Stimulating Estradiol Research Articles

Nuclear type II [ 3H]estradiol binding site ligands: inhibition of ER-positive and ER-negative cell proliferation and c-Myc and cyclin D1 gene expression

These studies assessed the effects of 3,4-dihydroxybenzalacetone (ZN-1) and 1-(3,4-dihydroxyphenyl)-2-propanol (ZN-2) on MCF-7 cell proliferation. The compounds blocked [ 3H]estradiol binding to nuclear type II sites, but did not compete for [ 3H]estradiol binding to recombinant ERα or ERβ. ZN-1 and ZN-2 inhibited the proliferation of ERα and ERβ positive (MCF-7) and negative (MCF-10A) breast cells, further ruling out direct binding to ER in the mechanism of action of these compounds. Pre-loading type II sites with ZN-1 or ZN-2 reduced [ 3H]estradiol exchange, strongly suggesting the drugs were binding covalently. ZN-1 treatment resulted in complete occupancy of type II sites and sustained (9 days) inhibition of MCF-7 cell proliferation following its removal from the tissue culture medium. This cell growth inhibition was not due to non-specific toxicity, as the numbers of viable, attached cells per dish (determined by trypan blue dye exclusion) remained constant throughout this 9-day period and eventually reversed by day 19. ZN-2 effects on cell proliferation reversed more rapidly following discontinuation of treatment, a response consistent with the inability of the compound to totally block type II binding. Both ZN-1 and ZN-2 blocked estradiol stimulation of c-Myc and cyclin D1 gene expression in MCF-7 cells, two events that are clearly coupled to cell cycle progression. We suspect this may occur through ZN-1 or ZN-2 modification of nucleosome function and/or chromatin remodeling since nuclear type II sites are localized to a complex of histones H3 and H4 (Shoulars et. al, J Steroid Biochem. Mol. Biol. 96: 19–30, 2005).

PTHrP potentiating estradiol-induced vitellogenesis in sea bream ( Sparus auratus, L.)

In fish, vitellogenin is an important nutritional precursor protein produced solely in the liver and released into the blood where it binds calcium. In the gilthead sea bream ( Sparus auratus) 17β-Estradiol (E 2) plays an important role in the synthesis of vitellogenin, but also the pituitary hormones prolactin (PRL) and growth hormone (GH) can stimulate vitellogenin induction in fish. Considering the emerging involvement of PTHrP in fish calcium metabolism and the importance of calcium regulation in reproduction, we investigated the possible role of PTHrP in vitellogenesis. E 2-naïve and E 2-primed sea bream hepatocytes were used in an in vitro primary hepatocyte culture and stimulated with a recombinant sea bream PTHrP (sbPTHrP) to establish the contribution of sbPTHrP alone or in combination with E 2 to the regulation of hepatic vitellogenin synthesis. Hepatocytes stimulated solely with sbPTHrP were not affected in their vitellogenesis. However, in hepatocytes stimulated with E 2 in combination with sbPTHrP a higher vitellogenin production was seen than with E 2 alone. It is concluded that sbPTHrP has a potentiating effect on estradiol stimulation of vitellogenin production by sea bream hepatocytes. The sea bream provides a unique model where vitellogenesis regulation can be studied on E 2-naïve liver cells, both in vivo and in vitro.

Estradiol stimulates the biosynthetic pathways of breast cancer cells: Detection by metabolic flux analysis

Selective estrogen receptor (ER) modulators are highly successful breast cancer therapies, but they are not effective in patients with ER negative and selective estrogen receptor modulator (SERM)-resistant tumors. Understanding the mechanisms of estrogen-stimulated proliferation may provide a route to design estrogen-independent therapies that would be effective in these patients. In this study, metabolic flux analysis was used to determine the intracellular fluxes that are significantly affected by estradiol stimulation in MCF-7 breast cancer cells. Intracellular fluxes were calculated from nuclear magnetic resonance (NMR)-generated isotope enrichment data and extracellular metabolite fluxes, using a specific flux analysis algorithm. The metabolic pathway model used by the algorithm includes glycolysis, the tricarboxylic acid cycle (TCA cycle), the pentose phosphate pathway, glutamine catabolism, pyruvate carboxylase, and malic enzyme. The pathway model also incorporates mitochondrial compartmentalization and reversible trans-mitochondrial membrane reactions to more accurately describe the role of mitochondria in cancer cell proliferation. Flux results indicate that estradiol significantly increases carbon flow through the pentose phosphate pathway and increases glutamine consumption. In addition, intra-mitochondrial malic enzyme was found to be inactive and the malate-aspartate shuttle (MAS) was only minimally active. The inactivity of these enzymes indicates that glutamine is not oxidized within mitochondria, but is consumed primarily to provide biosynthetic precursors. The excretion of glutamine carbons from the mitochondria has the secondary effect of limiting nicotinamide adenine dinucleotide (NADH) recycle, resulting in NADH buildup in the cytosol and the excretion of lactate. The observed dependence of breast cancer cells on pentose phosphate pathway activity and glutamine consumption for estradiol-stimulated biosynthesis suggests that these pathways may be targets for estrogen-independent breast cancer therapies.

Relationships among Self-Rated Tanner Staging, Hormones, and Psychosocial Factors in Healthy Female Adolescents

Study Objective Females are more likely than males to suffer from various mood and pain disorders. However, this female predominance does not appear to develop until puberty. The purpose of the present study was to examine the associations among hormone concentrations, self-rated Tanner staging and dysmenorrhea, and to determine whether various psychosocial factors modulate these relationships. Design As part of a larger prospective study of the influence of puberty on laboratory pain response, Tanner ratings, estradiol and follicle stimulating hormone concentrations, dysmenorrhea and psychosocial parameters including depression, anxiety, somatization, and socioeconomic status were observed. Participants 124 healthy girls ages 8 to 18 were evaluated. Main Outcome Measure There were significant correlations between pubertal status utilizing self-rated diagrams of Tanner stage and hormonal markers of pubertal development, specifically estradiol and FSH. Tanner stage but not estradiol was correlated with presence of dysmenorrhea. There was no effect of body mass index (BMI), socioeconomic status (SES), anxiety, depression, or somatization on presence or absence of painful menses. There was no correlation between BMI or SES and age of menarche. However, Tanner stage was correlated with BMI but not with SES. Results and Conclusions Tanner self-staging is at least as accurate as estradiol and FSH measurements alone in evaluating healthy female adolescents' physical changes and menstrual pain. Tanner self-ratings are thus particularly useful in large, epidemiologic, or cross cultural studies where physical examination and blood sampling may not be feasible due to cost, access, or psychosocial barriers.

Repeated 28-day oral toxicity study of ketoconazole in rats based on the draft protocol for the “Enhanced OECD Test Guideline No. 407” to detect endocrine effects

We performed a 28-day repeated-dose toxicity study of ketoconazole, a widely used an antimycotic drug, based on the draft protocol of the "Enhanced OECD Test Guideline 407" (Enhanced TG407) to investigate whether ketoconazole has endocrine-mediated properties according to this assay. Seven-week-old SD rats were administered with ketoconazole daily by oral gavage at doses of 0, 6.25, 25 or 100 mg kg(-1) day(-1) for at least 28 days. The ketoconazole-treated male rats showed reduction of epididymis and accessory sex organ weights, spermatid retention in the seminiferous tubules, decrease of testosterone and increases of estradiol, luteinizing hormone (LH) and follicular stimulating hormone (FSH). A prolongation of the estrous cycle and increases of estradiol, LH and FSH were observed in the treated female rats. Thyroxin and triiodothyronine were decreased and thyroid-stimulating hormone was increased in both sexes; however, there were no compound-related microscopic lesions in the thyroid gland or changes in the thyroid weight. The endocrine-related effects of ketoconazole could be detected by the parameters examined in the present study based on the Organization for Economic Cooperation and Development (OECD) protocol, suggesting that the Enhanced TG407 protocol should be a suitable screening test for detection of endocrine-mediated effects of chemicals.

Expression of estrogen receptors‐α and ‐β in bladder cancer cell lines and human bladder tumor tissue

Estrogen receptors (ERs) are known to mediate important physiologic responses as well as the growth of some tumors in response to estradiol stimulation. In a previous study the selective ER modulator raloxifene was shown to induce apoptosis in an ERbeta-positive bladder cancer cell line. However, the expression of ERbeta in human bladder cancer has not been thoroughly investigated. ERalpha and ERbeta expression in 224 bladder tumor samples was evaluated using tissue microarray and immunohistochemistry. Levels of ERalpha and ERbeta protein and mRNA expression were determined in several bladder cancer cell lines using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of estradiol and antiestrogen treatments on RT4 bladder cancer cell growth was determined by cell proliferation assays. Analyses revealed that only 2 human bladder cancers weakly expressed ERalpha. In contrast, the expression of ERbeta was detected in 141 tumors (63%). ERbeta was expressed in 58% of WHO Grade 1 and 2 tumors, whereas 70% of Grade 3 tumors demonstrated expression (P = .085). Importantly, although only 53% and 55% of Ta and T1 tumors demonstrated ERbeta expression, 80% of T2, 81% of T3, and 75% of T4 tumors showed ERbeta expression. The differences in ERbeta expression between Ta/T1 and T2/T3/T4 tumors were found to be highly significant (P < .001). Metastatic transitional cell carcinomas had ERbeta expression (80%) comparable to that of muscle invasive bladder cancers. Western blot analysis detected ERbeta protein expression in each of the 5 bladder cancer cell lines tested, whereas no or very low levels of ERalpha were found. Quantitative RT-PCR revealed that higher levels of ERbeta than ERalpha mRNA were present in 5637, T-24, TSU-Pr1, and TCC-Sup bladder cancer cells, whereas ER-alpha mRNA levels were greater than ERbeta in RT4 cells. Treatment with 17beta-estradiol modestly increased RT4 cell growth, whereas the antiestrogens, 4-hydroxtamoxifen, raloxifene, or ICI 182,780 inhibited the growth of RT4 cells. ERbeta is the dominant receptor expressed in bladder cancer cell lines and in the majority of human bladder tumors. Moreover, the degree of ERbeta expression increases with increasing stage and grade of differentiation. Antiestrogens have an inhibitory effect on the growth of bladder cancer cells in vitro.

Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells

Estrogens and androgens influence many properties of breast cancer cells; hence, regulation of local estrogen and androgen levels by enzymes involved in steroid hormone biosynthesis and metabolism would impact signaling by these hormones in breast cancer cells. In this study, we show that the UDP-glucuronosyltransferase (UGT) enzyme UGT2B15, a member of the UGT family of phase II enzymes involved in the glucuronidation of steroids and xenobiotics, is a novel, estrogen-regulated gene in estrogen receptor (ER)-positive human breast cancer cells (MCF-7, BT474, T47D, and ZR-75). UGT2B15 is the only UGT2B enzyme up-regulated by estrogen, and marked estradiol stimulation of UGT2B15 mRNA levels is observed, in a time- and dose-dependent manner. UGT2B15 stimulation by estradiol is blocked by the antiestrogen ICI182,780, but not by the translational inhibitor cycloheximide, indicating that UGT2B15 is likely a primary transcriptional response mediated through the ER. UGT2B15 up-regulation is also evoked by other estrogens (propylpyrazoletriol, genistein) and by the androgen 5alpha-dihydrotestosterone working through the ER, but not by other steroid hormone receptor ligands. Western blot and immunocytochemical analyses with several UGT2B-specific antibodies we have designed and steroid glucuronidation assays indicate a large increase in both cellular UGT2B15 protein and enzyme activity after estrogen treatment. Due to the important role of UGT enzymes in forming conjugates between steroids and glucuronic acid, thereby inactivating them and targeting them for removal, the estrogen-induced up-regulation of UGT2B15 might have a significant moderating effect on estrogen and androgen concentrations, thereby reducing their signaling in breast cancer cells.

Feasibility study of Nestorone®–ethinylestradiol vaginal contraceptive ring for emergency contraception

Objective The Nestorone®/ethinylestradiol (NES/EE) vaginal ring is being developed as a regular contraceptive method by the Population Council. This ring is designed to release NES 150 μg/day and EE 15 μg/day during 1 year. Here, we report a Phase I clinical trial to determine the usefulness of this ring for emergency contraception. To that end, we tested the ability of this ring to interfere with ovulation when it is inserted during the follicular phase. Method Forty-eight women protected from the risk of pregnancy by nonhormonal methods were divided into three groups, which differed by the size of the dominant follicle at the time of ring insertion: 12–14 mm ( n=16), 15–17 mm ( n=18) and ≥18 mm ( n=14) diameter. The NES/EE ring was left in the vagina for 7 consecutive days, after which it was removed. The growth of the leading follicle and plasma levels of estradiol, progesterone (P), luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the ensuing 5 days after ring insertion were determined. Afterwards, steroid hormones were measured twice a week, until menses took place. All women had a control cycle before the ring cycle, and the range of maximum follicular diameter assigned to each volunteer was the same for the control and the ring cycle at the time when placebo was ingested or the ring inserted. Results During the 5-day period after ring insertion with follicles 12–17 mm, ovulation was absent in 25 of 34 cycles (p<.01 vs. control), and ovulatory dysfunction (absent, blunted or mistimed LH peak) occurred in 8 of the 9 remaining cycles (33/34 ovulatory processes altered; p<.005 vs. control). After ring insertion with follicles ≥18 mm in diameter, ovulation did not occur in 2 of 14 cycles or was dysfunctional in 7 of the 12 remaining cycles (9/14 ovulatory processes altered; p<.025 vs. control). Altogether, 87.5% of ring cycles (42/48) had either no ovulation or ovulatory dysfunction in the 5-day study period, in contrast to 39.6% (19/48 cycles) in control cycles (p<.001). Among follicles that failed to rupture within the 5-day study period, none ruptured later on in the ring-treated cycles, while 9 of 16 did so in control cycles. Sixty-two percent of ring-treated cycles were shorter than 24 days. Nausea, vaginal discharge and abdominal pain were the most frequently reported adverse events during ring use. Conclusion Interference with 87.5% of ovulatory processes, without ovulation occurring later in the cycle and shortening of cycle length, suggests the NES/EE ring may be used as an emergency contraceptive method, with the potential advantage of providing continuing contraception after it has performed its emergency function.

Serum C-reactive protein and plasma homocysteine levels are associated with hormone therapy use and other factors: a population-based study of middle-aged Australian-born women

Objective To investigate the associations between C-reactive protein (CRP), homocysteine levels, use of hormone therapy (HT) and other factors.Methods A 12-year prospective study of 438 Australian-born women (Melbourne Women's Midlife Health Project), who at baseline were aged 45–55 years, had menstruated in the previous 3 months and were not taking HT. Fasting blood was collected in the 11th follow-up year for CRP, homocysteine, estradiol and follicle stimulating hormone (FSH) levels. Physical measurements and face-to-face interviews obtained information on health and lifestyle variables.Results A total of 258 women (mean age 60 years) participated in the 11th follow-up year. Multiple regression analysis found that CRP levels were positively associated with body mass index (p < 0.001), HT use (p < 0.01), and negatively associated with statin use (p < 0.005) and exercising (p < 0.05). In postmenopausal women currently not using HT (n = 173) and after adjusting for body mass index, exercise and smoking, CRP was negatively associated with FSH levels (β = −0.32, p < 0.05). Homocysteine levels were positively associated with smoking (p < 0.001) and negatively associated with HT use (p < 0.05).Conclusion In middle-aged Australian-born women, HT use was associated with increased CRP and decreased homocysteine levels. High CRP levels were also associated with high relative weight, low exercise levels and no statin medication.

Expression of estrogen-regulated genes in ER positive primary and metastatic breast cancer patients: possible applications for personalized treatment

9696 Background: Breast cancer is characterized by an important histo-clinical heterogeneity. New prognostic and predictive factors are needed to allow an individualization of therapy for patients with ER positive breast cancer since approximately 50% respond to endocrine therapy. Methods: We have carried out a data mining exercise and identified genes up-regulated by estrogen (> two fold) and developed Real-Time quantitative RT-PCR using LightCycler to identify a panel of 26 genes that would enable us to determine individual profiles and tailor therapy on this basis. Results: Of the 26 genes analyzed, 9 (ERα, PR, EEIG-1, PS2, GREB 1a, 1b and 1c, GATA-3 and EP-3) were shown to be expressed preferentially in a panel of ERα positive breast cancer cell lines and responsive to estradiol stimulation. The fold increases were: ERα 1–3 (after 24h), PR 2–6 (after 48h), PS2 2.5–18 (after 48h), EEIG-1 1–8 (after 8h), GATA-3 1–20 (after 48h) and EP-3 3–10 (after 24h). The expression pattern of GREB 1a, 1b and 1c, was also examined but not quantitated. The expression of the 9 genes was analyzed using purified breast cancer cells from 22 primary tumors, 3 involved lymph nodes and 7 metastatic effusions. ERα and PR expression was confirmed in all the ERα/PR positive tumors. The primary patients were categorized on the basis of immunohistochemistry into three groups ER+/PR+, ER+/PR- and ER-/PR-. The greatest difference in expression was seen between the ER/PR+ and the ER/PR- patients for ER (p <0.0005), PS2 (p =0.0005), EEIG-1 (p <0.0005) and GATA-3 (p<0.0005). 3/7 of the metastatic patients did not express ERα or any of the ER target genes. This evidence is of particular interest and could explain the presence of acquired resistance to anti-endocrine therapy in those particularly patients and may be a useful tool in guiding treatment choice. Conclusions: Limited gene profiling in individual breast cancer patients may enable us to predict those patients who will benefit from targeted therapy. This individualized classification of primary tumors based on ex vivo analysis, may lead to more effective therapeutic decisions. No significant financial relationships to disclose.

Sufficient Progesterone-Priming Prior to Estradiol Stimulation Is Required for Optimal Induction of the Cervical Prostaglandin System in Pregnant Sheep at 0.7 Gestations1

The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106-108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121-123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE(2) receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.

No evidence of mutations in the P450 aromatase gene in patients with polycystic ovary syndrome

Etiology and inheritance pattern in polycystic ovary syndrome (PCOS) remain uncertain. Granulosa cells from follicles of women with PCOS have little, if any, aromatase (encoded by the CYP19 gene) activity; follicles contain low levels of estradiol, P450arom mRNA and aromatase stimulating bioactivity. Mice with targeted disruption of the CYP19 gene present cystic follicles. It has been proposed that chronic exposure to high levels of LH, because of aromatase deficiency, determines the development of ovarian cysts. Herein, we investigated if mutations in the CYP19 gene and/or its ovary promoter are causal in patients with PCOS. Twenty-five patients with PCOS and 50 control women were studied. PCR analysis of genomic DNA and complete sequence of all exons of the aromatase gene and its ovary promoter were performed. No heterozygous or homozygous mutant alleles were present in any of the patients studied. In the population studied, mutations of the P450arom gene or its promoter are not the cause of PCOS. However, these findings do not preclude the possible importance of an aromatase disorder in PCOS etiology. Variations in aromatase complex function could play a role in PCOS etiology, but the determinants of such variations might be located in other genes.

Modular effects of estradiol on ethanol-induced apoptosis in human intestinal epithelial cells

Objective. Epidemiological data indicate that females develop alcohol-induced liver disease (ALD) more rapidly and more severely than males. Though the contribution of gut-derived endotoxin to the onset and development of ALD suggests the loss of epithelial cell viability that results in impaired intestinal function due to alcohol exposure, the additional effects of female sex hormones on intestinal cell viability is not known. The aim of this study was to examine the influence of estradiol on the intestinal cell death induced by acute and low concentrations of ethanol in an in vitro system.Material and methods. Human intestinal epithelial Caco-2 cells were incubated with 0, 5, and 10% ethanol for 3 h. Estradiol stimulation, concentration of 3, 30, and 300 pg/ml occurred in the presence or absence of 10% ethanol for 2 h. Phosphatidylserine (PS) externalization, caspase-mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were quantified using flow cytometry.Results. Treatment with 10% ethanol markedly induced PS externalization, caspase activation, and DNA fragmentation after 2 h incubation. Whereas estradiol itself did not affect cell viability, physiological concentrations of estradiol enhanced PS externalization and DNA fragmentation induced by 10% ethanol, and these were remarkable at 300 pg/ml estradiol.Conclusions. Ethanol-induced apoptosis was potentiated by physiological concentrations of estradiol, especially at the higher level which is found only in females. Our data suggest that enhanced ethanol-induced intestinal epithelial cell apoptosis in the presence of estradiol could cause greater intestinal permeability, which allows endotoxin to enter the circulation and eventually results in more severe ALD in females.

The pharmacokinetics and pharmacodynamics of 17-d-norgestimate and ethinyl estradiol alone and in the presence of cilansetron 2 mg TID, in healthy females

Background/Aims To assess the effects of cilansetron (CIL) 2 mg on the blood concentrations of ethinyl estradiol (EE), 17-d-Norgestimate (NG), progesterone (P), lutenizing hormone (LH), and follicle stimulating hormone (FSH) in healthy females receiving an oral contraceptive(OC). Methods Design A double-blind, balanced, randomized, two-period, placebo-controlled, cross-over study. Setting This study was conducted in a phase I clinic. Participants Twenty (20) females receiving OC daily for the first 21 days of two consecutive menstrual cycles. Intervention CIL 2 mg or placebo was administered orally three times daily from days 14 to 21 of each cycle. Blood samples for the measurement of PK and PD parameters were obtained. Statistical analysis of the PK and PD parameters was conducted using an ANOVA. Results (see Table) Conclusions Although EE levels were lower during co-administration of CIL (2 mg tid), no PK/PD interaction between CIL and EE and NG was observed in healthy females. Clinical Pharmacology & Therapeutics (2005) 77, P56–P56; doi: 10.1016/j.clpt.2004.12.103 Treatment1 Mean (SD) LS Mean2 (A/B) 90% CI EE AUC0-24(hr*ng/ml) A 1.21 (0.55) 1.07 90.15 (85.76–94.76) B 1.32 (0.57) 1.18 - - Cmax (ng/ml) A 0.14 (0.06) 0.13 91.87 (84.76–99.58) B 0.16 (0.06) 0.14 - - NG AUC(hr*ng/ml) A 18.90 (5.75) 17.34 97.29 (91.48–103.47) B 19.47 (6.08) 17.82 - - Cmax (ng/ml) A 2.33 (0.89) 2.09 101.87 (90.09–115.20) B 2.29 (0.79) 2.06 - - 1 A=OC+CIL, B=OC+Placebo Least Squares Mean. No difference was observed in blood levels of P, LH and FSH in the presence of CIL

Induction of Uterine Prostaglandin H Synthase 2 by Estradiol Following Fetal Adrenalectomy

In sheep, fetal cortisol stimulates the conversion of progesterone to estradiol in late gestation initiating labor. It is unclear whether an intact fetal hypothalamic-pituitary-adrenal (HPA) axis is required to induce the estradiol-triggered subsequent endocrine changes including enhanced intrauterine prostaglandin (PG) synthesis associated with the onset of labor. We have shown that maternal estradiol administration stimulates PG H synthase (PGHS)-2 expressions in pregnant ovine intra-uterine tissues. The current study was undertaken to determine whether the fetal adrenal mediates estradiol's stimulation of the intrauterine PGHS-2 in pregnant sheep. Placenta, myometrium, and endometrium were collected from two groups of ewes at 123-127 d of gestational age (dGA) after fetal adrenalectomy and vehicle treatment (ADX; n = 5); or fetal ADX and maternal estradiol administration (5 mg twice a day for 2 d, ADX+E2, n = 5). PGHS-2 mRNA and protein were analyzed by Northern and Western Blot analyses in both groups and presented as the ratios to beta actin mRNA for Northern and G protein beta subunit for Western blot analysis. Fetal plasma cortisol was measured by radioimmunoassay. Data were analyzed by Student's t test. Fetal plasma cortisol levels were low in ADX and ADX+ E2 groups (<6 ng/mL). The cervix of all ADX+E2 treated ewes was dilated at necropsy. Three out of five ADX+ E2-treated ewes delivered within 48 h. The cervix was closed in all fetal ADX ewes at necropsy. PGHS-2 mRNA and protein increased (p < 0.05) in myometrium and endometrium, but not placenta in ADX+E2-treated ewes compared with ADX group. These data provide the first in vivo evidence for estradiol upregulation of intrauterine PGHS-2 in late gestation in the absence of an intact fetal HPA axis. Thus, the fetal adrenal is not required to mediate estradiol's stimulation of uterine PGHS-2 expression associated with the onset of labor.

Particularities of reproduction and oogenesis in teleost fish compared to mammals

Compared to mammals, teleost reproduction presents many original features. Reproductive strategies of species are diversified into numerous adaptations to a large variety of aquatic environments. This diversity may concern sexuality, spawning and parental behaviour, sensitivity to environmental factors, and specific features of gametogenesis such as the duration of vitellogenesis, and egg morphology. Sexuality presents a variety of natural modalities, from gonochorism to hermaphrodism. The absence of definitive arrest of body growth in the adult of most species gives a particular interest to the practical control of growth-reproduction interactions. Vitellogenesis, which represents an important metabolic effort for the maternal organism, involves the synthesis of vitellogenin, a specific glycolipo-phosphoprotein produced in the liver under estradiol stimulation, and its incorporation into oocytes by a receptor mediated process. Both estradiol synthesis in follicle cells and vtg uptake by vitellogenic follicles appear to be mainly controlled by FSH. Oocyte maturation is directly triggered by a progestin, or MIS (maturation inducing steroid) synthesised in follicle cells mainly under LH control, and acting through the non-genomic activation of a membrane receptor. Practical applications of some of these particularities result mainly from the external character of the fertilisation process and of embryonic development, which allows manipulating respectively egg chromosome stocks and sex differentiation. Moreover, the sensitivity of sex differentiation to exogenous factors favours the development of practical methods to control the sex of farmed populations. Finally, the sensitivity of reproductive mechanisms to xenobiotics has led to various kinds of bioassays for putative pollutants.

Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

Previously, we have identified an imperfect estrogen response element (rtERE) in the promoter of the rainbow trout vitellogenin gene. Although this ERE leads to a lower transcriptional activation, a better estradiol stimulation in vivo as compared to consensus ERE (EREcs) was observed. Here we examine the ability of recombinant human estrogen receptor alpha (rhERalpha) to bind DNA containing the EREcs or the natural imperfect rtERE, which contains three mismatches. At low salt concentration, whatever the ERE sequence, dissociation equilibrium constants of the specific rhERalpha-ERE complexes are similar (K(D) = 2 nM) with the same stoichiometry. As salt concentration increases from 80 to 200 mM KCl, the affinity of the rhERalpha-rtERE complex largely diminishes whereas that of rhERalpha-EREcs seems less affected. Hence the nature of the interactions stabilizing these complexes is different: more ionic in rhERalpha-rtERE as compared to rhERalpha-EREcs. Moreover, kinetic measurements showed that specific rhERalpha-ERE complexes exhibit shorter half-lives (few seconds) and that the rhERalpha-EREcs complex is more stable (33 s) than the complex that formed with rtERE (19.8 s), in accordance with equilibrium binding results. Finally, dynamic studies of rhERalpha have shown that the protein fluctuations are damped when the salt concentration increases or when bound to ERE and all the more with rtERE. The interplay of affinity, complex half-lives, and protein dynamics in the transcriptional regulation of estrogen receptor is discussed.

Neuroactive pregnanolone isomers during pregnancy.

The pregnanolone isomers (PI) allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one), isopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one), epipregnanolone (3beta-hydroxy-5beta-pregnan-20-one), progesterone, and estradiol were measured in 138 pregnant women. The sampling was carried out from the first through the 10th month of pregnancy. Gas chromatography-mass spectrometry analysis and RIA were used for the measurement of steroid levels. The ratios of individual PI were similar to those found previously around parturition: about 25:10:7:1 for allopregnanolone, pregnanolone, isopregnanolone, and epipregnanolone, respectively. All the PI showed a significant increase during pregnancy, which was more pronounced in the 3alpha-steroids. The results indicated changing ratios between 3alpha- and 3beta-PI and between 5alpha- and 5beta-PI throughout pregnancy. The constant allopregnanolone/isopregnanolone ratio found through pregnancy weakened the hypothesis of the role of isopregnanolone in the onset of parturition. The ratio of estradiol (stimulating uterine activity) to 5alpha-PI and epipregnanolone exhibited significant changes during pregnancy in favor of estradiol up to the sixth or seventh month, in contrast to the constant estradiol/pregnanolone ratio. A pregnancy-stabilizing role of pregnanolone, counterbalancing the stimulating effect of estradiol on the onset of parturition, was suggested.

FC‐10 Adult‐onset hair loss in Chesapeake Bay retrievers: a clinical and histological study

Hair loss in Chesapeake Bay retrievers has been increasingly recognized by breeders in recent years. Anecdotal reports suggest an endocrine disorder or follicular dysplasia as the underlying cause, but no scientific study has been done to investigate the underlying problem. A prospective study was carried out in collaboration with the American Chesapeake Club. Affected dogs were recruited into the study. Routine dermatological and hormonal (blood and urine) tests, and skin biopsies were performed. Ten dogs (age 1.5–10 years), seven females (two spayed) and three males (two neutered), were included in the study. All dogs had mild or severe hair loss affecting the lateral ventral chest, flanks, rump and thighs. Affected dogs were clinically healthy. Hormonal tests revealed normal thyroid hormone panels, insulin‐like growth factor‐1 levels, and urinary cortisol:creatinine ratios in samples collected for ten consecutive days. In six of 10 dogs, an adrenal hormone panel showed slight or moderate increased values pre‐ and/or post‐ACTH stimulation of cortisol (three of six), 17‐hydroxyprogesterone (five of six), androstenedione (three of six), estradiol (two of six) and progesterone (six of six). The major histopathologic changes resembled canine flank alopecia and follicular dysplasia with pronounced infundibular hyperkeratosis, mild follicular atrophy, and occasional melanin clumping with dystrophic hair shafts. Chesapeake Bay retrievers suffer from a type of hair loss that is likely related to an abnormal production of adrenal sex hormone. Further studies are currently underway to determine if there is a heritable basis for this disease and to evaluate therapeutic options. Funding: University of Pennsylvania.

Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes

Protein supplementation during in vitro maturation can profoundly affect both the rate and overall efficiency of the maturation procedure [Biol. Reprod. 66 (2002) 901]. The present study was conducted to assess the ability of different concentrations (1, 5, and 10%) of bovine follicular fluid (bFF) to support in vitro maturation of oocytes and subsequent developmental capacity. The bFF was derived either from competent follicles (>8 mm) obtained by transvaginal recovery following superovulation or from a pool of small follicles (2–5 mm) from abbatoir-derived ovaries. Bovine oocytes were cultured for 24 h in synthetic oviduct fluid medium (m-SOF) supplemented with polyvinylpyrrolidone. Following fertilization and embryo culture, more oocytes ( P<0.05) reached the blastocyst stage when oocytes were cultured with 5% bFF from competent follicles (41±3.7%) compared with bFF derived from small follicles (16±2.9%). Estradiol and recombinant human follicle stimulating hormone added to the competent bFF during maturation acted in synergy to increase blastocyst production rate ( P<0.05); this blastocyst production rate (57±1.2%) was higher than those obtained with the addition of these two hormones to bFF derived from small follicles (26±2.9%). The quality of blastocysts obtained was reflected by inner cell mass (51.30±3.5 and 25.50±3.7) and trophectoderm cell numbers (99.72±2.5 and 94.80±4.7) for bFF from competent and small follicles, respectively. In conclusion, follicular fluid originating from competent follicles increased the developmental competence of abbatoir-derived oocytes.