Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7.
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 μM 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.
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
Juan M
JM
Gallardo Bolaños
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
Carolina M
CM
Balao da Silva
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
Patricia
P
Martín Muñoz
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
Antolín
A
Morillo Rodríguez
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
María
M
Plaza Dávila
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
Heriberto
H
Rodríguez-Martínez
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
Inés M
IM
Aparicio
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
José A
JA
Tapia
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden.
Cristina
C
Ortega Ferrusola
Laboratory of Equine Reproduction and Equine SpermatologyFaculty of Veterinary Medicine, Veterinary Teaching HospitalDepartment of PhysiologyFaculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, 10003 Cáceres, SpainDepartment of Clinical and Experimental MedicineFaculty of Health Sciences, Linköping University, Linköping, Sweden fjuanpvega@unex.es.
Fernando J
FJ
Peña
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3 / Q000737:chemistry* / Q000378:metabolism; D053179:Caspase 7 / Q000737:chemistry* / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D005434:Flow Cytometry; D006736:Horses; D007124:Immunoenzyme Techniques; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055101:Semen Analysis; D012662:Semen Preservation; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism
D051057:Proto-Oncogene Proteins c-akt; D053148:Caspase 3; D053179:Caspase 7