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Osmoregulatory function of prolactin and prolactin-releasing peptide on gill cells of silver sea bream, Sparus sarba.January 2012 (has links)
催乳素是一種負責廣鹽性硬骨魚低滲環境適應的重要激素。最近發現的催乳素釋放肽被報導在魚類生理活動中參與了刺激催乳素釋放和滲透調節過程。本研究以黃錫鯛為實驗動物,旨在詮釋催乳素和催乳素釋放肽在廣鹽性硬骨魚中的滲透調節作用和調控機制。 / 實驗第一部份將黃錫鯛催乳素在大腸桿菌中過量表達並用鎳離子親和層析法將之純化。此重組黃錫鯛催乳素可被抗黃錫鯛催乳素抗體特異識別。將此重組催乳素注射入活體紅箭魚可導致其血漿鈉離子濃度和滲透壓顯著提高,從而證明了此催乳素的生物活性。在第二部份的研究中,黃錫鯛催乳素受體和催乳素釋放肽受體的全長編碼基因被克隆和分析。黃錫鯛催乳素受體和硬骨魚二型催乳素受體蛋白質相似性較高(可達47%至94%),但同魚類一型催乳素受體及其他高等脊椎動物催乳素受體同源性較低,相似性只有22%至37%。此催乳素受體廣泛表達在各種組織器官中,包括腦組織、腮、心臟、腸、腎、肌肉及性腺。黃錫鯛催乳素釋放肽受體屬於G-蛋白偶聯受體家族,它具備該家族成員的各種保守序列特徵。並且它含有同其他催乳素釋放肽受體一樣的PDZ域結合位點。組織表達分佈結果顯示此受體在中樞神經系統各部份及腮、心臟、肝臟和性腺中有表達。本研究最後一部份以黃錫鯛原代培養腮細胞為實驗對象,分別探究了腮細胞對於不同滲透壓力、催乳素和催乳素釋放肽處理的反應。在這些實驗中,我們檢測了腮細胞的死亡及凋亡狀況,并用實時定量PCR分析了一些滲透調節相關基因的表達狀況。另外,我們還採用了蛋白免疫印跡法檢測了腮細胞處於不同滲透壓條件下,其熱休克蛋白70的蛋白質豐度變化。我們的結果顯示:低滲壓力會引起腮細胞死亡包括誘導凋亡。催乳素和催乳素釋放肽處理均可以顯著降低低滲壓力所誘導的細胞死亡,並且催乳素還可以阻止一部份細胞進行凋亡。腮細胞中鈉鉀泵兩個亞基、熱休克同族蛋白70及催乳素受體的基因表達均被催乳素和催乳素釋放肽處理上調。但是水通道蛋白3的基因表達并未出現明顯的變化。當腮細胞暴露于高滲透壓力環境時,水通道蛋白3、囊性纤维化跨膜转运调节因子、鈉鉀泵兩個亞基和熱休克同族蛋白70的基因表達都明顯上調。與之相反,這些基因的表達在處於低滲環境的腮細胞中均被下調。 / 本研究提供了許多關於催乳素和催乳素釋放肽在魚類腮滲透調節過程中的詳細作用,并且首次報導了催乳素釋放肽可以直接作用于魚類滲透調節組織發揮功能從而參與滲透調節過程,而不只是如以往被認為的通過旁分泌或者自分泌方式刺激催乳素的表達來發揮生理功能。本研究的探索和發現使我們更加深刻地理解催乳素和催乳素釋放肽在廣鹽性硬骨魚鹽度適應過程中的調控機制。 / Prolactin (PRL) is well known as a crucial hormone responsible for fresh (hypoosmotic) water acclimation in euryhaline teleosts. The recently discovered prolactin-releasing peptide (PrRP) has been reported to take part in stimulating PRL release and affecting osmoregulatory processes in fish. This study aims to investigate the osmoregulatory effects and regulatory mechanisms of PRL and PrRP in a euryhaline teleost, silver sea bream (Sparus sarba). / First, silver sea bream prolactin (ssPRL) was over-expressed in E. coli by IPTG induction and purified by Ni-based immobilized metal ion affinity chromatography. This recombinant silver sea bream prolactin (rssPRL) was recognized by specific antibodies against ssPRL. Subsequently, its bioactivity was confirmed by in vivo injection to swordtails, which resulted in significant increase in plasma Na⁺ level and osmolality. In the second part, full-length cDNAs of silver sea bream PRL receptor (ssPRLR) and PrRP receptor (ssPrRPR) were cloned and characterized. ssPRLR shares high amino acid identities (47% to 94%) with teleost PRLR2s but low identities (22% to 37%) with piscine PRLR1s and higher vertebrate PRLRs. It is widely distributed in brain, gill, heart, gut, kidney, muscle and gonad. ssPrRPR belongs to G protein-coupled receptor (GPCR) family with all the conserved features of GPCRs, and possesses the special PDZ domain-binding motifs of other PrRPRs. Its expression was detected in the central nervous system, gill, heart, liver and gonad. Lastly, a primary gill cell culture of silver sea bream was developed and used as a tool for studying responses of gill cells following exposure to media of different osmotic stress, rssPRL and ssPrRP, respectively. Then, cytotoxicity and apoptosis assays were performed, and effects of these treatments on expression profiles of osmoregulatory genes were analyzed by real time PCR. Influence of osmotic stresses on protein abundance of heat shock protein 70s was examined by Western blot. The results showed that hypoosmotic challenge could induce cell death including apoptosis. Both PRL and PrRP treatment markedly decreased the induced cell death, and PRL treatment prevented some gill cells from apoptosis. Expression levels of Na⁺-K⁺-ATPase alpha (NKA-α) and beta (NKA-β) subunits, heat shock cognate 70 (HSC70) and PRLR were up-regulated in PRL- and PrRP- treated gill cells, however no significant effect on expression of aquaporin3 (AQP3) mRNA was apparent. After hyperosmotic exposure, expression levels of AQP3, cystic fibrosis transmembrane conductance regulator (CFTR), NKA-α, NKA-β, and HSC70 were significantly increased. In contrast, hypoosmotic exposure considerably down-regulated expression levels of these genes. / The present study represented considerable addition of details about the precise actions of PRL and PrRP in branchial osmoregulation. Furthermore, it first reported that PrRP can exert direct effects on fish osmoregulatory epithelia, instead of its oft presumed autocrine or paracrine action. All these efforts provide new insights into the control mechanisms of PRL and PrRP in euryhaline teleost during salinity acclimation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qu, Zhe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 108-142). / Abstracts also Chinese. / Title page --- p.i / Thesis committee --- p.ii / Acknowledgement --- p.iii / Abstract --- p.v / Abstract-Chinese version --- p.viii / Table of contents --- p.x / List of figures --- p.xiv / List of abbreviations --- p.xvi / Chapter Chapter 1 --- General introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.6 / Chapter 2.1 --- The role of gill in teleost osmoregulation --- p.7 / Chapter 2.1.1 --- An overview of fish osmoregulation --- p.7 / Chapter 2.1.2 --- The gill structure and epithelial ionocytes --- p.8 / Chapter 2.1.3 --- Osmoregulatory molecules in gill cells --- p.10 / Chapter 2.2 --- Piscine prolactin --- p.16 / Chapter 2.2.1 --- General features and distribution --- p.16 / Chapter 2.2.2 --- Prolactin receptor --- p.17 / Chapter 2.2.3 --- Prolactin and teleost osmoregulation --- p.18 / Chapter 2.3 --- Prolactin-releasing peptide in teleost --- p.21 / Chapter 2.3.1 --- Discovery and characteristics --- p.21 / Chapter 2.3.2 --- Distribution and its receptor --- p.22 / Chapter 2.3.3 --- Functions of PrRP --- p.23 / Chapter A. --- PrRP and prolactin release --- p.23 / Chapter B. --- PrRP and neuroendocrine stress response --- p.24 / Chapter C. --- Effects on food intake and energy metabolism --- p.26 / Chapter D. --- PrRP and cardiovascular system --- p.27 / Chapter Chapter 3 --- Production and characterization of recombinant silver sea bream (Sparus sarba) prolactin --- p.29 / Chapter Abstract --- p.30 / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.33 / Chapter 3.2.1 --- Experimental animals --- p.33 / Chapter 3.2.2 --- Construction of recombinant silver sea bream prolactin expression vector --- p.33 / Chapter 3.2.3 --- Large-scale production and purification of rssPRL --- p.34 / Chapter 3.2.4 --- SDS polyacrylamide gel electrophoresis and Western blotting analysis of rssPRL --- p.35 / Chapter 3.2.5 --- In vivo bioassay of rssPRL --- p.36 / Chapter 3.2.6 --- Statistical analysis --- p.37 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- Overexpression and purification of rssPRL --- p.38 / Chapter 3.3.2 --- Biological activity assay of rssPRL in vivo --- p.38 / Chapter 3.4 --- Discussion --- p.43 / Chapter Chapter 4 --- Molecular cloning and characterization of prolactin receptor (PRLR) and prolactin-releasing peptide receptor (PrRPR) in silver sea bream (Sparus sarba) --- p.46 / Chapter Abstract --- p.47 / Chapter 4.1 --- Introduction --- p.48 / Chapter 4.2 --- Materials and methods --- p.51 / Chapter 4.2.1 --- Fish and tissue samples --- p.51 / Chapter 4.2.2 --- Molecular cloning of full-length ssPRLR and ssPrRPR cDNA --- p.51 / Chapter 4.2.3 --- Multiple sequence alignments and phylogenetic analysis --- p.53 / Chapter 4.2.4 --- Tissue distribution of ssPRLR and ssPrRPR --- p.53 / Chapter 4.3 --- Results --- p.54 / Chapter 4.3.1 --- Cloning and characterization of silver sea bream PRLR and PrRPR cDNA --- p.54 / Chapter 4.3.2 --- Tissue distribution of ssPRLR and ssPrRPR mRNA --- p.56 / Chapter 4.4 --- Discussion --- p.65 / Chapter Chapter 5 --- Influences of osmotic stress and hormones on gill cell of silver sea bream (Sparus sarba) --- p.69 / Chapter Abstract --- p.70 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Methods and materials --- p.77 / Chapter 5.2.1 --- Fish and primary gill cell culture --- p.77 / Chapter 5.2.2 --- Experiments using gill cell culture --- p.78 / Chapter 5.2.3 --- Cell survival assay and apoptosis relevant detections --- p.79 / Chapter 5.2.4 --- Real time PCR analysis --- p.80 / Chapter 5.2.5 --- Western blot analysis of HSP70 --- p.82 / Chapter 5.2.6 --- Data processing and statistical analysis --- p.82 / Chapter 5.3 --- Results --- p.83 / Chapter 5.3.1 --- Hypoosmotic stress induced cell death --- p.83 / Chapter 5.3.2 --- Influences of PRL and PrRP on hypoosmotic challenge induced cell death --- p.84 / Chapter 5.3.3 --- Effects of hormones and osmotic stresses on expression levels of branchial genes --- p.84 / Chapter 5.3.4 --- Osmotic stresses induced changes in branchial protein amount of HSP70s --- p.85 / Chapter 5.4 --- Discussion --- p.94 / Chapter Chapter 6 --- General discussion and conclusions --- p.102 / References --- p.108
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Serotonin receptors in the regulation of prolactin release and some behaviors in the ratAlbinsson, Agneta. January 1995 (has links)
Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.
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Serotonin receptors in the regulation of prolactin release and some behaviors in the ratAlbinsson, Agneta. January 1995 (has links)
Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.
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Molecular characterization of the chicken prolactin receptor geneHui, Mei-yee, Angela., 許美儀. January 2004 (has links)
published_or_final_version / Zoology / Master / Master of Philosophy
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Immunohistochemical Localization of Prolactin Receptors Within the Equine OvaryOberhaus, Erin Lea 01 August 2012 (has links)
Prolactin receptors (PRLr) were detected in anestrous (n=3), winter cycling (n=2), follicular (n=3) and luteal phase (n=3) equine ovaries by IHC. Follicle stages evaluated were primordial, preantral and antral. Receptors were detected in all follicle stages and in CL. PRLr staining was not different (P > 0.05) between primordial and preantral, but was greater (P < 0.001) in antral follicles. Primordial follicles stained weakest in anestrous and follicular phase ovaries, followed by luteal phase ovaries and was most intense in winter cycling. Staining in preantral follicles was weakest in anestrus, followed by follicular phase and highest in winter cycling and luteal phase. Staining was most intense in antral follicles with no difference (P > 0.05) between any of the reproductive states. Oocytes and ovulation fossa also possessed PRLr. In conclusion, concentrations of PRLr are highest in large, antral follicles suggesting a mechanistic role for PRL around the time of ovulation.
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Cloning of prolactin receptor cDNA from Syrian golden hamster (Mesocricetus auratus).January 1996 (has links)
by Ng Yuen Keng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 141-148). / Table of contents --- p.1 / List of figures --- p.5 / List of tables --- p.12 / List of abbreviations --- p.13 / Abbreviation table for amino acids --- p.16 / Chapter Chapter 1 --- Literature Review --- p.17 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.2 --- The Hematopoietin/cytokine receptor superfamily --- p.20 / Chapter 1.3 --- The PRLR protein --- p.22 / Chapter 1.3.1 --- The receptor size --- p.22 / Chapter 1.3.2 --- Primary structure --- p.22 / Chapter 1.3.3 --- Structure of the extracellular domain --- p.26 / Chapter 1.3.4 --- Structure of the cytoplasmic domain --- p.30 / Chapter 1.3.5 --- Characteristics of specific PRL binding to PRLR --- p.32 / Chapter 1.5 --- The PRLR gene --- p.33 / Chapter 1.6 --- Heterogeneity of PRLR --- p.33 / Chapter 1.7 --- Signal transduction of PRLR --- p.35 / Chapter 1.7.1 --- JAK: a novel family of cytoplasmic protein tyrosine kinases --- p.35 / Chapter 1.7.2. --- Interaction between JAK2 and PRLR --- p.37 / Chapter 1.7.3 --- STAT proteins: mediators of PRL-dependent gene transcription --- p.37 / Chapter 1.7.4 --- Other signaling pathways of PRLR --- p.38 / Chapter 1.7.5 --- Future prospects on PRLR signaling --- p.38 / Chapter 1.8 --- Regulation of PRLR gene expression --- p.39 / Chapter 1.9 --- Objective of cloning the PRLR cDNA in male Syrian golden hamster --- p.42 / Chapter Chapter 2 --- PCR cloning of hamster PRLR cDNA fragment from adult male hamster liver --- p.44 / Chapter 2.1. --- Introduction --- p.44 / Chapter 2.2. --- Materials and Methods --- p.45 / Chapter 2.2.1 --- Primer design and PCR strategy --- p.45 / Chapter 2.2.2 --- Collection of liver --- p.46 / Chapter 2.2.3 --- Reverse transcription of polyadenylated RNA --- p.46 / Chapter 2.2.4 --- Nested PCR --- p.47 / Chapter 2.2.5 --- Southern analysis of the PCR products --- p.48 / Chapter 2.2.6 --- Subcloning of PCR product --- p.49 / Chapter 2.2.7 --- Sequence determination of the positive recombinant clone --- p.49 / Chapter 2.2.8 --- Sequence alignment and homology comparison --- p.50 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Nucleotide sequence alignment and primer design --- p.55 / Chapter 2.3.2 --- Nested PCR --- p.55 / Chapter 2.3.3 --- Subcloning of the PCR product --- p.56 / Chapter 2.3.4 --- Analysis of nucleotide and predicted amino acid sequences --- p.56 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3 --- Nucleotide sequence determination of the 5' and the 3' ends of hamster PRLR cDNA --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- Materials and Methods --- p.71 / Chapter 3.2.1 --- Collection of liver --- p.71 / Chapter 3.2.2 --- Total RNA preparation and poly (A) + RNA isolation --- p.72 / Chapter 3.2.3 --- Double stranded cDNA synthesis --- p.73 / Chapter 3.2.4 --- Adaptor ligation --- p.74 / Chapter 3.2.5 --- 5´ة and 3' RACE PCR --- p.74 / Chapter 3.2.6 --- Cloning of the RACE PCR products --- p.76 / Chapter 3.2.7. --- Sequence determination of the RA CE PCR products --- p.77 / Chapter 3.2.8. --- Sequence analysis of the RACE PCR products --- p.78 / Chapter 3 .2.9 --- Northern blot analysis of hamster PRLR mRNA in male hamster tissues --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.1.1 --- RNA preparation and double stranded cDNA synthesis --- p.79 / Chapter 3.3.2 --- RACE PCRfor the 5' and the 3' ends of hamster PRLR cDNA --- p.84 / Chapter 3.3.3 --- Cloning of the 5' and 3'RACE PCR products --- p.92 / Chapter 3.3.4 --- Sequence determination of the RACE PCR products --- p.92 / Chapter 3.3.5 --- Nucleotide sequence analysis of hamster PRLR full length cDNA --- p.101 / Chapter 3.3.6 --- Northern blot analysis of hamster PRLR --- p.101 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter 4 --- Attempts to study the PRLR gene expression in male hamster tissues --- p.113 / Chapter 4.1 --- Introduction --- p.113 / Chapter 4.2 --- Materials and Methods --- p.115 / Chapter 4.2.1 --- Collection of tissues --- p.115 / Chapter 4.2.2 --- Total RNA preparation and poly (A)+ RNA isolation --- p.116 / Chapter 4.2.3 --- Reverse Transcription --- p.116 / Chapter 4.2.4 --- Polymerase chain reaction for detecting the presence of hamster PRLR cDNA in various tissues --- p.117 / Chapter 4.2.5 --- Nested PCR for detecting heterogeneity in PRLR cDNA sizes in various tissues --- p.117 / Chapter 4.2.6 --- Analysis and quantitation of PCR products --- p.118 / Chapter 4.3 --- Results --- p.119 / Chapter 4.4 --- Discussion --- p.134 / Chapter Chapter 5 --- General Discussion --- p.137 / References --- p.141 / Appendices --- p.149 / Chapter I. --- "Stock solution preparation (Sambrook et al., 1989)" --- p.149 / Chapter II. --- List of primers --- p.152 / Primers for sequence determination --- p.152 / "Primer for first strand cDNA synthesis and 3' RACE PCR (Frohman et al., 1988 and Loh et al.,1989)" --- p.152 / "Primers for amplifying the actin cDNA fragment (Chan et al.,1995)" --- p.152 / Primers used for PCR-cloning and semi-quantitative analysis of hamster PRLR cDNA --- p.153 / Chapter III. --- "First strand cDNA synthesis primer, cDNA adaptor and adaptor primers used in the 5' and3' end sequence determinations of hamster PRLR cDNA" --- p.154 / Chapter IV. --- "Multiple cloning sites of the pCRII (Invitorgen), pUC 18 (Pharmacia) and pBluescript SK+ vectors (Clontech)" --- p.155 / Chapter VI. --- Nucleic acid molecular weight size markers --- p.158
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THE DYNAMICS OF THE GONADOTROPIN RECEPTOR POPULATION IN THE CORPUS LUTEUM OF THE RHESUS MONKEY (MACACA MULATTA) DURING THE MENSTRUAL CYCLECameron, Judy Lee January 1981 (has links)
The present investigation was designed to further our understanding of the interaction of pituitary (luteinizing hormone, LH) and placental (chorionic gonadotropin, CG) gonadotropins with the primate corpus luteum. Studies were performed (1) to characterize the LH/CG receptor population in the
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Molecular and functional characterization of the prolactin receptor, prolactin-releasing peptide receptor, and growth hormone-releasinghormone receptor genes in chickenWang, Ying, 王莹 January 2007 (has links)
published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy
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Expressão gênica da prolactina e seus receptores na hipófise e no útero de camundongo fêmea tratado com metoclopramida / Gene expression of prolactin and its receptors in the pituitary and uterus of the metoclopramide-treated female mouseAmaral, Vinícius Cestari do 05 July 2012 (has links)
INTRODUÇÃO: A prolactina é um hormônio polipeptídico, que possui reconhecida ação sistêmica, principalmente na fisiologia da reprodução, porém, seu desequilíbrio, em especial a hiperprolactinemia, é cada vez mais frequente na prática clínica. Apesar de ser um distúrbio relativamente comum, ainda existem dúvidas quanto aos efeitos moleculares da hiperprolactinemia no trato genital, particularmente no útero, e também na hipófise. O presente estudo teve por objetivo verificar os efeitos da hiperprolactinemia induzida pela metoclopramida na expressão gênica da prolactina e de seus receptores no útero e na hipófise de camundongo fêmea. MÉTODOS: Utilizaram-se 49 camundongos fêmeas (Wistar), randomicamente divididas em 7 grupos contendo 7 animais cada: 1) SS não ovariectomizadas que receberam solução salina (veículo); 2) M não ovariectomizadas tratadas com metoclopramida; 3) OSS ovariectomizadas tratadas com solução salina (veículo); 4) OM ovariectomizadas tratadas com metoclopramida; 5) OME ovariectomizadas tratadas com metoclopramida e 17-estradiol; 6) OMP ovariectomizadas tratadas com metoclopramida e progesterona micronizada; 7) OMEP ovariectomizadas tratadas com metoclopramida, 17-estradiol e progesterona micronizada. Após 50 dias os animais foram sacrificados sendo retirados o útero e a hipófise de cada animal para extração do ácido ribonucleico total, que foi utilizado para a síntese de ácido desoxirribonucleico complementar e avaliação da expressão gênica da prolactina e das diferentes isoformas de seus receptores, por reação em cadeia da polimerase em tempo real. RESULTADOS: Na hipófise, em animais não ovariectomizados, o tratamento com metoclopramida aumentou a expressão do gene que codifica a prolactina em relação ao tratamento apenas com o veículo. Nos animais castrados, a progesterona isoladamente ou associada ao estrogênio determinou o incremento do RNA mensageiro da prolactina em relação aos outros animais castrados que receberam outras combinações de tratamento. Este efeito foi semelhante ao da metoclopramida em animais com os ovários intactos. Em relação ao receptor de prolactina, o estrogênio e a progesterona, isoladamente, foram responsáveis pelo incremento da isoforma S2. No útero houve aumento na expressão de RNA mensageiro de prolactina após tratamento com metoclopramida ou com tratamento isolado ou combinado de estrogênio e progesterona. A ovariectomia determinou a redução da expressão das isoformas S1 e S2 do receptor de prolactina de todas as isoformas estudadas. Já o tratamento estroprogestativo determinou elevação da formas S3 e L do receptor, enquanto com a progesterona isoladamente causou apenas o incremento da forma L do receptor da prolactina no útero dos animais castrados. CONCLUSÕES: Nossos dados sugerem que o tratamento com metoclopramida altera de forma diferente a expressão de prolactina e de seus receptores quando se compara o resultado da hipófise em relação ao útero em camundongos fêmeas castrados e tratados com esteróides sexuais / INTRODUCTION: Prolactin is a polypeptide hormone with a recognized systemic action mainly on reproductive physiology. However, prolactin imbalance, particularly hyperprolactinemia, is increasingly more frequent in clinical practice. Although it is a comparatively common disorder, there are still doubts about the molecular effects of hyperprolactinemia on the genital tract especially in the uterus and the pituitary. The present study aimed at verifying the effects of metoclopramide-induced hyperprolactinemia on the gene expression of prolactin and its receptors in the uterus and pituitary of the female mouse. METHODS: Forty-nine female Wistar mice were randomized to 7 equal-sized groups as follows: 1) SS nonoophorectomized mice treated with saline solution (vehicle); 2) M nonoophorectomized mice treated with metoclopramide; 3) OSS oophorectomized mice treated with saline solution (vehicle); 4) OM oophorectomized mice treated with metoclopramide; 5) OME oophorectomized mice treated with metoclopramide and 17-estradiol; 6) OMP oophorectomized mice treated with metoclopramide and micronized progesterone; 7) OMEP oophorectomized mice treated with metoclopramide, 17-estradiol, and micronized progesterone. The animals were sacrificed 50 days after the end of the treatment, and the uterus and pituitary of each animal were removed for extraction of total ribonucleic acid, which was then used for synthesizing complementary deoxyribonucleic acid and for evaluating the gene expression of prolactin and the different isoforms of its receptors by the real-time polymerase chain reaction. RESULTS: In the pituitary of the nonoophorectomized mice, the treatment with metoclopramide against that with vehicle alone increased the expression of the prolactin-encoding gene. In the castrated animals, progesterone by itself or in conjunction with estrogen determined a raise in prolactin messenger RNA as opposed to the two other treatments with different combinations. This effect was similar to that produced by metoclopramide in animals with intact ovaries. Estrogen and progesterone, acting independently of each other, were responsible for the increase in the S2 isoform of the prolactin receptor. In the uterus, there was heightened expression of prolactin messenger RNA under the effect of the treatment with metoclopramide or with estrogen and/or progesterone. Oophorectomy caused a greater reduction in expression of the prolactin receptor S1 and S2 isoforms than in the other isoforms. However, the combined estrogen plus progesterone treatment led to an increase in the S3 and L forms of the receptor, while progesterone alone resulted solely in a higher expression of the L form of the prolactin receptor in the endometrium of the castrated mice. CONCLUSION: Our data suggest that metoclopramide treatment induces different changes in the expression of prolactin and its receptors according to whether the effect occurs in the pituitary or the uterus of castrated female mice treated with sex steroids
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Expressão gênica da prolactina e seus receptores na hipófise e no útero de camundongo fêmea tratado com metoclopramida / Gene expression of prolactin and its receptors in the pituitary and uterus of the metoclopramide-treated female mouseVinícius Cestari do Amaral 05 July 2012 (has links)
INTRODUÇÃO: A prolactina é um hormônio polipeptídico, que possui reconhecida ação sistêmica, principalmente na fisiologia da reprodução, porém, seu desequilíbrio, em especial a hiperprolactinemia, é cada vez mais frequente na prática clínica. Apesar de ser um distúrbio relativamente comum, ainda existem dúvidas quanto aos efeitos moleculares da hiperprolactinemia no trato genital, particularmente no útero, e também na hipófise. O presente estudo teve por objetivo verificar os efeitos da hiperprolactinemia induzida pela metoclopramida na expressão gênica da prolactina e de seus receptores no útero e na hipófise de camundongo fêmea. MÉTODOS: Utilizaram-se 49 camundongos fêmeas (Wistar), randomicamente divididas em 7 grupos contendo 7 animais cada: 1) SS não ovariectomizadas que receberam solução salina (veículo); 2) M não ovariectomizadas tratadas com metoclopramida; 3) OSS ovariectomizadas tratadas com solução salina (veículo); 4) OM ovariectomizadas tratadas com metoclopramida; 5) OME ovariectomizadas tratadas com metoclopramida e 17-estradiol; 6) OMP ovariectomizadas tratadas com metoclopramida e progesterona micronizada; 7) OMEP ovariectomizadas tratadas com metoclopramida, 17-estradiol e progesterona micronizada. Após 50 dias os animais foram sacrificados sendo retirados o útero e a hipófise de cada animal para extração do ácido ribonucleico total, que foi utilizado para a síntese de ácido desoxirribonucleico complementar e avaliação da expressão gênica da prolactina e das diferentes isoformas de seus receptores, por reação em cadeia da polimerase em tempo real. RESULTADOS: Na hipófise, em animais não ovariectomizados, o tratamento com metoclopramida aumentou a expressão do gene que codifica a prolactina em relação ao tratamento apenas com o veículo. Nos animais castrados, a progesterona isoladamente ou associada ao estrogênio determinou o incremento do RNA mensageiro da prolactina em relação aos outros animais castrados que receberam outras combinações de tratamento. Este efeito foi semelhante ao da metoclopramida em animais com os ovários intactos. Em relação ao receptor de prolactina, o estrogênio e a progesterona, isoladamente, foram responsáveis pelo incremento da isoforma S2. No útero houve aumento na expressão de RNA mensageiro de prolactina após tratamento com metoclopramida ou com tratamento isolado ou combinado de estrogênio e progesterona. A ovariectomia determinou a redução da expressão das isoformas S1 e S2 do receptor de prolactina de todas as isoformas estudadas. Já o tratamento estroprogestativo determinou elevação da formas S3 e L do receptor, enquanto com a progesterona isoladamente causou apenas o incremento da forma L do receptor da prolactina no útero dos animais castrados. CONCLUSÕES: Nossos dados sugerem que o tratamento com metoclopramida altera de forma diferente a expressão de prolactina e de seus receptores quando se compara o resultado da hipófise em relação ao útero em camundongos fêmeas castrados e tratados com esteróides sexuais / INTRODUCTION: Prolactin is a polypeptide hormone with a recognized systemic action mainly on reproductive physiology. However, prolactin imbalance, particularly hyperprolactinemia, is increasingly more frequent in clinical practice. Although it is a comparatively common disorder, there are still doubts about the molecular effects of hyperprolactinemia on the genital tract especially in the uterus and the pituitary. The present study aimed at verifying the effects of metoclopramide-induced hyperprolactinemia on the gene expression of prolactin and its receptors in the uterus and pituitary of the female mouse. METHODS: Forty-nine female Wistar mice were randomized to 7 equal-sized groups as follows: 1) SS nonoophorectomized mice treated with saline solution (vehicle); 2) M nonoophorectomized mice treated with metoclopramide; 3) OSS oophorectomized mice treated with saline solution (vehicle); 4) OM oophorectomized mice treated with metoclopramide; 5) OME oophorectomized mice treated with metoclopramide and 17-estradiol; 6) OMP oophorectomized mice treated with metoclopramide and micronized progesterone; 7) OMEP oophorectomized mice treated with metoclopramide, 17-estradiol, and micronized progesterone. The animals were sacrificed 50 days after the end of the treatment, and the uterus and pituitary of each animal were removed for extraction of total ribonucleic acid, which was then used for synthesizing complementary deoxyribonucleic acid and for evaluating the gene expression of prolactin and the different isoforms of its receptors by the real-time polymerase chain reaction. RESULTS: In the pituitary of the nonoophorectomized mice, the treatment with metoclopramide against that with vehicle alone increased the expression of the prolactin-encoding gene. In the castrated animals, progesterone by itself or in conjunction with estrogen determined a raise in prolactin messenger RNA as opposed to the two other treatments with different combinations. This effect was similar to that produced by metoclopramide in animals with intact ovaries. Estrogen and progesterone, acting independently of each other, were responsible for the increase in the S2 isoform of the prolactin receptor. In the uterus, there was heightened expression of prolactin messenger RNA under the effect of the treatment with metoclopramide or with estrogen and/or progesterone. Oophorectomy caused a greater reduction in expression of the prolactin receptor S1 and S2 isoforms than in the other isoforms. However, the combined estrogen plus progesterone treatment led to an increase in the S3 and L forms of the receptor, while progesterone alone resulted solely in a higher expression of the L form of the prolactin receptor in the endometrium of the castrated mice. CONCLUSION: Our data suggest that metoclopramide treatment induces different changes in the expression of prolactin and its receptors according to whether the effect occurs in the pituitary or the uterus of castrated female mice treated with sex steroids
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