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Regular Article |
and ß in the Baboon Fetal Ovary1
a Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23501-1980
b Department of Medical Nutrition, Karolinska Institute, S-14186 Huddinge, Sweden
c Departments of Obstetrics/Gynecology/Reproductive Studies and Physiology, Center for Studies in Reproduction, The University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and/or ERß were expressed in fetal ovaries obtained on Days 100 (n = 3) and 165–181 (n = 5) of baboon gestation (term = Day 184). On Day 100, ER protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERß protein was also detected by immunocytochemistry on Day 100 of gestation and was abundantly expressed in mesenchymal-epithelial cells in germ cell cords, lightly expressed in the germ cells, but was not detected in the surface epithelium. On Days 165–180 of gestation, ER expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ER expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERß expression was most intense in granulosa cells, especially in flattened granulosa cells, was weaker in mesenchymal-epithelial cells and nests of cells between follicles, and was absent in the surface epithelium. Using an antibody to the carboxy terminal of human ERß, ERß protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa on Day 100 and primarily 55 kDa on Day 180. The mRNAs for ER and ERß were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ER and ERß mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate.
estradiol receptor, ovary, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and/or ERß mRNAs and proteins are expressed in the baboon fetal ovary. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female baboons (Papio anubis) weighing 10–15 kg were housed individually in stainless steel cages in air-conditioned quarters and fed Purina monkey chow (Ralston Purina, St. Louis, MO) and fresh fruit and/or carrots daily and given water ad libitum. Females were paired with males for 5 days at the anticipated time of ovulation, and pregnancy was confirmed 30 days postovulation [14]. On Days 100 (n = 3) and 165–181 (n = 5) of gestation (term = Day 184), baboons were anesthetized with isoflurane, the placenta and fetus were delivered by cesarean delivery, and the fetus was killed with an overdose of sodium pentobarbital. Fetal ovaries were collected; one ovary was fixed in 10% buffered formalin [15], and the other ovary was snap-frozen and stored in liquid nitrogen. Ovarian tissue as well as endometrium were also collected from two adult baboons killed with sodium pentobarbital 72–24 h before anticipated ovulation, as judged by menstrual cycle history and daily examination of perineal turgescence [14]. The baboons were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication 85-23, 1985). The experimental protocol employed in this study was approved by the Institutional Animal Care and Use Committee of the Eastern Virginia Medical School.
Histology and Immunocytochemistry
Paraffin-embedded sections of baboon fetal ovaries were stained with hematoxylin and examined by light microscopy. The immunocytochemical detection of ER and ERß was determined essentially as described previously [16]. Briefly, representative sections (4 µm) of paraffin-embedded fetal and adult ovaries were mounted onto Superfrost microscope slides (Fisher Scientific Co., Arlington, VA) and heat fixed, and endogenous peroxidase was blocked with 0.4% H2O2 in methanol. Slides were microwaved in 10 mM sodium citrate buffer, pH 6 (Sigma Chemical Company, St. Louis, MO) at power level setting 9 (General Electric Model JE 1540 oven maximum power 900W; General Electric, Louisville, KY), for 15–20 min. After cooling for 30 min, sections were washed in PBS and preblocked with 5% normal goat serum (NGS) in PBS for 30 min at room temperature. Sections (n = 5–10 per ovary per animal) were then incubated 48 h (4°C) with a mouse monoclonal NCL-ER6 F11 antibody to the human ER (Vector Laboratories, Burlingame, CA) diluted 1:40 in 5% NGS, or with chicken polyclonal ERß 503 immunoglobulin (Ig) Y antibody to the human ERß protein (supplied by Dr. Jan-Åke Gustafsson) or rabbit polyclonal antibody PA1-313 to the C-terminal amino acids 467–485 of the human ERß protein (Affinity BioReagents, Inc., Golden, CO), each diluted 1:1000 in 5% NGS-PBS. Ovarian sections were then washed twice in PBS for 10 min and incubated with biotinylated goat anti-mouse IgG (ER), anti-chicken IgG, or anti-rabbit IgG (Vector), and then with the VectaStain Elite Kit (Vector). After a rinse in PBS, sections were stained with diaminobenzidine (DAB)-imidazole-H2O2 or DAB-nickel sulfate (0.250 g nickel sulfate, 2 mg DAB, and 8.3 µl 3% H2O2 in 10 ml 0.175 M acetate buffer, pH 6.0) as described by Hoffman et al. [17]. Tissue sections were then counterstained with Gill hematoxylin (DAB only), mounted in Permount (Fisher), and examined by light microscopy. Studies were also performed with ER antibody NCL-ER 6F11 and the rabbit ERß antibody incubated overnight (4°C) with recombinant human ER (Oncogene Research Products, Cambridge, MA) or ERß immunizing peptide (Affinity BioReagents), respectively, before application to tissue sections. Specificity for the chicken ERß antibody was previously confirmed in studies demonstrating an absence of nuclear signal in sections of rodent and human tissues incubated with antibody preabsorbed with excess human ERß protein [18, 19] and an inability of the antibody to detect ER protein on Western blot [18].
Western Immunoblot
Western blot analysis of ERß was performed using procedures developed in our laboratories [20]. Briefly, samples of frozen fetal ovaries were suspended in 1% cholic acid (Sigma), 0.1% SDS (Sigma), 1 mM EDTA in PBS (pH 7.4) containing 0.1 mg/ml PMSF, 10 µg/ml aprotinin, and 0.1 mg/ml soybean inhibitor (Sigma) and homogenized on ice. After addition of Laemmli buffer [21], samples were heated (100°C for 5 min), cooled, and then loaded (25 µg protein per lane) onto discontinuous 12% SDS-polyacrylamide minigels in electrophoresis chambers containing chilled 0.025 M Tris, 0.192 M glycine (Bio-Rad, Hercules, CA), and 0.1% SDS buffer (pH 8.3). Samples were electrophoresed and wet-transferred to Immobilon P (Millipore Corp., Bedford, MA), and membranes were blocked and then incubated with polyclonal antibody PA1-313 to ERß diluted to 10 µg/ml in buffer I (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20, and 0.05% IGEPAL CA-630) containing 1.5% BSA. Membranes were washed and then incubated with donkey anti-rabbit IgG horseradish peroxidase-conjugated second antibody (Amersham Life Sciences, Inc., Arlington Heights, IL) in buffer I containing 1.5% BSA. Samples were also incubated with ERß antibody that had been incubated overnight (4°C) with ERß immunizing peptide (Affinity BioReagents). After washing and application of enhanced chemiluminescent reagent (ECL; Amersham), membranes were placed in x-ray film cassettes containing Fugi Medical x-ray film (Fugi Medical Systems, Stamford, CT) and exposed in a dark room for 30–120 sec. The second antibody contributed no nonspecific bands at the concentrations employed.
Northern Analysis
Northern blot analysis was used to determine ER mRNA as described previously [7, 22]. Briefly, 15–30 µg poly(A)+ RNA isolated from fetal ovary and adult endometrium frozen in liquid nitrogen was denatured and size-fractionated by electrophoresis in 1.0% agarose gel containing 0.66 M formaldehyde and 20 mM MOPS. The RNA was transferred overnight onto a nylon membrane (Gene Screen, DuPont-New England Nuclear, Boston, MA), UV cross-linked, baked in a vacuum oven (80°C for 2 h), and prehybridized in buffer containing 50% formamide, 0.1% polyvinyl pyrrolidine, 0.1% BSA, 0.1% Ficoll, 2.5x SSPE (0.375 M NaCl, 0.025 M NaH2PO4-H2O and 2.5 mM EDTA-Na2, pH 7.4), 1.0% SDS, 10% dextran sulfate, and denatured salmon sperm DNA (100 µg/ml) for 24 h at 42°C before addition of labeled probe. The cDNAs for ER (1.9-kilobase [kb] insert of the pER7 plasmid generously supplied by Dr. G. Greene, University of Chicago, Chicago, IL) and ERß (1.8-kb insert of the human ERß gene, generously supplied by Dr. Jan Åke Gustafsson) were labeled with 50 µCi [-32P]dCTP (3000 Ci/mmol; Amersham) to a specific activity of approximately 109 dpm/µg DNA using the Random-Primed DNA labeling kit (Boehringer-Mannheim, Indianapolis, IN). Hybridization was performed in fresh buffer at 42°C for 23 h with 32P-labeled cDNA. After the membranes were washed under stringent conditions, they were exposed to Kodak X-AR film (Eastman Kodak, Rochester, NY) at -80°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On Day 100 (Fig. 1A) of gestation, the baboon fetal ovary was composed primarily of oocytes and mesenchymal-epithelial cells, which were enveloped in cordlike structures termed germ cell cords. Folliculogenesis, although initiated by Day 100 of gestation, was relatively minimal. This contrasts with the situation in late gestation (Fig. 1B), in which primordial follicles, composed of oocytes and granulosa cells, were abundant and interfollicular nests of mesenchymal-epithelial cells and oocytes were less abundant.
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ER Immunocytochemistry
On Day 100 of gestation, ER protein was detected in surface epithelium and mesenchymal-epithelial cells in germ cell cords (Fig. 2A) but not germ cells (i.e., oocytes). On Days 165–180 of gestation, expression of ER was still extensive in the surface epithelium (Fig. 2B) and in nests of mesenchymal-epithelial cells between follicles (i.e., interfollicular nests; Fig. 2C). However, compared with expression in interfollicular nests, ER protein expression was much lighter in granulosa cells, especially in primordial granulosa cells, and was not observed in all granulosa cells, particularly in those close to the outer cortex (Fig. 2D). In contrast to these observations in the fetal ovary, ER protein was detected only in granulosa cells of antral follicles, including the granulosa cells surrounding the oocyte, in the adult ovary (Fig. 2E). Specificity was confirmed by expression of receptor in nuclei and not in the cytoplasm and the absence of expression in sections of near-term fetal ovary incubated with primary antibody preabsorbed with ER recombinant protein (Fig. 2F).
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ERß Immunocytochemistry
ERß protein was also detected in baboon fetal ovary on Days 100 and 165–180 of gestation using the chicken polyclonal (Fig. 3) or the rabbit polyclonal (not shown) antibody. On Day 100 of gestation, ERß protein was localized to and extensively expressed in mesenchymal-epithelial cells in germ cell cords and only lightly expressed in the germ cells (i.e., oocytes) (Fig. 3, A and B). By Day 165 of gestation, ERß was localized and abundantly expressed in granulosa cells and in mesenchymal-epithelial cells in interfollicular nests (Fig. 3, C and D). ERß protein was also detected in adult baboon ovary, where it was abundantly expressed in granulosa cells of antral follicles (Fig. 3E) as well as the granulosa of preantral and less developed (e.g., primary) follicles and interstitial cells (Fig. 3F). ERß was not detected in the theca. Similar findings were noted using the rabbit polyclonal antibody PA1-313. Specificity was confirmed by the absence of staining in sections of near-term fetal ovary incubated without the polyclonal chicken antibody or with the rabbit polyclonal antibody preabsorbed with excess immunizing peptide to the ERß protein (data not shown).
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ERß by Western Immunoblot
Using the rabbit polyclonal antibody PA1-313, which recognizes the carboxy terminal of the human ERß, ERß protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa in the baboon fetal ovary on Day 100 of gestation but primarily 55 kDa on Days 165–181 of gestation and in adult ovary (Fig. 4). ERß was also detected in endometrium as a single band of 55 kDa (data not shown). Specificity was confirmed by the absence of signals for the 55- and 63-kDa isoforms using PA1-313 antibody preabsorbed with excess ERß peptide.
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ER and ERß mRNAs by Northern Blot
A 7-kb ER mRNA transcript was identified by Northern blot analysis in near-term fetal baboon ovaries and was identical to that in baboon endometrium (Fig. 5). The mRNA for ERß was also detected in fetal ovarian tissue primarily as a 7-kb transcript. After exposure of the latter membrane for an additional 10 days, ERß mRNA was also detected in baboon endometrium (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß are expressed in the primate fetal ovary as early as the first half of gestation and continue to be expressed throughout the remainder of gestation in association with the marked development of and follicular maturation within the fetal ovary in utero. Although ER has also been identified in the fetal mouse ovary [9, 10], and the mRNAs for ER and ERß have been found to be expressed in the midgestation human fetal ovary [23], the results of the current study further indicate that with development of the primate fetal ovary, localization of ER and ERß appears to be altered. Thus, at midgestation, both ERs are abundantly expressed in the mesenchymal-epithelial cells of the baboon fetal ovarian cortex. However, by term, ERß expression appears to be more extensive in granulosa cells of the majority of follicles both in the inner and more central regions of the fetal ovarian cortex, whereas ER is primarily expressed in cells between follicles (i.e., interfollicular nests), which presumably are the sites from which oocytes and presumptive granulosa emerge to form follicles. Moreover, very few granulosa cells expressed ER, particularly those with a more flattened shape. The factors regulating cell-specific expression of the ERs in the primate fetal ovary remain to be determined. In the baboon as in the human, placental estrogen production and secretion into the fetus is initiated early in gestation and increases throughout the course of pregnancy [11]. Moreover, we have shown that estrogen regulates key aspects of fetal-placental development and function, including fetal adrenal development [13]. Therefore, we suggest that estrogen may also play a role in regulating fetal ovarian development in the primate. Because the foundation for ovarian development in human and nonhuman primates occurs in utero [1], impairment of estrogen secretion may have profound effects on fetal ovarian development and thus reproductive function in adulthood. In support of this suggestion, ongoing studies in our laboratories have shown that fetal ovarian folliculogenesis is significantly altered in baboons deprived of estrogen during the second half of gestation [24].
The extent to which ERs were expressed in the fetal ovary contrasts with the more limited cellular expression of these receptors in the adult ovary. Thus, in the ovary of the adult baboon, ER was only detected, albeit in relatively abundant levels, in granulosa cells of antral follicles. Granulosa cells of antral follicles also expressed ERß; however, in contrast to ER, ERß was also expressed in the granulosa of preantral follicles. The results of the current study confirm our earlier work demonstrating expression of ER protein in nuclei of granulosa cells of antral follicles but not in the granulosa of primary and preantral follicles [7]. ER has also been localized to the granulosa cells of antral follicles in the human [6, 25, 26] and marmoset monkey [26] but apparently not the rhesus or cynomolgus monkey [27]. The NCL-ER6 F11 antibody used in the current study, as well as in a study by Saunders et al. [26], has been shown to be highly specific for ER and did not bind to human ERß on Western blot [26]. It is possible, therefore, that the difference between species reflects methodologic considerations, e.g., differences in antigen retrieval or the use of antibodies with differing levels of sensitivity. ERß has also been detected in the human and marmoset adult ovary [26] and localized to granulosa cells of antral as well as preantral follicles, consistent with observations of the current study in the adult baboon ovary. The localization of ERß protein in the baboon (present study) and human [26] correlates well with the site of expression of ERß mRNA as determined by in situ hybridization in adult ovary of the human [28] and cynomolgus monkey [8]. ER mRNA has also been detected by Northern blot in granulosa cells from women undergoing in vitro fertilization [29].
It is well established that ER and ERß can form homodimers as well as heterodimers [26, 30, 31]. Therefore, because both ERs appear to be expressed in the same cells in the fetal baboon ovary, particularly at midgestation, these cells theoretically should be capable of forming both and ß homodimers and/or ER:ERß heterodimers. The same is probably true for the adult ovary, particularly during the late follicular phase. Recently, Hall and McDonnell [32] showed that ERß, when present within a heterodimer, represses ER activity and thus sensitivity to estradiol. Moreover, both ER and ERß homodimers and heterodimers can have different affinities/specificities for other potential estrogen-like ligands [33] and may also cause differential gene activation [34, 35]. It is apparent, therefore, that the regulation of primate ovarian development and subsequent function in adulthood by estrogen is complex and may involve intricate interactions/cell signaling pathways coordinated by different receptor-receptor interactions. It is interesting, therefore, that with advancing gestation, there appeared to be a dissociation of ERß and ER expression in granulosa cells of primordial/primary follicles, although both receptors were still expressed in nests of cells between follicles. Whether these qualitative changes in cellular expression of ERß/ER are of physiologic relevance remains to be determined. Moreover, why and by what mechanism(s) granulosa cells of follicles regain expression of both ERs as they develop to the antral stage also remains to be studied.
The in-frame open reading frame of the DNA for the ERß receptor has two initiation codons that can lead to transcription and translation of at least two protein isoforms of the receptor, namely a long form composed of 530 amino acids with a molecular size of 63 kDa and a short form composed of 485 amino acids with a molecular size of 54 kDa [36, 37]. Various COOH terminal-truncated ERß isoforms have also been reported [38–40], and the protein contains potential posttranslation sites for phosphorylation and glycosylation [37]. Although both the short and long forms of ERß contain a functional activation function (AF)-2 ligand binding site, only the long form has a transactivation AF-1 region that is located in the N-terminal amino acid sequence [36]. Because the AF-1 site apparently regulates receptor transcriptional activity [32, 35], the biological activity of estrogen as mediated by ERß may also be dependent on which form(s) of ERß is expressed. The results of the current study indicate that in the late gestational fetal and adult baboon ovary and adult endometrium, the short form of the receptor predominates. Although granulosa cells obtained from ovaries of hormonally primed immature rats apparently express the long form [41, 42], the short form of ERß is expressed in ovaries of the adult human and marmoset [26] and rat [43]. In contrast, the midterm baboon fetal ovary expresses both the long and short forms of ERß. Thus, the results of the current study indicate that the nature of the ERß protein expressed by the fetal ovary changes with advancing gestation, suggesting that the ERß isoform expressed is not only tissue specific but also developmentally and/or hormonally regulated.
In summary, the results of the current study are the first to establish that the ER and ERß mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH U54 HD 36207 as part of the NICHD Specialized Cooperative Centers Program in Reproduction Research. 
2 Correspondence: Gerald J. Pepe, Department of Physiological Sciences, Eastern Virginia Medical School, 700 Olney Rd., P.O. Box 1980, Norfolk, VA 23501-1980. FAX: 757 624 2269; pepegj{at}evms.edu
Accepted: November 6, 2001.
Received: August 28, 2001.
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and ß in the baboon fetal adrenal gland. Endocrinology 1999; 140:5953-5961 and ß in the rodent mammary gland. Proc Natl Acad Sci U S A 2000; 97:337-342) and beta (ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol Metab 1997; 82::3509-3512 and -ß and androgen receptor in the ovaries of marmosets and humans. Biol Reprod 2000; 63:1098-1105) and beta (ER-ß) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down regulation of ER-ß in neoplastic tissues. J Clin Endocrinol Metab 1998; 83:1025-1028. Mol Endocrinol 1997; 11:1486-1496 transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 1999; 140:5566-5578 or estrogen receptor-ß. Endocrinology 1999; 140:800-804 receptor chimeras. Endocrinology 1998; 139:4513-4522This article has been cited by other articles:
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