Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Includes bibliographical references. Also available online.

Frankfurt (Main), Univ., Diss., 2003.

Lactation is the final stage of reproduction in mammals and is characterized by chronically elevated prolactin and suppressed luteinizing hormone. The neuroendocrine regulation of prolactin and luteinizing hormone during lactation are not fully understood. In the hypothalamic arcuate nucleus is a population of neurons known as KNDy neurons because they co-express the neuropeptides Kisspeptin, Neurokinin B and Dynorphin. These neurons are known to project to gonadotropin-releasing hormone cell bodes in the preoptic area and nerve terminals in the median eminence, which regulate the secretion of luteinizing hormone, and to dopaminergic tuberoinfundibular neurons in the arcuate nucleus, which are known to regulate prolactin. Because KNDy neurons project to neuronal populations known to regulate both prolactin and luteinizing hormone, the general hypothesis for these studies is that neuropeptides Kisspeptin, Neurokinin B and Dynorphin play a role in regulating these hormones or are regulated by these hormones during lactation. In a model of lactating rats deprived of their pups for 24 hours, intracerebroventricular injection of an endogenous Kisspeptin receptor ligand, Kp-10, modestly increased prolactin secretion and markedly increased luteinizing hormone secretion. Neither Neurokinin B nor the Neurokinin B receptor agonist, Senktide, had a significant effect on either hormone in this rat model. Dynorphin and U-50,488, a kappa opioid receptor agonist, robustly increased prolactin although no changes were measured in luteinizing hormone levels. In this model of 24-hour pup-deprived lactating rats, prolactin was responsive to kappa opioid receptor agonists and luteinizing hormone was responsive to Kisspeptin receptor agonists. In a second set of experiments, sense and anti-sense in situ hybridization probes were developed for Kiss1, the Kisspeptin gene, and Tac2, the gene encoding Neurokinin B. It was confirmed that the cDNA sequences cloned for these mRNAs were correct and were highly homologous to published rat mRNA sequences. In situ hybridization was performed using the Kiss1 and Tac2 probes, as well as a probe for Pdyn, which encodes Dynorphin. No specific cytoplasmic signal was observed using any of the three sense probes. With the anti-sense probes, clusters of reduced silver grains representing Kiss1, Tac2 and Pdyn mRNAs were observed in the arcuate nucleus, lateral to the third ventricle and superior to the median eminence. These expression patterns were consistent with the published literature. Also, the expression patterns for all three neuropeptides were similar to each other, suggesting that many of the arcuate nucleus neurons lateral to the third ventricle and superior to the median eminence are KNDy neurons.

Important physiological changes occur during lactation to allow for nourishment of the offspring. Specific neuronal groups within the arcuate nucleus of the hypothalamus influence prolactin (PRL) secretion, metabolism and fertility during lactation. Our overall goal was to identify gene expression changes in the arcuate nucleus during lactation and examine the roles of PRL and ovarian hormones in regulating expression of select genes. We evaluated transcriptome changes in the arcuate nucleus during lactation using RNA-sequencing. Thirty-seven differentially expressed genes, including neuropeptides, signaling molecules, receptors and enzymes, were identified between suckled and pup-deprived groups. Selected genes were evaluated by qRT-PCR in ovary-intact and ovariectomized lactating models, which included non-lactating, suckled and 24hr pup-deprived lactating groups. The mRNA expression of tyrosine hydroxylase (Th), kisspeptin (Kiss1), and neurokinin B (Tac3) was decreased, whereas mRNA expression of proenkephalin (Penk), parathyroid hormone 2 receptor (Pth2r), insulin-like growth factor binding protein 3 (Igfbp3), membrane progesterone receptor beta (Paqr8), suppressor of cytokine signaling 2 (Socs2) and cytokine-inducible SH2 domain-containing protein (Cish) was increased in suckled lactating rats. In 24hr pup-deprived dams, mRNA expression of Pth2r, Igfbp3, Paqr8, Socs2 and Cish was decreased and Th was increased, as compared to suckled rats. The mRNA expression of Kiss1 and Tac3 was increased and Penk was decreased after 72hr, but not 24hr, pup deprivation suggesting gene expression of these neuropeptides is slow to return to non-lactating levels after removing the suckling stimulus. Tyrosine hydroxylase (TH) protein and enkephalin (ENK) peptide expression was examined by immunohistochemistry. Lactating rats had increased ENK in the median eminence and decreased TH in the median eminence and arcuate nucleus as compared to virgin ovariectomized rats. ENK co-localization with TH in the arcuate nucleus was more predominant in lactating rats. Penk, Igfbp3, Pth2r, Cish, and Socs2 mRNA expression was decreased after 72hr bromocriptine treatment in suckled rats, suggesting that these genes are PRL-regulated. In contrast, gene expression of Th, Tac3 and Kiss1 were increased and Paqr8 was decreased with 72hr pup-deprivation, but expression of these genes, were not altered with bromocriptine treatment, indicating that these genes are regulated by a non-PRL component of the suckling stimulus. The mRNA expression of Kiss1, Socs2 and Igfbp3 was increased and Penk was decreased in ovariectomized as compared to ovary intact lactating rats, suggesting that ovarian hormones influence the expression of these genes during lactation. Our data show gene expression changes in the arcuate nucleus that may contribute to increased PRL secretion (Th, Penk and Pth2r), decreased PRL receptor signaling (Cis and Socs2), reduced fertility (Kiss1 and Tac3), increased metabolism (Igfbp3) and support a role for progesterone membrane actions (Paqr8). The expression of some genes appeared to be selectively regulated by ovarian hormone input and/or PRL feedback.

Orientador: Luis Antonio Justulin Junior / Resumo: A próstata é uma glândula do sistema genital masculino que possui grande importância na fertilidade. Estudos clássicos evidenciaram que para que se inicie o desenvolvimento prostático é necessário a presença de andrógeno produzido pelos testículos fetais. Porém, além da estimulação androgênica, estudos apontam para outros hormônios que também atuam na próstata como a Prolactina (PRL). PRL é um hormônio que é principalmente secretado por células lactotróficas da hipofise anterior e está envolvido em muitos processos biológicos, incluindo lactação e reprodução. Assim, investigamos se a modulação da sinalização PRL altera a morfofisiologia da próstata ventral (VP) em ratos castrados. Os ratos Sprague Dawley adultos (n = 6) foram castrados e após 21 dias divididos em 10 grupos experimentais: Castrado Controle (CC): animais castrados que não receberam tratamento; Castrado+testosterona (T): animais castrados que receberam T (4mg / kg); Castrado+PRL (PRL): animais castrados recebendo PRL (0,3 mg / kg); Castrado+T+PRL (TPRL): animais castrados que receberam associação de T e PRL; e Castrado+BR (BR): animais castrados que receberam Bromocriptina (BR) (0,4mg / kg). Grupo Controle: animais intactos (CTR). Os animais foram tratados durante 3 ou 10 dias consecutivos. Ao fim dos tratamentos, os animais foram anestesiados, eutanizados e a prostata ventral (VP) foi removida, pesada e processada para análise histológica e Western blot. O peso corporal não se alterou entre os grupos experiment... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Depression and other mental disorders are the leading cause of disability worldwide and their burden has increased considerably over past decades. However, advances in psychopharmacology of psychiatric disorders are not in measure with this negativ trend. As a result, a large body of researchinpsychiatryandneurosciencestries to furtherourunderstanding of pathophysiologicalmechanismsunderlyingmooddisorders andothermentalillnesses in order to improve the efficacy of current treatments and to identify new therapeutic agents. According to current evidence, stress-related pathways and inflammation processes are directly involved in thedevelopment of depressive disorder andseveral other psychiatric conditions.Thestudy of the effects and consequences of stress exposure requires an interdisciplinary approach,taking into account specific aspects of the "inputs", such as chronic stress and traumatic experiences, and related psychological processes, with the crucial role of dissociation. Following these theoretical findings, the empirical research performed in two cohorts of inpatients with depressive disorder focused on immune and endocrine responses to stress and their relationship to psychopathological symptoms, specifically trauma-related symptoms, psychic and somatoform dissociation and depressive...

O presente estudo visou caracterizar a secreção de PRL e estudar suas interrelações com a PGFM durante a pré-luteólise, luteólise e pós-luteólise em éguas (Experimento 1); avaliar o efeito da inibição de PRL e PGF2α na luteólise e definir a sincronia entre PRL e PGFM em novilhas (Experimento 2); definir a sincronia entre PRL e PGFM em éguas (Experimento 3); e avaliar a constante estimulação da PRL durante o ciclo estral em éguas (Experimento 4). No experimento 1 em éguas, amostras de sangue foram coletadas durante as 24 h da préluteólise, luteólise e pós-luteólise. As concentrações de PRL e PGFM foram rítmicas, sendo a duração dos pulsos de PRL de 5 h, com intervalos de 7,5 h entre pulsos e 12 h entre picos. Durante a luteólise e pós-luteólise, os pulsos de PRL foram mais proeminentes, as concentrações de PRL durante um pulso de PGFM foram maiores no pico de PGFM e notouse uma maior sincronia entre picos de PRL e PGFM. No experimento 2 em novilhas, as secreções de PRL e PGF2α foram inibidas durante a luteólise. A inibição da PRL associou-se a maiores concentrações de P4 e LH, sem efeito sobre a PGFM. Entretanto, a inibição da PGF2α associou-se a uma queda nas concentrações de PRL. A mensuração da área do CL mostrou-se eficiente em detectar a luteólise. No experimento 3 em éguas, no verão e outono, inibiu-se a secreção de PGF2α e PRL no Dia 14. As concentrações de PGFM foram reduzidas com a inibição de PGF2α, mas não com a inibição da PRL. No verão, a inibição tanto de PRL quanto de PGF2α reduziu as concentrações de PRL. As concentrações de PGFM não diferiram entre o verão e o outono, enquanto que as concentrações de PRL foram menores no outono. No experimento 4 em éguas, estimulou-se a secreção de PRL a cada 8 h. Amostras de sangue foram coletadas a cada 12 h do Dia 13 até a ovulação e a cada hora por 12 h no Dia 14. A estimulação repetida da PRL não aparentou manter as concentrações de PRL elevadas após o Dia 14. Nas amostras a cada hora, concentrações de PRL atingiram um valor máximo 4 horas após a estimulação e os pulsos de PRL foram aumentados. O aumento na PRL não afetou a PGFM, P4 e fluxo sanguíneo do CL. Entretanto, a estimulação da PRL quebrou a sincronia entre PGFM e PRL. Estão contidos nessa dissertação o primeiro relato em éguas sobre a caracterização e ritmicidade de pulsos de PRL, sincronia entre pulsos de PRL e PGFM e maior atividade da PRL durante a luteólise e pós-luteólise. A inibição da PRL interferiu na secreção de P4 em novilhas, mas foi confundida pelo aumento de LH. A sincronia entre pulsos de PGFM e PRL representa um efeito positivo da PGF2α sobre a PRL, tanto em éguas quanto em novilhas. / The aim of the present study was to characterize the PRL secretion and study the relationship between PRL and PGFM during preluteolysis, luteolysis and postluteolysis in mares (Experiment 1); evaluate the effect of PRL and PGF2α inhibition on luteolysis and define the synchrony between PRL and PGFM in heifers (Experiment 2); define the synchrony between PRL and PGFM in mares (Experiment 3); and evaluate the frequent stimulation of PRL during the estrous cycle in mares (Experiment 4). On experiment 1 in mares, blood samples were collected during the 24 h of preluteolysis, luteolysis and postluteolysis. Concentrations of PRL and PGFM were rhythmic. Prolactin pulses had 5h of duration, interval of 7,5 h between pulses, and 12 h between peaks. Pulses of PRL were more prominent during luteolysis and postluteolysis. Concentrations of PRL during PGFM pulses differ during luteolysis and postluteolysis, and were greater at the peak of PGFM. The synchrony between peaks of PRL and PGFM was greater during luteolysis and postluteolysis. On experiment 2 in heifers, the secretion of PRL and PGF2α were inhibited during luteolysis. The PRL inhibition was associated with greater concentrations of P4 and LH. The inhibition of PGF2α was associated with a decrease on PRL concentrations, but no effect on PGFM was observed after PRL inhibition. The CL area measurement was an efficient method to target luteolysis. On experiment 3 in mares, in summer and autumn, secretion of PGF2α and PRL were inhibited on Day 14. The inhibition of PGF2α reduced PGFM concentrations. No effect on PGFM was observed after PRL inhibition. Concentrations of PGFM were not different between summer and autumn, and PRL concentrations were low in the autumn. In the summer, PRL inhibition reduced PGF2α concentrations. On experiment 4 in mares, PRL was stimulated every 8 h. Blood samples were collected every 12 h from Day 13 to ovulation, and every hour for 12 h on Day 14. The frequent stimulation on PRL did not appear to maintain higher concentrations of PRL after Day 14. On hourly samples, concentrations of PRL reached maximum value 4 h after stimulation and pulses of PRL were increased. The increase on PRL did not affect PGFM, P4, and blood flow of the CL. The synchrony between PGFM and PRL was partially disrupted by PRL stimulation. This was the first report on characterization and rhythm of PRL pulses, synchrony between PRL and PGFM pulses, and greater PRL activity during luteolysis and postluteolysis. The inhibition of PRL interfered with P4 secretion in heifers, but was confounded by the LH increase. In mares and heifers, the synchrony between PGFM and PRL pulses represents a positive effect of PGF2α on PRL.

Mammotropes were successfully identified in the anterior pituitary gland of Miniopterus schreibersii using immunocytochemical (ICC) staining at the light and electron microscopy level. Mammotropes were distributed throughout the gland, were polygonal in shape and during secretory activity contained numerous large secretory granules (350 - 800nm). Using double ICC labelling, prolactin and growth hormone were never co-localiszed and found in individual cells only. Plasma prolactin levels were successfully measured on a monthly basis using radioimmunoassay and monthly pituitary prolactin levels were quantified using morphometric analysis of immunogold ICC staining and densitometry with polyacrylamide gels. Seasonal changes in the ultrastructure of mammotropes, and pituitary and plasma prolactin concentrations in female Miniopterus schreibersii indicated that there was an increase in prolactin secretion during the second half of the period of delayed implantation and that prolactin secretion remained elevated during normal embryonic development and lactation. This suggests that prolactin may be part of the luteotropic and lactogenic complex, and that the hormone might be responsible for terminating the period of delayed implantation. The latter is supported by experiments, where exogenous prolactin initiated precocious implantation during early delayed implantation, and treatment with bromocryptine (which inhibits prolactin synthesis) retarded implantation. Activation of mammotropes to synthesise prolactin and an increase of plasma prolactin levels occurred shortly after the winter solstice (21 June), suggesting that increasing daylength may be the environmental cue, which terminates the period of delayed implantation in Miniopterus schreibersii.

Spinal muscle atrophy (SMA) is an autosomal recessive neurodegenerative disease which is characterized by the loss of α motor neurons from the anterior horn of the spinal cord, resulting in progressive muscle atrophy. The loss of functional Survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene is the cause of SMA. A potential treatment strategy for SMA is to upregulate levels of the SMN protein originating from the copy gene SMN2 which can compensate in part for the absence of the functional SMN1 gene. I have shown a novel therapeutic strategy for SMA treatment through the activation of the p38 pathway by the bacterial antibiotic anisomycin which stabilizes and increases SMN mRNA levels in vitro. Activation of the p38 pathway by anisomycin leads to cytoplasmic accumulation of HuR protein which binds to the 3’UTR of SMN transcript resulting in increased SMN levels. This opens up a novel potential therapeutic strategy for SMA. I have also identified and demonstrated a significant induction of SMN protein levels in vitro and in vivo upon treatment with FDA approved drug celecoxib, which also activates the p38 pathway. Celecoxib mitigates disease severity along with increasing the lifespan of SMA mice. Sodium valproate, trichostatin A and aclarubicin, all agents which effectively enhance SMN2 expression, have been recently shown to activate STAT5 in SMA-like mouse embryonic fibroblasts and human SMN2-transfected NSC34 cells. Given that prolactin is also known to activate the STAT5 signalling pathway, can cross blood brain barrier and is FDA approved, we elected to assess its impact on SMN levels. In this manner, I have demonstrated a significant induction in SMN mRNA and protein levels in neuronal NT2 and MN-1 cells upon treatment with prolactin. I have also demonstrated that activation of the STAT5 pathway by prolactin is necessary for this transcriptional upregulation of the SMN gene. I have found that prolactin treatment induces SMN expression in brain and spinal cord samples and that it ameliorates the disease phenotype, improving motor neuron function and increasing survival in the SMA mouse model. Presently there is no cure for SMA. This study will help in the identification and characterization of potential therapeutic compounds for the treatment of SMA.

Binding sites for prolactin have been identified and characterized in a plasma membrane enriched fraction isolated from livers of mature female rats. By chemical and enzymatic analysis the membrane preparation was shown to have slight contamination with nuclei and endoplasmic reticulum, while mitochondria were not detected. Sidedness analysis indicated that the membrane preparation was largely composed of inside-out vesicles. ¹²⁵I-oPRL prepared by the lactoperoxidase method had a specific activity of 40-60 μCi/μg. Competition studies using iodoprolactin indicated that iodination of the hormone did not affect its affinity for the receptor as compared to the native hormone. Binding of ¹²⁵I-oPRL was inhibited by prolactin from various species including ovine, bovine and rat prolactin while bGH, pACTH and AVP had no effect on binding. The binding of 125 I-oPRL was activated by both bivalent and monovalent cations - bivalent cations exerting a greater effect than monovalent cations. In the presence of 10 mM CaCl₂, binding of ¹²⁵I-oPRL was equal to the binding in the presence of the physiological concentration of NaCI. The association of ¹²⁵I-oPRL with the membrane was a time and temperature dependent process, being maximal at 37°. The dissociation of ¹²⁵I-oPRL was time and temperature dependent only with 150 mM NaCl at 37° while at all other temperatures and in the presence of 10 mM CaCl₂ dissociation was not.observed. The binding of ¹²⁵I-oPRL was strongly influenced by pH with an optimum observed at pH 6.5. Receptor activity was destroyed by pronase and phospholipase C, while neuraminidase increased binding. Treatment of the membranes by RNase and DNase did not effect the binding. Binding of ¹²⁵I-oPRL was inhibited by p-chloromercuribenzoic acid, dithiothreitol, and by brief exposure to high temperatures. Scatchard analysis of the binding of ¹²⁵I-oPRL to receptors indicates that prolactin has a high affinity for its receptor / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate