LHRH mRNA Research Articles

Neural control of the synthesis and release of luteinizing hormone-releasing hormone.

Preovulatory surges of luteinizing hormone (LH) depend upon neurotransmitter activation of neurons that secrete LH-releasing hormone (LHRH, gonadotropin-releasing hormone GnRH) and noradrenaline plays a pivotal role in this critical event. The interaction is amongst noradrenaline and other neurotransmitters such as GABA (gamma-aminobutyric acid), opiates, serotonin and excitatory amino acids (N-methyl-D-aspartate, NMDA) on LHRH neuronal activity are complex. GABA and opiates suppress the presynaptic release of noradrenaline but only GABA also directly affects the responsiveness of LHRH neurons to noradrenaline. Morphine induces the release of serotonin which either directly or indirectly via other neurotransmitters (e.g. dopamine) sensitizes LHRH neurons to the stimulatory effects of noradrenaline. NMDA rapidly induces LH release but whether this drug directly affects the activity of LHRH neurons is not known. The neuronal release of LHRH is modulated by the action of oestrogen on these various neurotransmitter systems. Antioestrogens, when placed into the medial preoptic area of otherwise completely oestrogenized rats, block LH surges; LHRH mRNA levels in such animals resemble those in 9-day castrated rats. Normally, LHRH message levels increase about the time of increased noradrenaline secretion just before the LH surge. NMDA rapidly releases LH and LHRH mRNA levels are significantly raised within 15 minutes and remain so over the next 45 minutes. Thus, it seems that stimuli which evoke LHRH release also increase LHRH mRNA transcription to replenish the hormone released during the LH surge.

Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54.

Loss-of-function mutations of the gene encoding GPR54, the putative receptor for the KiSS-1-derived peptide metastin, have been recently associated with hypogonadotropic hypogonadism, in both rodents and humans. Yet the actual role of the KiSS-1/GPR54 system in the neuroendocrine control of gonadotropin secretion remains largely unexplored. To initiate such analysis, the effects of KiSS-1 peptide on LH secretion were monitored using in vivo and in vitro settings under different experimental conditions. Central intracerebroventricular administration of KiSS-1 peptide potently elicited LH secretion in vivo over a range of doses from 10 pmol to 1 nmol. The effect of centrally injected KiSS-1 appeared to be mediated via the hypothalamic LHRH. However, no effect of central administration of KiSS-1 was detected on relative LHRH mRNA levels. Likewise, systemic (i.p. and i.v.) injection of KiSS-1 markedly stimulated LH secretion. This effect was similar in terms of maximum response to that of central administration of KiSS-1 and might be partially attributed to its ability to stimulate LH secretion directly at the pituitary. Finally, the LH-releasing activity of KiSS-1 was persistently observed after blockade of endogenous excitatory amino acid and nitric oxide pathways, i.e. relevant neurotransmitters in the neuroendocrine control of LH secretion. In summary, our results provide solid evidence for a potent stimulatory effect of KiSS-1 on LH release, acting at central levels (likely the hypothalamus) and eventually at the pituitary, and further document a novel role of the KiSS-1/GPR54 system as a relevant downstream element in the neuroendocrine network governing LH secretion.

Mechanisms for altered reproductive function in female rats following neonatal administration of raloxifene.

Raloxifene is a non-steroidal selective estrogen receptor modulator (SERM) that mimics estrogenic activity on bone density and blood lipid concentration without uterotropic actions. Previous data from our laboratory indicated that, as is the case for estrogen, neonatal administration of raloxifene disturbed normal differentiation of the hypothalamic circuitries governing the gonadotropic axis. In contrast, raloxifene did not act in the same way as estrogen does on the neuronal systems controlling sexual receptivity in the female rat. At present, however, the mechanisms for these organizing effects of raloxifene are not completely elucidated. To analyze this phenomenon, female rats were injected daily with raloxifene (50, 100, 250 or 500 microg/rat per day) between days 1 and 5 of age. On day 23, hypothalamic gonadotropin-releasing hormone (LHRH) mRNA expression was assessed, and pituitary and plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured in basal and LHRH-stimulated conditions. In addition, LH and FSH responses to ovariectomy were evaluated in raloxifene-treated females. Finally, we monitored the ability of neonatal administration of a potent LHRH agonist ([d-Ala(6),d-Gly(10)]-LHRH ethylamide; 0.01 microg/kg per 12 h on days 1-5) to counteract the effects of raloxifene. Our analyses demonstrated that prepubertal rats (23-day-old females) treated neonatally with raloxifene showed decreased hypothalamic LHRH mRNA expression levels, reduced pituitary content of LH and FSH, reduced basal and LHRH-stimulated LH secretion in vivo and in vitro, and decreased response to ovariectomy. In addition, adult females treated neonatally with raloxifene showed anovulation and reduced serum LH levels; these effects were not prevented by the simultaneous administration of a LHRH agonist. In conclusion, our data demonstrate that neonatal administration of raloxifene can disrupt the programming of hypothalamic-pituitary-ovarian axis function. Reduced LH secretion, under basal and LHRH-stimulated conditions and after ovariectomy, is probably related to decreased LHRH expression, reduced pituitary LH content and/or decreased pituitary responsiveness to hypothalamic LHRH.

Decrease in the level and mRNA expression of LH-RH and EGF receptors after treatment with LH-RH antagonist cetrorelix in DU-145 prostate tumor xenografts in nude mice.

Using radioligand binding, RT-PCR, and Southern blot analyses, we evaluated whether agonist [D-Trp6]LH-RH and antagonist Cetrorelix could affect the levels of receptors for LH-RH and EGF and expression of mRNA for these receptors in DU-145 human androgen-independent prostate cancers xenografted into nude mice. Radioligand binding studies showed the presence of specific high affinity receptors for LH-RH and EGF in DU-145 prostate tumors. Cetrorelix, but not [D-Trp6]LH-RH significantly inhibited tumor growth. The concentration of LH-RH receptors was reduced by 22% (p<0. 05) and 67% (p<0.01) after 4 weeks of treatment with [D-Trp6]LH-RH and Cetrorelix respectively. The concentration of EGF receptors fell by 48% (p<0.05) in the [D-Trp6]LH-RH group, whereas Cetrorelix led to a 66% reduction (p<0.01). The expression of LH-RH and EGF receptor mRNA was investigated by RT-PCR analysis followed by Southern blotting. Densitometric analysis of the developed bands showed that the antagonist Cetrorelix decreased the expression of LH-RH receptor mRNA by 55% (p<0.01) compared to control group while the 20% reduction after treatment with the LH-RH agonist was non-significant. Treatment with [D-Trp6]LH-RH and Cetrorelix also reduced the expression of EGF receptor mRNA by 35% and 68% respectively (both, p<0.01) compared to control group. In conclusion, these data demonstrate that growth inhibition of DU-145 prostate tumors induced by prolonged administration of LH-RH antagonist Cetrorelix is accompanied by a marked decrease in the concentration of LH-RH and EGF receptors as well as in their mRNA levels.

Reversal of Ethanol-Induced Testosterone Suppression in Peripubertal Male Rats by Opiate Blockade

Teenage drinking is a major problem in the United States, as well as abroad. Besides psychosocial implications, ethanol (EtOH) has detrimental effects on the reproductive system. Clinical problems associated with reduced reproductive hormones include osteoporosis, decreased muscle function, anemia, altered immune function, prostate involution, and decreased reproductive abilities. Education coupled with strategies aimed at preventing these deleterious consequences even in the face of continued EtOH intake is extremely important. We have tested the possibility that naltrexone, a drug currently used in patients to decrease alcohol craving, might also prevent the fall in the male hormone, testosterone, caused by EtOH exposure. Rats aged 35 days old (prepubertal), 45 days old (midpubertal), and 55 days old (late pubertal) were injected (intraperitoneally) with either saline, EtOH, naltrexone, or EtOH plus naltrexone. In the two older age groups, EtOH significantly suppressed testosterone, which was prevented by administration of naltrexone. In the youngest animals, there was no treatment effect presumably due to low basal levels of testosterone. EtOH similarly reduced luteinizing hormone (LH), but this suppression was not prevented by naltrexone. There was no consistent effect of any treatment on hypothalamic concentration of pro-LH releasing hormone (RH) (LHRH), LHRH, or on steady-state levels of LHRH mRNA. We conclude that, as animals progress through puberty, EtOH suppresses LH and testosterone. The testosterone decline can be prevented by opiate blockade with naltrexone, an effect primarily seen at gonadal level. Thus, naltrexone, a drug already used clinically to reduce EtOH intake, also has protective physiological effects on the endocrine system.

Brain region-specific regulation of luteinizing hormone-releasing hormone messenger ribonucleic acid in the male ferret: interactions between pubertal maturation and testosterone.

This study examined the regulation of LHRH messenger RNA (mRNA) during pubertal maturation and by testosterone in male ferrets. Prepubertal and postpubertal ferrets were either intact or were castrated and treated with daily injections of oil or 5 mg/kg testosterone propionate for 14 days. In situ hybridization for LHRH mRNA was performed using an 35S-labeled 48-base oligonucleotide complementary to the human LHRH-coding region. Computerized image analysis was performed on cells in the preoptic area, retrochiasmatic area, arcuate nucleus (ARC), and median eminence; cells were classified as labeled if the number of pixels representing silver grains over the cell was 5 or more times the number of background silver grain pixels. Both pubertal maturation of intact males and castration of prepubertal males resulted in an increase in the number of labeled cells in the ARC. These effects were not observed in any of the other three brain regions, suggesting that ARC LHRH-producing neurons are of primary importance in the presumed increase in LHRH release that occurs as a consequence of either pubertal maturation or castration of prepubertal males. Castration of adults did not increase the number of labeled cells in any brain area, but resulted in an increase in silver grains per labeled cell only in the preoptic area. Thus, LHRH mRNA is regulated during puberty primarily in the ARC, and the particular cell group in which LHRH mRNA is most strongly regulated by testosterone changes with pubertal maturation.

Luteinizing hormone-releasing hormone gene expression differs in young and middle-aged females on the day of a steroid-induced LH surge

LHRH mRNA levels were examined in young and middle-aged female rats at 4 times (10:00 h, 14:00 h, 18:00 h and 20:00 h) on the day of a steroid-induced LH surge by in situ hybridization with a digoxigenin-labeled riboprobe. Young, but not middle-aged females, exhibited dynamic temporal changes in the number of LHRH mRNA positive neurons detected in the organum vasculosum of the lamina terminalis–preoptic area (OVLT–POA) continuum. Specifically, fewer LHRH mRNA positive neurons were detected at 18:00 h compared with the number detected at 14:00 h and 20:00 h ( P<0.01) in the OVLT–POA of young females. All LHRH mRNA positive neurons present in 4 anatomically matched sections through the rostral POA of young and middle-aged animals were digitized for detailed computer-assisted analysis of the hybridization reaction product. The mean hybridization area ( P<0.00025) and integrated optical density per cell ( P<0.006) were reduced in middle-aged compared to young females consistent with a relative age-related decline in LHRH mRNA levels. Moreover, an age-related reduction in cellular and/or regional hybridization area was noted at each of the time points examined ( P<0.05– P<0.001). These data confirm earlier reports of dynamic changes in LHRH mRNA levels on the day of an LH surge. Furthermore, they support a role for age-related alterations in LHRH gene expression in the disruption of regular estrous cyclicity in middle-aged females.

Dual label in situ hybridization studies provide evidence that luteinizing hormone-releasing hormone neurons do not synthesize messenger ribonucleic acid for mu, kappa, or delta opiate receptors.

Abundant evidence suggests that opiatergic neurons play an important intermediary role in the regulation of LHRH release by ovarian steroids; however, it is unclear whether opiates communicate directly or indirectly with LHRH neurons. To investigate this issue, we used dual label in situ hybridization histochemistry to determine whether LHRH neurons synthesize messenger RNA (mRNA) for mu, kappa, and/or delta opiate receptors. For these studies, we examined both intact (n = 3) and ovariectomized, steroid-treated rats. Ten of the ovariectomized rats were implanted 1 week later (day 0) with SILASTIC brand (Dow Corning) capsules of estradiol. On the morning of day 2, half of the estradiol-treated rats were injected with 5 mg progesterone. All animals were killed at approximately 1530 h on day 2. We found that cells expressing mu, kappa, and delta opiate receptor mRNAs were in all sections that also contained LHRH neurons. In every case, LHRH neurons were seen to be surrounded by or in close proximity to cells containing mu, kappa, or delta mRNAs. However, regardless of steroid treatment, we found no neurons containing both LHRH mRNA and mRNAs encoding any of the three receptor subtypes. These results support the hypothesis that LHRH neurons are regulated indirectly by opiatergic neurons.

Effect of immune and metabolic challenges on the luteinizing hormone-releasing hormone neuronal system in cycling female rats: an evaluation at the transcriptional level.

The purpose of this study was to investigate the influence of immune (systemic endotoxin administration) and metabolic (fasting) challenges on LHRH neuronal activity and transcription in the organum vasculosum of the lamina terminalis/medial preoptic area as well as on the expression of the LHRH receptor (LHRH-R) in the anterior pituitary of cycling female rats. The reproductive stages of adult female rats (200-250 g; 14 h of light; lights on at 0600 h) were verified by daily vaginal smears taken every morning for a minimum of three or four cycles before the experiment. The acute-phase response was induced via an i.p. injection of lipopolysaccharide (LPS; 200 microg/100 g BW), whereas the metabolic challenge consisted of food deprivation for at least 48 h. Control and challenged rats were killed at specific times in the ovulatory cycle (1200, 1500, and 1800 h on proestrus and diestrous day 2). Frozen brains and pituitaries were mounted on a microtome, cut into 30-microm slices, and then processed for the detection of transcripts encoding either LHRH or LHRH-R by means of in situ hybridization histochemistry using intronic (heteronuclear RNA) and exonic [messenger RNA (mRNA)] riboprobes. Dual immunocytochemistry to detect Fos-immunoreactive (ir) nuclei in LHRH-ir perikarya and colocalization of LHRH mRNA with Fos protein during the day of proestrus were performed by using both in situ hybridization and immunocytochemistry techniques on the same brain sections. The percentage of LHRH-ir and LHRH-expressing neurons displaying positive Fos-ir nuclei during the afternoon of proestrus was significantly inhibited 3 h after endotoxin administration. Rats exhibited an increase in the levels of LHRH primary transcript in the organum vasculosum of the lamina terminalis/medial preoptic area structure at 1500 h on proestrus, a phenomenon significantly attenuated by LPS injection only at this phase of the estrous cycle. On the other hand, fasting did not affect LHRH neuronal activity or gene expression in intact cycling rats, but affected these cells in animals exhibiting a disruption of the ovulatory cycle. Interestingly, LPS caused a profound down-regulation of LHRH-R gene expression in the anterior pituitary throughout the entire estrous cycle. Although food deprivation provoked a more variable pattern of LHRH-R mRNA in cycling rats, the signal for this transcript in the adenohypophysis was deeply altered in those showing a perturbed cycle. These results provide evidence that immune challenge interferes with the LHRH system at both hypothalamic and pituitary levels, whereas alteration of that neuroendocrine system in food-deprived rats seems highly associated with the impairment of reproductive cyclicity.

Astrocyte-Neuron Interactions in Vitro: Role of Growth Factors and Steroids on LHRH Dynamics

The data here reviewed, obtained with in vitro models, indicate that growth factors and steroids play a significant role in astrocyte–neuron interactions. Different designs have been adopted: (1) GT1-1 cells (a cell line derived from a mouse hypothalamic LHRH-producing tumor) were cocultured with type 1 rat astrocytes; and (2) GT1-1 cells were exposed to the conditioned medium (CM) in which type 1 rat astrocytes had been grown for 24 h. LHRH release and mRNA LHRH levels were measured respectively in the medium and in cell homogenates, at different time intervals (LHRH release, by RIA; LHRH mRNA by Northern blot analysis). The data obtained show that type 1 astrocytes secrete in the medium TGFβ, which is able to modulate the release and the gene expression of LHRH in GT1-1 cells; and that one or more LHRH-degrading enzymes is/are present in the conditioned medium of type 1 astrocytes. A second part of the experiments have indicated that type 1 astrocytes are also able to affect, in different directions, the metabolism of testosterone and progesterone into their 5α-reduced metabolites occurring in the GT1-1 cells. In particular, it has been observed that the conversion of testosterone into DHT is decreased by the coculture with type 1 astrocytes, while the conversion of progesterone into DHP is increased by the same coculture conditions. Moreover, type 1 astrocytes are sensitive to steroid hormones, and in particular to the 5α-reduced metabolites of progesterone; this has been shown by analyzing the effects exerted by different steroids on the gene expression of the typical astrocyte marker GFAP.

Neural and glial-mediated effects of growth factors acting via tyrosine kinase receptors on luteinizing hormone-releasing hormone neurons.

It is becoming increasingly evident that the secretory activity of LHRH neurons is regulated not only by transsynaptic inputs but also by trophic molecules of glial and neuronal origin. The present experiments were undertaken to gain insights into the potential cell-cell mechanisms by which basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha), two growth factors produced in the hypothalamus, may affect LHRH neuronal function. Northern blot analysis showed that the LHRH-producing cell line GT1-7 contains the messenger RNA (mRNA) encoding the type 1 fibroblast growth factor receptor (FGFR-1) but not that encoding the epidermal growth factor (EGF) receptors, which mediates the biological actions of both TGF alpha and EGF. Ligand-induced receptor phosphorylation experiments demonstrated that GT1-7 cells possess biologically active FGFR-1s but not EGF receptors. Exposure of the cells to bFGF resulted not only in FGFR-1 tyrosine phosphorylation, but also in tyrosine phosphorylation of phospholipase C gamma, one of the initial enzymes in the intracellular signaling cascade initiated by FGFR activation. GT1-7 cells proliferated in response to this activation. Despite the presence of biologically active receptors, bFGF did not significantly stimulate release of the mature LHRH decapeptide. Instead, bFGF increased the steady-state levels of the mRNA encoding the LHRH precursor processing endoprotease PC2, with a time course comparable to that of phorbol esters, suggesting that, as shown in the companion paper, the actions of the growth factor on LHRH neurons involve facilitation of the initial step in LHRH prohormone processing. The increase in PC2 gene expression was not accompanied by changes in LHRH mRNA levels. Unlike these direct actions of bFGF on GT-1 cells, TGF alpha appears to act indirectly via astroglial intermediacy. Exposure of GT1-7 cells to TGF alpha or EGF failed to affect several parameters of cellular activity including LHRH release, LHRH and PC2 mRNA levels, and cell proliferation. In contrast, astrocyte culture medium conditioned by treatment with TGF alpha led to sustained stimulation of LHRH release with no changes in LHRH gene expression and a transient increase in PC2 mRNA levels. Although no definitive evidence for the presence of FGFR-1 in normal LHRH neurons could be obtained by either double immunohistochemistry or double in situ hybridization procedures, fetal LHRH neurons in primary culture responded to bFGF with neurite outgrowth. Thus, normal LHRH neurons may have an FGFR-1 content too low for detection by regular histochemical procedures, and/or detectable expression of the receptor may be confined to a much earlier developmental stage. The mitogenic effect of bFGF on GT1-7 cells supports this possibility and suggests a role for FGF in the cell proliferation events that precede acquisition of the LHRH neuronal phenotype. It appears that once this phenotype is established, bFGF may promote the differentiation of LHRH neurons. The results also suggest that the secretory capacity of LHRH neurons develops under a dual trophic influence, one on peptide processing exerted directly by bFGF on early neurons, and another on LHRH release, exerted by TGF alpha via the intermediacy of astroglial cells.

Influence of testosterone on LHRH release, LHRH mRNA and proopiomelanocortin mRNA in male sheep.

The mechanism whereby testosterone (T) reduces pulsatile LHRH and LH release is unknown. We tested the hypothesis that hypothalamic levels of LHRH mRNA decrease and proopiomelanocortin (POMC) mRNA increase coincident with reduced LHRH release induced by either long-term or short-term T treatment in male sheep. Experiment 1 examined the effect of long-term T exposure on LHRH and LH release and LHRH and POMC mRNA levels. Yearling Suffolk rams were castrated and assigned to one of four treatments: 1) castrated (n = 4); 2) castrated, portal cannula (n = 5); 3) castrated+T (n = 4) and 4) castrated+T, portal cannula (n = 4). T-treated males received ten 10-cm silastic T-implants immediately after castration. Surgical placement of devices for collecting hypophyseal-portal blood occurred 2 to 3 months after castration. Seven to 10 days after surgery, blood samples were collected at 10-min intervals for 8 h from portal cannulated males or for 5 h from non-cannulated males to assess pulsatile LHRH and/or LH release. Immediately after blood sample collection, hypothalamic tissue was collected for in situ measurement of LHRH or POMC mRNA. T-treatment decreased (P < 0.01) mean LHRH and LH and decreased (P < 0.01) LHRH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron, but decreased (P < 0.01) silver grain area per POMC neuron. Portal cannulation tended to decrease (P = 0.057) silver grain area per LHRH neuron without significantly affecting (P > 0.10) LHRH cell numbers while reducing (P < 0.01) silver grain area per POMC neuron and POMC cell numbers. A second experiment examined the effect of 72 h of T-infusion on LHRH and POMC mRNA levels. Castrated yearling males were assigned to receive either vehicle (n = 4) or T (768 ug/kg/day; n = 4). Blood samples were collected at 10 min intervals for 4 h prior to and during the final 4 h of infusion. Infusion of T decreased (P < 0.01) mean LH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron or LHRH cell numbers. T reduced (P < 0.01) silver grain area per POMC neuron without affecting (P > 0.10) POMC cell number. We reject our hypothesis and conclude that reduced LHRH or heightened POMC gene expression are not mechanisms whereby T reduces pulsatile LHRH release in male sheep.

Effects of estrogen and progesterone on luteinizing hormone-releasing hormone messenger ribonucleic acid levels: consideration of temporal and neuroanatomical variables

We examined temporal effects of estradiol (E2) and progesterone on cellular levels of LHRH messenger RNA (mRNA) in several brain regions. Female rats were ovariectomized and implanted with SILASTIC brand capsules of oil or E2 1 week later (day 0). Progesterone capsules were implanted on the morning of day 2. Using in situ hybridization histochemistry, we found that on day 2, E2 induced a complex temporal pattern of changes in LHRH mRNA levels. Levels in ovariectomized E2-treated animals were lower than control values in the morning, then increased before the LH surge and declined again as the surge waned. The magnitude of these changes was greatest in neurons of the rostral preoptic area/organum vasculosum of the lamina terminalis, but a similar pattern was detected in neurons of the medial preoptic area. No changes were seen in neurons of the diagonal band of Broca. Our finding that the effect of E2 on LHRH mRNA levels depends on the time of day and brain region examined largely reconciles discrepancies among previous studies. Progesterone triggered events that advanced the onset of and augmented the LH surge, but had no effect on LHRH mRNA levels. These findings support the hypothesis that the mechanism by which E2 induces region-specific changes in LHRH mRNA levels before the surge is separate from the progesterone-amplified mechanism that induces LHRH release.

Effects of estrogen and progesterone on luteinizing hormone-releasing hormone messenger ribonucleic acid levels: consideration of temporal and neuroanatomical variables.

We examined temporal effects of estradiol (E2) and progesterone on cellular levels of LHRH messenger RNA (mRNA) in several brain regions. Female rats were ovariectomized and implanted with SILASTIC brand capsules of oil or E2 1 week later (day 0). Progesterone capsules were implanted on the morning of day 2. Using in situ hybridization histochemistry, we found that on day 2, E2 induced a complex temporal pattern of changes in LHRH mRNA levels. Levels in ovariectomized E2-treated animals were lower than control values in the morning, then increased before the LH surge and declined again as the surge waned. The magnitude of these changes was greatest in neurons of the rostral preoptic area/organum vasculosum of the lamina terminalis, but a similar pattern was detected in neurons of the medial preoptic area. No changes were seen in neurons of the diagonal band of Broca. Our finding that the effect of E2 on LHRH mRNA levels depends on the time of day and brain region examined largely reconciles discrepancies among previous studies. Progesterone triggered events that advanced the onset of and augmented the LH surge, but had no effect on LHRH mRNA levels. These findings support the hypothesis that the mechanism by which E2 induces region-specific changes in LHRH mRNA levels before the surge is separate from the progesterone-amplified mechanism that induces LHRH release.

Effects of morphine on hypothalamic tyrosine hydroxylase mRNA levels in dopaminergic neurons and on preoptic DOPAC levels measured by microdialysis

Morphine not only suppresses norepinephrine-induced increases in LHRH mRNA levels but, in these same animals, it simultaneously amplifies norepinephrine (NE)-induced LH release. These observations suggest that NE may activate parallel mechanisms which independently increase LHRH mRNA levels and LHRH release and suggest that some of these effects could be mediated indirectly via morphine's action on different components of the hypothalamic dopamine (DA) system. Accordingly, in the present studies we examined the effects of morphine on various components of this dopamine system using as our index of altered DA neuronal activity, the changes which occur in tyrosine hydroxylase (TH) mRNA levels following morphine. As an ancillary index of changes which occur in dopamine neuronal activity, we measured, by microdialysis, the changes which occur in preoptic dihydroxyphenylacetic acid (DOPAC) levels after either subcutaneous injections or following microinfusions of morphine into the zona incerta (ZI). In a final study, we evaluated whether DA when given alone (icv infusion) or prior to icv NE would altered LH release. Single cell levels of TH mRNA in preoptic A15 and periventricular anterior hypothalamic A14 DA neurons were not affected by morphine 1, 5 and 24 h later. In contrast, within 1 h after morphine, TH mRNA levels in ZI A13 neurons were significantly elevated and they remained high at 5 and 24 h compared to controls. Morphine also resulted in a significant rise in TH mRNA levels in tuberoinfundibular DA neurons (TIDA) (A12) within 1 h and these levels remained high to 5 h. Thereafter, by 24 h, message levels had returned to control values. Morphine also resulted in a rapid rise in plasma prolactin (Prl) with peak values occurring at 20 min and then returning to baseline by 90 min. When morphine was given sc it resulted, within 15 min, in a rapid rise in preoptic DOPAC levels and these levels continued to rise such that they were 217% higher than pretreatment values by 105 min. Preoptic 5-hydroxyindoleacetic acid (5-HIAA) levels also increased by 25–75% after sc morphine. The microinfusion of morphine into ZI also resulted in elevated preoptic DOPAC but not 5-HIAA levels within 15 min. The icv infusion of DA alone had no effect on plasma LH whereas, NE (icv) produced a modest but significant increase in plasma LH. When DA was given 15 min prior to the infusion of NE, neither amplification nor inhibition of NE-induced LH release occurred. From these and other studies we conclude that the morphine-induced suppression of TIDA neuronal activity may allow NE to release greater amounts of LHRH from axon terminals in the median eminence. On the other hand, we doubt that the rise in preoptic DA (DOPAC) which occurs after morphine plays a role in augmenting the action of NE on LHRH release since icv DA does not alter the patterns or concentrations of plasma LH which occur after NE alone.

Morphine amplifies norepinephrine (NE)-induced LH release but blocks NE-stimulated increases in LHRH mRNA levels: comparison of responses obtained in ovariectomized, estrogen-treated normal and androgen-sterilized rats

In these studies we examined the temporal effects of intracerebroventricular (i.c.v.) infusions of norepinephrine (NE) on plasma LH and on LHRH mRNA levels in the organum vasculosum of the lamina terminalis (OVLT) and in neurons located in the rostral (r), middle (m) and caudal (c) preoptic areas (POA) of ovariectomized, estrogen-treated rats. Thereafter, we compared these responses to those which occur in androgen-sterilized rats (ASR). NE infusions not only increased plasma LH concentrations but within 1 h after NE, LHRH mRNA levels also were increased significantly in the OVLT and rPOA but not in the mPOA or cPOA. By 4 h, these message levels still were elevated in the OVLT and rPOA and they now also were significantly higher than control values in the mPOA and cPOA. While NE also increased LH secretion in ASR, the plasma LH concentrations obtained were markedly blunted compared to control values. Moreover, NE infusions did not alter single cell levels of LHRH mRNA in any region of the rostral hypothalamus. Previously, we have reported that morphine (s.c.) markedly amplifies NE-induced LH release and questioned whether these responses are accompanied by concomitant augmented increases in LHRH mRNA levels. Morphine alone did not affect basal LHRH mRNA or plasma LH levels. However, when rats were pretreated with morphine (−15 min) and NE was infused i.c.v. at 0 time, significant amplification of LH release occurred but, unexpectedly, morphine completely blocked NE-induced increases in LHRH mRNA levels in all of the neurons we examined. Morphine also amplified LH release in ASR but these responses were significantly less than those obtained in control rats. Morphine alone or combined with NE infusions had no effect on single cell LHRH mRNA levels in ASR. These data suggest that NE may activate parallel mechanisms which independently increase LHRH mRNA levels and those which evoke LHRH release.

Luteinizing hormone-releasing hormone gene expression in the bovine brain: anatomical localization and regulation by ovarian state.

Experiments were conducted to identify neurons in the bovine brain that express the LHRH gene and to determine whether LHRH mRNA levels are influenced by the ovaries. Two groups of postpubertal heifers were utilized: heifers killed during the mid-luteal phase of the estrous cycle (LUTEAL, n = 5) and heifers killed 14-16 wk following ovariectomy (OVX, n = 5). In situ hybridization was performed through use of a 32P-end-labeled deoxyoligonucleotide (59 mer) complementary to the human LHRH mRNA sequence. LHRH-expressing neurons were identified in the diagonal band of Broca, the preoptic area, and the anterior hypothalamus in a manner consistent with immunocytochemical localization. Reduced silver grains, proportional to LHRH mRNA content, were quantified (in pixels, 45x objective) with an image analysis system. Expected serum hormone concentration differences between endocrine states were confirmed by radioimmunoassay for progesterone (LUTEAL > OVX, p < 0.01) and for LH (OVX > LUTEAL, p < 0.01). Compared to the OVX group, LUTEAL heifers had 34% fewer LHRH-expressing neurons (p < 0.05); on the average, these neurons possessed 28% fewer pixels/cell (p < 0.01), indicating fewer copies of LHRH mRNA per cell. When the numbers of pixels in all labeled cells were totalled, LUTEAL animals had 57% fewer pixels (p < 0.05) than did the OVX females--probably reflecting a decrease in LHRH synthetic capacity in the LUTEAL animals. Therefore, during the mid-luteal phase of the bovine estrous cycle, ovarian steroid (i.e., luteal progesterone) suppression of LHRH release (as reflected by serum LH) is coincident with decreased LHRH mRNA in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoactivation enhances the concentration of luteinizing hormone-releasing hormone peptide and its gene expression in human peripheral T-lymphocytes.

An immunomodulatory role for LHRH was suggested when we reported the presence of immunoactive and bioactive LHRH and its mRNA in rat splenic and thymic lymphocytes. In this paper we report that human peripheral T-cells as well as its subsets CD4+ and CD8+ contained immunoactive and bioactive LHRH. Furthermore, analysis of phytohemagglutinin (PHA)-activated T-cell lysates for LHRH by RIA demonstrated that the mean concentration of LHRH in PHA-activated T-cells increased from 45 +/- 4.5 to 64 +/- 7 pg/10(6) cells after 24 h of culture and from 47 +/- 3.6 to 117 +/- 11.8 pg/10(6) cells (P < 0.01) after 48 h. While the LHRH concentration in PHA-activated cells increased over the last 24 h of culture h from 64 +/- 7 to 117 +/- 11.8 pg/10(6) cells (P < 0.001), there was no change in mean concentration of LHRH in T-cells kept in medium alone. In a preliminary study we found that fresh T-cells contain 20 +/- 1.4 pg pro-LHRH/10(6) cells, and PHA stimulation increased the pro-LHRH content similar to the increase in LHRH. As with unfractionated T-cells, a significant PHA-induced time-dependent enhancement of intracellular LHRH was noted in CD4+ and CD8+ T-cells. RNA extracted from lymphocytes was subjected to reverse transcription-polymerase chain reaction analysis using LHRH and histone-3.3, primers, the latter as an internal control. The polymerase chain reaction-generated data demonstrated that the relative amount of LHRH mRNA in cultured, but non-PHA-stimulated (resting), cells diminished dramatically between 5-24 h, but recovered by 48 h of culture. The relative amount of LHRH mRNA in PHA-stimulated cells revealed a markedly different pattern. LHRH message expression in PHA-activated cells increased slightly at 5 h of culture and was maximally stimulated by 24 h, but declined by 48 h of culture. The PHA activation-induced time-dependent enhancement of intracellular accumulation of LHRH peptide at 5 and 24 h was accompanied by increased LHRH message. However, the increased concentration of LHRH peptide at 48 h coincided with decreased LHRH message expression. The data from total protein synthesis in PHA-activated cells showed a progressive increase in protein synthesis, a pattern entirely similar to the changes in the cell content of LHRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoactivation enhances the concentration of luteinizing hormone- releasing hormone peptide and its gene expression in human peripheral T- lymphocytes

An immunomodulatory role for LHRH was suggested when we reported the presence of immunoactive and bioactive LHRH and its mRNA in rat splenic and thymic lymphocytes. In this paper we report that human peripheral T-cells as well as its subsets CD4+ and CD8+ contained immunoactive and bioactive LHRH. Furthermore, analysis of phytohemagglutinin (PHA)-activated T-cell lysates for LHRH by RIA demonstrated that the mean concentration of LHRH in PHA-activated T-cells increased from 45 +/- 4.5 to 64 +/- 7 pg/10(6) cells after 24 h of culture and from 47 +/- 3.6 to 117 +/- 11.8 pg/10(6) cells (P < 0.01) after 48 h. While the LHRH concentration in PHA-activated cells increased over the last 24 h of culture h from 64 +/- 7 to 117 +/- 11.8 pg/10(6) cells (P < 0.001), there was no change in mean concentration of LHRH in T-cells kept in medium alone. In a preliminary study we found that fresh T-cells contain 20 +/- 1.4 pg pro-LHRH/10(6) cells, and PHA stimulation increased the pro-LHRH content similar to the increase in LHRH. As with unfractionated T-cells, a significant PHA-induced time-dependent enhancement of intracellular LHRH was noted in CD4+ and CD8+ T-cells. RNA extracted from lymphocytes was subjected to reverse transcription-polymerase chain reaction analysis using LHRH and histone-3.3, primers, the latter as an internal control. The polymerase chain reaction-generated data demonstrated that the relative amount of LHRH mRNA in cultured, but non-PHA-stimulated (resting), cells diminished dramatically between 5-24 h, but recovered by 48 h of culture. The relative amount of LHRH mRNA in PHA-stimulated cells revealed a markedly different pattern. LHRH message expression in PHA-activated cells increased slightly at 5 h of culture and was maximally stimulated by 24 h, but declined by 48 h of culture. The PHA activation-induced time-dependent enhancement of intracellular accumulation of LHRH peptide at 5 and 24 h was accompanied by increased LHRH message. However, the increased concentration of LHRH peptide at 48 h coincided with decreased LHRH message expression. The data from total protein synthesis in PHA-activated cells showed a progressive increase in protein synthesis, a pattern entirely similar to the changes in the cell content of LHRH.

Expression of luteinizing hormone-releasing hormone mRNA in the human prostatic cancer cell line LNCaP.

Recent evidence suggests that LHRH or a LHRH-like peptide might be produced by human prostatic tumor cells. To test this hypothesis, we have studied whether a mRNA for LHRH is expressed in the human prostatic cancer cell line LNCaP, by means of the RT-PCR (reverse transcription-polymerase chain reaction) technique. For these experiments, mRNA was extracted from LNCaP cells, from rat hypothalami and from rat pituitaries, reverse transcribed to cDNA and amplified via the PCR utilizing a pair of oligonucleotide primers complementary to the LHRH cDNA. Following gel electrophoresis, a band of the expected size of 228 base pairs was found in LNCaP cells as well as in the rat hypothalamus, but not in the rat anterior pituitary. This 228 base pair band from LNCaP cells and from the rat hypothalamus specifically hybridized to a 32P-labeled LHRH oligonucleotide probe. The cDNA band obtained from LNCaP cells was subcloned into a plasmid vector, and the analysis of its sequence showed a complete match with the authentic human placental LHRH cDNA. These observations clearly demonstrate that a mRNA for LHRH is expressed in human prostatic cancer cells, and suggest that LHRH or a LHRH-like peptide may be produced by these cells. To test the hypothesis whether this material might act as a local growth regulating factor on tumor cell proliferation, LNCaP cells, grown in a steroid-free medium, were treated daily with a potent LHRH antagonist. After 9, 12 and 15 days, the treatment resulted in a significant increase of tumor cell proliferation. These data clearly suggest that the LHRH mRNA expressed in LNCaP cells is possibly translated into LHRH or a LHRH-like peptide which probably functions as a local growth inhibitory factor on prostatic tumor cell proliferation, by acting on LHRH receptors.