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

Abstract

20 Background 21 The population of stallion spermatozoa surviving thawing, experience, among other changes, 22 compromised mitochondrial functionality and accelerated senescence. It is known that the 23 stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm 24 senescence through increased production of reactive oxygen species. Rosiglitazone has proven 25 to enhance the glycolytic capability of stallion spermatozoa maintained in refrigeration. Objectives27 Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. Material and Methods29 Thawed sperm doses were washed and re-suspended in Tyrodes media, split sampled and 30 supplemented with 0 or 75 M rosiglitazone. After 1 and two hours of incubation, 31 mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Further 32 samples were incubated in presence of the Akt1/2 inhibitor, compound C (AMPK inhibitor) and 33 GW9662 (antagonist of the PPAR receptor). Results35 Rosiglitazone maintained Akt phosphorylated and reduced caspase 3 activation (p<0.01) that 36 was prevented by incubation in presence of the three inhibitors. Rosiglitazone also enhanced 37 mitochondrial functionality (p<0.01). Conclusion39 We provide, for the first time, evidences that the functionality of frozen stallion spermatozoa 40 can be potentially improved after thawing through the activation pro survival pathways, opening 41 new clues to improve current sperm biotechnologies. 42 43 44 45 INTRODUCTION 46 47The stallion spermatozoa can be stored in the liquid state for short periods, or frozen for long-48 term storage. Frozen thawed sperm has numerous advantages, however its wider use is still 49 constrained by a number of weaknesses [1]. Among them, the high stallion-to-stallion 50 variability and the insufficient standardization of the freezing and thawing procedures. While 51 cryopreservation induces mortality to on average 50% of the initial sperm population, the 52 surviving spermatozoa are not completely functional; on the contrary they experience 53 accelerated senescence that requires more intense and costly mare management for insemination 54 to compensate this reduced lifespan. Most of the studies on sperm cryopreservation have aimed 55 to increase the number of spermatozoa surviving the procedure, but studies aiming to improve 56 the quality of the surviving population are scarce. Although the changes induced by 57 cryopreservation have been extensively investigated, mostly focusing on cryopreservation 58 induced necrosis [2][3][4], few studies have addressed the physiology of spermatozoa surviving 59 freezing and thawing. There are not many studies that have tried to develop measures to 60 increase the quality of frozen sperm after thawing, with the exception of procedures to remove 61 dead and damaged spermatozoa from the cryopreserved sample [5][6][7]. The population of 62 spermatozoa surviving freezing and thawing, experience changes recently termed spermptosis 63 [8]. These change...

Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa

“…Overall current knowledge on 417 sperm metabolism, suggests species specific differences and a great metabolic 418 plasticity in the spermatozoa, which are able to adapt their metabolism to the 419 changing environments that they are exposed to on their travels to fertilize the 420 oocyte. Recent research using the strategy of intervention on the metabolic flexibility 421 of stallion spermatozoa seem promising [7,11,26,105,130], both in the development of 422 new extenders for long time liquid storage, and as an intervention for the 423 development of thawing extenders. In this particular aspect, current extenders in 424 use for stallion spermatozoa contain high concentrations of glucose, around 270-300 425 mM, these concentrations are far from being physiological, and may preclude long 426 term preservation of liquid semen.…”