BIOLOGY OF MALE AND FEMALE GAMETES
7:30 AM-10:00 AM
(181) Na+K+ATPase INVOLVEMENT IN SPERM CAPACITATION.
Buhr, Mary1, Anzar, Muhammad1, Kakuda, Nipa1, Baines, Shaun1, 1 University of Guelph, Guelph, CA
ABSTRACT- Intracellular calcium [Ca2+]i) increases during capacitation of bull sperm. Involvement of voltage-gated or enzyme-mediated channels has been hypothesized, as has IP3-mediated Ca2+ flux. Our work suggested cryopreservation reduced the fertilizing ability of bull sperm by damaging membranes thus altering regulation of Ca2+ movement. Cryopreservation also eliminated biochemical activity of Na+K+ATPase, specifically localised to the sperm head's plasma membrane. We currently hypothesize that cryopreservation alters the specific location of Na+K+ATPase, thereby affecting bull sperm function. Aliquots of fresh and frozen sperm from an ejaculate (n = 5 bulls) were evaluated for [Ca2+]i), motility and ability to undergo lyso-PC-induced acrosome reactions (AR). Indirect immunofluorescence of sperm using mouse anti-rabbit Na+K+ATPase 1 subunit antibody, followed by FITC conjugated anti-mouse IgG, showed uniform distribution of Na+K+ATPase over the acrosomal cap of alcohol-permeabilised, but not intact, fresh bull sperm. Cryopreservation caused patchy distribution of Na+K+ATPase, identical in intact or permeabilised sperm. In fresh sperm, ouabain, a specific inhibitor of Na+K+ATPase, caused a biphasic effect on Ca2+ uptake (monitored spectrofluorometrically using indo-1AM), immediately reducing uptake of 150 mM Ca2+, but increasing uptake of 200 mM Ca2+ (P < 0.0001); these effects were maintained for at least 12 min. Ouabain similarly immediately increased uptake of 200 mM Ca2+ by cryopreserved sperm, but this effect was eliminated by 12 min; no effect was seen with 150 mM Ca2+. Ouabain significantly increased the % of fresh, but not cryopreserved, sperm undergoing lyso-PC-induced AR compared to control after 45 min in capacitating buffer (27.0±8.1 vs 0.6±6.6%, P<0.01) but had no effect on any CASA-detected motility measures. These results suggest Na+K+ATPase may play a major role(s) in regulating the onset of capacitation and AR. (NSERC, the Semex Alliance, and OMAFRA provided financial support).
KEY WORDS: calcium, acrosome reaction, bull, motility