Implantation involves trophoblast attachment to, and penetration of, the endometrial epithelium. The factors that regulate these processes in human and non-human primates are poorly understood, largely due to the lack of suitable in vitro systems. We have recently found that macaque blastocysts attach to monolayers of MDCK epithelial cells. This in vitro implantation model offers advantages not frequently met in other systems. Attachment was followed by development of the inner cell mass (ICM) and penetration of the epithelial monolayer by trophoblasts. Other preliminary data show that expression of integrins by isolated macaque trophoblasts is regulated by contact with immobilized chemokines. Also, trophoblasts migrate in a haptotactic manner towards the chemokine, RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted). RANTES and other chemokines are present in both human and macaque endometrium. Our long-term objective is to understand the mechanisms that regulate trophoblast penetration of the endometrial epithelium. We suggest that a novel in vitro implantation model that utilizes macaque blastocysts and polarized MDCK cells would allow many important questions relating to the regulation of implantation to be addressed. In this R03 application we propose experiments that will validate this model. Validation criteria will include acquisition of a migratory trophoblast phenotype and development of an inner cell mass and extraembryonic mesoderm. In addition, we will use this system to test the hypothesis that interaction of blastocysts with immobilized chemokines on the epithelial surface results in trophoblast activation and the acquisition of a polarized migratory phenotype.
Specific Aim 1 uses immunocytochemistry, in situ hybridization, and quantitative image analysis to characterize the expression of migratory phenotypic markers (integrins, chemokine receptors) during trophoblast penetration of polarized MDCK cells.
Specific Aim 2 characterizes blastocyst development (as defined by the appearance of an amniotic cavity, appearance of an epiblastic plate, and differentiation of extraembryonic mesoderm) during co-culture with MDCK cells.
Specific Aim 3 will use neutralizing antibodies and immobilized recombinant chemokines to characterize the role of chemokines in regulating expression of trophoblast migratory phenotypic markers and blastocyst development. ? ?
| Vandevoort, Catherine A; Thirkill, Twanda L; Douglas, Gordon C (2007) Blastocyst-derived trophoblast stem cells from the rhesus monkey. Stem Cells Dev 16:779-88 |
| Thirkill, Twanda L; Lowe, Kimberly; Vedagiri, Hemamalini et al. (2005) Macaque trophoblast migration is regulated by RANTES. Exp Cell Res 305:355-64 |
