ATP and cAMP Assay

For measurements of intracellular ATP, the ejaculated sperm (2×10⁷ cells /ml) were incubated with or without BisII (1 µM) for 10 min. After washing with sperm extender, the sperm pellet was dissolved in an ATP assay reagent (“Cellno” ATP Assay reagent, TOYO B-Net Co., Ltd., Tokyo, Japan) and the fluorescent signal measured using ImageQuant™ LAS 500 (GE Healthcare Japan Corporation, Hino, Japan). Intracellular cAMP was detected using a cAMP Biotrak enzyme immunoassay kit (GE Healthcare) according to the manufacturer's instructions. The optical density of the solution, at 492 nm, was measured using a microplate reader (Tecan Japan Co., Ltd., Kawasaki, Japan).