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.

Cytotoxicity of the mistletoe lectins I, II and III towards six human breast cancer cell lines was assessed using the Mossman assay. In addition, binding of the three mistletoe lectins to the separated membrane glycoproteins of these cell lines, the binding and uptake of these lectins into the cells in tissue culture and the binding of the lectins to histological preparations of these cell lines were analysed. The results indicate that there are quantitative differences concerning the toxicity of these three lectins towards the different cell lines. Furthermore, the lectin binding pattern in the cell lines differed. In Western blots, several membrane glycoproteins were labelled by the lectins. Our results indicate subtle differences between the three lectins with regard to the parameters mentioned above; however, the toxicity of all three lectins from mistletoe is so similar that they all seem suitable for the construction of immunotoxins.

Granulosa cells were prepared from follicular aspirates obtained at oocyte collection for in-vitro fertilization (IVF) and maintained in culture. Substantial loss of cells from the culture surface occurred in the absence of gonadotrophin when cells were maintained on a thin layer of extracellular matrix (ECM) using a defined, serum-free medium. This cell loss was clearly and significantly reduced in the presence of human chorionic gonadotrophin (HCG) by days 4-6 of culture, and occurred in conjunction with loss of ECM. Analysis of culture medium by zymography using gelatin as substrate demonstrated the presence of metalloproteinases (MMP), MMP-9 (gelatinase B) appearing as the predominant band. Measurement of overall gelatinase activity in culture media revealed a progressive fall in gelatinase expressed on a per cell basis in media from HCG-treated cultures and this was less marked in controls. This suppression of gelatinase activity was consistent with an observed increase in production of tissue inhibitor of metalloproteinase-1 (TIMP-1) by HCG-treated cells, which was significant by days 6-8 of culture. We speculate that stabilization of the ECM may be an important aspect of HCG action in the corpus luteum.