[The effect of iron on peritoneal macrophage activity and sperm phagocytosis in rats]

Excessive activation of macrophages is considered to be the etiological factor of marital infertility. Peritoneal macrophages participate in the phagocytosis of menstrual detritus and sperm in the peritoneal cavity. Iron ingested by peritoneal macrophages could be responsible for their increased spermiophagy. This mechanism would operate in some gynecological diseases, particularly in endometriosis when the ectopic location of endometrial tissue in the pelvic cavity or oviduct becomes a source of cyclic menorrhagia into the peritoneal fluid. The aim of this study was to establish the effect of iron (Jectofer, Astra D, complex salt of Fe+3; 50 micrograms Fe+3/ml) on the morphology and phagocytic activity of LPS-activated peritoneal macrophages. Macrophages were cultured with iron in the presence or absence of iron chelator--Desferal (DFO) (Sigma; 500 micrograms/ml), using the method of Nechala and Hrudka [20]. The viability of cells was evaluated with the trypan blue exclusion test. Cells were washed twice, suspended in modified Dulbecco's medium, supplemented with 2% inactivated fetal calf serum and antibiotics, than transferred (1 x 10(6)) into a culture dish. Nonadherent cells were removed by repeated washing after 1 h incubation at 37 degrees C and 5% CO2. Macrophages were cultured in 1 ml medium with LPS (1 microgram/ml). After 2 or 24 h the macrophages were covered with the same number of rat epididymal sperm cells. Following 1.5 h of incubation, phagocytosis was assessed on the basis of the spermiophagic index (SPI). After 3.5 h of culture macrophages formed monolayers and groups of cells with intersecting sperm tails (Fig. 2). Increased sperm phagocytosis was observed in the macrophage culture exposed to iron for 3.5 h. SPI was significantly higher compared to control value (Fig. 1). The findings were confirmed with scanning and transmission electron microscopy. Macrophages cultured with iron for 3.5 h displayed features of activation, growing to considerable size and developing numerous elongated processes with which they surrounded spermatozoa. The cytoplasm was replete with endosomes containing spermatozoa (Fig. 3). Electron-dense structures could be seen in phagolysosomes. The presence of iron in these structures was confirmed by X-ray microanalysis (Fig. 6). In comparison, macrophages cultured in the presence of iron and iron chelator demonstrated diminished phagocytic activity (Fig. 1). After 24 h of culture macrophages formed cluster-like structures. Spermiophagy was still taking place outside such aggregates and macrophages had a normal appearance (Fig. 4). When iron was added to such culture very few macrophages and spermatozoa could be seen in the electron microscope (Fig. 5A). Iron-loaded macrophages underwent necrosis, their nucleus, plasma membrane and organelles displayed features of degeneration (Fig. 5B). SPI of macrophages exposed to iron for 24 h was significantly decreased as compared with control value (Fig. 1). The ultrastructure of macrophages exposed for 24 h to DFO only was not altered and the phagocytic activity was comparatively higher (Fig. 1). There was a great number of macrophages and spermatozoa forming giant aggregations. The present results suggest that iron enhances spermiophagy in 3.5 h culture. As phagocytic activity of macrophages was reduced by Desferal in 3.5 h culture, an iron chelator could be beneficial in endometriosis to reduce the iron content in the peritoneal cavity where a regular influx of new macrophages takes place.