The integrated action of oestrogen receptor isoforms and sites with

107

The integrated action of oestrogen
receptor isoforms and sites withprogesterone receptor in the gonadotrope modulates LH secretion:evidence from tamoxifen-treated ovariectomized rats
Jose C Garrido-Gracia*, Ana Gordon*, Carmina Bellido, Rafaela Aguilar, Inmaculada Barranco1,

Yolanda Millan1, Juana Martın de las Mulas1 and Jose E Sanchez-Criado

Departments of Cell Biology, Physiology and Immunology and 1Comparative Pathology, University of Cordoba, Cordoba, Spain

(Requests for offprints should be addressed to J E Sanchez-Criado who is now at Seccion de Fisiologıa, Facultad de Medicina, Avda. Menendez Pidal s/n, 14004

Cordoba, Spain; Email: [email protected])

*( J C Garrido-Gracia and A Gordon contributed equally to this work.)

Abstract

The specific role of each oestrogen receptor (ER) isoform

(a and b) and site (nucleus and plasma membrane) in LH

release was determined in ovariectomized (OVX) rats

injected over 6 days (days 15–20 after OVX) with a saturating

dose (3 mg/day) of tamoxifen (TX), a selective ERmodulator

with nuclear ERa agonist actions in the absence of oestrogen.

This pharmacological effect of TX was demonstrated by the

fact that it was blocked by the selective ERa antagonist

methyl-piperidino-pyrazole. Over the past 3 days of the 6-day

TX treatment, rats received either 25 mg/day oestradiol

benzoate (EB), 1.5 mg/day selective ERa agonist propyl-

pyrazole triol (PPT) and the selective ERb agonist

diarylpropionitrile (DPN), or a single 3 mg injection of the

antiprogestin onapristone (ZK299) administered on day 20.

Blood samples were taken to determine basal and

progesterone receptor (PR)-dependent LH-releasing

Journal of Endocrinology (2007) 193, 107–1190022–0795/07/0193–107 q 2007 Society for Endocrinology Printed in Great

hormone (LHRH)-stimulated LH secretion and to evaluate

LHRH self-priming, the property of LHRH that increases

gonadotrope responsiveness to itself. Blood LH concentration

was determined by RIA and gonadotrope PR expression by

immunohistochemistry. Results showed that i) EB and DPN

potentiated the negative feedback of TX on basal LH release;

ii) DPN reduced TX-induced PR expression; iii) EB and

PPT blocked TX-elicited LHRH self-priming and iv)

ZK299 reduced LHRH-stimulated LH secretion and blocked

LHRH self-priming. These observations suggest that

oestrogen action on LH secretion in the rat is exerted at the

classic ERa pool and that this action might be modulated by

both ERb and membrane ERa through their effects on PR

expression and action respectively.

Journal of Endocrinology (2007) 193, 107–119

Introduction

Luteinizing hormone (LH) secretion in the rat is dependent

on the activation of gonadotrope oestrogen receptors (ERs)

by ovarian-derived oestradiol-17b (E2; Fink 1988, 1995). Rat

gonadotropes express both isoforms of ER, the predominant

ERa (Lindzey et al. 2006) and ERb at nuclear level (Kuiper

et al. 1997, Mitchner et al. 1998, Vaillant et al. 2002,

Sanchez-Criado et al. 2005a). In addition, an ER which

appears to be the same protein as nuclear ERa is expressed at

the plasma membrane level (Bression et al. 1986, Razandi

et al. 1999, Toran-Allerand et al. 1999, Schmidt et al. 2000,

Kelly & Levin 2001, Simoncini & Genazzani 2003,

Toran-Allerand 2004, Levin 2005). Integration of E2 effects

at different ER pools in the gonadotrope (nuclear ERa and

ERb and membrane ERa) determines the cellular actions of

E2 on basal and preovulatory LH secretion and hence on

ovulation (Fink 1988, 2000). Since E2 activates the complete

ER orchestra in the gonadotrope, it is difficult to chart

separately the specific role of each ER pool on LH secretion

using either intact cyclic or ovariectomized (OVX) rats

treated with the cognate ligand. ERa and ERb isoform

knockout mice (Pelletier et al. 2003, Lindzey et al. 2006) are

not useful models for the study of ER isoforms interaction

either because one or the other ER isoform is lacking. The

discovery of ER subtype-selective ligands, such as the ERaagonist propylpyrazole triol (PPT; Stauffer et al. 2000), the

ERb agonist diarylpropionitrile (DPN; Meyers et al. 2001)

and the ERa-selective antagonist methyl-piperidino-pyrazole

(MPP; Sun et al. 2002, Harrington et al. 2003), affords useful

tools for dissecting the biology of ER subtypes at pituitary

level (Sanchez-Criado et al. 2004, 2006a) in OVX rats.

A ying–yang relationship between ERb and ERa on

LH secretion has been reported in 2-week OVX rats

treated with these ER-selective agonists (Sanchez-Criado

et al. 2004, 2006a).

DOI: 10.1677/JOE-06-0214Britain Online version via http://www.endocrinology-journals.org

J C GARRIDO-GRACIA, A GORDON and others . ER isoforms and sites modulate LH secretion108

Tamoxifen (TX), a type I oestrogen antagonist with

selective ER modulator (SERM) properties (McDonnell

1999, 2003, McDonnell et al. 2002, Smith & O’Malley 2004),

exhibits agonist activities in the gonadotrope of OVX rats.

These actions of TX include shrinkage of OVX-induced

hypertrophy, induction of progesterone receptor (PR)

mRNA and protein expression, and PR-dependent LH-

releasing hormone (LHRH) self-priming in vitro (Bellido et al.

2003). LHRH self-priming is a phenomenon in which the

magnitude of the LH response to the second of two equal

exposures of LHRH separated by an interval of 60 min is

significantly greater than the response to the first exposure to

LHRH (Fink 1988). Evidence derived from in vitro work

indicates that the agonist actions of TX in the rat gonadotrope

are exerted at the nuclear ERa level exclusively. Therefore,

incubated pituitaries from OVX rats injected over 3 days with

pharmacological doses of TX exhibit LHRH self-priming

(Sanchez-Criado et al. 2005b) and this agonistic effect of TX is

inhibited when E2 or the membrane-impermeable analogue

conjugated E2–BSA is added to the incubation medium.

Moreover, addition of the pure type II anti-oestrogen

ICI182 780 (Smith & O’Malley 2004) to the medium blocks

the rapid inhibitory action of E2 on TX-elicited LHRH

self-priming, whereas TX itself does not (Sanchez-Criado

et al. 2005b). One emerging explanation of these in vitro data is

that TX selectively binds nuclear ERa but not ERb(Tzuckerman et al. 1994, Bellido et al. 2003, Sanchez-Criado

et al. 2004, 2005a) and exhibits extremely low affinity for

membrane ERa in rat gonadotropes (Sanchez-Criado et al.

2005b). In addition, these data suggest that E2 inhibition of

TX-elicited LHRH self-priming is due to activation of the

plasma membrane ERa (Sanchez-Criado et al. 2005b, 2006b).

The present study was designed to ascertain whether the

in vitro inhibitory effects of both nuclear ERb-initiatedsignalling (Sanchez-Criado et al. 2004) and membrane ERa-initiated signalling (Sanchez-Criado et al. 2005b) upon the

LH secretory actions of the nuclear ERa-initiated signalling

were also operative in the whole animal. To this purpose,

three groups of in vivo experiments were conducted: the first,

to verify the effects of TX on LH secretion, pituitary PR

expression and LHRH self-priming in 2-week OVX rats; the

second, to evaluate the role of the different ER isoforms and

sites in LH release in gonadotropes with TX-activated nuclear

ERa using the cognate ligand E2 and the selective ERa and

ERb agonists PPT and DPN respectively and the third to

verify that TX acts through ERa by studying the effects of the

selective ERa antagonist MPP.

Materials and Methods

Animals, general conditions and surgery

Adult female Wistar rats weighing 190–210 g were used. Rats

were housed under a 14 h light:10 h darkness cycle (light on

at 0500 h) and 22G2 8C room temperature, with ad libitum


access to rat chow and tap water available ad libitum. Rats were

bilaterally OVX under ether anaesthesia at random stages of

the oestrous cycle and assigned to experimental groups

14 days later. At the time indicated in each experiment, a

right atrial cannula was implanted using a previously

described procedure (Harms & Ojeda 1974, Sanchez-Criado

et al. 1993), and rats received an i.v. injection of 20 IU

heparin/250 ml saline. At 0900 h (time 0) the following day,

the distal ends of the cannulae were attached to extension

tubing (P50; Adams, Parsippany, NJ, USA) to permit blood

sampling and LHRH administration. Rats were given an i.v.

bolus of 25 ng LHRH, and a second one 60 min later. Blood

samples (250 ml each) were taken at 0, 15, 60, 75 and 120 min.

At the end of the experiments, anterior pituitaries were

removed and dissected out for the determination of protein

and LH contents. On day 18 (experiment 1) and day 21

(experiments 2, 3, 4 and 5), vaginal smears were taken. All

experimental protocols were approved by the Ethical

Committee of the University of Cordoba, and experiments

were performed in accordance with the rules on laboratory

animal care and with international law on animal

experimentation.

Drugs and treatments

TX (Sigma) was injected at pharmacological/saturating doses

of 3 mg/day (Sanchez-Criado et al. 2004, 2005b, 2006b). The

selective ERa and ERb agonists PPT and DPN respectively

(Tocris Cookson Ltd, Avonmouth, UK; Stauffer et al. 2000,

Meyers et al. 2001) were injected at doses of 1.5 mg/day

(Sanchez-Criado et al. 2004). PPT has a 400-fold preference

for ERa and does not activate ERb (Stauffer et al. 2000).

DPN has a 70-fold higher binding affinity for ERb than for

ERa (Meyers et al. 2001). Oestradiol benzoate (EB; Sigma)

was injected at the pharmacological dose of 25 mg/day. Thepotent ERa-selective antagonist methyl-piperidino-pyrazole

(MPP; Tocris) was dissolved in DMSA/olive oil (1/14, v/v)

and injected at a dose of 1 mg/day. This compound displays

220-fold more affinity for ERa than for ERb (Sun et al. 2002,

Harrington et al. 2003). The PR antagonist onapristone

(ZK299; Schering, Berlin, Germany; Neef et al. 1984, Bellido

et al. 1999) was injected at a dose of 3 mg. All ER ligands and

ZK299 were given subcutaneously in 0.2 ml oil (Table 1).

Synthetic LHRH (Peninsula Laboratory, Inc., Merseyside,

UK) was dissolved in saline at a concentration of 125 ng/ml,

and 0.2 ml of this solution was injected intravenously. The

saturating doses of steroid receptor ligands used in the present

experiments were based on previously published studies

(Table 1).

Experiment 1: effect of TX on LH secretion, PR expression andLHRH self-priming in OVX rats

Rats were injected either with TX or with oil vehicle and EB

(negative and positive control groups respectively) on days

15–17 after OVX. Less than 0.4 ml blood was drawn by direct

www.endocrinology-journals.org

Table 1 List of steroid receptor ligands used indicating their action and relevant references

Action

nERa nERb mERa nPR References

NameTamoxifen (TX) C Tzukerman et al. (1994) and

Sanchez-Criado et al. (2005b)Oestradiol benzoate (EB) C C C Sanchez-Criado et al. (2004, 2006b)Propylpyrazole triol (PPT) C C Stauffer et al. (2000) and

Sanchez-Criado et al. (2004)Diarylpropionitrile (DPN) C Meyers et al. (2001) and

Sanchez-Criado et al. (2004)Methyl-piperidino-pyrazole (MPP) K K Sun et al. (2002) and Harrington et al.

(2003)Onapristone (ZK299) K Neef et al. (1984) and Bellido et al.

(1999)

nERa, nuclear oestrogen receptor a; nERb, nuclear oestrogen receptor b; mERa, membrane oestrogen receptor a; nPR, nuclear progesterone receptor;C, activation; K, blockade.

ER isoforms and sites modulate LH secretion . J C GARRIDO-GRACIA, A GORDON and others 109

left jugular venipuncture under light ether anaesthesia at

0900 h on days 15, 16 and 17. On the afternoon of day 17,

rats were implanted with atrial cannulae. Pituitary LH

secretory response to LHRH was studied on day 18.

Pituitaries from similarly treated rats (four rats/group) were

processed for PR expression on day 18 after OVX. Serum and

plasma samples were stored frozen until the LH RIAwas run.

Experiment 2: effects of EB, PPTand DPN on LH secretion andLHRH self-priming in TX-injected OVX rats

Ratswere injectedwithTX (control group) on days 15–20 after

OVX. Over the past 3 days (days 18–20 after OVX) of TX

treatment, ratswere additionally injectedwithEB, PPTorDPN.

Less than 0.4 ml blood was taken by jugular venipuncture at

0900 h on days 18–20. On the afternoon of day 20, rats were

implanted with atrial cannulae. The pituitary LH secretory

response to LHRH was studied on day 21. Serum and plasma

samples were stored frozen until the LH RIAwas run.

Experiment 3: effects of EB, PPTand DPN on PR expression inTX-injected OVX rats

Rats were injected with TX (control group) on days 15–20

after OVX. Over the past 3 days (days 18–20 after OVX) of

TX treatment, rats were additionally injected with EB, PPTor

DPN. At 0900 h on day 21 after OVX, four rats in each of the

four groups (TX, TXCEB, TXCPPTand TXCDPN) were

decapitated and their anterior pituitaries dissected out and

processed for PR immunoreactivity.

Experiment 4: effects of ZK299 on LH secretion and LHRHself-priming elicited by TX

The action of ligand-independent activation of PR on LH

secretion was evaluated in 6-day TX-injected OVX rats given


a single injection of 3 mg ZK299 at 0900 h on day 20.

Basal and LHRH-stimulated LH secretion and LHRH

self-priming were studied on day 21. Plasma samples were

stored frozen until the LH RIA was run.

Experiment 5: effects of MPP on LH secretion, PR expressionand LHRH self-priming in TX-injected OVX rats

Rats were injected daily from days 15 to 20 after OVX with

MPP, with or without TX injections on days 18–20 after OVX.

Control groups consisted of OVX rats injected with 0.2 ml oil

from days 15 to 20 and TX alone from days 18 to 20. Less than

0.4 ml blood was taken by jugular venipuncture at 0900 h on

days 15, 18, 19 and 20. On the afternoon of day 20, rats were

implanted with atrial cannulae. On day 21, the pituitary LH

secretory response to LHRH was studied. In addition,

pituitaries from similarly treated OVX rats (three rats/group)

were processed for PR immunoreactivity on day 21. Serum and

plasma samples were stored frozen until the LH RIAwas run.

Pituitary LH content determination

At the end of each experiment, either on day 18 or 21,

anterior pituitaries were removed and homogenized in 1 ml

RIA buffer and subjected to ultrasonic treatment. Samples

were centrifuged at 2800 g for 10 min and the supernatants

frozen at K20 8C until assayed by LH RIA. Pituitary LH

content was expressed as mg/mg protein. Pituitary protein

content was determined by the micro-turbidimetric method

using benzethonium chloride in alkali (Iwata & Nishikaze

1979). The sensitivity of the method was 40 mg/ml.

RIA of LH

Serumor plasma LHconcentrationsweremeasured in duplicate

by RIA using a double-antibody method with a kit supplied by



NIH (Bethesda, MD, USA) and a previously described

microassay method (Sanchez-Criado et al. 1993, Bellido et al.

1999). Rat LH-I-9 was labelled with 125I by the chloramine-T

method (Greenwood et al. 1963). The intra- and inter-assay

coefficients of variation were 8 and 9% respectively. Assay

sensitivity was 3.5 pg/tube. LH was expressed as ng/ml of the

reference preparation LH-rat-RP-3.

LHRH self-priming

The peak pituitary response of LH occurs after 15-min

administration of LHRH pulse (Sanchez-Criado et al. 2005b).

In the present experiments, LHRH self-priming was

evaluated as the percentage increase in LH secretion to the

second LHRH pulse (primed pituitary response) with respect

to the first LHRH pulse (LHRH-stimulated LH secretion or

unprimed pituitary response).

Immunohistochemistry of pituitary PR

The immunohistochemical study was performed on dewaxed

and rehydrated 3 mm thick tissue sections of formalin-fixed,

paraffin-embedded tissue samples. The commercial mouse

monoclonal anti-human PR antibody clone PR10A9, raised

against the recombinant hormone-binding domain of human

PR located on the C-terminal domain of PR (Immunotech,

Marseille, France), diluted in the ratio of 1:15 000, and the

avidin–biotin peroxidase complex (ABC) technique (Vector,

Burlingame, CA, USA) were used as described previously

(Sanchez-Criado et al. 2004). Tissue sections from similarly

processed samples of rat uterus and human breast carcinoma

were used as positive controls. The specificity of the PR

antibody was shown by the lack of staining after pre-

incubation of tissue sections of rat uterus and pituitaries from

OVX rats treated with EB with 10K9, 10K7 and 10K5 M of

the cognate ligand for 1 h at 37 8C. Substitution of the specific

primary antibody by mouse ascitic fluid at the same dilution as

the specific primary antibody in tissue sections of the cases

under study was used as negative control. Several dilutions of

the PR10A9 monoclonal antibody were tested and the

optimal dilution was established at 1:15 000, because it gave

the highest intensity of nuclear staining with the lowest

background staining in pituitary and uterus (Sanchez-Criado

et al. 2004). Nuclear counterstaining was performed with

Mayer’s haematoxylin in all cases. The amount of cells

immunoreactive to PR antibody was expressed as the number

of positive nuclei counted in five fields at a magnification of

40! (about 240 pituitary cells/field) in each pituitary. All

immunoreactive cells were considered to be gonadotropes

because they are the only pituitary cells expressing PR (Fox

et al. 1990, Sanchez-Criado et al. 2005a).

Figure 1 Serum LH concentrations in ovariectomized (OVX) ratsinjected on days 15, 16 and 17 after OVX with 0.2 ml oil, 25 mgoestradiol benzoate (EB) or 3 mg tamoxifen (TX). Values aremeansGS.E.M. of ten rats. aP!0.05 versus oil-injected rats. ANOVAand Student–Newman–Keuls multiple range test.

Statistical analysis

Statistical analysis was performed by ANOVA to check

for significant differences among groups. When significant


differences existed, it was followed by the Student–Newman–

Keuls multiple range test for inter-group comparison.

Significance was considered at the 0.05 level.

Results

Effects of treatment with different ER ligands on vaginal smears inOVX rats

OVX rats treated with the ER agonists EB, TX and TX plus

EB, PPT, DPN or TX plus the PR antagonist ZK299 showed

either nucleated or fully cornified epithelial cells in vaginal

smears. In contrast, OVX rats injected with oil displayed

vaginal smears predominantly infiltrated by leukocytes. The

ERa antagonist MPP, either alone or in combination with

TX, behaved as an ER agonist because it also induced

cornification of vaginal smears.

Effect of TX on LH secretion, gonadotrope PR expression andLHRH self-priming in OVX rats

Treatment of 2-week OVX rats with TX over 3 days had

similar (though less pronounced) effects to treatment with the

cognate ligand EB over the same period of time. Therefore,

TX reduced both basal serum/plasma LH concentration

(Figs 1 and 2) only on days 17 and 18 and pituitary LH

content (Table 2) on day 18 after OVX. In addition,

TX-induced LHRH self-priming, as the magnitude of the


Figure 2 Plasma LH concentrations in ovariectomized (OVX) ratsinjected on days 15, 16 and 17 after OVX with 0.2 ml oil, 25 mgoestradiol benzoate (EB) or 3 mg tamoxifen (TX). At 0900 h on day 18(time 0), rats received an i.v. bolus of 25 ng LHRH (first arrow), anda second one 60 min later (second arrow). Blood samples (250 mleach) were taken at 0, 15, 60, 75 and 120 min through a right atrialcannula implanted on the afternoon of day 17 after OVX. Values aremeansGS.E.M. of eight to ten rats. aP!0.05 versus LH values 15 minafter the first challenge with LHRH. bP!0.05 versus oil-injected rats.cP!0.05 versus LH concentration (time 0) in oil-injected rats.ANOVA and Student–Newman–Keuls multiple range test.

Table 2 Luteinizing hormone (LH)-releasing hormone (LHRH) self-priming and LH pituitary content (mg/mg protein) in ovariectomized(OVX) rats injected daily with 0.2 ml oil (oil-3), 25 mg oestradiolbenzoate (EB-3) and 3 mg tamoxifen (TX-3) over 3 days (days 15–17after OVX), or with 3 mg TX alone (TX-6) over 6 days (days 15–20 afterOVX)or incombinationwith25 mgEB (TX-6CEB), 1.5 mgPPT (TX-6CPPT) or 1.5 mg DPN (TX-6CDPN) injected ondays 18–20after OVX.Asingle 3 mg ZK299 injection was given on day 20 to TX-treated rats (TX-6CZK299). See legend of Figs 2 and 4 for additional details oftreatments. LHRH self-priming was expressed as the percentageincrease of peak LH response to a second 25 ng LHRH pulse (primedpituitary response) with respect to the LH peak response to the first25 ng LHRH pulse (unprimed pituitary response) after 1 h. Values aremeansGS.E.M. of eight to ten determinations

LHRH self-priming LH (mg/mg)

GroupsOil-3 101.9G8.9 54.6G3.1EB-3 152.4G13.7* 43.3G3.9*TX-3 146.4G7.7* 39.7G3.6*TX-6 143.7G3.4* 35.2G2.4*TX-6CEB 100.8G11.0 38.7G3.9*TX-6CPPT 103.7G10.6 38.0G4.2*TX-6CDPN 150.5G11.6* 37.7G4.3*TX-6CZK299 104.9G8.9 31.6G2.3*

*P!0.05 versus oil-injected rats. ANOVA and Student–Newman–Keulsmultiple range test.



LH response to the second LHRH challenge, was signi-

ficantly increased with respect to the LH peak response to the

first LHRH pulse (Fig. 2; Table 2). A similar magnitude of

LHRH self-priming was also observed in OVX rats treated

with EB (positive control group), but not in OVX rats

injected with oil vehicle (negative control group; Fig. 2;

Table 2). However, TX did not sensitize the pituitary to

LHRH, which contrasted with the pituitary sensitizing action

of EB to LHRH (Fig. 2).

Effect of EB, PPTand DPN upon the inhibitory effect of TX onbasal LH secretion and on TX-elicited LHRH self-priming inOVX rats

The inhibitory effect of a 3-day TX treatment on serum LH

concentration and pituitary LH content in OVX rats was

further enhanced after 6-day treatment of OVX rats with TX

(Fig. 3; Table 2). This dose-related inhibitory effect of TX on

basal LH levels was potentiated by the simultaneous

administration of either EB or the selective ERb agonist

DPN on days 18–20 after OVX (Figs 3 and 4). However,

administration of the selective ERa agonist PPT did not

Figure 3 Serum LH concentrations on days 18, 19 and 20 inovariectomized (OVX) rats injected daily on days 15–20 after OVXwith 3 mg tamoxifen (TX) alone, or in combination with 25 mgoestradiol benzoate (TXCEB), 1.5 mg PPT (TXCPPT) or 1.5 mgDPN (TXCDPN) injected over the past 3 days (days 18–20 afterOVX) of TX treatment. Values are meansGS.E.M. of ten rats.aP!0.05 versus TX-injected rats. ANOVA and Student–Newman–Keuls multiple range test.


Figure 4 Plasma LH concentrations in ovariectomized (OVX) rats injected on days 15–20 after OVX with 3 mg tamoxifen (TX) alone,or combined with 25 mg oestradiol benzoate (TXCEB), 1.5 mg PPT (TXCPPT) or 1.5 mg DPN (TXCDPN) on days 18, 19 and 20 after OVX.A single 3 mg ZK299 injection was given on day 20 to TX-treated rats (TXCZK). At 0900 h on day 21 (time 0), rats received an i.v. bolusof 25 ng LHRH (first arrow), and a second challenge 60 min later (second arrow). Blood samples (250 ml each) were taken at 0, 15, 60, 75 and120 min through a right atrial cannula implanted on the afternoon of day 20 after OVX. Values are meansGS.E.M. of eight to ten rats. aP!0.05versus LH levels 15 min after the first LHRH challenge. bP!0.05 versus LHRH-stimulated LH secretion in TX-injected rats. cP!0.05versus LH concentration (time 0) in TX-treated rats. ANOVA and Student–Newman–Keuls multiple range test.


potentiate this inhibitory effect of TX (Figs 3 and 4). No effect

on pituitary LH content was noted after treatment with EB,

PPTor DPN (Table 2). TX treatment of OVX rats over 6 days

(days 15–20 after OVX) induced LHRH self-priming which

was blocked by both EB and PPT, but not by DPN treatments

over 3 days (days 18–20 after OVX, Fig. 4; Table 2).

Effect of ZK299 on LH secretion and LHRH self-priming inTX treated in OVX rats

The blockade of PR activity with ZK299 given on day 20 to

6-day TX-treated OVX rats reduced basal and LHRH-

stimulated LH secretion and blocked LHRH self-priming on

day 21 (Fig. 4). The antiprogestagen had no effect on pituitary

LH content (Table 2).

Effect of EB, PPT and DPN upon TX-induced PR expressionin OVX rats

Immunoreactive products to PR antibody were detected in

the nuclei of gonadotropes in pituitaries from OVX rats


treated over 3 or 6 days with TX (Fig. 5). PR expression was

higher after 6 days of TX treatment. In these rats, PR

immunoreactivity was found in either hypertrophied or

shrunken gonadotropes (Fig. 5; upper panel). In contrast, oil-

injected OVX rats exhibited hypertrophied gonadotropes

lacking PR expression. The number of PR immunoreactive

cells observed in pituitaries from 6-day TX-treated OVX rats

was reduced by co-administration of the selective ERbagonist DPN over the past 3 days of TX treatment (Fig. 5;

lower panel), while the selective ERa agonist PPT and the

cognate ligand EB had no significative effect on the number of

PR immunoreactive cells (Fig. 5; lower panel).

Effect of MPP on TX agonist actions in OVX rats

The agonist actions of TX in OVX rats injected on days 18–20

after OVX included: reduction of basal LH serum/plasma

concentration (Fig. 6) and pituitary LH content (Table 3),

induction of LHRH self-priming (Fig. 7; Table 3) and PR

expression on day 21 (Fig. 8). MPP treatment blocked the

TX-elicited LHRH self-priming (Fig. 7; Table 3) and reduced



www.endocrinology-journals.org Journal of Endocrinology (2007) 193, 107–119

Table 3 LHRH self-priming and LH pituitary content (mg/mgprotein) in OVX rats injected over 6 days (days 15–20 after OVX)with 0.2 ml oil or 1.0 mg selective ERa antagonist MPP alone, orcombined with 3 mg TX on days 18–20 after OVX

LHRH self-priming LH (mg/mg)

GroupsOil 92.3G9.8 49.8G6.1MPP 107.8G11.1 36.1G3.2*TX 166.7G14.8* 30.6G4.2*MPPCTX 113.8G12.4 32.2G2.4*

See legend of Fig. 7 for additional details of treatments. LHRH self-priming wasexpressed as the percentage increase of peak LH response to a second 25 ngLHRH pulse (primed pituitary response) with respect to the LH peak responseto the first 25 ng LHRH pulse (unprimed pituitary response) after 1 h. Valuesare meansGS.E.M. of seven to eight determinations. *P!0.05 versusoil-injected rats. ANOVA and Student–Newman–Keuls multiple range test.

Figure 6 Serum LH concentrations on days 15, 18, 19 and 20 inovariectomized (OVX) rats injected on days 15–20 after OVX with0.2 ml oil or 1.0 mg MPP alone or in combination with 3 mg TX ondays 18–20. Values are meansGS.E.M. of eight rats. Serum LHconcentration on day 15 after OVX is the mean of 32 rats. *P!0.05versus oil-injected rats. ANOVA and Student–Newman–Keulsmultiple range test.


the TX-induced PR expression (Fig. 8). On the contrary, MPP

alone injected daily from days 15 to 20 after OVX reduced basal

LH serumconcentration andpituitary LHcontent (Figs 6 and 7;

Table 3), a negative feedback on LH secretion similar to the

agonist effect of TX (Figs 1–3) in OVX rats.

Discussion

The results of the present experiments indicate that TX has

ERa agonist actions in the gonadotrope of OVX rats.

Assuming that a 6-day TX treatment also prevented the action

Figure 5 Immunohistochemical progesterone receptor (PR) expression inshows representative examples of PR expression in OVX rats injected ooestradiol benzoate (EB-3) or 3 mg tamoxifen (TX-3). An example of thTX-treated rats is also shown (TX-6). Hypertrophied gonadotropes withgonadotropes expressing PR (black arrows) are shown. Avidin–biotin perwith Mayer’s haematoxylin, !40. The lower panel represents the numbeinjected daily over 3 days with 0.2 ml oil (oil-3), 25 mg EB (EB-3) or 3 mgalone (TX-6) or in combination, over the past 3-day TX treatment, withDPN (TX-6CDPN) and studied on day 21 after OVX. Values are meansGOVX rats. bP!0.05 versus TX-6. cP!0.05 versus TX-3. ANOVA and St


of other ER ligands at the nuclear ERa level (Sanchez-

Criado et al. 2005b), results show, in addition, that the

bimodal components of gonadotrope LH release with

TX-activated nuclear ERa are differentially affected by the

simultaneous activation of ER isoforms and sites as follows:

1) The inhibitory effect of TX on PR-independent basal LH

secretion was potentiated by activation of ERb with DPN

and EB but not by activation of membrane ERa with PPT.

2) The PR-dependent LH secretory surge elicited through

nuclear ERa was inhibited by activation of ERb with DPN

and EB, and membrane ERa with PPTand EB through their

effects on PR expression and action respectively.

The known in vitro agonist actions of TX on LH release

have been confirmed in vivo using long-term OVX rats. The

2-week OVX rat model was used because treatment of these

rats over 3 days with EB mimicks the endocrine events of pro-

oestrus through augmentation of the LHRH-releasing

pathway, induction of PR expression and induction of

PR-dependent LHRH self-priming (Bellido et al. 2003,

Sanchez-Criado et al. 2004) culminating in an ovulatory LH

surge (Legan & Tsai 2003). The administration of pharma-

cological/saturating dose of TX to these OVX rats decreased

both serum and pituitary LH levels in a negative feedback

manner, induced upregulation of PR expression in the

gonadotrope, and elicited LHRH self-priming. In addition, it

has also been confirmed in vivo that these agonist actions of

TX in the gonadotrope of OVX rats are due to the activation

the pituitary of 15-day ovariectomized (OVX) rats. The upper panelver 3 days (days 15–17 after OVX) with 0.2 ml oil (oil-3), 25 mge PR expression in the pituitaries of 6-day (days 15–20 after OVX)(white arrows) or without (arrowhead) PR expression and shrunkenoxidase complex immunohistochemical technique, counterstainingr of anterior pituitary cells expressing PR in pituitaries from OVX ratsTX (TX-3) and studied on day 18 after OVX, or over 6 days with TX

25 mg oestradiol benzoate (TX-6CEB), 1.5 mg PPT (TX-6CPPT) orS.E.M. of 20 fields (five fields!four rats). aP!0.05 versus oil-injectedudent–Newman–Keuls multiple range test.


Figure 7 Plasma LH concentrations on day 21 after ovariectomized (OVX) rats injected on days 15–20 after OVX with 0.2 ml oil or 1.0 mgMPPalone or with 3 mg TX on days 18–20. At 0900 h on day 21 (time 0), rats received an i.v. bolus of 25 ng LHRH (first arrow), and a secondchallenge 60 min later (second arrow). Blood samples (250 ml each) were taken at 0, 15, 60, 75 and 120 min through a right atrial cannulaimplanted on the afternoon of day 20 after OVX. Values are meansGS.E.M. of seven to eight rats. aP!0.05 versus LH levels after the first LHRHchallenge at time 0 in TX-injected OVX rats. bP!0.05 versus oil-injected rats. ANOVA and Student–Newman–Keuls multiple range test.


of nuclear ERa (Sanchez-Criado et al. 2005b). The

administration of the ERa-selective antagonist MPP to

TX-treated OVX rats both reduced TX-induced PR

expression to a minimum and abolished TX-elicited

LHRH self-priming. However, MPP did not behave as an

ERa antagonist exclusively. This is because MPP reduced

basal LH secretion and induced vaginal smears cornification

in the absence of TX treatment. Taken together, these

findings indicate that MPP behaved as a SERM.

The first and longer phase of LH release in the gonadotrope

is PR-independent basal LH secretion. For most of the

reproductive life of females, LH levels are kept within a

relatively low range by the PR-independent (Chappell et al.

1999) negative feedback of moderate levels of oestrogen (Fink

1988). Whereas activation of ERa with the selective agonist


PPTreduces LH secretion in OVX rats (Sanchez-Criado et al.

2004), PPT failed to reduce serum LH levels when

administered to TX-treated OVX rats, indicating a probable

competition of the selective ERa agonist for the same ER

isoform in which TX acts. In addition, the present data

showed that: i) activation of ERb either with DPN or EB

potentiated the TX-induced reduction of serum LH levels in

OVX rats; ii) activation of both ER isoforms with EB was

more effective in reducing serum LH levels than activation of

nuclear ERa alone with TX and iii) the potent selective ERaantagonist MPP administered alone reduced PR-independent

LH secretion in an agonistic manner. These findings

indicate that the negative feedback of oestrogen on LH

secretion is a genomic ERa effect potentiated by activation

of ERb.


Figure 8 Immunohistochemical progesterone receptor (PR)expression in the pituitary of 15-day ovariectomized (OVX) rats. Theupper panel shows representative examples of PR expression inOVX rats injected over 6 days (days 15–20 after OVX) with 0.2 mloil or 1.0 mg selective ERa antagonist MPPalone, or combined with3 mg tamoxifen (TX) on days 18–20 after OVX. Hypertrophiedgonadotropes with (white arrows) and without (arrowhead) PR andshrunken gonadotropes expressing PR (black arrows) are shown.The lower panel represents the number of anterior pituitary cellsexpressing PR in pituitaries from OVX rats injected with oil, MPP, TXor MPPCTX and studied on day 21 after OVX. Values are meansGS.E.M. of 15 fields (five fields!three rats). aP!0.05 versus oil-injected OVX rats. bP!0.05 versus TX-injected OVX rats. Seelegend of Fig. 5 for additional technical and morphological details.



The expression of PR in the gonadotrope is an oestrogen

effect of both E2 and TX which occurs simultaneously with

their negative feedback on LH secretion (Sanchez-Criado

et al. 2004, 2006a). Gonadotrope PR expression gives rise to a

complex phase: the PR-dependent LH surge elicited by an

acute rise in E2 levels (Chappell et al. 1999). The LH surge has

two components: 1) LHRH-stimulated LH secretion and 2)

the LHRH self-priming. The former occurs in an oestrogen

positive feedback manner, in which oestrogen increases

pituitary responsiveness to the releasing effects of LHRH

(Arimura & Schally 1971, Schuiling et al. 1999, Schwartz

2000) through activation of the nuclear ERa isoform

(Sanchez-Criado et al. 2004, Lindzey et al. 2006). Since

TX, neither in vitro (Sanchez-Criado et al. 2002, 2004) nor

in vivo (present results), sensitized the pituitary to the releasing

effects of LHRH, the role of the different ER pools in

LHRH-stimulated LH secretion cannot be directly

determined from the present experiments. However, two

findings strongly suggest that PR actions are involved

(Chappell et al. 1999, Sanchez-Criado et al. 2004) in ER

actions in TX-treated rats: i) activation of ERb with DPN

halved pituitary PR expression levels and ii) the complete

blockade of PR action with ZK299 decreased LHRH-

stimulated LH secretion. The absence of a significant effect of

activation of ERb with EB on TX-induced PR levels might

be due to PR synthesis-related gene transcription from the

large cytosolic pool of ER as EB activates the whole ER

orchestra (Levin 2001, 2005). This may have resulted in

overlapping ER functions in TX-treated OVX rats. Further-

more, it is also possible that the cognate ligand could have

partially displaced TX from nuclear ERa because of the

pharmacological doses used. Overall, the accumulated

evidence from presented and previous data (Sanchez-Criado

et al. 2004, 2006a) suggests that activation of ERb reduces

the efficiency of LHRH in stimulating LH release in

TX-treated rats.

The second component of PR-dependent LH surge

is oestrogen-dependent LHRH self-priming (Waring &

Turgeon 1980, 1992), the property of LHRH that increases

gonadotrope responsiveness to itself. It depends both on

de novo synthesis of priming proteins (Turgeon & Waring

1991) and on the oestrogen-induced upregulation of PR in

gonadotropes (Turgeon & Waring 1994). PR is a neuro-

endocrine integrator keystone in the LHRH self-priming

process (Turgeon & Waring 1991, 1994, Levine et al. 2001).

It has been observed that LHRH self-priming is a convincing

nuclear ERa-mediated effect (Sanchez-Criado et al. 2004).

Activation of ERa with PPT induces PR expression and

elicits LHRH self-priming in OVX rats, whereas activation of

ERb alone with DPN induces PR expression not followed by

LHRH self-priming (Sanchez-Criado et al. 2004). The fact

that administration of TX to OVX rats induced both PR

expression and LHRH self-priming further supports a nuclear

ERa agonist action of TX at the rat pituitary level. Although

DPN reduced PR expression levels in TX-treated rats, and

LHRH self-priming depends on PR, the remnant PR, about


Figure 9 Proposed integrated action of oestrogen receptor (ER) isoforms and sites withprogesterone receptor (PR) in the rat gonadotrope. E2, oestradiol-17b; P4, progesterone;mERa, plasma membrane ERa; nERa, nuclear ERa; nERb, nuclear ERb; LHRH, luteinizinghormone-releasing hormone; LH, luteinizing hormone; GnSI/AF, putative non-steroidalovarian gonadotrophin surge inhibiting/attenuating factor (Byrne et al. 1996); mis,membrane-initiated signalling; nis, nucleus-initiated signalling. 1. Activation of nERa bygranulosa cells E2 transcriptionally induces PR expression and the simultaneous activation ofERb modulates this action in a ying–yang mode. 2. The E2-dependent LHRH surge activatesPR in the absence of P4 in a ligand-independent manner. 3. P4 from luteinized granulosa cellsin response to LH phosphorylates and activates PR. 4. Ovarian E2 activation of mERastimulates intracellular phosphatases (Sanchez-Criado et al. 2006b), which results in non-transcriptional reduction of PR phosphorylation and decreased LHRH self-priming. 5. Theputative ovarian GnSI/AF decreases PR action through membrane-initiated signalling.Broken arrow indicates that PR activation is not the only cause involved in elicitingoestradiol-augmenting and LHRH self-priming factors.


www.endocrinology-journals.org Journal of Endocrinology (2007) 193, 107–119


one-half of that found in TX-treated rats, may have been

enough to elicit LHRH self-priming once activated in a

ligand-independent manner (Levine 1997, Blaustein 2004).

This finding rules out the possibility of an ERb involvement

in EB inhibition of TX-induced LHRH self-priming. The

finding that treatment with either EB or PPT, but not DPN,

blocked TX-elicited LHRH self-priming sharply contrasted

with the facilitatory action of both ERa agonists on LHRH

self-priming in the absence of TX treatment (Sanchez-Criado

et al. 2004). Moreover, ZK299 similarly annulled TX-elicited

LHRH self-priming. These facts suggest that both EB

and PPT acted on an ERa pool different from that bound

to TX resulting in inhibition of PR action (Sanchez-Criado

et al. 2006b).

On the basis of both the present in vivo and the previous

in vitro results (Sanchez-Criado et al. 2004, 2005a,b, 2006a,b),

one might hypothesize the following mechanism of E2 action

on the complete ER orchestra upon LH secretion in the

gonadotrope of the rat: 1) the PR-independent negative

oestrogen feedback may be exerted at the nuclear ERa and

ERb complementarily; 2) the positive feedback of oestrogen

on LHRH-stimulated LH secretion, a nuclear ERa action,

may be modulated by the inhibitory action of ERb on PR

expression (Fig. 9) and 3) the oestrogen-dependent LHRH

self-priming may be a nuclear ERa action modulated by

surface ERa-initiated signalling inhibition of PR action

(Fig. 9).

Acknowledgements

This study was subsidized by grants (BFU2005-01443 and

AGL2006-09016/GAN) from the DGICYT (Spain). The

authors are grateful to the National Hormone and Pituitary

Program (Baltimore, MD, USA) for the LH RIA kit and to

Teresa Recio for technical assistance. The authors declare that

there is no conflict of interest that would prejudice the

impartiality of this scientific work.

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Received in final form 15 January 2007Accepted 17 January 2007Made available online as an Accepted Preprint25 January 2007


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The integrated action of oestrogen receptor isoforms and sites with