Progression through the prophase of the first meiotic division can be obtained in culture by treatment of mouse spermatocytes with the serine/threonine phosphatase inhibitor okadaic acid. Chromosome condensation during this G2/M transition, involves the activation of The MAPK pathway, which causes the activation of Nek2 and the phosphorylation of the chromatin architectural protein Hmga2. In an effort to set up conditions to allow a spontaneous progression of mouse spermatocytes through meiosis, we have investigated the cell-cycle features of these cells cultured for 24 h with a rotary cell culture system in a humidified atmosphere in a thermostatic incubator to simulate a microgravity environment. Morphological analysis of nuclear squashes indicated a 2-fold increase in late-pachytene spermatocytes with highly condensed chromosomes, and a contemporaneous decrease of mid-pachytene cells with less condensed chromatin. Microgravity induced a 2-fold activation of the cyclinB-cdc2 complex, confirming at the molecular level that cell-cycle progression had occurred. Moreover, using immuno-kinase assays with specific substrates we have demonstrated that the meiotic progression obtained under microgravity conditions is accompanied by activation of the Erk1/p90Rsk2 pathway. These data indicated that activation of the MAPK pathway correlates with chromatin condensation even under conditions in which meiotic progression occurs spontaneously and is not induced by a drug. We suggest that culture under microgravity conditions might help to release the block that inhibits isolated spermatocytes from progressing through prophase at unit gravity, and to study the physiological events of germ cell differentiation in vitro. © 2004 Society for Reproduction and Fertility.
Meiotic progression of isolated mouse spermatocytes under simulated microgravity
Di Agostino S.;
2004-01-01
Abstract
Progression through the prophase of the first meiotic division can be obtained in culture by treatment of mouse spermatocytes with the serine/threonine phosphatase inhibitor okadaic acid. Chromosome condensation during this G2/M transition, involves the activation of The MAPK pathway, which causes the activation of Nek2 and the phosphorylation of the chromatin architectural protein Hmga2. In an effort to set up conditions to allow a spontaneous progression of mouse spermatocytes through meiosis, we have investigated the cell-cycle features of these cells cultured for 24 h with a rotary cell culture system in a humidified atmosphere in a thermostatic incubator to simulate a microgravity environment. Morphological analysis of nuclear squashes indicated a 2-fold increase in late-pachytene spermatocytes with highly condensed chromosomes, and a contemporaneous decrease of mid-pachytene cells with less condensed chromatin. Microgravity induced a 2-fold activation of the cyclinB-cdc2 complex, confirming at the molecular level that cell-cycle progression had occurred. Moreover, using immuno-kinase assays with specific substrates we have demonstrated that the meiotic progression obtained under microgravity conditions is accompanied by activation of the Erk1/p90Rsk2 pathway. These data indicated that activation of the MAPK pathway correlates with chromatin condensation even under conditions in which meiotic progression occurs spontaneously and is not induced by a drug. We suggest that culture under microgravity conditions might help to release the block that inhibits isolated spermatocytes from progressing through prophase at unit gravity, and to study the physiological events of germ cell differentiation in vitro. © 2004 Society for Reproduction and Fertility.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
