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Objective: Primitive neuroectodermal tumors (PNETs) are thought to be derived from early central nervous system precursors. Therefore, we hypothesized that the neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) and their receptors (TrkA, TrkB, and TrkC), which are involved in the proliferation, differentiation, and survival of neuronal cells, might be important in regulating tumor growth., Methods: Using ribonucleic acid (RNA) blotting and reverse transcription-polymerase chain reaction analysis, we investigated the expression of these ligands and their receptors in six PNET cell lines (Daoy, PFSK, D283 Med, UW288-1, CHP707m, and D341 Med). Neurotrophin protein levels were measured using enzyme-linked immunosorbent assay procedures. Receptor function was demonstrated by autophosphorylation. Induction of c-Fos expression and effects on cell proliferation were assessed after the addition of exogenous neurotrophin., Results: Three cell lines expressed messenger RNA for all neurotrophins, whereas the other three expressed two of the three neurotrophins. Neurotrophin protein levels were low. All cell lines expressed trkA messenger RNA. Five expressed the amino terminus of trkB, but three of these did not express the carboxyl terminus. All cell lines contained trkC messenger RNA, but the receptor was truncated in two cell lines. No cell line contained message for a receptor containing an insertion in the tyrosine kinase domain. The addition of neurotrophin to PNET cells resulted in phosphorylation of a protein that was immunoprecipitated with an anti-pan-Trk antibody. c-Fos expression and cell growth were increased by preincubation with neurotrophins, but only in the cell lines expressing the relevant full-length receptors., Conclusion: The expression of neurotrophins and neurotrophin receptors by PNET cell lines is variable. The presence of activated Trk receptors in these cell lines may be required for rapid growth, via an autocrine loop mechanism. This will require further investigation.

Allelic loss of 17p13.3 is observed in approximately 40% of medulloblastomas, suggesting the presence of a tumor suppressor gene in this region. Deletion mapping has defined a region of common loss flanking the telomeric marker D17S34, and a recent report delineated a 9-kb homozygous deletion within the D17S34 locus in one such tumor. Using cDNA selection, we have identified a transcript spanning this deletion, designated (HSA)RPH3AL (rabphillin-3A-like), based on its 77% overall amino acid identity with a recently cloned rat gene, (RNO)Rph3al (originally termed Noc2), a gene putatively involved in regulated endocrine exocytosis through its interactions with the cytoskeleton. We determined the exon-intron boundaries of RPH3AL and screened the coding region for mutations by direct sequencing in DNA extracted from 33 tumor samples with allelic loss of 17p13, including 10 medulloblastoma, 14 follicular thyroid cancer (FTC), and 9 ovarian cancer specimens. No mutations were identified. Thus, despite its location in a homozygously deleted 17p13.3 locus, it is unlikely that RPH3AL is a gene involved in the oncogenesis of medulloblastoma, FTC, or ovarian cancer., (Copyright 1999 Academic Press.)

The beta-catenin, glycogen synthase kinase 3beta (GSK-3beta), and adenomatous polyposis coli (APC) gene products interact to form a network that influences the rate of cell proliferation. Medulloblastoma occurs as part of Turcot's syndrome, and patients with Turcot's who develop medulloblastomas have been shown to harbor germ-line APC mutations. Although APC mutations have been investigated and not identified in sporadic medulloblastomas, the status of the beta-catenin and GSK-3beta genes has not been evaluated in this tumor. Here we show that 3 of 67 medulloblastomas harbor beta-catenin mutations, each of which converts a GSK-3beta phosphorylation site from serine to cysteine. The beta-catenin mutation seen in the tumors was not present in matched constitutional DNA in the two cases where matched DNA was available. A loss of heterozygosity analysis of 32 medulloblastomas with paired normal DNA samples was performed with four microsatellite markers flanking the GSK-3beta locus; loss of heterozygosity with at least one marker was identified in 7 tumors. Sequencing of the remaining GSK-3beta allele in these cases failed to identify any mutations. Taken together, these data suggest that activating mutations in the beta-catenin gene may be involved in the development of a subset of medulloblastomas. The GSK-3beta gene does not appear to be a target for inactivation in this tumor.

The hypothalamic content of LH releasing factor (RF), pituitary ACTH and pituitary and plasma LH and FSH were measured by radioimmunoassay from foetal Day 15 to postnatal Day 65. Bioassayable corticotrophin releasing activity was also measured during the postnatal period. Hypothalamic LH-RF was detectable as early as foetal Day 15, increasing gradually until postnatal Day 2 and then steeply between Days 5 and 16. The levels of LH-RF were similar in both male and normal female rats until Day 41, after which the increase which had been occurring from Day 16 continued in the male but not the female. In female rats treated with testosterone propionate neonatally ('androgenized females') the hypothalamic content of LH-RF at Day 9 was significantly less than that in the male or normal female, levels reaching those found in the latter two groups by Days 16-22. The lower level of LH-RF in the androgenized female was associated with pituitary gonadotrophin and plasma FSH levels which were lower than in the normal female until Day 30. In the normal female, vaginal opening was associated with a marked drop in hypothalamic LH-RF content and in pituitary LH and FSH, but in the androgenized female, vaginal opening occurred while hypothalamic LH-RF and pituitary LH levels were still rising. The peaks in pituitary FSH and LH and in plasma LH seen on Days 22, 30 and 41, respectively, in the normal female were each delayed by 8-9 days in the androgenized female. In all three types of animal there was a significant drop in plasma FSH between Days 50 and 65 which was associated with a significant increase in pituitary FSH in the male and a significant decrease in pituitary FSH in the androgenized female rats. The day 17 foetal pituitary gland also contained ACTH, and again levels of this hormone rose steeply between Days 5 and 9. In contrast to the gonadotrophins, there was a marked divergence between the pituitary content and concentration of ACTH: content rose while concentration remained relatively steady after Day 9. There was no major difference in pituitary ACTH levels between the three types of animal throughout the study; however, around Days 16 and 50, corticotrophin releasing activity was higher in males and androgenized females compared with that in normal females.

There was a drop of 56% in the hypothalamic content of Gn-RH in female voles 5 min after mating compared with that in unmated but receptive animals. This suggests that the surge of LH in vole plasma associated with reflex ovulation is evoked by a massive release of Gn-RH.

As determined by fluorescence histochemistry, the distribution of catecholamine-containing neurons in the hypothalamus of the female rabbit is similar to that seen in the rat. The fluorescence appearance of the hypothalamus was not appreciably different from normal at 0.25, 1, 4 or 24 h after mating, but in animals in which the synthesis of catecholamines was inhibited by the administration of the tyrosine hydroxylase inhibitor, H44/68, the number of fluorescent neurons seen in the nucleus periventricularis arcuatus following copulation was markedly reduced. However, the concentrations of luteinizing hormone (LH) in serum and in the pituitary glands of mated and unmated animals treated with H44/68 were not significantly different from those found in the corresponding control animals. This, together with the fact that ovulation followed mating in drug-treated rabbits suggests that a normal level of catecholamines in the tubero-infundibular system is not essential for the secretion of the amount of LH necessary for ovulation.

1. Previous studies on the effect of preoptic and median eminence stimulation on the immunoreactive LRF content of pituitary stalk blood from pro-oestrous rats have been extended. Stimulation of the suprachiasmatic nuclei and anterior hypothalamic area produced increments in LRF which were 66 and 18% respectively, of that produced by preoptic stimulation, and 38 and 9%, respectively, of that produced by stimulation of the median emience. Stimulation of the amygdala and hippocampus had no effect. 2. The LRF response was not affected significantly when preoptic stimulation was accompanied by stimulation of the hippocampus. 3. In animals subjected to section of the dorsal afferents of the diencephalon, the LRF response to preoptic stimulation was similar to that in intact rats. However, the facilitatory effect of oestrogen on the LRF response to preoptic stimulation was significantly reduced in the roof sectioned compared with intact animals. The post-operative resumption of oestrous cycles was delayed but not abolished by dorsal deafferentation.

The effects of sex steroid hormones on the responsiveness of the neural mechanism responsible for the secretion of LH-RF have been examined in the female rat. Responsiveness was determined at pro-oestrus by measuring the increments in immunoreactive LH-RF of pituitary stalk blood produced by electrical stimulation of the medial preoptic area or median eminence. Ovariectomy on the morning of dioestrus reduced the LH-RF response to preoptic stimulation while oestradiol benzoate (OB) or testosterone propionate (TP) administered immediately after ovariectomy significantly augmented the response. The facilitatory effect of TP was possibly due to its conversion to an aromatized derivative since 5alpha-dihydrotestosterone monobenzoate was ineffective. Progesterone did not facilitate preoptic responsiveness, and, when administered to animals ovariectomized at 12.00 h of pro-oestrus, reduced the LH-RF response at 18.00 h the same day. Stimulation of the median eminence produced a significantly greater increment in LH-RF than stimulation of the preoptic area. The facilitatory action of OB on the LH-RF response was less marked for median eminence compared with preoptic stimulation. The administration of ICI 46474 at 17.00 h of dioestrus did not reduce preoptic responsiveness on the morning of the next day, suggesting that this compound does not act as an 'antioestrogen' at the level of the preoptic area.

We have investigated whether the priming effect of LH-RF can be elicited by electrical stimulation of the medial preoptic area, or by i.v. infusion or multiple i.v. injections of the synthetic decapeptide. All experiments were carried out on animals anaesthetized with sodium pentobarbitone at 13.30 h. In pro-oestrous rats, the LH response to the second of two electrical stimuli, 15 min in duration and separated by 60 min, was significantly greater than the response to the first stimulus. When synthetic LH-RF was infused at a constant rate for 90 min, plasma LH increased gradually for the first 45-60 min after which it increased markedly. This enhanced secretion of LH did not occur in rats which were infused with the same total dose of LH-RF, either 15 or 75 ng/100 g body wt, over periods of 45 min or less. When a dose of 15 ng LH-RF/100 g body wt was administered in six divided doses by i.v. injections, each separated by 15 min, there was a marked increase in plasma LH after 75 min. The profile of the mean plasma LH concentration in rats subjected to preoptic stimulation for 90 min was similar to that in rats infused for 90 min with LH-RF, but the variation in response was much greater in the stimulated rats. These results indicate that the priming effect can be elicited by endogenous as well as synthetic LH-RF, and that whether LH-RF reaches the pituitary at a constant rate or in a pulsatile manner the factor is capable of significantly increasing the responsiveness of the gonadotrophs. The relevance of these findings with respect to the development of the spontaneous preovulatory LH surge is discussed. A priming effect could not be elicited by constant LH-RF infusion in dioestrous rats; this supports the view that steroid hormones, especially oestradiol-17phi, determine the magnitude of the effect. The LH response in male rats subjected to i.v. infusion of LH-RF was much lower than in females. Pre-treatment with oestradiol benzoate did not increase the response significantly, suggesting that this sex difference cannot be ascribed simply to low levels of plasma oestrogen in the male.

Hypothalamic corticotrophin releasing (CR) activity and LH-releasing factor (RF) content, and pituitary and plasma LH, FSH and ACTH were measured in adult male and female Wistar rats maintained under 14 h light per day. Hypothalamic LH-RF and pituitary and plasma hormones were estimated by radioimmunoassay while CR-activity was assessed by the amount of ACTH released from hemipituitaries in vitro. Two experiments were carried out on male animals. In the first, some of the animals were kept in a room, distant from the animal house, in which the lighting was reversed with respect to the external environment. In animals exposed to the reversed lighting regime, hypothalamic LH-RF content and pituitary gonadotrophin concentrations were significantly lower than the values in male rats kept in the animal house where they were in close proximity to female rats. In the second experiment, which was carried out on animals which had all been kept in the animal house, there was no significant differences between the LH-RF contents measured at 3-4 h intervals throughout the day. Pituitary LH and FSH contents, but not concentrations, were significantly increased at 12.00 h. There was little differences between the experiments in CR-activity, plasma ACTH concentrations and profiles of pituitary ACTH content and concentration. As expected there was a diurnal rhythm in plasma corticosterone concentrations (determined by competitive protein-binding assay) with the peak occurring between 15.00 and 18.00 h. The profiles of plasma and pituitary ACTH were similar to that of plasma corticosterone. Corticotrophin releasing activity dropped significantly between 12.00 and 16.00 h, but remained steady at the other times. In female rats there were no significant differences between hypothalamic LH-RF content throughout the 4-day cycle. During pro-oestrus the mean LH-RF content rose to teach a high level at 18.00 h at which time plasma LH concentration had risen sharply to a level consistent with the peak of the preovulatory surge. Plasma FSH concentration also rose significantly between 15.00 and 18.00 h of pro-oestrus. At metoestrus and dioestrus, plasma FSH levels were lower in the morning than in the evening. These results suggest that (1) there is no diurnal rhythm in hypothalamic LH-RF, (2) there may be a diurnal rhythm in pituitary gonadotrophin content in the male and in plasma FSH concentration on the days of metoestrus and dioestrus in the female, (3) if a surge of LH-RF does occur on the afternoon of pro-oestrus, the rate of LH-RF synthesis exceeds its release, and (4) the mechanism which regulates gonadotrophin secretion in the male may be affected by factors in the environment other than daylength. The results provide further evidence for the view that the diurnal rhythm of corticosterone secretion is under hypothalamo-hypophysial control.