The Mitochondria of Stallion Spermatozoa Are More Sensitive Than the Plasmalemma to Osmotic‐Induced Stress: Role of c‐Jun N‐terminal Kinase (JNK) Pathway

Abstract

Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ranging from 75 to 1500 mOsm/kg, and the effect on sperm membrane integrity and mitochondrial membrane potential was studied. Additionally, the effects of changes in osmolality from hyposmotic to isosmotic and from hyperosmotic to isosmotic solutions were studied (osmotic excursions). The cellular volume of stallion spermatozoa under isosmotic conditions was 20.4 6 0.33 mm 3 . When exposed to low osmolality, the stallion spermatozoa behaved like a linear osmometer, whereas exposure to high osmolalities up to 900 mOsm/kg resulted in decreased sperm volume. Although sperm membranes were relatively resistant to changes in osmolality, mitochondrial membrane potential decreased when osmolalities were low or very high (10.7 6 1.74 and 16.5 6 1.70 at 75 and 150 mOsm/kg, respectively, and 13.1 6 1.83 at 1500 mOsm/kg), whereas in isosmolar controls the percentage of stallion sperm mitochondria with a high membrane potential was 41.1 6 1.69 (P , .01). Osmotic excursions induced greater damage than exposure of spermatozoa to a given nonphysiologic osmolality, and again the mitochondria were more prone to damage induced by osmotic excursions than was the sperm plasma membrane. In search of intracellular components that could mediate these changes, we have detected for the first time the c-Jun N-terminal kinase 1/2 in stallion spermatozoa, which are apparently involved in the regulation of the viability of these cells.

The Membrane of the Mammalian Spermatozoa: Much More Than an Inert Envelope

“…At the molecular level, these changes affect to lipid–protein interactions and to the asymmetry and composition of phospholipids, both implicated in the loss of the selective permeability of the membranes (Mazur 1984). Finally, recent research in our laboratory demonstrated a greater effect of osmotic‐induced stress on mitochondria than on plasma membrane damage, with mitochondrial dysfunction appearing earlier than plasma membrane damage (Garcia et al. 2012).…”

“…Cellular damage potentially occurs owing to this double osmotic stress: the hyperosmotic stress during freezing is followed by the hypoosmotic insult at thawing. Experiments in our laboratory (Garcia et al. 2012) and others (Pommer et al.…”

“…Removing these cryoprotectants can be especially damaging if the cells experience cell volume increases sufficiently large to induce membrane damage or cell lysis (Morris et al. 2007; Garcia et al. 2012).…”

How Stallion Sperm Age In Vitro? Scenario for Preservation Technologies

“…Recent research in our laboratory has demonstrated that osmotic shock leads to a significant inactivation of the expression of phosphorylated c-Jun-N-terminal kinase pJNK2/3, a mitogenactivated protein kinase that is known to be activated in response to stress [42]. Molecular injuries such as inactivation of key proteins [42] or cleavage of procaspases [43] related to cryopreservation and ultrastructural damage in the sperm and mitochondrial membranes have been described in stallion spermatozoa [44]. Together with this physical effect on sperm membranes, cryopreservation imposes damages of a biochemical nature.…”

Flow Cytometry Probes to Evaluate Stallion Spermatozoa

“…The oxidation of the plasma membrane leads to increased membrane permeability [61] that can be monitored with YoPro-1 [14,24,26,32,62]. Oxidation of DNA can be monitored using antibodies against the oxidized form of guanine.…”