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 Death1

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.

Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through Akt phosphorylation and reduction of caspase 3

“…This is an interesting finding since opens new clues to develop strategies to improve the quality of frozen sperm after thawing. The prevalence of pro survival or pro-death pathways is probably linked to the redox status of the cell [13]. In this regard Akt can improve mitochondrial function in different cellular models, independently of transcriptional activity [55], supporting the proposing mechanism here described of enhanced mitochondrial function after PPARγ agonist treatment in spermatozoa.…”

“…Cryopreservation causes a dramatic depletion of intracellular thiols in spermatozoa leading to unstable redox status rapidly leading to oxidative stress and cellular senescence [57], this form of sperm demise is characterized by activation of caspase 3. Interestingly, this activation, as described in our experiment can be delayed, thus increasing the lifespan of the spermatozoa.…”

“…This protocol has been developed in the laboratory where the present research was conducted and has been extensively described in previous publications [12, 13, 15, 27]. In brief, spermatozoa (5 x 10 6 /mL) in 1 mL of PBS were stained with CellEvent 2 μM and 0.5 μM Hoechst 33342 and incubated 25’ in the dark at r.t. Then 0.3 μM of Eth-1 was added to each sample and after incubation for 5 additional minutes, then samples were immediately evaluated in the flow cytometer.…”

“…Files were exported as FCS files and analyzed using FlowjoV 10.4.1 Software (Ashland, OR, USA). Unstained, single-stained, and Fluorescence Minus One (FMO) controls were used to determine compensations and positive and negative events, as well as to set regions of interest as described in previous publications from our laboratory [15, 28, 29]…”

“…These changes basically represent an acceleration of the default apoptotic dead that most of the spermatozoa experience after ejaculation [9, 10]. In brief, osmotic shock induces membrane and mitochondrial damage [11], the mitochondrial damage causes impairment of redox regulation, leading to lipid, protein and DNA modifications in the spermatozoa, resulting in decreased motility and viability [8, 12, 13]. Other changes recently described in relation to cryopreservation include increased intracellular Na+ and membrane depolarization due to the compromised functionality of the Na+-K+ATPase pump [14].…”