RESUMO
BACKGROUND: The objective was to develop an experimental model for studying the differentiation of trophoblast and inner cell mass (ICM) during the early stages of implantation in primates. METHODS: Marmoset monkey blastocytes were used in these studies. Ovulation was timed by plasma progesterone assays in ovarian cycles initiated by administering a luteolytic agent to mating marmosets. Embryos were recovered from the uterus usually at the eight-cell stage and cultured in minimum essential medium containing fetal calf serum, insulin, and transferrin. The embryos that formed hatched blastocysts by about day 11 after ovulation were transferred for further development in Matrigel-coated culture chambers. After 2, 4, and 6 days of development, two blastocysts were processed at each interval and serially sectioned for light and electron microscopy. RESULTS: All blastocysts adhered to the Matrigel at their embryonic pole within 24 hours. Adherent polar cytotrophoblast was differentiating to syncytiotrophoblast at all time intervals, but syncytium was not detected in mural trophoblast until day 4 after attachment. By day 2 syncytial microvilli and processes had penetrated the Matrigel surface, whereas by days 4 and 6 cytotrophoblast that was differentiating to syncytiotrophoblast had invaded the matrix. Since all blastocysts maintained their structural integrity progressive differentiation of the ICM, endoderm and presumptive mesoderm was observed. A small amniotic cavity was observed at 2 days and by 6 days a distinct cavity separated polarized epiblast and amnion cells. Visceral and parietal endoderm were present at 2 days, and a completed primary yolk sac was observed by 4 days after attachment. In all blastocysts a basal lamina lined the inner surface of mural and polar trophoblast and the basal surface of the differentiating ICM. CONCLUSIONS: The developmental time sequence of the cultured blastocysts closely resembled the time frame reported for marmoset embryos implanting in utero. An effective model for studying trophoblast invasion and differentiation of embryonic germ cell layers has been established.
