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ARTICLES |
and -ß and Androgen Receptor in the Ovaries of Marmosets and Humans
a MRC Human Reproductive Sciences Unit, Edinburgh, EH3 9ET, United Kingdom
b Oxford Brookes University, Oxford, OX3 0BP, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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] and beta [ERß]) and androgen receptor (AR) to investigate the pattern of receptor expression in ovaries obtained from women and from a New World primate, the Common marmoset (Callthrix jacchus). On Western blots, three antibodies directed against different peptides within human ERß all recognized recombinant human (h) ERß but did not bind to recombinant hER. The ERß protein was extracted from human ovary and prostate and marmoset ovary. In marmoset and human ovaries, ERß protein was detected in the nuclei of granulosa cells in all sizes of follicle (both before and after formation of the antrum), and it was also detected in thecal cells, corpora lutea, surface epithelium, and stroma. In contrast, ER protein was not detected in the nuclei of granulosa cells in preantral follicles, was low/absent from stromal and thecal cells, but was expressed in granulosa cells of antral follicles and in the surface epithelium. The pattern of expression of AR protein more closely resembled that of ERß than ER. In conclusion, three independent antibodies have demonstrated convincingly that ERß is expressed in a wide range of cells in the primate ovary. Granulosa cells in preantral follicles could contain ERß:ß dimers. In antral follicles, however, ER is also expressed, and the formation of homo- or heterodimers containing ER may influence the pattern of gene activation within these cells.
androgen receptor, corpus luteum, estradiol receptor, granulosa cells, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) [5] and beta (ERß) [6], arising from different genes [7] have been described.
Identification of the sites of expression of ER and ERß in the human ovary and in nonhuman primate ovaries are essential to elucidate the role of estrogens in ovarian physiology. Immunohistochemical analyses of ER protein expression in human ovaries have been limited to studies using antisera that recognize ER. The ER protein has been detected in granulosa cells of antral follicles, but not in granulosa cells of primary follicles, follicles not expressing aromatase [8], the dominant follicle at the time of the LH surge, or in the corpora lutea (CL) [8, 9]. Messenger RNA for ER, however, has been reported in the CL [10]. Expression of AR protein in human granulosa cells does not mirror that of ER and, in addition to expression in antral follicles, is reported to persist in granulosa cells of dominant follicles and in the CL [8, 11]. In monkeys, ER protein has been detected in granulosa cells of antral follicles on sections of baboon ovaries [12], but not in those of rhesus or cynomolgus monkeys [13]. In the marmoset, immunolocalization of AR revealed that the highest expression occurred in the granulosa cells of healthy preantral/early antral follicles [14], but to our knowledge, immunolocalization of ER has not been reported.
Also to our knowledge, specific immunolocalization of ERß to sections of human and monkey ovaries has not been reported. Studies in which mRNA levels have been determined using in situ hybridization, RNase protection, and/or reverse transcription-polymerase chain reaction (RT-PCR) have reported that ERß is expressed in the human ovary [7, 15], and that ERß-positive cells include those of the surface epithelium [16] and CL [10]. The ERß protein has been localized to the granulosa cells and CL in the rat [17–20] and cow [21]. In situ hybridization of probes specific for ERß mRNA has been reported for follicles in the rat [6] and cynomolgus monkey [22], and Northern blots have been used to detect ERß mRNA in granulosa cells recovered from patients undergoing treatment involving in vitro fertilization [15].
In this study, we utilized three novel antibodies raised against peptides within different domains of human (h) ERß to examine expression of the intact receptor protein in tissue sections of ovaries from the Common marmoset (Callthrix jacchus) and humans. In addition, use of parallel sections from both marmoset and human ovaries has allowed us to compare the expression of ERß, ER, and AR proteins within individual ovarian follicles for the first time.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Marmoset tissues were collected during a period of 3 yr from captive-bred animals maintained in a colony that has been closed since 1973. Progression through the ovarian cycle was determined by monitoring blood progesterone levels as described elsewhere [23], and ovaries were obtained during the follicular phase (early, n = 3; mid, n = 2; late, n = 3) or the first half of the luteal phase (n = 3) of the cycle. Ovarian tissues were also obtained from women (n = 5) undergoing surgery for benign gynecological conditions. The protocol was approved by the Lothian Reproductive Medicine Ethics Committee, and the women gave informed consent. Marmoset tissues were fixed by immersion in 4% paraformaldehyde for 24 h, and those from women were fixed in neutral buffered formalin for 24 h. Both tissue sets were stored in 70% ethanol and processed into paraffin wax as described elsewhere [24].
Antibodies Specific for ER and AR
Estrogen receptor- was immunolocalized using a mouse monoclonal antibody raised against full-length recombinant human protein obtained from Novocastra (NCL-ER-6F11, Newcastle, UK) according to the methods described by Fisher et al. [25]. This antibody is specific to hER, and it does not bind to recombinant hERß on Western blots (Fig. 1A). Androgen receptors were detected using a rabbit polyclonal antibody (N20) raised against a peptide within the N-terminal domain of hAR obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
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Preparation of Antisera Directed Against ERß
Peptides chosen within the N-terminal (P3, CEARSKEHTLPVNRETLKRK) and hinge (P4, CAGKA-KRSGGHAPRVREL) domains of hERß [7, 26] were synthesized by Affiniti Reagents (Exeter, UK) and conjugated via the terminal cysteine residue to keyhole limpet hemocyanin. Conjugated peptide P3 was used to immunize rabbits (n = 4), whereas P4 was injected into a sheep. Immunizations and recovery of antisera were all carried out by Diagnostics Scotland (Carluke, Lanarkshire, UK). An additional peptide (P7, CSPAEDSKSKEGSQNPQSQ) specific for the ß1 form of hERß [27] was synthesized in the Centre for Proteins and Peptides (Oxford Brookes University, Oxford, UK) and conjugated to tuberculin. The P7 was used to immunize mice, and a monoclonal antibody was prepared according to standard methods described elsewhere [28, 29]. Positive clones were identified by ELISA using recombinant hERß (P2466; PanVera, Madison, WI).
Peptide Affinity Purification of Polyclonal Antibodies
Polyclonal antisera were affinity purified against the unconjugated form of the immunizing peptide as follows: Sera were mixed with 2x volume of 60 mM sodium acetate (pH 4) and then stirred vigorously during the addition of 0.75 ml of caprylic acid (Sigma, St. Louis, MO) per 10 ml of the original volume of antiserum. The resulting cloudy solution was centrifuged at 3000 x g for 30 min at 4°C. The supernatant was decanted carefully, filtered through Whatman No. 1 filter paper, and dialyzed extensively against PBS (pH 7.4; Sigma). Peptides were individually, covalently linked separately to Sulpholink coupling gel (catalog no. 20401; Pierce, Rockford, IL) according to the manufacturer's instructions. Briefly, unconjugated peptides P3 and P4, which were synthesized to include a terminal cysteine residue, were dissolved in coupling buffer (50 mM Tris, 5 mM EDTA [pH 8.5]) and convalently linked to the sulpholink beads by incubation with gel (2 mg/ml gel bed) for 60 min at room temperature. The gel bed was packed under gravity into poly-prep columns (BioRad, Hercules, CA), and unconjugated sites on the sepharose were blocked by incubation with 50 mM cysteine (Sigma) dissolved in coupling buffer. Thereafter, the columns were washed with 10 column volumes of 1 mM NaCl and five column volumes of 10 mM Tris/HCl (pH 7.6) containing 0.02% sodium azide and stored at 4°C.
Part-purified supernatants (30 ml, described earlier) were applied to affinity columns coupled to the immunizing peptide. After the solution was run through the column (retained as unbound fraction), columns were washed with two column volumes of 10 mM Tris (pH 7.5) and 10 column volumes of 10 mM Tris (pH 7.5) and 0.5 M NaCl. Bound antibodies were eluted using eight 0.9-ml aliquots of 100 mM glycine (pH 2.5); eluted antibodies were collected into Eppendorf tubes containing 0.1 ml of 1 M Tris (pH 9).
The presence of antibodies in the eluted fractions was determined using ELISA. Briefly, 96 well plates coated with a sulfhydryl-binding surface (Corning Costar Co., Cambridge, MA) were incubated with 100 µl of unconjugated peptide per well (5 µg/µl in PBS [pH 6.5], 1 mM EDTA) for 1 h at room temperature and then washed in PBS (pH 7). Unconjugated sites were blocked using nonfat dried milk powder (0.2% in PBS [pH 6.5], 1 mM EDTA) for 1 h, after which the solution was removed but the wells were not washed. A doubling dilution series (starting at 1:500) of samples eluted from peptide columns, column flow through, preimmune sera, and part-purified antibodies were prepared in PBS containing 10% normal porcine serum (Diagnostics Scotland). Samples (100 µl/well) were incubated in wells for 1 h, after which the plate was washed in PBS; bound antibodies were detected with peroxidase-conjugated second antibodies (anti-rabbit immunoglobulin [Ig] or anti-sheep Ig; Roche Diagnostics, Lewes, Sussex, UK). Samples eluted from the column containing the highest concentration of specific antibody (usually E2–E4) were pooled, desalted on P10 minicolumns (Amersham Pharmacia Biotech, St. Albans, Herts, UK), equilibrated in PBS (pH 7.2), and stored with the addition of 10% glycerol and 0.02% sodium azide.
Western Blotting
Recombinant hERß corresponding to short (
53 kDa) and long (59 kDa) forms of the receptor [26, 30] and recombinant hER were all obtained from PanVera. Tissue extracts of marmoset and human ovaries and human prostate were prepared by rapid homogenization of tissue in denaturing/loading buffer (50 mM Tris-HCl [pH 6.8], 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol, all from Sigma). Recombinant proteins (0.5 µg/lane) and tissue extracts (total protein, 100 µg) as well as prestained protein molecular weight markers (BioRad) were separated on denaturing minigels containing an acrylamide gradient from 4% to 20% (w/v) polyacrylamide (Novex, San Diego, CA). Gels were run in MOPS/SDS running buffer (50 mM 3-[N-morpholino] propane sulfonic acid [MOPS], 50 mM Tris base, 0.1% SDS, 1.025 mM EDTA [pH 7.7]) for 50 min at 200 V. Samples were blotted onto PVDA membrane (Millipore, Watford, UK) using transfer buffer (25 mM bicine, 25 mM Bis-Tris, 1.025 mM EDTA, 50 nM chorobutanol [pH 7.2], Novex) in the Novex minigel tank according to the manufacturer's instructions. Thereafter, membranes were blocked for 2–3 h at room temperature in 5% nonfat dried milk powder dissolved in Tris-buffered saline containing 0.05% Tween-20 (TBST). Membranes were incubated overnight with the primary antibodies (anti-hERß P4, 1:2000; anti-hERß P3, 1:200; anti-hERß P7, 1:500), all of which were diluted in TBST containing either 5% normal donkey serum (for rabbit anti-P3 and anti-P7) or 5% normal rabbit serum (for anti-P4). Bound antibodies were detected using appropriate second antibodies (sheep anti-rabbit Ig, rabbit anti-sheep Ig, rabbit anti-mouse Ig), and the enhanced chemiluminescence visualization system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Immunohistochemistry
Deparaffinized, slide-mounted sections were rehydrated, and after a brief wash in tap water, sections were subjected to heat-induced antigen retrieval [31] by placing slides in a pressure cooker (Tefal, Nottingham, UK) containing 2 L of near-boiling, 0.01 M citrate buffer (pH 6.0) heated on a halogen hot plate. The lid was sealed and set to high pressure, and boiling continued for 5 min after full pressure was obtained (indicated by steady hissing). The pressure cooker was then removed from the heat, the pressure released, and the cooker left to stand for a further 20 min. The lid was removed, and running cold water was added until the temperature equilibrated. Sections were incubated with 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase. After a wash in water, slides were transferred into Tris-buffered saline (TBS; 0.05 M Tris [pH 7.4], 0.85% saline) for 5 min and blocked for 30 min in normal rabbit serum (NRS, Diagnostics Scotland) diluted 1:4 in TBS containing 5% BSA (NRS/TBS/BSA) for anti-P4 and anti-P7 antibodies or normal swine serum (NSS, Diagnostics Scotland) diluted 1:4 in TBS containing 5% BSA (NSS/TBS/BSA) for anti-P3 antibodies. Slides were rinsed briefly in TBS, and an avidin biotin block was performed using reagents from Vector (Peterborough, UK) as follows: Eight drops of avidin were added to each 1 ml of TBS used. This mixture was incubated on sections for 15 min, which were then rinsed in TBS and washed in TBS for 5 min. Thereafter, eight drops of biotin were added per each 1 ml of TBS, and this mixture was incubated on sections for 15 min, rinsed in TBS, and then washed in TBS for a further 15 min. The anti-ERß antibodies were used at the following dilutions in either NRS/TBS/BSA, anti-P4 (sheep, code 36/40, 1:1000); NRS/TBS/BSA, anti-P7 (mouse, code M9, 1:20); or NSS/TBS/BSA, anti-P3 (rabbit, code 515A, 1:50). Antibodies directed against ER were diluted 1:20 in NRS/TBS/BSA, and those against AR were diluted 1:200 in NSS/TBS/BSA. All antibodies were incubated on sections overnight at 4°C. Sections were washed twice for 5 min each time in TBS and then incubated with the appropriate biotinylated secondary antibodies for anti-P4, rabbit anti-sheep (Vector), and for ER and anti-P7, rabbit anti-mouse, (DAKO, Cambridge, UK), both of which were diluted 1:500 in NRS/TBS/BSA or, for AR, swine anti-rabbit (DAKO), which was diluted 1:500 in NSS/TBS/BSA. Incubations lasted for 1 h, followed by two washes in TBS (5 min each). Thereafter, sections were incubated in ABC-HRP complex (DAKO) for 1 h and washed in TBS (twice for 5 min each time). Bound antibodies were then visualized by incubation with 3,3'-diaminobenzidine tetra-hydrochloride (liquid DAB, catalog no. K3468; DAKO). Sections were counterstained with hematoxylin. Images were captured into a Macintosh PowerPC computer using an Olympus Provis microscope (Olympus Optical Co., London, UK) equipped with a Kodak DCS420 camera (Eastman Kodak Co., Rochester, NY) and assembled using Photoshop 5 (Adobe, Mountain View, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies specific to ERß raised in rabbit, sheep, and mouse, respectively, against peptides chosen within three different domains (A, D, and F) of hERß [7] all recognized nuclear protein in sections of fixed marmoset tissues including ovary, prostate (discussed later), uterus, spleen, testis, heart, and lung (not shown). Comparison of the peptides chosen for immunization with the sequence of marmoset ERß (accession no. Y09372) revealed that the peptides were well conserved (P3, 18/20 identical amino acids; P4, 17/18; P7, 17/19) between human and marmoset. On Western blots, none of the antibodies bound to recombinant hER, but all three recognized both long (59 kDa) and short (53 kDa) forms of recombinant hERß (Fig. 1). In contrast, the commercial anti-ER antibody (Fig. 1A) recognized recombinant ER but not ERß. Protein extracts prepared from marmoset and human ovaries and human prostate all contained protein recognized by all three anti-ERß antisera, which migrated on the gels with an identical molecular size to that of the recombinant protein run in parallel lanes. The low level of ERß protein in the total protein samples of tissues run on the membranes meant that it was not possible to distinguish whether both long and short forms were present. On Western blots in other studies, the anti-P4 antibody recognized an approximately 60-kDa protein in extracts from rat epididymis, seminal vesicle. and urogenital sinus [32]. Peptide P7 is not conserved in rodent, and the monoclonal anti-P7 antibody does not recognize ERß extracted from rat ovaries on Western blots or stain nuclei in sections from rat ovary or prostate (not shown).
Pattern of Expression of ERß Protein in Marmoset and Human Ovaries
Using an antibody directed against the P3 peptide located within the A/B domain of hERß, receptor protein was localized to the nuclei of granulosa cells in marmoset ovaries during the early (Fig. 2, a and b), mid (Fig. 2c), and late (Fig. 2, d and e) stages of the follicular phase, and it was also present in some cells of the CL (Fig. 2f). Immunopositive cells were present in follicles both before (Fig. 2a) and after formation of an antrum (Fig. 2, a and c). Staining appeared to be absent from cells within an atretic follicle (labeled as At in Fig. 2c). We tried several times to obtain material from marmosets immediately before ovulation to see if expression of ERß protein is down-regulated, as has been reported to occur in the rat [20]. Obtaining such material proved to be impossible, however, because the process is not as easy to control as in rodents. Even so, in one sample (Fig. 2, d and e, labeled PO?), the expression of ERß protein did appear to be reduced compared with that of the granulosa cells of an adjacent follicle (Fig. 2e). Further samples will be required to confirm this preliminary finding.
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These results were confirmed and extended using a monoclonal antibody specific for the F domain of hERß1 [27] and a polyclonal antibody specific for a peptide in the hinge (D) domain. Receptor protein was localized to the nuclei in the granulosa cells of primary follicles in the marmoset ovary, which contained only one or two layers of cells surrounding the oocyte (Pedersen type 3a/3b, Fig. 2h, small arrows) as well as those in larger preantral follicles (stages 4/5) and large antral follicles (stage 7, Fig. 2, i and q, antrum labeled *). In human ovaries, granulosa cells of all follicle sizes examined (e.g., antral follicles in Fig. 2, j, k, and s) contained nuclear ERß protein. In both marmoset and human follicles, a clearly defined row of granulosa cells that were immunopositive for ERß was present adjacent to the basal lamina (arrowheads, Fig. 2, h, i, o, t, and s). Cells expressing ERß protein were detected in the theca (Fig. 2, b, h, i, k, q, and s) and CL (Fig. 2, i, l, r, and t) of both species using all three anti-hERß peptide antisera.
No immunopositive nuclei were detected in liver from marmosets (Fig. 2g) or human (Fig. 2m) or in any cell type when the primary antibody was not included (e.g., Fig. 2n, marmoset follicle). Figure 2o shows a small follicle in a marmoset ovary stained with antibody raised against P7; in Figure 2p, a closely adjacent section of the same follicle has been incubated with the same antibody preabsorbed with peptide P7. The same result was obtained with both other antibodies used when they were preabsorbed with the unconjugated form of the peptide against which they had been raised (not shown).
Differential Expression of ERß, ER, and AR Proteins in Marmoset and Human Follicles
A direct comparison of the pattern of expression of both ERß and ER and of AR was made using adjacent sections of marmoset and human ovaries (Fig. 3). In the marmoset, all three receptors were expressed in the granulosa cells of large antral follicles (*, stage 7, Fig. 3, a, b, and c). In medium-sized follicles of this species (stage 5b/6), expression of ERß and AR appeared to be more intense than that of ER, and in small follicles containing one or two layers of granulosa cells, no ER-immunopositive cell nuclei were present (Fig. 3b, small arrows). Granulosa cells of the small follicles expressed both ERß (Fig. 3a) and AR (Fig. 3c).
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The pattern of expression of the three receptors in the marmoset was mirrored by that in human ovaries, in which ERß and AR proteins were expressed in the granulosa cells of all follicle stages examined, including those with one or two layers of granulosa cells (Fig. 3, d and f, arrows), preantral follicles with multiple layers of granulosa cells (stage 4/5, Fig. 3, g and i), and antral follicles (*, Fig. 3, j and l). In contrast, ER-positive granulosa cells were not present in primary follicles (e.g., Fig. 3e, arrow), and the first faint immunopositive staining of a few granulosa cells was seen in preantral follicles containing multiple layers of cells surrounding the oocyte (Fig. 3h). Consistent with previous reports [8], ER-immunopositive granulosa cells were present in human antral follicles (*, Fig. 3k). In the human antral follicles, a clearly defined layer of granulosa cells adjacent to the basal lamina (arrowheads) expressed all three receptors (Fig. 3, j, k, and l). Expression of ER, ERß, and AR proteins was noted in thecal cells surrounding both multilayered preantral follicles (Fig. 3, g, h, and i) and antral follicles (Fig. 3, j, k, and l). The surface epithelium contained ER-positive nuclei (ERß, Fig. 3m; ER, Fig. 3o). The intensity of AR immunostaining of the surface layer appeared to be lower than that of the adjacent stroma (Fig. 3q).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The use of fixed, paraffin-embedded tissue combined with mild antigen retrieval has resulted in the maintenance of tissue structure and consistent staining results with three antibodies independently generated against peptides specific for the receptor. The distribution of the ERß protein in both human and marmoset tissues correlated with the reported expression of ERß mRNA in the rat [33], human [7], and cynomolgus monkey [22]. Immunopositive staining was specific to nuclei and detected in multiple cell types within the ovary, prostate (this study), lung, spleen, testis, uterus, and placenta (unpublished observations), but it was not detected in the liver. On Western blots, as expected, all three antibodies could recognize both long and short forms of recombinant ERß, because the peptides chosen for immunization were within the coding region of both forms, which differ only in the length of their N-terminal domain, and result from use of alternative start sites [30].
In the ovaries, both from women and the Common marmoset, a New World monkey that has been used as a primate model of human follicular development [34], the highest levels of ERß protein expression, based on the intensity of the immunohistochemical reaction, were observed in granulosa cells. Consistent with data from rats, expression in granulosa cells was not confined to those cells surrounding antral follicles but could be detected in primary follicles as well [18, 19]. This finding suggests that synthesis of ERß protein is independent of the stimulation of granulosa cells by gonadotropins [35]. In antral follicles, ERß appeared to be evenly distributed in granulosa cells throughout the follicle, and in the current series of samples, no evidence for higher levels of expression of ERß protein in mural granulosa cells were found compared to those within the cumulus. Studies using granulosa cells isolated from rats have shown that ERß is the dominant form of ER in these cells [36]. Mice in which the sequences encoding the DNA-binding domain of ERß have been removed, so that the protein cannot make direct contact with estrogen response elements within DNA (ßERKO, [37]), have been prepared. In these animals, follicular development still occurs, but ovulatory capacity is reduced. However as ER protein has not been immunolocalized to granulosa cells on tissue sections from rodent ovaries [17]. Therefore, we cannot conclude that the regulation of follicle development in these species will parallel that of primates.
We have confirmed that the antibody used to identify expression of ER in other studies [25] does not react with recombinant hERß protein. The pattern of expression of ER protein in fixed sections of human ovaries was in agreement with published findings [8, 9], confirming that expression of this ER subtype is related to follicular maturation. To our knowledge, immunolocalization of ER to the marmoset ovary has not been reported previously, that the pattern of ER protein expression in this primate species is in agreement with the findings in humans, being absent from granulosa cells of primary follicles, is notable.
Using immunohistochemistry on fixed sections, we found that CL in human, marmoset (this study), rat [17], and mouse (unpublished observations) all contain cells that express ERß protein within their nuclei. The synthesis of ERß protein by these cells is consistent with data from assays using RT-PCR to demonstrate mRNAs encoding both ERß and ER in the samples extracted from the CL of 22 human subjects [10] and from freshly isolated granulosa-lutein cells [16]. In addition, ERß was detectable in the nuclei of the theca and stroma.
In the present study, both ERß and ER were detected in the surface epithelium of human ovaries, which is consistent with the detection of mRNAs for both ER subtypes in cultured ovarian epithelial cells [16]. That approximately 90% of ovarian cancers originate from this cell type, and that several studies have already investigated the relative expression of ER and ERß mRNAs in normal ovaries, ovarian cancer cell lines, and both benign and malignant ovarian cancer samples [15, 38, 39], is notable. Two of those studies [15, 38] found that whereas expression of ERß mRNA was higher than ER in normal ovarian tissue, it was down-regulated in cancer cell lines and ovarian cancers. Lau et al. [39] used RT-PCR to compare levels of receptor mRNA expression in freshly isolated, nonmalignant surface epithelial cells with those in cancer cells lines and ovarian surface cells from women with serous adenocarcinoma. They found no differences between samples in the level of expression of ERß mRNA, but they did find a loss of expression of AR and variable expression of ER.
A number of studies on the primate ovary have immunolocalized AR to multiple cell types within the primate ovary [8, 11, 14]. Our own results show that AR is expressed in the granulosa cells of preantral and antral follicles, in theca, and in stroma of both human and marmoset ovaries. That Vendola et al. [40] demonstrated androgen treatment of rhesus monkeys resulted in increased numbers of primary follicles and increased expression of mRNAs encoding insulin-like growth factor I and its receptor consistent with the direct effects of androgens mediated via AR on primordial follicle growth is notable. Other studies have implicated androgens in the amplification of FSH-induced granulosa cell differentiation [14, 41], and experiments demonstrating that, in some cell types, estrogens can induce transcriptional activity via AR [42] may also be relevant to estrogen action within the primate ovary.
In vitro studies have demonstrated that ER and ERß can form both homo- and heterodimers [43, 44]. From the studies described earlier on the pattern of ER protein expression in human and primate ovaries, we would anticipate that granulosa cells of primary and secondary follicles could form ERß:ERß homodimers in the presence of ligand. However, in antral follicles, the coexpression of ER and ERß proteins could allow for the formation of both and ß homodimers or ER:ERß heterodimers. The potential to form heterodimers will also exist in the theca, stroma, and surface epithelium. Data suggest that ERß may function as a repressor of ER activity when it is present within a heterodimer and, therefore, result in decreased sensitivity to estradiol [45]. Additional complexity has been added by the finding that a variant form of hERß (ERß2/cx [46]) is expressed in multiple ovarian cell types ([27]; unpublished observations). This variant is reported not to bind estradiol but to heterodimerize with wild-type ERß (ERß1) or ER and to reduce gene transcription. Both ER and ERß homo- and heterodimers can have different affinities/specificities for some ligands [47], and they may also cause differential gene activation [48, 49]. In light of this information, the potential for the formation of receptor dimers containing ER and/or ERß within different ovarian cell types suggests that, in primates and humans, regulation of gene expression by estradiol during follicle maturation may be more complex than previously thought.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: P.T.K. Saunders, MRC Human Reproductive Sciences Unit, 37 Chalmers St., Edinburgh, EH3 9ET, UK. FAX: 44 131 228 5571; p.saunders{at}ed.ac.uk 
Accepted: May 18, 2000.
Received: January 13, 2000.
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 J. F. Couse, M. M. Yates, K. F. Rodriguez, J. A. Johnson, D. Poirier, and K. S. Korach The Intraovarian Actions of Estrogen Receptor-{alpha} Are Necessary to Repress the Formation of Morphological and Functional Leydig-Like Cells in the Female Gonad Endocrinology, August 1, 2006; 147(8): 3666 - 3678. [Abstract] [Full Text] [PDF]
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J. L Juengel, D. A Heath, L. D Quirke, and K. P McNatty Oestrogen receptor {alpha} and {beta}, androgen receptor and progesterone receptor mRNA and protein localisation within the developing ovary and in small growing follicles of sheep Reproduction, January 1, 2006; 131(1): 81 - 92. [Abstract] [Full Text] [PDF]
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S. F. Sneddon, N. Walther, and P. T. K. Saunders Expression of Androgen and Estrogen Receptors in Sertoli Cells: Studies Using the Mouse SK11 Cell Line Endocrinology, December 1, 2005; 146(12): 5304 - 5312. [Abstract] [Full Text] [PDF]
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 E.V. Younglai, Y.J. Wu, T.K. Kwan, and C.-Y. Kwan Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells Hum. Reprod., September 1, 2005; 20(9): 2383 - 2390. [Abstract] [Full Text] [PDF]
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 L. J. Spicer Effects of Estradiol on Bovine Thecal Cell Function In Vitro: Dependence on Insulin and Gonadotropins J Dairy Sci, July 1, 2005; 88(7): 2412 - 2421. [Abstract] [Full Text] [PDF]
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N. Atanassova, C. McKinnell, J. Fisher, and R. M Sharpe Neonatal treatment of rats with diethylstilboestrol (DES) induces stromal-epithelial abnormalities of the vas deferens and cauda epididymis in adulthood following delayed basal cell development Reproduction, May 1, 2005; 129(5): 589 - 601. [Abstract] [Full Text] [PDF]
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C. A Oliveira, G. A B Mahecha, K. Carnes, G. S Prins, P. T K Saunders, L. R Franca, and R. A Hess Differential hormonal regulation of estrogen receptors ER{alpha} and ER{beta} and androgen receptor expression in rat efferent ductules Reproduction, July 1, 2004; 128(1): 73 - 86. [Abstract] [Full Text] [PDF]
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C. Martin, M. Ross, K. E. Chapman, R. Andrew, P. Bollina, J. R. Seckl, and F. K. Habib CYP7B Generates a Selective Estrogen Receptor {beta} Agonist in Human Prostate J. Clin. Endocrinol. Metab., June 1,&n |
