Luteinized human granulosa cells are associated with endogenous basement membrane-like components in culture

Human granulosa cells (GC), prepared from follicular aspirates using a non-enzymic method, were maintained in culture on chamber slides in a defined medium without additional attachment factors or extracellular matrix (ECM), In this system, GC clustered to a limited extent and attached only loosely to the substratum necessitating medium replacement through repeated partial changes to avoid cell loss. Using this new culture system, cell size and progesterone production per cell increased, consistent with continuing luteinization. These processes were associated with maintenance and deposition of endogenous ECM components. Thus, pericellular heparan sulphate proteoglycan (HSPG) was clearly visible by immunocytochemistry around the luteinized GC after culture. Also progressive accumulation of laminin (particularly alpha(2)-, beta(1)- and gamma(1)-subunits) during culture was shown by Western blotting of GC extracts. Small patches of collagen IV, shown to be already present between freshly prepared GC, were maintained in culture. A clear effect of gonadotrophin on the maintenance of progesterone production in culture was paralleled by an apparent increased pericellular deposition of HSPG. To conclude, luteinization and maintenance of the GC-derived layer of the corpus luteum is likely to involve deposition and conservation of pericellular ECM components.