GLRB allelic variation associated with agoraphobic cognitions, increased startle response and fear network activation: a potential neurogenetic pathway to panic disorder.

The molecular genetics of panic disorder (PD) with and without agoraphobia (AG) are still largely unknown and progress is hampered by small sample sizes. We therefore performed a genome-wide association study with a dimensional, PD/AG-related anxiety phenotype based on the Agoraphobia Cognition Questionnaire (ACQ) in a sample of 1370 healthy German volunteers of the CRC TRR58 MEGA study wave 1. A genome-wide significant association was found between ACQ and single non-coding nucleotide variants of the GLRB gene (rs78726293, P=3.3 × 10-8; rs191260602, P=3.9 × 10-8). We followed up on this finding in a larger dimensional ACQ sample (N=2547) and in independent samples with a dichotomous AG phenotype based on the Symptoms Checklist (SCL-90; N=3845) and a case-control sample with the categorical phenotype PD/AG (Ncombined =1012) obtaining highly significant P-values also for GLRB single-nucleotide variants rs17035816 (P=3.8 × 10-4) and rs7688285 (P=7.6 × 10-5). GLRB gene expression was found to be modulated by rs7688285 in brain tissue, as well as cell culture. Analyses of intermediate PD/AG phenotypes demonstrated increased startle reflex and increased fear network, as well as general sensory activation by GLRB risk gene variants rs78726293, rs191260602, rs17035816 and rs7688285. Partial Glrb knockout mice demonstrated an agoraphobic phenotype. In conjunction with the clinical observation that rare coding GLRB gene mutations are associated with the neurological disorder hyperekplexia characterized by a generalized startle reaction and agoraphobic behavior, our data provide evidence that non-coding, although functional GLRB gene polymorphisms may predispose to PD by increasing startle response and agoraphobic cognitions.

Is prepulse modification altered by continuous theta burst stimulation? DAT1 genotype and motor threshold interact on prepulse modification following brain stimulation.

Previous studies suggest an inhibitory top-down control of the amygdala by the prefrontal cortex (PFC). Both brain regions play a role in the modulation of prepulse modification (PPM) of the acoustic startle response by a pre-stimulus. Repetitive transcranial magnetic stimulation (rTMS) can modulate the activity of the PFC and might thus affect PPM. This study tested the effect of inhibitory rTMS on PPM accounting for a genetic variant of the dopamine transporter gene (DAT1). Healthy participants (N = 102) were stimulated with continuous theta burst stimulation (cTBS, an intense form of inhibitory rTMS) or sham treatment over the right PFC. Afterwards, during continuous presentation of a background white noise a louder noise burst was presented either alone (control startle) or preceded by a prepulse. Participants were genotyped for a DAT1 variable number tandem repeat (VNTR) polymorphism. Two succeeding sessions of cTBS over the right PFC (2 × 600 stimuli with a time lag of 15 min) attenuated averaged prepulse inhibition (PPI) in participants with a high resting motor threshold. An attenuation of PPI induced by prepulses with great distances to the pulse (480, 2000 ms) was observed following active cTBS in participants that were homozygous carriers of the 10-repeat-allele of the DAT1 genotype and had a high resting motor threshold. Our results confirm the importance of the prefrontal cortex for the modulation of PPM. The effects were observed in participants with a high resting motor threshold only, probably because they received a higher dose of cTBS. The effects in homozygous carriers of the DAT1 10-repeat allele confirm the relevance of dopamine for PPM. Conducting an exploratory study we decided against the use of a correction for multiple testing.

In vitro effect of leptin on anterior pituitary cells LH secretory activity during early pregnancy in pig.

Leptin modulates reproductive activity but its potential influence on LH secretion from anterior pituitary (AP) cells during implantation period in pigs (days 14-16 of pregnancy) remained unexplored. This study focused on determination whether leptin affects basal and GnRH-induced LH secretion and intracellular accumulation and whether leptin receptor (OB-Rb) mRNA is expressed in the AP gland during implantation in pigs. Four individual AP glands were developed into separate primary cultures. 2×105 cells/ml were preincubated (72 h) and next, for 3.5 h, experimentally treated with GnRH (100 ng/ml), leptin (10-11, 10-9, 10-7, 10-6 M) alone, or given in respective combinations with GnRH. In the AP gland, OB-Rb mRNA expression was determined by real-time PCR method. Leptin activated LH secretion and its concentration-dependent effect was observed as stimulation shown in a full range tested (culture 1) and exhibited only at 10-6 M (culture 2). A pooled data analysis revealed that basal LH secretion increased at 10-9, 10-7 and 10-6 M, but GnRH-induced LH release decreased at 10-6 M. Leptin down-regulated GnRH-induced LH secretion in all cultures, but only culture 3 exhibited sensitivity for all concentrations tested. Basal LH accumulation was activated in culture 1 (at 10-11 M) and inhibited in culture 4 (at 10-9 M). In the presence of GnRH leptin up-regulated LH accumulation with individual culture leptin-sensitivity (culture 1-3), while down-regulated LH accumulation in culture 4. Obtained data indicate that OB-Rb mRNA is expressed in the AP gland and leptin alone and in combination with GnRH specifically modulates LH activity during early pregnancy in pigs.

Cellular redox homeostasis in endothelial cells treated with nonmodified and Fenton-modified nanodiamond powders.

Diamond nanoparticles find numerous applications in pharmacy, medicine, cosmetics, and biotechnology. However, possible adverse cellular effects of diamond nanoparticle cells have been reported, which may limit their use. The aim of this study was to compare the effect of nonmodified diamond nanoparticles (D) and diamond nanoparticles modified by the Fenton reaction (D+OH) on human umbilical cord endothelial cells (HUVEC-ST). We found that both D and D+OH show time- and concentration-dependent cytotoxicity, inducing apoptosis and necrosis of HUVEC-ST. Interaction with D and D+OH also induced changes in the production of reactive oxygen and nitrogen species and changes in the level of glutathione and activities of antioxidant enzymes in the cells. These data demonstrate that diamond nanoparticles may induce oxidative stress in human endothelial cells, which contributes to their cytotoxic effects seen at higher concentrations of D and D+OH.

Stimulation of production of reactive oxygen and nitrogen species in endothelial cells by unmodified and Fenton-modified ultradisperse detonation diamond.

In recent years, the development of nanotechnology opens up new prospects for biomedical applications of unmodified and chemically modified diamond nanoparticles (DNPs). The problem of biocompatibility of DNPs is thus of primary importance. The first step in the modification of DNPs is usually the introduction of -OH groups, which can bind other functional groups. One of the basic methods to introduce -OH groups onto DNPs is the Fenton reaction. The aim of this study was to compare the effect of unmodified DNPs and nanoparticles modified by the Fenton reaction on human endothelial cells. Ultradisperse diamond (UDD) was modified by the Fenton reaction introducing surface -OH groups. Immortalized human umbilical cord endothelial cells (HUVEC-ST) were incubated with 2-100 µg/mL nanopowders in the opti-MEM medium. For comparison, graphite powder (GRAF and GRAF+OH) was also employed. UDD and GRAF augmented generation of reactive oxygen species in the cells after 24 H incubation, estimated by oxidation of 2',7'-dichlorofluorescin diacetate (H2DCF-DA). Cellular production of nitric oxide, estimated with DAF-FM-DA (3-amino-4-aminomethyl 2',7'-dichlorofluorescein diacetate), was also affected by UDD and GRAF after 24 H. Fenton-modified OH, in contrast to unmodified diamond, decreased NO production. Detonation nanoparticles also affected the cellular content of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase). This article was published online on 5 February 2013. Errors in the byline and affiliation line were subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 18 April 2013.

Induction of apoptosis in human endothelial cells by nanodiamond particles.

Carbon nanoparticles are a promising material which finds application in different fields in industry and medicine. For medical applications, biocompatibility of nanoparticles is of critical importance because a lot of medical implants are coated by carbon coating. Our previous results showed that nanoparticles may induce increased production of ROS by the cells so we decided to checked if nanopowders can induce apoptosis. Apoptosis was quantified by double-staining with acridine orange and ethidium bromide. For comparison, we identified apoptotic cells with annexin V-FITC/propidium iodide. Our data demonstrate that treatment of the cells with diamond nanopowders may induce apoptosis and necrosis and this effect is dependent on the time of treatment and concentration of the nanopowders. The highest level of apoptotic cells was observed after incubation with Ultrananocrystalline Detonation Diamond (UDD) suggesting that the size is the main determinant of nanoparticle cytotoxicity.

Spectral and photophysical modifications of porphyrins attached to core-shell nanoparticles. Theory and experiment.

Plasmonic nanostructures, of which gold nanoparticles are the most elementary example, owe their unique properties to localized surface plasmons (LSP), the modes of free electron oscillation. LSP alter significantly electromagnetic field in the nanostructure neighborhood (i.e., near-field), which can modify the electric dipole transition rates in organic emitters. This study aims at investigating the influence of Au@SiO2core-shell nanoparticles on the photophysics of porphyrins covalently attached to the nanoparticles surface. Guided by theoretical predictions, three sets of gold nanoparticles of different sizes were coated with a silica layer of similar thickness. The outer silica surface was functionalized with either free-basemeso-tetraphenylporphyrin or its zinc complex. Absorption and emission bands of porphyrin overlap in energy with a gold nanoparticle LSP resonance that provides the field enhancement. Silica separates the emitters from the gold surface, while the gold core size tunes the energy of the LSP resonance. The signatures of weak-coupling regime have been observed. Apart from modified emission profiles and shortened S1lifetimes, Q band part intensity of the excitation spectra significantly increased with respect to the Soret band. The results were explained using classical transfer matrix simulations and electronic states kinetics, taking into account the photophysical properties of each chromophore. The calculations could reasonably well predict and explain the experimental outcomes. The discrepancies between the two were discussed.

The possible involvement of salsolinol and hypothalamic prolactin in the central regulatory processes in ewes during lactation.

Salsolinol, a dopamine-related compound and prolactin-producing cells were found in the ovine hypothalamus. This study was designed to test the hypothesis that salsolinol, acting from the CNS level, is able to stimulate pituitary prolactin release as well as prolactin mRNA expression in the anterior pituitary cells (AP) and in the mediobasal hypothalamus (MBH) in lactating ewes. The intracerebroventricular infusions of salsolinol in two doses, total of 50 ng or 5 µg, were performed in a series of five 10-min infusions at 20-min intervals. All infusions were made from 12:30 to 15:00 and the pre-infusion period was from 10:00 to 12.30 h. The prolactin concentration in plasma samples, collected every 10 min, was determined by radioimmunoassay; prolactin mRNA expression in AP and MBH tissues was determined by real-time PCR. The obtained results showed that salsolinol infused at the higher dose significantly (p < 0.001) increased plasma prolactin concentration in lactating ewes, when compared with the concentration noted before the infusion and with that in lactating controls. In lactating ewes, the relative levels of prolactin mRNA expression in the AP and MBH were up to twofold and fivefold higher respectively than in non-lactating ewes (p < 0.05). In our experimental design, salsolinol did not significantly affect the ongoing process of prolactin gene expression in these tissues. We conclude that in ewes, salsolinol may be involved, at least, in the process of stimulation of prolactin release during lactation and that hypothalamic prolactin plays an important role in the central mechanisms of adaptation to lactation.

Effect of warm-rearing and heat acclimation on pituitary-gonadal axis in male rats.

Plasma gonadotrophic and testicular hormones concentrations in both immature and adult male rats exposed to 34 degrees C of ambient temperature were determined. In vitro steroidogenic ability of interstitial cells from experimental rats was also studied. Four groups of rats (n = 45) were used. Warm-reared (WR) males were housed in 34 degrees C and control-reared rats in 20 degrees C from birth to adulthood. The other groups were acclimated to 34 degrees C [warm-acclimated (WA) group] or 20 degrees C [deacclimated (DA) group] as adults. Decreased body weight and testis weight (p < 0.05) was found in heat-exposed groups, but relative testis weight was unchanged in WA and increased (p < 0.05) in WR and DA males. Plasma luteinizing hormone (LH) concentration increased in WA and DA males. Increased (p < 0.05) follicle-stimulating hormone (FSH) and prolactin plasma levels were found in DA and WR groups respectively. WA males had decreased testosterone (T) and WR rats androstenedione (A(4)) plasma concentration (p < 0.05). Interstitial cells (43% of them were Leydig cells by 3beta-hydroxysteroid dehydrogenase activity) from heat-exposed males secreted less (p < 0.05) T compared with the control group when incubated without LH (basal conditions). Androstenedione secretion decreased (p < 0.05) in WA rats. Secretion of estradiol-17beta (E(2)) was higher in WR and lower in DA cells under basal conditions. Weaker responsiveness to LH was observed in WR cells. Androgen synthesis from pregnenolone by interstitial cells increased (p < 0.05) in the WA group. We concluded that heat exposure of neonatal and adult male rats caused different pituitary-testicular axis adjustments. It seemed that long-term heat exposure of neonatal rats is less deleterious concerning the activity of pituitary-testicular axis than heat acclimation of adults.

Gonadotropin-releasing hormone and kisseptin-10 regulate nuclear receptor subfamily 5 group a member 1/catenin beta 1/ nuclear receptor subfamily 0 group B member 1 activity in female rat anterior pituitary gland.

In this study, we tested the hypothesis that modulation of endogenous gonadotropin-releasing hormone (Gnrh) neuronal network activity alters the mRNA expression of nuclear receptor subfamily 5 group A member 1 (Nr5a1), through one of the component of Wnt pathway signaling - catenin beta 1 (Ctnnb1) (its co-activator), and its co-repressor nuclear receptor subfamily 0, group B member 1 (Nr0b1) in the female rat pituitary gland in vivo. Adult ovariectomized rats were given a serial infusion of Gnrh, kisspeptin-10, Gnrh + Gnrh antagonist (Antide), or kisspeptin-10 + kisspeptin antagonist (kisspeptin-234) into the third ventricle of the brain. The anterior pituitary and blood was used to mRNA and protein expression analysis. We demonstrated that Gnrh up-regulates Nr5a1 mRNA expression in the anterior pituitary and induces NR5A1 depletion in gonadotropes. Gnrh administration increased both Ctnnb1 mRNA expression and protein synthesis, and induced activation of cellular Ctnnb1 via translocation from the gonadotropes cytoplasm to nucleus. After kisspeptin-10 treatment, up-regulation of Nr0b1 mRNA and protein expression in the anterior pituitary was observed. These data indicate that Gnrh-neuron-mediated network activity alters Nr5a1 gene transcription and translation in gonadotrope cells and this effect may result from the changes induced in the Ctnnb1 and Nr0b1 gene/protein expression balance.

Leptin and long form of leptin receptor genes expression in the hypothalamus and pituitary during the luteal phase and early pregnancy in pigs.

Leptin is a polypeptide that plays a key role in the regulation of energy homeostasis and is also linked, among others, to mechanisms controlling reproductive processes. Data concerning the involvement of leptin in controlling reproductive functions at the level of hypothalamus and pituitary in the pig are limited. Therefore, in the present study, an expression of genes coding for leptin and long-form leptin receptor (Ob-Rb) was determined by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in the discrete areas of porcine hypothalamus (medial basal hypothalamus - MBH, preoptic area - POA, stalk median eminence - SME) and pituitary (anterior - AP and posterior/neural - NP parts) during the luteal phase of the cycle (days 10-12 and 14-16) and two early stages of pregnancy (days 14-16 and 30-32). Leptin gene expression in MBH was found to be higher in the mid- than in the late-luteal phase, whereas in other structures studied it remained unchanged during these periods. More pronounced differences were noted in expression of Ob-Rb gene, which was increased in MBH, AP and NP during the late-luteal phase in comparison to the mid-luteal one, whilst the relationship in the POA was reversed. In turn, during pregnancy, leptin gene expression in all tested areas of hypothalamus as well as Ob-Rb mRNA content in MBH were higher on days 30-32 than on days 14-16. In contrast, in the anterior pituitary, Ob-Rb gene expression was more pronounced on days 14-16 than during later stage of pregnancy. Comparison of leptin and Ob-Rb mRNA content in studied structures between the mid-luteal phase and days 14-16 of pregnancy revealed inhibition of leptin gene expression in almost all examined tissues (MBH, POA, SME, NP) during early pregnancy whereas Ob-Rb gene expression was inhibited in POA but stimulated in both parts of the pituitary during this stage. In summary, obtained results suggest an involvement of leptin in the regulation of hypothalamic-pituitary axis activity during both the luteal phase of the cycle and early pregnancy in pigs.

Pituitary galaninergic system activity in female rats: the regulatory role of gonadal steroids.

The well-recognized sensitivity of the galanin gene in the anterior pituitary gland to estrogen suggests that estrogen receptor activity may influence the galaninergic system through modulation of galanin receptor (GALR) gene expression. Here, we evaluated the following: (i) the effects of estrogen on GALR mRNA expression; (ii) the estrogen receptor subtype that is specifically involved in this activity; and (iii) the effects of progesterone in the absence or presence of estrogen on galanin concentration in anterior pituitary gland. In the first experiment, ovariectomized 4-month-old rats were pre-treated subcutaneously with 17ß-estradiol (3 x 20 µg), the ESR1 (ERα) agonist propyl pyrazole triol (PPT) (3 x 5 mg), and the ESR2 (ERß) agonist diarylpropionitrile (DPN) (3 x 0.5 mg). In the second experiment, 4-month-old ovariectomized females received daily subcutaneous injections of 17ß-estradiol (3 x 20 µg), progesterone (2 x 5 mg), or combined estradiol (3 x 20 µg) and progesterone (2 x 5 mg). Anterior pituitaries were excised the day after the final 17ß-estradiol injection (experiment I) and 1 hour after receiving the second progesterone dose. Relative GALR1, GALR2, and GALR3 mRNA expression was evaluated using quantitative real-time PCR, and pituitary galanin concentration was determined using a specific radioimmunoassay. The results revealed that estrogen predominantly induced a 5-fold increase in GALR3 gene transcription. To a lesser extent, 17ß-estradiol also increased GALR1 mRNA expression, but had no effect on GALR2 mRNA levels. The estrogen-induced increase in GALR3 gene expression occurred exclusively through ESR1 activation. The increase in GALR1 gene expression occurred through activation of both estrogen receptor subtypes, but the ESR2 subtype was predominantly involved. Furthermore, the results revealed that progesterone regulates the activity of the pituitary galaninergic system by facilitating estradiol-induced galanin synthesis in the female rat anterior pituitary gland.

MAOA gene hypomethylation in panic disorder-reversibility of an epigenetic risk pattern by psychotherapy.

Epigenetic signatures such as methylation of the monoamine oxidase A (MAOA) gene have been found to be altered in panic disorder (PD). Hypothesizing temporal plasticity of epigenetic processes as a mechanism of successful fear extinction, the present psychotherapy-epigenetic study for we believe the first time investigated MAOA methylation changes during the course of exposure-based cognitive behavioral therapy (CBT) in PD. MAOA methylation was compared between N=28 female Caucasian PD patients (discovery sample) and N=28 age- and sex-matched healthy controls via direct sequencing of sodium bisulfite-treated DNA extracted from blood cells. MAOA methylation was furthermore analyzed at baseline (T0) and after a 6-week CBT (T1) in the discovery sample parallelized by a waiting time in healthy controls, as well as in an independent sample of female PD patients (N=20). Patients exhibited lower MAOA methylation than healthy controls (P<0.001), and baseline PD severity correlated negatively with MAOA methylation (P=0.01). In the discovery sample, MAOA methylation increased up to the level of healthy controls along with CBT response (number of panic attacks; T0-T1: +3.37±2.17%), while non-responders further decreased in methylation (-2.00±1.28%; P=0.001). In the replication sample, increases in MAOA methylation correlated with agoraphobic symptom reduction after CBT (P=0.02-0.03). The present results support previous evidence for MAOA hypomethylation as a PD risk marker and suggest reversibility of MAOA hypomethylation as a potential epigenetic correlate of response to CBT. The emerging notion of epigenetic signatures as a mechanism of action of psychotherapeutic interventions may promote epigenetic patterns as biomarkers of lasting extinction effects.

Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or beta-endorphin in female rats.

The effects of gonadotropin-releasing hormone (GnRH), beta-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of alpha mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHbeta mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased alpha and LHbeta mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of beta-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous beta-endorphin was unable to exert its negative action on beta-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of beta-endorphin or other substances on the functions of the gonadotrophs.

Stimulation of luteinizing hormone subunit gene expression by pulsatile intracerebroventricular microinjection of galanin in female rats.

Although galanin, which exerts its effects both at the hypothalamic and pituitary level, has been implicated as an important neuroendocrine regulator of hypothalamic-pituitary-gonadal axis activity, there is a lack of data concerning its involvement in the regulation of gonadotropin subunit gene expression. To elucidate whether galanin can influence luteinizing hormone (LH) subunit mRNA content, as well as affect gonadotropin-releasing hormone (GnRH) receptor activity, a model based on pulsatile (one pulse per hour over 5 h) galanin (1 nM) microinjections directly into the third cerebral ventricle of ovariectomized (OVX) and/or oestrogen/progesterone-pretreated rats was used. Furthermore, to determine galanin effects on GnRH-induced LH subunit mRNA synthesis, a cocktail of 1 nM GnRH and 1 nM galanin was coadministered in a pulsatile manner to OVX/steroid primed rats. Subsequently, to obtain data concerning the role of galanin receptors in the regulation of pituitary alpha (common to LH, follicle-stimulating hormone, thyroid-stimulating hormone) and LHbeta subunit gene expression, OVX/oestrogen/progesterone rats received microinjections of 1 nM of the receptor antagonist galantide and 1 nM of galanin. In this case, both substances were administered separately, with a 30 min lag, according to which each galantide pulse always preceded a galanin pulse. Northern-blot analysis revealed that intracerebroventricular pulsatile galanin injections were effective in stimulation of both alpha and LHbeta subunit mRNA levels and that this effect was apparently steroid-dependent. Moreover, galanin also up-regulated GnRH receptor functional parameters (affinity and maximum binding capacity) but was ineffective in potentiating GnRH-induced accumulation of both subunit mRNAs. The results from the study also indicate that galanin acts through its own receptor(s) because a receptor antagonist, galantide, significantly reduced the stimulatory effect exerted by galanin on the expression of both LH subunit genes in vivo.

Increased LH and FSH release from the anterior pituitary of ovariectomized rat, in vivo, by copper-, nickel-, and zinc-LHRH complexes.

The effect of Cu2+, Ni2+, Zn2+ and their complexes with LHRH on the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) was estimated in in vivo experiments with the use of the method proposed by Ramirez and McCann. Ovariectomized, estradiol, and progesterone pretreated rats were injected intravenously either with LHRH alone, a metal ion alone, a mixture of metal and hormone, or a metal-LHRH complex. A metal alone or a mixture of it with LHRH did not affect gonadotropin release at all or no more than LHRH alone. However, the complex of Cu2+ with LHRH brought about a high release of LH and even higher release of FSH. This indicates that copper complex is more effective than metal-free LHRH. The nickel complex showed a similar although lesser effect. The zinc complex had similar potency to free LHRH though higher FSH-releasing ability was noticed. We conclude that copper-, nickel-, and zinc-LHRH complexes were more potent than the peptide hormone itself and promoted the FSH release in the ovariectomized, estradiol, and progesterone pretreated rats.

Binding of Cu2+, Zn2+, and Ni(2+)-GnRH complexes with the rat pituitary receptor.

Complex of copper with the gonadotropin-releasing hormone, GnRH, competed more efficiently for the GnRH receptor than native GVRH, while complexes of nickel with GnRH and zinc with GnRH had slightly lower affinity. Copper ion added to the incubation mixture inhibited the buserelin binding to the receptor.

In vivo effect of growth hormone on DNA synthesis and expression of milk protein genes in the rabbit mammary gland.

The aim of this work was to show whether growth hormone (GH) is able to directly induce growth and functional differentiation of the mammary gland. We have shown that i.m. injections of prolactin and to lesser extent injections of growth hormone increased DNA synthesis in the mammary gland of pregnant rabbits. Injections of pituitary and recombinant bovine growth hormone (GH), similarly to prolactin, could also induce the expression of milk protein genes--caseins alpha S1 and beta and whey acidic protein (WAP). However, in contrast to prolactin, growth hormone failed to induce the synthesis of casein proteins. Lactogenic hormones act through binding to receptors in target tissues. Prolactin receptors were shown to be abundant in the rabbit mammary glands but no specific binding sites for 125I-labelled GH have been found in membranes isolated from mammary glands of pregnant or lactating rabbits. The specificity of hormone binding was examined using unlabelled hormones as competitive inhibitors of 125I-labelled prolactin. Bovine and recombinant bovine growth hormone did not displace prolactin from its receptors, thus excluding the possibility of action of GH through lactogenic receptors. Our results support the hypothesis that GH may act directly on the mammary gland and independently from prolactin; however, the mechanism of its action is still unknown.

Biosynthesis of gonadotropins in vivo.

GnRH is potent stimulator of gonadotropin's alpha and beta chains synthesis in vivo. Stimulation of LH beta gene transcription requires pulsatile GnRH administration but the transcription of alpha subunit can be stimulated independently of GnRH mode of administration. Castration increases whereas in vivo estradiol and testosterone replacement decreases the rate of gene transcription of pituitary gonadotropin subunits. Thyroid hormones can enhance or diminish the pituitary levels of LH beta and FSH beta subunit mRNAs in female rats. Inhibin, activin and follistatin were shown to be potent regulators of FSH beta gene expression.

Daily GnRH and LH secretion in ewes is not modified by exogenous melatonin during seasonal anestrus.

The effect of central, short-term melatonin administration on daily GnRH and LH secretion was studied in ewes during seasonal anestrus. Melatonin, in a total dose of 32 micrograms and the vehicle were perfused for 4 hours into the mediobasal hypothalamus/median eminence (MBH/ME). The mean GnRH concentration during perfusion with melatonin decreased significantly (P < 0.05), as compared to the concentration during the preceding perfusion with the vehicle only. This change resulted from high variations in GnRH concentration noted during the initial phase of perfusion rather than from an action of melatonin. Melatonin perfused into the MBH/ME did not significantly affect LH secretion. A higher dose of melatonin and vehicle were also infused intracerebroventricularly (icv.) in either intact (300 micrograms for 3 hours) or ovariectomized (OVX) ewes (400 micrograms for 4 hours, 100 micrograms/100 microliters/h). In the intact animals, melatonin did not significantly affect LH secretion. Interestingly, melatonin significantly decreased (P < 0.05) the number of LH peaks in OVX ewes. These results demonstrate that melatonin delivered for a few hours directly into the central nervous system did not affect either daily hypothalamic GnRH release or pituitary LH secretion in intact ewes during seasonal anestrus, but did modify pulsatile LH secretion in ewes deprived of the negative feedback of estradiol.