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

Background The population of stallion spermatozoa surviving thawing, experience, among other changes, compromised mitochondrial functionality and accelerated senescence. It is known that the stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has proven to enhance the glycolytic capability of stallion spermatozoa maintained in refrigeration. Objectives Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. Material and Methods Thawed sperm doses were washed and re-suspended in Tyrodes media, split sampled and supplemented with 0 or 75 μM rosiglitazone. After 1 and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Further samples were incubated in presence of the Akt1/2 inhibitor, compound C (AMPK inhibitor) and GW9662 (antagonist of the PPARγ receptor). Results Rosiglitazone maintained Akt phosphorylated and reduced caspase 3 activation (p<0.01) that was prevented by incubation in presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (p<0.01). Conclusion We provide, for the first time, evidences that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation pro survival pathways, opening new clues to improve current sperm biotechnologies.

Oxidation-reduction potential was measured using RedoxSYS Diagnostic system (Englewood 136 CO, USA). This is a novel technology that measures in 4 min the static oxidation reduction 137 potential (sORP), measuring the potential of an electrochemical cell under static conditions, 138 followed by measuring the capacity of oxidation reduction potential (cORP), which is the total 139 amount of readily oxidizable molecules [26]. In brief, 30L of semen was loaded in the sample 140 port of the pre-inserted disposable sensor, the measurement beginning in this moment. After 4 141 minutes. The static oxidative-reduction potential (sORP) is provided in millivolts (mV).

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According to the manufacturer sORP are measured while applying a low oxidizing current 143 (1nA) to the sample. After allowing 1 min and 50 s for equilibration the reader measures twice 144 per second over 10 s the difference in potential between working and reference electrode in mV.

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Subsequently cORP are measured applying a linearly increasing oxidizing current until the 146 charge rapidly changes between and working and reference electrode, indicating that all readily oxidizable molecules are oxidized. The time until the charge changes is used to calculate the 148 number of electrons needed to cause charge changes and is reported in Coulomb (C).        improvements with the treatment (Fig 1 A and B.). Other concentrations of rosiglitazone tested 235 had no effect. of incubation (Fig.2 E), but there was a significant increase in 0 2 in supplemented samples 250 after 2 hours of incubation at 38ºC (Fig. 2 F). When the analysis was performed on a cell by cell 251 basis of the whole sperm population, the heat map generated after the t-SNE analysis showed 252 evident changes indicating that rosiglitazone increased mitochondrial activity (estimated as the 253 number of JC-1 aggregates) as compared with controls (Fig 2 a-b) in the whole sperm 254 population, although in different degrees. Also to identify the major source of 0 2  -, a heat map was generated for superimposing the APC channel over the JC-1/H33342 2D plot, showing 256 that the major production of 0 2 occurred in more active mitochondria (Fig, 2 c).

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Rosiglitazone does not modify the oxidation reduction potential (sORP)

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In order to discriminate if the increased production of superoxide is just caused by intense 261 mitochondrial activity [36], or is a sign of oxidative stress; the oxidation-reduction status of the 262 samples was investigated. No changes were observed in the static oxidation reduction potential 263 (sORP), or in the total antioxidant capacity in supplemented samples (Fig 3).  326 22] and equine spermatozoa. An important aspect of our experiment is that Akt can be maintained phosphorylated for longer periods in thawed sperm, suggesting that thawed sperm, 328 although being exposed to extremely stressful osmotic and temperature conditions during the 329 procedure, still retains ability to delay pro-death pathways. This is an interesting finding since

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In conclusion, thawed stallion spermatozoa can be improved post thaw through mechanisms that 358 maintain Akt phosphorylated, a process that may involve AMPK and PPAR activation.

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Moreover these findings may have practical application to improve the quality of thawed