No abstract

Human granulosa cells were maintained in culture with extracellular matrix in the presence or absence of human chorionic gonadotrophin (HCG) using a defined culture medium. Such cultures are maintained by gonadotrophin in a manner suggesting that features of 'luteal rescue' may be occurring in vitro. Western analysis of culture medium demonstrated that the granulosa cells produced tissue inhibitor of metalloproteinases (TIMP)-1 but not TIMP-2. The presence of TIMP-1 in cultured cells was also detected immunocytochemically. Immunoassay of TIMP-1 output revealed that HCG exposure for 7 days caused a 2-fold increase in TIMP-1 production versus control reaching maximum at approximately 1 ng HCG/ml. The sensitivity of this response to HCG was similar to that observed for stimulation of progesterone production. Delayed addition of HCG, from day 4 of culture, elicited increases in TIMP-1 which were evident within 24 hours, and were not explained by changes in cell replication or survival. Removal of HCG from cultures previously luteinized with HCG for 6 days resulted in a fall in TIMP-1 production. Thus TIMP-1 production by luteinized granulosa cells in culture is gonadotrophin dependent. We speculate that prolonged cellular function associated with 'luteal rescue' may result from increased extracellular matrix stability mediated by up-regulation of TIMP-1 production.

We have previously shown that human granulosa cells cultured on a thin layer of extracellular matrix (ECM) are lost from culture in the absence of gonadotrophin. We now examine the effect of plating ECM onto glass or plastic. Such a comparison revealed that loss of cells from control cultures was more rapid when ECM was on glass, whereas cultures maintained with human chorionic gonadotrophin (HCG) showed greater stability when ECM was on plastic. The dose range of HCG required for the effect on cell retention was similar to that required for stimulation of progesterone production. Electron microscopy of cells freshly released as clusters revealed that many cells appeared undamaged, and confocal microscopy of cells stained with propidium iodide showed an absence of fragmented nuclei. Taken together, this evidence suggests that apoptosis is not the cause of cell release. We conclude that cells are released from culture, not as a result of cell death, but via an active process suppressed by HCG.