Hum. Reprod.-2009-Gordon-2618-28

8/10/2019 Hum. Reprod.-2009-Gordon-2618-28

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ORIGINAL ARTICLE Reproductive endocrinology

Activation of estrogen receptor-a

induces gonadotroph progesterone

receptor expression and action

differently in young and middle-aged

ovariectomized rats

Ana Gordon1,3, Rafaela Aguilar1, Jose C. Garrido-Gracia1,

Silvia Guil-Luna2, Raquel Sanchez-Cespedes2, Yolanda Milla n2,

Juana Martn de las Mulas2, and Jose E. Sa nchez-Criado1

1Departments of Cell Biology, Physiology and Immunology, University of Cordoba, Cordoba, Spain 2Departments of Comparative Pathology,University of Cordoba, Cordoba, Spain

3Correspondence address. Section of Physiology, Faculty of Medicine, Avda. Menendez Pidal s/n, 14004 Cordoba, Spain. Tel: 34-957-

218283; E-mail: [email protected]

background:We attempted to define the effect of estrogen receptor (ER)a activation on gonadotroph progesterone receptor (PR)

expression (mRNA and protein) and action (GnRH-stimulated and GnRH self-priming) in short- and long-term ovariectomized (OVX) rats.

methods:Two weeks or 1 year after OVX, rats were injected over 3 days with 125 mg/kg of estradiol benzoate (EB), 7.5 mg/kg of the

selective ERaagonist propylpyrazole triol (PPT), or 15 mg/kg of the selective ER modulator tamoxifen (TX). Controls were given 0.2 ml oil.

The last day of ER analog treatment, half of the rats in each group received 25 mg/kg of progesterone (P). The next day, anterior pituitaries

were removed and analyzed for PR-AB mRNA and protein. Gonadotrophin secretion in incubated pituitaries was also measured.

results: (i) PR mRNA expression was higher in young than in middle-aged OVX rats although PR protein was absent in pituitaries from

both groups of OVX rats; (ii) activation of ERa reduced gonadotroph hypertrophy and increased PR mRNA and protein expression (EB .

PPT . TX) more efficiently in young than in middle-aged rats, (iii) ER agonists elicited GnRH-stimulated LH and FSH secretion in young but

only FSH secretion in middle-aged OVX rats, (iv) evaluated by peak LH concentrations, GnRH self-priming was observed in both groups of

OVX rats and (v) P down-regulated PR protein expression in young, and to a lesser extent, in middle-aged OVX rats, in close association

with PR-dependent GnRH self-priming.

conclusions: Middle-aged OVX rats exhibited clear-cut LH, but not FSH, secretory defects in pituitary sensitivity to estrogen and P.

Key words: progesterone receptor expression / gonadotrophin secretion / GnRH self-priming / tamoxifen / estrogen receptor-a

agonists

IntroductionOvarian cyclicity in mammals depends on the endocrine interaction of

the components of the hypothalamuspituitaryovary uterus axis

(Feder, 1981). Estral/menstrual cyclicity depends on negative and

positive feedback mechanisms. In terms of the ovarian positive feed-

back mechanism, estradiol (E2) is the main component acting

through estrogen receptor (ER)a and b isoforms on the hypothala-

muspituitary system in both rats (Fink, 1988), and women (Messinis,

2006). At the pituitary level, E2, sensitizes the pituitary to GnRH (Fink,2000) and induces progesterone receptor (PR)-dependent (Collins

and Hodgen, 1986; Batista et al., 1992; Waring and Turgeon, 1992)

GnRH self-priming (Fink, 1995). All this results in the pro-estrous

afternoon (Smith et al., 1975) or midcycle (Hoffet al., 1983; Knobil

and Hotchkiss, 1988) pre-ovulatory gonadotrophin surges (Fink,

1988, Messinis, 2006).

LH surge-dependent ovarian progesterone (P) secretion enhances

the positive E2 feedback on LH surge. Activation of E2-dependent

& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

For Permissions, please email: [email protected]

Human Reproduction, Vol.24, No.10 pp. 26182628, 2009

Advanced Access publication on July 2, 2009 doi:10.1093/humrep/dep237


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(Szabo et al., 2000; Turgeon and Waring, 2006) gonadotroph PR

during pro-estrous afternoon or midcycle increases the magnitude of

the LH surge in rats (Rao and Mahesh, 1986; Sanchez-Criadoet al.,

1990; Turgeon et al., 1999; Szabo et al., 2000) and women (Chang

and Jaffe, 1978; Hoffet al., 1983; Collins and Hodgen, 1986; Batista

et al., 1992; Brensing et al., 1993). In addition, ovarian P secreted

around the time of the LH surge once P activates its own receptor,

down-regulates PR in rats (Turgeon et al., 1999; Szabo et al., 2000;

Turgeon and Waring, 2000) and women (Messinis and Templeton,

1990; Gill et al., 2002) through degradation by the 26S proteasome

(Lange et al ., 2000). This results in a reduction in PR protein

expression and extinction of PR-mediated LH secretion,

GnRH-stimulated LH secretion and GnRH self-priming (Chappell

et al., 1999). Therefore, it seems clear that ovarian P is involved in

both the LH surge magnitude and termination in both rats (Turgeon

and Waring, 2000) and women (Dafopoulos et al., 2006).

Beginning around 10 months of age in female rat, or during midlife in

woman, the diminished ovarian follicular reserve is followed by a

period of transition characterized by irregular ovarian cycles. In the

rat, this period is characterized by extended phases of persistent

estrus before entering persistent diestrus, acyclicity and anovulation(Mandl, 1961; Scarbrough and Wise, 1990; Pellicer et al., 1995). In

women, menstrual irregularity (perimenopause) is followed by meno-

pause in which the low circulating E2 concentrations render the posi-

tive feedback mechanism inactive. The complete process of ovarian

senescence until the anovulation stage is not precisely defined and

the magnitude of the E2-exposed pituitary response to GnRH has

not been quantified. This disruption appears to be caused by

changes in the pituitaryovarian axis (Cooper et al., 1980), and an

altered sensitivity of the pituitary to ovarian steroids seems possible

(Nass et al., 1984).

Young and middle-aged ovariectomized (OVX) rats, besides being a

valid model for the study of the relationship between ovarian steroids

and pituitary function, may be considered surgical menopause andpostmenopause models, respectively (Alonso et al., 2006). Accord-

ingly, the aim of the present study was to determine the effect of

ERa activation on gonadotrope PR expression (mRNA and protein)

and action (GnRH-stimulated gonadotrophins secretion and GnRH

self-priming) in 2 weeks (young) and 1-year-old (middle-aged) OVX

rats.

Materials and Methods

Animals, surgery and general conditions

Adult female Wistar rats weighing 170+15 g were housed under a 14 h

light:10 h darkness cycle (light on at 0500 h) and 22+28C room tempera-ture, withad libitumaccess to rat chow and tap water. Rats were included

in the experiments after showing at least three consecutive 4-day regular

estrous cycles. Bilateral ovariectomy (OVX) was performed under light

ether anaesthesia at a random stage of the estrous cycle. All experimental

protocols were approved by the Ethical Commitee of the University of

Cordoba, and experiments performed in accordance with rules on labora-

tory animal care and international law on animal experimentation.

Experimental groups and treatments

Two weeks and 1 year after OVX, rats were sc injected over three days

with: (a) 0.2 ml oil; (b) 125 mg/kg estradiol benzoate (EB, Sigma Chemical

Co. St. Louis, MO, USA); (c) 7.5 mg/kg of the selective ERa agonist, pro-

pylpyrazole triol (PPT, Tocris Cookson Ltd, Avonmouth, UK) (Stauffer

et al., 2000); and (d) 15 mg/kg of the selective estrogen receptor modu-

lator (SERM), tamoxifen (TX, Sigma) (Bellido et al., 2003). On the last day

of each analog treatment half of the OVX rats in each group were given

25 mg/kg progesterone (P, Sigma). Doses of drugs and length of treat-

ments derive from the results of previous studies (Legan and Tsai, 2003;

Sanchez-Criado et al., 2004, 2006). At 0900 h the next day, rats were

decapitated; anterior pituitaries were removed and used for RTPCR ofPR mRNA and immunohistochemical analysis of PR protein. In addition,

hemipituitaries from the eight groups of rats were incubated under con-

trolled conditions for the study of gonadotrophins secretion parameters:

basal secretion of LH and FSH, GnRH-stimulated LH and FSH secretion

and GnRH self-priming.

Analysis RTPCR of PR mRNAThree anterior pituitaries/group were immediately frozen in liquid nitro-

gen and stored at 2808C until used for RNA analysis. RTPCR, opti-

mized for semiquantitative detection, was used to analyze relative

expression levels of PR mRNA in pituitaries from the experimental

groups. Total RNA was isolated from pituitary samples using the simple-

step, acid guanidinium thiocyanate-phenol-chloroform extraction method

(Chomczynski and Sacchi, 1986). Analysis of total mRNA expression

was carried out using a primer pair flanking a 326-bp coding area

common to both PR-A and -B isoforms, as described in detail elsewhere

(Szabo et al., 2000; Bellido et al., 2003). In addition, to provide an appro-

priate internal control, parallel amplification of a 249-bp of the S11 ribo-

somal protein mRNA was carried out in each sample under

previously-published conditions (Gordon et al., 2008). For amplification

of the targets, RT and PCR were run in two separate steps. PCR reactions

consisted of a first denaturing cycle at 978C for 5 min, followed by a vari-

able number of amplification cycles (28 cycles for PR-AB and 25 for

RP-S11) defined by denaturation at 968C for 30 s, annealing for 30 s,

and extension at 728C for 1 min. A final extension cycle of 728C for

15 min was included. Annealing temperatures were adjusted for each

target: 57.58

C for PR-AB and 588

C for RP-S11. These cycling conditionshad previously been optimized to ensure amplification of PR transcript

in the exponential phase of PCR (Bellido et al., 2003). Semiquantitative

data from RNA assays were expressed as mean+ SEM from at least

three independent determinations within each experimental group. In all

assays, liquid controls and reactions without RT were included, yielding

negative amplification (data not shown).

Immunohistochemistry of PR

The immunohistochemical study was performed in formalin-fixed, paraffin-

wax embedded, 3mm-thick tissue sections of three pituitaries/group of

rats. PR expression was analyzed using 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 1:15 000 for

18 h at 48C, and the avidinbiotin peroxidase complex (ABC) as

described elsewhere (Bellido et al., 2003; Sanchez-Criado et al., 2006,

Gordonet al., 2008). Dewaxed and rehydrated tissue sections were sub-

jected to high-temperature antigen retrieval by incubation with 0.01 M

citrate buffer, pH 6.0, at 958C for 8 min in a decloaking chamber.

Tissue sections were counterstained with Mayers hematoxylin. Tissue sec-

tions of formalin-fixed, paraffin-wax-embedded rat uterus were used as

positive controls. Substitution of the specific primary antibody by non-

immune mouse IgG1 (Affinity Bioreagents, Golden, CO, USA) in tissue

section of the pituitaries under study was used as negative control in

every assay. The number of cells immunoreactive to PR antibody was

Gonadotroph PR expression/action in young and middle-aged OVX rats 2619


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expressed as the number of positive nuclei counted in five fields at a mag-

nification 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 (Sanchez-Criado et al., 2005;

Garrido-Gracia et al., 2008).

Hemipituitary incubation protocol

Incubation of hemipituitaries was carried out, with minor modifications, as

described previously (Gordonet al., 2008). Briefly, halves of anterior pitui-

taries were incubated at 378C with constant shaking (60 cycles/min) in an

atmosphere of 95% O2 and 5% CO2. Each incubation vial contained 1 ml

of Dulbeccos modified medium (DMEM), withoutL-glutamine and phenol

red, containing glucose (4.5 g/l) and bovine serum albumin (BSA, 0.1% w/

v), pH 7.4. After 1 h of pre-incubation, hemipituitaries from the eight

groups of rats were incubated for 2 h with the same test substances

that were injected: medium alone, 1028 M E2 (Sigma), 1027 M PPT or

1027 M TX, with or without 1026 M P, in connation with the in vivotreat-

ment. Finally, 1028 M GnRH (LHRH, Peninsula Lab. Inc. Merseyside, UK)

was used to stimulate gonadotrophin secretion. The first GnRH challenge

lasted 15 min; fresh medium without GnRH was then added. Finally, 1 h

later, over a 15 min period fresh medium containing GnRH was added,

then removed and replaced by fresh medium for the last 45 min of incu-

bation. Medium was removed every 15 min for determination of

GnRH-stimulated gonadotrophin concentrations and GnRH self-priming.

Radioimmunoassay of LH and FSH

Concentrations of LH and FSH in incubation media were measured in

duplicate by radioimmunoassay using a double-antibody method with

kits supplied by NIH (Bethesda, MD, USA) and a previously-described

microassay method (Sanchez-Criado et al ., 1990). Rat LH-I-10 and

FSH-I-9 were labeled with 125I by the chloramine T method (Greenwood

et al., 1963). All media samples were assayed in the same assay. Intra-assay

coefficient of variation was 8 and 8.8%, and assay sensitivity was 3.75 and

20 pg/tube, respectively. LH and FSH concentrations were expressed as

nanogram/hemipituitary of the reference preparation LH-rat-RP-3 and

FSH-rat-RP2, respectively.

GnRH self-priming

GnRH self-priming is the property of GnRH that increases responsiveness

of the gonadotroph to itself (Fink, 2000). Self-priming was evaluated as the

percentage increase in peak gonadotrophin accumulation in the medium

during the second hour of incubation after 15 min GnRH exposure, with

respect to the peak gonadotrophin accumulation in the medium during

the first hour after a 15 min GnRH challenge.

Statistical analysis

Statistical analysis was performed by ANOVA to test for significant differ-

ences among groups. When significant differences existed, ANOVA was

followed by the Student NewmanKeuls multiple range test to

compare means. Significance was considered at the 0.05 level.

Results

Effects of ER ligands on pituitary PR-AB

mRNA expression in young and middle-agedOVX rats

Treatment with EB or PPT increased pituitary PR-AB mRNA

expression both in young and middle-aged OVX rats (Fig. 1, upper

panel). Treatment with TX was less effective than the cognate ER

ligand or the selective ERa agonist (Fig. l, lower panel). These ERa

agonistic effects were of lesser magnitude in middle-aged than in

young OVX rats.

Effects of ER ligands on gonadotroph

morphology and PR protein expression in

young and middle-aged OVX ratsPituitaries from both young- and middle-aged-OVX rats showed mor-

phological changes characterized by a slight or marked hypertrophy of

some pituitary cells. These cells had a large, clear cytoplasm with

central nuclei (Fig. 2A) or large, mostly single cytoplasmic vacuoles

and eccentric nuclei (the so-called signet ring cell) (Fig. 2D). Immuno-

histochemically, PR expression was not detected in pituitaries from

OVX rats of any age (Fig. 2A and D). All ER ligands increased the

number of PR-positive pituitary cells (gonadotrophs) and decreased

both the number and the size of hypertrophied gonadotrophs,

though to a differing degree. Thus, around 2030 PR-positive cells/

high power field were counted in EB-treated, young-OVX rats

(Fig. 2C) although the number of PR-positive cells/high power field

was lower in EB-treated, middle-aged-OVX rats (Fig. 2F). In addition,

the size of gonadotrophs, now easily identified by their expression of

PR (Fig. 2B and E), was smaller in ER ligand-treated young-OVX

(Fig. 2B) and middle-aged-OVX (Fig. 2E) rats than in non-treated

OVX rats (Fig. 2A and D). It is interesting to note that some

PR-positive cells of ER ligand-treated middle-aged-OVX rats remained

hypertrophied (Fig. 2E). These effects of ER ligands on both PR protein

expression and gonadotroph morphology were more intense in EB

treated rats and less intense in TX treated rats (Fig. 2C). Overall,

the number of PR-positive cells was significantly lower in middle-aged

OVX rats (Fig. 2C and F). Thus, middle-aged rats displayed incomplete

shrinkage of gonadotrophs and a lower expression of PR.

Effects of ER ligands on pituitary LH

secretion and GnRH self-priming in young

and middle-aged OVX rats

With respect to young OVX rats, pituitaries from middle-aged rats

showed a 2-fold increase in basal LH secretion (Fig. 3, upper panels,

Table I). Under the effects of ERa agonists, all groups of pituitaries

of young OVX rats, except pituitaries under the effect of TX,

responded to the first challenge of GnRH with a significant release

of LH (Fig. 3 left panels, Table I). No significant differences were

found in GnRH-stimulated LH secretion after treatment with ER

ligands in middle-aged OVX rats (Fig. 3 right panels, Table I). By con-

trast, both groups of pituitaries from OVX rats, young andmiddle-aged, exhibited GnRH self-priming after treatment with ER

ligands (Fig. 3, Table I). Overall, pituitaries from middle-aged rats

showed an absence of ERa sensitization of pituitaries to

GnRH-stimulated LH secretion.

Effects of ER ligands on pituitary FSH

secretion and GnRH self-priming in youngand middle-aged OVX rats

With respect to young OVX rats, pituitaries from middle-aged OVX

rats showed a 2-fold increase in basal FSH secretion (Fig. 4 upper

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panels, Table II). In contrast with LH secretion, all ER ligands sensitized

(EB . PPT .TX) the pituitaries of both young (Fig. 4, left panels)

and middle-aged OVX rats (Fig. 4, right panels) to secrete FSH in

response to GnRH. Also, the first challenge of GnRH failed to

prime the pituitary to the second GnRH challenge on FSH secretion

(Table II).

Effect of P on PR expression and action inyoung and middle-aged OVX rats

Administration of P did not affect pituitary PR-AB mRNA levels in any

experimental group of young or middle-aged OVX rats (Fig. 1A and

B). By contrast, P decreased the number of PR positive cells in all

experimental groups of OVX rats (Fig. 2C and F). PR protein

expression was absent in EB-, PPT- and TX-treated young OVX

rats, although P only reduced the number of PR positive cells in

middle-aged OVX rats. (Fig. 2F). Thus, middle-aged OVX rats had a

deficient P-induced PR down-regulation.

P significantly reduced LH secretion of hemipituitaries from young

and middle-aged OVX rats. In pituitaries from young OVX rats

treated with E2, P reduced and blocked GnRH-stimulated LH

secretion and GnRH self-priming, respectively (Fig. 3, Table I),

although in pituitaries from young OVX rats treated with PPT or

TX, P blocked both parameters of LH secretion (Fig. 3, Table I).

Figure 1 Expression of total PR AB mRNA in pituitaries from young and middle-aged OVX rats injected over 3 days with EB, selective ERa

agonist PPT, or selective ER modulator TX.

Controls were given 0.2 ml oil. The last day of ER analogs treatment, half of the rats in each group received P. All groups consisted of three pituitaries. Upper panel:

representative ethidium bromide-stained gel electrophoresis of PR AB and S-11 cDNA fragments amplified by semiquantitative RTPCR from total pituitary RNA

samples of the different experimental groups. Lower panel: semiquantitative data on the steady-state levels of total PR A B mRNA in the experimental groups. Relative

expression levels were obtained, in each sample, by normalization of absolute optical densities (OD) of the specific target to that of RP-S11 signal. Expression levels of PR

AB transcripts in young OVX rats given oil were taken as 100%, and the other values were normalized accordingly. Values are given as mean +SEM of three inde-

pendent determinations. a: P, 0.05 versus oil vehicle-injected controls, P, 0.05 versus the corresponding group of young OVX rats. ANOVA and Student

NewmanKeuls multiple range test.



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In middle-aged OVX rats, treatment with P blocked the GnRH

self-priming induced by EB, PPT and SERM TX (Fig. 3, Table I). In

sharp contrast, P had no effect on basal or GnRH-stimulated FSH

secretion either in controls or EB-, PPT- and TX-treated young or

middle-aged OVX rats (Fig. 4, Table II).

Discussion

Overall, the results showed that OVX in rats induced deficient gona-

dotroph response to GnRH, a reduction of PR mRNA, and absence of

PR protein expression. These functional effects of OVX are

Figure 2 PR expression in the anterior pituitary of OVX young ( AC) or middle-aged (DF) OVX rats.

(A) Micrograph of the anterior pituitary of young OVX rat injected with oil. No IR products are seen at the nuclear level and some pituitary cells are hypertrophied

(arrowheads). (D) Micrograph of the anterior pituitary of middle-aged OVX rat injected with oil. No IR products are seen at the nuclear level and gonadotrophs are

hypertrophied and vacuolated even to the extend of forming signet ring cells (arrowhead). (B) Micrograph of anterior pituitary of young OVX rat treated with (EB)

stained for PR. Several IR nuclei with deep brown (arrows) intensity are seen. (E) Micrograph of anterior pituitary of middle-aged OVX rat treated with (EB) stainedfor PR. Several IR nuclei with deep brown are seen, even in hypertrophied cells (arrows). ABC technique, nuclei counterstained with Mayers hematoxylin (40).

The lower panel represents the number of PR immunoreactive positive pituitary cells in young (C) or middle-aged (F) OVX rats. See legend of Fig. 1 for details of treat-

ment. Values are given as mean+ SEM of four pituitaries/group. a: P, 0.05 versus oil vehicle-injected controls, P, 0.05 versus the corresponding pair without P treat-

ment (PR down-regulation). ANOVA and StudentNewmanKeuls multiple range test.

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Figure 3 LH secretion from young (left panels) or middle-aged (right panels) OVX rats hemipituitaries.

Rats were injected over 3 days with EB, selective ERa agonist PPT, or selective ER modulator TX. Controls were given 0.2 ml oil. The last day of ER analogs treatment, half

of the rats in each group received P (broken line). Eight hemipituitaries from each group were incubated with medium alone, 1028 M E2 (Sigma), 1027 M PPT or 1027 M

TX, with or without 1026 M P, in connation with the in vivotreatment. GnRH pulses were 1 h apart. Mean values and statistical significance among groups (ANOVA and

StudentNewmanKeuls multiple range test) are given in Table I.



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accompanied by alterations in gonadotroph morphology ranging from

a very light hypertrophy in young rats to signet-ring cells in

middle-aged OVX rats. Also, the present results showed that acti-

vation of pituitary ERa with EB, PPT (Sanchez-Criado et al., 2004),

or TX (Sanchez-Criadoet al., 2002; Bellidoet al., 2003) induced gona-

dotroph shrinkage, pituitary GnRH responsiveness, gonadotroph PR

protein expression and GnRH self-priming. Finally, treatment with P

induced PR down-regulation and lack of action where PR has a func-

tion. Thus, pituitaries from young OVX rats treated with ERaagonists

exhibited a normal pro-estrous reproductive pituitary function (Bellidoet al., 2003; Sanchez-Criado et al., 2004, 2005, 2006). By contrast,

1-year deprivation of E in middle-aged OVX rats induced clear-cut

changes in pituitary sensitivity to sex steroids. Pituitaries from these

rats had: (1) increased basal release of LH and FSH, (2) lower

expression of PR mRNA and protein, (3) absence of ERa-induced sen-

sitization of pituitaries to secrete LH in response to GnRH and (4)

extremely vacuolated and hypertrophied gonadotropes (Garner and

Blake, 1981), which after treatment with ERa agonist, however,

were still able to express functional PR protein. The functionality of

these PR was evidenced by the presence of GnRH self-priming and

PR down-regulation (Lange et al., 2000).

Both ERa and ERb are expressed in the pituitary gonadotroph

(Mitchneret al., 1998; Sanchez-Criadoet al. 2005), and E2 activates

all ER isoforms. Although pituitary gonadotroph from middle-aged

OVX rats had a deficient response to ERa selective agonist, the fact

that EB and PPT induce the full response of PR-dependent LH

secretion parameters (Chappell et al ., 1999) in pituitaries from

young OVX rats indicated that the reported agonistic effects are

mediated by activation of ERa (Sanchez-Criado et al., 2004). More-

over, it has been reported that in the absence of E, TX acting

through intracellular ERa (Bellido et al., 2003; Sanchez-Criadoet al.,

2005) induces PR expression and GnRH self-priming without sensitiz-

ing effects on GnRH-stimulated LH secretion (Bellidoet al., 2003). The

administration of PR antagonists RU486 (Bellido et al., 2003) or

ZK299 (Sanchez-Criado et al., 2004; Garrido-Gracia et al., 2007)

blocks ERa-dependent GnRH self-priming. Thus, the presence of

ligand-dependent (Turgeon and Waring, 2006) or independent

(Waring and Turgeon, 1992) activation of gonadotrope PR is an absol-

ute requirement for the expression of priming proteins (De Koning

et al., 1976; Fink, 1995) responsible for the enhanced LH secretion

(Chappellet al., 1999). Apart from the enhancing action of P on pre-

ovulatory LH secretion, pro-estrous afternoon P from ovarian granu-

losa cells phosphorylates/activates the E-dependent PR (Sheridanet al., 1988) at Ser residues exclusively (Ort et al., 1992; Takimoto

and Horwitz, 1993). Moreover, phosphorylation at Ser294 signals

the PR protein for degradation by the 26S proteasome (Lange et al.,

2000). PR mRNA is degradated also by P, reaching minimum levels

at 6 h after treatment. However, its recovery begins quickly even in

the presence of P, in contrast with the PR protein (Turgeon and

Waring, 2000). This has been demonstrated in young OVX rats by

P-induced complete PR down-regulation and abolition of the

PR-dependent (Chappell et al., 1999) GnRH-stimulated LH secretion

and GnRH self-priming. The power of P-induced PR down-regulation

was reduced in middle-aged OVX rats.

Of interest were the results related to FSH secretion. First, the sen-

sitivity of the pituitaries to GnRH was high and similar in rats in both

age-groups; second, the FSH response to the second GnRH challenge

is of a similar magnitude to the response to the first GnRH challenge;

and third, FSH secretion, either basal, GnRH-stimulated or

GnRH-primed is independent of PR. All this is probably due, in the

complete absence of inhibin (Watanabe et al., 1990; Arai et al.,

1996), to a heightened sensitivity of the FSH releasing mechanism to

GnRH. In a previous article, we demonstrated that there was no

GnRH priming effect on FSH secretion and the blockade of PR with

the antiprogestagen ZK299 has no effect on PR-independent FSH

secretion (Sanchez-Criado et al., 2004).

.......................... ................................................. .................................................. .................................................

.............................................................................................................................................................................................

Table I LH response (nanogram/hemipituitary) to two GnRH pulses 1 h apart of pituitaries from young and middle-aged

OVX rats injected over 3 days with, EB, PPT or TX in combination or not with P on the last day of ER agonist treatments

and incubated with medium alone, E2, PPT or TX, respectively, in the presence or not of P

Treatment Basal LH secretion GnRH-stimulated LH secretion GnRH self-priming (%)

In vivo In vi tro Young OVX Middle-aged OVX Young OVX Middle-aged OVX Young OVX Middle-aged OVX

Oil DMEM 39.3+10.4 78.6+8.37d

55.2+7.4 90.1+22.61 91.7+7.5 76.1+8.2Oil P P 41.8+5.1 66.7+6.92d 42.9+4.7 96.5+12.61 83.8+8.4 85.0+9.1

EB E2 45.2+5.3 90.9+10.45d 133.7+13a 108.5+8.6 153.7+16.2c 129.3+12.8c

EBP E2P 32.7+2.9 87.8+10.20d 95.2+11.8

a,b 85.3+8.03 98.94+10.3 110.0+10.8

PPT PPT 35.9+5.9 88.3+9.57d 99.8+6.7a 108.9+8.40 151.8+14.8c 126.6+11.4c

PPTP PPTP 38.9+6.5 98.8+15.30d 55.1+12.6b 94.0+6.53 114.7+9.7 89.0+9.8

TX TX 45.3+7.0 78.9+7.40d 64.7+7.2 96.5+11.89 212.9+22.1c 159.0+16.9c

TXP TXP 35.2+2.7 87.5+5.29d 42.0+6.3 86.1+9.57 95.7+10.3 95.6+8.5

Values are means+ SEM of eight hemipituitaries. ANOVA and Student Newman Keuls multiple range test. GnRH self-priming Peak response to second GnRH pulse 100/peak LH

response to first GnRH pulse.aP, 0.05 vs. the corresponding basal LH secretion values.bP, 0.05 vs. the same group without P.cP, 0.05 versus oil.

dAll values of basal secretion of LH in middle-aged OVX rats were significantly ( P, 0.05) higher than those of young OVX rats.

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Figure 4 FSH secretion from young (left panels) or middle-aged (right panels) OVX rats hemipituitaries

See legend of Fig. 3 for complete details of treatments. Mean values and statistical significance among groups (ANOVA and StudentNewmanKeuls multiple range test)

are given in Table II.



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Bilateral OVX in rats produced the same endocrinological effects as

those seen after surgical menopause. In all cases, serum ovarian hor-

mones (E2 and inhibin) fell, and this was followed by elevated serum

gonadotrophins levels (Araiet al., 1996). Replacement therapy in sur-

gical menopause includes activation of ER by estrogen analogs and

SERMs such as TX. Surgical menopause differs from physiological

menopause (McKinlayet al., 1992) in the rapidity with which hormonal

withdrawal occurs and the substantial decrease in androgen levels fol-

lowing castration. Although different from the effects of natural repro-

ductive ovarian senescence (Sluijmer et al., 1995; Goldgerg and

Penzias, 1999), surgical menopause may offer an opportunity to inves-

tigate the effect of E2 and P on pituitary function (Houmard and Seifer,1999). Because the complete process of natural menopause lasts

about 1 year, the endocrinological changes are not precisely defined

and the magnitude of the E2-exposed pituitary response to GnRH

in post-menopausal women has not been quantified in a comparative

way with that of perimenopausal women (Weiss et al., 2004). Also,

the exact relationship between the period of diminished ovarian

reserve and the onset of menopause remains unknown, and an

altered sensitivity of the pituitary to E2 and P cannot be excluded

(Nasset al., 1984).

Besides extensive similarities between rats and women in the basic

regulatory mechanisms of gonadotrophin secretion during the ovarian

reproductive cycle, they also share similarities in the physiopathologi-

cal effects of ovarian senescence. Whereas in young OVX rats acti-

vation of pituitary ERa restored all pituitary reproductive functions

so far studied in the present experiments, middle-aged OVX rats

showed clear-cut changes in the sensitivity of the pituitary to E2 and

P. Middle-aged OVX rats showed incomplete shrinkage of hypertro-

phied gonadotrophs after ERaagonist treatment, absence of sensitiz-

ation of the LH releasing apparatus in the gonadotroph to GnRH, and

deficient P-induced PR down-regulation. Recent data in perimenopau-

sal women suggest the existence of pituitary insensitivity to E (Weiss

et al., 2004). Although the sensitivity of E2 has not been studied

thoroughly with the age after menopause in women, it has been

found that in post-menopausal women there is a reduced sensitivity

of the pituitary to GnRH compared with younger post-menopausal

women, despite increasing quantities hypothalamic GnRH (Lambalk

et al ., 1997). The reduced pituitary gonadotroph response to

ovarian steroids E2 and P, and the gonadotroph LH responsiveness

to GnRH may contribute to the inefficiency of ovulatory treatments

in middle-aged women (Van Looket al., 1977).

Acknowledgements

The authors are grateful to the National Hormone and Pituitary

Program (Baltimore, MD, USA) for the LH and FSH radioimmuno-assay kits. The authors declare that there is no conflict of interest

that would prejudice the impartiality of this scientific work.

Funding

This study was subsidized by grants (BFU2008-01443) from DGPTC,

Ministerio de Ciencia e Innovacion and P07-CVI-2559 from CICE-

Junta de Andalucia (Spain).

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Submitted on February 12, 2009; resubmitted on April 29, 2009; accepted onJune 9, 2009

2628 Gordon et al.

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