Antiobesity effects of coumestrol through expansion and activation of brown adipose tissue metabolism

Coumestrol is a dietary phytoestrogen with estrogen-mimicking characteristics. This study investigated the molecular mechanisms of antiobesity effects of coumestrol. Two weeks of coumestrol treatment reduced body weight and improved glucose tolerance of high-fat diet (HFD)-fed mice. Notably, coumestrol treatment reduced adiposity but expanded brown adipose tissue mass. In addition, coumestrol treatment induced up-regulation of brown adipocyte markers and lipolytic gene expression in adipose tissue. Mechanistically, coumestrol induced an increase in mitochondrial contents of brown adipose tissue, which was associated with up-regulation of adenosine monophosphate-activated protein kinase and sirtuin 1. In vitro knockdown of estrogen receptor 1 inhibited the effect of coumestrol on brown adipogenic marker expression, increase in mitochondrial contents and oxygen consumption rate in brown adipocytes. Furthermore, lineage tracing of platelet-derived growth factor receptor A-positive (PDGFRA+) adipocyte progenitors confirmed increased levels of de novo brown adipogenesis from PDGFRA+ cells by coumestrol treatment. In conclusion, our results indicate that coumestrol has antiobesity effects through the expansion and activation of brown adipose tissue metabolism.     

In vitro effect of KCA-098, a derivative of coumestrol,on bone resorption of fetal rat femurs

The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoline-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of ⁴⁵Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of ⁴⁵Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.     

Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: chalcone,isoflavone and isoflavanone precursors

Comparative feeding experiments in CuCl₂,- and UV-treated lucerne (Medicago sativa) seedlings have shown that 2′,4,4′-trihydroxychalcone-[carbonyl-¹⁴C] and formononetin-[Me-¹⁴C] but not 2′,4′-dihydroxy-4-methoxychalcone-[carbonyl- ¹⁴C] or daidzein-[4-¹⁴C] were incorporated into the phytoalexins demethylhomopterocarpin, sativan and vestitol, and also into 9-O-methylcoumestrol. The synthesis of 9-O-methylcoumestrol is greatly stimulated by this abiotic treatment but coumestrol production is not noticeably affected. Daidzein and the trihydroxychalcone were precursors of coumestrol. The results are interpreted in favour of a mechanism in which methylation is an integral part of the aryl migration process associated with the biosynthesisof 4′-methoxyisoflavonoids. Formononetin, 2′,7-dihydroxy-4′-methoxyisoflavone-[Me-¹⁴C], 7-hydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] and 2′,7-dihydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] were all excellent precursors of demethylhomopterocarpin, sativan, vestitol and 9-O-methylcoumestrol, and thus a metabolic grid may be involved in their biosynthetic origin.     

Biosynthesis of coumestrol in Phaseolus aureus

Feeding experiments with 4′,7-dihydroxyisoflavone-[4-¹⁴C] (daidzein), 2′,4′,7-trihydroxyisoflavone-[T] and (±)-4′,7-dihydroxyisoflavanone-[T] (dihydrodaidzein) in suspension cultures of mung bean (Phaseolus aureus Roxb.) roots have shown that daidzein is a better precursor of the coumestan coumestrol than is the trihydroxyisoflavone and that dihydrodaidzein can also be converted very efficiently. The results provide further evidence for the intermediacy of a pterocarp-6a-en in coumestrol biosynthesis, and also indicate the possible existence of a 'metabolic grid' of isoflavones and isoflavanones in P. aureus.     

Host-Pathogen Interactions : XXIII. The Mechanism of the Antibacterial Action of Glycinol, a Pterocarpan Phytoalexin Synthesized by Soybeans

The biochemical basis for the ability of the pterocarpan phytoalexin glycinol (3,6a,9-trihydroxypterocarpan) to inhibit the growth of bacteria was examined. Glycinol at bacteriostatic concentrations (e.g. 50 micrograms per milliliter) inhibits the ability of Erwinia carotovora to incorporate [³H]leucine, [³H]thymidine, or [³H]uridine into biopolymers. Exposure of Escherichia coli membrane vesicles to glycinol at 20 micrograms per milliliter results in inhibition of respiration-linked transport of [¹⁴C]lactose and [¹⁴C]glycine into the vesicles when either d-lactate or succinate is supplied as the energy source. The ability of E. coli membrane vesicles to transport [¹⁴C]α-methyl glucoside, a vectorial phosphorylation-mediated process, is also inhibited by glycinol at 20 micrograms per milliliter. Furthermore, exposure of membrane vesicles to glycinol (50 micrograms per milliliter) at 20°C results in the leakage of accumulated [¹⁴C]α-methyl glucoside-6-phosphate. The effects of the phytoalexins glyceollin, capsidiol, and coumestrol, and daidzein, a compound structurally related to glycinol but without antibiotic activity, upon the E. coli membrane vesicle respiration-linked transport of [¹⁴C]glycine and of [¹⁴C]α-methyl glucoside was also examined. Glyceollin and coumestrol (50 micrograms per milliliter), but not daidzein, inhibit both membrane-associated transport processes. These data imply that the antimicrobial activity of glycinol, glyceollin, and coumestrol are due to a general interaction with the bacterial membrane. Capsidiol (50 micrograms per milliliter) inhibits d-lactate-dependent transport of [¹⁴C]glycine but not vectorial phosphorylation-mediated transport of [¹⁴C]α-methyl glucoside. Thus, capsidiol's mechanism of antimicrobial action seems to differ from that of the other phytoalexins examined.     

A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation

One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc⁺⁺, Nod ^−/+ phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.     

Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4α. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism.     

Biosynthesis of hydroxyphaseollin and related isoflavanoids in disease-resistant soybean hypocotyls

Phenylalanine-U-¹⁴C and isoliquiritigenin-9-¹⁴C were readily incorporated into the antifungal pterocarpan hydroxyphaseollin in soybean hypocotyls that were inoculated with incompatible strains of the phytopathogenic fungus Phytophthora megasperma var. sojae. Hydroxyphaseollin accounted for over half of the phenylalanine and isoliquiritigenin incorporated into ethyl acetate soluble compounds. Daidzein, coumestrol, and sojagol were identified as major compounds which accumulated coordinately with hydroxyphaseollin and contained significant amounts of radioactivity from the labelled isoflavanoid precursors. Hydroxyphaseollin was not present in healthy soybean plants and was not detected until ca. 16 hr after inoculation with the fungus. The pterocarpan then accumulated rapidly between 16 and 48 hr after inoculation, while the greatest accumulations of daidzein, coumestrol, and sojagol occurred between 48 and 72 hr after inoculation, when hydroxyphaseollin accumulation had ceased. Although soybean hypocotyls contained the anthocyanin malvin, neither this compound nor any other flavone pathway product was observed to accumulate after fungus-inoculation. The results therefore indicate that the accumulation of hydroxyphaseollin in fungus-inoculated soybean hypocotyls involves the activation of isoflavanoid biosynthesis with 'direction' of metabolic intermediates to biosynthesis of the pterocarpan.     

Virus infection stimulates phyto-oestrogen production in pasture legume plants growing in grazed swards

Four field experiments were sown with AMV‐infected or healthy seed of burr medic (Medicago polymorpha) and grazed by sheep; two were sown with cv. Circle Valley and two with cv. Santiago. Seed‐infected plants acted as primary sources for virus spread by naturally occurring aphids. Insecticides and admixture with annual ryegrass (Lolium rigidum), a non‐host of AMV, suppressed virus spread to different extents in the plots sown with infected seed. Effects of the different amounts of virus spread obtained on overall concentration of the oestrogenic compound coumestrol (dihydroxycoumestan) in dry stems and pods, and on seed production were measured in the medic. With cv. Santiago, stem and pod coumestrol concentration values for plots sown with healthy seed were significantly smaller than those for all plots sown with infected seed regardless of whether they were sprayed. With cv. Circle Valley, the coumestrol values for stems from plots sown with healthy seed were significantly smaller than those for unsprayed plots sown with infected seed but not than those for sprayed plots or ones with grass admixtures, and there were no significant differences with pods. There was always a significant positive relationship between concentration of coumestrol in medic stems and percentage AMV infection of swards; this was also so with pods in two experiments. A linear model best fitted this relationship with cv. Circle Valley but a logarithmic model did so with cv Santiago. In glasshouse grown plants, the coumestrol content of dried medic shoots was increased 11 (cv. Circle Valley) and five (cv. Santiago) times by AMV infection. AMV increased mean coumestrol concentrations up to 256 ppm (field) and 237 ppm (glasshouse) in stems and 223 ppm in pods (field). Sowing healthy seed in new pasture swards was an effective strategy for minimising coumestrol accumulation in burr medic swards. Two, but not single, applications of a newer generation pyrethroid insecticide to swards in which AMV was spreading significantly diminished coumestrol accumulation but applying organophosphorus insecticide twice and carbamate insecticide repeatedly did not. Medic seed yields and individual seed weights were sometimes significantly increased by the treatments, suppression of AMV spread by regular carbamate sprays being sufficient to increase seed yield by 55%.     

Effects of phytoestrogen-coumestrol on lipid and carbohydrate metabolism in young ovariectomized rats may be independent of its estrogenicity

Two groups of young ovariectomized female rats received one of two treatments. The first group was fed coumestrol in lab chow (200 microg of coumestrol per day) for 14 days; the second group received coumestrol (40 mg/L) via perfusion medium. There was a significant increase (78% compared with the control group) in the uterine weight after coumestrol treatment, which supports the estrogen-like activity of coumestrol. Phytoestrogen diminished the liver and skeletal muscle glycogen contents by 18% and 29%, respectively, and increased the blood glucose level by 24%. Glycogenolytic activity of coumestrol was observed when it acted directly on the liver areas. Although phytoestrogen did not influence insulin and glucagon blood level, liver and to some degree muscle susceptibility to insulin (measured as hormone binding by insulin receptors) was decreased. Coumestrol increased the content of triglycerides in muscle by 113% and enhanced the liver lipid synthesis from glucose by 179%. Liver cholesterol concentration was increased both after coumestrol feeding (by 12%) and when it acted directly on the liver (by 16%). These observations suggest that coumestrol is in general anabolic with regard to lipid and catabolic within-carbohydrate metabolism of young ovariectomized female rats. Based on the results of this study, it is concluded that influence of coumestrol on lipid and carbohydrate metabolism of ovariectomized rats is in part not related to its estrogenic action.     

Topical Delivery of Coumestrol from Lipid Nanoemulsions Thickened with Hydroxyethylcellulose for Antiherpes Treatment

We have recently shown that coumestrol, an isoflavonoid-like compound naturally occurring in soybeans, alfafa, and red clover, inhibited Herpes Simplex Virus types 1 (HSV-1) and 2 (HSV-2) replication. In this study, we designed coumestrol formulations in an attempt to enable its topical delivery to mucosa tissues. Physicochemical and microscopic examinations suggested that coumestrol was efficiently incorporated in positively-charged nanoemulsions dispersed in a hydroxyethylcellulose gel. The higher coumestrol flux through excised porcine esophageal mucosa was detected from nanoemulsions composed by a fluid phospholipid (dioleylphosphocholine, DOPC) in comparison with that of a rigid one (distearoylphosphocholine, DSPC) in two mucosa conditions (intact and injured). Such results were supported by confocal fluorescence images. Furthermore, the low IC₅₀ values demonstrated an increasement in the antiviral inhibition against HSV-1 and HSV-2 after incorporation of coumestrol into nanoemulsions containing DOPC. Overall, coumestrol-loaded nanoemulsions proved to be beneficial for herpes simplex treatment.     

THE INFLUENCE OF COUMESTROL,ZEARALENONE, AND GENISTEIN ADMINISTRATION ON INSULIN RECEPTORS AND INSULIN SECRETION IN OVARIECTOMIZED RATS

ABSTRACT

The influence of phytoestrogens (genistein and coumestrol) and mycoestrogen (zearalenone) on insulin secretion, liver insulin receptors and some aspects of lipid and carbohydrate metabolism were investigated in this study. Ovariectomized rats were injected s.c. with the above mentioned compounds in the amount of 1?mg for three days. Coumestrol and zearalenone caused a significant increase in uterus weight, similar to the effects observed after estrone action, while this effect was not observed after the genistein injection. Blood insulin level was not changed after phyto- or mycoestrogen treatment. However, coumestrol and genistein significantly decreased the binding capacity of liver insulin receptors. These changes corresponded with alterations in glucose and free fatty acids profiles in blood, as well as with glycogen content in liver. The effects observed after genistein and coumestrol injections differed from those noticed in rats treated with zearalenone or estrone. On the basis of these results we conclude that metabolic effects of high doses of coumestrol and genistein in ovariectomized rats are partly mediated by changes in insulin sensitivity of the liver and that the action of plant estrogens on metabolism is, at least to the some degree, independent of their estrogen activity.     

3, 9-di-O-substituted coumestrols incorporating basic amine side chains act as novel apoptosis inducers with improved pharmacological selectivity

There is much interest in the use of phytoestrogens such as coumestrol in breast cancer intervention due to their antiestrogenic activity and multiple modes of tumor cell death. However, the clear beneficial effects of naturally occurring estrogen mimetic coumestrol remain controversial due to experimental evidence that it has been shown to stimulate MCF-7 cell proliferation via agonist effect on estrogen receptor at low concentration. Herein, to disconnect the ER interaction and apoptosis-specific mechanism of coumestrol, various 3, 9-di-O-substituted coumestrols (7a-7e) and their furan ring-opened analogs (5a-5e) were synthesized and assessed for antiproliferative properties. Attachment of a dimethylamine-containing side chain to 3-O of coumestrol led to the most promising compound 7e with improved antiproliferative activity (1.7-fold increase) against MCF-7 cells, decreased estrogen activity (>20 times weaker ERα binder) and a novel action to induce apoptosis. Mechanistic studies revealed that 7e is a tubulin polymerization inhibitor, which could arrest cell cycle at G2/M phase and induce apoptosis along with the decrease of mitochondrial membrane potential. In summary, such subtle modifications to the 3, 9-di-hydroxyl groups of coumestrol allow the generation of a novel apoptosis inducer with distinct pharmacological properties, providing an excellent starting point to future development of novel tumor-vascular disrupting agents targeting tubulin.     

Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen-rich plants: Potent endocrine disruptors inducing infertility

Phytoestrogens exist in plants that are present in forages fed to horses. They may compete with 17-β estradiol and influence the estrous cycle. Therefore, the objective was to determine whether coumestrol from clover-mixed pastures is present in mare's plasma after their ingestion (experiment I), and when this phytoestrogen was present in mare's plasma after ingestion (experiment II). The effect of a long-term ingestion of phytoestrogens on estrous cycle disruption was assessed (experiment III; clinical case). Experiment I was carried out in nonpregnant anestrous and cyclic Lusitano mares (n = 14) kept on clover and grass-mixed pastures, and supplemented with concentrate and hay or cereal straw. Blood and feedstuff were obtained from November to March. In experiment II, stabled cyclic Lusitano mares (n = 6) were fed for 14 days with increasing amounts of alfalfa pellets (250 g to 1 kg/day). Sequential blood samples were obtained for 8 hours after feed intake on Day 0 (control) and on Days 13 and 14 (1 kg/day alfalfa pellets). Experiment III mares were fed with a mixture of alfalfa and clover haylage for 5 months (group 1; n = 4) or for 9 months (group 2; n = 12). Estrous cycle was determined on the basis of plasma estradiol (E₂), progesterone (P₄), and ultrasound (experiment III). Concentrations of phytoestrogen coumestrol and its metabolite methoxycoumestrol were determined by high-performance liquid chromatography coupled with mass spectrometry. Phytoestrogens decreased in pasture from November until March (P < 0.01) (experiment I), but were always detected in mares' plasma. In experiment II, plasma-conjugated forms of coumestrol and methoxycoumestrol were higher on Days 13 and 14 than in control (P < 0.05). The highest concentrations of conjugated form of coumestrol were at 1.5 and 4 hours (P < 0.001), whereas its free forms peaked at 1 and at 3.5 hours after ingestion (P < 0.05). Methoxycoumestrol-conjugated form concentration was the highest at 1.5 and 5 hours (P < 0.001), whereas its free form peaked at 1 hour (P < 0.05) and at 1.5 hours (P < 0.001). Long-term intake of coumestrol caused lack of ovulation, uterine edema, and uterine fluid accumulation (experiment III). Coumestrol and methoxycoumestrol in both forms were higher in group 2 (while still ingesting haylage) than in group 1, after haylage withdrawal (P < 0.001). These data show that in the mare, coumestrol and its metabolite increase in blood after ingestion of estrogenic plants and can influence reproduction in mares as potent endocrine disruptors.     

Plant derived coumestrol phytochemical targets human skin carcinoma cells by inducing mitochondrial-mediated apoptosis,cell cycle arrest,inhibition of cell migration and invasion and modulation of m-TOR/PI3K/AKT signalling pathway

The current study was undertaken to investigate anticancer activity of coumestrol phytoestrogen against human skin cancer. MTT assay was performed for cell viability assessment and clonogenic assay for cell colony formation assessment. Apoptosis was analysed by Annexin V/FITC staining, AO/EB staining and western blotting assays. Effects on the m-TOR/PI3K/AKT signalling pathway were investigated by western blotting. Results indicated that coumestrol induced significant toxicity in human skin cancer cells in contrast to mouse skin cancer cells. The proliferation rate in normal skin cells remained almost intact. Annexin V-FITC and AO/EB staining assays indicated coumestrol induced cytotoxicity in skin cancer cells is mediated through apoptosis stimulation. The apoptosis in skin cancer cells was mediated through caspase-activation. Cell migration and invasion was inhibited by coumestrol in human skin cancer cells via inhibition of MMP-2 and MMP-9 expressions. Moreover, m-TOR/PI3K/AKT signalling pathway in SKEM-5 cells was blocked by coumestrol.     

Inducible isoflavonoids from the lima bean,Phaseolus lunatus

Phaseolus lunatus seedlings treated with aqueous cuprous chloride produced 25 isoflavonoids which were isolated and characterized by UV, mass and ¹H NMR spectroscopy as kievitone, isoferreirin, 5-deoxykievitone, kievitone hydrate, cyclokievitone, cyclokievitone hydrate, daidzein, 2′-hydroxydaidzein, genistein, 2′-hydroxygenistein, 2,3-dehydrokievitone, luteone, phaseollidin, 2-(γ,γ-dimethylallyl)-phaseollidin, coumestrol, psoralidin, 7,8,2′,4′-tetrahydroxyisoflavone, 5,7,8,2′,4′-penta hydroxyisoflavone, 2,3-dehydrokievitol, lunatone, 5-deoxykievitol, kievitol, 3′-(γ,γ-dimethylallyl)-kievitone, 4-(γ,γ-dimethylallyl)-phaseollidin and 2-(γ,γ)-dimethylallyl)-6a-hydroxy-phaseollidin. The last nine compounds have novel structures. Possible biogenetic routes for these isoflavonoids and the taxonomic implications of their occurrence are discussed.     

A new perfusion system for the measurement and characterization of potential rates of soil nitrification

The effect of two isoflavonoids, coumestrol and daidzein which are present in aseptically grown roots and root exudates of soybean, was tested on some rhizospheric microorganisms. It was found that coumestrol promotes the growth ofR. japonicum USDA 138 (about 30%) andR. leguminosarum (about 15%) whereas it inhibits the growth ofAgrobacterium tumefaciens (about 50%) andPseudomonas sp. (about 20%). The following microorganisms were unaffected by this molecule:R. japonicum W505,Agrobacterium radiobacter, Micrococcus luteus andCryptococcus laurentii. It was found that daidzein promotesR. japonicum USDA 138 growth (about 20%) and inhibitsPseudomonas sp. growth (about 20%); other microorganisms were unaffected. In addition, coumestrol favoured the formation of ‘coccoids’ cells byRhizobium japonicum USDA 138 which could be the infective state of this strain. It seems that this compound is able to help nodulation of soybean by aRhizobium strain. This result supports the work of Peterset al. (1986) and Redmondet al. (1986) who show that flavones present in plant exudates induces expression of nodulation genes in Rhizobium.     

An Isoflavonoid-Inducible Efflux Pump in Agrobacterium tumefaciens Is Involved in Competitive Colonization of Roots

Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, and ifeR), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P ≤ 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::Ω-Tc), confirmed the importance of ifeA for competitive root colonization. Exposure to the isoflavonoid coumestrol increased β-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the ifeA-gusA reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on A. tumefaciens in an environment where the inducing molecules are known to be present.     

Coumestrol antagonizes neuroendocrine actions of estrogen via the estrogen receptor alpha

The phytoestrogen coumestrol has estrogenic actions on peripheral reproductive tissues. Yet in the brain this compound has both estrogenic and anti-estrogenic effects. We used estrogen receptor alpha knockout mice (ERalphaKO) to determine whether coumestrol has estrogenic actions in mice and also if these effects are mediated by the classic ERalpha. Female wild-type (WT) and ERalphaKO mice were ovariectomized and treated with estradiol (E2), dietary coumestrol, both, or neither compound. Ten days later the animals were sacrificed, blood was collected, and brain tissues were perfused. Fixed brains were sectioned and immunocytochemistry was employed to quantify progesterone receptors (PR) in the medial preoptic (POA) and ventromedial nucleus of the hypothalamus (VMN). Plasma was assayed for luteinizing hormone (LH). Estrogen treatment induced PR immunoreactivity in both regions in brains of WT females. In ERalphaKO mice, lower levels of PR were induced. The stimulatory effects of E2 on PR were attenuated in the POA by cotreatment with coumestrol, and the same trend was noted in the VMN. WT ovariectomized females treated with E2 had low levels of LH, while LH was high in untreated females and even higher in ovariectomized females treated with coumestrol. ERalphaKO females in all treatment groups had high levels of LH. Taken together, the results show that coumestrol has anti-estrogenic actions in the brain and pituitary and that ERalpha mediates these effects.