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Endocrinology, doi:10.1210/en.2004-0548
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Endocrinology Vol. 145, No. 10 4693-4702
Copyright © 2004 by The Endocrine Society

Formation of Cystic Ovarian Follicles Associated with Elevated Luteinizing Hormone Requires Estrogen Receptor-ß

John F. Couse, Mariana M. Yates, Ryan Sanford, Abraham Nyska, John H. Nilson and Kenneth S. Korach

Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology (J.F.C., M.M.Y., R.S., K.S.K.), and Laboratory of Experimental Pathology (A.N.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; Department of Environmental and Molecular Toxicology, North Carolina State University (J.F.C.), Raleigh, North Carolina 27695; and School of Molecular Biosciences, Washington State University (J.H.N.), Pullman, Washington 99164

Address all correspondence and requests for reprints to: Dr. Kenneth S. Korach, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, MD B3-02, Research Triangle Park, North Carolina 27709. E-mail: korach{at}niehs.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stringent regulation of LH secretion from the pituitary is vital to ovarian function in mammals. Two rodent models of LH hypersecretion are the transgenic LHß-C-terminal peptide (LHßCTP) and estrogen receptor-{alpha} (ER{alpha})-null ({alpha}ERKO) mice. Both exhibit ovarian phenotypes of chronic anovulation, cystic and hemorrhagic follicles, lack of corpora lutea, interstitial/stromal hyperplasia, and elevated plasma estradiol and testosterone. Because ERß is highly expressed in granulosa cells of the ovary, we hypothesized the intraovarian actions of ERß may be necessary for full manifestation of phenotypes associated with LH hyperstimulation. To address this question, we generated female mice that possess elevated LH, but lack ERß, by breeding the LHßCTP and ERß-null (ßERKO) mice. A comparison of LHßCTP, {alpha}ERKO, and ßERKOLHCTP females has allowed us to elucidate the contribution of each ER form to the pathologies and endocrinopathies that occur during chronic LH stimulation of the ovary. {alpha}ERKO ovaries respond to elevated LH by exhibiting an amplified steroidogenic pathway characteristic of the follicular stage of the ovarian cycle, whereas wild-typeLHCTP and ßERKOLHCTP females exhibit a steroidogenic profile more characteristic of the luteal stage. In addition, the hemorrhagic and cystic follicles of the LHßCTP and {alpha}ERKO ovaries require the intraovarian actions of ERß for manifestation, because they were lacking in the ßERKOLHCTP ovary. In turn, ectopic expression of the Leydig cell-specific enzyme, Hsd17b3, and male-like testosterone synthesis in the {alpha}ERKO ovary are unique to this genotype and are therefore the culmination of elevated LH and the loss of functional ER{alpha} within the ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LH IS OBLIGATORY to at least two major functions of the mammalian ovary: 1) thecal cell production of androstenedione, the requisite precursor to estradiol synthesis; and 2) ovulation and corpus luteum formation. Given the profound influence of LH on ovarian function, proper regulation of LH secretion from the anterior pituitary is vital to female fertility. This is achieved by the endocrine feedback loops that compose the hypothalamic-pituitary-gonadal axis. It is generally believed that GnRH is secreted from the hypothalamus in a pulsatile manner and flows via the portal vessels to the anterior pituitary, where it stimulates the synthesis and secretion of LH as well as the second gonadotropin, FSH. LH and FSH then act upon the ovary to induce oocyte maturation and the concomitant process of estradiol synthesis. As the ovarian cycle progresses toward ovulation, plasma estradiol levels rise and feedback upon the hypothalamus to negatively regulate GnRH secretion and thereby lower plasma LH levels.

The detrimental effects of improper LH regulation on ovarian function are often described in the context of human polycystic ovarian syndrome, currently defined as chronic anovulation and hyperandrogenemia that is often accompanied by elevated plasma LH and/or polycystic ovaries (1). In recent years, two laboratory animal models of chronic LH hypersecretion have also become available for study. The first is LHßCTP mice, a transgenic model intentionally designed to possess elevated plasma LH levels via a transgene consisting of the coding sequences for the bovine LHß (Lhb) subunit driven by the pituitary-specific bovine glycoprotein-{alpha} (Cga) promoter (2). In addition, sequences encoding the C-terminal peptide (CTP) of the human choriogonadotropin-ß subunit were fused in-frame to the 3' terminus of the bovine Lhb transgene to increase the serum half-life of transgenic LH (2). As a result, LHßCTP female mice possess basal plasma LH levels 5- to 10-fold those of nontransgenic littermates (2). The second model of LH hypersecretion is estrogen receptor-{alpha} (ER{alpha})-null ({alpha}ERKO) female mice, which possess plasma LH levels 3- to 5-fold above normal due to the loss of ER{alpha}/estradiol-mediated negative feedback on the hypothalamic-pituitary-gonadal axis (3, 4). Adult females from both lines exhibit strikingly similar ovarian phenotypes, characterized by chronic anovulation, cystic and hemorrhagic follicles, a lack of corpora lutea, interstitial/stromal hyperplasia, and granulosa cell tumors (4, 5, 6, 7, 8). Furthermore, each exhibits extraordinarily high levels of plasma estradiol and testosterone, both of which are ovarian in origin (4, 5). Additional observations in the {alpha}ERKO ovary include increased expression of the gonadotropin receptors and steroidogenic enzymes (4, 7), all of which are attributable to the elevated plasma LH because they return to normal after treatment with a GnRH antagonist (3, 4).

An enduring question in characterizing the ovarian phenotypes of the LHßCTP and {alpha}ERKO mice and the effects of LH hypersecretion on the ovary is the potential intraovarian role that ERß may play. ERß is clearly the more highly expressed ER form in the ovary relative to ER{alpha} and is localized to the granulosa cells of growing follicles, whereas the latter is predominantly expressed in the thecal and interstitial compartments (9, 10). Female mice lacking ERß (ßERKO) exhibit an attenuated ovulatory response to the LH analog, human chorionic gonadotropin (11). Based upon these findings, we hypothesize that ERß is involved in the granulosa cell response to LH. If true, ERß may also be necessary for the ovarian manifestations that follow chronic LH hyperstimulation. To address this question, we crossed the LHßCTP and ßERKO lines to generate mice that possess elevated LH, but lack ERß. Herein we compare the ovarian and endocrine phenotypes of ßERKOLHCTP female mice with those of their wild-type (WT)LHCTP littermates as well as nontransgenic controls. Also included are analyses of {alpha}ERKO ovarian phenotypes as a model of LH hyperstimulation in the absence of ER{alpha}. Compared with WTLHCTP and {alpha}ERKO females, ßERKOLHCTP females failed to exhibit cystic and hemorrhagic follicles and possessed a less robust increase in testosterone synthesis. In contrast, the lack of ERß had a minimal effect on other phenotypes associated with LH hypersecretion, such as increased steroidogenic enzyme expression in the ovary and elevated plasma estradiol and progesterone levels. Furthermore, LH-induced ectopic expression of the Hsd17b3 gene, which encodes a Leydig cell-specific enzyme, was unique to the {alpha}ERKO ovary and was not observed in WTLHCTP or ßERKOLHCTP ovaries.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of ßERKOLHCTP mice
Animals were maintained in plastic cages under a 12-h light, 12-h dark schedule in a temperature-controlled room (21–22 C) and fed NIH 31 mouse chow and fresh water ad libitum. The ßERKOLHCTP colony was initially established at Charles River Laboratories (Wilmington, MA) and after approximately 1 yr was transferred to our facility at NIEHS. To generate founder animals possessing both a disrupted Esr2 (ERß) allele and the LHßCTP transgene, LHßCTP-positive males (C57BL/6xCF-1) from the original colony at Case Western Reserve University (Cleveland, OH) were bred with Esr2+/– (C57BL/6x129SJ) females from our NIEHS colony. Because LHßCTP-positive females are infertile (2, 12) and Esr2–/– (ßERKO) females are subfertile (11), generation of ßERKOLHCTP (Esr2–/–LHCTP) females required breeding ßERKO heterozygous males possessing the LHßCTP transgene (Esr2+/– LHCTP) with Esr2+/females. All Esr1–/– ({alpha}ERKO) females used in this study were of the C57BL/6 strain obtained from our colony at Taconic Farms (Germantown, NY). All animals were genotyped by PCR on DNA extracted from tail biopsy using the Wizard SV 96 Genomic DNA extraction kit (Promega Corp., Madison, WI). PCR determination of Esr1 and Esr2 gene disruption was carried out as previously described (4). PCR detection of the LHßCTP transgene used the following primers to produce a 290-bp amplimer indicative of the transgene: forward, 5'-CTGGAACATCTCCATCCTTG; reverse, 5'-AAGGGCTGAAACAAGATAAGATAA. All PCR results were evaluated by agarose gel electrophoresis.

All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were preapproved by the NIEHS institutional animal care and use committee. The average age of female mice used in this study was 147 d (SD = 67 d). In those experiments using the GnRH antagonist, antide (Sigma-Aldrich Corp., St. Louis, MO), animals were treated with 60 µg antide or vehicle (20% propylene in 0.85% saline) in a 0.1-ml volume, sc, at 1200–1300 h every 48 h for a total of six treatments as previously described (3); tissues were collected 24 h after the final treatment. In those experiments involving testosterone treatment, animals were implanted sc with a single 21-d release pellet containing 5 mg testosterone (Innovative Research of America, Sarasota, FL), or placebo and tissues were collected 21 d later. All mice were euthanized by carbon dioxide asphyxiation, whole blood was collected from the inferior vena cava and heparinized, and the plasma was stored at –70 C until assayed. Ovaries intended for RNA extraction were excised, trimmed of surrounding tissue, and immediately frozen on dry ice, followed by storage at –70 C. Ovaries intended for histological evaluation were immediately fixed in ice-cold 10% buffered formalin (for paraffin embedding) or ice-cold 4% paraformaldehyde [for OTC (Miles, Inc., Elkhart, IN) embedding and frozen sections].

Histological evaluation of ovarian phenotypes
For pathological evaluation, formalin-fixed ovaries were embedded in paraffin, sectioned, and stained with hematoxylin and eosin according to standard laboratory protocols. Pathological evaluation was carried out on multiple sections per ovary while the examiner remained blind of genotype or treatment until all data were compiled. Frozen OTC-embedded ovaries were cryosectioned and mounted for staining with Oil Red-O (Sigma-Aldrich Corp.) according to the protocol described by Boon and Drijver (13). All photomicrographs were collected using a BX-50 microscope and OLY-750 camera (Olympus, New Hyde Park, NY).

RNA isolation and gene expression assays
Total RNA was isolated from frozen tissues using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. All RNA samples were rid of contaminating DNA using the DNA-free reagents (Ambion, Inc., Austin, TX) according to the manufacturer’s protocol, followed by normalization to a concentration of 0.2 µg/µl in ribonuclease-free water. A semiquantitative RT-PCR approach was then used to assess the level of gene expression as previously described (4). For each sample, 1 µg RNA was used in a 25-µl cDNA reaction using random hexamers and the Superscript cDNA synthesis system (Invitrogen Life Technologies) according to the manufacturer’s protocol. PCRs were then prepared using the equivalent of 1 µl cDNA/reaction for each respective primer set (Table 1Go) in a 15-µl total reaction volume using PCR reagents and platinum Taq polymerase (Invitrogen Life Technologies) as previously described (4). PCR was carried out in a Thermo Multiblock System (Hybaid, Franklin, MA) with the following cycling conditions: 95 C for 30 sec, 58 C for 45 sec, and 72 C for 30 sec for 26–36 cycles depending on the level of gene expression. Primers for ribosomal protein L7 (Rpl7) were included in all reactions as an internal positive control and for normalization. All samples were then electrophoresed on an agarose gel (2% NuSieve/0.7% SeaKem; BMA Bioproducts, Rockland, ME) in 1x Tris-borate and EDTA, followed by immobilization to BrightStar nylon membrane (Ambion, Inc.) using the Royal Genie Blotter (Idea Scientific, Minneapolis, MN). All blots were then probed with a nested oligo specific to sequences internal to the PCR primers (Table 1Go) that was 5'-radiolabeled with [{gamma}-33P]ATP (Amersham Biosciences, Little Chalfont, UK) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Hybridization of blots was carried out in Rapid-Hyb buffer (Amersham Biosciences) with more than 3 x 106 cpm probe/ml overnight in a rotisserie hybridization oven (Hybaid) at 42 C, followed by washing according to the manufacturer’s protocol. Final semiquantitative RT-PCR blots were exposed to a PhosphorImager screen, and the data were analyzed with a Storm 860 and accompanying ImageQuant software (Molecular Dynamics, Sunnyvale, CA).


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  		TABLE 1. Oligonucleotide primers and probes used for semiquantitative RTPCR

 
Hormone RIAs
All hormone assays were carried out on plasma collected from individual animals. Plasma LH was assessed in singlicate at 100 µl plasma/animal using the Biotrak 125I RIA kit (Amersham Biosciences). Plasma progesterone, estradiol, and testosterone were assayed in singlicate per animal on 25-, 200-, and 50-µl aliquots, respectively, using the Active Progesterone RIA, Ultra-Sensitive Estradiol Double-Antibody RIA, and Active Testosterone RIA kits (Diagnostic Systems Laboratories, Webster, TX) according to the manufacturer’s protocol. All final assays were quantified using a Multi-Prias 2 {gamma}-counter (Packard, Downers Grove, IL). To avoid interassay variation, assays for each hormone were carried out in a single set-up, with the exception of LH. The following parameters apply to the above RIAs (least detectable concentration, intraassay coefficient of variation, and interassay coefficient of variation): LH, 0.8 ng/ml, 9.2%, 15.6%; estradiol, 2.2 pg/ml; 10.6%; not applicable; and testosterone, 0.8 ng/ml; 5.7%; not applicable.

Statistical analysis
All data were analyzed for statistical significance (P < 0.05) using JMP software (SAS Institute, Inc., Cary, NC). Datasets were first tested for homoscedasticity of variance using Levene’s test, and if they failed, data were log-transformed before additional statistical analysis. All datasets were then evaluated by one-way ANOVA, followed by the Tukey-Kramer highest significant difference post hoc test when applicable.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of ßERKOLHCTP mice
Because LHßCTP females are infertile (2, 12), all breeding required males to serve as the genetic contributor of the LHßCTP transgene. Multiple breeding pairs consisting of an Esr2+/–LHCTP male and an Esr2+/– female yielded a total of 137 offspring that exhibited the expected Mendalian distribution for Esr2 genotypes (1:2:1; 37 wild-type, 64 heterozygous, and 36 ßERKO) and the expected ratio of animals possessing the LHßCTP transgene (1:1; 73 negative and 64 positive). These data indicate independent segregation of the Esr2 gene and the LHßCTP transgene. No bias in sex ratio was observed. Eventually, additional breeding pairs consisting of transgenic ßERKO homozygous (Esr2–/–LHCTP) males and Esr2+/– females were included to increase the ratio of ßERKOLHCTP offspring.

Plasma hormone levels
WTLHCTP and ßERKOLHCTP females exhibited equally elevated plasma LH levels (Fig. 1Go), indicating that a lack of functional ERß had no influence on expression of the LHßCTP transgene or maintenance of elevated plasma LH levels. Also shown in Fig. 1Go are the average plasma levels for progesterone, testosterone, and estradiol for each of the four genotypes. All three gonadal steroids were significantly elevated in the WTLHCTP and ßERKOLHCTP females relative to their nontransgenic controls. However, plasma testosterone levels in the ßERKOLHCTP, although elevated vs. nontransgenic ßERKO, were significantly lower than those exhibited by WTLHCTP.



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  		FIG. 1. WTLHCTP and ßERKOLHCTP females exhibit increased plasma LH and sex steroid levels. Shown are the average (±SEM) plasma hormone levels for each of the four genotypes. Sample numbers were as follows (LH, progesterone, testosterone, and estradiol): WT control, 7, 2, 19, and 20; WTLHCTP, 12, 15, 29, and 29; ßERKO, 11, 2, 22, and 23; and ßERKOLHCTP, 9, 6, 15, and 16. Statistical analysis of progesterone levels was not performed because of the low sample number in the control groups. a, P < 0.05 vs. nontransgenic control of the same Esr2 (ERß) genotype; b, P < 0.05 vs. WTLHCTP.

 
Ovarian morphology and pathology
Our evaluation of ovaries from each of the four genotypes included an initial assessment of total ovarian weight and gross appearance. This was followed by histological evaluation of the interstitium/stroma compartments; the number of developing, atretic, and hemorrhagic follicles; the number of corpora lutea; and the presence of granulosa cell hyperplasia or tumors. These data are summarized in Table 2Go and illustrated in Figs. 2Go and 3Go. The estrous cycle of each of the animals was not determined before necropsy and tissue collection.


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  		TABLE 2. Summary of pathology observed in WT, WTLHCTP, ßERKO, and ßERKOLHCTP ovaries

 


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  		FIG. 2. ERß is required for the development of hemorrhagic and cystic follicles in the ovary during chronic LH stimulation. Shown are photographs of whole ovaries from WT (A), WTLHCTP (B), ßERKO (C), and ßERKOLHCTP (D) adult females. Note the dramatic increase in ovarian size when comparing WTLHCTP with the WT control as well as the multiple hemorrhagic and cystic follicles (indicated by arrows) in the former. ßERKO ovaries were grossly normal in appearance, although slightly smaller than the WT. ßERKOLHCTP ovaries were increased in size compared with the ßERKO, however not to the extent seen in WTLHCTP. Also note the absence of hemorrhagic and cystic follicles in the ßERKOLHCTP ovary. Shown are low power (x10) photomicrographs of ovaries from WT (E), WTLHCTP (F), ßERKO (G), and ßERKOLHCTP (H) adult females. Once again note the enlarged size and hemorrhagic follicle in the WTLHCTP ovary. ßERKO and ßERKOLHCTP ovaries exhibited some growing follicles and luteinized interstitial cell hyperplasia, but no hemorrhagic or cystic follicles.

 


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  		FIG. 3. WTLHCTP, ßERKO, and ßERKOLHCTP ovaries all exhibit luteinized interstitial cell hyperplasia. Shown is a representative control ovary (A), a WTLHCTP ovary (B) with grade 3 luteinized interstitial cell hyperplasia, a WTLHCTP ovary (C) with grade 4 luteinized interstitial cell hyperplasia (all at x2 magnification), and an affected portion of a WTLHCTP ovary (D; x20). Also shown is a representative ßERKOLHCTP ovary exhibiting grade 4 luteinized interstitial cell hyperplasia (E, x2; F, x20). G and H, WTLHCTP (G) and ßERKOLHCTP(H) ovaries stained with Oil Red-O for visualization of intracellular lipid deposits (stained red). I, Stacked bar graph shows the incidence of luteinized interstitial cell hyperplasia per grade for each of the four genotypes. Sample numbers were as follows: WT control, 41; WTLHCTP, 36; ßERKO, 31; and ßERKOLHCTP, 22.

 
Ovaries from nontransgenic WT females were largely unremarkable, with more than 70% exhibiting no abnormalities. In contrast, ovaries from adult WTLHCTP females exhibited multiple pathologies, similar to earlier descriptions of the LHßCTP model (2, 12). These included a significant increase in ovarian weight compared with nontransgenic controls (68.2 vs. 13.7 mg; P < 0.01) and the invariable presence of hemorrhagic and cystic follicles (81% vs. 2.5%; Fig. 2Go, compare A and B). In addition, WTLHCTP ovaries exhibited an increased incidence and severity of luteinized interstitial cell hyperplasia, a reduced number of healthy developing follicles, an increased number of atretic follicles, and granulosa cell luteinization (Table 2Go and Fig. 2Go, compare E and F). Luteinized interstitial cell hyperplasia in WTLHCTP ovaries consisted of diffuse or nodular collections of large cells exhibiting vacuoles and a foamy or eosinophilic cytoplasm (Fig. 3FGo). This phenotype was scored on a scale of 1–4, taking into consideration the extent and size of the cell aggregates. At lower grades (1 and 2), the cell collections were generally diffuse and limited to the medulla, whereas cells of the higher grades (3 and 4) exhibited a more nodular growth pattern and extended toward the cortex to occupy a larger proportion of the ovary. Less than 20% of nontransgenic WT ovaries exhibited luteinized interstitial cell hyperplasia, all of which was grade 3 or less. In contrast, almost 90% of the WTLHCTP ovaries exhibited luteinized interstitial cell hyperplasia, 80% of which was grade 4. As shown in Fig. 3GGo, Oil Red-O staining of WTLHCTP ovaries confirmed that the interstitial cytoplasmic vacuolation observed in the formalin-fixed sections was due to intracellular lipid droplets.

In contrast to nontransgenic WT ovaries, more than 90% of nontransgenic ßERKO ovaries exhibited abnormalities, the most prevalent of which were luteinized interstitial cell hyperplasia (77%), absence of corpora lutea (77%), reduced number of developing follicles (50%), and increased number of atretic follicles (71%; Table 2Go). Interestingly, the overall frequency of these abnormalities did not differ in the ßERKOLHCTP ovaries, although the proportion of ovaries exhibiting grade 4 luteinized interstitial cell hyperplasia rose from 10% in ßERKO to 60% in ßERKOLHCTP animals (Fig. 3IGo). Oil Red-O staining of ßERKOLHCTP ovaries also confirmed the presence of intracellular lipid droplets in the interstitial cell population of the ovary (Fig. 3HGo). However, ßERKOLHCTP females failed to exhibit three particular LH-induced abnormalities of the ovary that were prominent in WTLHCTP. First, average ovarian weight in the ßERKOLHCTP group was significantly increased compared with that in nontransgenic ßERKO littermates, but this increase was less than that in WTLHCTP animals (31.3 vs. 68.2 mg; P < 0.01; Table 2Go and Fig. 2Go, compare B and D). Secondly, hemorrhagic and cystic follicles were observed in only 18% (n = 4) of the ßERKOLHCTP females compared with 81% (n = 30) of the WTLHCTP animals (P < 0.001). As shown in Fig. 2DGo, ovaries from ßERKOLHCTP mice were of abnormal size and color, but undoubtedly lacked the hemorrhagic follicles characteristic of WTLHCTP ovaries. Finally, the incidence of granulosa cell hyperplasia was significantly reduced in ßERKOLHCTP (4.5%) compared with WTLHCTP (32%; P < 0.01).

Benign granulosa cell tumors were more prevalent in WTLHCTP and ßERKOLHCTP ovaries vs. nontransgenic controls of each respective genotype (Table 2Go). These tumors were distinguished by nodules of granulosa cells with moderate to scanty cytoplasm dependent on the degree of luteinization; were arranged in a follicular, solid, or trabecular pattern; and possessed few Call-Exner bodies. Interestingly, the only two malignant granulosa cell tumors observed among all four genotypes were in ßERKOLHCTP females, each of which was 240 d of age. These tumors were judged malignant due to increased atypia, an infiltrative growth pattern, and the presence of necrosis and hemorrhage (14).

The question arose as to whether the above-described ovarian phenotypes, such as the hemorrhagic and cystic follicles could be directly attributed to elevated LH or perhaps to the secondary increase in testosterone in the ovary. This is especially relevant in the ßERKOLHCTP, which exhibited plasma testosterone levels that were elevated, but lower than those in WTLHCTP animals (Fig. 1Go) and less than half those reported in {alpha}ERKO females (4). To address this issue, a separate experiment was conducted in which nontransgenic WT and ßERKO females as well as WTLHCTP and ßERKOLHCTP were implanted with slow release testosterone pellets for a period of 21 d. The average plasma testosterone level at necropsy among all testosterone pellet-implanted females was 9.5 ± 1.2 ng/ml, well above the already elevated levels innate to WTLHCTP and ßERKOLHCTP females. Nonetheless, none of the LHßCTP-associated ovarian phenotypes were observed in the testosterone-treated WT or ßERKO females, nor did the increased testosterone level exacerbate the incidence or appearance of LH-associated phenotypes in WTLHCTP or ßERKOLHCTP ovaries.

Ovarian gene expression
We previously reported that chronically elevated LH in the {alpha}ERKO female leads to increased expression of the gonadotropin receptors and the enzymes necessary for steroidogenesis in the ovary (4). To further characterize the effects of elevated LH on the steroidogenic capacity of WT vs. ßERKO ovaries, similar assays for expression of the following genes were carried out: FSH receptor (Fshr), LH receptor (Lhcgr), cytochrome P450 side-chain cleavage (Cyp11a), cytochrome P450 17{alpha}-hydroxylase/C17–20 lyase (Cyp17), 3ß-hydroxysteroid dehydrogenase/{Delta}5,{Delta}4-isomerase (Hsd3b1), cytochrome P450 aromatase (Cyp19), and 17ß-hydroxysteroid dehydrogenase types I, III, and VII (Hsd17b1, Hsd17b3, and Hsd17b7). Ovaries from {alpha}ERKO females were included in these assays to represent an environment of elevated LH in the absence of ER{alpha}. It is important to reiterate that {alpha}ERKO females retain normal expression of ERß in the ovary (7, 15). Furthermore, because we have previously shown that treatment of {alpha}ERKO females with a GnRH antagonist (antide) effectively reduces plasma LH levels to normal and subsequently returns aberrant expression of steroidogenic enzymes to wild-type levels (4), these experiments were repeated to include ßERKO females. Ultimately, this allows for comparison of WT and ßERKO ovaries in the presence of normal gonadotropin levels (control), decreased LH (antide), or increased LH (LHßCTP), as well as {alpha}ERKO ovaries in the presence of elevated LH (control), which is innate to this model, or decreased LH (antide).

As shown in Fig. 4Go, there were no significant differences in ovarian expression of Cyp11a, Hsd3b1, or Hsd17b1 among the different genotypes or treatments. Fshr levels also remained relatively unchanged in WT and ßERKO ovaries regardless of LH levels, but were elevated in untreated {alpha}ERKO ovaries and reduced after antide treatment, as previously reported (3). In contrast, Lhcgr and Cyp17 expression were increased in all genotypes possessing elevated LH, i.e. WTLHCTP, ßERKOLHCTP, and untreated {alpha}ERKO. The strong influence of elevated LH on Lhcgr and Cyp17 expression was illustrated by the dramatic decrease in the expression of each after antide treatment in {alpha}ERKO mice. Interestingly, Cyp17 expression in nontransgenic ßERKO ovaries was also elevated relative to that in WT ovaries. In contrast, elevated LH did not dramatically affect Cyp19 expression unless associated with a lack of ER{alpha}, as only {alpha}ERKO ovaries exhibited increased Cyp19 levels. The expression of Hsd17b7, a luteal cell-specific gene, was significantly reduced in nontransgenic ßERKO ovaries relative to WT, correlating with the paucity of corpora lutea in these ovaries (Table 2Go). However, both WTLHCTP and ßERKOLHCTP ovaries exhibited a dramatic increase in Hsd17b7 expression that was statistically significant in the latter. Because the estrous cycle of individual nontransgenic WT females was not evaluated before tissue collection, ovaries from these females may have possessed corpora lutea at the time of collection, thereby exaggerating the average Hsd17b7 levels in this group and prohibiting statistical significance compared with WTLHCTP. Interestingly, elevated LH levels in {alpha}ERKO animals did not lead to increased Hsd17b7 expression in the ovary.



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  		FIG. 4. ER genotype determines the ovarian gene expression pattern that occurs during chronic LH stimulation. Steroidogenic enzyme and gonadotropin receptor gene expressions were evaluated in ovaries collected from untreated (Control) WT (wt), ßERKO, and {alpha}ERKO females; GnRH antagonist (antide)-treated WT (wt), ßERKO, and {alpha}ERKO females; and transgenic (LHCTP) WT (wt) and ßERKO females. Left, Representative gene expression assays for the steroidogenic enzymes: Cyp11a, Hsd3b1, Cyp17, Cyp19, Hsd17b1, Hsd17b7, and Hsd17b3, and the gonadotropin receptor genes, Fshr and Lhcgr. Right, Bar graphs illustrating the average (±SEM) fold difference vs. WT control for those four genes that exhibited significant changes in expression among the different genotypes; Cyp17, Cyp19, Hsd17b7, and Lhcgr. All genes were normalized to Rpl7 expression as described in Materials and Methods. All data were generated from samples collected from individual animals (n ≥ 4 for all groups). a, P < 0.05 vs. WT control; b, P < 0.05 vs. nontransgenic control of the same ER genotype; c, P < 0.05 vs. WTLHCTP.

 
Finally, {alpha}ERKO females exhibit elevated levels of plasma testosterone and ectopic expression of the Hsd17b3 gene in the ovary (4). The Hsd17b3 gene is considered to be exclusively expressed in the Leydig cells of the testis under positive regulation of LH and encodes the enzyme necessary for the conversion of androstenedione to testosterone (16, 17, 18, 19). Ectopic expression of Hsd17b3 in the {alpha}ERKO ovary is directly related to elevated LH levels, because expression is abolished by antide treatment (Fig. 4Go). However, increased plasma LH in the WTLHCTP or ßERKOLHCTP females did not lead to Hsd17b3 expression in the ovary (Fig. 4Go). Furthermore, extended treatment of WTLHCTP and ßERKOLHCTP with testosterone did not induce Hsd17b3 expression in the ovary (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ßERKOLHCTP females exhibited elevated plasma LH levels comparable to those in the WTLHCTP group, indicating that ERß is not necessary for LHßCTP transgene expression. Consequently, any moderation in LH-induced ovarian and endocrine phenotypes in the ßERKOLHCTP female is presumably due to the lack of ERß and not to lower LH levels. Therefore, our successful generation of ßERKOLHCTP mice and the previously existing {alpha}ERKO model has allowed us to evaluate the contribution of each respective ER form to the ovarian phenotypes that occur during chronic LH hyperstimulation.

The studies reported herein clearly indicate that ovarian ER status influences the steroidogenic profile during chronic LH stimulation. Plasma estradiol levels were equally elevated in WTLHCTP and ßERKOLHCTP females, but were still lower than those reported in {alpha}ERKO, which range from 100 to more than 500 pg/ml (4). Although this disparity may be due to variation among background strains, we previously reported that mice lacking both ER forms ({alpha}ßERKO) also exhibit relatively normal plasma estradiol levels despite possessing elevated LH (4). The divergence in plasma estradiol levels observed among the genotypes was mirrored in the ovarian profiles of Cyp19 expression, in which {alpha}ERKO ovaries exhibited a 5-fold increase relative to WTLHCTP and ßERKOLHCTP. Therefore, the {alpha}ERKO environment of elevated LH and normal FSH (4) appears conducive to those ERß actions that may augment Cyp19 expression and estradiol synthesis in granulosa cells, congruent with reports of synergistic regulation of Cyp19 expression by estradiol and FSH (20, 21). The lower than expected Cyp19 expression in WTLHCTP ovaries despite the presence of ERß and elevated LH may be due to decreased plasma FSH levels, as Mikola et al. (22) reported in the LHßCTP female.

Despite a lack of increased Cyp19 expression in the ovary, WTLHCTP and ßERKOLHCTP females still exhibited above normal levels of plasma estradiol. Estradiol synthesis in granulosa cells requires both CYP19 to convert androstenedione to estrone as well as 17ß-hydroxysteroid dehydrogenase type I (HSD17B I) to convert estrone to estradiol (23, 24). Assays for Hsd17b1 expression in the ovaries of each respective ER and LHßCTP genotype indicated relatively normal expression, ruling this out as a cause of increased estradiol synthesis. However, similar assays for 17ß-hydroxysteroid dehydrogenase type VII (Hsd17b7), which also converts estrone to estradiol, indicated elevated expression in WTLHCTP and ßERKOLHCTP ovaries, but decreased levels in {alpha}ERKO. Hsd17b7 and Hsd17b1 are distinctly expressed during the ovarian cycle in the mouse, such that the former is unique to the corpus luteum of pregnancy, whereas the latter is limited to the granulosa cells of growing follicles (25). Therefore, our findings indicate that LH hyperstimulation leads to different steroidogenic cell types in the ovary depending on the functional ER forms present. In the absence of functional ER{alpha}, as in {alpha}ERKO, a predominantly granulosa cell population responds to chronic LH stimulation by producing excess estradiol via increased Cyp19 expression. In the presence of ER{alpha}, as in WTLHCTP and ßERKOLHCTP, LH hyperstimulation leads to an interstitial luteal-like cell population that synthesizes estradiol by means of increased Hsd17b7 expression. This is supported by a greater than 80% incidence of luteinized interstitial cell hyperplasia among WTLHCTP and ßERKOLHCTP ovaries. Additional indications that LH hyperstimulation in WTLHCTP and ßERKOLHCTP ovaries leads to a luteal-like cell population are the severely elevated plasma progesterone levels in WTLHCTP and ßERKOLHCTP females, but not in {alpha}ERKO females (8), and the considerable intracellular lipid content of interstitial cells of the WTLHCTP and ßERKOLHCTP ovaries, as indicated by Oil Red-O staining. Furthermore, these data agree with the report of prolonged luteal life span in the ovaries of LHßCTP females after mating with vasectomized males (6).

This disparity in the steroidogenic pathway used for estradiol synthesis between the {alpha}ERKO and WTLHCTP or ßERKOLHCTP ovaries may also be influenced by differences in the levels of circulating prolactin (PRL) between the genotypes. PRL is well known to facilitate the positive actions of LH on luteal cell steroidogenesis (26) as well as induce ER{alpha} expression in the corpus luteum (27). Although not assessed herein, Kero et al. (28) found that LHßCTP females show a 2-fold increase in plasma PRL. Therefore, it is plausible that spontaneous formation of luteal cells and subsequent Hsd17b7 expression in WTLHCTP and ßERKOLHCTP ovaries are due to the combined actions of increased LH and PRL on the ovary. This phenotype may be prevented in the {alpha}ERKO female, because the loss of ER{alpha} in the anterior pituitary results in significantly reduced Prl gene expression (4) and plasma PRL levels (29), but the loss of ERß has no effect (4). In fact, Bocchinfuso et al. (29) demonstrated that when plasma PRL levels in the {alpha}ERKO female are increased to normal by grafting a WT (ER{alpha}-positive) pituitary under the renal capsule, enormous luteal structures are formed in the ovary, and plasma progesterone levels increase to more than 5-fold of those in similarly treated WT females (29).

In addition to estradiol and progesterone, the plasma testosterone profile among the genotypes revealed another striking disparity. Plasma testosterone levels in WTLHCTP females were significantly elevated compared with those in WT control females, in agreement with previous descriptions of the LHßCTP female (12, 22). Plasma testosterone levels in ßERKOLHCTP females were also elevated above the ßERKO control level, yet were significantly lower than WTLHCTP levels. In contrast, {alpha}ERKO females exhibit plasma testosterone levels that are more than 2- and 8-fold the WTLHCTP and ßERKOLHCTP levels, respectively (4). This extraordinary capacity of {alpha}ERKO ovaries to synthesize testosterone is almost certainly due to ectopic expression of the Hsd17b3 gene, as previously described to be unique to {alpha}ERKO and {alpha}ßERKO ovaries among the ERKO lines (4). Hsd17b3 encodes the enzyme (17ß-HSD III) necessary to convert androstenedione to testosterone (30) and is considered to be exclusive to Leydig cells of the testis, where it is primarily regulated by LH (16, 17, 18, 19). Indeed, chronically elevated LH is the stimulus for Hsd17b3 expression in the {alpha}ERKO ovary, because we have previously shown (and repeated herein) that reducing LH via prolonged treatment with a GnRH antagonist completely abolishes any detectable expression (4). However, if ectopic expression of Hsd17b3 in the ovary is merely due to chronic LH stimulation, WTLHCTP and ßERKOLHCTP ovaries would be expected to exhibit similar expression. The lack of comparable Hsd17b3 expression in the WTLHCTP or ßERKOLHCTP ovaries indicates that this phenotype is unique to the {alpha}ERKO and is therefore the culmination of LH hyperstimulation and a lack of intraovarian ER{alpha} function. We previously reported that flutamide, an antiandrogen, is partially effective in reducing Hsd17b3 expression in the {alpha}ERKO ovary, suggesting that increased testosterone may also contribute to ectopic Hsd17b3 expression (4). However, in the current study we carried out the complementary experiment of prolonged testosterone treatment of WT, ßERKO, WTLHCTP, and ßERKOLHCTP females and found no induction of Hsd17b3 expression in the treated ovaries (data not shown). We can only speculate that Leydig-like cells may be present in the {alpha}ERKO ovary, because definitive data to indicate the ovarian cell type expressing Hsd17b3 is unavailable at this time. Nonetheless, this hypothesis is supported by recent reports of Leydig-like cells (31) and HSD17B3 expression (32) in the ovaries of Cyp19-null (ArKO) mice that also possess elevated LH and testosterone and presumably lack ER{alpha} action due to the absence of estradiol.

In the absence of Hsd17b3 expression in WTLHCTP and ßERKOLHCTP ovaries, why do these females still exhibit elevated testosterone levels relative to WT and ßERKO controls? A likely possibility is that LH hyperstimulation leads to increased thecal cell production of androgen precursors. In fact, Kero et al. (28) reported that LHßCTP females possess elevated plasma levels of androstenedione, the product of CYP17 activity and the immediate precursor of testosterone. Our finding of increased Cyp17 expression in WTLHCTP and ßERKOLHCTP ovaries also supports a phenotype of increased androstenedione synthesis. Therefore, it is possible that a milieu of increased androstenedione may provide for testosterone synthesis even in the absence of 17ß-HSD III, but via the estrogenic enzyme, 17ß-HSD I, in which the murine form is reported to convert androstenedione to testosterone as efficiently as estrone to estradiol (33). Therefore, we conclude that the greater capacity of the {alpha}ERKO ovary to synthesize testosterone is due to more efficient conversion of androstenedione via 17ß-HSD III, whereas the lower level of testosterone in WTLHCTP and ßERKOLHCTP females is due to the less efficient 17ß-HSD I.

One of the most striking phenotypes common to the LHßCTP and {alpha}ERKO ovaries is the invariable presence of hemorrhagic and cystic follicles that present at approximately 40 d of age and increase in severity thereafter (3, 7). The cystic follicles of the {alpha}ERKO, as previously described (3, 7), and the WTLHCTP described herein possess a mural granulosa layer one to several cells thick that exhibit indications of apoptosis, an enormous antrum often filled with blood and immune cells, a degenerating ovum if visible at all, and a hypertrophied theca. Prevention of this phenotype in the {alpha}ERKO ovary by reducing plasma LH levels via GnRH antagonist treatment (3) supports the hypothesis that the cystic and hemorrhagic follicles are a manifestation of chronic LH stimulation and not of the loss of intraovarian ER{alpha}. However, given the robust expression of ERß in granulosa cells, we hypothesized that ßERKOLHCTP females may be less susceptible to the formation of LH-induced cystic follicles than their WTLHCTP and {alpha}ERKO counterparts. Indeed, ßERKOLHCTP females exhibited a dramatic decrease in the incidence of cystic and hemorrhagic follicles vs. WTLHCTP, indicating that the intraovarian actions of ERß must contribute to cyst formation. The lack of hemorrhagic follicles in ßERKOLHCTP was not due to insufficient LH-receptor as Lhcgr expression was elevated in the ßERKOLHCTP ovary. A role for ERß in cyst formation is also supported by a decreased incidence of cystic and hemorrhagic follicles in the ovaries of {alpha}ßERKO females, which also possess elevated plasma LH but lack functional ERß as well (34). Prolonged testosterone treatment of ßERKOLHCTP as well as wild-type and ßERKO controls did not lead to cystic and hemorrhagic follicles, indicating this phenotype to be a direct effect of LH rather than due to LH-induced increases in ovarian testosterone.

Excluding the Lhcgr-null mouse for obvious reasons (35, 36), two other models that possess elevated plasma LH but fail to develop cystic and hemorrhagic follicles in the ovary are Fshb-null (FSHß subunit) (37) and Fshr-null (38, 39) mice, indicating that FSH signaling must also be obligatory to cystic follicle formation. Prolonged treatment of hypophysectomized rats with both FSH and LH also produces an increased number of cystic follicles of greater severity vs. either gonadotropin alone (40). Although categorical studies of FSH responsiveness in the ßERKO ovary have not been carried out, plasma FSH (4) and ovarian Fshr expression in the ßERKO female are relatively normal.

In light of our findings that ovarian cyst formation during LH hyperstimulation involves ERß, one may speculate that Cyp19-null (ArKO) females may also be less susceptible due to the absence of estradiol. ArKO females possess elevated levels of both LH and FSH and exhibit cystic and hemorrhagic follicles in the ovaries by 16–18 wk of age (31), a significant delay relative to WTLHCTP and {alpha}ERKO. ArKO ovaries continue to exhibit cystic and hemorrhagic follicles even when maintained on a soy-free diet, presumably void of exogenous estrogens that may act as ERß agonists (31). The possibility of ligand-independent actions of ERß notwithstanding, we must also consider that ERß may respond to endogenous ligands other than estradiol. Weihua et al. (41) recently proposed that 5{alpha}-androstane-3ß,17ß-diol, a metabolite of 5{alpha}-dihydrotestosterone, is the activating ligand for ERß in rat prostate cells. Therefore, 5{alpha}-androstane-3ß,17ß-diol synthesis in the ArKO ovary could provide for ligand-activated ERß, which in the context of elevated LH and FSH could lead to the formation of hemorrhagic and cystic follicles in this model.

Luteinized interstitial cell hyperplasia was a common ovarian phenotype in WTLHCTP and ßERKOLHCTP females. The most severe grade was recorded in 80% and 60% of the WTLHCTP and ßERKOLHCTP ovaries, respectively. These findings in WTLHCTP are consistent with previous descriptions of the LHßCTP ovary (12). Perhaps more surprising was that control ßERKO ovaries also exhibited a considerable degree of luteinized interstitial cell hyperplasia that qualitatively appeared no different from that in WTLHCTP and ßERKOLHCTP ovaries, although only 10% were of grade 4. Luteinized interstitial cell hyperplasia and hypertrophy in the rodent ovary are considered to be consequences of increased follicular atresia, in which the oocyte and surrounding granulosa cells die via apoptosis and the thecal cells survive and remain steroidogenic (42, 43). Therefore, the luteinized interstitial cell hyperplasia observed in the WTLHCTP, ßERKOLHCTP, and ßERKO ovaries is probably the result of comparable rates of follicle atresia and chronic anovulation (11, 12, 44). In the former two models, this is obviously due to chronically elevated LH, whereas the cause in the latter is less clear, but may be due to the loss of LH pulsatility (8).

In summary, our evaluation of WTLHCTP, ßERKOLHCTP, and {alpha}ERKO females has clarified the contribution of each ER form to the pathologies and endocrinopathies that occur during chronic LH stimulation of the ovary. The function of both ER forms is undoubtedly required for full manifestation of the effects of LH hyperstimulation. The primary defect in {alpha}ERKO appears be the lack of ER{alpha} in the hypothalamo-pituitary axis, which leads to elevated plasma LH but reduced PRL, resulting in an anovulatory ovary that exhibits an amplified steroidogenic pathway most characteristic of the follicular stage of the ovarian cycle. In contrast, WTLHCTP and ßERKOLHCTP females exhibit a heightened, but more balanced, LH to PRL ratio, leading to increased synthesis of both estradiol and progesterone, a phenotype more characteristic of the luteal stage of the ovarian cycle. In addition, two LH-induced phenotypes of the ovary were found to be dependent upon the actions of one particular ER form. The large hemorrhagic and cystic follicles so characteristic of WTLHCTP and {alpha}ERKO ovaries require the intraovarian actions of ERß for manifestation, because they were lacking in the ßERKOLHCTP ovary. In turn, LH-induced expression of Leydig cell-specific Hsd17b3 was unique to the ER{alpha}-null ovary.


   Footnotes
 
Abbreviations: CTP, C-Terminal peptide; ER, estrogen receptor; Hsd or HSD, hydroxysteroid dehydrogenase; PRL, prolactin; WT, wild-type.

Received April 29, 2004.

Accepted for publication June 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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