Biomedical subjects
Publications and source records attributed to N J MacLusky.
A double-label electron microscopic immunostaining procedure was employed to examine the interconnections of glutamic acid decarboxylase(GAD)- and LHRH-immunoreactive neurons in the medial preoptic area of the rat. The results provide ultrastructural evidence that GABA-ergic neurons establish symmetric (Gray II) synapses on LHRH neurons.
The pattern of periovulatory and luteal phase levels of serum estradiol (E2) and progesterone (P) were compared between 8 conception and 28 nonconception cycles of patients undergoing in vitro fertilization (IVF). Ten additional women served as control subjects and did not undergo follicular aspiration. Follicle growth was induced with an individualized Pergonal (human menopausal gonadotropin) regimen, and laparoscopy was performed 36 hours after human chorionic gonadotropin administration. The length of the luteal phase did not differ significantly among the three groups and was between 14 and 15 days in duration. When IVF conception cycles were compared with nonconception cycles, although no difference in the number of large follicles was observed (4.25 +/- 0.45 versus 3.6 +/- 0.25), the patterns of E2 and P differed significantly. Daily serum E2 levels tended to be higher in the periovulatory phase in conception cycles when compared with nonconception cycles, and were significantly (P less than 0.05) higher in the early, mid, and late luteal phases. Serum P levels were significantly higher (P less than 0.05) in conception cycles from the midluteal phase onward. A decline in both serum E2 and P in the midluteal phase in conception cycles suggested some degree of corpus luteum deficiency. It is suggested that high E2 levels in the periovulatory phase may be an indicator of better follicular development under human menopausal gonadotropin stimulation and that the deficiency observed in the late luteal phase is overcome with the establishment of pregnancy.
An exchange assay is described for the measurement of nuclear estrogen receptors (ERn) in microdissected brain regions. The distribution of ERn in the hypothalamus and amygdala of the rat 1 h after an injection of estradiol (E) is presented. Combining the exchange assay with a previously described method for measurement of cytosol estrogen receptors (ERc) in microdissected brain samples, gonadectomized male and female rats were compared for ERc and ERn. While ERc concentrations tended to be higher in females than in males in all regions of the hypothalamus, with a significant sex difference in the arcuate-median eminence, no sex difference in ERn concentrations was observed after E injection. These results suggest that ERc measurements alone are not sufficient to establish the capacity of the E receptor system: ERn measurements are also necessary to establish the relationship between receptor levels and physiologic estrogen responsiveness.
The production of 17 beta-estradiol and progesterone (Prog) by human granulosa-luteal cells obtained from 24 aspirated follicles of 11 women undergoing laparoscopy in an in vitro fertilization program was studied. Follicular growth was stimulated with an individualized human menopausal gonadotropin regimen begun on either day 1 (group I; n = 5) or day 3 (group II; n = 6) of the menstrual cycle, and laparoscopy was performed 36 h after hCG administration. Granulosa-luteal cells were cultured for 2 h in culture medium alone or in the presence of either pregnenolone (10(-7) M) or testosterone (10(-7) M). Aromatase activity was present in the granulosa-luteal cells, as evidenced by a significant (P less than 0.001) increase in E2 production in the presence of testosterone. The addition of pregnenolone did not augment Prog production. Granulosa-luteal cells derived from Group II patients produced significantly (P less than 0.001) more Prog than those derived from group I patients. In addition, group II granulosa-luteal cells associated with mature oocyte-coronacumulus complexes produced significantly (P less than 0.001) more Prog than those in group I. Fertilization and pregnancy correlated with Prog production, in that granulosa-luteal cells associated with oocytes that were fertilized produced significantly (P less than 0.001) less Prog than those associated with nonfertilized oocytes. Granulosa-luteal cells from the 2 patients in this series who conceived demonstrated a further significant (P less than 0.02) reduction in Prog production. It appears that administration of human menopausal gonadotropin early (day 1) in the follicular phase results in incomplete maturation of the granulosa cells. Furthermore, the optimal oocyte, in terms of successful fertilization, may be one derived from a follicle that has undergone appropriate stimulation resulting in adequate maturation, but has not surpassed that point.
Use of a microcomputer with the multiple exposure photography (MEP) method for rapid analysis of semen characteristics is described. The computer is programmed to calculate information fed from a digitizing tablet while scanning projected images of photographed sperm. In this way sperm concentration, percentage of motility, average velocity and frequency distribution of sperm velocity, as well as percentage of abnormal forms are immediately obtained. The method was found to be as accurate, less prone to error, and significantly less time consuming than the previous manual measuring method. It can greatly facilitate routine semen analysis in laboratories using the MEP method.
The cytosolic estrogen receptor (ERc) and progestin receptor (PRc) levels were measured in 56 human epithelial ovarian tumors. The maximum ERc content in a tumor sample was 163 fmol/mg cytosolic protein. Forty-six of the tumor samples were evaluable for clonogenic growth in soft agar, and 19 samples produced 15 or more colonies per 10(5) cells plated. Four of the samples that grew had ERc levels of greater than 30 fmol/mg cytosolic protein. No correlation, however, between growth in soft agar and ERc or PRc content was observed. The antiproliferative properties of the antiestrogen, tamoxifen, were studied. Although no decrease in colony formation was observed after a 1-hr exposure to 0.2 or 2 microM tamoxifen, continuous exposure of cells to 2 microM tamoxifen reduced clonogenicity in 8 of 18 solid ovarian carcinomas examined. The maximum diminution in colony formation was approximately 50% of that of the control and was seen in 2 tumor samples. Both tumors that displayed the maximum response to continuous tamoxifen treatment had ERc and PRc levels greater than 30 fmol/mg cytosolic protein. None of the 14 tumors with ERc levels less than or equal to 30 fmol/mg cytosolic protein exhibited a decrease in colony formation of more than 50%. Exposure of cells for 1 hr to the combination of doxorubicin and tamoxifen produced a significant antagonism of the individual doxorubicin or tamoxifen antiproliferative effects in 7 of 9 samples examined. These data suggest that in a subset of human ovarian epithelial carcinomas tamoxifen alone can have some direct antiproliferative action on the clonogenic cells. The maximum antiproliferative effect of tamoxifen observed was related to ERc content in ovarian tumors.
The mechanisms underlying the differences in uterotrophic potency between 2- and 4-hydroxyestrogens were explored. Doses of estradiol (E2)(10 micrograms/kg), 2-OHE2 (500 micrograms/kg) and 4-OHE2 (100 micrograms/kg) sufficient to induce near maximal cell nuclear estrogen receptor (ERn) binding were injected subcutaneously into 26 day old female rats. Uterine ERn concentrations declined more rapidly after 2-OHE2 than after E2 or 4-OHE2. E2 and 4-OHE2 both elicited a significant increase in uterine wet weight, measured at 24-36 hrs after injection. 2-OHE2 had no significant effect and neither synergized with nor antagonized the effects of simultaneously administered E2 or 4-OHE2. Under in vitro conditions at 25 degrees C, 2-hydroxyestrone (2-OHE1) and 2-OHE2 both dissociated from the receptors more rapidly than either their parent monophenolic estrogens or the corresponding 4-hydroxyestrogens. These results suggest that differences in estrogenic potency between 2- and 4-hydroxyestrogens may partly be a function of the dissociation kinetics of their estrogen receptor complexes.
In order to assess the relationship between tardive dyskinesia (TD) and baseline circulating concentrations of estradiol, prolactin and homovanillic acid, we studied 43 outpatient men and postmenopausal women on chronic antipsychotic medication. Serum estradiol did not correlate with severity of TD, antipsychotic medication dose, serum prolactin or plasma HVA. Multiple regression analysis indicated a significant relationship between plasma HVA and severity of TD in postmenopausal women. These findings support the hypothesis that estrogen might serve a protective role against neuroleptic-induced striatal dopamine pathology.
The effects of estradiol (E2) and its 2- and 4-hydroxylated metabolites on gonadotrophin regulation in the female rat brain were examined. Neonatal female rats were injected from day 1 through 5 with E2, 2-OHE2 and 4-OHE2, at doses of 0.1, 1 and 10 micrograms/day. At 2, 6 and 24 h after the last estrogen injection, some animals from each treatment group were killed and the concentration of estrogen receptors (ERn) in their brain cell nuclei determined. The remaining animals were allowed to mature. Their vaginal smear patterns were examined from 7 to 9 and from 15 to 17 weeks of age. They were then ovariectomized and tested for their capacity to exhibit a luteinizing hormone (LH) surge in response to estrogen and progesterone injections. In a parallel series of experiments, the affinities of the three test estrogens for alpha-fetoprotein (AFP) were determined from in vitro competition studies with fetal rat serum. All three estrogens increased brain cell nuclear ERn concentrations, measured at 2 h after the final injection. E2 was more potent in this respect than either 4-OHE2 or 2-OHE2. E2 and 4-OHE2 competed for binding to AFP to an approximately equal extent. 2-OHE2, however, was a much weaker competitor for AFP than either of the other two compounds. The neonatal E2 and 4-OHE2 treatments reduced the number of animals showing regular cyclic vaginal smears, at all three doses tested. In contrast, 2-OHE2 significantly affected vaginal cyclicity only at a dose of 10 micrograms/day.(ABSTRACT TRUNCATED AT 250 WORDS)
In order to understand the mechanism involved in the onset of labor in rats, a series of biochemical and morphological studies were done on pregnant rat myometrium, during the latter half of pregnancy and in the postpartum period. It was observed that plasma progesterone (P) levels gradually declined between day 15 and 20 of pregnancy as did cytosol (PRC) and nuclear P receptors (PRN). In contrast, nuclear estrogen receptors (ERN), both occupied and unoccupied, increased in number during this period of time. Between 24 to 6 hrs prior to labor, plasma P levels and PRN reached a nadir (day 15 v.s. day 21; 776 +/- 88.5 and 187.4 +/- 40.4 fmol/mg DNA, mean +/- S.E., p less than 0.01), while ERN showed an abrupt increase (day 21 v.s. day 22; 1710.6 +/- 61.1 and 3254.8 +/- 203.8 fmol/mg DNA, p less than 0.01), and remained high during labor. After delivery, ERN gradually decreased, but PRN showed a surge between 6 to 12 hrs postpartum in concert with the rise in plasma P levels due to ovulation. Electromicroscopic quantitation of gap junctions (gis) also showed a sharp increase in the number of gjs during labor (day 21 v.s. during labor; 2.0 +/- 0.4 and 17.9 +/- 1.5 gjs per 1,000mcm of plasma membrane, p less than 0.01). A large number of gjs were observed during the first 12 hrs postpartum, but they abruptly disappeared thereafter. These observations suggest that in rats, prepartum accumulation of ERN may facilitate the formation of gjs, which serve as a means for either electrical or chemical excitations to synchronize myometrial contractions and initiate/sustain labor. After delivery, on the other hand, P seems to down-regulate the number of gjs and induce rapid myometrial quiescence during postpartum ovulation which allows the myometrium to be receptive to fertilized ovum.
A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.
The effect of in vivo and in vitro estrogen treatment on 3-O-methyl-D-glucose (30MDG) uptake in isolated rat hepatocytes was examined. A 1-h preincubation of isolated hepatocytes with 17 beta-estradiol (E2) or 17 alpha-ethynyl estradiol stimulated uptake of 30MDG in a dose-dependent fashion. The response appeared to be sterospecific, in that 17 alpha-estradiol was approximately 100-fold less effective than its 17 beta isomer. By itself, the triarylethylene antiestrogen, nafoxidine, slightly increased hepatocyte 30MDG uptake, while in combination with estrogen, nafoxidine completely blocked the effects of both E2 and 17 alpha-ethynyl estradiol. The protein synthesis inhibitor, cycloheximide, inhibited basal 30MDG uptake and abolished the stimulatory effect of estrogen. Kinetic analysis indicated that the estrogen effect resulted from an increase in the Vmax of the 30MDG transport system, with no measurable change in its Km. In vivo estrogen treatment, using either estrogen injections or continuous release Silastic E2 capsules, produced an increase in hepatocyte 30MDG uptake of 52-86%. A similar (66%) increase was seen in pregnant animals between the 14th and 19th days of gestation. These findings demonstrate that estrogens exert a rapid stimulatory effect on hepatocyte hexose transport. This effect is qualitatively similar to the response to estrogen of the hexose transport systems in other estrogen target tissues, such as the uterus. In addition, the results provide further support for the concept that the effects of the triarylethylene antiestrogens are both tissue and end-point dependent. Although previous studies have shown that nafoxidine exerts estrogen-like effects on hepatic renin substrate production, the present data indicate that, with respect to hepatocyte 30MDG, nafoxidine is almost a pure estrogen antagonist.
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The ability of estradiol-17 alpha to serve as a substrate for estrogen-2/4-hydroxylase in rabbit hypothalamic tissue was determined and compared to that of estradiol-17 beta. Both 2- and 4-hydroxy metabolites of estradiol-17 alpha were formed by the hypothalamic tissue in vitro. The rates of formation of 2-hydroxyestradiol (2-OHE2)-17 alpha and -17 beta were similar as were their kinetic constants (Km and Vmax). In addition, 2-OHE2-17 alpha was shown to inhibit purified rat adrenal tyrosine hydroxylase with a potency comparable to that of 2-OHE2-17 beta, a finding similar to that reported by others with respect to catechol-o-methyltransferase. Since estradiol-17 alpha has a markedly reduced affinity for estrogen receptors compared with extradiol-17 beta, this steroid could be useful in studies designed to distinguish between receptor mediated effects of estrogens and effects that locally formed catechol estrogens may have through their direct interaction with catecholaminergic system in neural tissue.
This study describes the presence of a population of oestrogen receptors in cell nuclei from the pituitary gland and brain of untreated and oestradiol-treated ovariectomized rats. The receptors behaved as if they were not occupied by oestradiol. These 'unfilled' oestrogen receptors could be distinguished from occupied nuclear receptor sites on the basis of their ability to bind [3H]oestradiol at low temperatures (0-4 degrees C). Occupied receptors bound labelled [3H]oestradiol only under exchange conditions at an increased temperature (25 degrees C). Unfilled and occupied nuclear receptors were physicochemically similar in terms of sedimentation coefficients in sucrose density gradients containing 0.4 M-KCl (4-5S), equilibrium dissociation constants for reaction with [3H]oestradiol (0.2-0.6 nmol/l) and ligand specificity. In ovariectomized rats, unfilled receptors constituted more than 75% of the total nuclear receptor population. One hour after i.v. treatment with oestradiol (3.6 micrograms/kg), both total and unfilled nuclear receptor concentrations increased and then subsequently declined over the next 12 h. The increase in unfilled sites was, however, proportionately less than that occurring in the filled component; at 1 h after oestradiol injection unfilled sites constituted less than 20% of the receptors present in brain and pituitary cell nuclei. The physiological significance of unfilled nuclear oestrogen receptors remains unknown. The observations that they exist in various oestrogen target tissues and that their levels are influenced by oestradiol treatment suggest a possible role for these receptors in the mechanism of oestrogen action.
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The effects of primary and catechol oestrogens on the uterus of the immature rat were compared. Because differences between the in-vivo and in-vitro oestrogenic actions of catechol oestrogens on the secretion of LH had been observed, their effects on a peripheral target organ, the uterus, were examined under similar conditions. In-vivo effects were assessed by measurement of uterine weight, induction of uterine cytoplasmic progestogen receptors, and by histological examination. In-vitro actions were determined by measurement of oestrogen-specific induced protein. It was found that the uterotrophic effects in vivo of 4-hydroxyoestradiol were indistinguishable from those of oestradiol whereas 2-hydroxyoestradiol was only weakly oestrogenic and 2-hydroxyoestrone had no effect. However, in vitro, 2-hydroxyoestradiol was as effective as 4-hydroxyoestradiol or oestradiol in stimulating synthesis of uterine induced protein, and 2-hydroxyoestrone, although less potent than oestradiol, had a significant effect. These results were consistent with the observed effects on the secretion of LH. The differences between in-vivo and in-vitro uterotrophic properties of catechol oestrogens can be explained on the basis of known pharmacokinetic factors.