Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2

The highly selective fluorescent Ca2+ indicator 'quin 2' has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, 'quin 2/AM', which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean +/- S.E.M.) indicated a normal cytoplasmic free-calcium concentration, Ca2+]i, of 193 nM +/- 0.2 (n = 10) for ejaculated ram sperm, 175 nM +/- 3.9 (n = 10) for cauda epididymal boar sperm and 105 nM +/- 10 (n = 10) for the caput sperm. After cold shock ejaculated ram and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a Ca2+]i similar to control sperm. Sodium azide, propranolol and caffeine did not affect the Ca2+]i of ram and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rotenone and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of Ca2+]i, most likely resulting from release of mitochondrial calcium. The increased Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4-DNP and CCCP had no effect on Ca2+ )i. Addition of filipin to ram and boar sperm resulted in a large increase in Ca2+]i but addition of filipin to ionophore-treated sperm caused Ca2+]i to fall well below control levels.