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 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.
© 2014 Society for Reproduction and Fertility.
Publication Date: 2014-05-21 PubMed ID: 24850868DOI: 10.1530/REP-13-0191Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article presents a study on how a protein called AKT affects the viability and motion of stallion sperm, particularly focusing on how it could prevent certain types of cell death.
Aims of the study
- The primary aim of the study was to investigate the role of a protein called AKT (also known as protein kinase B or PKB or RAC), which is understood to regulate cell survival and prevent cell death (apoptosis), in stallion spermatozoa.
- The researchers sought to understand how AKT influenced the survival of sperm after ejaculation.
Methodologies
- The researchers conducted their experiments on stallion spermatozoa, using a specific inhibitor of AKT called SH5 at different concentrations.
- The spermatozoa were treated with this inhibitor and incubated in a specific medium known as Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol.
- Observations were made over a period of 4 hours, recording AKT’s phosphorylation (a process that activates the AKT).
Observations and findings
- Upon administration of SH5, the research team observed that the percentage of sperm showing AKT phosphorylation diminished, with the inhibition peaking after an hour of treatment.
- They noticed that the sperm cells spontaneously dephosphorylated during the incubation period, causing a lack of difference in AKT phosphorylation between SH5-treated sperm and control after 4 hours.
- The team found that inhibition of AKT led to a decrease in the proportion of motile sperm and sperm velocity.
- Furthermore, they found that AKT inhibition led to reduced membrane integrity, increased membrane permeability, and reduced mitochondrial membrane potential concomitantly with the activation of caspases 3 and 7, enzymes that play essential roles in programmed cell death.
- However, they found no effect on oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation upon inhibiting AKT.
Conclusions
- The study concludes that AKT plays an essential role in maintaining the integrity of the membrane of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7.
- By doing this, it prevents the progression of sperm to an incomplete form of apoptosis, thereby maintaining their viability and motility.
Cite This Article
APA
Gallardo Bolaños JM, Balao da Silva CM, Martín Muñoz P, Morillo Rodríguez A, Plaza Dávila M, Rodríguez-Martínez H, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.
(2014).
Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7.
Reproduction, 148(2), 221-235.
https://doi.org/10.1530/REP-13-0191 Publication
Researcher Affiliations
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
MeSH Terms
- Animals
- Apoptosis
- Blotting, Western
- Caspase 3 / chemistry
- Caspase 3 / metabolism
- Caspase 7 / chemistry
- Caspase 7 / metabolism
- Cell Proliferation
- Cells, Cultured
- Flow Cytometry
- Horses
- Immunoenzyme Techniques
- Male
- Membrane Potential, Mitochondrial
- Mitochondria / metabolism
- Phosphorylation
- Proto-Oncogene Proteins c-akt / antagonists & inhibitors
- Proto-Oncogene Proteins c-akt / metabolism
- Semen Analysis
- Semen Preservation
- Sperm Motility
- Spermatozoa / cytology
- Spermatozoa / metabolism
Citations
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