Expression and activation of N-methyl-D-aspartate receptor subunit-1 receptor subunits in gonadotrophin-releasing hormone neurones of young and middle-aged mice during the luteinising hormone surge.

Glutamate is an important excitatory neurotransmitter that stimulates the release of gonadotrophin-releasing hormone (GnRH) and participates in the generation of the luteinising hormone (LH) surge. To determine the mechanisms of action of glutamate and possible changes in the glutamatergic input to GnRH neurones during reproductive ageing, we measured the expression and activation of the mandatory N-methyl-D-aspartate receptor subunit-1 (NMDAR1) in GnRH neurones of young and middle-aged mice prior to and during a steroid-induced LH surge. The results show that, in young animals, approximately 55% of all GnRH neurones contain immunoreactive NMDAR1 protein and this percentage does not change during the day of the LH surge. In approximately 10% of the GnRH neurones, NMDA receptor protein is phosphorylated at Ser 890 prior to the surge, whereas, in approximately 55% of the GnRH neurones, NMDAR1 subunits are phosphorylated during the LH surge. Activation of NMDAR1 receptor subunits stimulates the calcium-calmodulin-kinase IV (CaMK IV). pathway, which leads to the translocation of CaMK IV into the nucleus where this enzyme can phosphorylate the cAMP response element-binding protein (CREB) and CREB-binding protein. We show that, in young animals, approximately 20% of the GnRH neurones contain CaMK IV in their nuclei 7 h prior to the LH surge; this percentage increases to 60% at the beginning of the surge and decreases to approximately 40% some 2 h into the LH surge. In middle-aged animals, approximately 25% of the GnRH neurones contain NMDAR1 protein and only 10% of the GnRH neurones contain phosphorylated NMDAR1 protein prior to and during the surge; however 2 h after the peak of the surge, 20% of the GnRH neurones contain phosphorylated NMDAR1 subunits. Similarly, 20% of GnRH neurones contain nuclear CaMK IV and this percentage does not change during the day of the LH surge. The results suggest that, in the young animal, glutamatergic innervation of GnRH neurones during the LH surge causes the activation and phosphorylation of NMDAR1 receptor subunits which results in the translocation of CaMK IV into the nucleus. However, both, the expression and activation of NMDAR1 receptor subunits are greatly reduced in the middle-aged animals, which could result in the absence of LH surges.

Expression of AMPA receptor subunits (GluR1-GluR4) in gonadotrophin-releasing hormone neurones of young and middle-aged persistently oestrous rats during the steroid-induced luteinising hormone surge.

Glutamate provides excitatory input to gonadotrophin-releasing hormone (GnRH) neurones and elicits a response indicative of AMPA receptors. To determine if and which AMPA subunits are expressed by GnRH neurones, we conducted triple-label immunohistochemistry and confocal analyses on tissue obtained at 08.00, 12.00, 16.00 and 20.00 h from young and middle-aged, persistently oestrous (MA-PE) rats that were ovariectomised and primed with oestrogen and progesterone to induce a luteinising hormone (LH) surge. Each AMPA subunit was found in GnRH neurones, but in different patterns across the diurnal cycle, which were influenced by age. GluR1 expression increased earlier in young rats and the percentage of Fos-positive GnRH neurones expressing GluR1 rose significantly and was sustained from 12.00-16.00 h. GluR1 expression was delayed in MA-PE rats and the percentage of Fos-positive GnRH neurones expressing GluR1 peaked at 20.00 h. GluR2 expression in GnRH neurones did not change over time and was not affected by age; however, the percentage of Fos-positive GnRH neurones expressing GluR2 increased earlier and was sustained from 08.00-16.00 h in young rats whereas, in MA-PE rats, this percentage peaked at 20.00 h. GluR3 expression also increased earlier in young rats and peaked at 12.00 h but was delayed in MA-PE rats and peaked at 20.00 h. The number of Fos-positive GnRH neurones that coexpressed GluR3 peaked at 12.00 h in young rats but showed little change from 12.00-20.00 h in MA-PE rats. GluR4 expression was maintained at higher levels at 08.00 and 12.00 h in young rats; although the percentage of Fos-positive GnRH neurones expressing GluR4 peaked at 12.00 h in young rats, it showed little change in MA-PE rats. In summary, our data show that a higher proportion of Fos-positive GnRH neurones coexpressed AMPA receptor subunits in young rats and the expression, particularly of GluR1 and GluR2, was increased and sustained throughout the surge, whereas GluR3 and GluR4 expression peaked just before. In MA-PE rats, the rate of expression of GluR subunits and Fos in GnRH neurones was altered in a manner that may explain the delay and attenuation of the LH surge.

Identification of neurones in the female rat hypothalamus that express oestrogen receptor-alpha and vesicular glutamate transporter-2.

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)-alpha and ER-beta. Evidence suggests that oestrogen-receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin-releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor-expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter-2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERalpha mRNA-(35S-labelling) and VGLUT2 mRNA-(digoxigenin-labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERalpha mRNA are consistent with previous data in the literature. Dual-labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 +/- 3.4% of the ERalpha mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERalpha expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen-receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones.

Characterization of a polyclonal antibody to human pituitary tumor transforming gene 1 (PTTG1) protein.

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His(6) tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1-GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1-GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1-GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors.

Presence of luteinizing hormone-releasing hormone fragments in the rhesus monkey forebrain.

Previously, we have shown that two types of luteinizing hormone-releasing hormone (LHRH) -like neurons, "early" and "late" cells, were discernible in the forebrain of rhesus monkey fetuses by using antiserum GF-6, which cross-reacts with several forms of LHRH. The "late" cells that arose from the olfactory placode of monkey fetuses at embryonic days (E) 32-E36, are bona fide LHRH neurons. The "early" cells were found in the forebrain at E32-E34 and settled in the extrahypothalamic area. The molecular form of LHRH in "early" cells differs from "late" cells, because "early" cells were not immunopositive with any specific antisera against known forms of LHRH. In this study, we investigated the molecular form of LHRH in the "early" cells in the nasal regions and brains of 13 monkey fetuses at E35 to E78. In situ hybridization studies suggested that both "early" and "late" LHRH cells expressed mammalian LHRH mRNA. Furthermore, "early" cells predominantly contain LHRH1-5-like peptide and its cleavage enzyme, metalloendopeptidase E.C.3.4.24.15 (EP24.15), which cleaves LHRH at the Tyr5-Gly6 position. This conclusion was based on immunocytochemical labeling with various antisera, including those against LHRH1-5, LHRH4-10, or EP24.15, and on preabsorption tests. Therefore, in primates, a group of neurons containing mammalian LHRH mRNA arises at an early embryonic stage before the migration of bona fide LHRH neurons, and is ultimately distributed in the extrahypothalamic region. These extrahypothalamic neurons contain LHRH fragments, rather than fully mature mammalian LHRH. The origin and function of these neurons remain to be determined.

Neural signals that regulate GnRH neurones directly during the oestrous cycle.

GnRH, produced by a loose network of neurones in the basal forebrain, is the primary brain signal responsible for the release of LH and FSH from the anterior pituitary gland. The ovarian steroid hormone oestradiol feeds back at both the central nervous system and the anterior pituitary to regulate the patterns of release of GnRH and the gonadotrophins. Although recent evidence indicates that oestradiol may act directly on some GnRH neurones through classical genomic mechanisms, data from published studies have demonstrated that neurotransmission of afferent neuronal systems that are receptive to oestradiol is necessary to drive reproductive cyclicity. Many classical neurotransmitters and neuropeptides alter GnRH neuronal activity, through direct and sometimes indirect actions. This review focuses on the neurotransmitters that regulate GnRH neurones by binding to and activating specific membrane receptors that are expressed in GnRH neurones. These include the catecholamines, gamma-aminobutyric acid, glutamate, neuropeptide Y, neurotensin, beta-endorphin and vasoactive intestinal polypeptide. On the basis of recent molecular and neuroanatomical evidence, it is proposed that oestradiol influences the activity of these neurotransmitter and neuropeptide systems within the GnRH network to drive reproductive cyclicity.

Expression of multidrug resistance-associated protein 2 in small intestine from pregnant and postpartum rats.

We analyzed the expression of multidrug resistance-associated protein 2 (mrp2) in the small intestine of control female rats and in rats during late pregnancy (19-20 days of pregnancy) and lactation (2-4, 10-14, and 21 days after delivery). Western blot analysis was performed on brush-border membranes prepared from different regions of the small intestine. Expression of mrp2 was maximal in the proximal segments for all experimental groups, was preserved in pregnant rats, and increased by 100% in postpartum rats by late lactation with respect to control animals. Northern blot analysis of mrp2 mRNA revealed a positive correlation with protein levels. Transport of S-glutathione-dinitrophenol (DNP-SG) from the intestinal cell to the lumen was analyzed in the everted intestinal sac model. Secretion of DNP-SG was not altered in pregnant rats but increased in lactating animals by late lactation. Intestinal mrp2 mRNA, protein, and transport activity are increased in lactating rats, suggesting that this may represent an adaptive mechanism to minimize the toxicity of dietary xenobiotics in response to increased postpartum food consumption.

Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus.

The excitatory amino acid neurotransmitter glutamate participates in the control of most (and possibly all) neuroendocrine systems in the hypothalamus. This control is exerted by binding to two classes of membrane receptors, the ionotropic and metabotropic receptor families, which differ in their structure and mechanisms of signal transduction. To gain a better understanding about the precise sites of action of glutamate and the subunit compositions of the receptors involved in the glutamatergic neurotransmission in the hypothalamus and septum, in situ hybridization was used with 35S-labeled cRNA probes for the different ionotropic receptor subunits, including glutamate receptor subunits 1-4 (GluR1-GluR4), kainate-2, GluR5-GluR7, N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), and NMDAR2A-NMDAR2D. The results showed that subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-preferring, kainate-preferring, and NMDA-preferring receptor subunits are distributed widely but heterogeneously and that the GluR1, GluR2, kainate-2, NMDAR1, NMDAR2A, and NMDAR2B subunits are the most abundant in the hypothalamus. Thus, GluR1 subunit mRNA was prominent in the lateral septum, preoptic area, mediobasal hypothalamus, and tuberomammillary nucleus, whereas kainate-2 subunit mRNA was abundant in the medial septum-diagonal band, median and anteroventral preoptic nuclei, and supraoptic nuclei as well as the magnocellular portion of the posterior paraventricular nucleus. Regions that contained the highest levels of NMDAR1 subunit mRNA included the septum, the median preoptic nucleus, the anteroventral periventricular nucleus, and the supraoptic and suprachiasmatic nuclei as well as the arcuate nucleus. Together, the extensive distribution of the different GluR subunit mRNAs strengthen the view that glutamate is a major excitatory neurotransmitter in the hypothalamus. The overlap in the distribution of the various subunit mRNAs suggests that many neurons can express GluR channels that belong to different families, which would allow a differential regulation of the target neurons by glutamate.

Expression of estrogen receptor-alpha and c-Fos in adrenergic neurons of the female rat during the steroid-induced LH surge.

Epinephrine is an important neurotransmitter that is synthesized in relatively few neurons of the medullary regions C1-C3. Epinephrine is involved, among others in the control of most neuroendocrine systems, such as corticotropin releasing hormone-, gonadotropin releasing hormone- and oxytocin/vasopressin-containing neurons as part of complex feedback loop systems that often include interactions with the gonadal or adrenal steroid hormones. In order to determine if the interactions between gonadal steroid hormones with the adrenergic neurons are direct or involve steroid-receptive interneurons that in turn innervate the adrenergic neurons, dual immunohistochemistry was applied to identify if estrogen receptor-alpha (ERalpha) protein was expressed by adrenergic, phenylethanolamine-N-methyl transferase (PNMT)-positive neurons and if estradiol can activate these neurons as determined by the transient expression of the transcription factor c-Fos. The results show that an average of 22% of all PNMT neurons in the C1 region, 38% in C2 and 42% in the C3 region express estrogen receptor-alpha protein with the highest numbers of dual labeled neurons in the central levels of the C1-C3 regions. Overall, the percentages of dual labeled PNMT/ERalpha neurons did not change during the steroid-induced LH surge. In contrast, the percentage of c-Fos expressing PNMT neurons changed significantly during the LH surge. Thus, c-Fos immunoreactivity was highest in all three regions at 1200 h with 69% of the PNMT neurons in C1, 60% in C2 and 79% in C3 co-expressing c-Fos. C-Fos expression was lowest before and after the surge with 39% of the PNMT neurons in the C2 region containing c-Fos at 0800 h, 52% c-Fos-positive PNMT neurons in C1 and 54% in area C3. The results show that many adrenergic neurons are direct targets for estradiol and that most PNMT neurons in the brainstem are activated during the initiation of the steroid-induced LH surge which suggests that epinephrine is one of the triggers that stimulates GnRH release during the surge.

Localization of serotonin(5A) receptors in discrete regions of the circadian timing system in the Syrian hamster.

Endogenous serotonin and serotonergic drugs influence many aspects of circadian rhythms, including phase shifts, onset of locomotor activity, and period length and integrity of rhythms during exposure to constant light. The receptor subtype(s) mediating all of these circadian effects of serotonin has (have) not been identified. Immunoreactivity for the serotonin(5A) (5-HT(5A)) receptor has recently been identified in the rat suprachiasmatic nucleus (SCN). In this study, we investigated the distribution of the 5-HT(5A) receptors in four neural components of the circadian timing system (the SCN, the intergeniculate leaflet, and the median and dorsal raphe nuclei), in the Syrian hamster. Single and dual immunohistochemistry were conducted using an affinity-purified rabbit antibody generated against a peptide sequence unique to the 5-HT(5A) receptor, guinea pig anti-5-HT antisera and guinea pig anti-GABA antisera. For single labeling, immunoreactivity was visualized using DAB-nickel as the chromagen. All four regions showed strong, yet distinct, immunoreactivity for the 5-HT(5A) receptor. No specific labeling was present in the absorption or omission controls. For double labeling, immunoreactivity was visualized using immunofluorescence with Cy5- and FITC-labeled second antibodies followed by confocal microscopy. In the raphe nuclei, 5-HT-immunoreactivity and 5-HT(5A)-immunoreactivity were co-localized in cell bodies and axons. GABA-immunoreactive fibers surrounded some of the 5-HT(5A) receptor-immunoreactive cell bodies in the raphe nuclei. In conclusion, the 5-HT(5A) receptors are localized within several important neuroanatomical substrates of the circadian timekeeping system, and within the raphe nuclei, appear to be present on serotonin neurons. These findings suggest that some of the circadian effects of 5-HT may be mediated by the 5-HT(5A) receptor, which may function as a presynaptic autoreceptor.

Expression of ionotropic glutamate receptor subunit mRNAs in the hypothalamic paraventricular nucleus of the rat.

The hypopthalamic paraventricular nucleus (PVN) coordinates multiple aspects of homeostatic regulation, including pituitary-adrenocortical function, cardiovascular tone, metabolic balance, fluid/electrolyte status, parturition and lactation. In all cases, a substantial component of this function is controlled by glutamate neurotransmission. In this study, the authors performed a high-resolution in situ hybridization analysis of ionotropic glutamate receptor subunit expression in the PVN and its immediate surround. N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B mRNAs were expressed highly throughout the PVN and its perinuclear region as well as in the subparaventricular zone. NMDAR2C/2D expression was limited to subsets of neurons in magnocellular and hypophysiotrophic regions. In contrast with NMDA subunit localization, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate)-preferring and kainate (KA)-preferring receptor subunit mRNAs were expressed heterogeneously in the PVN and surround. Glutamate receptor 1 (GluR1) mRNA labeling was most intense in preautonomic subregions, whereas GluR2, GluR4, GluR5, and KA2 were expressed in hypophysiotrophic cell groups. It is noteworthy that GluR5 mRNA expression was particularly robust in the dorsolateral region of the medial parvocellular PVN, suggesting localization in corticotropin-releasing hormone neurons. All four AMPA subunits and GluR6 and GluR7 mRNAs were expressed highly in the perinuclear PVN region and the subparaventricular zone. These data suggest the capacity for multifaceted regulation of PVN function by glutamate, with magnocellular neurons preferentially expressing NMDA subunits, preautonomic neurons preferentially expressing AMPA subunits, and hypophysiotrophic neurons preferentially expressing KA subunits. Localization of all species in the perinuclear PVN suggests that glutamate input to the immediate region of the PVN may modulate its function, perhaps by communication with local gamma-aminobutyric acid neurons.

Expression and localization of multidrug resistant protein mrp2 in rat small intestine.

The expression of multidrug resistance-associated protein isoform 2 (mrp2), the ATP-dependent export pump that mediates the transport of glucuronic acid-, glutathione-, and sulfate-conjugated derivatives, was studied in rat small intestine. The small intestine was divided into nine equal segments, and mrp2 content was analyzed in homogenate and brush border membrane preparations by Western analysis. mrp2 protein was present mainly in brush border membrane of the proximal segments and gradually decreased from jejunum to the distal ileum. We also analyzed the content of mrp2 in three different populations of proximal enterocytes obtained from the upper and lower villus and the crypt regions. The export pump was mainly expressed in the villus cells and to a lesser degree in the crypt cells of the epithelium. Immunohistochemical analysis performed in duodenum, jejunum, and ileum confirmed in situ the Western blot findings. Analysis of mRNA encoding mrp2 in proximal and distal segments revealed a similar content in both regions, whereas distribution along the villus-crypt axis was similar to the protein gradient. Because conjugating enzymes are distributed similarly to mrp2, we conclude that they may act coordinately to contribute to first-pass metabolism of drugs and other xenobiotics in the proximal small intestine.

Evidence that members of the TGFbeta superfamily play a role in regulation of the GnRH neuroendocrine axis: expression of a type I serine-threonine kinase receptor for TGRbeta and activin in GnRH neurones and hypothalamic areas of the female rat.

The present study was designed to determine whether transforming growth factor (TGF)beta and/or activin participate in the regulation of the gonadotropin releasing hormone (GnRH) neuroendocrine axis in vivo. Single-label in situ hybridization histochemistry was used to determine the anatomical distribution of a TGFbeta and activin type I receptor (B1) mRNA, in the adult female rat hypothalamic areas that are known to be important sites for the regulation of reproduction. Dual-label in situ hybridization histochemistry was performed to determine whether B1 mRNA was expressed in GnRH neurones. The results of these studies revealed an extensive distribution of B1 mRNA in the hypothalamic regions, including diagonal bands of Broca, preoptic area, arcuate nucleus and median eminence. In the median eminence, B1 mRNA was detected in tanycytes and in the endothelial cells of the pituitary portal blood capillaries. Dual-label in situ hybridization histochemistry showed that 31+/-5% of GnRH neurones expressed B1 mRNA, thus providing evidence that TGFbeta and/or activin can act directly on GnRH neurones to modulate their activity. Taken together, these data provide morphological arguments in favour of a participation of TGFbeta and/or activin in the regulation of reproduction at the hypothalamic level.

Growth-associated protein-43 messenger ribonucleic acid expression in gonadotropin-releasing hormone neurons during the rat estrous cycle.

We have shown previously at the ultrastructural level that morphological changes occur in the external zone of the median eminence allowing certain GnRH nerve terminals to contact the pericapillary space on the day of proestrus. The present study was designed to determine whether the intrinsic determinant of neuronal outgrowth, growth-associated protein-43 (GAP-43), was expressed in GnRH neurons of adult female rats, and whether its expression varied throughout the estrous cycle. To accomplish this, we perfusion-fixed groups of adult female rats at 0800 and 1600 h on diestrous day 2 (diestrous II), at 0800 h and 1600 h on proestrus, and at 0800 and 1600 h on estrus (n = 4 rats/group) and used double labeling in situ hybridization and quantification to compare the levels of GAP-43 messenger RNA (mRNA) in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense complementary RNA (cRNA) probe labeled with the hapten digoxigenin, whereas the GAP-43 cRNA probe was labeled with 35S and detected by autoradiography. In addition, GAP-43 protein was identified with immunohistochemistry in the median eminence. The results show that many GnRH neurons expressed GAP-43 mRNA and that GAP-43 protein was present in many GnRH axon terminals in the outer layer of the median eminence. The number of GnRH neurons expressing GAP-43 mRNA was significantly higher on proestrus (64 +/- 5%) than on diestrous II (40 +/- 2%; P < 0.001) or on estrus (45 +/- 8%; P < 0.05), and the GAP-43 mRNA levels in GnRH neurons also varied as a function of time of death during the estrous cycle. The GAP-43 mRNA levels in GnRH neurons were higher on proestrus and estrus than on diestrous II (P < 0.05). These data show that 1) GAP-43 is expressed in adult GnRH neurons; 2) GAP-43 mRNA expression in GnRH neurons fluctuates during the estrous cycle; and 3) GAP-43 mRNA content in GnRH neurons is highest on the day of proestrus, before and during the onset of the LH surge. These observations suggest that the increased GAP-43 mRNA expression in GnRH neurons on the day of proestrus could promote the outgrowth of GnRH axon terminals to establish direct neurovascular contacts in the external zone of the median eminence and thus facilitate GnRH release into the pituitary portal blood.

Kainate receptor subunit-positive gonadotropin-releasing hormone neurons express c-Fos during the steroid-induced luteinizing hormone surge in the female rat.

During the preovulatory and estradiol-progesterone-induced GnRH-LH surge, a subpopulation of GnRH neurons transiently expresses the transcription factor c-fos, which is a useful marker of cell activation. To further characterize this subpopulation of GnRH neurons, multiple immunohistochemical procedures were applied to visualize GnRH, c-Fos, KA2, GluR5, GluR6, and GluR7 receptor subunits during different phases of the estrogen-progesterone-induced LH surge. The results show that the LH surge begins at 1400 h and peaks at 1600 h before returning to baseline late in the evening. At 1400 h, about 50% of the GnRH neurons contained c-Fos, and this percentage remained high at 65% at 1600 and 2000 h. During the surge, 50% of the c-Fos-positive GnRH neurons contained KA2 receptor subunit protein at 1400 h, 65% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 1600 h, and 50% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 2000 h. As KA2 subunits require other kainate-preferring subunits to form functional receptor channels, we examined GnRH neurons for the presence of GluR5, GluR6, and GluR7 messenger RNA (mRNA) and protein. The results show that the KA2-containing GnRH neurons also contain GluR5 receptor subunit mRNA and protein, and that these GnRH neurons are c-Fos positive during the steroid-induced LH surge. To determine whether administration of kainate is sufficient to induce c-Fos in GnRH neurons, steroid-primed animals received iv injections of subseizure-inducing amounts of kainic acid and were processed for immunohistochemistry and in situ hybridization. The results show that kainic acid causes a significant increase in circulating LH; however, it does not induce c-Fos in GnRH neurons, nor does it cause an increase in GnRH mRNA. Together, the results suggest that a large subset of GnRH neurons expresses KA2 as well as GluR5 receptor subunits, which would allow the formation of functional glutamate receptor channels, and that this subset of GnRH neurons is activated during the steroid-induced LH surge.

Evidence for expression of galanin receptor Gal-R1 mRNA in certain gonadotropin releasing hormone neurones of the rostral preoptic area.

Previous studies have shown that galanin plays an important role in the regulation of gonadotropin releasing hormone (GnRH) release. At present, it is not known if this role is exerted by direct or indirect interactions between galanin producing neurones and GnRH neurones. The objective of this study was to determine whether or not GnRH neurones could express galanin receptor Gal-R1 mRNA. Dual in-situ hybridization experiments were carried out with digoxigenin-labelled cRNA probes encoding GnRH in combination with 35S-labelled riboprobe encoding the galanin receptor Gal-R1. In order to detect possible variations in the expression of the Gal-R1 mRNA under different physiological conditions, male rats, intact female rats throughout the phases of the oestrous cycle, ovariectomized (OVX) and steroid-treated rats were analysed. The results show that many cells expressing Gal-R1 mRNA were present throughout the preoptic area. Gal-R1 mRNA-expressing cells were observed in very close proximity with GnRH neurones. In the female rat, some GnRH neurones located in the rostral preoptic area/vascular organ of the lamina terminalis expressed Gal-R1 mRNA. These double-labelled cells were observed at all times of the oestrous cycle, except during diestrus at 08.00 h and pro-oestrus at 18.00 h. Conspicuously, at oestrus 1800 h, we found that 21.6% of rostral GnRH neurones expressed the Gal-R1 mRNA. In addition, dual-labelled GnRH neurones were seen in OVX animals but not in oestrogen plus progesterone-treated ones. In the male rat, colocalization of GnRH mRNA and Gal-R1 receptor mRNA was not observed. In the medial preoptic area, no double-labelled GnRH neurones were detected regardless of the endocrine conditions. These results suggest that, in addition to a possible indirect action of galanin on GnRH cells via neurones located at close proximity, the effects of galanin on GnRH can be mediated by direct activation of galanin receptors in rostral GnRH neurones. This study also shows that expression of Gal-R1 mRNA in GnRH cells is influenced by the levels of circulating gonadal steroids.

Molecular cloning and characterization of the tumor transforming gene (TUTR1): a novel gene in human tumorigenesis.

We cloned and sequenced the cDNA of a potent tumor transforming gene (TUTR1) from human testis and determined its primary structure. The TUTR1 cDNA is composed of 656 nucleotides and encodes a novel protein of 202 amino acids. The predicted TUTR1 protein is extremely hydrophilic and contains two proline-rich motifs at its C-terminus. Northern blot analysis of the mRNA from various human tissues and tumors revealed that TUTR1 mRNA is highly expressed in tumors of the pituitary gland, adrenal gland, ovary, endometrium, liver, uterus, and kidney as well as in cell lines derived from tumors of the pituitary, breast, endometrium, and ovary. With the exception of the testis, the levels of TUTR1 mRNA were either very low or undetectable in normal human tissues. Overexpression of TUTR1 in mouse fibroblasts (NIH 3T3) cells resulted in an increase in cell proliferation, induced cellular transformation in vitro, and promoted tumor formation in nude mice. These results suggest that TUTR1 is a novel and potent transforming gene, which may be involved in tumorigenesis in numerous different human tumors.

Galanin within the normal and hyperplastic anterior pituitary gland: localization, secretion, and functional analysis in normal and human growth hormone-releasing hormone transgenic mice.

Studies evaluating estrogen-induced anterior pituitary tumors revealed a strong direct correlation between expression of the peptide galanin and tumor growth. To evaluate further the potential roles of galanin in the hyperplastic pituitary, we used a model of estrogen-independent anterior pituitary tumor formation, the male human GH-releasing hormone (hGHRH) transgenic mouse. Pituitaries of hGHRH transgenic mice are characterized by a hyperplasia of somatotrophs and contain markedly elevated levels of galanin. We examined the population of galanin-producing pituitary cells in 4- to 6-month-old male hGHRH transgenic mice and their nontransgenic siblings. The percentage of galanin-containing pituitary cells was significantly increased within the anterior pituitaries of hGHRH transgenic mice. By using the cell immunoblot assay we found that the basal secretion of galanin and GH from individual pituitary cells of hGHRH transgenic mice was significantly greater than that from pituitary cells of nontransgenic mice. By modifying the cell immunoblot assay, we determined that somatotrophs from both hGHRH transgenic and normal mice that were positive for galanin immunoreactivity secreted significantly greater amounts of GH than those somatotrophs devoid of galanin immunoreactivity. Moreover, immunoneutralization of galanin significantly decreased GH secretion from pituitary cells obtained from hGHRH transgenic mice. Thus, we now show that the increased levels of galanin peptide within the hyperplastic pituitaries of hGHRH transgenic mice are due to an increase in the population of cells containing galanin, and that galanin participates in the augmented secretion of GH from hyperplastic proliferating pituitary cells.

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection.

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post-transection loss of target-derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6-72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF-like activity was determined in DRG, in 5-mm nerve segments proximal and distal to the transection site and in a corresponding 5-mm segment of the contralateral nonoperated nerve. NGF-like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF-like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P=0.001) 2-fold increase in DRG NGF-like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF-like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF-specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF-treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF-treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity.

Identification of kainate-preferring glutamate receptor subunit GluR7 mRNA and protein in the rat median eminence.

In situ hybridization and immunohistochemistry were used to determine the presence of kainate-preferring glutamate receptor subunits GluR6 and GluR7 mRNA and protein in the median eminence of the rat. The results show that most tanycytes lining the ventral third ventricle and many astrocytes within the median eminence contain the GluR7 receptor subunit mRNA but not the GluR5 and GluR6 receptor subunit mRNA. Immunohistochemical stainings show that the GluR6/7 receptor protein was localized to tanycytic cell bodies, their basal processes and to many other astrocytes in different layers of the median eminence. The results suggest that glutamate can act directly on the glial cells in the median eminence by binding to the GluR7 subunit which may be important for the control of the secretion of releasing and inhibiting hormones from axon terminals in the external layer. In order to determine if these receptor subunits are functional, kainic acid was injected and c-fos expression monitored. Results show that kainic acid induced c-fos synthesis in most of these glial cells.