Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro

The recent isolation of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor (GnRHR) allows studies of the regulation of the synthesis of the GnRHR and its relationship to reproductive function. Analyses of the regulation of GnRHR mRNA levels in the rat pituitary in vivo revealed a progressive increase in levels to 2.0 +/- 0.2-fold after ovariectomy (OVX) and 5.2 +/- 1.3-fold after castration (CAST) (21 days post-operative), compared to intact adult female and male controls, respectively. Replacement therapy with 17 beta-estradiol benzoate in 21-day post-OVX female rats resulted in a marked decrease in GnRHR mRNA levels by 7 days, compared to controls. In contrast, therapy with testosterone propionate in 21-day post-CAST male rats resulted in only a modest decrease in GnRHR mRNA levels. Thus, manipulation of the reproductive endocrine system in vivo results in alterations in GnRHR synthesis at the pretranslational level, which parallel known changes in cell surface gonadotropin-releasing hormone (GnRH) binding activities. The treatment of superfused primary monolayer cultures of rat pituitary cells with hourly pulses of GnRH (10 nM, 6 min/h) resulted in a marked increase in GnRHR mRNA levels (12.8 +/- 4.3-fold compared to untreated cells). In contrast, treatment of cultured cells with continuous GnRH caused no change in GnRHR mRNA levels. These in vitro data show homologous regulation of GnRHR gene expression by GnRH, and suggest that the changes in GnRHR gene expression observed in vivo may be attributable at least in part to changes in the pattern of hypothalamic GnRH secretion.     

Effects of thyroid hormone on embryonic oligodendrocyte precursor cell development in vivo and in vitro

The oligodendrocyte precursor cell divides a limited number of times before terminal differentiation. The timing of differentiation depends on both intracellular mechanisms and extracellular signals, including mitogens that stimulate proliferation and signals such as thyroid hormone (TH) and retinoic acid (RA) that help trigger the cells to stop dividing and differentiate. We show here that, both in vivo and in vitro, TH is required for the normal development of rodent optic nerve oligodendrocytes, although in its absence some oligodendrocyte development still occurs, perhaps promoted by signals from axons. We also demonstrate that TH from both mother and pup plays a part in oligodendrocyte development in vivo. Finally, we show that precursors in embryonic nerve cultures differ from those in postnatal cultures in two ways: they respond much better to TH than to RA, and they respond more slowly to TH, suggesting that oligodendrocyte precursor cells mature during their early development.     

Use of a long-acting gonadotropin-releasing hormone agonist for treatment of steroid cell tumors of the ovary

OBJECTIVE: To report a complete serologic response in a 50-year-old women who received long-acting gonadotropin-releasing hormone agonist (GnRH-A) therapy for steroid cell tumor of the ovary, not otherwise specified. DESIGN: Case report. SETTING: University hospital-based reproductive biology unit. PATIENT(S): A 50-year-old female patient exhibited persistent elevation of T (>2.0 ng/mL) after surgery for steroid cell tumor of the ovary, not otherwise specified, stage IIA for 3 months. This elevation suggested the presence of some residual active tumor. INTERVENTION(S): All tumor evaluations, including those for tumor markers, a thorough physical examination, imaging studies, and evaluations of nuclear medicine studies were negative except for elevated serum T levels. The patient was treated with GnRH-a between the fourth month and sixth month postoperatively. MAIN OUTCOME MEASURE(S): Serum levels of T and tumor survey. RESULT(S): The serum T levels returned to normal limits after administration of the first dose of GnRH-a. Follow-up of tumor survey was negative. The patient was alive and free of disease 26 months after treatment with GnRH-a. CONCLUSION(S): GnRH-a may be an alternative choice as adjuvant therapy for managing a persistent or recurrent hormone-producing steroid cell tumor of the ovary.     

Use of a gonadotropin-releasing hormone agonist in the evaluation of postmenopausal virilization due to ovarian hyperthecosis. A case report

BACKGROUND: Hyperthecosis in a postmenopausal woman is a very rare cause of virilization, and only five cases have been reported previously. CASE: A woman presented with a nine-year history of increasing hirsutism and a mild virilization beginning in the perimenopausal period. Initial androgen metabolite concentrations suggested attenuated late-onset adrenal hyperplasia, but a trial of dexamethasone treatment was ineffective. Subsequent use of leuprolide acetate resulted in a biochemical and clinical improvement in the signs and symptoms. CONCLUSION: This case is unique because gonadotropin-releasing hormone agonist administration was utilized as both a diagnostic and therapeutic modality.     

Effects of long-term testosterone, gonadotropin-releasing hormone agonist, and pimozide treatments on gonadotropin II levels and ovarian development in juvenile female striped bass (Morone saxatilis)

The ability of the juvenile female reproductive axis to respond to hormonal stimulation was investigated in a Perciform fish, the striped bass (Morone saxatilis) using various combinations of testosterone (T), GnRH agonist (GnRHa), and pimozide. A long-term treatment with T alone, or T in combination with GnRHa, increased pituitary gonadotropin II (GtH II) levels 2- and 3-fold, respectively, suggesting that T and GnRHa each stimulate GtH II accumulation. Release of the accumulated GtH II could be induced only by high doses of GnRHa in combination with T, indicating that GtH II synthesis and release require different levels of GnRH stimulation. The addition of the dopamine antagonist pimozide did not affect pituitary and plasma GtH II levels but, in response to an additional acute GnRHa challenge, inhibited the release of GtH II. Although ovarian development was slightly stimulated by a combined T and GnRHa treatment, vitellogenesis was generally not initiated. The present study demonstrated that the juvenile striped bass pituitary is responsive to hormonal stimulation, resulting in elevated levels of GtH II in the pituitary and plasma. However, increased plasma levels of GtH II did not result in precocious puberty, suggesting that additional factors are required for the initiation of ovarian development in this teleost.     

Suppression of cell proliferation and induction of apoptosis in uterine leiomyoma by gonadotropin-releasing hormone agonist (leuprolide acetate)

Cell proliferation and apoptosis in uterine leiomyoma were investigated during therapy with GnRH agonist (GnRHa). Patients with uterine leiomyomas were injected with 3.75 mg GnRHa (depot leuprolide acetate) at intervals of 4 weeks and underwent hysterectomy or myomectomy at the 2nd, 4th, 8th, 12th, or 16th week of GnRHa therapy. Tissue sections of leiomyomas from these patients and from control patients (control patients received no GnRHa therapy) were stained with the Ki-67 antibody or by an in situ DNA 3'-end labeling method, and numbers of Ki-67 immunostained cells and DNA 3'-end-labeled cells per cm2 were examined as indices of cell proliferation and apoptosis, respectively. The number of Ki-67 immunostained cells/cm2 in leiomyomas at the 2nd week of the GnRHa therapy was comparable with that of control patients. However, it decreased to a level less than one forth that of control patients at the 4th week, and it remained at similar low levels at the 8th, 12th, and 16th week. The number of DNA 3'-end-labeled cells/cm2 in leiomyomas of control patients and in leiomyomas at the 2nd, 8th, 12th, and 16th weeks of GnRHa therapy were at low levels but, at the 4th week, was at an extremely high level (about 5 times more than that of control patients). The present results indicate that GnRHa therapy suppresses cell proliferation and causes a transient increase in apoptosis in uterine leiomyomas.     

Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines

The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.     

三种缝线材料对人肺腺癌细胞A549增殖和细胞周期的影响

Objective: The interaction of cell and medical biomaterial is one of the significant factors to affect clinical application of medical biomaterial. This research is to investigate three of suture lines how to affect the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro. Methods: Three of suture lines were respectively cultivated with lung adenocarcinoma cell A549, after of 72 hours, we detected absorptions of each group by MTT method in order to reflect the proliferation of lung adenocarcinoma cell A549, and also examined percentage of G1 period cells and S period cells of each group by flow cytometry. Results: Different of suture lines had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P ＜ 0.05). The effect of absorbent suture line was the strongest on the proliferation and cell cycle of lung adenocarcinoma cell A549, the effect of chorda serica chirurgicalis was medium, and the effect of slide wire was poor. Different length of each suture line had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P ＜ 0.05).Conclusion: Three of suture line materials have different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549, with dose-effect relationship.     

In vitro and in vivo effects of cisplatin and etoposide in combination on small cell lung cancer cell lines

The effects of cisplatin (CDDP) and etoposide (ETP) in combination were evaluated in vitro and in vivo using small cell lung cancer cell lines. The combination effects in vitro were investigated using isobologram analysis. Used together, CDDP and ETP showed a synergistic effect against cell growth on only 1 cell line (SBC-3), additive effects on 6 (SBC-2, SBC-5, Lu130, Lu134AH, Lu135T and H69) and an antagonistic effect on 1 (SBC-1). In the in vivo experiment, nude mice were inoculated with SBC-1, SBC-3 and SBC-5 cells. Two or 5 mg/kg CDDP and 10 or 30 mg/kg ETP were administered intraperitoneally alone and simultaneously in combination to nude mice. The in vivo effects of the combination were determined by comparing the observed growth ratio in mice treated with the combination with the expected value of this ratio calculated based on the assumption that the effects of the drugs were simply additive. According to this definition, synergistic effects were observed against all 3 tumors. Thus, the in vivo and in vitro effects differed. The toxicity of the combination therapy, which was analyzed by estimating the body weight change of mice, was no higher than that of CDDP or ETP alone. These results suggest that the excellent clinical effects of CDDP and ETP combination therapy may be attributable not to drug interaction at the cellular level but to the feasibility of combined use of them at full doses without overlapping side effects.     

Effect of hypophysectomy on immunocytochemically demonstrated gonadotropin-releasing hormone in the rat brain

The objective was to determine the effect of hypophysectomy on the store of gonadotropin-releasing hormone (GnRH) in certain parts of the brain as revealed by immunocytochemistry. The antiserum used was prepared against synthetic GnRH conjugated with limpet hemocyanin. No change was observed in the store of GnRH in the organum vasculosum of the lamina terminalis or in the cephalic segment of the median eminence GnRH was depleted severely from the central and caudal (junction with the infundibular stem) segments of the median eminence. GnRH was not found in the axons of magnocellular neurons that regenerate during repair of the median eminence-pituitary stalk after hypophysectomy.     

Effects of recombinant neutral endopeptidase (EC 3.4.24.11) on the growth of lung cancer cell lines in vitro and in vivo

Many lung cancers are stimulated by an autocrine/paracrine system of neuroendocrine peptide hormones. Attempts to block this autocrine growth pathway by interactions with specific ligand-receptor binding using monoclonal antibodies and peptide-specific antagonists have been largely unsuccessful because of the heterogeneity of hormone production and receptor expression. In the normal lung, neutral endopeptidase (NEP; CD10, CALLA, enkephalinase, and EC 3.4.24.11) plays a physiological role in degrading biologically active peptides, including all peptides implicated in autocrine growth stimulation of lung cancer. Cigarette smoke decreases the activity of NEP, indicating that the lack of NEP contributes to the dysregulation of the peptide autocrine system. The cloning of the human NEP gene allowed for production of sufficient quantities of recombinant NEP (rNEP) to evaluate its role in inhibiting the growth of lung cancer cells. In this study, we evaluated the ability of rNEP to inactivate the peptides involved in lung cancer signal transduction and to inhibit the growth of lung cancer cells as well as normal lung cells in vitro and in vivo in athymic nude mice. We showed that the growth inhibition of lung cancer cells by rNEP was related to the dose and schedule. Continuous exposure to high doses was required for growth inhibition. These studies confirm the importance of NEP in this autocrine pathway.     

Gonadotropin and gonadal steroid release in response to a gonadotropin-releasing hormone agonist in Gqalpha and G11alpha knockout mice

In this study, we used mice lacking the G11alpha [G11 knockout (KO)] or Gqalpha gene (Gq KO) to examine LH release in response to a metabolically stable GnRH agonist (Buserelin). Mice homozygous for the absence of G11alpha and Gqalpha appear to breed normally. Treatment of (5 wk old) female KO mice with the GnRH agonist Buserelin (2 microg/100 microl, sc) resulted in a rapid increase of serum LH levels (reaching 328 +/- 58 pg/25 microl for G11 KO; 739 +/- 95 pg/25 microl for Gq KO) at 75 min. Similar treatment of the control strain, 129SvEvTacfBr for G11 KO or the heterozygous mice for Gq KO, resulted in an increase in serum LH levels (428 +/- 57 pg/25 microl for G11 KO; 884 +/- 31 pg/25 microl for Gq KO) at 75 min. Both G11 KO and Gq KO male mice released LH in response to Buserelin (2 microg/100 microl of vehicle; 363 +/- 53 pg/25 microl and 749 +/- 50 pg/25 microl 1 h after treatment, respectively). These values were not significantly different from the control strain. In a long-term experiment, Buserelin was administered every 12 h, and LH release was assayed 1 h later. In female G11 KO mice and control strain, serum LH levels reached approximately 500 pg/25 microl within the first hour, then subsided to a steady level (approximately 100 pg/25 microl) for 109 h. In male G11 KO mice and in control strain, elevated LH release lasted for 13 h; however, LH levels in the G11 KO male mice did not reach control levels for approximately 49 h. In a similar experimental protocol, the Gq KO male mice released less LH (531 +/- 95 pg/25 microl) after 13 h from the start of treatment than the heterozygous male mice (865 +/- 57 pg/25 microl), but the female KO mice released more LH (634 +/- 56 pg/25 microl) after 1 h from the start of treatment than the heterozygous female mice (346 +/- 63 pg/25 microl). However, after the initial LH flare, the LH levels in the heterozygous mice never reached the basal levels achieved by the KO mice. G11 KO mice were less sensitive to low doses (5 ng/per animal) of Buserelin than the respective control mice. Male G11 KO mice produced more testosterone than the control mice after 1 h of stimulation by 2 microg of Buserelin, whereas there was no significant difference in Buserelin stimulated testosterone levels between Gq KO and heterozygous control mice. There was no significant difference in Buserelin stimulated estradiol production in the female Gq KO mice compared with control groups of mice. However, female G11 KO mice produced less estradiol in response to Buserelin (2 microg) compared with control strain. Although there were differences in the dynamics of LH release and steroid production in response to Buserelin treatment compared with control groups of mice, the lack of complete abolition of these processes, such as stimulated LH release, and steroid production, suggests that these G proteins are either not absolutely required or are able to functionally compensate for each other.     

Effect of constant administration of a gonadotropin-releasing hormone agonist on reproductive activity in mares: induction of ovulation during seasonal anestrus

The potential of a gonadotropin-releasing hormone (GnRH) agonist (goserelin acetate), delivered constantly for 28 days via a subcutaneous depot, to induce ovulation in seasonally anestrous mares, was investigated. Two experiments were conducted, in which a range of doses (30 to 240 micrograms/mare/d) was examined. Mares were selected on the basis of lack of substantial follicular development (follicle diameter < 20 mm determined ultrasonically) and low serum concentrations of luteinizing hormone (LH) and progesterone. Constant administration of the GnRH agonist-induced ovulation in anestrous mares, but a dose-response relation was not observed. Furthermore, with identical doses tested in consecutive or alternate years, considerable variation was observed in the ovulatory response. In general, ovulation in all treated mares was accompanied by increased circulating concentrations of LH and a decrease in follicle-stimulating hormone values. Ovulation was preceded by an increase in estradiol and LH concentrations. In mares in which ovulation did not occur, concentration of LH increased during agonist treatment, whereas that of follicle-stimulating hormone either increased or did not change. It was concluded that constant administration of GnRH agonists may induce ovulation in mares during seasonal anestrus; however, percentage of mares ovulating and the lack of reproducibility of effect indicate that this approach is inappropriate for use as a reliable method to manipulate breeding activity in commercial broodmares.     

Usefulness of Doppler ultrasound in predicting the effect of gonadotropin-releasing hormone agonist (GnRHa) on myoma uteri

This paper shows the value of Doppler ultrasound of the uterine blood flow in predicting the effect of GnRHa on myoma uteri. Thirty-eight patients with myoma uteri were divided into two groups by Doppler ultrasound before treatment: a group with positive arterial blood flow in or around the myoma nodule, and another group with negative arterial blood flow. Histological examinations which were performed in thirty patients demonstrated that the myoma in the negative blood flow group showed higher hyalinization with poor vascularization. In fifteen patients treated with GnRHa (Buserelin 900 micrograms/day), a significant increase (p < 0.01) in the resistance index of the uterine arteries was induced and suppressed the serum estradiol concentration in all cases during GnRHa therapy. The size of the myoma nodules also decreased in all 6 patients in the positive blood flow group, but in only 3 of the 9 patients in the negative blood flow group during GnRHa therapy. These results indicated that Doppler assessments of the arterial blood flow in myoma would be useful in predicting the effect of GnRHa on myoma uteri.     

Effects of oxytocin on in vitro ovarian contractility during the estrous cycle of the rat

Necrotizing skin lesions developed in a man with chronic ulcerative colitis. No evidence of intrinsic disease of medium or small-sized vessels was found. A circulating cryofibrinogen was thought to be responsible for in situ thrombosis leading to skin infarctions. Sodium warfarin in a daily dose of 2.5 to 5 mg appears to have thwarted progression of developing lesions and the occurrence of new ones.     

Reducing the dose of gonadotrophin-releasing hormone agonist on starting ovarian stimulation: effect on ovarian response and in-vitro fertilization outcome

The aim of this study was to examine if lowering the dose of gonadotrophin-releasing hormone agonist (GnRHa) on starting ovarian stimulation could be beneficial in in-vitro fertilization (IVF) programmes. A total of 64 normally ovulating patients entering an IVF programme were randomized to receive GnRHa (nafarelin acetate/Synarel) as an intranasal spray commencing in the midluteal phase, either at a dosage of 200 microg three times daily until the day of human chorionic gonadotrophin (HCG) administration, or to be reduced to 200 microg twice daily as ovarian stimulation was initiated. Patients in both groups were below 35 years with a body mass index below 30. All patients received three ampoules of Metrodin HP per day. Blood samples were taken on the day of HCG administration to measure luteinizing hormone (LH), oestradiol, and progesterone. LH and oestradiol were found to be significantly higher in the lower Synarel dose group. Our results show that reducing the GnRHa dose during ovarian stimulation in IVF might be beneficial in terms of significantly more oocytes recovered, and significantly greater number of embryos available for transfer and freezing, with no incidence of premature luteinization.     

The effect of dopamine on neurohypophysial hormone release in vivo and from the rat neural lobe and hypothalamus in vitro

1. The rat hypothalamus (containing the supra-optic nuclei, paraventricular nuclei, median eminence and proximal pituitary stalk) has been incubated in vitro and shown to be capable of releasing the neurohypophysial hormones, oxytocin and arginine vasopressin, at a steady basal rate about one twentieth that of the rat neural lobe superfused in vitro. 2. The hypothalamus and neural lobe in vitro released both hormones in a similar arginine vasopressin/oxytocin ratio of about 1-2:1. However, when release was expressed relative to tissue hormone content, the hypothalamus was shown to release about three times as much arginine vasopressin and six times as much oxytocin as the neural lobe. 3. Dopamine in a concentration range of 10(-3)-10(-9)M caused graded increases in hormone release from the hypothalamus in vitro to a maximum fivefold increase over preceding basal levels. The demonstration that apomorphine also stimulated hormone release whereas noradrenaline was relatively ineffective suggested that a specific dopamine receptor was involved. A separate cholinergic component in the release process was indicated by the finding that acetylcholine stimulated release to a maximum fivefold increase in concentrations of 10(-3)-10(-9)M. 4. The fact that the isolated hypothalamus can be stimulated by dopamine and acetylcholine to release increased amount of oxytocin and arginine vasopressin raises the question of the origin and fate of the hormones released in this way. The possibility that they could be released into the hypophysial portal circulation from median eminence to affect the anterior lobe of the pituitary is discussed. 5. In similar doses, both dopamine and noradrenaline injected into the lateral cerebral ventricles of the brain of the anaesthetized, hydrated, lactating rat caused the release of arginine vasopressin and oxytocin. Apomorphine release both hormones but at a higher dose level and to less effect than the catecholamines. 6. The hormone release induced in vivo by dopamine could be prevented by the prior administration of haloperidol or phentolamine and these antagonists were equally effective in blocking the hormone release due to noradrenaline. The involvement of a specific dopamine receptor was more clearly implicated by the use of pimozide which completely inhibited the hormone release due to dopamine and apomorphine but not that due to noradrenaline. 7. It is suggested that the release of neurohypophysial hormones can be stimulated via a dopaminergic nervous pathway in addition to a cholinergic one. The possibility that the osmoreceptor mechanism for the release of antidiuretic hormone from the neural lobe of the pituitary may involve such a dopaminergic pathway is discussed.