The type and differentiation of cells in vitro from unilaminar and bilaminar blastocysts of two marsupials, Antechinus stuartii and Sminthopsis macroura

The type and ability to differentiate in vitro of cells found in blastocysts of two marsupials were examined. Thirteen unilaminar blastocysts on Day 7 to Day 12 of gestation and 24 bilaminar blastocysts on Day 16 and Day 18 of gestation were collected from 11 brown antechinus, Antechinus stuartii. A total of of 77 unilaminar blastocysts on Day 5 and Day 6 of gestation and a total of 61 bilaminar blastocysts on Day 6 and Day 7 of gestation were collected from 40 stripe-faced dunnarts, Sminthopsis macroura. Pluriblast and trophoblast cells, confined to separate hemispheres, were found in unilaminar and bilaminar blastocysts, establishing that the blastocyst epithelium was not a protoderm. Hypoblast cells were found only in bilaminar blastocysts. Pluriblast and hypoblast cells did not differentiate or proliferate in up to eight weeks in culture. A small number of trophoblast cells transformed to a multinucleate state but the remainder did not proliferate or differentiate further. The presence of murine leukaemia inhibitory factor or medium conditioned by exposure to marsupial fibroblast feeder layer was not required for the maintenance of an undifferentiated state. Differentiation, proliferation and attachment of cells were not influenced by the presence of the yolk mass or the egg coats in culture. The time taken for attachment of dissociated cells varied significantly between cultures with no substrate and with collagen, fibronectin or laminin (P = 0.001, ANOVA) but did not vary significantly between substrates. The substrates did not influence the state of differentiation of the cells.