Chronic E2 Treatment Research Articles

Estradiol improves pulmonary hemodynamics and vascular remodeling in perinatal pulmonary hypertension.

Partial ligation of the ductus arteriosus (DA) in the fetal lamb causes sustained elevation of pulmonary vascular resistance (PVR) and hypertensive structural changes in small pulmonary arteries, providing an animal model for persistent pulmonary hypertension of the newborn. Based on its vasodilator and antimitogenic properties in other experimental studies, we hypothesized that estradiol (E(2)) would attenuate the pulmonary vascular structural and hemodynamic changes caused by pulmonary hypertension in utero. To test our hypothesis, we treated chronically instrumented fetal lambs (128 days, term = 147 days) with daily infusions of E(2) (10 microg; E(2) group, n = 6) or saline (control group, n = 5) after partial ligation of the DA. We measured intrauterine pulmonary and systemic artery pressures in both groups throughout the study period. After 8 days, we delivered the study animals by cesarean section to measure their hemodynamic responses to birth-related stimuli. Although pulmonary and systemic arterial pressures were not different in utero, fetal PVR immediately before ventilation was reduced in the E(2)-treated group (2.43 +/- 0.79 vs. 1.48 +/- 0.26 mmHg. ml(-1). min, control vs. E(2), P < 0.05). During the subsequent delivery study, PVR was lower in the E(2)-treated group in response to ventilation with hypoxic gas but was not different between groups with ventilation with 100% O(2). During mechanical ventilation after delivery, arterial partial O(2) pressure was higher in E(2) animals than controls (41 +/- 11 vs. 80 +/- 35 Torr, control vs. E(2), P < 0. 05). Morphometric studies of hypertensive vascular changes revealed that E(2) treatment decreased wall thickness of small pulmonary arteries (59 +/- 1 vs. 48 +/- 1%, control vs. E(2), P < 0.01). We conclude that chronic E(2) treatment in utero attenuates the pulmonary hemodynamic and histological changes caused by DA ligation in fetal lambs.

Cerebral vasodilating capacity during forebrain ischemia: effects of chronic estrogen depletion and repletion and the role of neuronal nitric oxide synthase.

The effects of chronic 17beta-estradiol (E2) depletion, via ovariectomy (OVX), and its repletion, on cortical cerebral blood flow (CBF) and EEG activities during forebrain ischemia, as well as post-ischemic recovery and neuropathology, were assessed and compared with results obtained in normal female rats. We also examined whether neuronal nitric oxide synthase (nNOS) activity is affected by OVX and E2 replacement and whether NOS-derived NO supports vasodilation during ischemia. OVX females displayed a significantly lower CBF during ischemia (10% of baseline) than did normal females (23% of baseline). In OVX rats, given chronic low-dose E2 treatment (0.1 mg kg(-1) day(-1)), intra-ischemic CBF was similar to normal females (25% of baseline). However, at supraphysiologic E2 doses (> or = 0.5 mg kg(-1) day(-1)), that benefit was diminished or lost. Intra-ischemic EEG power reductions and post-ischemic survival rates, neurological dysfunction, and histopathology displayed similar relative differences among groups as the CBF findings. Intra-ischemic CBF was reduced by nNOS inhibition, with ARL 17477, in normal and low-dose E2-treated OVX rats (4-8% baseline). The repressed intra-ischemic vasodilating function in OVX rats may be due to reductions in nNOS activity, because untreated OVX rats showed a 50% lower cortical nNOS activity than that in normal rats and in rats treated with low or high dose (5 mg kg(-1) day(-1)) E2. However, the inability to restore vasodilating function despite normalization of nNOS activity indicates that another mechanism is responsible for the repression of vasodilatory function in the high-dose group. These findings suggest that E2, at levels within the physiological range, promotes ischemic neuroprotection via improving vasodilating capacity. One possible mechanism may relate to E2 enhancing brain nNOS expression and activity.

Impact of chronic treatment of ewes with estradiol-17beta or progesterone on oxytocin receptor gene transcription and ovarian oxytocin secretion.

Three experiments were conducted, the objectives of which were to 1) examine the effects of exogenous estradiol (E2) and progesterone (P4) on uterine concentrations of oxytocin receptors (OTR) and OTR mRNA, as well as the effect of exogenous P4 on progesterone receptors (PR) during the late luteal phase of the cycle, and 2) ascertain whether chronic E2 treatment of ewes during the cycle would alter prostaglandin F2alpha (PGF2alpha)-induced secretion of luteal oxytocin (OT). In experiment 1, 15 ewes were assigned to a control (n = 5; 2 ml corn oil [CO] s.c. on Days 4-14 of the estrous cycle) and two treatment groups (n = 5 each) receiving either 250 microg E2 s.c. (Days 4-14) or 10 mg P4 s.c. (CO on Days 4-10 and P4 on Days 11-14). Endometria and corpora lutea were removed on Day 15 of the cycle. Mean luteal weights were greater in treated than in control ewes (p < 0.05). Endometrial concentrations of OTR and OTR mRNA were significantly greater in control than in E2- or P4-treated ewes. In experiment 2, five ewes each were treated s.c. with CO or 10 mg P4 on Days 11-14 of the cycle; endometria were then removed on Day 15 for PR assay. Endometrial concentration of PR did not differ between groups. Experiment 3 consisted of 20 ewes assigned to four groups in a 2 x 2 factorial arrangement. Treatment consisted of two dosages of E2 (0 or 250 microg/day) in 2 ml CO and two dosages of PGF2alpha analogue (0 or 125 microg Estrumate). All ewes were injected s.c. with E2 or CO for 11 days as described for experiment 1. On Day 15, all ewes received an i.v. injection of PGF2alpha or saline (Time 0); then jugular blood was collected at frequent intervals for analysis of serum concentrations of OT. PGF2alpha induced a release of OT in control and E2-treated ewes (p < 0.05) compared to the value in saline-treated ewes. Collectively, these data suggest that in cycling ewes, exposure of the uterus to increased concentrations of E2 or P4 causes down-regulation of OTR as a consequence of suppression of the OTR gene. Chronic E2 treatment of ewes during the cycle does not act directly on the ovary to alter the stores of luteal OT.

The role of kidney infringements and blood microcirculation in pathogenesis of molibdenous

The purpose of this study was to test whether the insulin sensitivity, lipid metabolism and the susceptibility of the heart to ischemia/reperfusion injury are modulated by the chronic estrogen status. Rats were ovariectomized (OVX), not ovariectomized (sham) or ovariectomized and treated with subcutaneous 17 -estradiol (30 μg/kg/day, OVX+E2) (n=14–17 per group). Within 3 months after operation, body weight, the serum levels of estrogen, glucose, insulin, total cholesterol (T-chol), HDL-chol, LDL-cholesterol (LDL-chol), triglycerides (TG) and lipoprotein a (Lp(a)) were monitored. Three months after operation, hearts of partial rats (n=6–8 per group) were isolated and allowed an initial 20-min stabilization period, and then cardiac function was recorded and creatine kinase (CK) release in the coronary effluent was measured after 4 h of hypothermic ischemia in isolated rat hearts. The experimental results showed that from 2 weeks after ovariectomy to the end of the study, body weights of OVX were significantly higher compared with the other two groups (p<0.05). On weeks 5 and 9, insulin level of OVX was significantly higher than that of the other two groups (p<0.05), whereas it was not different among the three groups on weeks 12 and 13 (p>0.05). Blood glucose on week 13 was significantly higher in OVX (p<0.05). Consequently, Insulin Sensitivity Index (ISI) of OVX was lower than that of the other two groups on weeks 5 and 9 (p<0.05), but not on weeks 12 and 13. Serum values for T-chol, HDL-chol and LDL-chol were not significantly different among the three groups within the observing period. On week 13, TG level in ovariectomized group was significantly lower than in the sham- and E2-treated groups (p<0.05). Compared with sham, Lp(a) level was slight increased in OVX rats (p<0.05), while it was further increased in E2-treated rats (p<0.05). Cardiac function (left ventricular pressure (LVP) and ±dp/dtmax) of hearts removed from OVX rats was depressed, and CK release was markedly increased (p<0.05). However, treatment with E2 significantly improved cardiac function, as shown by increasing left ventricular pressure,+dp/dtmax and −dp/dtmax, and decreased CK release. In conclusion, chronic E2 treatment has some beneficial effects on cardiovascular disease (CVD), which come from the results of improvement of insulin sensitivity and post-ischemia cardiac function. However, the mechanism did not include changes in lipids and lipoproteins. The change in Lp(a) level shows that estrogen does not confer cardiovascular protection and may increase the risk of stroke.

Myocardial Protection of Contractile Function After Global Ischemia by Physiologic Estrogen Replacement in the Ovariectomized Rat

The role of physiologic estrogen levels (pg) on post-ischemic myocardial function was studied in the isolated working rat heart without (n=28, experiment No. 1) or with (n=15, experiment No. 2) preconditioning. For experiment No. 1, female ovariectomized rats were treated with placebo (n=19) or 17β-estradiol (E2,n=9; chronic E2), and 14 days later hearts were removed and perfused with modified Krebs–Henseleit bufferin vitro. In nine placebo-treated rats, E2 was administered at 20 min prior to ischemia (acute E2). The hearts were subjected to 15 min of global ischemia and 20 min of reflow. In experiment No. 2, ovariectomized rats were treated with placebo (n=8) or 17β-E2 (chronic E2,n=7). In this experiment, hearts were first preconditioned and then subjected to 20 min of sustained ischemia followed by 20 min of reflow. Global ischemia was produced by clamping the aorta and restricting left atrial flow so that coronary flow was reduced to zero; hemodynamics were continuously monitored throughout the study. Aortic flow, coronary flow, cardiac output, and dP/dtwere assessed at baseline, at the end of the ischemic period, and during reflow. The severity of ischemia was measured by post-ischemic release of lactate, lactate dehydrogenase, and creatine kinase and was similar among all groups in each study. In experiment No. 1, recovery of aortic flow, cardiac output, and dP/dtfollowing reperfusion, was significantly improved in rats treated with chronic E2 (P<0.05); acute E2 had no significant benefit. Post-ischemic recovery of coronary flow was not significantly affected. In experiment No. 2, chronic E2 treatment also significantly improved post-ischemic recovery of cardiac function in preconditioned hearts when compared with controls (P<0.05). In summary, E2 replacement in ovariectomized rats improves contractile function following global ischemia and reflow; cardioprotection by estrogen was observed over and above that conferred by ischemic preconditioning. Since the cardioprotective effect of E2 was independent of a significant improvement in coronary flow a direct effect of the hormone on the cardiac myocytes is postulated.

INTRAUTERINE TREATMENT WITH ESTRADIOL IMPROVES HEMODYNAMICS AND VASCULAR REMODELING IN AN OVINE MODEL OF PULMONARY HYPERTENSION. † 1567

Decreased endothelial nitric oxide synthase (eNOS) may contribute to the high pulmonary vascular resistance (PVR) which characterizes persistent pulmonary hypertension of the newborn (PPHN). In experimental PPHN produced by ductus arteriosus (DA) ligation in fetal lambs, eNOS mRNA, protein, and activity are reduced. Because E2 upregulates eNOS in several experimental settings, we hypothesized that estradiol treatment would attenuate the hemodynamic and histologic changes of pulmonary hypertension (PH), and that this effect may be mediated by upregulation of eNOS. We studied the effects of chronic E2 treatment of late gestation (126-128 d; term=147 d) fetal lambs. Immediately following DA ligation and placement of catheters in the main pulmonary artery (MPA) and aorta, fetal lambs were treated with daily infusion of E2 (10 mcg; n=6) or saline (ctrl; n=5) in the MPA. Eight days after the initial surgery, a left pulmonary artery (LPA) flow transducer was placed and lambs were then delivered and mechanically ventilated with FiO2=1.0. Mean pulmonary artery pressure inutero was not different between groups(73±7 vs. 79±3 mm Hg, day 8; E2 vs. ctrl). At delivery, E2 animals had lower PVR, better oxygenation, and higher LPA flow(table; *=p<0.05, E2 vs. ctrl).

Chronic estrogen treatment in male rats reveals mammosomatotropes and allows inhibition of prolactin secretion by somatostatin.

Previous in vivo studies demonstrated that estrogen treatment of male rats allows somatostatin (SRIF) to inhibit PRL release. The objective of this study was to determine whether chronic estrogen (E2) treatment of male rats can induce the conversion of somatotropes to mammosomatotropes. In situ hybridization and reverse hemolytic plaque assay were used to evaluate the effects of E2 treatment on GH and PRL messenger RNA (mRNA) content and hormone secretion in individual pituitary cells. Male rats were implanted for 2-6 weeks with placebo or estradiol-containing pellets (5mg/90-day release). Pituitaries were removed and prepared for reverse haemolytic plaque assay to determine PRL and GH secretion. This was followed by in situ hybridization using 35S-labeled riboprobes for PRL and GH mRNA. Chronic E2 treatment increased both the percentage of pituitary cells that secreted PRL and the amount of PRL secreted per cell. Concomitantly, there was a decrease in both the percentage of GH-secreting cells and that amount of GH secreted per cell. In situ hybridization demonstrated that E2 treatment increased PRL mRNA while decreasing GH mRNA in single pituitary cells. Significantly, in control male rat pituitary cell cultures, no PRL-secreting cells were positive for GH mRNA. In contrast, after chronic E2 treatment, 10% of PRL-secreting cells contained GH mRNA. In the control pituitary cell cultures, SRIF had no effect on PRL release, but SRIF significantly inhibited PRL release from pituitary cell cultures prepared from E2-treated male rats. These studies demonstrate that the adult pituitary preserves plasticity and, under the appropriate steroid milieu, allows conversion of somatotropes to mammosomatotropes.

Physiological oestradiol administration does not change the biological to immunological ratio of LH in serum from post-menopausal women.

We investigated the effect of chronic physiological concentrations of oestradiol on the bioactivity (B) and immunoactivity (I) of LH in serum using radioimmunoassay (RIA) and a sensitive immunoradiometric (IRMA) assay. Twelve post-menopausal, hysterectomized women received a 50-mg subcutaneous E2 implant, in the absence of progestogen treatment. Pretreatment serum samples were obtained on two occasions for measurement of E2 and FSH by radioimmunoassay and for measurement of LH by bioassay (BIO), immunoradiometric assay and radioimmunoassay. Further serum samples were obtained monthly for 6 months for assay as above. The serum E2 level (basal levels < 70 pmol/l) rose following implant (P < 0.01) peaking at 1 month (mean 454, range 356-580 pmol/l) and remaining within premenopausal physiological levels for 6 months. There were falls in FSH, LH-RIA, LH-IRMA and LH-BIO (P < 0.001). There was no change in the B:I ratio for B:I-RIA or B:I-IRMA for LH which were 1.8 and 3.3, respectively pretreatment and 1.7 and 2.9 after 1 month's treatment. The treatment of post-menopausal women with 50-mg E2 subcutaneous implants produces physiological premenopausal E2 levels for at least 6 months. There is no change in the B:I ratio of LH using either the RIA or IRMA during chronic E2 treatment.

Stimulation of angiotensinogen mRNA levels in rat pituitary by estradiol.

Angiotensin II (ANG II) is a putative paracrine hormone in the anterior pituitary. Angiotensinogen mRNA, however, is not detectable by Northern blot hybridization, suggesting that ANG II may not be synthesized within the pituitary. An alternative explanation may be that angiotensinogen gene activity is low under normal conditions, with angiotensinogen mRNA being below the level of detection. Utilizing a sensitive solution hybridization method, we sought to determine whether angiotensinogen mRNA could be detected in pituitaries from normal male rats or ovariectomized (OVX) rats treated with estradiol (E2) for 4 days. Very low levels of angiotensinogen mRNA were detected from male or OVX rat pituitaries, but E2 treatment resulted in a marked dose-dependent increase in pituitary angiotensinogen mRNA levels. Levels of angiotensinogen within the pituitary were not significantly different after the E2 treatment. Angiotensinogen mRNA levels in liver and brain were much higher than in the pituitary but were not altered significantly by the chronic E2 treatment. These results are consistent with the local synthesis of angiotensinogen in the pituitary and further suggest that pituitary angiotensinogen gene transcription is regulated by estrogen.

Chronic estradiol treatment alters central cholinergic function in the female rat: effect on choline acetyltransferase activity, acetylcholine content, and nicotinic autoreceptor function

The purpose of the present study was to determine the effects of chronic (2-month) estradiol (E2) treatment on cholinergic function in the frontal cortex, hippocampus and hypothalamus of the female rat. Chronic E2 treatment selectively increased choline acetyltransferase (ChAT) activity in the frontal cortex and decreased ChAT activity in the hypothalamus: hippocampal ChAT activity was not significantly changed. The decrease of ChAT activity in the hypothalamus was parallelled by a significant reduction in the content and release (basal and evoked) of acetylcholine (ACh) in this structure. Neither the content nor the release of ACh was altered in the frontal cortex. In the hippocampus, there was a significant increase in spontaneous ACh release; however, hippocampal ACh content and evoked ACh release were not changed. Chronic E2 treatment also altered nicotinic binding sites in these same regions as determined by saturation analysis of [ 3H]methylcarbamylcholine (MCC) to membranes. There was a decrease in the affinity and an increase in the density of [ 3H]MCC binding sites in hippocampal membranes and an increase in the density of [ 3H]MCC binding sites was observed in hypothalamic membranes. These alterations were parallelled by changes in nicotinic autoreceptor function within these two structures. In the hippocampus, the increase in spontaneous ACh release induced by MCC in control animals was no longer apparent after chronic treatment with E2. In hypothalamus, higher concentrations of the nicotinic agonist were required to increase spontaneous ACh release in slices from E2-treated rats as compared to control rats. Taken together, these results suggest that chronic E2 treatment decreases presynaptic cholinergic function in the female rat hippocampus and hypothalamus.

Estradiol feedback inhibition of luteinizing hormone concentrations in the anestrous sow2

The suppressive effects of exogenous 17 beta-estradiol (E2) on LH concentrations in sows that remained anestrus following weaning and in those that returned to estrus were evaluated. Four anestrous and four cyclic sows were treated subcutaneously with silastic implants containing E2 at 13 d after ovariectomy (d 0). Three anestrous and six cyclic sows received silastic implants without E2. Blood was collected at 6-h intervals from d -1 to d 12 and at 15-min intervals for 8 h on d -1, 2, 7 and 12. Sows were treated with 1 microgram GnRH/kg BW at the completion of each 8-h frequent sampling period. Blood was collected at intervals of 10 to 30 min for 3 h after GnRH treatment. Concentrations of E2 remained less than 5 pg/ml in sham-treated sows and were between 20 and 25 pg/ml in E2-treated females. Pulsatile LH concentrations was similar between anestrous and cyclic sows prior to implant treatment. Sham-treated anestrous sows had greater (P less than .05) pulse frequency and mean LH concentrations than E2-treated anestrous sows on d 2, 7 and 12. Differences in pulsatile LH concentrations between E2-treated and sham-treated cyclic sows were not detected. Pulse frequency was less (P less than .05) in E2-treated anestrous sows than in E2-treated cyclic sows on d 7 and 12. Peak LH concentrations were greater (P less than .05) in E2-treated cyclic sows than in E2-treated anestrous sows at each GnRH challenge. These results suggest that the hypothalamo-hypophyseal axis is more sensitive to the negative feedback effects of E2 in anestrous sows than in cyclic sows. In addition, chronic E2 treatment reduces pituitary responsiveness to GnRH to a greater extent in anestrous than in cyclic sows. Failure to return to estrus in swine may be due, at least in part, to an increased sensitivity of the hypothalamo-hypophyseal axis to the negative feedback effect of estradiol.

Progesterone Implants Extend the Capacity for 4-Day Estrous Cycles in Aging C57BL/6J Mice and Protect Against Acyclicity Induced by Estradiol1

The onset of age-related acyclicity in rodents can be accelerated or attenuated by chronic treatment with estradiol (E2) or progesterone (P4), respectively. Because of the physiological effects of exogenous E2 and P4 on age-like reproductive changes, we sought to demonstrate if P4 could antagonize the acceleration of acyclicity by E2 in C57BL/6J mice. Mice were treated for 6 or 12 wk with P4 and/or orally administered E2. Twelve but not six weeks of E2 caused permanent acyclicity, whereas P4-treated mice had more 4-day cycles than did controls in both studies. The combination of E2 and P4 also caused a striking increase in 4-day cycles after treatment even greater than that from the P4 treatment alone. Thus, P4 transiently increases cycle regularity with a greater incidence of 4-day cycles and antagonizes the premature onset of acyclicity caused by chronic E2 treatment.

Effect of chronic or cyclic exposure to estradiol on the humoral immune response and the thymus.

Estradiol (E2) is known to enhance humoral immunity. Previous studies examining serum immunoglobulin levels on days within the estrous cycle or in the ovariectomized, chronically E2-implanted rat have determined a positive correlation between serum E2 concentrations and antibody responsiveness. No previous study has investigated the effect of fluctuating E2 concentrations, as during the estrous cycle, throughout the course of a specific humoral immune response. Therefore, in the present study we addressed the influences of cyclic and tonic E2 exposure on the anti-fluorescein response in the rat. Ovariectomized rats were submitted to a 2 x 2 factorial design of chronic E2 implants and 4-day E2 injection (and vehicle control) regimens. Anti-fluorescein antibody production and thymic weights were measured. Animals given a 4-day cycle of E2 showed significantly (p less than 0.001) greater anti-fluorescein responses than did animals given tonic or no E2 replacement. Animals which were given implants, with or without cyclic E2 injections, experienced significant (p less than 0.001) thymic involution relative to those without implants. The 4-day E2 injections alone did not lead to thymic atrophy. These results indicate that while chronic E2 treatment induced thymic atrophy, cyclic exposure to E2 exerted a greater immunoenhancing influence than did tonic E2 exposure.

Pituitary and hypothalamic glucose-6-phosphate dehydrogenase: effects of estradiol and age in C57BL/6J mice.

Activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH) were measured in the mediobasal hypothalamus and pituitary gland of C57BL/6J mice throughout their lifespan. Activities of pituitary G6PDH, pituitary 6-PGDH, and hypothalamic G6PDH increase with age in female mice, assayed 7 days after ovariectomy, but not in intact females or males. Pituitary G6PDH specific activity is increased by middle-age (13-14 months) in females, before the onset of acyclicity, and remains elevated throughout the acyclic phases of persistent vaginal cornification and persistent diestrus. This increase in activity is ovary dependent, because it can be prevented by long term (12-month) ovariectomy. The increased activity is not linked to pituitary tumorigenesis and does not result from trapped blood cells, as evaluated by studies with 51Cr-labeled erythrocytes. Responses to physiological levels of estradiol (E2) were analyzed with a graded series of chronic polyethylene implants or after a single sc injection in ovariectomized mice aged 5-22 months. The responsiveness of pituitary G6PDH to E2 is not altered during aging. Young cycling (6 months old) and older acyclic mice (19 months old) displaying persistent vaginal cornification show equivalent increases of about 100% in G6PDH specific activity after chronic E2 treatment and similar time courses of induction after a single E2 injection. Pituitary G6PDH is maximally induced (30% increase) by 48 h after E2 injection in all age groups. In addition, the rates of decline in pituitary G6PDH specific activity after ovariectomy are similar in young and older mice (half-life, 4 days). The specific activity of G6PDH in the mediobasal hypothalamus and blood is unaffected by E2 administration. The relatively low doses of E2 used here fail to alter 6-PGDH specific activity in pituitary or brain. These findings indicate that female reproductive senescence in mice is not associated with generalized losses of sensitivity and responsivity to E2 throughout the neuroendocrine axis.

Estrogen preferentially stimulates lactosaminoglycan-containing oligosaccharide synthesis in mouse uteri.

The effects of steroid hormones on the synthesis of lactosaminoglycan (LAG)-containing oligosaccharides by mouse uteri are reported. The uterine LAG-containing oligosaccharides were degraded partially by Pseudomonas endo-beta-galactosidase, releasing an oligosaccharide of the apparent structure: Gal beta----N-acetylglucosaminyl(----N-acetylgalactosaminyl)beta 1,3----galactose. A larger fraction of the LAG-containing oligosaccharides bound to pokeweed mitogen than to Datura stramonium lectin, suggesting the presence of highly branched structures. LAG-containing oligosaccharides were resistant to sequential digestion with Pronase, nitrous acid, hyaluronidase, and chondroitinase ABC. These polysaccharides exhibited a Gal:GlcNAc:GalNAc ratio of approximately 1.0:1.0:0.3 and were not fucosylated. The ion-exchange behavior of the LAG-containing oligosaccharides before and after mild acid hydrolysis indicated the presence of sialic acid residues. The LAG-containing glycopeptides were highly resistant to beta-elimination but were released quantitatively by hydrazinolysis, demonstrating an N-linkage to protein. Binding to pokeweed mitogen was markedly enhanced following release of these oligosaccharides from peptides by hydrazinolysis, suggesting that peptide-bound oligosaccharides were partially inaccessible to the lectin. Molecular exclusion chromatography of the oligosaccharides released by hydrazinolysis revealed a broad distribution ranging from Mr 4,000 to 15,000 with a median Mr of approximately 8,000. We extended the above observations by determining how the steroid hormones 17-beta-estradiol (E2) and progesterone affected synthesis of the LAG-containing oligosaccharides in ovariectomized mice. Generally, E2 and a number of E2 agonists stimulated glycoconjugate synthesis; however, chronic E2 treatment or combined treatment with E2 plus progesterone caused the synthesis of most glycosaminoglycans to return to basal levels. In contrast, E2 either alone or in combination with progesterone stimulated synthesis of LAG-containing oligosaccharides in preference not only to glycosaminoglycans but also to other classes of N-linked oligosaccharides. This effect was apparent during both priming and nidatory E2 treatments. Collectively, these data provide the first demonstration of LAG-containing oligosaccharides in uteri and for the hormonally regulated synthesis of lactosaminoglycans. In addition, this is the first demonstration of the ability of steroid hormones to induce the synthesis of certain types of N-linked oligosaccharides in preference to others in the same tissue.

Bromocryptine Prevents the Decline in Tuberoinfundibular Neuronal Release of Dopamine after Removal of Chronic Estrogen Treatment

Prolonged exposure to estradiol 17-beta (E2) in rats has been shown to decrease dopamine (DA) synthesis in and release from tuberoinfundibular dopaminergic (TIDA) neurons in Fischer 344 rats. The objective of the present study was to determine whether inhibition of the E2-induced increase in anterior pituitary (AP) weight and prolactin (PRL) secretion by concomitant administration of the dopaminergic agonist, bromocryptine, could prevent the decrease in TIDA neuronal function produced by chronic E2 administration. TIDA neuronal function was evaluated by in vitro superfusion and electrical stimulation of median eminence (ME) tissue after allowing for accumulation of [3H]dopamine (DA). The effect of chronic E2 and/or bromocryptine treatment on catecholamine content in tuberohypophyseal neurons in the neurointermediate lobe was also measured to determine whether increased pituitary size possibly damaged the tuberohypophyseal neurons. Treatment with E2 for 30 days significantly increased AP weight, serum PRL concentration, and AP PRL and DNA content over values in non-E2-treated controls. When bromocryptine was injected daily during E2 treatment, bromocryptine completely inhibited the E2-induced increase in serum PRL and AP DNA content, and AP weight was only moderately increased. The evoked release of 3H at the end of the 30-day E2 treatment was reduced during electrical stimulation and there was no augmented release of 3H from the ME tissue after 10 microM nomifensine infusion in E2-treated rats and in rats given both bromocryptine and E2. However, neurointermediate lobe DA content was diminished only in E2-treated rats and not in animals given bromocryptine together with E2. When all treatments were discontinued for 30 days, animals previously given only E2 showed sustained increases in AP weight, serum PRL levels, and AP PRL and DNA content, but reduced stimulation-evoked release of 3H, absence of response to nomifensine, and reduced neurointermediate lobe DA and norepinephrine content when compared with values in non-E2-treated controls. After withdrawal of E2 treatment for 30 days, animals previously given bromocryptine and E2 together were not different from control animals in any of the parameters measured. These results suggest that the decline in TIDA neuronal release of DA induced by chronic E2 treatment was at least partly exerted via the marked hyperprolactinemia and/or by compression of the medial basal hypothalamus by the enlarged AP.

Secretion of ovine luteinizing hormone in vitro: differential positive control by 17 beta-estradiol and a preparation of porcine ovarian inhibin.

17 beta-Estradiol (E2) and an enriched preparation of porcine ovarian inhibin can positively regulate LH secretion in two different ways in ovine pituitary cell cultures. One major effect of these agents is to increase the sensitivity of cultures to low levels of LHRH or LHRH analogs. The second effect is to increase culture responsiveness to high levels of LHRH or its analogs. Prolonged treatment with 1 nM E2, for instance, increased pituitary sensitivity to D-Lys6-LHRH by 10-fold, since it lowered the ED50 of D-Lys6-LHRH from approximately 400 pM to about 40 pM; the maximum LH secretory response to D-Lys6-LHRH (1-100 nM) was not changed, however. In contrast, short term treatment with an E2-free preparation of porcine ovarian inhibin could increase, by 350%, normal LH responsiveness to high levels of D-Lys6-LHRH (1-100 nM), but had no effect on sensitivity; the ED50 remained unchanged. Chronic treatment with inhibin eventually increased sensitivity, and interestingly, a short treatment with E2 (6 h) caused a major increase in both sensitivity and responsiveness, but the responsiveness component usually disappeared between 6 and 48 h of chronic E2 treatment. Treatment with both E2 and the inhibin preparation caused the greatest increase in pituitary sensitivity to D-Lys6-LHRH (ED50 decreased to approximately 30 pM), but the increased responsiveness to D-Lys6-LHRH (1-100 nM) declined with time as with E2-only treatment. The different actions of E2 and the ovarian inhibin preparation seen to perturb different aspects of LH secretion. Therefore, study of these phenomena may uncover novel molecular details governing LHRH-stimulated LH release. Furthermore, these two types of LH secretory control may be physiologically important, at least in sheep, and perhaps in other animals as well.

Effects of estradiol-17beta on LH-RH/FSH-RH-induced, and spontaneous, LH release in prepubertal female pigs.

The effect of estrogen (E2) on pituitary response to LH-RH/FSH-RH (LH-RH) was examined in immature female pigs (gilts). Eight gilts (9-10 weeks old) received iv injections of 2.5, 5.0, 10.0, 20.0, and 40.0 mug LH-RH, beginning 4 days after placement of indwelling jugular cannulae. Blood samples were drawn at 5-min intervals following LH-RH injection and plasma LH was determined by RIA and expressed as ng R786-3/ml. Pituitary responses were expressed as the increment in LH levels above baseline (delta LH). Pretreatment LH levels were 0.45-1.25 ng/ml. Responses to LH-RH (delta LH plus or minus SE) ranged from 3.7 plus or minus 0.6 (at 5 min) to 8.4 plus or minus 1.4 ng/ml (at 20 min) for 2.5 and 40 mug doses, respectively. Three additional prepubertal gilts were cannulated and implanted with E2. After 3 days plasma estrogens rose from approximately 30 pg/ml to 289 plus or minus 34 pg/ml and the dose-response studies were repeated. Prestimulation LH concentration was consistently below 0.4 ng/ml and delta LH levels ranged from 1.4 plus or minus 0.5 (at 10 min) to 5.7 plus or minus 1.3 ng/ml (at 20 min) for the 2.5 and 40.0 mug doses, respectively. Analysis of the regression equations for delta LH on log LH-RH dose showed that the adjusted means for LH response were significantly lower (P less than 0.02) in E2 implanted animals. However, the regression coefficients were not different (P greater than 0.5). On the basis of these and other analyses, it is concluded that chronic E2 treatment depresses both basal LH secretion and pituitary responsiveness, but that pituitary sensitivity to LH-RH is not modified. Several nonimplanted females showed bursts of LH release subsequent to the LH-RH induced peak, but multiple peaks were never observed in E2 implanted gilts. An explanation was sought and 7 of the nonimplanted gilts were used to study spontaneous LH release by determining plasma LH at 10-min intervals for 20 h. During this period spontaneous episodes of LH release were observed in control animals with a mean frequency of 1.3 LH peaks/h. When 4 animals were given 5 mug E2 iv and then sampled for 12 h, episodic secretion was suppressed and the latency to the first spontaneous LH release was 9.6 plus or minus 0.8 h, significantly greater (P less than 0.02) than the latency to the first peak after saline injections (4.2 plus or minus 1.5 h) in the controls. It is suggested that "multiple" LH peaks occurring after initial LH-RH stimulation may represent the superimposition of spontaneous episodic release upon the induced response.