Estrogen Receptor- Expression in Relation to the Expression of ...

Download PDF
3 downloads 0 Views 850KB Size Report
Comment
Changes in mRNA expression for estrogen receptor (ER ) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in ...
BIOLOGY OF REPRODUCTION 63, 1747–1755 (2000)

Estrogen Receptor-b Expression in Relation to the Expression of Luteinizing Hormone Receptor and Cytochrome P450 Enzymes in Rat Ovarian Follicles1 Bagna Bao,3,4 Narender Kumar,4 Russell M. Karp,4 H. Allen Garverick,5 and Kalyan Sundaram2,4 Center for Biomedical Research,4 The Population Council, New York, New York 10021 Department of Animal Sciences,5 University of Missouri-Columbia, Columbia, Missouri 65211 ABSTRACT

INTRODUCTION

Changes in mRNA expression for estrogen receptor (ERb) 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 ERb, LHr, cytochrome P450 side-chain cleavage enzyme (P450scc), 17a-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 mm), small (101–275 mm), medium (276–450 mm), and large (451–850 mm). Atretic follicles were divided into medium (276–450 mm) or large follicles (451–850 mm). A low level of ERb 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 ERb 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 ERb mRNA was observed in granulosa cells during follicular development. The increased expression of ERb 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 ERb 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.

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 3b-hydroxysteroid dehydrogenase (3bHSD) in theca or granulosa cells (D4-pathway) or to dehydroepiandrosterone (DHEA) by 17 a -hydroxylase (P450c17) in theca cells (D5-pathway). In the theca cells progesterone and DHEA are metabolized to androstenedione by P450c17 and 3b-HSD, respectively. Androstenedione is converted to estradiol (E 2) 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 E 2 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 E 2 and FSH [1, 16]. The action of E 2 on its target tissues is mediated via classical estrogen receptors (ER) now referred to as ERa. The expression of ERa in the ovarian cells has not been demonstrated convincingly [17–20]. Recent discovery of a new form of ER (designated as ERb) has led to the reexamination of estrogen action on its target tissues [21, 22]. The expression of ERb mRNA and protein has been demonstrated in rat ovarian granulosa cells [20, 23, 24]. This suggested that the action of E 2 on folliculogenesis and/or steroidogenesis in the ovary may be mediated via ERb. The expression of ERb 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 ERb mRNA in granulosa and theca cells during follicular development by in situ hybridization; and 2) relate ERb mRNA expression in granulosa cells to the expression of mRNAs for LH receptor (LHr),

estradiol receptor, follicle, follicular development, granulosa cells, ovary, theca cells This work was supported by cooperative agreement from the U.S. Agency for International Development (Cooperative Agreement CCP-A-00-9400013-04). 2 Correspondence: Kalyan Sundaram, Center for Biomedical Research, The Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 7678; e-mail: [email protected] 3 Current address: Ares-Advanced Technology Inc., 280 Pond Street, Randolph, MA 02368. 1

Received: 21 February 2000. First decision: 17 March 2000. Accepted: 30 June 2000. Q 2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

1747

1748

BAO ET AL.

TABLE 1. Properties of oligonucleotide primers used for PCR. Target mRNA

Size of PCR products (bp)

Sense (S) or antisense (AS)

ERb

558

ERa

344

AR

291

LHr

273

P450scc

540

P450c17

299

P450arom

271

StAR

404

S AS S AS S AS S AS S AS S AS S AS S AS

Primer sequence (location) 59-TAGGCACCCAGGTCTGCAAT-39 (49–68) 59-ACCAAGGACTCTTTTGAGGT-39 (603–584) 59-AATTCTGACAATCGACGCCAG-39 (681–701) 59-GTGCTTCAACATTCTCCCTCCTC-39 (1025–1003) 59-CCCATCGACTATTACTTC-39 (1623–1640) 59-TTCTCCTTCTTCCTGTAG-39 (1913–1906) 59-AGAGTGATTCCCTGGAAAGGA-39 (300–320) 59-TCATCCCTTGGAAAGCATTC-3′ (572–553) 59-AGAAGCTGGGCAACATGGAGTCAG-39 (286–309) 59-TCACATCCCAGGCAGCTGCATGGT-39 (831–808) 59-TCATCAAGAAGGGAAAAGAA-39 (299–318) 59-TGAAGCAGATAGCACAGATG-39 (592–573) 59-TGCACAGGCTCGAGTATTTCC-39 (1799–1820) 59-ATTTCCACAATGGGGCTGTCC-39 (20667–2047) 59-CTCAACAACCAAGGAAGGCTGG-39 (340–360) 59-GCAGGTGGGGCCGTGTTCAGC-39 (721–741)

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 2708C until sectioned.

References [26] [27] [28] [29] [30] [31] [30] [32]

Oligonucleotide Primers, RT-PCR, and Cloning of cDNA to Vector

Primers for rat ERb, ERa, 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 ml of synthesized cDNA was added to the PCR reaction mixture (GeneAmp PCR kit; PerkinElmer, Norwalk, CT) containing both antisense and sense primers (200 ng/tube) and amplified for 30 cycles by incubation at 958C for 1.5 min, 568C for 2 min, 728C for 4 min, and a final incubation at 728C for 5 min. The thermocycler 2400 (Perkin-Elmer) was used for both RT and PCR. After reaction was completed, 10 ml 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, ERa, ERb, and AR are shown in Figure 1. The PCR product (cDNA) for each gene was purified and cloned into pCR-script Amp SK(1) cloning vector using the procedures described in the pCR-Script Amp SK(1) 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

FIG. 1. Amplified PCR products for LHr, P450scc, StAR, P450c17, P450arom, ERa, ERb, and AR from total mRNA isolated from rat ovary (see Materials and Methods for details).

Both antisense and sense [ 35 S]UTP-labeled cRNA probes were transcribed from linearized cDNA templates using a transcription kit (Stratagene). The cRNA probes

ERb IN RAT OVARIAN GRANULOSA CELLS

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 2708C, 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 23 saline-sodium citrate (SSC: 13 SSC 5 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 23 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 3 107 cpm/ml. Hybridization was performed using 100 ml diluted probe/slide in a humidified oven at 558C for 20 h. After hybridization, slides were washed twice by shaking in 23 SSC (45–528C) for 15 min at room temperature and treated with RNase-A (50 mg/ml in 23 SSC) for 1 h at 378C. Slides were then washed at 558C in 23 SSC containing 0.1% b-mercaptoethanol (BME) for 15 min, 13 SSC/0.1% BME for 15 min, 13 SSC/50% formamide/0.1% BME for 30 min, and twice in 0.13 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, ERb, ERa, and AR and 3 days for enzymes and StAR at 48C. 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 (mm) 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 mm), small (101–275 mm), medium (276–450 mm), and large (451–850 mm) follicles. Very small follicles were primary follicles up to five layers of granulosa cells. When this class of follicles approached 100 mm 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 mm) and large (451–850 mm) follicles. From each block, 12 or more follicles (two or more follicles for each size group) were chosen to evaluate the expression of ERb 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].

1749

For each follicle, four fields at roughly 908 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 ERb mRNA expression, and the expression levels of other genes. Analyses were performed using SAS [35] and results reported as mean 6 SEM. RESULTS

Characteristics of Follicles

Morphologically, healthy follicles ranging in size from 40 to 850 mm, were classified as very small (40–100 mm, n 5 20), small (101–275 mm, n 5 12), medium (276–450 mm, n 5 12), and large (451–850 mm, n 5 12) follicles. Atretic follicles were divided into medium (276–450 mm, n 5 12), and large (451–850 mm, n 5 12) follicles. Detection of AR, ERa, and ERb mRNA in Rat Ovary by RT-PCR and In Situ Localization

Expression of AR, ERa, and ERb 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 ERa mRNA expression was localized in theca and interstitial stroma cells but not in granulosa cells (Fig. 2, C and D). Although both AR and ERa 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 ERb mRNA was localized in both granulosa and theca cells (Fig. 3). Expression levels of ERb mRNA in granulosa cells were stage specific (Fig. 3, A–N); therefore, the relative levels of ERb mRNA in granulosa cells were quantified. In theca cells, a low level of ERb mRNA was expressed in only some of the healthy medium and large follicles (Fig. 3, M–O). Thus, the relative intensity of ERb mRNA expression in theca cells was not quantified. 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).

1750

BAO ET AL.

FIG. 2. In situ localization of AR and ERa mRNA in cryosections of rat ovary. A, B) Brightfield and darkfield views of a cross section of ovary hybridized to AR antisense probe, showing hybridization signals in granulosa cells (G) and corpus luteum (CL). C, D) Brightfield and darkfield views of a cross section of ovary hybridized to ER antisense probe, showing hybridization signals in theca (T) and interstitial cells (IT). G, Granulosa cells; CL, corpus luteum; T, theca cells; IT, interstitial cells. Bar 5200 mm.

In addition, mRNAs for LHr, P450scc, P450arom, and StAR were also localized in luteal tissues (data not shown).

P450scc, and P450arom mRNA was low to undetectable in granulosa cells of atretic follicles (Fig. 5, B–D).

ERb mRNA Expression in Granulosa Cells

Changes in the Expression of LHr, P450scc, P450c17, and StAR mRNA in Theca Cells

In healthy follicles, mRNA for ERb was first detected in granulosa cells of very small follicles (Fig. 3, A and B). The expression of ERb 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 ERb mRNA further increased (P , 0.01) to the highest level in mediumsized 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 ERb mRNA than atretic follicles (Fig. 5A). In atretic follicles, a low level of ERb mRNA was detected in granulosa cells of large follicles but not in medium-sized follicles (Figs. 3, I–L, and 5A). 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,

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 mm 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 c FIG. 3. In situ hybridization of ERb mRNA in cryosections of rat follicles. A, B) Brightfield and darkfield views of very small follicles (arrows), 50 and 100 mm, respectively, showing specific low levels of hybridization in granulosa cells. C, D) Bright- and darkfield views of a small healthy follicle (235 mm) indicating intense hybridization in granulosa cells. E, F) Bright- and darkfield views of a medium healthy follicle (350 mm) indicating intense hybridization in granulosa cells. G, H) Bright- and darkfield views of a large healthy follicle (750 mm) indicating intense hybridization in granulosa cells. I, J) Bright- and darkfield views of a large early atretic follicle (700 mm) indicating reduced hybridization in granulosa cells. K, L) Bright- and darkfield view of a late atretic follicle (450 mm actual size) indicating no hybridization in granulosa cells. M, N) Bright- and darkfield views of a large follicle (750 mm); same follicle shown in G and H hybridized to the sense probe, showing no specific hybridization in either the theca or the granulosa cells. O) Magnified view of left bottom site of H showing low level hybridization in theca interna layers. G, Granulosa cells; T, theca layers. Bar 5 100 mm.

ERb IN RAT OVARIAN GRANULOSA CELLS

1751

1752

BAO ET AL.

FIG. 4. Representative photomicrographs of in situ localization of LHr, P450scc, StAR, P450arom, and P450c17 mRNAs in cryosections of rat ovarian follicles. A, B) Bright- and darkfield views of a large follicle showing specific hybridization of LHr mRNA in theca and granulosa cells. C, D) Brightand darkfield views of a large follicle showing specific hybridization of P450scc mRNA in theca and granulosa cells. E, F) Bright- and darkfield views of a large follicle showing specific hybridization of StAR mRNA in theca cells. G, H) Bright- and darkfield views of a large follicle indicating hybridization of P450arom mRNA in granulosa cells. I, J) Bright- and darkfield view of a large follicle indicating hybridization of P450c17 mRNA in theca cells. Bar 5 50 mm.

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 ERb mRNA in granulosa cells was strongly correlated with the expression of mRNAs for LHr (r 5 0.44; P , 0.01), P450scc (r 5 0.57; P , 0.01), P450c17 (r 5 0.46: P , 0.01), and StAR (r 5 0.45; P , 0.01) in theca cells. There was no correlation between expression of ERb mRNA and expression of mRNAs for LHr (r 5 0.21; P 5 0.12) and P450scc (r 5 0.21; P 5 0.10) in granulosa cells. A slight correlation was observed between expression of ERb and P450arom in granulosa cells (r 5 0.23; P 5 0.07). DISCUSSION

In the present study, changes in the expression of ERb 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 ERb and the expression of LHr, StAR, P450scc, and P450arom in granulosa cells was observed, the high level expression of ERb 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 E 2 may have a role in the initiation of the expression of mRNA for LHr and steroidogenic enzymes mediated via ERb. The role of E 2 in the ovary has been difficult to evaluate because the expression of ERa has not been demonstrated consistently in ovarian cells [20]. The cloning of ERb in the rat prostate and ovary suggested its possible role in estrogen action. Localization of ERb mRNA in the rat ovarian granulosa cells using in situ hybridization has been reported [21, 26]. Others have shown the expression of ERb 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 ERb mRNA in ovarian cells using in situ hybridization. In ad-

dition, stage-specific expression of ERb in granulosa cells of developing follicles was demonstrated for the first time. The increased expression of ERb 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 ERb 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 ERb 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 ERb 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 ERb over ERa in the rat ovary and the stage-specific expression of ERb in granulosa cells during follicular development imply a role for E 2 action on granulosa cell proliferation and differentiation via ERb. 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 E 2. It has been reported

ERb IN RAT OVARIAN GRANULOSA CELLS

1753

FIG. 5. Quantitative analysis of expression of mRNAs for ERb, LHr, P450scc, P450c17, P450arom, and StAR in rat ovarian granulosa and/or theca cells during follicular development. Intensity of hybridization was expressed as the percentage of pixels within a given marked area that was above a preset gray threshold level (see Materials and Methods for details). Bars carrying different letters differ within each panel (P , 0.05).

TABLE 2. Correlation among size and expression of mRNAs for ERb, LHr, StAR, P450scc, P450c17, and P450arom within healthy follicles.* Size Size ERb-gc LHr-tc LHr-gr StAR-tc P450scc-tc P450scc-gr P450c17-tc

ERb-gc

LHr-tc

LHr-gr

StAR-tc

P450scc-tc

P450scc-gr

P450c17-tc

P450arom-gc

0.14

0.64† 0.44†

0.65† 0.21 0.89†

0.50† 0.45† 0.80† 0.64†

0.50† 0.57† 0.85† 0.77† 0.69†

0.52† 0.21 0.77† 0.86† 0.57† 1.00†

0.58† 0.46† 0.91† 0.79† 0.76† 0.87† 0.76†

0.67† 0.23 0.92† 0.95† 0.72† 0.77† 0.83† 0.85†

* For analysis of correlation coefficients, all healthy follicles, categorized small, medium, and large were included. ERb-gc, ERb mRNA in granulosa cells; LHr-tc, LHr mRNA in theca cells; LHr-gr, LHr mRNA in granulosa cells; StAR-tc, StAR mRNA in theca cells; P450scc-tc, P450scc mRNA in theca cells; P450scc-gr, P450scc mRNA in granulosa cells; P450c17-tc, P450c17 mRNA in theca cells; P450arom-gc, P450arom mRNA in granulosa cells. † P , 0.01

1754

BAO ET AL.

earlier that E 2 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 E 2 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 ERb, 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 ERb mRNA in the theca cells was nondetectable. However, the expression of ERb 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 E 2 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 ERb, LHr, steroidogenic enzymes, and StAR. Theca cells associated with atretic follicles showed low level of expression of ERb, 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 ERb mRNA was observed in granulosa cells. The high expression of ERb mRNA with concomitant initiation of LHr, P450scc, and P450arom mRNA expression in granulosa cells of medium-sized follicles may signify a role for E 2 in follicular development. In addition, strong correlation between ERb 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. REFERENCES 1. Richards JS. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol Rev 1980; 60:51–89. 2. Nurst BS, Leslie KK. The role of estrogen in follicular development. Mol Hum Reprod 1997; 3:643–645. 3. Turner RT, Rigges BL, Spelsberg TC. Skeletal effects of estrogens. Endocr Rev 1994; 15:275–300. 4. Ciocca DR, Vargas-Roig LM. Estrogen receptors in human nontarget tissues: biological and clinical implications. Endocr Rev 1995; 16:35– 61. 5. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effect of estrogen. FASEB J 1996; 10:615–624. 6. Shupnik MA. Gonadotropin gene modulation by steroids and gonadotropin-releasing hormone. Biol Reprod 1996; 54:279–286. 7. Katzenellenbogen BS, Nardulli AM, Read LD. Estrogen regulation of proliferation and hormonal modulation of estrogen and progesterone

8. 9. 10. 11. 12. 13. 14. 15.

16.

17. 18.

19.

20. 21.

22. 23. 24. 25. 26. 27. 28. 29. 30.

31.

receptor biosynthesis and degradation in target cells. Prog Clin Biol Res 1990; 322:201–211. Roberts AJ, Skinner MK. Mesenchymal-epithelial cell interactions in the ovary: estrogen-induced theca cell steroidogenesis. Mol Cell Endocrinol 1990; 72:R1–R5. Drummond AE, Findlay JK. The role of estrogen in folliculogenesis. Mol Cell Endocrinol 1999; 151:57–64. Stocco DM, Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 1996; 17:221–244. Miller WL. Molecular biology of steroid hormone biosynthesis. Endocr Rev 1988; 9:295–318. Hillier SG, Whitelaw PF, Smyth CD. Follicular oestrogen synthesis: the ‘‘two-cell, two-gonadotrophin’’ model revisited. Mol Cell Endocrinol 1993; 100:51–54. Hsueh AJ, Adashi EY, Jones PB, Welsh TM Jr. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 1984; 5:76–127. Richards JS, Jahnsen T, Hedin L, Lifka J, Ratoosh S, Durica JM, Goldring NB. Ovarian follicular development: from physiology to molecular biology. Recent Prog Horm Res 1987; 43:231–270. Martel C, Labrie C, Coue¨t J, Dupont E, Trudel C, Luu-The V, Takahashi M, Pelletier G, Labrie F. Effects of human chorionic gonadotropin (hCG) and prolactin (PRL) on 3b-hydroxy-5-ene-steroid dehydrogenase/D5-D4 isomerase (3b-HSD) expression and activity in the rat ovary. Mol Cell Endocrinol 1990; 72:R7–R13. Segaloff DL, Wang H, Richards JS. Hormonal regulation of luteinizing hormone/chorionic gonadotropin receptor mRNA in rat ovarian cells during follicular development and luteinization. Mol Endocrinol 1990; 4:1856–1865. Hild-Petito S, Stouffer RL, Brenner RM. Immunocytochemical localization of estradiol and progesterone receptors in the monkey ovary throughout the menstrual cycle. Endocrinology 1988; 123:2896–2905. Chandrasekher YA, Melner MH, Naglla SR, Stouffer RL. Progesterone receptor, but not estrogen receptor, messenger ribonucleic acid is expressed in luteinizing granulosa cells and corpus luteum in rhesus monkeys. Endocrinology 1994; 135:307–314. Revelli A, Pacchioni D, Cassoni P, Bussolati G. In situ hybridization study of messenger RNA for estrogen receptor and immunohistochemical detection of estrogen and progesterone receptors in the human ovary. Gynecol Endocrinol 1996; 10:177–186. Byers M, Kuiper GG, Gustafsson JA, Park-Sarge OK. Estrogen receptor-b mRNA expression in rat ovary: down regulation by gonadotropins. Mol Endocrinol 1997; 11:172–182. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA. Comparison of ligand binding specificity and transcript tissue distribution of estrogen receptor alpha and beta. Endocrinology 1997; 138:863–870. Nilsson S, Kuiper G, Gustafsson TA. ERb: a novel estrogen receptor offers the potential for new drug development. Trends Endocrinol Metab 1998; 9:387–395. Saunders PTK, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ERb) in multiple rat tissues visualized by immunohistochemistry. J Endocrinol 1997; 154:R13–R16. Sar M, Welsch F. Differential expression of estrogen receptor-b and estrogen receptor-a in the rat ovary. Endocrinology 1999; 140:963– 971. Freeman ME. The neuroendocrine control of ovarian cycle of the rat. In: Knobil E, Neill IJ (eds.), The Physiology of Reproduction, vol. 2. New York: Raven Press; 1994: 613–758. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A 1996; 93:5925–5960. Koike S, Sakai M, Muramatsu M. Molecular cloning and characterization of rat estrogen receptor cDNA. Nucleic Acids Res 1987; 15: 2499–2513. Chang C, Kokontis J, Liao S. Structural analysis of complementary DNA and amino acid sequence of human and rat androgen receptors. Proc Natl Acad Sci U S A 1988; 85:7211–7215. Tsai-Morris CH, Buczko E, Wang W, Xie XZ, Dufau ML. Structural organization of rat luteinizing hormone (LH) receptor gene. J Biol Chem 1991; 266:11355–11359. Orly J, Eei Z, Greenberg NM, Richards JS. Tyrosine kinase inhibitor AG18 arrests follicle stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. Endocrinology 1994; 134:2336–2346. Nason TF, Han XG, Hall PF. Cyclic AMP regulates expression of rat

ERb IN RAT OVARIAN GRANULOSA CELLS

32.

33.

34.

35. 36.

37.

gene for steroid 17a-hydroxylase/C17–20 lyase P450 (CYP17) in rat leydig cells. Biochim Biophys Acta 1992; 1171:73–80. Clark BJ, Wells J, King SB, Stocco DM. The purification, cloning and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse leydig cells—characterization of steroidogenic acute regulatory protein (StAR). J Biol Chem 1994; 269:28314– 28322. Bao B, Garverick HA, Smith GW, Smith MF, Salfen BE, Youngquist RS. Changes in messenger RNA encoding LH receptor, cytochrome P450 side chain cleavage, and aromatase are associated with recruitment and selection of bovine ovarian follicles. Biol Reprod 1997; 56: 1158–1168. Bao B, Garverick HA, Smith GW, Smith MF, Salfen BE, Youngquist RS. Expression of messenger RNA encoding 3b-hydroxysteroid dehydrogenase/D5-D4 isomerase during recruitment and selection of bovine ovarian follicles: identification of dominant follicles by expression of 3b-HSD mRNA within the granulosa cell layer. Biol Reprod 1997; 56:1446–1473. SAS Institute Inc. SAS/STAT User’s Guide, version 6, vol. 2, 4th ed. Cary, NC: Statistical Analysis System Institute Inc.; 1989: 846. Camp TA, Rahal JO, Mayo KE. Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary. Mol Endocrinol 1991; 5: 1405–1417. Xu ZZ, Garverick HA, Smith GW, Smith MF, Hamilton SA, Youngquist RS. Expression of follicle stimulating hormone and luteinizing hormone receptor messenger ribonucleic acids in bovine follicles during the first follicular wave. Biol Reprod 1995; 53:951–957.

1755

38. Bao B, Garverick HA. Expression of steroidogenic enzyme and gonadotropin receptor genes in bovine follicles during ovarian follicular wave. A review. J Anim Sci 1998; 76:1903–1921. 39. Kim I, Greenwald GS. Effects of estrogens on follicular development and ovarian and uterine estrogen receptors in the immature rabbit, guinea pig and mouse. Endocrinol Jpn 1987; 34:871–878. 40. Chakravorty A, Mahesh VB, Mills TM. Regulation of follicular development by diethylstilbestrol in ovaries of immature rats. J Reprod Fertil 1991; 92:307–321. 41. Kawashima M, Greenwald GS. Comparison of follicular estrogen receptor in rat hamster and pig. Biol Reprod 1993; 47:172–179. 42. Wang XN, Greenwald GS. Hypophysectomy of the cyclic mouse. II. Effects on folliculogenesis, oocyte growth, and follicle-stimulating hormone and human chorionic gonadotropin receptors. Biol Reprod 1993; 48:585–594. 43. Wang XN, Greenwald GS. Hypophysectomy of the cyclic mouse. I. Effects on follicle-stimulating hormone (FSH) and luteinizing hormone on folliculogenesis, FSH and human chorionic gonadotropin receptors, and steroidogenesis. Biol Reprod 1993; 48:595–605. 44. Amsterdam A, Gold RS, Hosokawa K, Yoshida Y, Sasson R, Jung Y, Kotsuji F. Crosstalk among multiple signaling pathways controlling ovarian cell death. Trends Endocrinol Metab 1999; 10:255–262. 45. Goodman SB, Kugu K, Chen SH, Preutthipan S, Tilly JL, Dharmarajan AM. Estradiol mediated suppression of apoptosis in the rabbit corpus luteum is associated with a shift in expression of bc1-2 family members favoring cellular survival. Biol Reprod 1998; 59:820–827. 46. Peluso JJ. Placing progesterone in the apoptotic pathway. Trends Endocrinol Metab 1997; 8:261–266.