Produção de progesterona pelas células luteínicas esteroidogênicas bovina cultivadas e co-cultivadas in vitro
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Data
2016-01-22
Autores
Destro, Flavia Caroline [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo geral do trabalho foi caracterizar a produção de progesterona (P4) pelas células do corpo lúteo (CL) bovino submetidas a diferentes meios de cultivo e cocultivo in vitro. Para tanto foram realizados três experimentos descritos em dois artigos. No artigo I, CLs pertencentes à fase média (10-12 dias, n=5) foram coletados e processados em laboratório. As LCs foram cultivadas por 07 dias em atmosfera umida a 5% de CO2, com ou sem a adição de 10% de soro fetal bovino (SFB), com os respectivos tratamentos: CONTROLE; CONA (10 μg/mL); LH (100 μg/mL); CONALH; LHPGFβα (10 ng/mL); CONALHPGFβα. Amostras de meio de cultivo foram coletadas nos D1 e D7 para posterior dosagem de P4. Valores com P<0,05 foram considerados diferentes estatisticamente. O cultivo de LCs na presença de CONA diminuiu a capacidade secretória de P4 das LCs e este efeito foi revertido pela adição de LH no meio de cultivo no D1, mas não no D7. A ação supressora da CONA foi mais evidente no cultivo que não empregaram o SFB. No artigo II, No Exp. 1, foram utilizados CLs pertencentes à fase média (10-12 dias, n=4). No laboratório os CLs foram processados e as LCs foram cultivadas por 07 dias em atmosfera umida a 5% de CO2. As LCs receberam os respectivos tratamentos com ou sem LH: Controle; PGF2α (10 ng/mL); PGF2α (100 ng/mL); PGF2α (354,5 ng/mL). As amostras que receberam LH apresentaram maior produção de P4, e a administração de diferentes doses de PGFβα não mostrou alteração na secreção de P4. No Exp. β, foram utilizados CLs pertencentes à fase média (10-12 dias, n=4). No laboratório os CLs foram processados e as LCs, células luteínicas endoteliais (ECs) e células imunes (ICs) foram isoladas e cocultivadas por 07 dias em atmosfera umida a 5% de CO2. Os grupos foram divididos com ou sem PGF2α em: Controle: LC no fundo da placa; Controle: EC no fundo da placa; Co-cultivo: LC+EC+IC no fundo da placa; LC (fundo da placa) + EC+IC (interior do inserto); EC (fundo da placa) + LC+IC (interior do inserto). Não foi observado variação na produção de P4, quando adicionado PGF2α no cultivo e cocultivo das células do CL. Em ambos os experimentos, amostras de meio de cultivo foram coletadas nos D1 e D7 para posterior dosagem de P4. Valores com P<0,05 foram considerados diferentes estatisticamente. Conclui-se que o cultivo e co-cultivo in vitro das células do CL bovino são ferramentas alternativas para o estudo da função luteínica, contudo há a necessidade de maiores ajustes metodológicos a fim de se mimetizar processos fisiologicos relacionados a esteroidogênese e a luteólise.
The general objective of this study was to characterize the production of progesterone (P4) by the cells of the bovine corpus luteum (CL) subjected to different culture and coculture media in vitro. To this end, three experiments described in two articles were conducted. Article I: CLs belonging to the middle phase (10-12 days, n=5) were collected and processed in the laboratory. The luteal cells (LCs) were cultured for 07 days in a humid atmosphere of 5% CO2, with or without the addition of 10% fetal bovine serum (FBS), with their respective treatments: CONTROL; CONA (concanavalin-A) (10 μg/mL); LH (100 μg/mL); CONALH; LHPGFβα (10 ng/mL); CONALHPGFβα. Samples of culture medium were collected on D1 and D7 for further P4 measurement. P values <0.05 were considered statistically different. Culture of LCs in the presence of CONA decreases the ability of LC to secrete P4, and this effect was reversed by addition of LH in the culture medium in D1, but not in D7. The suppressive action was more evident in CONA cultures without FBS. In Article II, Experiment 1, CLs of middle phase of the estrous cycle (10-12 days, n = 4) were used. In the laboratory, the CLs were processed and the LCs were cultured for 07 days in a humidified atmosphere of 5% CO2. The LCs received the respective treatments with or without LH (100 μg/mL): Control; PGFβα (10 ng/mL); PGFβα (100 ng/mL); PGFβα (354,5 ng/mL). The samples that received LH produced more P4, and the administration of different doses of PGFβα showed no change in P4 secretion. In Experiment β, CLs of middle phase of the estrous cycle (10-12 days, n = 4) were used. In the laboratory, the CLs were processed and the LCs, luteal endothelial cells (ECs) and immune cells (ICs) were isolated and co-cultured for 07 days in a humidified atmosphere of 5% CO2. The groups were divided with or without PGFβα: Control: LC on bottom of the plate; Control: EC on bottom of the plate; Co-culture: LC + EC + IC on bottom of the plate; LC (bottom of the plate) + EC + IC (inside the insert); EC (bottom of the plate) + LC + IC (inside the insert). It was not observed variation in the production of P4, when added PGFβα in culture and co-culture of cells from CL. In both experiments, samples of culture medium were collected on D1 and D7 for further P4 measurement. P values <0.05 were considered statistically different. We conclude that the in vitro culture and co-culture of bovine CL cells are alternative tools for the study of luteal function, but there is a need for further methodological adjustments in order to mimic physiological processes related to steroidogenesis and luteolysis.
The general objective of this study was to characterize the production of progesterone (P4) by the cells of the bovine corpus luteum (CL) subjected to different culture and coculture media in vitro. To this end, three experiments described in two articles were conducted. Article I: CLs belonging to the middle phase (10-12 days, n=5) were collected and processed in the laboratory. The luteal cells (LCs) were cultured for 07 days in a humid atmosphere of 5% CO2, with or without the addition of 10% fetal bovine serum (FBS), with their respective treatments: CONTROL; CONA (concanavalin-A) (10 μg/mL); LH (100 μg/mL); CONALH; LHPGFβα (10 ng/mL); CONALHPGFβα. Samples of culture medium were collected on D1 and D7 for further P4 measurement. P values <0.05 were considered statistically different. Culture of LCs in the presence of CONA decreases the ability of LC to secrete P4, and this effect was reversed by addition of LH in the culture medium in D1, but not in D7. The suppressive action was more evident in CONA cultures without FBS. In Article II, Experiment 1, CLs of middle phase of the estrous cycle (10-12 days, n = 4) were used. In the laboratory, the CLs were processed and the LCs were cultured for 07 days in a humidified atmosphere of 5% CO2. The LCs received the respective treatments with or without LH (100 μg/mL): Control; PGFβα (10 ng/mL); PGFβα (100 ng/mL); PGFβα (354,5 ng/mL). The samples that received LH produced more P4, and the administration of different doses of PGFβα showed no change in P4 secretion. In Experiment β, CLs of middle phase of the estrous cycle (10-12 days, n = 4) were used. In the laboratory, the CLs were processed and the LCs, luteal endothelial cells (ECs) and immune cells (ICs) were isolated and co-cultured for 07 days in a humidified atmosphere of 5% CO2. The groups were divided with or without PGFβα: Control: LC on bottom of the plate; Control: EC on bottom of the plate; Co-culture: LC + EC + IC on bottom of the plate; LC (bottom of the plate) + EC + IC (inside the insert); EC (bottom of the plate) + LC + IC (inside the insert). It was not observed variation in the production of P4, when added PGFβα in culture and co-culture of cells from CL. In both experiments, samples of culture medium were collected on D1 and D7 for further P4 measurement. P values <0.05 were considered statistically different. We conclude that the in vitro culture and co-culture of bovine CL cells are alternative tools for the study of luteal function, but there is a need for further methodological adjustments in order to mimic physiological processes related to steroidogenesis and luteolysis.
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Palavras-chave
Corpo lúteo, Cultivo celular, Concanavalina-A, Prostaglandina F2α, Progesterona, Corpus luteum, Cell culture, Concanavalin-A, Prostaglandin Fβα, Progesterone