Serge Belisle

Hypothalamic-pituitary axis during reproductive aging in mice

We correlated the content of hypothalamic (HT) GnRH and pituitary (PT) GnRH receptor sites with PT and plasma gonadotropin levels throughout aging in C57BL/6J mice. Female mice of 4-6 months (young), 10-12 months (middle-age) or 15-18 months (old) of age were studied either intact or 15 days post-ovariectomy (OVX) with or without E2 therapy. In intact mice, HT GnRH content increased twofold during aging while GnRH receptor sites in PT remained unchanged. PT content of both FSH and LH gradually rose during aging while plasma concentration rose even more. OVX resulted in a significant decrease in both HT GnRH content and PT receptor sites and no age difference was observed. OVX also resulted in a significant increase in both PT content and plasma levels of gonadotropin in young and middle-age mice while old mice showed a blunted response. After E2 therapy for 7 days, HT GnRH content and PT GnRH receptor sites returned to normal levels in all age groups. By contrast, E2 therapy resulted in no change in PT content of FSH:LH in any age group. Whereas plasma FSH:LH levels returned to intact levels in young mice, they remained elevated to OVX levels in middle-age and old ones. Our results demonstrate an age related dichotomy in the PT production of FSH:LH unrelated to changes in either HT GnRH content or its PT receptor sites, thus suggesting cellular defects in post-receptor binding events within the pituitary.

Endocrine aging in C57 BL mice—II. Dynamics of estrogen receptors in the hypothalamic-pituitary axis

Specific cytosolic and nuclear binding sites for estrogens were measured in the hypothalamic-pituitary axis (HPA) of young (4-8 months) and old (16-18 months) C57 BL mice in order to determine any age-related alteration in hormone-receptor interaction. Our results indicated no age differences in the affinity (KD = 0.89 +/- 0.03 (SEM) vs 1.09 +/- 0.2 X 10(-9) M), the specificity, the sedimentation profile (6 s) or in the number (98.9 +/- 4.9 vs 84.4 +/- 2.3 fmol/mg protein) of unoccupied estrogen binding sites in the cytosols. Estradiol administration to young mice induced a complete translocation of cytosolic estrogen receptors to the nucleus, and two types of nuclear binding sites were observed: Type I were specific for estrogens with high affinity (KD = 0.51 +/- 0.06 X 10(-9) M) and low binding capacity (115.1 +/- 22.7 fmol/mg DNA) and sedimented in the 4.0 s area, while Type II binding sites showed a much higher capacity and lower affinity for R2858. HPA nuclear suspensions of aged untreated mice showed undetectable (less than 50 fmol/mg DNA) levels of nuclear estrogen receptors and E2 pre-treatment resulted in a significant increase in both types of binding sites. While no significant changes in the physicochemical characteristics of these nuclear receptors were observed, when compared to young animals, aging was manifested by a translocation defect in the HPA of C57 BL mice. These results suggest aging changes in the endocrine regulating centers of the brain with defective activation of estrogen receptors.

Dynamics of LHRH binding to human term placental cells from normal and anencephalic gestations

In order to improve our knowledge on human placental hCG production, we studied the binding of an LHRH agonist (N-Ac-Pro1,D-Leu6)-LHRH to third trimester intact placental cells from normal and anencephalic fetuses. In normal pregnancies, specific and saturable binding was found for both LHRH and its analogs with two classes of binding sites. Association constants were 4.7 +/- 2.2 (mean +/- SEM) X 10(5) M-1 for the low affinity sites and 1.7 +/- 0.8 X 10(8) M-1 for the higher affinity sites (P less than 0.01), and the estimated number of sites was 1.71 +/- 0.52 nmol/mg of cell protein and 2.79 +/- 0.54 pmol/mg of cell protein, respectively. Preincubation with increasing concentrations of LHRH agonist induced a progressive decrease in specific binding sites and manifested by a reduction in hCG production which paralleled the concentration of the agonist in preincubation buffer. Studies with placental cells from three anencephalic fetuses showed a decreased binding capacity for LHRH and its agonist, when compared to normal trophoblastic cells, as well as a reduced capacity to produce hCG. Our results suggest that mechanisms dependent upon LHRH binding to its receptor are required for placental hCG production in normal pregnancies. Furthermore our investigation suggests a role for the endocrine feto-placental milieu in the manifestation of these placental LHRH binding sites.

Human Placental Lhrh Receptor: Agonist and Antagonist Labeling Produces Differences in the Size of the Non-Denatured, Solubilized Receptor

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kavs indifferent from each other but significantly different from the Kavs of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Effect of ACTH on cholesterol and steroid synthesis in adrenocortical tissues

Previous studies have established that under normal conditions, adrenal HMG-CoA reductase activity is higher in hamsters than in rats and humans. The hamster reductase activity follows a diurnal rhythm corresponding to that of plasma ACTH and glucocorticoids [Endocrinology 107 (1980) 215] but not to that of aldosterone. ACTH treatments to hamsters increased reductase activity after a latency of 60 min; this enhancement was prevented by cycloheximide [J. steroid Biochem. 24 (1986) 325]. Immunotitration and immunoblotting studies confirmed that ACTH caused an increase in reductase protein synthesis. In rats, long-term (1-9 days) and short-term (3 h) treatments with ACTH also induced increase in adrenal HMG-CoA reductase activity and reductase protein. In the presence of iodoacetamide and inhibitors of proteolytic enzyme, a main specific band of enzyme was evinced in the area of 102 +/- 6 kDaMr, by Western blotting, for both hamster homogenate and microsomal preparations (Endocrinology, 120 (1987]. Similarly Mr values were found with rat adrenal preparations. The concentration of mRNA, analyzed using the c-DNA pRed-10 coding for the Chinese hamster ovary reductase, was increased in adrenals of hamsters treated with ACTH. The reductase mRNA levels also fluctuated during the day in parallel with those of reductase activity and reductase protein. In conclusion, these results indicate that ACTH and other conditions inducing a change in hamster adrenal HMG-CoA reductase activity provoke parallel changes in reductase mRNA and reductase protein content. ACTH acts on the adrenal reductase of species synthesizing large as well as small quantities of cholesterol, thus indicating the general importance of this hormonal control.

Endocrine aging in CBA mice: Characterization of uterine cytosolic and nuclear sex steroid receptors

We examined the evidence of alterations with age in sex steroid hormone/receptor interaction in the uteri of CBA mice. The mean affinity for estrogens and progesterone that we observed in uterine cytosols and nuclei of old (16-18 months) mice were no different than those observed in young (4-8 months) animals. Whereas the total number of unoccupied estrogenic receptor sites in old uteri did not differ significantly from that observed in uteri of young mice, aging was accompanied by a 3-fold and significant decrease in mean nuclear binding sites when standardized as mg of DNA (p less than 0.05). Contrary to estrogen receptors, aging uteri showed a 3-fold and significant decrease in the total number of uterine progesterone receptor concentration (p less than 0.05) which was manifested by a decrease of progesterone binding sites in both the cytosol and the nucleus. These results suggest that aging CBA mice present a profile of altered hormone-receptor interaction which is only partly dependent on ovarian steroids.

Adrenal Androgen Production in Hyperprolactinemic States

In order to better define the kinetics of the adrenal androgens in hyperprolactinemic states, we have studied 10 patients suspected of having a pituitary prolactinoma. Compared with the levels in 10 healthy control women (normal range: N), no significant differences in the mean (+/- standard error) plasma concentrations of cortisol (F), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DS), androstenedione (A), and testosterone (T) were found: F, 22.7 +/- 2.6 micrograms/100 ml (N = 10 to 25); DHEA, 7.5 +/- 1.4 ng/ml (N = 3.0 to 12.5); DS, 1.7 +/- 0.3 micrograms/ml (N = 1.1 to 3.6); A, 2.2 +/- 0.3 ng/ml (N = 0.5 to 3.5); T, 30.5 +/- 6.4 micrograms/100 ml (N = 20 to 80). Using constant infusions of unlabeled steroid, the metabolic clearance rates (MCR) of the adrenal androgens DHEA and DS were found to be 1282.6 +/- 342.6 liters/day and 5.6 +/- 1.4 liters/day, respectively, which were no different from the MRC of 1689.4 +/- 364.2 liters/day and 6.8 +/- 1.9 liters/day, respectively, found in the normal control women. Medical therapy with a dopaminergic agent in three of these patients reduced mean circulating levels of prolactin from 81.1 +/- 4.7 ng/ml to less than 15 ng/ml but did not change any of our results. It is concluded that, in the study group, hyperprolactinemia exerted minimal trophic effects on the production rates of adrenal androgens.

Hormonal regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase mRNA in the rat adrenal gland

We studied the effect of ACTH on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase enzyme. Reductase activity and reductase mass were enhanced by 22- and 6.2-fold respectively in one series of experiments, whereas in another the levels of reductase activity, reductase mass, and reductase mRNA were increased 6.6-, 3.6- and 2.2-fold respectively, following daily administration of exogenous ACTH for 3 days. Daily injection of 4-aminopyrazolopyrimidine (4-APP) to rats for 3 days increased circulating ACTH level 5.4-fold, whereas adrenal HMG-CoA reductase activity, reductase mass and reductase mRNA levels were greatly increased 36-, 10- and 16-fold, respectively. To counteract the effect of elevated plasma ACTH, dexamethasone acetate (Dex) was administered to 4-APP treated rats. At 3 h post Dex administration, plasma ACTH and corticosteroids levels were effectively decreased by 58 and 59%, respectively. The levels of adrenal HMG-CoA reductase mRNA, reductase activity and reductase mass were also diminished by 38, 31 and 40%, respectively. Our results show that rat adrenal HMG-CoA reductase can respond rapidly to hormonal changes, presumably through variations in circulating ACTH levels.

Effect of ACTH suppression on adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in 4-aminopyrazolopyrimidine-treated rats

4-Aminopyrazolopyrimidine (4-APP) treatments to rats for 3 days induced 2-fold increase of circulating ACTH and 11-fold increase of adrenal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA compared to NaCl-treated controls. This in vivo model was used to study the effect of the suppression of ACTH secretion on the adrenal HMG-CoA reductase mRNA level. Dexamethasone (Dex) administration to 4-APP-treated rats caused a rapid and parallel decline of the levels of plasma ACTH and adrenal HMG-CoA reductase mRNA to 50% within 2.5 h, whereas the free and esterified cholesterol content was increased 5 and 9.4 times respectively. These changes could be counteracted by the co-administration of ACTH with Dex. Aminoglutethimide (AG) administration to 4-APP-treated rats, which increased the adrenal esterified cholesterol content (7.5 times), decreased the HMG-CoA reductase mRNA level (44%), despite plasma ACTH level remaining elevated. Moreover, the participation of newly synthesized protein(s) in the lowering of adrenal HMG-CoA reductase mRNA level induced by ACTH suppression is suggested by the fact that cycloheximide (Cyclo), when co-administered with AG, completely blocked the decrease of HMG-CoA reductase mRNA level, despite the plasma ACTH level decreasing by 68% and the free and esterified cholesterol content increasing 3.9 and 12.3 times, compared to 4-APP-treated rats. Furthermore, the specificity of these effects was established by the fact that the beta-actin mRNA level was not affected by the administration of either Dex, AG, Cyclo, or AG + Cyclo to 4-APP-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of D2-dopamine receptors in human term placenta.

We studied the binding of [3H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37 degrees C and was reversed (t1/2 approximately 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [3H]-spiperone (0-25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 +/- 2 nM and a maximal binding capacity (Bmax) of 222 +/- 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D2-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 +/- 2 and 56 +/- 22 nM respectively. Dopamine inhibited [3H]-spiperone binding with a Ki of 570 +/- 50 microM whereas Schering 23390 (D1 antagonist) and propranolol (beta-adrenergic antagonist) were without effect. The binding was also inhibited by 100 microM GTP gamma S (38 +/- 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D2-dopamine receptors in human placenta.