Oestrogen receptor α AF-1 and AF-2 domains have cell population-specific functions in the mammary epithelium.

Oestrogen receptor α (ERα) is a transcription factor with ligand-independent and ligand-dependent activation functions (AF)-1 and -2. Oestrogens control postnatal mammary gland development acting on a subset of mammary epithelial cells (MECs), termed sensor cells, which are ERα-positive by immunohistochemistry (IHC) and secrete paracrine factors, which stimulate ERα-negative responder cells. Here we show that deletion of AF-1 or AF-2 blocks pubertal ductal growth and subsequent development because both are required for expression of essential paracrine mediators. Thirty percent of the luminal cells are ERα-negative by IHC but express Esr1 transcripts. This low level ERα expression through AF-2 is essential for cell expansion during puberty and growth-inhibitory during pregnancy. Cell-intrinsic ERα is not required for cell proliferation nor for secretory differentiation but controls transcript levels of cell motility and cell adhesion genes and a stem cell and epithelial mesenchymal transition (EMT) signature identifying ERα as a key regulator of mammary epithelial cell plasticity.

SERMs have substance-specific effects on bone, and these effects are mediated via ERαAF-1 in female mice.

The bone-sparing effect of estrogens is mediated primarily via estrogen receptor (ER)α, which stimulates gene transcription through activation function (AF)-1 and AF-2. The role of ERαAF-1 for the estradiol (E2) effects is tissue specific. The selective ER modulators (SERMs) raloxifene (Ral), lasofoxifene (Las), and bazedoxifene (Bza) can be used to treat postmenopausal osteoporosis. They all reduce the risk for vertebral fractures, whereas Las and partly Bza, but not Ral, reduce the risk for nonvertebral fractures. Here, we have compared the tissue specificity of Ral, Las, and Bza and evaluated the role of ERαAF-1 for the effects of these SERMs, with an emphasis on bone parameters. We treated ovariectomized (OVX) wild-type (WT) mice and OVX mice lacking ERαAF-1 (ERαAF-1(0)) with E2, Ral, Las, or Bza. All three SERMs increased trabecular bone mass in the axial skeleton. In the appendicular skeleton, only Las increased the trabecular bone volume/tissue volume and trabecular number, whereas both Ral and Las increased the cortical bone thickness and strength. However, Ral also increased cortical porosity. The three SERMs had only a minor effect on uterine weight. Notably, all evaluated effects of these SERMs were absent in ovx ERαAF-1(0) mice. In conclusion, all SERMs had similar effects on axial bone mass. However, the SERMs had slightly different effects on the appendicular skeleton since only Las increased the trabecular bone mass and only Ral increased the cortical porosity. Importantly, all SERM effects require a functional ERαAF-1 in female mice. These results could lead to development of more specific treatments for osteoporosis.

The role of total and cartilage-specific estrogen receptor alpha expression for the ameliorating effect of estrogen treatment on arthritis.

INTRODUCTION: Estrogen (E2) delays onset and decreases severity of experimental arthritis. The aim of this study was to investigate the importance of total estrogen receptor alpha (ERα) expression and cartilage-specific ERα expression in genetically modified mice for the ameliorating effect of estrogen treatment in experimental arthritis. METHODS: Mice with total (total ERα-/-) or cartilage-specific (Col2α1-ERα-/-) inactivation of ERα and wild-type (WT) littermates were ovariectomized, treated with E2 or placebo, and induced with antigen-induced arthritis (AIA). At termination, knees were collected for histology, synovial and splenic cells were investigated by using flow cytometry, and splenic cells were subjected to a T-cell proliferation assay. RESULTS: E2 decreased synovitis and joint destruction in WT mice. Amelioration of arthritis was associated with decreased frequencies of inflammatory cells in synovial tissue and decreased splenic T-cell proliferation. E2 did not affect synovitis or joint destruction in total ERα-/- mice. In Col2α1-ERα-/- mice, E2 protected against joint destruction to a similar extent as in WT mice. In contrast, E2 did not significantly ameliorate synovitis in Col2α1-ERα-/- mice. CONCLUSIONS: Treatment with E2 ameliorates both synovitis and joint destruction in ovariectomized mice with AIA via ERα. This decreased severity in arthritis is associated with decreased synovial inflammatory cell frequencies and reduced splenic T-cell proliferation. ERα expression in cartilage is not required for estrogenic amelioration of joint destruction. However, our data indicate that ERα expression in cartilage is involved in estrogenic effects on synovitis, suggesting different mechanisms for the amelioration of joint destruction and synovitis by E2.

Estrogen receptor-alpha promotes alternative macrophage activation during cutaneous repair.

Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERß, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.

Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions.

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2(0)) provided selective loss of function of nuclear ERα actions. In ERα-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2(0), whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.

Prevention of obesity and insulin resistance by estrogens requires ERα activation function-2 (ERαAF-2), whereas ERαAF-1 is dispensable.

The beneficial metabolic actions of estrogen-based therapies are mainly mediated by estrogen receptor α (ERα), a nuclear receptor that regulates gene transcription through two activation functions (AFs): AF-1 and AF-2. Using mouse models deleted electively for ERαAF-1 (ERαAF-1°) or ERαAF-2 (ERαAF-2°), we determined their respective roles in the actions of estrogens on body composition and glucose homeostasis in response to either a normal diet or a high-fat diet (HFD). ERαAF-2° males and females developed accelerated weight gain, massive adiposity, severe insulin resistance, and glucose intolerance--quite reminiscent of the phenotype observed in mice deleted for the entire ERα protein (ERα(-/-)). In striking contrast, ERαAF-1° and wild-type (wt) mice shared a similar metabolic phenotype. Accordingly, 17ß-estradiol administration regulated key metabolic genes in insulin-sensitive tissues and conferred a strong protection against HFD-induced metabolic disturbances in wt and ERαAF-1° ovariectomized mice, whereas these actions were totally abrogated in ERαAF-2° and ERα(-/-) mice. Thus, whereas both AFs have been previously shown to contribute to endometrial and breast cancer cell proliferation, the protective effect of estrogens against obesity and insulin resistance depends on ERαAF-2 but not ERαAF-1, thereby delineating new options for selective modulation of ERα.

Estradiol promotes functional responses in inflammatory and steady-state dendritic cells through differential requirement for activation function-1 of estrogen receptor α.

17ß-Estradiol (E2) has been shown to regulate GM-CSF- or Flt3 ligand-driven dendritic cell (DC) development through estrogen receptor (ER) α signaling in myeloid progenitors. ERα regulates transcription of target genes through two distinct activation functions (AFs), AF-1 and AF-2, whose respective involvement varies in a cell type- or tissue-specific manner. In this study, we investigated the role of ERα AFs in the development and effector functions of inflammatory DCs, steady-state conventional DCs, and plasmacytoid DCs (pDC), using mouse lacking either AF-1 or AF-2. In agreement with previous works, we showed that E2 fostered the differentiation and effector functions of inflammatory DCs through ERα-dependent upregulation of IFN regulatory factor (IRF)-4 in GM-CSF-stimulated myeloid progenitors. Interestingly, whereas AF-1 was required for early IRF-4 upregulation in DC precursors, it was dispensable to enhance IRF-4 expression in differentiated DCs to a level compatible with the development of the more functional Ly6C(-) CD11b(+) DC subset. Presence of E2 had no effect on progenitors from either knock-in mice with 7-aa deletion in helix 12 of ERα, lacking AF-2, or ERα(-/-) mice. By contrast, in Flt3 ligand-driven DC differentiation, activation of AF-1 domain was required to promote the development of more functionally competent conventional DCs and pDCs. Moreover, lack of ERα AF-1 blunted the TLR7-mediated IFN-α response of female pDCs in vivo. Thus, our study demonstrates that ERα uses AF-1 differently in steady-state and inflammatory DC lineages to regulate their innate functions, suggesting that selective ER modulators could be used to target specific DC subsets.

The role of activation functions 1 and 2 of estrogen receptor-α for the effects of estradiol and selective estrogen receptor modulators in male mice.

Estradiol (E2) is important for male skeletal health and the effect of E2 is mediated via estrogen receptor (ER)-α. This was demonstrated by the findings that men with an inactivating mutation in aromatase or a nonfunctional ERα had osteopenia and continued longitudinal growth after sexual maturation. The aim of the present study was to evaluate the role of different domains of ERα for the effects of E2 and selective estrogen receptor modulators (SERMs) on bone mass in males. Three mouse models lacking either ERαAF-1 (ERαAF-1(0)), ERαAF-2 (ERαAF-2(0)), or the total ERα (ERα(-/-)) were orchidectomized (orx) and treated with E2 or placebo. E2 treatment increased the trabecular and cortical bone mass and bone strength, whereas it reduced the thymus weight and bone marrow cellularity in orx wild type (WT) mice. These parameters did not respond to E2 treatment in orx ERα(-/-) or ERαAF-2(0). However, the effects of E2 in orx ERαAF-1(0) [corrected] were tissue-dependent, with a clear response in cortical bone parameters and bone marrow cellularity, but no response in trabecular bone. To determine the role of ERαAF-1 for the effects of SERMs, we treated orx WT and ERαAF-1(0) mice with raloxifene (Ral), lasofoxifene (Las), bazedoxifene (Bza), or vehicle. These SERMs increased total body areal bone mineral density (BMD) and trabecular volumetric BMD to a similar extent in orx WT mice. Furthermore, only Las increased cortical thickness significantly and only Bza increased bone strength significantly. However, all SERMs showed a tendency toward increased cortical bone parameters. Importantly, all SERM effects were absent in the orx ERαAF-1(0) mice. In conclusion, ERαAF-2 is required for the estrogenic effects on all evaluated parameters, whereas the role of ERαAF-1 is tissue-specific. All evaluated effects of Ral, Las and Bza are dependent on a functional ERαAF-1. Our findings might contribute to the development of bone-specific SERMs in males.

Estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance.

Pathway-selective insulin resistance where insulin fails to suppress hepatic glucose production but promotes liver fat storage may underlie glucose and lipid abnormalities after menopause. We tested the mechanisms by which estrogen treatment may alter the impact of a high-fat diet (HFD) when given at the time of ovariectomy (OVX) in mice. Female C57BL/6J mice underwent sham operation, OVX, or OVX with estradiol (E2) treatment and were fed an HFD. Hyperinsulinemic-euglycemic clamps were used to assess insulin sensitivity, tracer incorporation into hepatic lipids, and liver triglyceride export. OVX mice had increased adiposity that was prevented with E2 at the time of OVX. E2 treatment increased insulin sensitivity with OVX and HFD. In sham and OVX mice, HFD feeding induced fatty liver, and insulin reduced hepatic apoB100 and liver triglyceride export. E2 treatment reduced liver lipid deposition and prevented the decrease in liver triglyceride export during hyperinsulinemia. In mice lacking the liver estrogen receptor α, E2 after OVX limited adiposity but failed to improve insulin sensitivity, to limit liver lipid deposition, and to prevent insulin suppression of liver triglyceride export. In conclusion, estrogen treatment may reverse aspects of pathway-selective insulin resistance by promoting insulin action on glucose metabolism but limiting hepatic lipid deposition.

Estrogen receptor-α is required for the osteogenic response to mechanical loading in a ligand-independent manner involving its activation function 1 but not 2.

Estrogen receptor-α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene-targeted mouse models with (1) a complete ERα inactivation (ERα(-/-) ), (2) specific inactivation of activation function 1 (AF-1) in ERα (ERαAF-1(0) ), or (3) specific inactivation of ERαAF-2 (ERαAF-2(0) ) were subjected to axial loading of tibia, in the presence or absence (ovariectomy [ovx]) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα(-/-) mice displayed a severely reduced osteogenic response to loading with changes in cortical area (-78% ± 15%, p < 0.01) and periosteal BFR (-81% ± 9%, p < 0.01) being significantly lower than in wild-type (WT) mice. ERαAF-1(0) mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area -40% ± 11%, p < 0.05 and periosteal BFR -41% ± 8%, p < 0.01), whereas the periosteal osteogenic response to loading was unaffected in ERαAF-2(0) mice. Mechanical loading of transgenic estrogen response element (ERE)-luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE-mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand-independent manner involving AF-1 but not AF-2.

Estrogen dependent activation function of ERß is essential for the sexual behavior of mouse females.

We previously generated and characterized a genuine estrogen receptor (ER) ß-null mouse line (named ERß(ST)(L-/L-)) and showed that ERß(ST)(L-/L-) mice were sterile, due to an ovulation impairment in females and to an unknown reason in males, as their reproductive organs and spermatozoid motility appeared normal. We report here an assessment of the sexual behavior of ERß(ST)(L-/L-) null mice. We found that ERß(ST)(L-/L-) males display mildly impaired sexual behavior and that ERß(ST)(L-/L-) females are significantly less receptive and less attractive than wild-type (WT) females. Decreased attractivity is also exhibited by ERßAF2(0) but not by ERßAF1(0) mutant females (females devoid of either AF2 or AF1 activation function of ERß). Interestingly, by using an odor preference test, we have determined that the low attractiveness of ERß(ST)(L-/L-) and ERßAF2(0) females is related to a deficiency of a volatile chemosignal.

Insulin-like growth factor-1 promotes wound healing in estrogen-deprived mice: new insights into cutaneous IGF-1R/ERα cross talk.

Although it is understood that endogenous IGF-1 is involved in the wound repair process, the effects of exogenous IGF-1 administration on wound repair remain largely unclear. In addition, the signaling links between IGF-1 receptor (IGF-1R) and estrogen receptors (ERs), which have been elucidated in other systems, have yet to be explored in the context of skin repair. In this study, we show that locally administered IGF-1 promotes wound repair in an estrogen-deprived animal model, the ovariectomized (Ovx) mouse, principally by dampening the local inflammatory response and promoting re-epithelialization. Using specific IGF-1R and ER antagonists in vivo, we reveal that IGF-1-mediated effects on re-epithelialization are directly mediated by IGF-1R. By contrast, the anti-inflammatory effects of IGF-1 are predominantly via the ERs, in particular ERα. Crucially, in ERα-null mice, IGF-1 fails to promote healing, and local inflammation is increased. Our findings illustrate the complex interactions between IGF-1 and estrogen in skin. The fact that IGF-1 may compensate for estrogen deficiency in wound repair, and potentially other contexts, is an important consideration for the treatment of postmenopausal pathology.

Stromal estrogen receptor-α promotes tumor growth by normalizing an increased angiogenesis.

Estrogens directly promote the growth of breast cancers that express the estrogen receptor α (ERα). However, the contribution of stromal expression of ERα in the tumor microenvironment to the protumoral effects of estrogen has never been explored. In this study, we evaluated the molecular and cellular mechanisms by which 17ß-estradiol (E2) impacts the microenvironment and modulates tumor development of ERα-negative tumors. Using different mouse models of ER-negative cancer cells grafted subcutaneously into syngeneic ovariectomized immunocompetent mice, we found that E2 potentiates tumor growth, increases intratumoral vessel density, and modifies tumor vasculature into a more regularly organized structure, thereby improving vessel stabilization to prevent tumor hypoxia and necrosis. These E2-induced effects were completely abrogated in ERα-deficient mice, showing a critical role of host ERα. Notably, E2 did not accelerate tumor growth when ERα was deficient in Tie2-positive cells, even in mice grafted with wild-type bone marrow. These results were extended by clinical evidence of ERα-positive stromal cell labeling in the microenvironment of human breast cancers. Together, our findings therefore show that E2 promotes the growth of ERα-negative cancer cells through the activation of stromal ERα (extra-hematopoietic Tie-2 positive cells), which normalizes tumor angiogenesis and allows an adaptation of blood supply to tumors, thereby preventing hypoxia and necrosis. These findings significantly deepen mechanistic insights into the impact of E2 on tumor development with potential consequences for cancer treatment.

Estradiol increases hematopoietic stem and progenitor cells independent of its actions on bone.

Hematopoietic stem and progenitor cells reside in vascular and endosteal niches in the bone marrow. Factors affecting bone remodeling were reported to influence numbers and mobilization of hematopoietic stem cells. We therefore analyzed the effects of estradiol acting anabolic on bone integrity. Here we observe that estradiol increases progenitor cell numbers in the vascular but not in the endosteal compartment independent of its estrogen receptor α-dependent anabolic bone effects. Hematopoietic progenitors capable of reconstituting lethally irradiated mice are increased by enhanced cell cycle entry, leading to a diminished long-term reconstitution potential after serial transplantation. We demonstrate that estradiol action on stromal cells potently favors hematopoietic progenitor/stem cell frequency accompanied by enhanced expression of cell adhesion molecules. Finally, estradiol treatment enhances retention of hematopoietic stem cells in the vascular niche of the bone marrow. We describe for the first time the mechanism of estrogen action on hematopoietic stem and progenitor cells.

Estrogen receptor-α expression in neuronal cells affects bone mass.

It has generally been assumed that bone mass is controlled by endocrine mechanisms and the local bone environment. Recent findings demonstrate that central pathways are involved in the regulation of bone mass. Estrogen is involved in the regulation of bone homeostasis and the CNS is also a target for estrogen actions. The aim of this study was to investigate in vivo the role of central estrogen receptor-α (ERα) expression for bone mass. Nestin-Cre mice were crossed with ERα(flox) mice to generate mice lacking ERα expression specifically in nervous tissue (nestin-ERα(-/-)). Bone mineral density was increased in both the trabecular and cortical bone compartments in nestin-ERα(-/-) mice compared with controls. Femoral bone strength was increased in nestin-ERα(-/-) mice, as demonstrated by increased stiffness and maximal load of failure. The high bone mass phenotype in nestin-ERα(-/-) mice was mainly caused by increased bone formation. Serum leptin levels were elevated as a result of increased leptin expression in white adipose tissue (WAT) and slightly increased amount of WAT in nestin-ERα(-/-) mice. Leptin receptor mRNA levels were reduced in the hypothalamus but not in bone. In conclusion, inactivation of central ERα signaling results in increased bone mass, demonstrating that the balance between peripheral stimulatory and central inhibitory ERα actions is important for the regulation of bone mass. We propose that the increased bone mass in nestin-ERα(-/-) mice is mediated via decreased central leptin sensitivity and thereby increased secretion of leptin from WAT, which, in turn, results in increased peripheral leptin-induced bone formation.

From in vivo gene targeting of oestrogen receptors to optimization of their modulation in menopause.

The ancestral status of oestrogen receptor (ER) in the family of the steroid receptors has probably contributed to the pleiotropic actions of oestrogens, and in particular, that of 17ß-oestradiol (E2). Indeed, in addition to their well-described role in sexual development and reproduction, they influence most of the physiological processes. The pathophysiological counterpart of these actions includes prevention of osteoporosis, atheroma and type 2 diabetes, and also the promotion of uterus and breast cancer growth. Thus, the major challenge consists in uncoupling some beneficial actions from other deleterious ones, that is, selective ER modulation. Tamoxifen and raloxifene are already used, as they prevent the recurrence of breast cancer and mimic oestrogen action mainly on bone. Both E2 and tamoxifen exhibit a proliferative and, thus, a protumoural action on the endometrium. Activation of ERα and ERß regulates target gene transcription (genomic action) through two independent activation functions, AF-1 and AF-2, but can also elicit rapid membrane-initiated steroid signals. In the present review, we attempted to summarize recent advances provided by the in vivo molecular 'dissection' of ERα, allowing the uncoupling of some of its actions and potentially paving the way to optimized selective ER modulators.

Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing.

17ß-Estradiol (E2) regulates estrogen receptor-α (ERα) target gene transcription through the two independent activation functions (AFs), AF1 and AF2, located in the N-terminal and ligand binding domain of ERα, respectively. We previously reported that ERα is required for the E2 atheroprotective action as well as for its accelerative action on endothelial healing, but its AF1 function is dispensable. Here, we investigated the role of ERαAF2 in these two major beneficial actions of E2 by electively targeting ERαAF2 (named ERαAF2(0)). Our results prove four points. (i) Compared with WT ERα, the ability of ERαAF2(0) to stimulate the C3 complement or the estrogen response element-thymidine kinase promoter in two cell lines was dramatically decreased, confirming the importance of AF2 in the E2-induced transcriptional activity of ERα. (ii) The uterotrophic action of E2 was totally absent in ERαAF2(0) mice, showing the crucial role of ERαAF2 in E2-induced uterus hyperplasia. (iii) ERαAF2 was dispensable for the accelerative action of E2 on endothelial healing, underlining the functionality of ERαAF2(0) in vivo. (iv) Finally, the atheroprotective effect of E2 was abrogated in ERαAF2(0) LDL-r(-/-) mice. Thus, whereas ERαAF1 and ERαAF2 are both required for the uterotrophic action of E2, we show that only ERαAF2 is necessary for its atheroprotective effect.

Identification of estradiol/ERα-regulated genes in the mouse pituitary.

Estrogen acts to prime the pituitary prior to the GnRH-induced LH surge by undiscovered mechanisms. This study aimed to identify the key components that mediate estrogen action in priming the pituitary. RNA extracted from the pituitaries of metestrous (low estrogen) and proestrus (high estrogen) stage mice, as well as from ovariectomized wild-type and estrogen receptor α (ERα) knockout mice treated with 17ß-estradiol (E(2)) or vehicle, was used for gene expression microarray. Microarray data were then aggregated, built into a functional electronic database, and used for further characterization of E(2)/ERα-regulated genes. These data were used to compile a list of genes representing diverse biological pathways that are regulated by E(2) via an ERα-mediated pathway in the pituitary. This approach substantiates ERα regulation of membrane potential regulators and intracellular vesicle transporters, among others, but not the basic components of secretory machinery. Subsequent characterization of six selected genes (Cacna1a, Cacna1g, Cited1, Abep1, Opn3, and Kcne2) confirmed not only ERα dependency for their pituitary expression but also the significance of their expression in regulating GnRH-induced LH secretion. In conclusion, findings from this study suggest that estrogen primes the pituitary via ERα by equipping pituitary cells with critical cellular components that potentiate LH release on subsequent GnRH stimulations.

Genistein impairs early testosterone production in fetal mouse testis via estrogen receptor alpha.

The widespread consumption of soy-based products raises the issue of the reproductive toxicity of phytoestrogens. Indeed, it is well known that genistein, an isoflavone found in soybeans and soy products, mimics the actions of estrogens and that the fetal testis is responsive to estrogens. Therefore we investigated whether genistein could have deleterious effects on fetal testis. Using organ cultures of fetal testes from wild type and ERα or ERß knock-out mice we show that genistein inhibits testosterone secretion by fetal Leydig cells during early fetal development (E12.5), within the "masculinization programming window". This effect occurs through an ERα-dependent mechanism and starting at 10 nM genistein, a concentration which is compatible with human consumption. No effect of genistein on the number of gonocytes was detected at any of the studied developmental stages. These results suggest that fetal exposure to phytoestrogens can affect the development and function of the male reproductive system.

Amino acid-dependent activation of liver estrogen receptor alpha integrates metabolic and reproductive functions via IGF-1.

Throughout evolution, organisms have devised strategies to limit fertility in case of prolonged starvation. In mammals, the liver plays a central role in the orchestration of mechanisms allowing for the maintenance of energy homeostasis. We here demonstrate that dietary amino acids regulate the transcriptional activity of hepatic estrogen receptor alpha (ERα) through an mTOR-dependent mechanism. As a result of ERα activation, hepatic IGF-1 mRNA and blood IGF-1 are increased. Conversely, calorie restriction or selective ablation of ERα in the liver decrease blood IGF-1 to levels inadequate for the correct proliferation of the lumen epithelium in the uterus and the progression of the estrous cycle. We propose that the liver acts as critical mediator of energetic and reproductive functions responsible for the blockade of the estrous cycle in case of protein scarcity. Our findings may provide novel insights to understand the cause of selected forms of infertility and metabolic alterations in women after menopause.