Gonadoliberin (GnRH) and its copper complex (Cu-GnRH) enzymatic degradation in hypothalamic and pituitary tissue in vitro.

The amount of decapeptide decapeptide gonadoliberin (GnRH) that reaches pituitary gland depends not only on transcriptional, translational and posttranslatonal processes but also on the extent of degradation exerted by specific proteolytic enzymes. The copper-gonadoliberin (Cu-GnRH) complex preserves the native GnRH amino acid sequence but contains Cu(2+) ion bound to the nitrogen atom at the imidazole ring of the His(2). The aim of this study was to determine whether GnRH and Cu-GnRH molecules differ in their susceptibility to proteolysis in male rat hypothalamic and pituitary tissue in vitro. RIA was applied for a time-dependent study based on 0-90 min incubations at 30°C of exogenous peptide (2.5 µg GnRH or Cu-GnRH) in respective hypothalamic/pituitary supernatant and pellet fractions. To compare the protective effect of bacitracin, a competitive PEP inhibitor, incubations were made with (125 µg/sample) or without an inhibitor. In the second experiment 100 µg of GnRH or Cu-GnRH were incubated for 5 h at 37°C in hypothalamic and pituitary tissue in vitro and then HPLC analysis was applied both to characterize the elution pattern of GnRH and Cu-GnRH degradation products as well as to determine their AA composition. In both tissues, Cu-GnRH remained more resistant to enzymatic degradation and fully protected in the presence of bacitracin. In conclusion, the obtained data suggest that copper ion changed GnRH conformation and significantly modified its physiological properties due to a hindered endopeptidases access to specific AA bonds. Therefore, the Cu-GnRH complex might be considered as GnRH analog potentially able to prolong the occupation of a GnRH receptor at the gonadotrope cells.

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.

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.

Prolactin binding analysis and immunohistochemical localization of prolactin receptor in porcine ovarian cells.

In the present study we searched for prolactin receptor (PRL-R) in porcine ovarian theca tissue (Tc) of small, medium and large follicles, as well as in early corpus luteum (ECL). The objectives of this investigation were: 1) comparison of the direct effect of PRL action on progesterone (P4) and estradiol (E2) secretion from Tc and ECL cells in culture with adequate effects caused by luteinizing hormone (LH). 2) detection of the presence and distribution of PRL-R in thecal tissue of porcine follicles and in ECL. Tissues were cultured as monolayers either in control M199 medium with calf serum or in medium either with PRL (100 ng/ml) or with LH (100 ng/ml). After 2 days in vitro cultured media were assayed for steroid concentrations by radioimmunoassays. Content and distribution of PRL-R were evaluated by Scatchard analysis and by an immunohistochemical assay. Separated theca layers as well as fragments of ECL were excised on dry ice, homogenized, and incubated with [125I]-PRL. PRL stimulated P4 secretion from Tc 10-fold versus controls. LH stimulated P4 secretion only 2.5-fold. E2 secretion was stimulated by PRL 2.7-fold and by LH 2.4-fold. LH enhanced P4 secretion from ECL cells by 18% while PRL increased P4 secretion by as much as 73%. Femtomol amounts of PRL-R protein were detected in theca tissues of medium and large follicles and also in ECL, which was in accordance with immunohistochemical results. The results showed for the first time the presence of PRL-R in porcine Tc and ECL.

GnRH pulsality and the differential activation of the rat luteinizing hormone subunit genes in the anterior pituitary gland.

Detailed studies have been focused on the mechanisms by which the rat alpha and LHbeta genes are differentially regulated by GnRH and indicate that differential sensitivity to the second messenger exists in a physiological context. Differential signaling from the GnRH receptor may be a mechanism for preferential regulation of luteinizing hormone subunit gene transcription; however which of these genes are specifically regulated by PKC or calcium and how GnRH pulsatility could preferentially activate individual pathways of second messengers within gonadotrope cells remain unclear. Several transcription factors that have profound effects on basal and/or GnRH-stimulated LHbeta gene promoter activity have been identified: SF-1, Egr-1, Sp-1. A model explaining possible interactions among them in mediating GnRH responsiveness of the LHbeta gene has been proposed: Sp1, SF-1 and Egr-1 form a tripartite GnRH response element which is sensitive to the spacing changes between the upstream Sp1 binding sites and the downstream SF-1/Egr-1 binding elements and SF-1 plays a critical role in integrating the effects of Sp1 and Egr-1. GnRH responsive element located on LHbeta gene promoter in position between -495 to -342 has been identified. At 3'-end of the promoter three Sp-1 binding sites have been identified: position -416, sequence: GGGGGCTGGG and two sites almost completely overlapping, position -403, sequence; GGGGCGGCGCCCA while at the 5'region of the promoter one Sp-1 binding site exists: position -450, sequence: ACCACACCCATTTTTGG. The 5'Sp1 site overlaps a CArG box (at -443 to -434, sequence: CCATTTTTGG) which seems to be essential in LHbeta gene sensitivity for pulsatile GnRH stimulation.

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.

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.

Ovarian LH/hCG receptors and plasma level of LH,17-beta estradiol and progesterone in gonadotropin--induced PCO syndrome in rats.

The aim of this work was to investigate the morphologic changes, LH/hCG receptor content in the ovaries and plasma levels of LH, progesterone and estradiol of hypo--and hyperthyroid rats injected with PMSG and hCG. The hypothyroid state was induced by thyreoidectomy (Tr-X) and the hyperthyroid condition by injections of 40 micrograms L-thyroxine daily during 21 days (T4). Gonadotropins were injected during 14 days in daily doses: PMSG--5 i.u. and hCG--10 i.u. The following 8 groups (n = 10-20) were established: control (euthyroid, no treatment), Tr-X, PMSG + hCG, Tr-X + hCG, Tr-X + PMSG, Tr-X + PMSG + hCG, T4 and T4 + PMSG. At the end of experiments rats were sacrificed, ovaries weighed, macroscopically inspected and concentration of LH/hCG receptors was estimated. In blood plasma the level of LH, progesterone and 17-beta estradiol was also analysed. The experiments showed that injections of PMSG alone, or PMSG + hCG in eu-or hypothyroid rats, appear the most effective in induction of PCO syndrome in rats. Low levels of thyroid hormones sensitized the ovaries to gonadotropin action, but a hyperthyroid status diminished or inhibited this response. Thyroid function is also essential in production of LH/hCG receptors in the ovaries. In hypothyroid animals the amount of these receptors was greatly increased, while in hyperthyroid animals they decreased. The level of plasma LH, progesterone, and estradiol showed insignificantly differences and various inconsiderable deviations from norm. These differences were not dependent on large doses of gonadotropins, altered thyroid function, or on cystic or luteinizing changes in the ovary.

Effects of age and sex on basal and dopamine-inhibited in vitro prolactin release, and dopamine receptor binding, by the rat adenohypophysis.

We measured basal and dopamine-inhibited pituitary cell prolactin (PRL) release in vitro, and dopamine receptor binding in pituitary homogenates, from intact male and female Wistar rats of varying ages. During 48-72 hours in culture, the baseline secretion rate of PRL from pituitary cells of old (24 months) male rats was less than one-half that from cells of mature (6 months) male rats, whereas the corresponding basal secretion rate of PRL from cells of old female rats was nearly 3-fold greater than that from cells of mature female rats (p < 0.001). After in vitro exposure to various concentrations of dopamine (10(-10)M to 10(-6)M), PRL secretion decreased from pituitary cells of both mature and old rats (p < 0.001). However, repeated measures analysis of variance revealed an age-related dose-dependent decrease in the magnitude of dopamine-inhibited PRL release from cells of both male and female rats (p < 0.001). Dopamine receptor number did not differ with age (3-25 months), but was 2 to 3-fold greater in female than in male rats (p < 0.01). Receptor affinity was decreased with age only in female rats, and was greater in female than in male rats (p < 0.05). These data suggest that the decrease in dopamine-inhibited PRL release from pituitary cells of old male and female rats is not due to altered pituitary dopamine receptor binding, despite certain sex differences.

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.

Prolactin and LH release in response to vasoactive intestinal peptide (VIP) administration in spontaneously hypertensive and normotensive rats.

UNLABELLED: Vasoactive intestinal peptide (VIP) was injected intravenously at a dose of 10 micrograms in spontaneously hypertensive and normotensive Wistar-Kyoto rats. In order to evaluate the hemodynamic and hormonal effects of this peptide, the mean arterial pressure, heart rate as well as a serum rLH and rPRL levels, the contents of LH-RH in hypothalamus and the content of LH in pituitary tissue were determined. The same procedure was applied in rats receiving placebo. Serum rPRL concentration was measured additionally after combined administration of VIP+dopamine. VIP injection produced a decrease in mean arterial pressure and an increase in heart rate in both spontaneously hypertensive and normotensive rats. Serum rPRL concentration was significantly increased at 10 minutes after injection. The combined therapy (VIP+dopamine) partially inhibited this response. Serum rLH concentration, the content of LH-RH in hypothalamic tissue as well as the content of pituitary LH after VIP injection in spontaneously hypertensive and normotensive rats did not differ from the values obtained for the control group. CONCLUSIONS: 1. VIP injection produced the dramatic hypotensive effects in hypertensive rats; 2. A marked increase in PRL concentration in response to VIP was partially inhibited by dopamine in hypertensive and normotensive rats; 3. VIP injection did not change LH-RH and LH release in both hypertensive and normotensive rats.

Reduction of motor behavioural deficits in senescence via chronic prolactin or estrogen administration: time course and putative mechanisms of action.

The effects of chronic estrogen (E2), rat prolactin (rPRL), modified ovine prolactin (mPRL) administration on motor behavior (inclined screen performance) and striatal dopamine (DA) (D2subtype) receptor concentrations were examined in senescent (greater than 24 months of age) female rats, mPRL possesses no lactotrophic activity. Administration of either E2 or rPRL was effective in improving both inclined screen performance (increased time that the animal could remain on the screen by 95 and 413 s, respectively, compared to highest pre-injection performance) and striatal D2 receptor concentrations (14 and 20% respectively). These were indications, however, from separate analyses that improvements in inclined screen performance were seen prior to any increases in striatal D2 receptor concentrations. These early performance increases seemed instead to be the result of improved muscarinic receptor control over striatal DA autoreceptor function. Later improvements in inclined screen performance (at 6-7 days after the E2 injections were begun) were more dependent on increased striatal DA receptor concentrations. A second set of experiments which involved the injection of E2 into senescent male as well as female rats indicated that there were no sex differences in improvements in inclined screen performance, and that once the E2 injections were discontinued, performance returned to preadministration levels. The results are discussed in terms of two important processes that may be involved in mediating enhanced inclined screen performance following E2 administration: (1) enhancement of muscarinic receptor regulation of DA autoreceptor function; and (2) increases in striatal DA receptor density.

Effect of aging on concentrations of D2-receptor-containing neurons in the rat striatum.

Striatal D2 dopamine receptor concentrations were shown to decrease 30-35% during the lifespan of Wistar rats as assessed both radiochemically and autoradiographically. Binding densities and degree of age-change varied within the striatum; the latter ranging from 17 to 44% in 4 different regions. Overall neuronal loss during aging was 19%, and also varied considerably within the different striatal regions. Thus, it appears that neuronal loss may account for up to roughly half of the striatal D2 receptor loss during aging.

Impaired down-regulation of pituitary dopamine receptors by estradiol in aged rats.

Administration of 17 beta-estradiol to mature (6-12 months) rats results in a more than 50% reduction in pituitary dopamine receptor concentrations, without affecting binding affinity. In contrast, when the same manipulation is performed on senescent (24-25 months) rats, negligible change in receptor concentration occurs. These results suggest that age-related increases in estrogen-stimulated prolactin release are not due to decreased dopaminergic inhibition at the receptor level.

Effect of prolactin on the diurnal changes in immune parameters and plasma corticosterone in white leghorn chickens.

The effect of PRL on the diurnal changes in peripheral lymphocyte and granulocyte number, anti-sheep red blood cells, and natural anti-rabbit red blood cells serum agglutinins titre as well as plasma corticosterone concentration was examined in White Leghorn cockerels, immunized twice with sheep red blood cells. PRL was administered for six consecutive days at 4 or 8 h after light onset. Control birds were treated at the same times with hormone solvent alone. Immunized non-treated birds served as an additional control group. PRL injections influenced markedly the diurnal changes in all parameters examined. The effect of PRL administration on the diurnal changes in lymphocyte and granulocyte number and natural anti-rabbit red blood cells serum agglutinins consisted in elimination of the influence of solvent injections. In those cases where the solvent injections did not alter the pattern of the diurnal changes, i.e. in anti-sheep red blood cells serum agglutinins and in plasma corticosterone concentration, the pattern was modified by PRL injections. PRL administration affected also the correlations between the diurnal changes in plasma corticosterone concentration and those in lymphocyte number and anti-sheep red blood cells agglutinin titre. This suggests that the role of PRL in the regulation of the diurnal variations of immunity in chickens may be realized either directly, via its receptors in immune system or by its influence on plasma corticosterone concentration.

Effect of aging on striatal dopamine receptor subtype recovery following N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline blockade and relation to motor function in Wistar rats.

Recovery of D1- and D2-dopamine receptors in Wistar rat corpora striata were assessed following N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ blockade). Absolute recovery declines during senescence approximately 25 and 40% for the D1- and D2-subtypes, respectively. Net biosynthetic reductions are comparable to the overall age-related decreases in receptor concentrations for this rat strain. EEDQ administration also induces catalepsy behavior and impairs ability of animals to remain on an inclined screen. Recovery of inclined screen performance is also reduced with age, but is not strictly proportional to recovery of receptor concentrations.