title: Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death.
abstract: Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility, velocity, and membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, and mitochondrial membrane potential) were assessed before and after cryopreservation. Changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as dramatic depletion of intracellular thiols to less than half of the initial values (P < 0.001) postthaw. Interestingly, very high and positive correlations were observed among thiol levels with sperm functionality postthaw: total motility (r = 0.931, P < 0.001), progressive motility (r = 0.904, P < 0.001), and percentage of live spermatozoa without active caspase 3 (r = 0.996, P < 0.001). In contrast, negative correlations were detected between active caspase 3 and thiol content both in living (r = -0.896) and dead (r = -0.940) spermatozoa; additionally, 4-hydroxynonenal levels were negatively correlated with thiol levels (r = -0.856). In conclusion, sperm functionality postthaw correlates with the maintenance of adequate levels of intracellular thiols. The accelerated senescence of thawed spermatozoa is related to oxidative and electrophilic stress induced by increased production of 4-hydroxynoneal in thawed samples once intracellular thiols are depleted.
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Martin Muñoz
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Ortega Ferrusola
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Vizuete
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Plaza Dávila
authors.affiliation: Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
authors.forename: Heriberto
authors.initials: H
authors.lastname: Rodriguez Martinez
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain fjuanpvega@unex.es.
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Peña
mesh_terms: D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism*; D053179:Caspase 7 / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D015925:Cryopreservation; D006736:Horses; D042541:Intracellular Space / Q000378:metabolism; D015227:Lipid Peroxidation / Q000502:physiology; D008297:Male; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D012662:Semen Preservation / Q000379:methods; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D053148:Caspase 3; D053179:Caspase 7; C027576:4-hydroxy-2-nonenal
keywords: ROS; cryopreservation; donkeys; equids (horses; flow cytometry; semen; sperm; sperm senescence; spermatozoa; stallion; thiols; zebras)