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Regular Article |
a Center for Biomedical Research, The Population Council, New York, New York 10021
b Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
ABSTRACT
Changes in mRNA expression for estrogen receptor (ERß) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in granulosa and theca cells from proestrous rat ovarian follicles. Of the 30 ovaries harvested from 15 adult rats, 24 were processed for in situ hybridization, and the remaining were used for reverse transcription-polymerase chain reaction. Messenger RNAs for ERß, LHr, cytochrome P450 side-chain cleavage enzyme (P450scc), 17
-hydroxylase (P450c17), aromatase (P450arom), and steroidogenic acute regulatory protein (StAR) were localized in cross sections of ovaries by in situ hybridization and quantified in granulosa and theca cell layers by a computer-image analyzing system. Ovarian follicles were classified as healthy or atretic. Healthy follicles were divided into four size groups: very small (40–100 µm), small (101–275 µm), medium (276–450 µm), and large (451–850 µm). Atretic follicles were divided into medium (276–450 µm) or large follicles (451–850 µm). A low level of ERß mRNA expression was first detected in granulosa cells of very small healthy follicles, and the expression increased progressively up to medium-sized follicles. The expression of ERß mRNA was highest (P < 0.01) in medium-sized follicles that was followed by a decrease (P < 0.01) in large follicles. Messenger RNAs for LHr, P450scc, and P450arom were first detected in granulosa cells of medium-sized healthy follicles, while mRNAs for LHr, P450scc, P450c17, and StAR were first detected in theca cells associated with very small follicles. The highest expression of LHr, P450scc, P450c17, P450arom, and StAR was seen in granulosa and/or theca cells of large healthy follicles. In atretic follicles, level of gene expression was relatively low in both granulosa and theca cells. In conclusion, stage-specific expression of ERß mRNA was observed in granulosa cells during follicular development. The increased expression of ERß and a concomitant initiation of LHr, P450scc, and P450arom expression in granulosa cells of medium follicles may signify a role for estrogen in follicular development. Also, a strong correlation between ERß mRNA expression in granulosa cells, and the expression of mRNAs for LHr, P450scc, P450c17, and StAR in theca cells associated with growing follicles suggests a possible role for estrogen in steroidogenesis.
estradiol receptor, follicle, follicular development, granulosa cells, ovary, theca cells
INTRODUCTION
Estrogen has an obligatory role in the normal functioning of the female reproductive system [1, 2]. In addition, estrogen is known to influence the proliferation and differentiation of some nonreproductive tissues [3–5]. The action of estrogen on the female reproductive system is mediated via its ability to regulate the synthesis and secretion of pituitary gonadotropins [6], its direct proliferative effect on the uterine and breast tissue [7], and its possible intraovarian role to regulate steroidogenesis and folliculogenesis [8, 9].
The cyclic production of estradiol and progesterone by the ovary regulates the estrus cycle in rodents and menstrual cycle in the primates. The first step in the biosynthesis of steroids in the ovary is the transport of cholesterol from outer to inner mitochondrial membranes regulated by steroidogenic acute regulatory protein (StAR) [10]. Cholesterol is then converted to pregnenolone by cytochrome side-chain cleavage enzyme (P450scc) in theca and/or granulosa cells [11, 12]. Pregnenolone is metabolized to either progesterone by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in theca or granulosa cells (
4-pathway) or to dehydroepiandrosterone (DHEA) by 17-hydroxylase (P450c17) in theca cells (5-pathway). In the theca cells progesterone and DHEA are metabolized to androstenedione by P450c17 and 3ß-HSD, respectively. Androstenedione is converted to estradiol (E2) by cytochrome P450 aromatase (P450arom) in granulosa cells. The expression of StAR and steroidogenic enzymes involved in steroid biosynthesis in the ovary is regulated in a timely and cell-specific manner by LH, FSH, and prolactin, as well as other factors [13–15].
An intraovarian role for E2 has been proposed; however, its mode of action is not well understood. For example, LH receptor mRNA expression in rat granulosa cells has been shown to be stimulated by E2 and FSH [1, 16]. The action of E2 on its target tissues is mediated via classical estrogen receptors (ER) now referred to as ER. The expression of ER in the ovarian cells has not been demonstrated convincingly [17–20]. Recent discovery of a new form of ER (designated as ERß) has led to the reexamination of estrogen action on its target tissues [21, 22]. The expression of ERß mRNA and protein has been demonstrated in rat ovarian granulosa cells [20, 23, 24]. This suggested that the action of E2 on folliculogenesis and/or steroidogenesis in the ovary may be mediated via ERß. The expression of ERß in granulosa cells during different stages of follicular development has not been characterized.
The present study was undertaken to 1) characterize the changes in expression of ERß mRNA in granulosa and theca cells during follicular development by in situ hybridization; and 2) relate ERß mRNA expression in granulosa cells to the expression of mRNAs for LH receptor (LHr), P450scc, P450c17, P450arom, and StAR in theca and/or granulosa cells during follicular development.
MATERIALS AND METHODS
Animals, Tissue Collection, and Tissue Preparation
Twenty adult female Sprague-Dawley rats were purchased from Charles River Laboratories (NY). The rats were housed (light: 0600–1800 h) at the Laboratory Animal Research Center of the Rockefeller University according to the NIH guidelines outlined in the Guide for the Care and Use of Laboratory Animals. Stages of estrous cycles were monitored by examining vaginal smears daily [25]. Rats exhibiting three consecutive 4- to 5-day estrous cycles were killed on the day of proestrus (1400 h) to obtain follicles at different stages of development. Twenty-three ovaries from 12 rats were processed for in situ hybridization, and 6 ovaries collected from 3 rats were used for reverse transcription-polymerase chain reaction (RT-PCR) and generation of probes for in situ hybridization. For in situ hybridization, four ovaries from different rats were placed in a small container (block) containing Tissue Prep (Fisher Chemicals, St. Louis, MO). The orientation of ovaries within the block was adjusted so that the maximum cross sections from the middle plane of the ovaries would be obtained to evaluate ovarian follicular size and morphology. Six blocks were prepared and frozen over liquid nitrogen and stored at -70°C until sectioned.
Oligonucleotide Primers, RT-PCR, and Cloning of cDNA to Vector
Primers for rat ERß, ER, androgen receptor (AR), LHr, StAR, P450scc, P450c17, and P450arom were synthesized based on published reports [26–32]. Table 1 summarizes the anticipated size of PCR products, sense and antisense sequences, and locations of primers. Total RNA from the whole ovary was isolated using a micro RNA isolation kit (Stratagene, La Jolla, CA). Five micrograms of total RNA was reverse transcribed using an RT-PCR kit (Stratagene). For PCR amplification, 1 µl of synthesized cDNA was added to the PCR reaction mixture (GeneAmp PCR kit; Perkin-Elmer, Norwalk, CT) containing both antisense and sense primers (200 ng/tube) and amplified for 30 cycles by incubation at 95°C for 1.5 min, 56°C for 2 min, 72°C for 4 min, and a final incubation at 72°C for 5 min. The thermocycler 2400 (Perkin-Elmer) was used for both RT and PCR. After reaction was completed, 10 µl of PCR product was loaded on a 1% agarose gel containing ethidium bromide. The results with the anticipated size for LHr, P450scc, StAR, P450c17, P450arom, ER, ERß, and AR are shown in Figure 1.
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The PCR product (cDNA) for each gene was purified and cloned into pCR-script Amp SK(+) cloning vector using the procedures described in the pCR-Script Amp SK(+) cloning kit or pCR-Script Amp electroporation-competent cell cloning kit (Stratagene). After subcloning (ligation of cDNA to vector) and transformation, the presence of the cDNA insert within the vector was confirmed by double restriction digestion. Sequences of inserts were confirmed by DNA sequencing. Orientation of insert within the vector (plasmid) was determined by running PCR reactions in which KS primer, which is contained in the multicloning site of the vector, was combined with either the antisense or sense primer of the particular gene. The plasmid was linearized with a single restriction enzyme to generate sense and antisense cDNA templates for in vitro transcription [33, 34].
In Situ Hybridization
Both antisense and sense [35S]UTP-labeled cRNA probes were transcribed from linearized cDNA templates using a transcription kit (Stratagene). The cRNA probes were purified by centrifugation on a Sephadex G-50 column and used for hybridization within 1 wk.
Procedures for in situ hybridization were described previously [33, 34]. Fourteen-micron sections of frozen ovaries were air dried and stored at -70°C, in desiccated, air-tight boxes until fixation and hybridization (within 1 mo). Before hybridization, sections were fixed in 4% formaldehyde in 0.01 M PBS for 5 min, washed in 2x saline-sodium citrate (SSC: 1x SSC = 0.3 M NaCl and 0.03 M sodium citrate, pH 7.0), acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min, then rinsed in 2x SSC, and dehydrated in increasing concentrations of ethanol. Slides were then dipped in chloroform for 5 min, 100% ethanol for 2 min, 95% ethanol for 2 min, and air dried.
For hybridization, the labeled probes were diluted in hybridization buffer to about 2 x 107 cpm/ml. Hybridization was performed using 100 µl diluted probe/slide in a humidified oven at 55°C for 20 h. After hybridization, slides were washed twice by shaking in 2x SSC (45–52°C) for 15 min at room temperature and treated with RNase-A (50 µg/ml in 2x SSC) for 1 h at 37°C. Slides were then washed at 55°C in 2x SSC containing 0.1% ß-mercaptoethanol (BME) for 15 min, 1x SSC/0.1% BME for 15 min, 1x SSC/50% formamide/0.1% BME for 30 min, and twice in 0.1x SSC/BME for 15 min. The slides were then dehydrated with increasing concentrations of ethanol, air dried, dipped in Kodak NTB-2 emulsion (1:2 diluted), and exposed for 14 days for LHr, ERß, ER, and AR and 3 days for enzymes and StAR at 4°C. The slides were then developed, counterstained with hematoxylin and eosin, and mounted for microscopic examination. For each block containing four ovaries, one section was hybridized with the antisense probe, and the other with the sense probe. All slides were processed in a single hybridization experiment to minimize variations. A total of 22 sections or slides per ovary were collected (see next paragraph).
Classification of Follicles
Cross sections of ovarian follicles were examined using a microscopic-computer imaging system (Bioquant image analysis system; R & M Biometrics Inc., Nashville, TN). Maximum size (µm) of follicle including theca layers was determined by examining 22 adjacent cross sections collected from the middle plane of ovary. Follicles were morphologically classified as healthy or atretic [33, 34]. Healthy follicles were grouped into very small (40–100 µm), small (101–275 µm), medium (276–450 µm), and large (451–850 µm) follicles. Very small follicles were primary follicles up to five layers of granulosa cells. When this class of follicles approached 100 µm in size, theca interna layers started to appear. Small follicles were follicles with theca interna exhibiting the beginning of antrum formation (early antral follicles). Medium-sized follicles had well-developed antrum with oocyte migrated toward the side of the follicle. Large follicles were those with characteristics of preovulatory follicles. Atretic follicles were grouped into medium (276–450 µm) and large (451–850 µm) follicles. From each block, 12 or more follicles (two or more follicles for each size group) were chosen to evaluate the expression of ERß and other genes. A total of 80 follicles (56 healthy and 24 atretic follicles) were studied.
Image Analysis
Hybridization intensity was quantified using the Bioquant image analysis system as described earlier [33, 34]. For each follicle, four fields at roughly 90° angles were measured for the sections hybridized to the antisense probe and sense probes. The quantification of the expression of different mRNAs was done in the adjacent sections of the same follicle (ovary). For each of the mRNAs the expression was quantified in 12 follicles. Specific hybridization intensity was defined as the average hybridization intensity for a section hybridized to the antisense probe minus the average hybridization intensity for the section hybridized to the sense probe. Intensity of hybridization was expressed as the percentage of pixels within a given marked area that was above a preset gray threshold level.
Statistical Analysis
Analysis of variance (General Linear Model) was used to test the effects of stage of follicular development (size group) and follicular health on the various parameters measured. Duncan's multiple-range test was used to compare means. Pearson correlation was used to establish relationships between level of ERß mRNA expression, and the expression levels of other genes. Analyses were performed using SAS [35] and results reported as mean ± SEM.
RESULTS
Characteristics of Follicles
Morphologically, healthy follicles ranging in size from 40 to 850 µm, were classified as very small (40–100 µm, n = 20), small (101–275 µm, n = 12), medium (276–450 µm, n = 12), and large (451–850 µm, n = 12) follicles. Atretic follicles were divided into medium (276–450 µm, n = 12), and large (451–850 µm, n = 12) follicles.
Detection of AR, ER, and ERß mRNA in Rat Ovary by RT-PCR and In Situ Localization
Expression of AR, ER, and ERß mRNA was detected in the whole ovary by RT-PCR (Fig. 1). Using in situ techniques, AR mRNA was localized in granulosa, luteal, and interstitial stroma cells (Fig. 2, A and B). The ER mRNA expression was localized in theca and interstitial stroma cells but not in granulosa cells (Fig. 2, C and D). Although both AR and ER mRNA were localized in particular cell types of the ovary, changes in their expression were not examined in this study due to high variability. The ERß mRNA was localized in both granulosa and theca cells (Fig. 3). Expression levels of ERß mRNA in granulosa cells were stage specific (Fig. 3, A–N); therefore, the relative levels of ERß mRNA in granulosa cells were quantified. In theca cells, a low level of ERß mRNA was expressed in only some of the healthy medium and large follicles (Fig. 3, M–O). Thus, the relative intensity of ERß mRNA expression in theca cells was not quantified.
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Detection of LHr, P450scc, P450c17, P450arom, and StAR mRNA in Rat Ovary by RT-PCR and In Situ Hybridization
Messenger RNAs for LHr, P450scc, P450c17, P450arom, and StAR were detected in ovarian tissue by RT-PCR (Fig. 1). In situ localization results indicated that the mRNAs for LHr (Fig. 4, A and B) and P450scc (Fig. 4, C and D) were localized in both granulosa and theca cells while expression of mRNAs for P450c17 (Fig. 4, I and J) and StAR (Fig. 4, E and F) was localized mostly in theca cells. The P450arom mRNA was localized in granulosa cells (Fig. 4, G and H). In addition, mRNAs for LHr, P450scc, P450arom, and StAR were also localized in luteal tissues (data not shown).
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ERß mRNA Expression in Granulosa Cells
In healthy follicles, mRNA for ERß was first detected in granulosa cells of very small follicles (Fig. 3, A and B). The expression of ERß mRNA was quantified by measuring intensity of hybridization and showed significant increase (P < 0.01) in small follicles compared to very small follicles (Figs. 3, C, D, and 5A). The levels of ERß mRNA further increased (P < 0.01) to the highest level in medium-sized follicles (Figs. 3, E, F, and 5A) and then declined (P < 0.01) in large follicles to levels seen in small follicles (Figs. 3, G, H, and 5A). Regardless of the size of follicles, healthy follicles expressed greater (P < 0.05) levels of ERß mRNA than atretic follicles (Fig. 5A). In atretic follicles, a low level of ERß mRNA was detected in granulosa cells of large follicles but not in medium-sized follicles (Figs. 3, I–L, and 5A).
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Changes in the Expression of LHr, P450scc, and P450arom mRNA in Granulosa Cells
Levels of mRNAs for LHr, P450scc, and P450arom expression were quantitatively analyzed (by measuring intensity of hybridization) in granulosa cells of ovarian follicles (Fig. 5B for LHr, Fig. 5C for P450scc, and Fig. 5D for P450arom). By in situ localization, they were undetectable in granulosa cells of very small and small healthy follicles (Fig. 5, B–D) but were first detected in granulosa cells of medium-sized healthy follicles. The levels of expression then increased (P < 0.01) dramatically to the highest levels in large healthy follicles (Fig. 5, B–D). Expression of LHr, P450scc, and P450arom mRNA was low to undetectable in granulosa cells of atretic follicles (Fig. 5, B–D).
Changes in the Expression of LHr, P450scc, P450c17, and StAR mRNA in Theca Cells
Measurable expression was first seen when follicles grew from very small to small follicles. The intensity of expression was not measured in very small follicles because not all of them expressed these mRNAs. The levels of mRNAs for LHr, StAR, P450scc, and P450c17 in the theca cells of follicles >101 µm are shown in Figure 5 (Fig. 5E for StAR; Fig. 5F for LHr; Fig. 5G for P450scc; Fig. 5H for P450c17). The expression of these genes in theca cells increased (P < 0.01) with increasing follicular size. Highest expression was in large healthy follicles (Fig. 5, E–H), and least expression was in theca cells of atretic follicles (P < 0.01).
Correlation Analysis
Correlation analysis of different genes quantified in the same follicles (Table 2), excluding the very small follicles, showed that expression of ERß mRNA in granulosa cells was strongly correlated with the expression of mRNAs for LHr (r = 0.44; P < 0.01), P450scc (r = 0.57; P < 0.01), P450c17 (r = 0.46: P < 0.01), and StAR (r = 0.45; P < 0.01) in theca cells. There was no correlation between expression of ERß mRNA and expression of mRNAs for LHr (r = 0.21; P = 0.12) and P450scc (r = 0.21; P = 0.10) in granulosa cells. A slight correlation was observed between expression of ERß and P450arom in granulosa cells (r = 0.23; P = 0.07).
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DISCUSSION
In the present study, changes in the expression of ERß mRNA in relation to the expression of LHr, StAR, and some steroidogenic enzymes in rat ovarian follicles and theca cells were examined. Even though no direct correlation between ERß and the expression of LHr, StAR, P450scc, and P450arom in granulosa cells was observed, the high level expression of ERß mRNA in the medium follicles was associated with the beginning of the expression of LHr, StAR, P450scc, and P450arom in these follicles. This may suggest that E2 may have a role in the initiation of the expression of mRNA for LHr and steroidogenic enzymes mediated via ERß.
The role of E2 in the ovary has been difficult to evaluate because the expression of ER has not been demonstrated consistently in ovarian cells [20]. The cloning of ERß in the rat prostate and ovary suggested its possible role in estrogen action. Localization of ERß mRNA in the rat ovarian granulosa cells using in situ hybridization has been reported [21, 26]. Others have shown the expression of ERß mRNA in ovarian tissue and/or granulosa cells by RT-PCR and Northern blot [20]. Also, the presence of ER protein was demonstrated in granulosa, theca, and luteal cells [23, 24]. The present study confirmed the expression of ERß mRNA in ovarian cells using in situ hybridization. In addition, stage-specific expression of ERß in granulosa cells of developing follicles was demonstrated for the first time. The increased expression of ERß mRNA in the granulosa cells with follicular growth may have a direct or indirect (stimulation of steroidogenesis in the theca cells) role in the growth of follicles.
In the second part of the study, the expression profiles of LHr, P450scc, P540arom, P450c17, and StAR were assessed by in situ hybridization in granulosa and theca cells during follicular growth. The expression of LHr, P450scc, and P450arom mRNA was first detected in the granulosa cells during the transition of small- to medium-sized follicles. The levels of expression of these genes then increased dramatically to high levels in large follicles. This suggests that the high expression of ERß mRNA during transition from small- to medium-sized follicles may have been responsible for the initiation of the expression of LHr, P450scc, and P450arom in the granulosa cells of medium follicles. The beginning of ERß mRNA expression in granulosa cells of very small follicles coincides with the expression of FSH receptors in granulosa cells [36–38]. In hypophysectomized or immature female rats and mice, estrogen has been shown to stimulate preantral and early antral follicular growth [39–43]. Estrogen is also known to act synergistically with FSH to stimulate LHr expression in granulosa cells [1]. Thus, the significance of ERß mRNA expression in granulosa cells during early stages of follicular growth may be associated with granulosa cell proliferation and follicular recruitment. Strong evidence for the role of estrogen in follicular growth in the ovary has been provided recently. Knockout mice deficient in estrogen or ER are shown to be infertile due to a block in folliculogenesis prior to antrum formation [9]. However, the specific cell types in the ovary as the site of estrogen action have not been identified. The predominance of ERß over ER in the rat ovary and the stage-specific expression of ERß in granulosa cells during follicular development imply a role for E2 action on granulosa cell proliferation and differentiation via ERß.
The interaction between theca cells that surround ovarian follicles and granulosa cells is important for normal ovarian function. Androgens are produced by theca (stromal) cells in response to LH, while granulosa cells utilize androgens as a substrate for the production of E2. It has been reported earlier that E2 produced by granulosa cells could stimulate androgen production by theca cells [8]. It was demonstrated that estrogen-treated purified bovine theca cells secreted androgens in greater amounts compared to those treated with hCG. Combined treatment with E2 and hCG resulted in greater than additive response to the two hormones, suggesting that estrogen provides a local feedback loop in the follicle to stimulate steroidogenesis [8]. The expression of ERß, StAR, and steroidogenic enzymes was examined in theca cells during follicular growth. The expression of StAR mRNA in theca cells increased with increasing follicular size, reaching the highest level of expression in theca cells associated with large healthy follicles. In addition expression of LHr, P450scc, and P450c17 in theca cells was also highest in theca cells of large follicles. The expression of ERß mRNA in the theca cells was nondetectable. However, the expression of ERß mRNA in granulosa cells was strongly correlated with the expression of mRNA for LHr, P450scc, P450c17, and StAR in theca cells of growing follicles. This may suggest that E2 produced by granulosa cells may act on granulosa and/or theca cells via autocrine or paracrine pathways.
Only a few of the follicles in the ovary are destined to grow to complete maturation. The growth of the large number of follicles is arrested at different stages of development. This results in follicular atresia associated with early formation of characteristic pycnotic nuclei. The decrease in cell death may be the cause of ovarian pathology associated with ovarian cancer and polycystic ovary syndrome [44]. Estradiol is known to act as a survival factor in both corpus luteum and granulosa cells [45]. Progesterone was also suggested to maintain granulosa cell viability through nongenomic mechanisms [46]. In our studies, granulosa cells of atretic follicles showed little or no expression of mRNAs for ERß, LHr, steroidogenic enzymes, and StAR. Theca cells associated with atretic follicles showed low level of expression of ERß, whereas significant expression of LHr, steroidogenic enzyme, and StAR was observed in these cells. This suggests that theca cells of atretic follicles may have the capacity to synthesize steroid hormones.
In conclusion, stage-specific expression of ERß mRNA was observed in granulosa cells. The high expression of ERß mRNA with concomitant initiation of LHr, P450scc, and P450arom mRNA expression in granulosa cells of medium-sized follicles may signify a role for E2 in follicular development. In addition, strong correlation between ERß mRNA expression in granulosa cells of growing follicles and the expression of LHr, P450scc, P450c17, and StAR mRNAs in theca cells associated with follicles of increasing size suggests a possible role for estrogen in steroidogenesis.
FOOTNOTES
First decision: 17 March 2000.
1 This work was supported by cooperative agreement from the U.S. Agency for International Development (Cooperative Agreement CCP-A-00-94-00013-04). 
2 Correspondence: Kalyan Sundaram, Center for Biomedical Research, The Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 7678; kalyan{at}popcbr.rockefeller.edu
3 Current address: Ares-Advanced Technology Inc., 280 Pond Street, Randolph, MA 02368.
Accepted: June 30, 2000.
Received: February 21, 2000.
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