Culture systems are available for human granulosa cells (GCs) that perpetuate luteinization. The ... more Culture systems are available for human granulosa cells (GCs) that perpetuate luteinization. The present study examines the plating density effects and long-term serum-free culture on the in vitro dynamics differentiation of luteinizing human GCs. Cells were cultured in serum-free a-minimum essential medium (a-MEM) or serum-based tissue culture medium (TCM). The time course of GCs morphology and secretion of estradiol (E2), progesterone (P4), and relaxin were analyzed after 48, 96, and 144 hours of culture. Other functional markers as follicle-stimulating hormone/luteinizing hormone receptors and steroidogenic enzymes were investigated at the end of culture. The morphology of an a-MEM cell rather than a TCM cell resembles more closely that seen in vivo. Compared to TCM cultures, a-MEM cells secreted 93.7% and 87.2% more E2 and approximately 7% and 17% of the amount of P4 when cultured at densities of 2 104 or 4 104 cells/well, respectively. Relaxin secretion was significantly reduced in a-MEM cultures. a-MEM cells were estrogenic and expressed the CYP19 gene. Levels of CYP17 increased about 8-fold in a-MEM cells above the levels found in TCM cells. Our results reveal new insights into human GCs differentiation in vitro and demonstrate the critical importance of the culture system and cell-plating density on the establishment of estrogenic or progestogenic GC phenotypes.
oObjetivo: identificar produc¸ão de oócitos em mamíferos adultos com o uso de camundongocomo mode... more oObjetivo: identificar produc¸ão de oócitos em mamíferos adultos com o uso de camundongocomo modelo experimental.Método: empregamos a técnica de imuno-histoquímica em cortes de ovários de camundon-gos Balb-c (45 dias de idade) com o uso de anticorpo específico para marcac¸ão de célulasgerminativas. Como controles positivos da reac¸ão, usamos cortes de testículos de camun-dongos.Resultados: as células germinativas (espermatogônias, espermatócitos e espermátides) doscontroles positivos sofreram marcac¸ão, enquanto células não pertencentes a essa linhagem(células de Leydig e de Sertoli) mostraram negatividade de reac¸ão; nos cortes ovarianosobservou-se marcac¸ão de oócitos de folículos em diferentes estágios de maturac¸ão, mashouve também marcac¸ão de células não englobadas pela estrutura folicular.Conclusões: os achados sugerem que durante a puberdade ovários de camundongos fêmeascontêm células da linhagem germinativa em estágios anteriores à formac¸ão folicular, oque corrobora estudos anteriores; o trabalho é pioneiro no Brasil e progredirá para a com-pleta caracterizac¸ão de células com potencial oogênico em outras espécies de mamíferos.Resultados positivos poderão alterar o entendimento da biologia reprodutiva e abrir novasportas para o tratamento de infertilidade.
Culture systems are available for human granulosa cells (GCs) that perpetuate luteinization. The ... more Culture systems are available for human granulosa cells (GCs) that perpetuate luteinization. The present study examines the plating density effects and long-term serum-free culture on the in vitro dynamics differentiation of luteinizing human GCs. Cells were cultured in serum-free a-minimum essential medium (a-MEM) or serum-based tissue culture medium (TCM). The time course of GCs morphology and secretion of estradiol (E2), progesterone (P4), and relaxin were analyzed after 48, 96, and 144 hours of culture. Other functional markers as follicle-stimulating hormone/luteinizing hormone receptors and steroidogenic enzymes were investigated at the end of culture. The morphology of an a-MEM cell rather than a TCM cell resembles more closely that seen in vivo. Compared to TCM cultures, a-MEM cells secreted 93.7% and 87.2% more E2 and approximately 7% and 17% of the amount of P4 when cultured at densities of 2 104 or 4 104 cells/well, respectively. Relaxin secretion was significantly reduced in a-MEM cultures. a-MEM cells were estrogenic and expressed the CYP19 gene. Levels of CYP17 increased about 8-fold in a-MEM cells above the levels found in TCM cells. Our results reveal new insights into human GCs differentiation in vitro and demonstrate the critical importance of the culture system and cell-plating density on the establishment of estrogenic or progestogenic GC phenotypes.
oObjetivo: identificar produc¸ão de oócitos em mamíferos adultos com o uso de camundongocomo mode... more oObjetivo: identificar produc¸ão de oócitos em mamíferos adultos com o uso de camundongocomo modelo experimental.Método: empregamos a técnica de imuno-histoquímica em cortes de ovários de camundon-gos Balb-c (45 dias de idade) com o uso de anticorpo específico para marcac¸ão de célulasgerminativas. Como controles positivos da reac¸ão, usamos cortes de testículos de camun-dongos.Resultados: as células germinativas (espermatogônias, espermatócitos e espermátides) doscontroles positivos sofreram marcac¸ão, enquanto células não pertencentes a essa linhagem(células de Leydig e de Sertoli) mostraram negatividade de reac¸ão; nos cortes ovarianosobservou-se marcac¸ão de oócitos de folículos em diferentes estágios de maturac¸ão, mashouve também marcac¸ão de células não englobadas pela estrutura folicular.Conclusões: os achados sugerem que durante a puberdade ovários de camundongos fêmeascontêm células da linhagem germinativa em estágios anteriores à formac¸ão folicular, oque corrobora estudos anteriores; o trabalho é pioneiro no Brasil e progredirá para a com-pleta caracterizac¸ão de células com potencial oogênico em outras espécies de mamíferos.Resultados positivos poderão alterar o entendimento da biologia reprodutiva e abrir novasportas para o tratamento de infertilidade.
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Papers by Daniel Dentillo
plating density effects and long-term serum-free culture on the in vitro dynamics differentiation of luteinizing human GCs. Cells
were cultured in serum-free a-minimum essential medium (a-MEM) or serum-based tissue culture medium (TCM). The time
course of GCs morphology and secretion of estradiol (E2), progesterone (P4), and relaxin were analyzed after 48, 96, and 144
hours of culture. Other functional markers as follicle-stimulating hormone/luteinizing hormone receptors and steroidogenic
enzymes were investigated at the end of culture. The morphology of an a-MEM cell rather than a TCM cell resembles more
closely that seen in vivo. Compared to TCM cultures, a-MEM cells secreted 93.7% and 87.2% more E2 and approximately 7% and
17% of the amount of P4 when cultured at densities of 2 104 or 4 104 cells/well, respectively. Relaxin secretion was significantly
reduced in a-MEM cultures. a-MEM cells were estrogenic and expressed the CYP19 gene. Levels of CYP17 increased
about 8-fold in a-MEM cells above the levels found in TCM cells. Our results reveal new insights into human GCs differentiation in
vitro and demonstrate the critical importance of the culture system and cell-plating density on the establishment of estrogenic or
progestogenic GC phenotypes.
plating density effects and long-term serum-free culture on the in vitro dynamics differentiation of luteinizing human GCs. Cells
were cultured in serum-free a-minimum essential medium (a-MEM) or serum-based tissue culture medium (TCM). The time
course of GCs morphology and secretion of estradiol (E2), progesterone (P4), and relaxin were analyzed after 48, 96, and 144
hours of culture. Other functional markers as follicle-stimulating hormone/luteinizing hormone receptors and steroidogenic
enzymes were investigated at the end of culture. The morphology of an a-MEM cell rather than a TCM cell resembles more
closely that seen in vivo. Compared to TCM cultures, a-MEM cells secreted 93.7% and 87.2% more E2 and approximately 7% and
17% of the amount of P4 when cultured at densities of 2 104 or 4 104 cells/well, respectively. Relaxin secretion was significantly
reduced in a-MEM cultures. a-MEM cells were estrogenic and expressed the CYP19 gene. Levels of CYP17 increased
about 8-fold in a-MEM cells above the levels found in TCM cells. Our results reveal new insights into human GCs differentiation in
vitro and demonstrate the critical importance of the culture system and cell-plating density on the establishment of estrogenic or
progestogenic GC phenotypes.