Tsuguo Uemura

Electrophysiological correlates of pulsatile gonadotropin release in rats.

In order to reveal the electrophysiological correlates of pulsatile LH release in ovariectomized rats, changes in multiple unit activity (MUA) in the arcuate nucleus (ARC), the medial preoptic area, the ventromedial nucleus, the anterior hypothalamic area and other areas of the brain were recorded in lightly anesthetized female rats. Rats were ovariectomized at least 7 weeks prior to the experiment. 0.1 -ml blood samples were withdrawn from a cardiac catheter at 10-min intervals during the recording in order to measure the changes in the plasma LH concentration by radioimmunoassay. The LH concentration showed pulsatile changes under continuous intravenous infusion of thiopental sodium while cortical EEG was stable throughout the experiment. The firing rate in the middle part of the ARC (8 recordings) changed in parallel with the increase in the plasma LH concentration and was abruptly raised just prior to an increase in LH secretion. The number of MUA spike discharges per minute increased to 1.5-5 times that of the basal level. MUA started to increase 2-9 min prior to the LH rise and the high level of MUA lasted for 1-5 min. The firing rate in the ARC (14 recordings) and in other areas (54 recordings) recorded did not change periodically in parallel with the increase in LH. As these changes in MUA were localized in the ARC and were not reflected in cortical EEG, they might be specific local changes distinguished from generalized systemic effects involving the whole brain. These extreme increases in MUA seem to be due to the high activity either of neurons that secrete LH-RH or those that control the secretion of LH-RH.

Hyperprolactinemic recurrent miscarriage and results of randomized bromocriptine treatment trials

OBJECTIVE To evaluate hyperprolactinemia in the pathogenesis of recurrent spontaneous abortion. DESIGN Randomized trial. SETTING Miscarriage clinic, Yokohama City University Hospital, Yokohama, Japan. PATIENT(S) From a group of 352 women with recurrent spontaneous abortion, we identified 64 patients with a prolactin disorder that was not associated with any other etiologic abnormalities, including ovarian or endocrinologic disturbances such as luteal phase dysfunction, polycystic ovaries, hypersecretion of LH, galactorrhea, or thyroid hormone disorders. INTERVENTION(S) Restoration of prolactin levels with bromocriptine. MAIN OUTCOME MEASURE(S) Successful pregnancy (live birth). RESULT(S) The percentage of successful pregnancies was higher in the bromocriptine-treated group than in the group that was not treated with bromocriptine (85.7% versus 52.4%, P < .05). Serum prolactin levels during early pregnancy (5-10 weeks of gestation) were significantly higher in patients who miscarried (31.8-55.3 ng/mL) than in patients whose pregnancies were successful (4.6-15.5 ng/mL, P < .01 or P < .05). CONCLUSION(S) Appropriate circulating levels of prolactin may play an important role in maintaining early pregnancy, especially in cases of hyperprolactinemic recurrent miscarriage.

Effects of glucose and related substrates on the recovery of the electrical activity of gonadotropin-releasing hormone pulse generator which is decreased by insulin-induced hypoglycemia in the estrogen-primed ovariectomized rat.

We investigated the effect of glucose and its related substrates on the recovery of pulsatile luteinizing hormone (LH) secretion which was suppressed by insulin in estrogen-primed ovariectomized rats. We also examined the effect of glucose on the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator which was suppressed by insulin. The intravenous (i.v.) injection of insulin (5 units/rat) suppressed the pulsatile LH secretion for 3 h in estrogen-primed ovariectomized rats. This suppressive effect of insulin on the LH secretion was rapidly reversed by the i.v. injection of glucose and mannose but not by the injection of lactate and saline. Fructose could recover the LH secretion suppressed by insulin, but took a longer time than glucose did. By monitoring the electrical activity of the GnRH pulse generator, we found that i.v. injection of insulin suppressed the pulsatile LH secretion by decreasing the activity of the GnRH pulse generator. Again, the i.v. injection of glucose, but not saline, immediately recovered the decrease in the electrical activity of the GnRH pulse generator. Fructose could recover the activity of the GnRH pulse generator, but it took a longer time than glucose did. We suggest that glucose availability, but not simply a metabolic state such as the ATP level, is an essential factor for maintaining the electrical activity of the GnRH pulse generator which is responsible for pulsatile LH secretion.

Effect of Estriol on Bone Loss in Postmenopausal Japanese Women: A Multicenter Prospective Open Study

Objectives: To assess the effects of oral estriol on the bone mineral density (BMD) and bone metabolism in postmenopausal women.

Direct Effects of Gonadotropin-Releasing Hormone on the Ovary in Rats and Humans

In order to know whether gonadotropin-releasing hormone (GnRH)-like substances play a role as local regulators in the ovary, localization of the GnRH receptor, and the effects of GnRH agonist (GnRHa) on the cyclic AMP system in the rat ovary was studied. In addition, effects of GnRH antagonist on the rat ovary and effects of GnRHa on steroidogenesis in the human ovary were also examined. In the rat, localization of the GnRH receptor was observed to change with follicular development, and GnRHa decreased follicle-stimulating-hormone-stimulated cAMP production. On the other hand, GnRH antagonist showed the tendency to promote ovarian function. In the human ovary, GnRHa seemed to inhibit steroidogenesis directly, in vivo and in vitro. These data suggested that GnRH/GnRH-like substances play a role as local regulators in the ovary.

Immortalized Gonadotropin-Releasing Hormone Neurons (GT1-7 Cells) Exhibit Synchronous Bursts of Action Potentials

Although it has been assumed that synchronized firing of gonadotropin-releasing hormone (GnRH) neurons is necessary for pulsatile GnRH secretion, there is no clear evidence for this. In the present study we simultaneously recorded spontaneous action potentials from multiple cells. Immortalized GnRH neurons (GT1-7 cells) were cultured on a multi-electrode dish (MED) and action potentials recorded by an extracellular recording method. One to two weeks after the beginning of culture, spontaneous action potentials appeared, exhibiting bursts composed of 5–10 action potentials. Burst activity was intermittent and periodic with mean burst intervals of 13.3 s. Furthermore, burst activity was recorded almost simultaneously from several micro-electrodes, suggesting that electrical activities of GT1-7 cells were synchronized with each other. Periodic bursts were completely and reversibly blocked by 1–5 µM tetrodotoxin, indicating that voltage-dependent Na+ channels are involved in their generation. γ-Aminobutyric acid (GABA) given at a 10-µM concentration shortened inter-burst intervals, whereas 10 µM bicuculline lengthened them. Finally, the gap junctional blockers n-octyl alcohol (1 mM) and carbenoxolone (100 µM) reversibly blocked periodic burst activity. The present study provides direct evidence that the electrical activity of GT1-7 cells exhibits synchronous and periodic bursts composed of action potentials. In addition, endogenous GABA is involved in GT1-7 cells in determining burst frequency. Although the precise mechanism of synchronized burst activities needs to be clarified, gap junctional communications among GT1-7 cells are at least partially involved.

Effect of gonadotropin-releasing hormone agonist on the bone mineral density of patients with endometriosis

OBJECTIVE To examine changes in bone mineral density (BMD) of patients treated with GnRH agonist (GnRH-a) by dual-energy X-ray absorptiometry and to understand factors related to bone loss. DESIGN Prospective controlled trial examining BMD during and after GnRH-a therapy every 24 weeks for 18 months in patients with endometriosis compared with nontreated controls. SETTING Outpatients clinic at a university hospital and its affiliated outpatient clinic. PATIENTS Twenty-two patients with endometriosis as GnRH-a-treated group, 12 healthy women with normal menstrual cycle, and 7 patients with mild endometriosis as control group. INTERVENTIONS Patients were treated with a GnRH-a (buserelin acetate) at 900 micrograms/d by nasal spray for 24 weeks. RESULTS The significance of differences in change-rates at all measured points in both groups was assessed by analysis of variance. The interaction between treatment and period was significant, and only week 24 of the GnRH-a-treated group was significantly lower compared with baseline. The reduction rate of BMD was high in patients 33 years of age or younger compared with those who were 34 years of age or older. According to a multiple-regression model, the most important factor related to bone loss was the post-treatment serum levels of E2. CONCLUSION At the end of treatment, BMD was significantly lower than that of the control group, and the reduction rate was 3.4%. A factor related to bone loss was degree of ovarian suppression.

Mechanisms involved in the pituitary desensitization induced by gonadotropin-releasing hormone agonists

OBJECTIVE We investigated the mechanisms of desensitization induced by gonadotropin-releasing hormone agonist in the pituitary. STUDY DESIGN Effects of gonadotropin-releasing hormone agonist on the pituitary were studied in vitro and in vivo in the rat. In the clinical study serum luteinizing hormone was measured by radioimmunoassay with a polyclonal luteinizing hormone antibody (luteinizing hormone-radioimmunoassay) and by immunoradiometric assay with monoclonal luteinizing hormone antibodies (luteinizing hormone-immunoradiometric assay) during gonadotropin-releasing hormone agonist treatment. RESULTS In the in vitro study bead-attached pituitary cells that were desensitized with a continuous infusion of 10(-7)mol/L gonadotropin-releasing hormone responded to 50 mmol/L K+. In the in vivo study gonadotropin-releasing hormone binding sites and rat luteinizing hormone beta-messenger ribonucleic acid in the pituitary decreased during gonadotropin-releasing hormone agonist treatment, but serum levels of rat luteinizing hormone did not decrease. In addition, a disparity between luteinizing hormone-radioimmunoassay and luteinizing hormone-immunoradiometric assay was demonstrated during gonadotropin-releasing hormone agonist treatment. CONCLUSION Pituitary desensitization in response to gonadotropin-releasing hormone agonist may not be wholly receptor mediated and a nonreceptor process may be involved.

Effects of a gonadotropin releasing hormone agonist on oocyte maturation, fertilization, and embryonal development in mice

PurposeThe effects of a GnRH agonist (GnRHa) on oocyte quality were investigated by assessing the influence of GnRHa on oocytes, and fertilized oocytes were examined in vivo and in vitro. Administration of gonadotropin in conjunction with GnRHa induced a significantly greater degree of germinal vesicle breakdown, significantly higher rates of in vitro fertilization, and significantly faster development of the oocytes than with pregnant mare serum gonadotropin alone.ResultsThe hatching-success rate in the GnRHa treated hypophysectomized mice was higher than in control mice. The rate of in vitro fertilization was also higher in oocytes cultured in the presence of low doses of GnRHa and these effects were reversed by a GnRH antagonist.ConclusionOocytes obtained following ovarian stimulation with GnRHa were of higher quality than control oocytes, and the efficacy of GnRHa may be due in part to its direct action on the ovary.

NEURONAL SYNCHRONIZATION AND IONIC MECHANISMS FOR PROPAGATION OF EXCITATION IN THE FUNCTIONAL NETWORK OF IMMORTALIZED GT1-7 NEURONS : OPTICAL IMAGING WITH A VOLTAGE-SENSITIVE DYE

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage‐sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1–7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1–7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1–7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus‐evoked excitation was prevented by the voltage‐dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage‐dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L‐type) and flunarizine (T‐type>L‐type), but not apparently affected by &ohgr;‐agatoxin IVA (P‐ and Q‐type) and &ohgr;‐conotoxin MVIIA (N‐type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4‐aminopyridine (4‐AP) and prevented by apamin. These results suggest that voltage‐dependent Na+ channels and L‐ and T‐type Ca2+ channels are involved in the propagation of electrical signals in the GT1–7 neuronal network. Ionic mechanisms, through 4‐AP‐ or apamin‐sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.