O Grupo de Hormônios do Centro de Biotecnologia do IPEN desenvolveu várias linhagens de células de ovário de hamster chinês (CHO) modificadas geneticamente e comprovadamente eficientes na expressão de proteínas heterólogas, dentre elas a prolactina humana (hPRL) e os análogos antagonistas de prolactina (S179D-hPRL e G129R-hPRL). No entanto, todas as linhagens para expressão são cultivadas em monocamadas e dependentes da presença de soro fetal bovino (SFB) no meio de cultivo para um crescimento eficiente. As células em suspensão apresentam um grande interesse industrial-farmacêutico, tanto pela facilidade de cultivo e ampliação de escala, como pela produtividade volumétrica. Desenvolvemos um protocolo para adaptação de células CHO para o crescimento em suspensão e também processos de produção em frascos spinners. Nesse trabalho foi realizada a adaptação das linhagens produtoras de hPRL; S179D-hPRL e G129R-hPRL para o crescimento em suspensão e em meio livre de SFB. Realizamos também a produção em escala laboratorial com as três linhagens adaptadas, assim como a correspondente purificação e caracterização de quatro proteínas heterólogas, incluindo a prolactina humana glicosilada (G-hPRL). / The Hormone Group of the Biotechnology Center of IPEN has developed different cells lines of genetically modified chinese hamster ovary cells (CHO) for the expression of heterologus protein like human prolactin (hPRL) and its analogs/antagonists (S179D-hPRL and G129R-hPRL). All cell lines for expression are however cultured in monolayer culture dish and depend on fetal bovine serum (FBS) in the medium for an efficient growth. Cells in suspension show a great industrial-pharmaceutical interest, especially for the cultivation facility and scale enlargement as well as for volumetric productivity. We developed a protocol for adapting CHO cells to suspension growth, in spinner flasks. The adaption of our cell lines producing hPRL; S179D-hPRL and G129R-hPRL to suspension growth and in serum-free medium was obtained. We also carried out laboratory scale production with the three suspension-adapted culture line cells and the corresponding purification and characterization of four heterologous proteins, including glycosylated human prolactin (G-hPRL).

A prolactina é um neurohormônio que faz parte da superfamília das citocinas e está envolvida em inúmeros processos biológicos. Devido à sua ação endócrina, autócrina e parácrina, a prolactina está muitas vezes relacionada ao desenvolvimento de patogenias humanas como carcinomas e doenças autoimunes. Considerando-se que: a) diferença de 41% encontrada na sequência de aminoácidos da prolactina de camundongo em relação à humana, b) diferenças na glicosilação, fosforilação, e ligação ao receptor e, c) o fato que os modelos animais utilizados em ensaios in vivo com prolactina humana são geralmente ratos ou camundongos (modelos heterólogos), fica evidente que esses fatores podem interferir na interpretação dos resultados. Portanto, experimentos em sistemas homólogos seriam desejáveis. Esse trabalho descreve a obtenção pela primeira vez da mPRL no espaço periplásmico bacteriano, portanto na sua forma autêntica, ou seja, sem a metionina inicial, com expressão de 0,1 ± 13,2% g/mL/A600. Para isso um vetor de expressão baseado no promotor lPL foi construído e utilizado como promotor constitutivo, com ativação a 37° C. Um processo de fermentação em biorreator, com rendimentos de expressão de até 2,5 g/mL, e um processo de purificação com três etapas: concentração e purificação por hidrofobicidade (Phenyl Sepharose CL-4B) seguida por HPLC de fase reversa e HPSEC, foram também desenvolvidos. A mPRL purificada foi caracterizada por técnicas físico-químicas e biológicas em comparação com o padrão de referência da mPRL recombinante do Instituto Nacional de Saúde (NIH, EUA). A atividade biológica foi analisada e sua potência calculada foi de 33,9 ± 1,4 UI/mg. O mesmo vetor foi utilizado para a expressão do antagonista S177DmPRL, mas o baixo nível de expressão obtido inviabilizou a sua produção no espaço periplásmico de bactérias. Como alternativa, optamos por produzir essa proteína em células CHO e clones com expressão da ordem de 1 g/mL/dia foram obtidos. Com a expressão tanto da mPRL autêntica, como do S177D-mPRL, está aberto o caminho para o desenvolvimento dos estudos que envolvam modelos animais onde a mPRL e seu antagonista sejam fatores relevantes a serem considerados. / Prolactin is a neurohormone included in cytokine superfamily and involved in innumerous biological processes. Due to its endocrine, autocrine and paracrine action, it is, frequently, related to development of human pathologies, such as carcinomas and autoimmune diseases. Considering some factors: a) the difference of 41% between the amino acid sequence of mouse prolactin in relation to the human one, b) glycosylation, phosphorylation and receptor ligation differences, and c) the fact that the animal model used for in vivo assays with human prolactin are generally rats or mice (heterologous), it is evident that these factors might interfere in the interpretation of results. Therefore, experiments with homologous systems are desirable. This work describes, for the first time, the expression of mouse prolactin in the periplasmic space of bacteria, in its authentic form, i. e., without initial metionine, showing expression levels of 0.1 ± 13.2% g/mL/A600. For this purpose, an expression vector based on PL promoter was constructed and used as a constitutive form, with activation at 37° C. A fermentation process in bioreactor, with expression yields up to 2.5 g/mL, and purification process with three steps: concentration and purification by hydrophobicity (Phenyl Sepharose CL-4B) followed by reverse phase HPLC and HPSEC, were also developed. The mPRL was purified and characterized by chemo physical and biological techniques compared to the recombinant standard reference from the National Institute of Health (NIH, USA). Its biological activity was analyzed and the calculated potency was 33.9 ± 1.4 UI/mg. The same vector was used for the expression of S177D-mPRL antagonist, but the low expression level obtained makes it impracticable to produce this protein in the periplasmic space of bacteria. As an alternative, the S177D-mPRL was produced in CHO cells and clones with expression level of about 1 g/mL/day were obtained. The expression of both proteins, the authentic mPRL and the S177D-mPRL, opens the door for developing studies with animal models if mPRL and its antagonist might be considered relevant factors.

Objetivo: Foi investigada a hipótese da hiperprolactinemia modular a resposta inflamatória alérgica pulmonar em ratos machos e em fêmas lactantes sem tratamento de domperidona. Métodos: Em ratos machos, a hiperprolactinemia foi de curta duração (5 dias) induzida pela domperidona (5,1 mg.kg-1 por dia, i.p). A resposta alérgica foi gerada por sensibilização e desafios inalatórios com ovoalbumina. Foi feita contagem de leucócitos totais e diferenciados do lavado bronco alveolar (BAL), lavado medular femoral (BFL) e sangue; a percentagem de produção de muco e colageno no pulmão, níveis de corticosterona e prolactina e citocinas TNF-α, IL-4, IL-6, IL-10, em explantes de pulmão e IFNg no BAL, foram medidos. Pela citometria foram avaliadaos os receptores de prolactina; Resultados: Hiperprolactinemia de curta duração feita antes do desafio inalatório disminuiu a resposta alérgica pulmonar na contagem de leucócitos no lavado broncoalveolar. Esse tratamento reduziu a celularidade no BFL e a percentagem de muco e aumentou a expressão de citocinas IL-4, IL-6, IL-10, TNFα e da expressão do IFNg. Níveis altos de prolactina diminuiram o número de eosinófilos ao pulmão no BAL. Pela citometria revelou-se que além de ter menor número de granulócitos migrados ao pulmão, estes apresentaram maior expressão do número de receptores por granulócito para prolactina no grupo tratado com domperidona. Alterações similares foram reveladas em fêmeas lactantes como foi a diminuição nos leucócitos do BAL, e no número de células do BFL. O tratamento profilático diminuiu a resposta alérgica tanto no grupo hiperprolactinêmico como no grupo veículo. O tratamento feito após o desafio inalatório não evidenciou alterações relevantes nas variáveis medidas. Conclusões: A hiperprolactinemia de curta duração, feita após a sensibilização e antes da inalação diminui a resposta inflamatória no pulmão em ratos. Os resultados deste estudo demonstram que a hiperprolactinemia induzida antes do desafio antigênico diminue a inflamação alérgica pulmonar. Assim, é provável que a prolactina endógena tenha um papel relevante como um imunomodulador da asma. Este estudo aponta a possibilidade futura do uso da domperidona para pacientes asmáticos. Durante a primavera muitas espécies de mamíferos têm seus filhotes e ocorre abundância de fatores alergenos no ar. Logo, um fator endógeno que favoreça a proteção de fêmeas durante a lactação, tal como a hiperprolactinemia, tem elevado valor adaptativo / Objective: It was investigated if hyperprolactinemia has modulatory actions on lung allergic inflammatory response in male rats. Lactating female rats were tested for pulmonary allergy as well. Methods: In male rats, short-term (5 days) hyperprolactinemia was induced by domperidone (5.1 mg.kg-1 per day, ip). Allergic response was generated by sensitization and inhalation challenge with ovalbumin. Total and differential leukocytes bronchoalveolar lavage (BAL), femoral medullary lavage (BFL) and blood; the percentage of collagen and mucus production in the lungs, plasma levels of corticosterone and prolactin cytokines and TNF-α, IL-4, IL-6, IL-10, IFNg explants lung and BAL, were measured. Flow cytometry was used to evaluate prolactin receptor; Results: Short-term hyperprolactinemia made before the inhaled challenge reduced the pulmonary allergic response in white blood cell counts in BAL. This treatment reduced the cellularity in BFL and the percentage of mucus and increased expression of cytokines IL-4, IL-6, IL-10, TNFa and IFNg expression. High prolactin levels decreased the number of eosinophils to the lung in BAL. There were fewer granulocytes migrated to the lung. These granulocytes showed higher expression prolactin receptors in hyperprolactinemia animals. Similar changes were revealed in lactating females. In these animals, there was a reduction in BAL leukocyte, and the number of cells BFL. Prophylactic treatment decreased the allergic response in both hyperprolactinemic and vehicle groups. The treatment made after inhalational challenge did not induce significant changes in the variables measured in this study. Conclusions: Short-term hyperprolactinemia, made after sensitization and before inhalation, decreases the inflammatory response in the lung of rats. The results of this study demonstrate that hyperprolactinemia, induced before antigen challenge, decreases pulmonary allergic inflammation. Thus, it is probable that the endogenous prolactin has an important role as an immunomodulator of asthma. This study points out the prospect of a future use of domperidone for asthmatic patients. For various mammalian species, parturition occurs during springtime. Pollen in the air might be an abundant allergic factor during springtime. Thus, protecting lactating females against this type allergy might have high adaptive value

Finally, the isolation and cloning of black sea bream SL receptor using PCR cloning and protein pull down assay were also attempted. Based on the PCR cloning results, the phylogenetic analysis of nonsalmonids fish GHR1 and SLR protein sequence, the GHR1 data of tissue distribution and effects of environmental salinity and fasting in tilapia, along with the results of far western blot, black sea bream GHR1 is probably a receptor for SL, however there is also a SL specific receptor in black sea bream. / In hormone treated primary cell culture of nonspawning black sea bream pituitary, 10-8 M E2 significantly increases SL mRNA level but 10-10 M, 10-9 M, 10-8 M of E2 inhibit GH mRNA level in female black seabream; 10-8 M E2 also inhibits SL and GH mRNA expression in bisexual black sea bream; 10-8 M MT inhibits SL mRNA expression in male black sea bream but any concentration of MT detected shows no significant effect on GH mRNA level. / Key words. somatolactin (SL), monthly changes, SL promoter, pit-1 and SL receptor / Somatolactin, SL, is a novel member of GH family of pituitary hormone only found in fish. It is considered to be a member of the GH gene family after gene duplication. Two types of SL, SL alpha and SL beta were identified, and SL 13 seems only in fresh water fish, such as goldfish, catfish, rainbow trout, eel and zebrafish. Black sea bream is a marine fish, and there is only SL alpha found from sequencing of over 100 SL cDNA clones. / The cDNAs encoding for transcription factor pit-1 variants were cloned and the transactivation of these Pit-1 isoforms on SL gene promoter were studied. Three variants of Pit-1 are first identified in fish. Pit-1b and Pit-1c can enhance SL promoter activity in Hepa-T1 cells respectively to about 2 fold and 12 fold, but pit-1a failed to activate the SL gene it in the same cells. All the three pit-1s of black sea bream couldn't reverse the inhibition of SL promoter in GH3 cells. The data suggest that N terminal 60 amino acid residues are critical in transactiation on SL promoter and SL promoter activity is possibly limited to fish SL secreting cells. / The SL gene promoter was obtained for gene regulation studies aiming to search for possible regulatory elements controlling the transcription of SL gene in black seabream. SL gene promoter is active in HepaT1 cells, but is inhibited in GH3 cells. Seven putative pit-1 response elements were confirmed with EMSA and super shift assay. / To study the physiological function of SL in black seabream, we initiated a study of monthly expressions of SL mRNA and gonadal somatic index (GSI) to determine whether SL is related to reproduction in black seabream, with GH mRNA levels were also detected for comparison. The results imply that function of SL is possibly related to early development of testis, while GH probably plays some roles in testis and ovary maturation. / by Tian, Jing. / "October 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4574. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 156-170). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Objetivo: Foi investigada a hipótese da hiperprolactinemia modular a resposta inflamatória alérgica pulmonar em ratos machos e em fêmas lactantes sem tratamento de domperidona. Métodos: Em ratos machos, a hiperprolactinemia foi de curta duração (5 dias) induzida pela domperidona (5,1 mg.kg-1 por dia, i.p). A resposta alérgica foi gerada por sensibilização e desafios inalatórios com ovoalbumina. Foi feita contagem de leucócitos totais e diferenciados do lavado bronco alveolar (BAL), lavado medular femoral (BFL) e sangue; a percentagem de produção de muco e colageno no pulmão, níveis de corticosterona e prolactina e citocinas TNF-α, IL-4, IL-6, IL-10, em explantes de pulmão e IFNg no BAL, foram medidos. Pela citometria foram avaliadaos os receptores de prolactina; Resultados: Hiperprolactinemia de curta duração feita antes do desafio inalatório disminuiu a resposta alérgica pulmonar na contagem de leucócitos no lavado broncoalveolar. Esse tratamento reduziu a celularidade no BFL e a percentagem de muco e aumentou a expressão de citocinas IL-4, IL-6, IL-10, TNFα e da expressão do IFNg. Níveis altos de prolactina diminuiram o número de eosinófilos ao pulmão no BAL. Pela citometria revelou-se que além de ter menor número de granulócitos migrados ao pulmão, estes apresentaram maior expressão do número de receptores por granulócito para prolactina no grupo tratado com domperidona. Alterações similares foram reveladas em fêmeas lactantes como foi a diminuição nos leucócitos do BAL, e no número de células do BFL. O tratamento profilático diminuiu a resposta alérgica tanto no grupo hiperprolactinêmico como no grupo veículo. O tratamento feito após o desafio inalatório não evidenciou alterações relevantes nas variáveis medidas. Conclusões: A hiperprolactinemia de curta duração, feita após a sensibilização e antes da inalação diminui a resposta inflamatória no pulmão em ratos. Os resultados deste estudo demonstram que a hiperprolactinemia induzida antes do desafio antigênico diminue a inflamação alérgica pulmonar. Assim, é provável que a prolactina endógena tenha um papel relevante como um imunomodulador da asma. Este estudo aponta a possibilidade futura do uso da domperidona para pacientes asmáticos. Durante a primavera muitas espécies de mamíferos têm seus filhotes e ocorre abundância de fatores alergenos no ar. Logo, um fator endógeno que favoreça a proteção de fêmeas durante a lactação, tal como a hiperprolactinemia, tem elevado valor adaptativo / Objective: It was investigated if hyperprolactinemia has modulatory actions on lung allergic inflammatory response in male rats. Lactating female rats were tested for pulmonary allergy as well. Methods: In male rats, short-term (5 days) hyperprolactinemia was induced by domperidone (5.1 mg.kg-1 per day, ip). Allergic response was generated by sensitization and inhalation challenge with ovalbumin. Total and differential leukocytes bronchoalveolar lavage (BAL), femoral medullary lavage (BFL) and blood; the percentage of collagen and mucus production in the lungs, plasma levels of corticosterone and prolactin cytokines and TNF-α, IL-4, IL-6, IL-10, IFNg explants lung and BAL, were measured. Flow cytometry was used to evaluate prolactin receptor; Results: Short-term hyperprolactinemia made before the inhaled challenge reduced the pulmonary allergic response in white blood cell counts in BAL. This treatment reduced the cellularity in BFL and the percentage of mucus and increased expression of cytokines IL-4, IL-6, IL-10, TNFa and IFNg expression. High prolactin levels decreased the number of eosinophils to the lung in BAL. There were fewer granulocytes migrated to the lung. These granulocytes showed higher expression prolactin receptors in hyperprolactinemia animals. Similar changes were revealed in lactating females. In these animals, there was a reduction in BAL leukocyte, and the number of cells BFL. Prophylactic treatment decreased the allergic response in both hyperprolactinemic and vehicle groups. The treatment made after inhalational challenge did not induce significant changes in the variables measured in this study. Conclusions: Short-term hyperprolactinemia, made after sensitization and before inhalation, decreases the inflammatory response in the lung of rats. The results of this study demonstrate that hyperprolactinemia, induced before antigen challenge, decreases pulmonary allergic inflammation. Thus, it is probable that the endogenous prolactin has an important role as an immunomodulator of asthma. This study points out the prospect of a future use of domperidone for asthmatic patients. For various mammalian species, parturition occurs during springtime. Pollen in the air might be an abundant allergic factor during springtime. Thus, protecting lactating females against this type allergy might have high adaptive value

O Grupo de Hormônios do Centro de Biotecnologia do IPEN desenvolveu várias linhagens de células de ovário de hamster chinês (CHO) modificadas geneticamente e comprovadamente eficientes na expressão de proteínas heterólogas, dentre elas a prolactina humana (hPRL) e os análogos antagonistas de prolactina (S179D-hPRL e G129R-hPRL). No entanto, todas as linhagens para expressão são cultivadas em monocamadas e dependentes da presença de soro fetal bovino (SFB) no meio de cultivo para um crescimento eficiente. As células em suspensão apresentam um grande interesse industrial-farmacêutico, tanto pela facilidade de cultivo e ampliação de escala, como pela produtividade volumétrica. Desenvolvemos um protocolo para adaptação de células CHO para o crescimento em suspensão e também processos de produção em frascos spinners. Nesse trabalho foi realizada a adaptação das linhagens produtoras de hPRL; S179D-hPRL e G129R-hPRL para o crescimento em suspensão e em meio livre de SFB. Realizamos também a produção em escala laboratorial com as três linhagens adaptadas, assim como a correspondente purificação e caracterização de quatro proteínas heterólogas, incluindo a prolactina humana glicosilada (G-hPRL). / The Hormone Group of the Biotechnology Center of IPEN has developed different cells lines of genetically modified chinese hamster ovary cells (CHO) for the expression of heterologus protein like human prolactin (hPRL) and its analogs/antagonists (S179D-hPRL and G129R-hPRL). All cell lines for expression are however cultured in monolayer culture dish and depend on fetal bovine serum (FBS) in the medium for an efficient growth. Cells in suspension show a great industrial-pharmaceutical interest, especially for the cultivation facility and scale enlargement as well as for volumetric productivity. We developed a protocol for adapting CHO cells to suspension growth, in spinner flasks. The adaption of our cell lines producing hPRL; S179D-hPRL and G129R-hPRL to suspension growth and in serum-free medium was obtained. We also carried out laboratory scale production with the three suspension-adapted culture line cells and the corresponding purification and characterization of four heterologous proteins, including glycosylated human prolactin (G-hPRL).

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.

The pituitary hormone prolactin (Prl) is essential for alveolar morphogenesis and plays a role in breast carcinogenesis, however the mechanism that underlies these actions remains to be defined. Alterations in serum Prl provide the primary endocrine signal regulating developmental events in the mammary gland in sexually mature mammals. Prl production and post-translational phosphorylation by the pituitary is regulated by the neuropeptide Galanin (Gal) in response to hypothalamic signals integrating neuronal and endocrine inputs. Prl exerts its effects on the mammary epithelium in two ways, indirectly by modulation of the systemic hormonal environment, for example the release of progesterone from the corpus luteum, and directly by binding to Prl receptors (Prlr) within the mammary epithelium. Prl binding to Prlr initiates signalling predominantly via activation of the Jak2/Stat5 pathway, leading to altered patterns of gene transcription. One of these target genes is the ets transcription factor Elf5, which is required by the epithelium for alveolar morphogenesis. This thesis aims to further our understanding of the mechanisms by which prolactin exerts its influence on the mammary gland during alveolar morphogenesis and carcinogenesis. Transcript profiling revealed a lactation signature of 35 genes in Prlr+/- mice, Gal-/- mice and mice treated with a Prl mutant (S179D) that mimics phosphorylated Prl. We discovered that the majority of changes in gene expression were produced by prolactin rather than by Gal. The action of Gal was predominantly via modulation of Prl phosphorylation and release, as its effects were very similar to that of S179D. Knockout of Elf5 phenocopied knockout of Prlr, resulting in failure of alveolar morphogenesis and reduced expression of milk and lipid synthesis genes. Forced Elf5 expression at puberty resulted in aberrant differentiation of the terminal end buds and milk protein synthesis during ductal morphogenesis. Re-expression of Elf5 in Prlr-/- mammary epithelial cells completely rescued alveolar morphogenesis. These observations indicate that Elf5 is a master regulator of alveolar morphogenesis downstream of the Prlr. Loss of mammary epithelial Prlr resulted in reduced proliferation of low-grade neoplastic lesions resulting in increased tumour latency in the C3(1)/SV40T model of mammary carcinogenesis. There was no change in the growth rate, proliferation nor the morphology of tumours in Prlr-/-/C3(1)/SV40T transplants, thus Prl acts early in carcinogenesis to drive the proliferation of pre-invasive lesions resulting in faster progression to cancer.

Maternal behavior in rodents is dependent, at least in part, on prolactin acting in the brain. Pheromones carried by male mouse major urinary proteins lower serum prolactin levels in female mice. Therefore, we hypothesized that virgin female C57BL/6J mice housed in split cages, where they had pheromonal but not physical contact with a male, would show suppressed maternal behavior. Contrary to our hypothesis, we found split-cage housed females were significantly faster to retrieve 3 foster pups on the first and second day of maternal behavior testing compared to mice housed in individual cages. The advancement in maternal behavior was replicated when virgin females were simply exposed to male mouse urine-soaked bedding. Ovariectomising the mice, to remove the influence of steroid hormones, prior to placement in the split cages, prevented the pheromonal advancement of maternal behavior. The data infer that an ovarian steroid-dependent action of male mouse pheromones primes virgin female mice to express maternal behavior more rapidly when mouse pups are introduced. This effect required greater than 14 days exposure to male pheromones. Male mouse pheromones are reported to suppress prolactin secretion. However, serum prolactin levels in split-caged housed females, where they had pheromonal but not physical contact with a male, were only briefly lowered and became significantly elevated from 24 hours until 72 hours of pheromonal contact. Despite the early increases in prolactin after pheromone exposure, levels were significantly lower in the pheromone-exposed females when maternal behavior was tested after 21 days. It has been previously reported that prolactin is important in the onset of maternal behavior, but is not required for the ongoing maintenance of maternal behavior. We hypothesised that the hyperprolactinemia observed in the first 24-72 hours of pheromonai exposure had subsequently led to the enhanced maternal behavior. To test this we injected a group of individually-housed mice with slow release prolactin for 48 hours to simulate the period of hyperprolactinemia, and blocked prolactin secretion in a group of split-caged housed females with bromocriptine, and tested their maternal behavior 18 days later. The mice injected with prolactin had enhanced maternal behavior, compared to controls injected with a placebo. By contrast, bromocriptine inhibition of prolactin secretion completely prevented the pheromonal enhancement of maternal behavior. This suggests that the pheromonal advancement of maternal behavior is specifically mediated by a 48-hour period of sustained hyperprolactinemia. It has been previously shown that pregnancy increases neurogenesis in the subventricular zone in a prolactin-dependent manner. Therefore, as the male pheromone-induced advancement of maternal behavior is prolactin-dependent and takes some time to occur, we hypothesized that long-term pheromonal contact initiates mitogenesis in the subventricular zone. Split-caged housed mice showed a significant increase in BrdU-labeled cells in the subventricular zone after 7 days of contact which reduced to baseline levels by 14 days of contact. The mice injected with BrdU on day 7 of contact and killed 21 days later showed a significant increase in labeled cells in the accessory olfactory bulb compared to controls. The data suggest that male mouse pheromones initiate mitogenesis in the subventricular zone of virgin C57B6 mice, in an exposure-dependent manner, and that these cells travel via the rostral migratory stream to the accessory olfactory bulb. As with the effect on maternal behavior, the pheromone-induced increase in neurogenesis was steroid- and prolactin-dependent. During pregnancy and lactation in rodents, prolactin receptor expression is increased in the MPOA, an adaptive change, which could lead to an increased neuronal response to serum prolactin levels, which are high just prior to parturition, and consequently could underlie the enhanced maternal responses seen in late pregnancy and after parturition. It is known that systemic prolactin can access the brain, but it is also possible that there could be local synthesis of brain prolactin acting in an autocrine or paracrine manner. Therefore we hypothesized that the pheromonal-induced changes in maternal behavior are being mediated by altered prolactin receptor expression/sensitivity and/or increased production of brain prolactin. Using RT-PCR to measure levels of prolactin receptor and prolactin mRNA, we found changed expression of the 3 short forms and the long form of prolactin receptor mRNA in the arcuate nucleus, paraventricular nucleus, bed nucleus of the stria terminalis, and MPOA with either exposure to male pheromones or pups. We also found changes in prolactin mRNA in the MPOA and paraventricular nucleus after exposure to pups or male pheromones. The data suggest that altered levels of expression of the receptor, coupled with local production of brain prolactin acting in an autocrine or paracrine manner, may cause a net change in prolactin cell signaling, which leads to adaptive responses which ensure reproductive success. There is extensive evidence that dopamine is a key neurotransmitter mediating maternal behavior. In addition, there is some evidence that serotonin may also be involved in regulating maternal behavior. Therefore, we hypothesised that the pheromonal-induced changes in maternal behavior would be associated with increased dopaminergic and/or serotonergic neuronal activity in the MPOA and other areas of the brain implicated in maternal behavior expression. Using HPLC to measure levels of dopamine and serotonin and their respective metabolites, we found a significant increase in serotonergic and dopaminergic neuronal activity in the MPOA of virgin female C57BL/6J mice after 24 hours of pheromonal contact. The neuronal activity returned to basal levels after exposure to pups. The data suggest that male mouse pheromones increase serotonergic and dopaminergic neuronal activity in the MPOA, but that dopamine and serotonin levels are tightly regulated within strict parameters dependent on what physical stimuli the female is receiving. Changes in prolactin levels are associated with altered responses to anxiety. There is an increased risk of anxiety and depression with sustained periods of hyperprolactinemia, and in the postpartum period, where there are fluctuations in prolactin levels, there is an increased risk of mood disorders. As pheromones change both serum and brain prolactin levels and prolactin modulates anxiety, we hypothesised that female mice exposed to pheromones would show altered behavioral responses to a standardized test of anxiety. We found that male pheromone-exposed mice showed decreased levels of anxiety on an elevated plus maze compared to individually housed controls. Female mice exposed to female pheromones displayed 2 disparate responses to the plus maze. One female from each cage showed increased anxiety, while her cage-mate showed decreased anxiety, yet both groups of female mice showed impaired maternal behavior. We infer, that in this model, male pheromones decrease anxiety, but anxiety and expression of maternal behavior are not directly correlated. The major signal transduction pathway activated by prolactin binding to its receptors in the brain is the JAK/STAT signalling pathway, and in some neurons, in particular, the STAT5B pathway. The expression of prolactin and its receptor affect maternal behavior in mice. Therefore, we hypothesised that if the JAK/STAT STAT5B pathway is involved in maternal behavior, then STAT5B-deficient mice would have altered maternal behavior. We found that there were no significant differences in expression of full maternal behavior between the STAT5B-deficient mice and wild-type controls. The data suggest that STAT5B is not required for normal expression of maternal behavior. We propose that the prolactin-mediated pheromonal increase in neurogenesis, alteration in monoamine synthesis, and alteration of prolactin and prolactin receptor mRNA levels facilitate expression of enhanced maternal behavior. We further propose that the pheromonal decrease in anxiety does not mediate enhanced maternal behavior. In addition, we propose that prolactin does not mediate maternal behavior through STAT5B. While pheromones have previously been reported to exert powerful actions on the reproductive system, these results demonstrate for the first time that male pheromones potentially complement the prolactin-mediated establishment of maternal behavior.

A series of trials was carried out to examine the role of prolactin (PRL) in the control of lactogenesis in New Zealand Romney x Border Leicester ewes. In addition, a study was made of differences in milk yields and plasma PRL concentrations between spring- and autumn-lambing ewes.Daily subcutaneous injections of 2 mg CB154 inhibited PRL secretion and delayed lactogenesis. There were no consistent effects on plasma progesterone or insulin concentrations. CB154 treatment was more effective in reducing milk yield in twin-bearing than in single-bearing ewes when used for 20 days than for 9 days prepartum. The differential effects on milk yield cannot be explained by corresponding effects on plasma PRL or insulin concentrations. Circulating PRL during the period 20 to 10 days prepartum may have an important effect on milk yield in twin- but not single-bearing ewes.Subcutaneous injections of 0.5 mg/kg live weight oPRL, administered on 2 consecutive days peripartum, to ewes treated with CB154 for 7 days prepartum, resulted in milk yields similar to those in control ewes and significantly (P<0.01) greater than those in ewes treated with CB154 alone. This indicated that oPRL prevented the CB154-induced reduction of milk yields and has established that the effect of CB154 on lactogenesis is mediated through suppression of PRL secretion and not by effects on some other hormone.Injection of 10 mg oPRL directly into one mammary gland (via the teat duct) increased milk yields relative to the contralateral, bicarbonate-treated gland in CB154-treated ewes. The intramammary oPRL injection did not raise circulating PRL concentrations. Furthermore, the milk yields of bicarbonate-treated glands in ewes treated with bicarbonate only, did not differ from those of bicarbonate-treated glands in ewes treated with oPRL in the contralateral gland, demonstrating that there were no effects of oPRL, transferred via the circulation from the treated gland, on the contralateral gland. Glands treated with oPRL produced 15% (P<0.05) more milk than the bicarbonate-treated glands during the first 8 days of lactation and the difference was maintained throughout the 8-week lactation period, indicating that the oPRL had effected a permanent change in the ability of the gland to produce milk. It is concluded that PRL acts directly on the mammary gland without the need for a putative intermediate hormone, and that intramammary PRL concentrations during lactogenesis may have long-lasting effects on lactation.The possibility was examined that dietary differences were responsible for seasonal differences in plasma PRL concentrations, milk yields, milk composition, lamb birthweight and lamb growth rate, observed in earlier trials. Mean plasma PRL levels were significantly (P<0.01) higher in spring- (192±38 ng/ml) than in autumn- (71±17 ng/ml) lambing ewes housed indoors under constant photoperiod (18L:6D) and fed the same diet. Milk yields were also significantly (P<0.05) higher in the spring- (2041±114 g/d) than in the autumn- (1563±109 g/d) lambing ewes over the 8 day lactation. Lamb growth rates (adjusted for birthweight, birthrank and sex of lamb) from birth to 8 weeks of age were significantly (P<0.001) higher in spring (282±12 g/d) than in autumn (225±15 g/d). The seasonal differences were confounded with corresponding differences in ewe live weight and it was not possible to determine whether dietary differences contributed significantly to the differences observed.Two routes of oPRL supplementation were used to test the effectiveness of elevating peripheral or local levels of PRL in autumn-lambing ewes which, based on previous results, were expected to have low plasma PRL concentrations and milk yields relative to spring-lambing ewes. Administration of 10 mg supplementary oPRL directly into the gland or subcutaneous injection of 0.5 mg/kg oPRL did not increase the milk yields, or change the composition of milk, compared to controls. These results suggest that the circulating level of PRL, and the intramammary concentration of PRL, in autumn-lambing ewes are not limiting lactogenesis. Because the plasma prolactin concentration in the ewes was unexpectedly high, it was not possible to reach firm conclusions regarding possible effects of supplementary oPRL in ewes with naturally low plasma PRL concentrations. Nevertheless, the results indicate that raising the intramammary concentration of PRL around the time of parturition, in ewes with circulating PRL levels characteristic of normal spring-lambing ewes, does not enhance lactogenesis.It is concluded that PRL is important to the complete initiation of lactogenesis in ewes, that it acts directly on the gland and that it is necessary for establishing the maximum potential of the gland to secrete milk.