EFFECTS OF CALCIUM, MAGNESIUM, POTASSIUM AND BORON ON SPERM CELLS ISOLATED FROM POLLEN OF ZEA-MAYS L

Our previous studies showed that Brewbaker and Kwack salts, which have been widely used in pollen germination and sperm isolation, are not a ppropriate for the maintenance of isolated maize (Zea mays L.) sperm c ells. In the present study, we have characterized the effects of each BKS component salt on the integrity of isolated sperm cells using hema cytometry. At 0.01 and 0.1 mM, there were no differences in cell numbe r between control and any salt-treated cells except a 22% decrease wit h 0.1 mM MgSO4 at 48 h. At the 1 mM level, cell number decreased with time in the presence of Ca(NO3)(2) and MgSO4, with loss of integrity o f most cells at 48 h, while KNO3 and H3BO3 had little or no effect. Fu rther characterization of calcium-induced reduction in cell integrity using flow cytometry showed that depletion of possible residual free c alcium by addition of EGTA to the suspension medium improved cell long evity and viability. Exposure of isolated sperm cells to 1 mM calcium had no effect on cell integrity and viability in 5 h; however, only 12 % of cells remained intact at 24 h. The reduction in cell integrity wa s hastened when cells were pretreated with the calcium ionophore A2318 7 prior to exposure to 1 mM calcium, with a 54% reduction in cell numb er at 1 h and complete cell lysis at 24 h. However, depletion of cytos olic free calcium by pretreatment of cells with the calcium ionophore followed by resuspension in the presence of EGTA resulted in rapid red uction of cell integrity as well. These results collectively suggest t hat maize sperm cells are sensitive to exogenous free calcium; however , a certain level of cytosolic free calcium is necessary for maintenan ce of integrity. Mechanisms of calcium-induced reduction in cell integ rity are discussed along with possible roles of the sensitivity of spe rm cells to calcium in fertilization.