Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7

Abstract

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 mM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis.Free Spanish abstract A Spanish translation of this abstract is freely available at

Profiling signaling proteins in human spermatozoa: biomarker identification for sperm quality evaluation

“…Recently it was suggested that phosphorylated Akt maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which preserves sperm vitality (34). Here we report for the first time a negative correlation between phosphorylated Akt (pT308) levels and the percentage SDF, which supports a role of Akt in sperm survival.…”

Flow Cytometry Probes to Evaluate Stallion Spermatozoa

“…Detection of more subtle changes in the sperm membrane requires the use of other probes. Fluidity of sperm membranes can be assessed with merocyanine 540 [24], and subtle increases in the permeability of the sperm plasma membrane can be detected with YoPro-1 [25,26]. Yo-Pro-1 is routinely used in the authors' laboratory in combination with PI; H33342 is also incorporated to sort debris.…”

“…Recently, mitotracker dyes [35,36] have become available in multiple colors and provide colorful alternatives to be used in multicolor experiments. Both JC-1 and Mitotracker deep red have been recently used in our laboratory [25]. These probes measure different aspects of mitochondrial function; JC-1 reflects mitochondrial membrane potential, while mitotracker deep red passively diffuses across membranes and binds to thiols in active mitochondria.…”

“…Depending of the presence of pro-survival factors, caspase 3 remains inactive due to the phosphorylation of protein kinase B (PKB or Akt) [25]. If pro-survival factors are lost or oxidative stress reaches a threshold, caspase 3 is activated and sperm senescence and death are triggered [58].…”

Advances in sperm proteomics: best-practise methodology and clinical potential

“…Possibly also new data on the heterogeneity of the individual sperm cells or of sperm cell subpopulations within a sample will be available. Toward this end, it may be useful the use of flow cytometry sorting of the different cells based on specific modifications [105].…”