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

26182628, 2009
Advanced Access publication on July 2, 2009 doi:10.1093/humrep/dep237

ORIGINAL ARTICLE Reproductive endocrinology

Ana Gordon 1,3, Rafaela Aguilar 1, Jose C. Garrido-Gracia 1,

Silvia Guil-Luna 2, Raquel Sanchez-Cespedes2, Yolanda Millan2,
Juana Martn de las Mulas 2, and Jose E. Sanchez-Criado 1
1

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

Correspondence address. Section of Physiology, Faculty of Medicine, Avda. Menendez Pidal s/n, 14004 Cordoba, Spain. Tel: 34-957218283; E-mail: anagordonbc@gmail.com

background: We attempted to dene 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 ERa agonist 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 efciently 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

Introduction
Ovarian cyclicity in mammals depends on the endocrine interaction of
the components of the hypothalamus pituitaryovaryuterus axis
(Feder, 1981). Estral/menstrual cyclicity depends on negative and
positive feedback mechanisms. In terms of the ovarian positive feedback mechanism, estradiol (E2) is the main component acting
through estrogen receptor (ER)a and b isoforms on the hypothalamuspituitary 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 (Hoff et 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: journals.permissions@oxfordjournals.org

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

Activation of estrogen receptor-a

induces gonadotroph progesterone
receptor expression and action
differently in young and middle-aged
ovariectomized rats

2619

(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-Criado et al.,
1990; Turgeon et al., 1999; Szabo et al., 2000) and women (Chang
and Jaffe, 1978; Hoff et 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 menopause in which the low circulating E2 concentrations render the positive feedback mechanism inactive. The complete process of ovarian
senescence until the anovulation stage is not precisely dened and
the magnitude of the E2-exposed pituitary response to GnRH has
not been quantied. This disruption appears to be caused by
changes in the pituitary ovarian 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 and
postmenopause models, respectively (Alonso et al., 2006). Accordingly, 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.

Co. St. Louis, MO, USA); (c) 7.5 mg/kg of the selective ERa agonist, propylpyrazole triol (PPT, Tocris Cookson Ltd, Avonmouth, UK) (Stauffer
et al., 2000); and (d) 15 mg/kg of the selective estrogen receptor modulator (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 treatments 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 RT PCR of
PR mRNA and immunohistochemical analysis of PR protein. In addition,
hemipituitaries from the eight groups of rats were incubated under controlled 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 RT PCR of PR mRNA

Three anterior pituitaries/group were immediately frozen in liquid nitrogen and stored at 2808C until used for RNA analysis. RT PCR, optimized 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 simplestep, acid guanidinium thiocyanate-phenol-chloroform extraction method
(Chomczynski and Sacchi, 1986). Analysis of total mRNA expression
was carried out using a primer pair anking 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 appropriate internal control, parallel amplication of a 249-bp of the S11 ribosomal protein mRNA was carried out in each sample under
previously-published conditions (Gordon et al., 2008). For amplication
of the targets, RT and PCR were run in two separate steps. PCR reactions
consisted of a rst denaturing cycle at 978C for 5 min, followed by a variable number of amplication cycles (28 cycles for PR-AB and 25 for
RP-S11) dened by denaturation at 968C for 30 s, annealing for 30 s,
and extension at 728C for 1 min. A nal extension cycle of 728C for
15 min was included. Annealing temperatures were adjusted for each
target: 57.58C for PR-AB and 588C for RP-S11. These cycling conditions
had previously been optimized to ensure amplication 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 amplication (data not shown).

Immunohistochemistry of PR

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 temperature, with ad libitum access 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 laboratory 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

The immunohistochemical study was performed in formalin-xed, parafnwax embedded, 3 mm-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 avidin biotin peroxidase complex (ABC) as
described elsewhere (Bellido et al., 2003; Sanchez-Criado et al., 2006,
Gordon et al., 2008). Dewaxed and rehydrated tissue sections were subjected 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 sections of formalin-xed, parafn-wax-embedded rat uterus were used as
positive controls. Substitution of the specic primary antibody by nonimmune mouse IgG1 (Afnity 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

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

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

2620
expressed as the number of positive nuclei counted in ve elds at a magnication of 40 (about 240 pituitary cells/eld) 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

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
coefcient 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 rst hour after a 15 min GnRH challenge.

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 rats
Pituitaries from both young- and middle-aged-OVX rats showed morphological 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). Immunohistochemically, 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 20 30 PR-positive cells/
high power eld were counted in EB-treated, young-OVX rats
(Fig. 2C) although the number of PR-positive cells/high power eld
was lower in EB-treated, middle-aged-OVX rats (Fig. 2F). In addition,
the size of gonadotrophs, now easily identied 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 signicantly 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

Results

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 rst challenge of GnRH with a signicant release
of LH (Fig. 3 left panels, Table I). No signicant 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 contrast, both groups of pituitaries from OVX rats, young and
middle-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 PR-AB

mRNA expression in young and middle-aged
OVX rats

Effects of ER ligands on pituitary FSH

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

Treatment with EB or PPT increased pituitary PR-AB mRNA

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

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

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

Statistical analysis
Statistical analysis was performed by ANOVA to test for signicant differences among groups. When signicant differences existed, ANOVA was
followed by the Student Newman Keuls multiple range test to
compare means. Signicance was considered at the 0.05 level.

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

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

described previously (Gordon et al., 2008). Briey, halves of anterior pituitaries 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 modied medium (DMEM), without L-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 vivo treatment. Finally, 1028 M GnRH (LHRH, Peninsula Lab. Inc. Merseyside, UK)
was used to stimulate gonadotrophin secretion. The rst 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 incubation. Medium was removed every 15 min for determination of
GnRH-stimulated gonadotrophin concentrations and GnRH self-priming.

Gordon et al.

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

2621

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 amplied by semiquantitative RT PCR from total pituitary RNA
samples of the different experimental groups. Lower panel: semiquantitative data on the steady-state levels of total PR AB mRNA in the experimental groups. Relative
expression levels were obtained, in each sample, by normalization of absolute optical densities (OD) of the specic 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 independent determinations. a: P , 0.05 versus oil vehicle-injected controls, P , 0.05 versus the corresponding group of young OVX rats. ANOVA and Student
Newman Keuls multiple range test.

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 rst 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 in

young 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
decient P-induced PR down-regulation.
P signicantly 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).

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

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.

2622

Gordon et al.

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

Figure 2 PR expression in the anterior pituitary of OVX young (A C) or middle-aged (D F) 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) stained
for 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 treatment. 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 treatment (PR down-regulation). ANOVA and Student Newman Keuls multiple range test.

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 decient gonadotroph response to GnRH, a reduction of PR mRNA, and absence of
PR protein expression. These functional effects of OVX are

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

2623

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

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 vivo treatment. GnRH pulses were 1 h apart. Mean values and statistical signicance among groups (ANOVA and
Student NewmanKeuls multiple range test) are given in Table I.

2624

Gordon et al.

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

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

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

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

In vivo

Young OVX

Young OVX

Young OVX

..........................
In vitro

Middle-aged OVX

GnRH-stimulated LH secretion

Middle-aged OVX

GnRH self-priming (%)

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

Oil P

41.8 + 5.1

66.7 + 6.92d

42.9 + 4.7

96.5 + 12.61

83.8 + 8.4

EB

E2

45.2 + 5.3

90.9 + 10.45

133.7 + 13

108.5 + 8.6
a,b

76.1 + 8.2
85.0 + 9.1
c

153.7 + 16.2

129.3 + 12.8c

EBP

E2P

32.7 + 2.9

87.8 + 10.20

95.2 + 11.8

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
TXP

TX
TXP

38.9 + 6.5

98.8 + 15.30

45.3 + 7.0

64.7 + 7.2

96.5 + 11.89

212.9 + 22.1

159.0 + 16.9c

42.0 + 6.3

86.1 + 9.57

95.7 + 10.3

95.6 + 8.5

35.2 + 2.7

78.9 + 7.40
87.5 + 5.29

55.1 + 12.6

94.0 + 6.53

114.7 + 9.7

89.0 + 9.8
c

Values are means + SEM of eight hemipituitaries. ANOVA and Student NewmanKeuls multiple range test. GnRH self-priming Peak response to second GnRH pulse  100/peak LH
response to rst GnRH pulse.
a
P , 0.05 vs. the corresponding basal LH secretion values.
b
P , 0.05 vs. the same group without P.
c
P , 0.05 versus oil.
d
All values of basal secretion of LH in middle-aged OVX rats were signicantly (P , 0.05) higher than those of young OVX rats.

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 activation of pituitary ERa with EB, PPT (Sanchez-Criado et al., 2004),
or TX (Sanchez-Criado et al., 2002; Bellido et al., 2003) induced gonadotroph 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 function. Thus, pituitaries from young OVX rats treated with ERa agonists
exhibited a normal pro-estrous reproductive pituitary function (Bellido
et 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 sensitization 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
(Mitchner et al., 1998; Sanchez-Criado et al. 2005), and E2 activates
all ER isoforms. Although pituitary gonadotroph from middle-aged
OVX rats had a decient 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). Moreover, it has been reported that in the absence of E, TX acting
through intracellular ERa (Bellido et al., 2003; Sanchez-Criado et al.,
2005) induces PR expression and GnRH self-priming without sensitizing effects on GnRH-stimulated LH secretion (Bellido et 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 absolute requirement for the expression of priming proteins (De Koning
et al., 1976; Fink, 1995) responsible for the enhanced LH secretion
(Chappell et al., 1999). Apart from the enhancing action of P on preovulatory LH secretion, pro-estrous afternoon P from ovarian granulosa cells phosphorylates/activates the E-dependent PR (Sheridan
et 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 sensitivity 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 rst 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).

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

TX

PPTP

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

2625

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

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 signicance among groups (ANOVA and Student NewmanKeuls multiple range test)
are given in Table II.

2626

Gordon et al.

Table II FSH response (nanogram/hemipituitary) to two GnRH pulses 1 h apart of pituitaries from young OVX rats
injected over 3 days with oil, EB, PPT or TX in combination or not with P 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 FSH secretion

GnRH-stimulated FSH secretion

GnRH self-priming (%)

In vivo

Young OVX

Young OVX

Young OVX

..........................
In vitro

.................................................
Middle-aged OVX

.................................................
Middle-aged OVX

.................................................
Middle-aged OVX

.............................................................................................................................................................................................
Oil

DMEM

OilP

7.4 + 2.2

27.8 + 2.4b

12.1 + 4.4

22.5 + 4.4

115.9 + 8.2

116.1 + 11.2

10.0 + 3.8

21.2 + 3.3b

14.0 + 3.4

25.2 + 4.6

114.3 + 7.9

95.2 + 8.2

E2

6.9 + 0.8

26.2 + 4.2

34.8 + 2.7

118.6 + 8.9

111.8 + 12.8

EBP

E2P

9.7 + 2.9

28.3 + 2.1b

27.6 + 1.9a

37.0 + 2.9a

128.6 + 11.5

111.6 + 10.6

PPT

PPT

13.5 + 3.7

22.3 + 4.9b

30.5 + 1.8a

32.4 + 6.1a

114.8 + 11.2

104.1 + 9.1

PPTP

PPTP

15.1 + 3.9

25.0 + 3.8b

27.2 + 3.8a

37.9.2 + 3.2a

104.6 + 12.3

104.4 + 14.0

30.9 + 3.5

101.5 + 9.6

103.4 + 11.5

34.0 + 3.4a

121.5 + 13.1

101.6 + 9.6

TX

TX

9.9 + 1.5

20.2 + 5.9

TXP

TXP

8.4 + 2.0

22.2 + 2.3b

30.1 + 1.6

18.0 + 2.9

16.8 + 2.2a

Values are means + SEM of eight hemipituitaries. ANOVA and Student NewmanKeuls multiple range test. GnRH self-priming Peak response to second GnRH pulse  100/peak LH
response to rst GnRH pulse.
a
P , 0.05 versus the corresponding basal FSH secretion.
b
All values of basal secretion of FSH in middle-aged OVX rats were signicantly (P , 0.05) higher than those of young OVX rats.

Bilateral OVX in rats produced the same endocrinological effects as

those seen after surgical menopause. In all cases, serum ovarian hormones (E2 and inhibin) fell, and this was followed by elevated serum
gonadotrophins levels (Arai et al., 1996). Replacement therapy in surgical menopause includes activation of ER by estrogen analogs and
SERMs such as TX. Surgical menopause differs from physiological
menopause (McKinlay et al., 1992) in the rapidity with which hormonal
withdrawal occurs and the substantial decrease in androgen levels following castration. Although different from the effects of natural reproductive ovarian senescence (Sluijmer et al., 1995; Goldgerg and
Penzias, 1999), surgical menopause may offer an opportunity to investigate 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 dened
and the magnitude of the E2-exposed pituitary response to GnRH
in post-menopausal women has not been quantied 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
(Nass et 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 physiopathological effects of ovarian senescence. Whereas in young OVX rats activation 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 hypertrophied gonadotrophs after ERa agonist treatment, absence of sensitization of the LH releasing apparatus in the gonadotroph to GnRH, and
decient P-induced PR down-regulation. Recent data in perimenopausal 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 inefciency of ovulatory treatments
in middle-aged women (Van Look et al., 1977).

Acknowledgements
The authors are grateful to the National Hormone and Pituitary
Program (Baltimore, MD, USA) for the LH and FSH radioimmunoassay kits. The authors declare that there is no conict of interest
that would prejudice the impartiality of this scientic work.

Funding
This study was subsidized by grants (BFU2008-01443) from DGPTC,
Ministerio de Ciencia e Innovacion and P07-CVI-2559 from CICEJunta de Andalucia (Spain).

References
Alonso R, Marn F, Gonzalez M, Guelmes P, Bellido C, Hernandez H,
Marn R, Daz M, Sanchez-Criado JE. The hypothalamus pituitary
ovarian axis as a model system for the study of SERM: An overview
of experimental and clinical studies. In: Cano A, Calaf i Alsina J,
Duenas-Diez JL (eds). Selective Estrogen Receptor Modulators. New
Brand of Multitargeted Drugs. Berlin, Germany: Springer-Verlag, 2006,
103 139.
Arai K, Watanabe G, Taya K, Sasamoto S. Roles of inhibin and estradiol in
the regulation of follicle-stimulating hormone and luteinizing hormone
secretion during the estrous cycle of the rat. Biol Reprod 1996;
55:127 133.
Batista MC, Cartledge TP, Zellmer AW, Nieman LK, Merriam GM,
Loriaux DL. Evidence for a critical of progesterone in the regulation

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

EB

2627

of the midcycle gonadotropin surge and ovulation. J Clin Endocrinol

Metab 1992;74:565 570.
Bellido C, Martn de las Mulas J, Tena-Sempere M, Aguilar R, Alonso R,
Sanchez-Criado JE. Tamoxifen induces gonadotropin-releasing
hormone self-priming through an estrogen-dependent progesterone
receptor expression in the gonadotrope of the rat. Neuroendocrinology
2003;77:425 535.
Brensing KA, Smyczek B, Waibel Treber S, Wildt L, Leyendecker G.
Luteinizing hormone (LH) pulsatility during the estradiol- and
estradiol/progesterone-induced LH surge in the human female. Hum
Reprod 1993;8:72 76.
Chang RJ, Jaffe RB. Progesterone effects on gonadotropin release in
women pretreated with estradiol. J Clin Endocrinol Metab 1978;
47:119 125.
Malley BW, Levine JE.
Chappell PE, Schneider JS, Kim P, Xu M, Lydon JP, O
Absence of gonadotropin surges and gonadotropin-releasing hormone
self-priming in ovariectomized (OVX), estrogen (E2)-treated,
progesterone receptor knockout (PRKO) mice. Endocrinology 1999;
140:3653 3658.
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem
1986;162:156 159.
Collins RL, Hodgen GD. Blockade of the spontaneous midcycle
gonadotropin surge in monkeys by RU486: a progesterone antagonist
or agonist? J Clin Endocrinol Metab 1986;63:1270 1276.
Cooper RL, Conn PM, Walker RF. Characterization of the LH surge in
middle-aged female rats. Biol Reprod 1980;23:611 615.
Dafopoulos K, Mademtzis I, Vanakara P, Kallitsaris A, Stamatiou G,
Kotsovassilis C, Messinis IE. Evidence that termination of the
estradiol-induced luteinizing hormone surge in women is regulated by
ovarian factor. J Clin Endocrinol Metab 2006;91:641 645.
De Koning J, van Dieten JM, van Rees GP. LHRH-dependent synthesis of
protein necessary for LH release from rat pituitary glands in vitro. Mol
Cell Endocrinol 1976;5:151 160.
Feder HH. Estrous cyclicity in mammals. In: Adler NT (ed).
Neuroendocrinology of Reproduction: Physiology and Behavior New York,
USA: Plenum Press, 1981, 279 348.
Fink G. Gonadotropin secretion and its control. In: Knobil E, Neill J (eds).
The Physiology of Reproduction. New York: Raven Press, Ltd, 1988,
1349 1378.
Fink G. The self-priming effect of LHRH: a unique servomechanism and
possible cellular model for memory. Front Neuroendocrinol 1995;
16:183 190.
Fink G. Neuroendocrine regulation of pituitary function. In: Conn PM,
Freeman ME (ed). Neuroendocrinology in Physiology and Medicine.
Totowa, NJ: Humana Press Inc, 2000, 107 133.
Garner LL, Blake E. Ultrastructural, immunocytochemical study of the LH
secreting cell of the anterior pituitary gland: changes occurring after
ovariectomy. Biol Reprod 1981;24:461 474.
Garrido-Gracia JC, Gordon A, Bellido C, Aguilar R, Barranco I, Millan Y,
Martn de las Mulas J, Sanchez-Criado JE. The integrated action of
estrogen receptor isoforms and sites with progesterone receptor in
the gonadotrope modulates LH secretion: evidence from
tamoxifen-treated ovariectomized rats. J Endocrinol 2007;193:107 119.
Garrido-Gracia JC, Gordon A, Aguilar R, Monterde JG, Blanco A, Martn
de las Mulas J, Sanchez-Criado JE. Morphological effects of estradiol
17b, and selective estrogen receptor a and b agonist on luteinising
hormone-secreting cells in tamoxifen-treated ovariectomised rats.
Histol Histopathol 2008;23:1453 1463.
Gill S, Sharpless JL, Rado K, Hall JE. Evidence that GnRH decreases with
gonadal steroid feedback but increases with age in postmenopausal
women. J Clin Endocrinol Metab 2002;87:2290 2296.

Goldgerg RP, Penzias AS. Premature ovarian failure and surgical

menopause. In: Seifer DB, Kennard EA (eds). Contemporary
Endocrinology: Menopause, Endocrinology and Management. Totowa, NJ:
Humana Press Inc, 1999, 125 137.
Gordon A, Garrido-Gracia JC, Aguilar R, Bellido C, Velasco JA, Milan Y,
Tena-Sempere M, Martn de las Mulas J, Sanchez-Criado JE. The
ovary-mediated FSH attenuation of the LH surge in the rat involves a
decreased gonadotroph progesterone receptor (PR) action but not
PR expression. J Endocrinol 2008;196:583 592.
Greenwood FC, Hunter WM, Glover JS. The preparation of 131I-labeled
human growth hormone of high specic radioactivity. Biochem J 1963;
89:114 123.
Hoff JD, Quigley ME, Yen SSC. Hormonal dynamics at midcycle: a
reevaluation. J Clin Endocrinol Metab 1983;57:792 796.
Houmard BS, Seifer DB. Predicting the onset of menopause. In: Seifer DB,
Kennard EA (eds). Contemporary Endocrinology: Menopause,
Endocrinology and Management. Totowa, NJ: Humana Press Inc, 1999,
1 19.
Knobil E, Hotchkiss J. The menstrual cycle and its neuroendocrine control.
In: Knobil E, Neill J (eds). The Physiology of Reproduction. New York:
Raven Press, Ltd, 1988, 1971 1994.
Lambalk CB, de Boer L, Schoute E, Popp-Snyders C, Schoemaker J.
Post-menopausal and chronological age have divergent effects on
pituitary and hypothalamic function in episodic gonadotrophin
secretion. Clin Endocrinol 1997;46:439 443.
Lange CA, Shen T, Horwitz KB. Phosphorylation of human
progesterone receptors at serine-294 by mitogen-activated protein
kinase signals their degradation by the 26S proteasome. PNAS 2000;
97:1032 1037.
Legan SJ, Tsai HW. Estrogen receptor-alpha and -beta immunoreactivity in
gonadotropin-releasing hormone neurones after ovariectomy and
chronic exposure to estradiol. J Neuroendocrinol 2003;15:1164 1170.
Mandl AM. Cyclical changes in the vaginal smears of senile nulliparous and
multiparous rats. J Endocrinol 1961;22:257 268.
Messinis IE. Ovarian feedback, mechanism of action and possible clinical
implications. Hum Reprod Update 2006;12:557 571.
Messinis IE, Templeton AA. Effects of supraphysiological concentrations of
progesterone on the characteristics of the estradiol-induced
gonadotropin surge in women. J Reprod Fertil 1990;88:513 519.
McKinlay SM, Brambilla DJ, Posner. The normal menopause transition. Am
J Hum Biol 1992;4:37 46.
Mitchner NA, Garlick C, Ben-Jonathan N. Cellular distribution and gene
regulation of estrogen receptors a and b in the rat pituitary gland.
Endocrinology 1998;139:3976 3983.
Nass TE, LaPolt PS, Judd HL, Lu JKH. Alterations in ovarian steroid and
gonadotrophin secretion preceding the cessation of regular estrous
cycles in ageing female rats. J Endocrinol 1984;100:43 50.
Ort E, Bodwell JE, Munck A. Phosphorylation of steroid hormone
receptors. Endocr Rev 1992;13:105 128.
Pellicer A, Simon C, Remohi J. Effects of aging on the female reproductive
system. Hum Reprod 1995;10:77 83.
Rao IM, Mahesh VB. Role of progesterone in the modulation of the
preovulatory surge of gonadptropins and ovulation in the pregat
mares serum gonadotropin-primed immature rat and the adult rat.
Biol Reprod 1986;35:1154 1161.
Sanchez-Criado J, Bellido C, Galiot F, Lopez FJ, Gaytan F. Apossible dual
mechansm of the anovulatory action of antiprogesterone RU486 in
the rat. Biol Reprod 1990;42:877 886.
Sanchez-Criado JE, Guelmes P, Bellido C, Gonzalez M, Hernandez G,
Aguilar R, Garrido-Gracia JC, Bello AR, Alonso R. Tamoxifen but not
other selective estrogen receptor modulators antagonizes estrogen
actions on lteinizing hormone secretion while inducing gonadotropin-

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

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

2628

Stauffer SR, Coletta CJ, Tedesco L, Nishiguchi G, Carlson K, Sun J,

Katzenellenbogen BS, Katzenellenbogen JA. Pyrazole ligands:
structure-afnity/activity relationships and estrogen receptor-aselective agonist. J Med Chem 2000;43:4934 4947.
Szabo M, Kilen SM, Nho SJ, Schwartz NB. Progesterone receptor A and B
messenger ribonucleic acid levels in the anterior pituitary of rats are
regulated by estrogen. Biol Reprod 2000;62:95 102.
Takimoto GS, Horwitz KB. Progesterone receptor phosphorylation.
Complexities in dening a functional role. TEM 1993;4:1 7.
Turgeon JL, Waring DW. Progesterone regulation of the progesterone
receptor in rat gonadotrope. Endocrinology 2000;141:3422 3429.
Turgeon JL, Waring DW. Differential expression and regulation of
progesterone receptor isoforms in rat and mouse pituitary cells and
LbT2 gonadotropes. J Endocrinol 2006;190:837 846.
Turgeon JL, Van Patten M, Shyamala G, Waring DW. Steroid regulation of
progesterone receptor expression in cultured rat gonadotropes.
Endocrinology 1999;140:2318 2325.
Van Look PFA, Lothian H, Hunter WM, Michie EA, Baird DT.
Hypothalamic-pituitary ovarian function in perimenopausal women.
Clin Endocrinol 1977;7:13 31.
Waring DW, Turgeon JL. A pathway for luteinizing hormone
releasing-hormone self-potentiation: cross-talk with the progesterone
receptor. Endocrinology 1992;130:3275 3282.
Watanabe G, Taya K, Sasamoto S. Dynamics of ovarian inhibin
secretion during the estrous cycle of the rat. J Endocrinol 1990;
126:151 157.
Weiss G, Skurnick JH, Goldsmith LT, Santoro NH, Park SJ. Menopause
and hypothalamic-pituitary sensitivity to estrogen. JAMA 2004;
292:2991 2996.
Submitted on February 12, 2009; resubmitted on April 29, 2009; accepted on
June 9, 2009

Downloaded from http://humrep.oxfordjournals.org/ at Universidad Arturo Prat on December 22, 2014

releasing hormone self-priming in the rat. Neuroendocrinology 2002;

76:203 213.
Sanchez-Criado JE, Martn de las Mulas J, Bellido C, Tena-Sempere M,
Aguilar R, Blanco A. Biological role of pituitary estrogen receptors
ERa and ERb on progesterone receptor expression and action and
on gonadotropin and prolactin secretion in the rat. Neuroendocrinology
2004;79:247 258.
Sanchez-Criado JE, Martn de las Mulas J, Bellido C, Aguilar R,
Garrido-Gracia JC. Gonadotrope estrogen receptor-a and -b and
progesterone receptor immunoreactivity after ovariectomy and
exposure to estradiol benzoate, tamoxifen or raloxifene in the rat:
correlation with LH secretion. J Endocrinol 2005;184:59 68.
Sanchez-Criado J, Martn de las Mulas J, Bellido C, Navarro VM, Aguilar R,
Garrido-Gracia JC, Malagon MM, Tena-Sempere M, Blanco A.
Gonadotropin-secreting cells in ovariectomized rats treated with
different estrogen receptor ligands: a modulatory role of ERb in the
gonadotrope? J Endocrinol 2006;188:167 177.
Scarbrough K, Wise PM. Age-related changes in pulsatile luteinizing hormone
release precede the transition to estrous acyclicity and depend upon
estrous cycle history. Endocrinology 1990;126:884890.
Sheridan PL, Krett NL, Gordon JA, Horwitz KB. Human progesterone
receptor transformation and nuclear down-regulation are independent
of phosphorylation. Mol Endocrinol 1988;2:1329 1342.
Sluijmer AV, Heineman MJ, de Jong FH, Evers JL. Endocrine activity of the
postmenopusal ovary: the effects of pituitary down-regulation and
oophorectomy. J Clin Endocrinol Metab 1995;80:2163 2167.
Smith MS, Freeman ME, Neill JD. The control of progesterone secretion
during the estrous cycle and early pseudopregnancy in the rat:
prolactin, gonadotropin and steroid hormone levels associated with
rescue of the corpus luteum of pseudopreganancy. Endocrinology
1975;96:219 226.

Gordon et al.