An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm

In vitro incubation of epididymal and vas deferens sperm with Mn²⁺ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn²⁺ and Mg²⁺) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry.

Incubation with Mn²⁺/Ca²⁺ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg²⁺/Ca²⁺ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn²⁺/Ca²⁺ or Mg²⁺/Ca²⁺, SCF generated DNA fragments of 33-194 Kb, compatible with the size of one or multiple toroids. Besides, Mn²⁺/Ca²⁺ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg²⁺/Ca²⁺ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn²⁺/Ca²⁺ or Mg²⁺/Ca²⁺ treatments.

Mn²⁺/Ca²⁺ and Mn²⁺/Ca²⁺ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.