Dibutyl phthalate disrupts [Ca2+]i, reactive oxygen species, [pH]i, protein kinases and mitochondrial activity, impairing sperm function

Abstract

To explore the mechanism of sperm dysfunction caused by dibutyl phthalate (DBP), the effects of DBP on intracellular [Ca2+] and [pH], reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, phosphorylation of protein kinase A (PKA) substrate proteins and phosphotyrosine (p-Tyr) proteins, sperm motility, spontaneous acrosome reaction, and tail bending were examined in mouse spermatozoa. At 100 µg/mL, DBP significantly increased tail bending and [Ca2+]i. Interestingly, DBP showed biphasic effects on [pH]i. DBP at 10–100 µg/mL significantly decreased sperm motility. Similarly, Ca2+ ionophore A23187 decreased [pH]i sperm motility, suggesting that DBP-induced excessive [Ca2+]i decreased sperm motility. DBP significantly increased ROS and LPO. DBP at 100 µg/mL significantly decreased mPTP closing, MMP, and ATP levels in spermatozoa, as did H2O2, indicative of ROS-mediated mitochondrial dysfunction caused by DBP. DBP as well as H2O2 increased p-Tyr sperm proteins and phosphorylated PKA substrate sperm proteins. DBP at 1–10 µg/mL significantly increased the spontaneous acrosome reaction, suggesting that DBP can activate sperm capacitation. Altogether, DBP showed a biphasic effect on intracellular signaling in spermatozoa. At concentrations relevant to seminal ortho-phthalate levels, DBP activates [pH]i, protein tyrosine kinases and PKA via physiological levels of ROS generation, potentiating sperm capacitation. DBP at high doses excessively raises [Ca2+]i and ROS and disrupts [pH]i, impairing the mitochondrial function, tail structural integrity, and sperm motility.