PARENT SESSION
Capacitation & Acrosome Reaction

INVESTIGATION INTO THE EXISTENCE OF ADRENERGIC RECEPTORS ON BULL SPERM.

Way, Amy¹, Killian, Gary¹, ¹

ABSTRACT- Sperm are suspected to possess adrenergic receptors (R) based on their response to catecholamines in vitro. However, attempts to identify R using radioligand binding and fluorescently labeled antagonists have been unsuccessful. We recently reported that norepinephrine (NE) capacitated bull sperm in vitro. This study was conducted to determine if ejaculated bull sperm contain R. To visualize binding of catecholamines, sperm were incubated in BODIPY-FL CGP12177 (-agonist) or BODIPY-FL prazosin (-agonist) and evaluated by flow cytometry. Sperm were incubated with labeled agonist and increasing amounts of unlabeled agonist to define nonspecific binding. Capacitated sperm were evaluated in the event that R were masked until capacitation. Frozen, thawed preparations of washed sperm were used in a radioligand assay with 1 nM [4-³H]-propranolol HCl. Parallel sperm preparations were incubated with radioligand and isoproterenol HCl to define nonspecific binding. Binding was quantified by liquid scintillation. Western blots of solubilized sperm membrane protein, FCS (negative control) and cell lysate from DDT1 MF-2 cells (positive control) were probed with polyclonal rabbit anti-₁ adrenoreceptor antibody. Antibody binding was visualized using ECL. To test whether adrenergic antagonists blocked the effects of NE on sperm, washed sperm were incubated in ₁ (benoxathian HCL), ₂ (rauwolscine HCl), ₁ (CGP 20712A methanesulfate), and ₂ (ICI-118,551 HCl) antagonists with and without 10 ng/ml NE, then incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction in capacitated sperm. Additionally, capacitated sperm (10 g/ml heparin) were challenged to acrosome react by NE, each antagonist, and NE + each antagonist. Attempts to identify R using fluorescently conjugated agonists, [4-³H]-propranolol HCl and antibody against ₁-adrenergic receptor were unsuccessful. Incubating sperm with adrenergic antagonists gave ambiguous results, with the antagonists themselves often increasing the percentage of acrosome reactions. These studies suggest that the action of NE on sperm is not R-mediated. Although it is difficult to rule out a R-mediated action of NE without identifying an alternative mechanism, the negative findings of this study are supported by studies on sperm from other species.

KEY WORDS: catecholamines, adrenergic receptors