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. 2013 Apr 11;154(6):2174–2187. doi: 10.1210/en.2012-2256

The Absence of ER-β Results in Altered Gene Expression in Ovarian Granulosa Cells Isolated From In Vivo Preovulatory Follicles

April K Binder 1,*, Karina F Rodriguez 1,*, Katherine J Hamilton 1, Patricia S Stockton 1, Casey E Reed 1, Kenneth S Korach 1,✉
PMCID: PMC3740481  PMID: 23580569

Abstract

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-β is highly expressed in ovarian granulosa cells, and mice lacking ER-β are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in vitro conditions. Herein we isolated preovulatory granulosa cells from wild-type (WT) and ERβ-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of pregnant mare serum gonadotropin (mimicking FSH) and pregnant mare serum gonadotropin/human chorionic gonadotropin (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERβ-null ovaries. ERβ-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERβ in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ERβ-null granulosa cells; however, novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggest that granulosa cells from ERβ-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.

The follicle is the functional unit of the ovary and is essential for female reproduction. In response to signals that are not fully understood, primordial follicles are recruited for growth as granulosa cells proliferate, thus increasing from a single layer of cells to multiple layers surrounding the growing oocyte (1). Granulosa cells in secondary follicles differentiate and begin to express FSH receptor (Fshr) and aromatase (Cyp19a1), resulting in local estrogen production. Estrogen production is essential for follicle growth and ovulation, and synergism between FSH and estrogen results in further differentiation of granulosa cells into preovulatory granulosa cells, which express LH receptor (LHR; officially Lhcgr) (2, 3). The expression of Lhcgr allows the follicle to respond to the ovulatory surge of LH from the pituitary (reviewed in Reference 4). Successful ovulation is the result of orchestrated differentiation of the granulosa and theca layers, and the expression of critical genes at specific times and requires coordinated regulation of multiple signaling pathways (reviewed in Reference 5).

Estrogen receptor (ER)-β is highly expressed in the granulosa cells of the ovary, whereas ERα is expressed predominately in the theca cell compartment (6, 7). The ovaries of ERβ knockout mice (βERKO), although slightly smaller than those of the wild type (WT), appear normal with follicles at all stages of folliculogenesis, yet the mice are subfertile due to inefficient ovulation (8). After treatment with gonadotropins, βERKO ovaries exhibit reduced estradiol synthesis and fail to acquire sufficient LHR/Lhcgr in granulosa cells of preovulatory follicles (8, 9). This failure of βERKO preovulatory follicles to differentiate leads to a poor ovulatory response after a bolus of human chorionic gonadotropin (hCG) in vivo (8) or follicles cultured in vitro (9). In addition, preovulatory βERKO follicles express lower levels of LHR/Lhcgr and aromatase (Cyp19a1) compared with WT preovulatory follicles (9, 10).

Expression of ERβ is required for the synergistic effect of estradiol with FSH to induce LHR during folliculogenesis (8–10). Interestingly, transcripts that are thought to be expressed only in the theca cell compartment, such as ERα and Cyp17, appear to be aberrantly expressed in the βERKO preovulatory follicle, with ERα found to be 2.5-fold higher than WT, whereas Cyp17 is 2.5-fold lower than WT (10). Interpretation of these results, although significant, is difficult due to the heterogeneous cell types present in follicles. Many publications have successfully used granulosa cell culture to study the responses of these cells to both FSH and LH stimulus (11–13). Culture of isolated granulosa cells, although extremely useful to obtain highly enriched preparation of granulosa cells, originates from cells obtained from ovaries that are mechanically dissociated, resulting in cells from follicles at different stages of folliculogenesis. Furthermore, exposure of these cells in culture may alter gene expression such that it no longer reflects that of an intact follicle.

To compare the transcriptional profiles of theca and granulosa cells in preovulatory follicles of WT and ERβ-null granulosa cells, we used laser capture microdissection (LCM) to obtain pure preparations of each cellular compartment. The purpose of this study was to expand on the current knowledge regarding the role of ERβ in mediating regulation of genes involved in differentiation of granulosa cells in response to FSH and to the ovulatory surge of LH. Herein we focused on the gene expression profile of granulosa cells obtained exclusively from in vivo preovulatory follicles and the role of ERβ in response to gonadotropins. To our knowledge, this is the first time that such stringent conditions have been used for this purpose and the data obtained uncover novel targets for further study of the role of ERβ in folliculogenesis and ovulation.

Materials and Methods

Animals and treatments

All animal procedures were approved by the National Institutes of Health Animals Care and Use Committee and were performed in accordance with an approved National Institute of Environmental Health Sciences animal study proposal. In this study we used the Ex3βERKO mouse that has a deletion of exon 3 rather than the βERKO mouse, which has a Neo insertion in the same region. The strategy used to generate the Esr2 exon 3-null (Ex3βERKO) animals was similar to that used to generate the Ex3αERKO mouse (14) (see Supplemental Materials and Methods, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org).

Treatment and tissue collections

Ovaries of WT and Ex3βERKO prepubertal females (21 days) were collected 48 hours after treatment (ip injections) with 5 IU of pregnant mare serum gonadotropin (PMSG) (Sigma-Aldrich, St Louis, Missouri) or after 48 hours of PMSG followed by 4 hours of 5 IU hCG (Sigma-Aldrich). Ovaries were immediately embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, California), and blocks were stored at −80°C for LCM.

Laser capture microdissection

LCM was carried out on frozen ovarian sections cut using a cryostat (5 μm). Sections were stained using 1% cresyl violet acetate for 20 seconds. Granulosa cells were collected by LCM from WT and Ex3βERKO preovulatory follicles using an MMI CellCut laser-capture microscope (Molecular Machines and Industries, Haslett, Michigan). Only cells from large antral follicles were collected, and care was taken to avoid oocytes and cumulus cells, enriching for mural granulosa cells. The maximum amount of time allowed for LCM was 20 minutes. After collection, 50 μL of extraction buffer from the Arcturus Picopure RNA isolation kit (Applied Biosystems, Foster City, California) was added to each sample. The samples were incubated for 30 minutes at 42°C, centrifuged at 800 × g for 2 minutes and frozen at −80°C until used.

RNA extraction and microarray analysis

Samples in extraction buffer were thawed and pooled within genotype. Captured cells from 2-3 animals of the same genotype were pooled, with at least 3 pools collected per cell type. RNA quality and concentration were determined using N Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, California) and Qubit fluorometer (Life Technologies Co, Grand Island, New York).

Gene expression analysis was conducted using Affymetrix Mouse Genome 430 2.0 GeneChip arrays (Affymetrix, Santa Clara, California). Five nanograms of total RNA were amplified as directed in the NuGEN Ovation Pico WTA system protocol (San Carlos, California) and labeled using the NuGEN Encore Biotin module. Five micrograms of amplified biotin-amplified RNAs were fragmented and hybridized to each array for 18 hours at 45°C in a rotating hybridization chamber. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip hybridization, wash, and stain kit and user manual following protocol FS450-0004. Arrays were scanned in an Affymetrix Scanner 3000 and data were obtained using the GeneChip Command Console software (version 1.1). To verify consistency between pools, the average correlation coefficient (r) for each array/treatment group was calculated. Within the PMSG group, the r for the WT samples was 0.89 and for the βERKO samples was 0.85. For the hCG-treated groups, the r for the WT samples was 0.89, whereas the r for the βERKO samples was 0.87. All data have been deposited in the Gene Expression Omnibus (accession number GSE44651).

Data preprocessing, normalization, and error modeling was performed with the Rosetta Resolver system (version 7.2; Kirkland, Washington). To identify differentially expressed probes, an ANOVA was used to determine whether there was a statistical difference between the means of the WT and Ex3βERKO samples. Specifically, an ANOVA using the Benjamini-Hochberg false discovery rate multiple test correction was performed. Hierarchical clustering was performed using Entrez gene identifications with the agglomerative clustering algorithm and the cosine correlation similarity metric. In addition, a 2-fold change cutoff, P < .001 and a signal intensity of at least 100 in at least 1 sample were used. Pathway analysis was performed using ingenuity pathway analysis (IPA; Ingenuity Systems Inc, Redwood City, California).

Granulosa cell isolation and confirmation studies

Enriched populations of granulosa cells were isolated from pooled ovaries of 5-7 mice as previously described (15). Cells were frozen at −80°C until RNA isolation using TRIzol (Invitrogen, Carlsbad, California) according to the manufacturer's protocol. RNA quality and concentration were examined by spectrophotometry, and cDNA was reverse transcribed using the SuperScript first-strand synthesis system (Invitrogen). Primers used are shown in Supplemental Table 1, and data are shown as a ratio of gene of interest to pL7 expression as described previously (16).

Results

Isolation of cells from WT and Ex3βERKO ovaries using LCM

The Ex3βERKO ovary exhibits a significant decrease in the expression of mRNA for Esr2 compared with control ovaries (data not shown) and lacks expression of the ERβ protein assessed by immunohistochemistry, whereas the WT shows positive staining in granulosa cells (Supplemental Figure 1A). Ex3βERKO females are subfertile and do not respond to exogenous gonadotropin treatments to induce ovulation (Supplemental Figure 1, B and C), confirming that the lack of ERβ protein results in the phenotypes described previously (7, 8).

To obtain preovulatory granulosa cells, we used LCM to isolate target cells without disturbing the oocyte or cumulus cells as shown in the Figure 1 before (A) and after (B) isolation. Theca cells were also isolated from large preovulatory follicles (Figure 1C). To confirm isolation of pure cell populations, real-time PCR was performed using primers specific for Cyp19a1 (granulosa cells) or Cyp17 (theca cells) (Supplemental Figure 2). Cyp19a1 was confirmed to be granulosa cell specific, whereas Cyp17 has specific expression in theca cells demonstrating isolation of pure cell populations. Esr1 (ERα) is expressed predominately in theca cells in WT ovaries; however, increased Esr1 expression is observed in ERβ-null granulosa cells. Esr1 is also increased in the theca cells from ERβ-null ovaries, suggesting that loss of one ER leads to an increase in the other, implying compensatory mechanisms may exist. Moreover, Lhcgr expression is reduced in both granulosa and theca cells in the ERβ-null ovary (Supplemental Figure 2), as previously reported in whole ovary (8) and cultured follicles (10).

Figure 1.

Figure 1.

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Use of LCM to isolate preovulatory granulosa and theca cells. Immature WT and Ex3βERKO ovaries were frozen in optimum cutting temperature after exogenous gonadotropin stimulation. A, Representative ovarian section stained with 1% cresyl violet acetate prior to LCM isolation. B, Pure populations of mural granulosa cells from large preovulatory follicles were isolated. C, Theca cells were also isolated from large preovulatory follicles.

Attenuated response to FSH in preovulatory granulosa cells

Isolation of theca cells using LCM resulted in small amounts of RNA that were not of high enough quality to be used for subsequent microarray analysis; therefore, we chose to focus on granulosa cells. Granulosa cells were collected from WT or ERβ-null large antral follicles 48 hours after PMSG stimulation (Figure 1, A and B). After microarray analysis, 449 probes were differentially expressed between WT and ERβ-null granulosa cells (P < .001, > 2-fold, signal intensity > 100) and mapped to 414 annotated genes via IPA. Most of these genes show decreased expression in the ERβ-null granulosa cells (n = 342), whereas a small subset show increased expression (n = 66), suggesting that ERβ contributes to both the activation and repression of target genes downstream of FSHR activation.

The 414 differentially expressed genes included genes previously reported to be altered in βERKO granulosa cells such as Lhcgr (−3.6-fold), Comp (−28.6-fold), Lrp11 (−10.5-fold), Car14 (−4.2-fold), Mro (−3.2-fold) (15), and Nld2 (+8.4-fold) (17). The expression of Cyp19a1 (−1.9-fold; P = 8.7 × 10−6), Inhba (−1.3-fold; P = 6.8 × 10−6), and Prkar2b (−1.3-fold; P = 1 × 10−5) whose expression was previously reported to be down-regulated in ERβ-null granulosa cells (15) was found to be reduced; however, these genes were not included in the list generated for further analysis because the fold change was less than 2. Forty genes that show altered expression in ERβ-null granulosa cells can be found in Table 1 (20 genes with increased expression) and Table 2 (20 genes with decreased expression). Most the genes (n = 304) identified in our study have not previously been associated with ERβ expression. The complete list of genes (Supplemental Table 2) was used for the functional analyses described below.

Table 1.

Top 20 Genes With Increased Expression in Ex3βERKO vs WT PMSG Primed Preovulatory Granulosa Cells

Primary Sequence Name Sequence Description Fold Change ANOVA P Value
Tigd3 Tigger transposable element derived 3 19.7 1.4E-06
Trim61 Tripartite motif-containing 61 12.3 .00088
Ak7 Adenylate kinase 7 10.4 6.88E-08
Pou5f1 POU domain, class 5, transcription factor 1 9.5 .00028
Arnt2 Aryl hydrocarbon receptor nuclear translocator 2 8.9 .00022
Nid2 Nidogen 2 8.4 1.87E-14
Tmem182 Transmembrane protein 182 7.7 1.87E-14
Fam110c Family with sequence similarity 110, member C 7.2 .00003
Eya4 Eyes absent 4 homolog (Drosophila) 6.8 3.83E-06
Gm15698 Transcription elongation factor B (SIII), polypeptide 2 pseudogene 6.5 1.44E-11
Akap12 A kinase (PRKA) anchor protein (gravin) 12 5.8 2.03E-06
Kazald1 Kazal-type serine peptidase inhibitor domain 1 5.7 1.87E-14
Mbl2 Mannose-binding lectin (protein C) 2 5.0 1.94E-09
Fbxw16 F-box and WD-40 domain protein 16 4.5 .00097
Oog1 Oogenesin 1 4.3 .00072
Oosp1 Oocyte secreted protein 1 4.3 7.23E-08
Cnnm1 Cyclin M1 4.3 5.68E-12
Ooep Oocyte expressed protein homolog (dog) 4.3 .00006
Fbxw24 F-box and WD-40 domain protein 24 4.2 .00006
Ptgis Prostaglandin I2 (prostacyclin) synthase 4.2 1.87E-14
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Table 2.

Top 20 Genes With Reduced Expression in Ex3βERKO vs WT PMSG Primed Preovulatory Granulosa Cells

Primary Sequence Name Sequence Description Fold Change ANOVA P Value
Sfrp4 Secreted frizzled-related protein 4 −84.6 3.96E-07
Cadm3 Cell adhesion molecule 3 −32.8 1.13E-06
Comp Cartilage oligomeric matrix protein −28.6 1.95E-13
Perp PERP, TP53 apoptosis effector −28.0 1.87E-14
Ddah1 Dimethylarginine dimethylaminohydrolase 1 −16.0 1.87E-14
Susd4 Sushi domain containing 4 −15.9 1.53E-09
Thbs4 Thrombospondin 4 −10.6 1.87E-14
Lrp11 Low-density lipoprotein receptor-related protein 11 −10.5 1.87E-14
Chst15 Carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15 −10.4 8.97E-10
Epha5 Eph receptor A5 −10.0 2.09E-12
Arrdc4 Arrestin domain containing 4 −9.8 3.38E-10
Reln Reelin −9.7 0.00002
Ank1 Ankyrin 1, erythroid −9.1 5.68E-09
Me2 NAD-dependent malic enzyme, mitochondrial-like /// malic enzyme 2, NAD (+)-dependent, mitochondrial −9.0 2.01E-11
Ces1d Carboxylesterase 1D −8.4 1.64E-10
Mpp7 Membrane protein, palmitoylated7 (MAGUK p55 subfamily member 7) −8.3 1.86E-06
Cbs Cystathionine β-synthase −7.2 1.87E-14
Scgb3a1 Secretoglobin, family 3A, member 1 −7.2 .00011
Cyp11a1 Cytochrome P450, family 11, subfamily a, polypeptide 1 −6.3 7.28E-06
Cdh2 Cdh2, CDHN, Ncad, N-cadherin −6.2 1.87E-14
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The large number of genes that show altered expression validates previous reports that loss of ERβ leads to an attenuated FSH response (15). Analysis of the 414 genes by IPA demonstrates that the top molecular and cellular functions altered include lipid metabolism, small molecule biochemistry, and vitamin and mineral metabolism. Table 3 lists the top 5 molecular and cellular functions altered as well as the number of molecules identified as changed in our study that are included in these networks. The largest numbers of molecules identified in the analysis include functions that are important in metabolism and small molecule biochemistry. The inclusion of these functional groups is not surprising, given the reduced steroid synthesis and altered expression of steroidogenic enzymes observed in the ERβ-null ovary (10, 15). Evaluation of the top upstream regulators for these differentially expressed genes was performed in IPA. The data predicts the inhibition of inhibin A targets (14 genes) including Sfrp4, Lrp11 (Figure 2), and Comp (18); however, the expression of inhibin genes (Inha, Inhba, and Inhbb) are not reduced less than 2-fold. From IPA analysis, targets of progesterone (25 genes), prolactin (14 genes), and β-estradiol (53 genes) are implicated as top upstream regulators (Supplemental Table 3) of genes differentially expressed after PMSG stimulation in preovulatory granulosa cells.

Table 3.

Top Molecular and Cellular Functions Altered After PMSG Stimulation

Molecular and Cellular Functions P Value Molecules, n
Cellular compromise 1.35E-04 to 2.42E-02 9
Lipid metabolism 1.41E-04 to 2.42E-02 37
Small molecule biochemistry 1.41E-04 to 2.42E-02 54
Vitamin and mineral metabolism 1.41E-04 to 2.42E-02 20
Cellular movement 1.73E-04 to 2.42E-02 37
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Figure 2.

Figure 2.

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Confirmation of genes showing altered expression after PMSG stimulation. Granulosa cells were mechanically isolated from WT and Ex3βERKO ovaries 48 hours after PMSG stimulation. RNA was isolated and reverse transcribed, and real-time PCR was performed with primers specific for Akap12 (A), Pou5f1 (B), Arnt2 (C), Ahr (D), Trim61 (E), Lrp11 (F), Fam110c (G), Perp (H), Runx2 (I), and Reln (J). Data shown are a ratio of the gene of interest to Pl7 (used as a housekeeping gene), each ran in duplicate. Data were analyzed by an unpaired Student's t test. *, P < .05; **, P < 0,01; ***, P < .001.

Attenuated response to LH in preovulatory granulosa cells

Expression of ERβ is necessary for ovulation in vivo [Supplemental Figure 1C and Reference 8], and cultured follicles in vitro (9, 10). Although an attenuated response to PMSG has been described above, we were also interested in the in vivo response of ERβ-null granulosa cells isolated from the few large preovulatory follicles found in ERβ-null ovaries to an ovulatory signal. To this end, mice were treated with PMSG for 48 hours, injected with hCG, and ovaries collected for LCM 4 hours later.

From our microarray analysis, 1363 probes are differentially expressed in ERβ-null granulosa cells compared with WT cells (P < .001, intensity at least 100, > 2-fold difference) and mapped to 1258 genes using IPA. After the hCG treatment, there were similar numbers of genes that showed either increased (n = 576) or decreased (n = 665) expression in the ERβ-null preovulatory granulosa cells. Initial LH signaling in granulosa cells includes increased expression of several well-characterized genes, previously reported to be reduced in in vitro βERKO preovulatory follicles including Ptgs2 (−6.5-fold), Timp1 (−4.7-fold), Sult1e1 (−3.7-fold), and Pgr (−3 fold) (10) or increased in cultured granulosa cells, including Col11a1 (+9-fold) (15), are also observed in in vivo preovulatory granulosa cells. Additionally, expression of known LH targets including Areg (−6 fold) and Ereg (−3.2-fold) were found to be reduced in our microarray data set. Forty genes that show altered expression in ERβ-null granulosa cells downstream of LH can be found in Table 4 (20 genes with increased expression) and Table 5 (20 genes with decreased expression). The complete list of genes differentially expressed (Supplemental Table 4) were used for the functional analyses described below.

Table 4.

Top 20 Genes With Increased Expression in Ex3βERKO vs WT PMSG/hCG-Stimulated Preovulatory Granulosa Cells

Primary Sequence Name Sequence Description Fold Change ANOVA P Value
Scube1 Signal peptide, CUB domain, EGF-like 1 14.2 1.56E-14
Limch1 LIM and calponin homology domains 1 13.4 .00013
8030463A06Rik RIKEN cDNA 8030463A06 gene 13.1 7.88E-07
St8sia1 ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 12.7 6.59E-13
1110032F04Rik RIKEN cDNA 1110032F04 gene 12.7 1.564E-14
Cml3 Camello-like 3 12.2 .00015
Odz4 L0940E08–3 NIA mouse newborn kidney cDNA library (long) Mus musculus cDNA clone NIA:L0940E08 IMAGE:30003895 3′, mRNA sequence. 12.1 .00014
Rassf2 Ras association (RalGDS/AF-6) domain family member 2 11.4 .00006
Bcl2l1 BCL2-like 1 11.1 3.35E-11
Tdrd5 Tudor domain containing 5 11.0 1.56E-14
Kcnj3 Potassium inwardly rectifying channel, subfamily J, member 3 10.3 1.56E-14
Cbfa2t3 Core-binding factor, runt domain, α subunit 2, translocated to, 3 (human) 9.6 5.51E-11
Cnnm1 Cyclin M1 9.6 0.00002
Col11a1 Collagen, type XI, α1 9.0 0.00005
Mycl1 V-myc myelocytomatosis viral oncogene homolog 1, lung carcinoma derived (avian) 8.8 3.92E-10
Dnahc11 Dynein, axonemal, heavy chain 11 8.5 3.06E-14
Hpgd Hydroxyprostaglandin dehydrogenase 15 (NAD) 8.4 1.68E-12
Kcnq5 Potassium voltage-gated channel, subfamily Q, member 5 8.2 1.56E-14
Kcnt2 Potassium channel, subfamily T, member 2 8.2 .00002
Cldn11 Claudin 11 8.0 .00003
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Table 5.

Top 20 genes With Reduced Expression in Ex3βERKO vs. WT PMSG/hCG-Stimulated Preovulatory Granulosa Cells

Primary Sequence Name Sequence Description Fold Change ANOVA P Value
Abcb1b ATP-binding cassette, subfamily B (MDR/TAP), member 1B −31.3 .00029
Slc5a7 Solute carrier family 5 (choline transporter), member 7 −23.6 3.16E-09
Hsd17b7 Hydroxysteroid (17β) dehydrogenase 7 −20.9 .00003
Enpp2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 −18.3 6.52E-09
Cd93 CD93 antigen −15.9 .00001
Ptgfr Prostaglandin F receptor −15.3 4.08E-07
Enpp3 Ectonucleotide pyrophosphatase/phosphodiesterase 3 −13.3 3.26E-13
Emb Embigin −12.8 1.28E-10
Lrrn3 Leucine-rich repeat protein 3, neuronal −12.4 1.56E-14
Tchhl1 Trichohyalin-like 1 −12.1 4.19E-08
Ggct γ-Glutamyl cyclotransferase −11.5 3.36E-06
Elavl2 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B) −11.0 1.03E-10
Retn Resistin −10.6 3.30E-11
Gm2a GM2 ganglioside activator protein −10.6 .00044
Srxn1 Sulfiredoxin 1 homolog (S. cerevisiae) −10.0 1.17E-08
Adh7 Alcohol dehydrogenase 7 (class IV), μ- or σ-polypeptide −9.8 2.162E-11
Epha3 Eph receptor A3 −9.8 .00065
St8sia4 ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 4 −9.4 1.56E-06
Lrp1b Low density lipoprotein-related Protein 1B (deleted in tumors) −9.3 4.57E-09
Tspan13 Tetraspanin 13 −9.2 .0002
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A large number of genes show differential expression between ERβ-null and WT preovulatory granulosa cells after hCG stimulation. Additionally, similar numbers of genes show changes in expression, which cannot be explained solely due to decreased Lhcgr/LHR expression. IPA analysis indicates that the top molecular and cellular functions altered include those important for metabolism and cellular development. Table 6 lists the top 5 molecular and cellular functions altered and the number of molecules differentially expressed that are included in these networks. The largest numbers of molecules identified in the analysis include functions that are important in metabolism and small molecule biochemistry, similar to what is found after PMSG stimulation. In addition to genes important for steroid biosynthesis, genes that are involved in cholesterol homeostasis and biosynthesis, lipid synthesis, and various other metabolic processes are differentially expressed compared with WT.

Table 6.

Top Molecular and Cellular Functions Altered After hCG Stimulation

Main Molecular and Cellular Functions P Value Molecules, n
Cell death and survival 1.56E-07 to 8.13E-03 272
Lipid metabolism 4.08E-07 to 8.15E-03 122
Small molecule biochemistry 4.08E-07 to 8.15E-03 154
Vitamin and mineral metabolism 4.08E-07 to 8.01E-03 65
Cellular development 6.67E-07 to 7.91E-03 265
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Examination of upstream regulators via IPA suggested that the top upstream pathway affected was inhibition of progesterone signaling (30 genes) due to decreased expression of progesterone receptor targets including Klf4 (−2-fold), F3 (−3.2-fold), and Ier3 (−3-fold), which have been identified as LH-responsive genes in granulosa cells (19). Genes downstream of the CCAAT/enhancer binding proteins (CEBP)-α and -β are also predicted to be inhibited in ERβ-null granulosa cells. CEBPα targets (45 genes) and CEBPβ targets (36 genes) including Ptgfr (−15.3-fold), Prlr (−4.3-fold), and Abcb1b (−31-fold) show decreased expression. CEBPA/B are essential genes for rodent ovulation and luteinization (20), and due to the reduced ovulation observed in ERβ-null mice, it is not surprising that these pathways are predicted to be inhibited. Specificity protein-1 (43 genes), sterol regulatory element-binding protein-F1 (24 genes), and sterol regulatory element-binding protein-F2 (16 genes) are other transcription factors with predicted inhibition (Supplemental Table 5).

Confirmation of genes differentially expressed

Further confirmation using real-time PCR was done using isolated granulosa cells to compare expression of transcripts in vivo 48 hours after PMSG stimulation. Several genes were selected from Tables 1 and 2 to include genes that had either increased (Table 1) or decreased (Table 2) expression in ERβ-null preovulatory granulosa cells relative to WT. We examined genes that have been previously been described in the ovary [Akap12 (21), Trim61 (22), Fam110c (23), Gab2 (24), Ahr (25), Lrp11 (18), and Sfrp4 (26)] as well as those that have not been examined in the rodent ovary to our knowledge (Arnt2, Perp, and Reln) and a gene thought to be germ cell specific (Pou5f1) (27).

Expression of Esr2 was absent in ERβ-null granulosa cells, whereas Cyp19a1 expression was also reduced compared with WT granulosa cells, confirming previous reports in granulosa cells (8) and cultured follicles (10) (data not shown). Genes that were implicated in microarray data analysis as having reduced expression in ERβ-null cells were confirmed to be reduced in isolated granulosa cells from ERβ-null ovaries compared with WT after PMSG stimulation (Figure 2). This includes genes that have been implicated in cellular signaling pathways such as Lrp11 and Runx2. Genes that were implicated as having increased expression in ERβ-null granulosa cells were also confirmed, including Akap12, Arnt2, Trim61, Fam110c, and Pou5f1. Although most of the genes examined reconfirmed as expected, 3 genes show dissimilar responses, including Ahr (no difference), Perp (−1.5-fold compared with −28-fold, P = .16), and Reln (+1.5-fold compared with −2-fold, P > .01).

Further studies were done on isolated granulosa cells 4 hours after hCG treatment to confirm the findings of our microarray. Genes that have previously been reported to be LH responsive in the ovary were selected, including Hpgd (28), Fam110c (23), Abcb1b, Ptgfr (20), Srxn1 (also called Npn3), and Klf4 (19). Genes not previously reported to be expressed in the ovary (Scube1, Rassf2, and Megf10) were also examined. Genes that were implicated in our microarray data analysis as having increased expression were confirmed in isolated granulosa cells from ERβ-null ovaries compared with WT after PMSG stimulation (Figure 3). This includes genes that have been implicated in cellular signaling pathways, Hpgd and Rassf2. Genes that were implicated as having decreased expression were also confirmed, including Fam110c, Abcb1b, Klf4, Srxn1, Ptgfr, and Megf10. Additionally, Scube1 was found to have no difference in expression in isolated granulosa cells compared with 14-fold increase in LCM-isolated granulosa cells. Although most genes reconfirmed with our data sets, several genes did not. These differences suggest that cells isolated mechanically from the ovaries have different characteristics from those isolated using LCM, possibly due to the heterogeneity of the cell preparations.

Figure 3.

Figure 3.

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Confirmation of genes showing altered expression after PMSG/hCG stimulation. Granulosa cells were mechanically isolated from WT and Ex3βERKO ovaries after 48 hours of PMSG and 4 hours of hCG stimulation. RNA was isolated and reverse transcribed, and real-time PCR was performed with primers specific for Hpgd (A), Abcb1b (B), Rassf2 (C), Klf4 (D), Scube1 (E), Srxn1 (F), Fam110c (G), and Megf10 (H). Data shown are a ratio of the gene of interest to Pl7 (used as a housekeeping gene), each ran in duplicate. Data were analyzed by an unpaired Student's t test. ***, P < .001.

Discussion

We have previously shown that expression of ERβ is essential for appropriate expression of Lhcgr/LHR, which results in the reduced ovulatory capacity of the ERβ-null females and altered signaling mechanisms (8, 10). One caveat of our studies is that ERβ-null follicles will occasionally ovulate (both in vitro and in vivo) and are phenotypically indistinguishable from those that fail to respond to the ovulatory signal. When granulosa or follicle culture techniques are used, the final sample is an enriched preparation that also includes cells from various compartments (theca, granulosa, and oocytes). Because we are interested in the response of preovulatory follicles to FSH (PMSG) and LH (hCG), we obtained purified preovulatory granulosa cells using LCM. Previous work indicated that the βERKO ovary has a reduced number of large antral follicles after PMSG stimulation (9). Herein we have focused on LCM-isolated preovulatory granulosa cells after FSH and LH stimulation to examine the gene expression differences in granulosa cells found in follicles matched for stage of follicular development.

Loss of ERβ leads to altered steroidogenesis as evidenced by reduced estradiol (E2) accumulation in the serum of βERKO mice after PMSG stimulation in vivo (8) and in βERKO follicles cultured in vitro (9). Three of the top 5 molecular and cellular functions found to be altered after PMSG stimulation in ERβ-null preovulatory granulosa cells (Table 3) include genes that are involved in metabolic processes and small molecule biochemistry. In agreement with reduced E2 production, genes important for steroidogenesis, steroid metabolism, and secretion show altered expression in ERβ-null preovulatory granulosa cells. This includes genes important for estrogen metabolism including Cyp11a1 (29), Cyp1b1 (30), and Ahr (31). ERβ-null mice have the capacity of secreting the same maximal concentration of E2; however, the timing of steroid production is altered. Four hours after hCG stimulation, E2 metabolic genes were identified as having abnormal expression including Cyp19a1, Sult1e1, and Timp1 as described previously (10). Additional E2 metabolic genes, Adcyap1 and Runx2, were found to have reduced expression in the ERβ-null granulosa cells in this study. Adenylate cyclase activating polypeptide 1 (Adcyap1), a hypothalamic peptide regulates cAMP accumulation and E2 synthesis in rat granulosa cells (32), and Runt-related transcription factor 2 (Runx2) regulates E2 synthesis in skeletal muscle and was recently reported to be an LH target in granulosa cells (20). Both of these genes have altered expression in our analysis (Supplemental Table 2 and Figure 2), suggesting that altered E2 production is due to the misregulation of multiple genes in the ovaries of mice lacking ERβ.

LH stimulation leads to ovulation and terminal granulosa cell differentiation into luteal cells that secrete progesterone (P4) (reviewed in Reference 33), and genes that are implicated in P4 production are predicted by IPA to be decreased in both PMSG and hCG stimulated ERβ-null granulosa cells (Supplemental Table 3). Preovulatory granulosa cells in the absence of ERβ have decreased expression of genes implicated in P4 synthesis including Prlr (34), Smad2 (35), and Gdf9 (36) after PMSG treatment. Genes important in P4 synthesis also have altered expression after LH treatment in the ERβ-null ovary, including Prlr (34) and Hpdg (28). Hpgd is a catabolic enzyme that controls the first step of prostaglandin inactivation and was reported to be inhibited by hCG in rat granulosa cell cultures (28). Increased expression of Hpgd, Ptges, and reduced expression of Ptgs2, and 2 receptors, Ptger4 and Ptgfr, indicates that ERβ-null granulosa cells may also have altered prostaglandin metabolism. Prostaglandins are important for cumulus-oocyte complex (COC) expansion, which is necessary for ovulation and fertility (37). Interestingly, IPA predicts both an increase and decrease in prostaglandin metabolism based on the gene expression profile, suggesting that further examination of the role of prostaglandins in ERβ-null animals is necessary.

Aberrant expression of signaling molecules downstream of gonadotropins

Protein kinase A (PKA) and cAMP signaling downstream of FSH is essential for granulosa cell proliferation and follicle maturation. ERβ is involved in this signaling pathway because ERβ-null follicles have reduced PKA activity after FSH stimulation (15). In accordance, several components of the PKA signaling pathway are misregulated in ERβ-null preovulatory granulosa cells. A kinase anchoring proteins (AKAPs) act within cells to confine regulatory PKA subunits within the cell, and Akap12 was reported to be down-regulated in granulosa cells after FSH (21). Akap12 shows increased expression in ERβ-null granulosa cells downstream of FSH signaling, suggesting that the down-regulation of Akap12 after FSH treatment requires ERβ expression. The increased Akap12 expression may contribute to the sequestration of PKA regulatory units and contribute to the reduced accumulation of cAMP observed (15). Overexpression of a constitutively active PKA in granulosa cell cultures was able to stimulate increased expression of Cyp11a1, 3β-HSD, and Inhba to similar levels induced by FSH, whereas Cyp19a1 and Lhcgr need input from other signaling pathways (21). PKA is necessary but not sufficient for activation of all FSH target genes, and genes found to be differentially expressed in ERβ-null granulosa cells suggest other signaling pathways may be aberrantly expressed.

PKA can signal through multiple targets to activate other signaling molecules, including the phosphatidylinositol 3-kinase/AKT pathway. In this regard, recent work in rat granulosa cells demonstrates that GRB-associated binding protein 2, a recently identified AKAP, signals downstream of FSH leading to phosphatidylinositol 3-kinase/AKT activation (24). Our data suggests that Gab2 expression is reduced in ERβ-null preovulatory cells, providing a second signaling pathway that may be affected by the loss of ERβ. Future work is necessary to examine phosphorylation status of the signaling molecules in ERβ-null granulosa cells to further characterize the altered FSH response observed. The loss of ERβ contributes to reduced PKA signaling downstream of PMSG, suggesting that the granulosa cells are unable to fully differentiate and activate signaling pathways necessary for maximal response to LH.

Ovarian follicles are selected for proliferation and maturation from a primordial pool by unknown mechanisms. An attenuated cAMP response to PMSG or PMSG/hCG was observed in cells isolated from ewes during anestrous compared with cells isolated during the follicular phase of the estrous cycle (38). This suggests that superovulation may not be able to overcome the hierarchical stage of the follicle development completely, and we can hypothesize that loss of ERβ may alter the development of the granulosa cells prior to gonadotropin stimulation because altered cAMP signaling is also observed. The inability of most ovarian follicles to respond to exogenous gonadotropins and the aberrant transcriptional responses observed after gonadotropin stimulation support the idea that ERβ-null granulosa cells may not be properly differentiated.

Family with sequence similarity 110, member C (Fam110c), is a cytoskeletal protein that regulates cell proliferation in rat granulosa cells downstream of PMSG/hCG. It was found to have a temporal expression pattern because expression was initially increased 4 hours after hCG treatment and returned to basal levels after 24 hours in WT granulosa cells (23). Interestingly, Fam110c shows increased expression in PMSG stimulated ERβ-null granulosa cells and decreased expression after hCG stimulation (Figures 2 and 3). Fam110c is thought to control cell differentiation of granulosa to luteal cells by arresting cell cycle progression (23), and this altered regulation in ERβ-null granulosa cells provides further evidence that the cells may not be properly differentiated.

Expression of several genes that have previously been reported to be oocyte specific were identified in ERβ-null granulosa cells, even though careful attention was paid to not capture any cumulus cells or oocytes in the LCM isolation. These genes are found to be differentially expressed in ERβ-null cells, suggesting that loss of ERβ leads to increased expression of these genes. Interestingly, 4 genes that are targets of the homeobox family member reported to control oocyte-specific genes, newborn ovary homeobox protein, are predicted to be activated due to increased expression in ERβ-null cells. These genes, which include Gdf9 and Pou5f1 (also called Oct4) (27, 39), have increased expression in ERβ-null granulosa cells. Additional oocyte-specific markers were also increased in the ERβ-null granulosa cells including Oog1, Oosp1, and Ooep. The aberrant expression of germ cell-specific genes in ERβ-null granulosa cells supports the hypothesis that ERβ may be repressing the expression of oocyte-specific genes in the granulosa cells, essentially maintaining the granulosa cell phenotype.

The canonical WNT/β-catenin signaling pathway is activated downstream of FSH in granulosa cells (reviewed in References 40 and 41). WNT signals by binding to its receptor, Frizzled (Fzd), which leads to the stabilization of β-catenin and activation of target genes (42). In granulosa cells, β-catenin is important for Cyp19a1 expression through interactions with NR5A1 (43), whereas overexpression can lead to increased incidence of granulosa cell tumors (44). Expression of Fzd2 and Sfrp4, an inhibitor of Wnt signaling, is reduced in ERβ-null granulosa cells. Low density lipoprotein-receptor related proteins act as coreceptors for Frizzled in WNT signaling, and Lrp11 shows reduced expression in ERβ-null granulosa cells. Collectively this suggests aberrant WNT signaling may contribute to the altered gonadotropin response observed in ERβ-null cells.

Attenuation of signaling pathways downstream of hCG may partially be due to decreased expression of Lhcgr as previously discussed (10); however, other signaling pathways are also implicated. Mack et al (45) recently reported that IGF-I synergizes with cAMP to regulate ovulatory responsive genes including Areg, Ereg, Btc, and Il6 through nuclear factor-κB signaling. Furthermore, expression of Igf1r is reduced in ERβ-null granulosa cells, which may reduce the activity of IGF-I and its downstream targets in granulosa cells in addition to the reduced cAMP signaling pathway observed after hCG treatment of ERβ-null follicles (10).

As mentioned, LH induces the expression of epidermal growth factor (EGF)-like growth factors including Areg, Ereg, and Btc, which act to induce oocyte maturation and COC expansion (reviewed in Reference 46). Areg and Ereg have been reported to be reduced slightly in βERKO follicles (10); however, the genes were not found to be differentially expressed in whole ovarian samples (8). In preovulatory granulosa cells, Areg, and Ereg have reduced expression in ERβ-null ovaries, as does another EGF-like gene, Megf10. Multiple EGF-like domains 10 (Megf10) is a member of the EGF-like domain-containing family that is involved in cell adhesion and was recently reported to play a role in intracellular repulsion in retina neuronal cells (47). Megf10 was identified in a microarray analysis as being specifically expressed in a subtype of neuronal cells in the retina (47).

Our analysis also identified Has2 as a gene that has reduced expression in ERβ-null granulosa cells. Has2 is essential for the formation of the extracellular matrix during COC expansion (48). COC expansion is impaired in ERβ-null follicles (8), and Ptgs2 and Ptx3, genes implicated in COC expansion, also have reduced expression in ERβ-null preovulatory granulosa cells. ERK1/2 signaling and the transcription factor CEBPα/β are also necessary for COC expansion (20), and genes that are reported to be regulated downstream show reduced expression in ERβ-null preovulatory granulosa cells. This implies that even though the ERβ-null follicles selected for LCM isolations were able to reach the large antral stage of development, they still exhibit altered gene expression that contributes to the reduced ovulation and reduced fertility phenotype observed in these mice.

ERβ-null granulosa cells fail to respond to gonadotropin signaling to both increase and reduce gene expression, suggesting that ERβ may function as both a repressor and activator of target genes downstream of FSH and LH. Examination of promoters bound by ERβ is difficult due to the lack of a specific antibody for immunoprecipitation and Western blotting experiments (data not shown), making mechanistic chromatin immunoprecipitation studies for ERβ similar to those done with ERα in the uterus (49) impossible at this time. We have successfully isolated pure populations of preovulatory granulosa cells from both WT and ERβ-null animals after gonadotropin stimulation to examine global gene expression. Even as we focus on the in vivo response to FSH, we do not exclude the possibility that ERβ may be required for the proper differentiation of granulosa cells even prior to exposure to FSH. In this regard, components of the extracellular matrix were found to be aberrantly expressed in βERKO mice aged 13 days (17). The present study selected only follicles that were able to reach the large antral state of development potentially minimizing isolation of cells that were unable to proliferate completely in response to PMSG. Loss of ERβ affects the ability of granulosa cells to appropriately respond to gonadotropin stimulation. This observation parallels what is observed in humans undergoing in vitro fertilization that are poor responders and/or have luteinized unruptured follicle syndrome. The phenotypes observed in the ERβ-null granulosa cells provide a potential data set for comparison. The altered metabolic genes and signaling components identified in ERβ-null granulosa cells provides a plethora of additional mechanistic studies that can be done both in vivo and in vitro to further characterize and identify novel pathways impacted by the loss of ERβ in ovarian folliculogenesis.

Supplementary Material

Supplemental Data
supp_154_6_2174__index.html (1.4KB, html)

Acknowledgments

We thank Liwen Liu and Dr Wipawee Winuthayanon for technical assistance and discussion and Drs Wendy Jefferson and Miranda Bernhardt for critical review of this manuscript.

This work was supported by National Institutes of Health Grant Z01ES70065 and the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
AKAP
A kinase anchoring protein
CEBP
CCAAT/enhancer binding protein
COC
cumulus-oocyte complex
E2
estradiol
EGF
epidermal growth factor
ER
estrogen receptor
βERKO
ERβ knockout mice
hCG
human chorionic gonadotropin
IPA
ingenuity pathway analysis
LCM
laser capture microdissection
LHR
LH receptor
P4
progesterone
PKA
protein kinase A
PMSG
pregnant mare serum gonadotropin
WT
wild type.

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Supplemental Data
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supp_en.2012-2256_EN-12-2256SuppTab1.xlsx (10.8KB, xlsx)
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