The role of the bed nucleus of the stria terminalis in stress-induced inhibition of pulsatile luteinising hormone secretion in the female rat.

The bed nucleus of the stria terminalis (BNST) occupies a central position in the neural circuitry regulating the hypothalamic-pituitary-adrenocortical axis response to stress. The potential role of the BNST in stress-induced suppression of the gondotrophin-releasing hormone (GnRH) pulse generator, the central regulator of the reproductive system, was assessed by examining the effects of micro-infusion of corticotrophin-releasing factor (CRF) or its antagonist into the BNST on pulsatile luteinising hormone (LH) secretion or stress-induced inhibition of LH pulses, respectively. Ovariectomised oestrogen-treated rats were implanted chronically with bilateral cannulae in the dorsolateral BNST and i.v. catheters. CRF (25, 50 or 100 pmol in 200 nl of artificial cerebrospinal fluid) administered bilaterally into the BNST resulted in a dose-dependent decrease in LH pulse frequency, and induced Fos expression in glutamic acid decarboxylase immunostained neurones in the medial preoptic area. These results suggest that the activation of hypothalamic GABAergic neurones in response to intra-BNST administration of CRF may be involved in the suppression of LH pulses. Furthermore, administration of CRF antagonist (280 pmol astressin-B, three times at 20-min intervals) into the BNST effectively blocked the suppression of pulsatile LH secretion in response to restraint (1 h) but not hypoglycaemic (0.25 U insulin/kg, i.v.) stress. These data suggest that CRF innervation of the dorsolateral BNST plays a key, but differential, role in stress-induced suppression of the GnRH pulse generator.

Corticotrophin-releasing factor alters the timing of puberty in the female rat.

Puberty is a developmental process that is dependent upon activation of the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator. It is well established that the stress neuropeptide, corticotrophin-releasing factor (CRF), has a profound inhibitory action on GnRH pulse generator frequency. Although stress is known to affect the timing of puberty, the role of CRF is unknown. The present study aimed to test the hypothesis that CRF plays a critical role in the timing of puberty. On postnatal day (pnd) 28, female rat pups were chronically implanted with i.c.v. cannulae and received 14 days of administration of either CRF, CRF receptor antagonist (astressin-B) or artificial cerebrospinal fluid via an osmotic mini-pump. A separate group of rats served as nonsurgical controls. As a marker of puberty, rats were monitored for vaginal opening and first vaginal oestrus. Levels of CRF, CRF receptor types 1 and 2 (CRF-R1, CRF-R2) mRNA expression in micropunches of the medial preoptic area (mPOA), hypothalamic paraventricular nucleus (PVN) and arcuate nucleus (ARC) were determined across pubertal development; brain tissue was collected from a naive group of rats on pnd 14, 32, on the day of vaginal opening, and pnd 77 (Adult). Administration of CRF resulted in a delay in the onset of puberty, whereas astressin-B advanced pubertal onset. Additionally, CRF and CRF-R1 mRNA expression was reduced in the mPOA, but not ARC, at puberty. In the PVN, expression of CRF, but not CRF-R1 mRNA, was reduced at the time of puberty. These data support the hypothesis that CRF signalling may play an important role in modulating the timing of puberty in the rat.

Neonatal lipopolysaccharide exposure delays puberty and alters hypothalamic Kiss1 and Kiss1r mRNA expression in the female rat.

Immunological challenge experienced in early life can have long-term programming effects on the hypothalamic-pituitary-adrenal axis that permanently influence the stress response. Similarly, neonatal exposure to immunological stress enhances stress-induced suppression of the hypothalamic-pituitary gonadal (HPG) axis in adulthood, but may also affect earlier development, including the timing of puberty. To investigate the timing of the critical window for this programming of the HPG axis, neonatal female rats were injected with lipopolysaccharide (LPS; 50 microg/kg i.p.) or saline on postnatal days 3 + 5, 7 + 9, or 14 + 16 and monitored for vaginal opening and first vaginal oestrus as markers of puberty. We also investigated the effects of neonatal programming on the development of the expression patterns of kisspeptin (Kiss1) and its receptor (Kiss1r) in hypothalamic sites known to contain kisspeptin-expressing neuronal populations critical to reproductive function: the medial preoptic area (mPOA) and the arcuate nucleus in neonatally-stressed animals. We determined that the critical period for a significant delay in puberty as a result of neonatal LPS exposure is before 7 days of age in the female rat, and demonstrated that Kiss1, but not Kiss1r mRNA, expression in the mPOA is down-regulated in pre-pubertal females. These data suggest that the mPOA population of kisspeptin neurones play a pivotal role in controlling the onset of puberty, and that their function can be affected by neonatal stress.

Down-regulation of hypothalamic kisspeptin and its receptor, Kiss1r, mRNA expression is associated with stress-induced suppression of luteinising hormone secretion in the female rat.

Identification of kisspeptin (Kiss1) and its G protein-coupled receptor 54 (Kiss1r) as an essential component of the hypothalamic-pituitary-gonadal (HPG) axis controlling gonadotrophin secretion raises the possibility that kisspeptin-Kiss1r signalling may play a critical role in the transduction of stress-induced suppression of reproduction. We examined the effects of: (i) three different stressors, known to suppress pulsatile luteinising hormone (LH) secretion; (ii) corticotrophin-releasing factor (CRF); and (iii) corticosterone on Kiss1 and Kiss1r expression in key hypothalamic sites regulating gonadotrophin secretion: the medial preoptic area (mPOA) and arcuate nucleus (ARC). Ovariectomised oestrogen-replaced rats were implanted with i.v., subcutaneous or i.c.v. cannulae. Blood samples were collected at 5-min intervals for 5-6 h for detection of LH. Quantitative reverse transcriptase-polymerase chain reaction was used to determine Kiss1 and Kiss1r mRNA levels in brain punches of the mPOA and ARC collected 6 h after restraint, insulin-induced hypoglycaemia or lipopolysaccharide stress, or after i.c.v. administration of CRF, or acute or chronic subcutaneous administration of corticosterone. We observed down-regulation of at least one component of the kisspeptin-Kiss1r signalling system by each of the stress paradigms within the mPOA and ARC. CRF decreased Kiss1 and Kiss1r expression in both the mPOA and ARC. Both acute and chronic stress levels of corticosterone resulted in a concomitant decrease in Kiss1 and an increase in kiss1r mRNA expression in the mPOA and ARC. This differential regulation of Kiss1 and Kiss1r might account for the lack of effect corticosterone has on pulsatile LH secretion. Considering the pivotal role for kisspeptin-Kiss1r signalling in the control of the HPG axis, these results suggest that the reduced Kiss1-Kiss1r expression may be a contributing factor in stress-related suppression of LH secretion.

The hormonal control of uterine luminal fluid secretion and absorption.

The secretion of uterine luminal fluid initially provides a transport and support medium for spermatozoa and unimplanted embryos, while the absorption of uterine luminal fluid in early pregnancy results in the closure of the lumen and allows blastocysts to establish intimate contact with the uterine epithelium. We have established an in vivo perfusion technique of the lumen to study the hormonal control of the events in the peri-implantation period. Fluorescein-labelled dextran was included in the perfusion medium to monitor fluid movements and the concentrations of Na(+) and CI(-) ions in the effluent were monitored. Using an established regimen of steroid treatment of ovariectomized rats mimicking early pregnancy, oestradiol caused fluid secretion, while progesterone resulted in an amiloride-sensitive fluid absorption. Fluid absorption peaked at about the expected time of implantation. The effect of progesterone could be inhibited by treatment with a high dose of oestradiol, by the anti-progestin RU486, and by the presence of an intra-uterine contraceptive device. Studies of expression of Na(+) and CI(-) channels (ENaC, CFTR) indicated that these channels were subject to tissue-specific regulation within the uterus, but more work is required to determine their role and the factors controlling their abundance and localization in early pregnancy.

17beta-Estradiol and environmental estrogens significantly affect mammalian sperm function.

BACKGROUND: Compounds with estrogenic activity can affect reproductive function in mammals. This study investigated possible effects of 17beta-estradiol (E(2)) and three weakly estrogenic environmental estrogens on mammalian sperm capacitation and fertilizing ability in vitro. METHODS: Uncapacitated and capacitated mouse sperm suspensions were incubated for 30 min in the presence of E(2), genistein (Gen), 8-prenylnaringenin (8-PN) and nonylphenol (NP), and then assessed using chlortetracycline (CTC) fluorescence analysis. In addition, treated uncapacitated sperm suspensions were tested for changes in fertilizing ability. RESULTS: In uncapacitated cells, E(2) at >or=1 micromol/l and Gen, 8-PN and NP at >or=0.001 micromol/l, significantly stimulated capacitation and acrosome reactions. Hydroxytamoxifen (an estrogen antagonist) did not inhibit responses to any of these compounds. In capacitated cells, E(2) had no effect, but the other three compounds significantly stimulated acrosome reactions. Added to uncapacitated suspensions, 10 micromol/l E(2), 0.1 micromol/l Gen and 0.1 micromol/l 8-PN all significantly stimulated sperm fertilizing ability ( approximately 76% oocytes fertilized) compared with untreated control sperm ( approximately 36%). CONCLUSIONS: This study provides the first evidence that E(2) and environmental estrogens can significantly stimulate mammalian sperm capacitation, acrosome reactions and fertilizing ability, with the environmental estrogens being much more potent than E(2). The inability of hydroxytamoxifen to block these responses suggests that classical estrogen receptors may not be involved. Whether these responses have effects on fertility in vivo remains to be determined, along with the mechanisms of action involved.

Assessment of estrogenic activity in some common essential oil constituents.

Estrogenic responses have not only been associated with endocrine function, but also with cognitive function. Several studies have indicated that estrogen replacement therapy has favourable effects on cognition, and may have potential in the prevention and treatment of Alzheimer's disease. Thus, ligands for the estrogen receptor, that have a better efficacy and adverse-effect profile than drugs currently available, require investigation. This study was undertaken to investigate the potential estrogenic activity of a number of essential oil constituents. Initially, estrogenic activity was determined by a sensitive and specific bioassay using recombinant yeast cells expressing the human estrogen receptor. At high concentrations, estrogenic activity was detected for citral (geranial and neral), geraniol, nerol and trans-anethole, while eugenol showed anti-estrogenic activity. Molecular graphics studies were undertaken to identify the possible mechanisms for the interaction of geranial, neral, geraniol, nerol and eugenol with the ligand-binding domain of the estrogen alpha-receptor, using the computer program HyperChem. Citral, geraniol, nerol and eugenol were also able to displace [(3)H]17beta-estradiol from isolated alpha- and beta-human estrogen receptors, but none of these compounds showed estrogenic or anti-estrogenic activity in the estrogen-responsive human cell line Ishikawa Var I at levels below their cytotoxic concentrations, and none showed activity in a yeast screen for androgenic and anti-androgenic activity. The potential in-vivo estrogenic effects of citral and geraniol were examined in ovariectomized mice, but neither compound showed any ability to stimulate the characteristic estrogenic responses of uterine hypertrophy or acute increase in uterine vascular permeability. These results show that very high concentrations of some commonly used essential oil constituents appear to have the potential to interact with estrogen receptors, although the biological significance of this is uncertain.

Progesterone stimulation of fluid absorption by the rat uterine gland.

The purpose of this study was to test the hypothesis that the steroid environment affects fluid absorption by the uterine glands. Laser scanning confocal microscopy of the distribution of an extracellular marker (fluorescein isothiocyanate-labelled dextran) within rat uterine glands showed that the endometrial glands change their fluid handling characteristics under different hormonal conditions. Under progesterone dominance, the glands showed an amiloride-sensitive dextran accumulation indicating sodium-dependent fluid absorption; however, this was absent in the oestrogen-dominated state. The rate of fluid uptake in the progesterone-stimulated gland opening was estimated to be approximately 1 x 10(-4) cm s(-1), requiring a suction pressure of between 10 and 20 mm Hg at the mucosal surface. This study provides the first direct evidence of fluid absorption by the uterine glands. Such absorption may provide the mechanism for closure of the uterine lumen and immobilization of the blastocyst necessary for implantation.

Comparison of the oestrogenic effects of infant milk formulae, oestradiol and the phytoestrogen coumestrol delivered continuously in the drinking water to ovariectomised mice.

The potential oestrogenic effects of infant milk formulae, coumestrol and oestradiol delivered in the drinking water were investigated in ovariectomised mice. None of the infant formulae tested (three soya, two cow's milk) produced any uterotrophic or mitotic responses in the reproductive tract, although the soya milks displayed weak oestrogenic activity in vitro. Studies of the interactions between coumestrol and oestradiol were undertaken to investigate claims that phytoestrogens may act as oestrogen antagonists. The responses to coumestrol (100 g/ml drinking water) and 17-oestradiol (100 ng/ml) given separately were similar. Combined administration begun simultaneously produced only additive effects on uterine weight and cell proliferation in the vagina and uterus. While pretreatment with coumestrol for 24 h reduced the mitotic response of the uterus 48 h after placement of an oestradiol implant, the uterine weight increase was unaffected and the apparent reduction in mitoses reflected the natural fluctuations in the underlying cycle of cell proliferation. These studies indicate that coumestrol acts as a typical oestrogen and shows only additive effects with oestradiol. The results also indicate that infant soya milk formulae do not constitute a large enough source of oestrogenic compounds to invoke oestrogenic effects in the reproductive tract of mature mice.

Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat.

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans. The aims of this study were 2-fold: 1) to determine in vivo whether genistein and coumestrol act on the GnRH pulse generator to suppress hypothalamic multiunit electrical activity volleys and associated LH pulses and/or on the pituitary to suppress the LH response to GnRH; and 2) to examine the effect of these phytoestrogens on GnRH-induced pituitary LH release in vitro and to determine whether estrogen receptors are involved. Wistar rats were ovariectomized and chronically implanted with recording electrodes and/or indwelling cardiac catheters, and blood samples were taken every 5 min for 7--11 h. Intravenous infusion of coumestrol (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) resulted in a profound inhibition of pulsatile LH secretion, a 50% reduction in the frequency of hypothalamic multiunit electrical activity volleys, and a complete suppression of the LH response to exogenous GnRH. In contrast, both genistein (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) and vehicle were without effect on pulsatile LH secretion. Coumestrol (10(-5) M; over 2 or 4 h) suppressed GnRH-induced pituitary LH release in vitro, an effect blocked by the antiestrogen ICI 182,780. It is concluded that coumestrol acts centrally to reduce the frequency of the hypothalamic GnRH pulse generator. In addition, the inhibitory effects of coumestrol on LH pulses occur at the level of the pituitary by reducing responsiveness to GnRH via an estrogen receptor-mediated process.

The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids.

The female flowers of the hop plant have long been used as a preservative and a flavoring agent in beer, but they are now being included in some herbal preparations for women for "breast enhancement." This study investigated the relative estrogenic, androgenic and progestogenic activities of the known phytoestrogen, 8-prenylnaringenin, and structurally related hop flavonoids. 6-Prenylnaringenin, 6,8-diprenylnaringenin and 8-geranylnaringenin exhibited some estrogenicity, but their potency was less than 1% of that of 8-prenylnaringenin. 8-Prenylnaringenin alone competed strongly with 17ss-estradiol for binding to both the alpha- and ss-estrogen receptors. None of the compounds (xanthohumol, isoxanthohumol, 8-prenyl-naringenin, 6-prenylnaringenin, 3'-geranylchalconaringenin, 6-geranylnaringenin, 8-geranylnaringenin, 4'-O:-methyl-3'-prenylchalconaringenin and 6,8-diprenylnaringenin) nor polyphenolic hop extracts showed progestogenic or androgenic bioactivity. These results indicate that the endocrine properties of hops and hop products are due to the very high estrogenic activity of 8-prenylnaringenin and concern must be expressed about the unrestricted use of hops in herbal preparations for women.

Identification of a potent phytoestrogen in hops (Humulus lupulus L.) and beer.

The female flowers of the hop plant are used as a preservative and as a flavoring agent in beer. However, a recurring suggestion has been that hops have a powerful estrogenic activity and that beer may also be estrogenic. In this study, sensitive and specific in vitro bioassays for estrogens were used for an activity-guided fractionation of hops via selective solvent extraction and appropriate HPLC separation. We have identified a potent phytoestrogen in hops, 8-prenylnaringenin, which has an activity greater than other established plant estrogens. The estrogenic activity of this compound was reflected in its relative binding affinity to estrogen receptors from rat uteri. The presence of 8-prenylnaringenin in hops may provide an explanation for the accounts of menstrual disturbances in female hop workers. This phytoestrogen can also be detected in beer, but the levels are low and should not pose any cause for concern.

Competitive binding of xenobiotic oestrogens to rat alpha-fetoprotein and to sex steroid binding proteins in human and rainbow trout (Oncorhynchus mykiss) plasma.

The ability of a variety of "environmental oestrogens" to compete with radiolabelled steroids to rat alpha-fetoprotein (AFP) and to sex steroid binding proteins was investigated in human and rainbow trout (Oncorhynchus mykiss) plasma. For [3H]oestradiol binding to AFP, diethylstilbestrol and 4-nonylphenoxyacetic acid showed significant competition at concentrations about 100-fold greater than oestradiol (relative binding affinities approximately 1% c.f. oestradiol). All other compounds (phytooestrogens: coumestrol, daidzein, genistein; others: 4-nonylphenol, 4-tert-octylphenol, 4-nonylphenoldiethoxylate, 4-tert-butylphenol, bisphenol-A (Bis-A), bis(2- ethylhexl)phthalate, dioctylphthalate, dibutyl phthalate, 2, 4'DDT (op' enantiomer), 2,4'-DDE (mixed enantiomers), kepone) showed only very weak or no competition (relative binding affinities <<0.1% c.f. oestradiol). The situation for both human and fish plasma was very similar, with only very high concentrations (>>1000 fold more than the natural ligand) of a few of the compounds showing any ability to displace the natural ligand. These results suggest that environmental oestrogenic agents are unlikely to produce biological effects by displacing endogenous steroids from plasma steroid binding proteins unless they are present in very high concentrations.

Effects of platelet-derived endothelial cell growth factor/thymidine phosphorylase, substrate, and products in a three-dimensional model of angiogenesis.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is associated with angiogenesis and the progression of human breast and ovarian cancers. The aim of this study was to obtain information about the possible mechanisms of PD-ECGF/TP activity in an established three-dimensional model of angiogenesis. The plan was to study the effects of the enzyme, substrate, products, and further metabolites on the formation and rate of microvessel growth from cultured segments of rat aorta in serum-free media. The end-points were the number and length of microvessels compared with controls after 4, 7, 11, and 14 days in culture. Thymidine (10 to 1000 mumol/L), thymidine-5'-monophosphate (1000 mumol/L), and 2'-deoxy-D-ribose-1-phosphate (1000 mumol/L) inhibited the number of microvessels produced. Conversely PD-ECGF/TP (50 to 100 ng/ml) and beta-amino-iso-butyric acid (1000 mumol/L--a metabolite of thymine) had a significant stimulatory effect (P < 0.05, P < 0.01, P < 0.001 respectively on culture day 11). PD-ECGF (10 ng/ml), beta-amino-iso-butyric acid (1000 mumol/L), and 2-deoxy-D-ribose (100 to 1000 mumol/L) significantly (P < 0.001, P < 0.01, P < 0.01, respectively) stimulated microvessel elongation by day 11. We conclude that PD-ECGF/TP may affect angiogenesis by changing the relative concentrations of pyrimidine-based compounds and their metabolites in interstitial fluid surrounding endothelial cells. Drugs that inhibit PD-ECGF/TP activity may therefore delay abnormal angiogenesis and the progression of various cancers.

Relative potency of xenobiotic estrogens in an acute in vivo mammalian assay.

The in vivo effects of xenoestrogens are of interest in relation to their potential health risks and/or beneficial effects on humans and animals. However, the apparent in vivo potency of the examined response can be confounded by a short half-life, and the metabolism of estrogens is very dependent on the nature of conversion and/or inactivation. To minimize such variables, we examined the estrogenic potency of a range of xenoestrogens in an acute in vivo assay--the stimulation of increased uterine vascular permeability in ovariectomized mice 4 hr after subcutaneous administration. While estradiol (E 2 ) and estriol (E 3 ; a relatively weak natural estrogen) readily induced vascular responses [median effective dose (ED 50 ) <10 -9 mol], much higher amounts of xenoestrogens were required. Bisphenol A was about 10,000-fold less potent than E 2 and E 3 , and octylphenol and nonylphenol were about 100,000-fold less potent; dioctyl phthalate, benzyl butyl phthalate, dibutyl phthalate, and trichlorinated biphenol produced no effect. Coumestrol was the most active phytoestrogen, with an ED 50 between 10 -6 and 10 -7 mol; genistein was about 10-fold less potent than coumestrol, and neither daidzein nor formononetin produced any marked effect, even at doses up to 10 -5 mol. All increases in vascular permeability could be blocked by the pure antiestrogen ICI 182,780. There was no evidence that any of the compounds could act as an antiestrogen in this assay or that they could exert synergistic effects in combination. These results indicate that even short-term exposure to most of the xenobiotic estrogens can induce typical estrogenic effects in vivo , but their estrogenic potency is very weak even when assessed in an acute response.

Minimal progesterone support required for the maintenance of pregnancy in mice.

A study of the degree of progesterone support required for the maintenance of various stages of pregnancy was undertaken in mice. Mated females were ovariectomized at various stages of pregnancy and progesterone and oestradiol support provided by s.c. Silastic implants with known release characteristics. In the earliest stages of pregnancy (days 1-5), very low concentrations of progesterone (<25% of normal physiological values) were sufficient to maintain pre-implantation stages and allow implantation. In the immediate post-implantation period (days 5-9), the development of implantation sites and decidualization required considerably higher progesterone support. In mid-pregnancy (days 11-14), progesterone alone could not maintain pregnancy unless present in very high amounts; however, the presence of oestradiol during this period lowered the progesterone requirements to well within the physiological range. This effect of oestradiol started on day 11 but required the level of oestradiol support to be kept within strictly defined limits, with high concentrations inducing abortion. Progesterone alone was able to maintain pregnancy from day 15. These results indicate that the minimal progesterone support required for pregnancy in mice varies considerably at different stages of pregnancy and is at least partly modulated by oestradiol.

Relaxin and decidualization in mice: a reappraisal.

The nature of the physiological stimulus inducing decidualization in the endometrium is unknown. In this study we attempted to verify a recent report that relaxin can induce decidualization in intact mice primed with a high dose of estradiol valerate (5 micrograms) and a low dose (10 micrograms) of medroxyprogesterone acetate. In our study, neither s.c. nor intrauterine relaxin, nor intraluminal arachis oil, (an established deciduogenic stimulus) were able to induce decidualization. In addition, while oil was able to induce decidualization (increased uterine weight, and positive Pontamine Sky Blue and stromal alkaline phosphatase reactions) in ovariectomized mice treated with a regimen of estradiol and medroxyprogesterone acetate designed to produce optimum uterine sensitivity, no decidualization occurred in response to either s.c. or intraluminal relaxin. This study fails to provide any support for a role for relaxin as a deciduogenic stimulus.

Potent inhibitory effects of steroids in an in vitro model of angiogenesis.

The regulation of angiogenesis in the ovarian follicle and corpus luteum is unclear. Steroids are produced at very high concentrations in these tissues and we therefore examined the effect of steroids on angiogenesis in vitro. Explants of rat aorta were embedded in collagen gel and cultured in serum-free medium. Capillary-like microvessels were produced from the explants and microvessel number and length were measured in the presence and absence of steroids. At a concentration of 10 micrograms/ml, cortisol, progesterone, 17 alpha-hydroxyprogesterone and medroxyprogesterone acetate produced degeneration of microvessels after 7 days of steroid treatment (P < 0.01). Androstenedione and tetrahydro-S-(11-deoxytetrahydrocortisol) (tetrahydro S) produced degeneration at a slower rate: androstenedione inhibited microvessel growth after 11 days (P < 0.01) and tetrahydro S after 14 days (P < 0.05). Oestriol had no effect on microvessels; oestrone had a slow degenerative effect with significant inhibition seen after 14 days (P < 0.01). Oestradiol-17 beta at a concentration of 10 micrograms/ml completely inhibited microvessel growth from the explant cultures (P < 0.01) while at 1 microgram/ml it caused degenerative effects on growing microvessels. The effects of oestradiol and cortisol were reversible on removal of steroid-containing medium and replacement with 10% serum. We conclude that oestradiol may modulate angiogenesis in tissues in which the steroid concentration is high.

The minimum requirements for oestradiol to induce uterine sensitivity for implantation and decidualization in mice.

Steroid-containing s.c. silastic capsules with known physiological release characteristics were used to study the control of implantation and the induction of uterine sensitivity in ovariectomized mice. In mated, ovariectomized animals maintained on progesterone, the implantation sites were detected after approximately 12 h of oestradiol exposure, and alkaline phosphatase activity at the implantation sites developed within 21 h. Implantation could be induced in > 60% of animals by 4 h exposure to an oestradiol implant, in which time approximately 1.6 ng oestradiol would have been delivered. Continuous delivery of a low dose of oestradiol near the threshold for implantation induced a full complement of implantation sites in the responding animals. The sensitivity of implantation to low amounts of oestradiol suggests that this response is at least as sensitive as either the induction of vaginal cornification or the stimulation of uterine weight. The minimum time for the induction of uterine sensitivity when oestradiol and progesterone treatment were started simultaneously was approximately 36 h. The use of slow-release oestradiol-containing capsules provides a good model to investigate the roles of oestradiol in initiating and defining the 'implantation window'.