Concentrations Of 17beta-estradiol Research Articles

Oestradiol or genistein rescues neurons from amyloid beta‐induced cell death by inhibiting activation of p38

Oestrogenic compounds have been postulated as neuroprotective agents. This prompted us to investigate their mechanism action in neurons in primary culture. Cells were pretreated with physiological concentrations of 17-beta estradiol (0.2 nm) or with nutritionally relevant concentrations of genistein (0.5 microm), and 48 h later treated with 5 microm of amyloid beta (Abeta) for 24 h. We found that Abeta increased oxidative stress, measured as peroxide levels or oxidized glutathione/reduced glutathione ratio, which in turn, caused phosphorylation of p38 MAP kinase. Amyloid beta subsequently induced neuronal death. Inhibiting the MAP kinase pathway prevented cell death, confirming the role of p38 in the toxic effect of Abeta. All these effects were prevented when cells were pretreated for 48 h with oestradiol or genistein. Therefore, oestrogenic compounds rescue neurons from Abeta-induced cell death by preventing oxidative stress, which in turn inhibits the activation of p38, protecting neurons from cell death. Because hormone replacement therapy with oestradiol could cause serious setbacks, the potential therapeutic effect of phyto-oestrogens for the prevention of Abeta-associated neurodegenerative disorders should be more carefully studied in clinical research.

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.

Follicular fluid nitric oxide (NO) concentrations in stimulated cycles: the relationship to embryo grading

The microenvironment of the ovarian follicle is vital for normal oocyte development, folliculogenesis, and timely ovulation. We investigated the concentration of nitric oxide (NO) in follicular fluid, collected during oocyte retrieval after in-vitro fertilization and embryo transfer (IVF-ET) and its relationship to oocyte and embryo grading. A total of 53 follicular fluid samples were obtained from 15 patients undergoing IVF-ET program, oocyte retrieval by transvaginal guidance was per formed approximately 34-35 h after the hCG administration in stimulated cycles. Using a modified grading system the samples were divided in Group 1 with very few fragments in the cytoplasm with equal size blastomeres (best embryos) and Group 2 with significant or severe fragmentation or blastomeres of distinctively unequal size. Follicular NO was measured as nitrite/nitrate. The mean concentration of NO follicular fluid is significantly higher in Group 2 than in Group 1 (57.54 +/- 12.67 nmol/mg vs. 42.43 +/- 16.32 nmol/mg). Using the correlation analysis, we observed a direct correlation between follicular NO and embryo grading (r = 0.61; P < 0.001) and an inverse correlation between follicular NO and serum 17beta-estradiol (r = -0.28; P < 0.05). High levels of NO in human follicles may be detrimental. The inverse correlation found between NO and serum concentrations of 17beta-estradiol may be explained as a regulation of estradiol on maturation process of the oocytes and embryos in stimulated cycles through NO mediation.

Influence of Estrogen and Xenoestrogens on Basolateral Uptake of Tetraethylammonium by Opossum Kidney Cells in Culture

The sex steroid hormone estrogen down-regulates renal organic cation (OC) transport in animals, and it may contribute to sex-related differences in xenobiotic accumulation and excretion. Also, the presence of various endocrine-disrupting chemicals, i.e., environmental chemicals that possess estrogenic activity (e.g., xenoestrogens) may down-regulate various transporters involved in renal accumulation and excretion of xenobiotics. The present study characterizes the mechanism by which long-term (6-day) incubation with physiological concentrations of 17beta-estradiol (E(2)) or the xenoestrogens diethylstilbestrol (DES) and bisphenol A (BPA) regulates the basolateral membrane transport of the OC tetraethylammonium (TEA) in opossum kidney (OK) cell renal cultures. Both 17beta-E(2) and the xenoestrogen DES produced a dose- and time-dependent inhibition of basolateral TEA uptake in OK cell cultures, whereas the weakly estrogenic BPA had no effect on TEA uptake. Treatment for 6 days with either 1 nM 17beta-E(2) or DES reduced TEA uptake by approximately 30 and 40%, respectively. These effects were blocked completely by the estrogen receptor antagonist ICI 182780 (Faslodex, fulvestrant), suggesting that these estrogens regulate OC transport through the estrogen receptor, which was detected (estrogen receptor alpha) in OK cell cultures by reverse transcription-polymerase chain reaction. The J(max) value for TEA uptake in 17beta-E(2)- and DES-treated OK cell cultures was approximately 40 to 50% lower than for ethanol-treated cultures, whereas K(t) was unaffected. This reduction in transport capacity was correlated with a reduction in OC transporter OCT1 protein expression following treatment with both agents.

Ultrasonographic Characteristics of Ovulatory Follicles and Associated Endocrine Changes in Cyclic Ewes Treated with Medroxyprogesterone Acetate (MAP)‐releasing Intravaginal Sponges and Equine Chorionic Gonadotropin (eCG)

The aim of this study was to assess the ultrasonographic characteristics of ovulatory follicles in cyclic Western White Face ewes (December) that had received intravaginal sponges containing medroxyprogesterone acetate (MAP; 60 mg) for 12 days, with or without an injection of 500 IU of equine chorionic gonadotropin (eCG) at sponge removal. We hypothesized that quantitative echotextural attributes of the follicles in ewes treated only with MAP would differ from those in MAP/eCG-treated ewes, reflecting the increased antral follicular growth and secretory function under eCG influence. Digital images of ovulatory follicles obtained at 0 and 24 h after MAP sponge removal and at 24 h before ovulation in the eCG-treated (five ewes, 13 follicles) and control (six ewes, 9 follicles) animals, were subjected to computerized analyses. The mean diameter of ovulatory follicles increased (p < 0.001) 24 h after eCG treatment. The mean pixel intensity and heterogeneity of the follicular antrum (p < 0.001), as well as mean pixel intensity of the follicular wall and perifollicular ovarian stroma (p < 0.05), were greater in eCG-treated animals compared with control ewes 24 h after sponge removal and at 24 h before ovulation. Mean serum concentrations of oestradiol-17beta tended to increase (p = 0.06) 24 h after eCG treatment and the eCG-treated ewes exceeded (p < 0.05) control animals in progesterone concentrations from days 9-15 after ovulation. Our results support the hypothesis that large antral follicles in eCG-treated ewes exhibit distinctive echotextural characteristics. Follicular image attributes in eCG-treated ewes appear to be indicative of the changes in follicular morphology and secretory activity caused by the administration of the exogenous gonadotropin, which has both FSH- and LH-like activities.

Involvement of Plasma Progesterone, Oestradiol‐17β and Cortisol in Ovulatory Response to Gonadotropin‐releasing Hormone in Dairy Cows with Cystic Follicles

The aim of the present study was to examine the plasma concentrations of progesterone, oestradiol-17beta and cortisol in the cows with cystic follicle and to examine its relationship with the ovulatory response to gonadotropin-releasing hormone (GnRH). Eighty-five post-partum Holstein-Friesian cows with cystic follicles regardless of the presence of corpus luteum were studied. Follicular size, presence of corpus luteum and occurrence of ovulation were checked by palpation per rectum. Blood collection and palpation per rectum were conducted on days 0, 7 and 14. Gonadotropin-releasing hormone was administered at day 7. Plasma concentrations of progesterone, oestradiol-17beta and cortisol were determined. Progesterone concentrations of <3.2, 3.2-4.8 and >4.8 nmol/l were defined as low, intermediate and high, respectively. Sixty-three (74.1%) of 85 cows showed low (<3.2 nmol/l =1 ng/ml) progesterone concentrations on day 0. Only 40 (47.1%) of them showed low-low pattern of progesterone at days 0 and 7. In 27 (31.8%) of them, progesterone concentration had increased by day 14. Of 22 cows having high progesterone concentration (>/=4.8 nmol/l) on day 0, corpus luteum was not detected in 18 cows (21.2%). Only in 10 cows, cystic follicle disappeared after GnRH administration. However, only one of 27 cows in which progesterone pattern was low-low-high at days 0, 7 and 14 experienced ovulation of the cystic follicle. Significantly lower oestradiol-17beta concentration was found on day 7 in cows showing a low-low-low pattern than a low-low-high pattern of progesterone (43.0 +/- 4.6 vs 55.8 +/- 2.8 pmol/l, p < 0.05). There was no significant difference in cortisol concentration on any days (days 0, 7 and 14) between cows showing a low-low-low and low-low-high pattern of progesterone. These results suggest that approximately one-fifth of cows diagnosed to have ovarian cysts possess luteal cysts and that a high oestradiol-17beta concentration at the time of GnRH administration is involved in the subsequent ovulation of the follicle, although ovulated follicle may not be cystic.

Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, triggers hypersensitive reactions when expressed in plants. To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells, we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the beta-estradiol-inducible promoter. The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on beta-estradiol concentrations applied. Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 micromol/L beta-estradiol are 5.0-and 5.5-fold of the control cells. Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str, two key genes in terpenoid indole alkaloid biosynthetic pathway of Catharanthus roseus cells, and stimulates the accumulation of defense-related protein PR1 in the cells, showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway. Thus, our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

Post‐mortem Examination of Sows Genital Organs Culled for Reproductive Disturbances and Immunohistochemical Studies on ERα and PR A Receptors in the Anoestral Sows Uterus

The aim of present study was post-mortem examination of ovaries, uterus and plasma oestradiol-17beta (E2) and progesterone (P4) concentrations in the blood of sows with reproductive disturbances and the distribution of oestradiol receptor (ERalpha), as well as progesterone (PR-A) in the anoestrous sows uteri. Reproductive organs of 150 crossbred sows (Lithuanian White x Danish Landrace) culled for the reasons of reproductive disturbances, were collected in local abattoir over a period of 3 months (September-November). Organs were assessed to determine the stage of the oestrous cycle or anoestrus and their development. Blood samples were collected for E2 and P4 analysis from the jugular vein 1 h prior to slaughter. For this study uterine samples only from pathological anoestrous sows were subjected to immunohistochemical staining to assess the distribution of ERalpha and PR-A in surface epithelium, subepithelial connective tissue, glandular epithelium and myometrium. Macroscopic examination of the ovaries showed that 68.7% sows had active cycling ovaries, 26.6% sows were anoestrus, ovaries were small without CL, and 4.7% of sows had multiple follicular cysts. In anoestrous sows (n = 27) the number and intensity of the nuclear staining of ERalpha varied between different uterine tissue compartments. The highest number (>80%) and the strongest intensity (+++) of positively stained cells for ERalpha was seen in myometrium and glandular epithelium. In other uterine wall compartments the number and intensity of positively stained for ERalpha nuclei was lower (+/++). The PR-A was absent from all tissue compartments. The intensity of the nuclear staining for ERalpha varied not only between the different uterine compartments but also between the sows. The 11.1% of the sows presented ERalpha in surface epithelium, 74.1% of the sows in glandular epithelium and 63.0% of sows in the myometrium.

Patterns of late embryonic and fetal mortality and association with several factors in sheep1

Embryonic and fetal mortality reduce lambing rates and litter sizes, thus contributing to economic losses in the sheep industry. In the current study, the timing of late embryonic and fetal loss in ewes and the factors with which these losses were associated were examined. Ewes lambing and lambs born were compared with pregnancy diagnosis and counts of embryos by ultrasonography near d 25, 45, 65, or 85 of gestation. Approximately 19.9% of the ewes experienced late embryonic loss, fetal loss, or both; and 21.2% of the embryos or fetuses were lost from d 25 to term. Potential offspring were lost throughout gestation; 3.7% of embryos from d 25 to 45, 4.3% of fetuses from d 45 to 65, 3.3% from d 65 to 85, and 11.5% from d 85 to parturition; thus, approximately 3 to 4% of the potential offspring were lost for each 20-d period of pregnancy beyond d 25. A greater proportion of ewes lost one (36.7%) rather than all (20.5% single; 3.8% multiple) embryos or fetuses. The patterns of loss were similar in ewes mated during the anestrous season and the transitional period and did not vary with service period within breeding season or method of synchronization of estrus. Late embryonic or fetal losses were not related to the temperature-humidity index. Maternal serum collected near d 25, 45, 65, or 85 of gestation was assayed for concentrations of progesterone, estradiol-17beta , and vascular endothelial growth factor (VEGF). The proportions of embryos or fetuses lost were associated with breed type (P < 0.05), as were concentrations of progesterone (P < 0.01), estradiol (P < 0.05), and VEGF (P < 0.01). The relationships of loss or retention of pregnancy to hormonal variables at the 4 stages studied were limited. Complete and partial losses increased rapidly as maternal progesterone at d 25 decreased below 2 ng/mL (P < 0.05). Survival of fetuses within a litter from d 25 to 65 was greater for ewes with medium concentrations of VEGF near d 25 and from d 65 to parturition was greater for ewes with high concentrations of VEGF near d 45 (P < 0.05). In summary, late embryonic or fetal losses occurred from d 25 throughout gestation and varied with breed type and with concentrations of progesterone in maternal serum on d 25.

Evidence for estrogenic endocrine disruption in an offshore flatfish, the dab (Limanda limanda L.)

Dab (Limanda limanda) caught in UK offshore waters show evidence of being exposed to estrogenic endocrine disrupters at a relatively low level. Two of 449 males caught between June and July 2005 had markedly elevated levels of vitellogenin (VTG; 21 and 750 microg/ml) and the remainder ranged from <0.01 to 8.6 microg/ml. Omitting the two outliers, there was a very significant positive relationship with the mass of individual males (a feature noted in previous studies on cod). Mean VTG concentrations in males differed significantly between sites. The site with the highest mean (1.6 microg/ml) was North East of the Dogger Bank and the site with the lowest (0.04 microg/ml) was in Cardigan Bay. Mean VTG concentrations in all North Sea fish were significantly higher than English Channel and Irish Sea fish, but this difference disappeared when fish mass was taken into account. VTG concentrations showed no relationship to water depth, stage of sexual maturity or age of the males. Sixty selected male plasmas were assayed for 17beta-estradiol but only two had measurable amounts (assay limit 0.04 ng/ml). Despite being the start of summer, the gonads of many of the males and females (especially those caught in the North Sea) showed signs of sexual maturity (presence of sperm in males and vitellogenic eggs in females). Many females had high VTG concentrations (up to 14 mg/ml) and 78 out of 80 had measurable concentrations of 17beta-estradiol. The cause of elevated VTG levels in male dab is unknown. As seen in cod, the presence of affected males does not appear to be linked to proximity to land or to known point sources of endocrine disrupters. However, our data, showing that larger fish are more likely to have elevated VTG concentrations, suggests a gradual accumulation by marine fish, probably through feeding, of persistent (probably relatively weak) estrogenic compounds.

Deficient Proliferation and Apoptosis in the Granulosa and Theca Interna Cells of the Bovine Cystic Follicle

The aim of the present study was to examine the frequencies of cell proliferation and death of granulosa and theca interna layers during development of cystic follicles in order to understand the mechanisms of cystic follicle formation. Paraffin sections of cystic follicles were immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 in order to observe proliferating and apoptotic cells, respectively. The concentrations of estradiol-17beta and progesterone in the follicular fluid of these follicles were measured by ELISA. The granulosa and theca interna layers contained both PCNA- and caspase-3-positive cells, although their numbers were limited. There was significant negative correlation between the estradiol-17beta and progesterone concentrations in the follicular fluid. Regression analysis revealed no significant correlation, except for that between the PCNA-positive cells in the theca interna and the caspase-3-positive cells in the granulosa layer. These results indicate that the granulosa and theca interna cells of the cystic follicle show weak proliferative activity and low apoptotic frequency; this implies that the cystic follicle grows slowly and then maintains a static condition without degeneration, which leads to long-term persistence of the follicle.

Effects of 3-Methyl-4-Nitrophenol in Diesel Exhaust Particles on the Regulation of Reproductive Function in Immature Female Japanese Quail (Coturnix japonica)

In a previous study, we found that 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust particles and also a degradation product of the insecticide fenitrothion, exhibits reproductive toxicity in the adult male Japanese quail. The present study investigated the toxicity of PNMC in the female Japanese quail and its ability to influence reproduction in immature females. The quail (21-day-old) were injected intramuscularly (im) with PNMC at doses 0.1, 1 or 10 mg/kg body weight daily for 3 days. There was no significant difference in body growth between the PNMC-administered and control birds. However, the weights of the oviducts were significantly lower in the PNMC-treated birds at all doses. Furthermore, the plasma concentrations of luteinizing hormone (LH) and estradiol-17 beta were significantly decreased with 1 and 10 mg/kg of PNMC. These findings suggest that PNMC might influence the hypothalamo-pituitary-gonadal axis with decreasing in secretion of GnRH, LH and ovarian steroid hormones and subsequently disturb growth of the reproductive organs of immature female quail. This study indicates that PNMC induces reproductive toxicity at the central level and disrupts reproductive function in the immature female quail.

Effects of L‐carnitine supplementation in pregnant sows on plasma concentrations of insulin‐like growth factors, various hormones and metabolites and chorion characteristics

Previous studies have shown that supplementation of sow diets with L-carnitine increases the body weight of piglets at birth. This study was conducted to elucidate the reasons for this phenomenon. Three experiments with 24 (experiment 1), 40 (experiment 2) and 12 (experiment 3) sows were conducted. In all three experiments, sows were allotted to two groups which had free access to a nutritionally adequate diet. Sows of one group were supplemented with 125 mg L-carnitine/day during pregnancy; sows of the other group (control group) did not receive L-carnitine. In experiment 1, plasma samples were collected at day 95 of pregnancy, in experiment 2 plasma samples were collected at days 80 and 100 of pregnancy. In experiment 3, chorions of the sows were collected at parturition. L-carnitine-treated sows had higher plasma concentrations of total L-carnitine than control sows (p < 0.05). The number of piglets born and weights of litter and individual piglets at birth were not different between both groups in all three experiments. L-carnitine-treated sows had higher plasma concentrations of insulin-like growth factor-I (IGF-I) on day 80 of pregnancy (experiment 2, p < 0.05) and on day 95 (experiment 1, p < 0.10), and a higher plasma concentration of IGF-II on day 80 (experiment 2, p < 0.05) than control sows. Moreover, sows supplemented with L-carnitine had heavier chorions (+22%, p =0.10) with greater amounts of protein (+45%, p < 0.05) and DNA (+38%, p < 0.10) and a higher protein concentration of glucose transporter-1 (+62%, p < 0.05). Plasma concentrations of 17beta-oestradiol, progesterone and thyroid hormones as well as concentrations of urea and total free amino acids were not different between both groups of sows. Plasma concentrations of non-esterified fatty acids, ketone bodies, triacylglycerols and cholesterol were also largely indifferent between both groups of sows. In conclusion, this study shows that L-carnitine has less influence on lipid metabolism and utilization of nitrogen in pregnant sows but increases their plasma concentrations of IGFs. This in turn may enhance development of the placentae and the intrauterine nutrition of the fetuses. This may be the reason for increased birth weights observed in recent studies in sows supplemented with L-carnitine.

17β-Estradiol Antagonizes the Down-Regulation of Endothelial Nitric-Oxide Synthase and GTP Cyclohydrolase I by High Glucose: Relevance to Postmenopausal Diabetic Cardiovascular Disease

In postmenopausal women, the risk of diabetic cardiovascular disease drastically increases compared with that of men or premenopausal women. However, the mechanism of this phenomenon has not yet been clarified. We hypothesized that the beneficial effects of estrogen on endothelial function may be relevant to protection against hyperglycemia-induced vascular derangement. Bovine aortic endothelial cells were incubated for 72 h in the presence and absence of the physiological concentration of 17beta-estradiol (17beta-E2) under normal and high-glucose conditions. The presence of 17beta-E2 significantly counteracted the reduction in basal nitric oxide production under high-glucose conditions. This finding was associated with the recovery of endothelial nitric-oxide synthase (eNOS) protein expression, tetrahydrobiopterin (BH4) levels, and the activity and gene expression of GTP cyclohydrolase I (GTPCH-I), a rate-limiting enzyme for BH4 synthesis. Both the gene transfer of estrogen receptor alpha using adenovirus and treatment with the protein kinase C inhibitor bisindolylmaleimide I significantly enhanced the effects of 17beta-E2 treatment under high-glucose conditions, whereas these effects were abolished by the estrogen receptor antagonist ICI 182,780 (faslodex). Transfection of small-interfering RNA targeting eNOS resulted in a marked reduction in GTPCH-I mRNA under both normal and high-glucose conditions, but this reduction was strongly reversed by 17beta-E2. These results suggest that the activation of ERalpha with 17beta-E2 can counteract high-glucose-induced down-regulation of eNOS and GTPCH-I in endothelial cells. Therefore, estrogen deficiency may result in an exaggeration of hyperglycemia-induced endothelial dysfunction, leading to the development of cardiovascular disease in postmenopausal diabetic women.

Inhibition of leiomyoma cell proliferation in vitro by genistein and the protein tyrosine kinase inhibitor TKS050

To determine the potency of TKS050, a new epidermal growth factor receptor (EGFR) inhibitor and genistein, a naturally occurring protein tyrosine kinase inhibitor, to inhibit leiomyoma cell proliferation in vitro. Establishment of paired cultures of leiomyoma and normal myometrial samples. University clinical research laboratory. Hysterectomy specimens from premenopausal women affected by uterine leiomyomas. The suppressive effect of TKS050 and genistein on the cells, before and after steroidal hormone treatment, was examined. Cell proliferation, recovery after treatment, cell cycle analysis, and immunochemical analysis of relevant proteins were performed. TKS050 (2 micromol/L) and genistein (50 micromol/L) completely suppressed leiomyoma cell proliferation, and the cells did not recover after cessation of treatment. TKS050 induced cell cycle arrest and apoptosis in a dose- and time-dependent manner. Cells accumulated in the G(0)/G(1) phase of the cell cycle at the expense of the S and G(2)+M phases. Treatment of cells with TKS050 resulted in a dose-dependent inhibition of EGFR autophosphorylation and of phosphorylated signal transducer and activator of transcription 3 (Stat3). Genistein inhibited the phosphorylated Stat3 but did not affect EGFR autophosphorylation. The inhibitory effects of TKS050 or genistein were unaffected by the presence of physiologic concentrations of estradiol-17beta. Leiomyoma cell growth is effectively blocked by TKS050 and genistein. The inhibitory action of newly developed and natural inhibitors derived from diet may be useful as a possible alternative therapy for leiomyomas.

Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17β-estradiol

Estrogens are powerful mitogens that play a critical role in the onset of breast cancer and its progression. About two-thirds of all breast cancers are estrogen receptor (ER)+ at the time of diagnosis, and the ER expression is the determinant of a tumor phenotype associated with hormone responsiveness. The molecular basis of the relationship between ER expression, (anti)hormonal responsiveness, and breast cancer prognosis is still unknown. To identify the proteins affected by the presence of the hormone we used 2-D-PAGE-based bottom-up proteomics for the study of the proteome of MCF-7 cells of estrogen-responsive breast carcinoma exposed to a mitogenic concentration of 17beta-estradiol (E2) for 12, 18, 24, and 30 h. Differential expression analysis showed significant changes for 12 proteins. These include ezrin-radixin-moesin-binding phosphoprotein of 50 kDa which was previously shown to be directly regulated by E2. Expression profiles of other proteins already implicated in the progression of breast cancer, such as stathmin, calreticulin, heat shock 71 kDa, alpha-enolase are also described. Moreover, it is observed that different unexpected proteins, translation factors, and energetic metabolism enzymes are also influenced by the presence of the hormone.

Excretion Patterns of Fecal Progestagens, Androgen and Estrogens During Pregnancy, Parturition and Postpartum in Okapi (Okapia johnstoni)

The aim of the present study was to establish a simple method to monitor ovarian activity and non-invasively diagnose pregnancy in okapi (Okapia johnstoni). The feces of a female okapi were collected daily or every 3 days for 28 months. Steroids in lyophilized feces were extracted with 80% methanol, and the fecal levels of immunoreactive progestagens (progesterone and pregnanediol-glucuronide), androgen (testosterone), and estrogens (estradiol-17beta and estrone) were determined by enzyme immunoassays with commercially available antisera. Using the progesterone profiles, the durations of the luteal phase, follicular phase, and estrous cycle were determined to be 11.1 +/- 0.4, 5.3 +/- 0.6, and 16.5 +/- 0.7 days (n=22), respectively. Fecal levels of immunoreactive progesterone, pregnanediol glucuronide, and testosterone gradually increased from early pregnancy and peaked several months before parturition. More pregnanediol glucuronide was excreted in feces than progesterone during late pregnancy, but not during the estrous cycle. Although the fecal concentrations of immunoreactive estradiol-17beta and estrone change a little throughout pregnancy and non-pregnancy, they rose sharply and temporarily on the day following parturition. The present study indicates that fecal assays with commercial antisera for progesterone and pregnanediol glucuronide are useful for evaluating luteal activity and diagnosing pregnancy and indicates that estrogens might have some role as a trigger of parturition.

Environmental Estrogens Induce Mast Cell Degranulation and Enhance IgE-Mediated Release of Allergic Mediators

BackgroundPrevalence and morbidity of allergic diseases have increased over the last decades. Based on the recently recognized differences in asthma prevalence between the sexes, we have examined the effect of endogenous estrogens on a key element of the allergic response. Some lipophilic pollutants have estrogen-like activities and are termed environmental estrogens. These pollutants tend to degrade slowly in the environment and to bioaccumulate and bioconcentrate in the food chain; they also have long biological half-lives.ObjectivesOur goal in this study was to identify possible pathogenic roles for environmental estrogens in the development of allergic diseases.MethodsWe screened a number of environmental estrogens for their ability to modulate the release of allergic mediators from mast cells. We incubated a human mast cell line and primary mast cell cultures derived from bone marrow of wild type and estrogen receptor α (ER-α )–deficient mice with environmental estrogens with and without estradiol or IgE and allergens. We assessed degranulation of mast cells by quantifying the release of β -hexosaminidase.ResultsAll of the environmental estrogens tested caused rapid, dose-related release of β -hexosaminidase from mast cells and enhanced IgE-mediated release. The combination of physiologic concentrations of 17β -estradiol and several concentrations of environmental estrogens had additive effects on mast cell degranulation. Comparison of bone marrow mast cells from ER-α –sufficient and ER-α –deficient mice indicated that much of the effect of environmental estrogens was mediated by ER-α .ConclusionsOur findings suggest that estrogenic environmental pollutants might promote allergic diseases by inducing and enhancing mast cell degranulation by physiologic estrogens and exposure to allergens.

Suppression of Follicle Wave Emergence in Cyclic Ewes by Supraphysiologic Concentrations of Estradiol-17beta and Induction with a Physiologic Dose of Exogenous Ovine Follicle-Stimulating Hormone1

Follicle waves are preceded by follicle-stimulating hormone (FSH) peaks in ewes. The purpose of the present study was to see whether estradiol implant treatment would block FSH peaks to create a model in which the effect of the timing and mode of FSH peaks could be studied by ovine FSH (oFSH) injection. In Experiment 1, 10 ewes received estradiol-17beta implants on Day 4 after ovulation (Day 0, day of ovulation); five ewes received large implants, and five ewes received small implants. Five control ewes received empty implants. In Experiment 2, 12 ewes received large implants on Day 4. On Day 9, six ewes received oFSH twice, 8 h apart (0.5 microg/kg; s.c.). Implants were left in place for 10 days in both experiments. In both studies, ovarian ultrasonography and blood sampling was done daily. In Experiment 1, estradiol concentrations were significantly higher in ewes with large implants (10.4 +/- 0.7 pg/ml) compared with controls (3.9 +/- 0.7 pg/ml) and ewes with small implants (5.4 +/- 0.7 pg/ml; P < 0.001). A significant reduction was found in mean FSH peak concentration (31%; P < 0.05) and FSH peak amplitude (45%; P < 0.05) in ewes with large implants compared with controls. Mean and basal FSH concentrations were unaffected by the large implants. The large implants halted follicle-wave emergence between Day 0 and 8 after implant insertion. The small follicle pool (2-3 mm in diameter) was unaffected by the large implants. When oFSH was injected into ewes with large implants, a follicle wave emerged 1.5 +/- 0.5 days after injection; however, in ewes given saline alone, a follicle wave emerged 4.8 +/- 0.8 days after injection (P < 0.01). We concluded that truncation of FSH peaks by estradiol implants prevented follicle-wave emergence, but injection of physiologic concentrations of oFSH reinitiated follicle-wave emergence.

Effect of ormeloxifene on ovariectomy-induced bone resorption, osteoclast differentiation and apoptosis and TGF beta-3 expression

Effect of ormeloxifene, a multifunctional selective estrogen receptor modulator, on prevention of ovariectomy-induced bone resorption in retired breeder female rats, osteoclastogenesis using bone marrow cells from adult Balb/c mice cultured in presence of M-CSF and RANKL, osteoclast apoptosis using terminal deoxynucleotidyl transferase fragment end labeling and TGF beta-3 expression were investigated. Raloxifene, a benzothiophene reported to mimic effects of estrogen in bone, and estradiol were used for comparison. Ormeloxifene (10 −6 and 10 −8 M) significantly inhibited osteoclastogenesis ( P < 0.001 versus vehicle control) as evidenced by lower number of TRAP-positive osteoclasts in bone marrow cultures and caused apoptosis of osteoclasts. The effect was almost equivalent to that observed in presence of estradiol-17 beta, except that significant number of cells undergoing apoptosis was evident even at 10 −9 M concentration of estradiol-17 beta ( P < 0.001). Raloxifene, though inhibited osteoclastogenesis at much lower concentrations (10 −8 to 10 −12 M; P < 0.001), failed to cause apoptosis of osteoclasts at any of the concentrations used. While ormeloxifene, raloxifene and ethynylestradiol significantly prevented ovariectomy-induced bone loss in vivo in retired breeder female rats, prevention of ovariectomy-induced decrease in BMD and trabecular network of proximal tibia, calcium and phosphorus levels in femur and tibia and prevention of ovariectomy-induced down-regulation of TGF beta-3 expression in lumbar vertebrae was of lower order in raloxifene- than ormeloxifene- or ethynylestradiol-supplemented females. Both the SERMs, however, produced considerable estrogenic effects at the uterine level as evidenced by increase in weight, total and endometrial area and luminal epithelial cell height; the effect being generally greater in raloxifene- than ormeloxifene-treated rats. Findings demonstrate that inhibition of estrogen-deficiency osteoporosis by ormeloxifene, as in case of estradiol, was mediated via inhibition of osteoclastogenesis, apoptosis of osteoclasts and up-regulation of TGF beta-3 expression. Raloxifene, though effective in inhibiting osteoclastogenesis in vitro at much lower concentrations, was not only less potent in preventing ovariectomy-induced bone loss in retired breeder female rats in vivo but also appeared to have a different mechanism of action than ormeloxifene and estradiol.