Decreased angiotensin receptor 1 expression in ± AT1 Knockout mice testis results in male infertility and GnRH reduction.

BACKGROUND: This study aimed to detect the effect of angiotensin receptor 1 (AT1) knock out (KO) on spermatogenesis and hypothalamic-pituitary-gonadal (HPG) axis hormone expression. METHODS: Normal C57BL/6 male mice were used as control group or treated with angiotensin receptor blocker, in addition heterozygous ± AT1KO mice were generated. After caged at a ratio of 2 to 1 with females, pregnancy rates of female mice were determined by detection of vaginal plugs. Deformity rate of spermatozoa was evaluated by eosin staining and morphology evaluation. The AT1 mRNA expression in the testes of male ± AT1KO mice was detected by quantitative real-time polymerase chain reaction (QRT-PCR). Serum GnRH level was determined by ELISA. RESULTS: Compared to control, ± AT1KO mice showed reduced expression of AT1 in testes, pituitary and hypothalamus. In addition, decreased level of GnRH, but not follicle stimulating hormone (FSH) or luteinizing hormone (LH), in ± AT1KO mice was detected. Treatment with angiotensin receptor blocker (ARB) did not have significant effects on HPG hormones. ± AT1KO mice exhibited male infertility and significant abnormality of sperm morphology. CONCLUSION: Reduced AT1 knockout resulted in male infertility, potentially by inducing abnormal spermatogenesis. Both testis and HPG axis signaling may be involved.

Erythropoietin-producing hepatocellular receptor A7 restrains estrogen negative feedback of luteinizing hormone via ephrin A5 in the hypothalamus of female rats.

We have previously shown that systemic injection of erythropoietin-producing hepatocellular receptor A7 (EPHA7)-Fc raises serum luteinizing hormone (LH) levels before ovulation in female rats, indicating the induction of EPHA7 in ovulation. In this study, we aimed to identify the mechanism and hypothalamus-pituitary-ovary (HPO) axis level underlying the promotion of LH secretion by EPHA7. Using an ovariectomized (OVX) rat model, in conjunction with low-dose 17ß-estradiol (E2) treatment, we investigated the association between EPHA7-ephrin (EFN)A5 signaling and E2 negative feedback. Various rat models (OVX, E2-treated OVX, and abarelix treated) were injected with the recombinant EPHA7-Fc protein through the caudal vein to investigate the molecular mechanism underlying the promotion of LH secretion by EPHA7. Efna5 was observed strongly expressed in the arcuate nucleus of the female rat by using RNAscope in situ hybridization. Our results indicated that E2, combined with estrogen receptor (ER)α, but not ERß, inhibited Efna5 and gonadotropin-releasing hormone 1 (Gnrh1) expressions in the hypothalamus. In addition, the systemic administration of EPHA7-Fc restrained the inhibition of Efna5 and Gnrh1 by E2, resulting in increased Efna5 and Gnrh1 expressions in the hypothalamus as well as increased serum LH levels. Collectively, our findings demonstrated the involvement of EPHA7-EFNA5 signaling in the regulation of LH and the E2 negative feedback pathway in the hypothalamus, highlighting the functional role of EPHA7 in female reproduction.

Roles of copper in neurokinin B and gonadotropin-releasing hormone structure and function and the endocrinology of reproduction.

Copper is a metal ion present in all organisms, where it has well-known roles in association with proteins and enzymes essential for cellular processes. In the early decades of the twentieth century copper was shown to influence mammalian reproductive biology, and it was subsequently shown to exert effects primarily at the level of the pituitary gland and/or hypothalamic regions of the brain. Furthermore, it has been reported that copper can interact with key neuropeptides in the hypothalamic-pituitary-gonadal axis, notably gonadotropin-releasing hormone (GnRH) and neurokinin B. Interestingly, recent phylogenetic analysis of the sequences of GnRH-related peptides indicates that copper binding is an evolutionarily ancient property of this neuropeptide family, which has been variously retained, modified or lost in the different taxa. In this mini-review the metal-binding properties of neuropeptides in the vertebrate reproductive pathway are reviewed and the evolutionary and functional significance of copper binding by GnRH-related neuropeptides in vertebrates and invertebrates are discussed.

Effects of High-Glucose and High-Fat Condition on Estrogen Receptor- and Sexual Precocity-Related Genes in GT1-7 Cells.

BACKGROUND This study was designed to investigate the effect of high-glucose and high-fat condition on estrogen receptor- and sexual precocity-related genes in GT1-7 cells. MATERIAL AND METHODS In this study, CCK8 was used to detect cell viability, and TUNEL assay was used to detect apoptosis levels of GT1-7 cells after treatment with glucosamine and palmitate. The expression level of GnRH was measured by ELISA and RT-qPCR. RT-qPCR and Western blot were used to detect the expression of ERß, CD36, and GPR54 in GT1-7 cells, and the expression of ERß was detected using immunohistochemistry analysis. Finally, after adding the intervening drug tamoxifen to GT1-7 cells, the expression level of GnRH was measured by ELISA and Western blot analysis was used to detect the expression of GPR54 and GnRH. RESULTS GnRH secretion in the high-fat and high-glucose group increased continuously over time and peaked at 18 h, and GnRH gene expression peaked at 12 h. High-fat and high-glucose conditions also significantly increased the levels of estrogen receptors ß (ERß), fatty acid translocase protein (CD36), and G Protein-Coupled Receptors 54 (GPR54) in GT1-7 cells. After estrogen receptors ß (ER) was inhibited, GnRH secretion and GPR54 expression were decreased at 12 h and 18 h. CONCLUSIONS Our study demonstrates that high-glucose and high-fat conditions promote the secretion of GnRH and ER and the expression of genes related to sexual precocity in GT1-7 cells.

The advancement of the onset of vaginal opening in female rats subjected to chronic testosterone treatment occurs independently of hypothalamic Kiss1 and RFRP expression.

OBJECTIVE: The neonatal and/or prepubertal androgen milieu affects sexual maturation. In rodents, neonatal chronic testosterone treatment, which is used as a model of polycystic ovary syndrome (PCOS), results in the onset of vaginal opening occurring earlier in the pubertal period. DESIGN: In the present study, the changes in hypothalamic Kiss1 (a gonadotropin-releasing hormone (GnRH)-stimulating factor) and RF-amide related peptide (RFRP; a GnRH inhibitory factor) mRNA expression induced by testosterone treatment were examined in order to clarify whether these factors are involved in the testosterone-induced acceleration of sexual maturation. RESULTS: The onset of vaginal opening occurred earlier and uterine weight was increased in female rats subjected to chronic (from postnatal day 23 to day 31) testosterone treatment. Contrary to our expectations, the rats' hypothalamic Kiss1 and Kiss1 receptor mRNA levels were not changed, and their serum luteinizing hormone (LH) levels were decreased. Although hypothalamic RFRP mRNA expression was decreased in the testosterone-treated rats, this change was not reflected in their serum LH levels. CONCLUSIONS: These results indicate that the advancement of sexual maturation observed in chronic testosterone-treated rats might be caused by a peripheral, rather than a central, mechanism.

Effects of amlodipine administration and withdrawal on rat pituitary gonadotropins.

OBJECTIVE: The study was aimed to observe the effect of amlodipine on rat pituitary gonadotropins after amlodipine administration and withdrawal. METHODS: It was an experimental study done at Army Medical College, National University of Sciences and Technology, Islamabad, Pakistan from 2009-2010. Sixty adult male rats were divided into groups A and B. Each group was further subdivided into two subgroups of 15 rats each; A1 (control), A2 (control recovery), B1 (amlodipine-treated) and B2 (amlodipine recovery). Amlodipine, 0.04 mg/kg body weight daily for fifty days was given by means of gavage to groups B1 and B2. Groups A1 and A2 were given vehicle (0.5 ml distilled water). After 50 days, rats in groups A1 and B1 were sacrificed and their serum LH and FSH levels were measured by Enzyme Immunoassay method. Vehicle and amlodipine were withdrawn in groups A2 and B2, respectively, and the rats were left for recovery to take place for another fifty days. The above procedure was adopted for the measurement of LH and FSH levels in the recovered rats. RESULTS: Amlodipine administration for 50 days resulted in a significant rise in serum LH (p < 0.01) whereas serum FSH remained unchanged (p ≥ 0.05). Serum LH in amlodipine-treated rats returned to normal after amlodipine withdrawal (p ≥ 0.05). CONCLUSION: Amlodipine causes a reversible increase in serum LH but it has no effect on serum FSH (Fig. 2, Ref. 17).

Kisspeptin inhibits high-voltage activated Ca2+ channels in GnRH neurons via multiple Ca2+ influx and release pathways.

Kisspeptin plays an important role in puberty and subsequent fertility by activating its receptor, G-protein-coupled receptor 54 (GPR54), and increasing cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and gonadotropin-releasing hormone (GnRH) secretion in GnRH neurons. Yet the mechanism by which kisspeptin increases [Ca(2+)](i) in GnRH neurons remains to be fully elucidated. In other neurons, voltage-gated Ca(2+) channel (VGCC) activity has been shown to be inversely related to [Ca(2+)](i). We used whole-cell patch-clamp recording to examine the effects of kisspeptin-10 (KP-10) on VGCC activity evoked by step depolarizations in GnRH neurons in brain slices from pubertal male GnRH-green fluorescent protein transgenic mice. Prolonged (>30 s) KP-10 application inhibited Ca(2+) currents. The GPR54 antagonist peptide 234, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, substitution of Ba(2+) for Ca(2+), the calmodulin antagonists calmidazolium and trifluoperazine, the phospholipase C inhibitor edelfosine, the canonical transient receptor potential (TRPC) channel and inositol 1,4,5-trisphosphate receptor (IP(3)R) antagonist 2-APB, the TRPC channel antagonist BTP2 and the endoplasmic reticulum Ca(2+)-ATPase blocker cyclopiazonic acid each prevented inhibition. The IP(3)R antagonists caffeine (10 µM), heparin and intracellular 2-APB prevented inhibition to a lesser extent. The ryanodine receptor (RyR) antagonists ryanodine and dantrolene prevented inhibition, and the RyR agonist caffeine (30 mM) mimicked the effects of KP-10 on Ca(2+) currents. Our results suggest that kisspeptin induces Ca(2+) influx through TRPC channels and Ca(2+) release via IP(3)Rs and RyRs, and that this is followed by Ca(2+)/CaM-dependent inhibition of VGCCs.

Brain and testicular metabonomics revealed the protective effects of Guilingji on senile sexual dysfunction rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Guilingji (GLJ), which has been used to treat male diseases in China for centuries, contains 28 Chinese herbs and was previously established as an effective treatment for male sexual dysfunction. However, its mechanism of action remains unclear. AIM OF THE STUDY: To explore the efficacy and mechanism of action of GLJ in improving senile sexual dysfunction (SSD) in aging rats. MATERIALS AND METHODS: An aging rat model of SSD was induced by the subcutaneous injection of d-galactose (300 mg⋅kg-1) and used to analyse the effects of GLJ (different concentrations of 37.5, 75, and 150 mg⋅kg-1) on the mating of aging rats. At the end of the 8th week, histopathological analysis of testicular tissues, assessment of the hypothalamic-pituitary-gonadal (HPG) axis hormone levels in serum or brain, and metabonomics analysis of the brain and testicular tissue with liquid chromatography-mass spectrometry was performed to explore the mechanism of action of GLJ. RESULT: After treatment with GLJ, the mount and ejaculation latency levels were increased in the treatment group than those in model group (P < 0.05), moreover, the testicular morphology was improved. Gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) levels in rats were also improved significant (P < 0.05) compared with those in the model group. Furthermore, the metabonomics results in the testicular and brain tissue showed that GLJ improved SSD by adjusting amino acid and lipid metabolism. CONCLUSION: This study integrated the complementary metabolic profiles of the target tissues. GLJ might affect SSD rats by regulating amino acid and lipid metabolism and may modulate sensitivity to the signaling pathway in the HPG axis. This study provides an essential basis for the broad clinical application of GLJ.

Effect of the organochlorine pesticide endosulfan on GnRH and gonadotrope cell populations in fish larvae.

Endocrine-disrupting chemicals can influence the hypothalamus-pituitary-gonad axis and possibly affect reproduction in vertebrates. We analyzed the effect of 30-day endosulfan (ES) exposure in sexually undifferentiated larvae of the cichlid fish Cichlasoma dimerus. The number, area, mean cytoplasmic and nuclear diameter, and mean cytoplasmic optical density of gonadotropin-releasing hormone (GnRH) I, II, and III immunoreactive (ir-) neurons and ß follicle-stimulating hormone (ßFSH) ir-cells were measured. Animals exposed to the highest ES concentration (0.1 µg/l) showed a decrease in GnRH I nucleus/cytoplasm area ratio upon exposure. Nuclear area and mean nuclear diameter of ßFSH ir-cells was higher in ES treated fish. ßFSH nucleus/cytoplasm area ratio was high in exposed animals, and animals exposed to 0.1 µg/l ES showed smaller mean cytoplasmic optical density. These findings suggest that ES affects GnRH I and ßFSH protein synthesis/release. However, these responses seem to be insufficient to affect gonadal differentiation at this stage of development.

Cellular and molecular features of EDC exposure: consequences for the GnRH network.

The onset of puberty and the female ovulatory cycle are important developmental milestones of the reproductive system. These processes are controlled by a tightly organized network of neurotransmitters and neuropeptides, as well as genetic, epigenetic and hormonal factors, which ultimately drive the pulsatile secretion of gonadotropin-releasing hormone. They also strongly depend on organizational processes that take place during fetal and early postnatal life. Therefore, exposure to environmental pollutants such as endocrine-disrupting chemicals (EDCs) during critical periods of development can result in altered brain development, delayed or advanced puberty and long-term reproductive consequences, such as impaired fertility. The gonads and peripheral organs are targets of EDCs, and research from the past few years suggests that the organization of the neuroendocrine control of reproduction is also sensitive to environmental cues and disruption. Among other mechanisms, EDCs interfere with the action of steroidal and non-steroidal receptors, and alter enzymatic, metabolic and epigenetic pathways during development. In this Review, we discuss the cellular and molecular consequences of perinatal exposure (mostly in rodents) to representative EDCs with a focus on the neuroendocrine control of reproduction, pubertal timing and the female ovulatory cycle.

Semen Modulates the Expression of NGF, ABHD2, VCAN, and CTEN in the Reproductive Tract of Female Rabbits.

Semen changes the gene expression in endometrial and oviductal tissues modulating important processes for reproduction. We tested the hypothesis that mating and/or sperm-free seminal plasma deposition in the reproductive tract affect the expression of genes associated with sperm-lining epithelium interactions, ovulation, and pre-implantation effects (nerve growth factor, NGF; α/ß hydrolase domain-containing protein 2, ABHD2; C-terminal tensin-like protein, CTEN or TNS4; and versican, VCAN) in the period 10-72 h post-mating. In Experiment 1, does (n = 9) were treated with gonadotropin-releasing hormone (GnRH) (control), GnRH-stimulated, and vaginally infused with sperm-free seminal plasma (SP-AI), or GnRH-stimulated and naturally mated (NM). In Experiment 2, does (n = 15) were GnRH-stimulated and naturally mated. Samples were retrieved from the internal reproductive tracts (cervix-to-infundibulum) 20 h post-treatment (Experiment 1) or sequentially collected at 10, 24, 36, 68, or 72 h post-mating (Experiment 2, 3 does/period). All samples were processed for gene expression analysis by quantitative PCR. Data showed an upregulation of endometrial CTEN and NGF by NM, but not by SP-AI. The findings suggest that the NGF gene affects the reproductive tract of the doe during ovulation and beyond, influencing the maternal environment during early embryonic development.

Clinical Pharmacology of Elagolix: An Oral Gonadotropin-Releasing Hormone Receptor Antagonist for Endometriosis.

The clinical pharmacology of elagolix was extensively evaluated in clinical studies in healthy subjects and in women with endometriosis. Elagolix pharmacokinetics (PK) show significant population variability, however they are minimally affected by patients' baseline characteristics and demographics, except for clinically relevant extrinsic and intrinsic factors such as coadministrated strong organic anion transporting polypeptide (OATP) 1B1 inhibitors and severe hepatic impairment, which are contraindications for the use of elagolix. These studies enabled a comprehensive understanding of elagolix mechanism of action and the downstream pharmacodynamic (PD) effects on gonadotropin and ovarian hormones, as well as full characterization of the PK/PD (PKPD) relationships of elagolix at various dosages, including the approved 150 mg once daily and 200 mg twice daily dosing regimens for the management of moderate to severe pain associated with endometriosis. Several model-based analyses have contributed to understanding of the benefit-risk profile of elagolix in patients with endometriosis, through characterization of the exposure relationship with responder rates, with changes in bone mineral density over time, as well as the interaction with coadministered drugs. Collectively, these studies and analyses served as supportive evidence for the effectiveness of the approved dosages and provided general dosing instructions of the first approved oral gonadotropin-releasing hormone receptor antagonist.

CSF1R inhibitor PLX5622 and environmental enrichment additively improve metabolic outcomes in middle-aged female mice.

As the elderly population grows, chronic metabolic dysfunction including obesity and diabetes are becoming increasingly common comorbidities. Hypothalamic inflammation through CNS resident microglia serves as a common pathway between developing obesity and developing systemic aging pathologies. Despite understanding aging as a life-long process involving interactions between individuals and their environment, limited studies address the dynamics of environment interactions with aging or aging therapeutics. We previously demonstrated environmental enrichment (EE) is an effective model for studying improved metabolic health and overall healthspan in mice, which acts through a brain-fat axis. Here we investigated the CSF1R inhibitor PLX5622 (PLX), which depletes microglia, and its effects on metabolic decline in aging in interaction with EE. PLX in combination with EE substantially improved metabolic outcomes in middle-aged female mice over PLX or EE alone. Chronic PLX treatment depleted 75% of microglia from the hypothalamus and reduced markers of inflammation without affecting brain-derived neurotrophic factor levels induced by EE. Adipose tissue remodeling and adipose tissue macrophage modulation were observed in response to CSF1R inhibition, which may contribute to the combined benefits seen in EE with PLX. Our study suggests benefits exist from combined drug and lifestyle interventions in aged animals.

Internally cationic polyamidoamine PAMAM-OH dendrimers for siRNA delivery: effect of the degree of quaternization and cancer targeting.

A novel cancer targeted, internally cationic and surface neutral polyamidoamine (PAMAM) dendrimer, was designed, synthesized, and evaluated as a nanocarrier for the targeted intracellular delivery of siRNA. The dendrimer contained a synthetic analog of Luteinizing hormone-releasing hormone as cancer targeting moiety. The proposed delivery system possesses the following advantages: (1) internal cationic charges for complexation with siRNA and enhanced siRNA protection; (2) low cytotoxicity; (3) lesser degree of quaternization offering free tertiary amines for potential proton sponge effect; and (4) targeting specifically to cancer cells for enhancing siRNA uptake and efficiency and potential limitation of adverse side effects of chemotherapy on healthy organs. Both nontargeted and targeted dendrimer-siRNA complexes formed compact nanometer size spherical particles, exhibited very low cytotoxicity even at the higher concentration, and efficiently penetrated cancer cells in vitro. However, only the targeted dendrimer-siRNA complex was able to substantially decrease the expression of a targeted BCL2 gene.

Vasopressin regulates hypothalamic GnRH synthesis: Histomorphological evidence in hypothalamus and biological effects in GT1-7 cells.

AIMS: To investigate the direct histomorphological clues and observe the biological effects of VP acting on gonadotropin-releasing hormone (GnRH) secretion. MAIN METHODS: Immunofluorescence was conducted to investigate the expressions of GnRH and VP in experimental left varicocele (ELV) rats and ELV repair rats. The colocalization of GnRH and VP was observed by electron microscopy immunohistochemistry. The protein-protein interaction between GnRH and VP was tested by co-immunoprecipitation (co-IP) and the proximity ligation assay (PLA). The effects of intracellular and extracellular VP on GnRH and relative transcription factors (Oct-1, Otx2, Pbx1b and DREAM) were respectively evaluated in VP overexpressed and VP treated GT1-7 cells. KEY FINDINGS: Both hypothalamic GnRH and VP decreased in ELV rats and recovered by ELV repair. The overlapped immunolocalizations of GnRH and VP mainly distributed in the lateral part of the arcuate nucleus (ArcL) and median eminence (ME) with a Manders' overlap coefficient of 0.743 ±â€¯0.117. Immunoreactive substances of GnRH and VP existed in the same and adjacent terminals. VP overexpression did not cause any significant effects on the expressions of GnRH and Oct-1, as well as GnRH promoter activity. While 50-200 pg/ml VP treatments increased GnRH mRNA levels in a dose- and time-dependent manner in GT1-7 cells. Additionally, 200 pg/ml VP triggered a marked promotion of expressions of GnRH, Oct-1, Oxt2 Pbx1b and DREAM, as well as GnRH promoter activity (P < 0.05). SIGNIFICANCE: The results reveal the colocalization and interaction of VP and GnRH, which will be conducive to explain the effects and mechanisms of VP acting on reproduction.

Obestatin may affect the GnRH/KNDy gene network in sheep hypothalamus.

The effects of obestatin on gonadotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the effect of intracerebroventricular infusions of obestatin on the gonadotrophin-releasing hormone (GnRH) mRNA and protein expressions as well as on KNDy mRNA and kisspeptin (Kiss) peptide expressions in peripubertal female sheep. Animals were randomly divided into two groups: the control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h-1). The series of four 1-h infusions per day during three consecutive days were performed. After the end of the experiment parts of sheep brains were fixed in situ for immunohistochemical analysis, while the remaining brains were frozen for Real Time qPCR analysis. Substantial changes in the activity of the GnRH and KNDy gene network were observed in obestatin-infused sheep. In those animals an increase of GnRH mRNA expression in the preoptic area, a decrease of GnRH mRNA expression in the median eminence and an increase of GnRH immunoreactivity in the median eminence were found. Moreover, changes in the KNDy mRNA expression in mediobasal hypothalamus as well as decrease Kiss expression in arcuate nucleus and median eminence were observed. It was revealed that obestatin affects the GnRH and KNDy gene network as well as Kiss at the level of mRNA and protein expression. Thereby, it can be concluded that obestatin participates in the mechanism modulating gonadotrophic axis activity at the central level in peripubertal female sheep.

Noradrenergic control of GnRH release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study investigates the influence of alpha(1)-adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates alpha(1)-adrenoreceptor-GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media-alpha (MEM-alpha) at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to specific alpha(1)-adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1-10 mm). The alpha(1)-adrenoreceptor agonist (10 mm) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, alpha(1)-adrenoreceptor agonist (10 mm)-induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus-pituitary sequential double-chamber perifusion. Only after exposure of hypothalamic slices to alpha(1)-adrenoreceptor agonist (10 mm), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 +/- 12.3-36.2 +/- 21.6 ng/ml) confirming that the alpha(1)-adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.

Neurological improvement in patients with chronic spinal cord injury treated with leuprolide acetate, an agonist of GnRH.

It has been reported that gonadotropin­releasing hormone (GnRH), and its analogue leuprolide acetate (LA), have neurotrophic properties; particularly in the regeneration of injured spinal cord in animal models and in the case of a patient with spinal cord injury (SCI). The aim of this study was to establish whether treatment with LA improves sensitivity, motor activity and independence in patients with chronic SCI. Patients were treated LA once a month for six months. They were evaluated at the beginning and at the end of treatment; using a sensitivity and motor impairment scale, according to the American Spinal Injury Association (ASIA), and grade of independence scale; employing the spinal cord independence measure (SCIM). Statistical analysis showed a significant improvement in the ASIA sensory score and the SCIM score when comparing the initial versus final evaluation after six months of LA administration. Some patients showed an increase in frequency of bowel movements. Treatment with LA induces improvements in sensitivity, motor activity and independence in patients with chronic SCI. One advantage of this protocol is that it is a non-invasive method of easy and safe application, with few side effects.

LHRH antagonist attenuates the effect of fluoxetine on marble-burying behavior in mice.

Leuprolide--a luteinizing hormone-releasing hormone (LHRH) agonist, dose dependently (100, 200 and 300 microg/kg, s.c.) inhibited marble-burying behavior in mice, which was comparable to that of fluoxetine (10 and 15 mg/kg, i.p.)--a drug used in the treatment of obsessive-compulsive disorder. Co-administration of sub-effective dose of leuprolide (50 microg/kg) and fluoxetine (5 mg/kg) significantly inhibited marble-burying-behavior. Pre-treatment with parachlorophenylalanine [300 mg/kg, i.p. (x3 days)]--a serotonin depleting agent, reversed the effect of fluoxetine, whereas partially attenuated the effect of leuprolide. Further, LHRH antagonist pre-treatment (2.5 microg/mouse, s.c.) completely blocked the effect of leuprolide and reduced the effect of fluoxetine. Motor activity remained unaffected after all treatments. In conclusion, the findings suggest that fluoxetine also implicates LHRH in its anti-compulsive effect.

The effect of intracerebroventricular infusions of leptin on the immunoreactivity of neuropeptide Y and gonadotrophin releasing hormone neurons in the hypothalamus of prepubertal sheep in conditions of short fasting.

In the study we evaluated the effects of infusion of exogenous leptin to the third ventricle of the brain on the expression of immunoreactive (ir) neuropeptide Y (NPY) neurons in the hypothalamus and ir gonadotrophin releasing hormone (GnRH) nerve terminals in the median eminence of prepubertal lambs in the conditions of short fasting. Merino female sheep (n=16) were randomly divided into four groups, two fed with standard feeds and two fasted for 72 h. One standard and one fasted groups were infused with Ringer saline (controls), remaining standard and fasted groups with leptin (25 microg/120 microl/h), for 4 h during three consecutive days, and then slaughtered. Ir NPY and ir GnRH were localized by immunohistochemistry using specific polyclonal antibodies. Detection of both hormones was followed by the image analysis and expressed as the percent area stained and integral density of immunostaining. In the hypothalami from all groups the ir NPY perikarya and varicose nerve fibers were localized in three distinct sub-areas, in the arcuate (ARC), paraventricular and periventricular nuclei. In fasted sheep the percent area and integral density for immunoreactivity of NPY increased significantly (P<0.001) in three sub-areas compared to the standard-fed animals. Leptin infusion lowered the both parameters (P<0.001) but solely in the ARC NPY population of fasted sheep. The percent area and integral density of immunostaining for ir GnRH in fasted sheep revealed the augmentation (P<0.001) compared to standard-fed sheep. Leptin infusions diminished (P<0.001) both parameters in fasted, without effects in standard-fed lambs. In conclusion, the enhanced by fasting immunoreactivity of the ARC NPY perikarya and varicose nerve fibers and restrained immunoreaction of GnRH terminals in the median eminence were reversed by exogenous leptin. It is suggested that leptin can affect GnRH release via ARC NPY neurons in conditions of deficit of nutrients in prepubertal, female lambs.