Calcium requirement and time course of capacitation of goat spermatozoa assessed by chlortetracycline assay.
We standardized chlortetracycline fluorescent assay for studies of calcium requirement and time course of capacitation of goat spermatozoa. Three distinct fluorescent patterns were easily detected in goat spermatozoa incubated under capacitating conditions. Categorised according to nomenclature reported earlier, these are: 'F' with bright fluorescence in the postacrosomal region, characteristic of uncapacitated acrosomal-intact cells; 'B' with bright fluorescence on the anterior portion of the head and dark band in the postacrosomal region, characteristic of capacitated, acrosome-intact cells; 'AR' with lack of fluorescence on the head characteristic of acrosome-reacted cells. A close correspondence 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 increased with increase in calcium concentration; at 1.0 mmol l-1 a peak representing 65-70% capacitated cells accumulated in 4 h. At higher concentrations, '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 studies 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 to 'B' cells was noticed. However, at no time were 'AR' cells found exclusively 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 (HCO3-) and organic phosphates (beta-glycerophosphate, glucose-6-phosphate) were unable to replace phosphate. The reason for their failure for the incidence of capacitation was traced to low uptake of calcium by goat 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.
Kaul
G
G
Department of Biochemistry, National Dairy Research Institute, Karnal, India.
Singh
S
S
Gandhi
K K
KK
Anand
S R
SR
D000177:Acrosome / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D002751:Chlortetracycline; D004533:Egtazic Acid / Q000494:pharmacology; D006041:Goats; D007700:Kinetics; D008297:Male; D008634:Mersalyl / Q000494:pharmacology; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010710:Phosphates / Q000494:pharmacology; D013075:Sperm Capacitation / Q000187:drug effects*; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D010710:Phosphates; D013439:Sulfhydryl Reagents; D004533:Egtazic Acid; D008634:Mersalyl; D002118:Calcium; D002751:Chlortetracycline