Gonadotropin-releasing hormone desensitization preferentially inhibits expression of the luteinizing hormone beta-subunit gene in vivo.

In this study we investigated changes in steady state cytoplasmic mRNA levels for LH subunits in pituitaries of male rats desensitized by continuous infusion of GnRH in vivo. Seven days of GnRH infusion (340 micrograms/day) reduced (P less than 0.01) LH beta mRNA levels in intact adult male rats and prevented the LH beta mRNA rise observed after castration. In contrast, common alpha mRNA doubled (P less than 0.05) in intact rats, and the elevated alpha mRNA after 7 days castration was unchanged. Serum and pituitary LH levels were suppressed below values of intact controls. Fourteen days of GnRH infusion (290 micrograms/day) further reduced LH beta mRNA levels in both intact and castrated male rat pituitaries. alpha mRNA levels in intact rat pituitaries were unchanged by 14 days of GnRH infusion, while in castrated rats there was a 23% (P less than 0.05) decrease, though values were still twice those of intact controls. As at 7 days, serum and pituitary LH were suppressed. Infusion of a superagonist analog (Buserelin) at a dose of 14 micrograms/day for 28 days reduced LH beta mRNA to 15% of intact control values in both castrated and intact rats. Common alpha mRNA was significantly (P less than 0.05) increased in intact rats and reduced by 13% (P less than 0.05) in castrates by superagonist infusion. These results were similar to those produced by 20- to 30-fold higher doses of native GnRH. GnRH and agonist analog effects were specific since no changes were observed in other mRNA species (GH, PRL, actin). These results indicate that in GnRH-desensitized gonadotropes LH beta gene expression is inhibited, and this may largely explain the reduced LH biosynthesis. However, there is a differential effect of continuous GnRH or agonist analog treatment on LH subunit gene expression, with a time-dependent stimulation of common alpha gene expression in intact rats. This may be caused by a stimulatory interaction between GnRH and progestagens at the level of the gonadotrope. Thus, common alpha gene expression is less tightly coupled than that of LH beta to GnRH action.

Gonadotropin-releasing hormone is required for enhanced luteinizing hormone subunit gene expression in vivo.

Pre- and postcastration changes in LH beta and common alpha mRNAs were correlated with pituitary and serum LH levels in two different species after abolition of pituitary stimulation by GnRH. A GnRH antagonist (GnRH-ANT) was used to block gonadotroph GnRH receptors in male rats, and a GnRH antiserum (GnRH-AS) was used to inhibit GnRH stimulation of female and male mouse and male rat pituitaries. The postcastration increases in LH beta and common alpha mRNA levels (2- and 3.5-fold, respectively) were abolished in male rats after 7 days of continuous GnRH-ANT infusion. The postcastration increases in LH beta and common alpha mRNA in female (1.9- and 2.2-fold respectively) and male mice (1.4- and 3.6-fold, respectively) were also prevented after daily sc injection of GnRH-AS, as were the rises in LH beta (3-fold) and common alpha (4-fold) in castrated male rats. The pituitary LH content (postgonadectomy) was no different from intact control levels in all experimental animals regardless of treatment, while the increase in serum LH concentration in rats (7- and 8-fold) and in female (4.8-fold) and male mice (9.8-fold) was prevented by both GnRH-ANT and GnRH-AS administration. In intact rats treated with GnRH-ANT the LH beta mRNA level decreased (57%) while the common alpha mRNA level was unaffected after 7 days. Neither pituitary nor serum LH levels were altered in intact rats or mice after appropriate treatments. We conclude that endogenous GnRH is required for the postcastration rise of both LH beta and common alpha-subunit mRNA levels in rats and mice.

Direct interaction with target-derived glia enhances survival but not differentiation of human fetal mesencephalic dopaminergic neurons.

Regulation of the developing nervous system involves attraction, guidance and modification of innervating neurons by target cells through diffusible and membrane-related factors. The trophic effects from specific cell types remain to be investigated and characterized. In a series of experiments in which human fetal mesencephalic dopaminergic cells were co-cultured with target or non-target neurons or glial cells in direct or contiguous contact, we demonstrate that striatal glial cells (target-derived glia) can enhance dopaminergic neuron survival by up to 400% compared to either non-target cell co-cultures or mesencephalic controls. When in direct contact with striatal neurons, a greater proportion of dopaminergic neurons had a more differentiated morphology. The enhancement of dopaminergic neuron survival by target-derived glia appears to be mediated both by direct contact, possibly through target membrane-specific phenomena, and by diffusible substances, whereas non-target glia appear to exert the trophic effects predominantly through the latter mechanism. The finding that target neurons influence mainly dopaminergic neuron differentiation and target glia their survival indicates multiple, target cell type-specific regulation of innervating neuron development. These findings also have relevance to the establishment of neuronal cultures for neural transplantation.

Homologous ligand regulation of gonadotrophin-releasing hormone receptors in vivo: relationship to gonadotrophin secretion and gonadal steroids.

A single injection of gonadotrophin-releasing hormone (GnRH) (60 ng s.c., 42.9 nmol) induced biphasic GnRH receptor regulation in normal intact adult female mice. A transient 22% receptor decrease occurred 30-60 min after injection of GnRH when peak serum decapeptide concentrations were reached (137 +/- 41 (S.E.M.) ng/l). This GnRH receptor decrease occurred shortly after the peak serum LH values at 15-30 min. The subsequent rapid (within 1 h) return of GnRH receptor levels to normal suggested transient receptor occupancy by GnRH rather than true receptor loss. At 8 h after injection of GnRH a significant 35% increase in GnRH receptors was consistently observed, when serum GnRH levels were undetectable and serum LH had returned to basal levels. This receptor increase was not due to increased receptor affinity, and was prevented by a non-specific protein synthesis inhibitor. Ovariectomy, which caused a 50% fall in GnRH receptors (59.4 +/- 4.9 fmol/pituitary gland in intact controls; 26.9 +/- 2.6 in ovariectomized mice) abolished the induction by GnRH of its own receptors, although the initial transient decrease occurred over the period of the acute serum LH and FSH rise. Despite a 50% reduction in GnRH receptors in ovariectomized mice, increased serum gonadotrophin levels and responsiveness to GnRH were maintained, indicating dissociation between receptor changes and gonadotrophin levels. No GnRH receptor up-regulation was observed 8 h after a single GnRH injection (60 ng s.c.) in either intact or orchidectomized normal male mice. However, the same treatment doubled GnRH receptors in GnRH-deficient (hpg) female mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic adenosine nucleotides and growth hormone-releasing factor increase cytosolic growth hormone messenger RNA levels in cultured rat pituitary cells.

The cellular mechanisms involved in GH biosynthesis have been investigated by the measurement of steady-state levels of cytosolic GH messenger RNA (mRNA) in primary cultures of rat pituitary cells using an RNA-complementary DNA (cDNA) hybridization assay. Growth hormone mRNA-cDNA hybridization increased in a linear manner with increasing cytosol concentration. Cellular GH mRNA levels rose by an average of 2.4-fold (range, 1.6-3.3; n = five experiments) after exposure to GH-releasing factor (GRF(1-40); 10 nmol/l) for 3 days. Treatment with GRF increased the release of GH into the culture medium, and depleted the cellular GH content by 40%. Total GH (in the medium plus cells) after GRF treatment increased by between 1.5- and 3.8-fold, a magnitude similar to the increase in GH mRNA levels. Treatment of cells with dibutyryl adenosine 3':5'-cyclic monophosphate (1 mmol/l) or forskolin (5 mumol/l) increased the levels of cytosolic GH mRNA by between 1.6- and 4.7-fold. These agents increased GH release into the medium, depleted cellular GH content and increased total GH in the system to the same extent as GRF (10 nmol/l). These data demonstrate that cyclic adenosine nucleotides may mediate the GRF induction of GH gene transcription. In addition, we have shown that increases in the levels of cellular GH mRNA are reflected by increased GH biosynthesis, suggesting that the regulation of hormone gene transcription is one cellular site for the control of hormone biosynthesis and, ultimately, hormone available for release.

Blockade of rat testicular gonadotropin releasing hormone (GnRH) receptors by infusion of a GnRH antagonist has no major effects of Leydig cell function in vivo.

The purpose of this study was to examine the physiological functions of the gonadotropin releasing hormone (GnRH) receptors present in the rat testis. The receptors were blocked in situ by infusing one testis of adult rats for 7 days with 10-100 ng/h of a potent GnRH antagonist (N-Ac-Ala1, D-p-Cl-Phe2, D-Trp3,6-GnRH) using Alzet osmotic minipumps. The contents of the pump were delivered to the testis through a cannula perforating, and fixed, to the tunica albuginea. A plastic cannula alone was attached to the contralateral testis, to act as a control. Infusion of the antagonist resulted in a dose-dependent decrease of testicular GnRH receptors, up to 90%. Some of the antagonist also occupied GnRH receptors in the contralateral testis and pituitary, but these effects were always clearly less than in the infused testis. None of the doses used affected circulating levels of gonadotropins, prolactin (Prl) or testosterone. However, when the endocrine parameters of the two testes were compared, the 100 ng/h dose of the antagonist resulted in a significant (P less than 0.01-0.05) 16-32% decrease in the testicular content of testosterone, and LH, FSH and lactogen receptors. Similar effects (inhibition of the same parameters by 22-42%) were observed when immature (30-day-old) male rats were treated for 1 week with intratesticular infusions of the antagonist. It is inferred from these observations that, in physiological circumstances, testicular GnRH receptors may mediate stimulatory effects of Leydig cell LH and lactogen receptors, and testosterone synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Homologous ligand induction of pituitary gonadotrophin-releasing hormone receptors in vivo is protein synthesis dependent.

This study demonstrates that a single subcutaneous injection of gonadotrophin-releasing hormone (GnRH) (60 ng) to GnRH-deficient (hpg) male mice causes a doubling of pituitary GnRH receptors (GnRH-R). No change in GnRH-R occurs during the time of LH release (15-60 min) or up until 4 h post-GnRH. Between 4 and 12 h there is a progressive increase in GnRH-R, which is still apparent 24 h later. No induction of GnRH-R occurs after the same treatment of intact adult normal mice. The same degree of GnRH-R induction occurs 12 h after a single GnRH injection (60 ng) to orchidectomized hpg male mice, indicating that this effect is mediated by a direct action of GnRH on the pituitary gonadotroph, rather than being secondary to stimulation of some gonadal product. Homologous ligand GnRH-R induction in hpg mouse pituitaries in vivo is prevented by prior treatment with cycloheximide, a non-specific protein synthesis inhibitor. Cycloheximide alone had no effect on GnRH-R in normal male mice but when combined with GnRH caused a 40% depletion of receptors, implying ligand-induced receptor loss without subsequent replenishment. The similarity between the extent, time-course, and dependence on protein synthesis of GnRH induction of its own receptors in vivo and in cultured pituitary cells in vitro indicates that the hpg mouse pituitary behaves like an in vivo pituitary cell culture system in this respect. Similarity of data derived from this in vivo model provides direct support for the view that in vitro studies on the cellular mechanism of GnRH action can be physiologically relevant to the intact animal.

Influence of needle lumen size on viability of injected dispersed and clumped foetal brain cells.

Foetal tissue from cortex, striatum, cerebellum and mesencephalon of human foetuses of gestational age 18 weeks have been examined for cell viability pre- and postpassage through needles of varying dimensions. Fully dispersed cells have less viability than cell clumps, but needle lumenar sizes employed in this study do not appear to have any significant influence on viability as measured by the vital staining method. There is a tendency of narrower needles to adversely affect viability of both dispersed and clumped cells; the vulnerability of component cells, i.e. neurones or glia has not been determined.

Phenotypic plasticity of 'mature' human fœtal mesencephalic dopaminergic neurons and glial cells.

Human mesencephalic neurons from the second trimester have been cultured and characterised. Fresh, non-cultured cells were rounded and without processes post-dispersion but in culture differentiated with neurite outgrowth when treated with 2 mMdibutyryl cyclic AMP in the absence of serum. This morphological differentiation could be reversed by the addition of the serine protease, prothrombin. Immunocytochemical staining for dopamine, tyrosine hydroxylase, neuron-specific enolase and glial fibrillary acid protein, demonstrated that dopaminergic, non-dopaminergic and glial elements responded similarly. The conditioned medium contained quantifiable levels of catecholamines as measured by HPLC. These findings are relevant to both developmental neurobiology and clinical neural transplantation, evidencing the considerable plasticity and functional integrity of mesencephalic cells in the second trimester as well as the influence of environmental factors.

Recovery of cellular activity in cryopreserved human foetal brain tissue.

Since long-term cryopreservation can cause losses in neural tissue viability and function a prerequisite would be the ability to monitor and promote functional recovery in donor tissue intended for neural transplantation. Rapid assessment of cryopreserved tissue's functional status prior to grafting is presently difficult in a clinical setting. A convenient indicator of functional status may be the level of DNA synthesis activity taking place in the tissue. Using immunocytochemical detection of incorporated bromodeox-yuridine we have quantified and compared DNA synthesis activity (expressed as proliferative capacity (PC)) in human foetal mesencephalic, striatal, cortical and cerebellar tissue before and after a 275-376 day storage in liquid nitrogen. There was a post-storage reduction in viability of 48-73% and in PC of 26-59%; the higher the PC before storage the greater the reduction after. Incubation of cryopreserved tissue with fetal calf serum resulted in 2-4-fold higher PC levels than serum-untreated controls and reached 80% of fresh tissue levels in mesencephalic cells after 3-4 h incubation. Assuming that quantification of proliferative activity is a practical indicator of the tissue's functional status, these findings suggest that treatment of the tissue with serum can largely restore the lost function caused by cryopreservation.

The selective viability of human foetal brain cells.

The influence of various factors upon the survival of human foetal neurones has been examined. The viability of several brain structures was assessed using ethidium bromide and acridine orange fluorescence in both 'intact' and mechanically dispersed tissue. Striatum was least vulnerable to dissociation while cortex, mesencephalon, pons, cerebellum and cord were more vulnerable to a greater or lesser extent. Material can be preserved in vitro with greater viability in the undissociated rather than dissociated state. The effects of other factors including foetal age upon viability are discussed.

Susceptibility of human foetal brain tissue to cool- and freeze-storage.

Human second trimester foetal brain tissue was stored for a period of 1-6 weeks under various conditions in an attempt to evaluate factors influencing its susceptibility (cell loss) and survivability. Post-storage viability of mesencephalon, striatum, cerebellum and occipital cortex was assessed by a protocol combining vital staining with cell density counts so that tissue viability and cell loss could be evaluated simultaneously; tissue survivability was evaluated by cell culture. A significant amount of cell loss occurred after 24 h storage at room temperature, after one week at 4 degrees C and by two weeks at -20 degrees C in all structures; storage at -196 degrees C resulted in 17-21% cell loss at the end of a 6 week period. At -20 degrees C the cryoprotective effect of 20% FCS was equivalent to that of 15% FCS + 7% DMSO combined, suggesting potential use of serum in replacement of chemical additives. The procedure for removal of DMSO was critical to cell viability and survivability: single step dilution led to 27-39% greater cell loss than slow, multi-step dilutions. In comparison to fresh, non-stored tissue, immunocytochemical characterization of in vitro propagated stored tissue revealed no changes in the populations of major constituent cell types including neurones, dopaminergic neurones, glial and fibroblast cells. These results provide information on possible conditions under which transplant tissue can be satisfactorily stored depending on the prevailing requirements.

Enhanced in vitro survival and growth of foetal human mesencephalic dopaminergic neurones on laminin and collagen: implications for cell banking.

Culture of second trimester mesencephalic cells on laminin and collagen substrata has been investigated in an attempt to ascertain the effects of these extracellular matrix components on survival and growth of central dopaminergic (DA) neurones. There were 156.8-186.4% more cells attached to laminin and collagen than poly-D-lysine 6 h post-plating. By 24 h there was statistically no significant difference in the total number of cells attached to the three substrate but in terms of cell type-specific survival the proportion of mesencephalic DA neurones surviving on laminin and collagen substrata after 7 days in culture increased significantly compared with poly-D-lysine (1.4-1.6% versus 0.4% of the total cellular population), an effect augmented by bFGF treatment, which led to levels of 2% or more, with a concomitant decrease in the proportion of attritic DA neurones. These results indicate a critical requirement for ECM proteins in the survival and growth of in vitro-propagated central DA neurones at the time of plating and throughout the culture period. They also imply survival-enhancing interactions of ECM proteins and neurotrophic factors in developmental neuronal regulation and provide paradigms for obtaining high yields of these cells for neural transplantation cell banks.

Rapidly growing histologically benign meningiomas: cell kinetic and deoxyribonucleic acid ploidy features: report of three cases.

We present three cases of histologically benign meningiomas with a rapid and known time course to the development of symptoms. Tumor doubling time calculated from sequential computed tomographic scans and computer-assisted image analysis of proliferating cell nuclear antigen reactivity suggested rapid growth. Feulgen staining indicated deoxyribonucleic acid aneuploidy. Tests for progesterone and estrogen receptor immunoreactivity were negative. These cases are noteworthy for their uncharacteristically rapid growth in the absence of histological evidence of atypia.

Correlation of proto-oncogene expression and proliferation and meningiomas.

Proliferation and proto-oncogene expression in 19 meningiomas of typical and atypical histology were analyzed in an attempt to understand the mechanism of growth that characterizes the neoplastic process in these tumors. Proliferation was estimated as the proliferative index by the enumeration of S-phase cells in imprints of tumor tissue exposed to bromodeoxyuridine in vitro, and the gene expression of c-myc, c-fos, c-src, c-H-ras, N-myc, acidic and basic fibroblast growth factor, insulin-like growth factors I and II, platelet-derived growth factor-alpha, and epidermal growth factor was quantified by messenger ribonucleic acid dot-blot hybridization assay. Atypical and malignant tumors had significantly higher proliferative indexes than did their nonmalignant counterparts. Levels of c-myc and c-fos messenger ribonucleic acid were elevated more than fivefold in 72 and 78% of the tumors, respectively, relative to the lowest levels detected in the series. Levels of growth factor messenger ribonucleic acid were sporadically elevated; 37 to 44% of tumors had more than fivefold enhanced levels of acidic and basic fibroblast growth factor. Positive correlations between proliferation and proto-oncogene/growth factor expression were found for c-myc in atypical/malignant tumors and for epidermal growth factor in fibroblastic meningiomas. Deregulated expression of c-myc and c-fos common to both typical and atypical tumors suggests that these are early events in the meningioma tumor process that may disturb the control of cell differentiation and together with fibroblast growth factors are likely to endow the transformed cell with a selective growth advantage by reducing the requirement for exogenous mitogens and by providing a niche for the growth of the tumor clone. Positive correlation of c-myc levels with proliferation in atypical/malignant meningiomas implies that this is a feature of malignancy and indicates continued disruption of the negative regulation of proto-oncogene expression, perhaps by tumor suppressor gene losses, during the course of tumor progression.

Hitchcock's experimental series of foetal implants for Parkinson's disease: co-grafting ventral mesencephalon and striatum.

Following 4 previous experimental series of foetal implants (mesencephalon) to treat patients with Parkinson's disease subjects (N7) in the fifth series were treated with co-grafts of foetal mesencephalon and striatum implanted stereotactically into the caudate nucleus bilaterally. The clinical outcome, better than in the previous series, included improvements lasting through 18 months follow-up in activities of daily living, clinical neurological motor examination, timed motor tasks, and dyskinesia-with reduction in the patients' need for dopaminergic medication.

A cancer research UK pharmacokinetic study of BPA-mannitol in patients with high grade glioma to optimise uptake parameters for clinical trials of BNCT.

This paper describes results to-date from a human pharmacokinetic study which began recruitment in December 2007. Results are presented for a single patient recruited in December 2007. A second patient was recruited in July 2008 but detailed data are not available at the time of writing. The trial is an open-label, non-comparative, non-therapeutic study of BPA-mannitol in patients with high-grade glioma, who will be undergoing stereotactic brain biopsy as part of the diagnostic process before definitive treatment. The study investigates the route of infusion (intra-venous (IV) or intra-carotid artery) and in each case will assess the effect of administration of mannitol as a blood-brain barrier disrupter. All cohorts will receive a 2 h infusion of BPA-mannitol, and for some cohorts an additional mannitol bolus will be administered at the beginning of this infusion. Measurements are made by inductively coupled plasma mass spectrometry (ICP-MS) of (10)B concentration in samples of blood, urine, extra-cellular fluid in normal brain (via a dialysis probe), brain tissue around tumour and tumour tissue. Additional analysis of the tumour tissue is performed using secondary ion mass spectrometry (SIMS). The first patient was part of the cohort having intra-venous infusion without mannitol bolus. No serious clinical problems were experienced and the assay results can be compared with available patient data from other BNCT centres. In particular we note that the peak (10)B concentration in blood was 28.1 mg/ml for a total BPA administration of 350 mg/kg which is very consistent with the previous experience with BPA-fructose reported by the Helsinki group.

Rapid detection of gene expression.

Conventional methods of in situ hybridization are not only complex, laborious and lengthy, tending to produce poor cellular resolution but are also inherently hazardous. We report a new method that overcomes many of these difficulties. Denovo attachment of a non-isotopic ligand, viz. 5-bromo-2'-deoxyuridine (BrdU) to oligonucleotide probes guaranteed the production of labelled probes of 100% specific activity that gave consistent, reproducible results, within hours, in a rapid in situ hybridization (ISH) technique. The method is demonstrated by localization of insulin-like growth factor II (IGF-II) mRNA in human intracranial tumours and foetal brain. Five of 10 meningiomas expressed IGF-II mRNA to an extent of 1% of their tumour cells and two of three pituitary adenomas exhibited a less than 0.2% IGF-II mRNA-positive cell population. IGF-II may be important in the progression and/or maintenance of these tumours. Foetal brain showed no hybridization. The method's simplicity, rapidity and reliability commends its use in a variety of research and diagnostic applications and the BrdU-labelled probes can be used in any hybridization technique.