The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions.
Abstract: Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at -196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an 'autophagy-like' mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.
Publication Date: 2016-03-02 PubMed ID: 26932581DOI: 10.1017/S1751731116000240Google Scholar: Lookup
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- Journal Article
Summary
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This study delves into the impact of sperm storage methods like cooling and freezing on stallion sperm cells. It notably explores the process of autophagy (cell death mechanism), triggered under stressful storage conditions, and the role of a specific protein, LC3, within it. The research also investigates how manipulation of LC3 processing might enhance the quality of preserved semen.
Understanding autophagy and LC3 processing in sperm storage
- The focus of the study is on autophagy, a mechanism involving cell death, which hasn’t been extensively researched in relation to sperm cell storage. The researchers are particularly interested in the processing of a protein called LC3 that plays a key role in autophagy.
- In autophagy, LC3 protein in its form LC3-I is lipidated (combines with fats) to convert into LC3-II. The researchers used the LC3-II/LC3-I ratio as a way to measure autophagy activation.
- The primary objective of the study was to examine if LC3 processing takes place during sperm storage involving cooling, freezing and related stressful environments.
Investigating modulation of LC3 processing
- Apart from investigating whether LC3 processing occurs, the study secondarily aimed to see if this transformation process could be modulated to possibly improve the quality of preserved sperm.
- The modulation of LC3-I to LC3-II was explored with known substances that either activate (STF-62247 and rapamycin) or inhibit (chloroquine and 3-MA) autophagy.
Findings of the study
- The findings showed that the conversion of LC3-I to LC3-II significantly increased during cooling, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress, temperature changes).
- The autophagy activators increased the LC3-II/LC3-I ratio and decreased sperm viability, while the inhibitors of autophagy kept LC3 processing in check and preserved viable cells after 2 hours of incubation at 37°C.
- Long-term storage options like refrigeration and freezing in the presence of autophagy inhibitors resulted in better survival of the sperm cells.
Conclusions and implications
- The study concludes that an ‘autophagy-like’ mechanism may play a role in determining sperm viability during cryopreservation of stallion semen.
- Being able to modulate autophagy during sperm storage could potentially lead to enhanced semen quality and, consequently, better fertility rates.
Cite This Article
APA
Aparicio IM, Martin Muñoz P, Salido GM, Peña FJ, Tapia JA.
(2016).
The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions.
Animal, 10(7), 1182-1191.
https://doi.org/10.1017/S1751731116000240 Publication
Researcher Affiliations
- 1Cell Physiology Research Group,Department of Physiology,University of Extremadura,Caceres 10003,Spain.
- 2Veterinary Teaching Hospital,Laboratory of Spermatology,University of Extremadura,Caceres 10003,Spain.
- 1Cell Physiology Research Group,Department of Physiology,University of Extremadura,Caceres 10003,Spain.
- 2Veterinary Teaching Hospital,Laboratory of Spermatology,University of Extremadura,Caceres 10003,Spain.
- 1Cell Physiology Research Group,Department of Physiology,University of Extremadura,Caceres 10003,Spain.
MeSH Terms
- Animals
- Apoptosis
- Autophagy / physiology
- Cryopreservation / veterinary
- Flow Cytometry
- Gene Expression Regulation / physiology
- Horses / physiology
- Male
- Microtubule-Associated Proteins / genetics
- Microtubule-Associated Proteins / metabolism
- Protein Isoforms
- Semen / physiology
- Semen Analysis / veterinary
- Spermatozoa / physiology
- Stress, Physiological
- Time Factors
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