CALCIUM REQUIREMENT AND TIME-COURSE OF CAPACITATION OF GOAT SPERMATOZOA ASSESSED BY CHLORTETRACYCLINE ASSAY

We standardized chlortetracycline fluorescent assay for studies of cal cium requirement and time course of capacitation of goat spermatozoa. Three distinct fluorescent patterns were easily detected in goat sperm atozoa incubated under capacitating conditions. Categorised according to nomenclature reported earlier, these are: 'F' with bright fluoresce nce in the postacrosomal region, characteristic of uncapacitated acros omal-intact cells; 'B' with bright fluorescence on the anterior portio n of the head and dark band in the postacrosomal region, characteristi c of capacitated, acrosome-intact cells; 'AR' with lack of fluorescenc e on the head characteristic of acrosome-reacted cells. A close corres pondence was observed when the results of CTC assay were compared with those obtained by transmission electron microscopy. Goat spermatozoa were not capacitated when calcium was omitted from the medium and 80% had CTC fluorescence of 'F' type. The size of 'B' cell population incr eased with increase in calcium concentration; at 1.0 mmol l(-1) a peak representing 65-70% capacitated cells accumulated in 4 h. At higher c oncentrations, 'AR' cells were found along with 'B' cells and the two cell types were in equal proportions at 1.71 mmol l(-1). Time course s tudies revealed a 2 h incubation period at 1.0 mmol l(-1) and 1 h at 2 mmol l(-1) calcium concentration before transformation of 'F' cells t o 'B' cells was noticed. However, at no time were 'AR' cells found exc lusively pointing to an equilibrium between the two sperm populations. Goat spermatozoa were also not capacitated when phosphate was omitted from the medium. Permeant anions (NO3- SCN-), permeant weak acid (HCO 3-) and organic phosphates (beta-glycerophosphate, glucose-6-phosphate ) were unable to replace phosphate. The reason for their failure for t he incidence of capacitation was traced to low uptake of calcium by go at spermatozoa. In the presence of phosphate, a 6-8-fold increase was measured over the calcium uptake when phosphate was omitted (2-4 nmol l(-1) 10(8) cells(-1)). Mersalyl inhibited the calcium uptake by goat spermatozoa as well as its capacitation most likely by inhibiting the calcium phosphate transporter located in the sperm plasma membrane.