O estradiol (E2) é requerido para a luteólise de fêmeas bovinas e injeções de E2 estimulam a liberação de prostaglandina F2α (PGF2α). É possível que o E2 estimule a síntese e/ou a atividade de moléculas envolvidas na cascata geradora de PGF2α, como enzimas e receptores para outros ligantes. O cálcio é um conhecido cofator para a proteína quinase C e fosfolipase A2, enzimas envolvidas na produção PGF2α. O objetivo geral desse estudo foi investigar os mecanismos endócrinos e moleculares estimulados pelo E2 durante a luteólise. No experimento 1, vacas holandesas não lactantes foram tratadas com 3mg de E2 nos dias 13 (n=2), 15 (n=2), 17 (n=3) e 19 (n=5) do ciclo estral e a produção de PGFM (metabólito plasmático da PGF2α) mensurada por radioimunoensaio. Concluiu-se que a administração de E2 no 17º dia do ciclo estral constitui um modelo experimental adequado para determinar os mecanismos envolvidos na síntese de PGF2α. O experimento 2, foi realizado para investigar o papel do E2 na cascata produtora de PGF2α. Novilhas cruzadas de corte, cíclicas, não lactantes, foram pareadas no dia 17 de um ciclo estral sincronizado, injetadas com 0 (n=6) ou 3mg de E2 (n=7) e abatidas 2 horas após. Explantes endometriais foram tratados com os seguintes estimuladores da síntese de PGF2α: ionóforo de cálcio (CI), melitina ou ocitocina. Explantes foram incubados em quadruplicata e amostras de meio foram coletadas imediatamente e 60 minutos após o início da cultura. As concentrações de PGF2α foram mensuradas por radioimunoensaio. Explantes endometriais tratados in vitro com CI tiveram um incremento na síntese de PGF2α de 48,41% quando foram previamente tratados com o E2 (P≤0,01). No experimento 3, células endometriais bovinas (células BEND) foram tratadas com 0, 10-7, 10-6 ou 10-5M CI por 12h em triplicata, em três experimentos independentes. As concentrações de 10-6 e 10-5M de CI estimularam a produção de PGF2α em comparação às outras concentrações (P≤0,05). No experimento 4, células BEND receberam 0 ou 10-13M E2 e 0 ou 10-6M CI em um arranjo fatorial 2 x 2, durante 12h em triplicata, em três experimentos independentes. A produção de PGF2α foi de 33,1; 32,5; 92,4 e 145,6 (EPM: 21,8) pg/mL para células não tratadas, tratadas com E2, CI e E2 + CI, respectivamente. Houve uma tendência do tratamento com CI estimular a produção de PGF2α (P≤0,08), entretanto, na presença de E2 o CI estimulou significativamente a síntese de PGF2≤ (P≤0,01). Conclui-se que em fêmeas bovinas o E2 potencializou os efeitos do CI na síntese de PGF2α endometrial. Propõe-se que o E2 ativa a síntese de enzimas que, estimuladas pelo cálcio, atuam na síntese de PGF2α endometrial / Estradiol (E2) is required for luteolysis in bovine female and E2 injections stimulate prostaglandin F2α (PGF2α) release. It is possible that E2 stimulates synthesis and/or activity of molecules in the cascade of PGF2α production, such as enzymes and receptors to other ligands. Calcium is a known cofactor for protein kinase C and phospholipase A2, enzymes involved in PGF2α production. The main goal of this study was to investigate endocrine and molecular mechanisms stimulated by E2 during luteolysis. In experiment 1, non-lactating Holstein cows were treated with 3mg E2 on days 13 (n=2), 15 (n=2), 17 (n=3) or 19 (n=5) of the day estrous cycle and production PGFM (a PGF2α plasma metabolite) was evaluated by radioimmunassay. It was concluded that E2 administration on day 17 of the cycle was an adequate experimental model to determine mechanisms involved in endometrial PGF2α synthesis. Experiment 2 was designed in order to investigate the role of E2 in the enzymatic cascade of PGF2α production. Cyclic, cross-bred beef heifers were paired on day 17 of a synchronized estrous cycle, injected with 0 (n=6) or 3mg of E2 (n=7) and slaughtered after two hours. Endometrial explants were treated with stimulators of the cascade of PGF2α synthesis, i.e., calcium ionophore (CI), melittin or oxytocin. Explants were incubated in quadruplicate and medium samples were collected immediately and 60min after to begin culture. The concentrations PGF2α were measured by radioimmunoassay. Endometrial explants in vitro treatment with CI production the PGF2α was 48,41% higher in from cows treated with E2 (P≤0,01). In experiment 3, bovine endometrium cells (BEND cells) were treated with 0, 10-7, 10-6 or 10-5M CI for 12h in triplicate, in three independent experiments. The 10-6 and 10-5M concentrations of CI stimulated production of PGF2α in comparison to other concentrations (P≤0,05). In experiment 4, BEND cells received 0 or 10-13M E2 and 0 or 10-6M CI in a 2 x 2 factorial arrangement, for 12h in triplicate cultures in three independent experiments. Production of PGF2α was 33.1, 32.5, 92.4 and 145.6 (pooled SEM: 21.8) pg/mL for cells treated with nothing, E2, CI and E2 + CI, respectively. Treatment with CI alone tended to stimulate PGF2α production (P≤0,08), however, in the presence of E2, CI significantly stimulated PGF2α synthesis (P≤0,01). It was concluded that in bovine female the E2 improved the CI effects in endometrial PGF2α synthesis. It proposes that E2 active the enzyme synthesis that, calcium stimulated, actuate in endometrial PGF2α synthesis

O estradiol (E2) é requerido para a luteólise de fêmeas bovinas e injeções de E2 estimulam a liberação de prostaglandina F2α (PGF2α). É possível que o E2 estimule a síntese e/ou a atividade de moléculas envolvidas na cascata geradora de PGF2α, como enzimas e receptores para outros ligantes. O cálcio é um conhecido cofator para a proteína quinase C e fosfolipase A2, enzimas envolvidas na produção PGF2α. O objetivo geral desse estudo foi investigar os mecanismos endócrinos e moleculares estimulados pelo E2 durante a luteólise. No experimento 1, vacas holandesas não lactantes foram tratadas com 3mg de E2 nos dias 13 (n=2), 15 (n=2), 17 (n=3) e 19 (n=5) do ciclo estral e a produção de PGFM (metabólito plasmático da PGF2α) mensurada por radioimunoensaio. Concluiu-se que a administração de E2 no 17º dia do ciclo estral constitui um modelo experimental adequado para determinar os mecanismos envolvidos na síntese de PGF2α. O experimento 2, foi realizado para investigar o papel do E2 na cascata produtora de PGF2α. Novilhas cruzadas de corte, cíclicas, não lactantes, foram pareadas no dia 17 de um ciclo estral sincronizado, injetadas com 0 (n=6) ou 3mg de E2 (n=7) e abatidas 2 horas após. Explantes endometriais foram tratados com os seguintes estimuladores da síntese de PGF2α: ionóforo de cálcio (CI), melitina ou ocitocina. Explantes foram incubados em quadruplicata e amostras de meio foram coletadas imediatamente e 60 minutos após o início da cultura. As concentrações de PGF2α foram mensuradas por radioimunoensaio. Explantes endometriais tratados in vitro com CI tiveram um incremento na síntese de PGF2α de 48,41% quando foram previamente tratados com o E2 (P≤0,01). No experimento 3, células endometriais bovinas (células BEND) foram tratadas com 0, 10-7, 10-6 ou 10-5M CI por 12h em triplicata, em três experimentos independentes. As concentrações de 10-6 e 10-5M de CI estimularam a produção de PGF2α em comparação às outras concentrações (P≤0,05). No experimento 4, células BEND receberam 0 ou 10-13M E2 e 0 ou 10-6M CI em um arranjo fatorial 2 x 2, durante 12h em triplicata, em três experimentos independentes. A produção de PGF2α foi de 33,1; 32,5; 92,4 e 145,6 (EPM: 21,8) pg/mL para células não tratadas, tratadas com E2, CI e E2 + CI, respectivamente. Houve uma tendência do tratamento com CI estimular a produção de PGF2α (P≤0,08), entretanto, na presença de E2 o CI estimulou significativamente a síntese de PGF2≤ (P≤0,01). Conclui-se que em fêmeas bovinas o E2 potencializou os efeitos do CI na síntese de PGF2α endometrial. Propõe-se que o E2 ativa a síntese de enzimas que, estimuladas pelo cálcio, atuam na síntese de PGF2α endometrial / Estradiol (E2) is required for luteolysis in bovine female and E2 injections stimulate prostaglandin F2α (PGF2α) release. It is possible that E2 stimulates synthesis and/or activity of molecules in the cascade of PGF2α production, such as enzymes and receptors to other ligands. Calcium is a known cofactor for protein kinase C and phospholipase A2, enzymes involved in PGF2α production. The main goal of this study was to investigate endocrine and molecular mechanisms stimulated by E2 during luteolysis. In experiment 1, non-lactating Holstein cows were treated with 3mg E2 on days 13 (n=2), 15 (n=2), 17 (n=3) or 19 (n=5) of the day estrous cycle and production PGFM (a PGF2α plasma metabolite) was evaluated by radioimmunassay. It was concluded that E2 administration on day 17 of the cycle was an adequate experimental model to determine mechanisms involved in endometrial PGF2α synthesis. Experiment 2 was designed in order to investigate the role of E2 in the enzymatic cascade of PGF2α production. Cyclic, cross-bred beef heifers were paired on day 17 of a synchronized estrous cycle, injected with 0 (n=6) or 3mg of E2 (n=7) and slaughtered after two hours. Endometrial explants were treated with stimulators of the cascade of PGF2α synthesis, i.e., calcium ionophore (CI), melittin or oxytocin. Explants were incubated in quadruplicate and medium samples were collected immediately and 60min after to begin culture. The concentrations PGF2α were measured by radioimmunoassay. Endometrial explants in vitro treatment with CI production the PGF2α was 48,41% higher in from cows treated with E2 (P≤0,01). In experiment 3, bovine endometrium cells (BEND cells) were treated with 0, 10-7, 10-6 or 10-5M CI for 12h in triplicate, in three independent experiments. The 10-6 and 10-5M concentrations of CI stimulated production of PGF2α in comparison to other concentrations (P≤0,05). In experiment 4, BEND cells received 0 or 10-13M E2 and 0 or 10-6M CI in a 2 x 2 factorial arrangement, for 12h in triplicate cultures in three independent experiments. Production of PGF2α was 33.1, 32.5, 92.4 and 145.6 (pooled SEM: 21.8) pg/mL for cells treated with nothing, E2, CI and E2 + CI, respectively. Treatment with CI alone tended to stimulate PGF2α production (P≤0,08), however, in the presence of E2, CI significantly stimulated PGF2α synthesis (P≤0,01). It was concluded that in bovine female the E2 improved the CI effects in endometrial PGF2α synthesis. It proposes that E2 active the enzyme synthesis that, calcium stimulated, actuate in endometrial PGF2α synthesis

Orientador: José Luiz Moraes Vasconcelos / Resumo: O objetivo deste estudo foi avaliar se vacas Nelore, que receberam duas doses de PGF2α em protocolos de IATF a base de progesterona e estradiol, teriam melhor fertilidade em comparação a uma dose de PGF2α. Foram realizados 2 experimentos em que os animais receberam o protocolo de IATF: dispositivo intravaginal de P4 (CIDR) e 2,0 mg benzoato de estradiol (i.m., 2 ml Gonadiol®, Zoetis), no d-11, dinoprost trometamina (PGF2α; i.m., 2,5 ml Lutalyse®, Zoetis) no d-4, retirada do dispositivo de P4, 0,6 mg de cipionato de estradiol (i.m., 0,3 ml ECP®, Zoetis) e 300 UI de eCG (i.m., 1,5 ml Novormon®, Zoetis) no d-2, e IATF no d0. No experimento 1, as vacas (n=1.039) foram divididas aleatoriamente para receber uma ou duas doses de PGF2α, sendo a primeira no d-4 e a segunda no d-2. No experimento 2, foram avaliadas 1.051 fêmeas Nelore, permanecendo para as análises deste estudo apenas fêmeas que não apresentaram um CL no d-4 (momento da primeira PGF2α; n=934), os tratamentos foram os mesmos descritos no experimento 1. Foi fixado um dispositivo para avaliar a expressão do cio (Estrotect®) no d-2, avaliado o diâmetro folicular no d0, as colheitas de sangue foram realizadas no d0 e d7, para dosagem de P4. O diagnóstico de gestação (DG) foi realizado 30 dias após IA. No experimento 1, a taxa de prenhez foi maior para os animais do grupo 2PG (54,5% vs. 46,6%; P = 0,01) em relação a 1PG. No experimento 2, a taxa de sincronização e a taxa de prenhez foram maiores no grupo 2PG, (81,4% vs. 72,1... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate whether Nelore cows that received two doses of PGF2α in progesterone and estradiol-based TAI protocols would have better fertility compared to one dose of PGF2α. Two experiments were performed in which the animals received an TAI protocol: intravaginal P4 device (CIDR) and 2.0 mg estradiol benzoate (i.m., 2 ml Gonadiol®, Zoetis) at d-11, dinoprost tromethamine (PGF2α; i.m., 2 , 5 ml Lutalyse®, Zoetis) at d-4, P4 device withdrawal, 0.6 mg estradiol cypionate (i.m., 0.3 ml ECP®, Zoetis) and 300 IU eCG (i.m., 1.5 ml Novormon®, Zoetis) at d-2, and AI at d0. In experiment 1 the animals (n = 1,039) were randomly assigned to receive one or two doses of PGF2α, the first on d-4 and the second on d-2. In experiment 2, 1,051 Nelore cows were used, and remaining for the analysis of this study only females without CL on d-4 (moment of first PGF2α; n=934), the treatments were the same as described in experiment 1. A device was fixed to evaluate estrus expression (Estrotect®) at d-2, measured follicular diameter at d0, blood samples were collected at d0 and d7 for P4 dosage. The pregnancy diagnosis was made 30 days after AI. In experiment 1, the pregnancy rate was higher for animals in group 2PG (54.5% vs. 46.6%; P = 0.01) compared to 1PG. In experiment 2, synchronization rate and pregnancy rate were higher in group 2PG (81.4% vs. 72.1%; [P = 0.01] and 55.5% vs. 45.6%; [P = 0.04], respectively) only in animals with BCS < 5.0 (scale from 1 to 9). In anim... (Complete abstract click electronic access below) / Mestre

No / The pharmacology of bimatoprost, a synthetic prostaglandin-amide, was examined in prostaglandin F2¿ (PGF2¿)-sensitive preparations. Bimatoprost potently contracted the rabbit isolated uterus (pEC50=7.92±0.16). In contrast, bimatoprost exhibited weak excitatory activity in human myometrium from pregnant and nonpregnant donors, mouse uterus, rat uterus, and endothelium-intact rabbit jugular veins, and did not stimulate DNA synthesis in mouse fibroblasts. The possibility that the effects of bimatoprost may reflect partial agonism at prostanoid FP receptors was examined and the contractile effects of full agonists, 17-phenyl PGF2¿ (FP) and U-46619 (TP, a control), were determined in the absence and presence of 1 ¿M bimatoprost on the mouse uterus. Analyses of the agonist¿agonist functional studies showed no antagonism, indicating that bimatoprost is not a partial agonist. Bioassay metabolism studies of bimatoprost and latanoprost (FP receptor agonist prodrug) in the rabbit uterus were conducted using recipient mouse uterus. Results indicated that the potent responses to bimatoprost in the rabbit uterus are produced by the intact molecule and not by its putative free acid metabolite, 17-phenyl PGF2¿. Some hydrolysis of latanoprost to latanoprost free acid appears to have occurred in the rabbit uterus, according to biological detection. The pharmacology of bimatoprost could not be explained by its interaction with known prostanoid FP receptors and was independent of species-, tissue-, or preparation-related factors. The potent contractile effects of bimatoprost in the rabbit uterus provide further pharmacological evidence for the presence of a novel receptor population that preferentially recognises bimatoprost.

No / Human saphenous vein endothelial cells (HSVECs) derived from primary cultures of adult human veins constitute an excellent in vitro model for studying human endothelial metabolism. In this study we report the14C-labelled prostanoid profile of HSVECs under resting and stimulated conditions and the effect of the n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid on them. Results indicate that HSVECs while under resting conditions produce mainly prostaglandin F2 ¿(PGF2 ¿). After stimulation with calcium ionophore A23187, the cells were found to synthesise PGI2, PGE2and PGF2¿as major products and thromboxane B2and PGD2as minor products. Production of14C-labelled hydroxyeicosatetraenoic acids was not detected. Eicosapentaenoic acid was found to inhibit basal and stimulated prostanoid production whereas docosahexaenoic acid inhibited basal but strongly increased stimulated prostanoid production. These results may offer the basis for further studies aiming to investigate targets for pharmacological intervention in inflammatory conditions.