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General and Comparative Endocrinology 155 (2008) 857–868

Oestrogen receptor a and b in female rat pituitary cells:An immunochemical study

Miriam Gonzalez a,b, Ricardo Reyes a, Carmen Damas c, Rafael Alonso b, Aixa R. Bello a,*

a Cell Biology Section, University of La Laguna School of Biology and FICIC, 38230 La Laguna, Tenerife, Spainb Laboratory of Cellular Neurobiology, Department of Physiology, University of La Laguna School of Medicine

and Institute of Biomedical Technologies, Tenerife, Spainc Department of Psychobiology, University of La Laguna School of Psychology, Tenerife, Spain

Received 31 July 2007; revised 17 October 2007; accepted 23 October 2007Available online 6 November 2007

Abstract

Estradiol is a critical factor in the anterior pituitary secretory activity of mammalian females. Previous reports have demonstrated thepresence of oestrogen receptor alpha (ERa) and beta (ERb) in specific anterior pituitary cells from ovariectomized rats, as well as in thewhole anterior pituitary at particular stages of the rat oestrous cycle. However, the ERa and ERb distribution patterns in specific hor-mone producing cells of the anterior pituitary during the oestrous cycle remain to be clarified. The purpose of this study was to determinethe cellular and subcellular distribution of both ER-subtypes during the rat oestrous cycle, using immunochemistry at light- and electron-microscope levels.

ERa-immunoreactive (ir) cells mainly corresponded to PRL-ir cells and, to a lesser extent, to TSH-, FSH- and GH-ir cells. ERb-ircells corresponded to a few GH-, PRL- and FSH-ir cells, whichever the phase of the cycle. ERa-ir was found either in the cytoplasmand/or the nucleus, depending on the phase of the oestrous cycle, while ERb-ir was always detected in the cytoplasm. Both ER-subtypeswere immunoreactives inside the rough endoplasmic reticulum (RER), secretory vesicles (SV) and free in the cytosol. The highest numberof ERa-ir cells was consistently found at pro-oestrus midday and the lowest at metaoestrous, while the number of ERb-ir cells was low inall stages of the cycle.

These results indicate that the genomic actions of oestrogen in the anterior pituitary cells during the oestrous cycle are mediated byERa. However, the localization of ERa and ERb in the RER and SV suggest a different translational and/or post-translational pathway,which could be involved in non-genomic mechanisms.� 2007 Elsevier Inc. All rights reserved.

Keywords: Anterior pituitary; Estradiol; ERa; ERb; Immunohistochemistry; Immunocytochemistry; Oestrous cycle

1. Introduction

The anterior pituitary is a well-established target tissuefor oestrogens. In this respect, ovarian estradiol is a criticalfactor in the regulation of secretory activity and cellularproliferation of the anterior pituitary gland in the mamma-lian female. In differentiated lactotrophic cells, estradiolincreases PRL synthesis, storage and releases (Liebermanet al., 1978; Kiino and Dannies, 1981; Maurer, 1982),

0016-6480/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2007.10.007

* Corresponding author. Fax: +34 922 318311.E-mail address: abello@ull.es (A.R. Bello).

and promotes cell growth and proliferation (Liebermanet al., 1982; Hashi et al., 1996). In the same cell type, estra-diol is also able to modulate the inhibitory effect of dopa-mine on PRL secretion (Raymond et al., 1978) byregulating the number of D2 receptors in lactotrophs (Hei-man and Ben-Jonathan, 1982). In addition, oestrogen alsomodulates the stimulatory effect of GnRH by increasingthe number of its receptors on the same cells (Aiyeret al., 1974; Knobil, 1974; Drouin et al., 1976; Clarkeand Cummins, 1984).

The mechanisms underlying these diverse functions ofoestrogen are not fully understood. In general, these effectsoccur when the steroid binds to intracellular oestrogen

858 M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868

receptors (ERs), which then bind as dimers to highly con-served DNA sequences known as oestrogen response ele-ments (ERE) present in the regulatory regions of targetgenes (Beato et al., 1995). Traditionally, ERs act as nucleartranscription factors by modulating target genes throughcomplex interactions with coactivator or corepressor pro-teins, and proteins comprising basal transcriptionalmachinery (Beato, 1989; Pettersson et al., 1997). In addi-tion to these classic genomic mechanisms mediated byintracellular ERs, there is enough evidence supporting thatoestrogen could also exert rapid actions, typically initiatedat the cell surface, stimulating second messenger genera-tion, kinase and phosphatase activation, and calcium flux(Kelly and Levin, 2001).

In different cell models, oestrogens may bind plasmamembrane proteins comparable to classical nuclear oestro-gen receptor (Razandi et al., 1999; Marin et al., 2006, 2007;Pedram et al., 2006; Morales et al., 2007), although somestudies have identified nonclassical receptors, such as Gprotein-coupled receptor (GPR30) (Filardo et al., 2002;Revankar et al., 2005; Thomas et al., 2005). In pituitarycells, a membrane ER involved in rapid oestrogen-inducedPRL secretion has been described (Pappas et al., 1994;Norfleet et al., 2000; Christian and Morris, 2002; Bulayevaet al., 2005; Watson et al., 2005).

Until recently, oestrogenic effects were thought to bemediated by a single ER, now referred to as ERa. How-ever, the discovery of a second receptor protein, ERb (Kui-per et al., 1996), raised new questions regarding differentialER cellular and subcellular distribution, cell type localiza-tion, and their role in pituitary function. These two ER-subtypes are encoded by two different genes (Kuiperet al., 1996). Their protein sequences show considerablesimilarity in the DNA-binding domain (>95% amino acididentity) and in part of the ligand-binding domain (55%amino acid identity), but they differ considerably in theirN-terminal A/B domains and transactivation region AF-1(Kuiper et al., 1996, 1997). Although both ER subtypesare found in the anterior pituitary cells of adult rats,ERa is predominant (Mitchner et al., 1998; Pelletieret al., 2000; Schreihofer et al., 2000).

Conflicting data have been published regarding theidentification of ERa- and/or ERb-immunopositive hor-mone producing cells in the pituitary gland probablybecause of the different techniques used. Thus, in the ear-lier studies, ER was detected in gonadotrophs, lacto-trophs, somatotrophs and thyrotrophs, using in vitro

autoradiography (Keefer et al., 1976), and in gonado-trophs, lactotrophs and somatotrophs using ultrastructur-al immunocytochemistry (Morel et al., 1981). After thediscovery of ERb, Mitchner et al. (1998) used combinedimmunohistochemistry and in situ hybridization to studythe specific ER subtypes in hormone-producing cells inthe anterior pituitary of ovariectomized rats. ERa andERb expression was found in lactotrophs, corticotrophs,folliculo-stellate cells and gonadotrophs. However, thereis no information, in vivo, about the specific anterior

pituitary cells expressing ERa and/or ERb during theoestrous cycle in the intact female rat. Interestingly, thereare no data, so far, about the subcellular distribution ofERa and ERb in anterior pituitary cells of female rats atultrastructural level.

The purpose of this research was therefore to demon-strate, in vivo, to identify the presence of ERa- and ERb-ir in specific hormone producing cells of the rat anteriorpituitary gland, and to show their cellular and subcellularlocalization during the different phases of the oestrouscycle.

2. Materials and methods

2.1. Animals and environment

Adult female Sprague-Dawley rats (200–250 g) were caged in groups of3–5 with a 14:10 light:dark cycle in an air-conditioned room (23 ± 1C),and given free access to commercial rat food and tap water. Animals wereleft undisturbed for at least 1 week before the experiments began, and trea-ted in accordance with the accepted guidelines for the care and use of lab-oratory animals in the European Community. Four to five rats wereanalysed for the different experimental groups.

Vaginal smears were examined daily, and only rats showing at leasttwo consecutive 4-day oestrous cycles were used. The animals were killedat different times during the phases of the cycle, based on the hormone lev-els described by Freeman (Freeman, 1994): at 09.00 h in dioestrus, chosenbecause the plasmatic estradiol begins to rise. At 09.00 h in pro-oestrus,when estradiol levels reach a peak. At 14.00 h in pro-oestrus, when estra-diol levels reach a plateau and the circulating LH and FSH begin toincrease rapidly. At 17.00 h in pro-oestrus, at this time, estradiol levels fallrapidly and those of PRL, LH and FSH reach a peak. At 09.00 h in oes-trus, when a secondary rise in FSH begins and circulating levels of PRLand LH reach baseline. At 17.00 h in oestrus, when circulating FSH con-centration reaches baseline. At 09.00 h in metaoestrus; the serum levels ofestradiol, FSH, LH and PRL are basal.

2.2. Antibodies

For ERa and ERb, a commercial rabbit polyclonal IgG anti-ERaserum (H184, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA),directed against the N-terminus of human ERa, and commercial goatpolyclonal IgG anti ERb serum (Y-19, Santa Cruz BiotechnologyInc., Santa Cruz, CA, USA), against the N-terminus of mouse ERb,were used.

For identification of specific hormone-producing, polyclonal antiseraprovided by Dr. G Tramu, Bordeaux, France, were used. The antiserawere developed in rabbits against ACTH (1-24), from synthetic antigen(CIBA, Basel, Switzerland), against human bTSH, human bFSH,human GH and rat PRL hormones from synthetic antigen (ChemiconInternational, Los Angeles, CA, USA). Their immunological propertieshave been described previously elsewhere (Dubois, 1972a, 1972b; Tramuand Dubois, 1977; Hemming et al., 1986; Jamali and Tramu, 1999).

2.3. Immunocytochemistry at light microscopic level

The animals were anaesthetised using pentobarbital (50 mg/kg) andfixed by intracardiac perfusion of sodium phosphate buffer (pH 7.4,0.1 M) containing 4% paraformaldehyde and 0.2% picric acid. Anteriorpituitaries were quickly dissected out, postfixed in the same fixative foran additional 2 h, and then immersed overnight in sodium veronal buffer(pH 7.4, 0.1 M) containing 20% sucrose. Pituitaries were embedded inTissue-Tek, frozen in isopentane cooled with liquid nitrogen, and cryo-stat 8–10 lm horizontal sections were obtained and collected on gela-

M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868 859

tine-coated slides. Sections were allowed to dry then rehydrated insodium veronal buffer, which was used for all further incubations andwashes.

The indirect immunocytochemical procedure was carried out by incu-bating the sections overnight at room temperature with ERa (1:250) orERb (1:250) antisera and afterwards, the peroxidase activity was devel-oped using Tris–HCl buffer (pH 7.6, 0.05 M) containing 0.04% 4-chloro-1-naphthol (SigmaR, UK) and 0.01% hydrogen peroxide. The specificityof the immunostaining was tested by replacing the specific antiserum bynormal serum, omitting one step of the reaction or following preabsorp-tion of the antiserum with the corresponding antigen.

To characterize the pituitary cell type as ER-immunoreactive, doubleimmunostaining procedure was used, where the second antibodies wereagainst PRL (1:1000), bTSH (1:1000), bFSH (1:800) GH (1:1200) andACTH 1-24 (1:1000). Peroxidase activity was developed using 0.04%diethyl-carbazol and 0.01% hydrogen peroxide in acetate buffer (pH 5,0.05 M).

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2.4. Immunocytochemistry at electron microscopic level

Animals were anaesthetised with pentobarbital (50 mg/kg) and fixedby intracardiac perfusion of sodium phosphate buffer (pH 7.4, 0.1 M) con-taining 4% paraformaldehyde and 0.05% glutaraldehyde. Rats were killedby decapitation and their pituitaries removed rapidly, cut into small piecesand fixed by immersion in the same fixative for 1h. The tissues were dehy-drated through increasing concentrations of ethanol (40–100%) andembedded in Spurr’s resin. The tissues were processed for postembeddingultrastructural analysis using immunolabelled gold. Ultra-thin sections(600–800 ) were obtained using the Reichert Ultracut S., collected onnickel grids and treated with normal goat serum (1/30) for 20 min. Thesections were incubated 5 h at room temperature in ERa antiserum(H184 antibody, Santa Cruz Biotechnology) (1/200) or ERb antiserum(Y-19, antibody, Santa Cruz Biotechnology) (1/250). The day after, thesections were washed in phosphate buffer (pH 7.6, 0.1 M) and afterwards,in Tris–HCl buffer (pH 7.6, 0.1 M), then incubated at room temperaturefor 1 h in goat anti-rabbit IgG conjugated with 6 nm gold spheres or insheep anti-goat IgG conjugated with 10 nm gold spheres. The sectionswere washed in Tris–HCl buffer and in distilled water, and finally, stainedwith uranyl acetate and examined with a JEM-1010 transmission electronmicroscope (JEOL). The specificity of the immunocytochemistry wastested by replacing the specific antiserum by normal serum.

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Fig. 1. Number of ERa-labelled pituitary cells per unit area in differentphases of the oestrous cycle, as determined by immunohistochemistry andcomputerized image analysis. Pituitaries were collected in the morning, at09.00 h, of metoestrus (M), dioestrus (D), pro-oestrus (PMo) and oestrus(OM), as well as in the midday of pro-oestrus, at 14.00 h (PMi) and in theafternoon of oestrus, at 17.00 h (OA). Each value represents means ± SD.aP < 0.001 vs PMi.

2.5. Western blotting

Blocks from unfixed female adult rat anterior pituitary lobe anduterus, at dioestrus (9.00 h) and pro-oestrus (14.00 h), were dissected outand frozen immediately. For whole tissue extracts, each sample was sepa-rately freeze-dried and homogenized at 4 �C in a TEXT buffer with proteininhibitors (65 mM Tris–HCl, 10% glycerol, 10% b-mercaptoethanol, 2.3%SDS, pH 6.8). Homogenized extracts were centrifuged at maximum speedfor 10 min. The supernatant was collected and the protein concentrationwas measured in a microplate reader using the bicinchoninic acid/cupricsulphate protocol.

Forty micrograms of proteins from each sample were separated onSDS–PAGE (12.5% polyacrylamide gels) and then transferred to poly-vinylidine difluoride membranes (Biorad, Hercules, CA) by electroblot-ting. Membranes were incubated with 5% non-fat powdered milkdissolved in TBS for 1 h to block non-specific binding sites. Afterwards,membranes were rinsed in TBS-Tween 20 and incubated overnight withthe polyclonal antibodies against ERa (1:250 dilution. H184 antibody,Santa Cruz Biotechnology) and ERb (1:250 dilution. Y-19 antibody,Santa Cruz Biotechnology). The membranes were treated with theanti-rabbit or anti-goat IgG-peroxidase-conjugated secondary antibod-ies, in each case for 1 h. Finally, the immunoreactive protein wasdetected by chemiluminescence (ECL; Amersham Biosciences, Bucking-hamshire, UK). The same blots were stripped and probed with an anti-a tubulin to ensure equal loading.

2.6. Image analysis

For quantitative analysis, anterior pituitary sections were examined bylight microscopy (Leitz Laborlux S, Germany), digitalized through a cam-era and the immunoreactive cells per unit area (200 lm2) were counted byusing Leica Q-Win software. Measurements were taken of 4–5 pituitariesfrom each group and the result of the 9–20 measurements was averaged togive the overlook for that animal. The percentages of PRL, TSH, FSH,GH and ACTH colocalized with ERa or ERb were calculated for 100–150 cells.

2.7. Statistical analysis

Data are expressed as means ± SEM. Statistical analysis was per-formed using a parametric one-way ANOVA with a Tukey post hoc to testfor significant differences among groups. Differences between means aregiven from a level of P < 0.05.

3. Results

3.1. ERa and ERb immunoreactivity in pituitary gland

during oestrous cycle

Numerous ERa-ir cells were found all over the anteriorpituitary lobe. Immunoreactivity was present in differenthormone producing cells either in the cytoplasm and/orin the nucleus, depending on the stage of the cycle. Theweakest immunoreactivity for ERa was detected in veryfew anterior lobe cells during metaoestrus (�16 ERacells/200 lm2) (Fig. 1). In dioestrus, a high number ofERa-ir cells were detected in the whole anterior lobe

860 M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868

(�25 ERa cells/200 lm2), which went on increasing duringpro-oestrus, reaching the highest levels of the cycle at mid-day of pro-oestrus (�55 ERa cells/200 lm2) (Fig. 1). Onthe morning of oestrus, the number and intensity of immu-nopositive cells began to decrease (�40 ERa cells/200 lm2), reaching low values on the oestrus afternoon(17.00) (�20 ERa cells/200 lm2) (Fig. 1).

During metaoestrus, the immunostaining for ERa waspredominantly cytoplasmic, in cells near the centre of theanterior pituitary gland (Fig. 2A). In dioestrus (Fig. 2Band C), on the morning of pro-oestrus (09.00) (Fig. 2Dand E) and on the afternoon of oestrus (17.00), the numberof ERa-ir cells was higher than that of metoestrus and theimmunostaining was more intense in the cytoplasm than inthe nucleus. On the midday of pro-oestrus (14.00) and onthe morning of oestrus (09.00), the immunoreactivity for

Fig. 2. Photomicrographs of anterior pituitary sections labelled for ERa and Emetoestrus, showing cytoplasmic immunostaining (A). An increased number of(D and E), and the immunoreactivity was mainly located in cytoplasm. Strong Eat midday of pro-oestrus (F and G) and in oestrus morning (H and I). Strong ifew clustered cells, whichever the phase of oestrous cycle (J and K). Arrows pnuclei Magnifications; X300 (B and F), X310 (A, D, and J), X350 (H) and X

ERa was located exclusively in nuclei (Fig. 2F, G and H,I, respectively).

ERb immunoreactivity was observed in only a few cellsgrouped in clusters. The immunoreactivity was alwaysintense and located in cytoplasm (Fig. 2J and K). Thisreceptor did not undergo variation at any of the studiedstages. The number of ERb-ir cells was steadily maintainedduring the cycle (�9 ERb cells/200 lm2).

3.2. Western blot analysis

Western blotting performed on whole tissue extractsfrom adult female rat anterior pituitary gland and uterusshowed the presence of ERa and ERb proteins at dioes-trus (A) and pro-oestrus midday (B). The anti-ERa anti-serum recognized an immunoreactive band at 67 kDa in

Rb during the oestrous cycle. Few ERa-ir cells were observed at 09.00 inERa-ir cells were detected at 09.00 in dioestrus (B and C) and pro-oestrusRa staining in the nuclei of numerous anterior pituitary cells was observed

mmunoreactivity for ERb was identified exclusively in the cytoplasms of aoint to immunoreactive cytoplasm and arrowheads mark immunopositive430 (C, E, G, I, and K).

M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868 861

the anterior pituitary, the reported molecular weight offull length ERa (Furlow et al., 1990), which comigrateswith a protein of identical mobility highly expressed inthe uterus (Fig. 3). Using a specific antibody to ERb,a 55 kDa band was detected, corresponding to the pre-dicted molecular weight of full length ERb (Groheet al., 1998) (Fig. 3). Western blots confirmed higher lev-els of ERa than ERb in whole tissue extracts from ante-rior pituitary at the different stages of the cycle studied.On the other hand, the ERa levels were higher at pro-oestrus midday than at dioestrus in the anterior pituitaryglands, whereas anterior pituitary ERb levels where sim-ilar in both phases (Fig. 3).

3.3. Ultrastructural localization of ERa and ERb

At the electron-microscopic level, the immunogold tech-nique revealed that ERa was located mainly in the cyto-plasm of anterior pituitary cells at dioestrus. At thisstage, gold particles were located in several regions of thecytoplasm, as free in the cytosol, in the rough endoplasmicreticulum membrane, and inside secretory vesicles (Fig. 4A,

A B A B

UterusPituitary

ER

ER

-tubulin

Fig. 3. Western blot analysis of anterior pituitary gland protein extracts fromextracts from pituitary of female rats on dioestrus (A) and in the midday operformed using two polyclonal antibodies, H184 against ERa and Y-19 aapproximately 67 kDa and Y-19 gave a very weak band at approximately 55 kmidday of pro-oestrus (A) and in dioestrus (B) were used. The same blots werrelative expression of ERa and ERb was quantified by densitometry analysis omeans ± SD. One asterisk indicates differences at P < 0.001.

D, and E). However, on the afternoon of oestrus the immu-nogold for ERa was located mainly in secretory vesicles(Fig. 4C), and at midday of pro-oestrus (14.00) was locatedexclusively in nuclei (Fig. 4B).

The immunogold for ERb was detected in severalregions in the cytoplasm, inside secretory vesicles, inRER and free in the cytosol, whichever the cycle phase(Fig. 5).

3.4. Characterization of ERa and ERb immunoreactive cell

types

To determine which pituitary hormone producing-cellsare immunostained for ERa or ERb, double-label immu-nohistochemistry was performed in samples at midday ofpro-oestrus, when the number of ERa-ir cells was highestand the immunoreactivity for ERa was localized innuclei.

ERa was present in cells that were also PRL, GH, TSHor FSH-ir, while it was not detected in ACTH-ir cells(Fig. 6). The highest incidence of immunoreactivity forERa was found in lactotrophs, �58% of lactotrophs also

Uterus

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adult female rats probed for ERa and ERb. Forty micrograms of proteinf pro-oestrus (B) were processed for SDS–PAGE. Western blotting wasgainst ERb. The H184 antibody recognized one very intense band atDa. As a positive control for ERa, protein extracts from uterus of rats ate stripped and probed with an anti-a tubulin to ensure equal loading. Thef three samples and normalized to a-tubulin signals. Values correspond to

Fig. 4. Electron micrograph showing immunogold labelling for ERa. (A) Pituitary cell at dioestrus (09.00) showing immunogold label inside RER,vesicles and free in cytosol. (B) Pituitary cell at pro-oestrus (14.00); ERa-ir is mostly in nucleus. (C) Pituitary cell at oestrus (17.00); ERa-ir is detectedmainly inside vesicles. (D) Higher magnification of A, showing ERa-ir inside the RER. (E) Higher magnification of C in which the receptor is detectedinside the vesicle. Immunogold labelling is shown by arrows. N, nucleus; SV, secretion vesicles; RER, rough endoplasmic reticulum. Magnifications:25,000· (B), 28,200· (C), 30,200· (A), 60,000· (D and E).

862 M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868

being immunopositive for ERa with respect to total lacto-trophs (Fig. 7). There were fewer instances of immunoreac-tivity for ERa in somatotrophs and gonadotrophs,showing �15% of GH-ir cells or FSH-ir cells that were alsoERa-ir compared to the (ir) cells. The lowest level of immu-noreactivity for ERa was observed in thyrotrophs, with�26% of TSH-ir cells being also ERa-ir.

ERb-ir was present in cells that were also PRL, GHor FSH-ir (Fig. 6), and it was never observed in eithercorticotrophs or thyrotrophs. The population of ERb-ircells was invariable during the studied reproductive per-iod and their number did not significantly change atany stage of the cycle. At midday of pro-oestrus, theincidence of immunoreactivity for ERb in somatotrophs(�3.4% of GH-ir cells), gonadotrophs (�2.4% of FSH-ir cells) and lactotrophs (�2.8% of PRL-ir cells) wasalways low (Fig. 8).

4. Discussion

Earlier studies without a specific ER subtype reportedER distribution in the pituitary cells using autoradiogra-phy (Keefer et al., 1976), immunocytochemistry (Morelet al., 1981, Yamashita) or in situ hybridization (Pelletier,1988). After the discovery of ERb, Mitchner et al. studiedERa and ERb distribution in the pituitary of ovariecto-mized estradiol-treated rats (Mitchner et al., 1998).

In this study, we used immunochemical procedures todetermine the presence, cellular distribution and subcellu-lar localization of ERa and ERb in intact female adultrat pituitary during the oestrous cycle.

The major findings of our study are as follows: First,whereas in the pituitary cells of female rat during the oes-trous cycle, ERa-ir is the most abundant, ERb is also pres-ent in PRL, FSH and GH-ir cells. Second, ERb-ir was

Fig. 5. Electron micrograph showing immunogold labelling for ERb. (A) Pituitary cell at dioestrus (09.00) showing immunogold label inside RER,vesicles and free in cytosol. The nucleus is ERb negative. (B) High magnification showing ERb-ir inside the RER in a pituitary cell at pro-oestrus (14.00);(C) High magnification in which the receptor is detected inside the vesicle of a pituitary cell at oestrus (17.00). Immunogold labelling is shown by arrows.N, nucleus; SV, secretion vesicles; RER, rough endoplasmic reticulum. Magnifications: 23,000· (A), 45,000· (C and D).

M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868 863

always detected in the cytoplasm, while, in contrast, ERawas found in both the cytoplasm and the nucleus, or onlyin nuclei, depending on the stage of the oestrous cycle.Third, ERa was not only detected in the nucleus and freein the cytosol, but also inside RER and inside the secretoryvesicles, while ERb was detected in the cytoplasm which-ever the stage of the cycle, inside secretory vesicles, RER,and free in the cytosol. Fourth, the greatest populationof ERa was PRL-ir cells.

Our data on the cellular distribution of ERa supportprevious reports in which oestrogen binding was demon-strated by in vitro autoradiography to be located withingonadotrophs, lactotrophs, somatotrophs and thyrotrophs

(Keefer et al., 1976). Morel et al. (1981), using immunocy-tochemistry at ultrastructural level, observed ER-ir ingonadotrophs, lactotrophs and somatotrophs in the ante-rior pituitary of 29-day-old female rats. Besides this,Mitchner et al. (1998) also demonstrated ERa expressionin lactotrophs, corticotrophs, folliculo-stellates andgonadotrophs by using combined immunohistochemistryand in situ hybridization in the anterior pituitary of ovari-ectomized rats. Using immunohistochemical techniques,we have detected the ERa protein in PRL-, ACTH-, GH-and FSH-ir cells. The differences observed among the pres-ent work and other previous studies can be explained sincethe circulating oestrogen levels affect mRNA expression

Fig. 6. Double immunohistochemical staining showing ERa (red nuclei) or ERb (red cytoplasm) that were also labelled for the pituitary hormones (bluecytoplasm) performed in anterior pituitary glands of rats at midday of pro-oestrus. Numerous lactotrophs and somatotrophs showed immunoreactivityfor ERa (A, B and E, F, respectively). A few thyrotrophs and gonadotrophs were also ERa-ir (C, D and G, H). However no corticotrophs wereimmunopositive for ERa (I and J). Very few lactotrophs (K and L), somatotrophs (M and N) and gonadotrophs (O and P) were immunoreactive for ERb.Magnifications: 270· (I and K), 300· (C), 320· (E and G), 340· (A, M, and O), 500· (L, N, and P), 520· (J) and 650· (B, D, F, and H).

864 M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868

differently to that of the protein. Moreover some studieshave shown (Alarid et al., 1999; Sanchez-Criado et al.,2005) that the high levels of estradiol can induce a fall inthe ERa protein without altering mRNA levels. Ovarianfactors, unexpressed in ovariectomized animals, are alsoimportant in the modulation of ER expression. In spiteof these technical differences, our results coincide withMitchner et al. (1998) as far as the low percentage ofgonadotrophs expressing ERa which seems to confirmthe low expression of ERa in rat gonadotrophs comparedto other species: human 70% or ovine 72% (Tobin et al.,

2001). This would confirm that the main action of oestro-gen affecting rat gonadotrophs is via the hypothalamus.Mitchner et al. (1998) similarly observed the maximal per-centage of cells expressing ERa in lactotrophs, whereas inboth humans and sheep the percentage of lactotrophsexpressing ERa was lower than that of gonadotrophs.

In contrast, immunoreactivity for ERb was observed infewer cell types than those for ERa, and this receptorsubtype was expressed in lactotrophs, gonadotrophs andsomatotrophs but not in thyrotrophs. However, Mitchneret al. (1998) reported ERb expression in the same cells as

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Fig. 7. Percentage of specific pituitary cell types; lactotrophs, gonado-trophs, thyrotrophs and somatotrophs immunostained for ERa withrespect to the total corresponding hormone-ir cells at midday of pro-oestrus, as determined by double-label immunohistochemistry and com-puterized image analysis. Pituitaries were collected at 14.00 h of pro-oestrus midday. Each value represents means ± SD of 100–150 total cells.One asterisk indicates differences at P < 0.001.

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5

6

Fig. 8. Percentage of specific pituitary cell types; lactotrophs, gonado-trophs and somatotrophs immunostained for ERb with respect to the totalcorresponding hormone-ir cells at midday of pro-oestrus, as determinedby double-label immunohistochemistry and computerized image analysis.Pituitaries were collected at 14.00 h of pro-oestrus midday. Each valuerepresents means ± SD of 100–150 total cells. No significant differencesamong the three groups were detected.

M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868 865

ERa-ir, so that lactotrophs, corticotrophs, folliculo-stel-lates and gonadotrophs were also ERb mRNA positive.It has been suggested that an important physiological roleof ERb is to modulate ERa-mediated gene transcriptionby inhibiting it in the presence of ERa, and partially

replacing ERa in its absence (Scully et al., 1997; Vaillantet al., 2002).

A greater expression of ERa than ERb in anterior pitu-itary cells of adult female rats has been reported by severalauthors (Mitchner et al., 1998; Wilson et al., 1998; Vaillantet al., 2002). We did not detect any significant changes inERb-ir at any phase studied, in accordance with severalarticles in which ERb mRNA levels remain relatively con-stant (Mitchner et al., 1998; Vaillant et al., 2002). Sanchez-Criado et al. (2005) also found that the immunohistochem-ical expression of ERb in gonadotrophs was not oestrous-cycle dependent nor was it influenced by different SERMs,although Childs et al. (2001) found a peak expression ofERb-ir during pro-oestrus and Schreihofer et al. (2000)detected a significant ERb mRNA level decrease in pro-oestrus morning.

We also show that the expression of ERa protein in theanterior pituitary varies during the oestrous cycle, with thelowest expression at metoestrus. Expression levels of ERaprotein increased during dioestrus, reaching the highestvalues in midday pro-oestrus, suggesting that this peakmay facilitate the positive feedback before the LH andPRL surges.

The variations in ERa levels during the cycle have alsobeen observed in previous studies with frozen pituitaryglands in which the mRNA levels were the highest duringpro-oestrus, significantly decreasing in oestrus and metoes-trus and rising again in dioestrus (Vaillant et al., 2002).Using an in vitro immunocytochemical method, Childset al. (2001) found the highest levels of ERa protein indioestrus rats, stayed relatively high in pro-oestrus, signif-icantly decreasing during oestrus and increasing again bymetoestrus. These data show that at stages where the oest-rogen levels are high, so are those of ERa, therefore thepattern of ERa expression in the pituitary during the cyclecould be positively related to circulating estradiol levels.Between pro-oestrus midday and oestrus morning we alsodetected ERa protein limited to the nuclei, mainly in lacto-troph cells.

Positive influence of ER on gonadotropin secretionmay only occur at the level of the pituitary in mouse(Lindzey et al., 2006). However the scarce FSH-ir cellsexpressing ERa observed in rat suggests that the responseof gonadotrophs to oestrogen happens not only at pitui-tary level but rather is mediated by interrelated pituitary,ovarian and hypothalamic factors like GnRH, able toupregulate its own receptors in rat (Frager et al., 1981;Naftolin et al., 2007). This interrelation is necessary forregulation the gonadotropic hormones synthesis andsecretion in both positive and negative feedback. In addi-tion, our results suggest the action of ERa on pituitarycells is genomic for positive feedback and non-genomicfor negative, as can be deduced from the changesobserved at subcellular level, whether nuclear or cytosolic.The high percentage of PRL-ir cells that express ERa inthe rat suggests that the regulation of lactotrophs by oest-rogen occurs mainly at pituitary level. This action

866 M. Gonzalez et al. / General and Comparative Endocrinology 155 (2008) 857–868

involves the transcription of PRL genes necessary for thepreovulatory surges, or in the modulation of transcriptionof other key genes involved in lactotroph proliferation,given that only the lactotrophs among the anterior pitui-tary cells show increased proliferation rates on the morn-ing of oestrus (Hashi et al., 1996). In fact, this hypothesisis supported by Scully et al. (1997), who reported resultsobtained by analysis of ERa gene-disrupted mice, reveal-ing a marked reduction in PRL mRNA and a decrease inlactotrophs.

In addition to a cytoplasmic/nuclear ER localization,responsible for classical genomic action, increasing evi-dence for non-genomic effects of oestrogen in different celltypes has accumulated, these being attributable to mem-brane-localized ERs. Plenty of data support the idea thatplasma-membrane localized ER is very similar to the clas-sical nuclear ERs. The extranuclear localization of ERa indendritic spines and axon terminals of hippocampal neu-rons and in glial processes has been shown by ultrastructur-al techniques (Milner et al., 2001). Using a battery ofantibodies to multiple epitopes of the nuclear ERa, plasmamembrane ERs were identified in several cell types (Pappaset al., 1995; Norfleet et al., 1999; Marin et al., 2006). Post-translational ER modifications can contribute to mem-brane localization, as in palmitoylation (Levin, 2005; Mar-ino et al., 2006).

This membrane form of ERa is shown to be involved inthe rapid non-genomic response to oestrogen in pituitarycells (Norfleet et al., 2000). This plasma membrane ERhas been identified in a rat pituitary tumour cell-line andshows immunological similarity to ERa (Pappas et al.,1995; Watson et al., 1999, 2005; Norfleet et al., 1999). Fur-thermore, several groups have identified membrane ERa inthe same cell-lines in association with rapid oestrogen-induced PRL release (Pappas et al., 1994; Norfleet et al.,2000; Christian and Morris, 2002; Bulayeva et al., 2005;Watson et al., 2005).

In pituitary cells, only Morel et al. (1981) reported ultra-structural localization of ER-ir, locating it inside nucleusand cytoplasm. To our knowledge, there are no previousstudies that have detected both proteins (ERa and ERb)ultrastructurally in pituitary cells.

There are a few studies on the detection of oestrogenreceptor in microsomal fraction in rabbit uterus (Monjeand Boland, 1999; Govind and Thampan, 2003). How-ever, we know of none that have detected both proteins(ERa and ERb) ultrastructurally inside RER lumen. Wedescribe for the first time in pituitary cells at ultrastruc-tural level that both ERs have extranuclear localizationsites, being immunodetected inside endoplasmic reticu-lum, secretion vesicles and free in cytosol. Althoughthe functional significance of these sites remains elusive,it could be involved in novel signal transduction mecha-nisms associated with endoplasmic reticulum, in view ofthe existence of several transcription factors as well asprotein kinases associated with it. On the other hand,the localization of both ERs in endoplasmic reticulum

and secretion vesicles may suggest the other translationpathway, which leads to the synthesis of an ERa isoformresponsible for non-genomic responses during the nega-tive feedback.

While regulation may vary among species, observationsby Glidewell-Kenney et al. (2007) in mice using knockoutand knockin models demonstrate a non-genomic role ofERa in regulating gonadotrophic hormones during nega-tive feedback and a genomic role in positive, with bothhypothalamus and pituitary being involved. Takentogether, our observations on the expression of ERa pro-tein in FSH-ir cells during the cycle suggest a similar actionon these cells in the rat pituitary.

In conclusion, the actions of oestrogen on pituitary cellsduring the oestrous cycle are probably mediated by ERa.The ultrastructural results suggest a Golgi-dependent path-way could exist for ER translation. A relationship is evi-dent between the quantity of ERa staining, its cell typedistribution, the subcellular location and the differentphases of the cycle. In contrast, ERb is maintainedunchanged. These results suggest that oestrogen hasERa-mediated genomic and non-genomic action on thepituitary cells during the oestrous cycle.

Acknowlegments

This work was supported by Grants SAF2004-08316-C02-01 (Ministerio de Educacion y Ciencia, Spain), 87/05from FUNCIS (Fundacion Canaria de Investigacion y Sa-lud) and FICIC (Fundacion del Instituto Canario de Inves-tigacion del Cancer). M.G. held research fellowships fromConsejerıa de Industria, Comercio y Nuevas Tecnologıas(Gobierno de Canarias).

We thank Marıa Encarnacion Perez and Juan Luis Gon-zalez for electron microscopy technical support, and Ger-ard Tramu for hormone antisera.

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