Cryopreservation-induced alterations in boar spermatozoa mitochondrial function are related to changes in the expression and location of midpiece mitofusin-2 and actin network

The authors analyzed changes in mitochondrial activity of boar semen during a standard cryopreservation protocol. For this purpose, mitochondrial activity was evaluated simultaneously with the rhythm of mitochondrial formation of reactive oxygen species (mROS) through a double MitoTracker Red/proxylfluorescamine stain. Moreover, we analyzed changes in the expression and location of two key regulatory elements of mitochondrial function, namely mitofusin-2 (Mfn2) and actin, during the freezing-thawing protocol. Our results indicate that mitochondrial activity and mROS formation decreased during cyropreservation, with an initial decrease during the cooling phase of the protocol. This decrease was accompanied by an increase in the amount of solubilized Mfn2, which was concomitant with a progressive extension of Mfn2 location from the apical zone of the midpiece to the whole midpiece. Simultaneously, cryopreservation induced a decrease in solubilized actin, which was concurrent with significant changes in the midpiece actin location. The observed changes in the expression and location of both Mfn2 and actin were already present after the cooling phase of the cryopreservation protocol. Our results suggest that freezing-thawing impaired mitochondrial function. This impairment was concomitant with a decrease in the mitochondrial capacity to synthesize mROS. This impairment is attributed to changes in mitochondrial volume as a result of alterations in the expression and location of both Mfn-2 and the actin network. Finally, the alterations of mitochondrial function induced by the cryopreservation protocol were already apparent at the cooling phase. This observation indicates that the cooling phase is a crucial stage in which mitochondrial alterations occur during cryopreservation.