Abstract
Extracellular calcium at millimolar concentrations inhibits collective motility of ejaculated ram spermatozoa. In untreated cells, or when motility was made dependent upon glycolytic activity, there is very small inhibition, but when motility was made dependent upon mitochondrial respiration there is very high inhibition in motility by increasing extracellular Ca2+ concentration. Quercetin, which inhibits (Ca2+ + Mg2+)-ATPase activity in isolated plasma membranes, also inhibits motility mainly in cells that have been made dependent upon glycolytic activity, but there is also inhibition in untreated cells. When motility was made dependent upon mitochondrial activity, there is no inhibition but rather some stimulation in motility by quercetin. The inhibitory effect of quercetin is enhanced by increasing Ca2+ concentration in the medium. Quercetin also inhibits uptake of calcium into the cells, in a mechanism by which a calcium channel is involved. This inhibition is high only when the glycolysis is inhibited in the cells. The rate of glycolysis is decreased by quercetin or ouabain, but their effects on motility are quite different. Based on these data, it appears that the plasma membrane (Ca2+ + Mg2+)-ATPase or the Ca2+ pump have a functional role in the regulation of spermatozoa motility. This motility regulation is functioning through mechanisms which include glycolytic activity and maintenance of intracellular calcium concentrations.
Highlights
It is shown in Fig. 1that under conditions by which the cells were exposed to antimycin A, which inhibits mitochondrial electron transport, there is a small effect of Ca" on motility
Since ATP-dependent calcium uptake inisolated plasma membrane antimycin A causes above 90% inhibition of calcium uptake; we can exclude any involvement of into thecells,we suggest that about 90% of the calcium uptake this calcium pump in thedescribed inhibitory effect of D-600 has gone into the mitochondria and only about 10%into the on calcium uptake into intact cells
We demonstrate here for the first time the effect of extracellular calcium concentrationsandATPaseinhibitors on collective motility of ram spermatozoa
Summary
The fresh semen was immediately diluted with calcium-free Krebs-Ringer buffer solution (pH 7.2) containing 20 mM sodiumphosphate and 28 mM fructose [21]. Extracellular Ca” strongly affects motility of intact hamster [4,5], mouse [5], and rat( 6 )sperm pellet was resuspended in Krebs-Ringer buffer to reach a final concentration of 4 X IOs cefis/ml. Aebxotruatcleol3~-ulartPioiaPnsrmeapanadmrasetuimocnbroroasfenSegpvreaerdsmiicePinetsh, m a Membranes and Uptakeof Calciumwere prepared by differential centrifugaas we described previously [18] Measurements of Lactate Production-Washed cells were suspended in Krebs-Ringer buffer (pH 7.2) containing 20 mM sodium phosphate and 28 mM frucose, and were incubated a t 37 "C. Calcium Uptake by Intact Cells-Calcium uptake by intact spermatozoa was measured in Krebs-Ringer buffer (pH 7.2) containing 20 mM sodium phosphate, 0.2 mM CaCI2,and 10 pCi of *CaCI,.
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