Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations.
Boar spermatozoa loaded with the Ca2+ probe fluo-3 were incubated in various Tyrode's-based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two-colour flow cytometry; propidium iodide was included in the media to detect membrane-damaged ("dead") cells. If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonate-mediated increase in cell death took place in the absence of external Ca2+. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo-3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it. The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times.
Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, United Kingdom.
R A
RA
Harrison
B
B
Mairet
N G
NG
Miller
D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology*; D003994:Bucladesine / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D016923:Cell Death; D005576:Colforsin / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D003839:Deoxyadenosines / Q000494:pharmacology; D004734:Energy Metabolism; D005307:Fertilization in Vitro; D005434:Flow Cytometry; D005452:Fluoresceins; D006531:HEPES / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D010208:Papaverine / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D013552:Swine
D001639:Bicarbonates; D003470:Culture Media; D003839:Deoxyadenosines; D005452:Fluoresceins; D007546:Isoquinolines; C053897:5(6)-carboxy-2',7'-dichlorofluorescein; D005576:Colforsin; D012710:Serum Albumin, Bovine; D002110:Caffeine; D003994:Bucladesine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D010208:Papaverine; D000242:Cyclic AMP; D006531:HEPES; D002118:Calcium