Toxic effects of Pb2+ entering sperm through Ca2+ channels in the freshwater crab Sinopotamon henanense

Lead (Pb) is a heavy metal that can damage animal sperm. To study the effects of Pb on calcium homeostasis and calcium channel in the sperm of freshwater crab Sinopotamon henanense, the induction of acrosome reaction (AR) and acrosin activity were investigated when crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8 mg/L) for 3, 5 and 7 d separately. Fluorescent probe Fluo-3/AM was loaded into the sperm, and [Ca2+] in the sperm was measured by fluorescence microscopy and using microplate reader. The calmodulin (CaM) concentration was measured by ELISA method. Verapamil (VRP), a calcium channel blocker, was used to evaluate whether Pb can enter the sperm through calcium channels leading to sperm damage. After sperm were exposed at 50 μg/L VRP, 100 μg/L Pb, 50 μg/L VRP + 100 μg/L Pb, 1000 μg/L Pb and 50 μg/L VRP + 1000 μg/L Pb for 1 h in vitro，sperm quality parameters (sperm survival and sperm DNA integrity) and levels of parameters indicating oxidative stress (protein carbonylation [PCO] and malondialdehyde [MDA]) were measured. Our data showed that Pb reduced the induction of acrosome reaction (AR), down-regulated the acrosin activity, decreased the intracellular concentration of Ca2+ and elevated CaM concentration. Compared to controls, Pb alone induced significant stress, as reflected by decreasing sperm survival and sperm DNA integrity, and increasing PCO and MDA contents. In the presence of VRP, 100 μg/L Pb-induced stresses were reduced, all the measured parameters in the sperm exposed at 100 μg/L Pb returned to control levels. Our results indicate that Pb enters the sperm of the crab S. henanense through calcium channels, the inhibition of which blocks Pb-induced stresses such as sperm quality decline and oxidative damage.