MilliporeSigma

Analysis of trophoblast giant cell steroidogenesis in primary cultures.

Methods in molecular medicine (2006-03-04)
Noa Sher, Joseph Orly
ABSTRACT

Placental progesterone synthesis in humans prevents abortion of the fetus by maintaining uterine quiescence and low myometrial excitability. In rodents, a transient steroidogenic output is observed in the trophoblast giant cells during mid-pregnancy. Although the exact role of this locally produced progesterone is not clear, rodent trophoblast giant cells are an important cell model for studying the regulation of placental steroidogenesis. This chapter describes the methods we developed to analyze the regulation of genes involved in progesterone biosynthesis in miniature cultures of primary trophoblast cells from rodents. These genes include cholesterol side chain cleavage cytochrome P450 (P450scc) and its accessory proteins, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD). To obtain giant cells, uterine implantation sites are sliced in half, and the trophoblast giant cell layers are separated from the surrounding decidua by scraping. Cells can subsequently be separated by gentle enzymatic digestion with trypsin, or collagenase, and plated for further study in vitro. This chapter provides instructions, insights, and comments instrumental for performing in situ visualization of giant cell mRNA and proteins, analyzing enzyme activities, and conducting promoter analyses with a limited number of cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Dextran sulfate sodium salt from Leuconostoc spp., for molecular biology, average Mw >500,000 (dextran starting material), contains 0.5-2% phosphate buffer