Katherine J. Hamilton

An estrogen receptor-α knock-in mutation provides evidence of ligand-independent signaling and allows modulation of ligand-induced pathways in vivo

Estrogen-nonresponsive estrogen receptor-alpha (ERalpha) knock-in (ENERKI) mice were generated to distinguish between ligand-induced and ligand-independent ER-alpha actions in vivo. These mice have a mutation [glycine 525 to leucine (G525L)] in the ligand-binding domain of ERalpha, which significantly reduces ERalpha interaction with and response to endogenous estrogens, whereas not affecting growth factor activation of ligand-independent pathways. ENERKI mice had hypoplastic uterine tissues and rudimentary mammary gland ductal trees. Females were infertile due to anovulation, and their ovaries contained hemorrhagic cystic follicles because of chronically elevated levels of LH. The ENERKI phenotype confirmed that ligand-induced activation of ERalpha is crucial in the female reproductive tract and mammary gland development. Growth factor treatments induced uterine epithelial proliferation in ovariectomized ENERKI females, directly demonstrating that ERalpha ligand-independent pathways were active. In addition, the synthetic ERalpha selective agonist propyl pyrazole triol (PPT) and ER agonist diethylstilbestrol (DES) were still able to activate ligand-induced G525L ERalpha pathways in vitro. PPT treatments initiated at puberty stimulated ENERKI uterine development, whereas neonatal treatments were needed to restore mammary gland ductal elongation, indicating that neonatal ligand-induced ERalpha activation may prime mammary ducts to become more responsive to estrogens in adult tissues. This is a useful model for in vivo evaluation of ligand-induced ERalpha pathways and temporal patterns of response. DES did not stimulate an ENERKI uterotrophic response. Because ERbeta may modulate ERalpha activation and have an antiproliferative function in the uterus, we hypothesize that ENERKI animals were particularly sensitive to DES-induced inhibition of ERalpha due to up-regulated uterine ERbeta levels.

Estrogen hormone physiology: Reproductive findings from estrogen receptor mutant mice

Estrogen receptors (ERs) play a crucial role in reproduction and normal physiology. The two sub-types of ER (ERα and β) are expressed in various levels in different tissues and selective cell types. Gene targeting technology allowed us to produce lines of mice with disrupted ERα (αERKO) and ERβ genes (βERKO) as well as a compound αβERKO in the whole body. Male and female αERKO mice are infertile. Estrogen, EGF and IGF-1 treatments failed to induce uterine growth and DNA synthesis in αERKO uteri. αERKO females are infertile due to hypoplastic uteri and hyperemic ovaries with no corpora lutea due to persistent LH stimulation from loss of negative feedback. αERKO males are infertile, with testicular atrophy and seminiferous tubule dysmorphogenesis producing decreased spermatogenesis and inactive sperm. βERKO females show arrested folliculogenesis and subfertility. Ovarian analyses indicate differential gene expression related to ovulatory stimulation deficits including lack of LH, PR, Cyp19 and Cox2 expression. A unique ovarian phenotype is found only in αβERKO females showing transdifferentiation of granulosa cells to Sertoli cells. We describe here several novel mouse models which possess ERα gene modification. To understand ERα function in uterine endometrial epithelial cells, we generated a tissue selective ERα gene disrupted mouse model, the uterine epithelial-specific ERα knockout (UtEpiαERKO). To understand the physiological role of ERα functional domains, we generated a mouse model with a mutation in the ligand dependent transcription activation domain of ERα (AF2ERKI). Findings from the ERα mutant mice suggest that the absence of functional ERα is not lethal and results in significant endocrine effects and altered physiological processes.

Estrogen receptor α AF-2 mutation results in antagonist reversal and reveals tissue selective function of estrogen receptor modulators

The estrogen receptor (ER) is a ligand-dependent transcription factor containing two transcriptional activation domains. AF-1 is in the N terminus of the receptor protein and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. Two point mutations of leucines 543 and 544 to alanines (L543A, L544A) in helix 12 minimized estrogen-dependent transcriptional activation and reversed the activity of the estrogen antagonists ICI182780 (ICI) and tamoxifen (TAM) into agonists in a similar manner that TAM activated WT ERα through AF-1 activation. To evaluate the physiological role of AF-1 and AF-2 for the tissue-selective function of TAM, we generated an AF-2–mutated ERα knock-in (AF2ERKI) mouse model. AF2ERKI homozygote female mice have hypoplastic uterine tissue and rudimentary mammary glands similar to ERα-KO mice. Female mice were infertile as a result of anovulation from hemorrhagic cystic ovaries and elevated serum LH and E2 levels, although the mutant ERα protein is expressed in the AF2ERKI model. The AF2ERKI phenotype suggests that AF-1 is not activated independently, even with high serum E2 levels. ICI and TAM induced uterotropic and ER-mediated gene responses in ovariectomized AF2ERKI female mice in the same manner as in TAM- and E2-treated WT mice. In contrast, ICI and TAM did not act as agonists to regulate negative feedback of serum LH or stimulate pituitary prolactin gene expression in a different manner than TAM- or E2-treated WT mice. The functionality of the mutant ERα in the pituitary appears to be different from that in the uterus, indicating that ERα uses AF-1 differently in the uterus and the pituitary for TAM action.

Estrogen/Estrogen Receptor Alpha Signaling in Mouse Posterofrontal Cranial Suture Fusion

Background While premature suture fusion, or craniosynostosis, is a relatively common condition, the cause is often unknown. Estrogens are associated with growth plate fusion of endochondral bones. In the following study, we explore the previously unknown significance of estrogen/estrogen receptor signaling in cranial suture biology. Methodology/Principal Findings Firstly, estrogen receptor (ER) expression was examined in physiologically fusing (posterofrontal) and patent (sagittal) mouse cranial sutures by quantitative RT-PCR. Next, the cranial suture phenotype of ER alpha and ER beta knockout (αERKO, βERKO) mice was studied. Subsequently, mouse suture-derived mesenchymal cells (SMCs) were isolated; the effects of 17-β estradiol or the estrogen antagonist Fulvestrant on gene expression, osteogenic and chondrogenic differentiation were examined in vitro. Finally, in vivo experiments were performed in which Fulvestrant was administered subcutaneously to the mouse calvaria. Results showed that increased ERα but not ERβ transcript abundance temporally coincided with posterofrontal suture fusion. The αERKO but not βERKO mouse exhibited delayed posterofrontal suture fusion. In vitro, addition of 17-β estradiol enhanced both osteogenic and chondrogenic differentiation in suture-derived mesenchymal cells, effects reversible by Fulvestrant. Finally, in vivo application of Fulvestrant significantly diminished calvarial osteogenesis, inhibiting suture fusion. Conclusions/Significance Estrogen signaling through ERα but not ERβ is associated with and necessary for normal mouse posterofrontal suture fusion. In vitro studies suggest that estrogens may play a role in osteoblast and/or chondrocyte differentiation within the cranial suture complex.

Diethylstilbestrol (DES)-Stimulated Hormonal Toxicity is Mediated by ERα Alteration of Target Gene Methylation Patterns and Epigenetic Modifiers (DNMT3A, MBD2, and HDAC2) in the Mouse Seminal Vesicle

Background: Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes. Objectives: We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression. Methods: We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice. Results: The DNA methylation status at four specific CpGs (–160, –237, –306, and –367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (–449 and –459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers—DNMT3A, MBD2, and HDAC2—increased in the SV of DES-exposed WT mice. Conclusion: DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV. Citation: Li Y, Hamilton KJ, Lai AY, Burns KA, Li L, Wade PA, Korach KS. 2014. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle. Environ Health Perspect 122:262–268;u2002http://dx.doi.org/10.1289/ehp.1307351

Estrogen Receptor β Is Required for Optimal cAMP Production in Mouse Granulosa Cells

Granulosa cells of preovulatory follicles differentiate in response to FSH, and this differentiation is augmented by estradiol. We have previously shown that FSH-mediated granulosa cell differentiation requires functional estrogen receptor-beta (ERbeta) by demonstrating that the granulosa cells of ERbeta(-/-) FSH-treated mice are unable to maximally induce expression of the LH receptor (an indicator of granulosa cell differentiation) compared with ERbeta(+/+) controls. As a result, FSH-primed ERbeta(-/-) granulosa cells exhibit a reduced response to a subsequent ovulatory dose of LH. In this study, we further characterized the attenuated response of ERbeta(-/-) granulosa cells to stimulation by LH and FSH using isolated mouse granulosa cells and primary granulosa cell cultures. We observed a 50% reduction in cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to LH compared with ERbeta(+/+) controls. We also observed an attenuated genomic response in granulosa cells isolated from FSH-primed ERbeta(-/-) mice compared with ERbeta(+/+) controls. Our data indicate that this attenuated response may result from inadequate levels of cAMP, because cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to forskolin were approximately 50% lower than in ERbeta(+/+) granulosa cells. Phosphorylation of cAMP regulatory element binding protein, an indicator of protein kinase A activity, was also reduced in FSH-treated ERbeta(-/-) granulosa cells compared with ERbeta(+/+) controls. These are the first data to indicate that ERbeta plays a role in the induction of the cAMP pathway in mouse granulosa cells and that disruption of proper ERbeta signaling associated with this pathway may cause negative effects on ovulation and fertility.

Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as tethering. Evidence for tethering is based on in vitro studies and a widely used KIKO mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the EAAE ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

Insufficient Luteinizing Hormone-Induced Intracellular Signaling Disrupts Ovulation in Preovulatory Follicles Lacking Estrogen Receptor-β

Gonadotropin-stimulated estrogen receptor-beta (ERbeta)-null preovulatory follicles exhibit submaximal estradiol production, insufficient acquisition of LH receptor, and attenuated expression of essential ovulatory genes. These observations lead to low ovulatory rates compared with wild-type (WT) follicles. We hypothesize that insufficient LH receptor results in reduced cAMP production after an ovulatory stimulus. Individual preantral follicles were cultured with FSH for 4 d and then induced to ovulate with a single dose of human chorionic gonadotropin (hCG). cAMP levels 1 h after hCG were 50% lower in ERbeta-null than WT follicles. To determine whether the lack of LH receptor, and resulting lack of cAMP, could be bypassed by direct activation of adenylyl cyclase, WT and ERbeta-null follicles were induced to ovulate with forskolin. Ten micromolar forskolin doubled the ovulatory rate of ERbeta-null follicles compared with treatment with hCG ( approximately 50 vs. 25%, respectively). In WT follicles, 10 microm forskolin reduced the ovulation rate compared with hCG (14 vs. 83%, respectively), indicating that high doses of forskolin inhibited WT ovulation. A 10 microm concentration of forskolin induced cAMP levels in ERbeta-null follicles that were comparable to levels produced in WT follicles after hCG and either partially or completely rescued the attenuated expression of LH-responsive genes. These data indicate that direct activation of adenylyl cyclase, resulting in increased production of cAMP, partially rescues the ovulatory response of ERbeta-null follicles, suggesting that insufficient LH receptor and low cAMP levels contribute to their poor ovulatory rates. We also determined that ERbeta-null ovaries exhibit an alteration in the activation of ERK1/2. Our evaluation of the ERbeta-null ovarian phenotype indicates that ERbeta plays a role in facilitating folliculogenesis. We show that expression of ERbeta in preovulatory follicles is required for adequate cAMP production and propose that an optimal level of cAMP is required for hCG-stimulated ovulation.

The absence of ER-β results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles.

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.

Ex3αERKO male infertility phenotype recapitulates the αERKO male phenotype

Disruption of the Esr1 gene encoding estrogen receptor α (ERα) by insertion of a neomycin resistance gene (neo) into exon 2 (αERKO mice) was shown previously to cause infertility in male mice. While full-length ERα protein was not expressed in αERKO mice, alternative splicing resulted in the low-level expression of a truncated form lacking the N-terminus A/B domain and containing the DNA- and ligand-binding domains. Thus, it was unclear whether the reproductive phenotype in αERKO males was only due to the lack of full-length ERα or was affected by the presence of the variant ERα isoform. The present study examined male mice with deletion of exon 3 of Esr1 gene, lacking the DNA-binding domain, and null for ERα (Ex3αERKO). Dilation of some seminiferous tubules was apparent in male Ex3αERKO mice as early as postnatal day 10 and was pronounced in all tubules from day 20 onward. At 6 weeks of age, sperm numbers and sperm motility were lower in Ex3αERKO mice than in wild-type (WT) mice, and the rete testis and efferent ductules were dilated. Mating studies determined that adult Ex3αERKO males were infertile and failed to produce copulatory plugs. Serum testosterone levels and Hsd17b3 and Cyp17a1 transcript levels were significantly higher, but serum estradiol, progesterone, LH, and FSH levels and Cyp19a1 transcript levels were not significantly different from those in WT mice. These results confirm and extend those seen in other studies on male mice with deletion of exon 3 of Esr1 gene. In addition, the reproductive phenotype of male Ex3αERKO mice recapitulated the phenotype of αERKO mice, strongly suggesting that the αERKO male infertility was not due to the presence of the DNA-binding domain in the truncated form of ERα and that full-length ERα is essential for maintenance of male fertility.