor antagonist, did not affect secretion of progesterone, PGE,or PGF2a in vitro by day-11 CL of the estrous cycle or day-90 CL of pregnancy. MER-25decreased plasma progesterone in baboons during pregnancy [18] and the decline appearedto be of placental origin [18,42]. Since treatment with MER-25 on day-90 ovine CL ofpregnancy did not affect secretion of progesterone in vitro, the action of estrogen onbiosynthesis of progesterone may be regulated at a place other than an ovarian site. Themajority of receptors for estradiol-17b are on ovine large luteal cells [14] and the presenceof estrogen receptors and its messenger RNA in the sheep ovary suggests an autocrine/paracrine role of estradiol-17b and its receptor in the regulation of ovarian folliculardevelopment [43]. In the present study, no evidence of an estradiol-17b regulatory mech-anism was detected. The role of estrogen on the regulation of progesterone production needsfurther examination.

In summary, it is concluded that PGE2, not LH, regulates luteal secretion of progesteroneby mid-pregnant ovine CL. Indomethacin decreased secretion of progesterone and PGE byday-90 CL of pregnancy. In addition, PGE, not LH, in combination with indomethacinovercomes indomethacin-induced decreases in progesterone secretion. Day 8 CL of theestrous cycle do not secrete detectable quantities of PGE. However, PGE was secreted indetectable quantities by day-11 of the estrous cycle. It is concluded that by day-11 post-estrus, ovine CL are capable of producing PGE. Treatment by trilostane, mifepristone, orMER-25 does not affect secretion of progesterone, PGE, or PGF2a by CL slices fromnon-pregnant ewes on day-11 of the estrous cycle or pregnant ewes at day-90 of gestation.Although specific non-genomic, membrane-localized binding sites for progesterone havebeen suggested to play a role in the mediation of autocrine and/or paracrine actions ofprogesterone in the bovine CL [40], the use of progesterone and estradiol-17b receptorantagonists, respectively, and an inhibitor of the steroid biosynthetic pathway, did notdemonstrate this effect in mature ovine CL of the estrous cycle or mid-pregnancy.

Acknowledgments

The authors gratefully acknowledge the assistance of Mr. Alan Umaki and ArmandUnabia, Waialee Livestock Research Farm for care, estrous detection, and mating of sheepused in these studies. The antiserum for the progesterone assay was kindly supplied by Dr.R.L. Butcher, West Virginia University, antisera for PGF2a assays was kindly provided byDr. L. Levine, Brandeis University, Waltham, MA and antisera for PGE assays was kindlysupplied by Dr. N. Mason, Lily Research Laboratories, Indianapolis, IN. Also, the authors


acknowledge the support of Mr. Bruce Robinson, Niihau Ranch. The authors also want tothank Mr. Eric Johnson, Hawaii Mega-Cor Inc., Honolulu, HI for providing surgical sup-plies. The authors want to thank Mr. Wayne Toma for assistance with statistical analysis.

This paper is Journal Series No. 4519 of the Hawaii Institute of Tropical Agriculture andHuman Resources. This research was supported in part by USDA-CSREES Special GrantNo. CSREES 98–34135-6783 administered by the Pacific Basin Advisory Group and HatchProject 259 (USDA Regional Research Project W-112).

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effects of indomethacin, luteinizing hormone (lh), prostaglandin e2 (pge2), trilostane,...

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