Tamoxifen decreases the estradiol induced progesterone receptors by interfering with nuclear estrogen receptor accumulation

The retention time of the estrogen receptor in the nucleus of target cells after antiestrogen treatment has been shown to be longer than after estradiol. This paper describes the accumulation of nuclear estrogen receptors and the obtention of estrogenic responses (i.e. synthesis of cytosolic progesterone receptors and DNA) in the rat uterus after tamoxifen treatment in the presence or absence of estradiol. One-week ovariectomized adult rats were implanted with a silicone elastomer capsule containing corn oil or 25 micrograms estradiol/capsule (0 h). 48 h after implantation rats were injected with corn oil or 2 mg tamoxifen/kg and decapitated at 72, 96 or 120 h after implantation. In parallel experiments the implants were removed just before the injections of tamoxifen or oil. Tamoxifen injected into rats implanted with oil increased both the occupied nuclear receptors and the progesterone receptors at 96 h. In rats implanted with estradiol, tamoxifen did not increase the occupied nuclear receptors and decreased the levels of progesterone receptor and DNA at 96 h. In rats whose estradiol implants were removed at 48 h tamoxifen did not change the level of occupied nuclear receptors at 72 h but it increased them abruptly at 96 and 120 h. In these rats progesterone receptors decreased at 72 h but they increased at 96 and 120 h, and DNA decreased at 120 h to a lower level than before implantation. The results suggest that when estradiol is acting, tamoxifen is not able to increase the level of occupied estrogen receptor and it acts as an antiestrogen by decreasing the high level of progesterone receptors previously induced by estradiol. When estradiol is not acting tamoxifen behaves as a partial estrogen agonist by inducing progesterone receptors. However, the antiestrogenic action of tamoxifen on the rat uterus DNA does not seem to be affected by estradiol.     

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.     

卵巢激素调节大鼠胃运动及感觉功能的机制

女性患者在孕期及月经周期的黄体期常有腹痛、腹胀及腹泻等胃肠道功能紊乱的症状.本文探讨雌二醇（estradiol benzoate,EB）和孕酮（progesterone,P4）对卵巢切除大鼠血浆胆囊收缩素（cholecystokinin,CCK）及胃组织内胆囊收缩素受体A（CCKA）、血浆降钙素基因相关肽（calcitonin gene-related peptide,CGRP）及胃组织内其受体表达水平的影响,以期阐明卵巢激素调节胃肠道运动及感觉功能的机制.给予卵巢切除大鼠EB和P4替代治疗,用放射免疫分析法测定血浆CCK、CGRP的浓度,用Western blot法检测胃组织内CCKA受体的表达量,用125I-CGRP放射配体结合分析法测定胃组织内CGRP受体的表达量.EB可以升高血浆CCK的浓度,同时引起胃组织内CCKA受体表达增高.P4对血浆CCK的浓度以及胃组织内CCKA受体的表达无明显影响,但P4可以升高血浆CGRP的浓度,上调胃组织内CGRP受体的活性.EB、P4联合作用升高血浆CCK、CGRP的浓度,增加胃内CCKA、CGRP受体的表达.因此EB通过促进CCK的分泌以及上调胃内CCKA受体的表达,抑制胃排空;而P4可以通过增加CGRP的释放上调胃内CGRP受体的活性,从而增加肠神经系统对外来刺激的敏感性.结果提示,可以利用CCKA、CGRP受体的拈抗剂治疗女性患者中与月经周期有密切关系的胃肠道功能紊乱症状,如腹胀、早饱、腹痛等.     

Effect of estrogens on β-hydroxy-β-methylglutaryl coenzyme a reductase activity and cholesterol levels

The effect of estrogens on hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity and cholesterol in serum and liver of ovarietcomized rats on normal diet, 2% cholestyramine diet or 2% cholesterol diet was investigated. Estrogen administration to ovariectomized rats on normal diet resulted in increased reductase activity and was correlated with decreased serum cholesterol and increased liver cholesterol levels wlth mestranol (ME), ethinyl estradiol (EE) and estradiol benzoate (EB, 250 μg) but increased serum and liver cholesterol levels with 25 μg and 100 μg EB administration. The increased stimulation of reductase activity by estrogen administration was absolished when rats were fed a 2% cholesterol diet. Cholestyramine feeding markedly increased reductase activity in livers of ovariectomized rats. These studies show that estrogens are not absolutely required for the stimulation of reductase activity and therefore is consistent with the model in which cholesterol functions as a feedback repressor of reductase activity.     

Estrogen potentiates the stereotypy induced by dopamine agonists in the rat

Repeated weekly treatment with 100 μg/kg of estradiol benzoate (EB) to ovariectomized female rats intensified the stereotypy induced by the dopamine agonists amphetamine and apomorphine. A similar effect on amphetamine-stereotypy was produced 48 hours after a single injection of 10 μg/kg of EB. The fact that EB failed to increase blood or brain levels of either ³H-amphetamine or ³H-apomorphine suggests that these behavioral effects were not due to altered peripheral drug metabolism or uptake into the brain. The enhancement of stereotypy produced by EB is viewed as one manifestation of a more complex modulatory influence of estrogen on DA function.     

Effects of ovarian hormones on the behavior of captive Macaca fascicularis

To examine the effects of ovarian hormones on the behavior of female Macaca fascicularis and their male partners, daily 1-hr behavior tests were conducted while ovariectomized females were (1) untreated, (2) given estradiol benzoate (EB) (5 μg subcutaneously [s.c.]/day), (3) given estradiol benzoate together with increasing doses of progesterone (P) (5 mg, 10 mg, and 20 mg. s.c./day), and (4) given testosterone propionate (TP) (0.25 mg s.c./day) (six pairs, 540 tests). Weekly blood samples were analyzed by radioimmunoassay for plasma hormone levels (81 samples). Estrogen treatment produced plasma estradiol levels similar to those of intact females during the late follicular phase of the menstrual cycle. Additional progesterone at the lowest dose produced plasma progesterone levels similar to or somewhat higher than those during the midluteal phase, while higher doses produced supraphysiological levels. Androgen treatment resulted in plasma levels well above the physiological range. Hormone treatments produced highly significant effects on the sexual, social, and aggressive interactions of the pairs. As in rhesus monkeys, estrogen increased male and female sexual activity, and increasing doses of additional progesterone reversed these effects. Unlike in rhesus monkeys, testosterone propionate increased both female sexual motivation (invitations) and also male sexual activity and ejaculatory performance. The direction of the hormone-dependent changes in grooming and aggressive interactions confirmed earlier results with intact females and indicated that aggressive interactions and male grooming times were highest, and female grooming times were lowest, when copulatory activity was at its height.     

Cyclic estradiol treatment phasically potentiates endogenous cholecystokinin's satiating action in ovariectomized rats.

The influence of ovarian cycling and of exogenous estradiol on the cholecystokinin (CCK) satiety-signalling system was investigated in intact and ovariectomized Long-Evans rats, respectively. Intraperitoneal injection of 1 mg/kg devazepide, the most potent and selective CCK(A) receptor antagonist, increased test meal size during estrus, but not during diestrus, confirming the influence of hypothalamic-pituitary-gonadal function on CCK satiety in intact rats. Devazepide was then tested in ovariectomized rats that received chronic cyclic estradiol (2 microg estradiol benzoate on Tuesday and Wednesday each week) or oil treatment. Devazepide did not increase meal size in estradiol-treated rats on Tuesday, prior to estradiol treatment, compared to oil-treated rats, but did selectively increase meal size on Friday, late in the estradiol replacement cycle, compared to Tuesday, early in the cycle. These results suggest that a phasic potentiation of the endogenous CCK satiety-signalling system is part of the mechanism for the decrease in meal size in female rats during estrus.     

Estrogen upregulates hepatic apolipoprotein M expression via the estrogen receptor

Apolipoprotein M (apoM) is present predominantly in high-density lipoprotein (HDL) in human plasma, thus possibly involved in the regulation of HDL metabolism and the process of atherosclerosis. Although estrogen replacement therapy increases serum levels of apoAI and HDL, it does not seem to reduce the cardiovascular risk in postmenopausal women. Therefore, we investigated the effects of estrogen on apoM expression in vitro and in vivo. HepG2 cells were incubated with different concentrations of estrogen with or without the estrogen receptor antagonist, fulvestrant, and apoM expression in the cells was determined. Hepatic apoM expression and serum levels of apoM were also determined in normal and in ovariectomized rats treated with either placebo or estradiol benzoate, using sham operated rats as controls. Estrogen significantly increased mRNA levels of apoM and apoAI in HepG2 cell cultures in a dose- and time-dependent manner; the upregulation of both apolipoproteins was fully abolished by addition of estrogen receptor antagonist. In normal rats, estrogen treatment led to an increase in plasma lipid levels including HDL cholesterol, a marked upregulation of apoM mRNA and a significant increase in serum levels of apoM. The same pattern of regulation was found in ovariectomized rats treated with estrogen. Thus, estrogen upregulates apoM expression both in vivo and in vitro by mechanism(s) involving the estrogen receptor.     

Induction of sexual receptivity in the female lizard, Anolis carolinensis: Effects of estrogen and the antiestrogen CI-628

Daily behavioral testing revealed that there is a latency period of at least 48 hr from the administration of a single injection of estradiol benzoate (EB) to the first significant increase in female sexual receptivity in the ovariectomized female lizard, Anolis carolinensis. This latency period did not vary with dosage of EB used in these experiments (i.e., 0.8, 1.4, and 4.0 μg) nor with method of injection (subcutaneous vs intraperitoneal for dose of 1.4 μg EB). Following a single EB injection, female sexual receptivity increased after the 48-hr latency period, reached an observed peak from Day 3 to Day 6, and thereafter declined to pretreatment levels by Day 19. Although both 1.4 and 4.0 μg of EB produced higher levels of female sexual receptivity than did treatment with 0.8 μg of EB, results obtained with 4.0 μg EB did not differ from those obtained with 1.4 μg EB. Administration of the nonsteroidal antiestrogen CI-628 (80 μg) at either 4 or 24 hr following a single subcutaneous injection of 1.4 μg EB significantly reduced subsequent female sexual receptivity. These results suggest that there is a critical length of time during which estrogen must act on the brain and support the concept of an estrogen “maintenance” effect during this priming period.     

Effect of prolactin and estradiol on rat striatal dopamine receptors

Chronic estrogen treatment has been found to increase the level of rat striatal dopamine receptors. Since it is well known that estrogen treatment increases circulating prolactin levels, we have investigated the possibility that the stimulatory effect of estrogens on dopamine receptors is exerted via prolactin. Ovariectomized female or intact male rats were implanted with three adenohypophyses under the kidney capsule or treated with 17 β-estradiol (10 μg, twice daily) for 2 weeks. In animals of both sexes, the pituitary-implanted and estradiol-treated rats showed higher levels of [³H]spiperone binding to striatal dopamine receptors. This effect of estradiol or pituitary implants on dopamine receptors was further investigated in ovariectomized rats. The pituitary-implanted and estradiol-treated rats had elevated plasma prolactin levels and an increased density of striatal dopamine receptors without alteration of their affinity. The role of the pituitary in the effect of estradiol was next investigated using hypophysectomized female rats treated with 17 β-estradiol (10 μg, twice daily), o-prolactin (500 μg, twice daily) or bearing three anterior pituitary implants. The implants as well as the treatment with estradiol or prolactin increased the level of striatal dopamine receptors in hypophysectomized rats while, as expected, the estradiol-treated animals did not have elevated plasma prolactin levels. The present data indicate that high prolactin levels lead, as observed with chronic estradiol treatment, to an increased density of striatal dopamine receptors. However, the effect of estradiol may not be explained exclusively by increased prolactin levels since a similar stimulatory effect is observed in hypophysectomized animals.     

Resistin mRNA levels are downregulated by estrogen in vivo and in vitro

Resistin, a hormone secreted by adipocytes, is suggested to be an important link between obesity and diabetes. The aim of this study was to evaluate the regulatory effect of estrogen on adipocyte resistin gene expression in ovariectomized (OVX) rats and in isolated rat adipocytes in vitro. Subcutaneous injection of estradiol benzoate reduced resistin mRNA levels in adipocytes isolated from the inguinal, parametrial, perirenal, retroperitoneal, or periovarian fat deposits of OVX rats, while an in vitro study showed that estradiol treatment decreased resistin mRNA levels in cultured rat periovarian fat adipocytes. Results of Western blotting analysis also showed that estrogen decreased adipose resistin contents in vivo and in vitro. These data suggest that estrogen is a pivotal negative regulator of resistin gene expression.     

Effect of serum estradiol and leptin levels on thyroid function, food intake and body weight gain in female Wistar rats

We evaluated the interplay among estrogen, leptin and thyroid function in the regulation of body mass in female rats. Adult female rats were divided into four groups: control (C, sham-operated), ovariectomized (OVX), ovariectomized treated with estradiol benzoate (Eb) 0.7 or 14 μg/100 g bw per day, during 21 days. OVX led to an increase in body mass, food intake and food efficiency (change in body mass as function of the amount of food ingested) which were normalized by the lower Eb dose, and decreased significantly when the higher dose was given. Serum leptin levels were increased more than two-fold in all ovariectomized groups. Serum T4 levels of the Eb treated OVX were significantly lower than in the controls. Serum T3 and TSH were unaffected by OVX or by Eb treatment. Uterine type 2 iodothyronine deiodinase (D2) activity changed in parallel with serum estradiol: decreased after OVX, returned to control levels after the lower E2 treatment, and increased significantly after the high Eb dosage. The hypothalamic D2 activity was reduced around 30% in all castrated groups, treated or not with estrogen, whereas in the brown adipose tissue the enzyme was not changed. Interestingly, although estrogen-treated OVX rats had lower body weight, serum leptin was high, suggesting that estrogen increases leptin secretion. Our results show that estradiol is necessary for the hypothalamic action of leptin, since the increase in leptin levels observed in all ovariectomized rats was associated with a decrease in food intake and food efficiency only in the rats treated with estrogen.     

Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat

The effects of estrogen on proenkephalin (PE) gene expression were measured in neurons of the ventromedial hypothalamus. Slot blot hybridization analysis indicates that the levels of PE mRNA in the ventromedial hypothalamus of ovariectomized rats increase 3.1-fold after 2 weeks of estrogen replacement. In situ hybridization reveals that the estrogen-inducible enkephalinergic neurons are located in the ventrolateral aspect of the ventromedial nucleus, a subnucleus known to contain many estrogen-concentrating neurons. The increase in PE mRNA levels is due to both a 63% increase in the number of detectable PE mRNA-containing neurons and a 2.0-fold increase in the levels of PE mRNA per enkephalinergic neuron (1.63 x 2.0 = 3.3-fold overall induction). This estrogen-regulated enkephalinergic cell group may represent part of the neural network mediating estrogen's effects on reproductive behavior and/or other neuroendocrine processes.     

Estradiol-mediated suppression of CYP1B1 expression in mouse MA-10 Leydig cells is independent of protein kinase A and estrogen receptor

Estrogens have multifaceted roles in mammalian testis. In the present study, we focused on estradiol as a potential regulator of testicular cytochrome P450 1B1 (CYP1B1) expression and investigated the possible mechanisms involved in the estradiol-mediated suppression. CYP1B1 protein levels were measured in the testes of rats that were treated with 17β-estradiol benzoate (1.5 mg/kg) at different stages of development. In addition, CYP1B1 mRNA levels were measured in mouse MA-10 Leydig tumor cells treated with (a) various concentrations of 17β-estradiol benzoate, (b) 17β-estradiol benzoate in the presence of exogenous luteinizing hormone (LH), or (c) 17β-estradiol benzoate in the presence of ICI 182,780, a competitive steroidal antagonist of estrogen receptors (ERs). Treatment of neonatal, pubertal, or adult rats with 17β-estradiol benzoate was associated with a reduction of approximately 90% in testicular CYP1B1 protein content compared to age-matched controls. Treatment of MA-10 cells with 17β-estradiol benzoate (10-500 nM) produced a concentration- and time-dependent decrease in CYP1B1 mRNA levels, but had no effect on LH receptor mRNA levels or on protein kinase A (PKA) activity. However, 17β-estradiol benzoate (10-500 nM), regardless of the concentration tested, failed to attenuate the LH-elicited increase in CYP1B1 mRNA or PKA activity in MA-10 cells that were co-treated with LH and estradiol. Similarly, ICI 182,780 (10-1000 μM) did not reverse the suppressive effect of estradiol on CYP1B1 mRNA expression in MA-10 cells co-treated with estradiol and ICI 182,780. The results indicate that downregulation of testicular CYP1B1 by estradiol was independent of PKA activity and was not mediated by ERs in MA-10 cells.     

电针对大鼠脑内雌激素受体蛋白及其mRNA表达的影响

本文应用放射免疫分析（ＲＩＡ）、ＲＮＡ点杂交和Ｎｏｒｔｈｅｒｎ ｂｌｏｔ、单克隆抗体免疫组织化学和计算机图像处理技术研究电针对切除卵巢大鼠脑内雌激素受体（ＥＲ）蛋白和ｍＲＮＡ表达的影响。以探讨针刺作用的分子生物学机理。结果表明,切除卵巢可导致血雌二醇（Ｅ２）水平降低,动物脑内ＥＲ蛋白和ｍＲＮＡ的表达增强;电针实验穴位后,去卵巢大鼠血的Ｅ２含量明显增加,脑内ＥＲ蛋白和ｍＲＮＡ表达受到明显抑制。正常大     

Cyclic estradiol treatment normalizes body weight and restores physiological patterns of spontaneous feeding and sexual receptivity in ovariectomized rats

Hypothalamic-pituitary-gonadal axis function strongly influences feeding and body weight in cycling females in many species. To test the sufficiency of cyclic variations in plasma estradiol to reproduce normal patterns of spontaneous feeding, food intake, and body weight, ovariectomized Long-Evans rats were subcutaneously injected every fourth day with 2 microg estradiol benzoate or with the oil vehicle alone. Cyclic estradiol treatment completely normalized the trajectory of body weight gain and total food intake through seven treatment cycles. The hyperphagia of ovariectomized rats was expressed as an increase in spontaneous meal size. Meal frequency decreased, but not enough to compensate for the increase in meal size. Estradiol treatment normalized both parameters. In addition, cyclic estradiol treatment produced a further phasic decrease in meal size (and increase in meal frequency) and a decrease in food intake during the second night after injection. This phasic change is similar to the feeding changes occurring during estrus in intact rats. Sexual receptivity was measured during the eighth estradiol treatment cycle, 4 h after injection of 0.5 mg progesterone. Lordosis scores at the time of the treatment cycle modeling estrus were maximal, and scores at the time modeling diestrus were slightly increased over those of rats that did not receive estradiol. Finally, plasma estradiol levels, measured during the ninth treatment cycle, revealed a near-normal cyclic pattern of plasma estradiol levels. These results provide the first demonstration that the induction of a cyclic, near-physiological pattern of plasma estradiol is sufficient to maintain normal levels of body weight, spontaneous feeding patterns, total food intake, and (together with progesterone) sexual receptivity in ovariectomized rats.     

Effect of estradiol benzoate and clomiphene on tyrosine hydroxylase activity and on luteinizing hormone and prolactin levels in ovariectomized rats

The effects of estradiol benzoate (EB) on tyrosine hydroxylase (TH) activity in the medial basal hypothalamus (MBH) and on plasma levels of luteinizing hormone (LH) and prolactin were studied in long-term ovariectomized rats. Administration of 10 μg EB produced significant elevation of TH activity on Days 1 and 3 following injection. LH levels were significantly lower than controls throughout the three day treatment period, although there was a significant increase from Day 1 to Day 2. TH activity and LH levels were inversely related throughout the experimental period. Clomiphene (15 μg/rat/day), a purported estrogen antagonist, was administered over a period of three days to control and EB-treated rats to determine whether the effect of EB on plasma LH levels was causally related to changes in TH activity. In rats receiving both EB and clomiphene, TH activity was lower and plasma LH was higher than after EB alone. The results support the hypothesis that the feedback effects of estradiol on LH release involve an action on the tuberoinfundibular dopaminergic (TIDA) neurons of the MBH and that clomiphene can oppose the inhibitory effect of estradiol on LH release by directly inhibiting TIDA neuron activity. Furthermore, EB-induced release of prolactin does not appear to involve detectable changes in the activity of TIDA neurons.     

Local intracerebral implants of estrogen masculinize some aspects of the zebra finch song system

The song system of zebra finches is sexually dimorphic: the volumes of the song control nuclei and the neurons within these nuclei are larger in males. The song system of hatching female zebra finches is masculinized by systemic treatment with estrogen. We investigated the locus of this estrogen action by using microimplants of estradiol benzoate (EB). We implanted female zebra finch nestlings 10–13 days old with Silastic pellets containing approximately 2 μg EB at one of several sites: near the higher vocal center (HVC), in the brain distant from HVC, or in the periphery either under the skin of the breast or in the peritoneal cavity. Controls were either unimplanted or implanted near HVC with Silastic pellets without hormone. The brains were fixed by perfusion at 60 days, and the volumes of the song control regions as well as the sizes of individual neurons were measured. Neurons in HVC were lerger (more masculine) in the HVC-implanted group than in other groups, which did not differ among themselves. The size of neurons in the robust nucleus of the archistriatum (RA) and the lateral magnocellular nucleus ofthe neostriatum (lMAN) were inversely correlated with the distance of the EB pellet to HVC; neurons in RA and lMAN were larger when the EB pellets were closer to HVC. This result suggests that implants near HVC were at or near a site of estrogen action. To our knowledge, this is the first demonstration that localized brain implants of estrogen cause morphological masculinization in any species. 1994 John Wiley & Sons, Inc.     

The Effects of Estrogen and Progesterone on the Blood Levels of Glucose,Non-Esterified Fatty Acids and Cholesterol in Ovariectomized Sheep

The effects of estrogen and progesterone on the blood levels of glucose, non-esterified fatty acids and cholesterol in ovariectomized sheep. The effects of estradiol benzoate and progesterone on blood glucose, NEFA and cholesterol were studied in ovariectomized sheep. Intramuscular injection of 2.5 mg estradiol benzoate gave biphasic changes in NEFA. After 2 hrs. NEFA was decreased, but thereafter an increase occurred and maximum levels were reached after 24 hrs. Blood glucose was significantly increased from 12 to 48 hrs. after the injection. Serum cholesterol was lowered after 24 hrs., but thereafter the level increased. Maximum values were obtained after 120 hrs. Progesterone at the same dose did not change any of the measured parameters. Simultaneous administration of estradiol benzoate and progesterone gave similar responses as estradiol benzoate alone. Blood glucose and NEFA were followed during heat in a lactating cow. Both parameters increased after ovulation. Since NEFA was increased during so long time after the injection of estradiol benzoate, the mechanism behind this effect was discussed. No lipolytic hormone has been reported to give a response of this duration. Estrogen is known to increase plasma GH, and since GH is strongly lipolytic in sheep it seemed possible that the elevated NEFA levels were caused by increased GH secretion. There is now evidence that also estrogen-induced changes in serum cholesterol are pituitary dependant. It was therefore considered possible that all the noted metabolic changes were mediated by the pituitary.