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a Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23501-1980
b Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, The Center for Studies in Reproduction, 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 ß in mesenchymal-epithelial cells (i.e., pregranulosa) as early as midgestation. Therefore, the current study determined whether estrogen regulates fetal ovarian follicular development. Pregnant baboons were untreated or treated with the aromatase inhibitor CGS 20267, or with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100–164 (term = Day 184). On Day 165, baboon fetuses were delivered by cesarean section and the number of total follicles and interfollicular nests consisting of oocytes and mesenchymal-epithelial cells in areas (0.33 mm2) of the outer and inner cortices of each fetal ovary were quantified using image analysis. Maternal and umbilical serum estradiol levels were decreased by >95% with CGS 20267. Treatment with CGS 20267 and estrogen restored maternal estradiol to normal and fetal estradiol to 30% of normal. Although fetal ovarian weight was unaltered, the mean number of follicles ± SEM/0.33 mm2 in the inner (59.0 ± 1.7) and outer (95.3 ± 2.4) cortical regions of fetal ovaries in untreated animals was 35%–50% lower (P < 0.01) in estrogen-depleted baboons (25.9 ± 1.4, inner cortex; 62.5 ± 2.7, outer cortex) and was restored to normal by treatment with CGS 20267 and estrogen. In contrast, the number of interfollicular nests was 2-fold greater (P < 0.01) in fetal ovaries of estrogen-suppressed animals, a change that was prevented by treatment with estrogen. In summary, fetal ovarian follicular development was significantly altered in baboons in which estrogen was depleted during the second half of gestation and restored to normal by estradiol. We propose that estrogen plays an integral role in regulating, and perhaps programming, primate fetal ovarian development.
estradiol, follicle, oocyte development, ovary, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well established that estrogen exerts profound effects on various aspects of ovarian function in adult rats [4]. During human and baboon pregnancy, placental production and secretion of estradiol into the maternal and fetal circulations increases with advancing gestation [5]. Moreover, we have shown that estrogen regulates several aspects of fetal-placental function [6], including placental expression [7] and localization [8] of the 11ß-hydroxysteroid dehydrogenase enzymes that control cortisol-cortisone metabolism and thus maturation of the fetal pituitary-adrenocortical axis [9]. Estrogen receptor protein is expressed in granulosa cells of antral follicles in the ovaries of adult humans [10], baboons [11], and cynomolgus monkeys [12]. Moreover, we recently demonstrated that estrogen receptors (ER) and ß (ERß) are expressed in the baboon fetal ovary during mid and late gestation [13]. Therefore, in the current study, we used a highly specific aromatase inhibitor and the baboon as a model to study the endocrinology of human fetal development to determine whether estrogen regulates fetal ovarian folliculogenesis and thus the ovulatory pool of follicles for adult life in primates.
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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, carrots, or both, and water ad libitum. Females were paired with males for 5 days at the anticipated time of ovulation, and pregnancy was subsequently confirmed by palpation and ultrasonography. Pregnant baboons were either untreated (n = 6) or treated (n = 6) with a highly specific aromatase inhibitor, CGS 20267 (Letrozole; 4,4-[1,2,3-triazol-1yl-methylene] bis-benzonitrite, Norvartis Pharma AG, Basel, Switzerland; 2 mg/day), administered s.c. on Days 100–165 (term = Day 184) as described previously [14]. Additional animals (n = 4) were injected with CGS 20267 (2 mg/day) plus estradiol benzoate (1–3 mg/day) on Days 100–165 of gestation. Blood samples (3–5 ml) were obtained at 1- to 4-day intervals between Days 85 and 165 of gestation via a maternal saphenous vein after sedation with an i.m. injection of ketamine-HCl (10 mg/kg body weight; Parke-Davis, Detroit, MI). On Day 165 of gestation, baboons were sedated with ketamine, anesthetized with isoflurane, and after obtaining maternal and umbilical blood samples, the placenta and the fetus were delivered by cesarean section, and the fetus was killed with an overdose of sodium pentobarbital. Fetal ovaries were excised, trimmed of fat, and weighed. One ovary was fixed in 10% buffered formalin and embedded in paraffin, and the other ovary was stored in liquid nitrogen. Ovaries were also obtained from three baboon fetuses delivered by cesarean section on Day 100 of gestation. 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 Institutional Animal Care and Use Committee of the Eastern Virginia Medical School approved the experimental protocol employed in this study.
Radioimmunoassays
Maternal and umbilical venous serum samples were stored at -20°C until measurement by radioimmunoassay of estradiol and testosterone using an automated chemiluminescent immunoassay system (Immulite; Diagnostic Products Corp., Los Angeles, CA) as described previously for estradiol [14] or for androstenedione using a solid phase 125I radioimmunoassay kit (Coat-A-Count, Diagnostic Products). The Immulite assay for estradiol was previously shown to be highly specific for estradiol and did not detect (<1%) estradiol benzoate. The antibodies for testosterone and androstenedione assays were also highly specific and exhibited minimal (<1%) cross-reactivity with cortisol, dehydroepiandrosterone (DHA), DHA-sulfate, progesterone, and corticosterone.
Immunocytochemistry
The immunocytochemical detection of ER and ERß was determined essentially as described previously [13]. Briefly, paraffin-embedded sections (4 µm) of fetal 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. Sections were incubated for 48 h at 4°C with the mouse monoclonal NCL-ER6 F11 antibody to the human ER (Vector Laboratories, Burlingame, CA) diluted 1:40 in 5% normal goat serum (NGS) in PBS, or with the chicken polyclonal antibody to the human ERß protein (generously supplied by Dr. Jan-Åke Gustafsson) and 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 immunoglobulin G (ER) or anti-chicken immunoglobulin G and then with the VectaStain Elite Kit (Vector). After rinsing in PBS, sections were stained for ERß with diaminobenzidine (DAB)-imidazole-H2O2 and for ER with DAB-nickel sulfate (0.250 g nickel sulfate, 2 mg DAB, and 8.3 µl of 3% H2O2 in 10 ml of 0.175 M acetate buffer, pH 6.0) as described by Hoffman et al. [15].
Histology and Image Analysis
Representative sections (4 µm) of paraffin-embedded fetal ovaries were mounted onto Superfrost microscope slides (Fisher), stained with hematoxylin and eosin, and examined by light microscopy using an Optiphot-2 microscope attached to a video-based Image-1 analysis system (Universal Imaging Corp., West Chester, PA). A minimum of 10 areas (0.33 mm2) from both the outer (close to the surface epithelium) and inner (adjacent to the medullary region) cortex of 20–40 randomly selected sections of each fetal ovary were examined. The average number of total follicles, including primordial (one layer of flattened or combined flattened and cuboidal granulosa) and primary-like (one layer of cuboidal granulosa) follicles, as well as the number of interfollicular nests per 0.33 mm2 were calculated for each animal, and the data were expressed as an overall mean (±SEM). The applicability of this sampling procedure was confirmed in an additional experiment in which the number of follicles were determined in 10 areas (0.33 mm2) from both the outer and inner cortices of approximately every 8th to 10th section of an entire fetal ovary obtained from an untreated baboon and from a baboon treated with CGS 20267.
Statistics
Data are expressed as an overall mean (±SEM) and were analyzed by ANOVA with post hoc comparisons of the means by the Student-Newman-Keuls multiple comparison tests.
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RESULTS |
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As previously demonstrated [14], maternal serum estradiol levels rose from approximately 1 ng/ml on Days 85–120 of gestation to 2.5–3.0 ng/ml by Day 165 (Fig. 1A). Within 48–72 h of the onset of CGS 20267 treatment on Day 100, maternal serum estradiol levels decreased to and remained at approximately 0.1 ng/ml. The pattern of serum estradiol in baboons treated with CGS 20267 plus estradiol benzoate was similar to that in controls, however, absolute levels were slightly greater than normal. As seen in Figure 1B, mean (±SEM) estradiol levels in umbilical venous serum on the day of delivery in untreated baboons (0.59 ± 0.13 ng/ml) were also significantly (P < 0.01) reduced by administration of CGS 20267 (0.04 ± 0.01 ng/ml) and restored (0.19 ± 0.08 ng/ml) to 30% of normal in baboons treated with CGS 20267 and estradiol benzoate. The restoration of maternal but not fetal serum estradiol to normal in baboons treated with CGS 20267 and estrogen presumably reflects the fact that placental estradiol is preferentially secreted into the maternal circulation during primate pregnancy [5] and that estradiol benzoate was injected into and thus distributed to all maternal tissues rather than originating exclusively in the placenta. As seen in Table 1, serum testosterone and androstenedione concentrations were higher (P < 0.05) in both the maternal saphenous and the umbilical veins in baboons treated with CGS 20267. Testosterone and androstenedione levels in the umbilical vein remained elevated in baboons that received CGS 20267 and estrogen (Table 1). Although levels of both androgens in maternal saphenous serum were reduced by treatment with CGS 20267 and estrogen, values were not entirely restored to normal (Table 1).
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Effects of Estrogen on Fetal Ovarian Maturation
As previously demonstrated [13], the baboon fetal ovary at midgestation (Day 100) primarily consisted of oogonia/oocytes and mesenchymal-epithelial cells enveloped in cord-like structures termed germ cell cords (Fig. 2A). Although folliculogenesis began at midgestation, it was minimal. This contrasts with late gestation (Day 165), when the fetal ovary consisted almost exclusively of primordial follicles, whereas interfollicular nests of mesenchymal-epithelial cells and oocytes were less abundant both in the outer (Fig. 2B) and inner cortical regions (Fig. 2D). In estrogen-suppressed baboons, fetal ovarian development appeared to be markedly altered. Thus, in both the outer (Fig. 2C) and inner (Fig. 2E) cortices, there appeared to be fewer follicles and more interfollicular nests. Moreover, the ovarian surface epithelium/capsule, which was well developed in untreated baboons (Fig. 2B), appeared to be incompletely differentiated in the majority of ovaries from estrogen-deprived fetuses (Fig. 2C). In baboons in which estrogen was restored, fetal ovarian histology (Fig. 2, F and G) appeared to be similar to that of untreated controls.
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The effects of estrogen deprivation on fetal ovarian development were quantified using image analysis. Thus, as shown in Figure 3A, the mean number of follicles ± SEM/0.33 mm2 in the outer cortical region of fetal ovaries of untreated baboons (95.3 ± 2.4) was significantly (P < 0.01) reduced in baboons treated with CGS 20267 (62.5 ± 2.7) and restored (P < 0.01) to normal in baboons treated with CGS 20267 plus estradiol (96.7 ± 6.0). Similarly, the number of follicles in the inner cortical region of fetal ovaries of untreated baboons (59.0 ± 1.7) was significantly (P < 0.01) lower in animals depleted of estrogen by CGS 20267 (25.9 ± 1.4) and restored to normal in baboons treated with CGS 20267 and estradiol (64.9 ± 5.2, Fig. 3B). In contrast, the number of interfollicular nests in the outer cortex of ovaries of untreated fetal baboons (3.1 ± 0.6, Fig. 4A) was higher (P < 0.01) in estrogen-depleted baboons (9.5 ± 0.8) and restored (P < 0.05) to normal in animals treated with CGS 20267 and estradiol (5.2 ± 0.4). A similar pattern was also observed in the inner cortical region in which the number of interfollicular nests in fetal ovaries of untreated baboons (5.8 ± 0.2) was 2-fold greater (P < 0.05) with CGS 20267 treatment (12.0 ± 2.0), an effect partially prevented by treatment with CGS 20267 and estrogen (9.6 ± 0.9).
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As seen in Figure 5, in fetal ovaries of untreated baboons, the number (0.6 ± 0.08) of primary-like follicles (i.e., an oocyte surrounded by a single layer of cuboidal granulosa cells) was minimal and accounted for less than 1% of all follicles counted. However, the number of primary-like follicles was higher (P < 0.05) in ovaries of estrogen-depleted baboon fetuses (2.3 ± 0.2) and accounted for approximately 6% of all follicles counted. In baboons treated with CGS 20267 and estradiol benzoate, the number of primary-like follicles (1.5 ± 0.1) was restored (P < 0.05) to a value intermediate between that in fetal ovaries of baboons untreated or treated with CGS 20267.
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Despite alterations in the cellular structure of the ovary, fetal ovarian weights as well as fetal body and pituitary weights were not altered by treatment with CGS 20267 or CGS 20267 plus estradiol (Table 1).
Effects of Estrogen on Fetal Ovarian ER and ERß Expression
Both ER and ERß were expressed in baboon fetal ovaries on Days 100 and 165 of gestation. ER protein was detected in surface epithelium and mesenchymal-epithelial cells but not in oogonia/oocytes at midgestation (Fig. 6A). By late gestation (Fig. 6B), ER was still highly expressed in the surface epithelium and mesenchymal-epithelial cells between follicles (i.e., interfollicular nests) but was only lightly or not expressed in granulosa cells of follicles. The latter results also indicate that the nests are a discrete group of cells and are not components of follicles resulting from the cutting of sections adjacent to or just into a follicle. As seen in Figure 6E, ERß protein expression was also localized to mesenchymal-epithelial cells at midgestation and abundantly expressed in granulosa cells and mesenchymal-epithelial cells in interfollicular nests on Day 165 of gestation (Fig. 6F). In baboons in which estrogen levels were suppressed by treatment with CGS 20267, the site and apparent expression levels of ER (Fig. 6C) and ERß (Fig. 6G) were similar to those in fetal ovaries of baboons untreated (Fig. 6, B and F) or treated with CGS 20267 and estrogen (Fig. 6, D and H). Specificity for these antibodies was confirmed in previous experiments [13] demonstrating absence of signal in cytoplasm and in sections of near term fetal ovary incubated with antibody (ER) preabsorbed with excess immunizing peptide or without primary (ERß) antibody.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ohno and Smith [17] originally suggested that in humans, many oocytes regress during the early stages of meiosis unless they are enveloped by granulosa cells. Thus, the availability of oocytes may be severely compromised in the fetal ovary of estrogen-deprived baboons. Because development of the ovulatory pool of follicles by birth is considered vital to establishing the foundation for normal reproductive function in adults [2, 3], we further propose that the ovulatory pool (i.e., the number) of follicles available for normal reproductive function in adulthood is dependent on the presence of estrogen in utero. Although it remains to be determined whether depriving the fetus of estrogen during development compromises adult ovarian function, including the onset of puberty, follicular maturation/ovulation, the timing of menopause, or all these, we suggest that the baboon provides an excellent model for performing such clinically relevant studies.
It remains to be determined whether estrogen elicits its effects directly on the ovary or indirectly via actions on the fetal hypothalamic-pituitary axis, or both. A direct action of estrogen on the ovary is supported by the fact that as early as midgestation the baboon fetal ovary expresses both ER and ERß [13] in the mesenchymal-epithelial cells that apparently emerge from their environment and surround meiotically active oocytes to form a primordial follicle. In addition, the placental production and fetal serum levels of estradiol increase over the course of primate pregnancy [5, 18]. Moreover, mice with targeted disruption of the genes for ER [19, 20] or ERß [21] exhibit infertility and abnormalities in ovarian function such as premature loss of follicles and anovulation [21]. In addition, in mice that lack both estrogen receptor genes [22, 23], ovaries undergo follicle transdifferentiation to structures resembling seminiferous tubules, including Sertoli-like cells. Thus, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual estrogen receptor knockout animals, indicating that both receptors are required for the maintenance of germ cells and somatic cells in the postnatal ovary [22]. Targeted disruption of the aromatase P-450 gene required for conversion of androgens to estrogen also resulted in ovarian dysfunction that included anovulation and precocious depletion of ovarian follicles [24]. In a couple of reported cases of human pregnancy in which there was a mutation in the aromatase gene in both the placenta and fetus [25–27], the female neonates did not exhibit puberty due to polycystic ovaries secondary to hypergonadotropism. Nevertheless, these authors [27] proposed that estrogen synthesis in the blastocyst, fetus, and placenta was not essential for normal embryonic and fetal development [27]. However, in these aromatase-deficient patients, although maternal and fetal estrogen levels were extremely low, they remained in the 10-9 M range, which approximates the association constant for estrogen receptor binding. Therefore, as suggested previously [14], it appears that a considerable excess of estrogen exists during human pregnancy and that the requisite biological effects of estrogen are dependent on availability of one or more receptors and a level of estradiol sufficient to interact with the receptor. Indeed, in the current study, the ovarian dysfunction apparent in estrogen-depleted baboon fetuses was prevented in fetal baboons in which umbilical venous serum estradiol levels were restored to values that were considerably lower than those in untreated animals.
In humans [28, 29] and nonhuman primates [30, 31], fetal FSH and LH levels are very low in early gestation, they increase to a maximum at midgestation, and they decline steadily thereafter. Although fetal ovarian development appears to proceed relatively normally up to Weeks 32–34 of gestation in human anencephalic fetuses [32], the number of oogonia was lower and the number of atretic germ cells was higher in fetal ovaries of rhesus monkeys in which the fetal pituitary was removed at midgestation [33]. It has been suggested, therefore, that fetal pituitary gonadotropins may be required for fetal ovarian development in primates at some point during mid to late gestation [33]. Although FSH receptor binding has not been detected in human or rhesus monkey fetal ovaries at midgestation [34], preliminary data in our laboratories indicate that FSH receptor mRNA was detectable in the baboon fetal ovary at midgestation and that expression appears to be higher by term [35]. It remains to be determined whether fetal FSH levels, FSH receptor levels, or both are modified in estrogen-depleted baboons, and thus whether the effects of estrogen deprivation on fetal ovarian development also reflect an action of estrogen on the fetal hypothalamus-pituitary. In this regard, results of the current study showed that the number of primary-like follicles was higher in fetal ovaries of baboons treated with CGS 20267, perhaps reflecting increased follicle growth secondary to FSH stimulation. However, because the formation of a primordial follicle first entails envelopment of oocytes by the more round-shaped mesenchymal-epithelial cells (i.e., pregranulosa) and then flattening of these somatic cells (i.e., granulosa), it remains to be determined whether the primary-like follicles observed are truly primary or have not reached the primordial stage and thus are more immature. Studies designed to examine expression of specific markers that reflect development of follicles beyond the primordial stage [36, 37] are needed to answer this important question.
In estrogen target tissues, including those of uterus and breast, estrogen receptor expression is up-regulated by estrogen itself [23]. However, in the current study, based on immunocytochemical analyses, neither ER nor ERß expression in the fetal ovary of estrogen-deprived baboons appeared to be markedly different from that in ovaries of untreated animals. Therefore, estrogen may not regulate expression of its requisite receptors in the fetal ovary, however, the effects of estrogen on cell specific expression of both receptors needs to be determined using more quantitative methods.
Even though umbilical serum testosterone and androstenedione concentrations were higher in baboons treated with CGS 20267, androgen levels in the fetus remained elevated in baboons treated with CGS 20267 and estradiol, although ovarian maturation was normal. Furthermore, it appears that the baboon fetal ovary does not express the androgen receptor (unpublished results). Therefore, it is unlikely that the effects of estrogen deprivation on fetal ovarian maturation reflected an action of androgens.
In summary, ovarian development was significantly altered in baboon fetuses in which estrogen was depleted during the second half of gestation and restored to normal by treatment with estradiol. These observations, plus the fact that the fetal ovary expresses estrogen receptors and ß, support the suggestion that estrogen plays an integral role in regulating, and perhaps programming, primate fetal ovarian development and thus fertility in adulthood.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gerald J. Pepe, Department of Physiological Sciences, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501-1980. FAX: 757 624 2269; pepegj{at}evms.edu
Received: 31 January 2002.
First decision: 27 February 2002.
Accepted: 29 April 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß in the baboon fetal ovary. Biol Reprod 2000 66:1054-1060 and ß. Science 1999 286:2328-2331This article has been cited by other articles:
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P. Smith, T. L. Steckler, A. Veiga-Lopez, and V. Padmanabhan Developmental Programming: Differential Effects of Prenatal Testosterone and Dihydrotestosterone on Follicular Recruitment, Depletion of Follicular Reserve, and Ovarian Morphology in Sheep Biol Reprod, April 1, 2009; 80(4): 726 - 736. [Abstract] [Full Text] [PDF]
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4A), and on fetal body, ovarian, and pituitary weights in late baboon pregnancy.*
