Nitric Oxide and Superoxide Anion Production During Heparin-Induced Capacitation in Cryopreserved Bovine Spermatozoa

Abstract

The aim of this work was to quantify NO, O2− and ONOO− production during heparin-induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin-dependent capacitation, O2 uptake, and NO production. Conversely, O2− production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O2 consumption (5.9 ± 0.6 nmol/min × 107 cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 107 cells), and a five-fold increase in O2− production (1.3 ± 0.07 nmol/min × 107 cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O2− generation and the second-order rate constant of the reaction between these species. To conclude, heparin-induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O2 uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O2− production by a membrane-bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.