Transcriptomic analysis of the porcine anterior pituitary gland during the peri-implantation period.

The peri-implantation period is controlled by signals originating from hypothalamic-pituitary-ovarian axis, uterus and developing embryos. The transcriptomic activity of the anterior pituitary gland may be important for the control of the peri-implantation period. The aim of this study was to determine the alternations in the transcriptomic profile of porcine anterior pituitary gland during the peri-implantation period (days 15-16 of pregnancy) in comparison with established for the respective days of the oestrous cycle. Analysis using a microarray approach indicated that the 651 genes (fold-change ˂1.2; p ≤ .05) were differentially expressed (DEGs) in the anterior pituitary of pigs during the peri-implantation period when compared to cyclic females. Of these DEGs, 404 were upregulated and 247 downregulated. Analysis of occurred relationships among DEGs revealed that some of them are involved in steroid-response and oestrogen synthesis, FSH secretion, immune response, PPAR signalling pathway and the potential for DNA methylation. In conclusion, the altered transcriptomic profile of the porcine pituitary gland in pigs during the peri-implantation period indicates the role of embryos presence in the creation of transcriptomic activity of the pituitary gland in pigs.

Effects of intracerebroventricular infusions of ghrelin on secretion of follicle-stimulating hormone in peripubertal female sheep.

Reproduction depends on mechanisms responsible for the regulation of energy homeostasis and puberty is a developmental period when reproductive and somatic maturity are achieved. Ghrelin affects the activity of the hypothalamo-pituitary-gonadal axis under conditions of energy insufficiency. An in vivo model based on intracerebroventricular (i.c.v.) infusions was used to determine whether centrally administered acyl ghrelin affects transcriptional and translational activity of FSH in peripubertal lambs and whether ghrelin administration mimics the effects of short-term fasting. Standard-fed lambs received either Ringer-Lock (R-L) solution (120µL h-1) or ghrelin (120µL h-1, 100µg day-1). Animals experiencing a short-term (72h) fast were treated only with R-L solution. In each experimental group, i.c.v. infusions occurred for 3 consecutive days. Immunohistochemistry, in situ hybridisation and real-time reverse transcription quantitative polymerase chain reaction analyses revealed that short-term fasting, as well as exogenous acyl ghrelin administration to standard-fed peripubertal lambs, augmented FSHß mRNA expression and immunoreactive FSH accumulation. In addition to the effects of ghrelin on FSH synthesis in standard-fed animals, effects on gonadotrophin release were also observed. Acyl ghrelin increased the pulse amplitude for gonadotrophin release, which resulted in an elevation in mean serum FSH concentrations. In conclusion, the present data suggest that ghrelin participates in an endocrine network that modulates gonadotrophic activity in peripubertal female sheep.

Effects of Restraint Stress on Circulating Corticosterone and Met Enkephalin in Chickens: Induction of Shifts in Insulin Secretion and Carbohydrate Metabolism.

This study examined the effects of acute restraint stress in the presence or absence of naltrexone on the circulating concentrations of insulin, glucose, Met-enkephalin and corticosterone in 14-week-old chickens [design: 2 sex × 2 stress/non-stress × 2 +/- naltrexone]. In chickens (five male and five females per treatment) subjected to restraint for 30 min, there were increases in the plasma concentrations of corticosterone and Met-enkephalin. The plasma concentrations of insulin and glucose were also increased in the chickens during restraint. Moreover, there were increases in the plasma concentrations of insulin and glucose in the chickens. The patterns of expression of the proenkephalin gene (PENK) in both the anterior pituitary gland and the adrenal gland were very similar to that of plasma Met-enkephalin. There were relationships between the plasma concentrations of corticosterone, Met-enkephalin, insulin and glucose after 30 min of restraint. The effects of naltrexone treatment on both untreated and stressed chickens were also examined, with naltrexone attenuating the stress-induced increases in the plasma concentrations of corticosterone, Met-enkephalin and glucose but not in those of insulin. The present study demonstrates that stress increases insulin secretion in chickens but also induces insulin resistance.

The effect of valproate (VPA) treatment on inositol phosphates (IPs) accumulation in non-stimulated and GnRH-treated female rat anterior pituitary cells in vitro.

OBJECTIVE: Mechanism(s) responsible for VPA-induced effects on reproductive axis activity are not fully recognized. Previously we reported that VPA suppressed only GnRH-stimulated but not the basal LH release from rat anterior pituitary (AP) cells in vitro. Since the inhibitory effect of VPA was exerted only in GnRH-activated cells, potential VPA impact on GnRH-R-coupled IP3/PKC signaling could not be excluded. In this study the effect of VPA on IPs synthesis in non-stimulated and GnRH-treated rat AP cells was examined. MATERIAL AND METHODS: In the first experiment 5 × 105 cells/ml were incubated for 3h with VPA (10 nM-10 µM), PMA (100 nM), GnRH (100 nM), PMA (100 nM) + VPA (10 nM-10 µM), GnRH (100 nM) + VPA (10 nM-10 µM). In the second experiment cells were preincubated for 24h with 1µCi myo-[23 H]-inositol, then for 30 min with 10 mM LiCl and finally for 3hr with GnRH (100 nM) VPA (1 µM, 10 µM), GnRH (100 nM) + VPA (1 µM, 10 µM). LH concentration was measured by RIA and intracellular IPs accumulation by ion-exchange chromatography analysis. RESULTS: VPA diminished GnRH-stimulated LH release without affecting PMA-induced LH release at any dose tested. Moreover, VPA-induced increase of IPs accumulation occurred in both non-stimulated and GnRH-treated cells and intensity of cellular response was similar in both groups. CONCLUSION: VPA affects IP3/PKC pathway activity through its up-regulatory effect on IPs synthesis in AP cells. VPA-induced inhibition of GnRH-stimulated LH release from gonadotrope cells appears to be the result of still unrecognized cellular mechanism.

Valproate inhibits GnRH-induced gonadotropin release from anterior pituitary cells of male rat in vitro.

OBJECTIVE: Valproate (VPA) a potent antiepileptic drug has been claimed to induce reproductive disturbances in men. Long-term VPA treatment can affect sperm morphology and induce testicular atrophy in non-epileptic rats. It has been reported that VPA reduced testosterone secretion stimulated by hCG in isolated rat Leydig cells. These results suggest direct effect of VPA on testes in rats. However centrally mediated effects at hypothalamo-pituitary level can therefore not be excluded. This study focused on the dose and time-dependent effects of VPA on basal and GnRH-induced LH and FSH release from the primary anterior pituitary cells culture of male rats. MATERIAL AND METHODS: The dose-dependent effect of 10 nM-100 mM of VPA on basal LH release from anterior pituitary cells after 3h of incubation was examined. To determine the time-dependent effects on LH, FSH, TSH and PRL release short (3 h) and long-term (24 h) incubations in the presence of 10 nM, 100 nM and 1 µM of VPA were maintained.To assess whether VPA can affect GnRH-induced LH and FSH release, cells were incubated for 3 h with 10 nM, 100 nM and 1 µM of VPA in the presence of GnRH. The concentration of rLH, rFSH, rPRL and rTSH in incubation medium was determined by RIA method. RESULTS: VPA did not affect the basal LH, FSH, PRL and TSH release from the primary anterior pituitary cells culture of male rats. VPA in concentration 1µM significantly suppressed GnRH-induced LH secretion. However VPA at all tested doses diminished GnRH-induced FSH release. CONCLUSIONS: VPA may diminish gonadotropin release in vitro but this effect can only be achieved after GnRH-dependent specific receptor activation. Both gonadotropins differ in their pattern of response for increasing doses of VPA.

Exogenous orexin-A downregulates luteinizing hormone secretory activity in prepubertal female rats.

INTRODUCTION: Orexin-A is a neuropeptide synthesized in the lateral hypothalamus. Orexin-A immunoreactive fibres overlap distribution with GnRH neurons. In adult rats, orexin A is known to affect LH secretion via GnRH release modulation. Because data concerning the impact of orexin-A on the hypothalamo-pituitary axis activity are limited, we focused on the involvement of orexin-A and receptors of NPY in the modulation of LH release and LH subunit b (Lhb) mRNA expression in prepubertal female rats. MATERIAL AND METHODS: Forty immature female Wistar rats were divided into 4 groups and received 2 intracerebroventricular (icv) microinjections of: 1 - artificial cerebrospinal fluid (CSF) (controls); 2 - CSF followed by orexin A; 3 - selective NPY receptor antagonist (BIBP) followed by CSF; 4 - BIBP followed by orexin A. One hour after the last microinjection, all rats were decapitated. Trunk blood was collected, and serum was stored at -20°C for the LH RIA examination. The adenohypophysis was immediately excised, flash-frozen, and kept at -80°C for RNA extraction. Real-time PCR amplification was carried out, and relative Lhb gene expression was calculated. RESULTS: In comparison to the CSF-treated controls with a mean LH serum concentration of 0.40 ± 0.02 ng/mL, the mean LH serum level was diminished both after orexin-A (0.27 ± 0.01 ng/mL) and after BIBP (0.30 ± 0.02 ng/mL) icv microinjections. In the presence of BIBP, orexin-A more effectively inhibited LH release (0.20 ± 0.01 ng/mL) when compared to the BIBP-treated group. Orexin-A and BIBP exerted a consistent inhibitory effect on Lhb mRNA expression levels in the anterior pituitary gland. In comparison to the CSF-treated controls, orexin-A, and BIBP-treated females responded with, respectively, 35% and 40% reduction of Lhb mRNA expression. Orexin-A and BIBP co-administration evoked a further reduction of Lhb gene transcriptional activity. CONCLUSIONS: Orexin-A exerts a down-regulatory effect on LH synthesis and release in immature female rats. Considering that Y1R-oriented down-regulation of endogenous NPY activity did not reverse the suppressive effect of exogenous orexin-A, it might be suggested that NPY and orexin A systems can operate independently to affect gonadotropin activity in the anterior pituitary of the immature female rats.

Gonadotropin-releasing hormone-Cu complex (Cu-GnRH) transcriptional activity in vivo in the female rat anterior pituitary gland.

Unlike gonadotropin-releasing hormone (GnRH) analogues characterized by amino acid replacement in decapeptide primary structure, Cu-GnRH molecule preserves the native sequence but contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of His2. Cu-GnRH can operate via cAMP/PKA signalling in anterior pituitary cells, suggesting that it may affect selected gonadotropic network gene transcription in vivo. We analysed pituitary mRNA expression of Egr-1, Nr5a1, and Lhb based on their role in luteinizing hormone (LH) synthesis; and Nos1, Adcyap1, and Prkaca due to their dependence on cAMP/PKA activity. In two independent experiments, ovariectomized rats received intracerebroventricular pulsatile (one pulse/h or two pulses/h over 5 h) microinjections of 2 nM Cu-GnRH; 2 nM antide (GnRH antagonist) + 2 nM Cu-GnRH; 100 nM PACAP6-38 (PACAP receptor antagonist) + 2 nM Cu-GnRH. Relative expression of selected mRNAs was determined by qRT-PCR. LH serum concentration was examined according to RIA. All examined genes responded to Cu-GnRH stimulation with increased transcriptional activity in a manner dependent on pulse frequency pattern. Increased expression of Nr5a1, Lhb, Nos1, Adcyap1, and Prkaca mRNA was observed solely in rats receiving the complex with frequency of two pulses/h over 5 h. Egr-1 transcription was up-regulated for both applied Cu-GnRH pulsatile patterns. The stimulatory effect of Cu-GnRH on gene transcription was dependent on both GnRH receptor and PAC-1 activation. In conclusion, obtained results indicate that Cu-GnRH complex is a GnRH analogue able to induce both IP3/PKC and cAMP/PKA-dependent gonadotrope network gene transcription in vivo.

Effect of kisspeptin and RFamide-related peptide-3 on the synthesis and secretion of LH by pituitary cells of pigs during the estrous cycle.

Actions of kisspeptins (KISSs) and RFamide-related peptide-3 (RFRP-3) at the hypothalamus modulate female reproduction. The action of KISS and RFRP-3 in the pituitary gland of pigs during the estrous cycle has not yet been delineated. The present study was conducted to assess the effect of KISS and RFRP-3 on relative abundance of αGSU and ßLH mRNA transcript and LH secretion in vitro by pituitary cells of gilts during the estrous cycle. The cells were isolated from gilts on Days 2-3, 10-12, 15-16 and 19-20 of the estrous cycle and cultured in vitro without inclusion of GnRH (control) or with GnRH (100 ng/ml), KISS (10-6 M, 10-7 M) and RFRP-3 (10-6 M, 10-7 M) alone or in combination. The relative abundance of α-GSU and ß-LH mRNAs was examined. Treatment with KISS increased the synthesis and/or secretion of LH by pituitary cells and RFRP-3 inhibited the synthesis and secretion of LH in the presence of GnRH on Days 10-12 and 15-16 of the estrous cycle. The synthesis and secretion of LH was greater when there was treatment with KISS and GnRH during the late follicular phase. Treatments with KISS and RFRP-3 affected the synthesis and/or secretion of LH during the luteal phase and luteolysis. In conclusion, KISS and RFRP-3 apparently affects the synthesis and secretion of LH by pituitary cells of estrous cyclic pigs. There appears to be a greater effect of KISS in modulation of LH secretion than RFRP-3 in pigs.

Differential binding of mammalian and salmon GnRHs with rat and carp pituitary receptors.

OBJECTIVES: Receptor binding of GnRH is connected with the stimulation of pituitary gonadotropic cells leading to both the release and biosynthesis of gonadotropins. The binding is connected with the conformational changes in the receptor which induce the specific intracellular signalisation. The study of fish GnRHs and their receptors may give us new knowledge of the complex interplay of different mechanisms involved in neuroendocrine regulation of reproduction. METHODS: Receptor binding of both mGnRH and sGnRH were compared by the study utilizing the displacement method with mGnRH or sGnRH as radioactive tracers. Incubation was performed at 2 degrees C to avoid ligand degradation. RESULTS: The comparative binding of mGnRH and sGnRH with GnRH receptors from the female rat pituitary and female carp pituitary was studied. At the 50% of displacement, the binding of sGnRH to the rat pituitary receptor was very small and in comparison to the binding of mGnRH (100%) was in the range 2-15%. However, the binding of mGnRH to carp pituitary receptors is small in comparison with the binding of sGnRH (100%) and was in the range 5-20%. CONCLUSION: The results demonstrated the differences in binding of different GnRHs to the receptor in rats and carp. This suggests that the structures of GnRH and its receptor undergo co-evolution in different classes of animals.

Effect of the polymorphism in 5' UTR region of pig prolactin gene on prolactin gene expression and reproduction performance in the female pig.

OBJECTIVES: The recent genetics and molecular biology progress seems to be a fascinating challenge for the interdisciplinary studies on the effects of genetic changes in gene structure that causes the modification of physiological functions of many important proteins including hormones. Pig prolactin is one of the interesting hormones for this study. AIM OF THE STUDY: The aim of the study was to analyze the mutation in 5'UTR region of the pig prolactin (PRL) gene and to evaluate the effect of this polymorphism on changes in plasma prolactin concentration. RESULTS: It was found that only two individual groups of animals differed by the genotype in examined PRL gene locus - homozygote C/C and heterozygote C/T. PRL plasma concentration was 38.4 ng/ml (for C/T animals) or 42.7 ng/ml (for C/C animals). Animals with C/C genotyped exhibited a tendency to elevate PRL concentration as compared to the C/T group (p< 0.07). CONCLUSIONS: This research combines the genetic, molecular and, in vivo, physiological study which allows focus on the possible relationship between the gene polymorphism and physiological status of animal.

Development of real-time PCR assays in the study of gonadotropin subunits, follistatin and prolactin genes expression in the porcine anterior pituitary during the preovulatory period.

OBJECTIVES: Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells. MATERIAL AND METHODS: Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture. RESULTS: The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control. CONCLUSIONS: Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes.

Single base substitution in growth hormone receptor gene influences the receptor density in bovine liver.

OBJECTIVES: Nucleotide sequence polymorphisms in the coding gene regions may influence the biological properties of proteins encoded by a gene. The A/T substitution in exon 8 of the growth hormone receptor (GHR) gene results in changed amino acid sequence 279 (Phe/Tyr) in the transmembrane domain of the receptor protein and therefore could influence its functional parameters. We searched for the relationship between the A/T nucleotide polymorphism in the GHR gene the receptor binding capacity and dissociation constant. METHODS: Nucleotide sequence variations in the exon 8 (coding for the transmembrane domain of the receptor) of the bovine GHR gene and in fragments of adjacent introns were analysed using PCR-SSCP and sequencing techniques. GH receptor binding capacity (Bmax) and dissociation constant (Kd) for GHR were determined by the Scatchard analysis in livers of ten bulls carrying the AA or AT GHR genotypes. RESULTS: Two single nucleotide polymorphisms (SNPs) were identified--the C/T transition in intron 8 at position 863+32 and the A/T transversion in exon 8 at position 836, the latter resulting in Phe/Tyr amino acid substitution in the receptor protein. The results showed significant differences in the GHR binding capacity between these genotypes. Bmax was significantly greater (p< or =0.01) in bulls carrying TT genotype of GHR in comparison to those with the AT genotype. No significant differences in the dissociation constants (Kd) were found. CONCLUSION: Our results demonstrated that single base substitution in the transmembrane domain encoding region of GH receptor gene may influence the physiological properties of the receptor.

Modifications of Western-type diet regarding protein, fat and sucrose levels as modulators of steroid metabolism and activity in liver.

The aim of the study was to evaluate whether the modification of the Western-type diet (high-fat, high-sucrose diet rich in saturated fatty acids) considering macronutrients content would influence hepatic metabolism and activity of steroids. For 3 weeks Wistar rat were fed the Western-type diet (21% fat, 35% sucrose, 19% protein, lard) and its modifications regarding dietary protein (10 and 19%), fat (5 and 21%) and sucrose (0 and 35%) levels. The steroid 5α-reductase type 1 (Srd5a1) and androgen receptor (Ar) gene expression as well as testosterone (T) conversion towards 5α-reduced derivatives in liver were positively correlated with body weight gain. The Western-type diets with decreased protein content regardless of the sucrose level exerted the most negative effect on the antioxidant system decreasing catalase (Cat), sodium dismutase (Sod1) and glutathione peroxidase (Gpx1) gene expression as well as Cat and Gpx activity and total antioxidant status, simultaneously intensifying lipid peroxidation. The impaired antioxidant system was accompanied by decreased level of hepatic T metabolism towards estrogens: 17ß-estradiol (E2) and estriol, and increased estrogen receptor type 1 (Esr1) gene expression. Liver Esr1 mRNA level was differently correlated with T (positively) and E2 (negatively) plasma levels. Whereas the fat reduction in Western-type diet restored the plasma proportion between T and E2. In conclusion it could be stated that Western-type diet modification relating to protein, sucrose and fat content can influence hepatic steroid metabolism and activity; however the estrogens and androgens metabolism in liver would be connected with impairment of liver function or catabolic activity, respectively.

Growth hormone cell phagocytosis in adenohypophysis of mosaic mice: morphological and immunocytochemical electron microscopy study.

An electron microscopy immunocytochemical study was performed to determine the expression pattern of growth hormone (GH) in mosaic mutant mice adenohypophysis. In normal condition GH was restricted to the secretory granules of all growth hormone cells. Mosaic mice adenohypophysis contained growth hormone cells which have distinctive GH labeled secretory granules at the level seen in control animals. Ultrastructurally, some GH cells of mosaic mice presented abnormalities, but labeling intensity of secretory granules in these cells was always comparable to the basal condition. The striking findings presence of two forms (simple and activated) of folliculo-stellate cells (FS) in close association trough gap or tight junction with GH cells localized especially near the perivascular space. Frequently, in cytoplasm of FS cells, large clusters containing fragments of GH labeled cell were present. Additionally, the existence of large intracellular, electron-lucent spaces, with remnant cellular material in parenchyma of mosaic mutant mice adenohypophysis could suggest intensive process of GH-cell destruction. Our electron microscopy immunocytochemical results provide evidence for loss of GH cells in mosaic mice by phagocytosis. We suppose that impaired body growth observed in mosaic mutant male rats may be, at least partially, a consequence of an alteration in somatotropic axis activity. Loss of GH cells in mosaic mice by phagocytosis supported by FS cells may contribute to this effect.

Effect of early thermal experience on pituitary-gonadal axis in female rats.

The reproductive system fully develops during postnatal stages of life, and as such it may be susceptible to environmental cues such as high temperature. Thus, the purpose of the study was to compare how exposure to 34 degrees Celsius ambient temperature affects pituitary-gonadal axis of immature and adult female rats. Three groups of females at proestrous or metaestrous (n=38) were used in the study. The females were housed in ambient temperature of 34 degrees Celsius (WR group, n=10) or room temperature (CR group, n=16) from birth to adulthood. The females from the third group were acclimated to 34 degrees Celsius as adults (WA group, n=12). In the WR group the onset of puberty was delayed in comparison to the CR group. The plasma PRL level was lower during proestrous and higher during metaestrous in the WR group compared to the CR group. During metaestrous, lower FSH and higher progesterone (P(4)) plasma concentrations were found in the WR females. No changes in LH and oestradiol (E(2)) plasma concentrations were demonstrated. Higher in vitro E(2) and lower P(4) secretions under FSH stimulation were observed in the WR follicles compared to those of the CR group. The WR group also demonstrated higher basal and LH-stimulated luteal in vitro secretion of P(4) than controls. Plasma LH and FSH concentrations during metaestrous were higher in WA females than in the WR group, but PRL level was lower. Follicles of the WA group were unresponsive to FSH with respect to steroid secretion. In addition, LH stimulated luteal E(2) secretion in this group. P(4) release by luteal cells was lower in the WA than in the WR group. We concluded that WR females differ from WA in reproductive system adjustments to rearing temperature and that early thermal experience is more effective in antagonizing the effect of high temperature than acclimation of adult females.

LH release by Cu and Ni salts and metal-GnRH complexes, in vitro.

The present studies were undertaken to examine the effect of copper and nickel salts and their complexes with GnRH on LH release from the pig anterior pituitary cells in vitro. The potency of Cu-GnRH and Ni-GnRH binding to GnRH receptors with iodinated GnRH as a radioactive tracer was also verified. The incubation of pig pituitary cells with Cu and Ni acetate salts showed no effect of the studied ions on LH release at any concentration used. However, nickel salt at a lower dose (10(-10) and 10(-9) M) tended to decrease LH output. By contrast, the native GnRH as well as its metal complexes significantly stimulated LH release after three hours of treatment and Cu-GnRH was found to be the most effective. The results showed that Cu and Ni complexes with GnRH but not their acetate salts are effective in LH release from pig pituitary cells collected from adult female pigs.

Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior pituitary cells in vitro.

The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu(2+) ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine(2). A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 µM antide, a GnRH antagonist; 0.1 µM cetrorelix, a GnRH receptor antagonist; 0.1 µM PACAP6-38, a PAC-1 receptor antagonist; 2 µM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 µM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 µM thapsigargin; 5 µM A23187, a Ca(2+) ionophore; or 10 µg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 µM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca(2+)-dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro.

Vasoactive intestinal peptide modulates luteinizing hormone subunit gene expression in the anterior pituitary in female rat.

The direct monosynaptic pathway which exists between vasoactive intestinal peptide (VIP) and GnRH neurons in the hypothalamic preoptic area provides a neuroanatomical background for the modulatory effects of VIP exerted on GnRH neurons activity. Though central microinjection of VIP revealed its involvement in the modulation of LH release pattern, there is a lack of data concerning a possible VIP influence on the alpha and LHbeta subunit gene expression in the pituitary gland. Using a model based on intracerebroventricular pulsatile peptide(s) microinjections (1 pulse/h [10 microl/5 min] over 5 h) the effect of exogenous VIP (5 nM dose) microinjection on subunits mRNA content in ovariectomized/oestrogen-pretreated rats was studied. Subsequently, to obtain data concerning the involvement of GnRH and VIP receptor(s) in the regulation of alpha and LHbeta subunit mRNA expression, OVX/estrogen-primed rats received a pulsatile microinjections of 5 nM VIP with 3 nM antide (GnRH receptor antagonist) or 5 nM VIP with 15 nM VIP 6-28 (VIP receptor antagonist). In this case, substances were given separately with a 30 min lag according to which each antagonist pulse preceded a VIP pulse. Northern-blot analysis revealed that VIP microinjection resulted in a decreased alpha and LHbeta mRNA content in pituitary gland and this effect was dependent on GnRH receptor activity. Moreover, obtained results indicated that centrally administered VIP might operate through its own receptor(s) because a receptor antagonist, VIP 6-28, blocked the inhibitory effect of VIP exerted on both LH subunit mRNA content and LH release.

Cobalt complex with GnRH stimulates the LH release and PKA signaling pathway in pig anterior pituitary cells in vitro.

Metal complexes with GnRH were shown to interact with GnRH receptors in pituitary cells. In the present study we examined the effects of GnRH and its cobalt complex form (Co-GnRH) on LH secretion and generation of second messengers, namely inositol phosphates (IPs) and cAMP, in porcine pituitary cells in vitro. The cells were obtained from gilt pituitary at the pre-ovulatory phase of estrous cycle and cultured for 72 h before challenge with GnRH or Co-GnRH. Both substances induced a significant increase in LH release that was detectable after 60 min (P<0.05) of treatment, with the Co-GnRH complex being more efficient than GnRH at 180 min (P<0.01). GnRH and Co-GnRH were equally effective at 10(-8)M (P<0.01), however, at the lowest (10(-9)M) as well as the highest (10(-7)M) concentrations tested, Co-GnRH was more potent than its native counterpart (P<0.01). Interestingly, Co-GnRH revealed twice more efficient than GnRH at stimulating cAMP production, an effect which was detectable in cells after 1h-incubation (P<0.001). In contrast, while native GnRH induced a rapid increase (P<0.05) in IPs no such effect of Co-GnRH was observed. These data demonstrate that Co-GnRH and GnRH differentially effect on the signaling pathway in porcine gonadotropes and suggest that in these cells, the releasing action of Co-GnRH results from the mediation via the cAMP/protein kinase A second messenger system.

Different signaling in pig anterior pituitary cells by GnRH and its complexes with copper and nickel.

Gonadotropin releasing hormone (GnRH) is an essential factor in the regulation of synthesis and release of pituitary gonadotropins. After binding to specific receptors and coupling with G proteins, it triggers the intracellular signaling involving the synthesis of inositol phosphates and diacylglycerol. Previously we have showed that certain metal complexes with GnRH, i.e. copper (Cu-GnRH) and nickel (Ni-GnRH) are able to bind to the GnRH receptors. The intracellular signalling of these complexes, however, has not been yet elucidated. In this experiment, the ability of the Cu-GnRH and Ni-GnRH complexes to modulate cAMP synthesis and phosphoinositols formation in the pig anterior pituitary cells in vitro was studied. The native GnRH and its metal complexes stimulated the luteinizing hormone (LH) release, but only the effect of Cu-GnRH was found to be a dose-dependent. The metal complexes did not significantly influence inositol phosphates accumulation, while their effect on cAMP synthesis was significantly more potent than that of GnRH alone. We conclude that the Cu-GnRH and Ni-GnRH complexes increase LH release in the porcine pituitary cells although their intracellular signaling is different from that of the native GnRH. It seems that metal complexes with GnRH deserve more attention in further studies.