The effect of glutathione and buserelin on the stereological parameters of the hypothalamus in the cyclophosphamide-treated mice.

INTRODUCTION: New anticancer drugs have increased the survival and fertility rates in young patients. These drugs (i.e., cyclophosphamide; Cyc) have some side effects on the hypothalamus and fertility. One possible chemical for reducing these side effects is thiol or GnRH agonist. This study aimed to evaluate the capability of these agents for reducing the cyclophosphamide effects on the hypothalamus. METHODS: Sixty-three female mice were randomly assigned into seven groups. All groups including the control group had free access to water and mouse chow ad libitum. The sham group received normal saline. The Glu and Bus groups received glutathione (Glu) and buserelin (Bus) daily for 16 days, while the Cyc group received only cyclophosphamide as a single dose; the Cyc + Glu and Cyc + Bus groups, in addition to cyclophosphamide, received glutathione and buserelin, respectively. The volume of the hypothalamus, its neuron number, and dead neurons were evaluated using stereological methods. RESULTS: There was no significant difference in the evaluated stereological parameters between the control and sham groups. However, the animals which received Cyc showed a decrease in the volume of the hypothalamus and its neuron number and density and an increase in cell death as compared with the control group. The treatment of the mice that received Cyc with Glu or Bus prevented these changes. CONCLUSION: This study showed that both GnRH agonist and thiol preserved or improved structural changes in the hypothalamus caused by cyclophosphamide in mice, suggesting that using thiol and especially GnRH agonist along with chemotherapy drugs may have protective effects on fertility.

Antiproliferative effect of Scutellaria barbata D. Don. on cultured human uterine leiomyoma cells by down-regulation of the expression of Bcl-2 protein.

Scutellaria barbata D. Don (Lamiaceae; SB) inhibited the growth of leiomyomal cells (LM). A time-dependent antiproliferative effect was noted when 10(-5) m buserelin, gonadotrophin-releasing hormone (GnRH) agonist or 20-40 microg/mL SB was added. The inhibition of cell growth decreased with the addition of the PKC activator (12-O-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of SB, and the decreases in the viable cells caused by the addition of SB were reversed completely by pretreatment with a protein kinase C (PKC) inhibitor (calphostin C). The findings suggest that SB inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied by PKC activation. Next, the study investigated the effect of SB on fetal development for toxicity. Pregnant Sprague-Dawley rats, from gestation day 6-15, were administered 20 g/L or 50 g/L SB in the drinking water and then killed on day 20. No maternal toxicity was observed, however, embryonic loss in the treatment groups was double that of the controls (p < 0.05). No gross morphologic malformations were seen in the treated fetuses. Fetuses exposed to SB were found to be significantly heavier than the controls, an effect that was greater in female fetuses and was not correlated with increased placental size. The results suggest that the SB had no toxicity and that in utero exposure to SB resulted in increased early embryo loss with increased growth in surviving fetuses. On the other hand, Western blot analyses revealed that Bcl-2 protein of a 26 kDa was abundant in leiomyomal cells, but not in normal myometrial cells. The addition of progesterone (100 ng/mL) resulted in a striking increase in Bcl-2 protein expression in the cultured leiomyoma cells. However, the addition of SB (20 microg/mL) resulted in a significant reduction in Bcl-2 protein expression in the cells. The results indicated that human uterine leiomyomal cells express Bcl-2 protein and progesterone enhances its expression, however, SB reduces the expression of Bcl-2 protein in human uterine leiomyoma cells.

Down-regulation of proliferation and up-regulation of apoptosis by gonadotropin-releasing hormone agonist in cultured uterine leiomyoma cells.

OBJECTIVE: To elucidate the direct effects of gonadotropin-releasing hormone agonist (GnRHa) on the growth of human uterine leiomyoma cells, cell proliferation and apoptosis in cultured leiomyoma cells treated with GnRHa were investigated. METHODS: Isolated leiomyoma cells were subcultured in DMEM supplemented with 10% FBS for 5 days and stepped down to serum-free conditions for an additional 6 days in the presence or absence of graded concentrations of GnRHa (10(-9) mol/l to 10(-12) mol/l). The effects of GnRHa on the number of viable cells, expression of proliferating cell nuclear antigen (PCNA), Fas and Fas ligand, and apoptosis in cultured leiomyoma cells were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) assay, immunocytochemical analysis, Western blot analysis and TUNEL assay respectively. RT-PCR was performed to detect the expression of GnRH receptor mRNA in cultured leiomyoma cells. RESULTS: Treatment with GnRHa resulted in a decrease in the number of cultured viable leiomyoma cells assessed by MTT assay in a dose-dependent manner compared with that in control cultures (P<0.01). The growth inhibition of cultured leiomyoma cells treated with GnRHa in concentrations higher than 10(-10) mol/l was associated with the suppression of the proliferative potential characterized by a decrease in PCNA-positive rate of the cultured cells (P<0.01) and an increase in the apoptosis-positive rate assessed by TUNEL assay (P<0.05 and P<0.01). GnRHa markedly increased the expression of Fas and induced the expression of Fas ligand in the cultured leiomyoma cells on the basis of Western blot analysis. These direct effects of GnRHa on the number of viable cultured leiomyoma cells, PCNA-positive rate, apoptosis-positive rate and Fas/Fas ligand expression in the cultured leiomyoma cells were only attained after the 4-day treatment. RT-PCR analysis revealed that GnRH receptor mRNA was expressed in cultured leiomyoma cells. CONCLUSIONS: The present results demonstrate that GnRHa directly inhibits the growth of human uterine leiomyoma cells by suppressing cell proliferation and inducing apoptosis, which might be associated with the increase in Fas expression and the induction of Fas ligand expression in the cells.

Conservative management for perimenopausal women with uterine leiomyomas using Chinese herbal medicines and synthetic analogs of gonadotropin-releasing hormone.

The effects of a long-term intranasal administration of each of the gonadotropin-releasing hormone analogs, buserelin and nafarelin on uterine leiomyomas after conservative treatment using Chinese herbal medicines, Keishi-bukuryo-gan and Shakuyaku-kanzo-to were investigated in 30 perimenopausal women with leiomyomas. Hypermenorrhea and/or dysmenorrhea as a chief complaint was moderately improved by the treatment using Chinese herbal medicines in more than 60% of the patients with less than fist-sized leiomyomas, but not the over fist-sized. Afterwards, continuous treatment using analogs produced a long-term reduction in leiomyomas (less than 60%) along with decreases in the serum levels of luteinizing hormone, follicle-stimulating hormone, estradiol, and the tumor marker CA-125, and adverse effects including slight boneloss. Long-term treatment using Chinese herbal medicines and gonadotropin-releasing hormone analogs for the management of uterine leiomyomas could be beneficial for patients a few years before menopause, though possible side effects of this treatment should be monitored.

The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to Gs- and G(q/11)-mediated signal transduction pathways: evidence from loop fragment transfection in GGH3 cells.

The GnRH receptor (GnRH-R) belongs to the rhodopsin/beta-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane-proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH(3)1' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both Gs and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other Gs-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH(3)1' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH(3)1' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the alpha1B-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach-muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the Gs protein). Transfection of loop 3i from the D1A -dopamine receptor (coupled to the Gs protein) produced a selective attenuation (40%) in Gs-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and alpha2A-adrenergic receptors). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH(3)1' cell line, this loop is also involved in signal transduction mediated through the Gs protein pathway.

Evidence for autocrine inhibition of gonadotropin-releasing hormone (GnRH) gene transcription by GnRH in hypothalamic GT1-1 neuronal cells.

To examine whether an ultrashort feedback mechanism of gonadotropin-releasing hormone (GnRH) operates at the level of gene transcription, we studied the effects of GnRH analogs on GnRH promoter activity and GnRH mRNA level in hypothalamic GT1-1 neuronal cells. Treatment of GT1-1 cells with buserelin, a GnRH agonist, or native GnRH for 24 h significantly decreased GnRH promoter activity and its mRNA level, whereas that with GnRH antagonists, antide or [D-Phe2,D-Ala6]-GnRH, showed no effect. The inhibitory effects of buserelin on GnRH gene transcription and GnRH mRNA level were dose-related, and a significant inhibition was observed in cells treated with buserelin at concentrations higher than 0.1 microM. Time-course experiments showed that significant decreases in GnRH promoter-driven luciferase activity and GnRH mRNA level were observed within 12 h and sustained up to 48 h. Moreover, treatment with GnRH agonist for 12 h significantly decreased the transcription rate of the mouse GnRH gene, as revealed by nuclear run-on transcription assay. The promoter analysis with the 5'-deletional constructs demonstrated that cis-acting elements important for GnRH autoregulation by GnRH agonist reside within -854 bp upstream from the transcription start site. These data clearly demonstrate that GnRH can exert autocrine regulation at the level of GnRH gene transcription.

Gonadotropin releasing hormone modulates gamma-aminobutyric acid-evoked intracellular calcium increase in immortalized hypothalamic gonadotropin releasing hormone neurons.

To examine the functional role of calcium signaling in the interactive modulation of gonadotropin releasing hormone (GnRH) neurons by gamma-aminobutyric acid (GABA) and GnRH itself, we analyzed the intracellular calcium level ([Ca2+]i), using fura-2AM fluorescent dye in immortalized hypothalamic GT1-1 cells. GT1-1 cells showed spontaneous [Ca2+]i oscillations, which were dependent on extracellular Ca2+ level, L-type Ca2+ channel and SK-type K+ channel. When GABA or a specific GABAA type receptor agonist, muscimol was applied to the media, [Ca2+]i rapidly increased through L-type Ca2+ channel in a dose-dependent manner, and subsequently decreased below the basal level without any oscillation. However, a specific GABAB type receptor agonist, baclofen showed no effect. On the other hand, application of GnRH or its potent agonist buserelin, rapidly abolished the spontaneous [Ca2+]i oscillations. Interestingly, a prior treatment with buserelin abolished GABA-evoked increase in [Ca2+]i in a noncompetitive manner. Since buserelin also blocked K(+)-evoked increase in [Ca2+]i, we suggest that GnRH may block spontaneous [Ca2+]i oscillation through modulating the L-type [Ca2+]i channel activity. These results show that GABAergic agents may exert both stimulatory and inhibitory controls over the GnRH neuronal activity, and GnRH can block the stimulatory effect of GABA, implicating the possible existence of an ultrashort feedback circuit.

Evidence to suggest that gonadotropin-releasing hormone inhibits its own secretion by affecting hypothalamic amino acid neurotransmitter release.

The mediobasal hypothalamus of rats contains gonadotropin-releasing hormone (GnRH) receptors. These hypothalamic neurons also express the GnRH corresponding gene. Under these circumstances, the possibility exists that these GnRH receptors could be localized in other neurons, which are GnRH-receptive, unknowing the neurotransmitter quality. Therefore, we studied the in vitro effects of the GnRH agonist buserelin on GnRH, glutamate, gamma-amino-butyric acid (GABA) and taurine release from explanted superfused hypothalami of untreated and buserelin-pretreated (down-regulated) male rats. When buserelin was added to the superfusion medium it inhibited promptly the release of GnRH and the excitatory amino acid neurotransmitter glutamate, but stimulated the release of the inhibitory neurotransmitters, GABA and taurine. Hypothalamic release of GnRH from hypothalami collected from buserelin-treated (30 micrograms/100 g b.w. twice daily for 4 days) male rats released significantly less GnRH, glutamate and more GABA and taurine. The inhibitory effect of buserelin was maintained when the superfusion medium continuously contained the GnRH analog. When superfusion of hypothalami from buserelin-pretreated animals was performed in the absence of buserelin, GnRH and glutamate release increased significantly within 45-60 min, whereas GABA and taurine release decreased at this time point. When buserelin was added to the superfusion medium 2 h after buserelin-free superfusion, GnRH and glutamate release decreased whereas GABA and taurine release increased instantaneously. Buserelin-treated rats showed significantly low values of LH and testosterone than the untreated rats. These results suggest that GnRH receptors may not only be present in GnRH axon terminals in the median eminence, but also on glutamatergic, GABAergic and taurinergic neurons by which GnRH may exert an autoinhibitory ultrashort loop feedback on its own secretion. This effect appears to be connected with glutamatergic, GABAergic and taurinergic neurons.

Gonadotropin-releasing hormone (GnRH)-receptor coupling to inositol phosphate and prolactin production in GH3 cells stably transfected with rat GnRH receptor complementary deoxyribonucleic acid.

This study examines the relation between inositol phosphate (IP) production and PRL release in four GGH3 cell lines (GGH(3)1', GGH(3)2', GGH(3)6', and GGH(3)12'; lactotropic GH3 cells that have been stably transfected with rat GnRH receptor complementary DNA). Production of IPs is an early response of GGH3 cells to a GnRH agonist, measurable at 15-30 min and maximal at 60 min after treatment with Buserelin in [3H]inositol preloaded cells. In contrast, PRL release, which requires protein synthesis, is not measurable until 1-3 h and total cAMP production is not measurable until about 24 h (3). In one of the lines studied (GGH(3)2'), PRL was also released in response to TRH. Measurable expression of the PRL gene requires 1-2 days (2). All four lines produced IPs robustly after treatment with Buserelin, although the IP response to TRH is minimal in all lines, being the best in the GGH(3)2' line. Pretreatment of cells with cholera toxin (CTX) or pertussis toxin (PTX) attenuated TRH-induced IP production in GGH(3)1', GGH(3)2', or GGH(3)12' cells. No effect of CTX or PTX is measurable in GGH(3)6' cells in terms of TRH stimulation of IP production. In contrast, both toxins augment Buserelin-stimulated IP production in GGH(3)1' and GGH(3)6' cells, but have no action in the other two lines. Both CTX and PTX inhibit Buserelin-stimulated PRL production. This study suggests that IP production is the earliest measurable response of GGH3 cells to a GnRH agonist, although this event does not appear to be coupled to Buserelin-stimulated PRL release. Further, the studies with toxins suggest that Buserelin and TRH appear to regulate IP production by different mechanisms.

Functional and morphological characterization of four cell lines derived from GH3 cells stably transfected with gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid.

Four cell lines, stably transfected with rat GnRH receptor complementary DNA, have been prepared from the lactotropic GH3 cell line. All four lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, characteristic of high protein synthetic activity, although secretory granules are virtually absent; the rough endoplasmic reticulum (rER) cisternae were short and straight. Instances were observed in which the ER reaches to the plasma membrane, suggesting a possible nongranular secretory route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depending on the cell line) after exposure to 10 nM GnRH; receptors then recovered (2-7 h). This pattern is reminiscent of the GnRH receptor in the primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer6-des-Gly10-Pro9-ethylamide-GnRH), an event that was inhibited by all three major classes of Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines, and diltiazem); in contrast, GnRH-stimulated LH release from pituitary-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, although the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 microgram/ml Buserelin than to 1 microgram/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.

Evidence that signalling pathways by which thyrotropin-releasing hormone and gonadotropin-releasing hormone act are both common and distinct.

TRH and GnRH receptors are each coupled to G proteins of the Gq/11 family. Activation of each of these receptors by their respective ligands results in the stimulation of phospholipase C activity, leading to calcium mobilization and protein kinase C activation. Thus, the effects of TRH and GnRH may be mediated through the same intracellular signal transduction pathway. To compare responses to TRH and GnRH directly within one cell type, we have stably transfected the rat pituitary GH3 lactotrope cell line, which expresses the endogenous TRH receptor, with an expression vector containing rat GnRH receptor cDNA. Transfected cells specifically bound GnRH with high affinity and responded to GnRH stimulation with an increase in PRL mRNA levels, analogous to their response to TRH stimulation. Stably transfected GH3 cells, which were then transiently transfected with luciferase reporter constructs containing either the PRL or the glycoprotein hormone alpha-subunit promoter, responded to either GnRH or TRH stimulation with an increase in luciferase activity in a time- and dose-dependent fashion. The stimulatory effects of maximally effective concentrations of TRH and GnRH were additive on PRL, but not alpha-subunit, gene expression. These data, coupled with evidence of cross-desensitization of alpha-subunit, but not PRL, promoter activity stimulation by TRH and GnRH, suggest that there may be differences in the signal transduction pathways activated by TRH and GnRH receptors in the regulation of PRL and alpha-subunit gene expression.

[The effects of intranasal application of low dose buserelin in women with hypothalamic amenorrhea].

Low doses of the Gn-RH agonist (buserelin, 30 micrograms) were given intranasally to 14 women with clomiphene ineffective hypothalamic amenorrhea three times daily for three weeks in order to study pituitary responses and to induce follicular maturation and ovulation. Clomiphene ineffective hypothalamic amenorrhea patients were classified into two groups by LH-RH stimulation test before the treatment. Group 1 was defined as having basal serum LH and FSH levels lower than 1.5 mIU/ml, LH and FSH peaks lower than 3mIU/ml by LH-RH stimulation test. Group 2 consisted of cases other than those in Group 1. While a significant increase in basal LH and FSH (p less than 0.01, p less than 0.001) and improvement in pituitary response to LH-RH stimulation test were observed in group 1, the basal levels of LH and FSH did not increase significantly and pituitary response to a LH-RH stimulation test was decreased in group 2. It is suggested that pituitary priming occurred in group 1 and pituitary desensitization occurred in group 2. None of 14 patients showed signs of follicular maturation during or after the treatment. The results demonstrated that the biphasic pituitary response to intranasal buserelin spray and the limit of its therapeutic use for the treatment of hypothalamic amenorrhea.

Experience with a potent LH-RH agonist, buserelin, alone and in combination with testosterone for antispermatogenic activity, reversibility and toxicity in langur monkey.

Chronic intermittent treatment of LH-RH superagonist Buserelin alone or in combination with testosterone enanthate were given to adult male langurs for 90 days to evaluate antispermatogenic activity of alone and combination therapy, maintenance of normal androgenicity, possible toxic effects of agonist treatment, related side effects of testosterone supplementation and complete reversibility of the procedure. A gradual decrease in sperm count was recorded in both treatment groups, along with reduced motility and vitality of the spermatozoa. In the combination group, oligospermia was achieved in 4 out of 5 animals, whereas, only 2 animals became oligospermic in the agonist alone group. Significant decrease in serum testosterone levels along with impaired libido and other testosterone withdrawal symptoms were observed in the Buserelin alone group, conversely normal testosterone levels and libido were observed in the combination group. An elevation in haematological variables and serum total protein concomitant with a slight gain in body weight of the animals were recorded in the combination group; these changes were not encountered in the agonist alone group. Reversibility of all the altered parameters to control range was observed in both treatment groups following 75 to 90 days of treatment withdrawal.

Maturation of the hypothalamic control of pulsatile gonadotropin-releasing hormone secretion at onset of puberty: II. Reduced potency of an inhibitory autofeedback.

At the time of onset of puberty in the male rat, between 15 and 25 days of age, we have reported that pulsatile GnRH secretion by hypothalamic explants showed an increased frequency as indicated by the reduction of the mean interpulse interval from 66 to 40 min (P less than 0.05). This study aimed to evaluate whether these changes in GnRH secretion involved a self-regulatory mechanism. A 7.5-min exposure of explants obtained at 50 days to 0.1 microM superagonist D-TRP6-PRO9-N-Et-GnRH (GnRH-A) resulted in a delay of the next GnRH secretory pulse so that the mean interpulse interval increased from 35 to 67 min (P less than 0.001). In addition, after a 7.5-min exposure to GnRH-A, there was a 15-min period with absent or reduced release of GnRH in response to 50 microM veratridine, a depolarizing agent. A similar refractory period of 15 min was observed using explants obtained at 25 and 50 days whereas, at 15 days, the period of refractoriness lasted for 52.5 min. The inhibitory effect of GnRH-A on the subsequent response to veratridine occurred at similar concentrations of GnRH-A at the three studied ages and the inhibition was prevented using an antagonist of GnRH together with GnRH-A. The involvement of GnRH itself in an autofeedback mechanism was evaluated by studying the period of refractoriness separating two GnRH pulses elicited by 7.5-min exposures to veratridine. The initial responsiveness to veratridine was recovered after a refractory period of 52.5, 22.5, and 15 min when studied at 15, 25, and 50 days, respectively. While refractoriness occurred during repeated depolarization with K+ or veratridine, such an effect was not observed using N-methyl-D-aspartate (NMDA). During exposure to GnRH-A, the NMDA-induced release of GnRH was only reduced by 38% whereas veratridine-induced secretion showed a 94% reduction. Thus, exogenous activation of NMDA-sensitive receptors could bypass the inhibitory autofeedback. We conclude that: 1) pulsatile GnRH secretion is controlled by an inhibitory autofeedback involving NMDA sensitive receptors, 2) the increased frequency of pulsatile GnRH secretion at onset of puberty may be related to a reduced sensitivity of the hypothalamic pulse generator to an inhibitory autofeedback.

Incidence of severe ovarian hyperstimulation syndrome after GnRH agonist/HMG superovulation for in-vitro fertilization.

In 1673 treatment cycles stimulated with buserelin and HMG, for IVF, GIFT or ZIFT, the severe ovarian hyperstimulation syndrome (OHSS) occurred in 10 cycles (0.6%). Eight patients were hyperandrogenic and showed an increased ovarian response to HMG. After replacement of a maximum of three embryos or zygotes, seven women became pregnant. Three women had a multiple gestation. All patients recovered uneventfully with conservative treatment. Support with progesterone or continuation of the agonist during the luteal phase did not prevent OHSS, confirming that the ovulatory HCG dose is the most important factor in inducing this severe complication. Luteal supplementation with HCG and/or HCG production during implantation could exacerbate OHSS.

Regulation of the pituitary 5 alpha-reductase activity by gonadotropin releasing hormone and testosterone in the adult male rat.

Intact or castrated adult male rats were treated for nine days with GnRH (10 micrograms/day), the synthetic GnRH goserelin (100 micrograms/day) or the GnRH-antagonist Org 30276 (250 or 500 micrograms/day). In some series, 1 mg testosterone propionate was administered alone, or in combination with goserelin or Org 30276. The in vitro metabolism of [1 alpha,2 alpha-3H]testosterone by pituitary and hypothalamic homogenates was investigated in combination with the estimation of plasma concentrations of testosterone and gonadotropins. No qualitative or quantitative differences were observed in hypothalamic testosterone metabolism or in the pituitary 17 beta-hydroxysteroid dehydrogenase activity. Testosterone administration to intact male rats decreased the pituitary 5 alpha-reductase activity and LH, while administered to castrated rats, it was able to suppress totally the castration-induced increase of the 5 alpha-reductase activity and of the gonadotropin secretion. The drastic decrease of the plasma levels of testosterone, observed after a prolonged treatment with GnRH, goserelin or Org 30276 was not accompanied by an increased pituitary 5 alpha-reductase activity. Injected to castrated rats, it was observed that the castration-induced increase of the pituitary 5 alpha-reductase was further stimulated by GnRH, totally suppressed by goserelin and partially suppressed by Org 30276. Concomitant administration of goserelin or Org 30276 and testosterone propionate to castrated rats resulted in a further decrease of the pituitary 5 alpha-reductase activity, compared to the castrated, GnRH-analogue treated rats. These data indicate that the pituitary 5 alpha-reductase enzyme system is controlled by both direct steroidal and indirect GnRH-mediated mechanisms.

Buserelin. A review of its pharmacodynamic and pharmacokinetic properties, and clinical profile.

The gonadotrophin releasing hormone (GnRH) [luteinising hormone-releasing hormone (LHRH); gonadorelin] agonist buserelin is a promising new agent in the treatment of a variety of disorders in gynaecology and andrology, paediatrics and oncology. While a single dose of buserelin stimulates the release of pituitary gonadotrophins, multiple doses produce reversible pituitary desensitisation, and this specific blockade of gonadotrophin support to the gonads provides the basis for the drug's efficacy in conditions dependent on sex hormone secretion. Thus, buserelin provides comparable efficacy to orchidectomy or high dose estrogens in the treatment of hormone-sensitive prostate cancer and exhibits a lower incidence of adverse effects. During the early phase of treatment it may be particularly useful in combination with antiandrogens. Buserelin also appears promising in hormone-sensitive premenopausal breast cancer. Extensive studies have proven the value of buserelin in endometriosis, where it produces a transient remission with gradual recurrence of the disease on cessation of treatment. Surgical intervention is necessary in severe disease after buserelin-induced involution of the lesions. In patients with uterine leiomyoma, preliminary data suggest that buserelin may be beneficial in rendering surgery more conservative by reducing fibroid size, although it appears unlikely to preclude surgical intervention. The use of buserelin to induce a state of reversible hypogonadotrophism before administration of exogenous gonadotrophins is a promising strategy in the treatment of infertility associated with polycystic ovary syndrome and other conditions of infertility with underlying ovarian dysfunction; such a strategy also clearly enhances the efficiency of in vitro fertilisation programmes. Initial studies suggest its potential usefulness as a female contraceptive when administered intermittently in conjunction with a progestogen. Buserelin represents a first-line treatment of central precocious puberty. In endometriosis the adverse effect profile of buserelin is generally favourable, with hypoestrogenic effects such as hot flushes and vaginal dryness, and decreased libido, predominating. There is no apparent detrimental effect on lipid metabolism. The potential for adverse hypoestrogenic effects on bone mineral content with long term administration remains to be clarified. Thus, the GnRH agonist buserelin represents an advance in the treatment of a variety of gynaecological and andrological as well as paediatric and oncological conditions, infertility and other sex-hormone dependent conditions, with a low incidence of adverse treatment effects.

Luteinizing hormone-releasing hormone-binding sites in the rat thymus: characteristics and biological function.

The present study was designed to explore the effects of LHRH and its agonists on immune system function. As a first step, to identify a putative site of action, the very potent and stable LHRH agonist (LHRH-A), [D-Ser(TBU6)] des-Gly10-LHRH ethylamide (buserelin), was used as an iodinated ligand to characterize LHRH receptors in a membrane preparation of rat thymus, a key organ of the immune system. The effects of LHRH and LHRH-A were then investigated on the proliferative capacity of rat thymocytes exposed in vitro to a mitogen and on ornithine decarboxylase specific activity. In addition, to determine whether LHRH-A treatment in vivo might directly influence thymic function, we treated hypophysectomized (hypox) rats with a moderately high dose of LHRH-A for a period of 2 weeks, and thymocyte mitogenic capacity, thymus weight, and the histological and functional appearance of the thymus were then assessed. Specific binding of LHRH-A to rat thymic membrane preparations is a saturable process, depending on both time and temperature of incubation, but differs markedly from binding to the rat pituitary or ovarian LHRH receptor in its low binding affinity. Binding is optimal in the absence of chelating agents (EDTA) or divalent metal ions, and increases linearly with increasing protein concentration. Binding is specific for LHRH, LHRH-A, and antagonists. Both the C-terminal amide and N-terminal regions of the LHRH molecule were required for binding, and amino acid substitutions at position 6 markedly enhanced and at position 8 markedly reduced binding potencies in rat thymic tissue. A number of peptides, proteins, and other agents had no effect on the specific binding of LHRH-A to thymic membrane preparations. The binding affinity (Ka) of the membrane receptor of the rat thymus for the LHRH superagonist buserelin was 8.4 x 10(8) M-1, while a higher binding affinity (Ka = 2.8 x 10(9) M-1) was calculated for the ovarian LHRH-binding site. Preincubation of rat thymocytes with LHRH-A for 20 h induced a significant dose-dependent increase in the proliferative response to the mitogen Concanavalin-A, monitored by [3H]thymidine incorporation. Using native LHRH, it was also possible to elicit stimulatory effects on the same parameter, although much higher concentrations were required than with LHRH-A. Furthermore, simultaneous addition of a LHRH antagonist, abolished the LHRH effect on thymocytes. Ornithine decarboxylase specific activity under lectin stimulation was also significantly increased by LHRH-A in cultures of rat thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Pulsatile release of gonadotropin-releasing hormone (GnRH) from the rat hypothalamus in vitro: calcium and glucose dependency and inhibition by superactive GnRH analogs.

We have shown previously that the rat hypothalamus retains in vitro its capacity of generating pulsatile release of GnRH. The present study evaluated if pulsatile release of GnRH in vitro was influenced by metabolic conditions (calcium and glucose availability) and the possible self-regulatory role of GnRH in its pulsatile secretion. In the presence of a calcium-chelating agent (EGTA, 20 mM) or a calcium-channel blocker (D-600, 0.1 mM), the release of GnRH induced by a depolarization (veratridine, 50 microM) was markedly and reversibly decreased. In addition, frequency and amplitude of GnRH secretory pulses were significantly reduced (P less than 0.05). When glucose use was inhibited using 2-deoxyglucose (5.6 mM) the release of GnRH induced by veratridine and the frequency of GnRH pulses were also blunted (P less than 0.05). Superactive agonists of GnRH (Buserelin and D-TRP6-PRO9-N-ET, 10 nM) caused a prompt decrease of GnRH release in basal conditions and in the presence of veratridine. A significant inhibition (P less than 0.05) was observed using buserelin concentrations greater than 0.01 nM, whereas two GnRH analogs without biopotency (Leu8-GnRH, Des-gly10-N picolylamide GnRH, 100 nM) did not affect GnRH release. The two agonists of GnRH reduced by 43% to 66% (P less than 0.05) the occurrence of significant GnRH pulses. We conclude that, in vitro, the hypothalamic neuronal circuitry resulting in GnRH pulsatile secretion is dependent on calcium and glucose availability and is sensitive to an ultrashort-loop inhibitory feedback mechanism.

On the short-loop feedback regulation of the hypothalamic luteinizing hormone releasing hormone 'pulse generator' in the rhesus monkey.

The characteristic electrophysiological manifestations of LHRH 'pulse generator' activity were unaffected by prolonged elevations in plasma LH concentrations achieved by the administration of a long-acting LHRH agonist. These findings do not lend support to the existence of a 'short-loop feedback' inhibition of LHRH 'pulse generator' activity by LH.