Biol Reprod. 2002 Jun;66(6):1862-8.
Autocrine role of transforming growth factor beta1 on rat granulosa cell proliferation.

Saragueta PE, Lanuza GM, Baranao JL.

Instituto de Biologia y Medicina Experimental-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,1428 Buenos Aires, Argentina. sarana.uba.ar

We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12021073&dopt=Abstract estradiol [PubMed - indexed for MEDLINE]<



Endocrinology. 2002 Jun;143(6):2284-92.
Estradiol-sensitive afferents modulate long-term episodic firing patterns of GnRH neurons.

Nunemaker CS, DeFazio RA, Moenter SM.

Department of Internal Medicine and National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22908, USA.

GnRH neurons comprise the final common pathway of an estrogen-sensitive pattern generator controlling fertility. To determine estradiol effects on GnRH neuron firing patterns, adult transgenic mice were ovariectomized (OVX), and half were treated with estradiol (OVX+E). One week later targeted single-unit extracellular recordings were made from GnRH neurons identified by green fluorescent protein expression. Estradiol markedly affected GnRH neuron firing patterns, increasing the percentage and duration of time these cells were quiescent (< or = 1 action current/min). Estradiol increased the interval between episodes of increased firing rate determined by Cluster analysis of recordings more than 45 min (OVX+E 38.8 +/- 7.2 min, OVX 16.7 +/- 2.1 min, n = 6 each). Possible mechanisms of estradiol modulation were examined by simultaneously blocking ionotropic secretion of gamma-aminobutyric acid and glutamatergic receptors. This treatment had no effect on cells from OVX mice (n = 10), indicating episodic firing of GnRH neurons is not driven by activation of these receptors. Receptor blockade eliminated estradiol effects on GnRH neurons in the midventral preoptic area (n = 7) but not elsewhere (n = 7). Individual GnRH neurons thus display episodic firing patterns at intervals previously reported for secretory pulses. Estradiol modulates episode frequency to exert feedback control; in a substantial subset of GnRH neurons, estradiol feedback is enforced via GABAergic and/or glutamatergic afferents.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12021193&dopt=Abstract estradiol




Ann Clin Biochem. 2002 May;39(Pt 3):184-93.
Hormone replacement therapy (oestradiol-only preparations): can the laboratory recommend a concentration of plasma oestradiol to protect against osteoporosis?

Armston A, Wood P.

Department of Chemical Pathology, Southampton University Hospital Trust, UK. anniearmstootmail.com

Target concentrations of plasma oestradiol to protect against osteoporosis have now been published in at least one textbook and are increasingly discussed by clinicians. A survey of the literature revealed that many reports do not give details of oestradiol assay methods, despite the fact that there is a wide range of biases. Plasma oestradiol measurements made after ingestion of equine oestrogen preparations are not valid, and for oral oestradiol and oestradiol valerate preparations a single untimed sample does not give a reliable estimate of the average exposure over 24 h. Oestradiol measurements in plasma from women using patch, gel or implant preparations could be useful provided that consideration is given to the time of sampling and to the assay method used, although target levels have not been established. Further studies are required in this area.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12038591&dopt=Abstract estradiol




Domest Anim Endocrinol. 2002 Jun;22(4):237-54.
Influence of gonadotropins on insulin- and insulin-like growth factor-I (IGF-I)-induced steroid production by bovine granulosa cells.

Spicer LJ, Chamberlain CS, Maciel SM.

Department of Animal Science, Oklahoma State University, Stillwater, OK 74078, USA. igf1lekstate.edu

To determine the effect of gonadotropins on insulin- and insulin-like growth factor (IGF-I)-induced bovine granulosa cell functions, granulosa cells from bovine ovarian follicles were cultured for 2 days in the presence of 10% fetal calf serum (FCS), and then cultured for an additional 2 days in serum-free medium with added hormones. In the presence of 0 or 1 ng/mL of insulin or IGF-I, FSH had little or no effect (P > 0.05) on estradiol production by granulosa cells from both small (1-5mm) and large (> or = 8mm) follicles. However, in the presence of > or = 3 ng/mL of insulin, FSH increased (P < 0.05) estradiol production by granulosa cells from small and large follicles such that the estimated dose (ED(50)) of insulin necessary to stimulate 50% of the maximum estradiol production was decreased by 2- to 3-fold from 22 to 28 ng/mL in the absence of FSH to 7-14 ng/mL in the presence of FSH. Similarly, in the presence of > or = 3 ng/mL of IGF-I, FSH increased (P< 0.05) estradiol production by granulosa cells from small and large follicles such that the ED(50) of IGF-I for estradiol production was decreased by 4- to 5-fold from 25 to 36 ng/mL in the absence of FSH to 5-6 ng/mL in the presence of FSH. In the presence of FSH, the maximal effect of insulin on estradiol production was much greater than that of IGF-I (137- versus 12-fold increase) and were not additive; when combined, 100 ng/mL of IGF-I completely blocked the stimulatory effect of 100 ng/mL of insulin. In the absence of FSH, the maximal effect of insulin and IGF-I on estradiol production was similar. Concomitant treatment with 30 ng/mL of LH reduced (P<0.05) insulin-stimulated estradiol production by 5




J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Jul 5;774(1):33-8.
Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics.

Alkharfy KM, Frye RF.

Department of Pharmaceutical Sciences, University of Pittsburgh, 807 Salk Hall, Pittsburgh, PA 15261, USA.

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12052720&dopt=Abstract estradiol




Int J Pharm. 2002 Jun 20;240(1-2):55-66.
Iontophoretic estradiol skin delivery and tritium exchange in ultradeformable liposomes.

Essa EA, Bonner MC, Barry BW.

Drug Delivery Group, School of Pharmacy, University of Bradford, BD7 1DP, Bradford, UK.

This work evaluated the in vitro transdermal iontophoretic delivery of tritiated estradiol from ultradeformable liposomes compared with saturated aqueous solution (control). Effects of current density and application time on tritium exchange with water were also determined. Penetration studies used three Protocols. Protocol I involved occluded passive steady state estradiol penetration from ultradeformable liposomes and control. The effect of current densities on drug penetration rates was also assessed (Protocol II). In Protocol III, three consecutive stages of drug penetration (first passive, iontophoresis and second passive) through the same human epidermal membranes were monitored. Such an experimental design investigated the possible effect of high current density (0.8 mA/cm(2)) on skin integrity. The tritium exchange study showed that extent of exchange correlated well with current density and time of application, with some shielding of estradiol by the liposomal structure. Liposomes enhanced estradiol passive penetration after occlusion. Protocol II showed that estradiol flux increased linearly with current density, although being delivered against electroosmotic flow. In Protocol III, reduction in flux of the second passive stage to near that of the first reflected a reversibility of the structural changes induced in skin by current.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12062501&dopt=Abstract estradiol




J Mol Endocrinol. 2002 Jun;28(3):165-75.
Comparative activity of pulsed or continuous estradiol exposure on gene expression and proliferation of normal and tumoral human breast cells.

Cavailles V, Gompel A, Portois MC, Thenot S, Mabon N, Vignon F.

Inserm Unit 540, Molecular and Cellular Endocrinology of Cancers, 60 rue de Navacelles, 34090 Montpellier, France.

Intranasal administration of hormone replacement therapy presents an original plasma kinetic profile with transient estrogen levels giving rise to the concept of pulsed therapy. To further understand the molecular effects of this new therapy, we have compared the effects of pulsed and continuous estradiol treatments on two critical aspects of estradiol action: gene expression and cell proliferation. Cells were stimulated with estradiol as 1-h pulsed or 24-h continuous treatments at concentrations such that the 24-h exposure (concentration x time) was identical in both conditions. In MCF7 cells, the transcriptional activity of estrogen receptors (ER) on a transiently transfected responsive estrogen response element-luciferase reporter construct was shown to be drastically (approximately 10-fold) and similarly stimulated after both treatments. Moreover, the increased mRNA expression of three representative estradiol-sensitive genes (pS2, cathepsin D, progesterone receptor), evaluated by Northern blot, was identical after 1-h pulse with 7 nM estradiol or continuous treatment with 0.29 nM estradiol with the same kinetic profile over 48 h. Proliferation was quantified by a histomorphometric method on primary cultures of human normal breast cells from reduction mammoplasties and using a fluorescence DNA assay in six human breast cancer cell lines which were ER positive or negative. After a 7-day treatment period, estradiol had no effect on the proliferation of the three ER negative cell lines (BT20, MDA MB231, SK BR3) but significantly stimulated the proliferation of the normal cells and of the three tumoral hormone-sensitive cell lines (MCF




Am J Physiol Gastrointest Liver Physiol. 2002 Jul;283(1):G27-36.
Modulatory effects of estrogen in two murine models of experimental colitis.

Verdu EF, Deng Y, Bercik P, Collins SM.

Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.

The association between oral contraceptives or pregnancy and inflammatory bowel disease is unclear. We investigated whether 17beta-estradiol modulates intestinal inflammation in two models of colitis. Female mice were treated with 17beta-estradiol alone or with tamoxifen, tamoxifen alone, 17 alpha-estradiol, or placebo. Dinitrobenzene sulfonic acid (DNB)- or dextran sodium sulfate (DSS)-induced colitis were assessed macroscopically, histologically, and by myeloperoxidase (MPO) activity. Malondialdehyde and mRNA levels of intercellular adhesion molecule-1 (ICAM-1), interferon-gamma (IFN-gamma), and interleukin-13 (IL-13) were determined. In DNB colitis, 17beta-estradiol alone, but not 17beta-estradiol plus tamoxifen, or 17 alpha-estradiol reduced macroscopic and histological scores, MPO activity and malondialdehyde levels. 17beta-Estradiol also decreased the expression of ICAM-1, IFN-gamma, and IL-13 mRNA levels compared with placebo. In contrast, 17beta-Estradiol increased the macroscopic and histological scores compared with placebo in mice with DSS colitis. These results demonstrate anti-inflammatory and proinflammatory effects of 17beta-estradiol in two different models of experimental colitis. The net modulatory effect most likely reflects a combination of estrogen receptor-mediated effects and antioxidant activity and may explain, in part, conflicting results from clinical trials.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12065288&dopt=Abstract estradiol




Neuroendocrinology. 2002 Jun;75(6):392-401.
Direct actions of estradiol on the anterior pituitary gland are required for hypothalamus-dependent lactotrope proliferation and secretory surges of luteinizing hormone but not of prolactin in female rats.

Yin P, Kawashima K, Arita J.

Department of Physiology, Yamanashi Medical University, Yamanashi, Japan.

Estradiol induces surges of prolactin (PRL) and luteinizing hormone (LH) secretion as well as lactotrope proliferation in female rats. We examined whether these hypothalamus-dependent events require the direct action of estradiol on the anterior pituitary gland by selective blockade of its peripheral actions, using ICI182,780 (ICI), an antiestrogen that cannot cross the blood-brain barrier. Injection of ICI into ovariectomized rats, at a dose of 250 microg/day for 4 days, almost completely inhibited estradiol-induced growth of the uterus, proliferation of lactotropes as determined by bromodeoxyuridine incorporation, and afternoon surges of LH secretion. However, ICI only partially inhibited estradiol-induced surges of PRL secretion and had no effect on estradiol-induced tonic inhibition of LH secretion even at the highest dose of 1,000 microg/day. The inhibitory effects of ICI found at 250 microg/day were attributable to its selective peripheral, but not central actions since ICI did not alter hypothalamic expression of progesterone receptors, an estradiol-dependent brain process. Estradiol-induced increases in the number of progesterone receptor-immunoreactive cells in the hypothalamic ventromedial nucleus and the medial preoptic area were not inhibited by this dose of ICI but were inhibited by 500 microg/day tamoxifen, an antiestrogen that can cross the blood-brain barrier. Treatment of cycling female rats with 250 microg/day ICI beginning from diestrus day 2 was also effective in blocking estrous lactotrope proliferation and preovulatory surges of LH secretion but not PRL secretion. Finally, in ovariectomized estradiol-treated pup-deprived lacta