Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E2) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E2, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E2- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E2, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.
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Sergio A Arispe, Betty Adams and Thomas E Adams
VK Pedchenko and W Imagawa
Proliferation and differentiation of mammary epithelia are regulated by the combined action of systemic hormones and locally derived paracrine growth factors. Keratinocyte growth factor (KGF) is a potential candidate stromal factor that may participate in the hormonal control of stromal/epithelial interactions. In this study, we have examined the in vivo effect of 17beta-estradiol (E) treatment on KGF expression in mammary glands of peripubertal (5-week-old) and mature (11-week-old) mice. Mice received subcutaneous injections of hormone after which KGF mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. E treatment caused a dose- and time-dependent increase in KGF mRNA levels in intact mice from both age groups. Neither 17alpha-estradiol nor progesterone injection affected KGF mRNA levels. Comparison of the relative expression of KGF in parenchyma-free fat pads and in intact glands demonstrated that the basal and E-dependent KGF mRNA levels did not require the presence of mammary epithelium. ELISA assay of KGF tissue content demonstrated that concomitantly with an up-regulation of mRNA, E treatment also increased KGF protein in mammary glands from peripubertal and mature mice. These data show that E treatment stimulates both KGF mRNA and protein expression in mammary stroma in vivo and raises the possibility that KGF has a role in E-regulated mammary gland development.
Y Feuermann, S J Mabjeesh, L Niv-Spector, D Levin and A Shamay
One of the roles of the endocrine system is to synchronize mammary function. Hormones, such as estrogen, progesterone, and prolactin act directly on the mammary gland. Metabolic hormones, such as GH, glucocorticoids, insulin, and leptin are responsible for coordinating the body’s response to metabolic homeostasis. Leptin has been shown to be an important factor in regulating the metabolic adaptation of nutrient partitioning during the energy-consuming processes of lactation. In the present study, we show that leptin is secreted from the mammary fat, and is regulated by prolactin. The expression of α-casein in a co-culture of epithelial cells and fat explants was enhanced by prolactin compared with that in epithelial cells cultured alone. Leptin antagonist abolished the effect of leptin on α-casein expression in mammary gland explants when exogenous leptin was not present in the medium. This finding supports our hypothesis that the antagonist abolishes the action of endogenous leptin secreted by the mammary adipocytes. These results lead us to the hypothesis that prolactin and leptin act in the bovine mammary gland, via mammary fat pad/adipocytes.
V K Turan, R I Sanchez, J J Li, S A Li, K R Reuhl, P E Thomas, A H Conney, M A Gallo, F C Kauffman and S Mesia-Vela
Several investigators have suggested that certain hydroxylated metabolites of 17β-estradiol (E2) are the proximate carcinogens that induce mammary carcinomas in estrogen-sensitive rodent models. The studies reported here were designed to examine the carcinogenic potential of different levels of E2 and the effects of genotoxic metabolites of E2 in an in vivo model sensitive to E2-induced mammary cancer. The potential induction of mammary tumors was determined in female ACI rats subcutaneously implanted with cholesterol pellets containing E2 (1, 2, or 3 mg), or 2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), 16α-hydroxyestradiol (16α-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16α-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (~30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2. In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16α-OH E2, and 4-OH E1 were inactive in inducing mammary tumors.
Darryl L Hadsell, Walter Olea, Nicole Lawrence, Jessy George, Daniel Torres, Takahashi Kadowaki and Adrian V Lee
Expression of insulin receptor substrates (IRS)-1 and -2 within the mammary gland was found to be high at mid-lactation and dramatically decreased with mammary involution. This observation supports the hypothesis that these proteins are induced in the mammary gland with lactogenesis and involved in normal milk synthesis. To test this hypothesis, lactation capacity, along with indices of mammary secretory cell glucose metabolism and cell signaling were compared in normal mice and mice carrying targeted mutations in either the Irs1 or Irs2 genes. Mammary IRS-1 and IRS-2 protein levels were increased within 1 day of parturition and reached maximal levels by 5 days post partum. Dams carrying germline mutations of Irs1 or Irs2 displayed reduced lactation capacity as assessed by weight gain of pup litters. The reduction was more dramatic in Irs1 −/− versus Irs2 −/− dams. Maternal body weight was also reduced in Irs1 −/− dams as well as in Irs1 +/− Irs2 +/− dams. The loss of IRS-1 had little impact on mammary gland expression of milk protein mRNAs, glucose transport, or on the abundance and subcellular localization of hexokinases I and II. The loss of IRS-1 was associated with a compensatory increase in insulin-induced IRS-2 phosphorylation; however, the loss of IRS-1 did also cause a reduction in insulin-dependent mammary gland-specific activation of Akt phosphorylation. These results support the conclusion that IRS-1 is important for insulin-dependent activation of Akt signaling within the lactating mammary gland, but that loss of this protein has only modest impact on normal milk synthesis, since related signaling proteins such as IRS-2 may act in compensatory fashion.
Maria Theresa E Montales, Omar M Rahal, Hajime Nakatani, Tsukasa Matsuda and Rosalia C M Simmen
Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and in rodent breast cancer models. Despite the suggested linkage between obesity and breast cancer, the local influence of bioactive dietary components on mammary adiposity for antitumor effects remains unknown. Herein, we report that post-weaning dietary exposure to soy protein isolate and its bioactive isoflavone genistein (GEN) lowered mammary adiposity and increased mammary tumor suppressor PTEN and E-cadherin expression in female mice, relative to control casein diet. To ascertain GEN's role in mammary adipose deposition that may affect underlying epithelial cell phenotype, we evaluated GEN's effects on SV40-immortalized mouse mammary stromal fibroblast-like (MSF) cells during differentiation into adipocytes. MSF cells cultured in a differentiation medium with 40 nM GEN showed reductions in mature adipocyte numbers, triglyceride accumulation, and Ppar γ (Pparg) and fatty acid synthase transcript levels. GEN inhibition of adipose differentiation was accompanied by increased estrogen receptor β (Er β (Esr2)) gene expression and was modestly recapitulated by ERβ-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN). Reduction of Er β expression by siRNA targeting increased Ppar γ transcript levels and stromal fibroblast differentiation into mature adipocytes; the latter was reversed by GEN but not by DPN. Conditioned medium from GEN-treated adipocytes diminished anchorage-independent mammosphere formation of human MCF-7 breast cancer cells. Our results suggest a mechanistic pathway to support direct regulation of mammary adiposity by GEN for breast cancer prevention.
Sarit Ben-Shmuel, Eyal J Scheinman, Rola Rashed, Zila Shen Orr, Emily J Gallagher, Derek LeRoith and Ran Rostoker
Obesity and type 2 diabetes (T2D) are associated with an increased risk of breast cancer incidence and mortality. Common features of obesity and T2D are insulin resistance and hyperinsulinemia. A mammary tumor promoting effect of insulin resistance and hyperinsulinemia was demonstrated in the transgenic female MKR mouse model of pre-diabetes inoculated with mammary cancer cells. Interestingly, in MKR mice, as well as in other diabetic mouse models, males exhibit severe hyperglycemia, while females display insulin resistance and hyperinsulinemia with only a mild increase in blood glucose levels. This gender-specific protection from hyperglycemia may be attributed to estradiol, a key player in the regulation of the metabolic state, including obesity, glucose homeostasis, insulin resistance, and lipid profile. The aim of this study was to investigate the effects of ovariectomy (including the removal of endogenous estradiol) on the metabolic state of MKR female mice and subsequently on the growth of Mvt-1 mammary cancer cells, inoculated into the mammary fat pad of ovariectomized mice, compared with sham-operated mice. The results showed an increase in body weight, accompanied by increased fat mass, elevated blood glucose levels, and hypercholesterolemia, in ovariectomized MKR mice. In addition, mammary tumor growth was significantly higher in these mice. The results suggest that ovarian hormone deficiency may promote impaired metabolic homeostasis in the hyperinsulinemic MKR female mice, which in turn is associated with an increased growth of mammary tumors.
E E Connor, D L Wood, T S Sonstegard, A F da Mota, G L Bennett, J L Williams and A V Capuco
Steroid receptors are key transcriptional regulators of mammary growth, development and lactation. Expression of estrogen receptors alpha (ERα) and beta (ERβ), progesterone receptor (PR), and estrogen-related receptor alpha-1 (ERRβ) have been evaluated in bovine mammary gland. The ERRα is an orphan receptor that, in other species and tissues, appears to function in the regulation of estrogen-response genes including lactoferrin and medium chain acyl-CoA dehydrogenase and in mitochondrial biogenesis. Expression of ERα, ERβ, PR and ERRα was characterized in mammary tissue obtained from multiple stages of bovine mammary gland development using quantitative real-time RT-PCR. Expression was evaluated in prepubertal heifers, primigravid cows, lactating non-pregnant cows, lactating pregnant cows and non-lactating pregnant cows (n=4 to 9 animals/stage). In addition, ERα, ERβ, PR and ERRα were mapped to chromosomes 9, 10, 15 and 29 respectively, by linkage and radiation hybrid mapping. Results indicated that expression of ERα, PR and ERRα was largely coordinately regulated and they were present in significant quantity during all physiological stages evaluated. In contrast, ERβ transcripts were present at a very low concentration during all stages. Furthermore, no ERβ protein could be detected in bovine mammary tissue by immunohistochemistry. The ERα and PR proteins were detected during all physiological states, including lactation. Our results demonstrate the presence of ERα, PR and ERRα during all physiological stages, and suggest a functional role for ERRα and a relative lack of a role for ERβ in bovine mammary gland development and lactation.
L Sivaraman, SG Hilsenbeck, L Zhong, J Gay, OM Conneely, D Medina and BW O'Malley
An early single full-term pregnancy induces a long-lasting protective effect against mammary tumor development in humans and rodents. This protective effect can be mimicked in rats by short-term administration of estrogen and progesterone hormones prior to carcinogen administration. The hormones of pregnancy are able to induce a proliferative block upon carcinogen challenge that is not observed in the age-matched virgin. We wished to determine whether carcinogen is needed to induce a paracrine-to-autocrine shift of proliferation in steroid receptor positive cells or if such a cell population already exists in the age-matched virgin mammary gland. Here we show that estrogen receptor positive (ER+) proliferating cells are rare in the developing mammary gland of the virgin rat but represent the majority of the proliferating cells in the mature (96-day-old) mammary gland of the virgin rat. As the majority of the proliferating cells before carcinogen challenge were ER positive, the ER+ proliferating cells in the mature mammary gland may represent the target cells for carcinogen-induced transformation. Importantly, prior exposure of the mammary gland to pregnancy levels of estrogen/progesterone blocked this positive association. This ability to block the proliferation of the ER+ cells may be one factor by which pregnancy induces protection against breast cancer.
MA Lawson, D Li, CA Glidewell-Kenney and FJ Lopez
Androgens have a profound effect on the hypothalamic-pituitary axis by reducing the synthesis and release of the pituitary gonadotropin LH. The effect on LH is partly a consequence of a direct, steroid-dependent action on pituitary function. Although androgen action has been well studied in vivo, in vitro cell models of androgen action on pituitary gonadotropes have been scarce. Recently, an LH-expressing cell line, LbetaT2, was generated by tumorigenesis targeted to the LH-producing cells of the mouse pituitary. The purpose of these studies was to determine the presence of androgen receptor (AR) and establish its function in this cell line. RT-PCR analysis indicated that the LbetaT2 cell line expresses AR mRNA. Transient transfection assays, using the mouse mammary tumor virus (MMTV) promoter, showed that a functional AR is also present. Testosterone (TEST), dihydrotestosterone (DHT), 7alpha-methyl-19-nortestosterone (MENT), and fluoxymesterone (FLUOXY) increased reporter gene activity in the rank order of potencies MENT>DHT> TEST>FLUOXY. Additionally, activation of MMTV promoter activity by DHT in LbetaT2 cells was diminished by the AR antagonists casodex and 2-hydroxy-flutamide, indicating that the effects of DHT are mediated through AR. In summary, these studies showed that the LbetaT2 cell line is a useful model for the evaluation and molecular characterization of androgen action in pituitary gonadotropes.